Research Article Periductal Mastitis: An Inflammatory Disease Related to Bacterial Infection and Consequent Immune Responses? Lu Liu, 1,2 Fei Zhou, 1 Pin Wang, 1,3 Lixiang Yu, 1 Zhongbing Ma, 1 Yuyang Li, 1 Dezong Gao, 1 Qiang Zhang, 1 Liang Li, 1 and Zhigang Yu 1 1 Department of Breast Surgery, e Second Hospital of Shandong University, Jinan, Shandong 250033, China 2 School of Medicine, Shandong University, Jinan, Shandong 250012, China 3 Department of Breast Surgery, e ird People’s Hospital of Chengdu, Chengdu, Sichuan 610031, China Correspondence should be addressed to Liang Li; [email protected] and Zhigang Yu; [email protected] Received 26 August 2016; Revised 20 November 2016; Accepted 6 December 2016; Published 15 January 2017 Academic Editor: Yona Keisari Copyright © 2017 Lu Liu et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Periductal mastitis (PDM) is a prolonged inflammatory disease, but the cause of PDM is poorly understood. In the present case control study, 87 PDM and 87 healthy controls were enrolled and the results were evaluated to identify the significant risk factors for PDM. To investigate the roles of bacterial infection and critical cytokines expression, 16S rRNA gene sequencing and bacterial culturing were conducted. We also measured the levels of interferon-, interleukin-12A, and interleukin-17A by semiquantitative immunohistochemistry method. In a multivariable logistic regression model, we identified overweight/obesity and late onset of menarche as independent risk factors for PDM. In contrast, age of first birth >27 years had a protective effect. With 16S rRNA gene sequencing, we confirmed bacterial infections were found in all PDM patients, but none of the control patients was positive on the gene expression of 16S rRNA. Our results also demonstrated significant increases of the IFN- and IL-12A expression in PDM, but there was no difference in IL-17A expression in these two groups. Taken together, this study suggests that reproductive factors and overweight/obesity are possible predisposing risk factors for PDM. Bacterial infection and the increased expression of some proinflammatory cytokines are associated with the pathogenesis of this disease. 1. Introduction Periductal mastitis (PDM) is histopathologically defined as a chronic inflammation of the breast, with dilation of the mammary duct, plasma cell infiltration, and abscess formation. A variety of different terms, including mammary ductal ectasia and plasma cell mastitis, have been used for this condition [1, 2]. e use of different terms probably reflects different stages in the disease process. In recent years, the morbidity rate of PDM has risen rapidly [3]. Moreover, in several studies, mammary ductal ectasia has been found to be responsible for 5–25% of all symptomatic breast conditions [4, 5]. e clinical presentations of PDM are not specific and are easily confused with breast carcinoma in imaging manifestations [6, 7]. us far, the etiology of PDM is still unknown. Several factors have been associated with an increased risk for this disease, including smoking, obesity, and diabetes mellitus. However, all of these stated risks are identified based on small case series reports and have not been confirmed epidemiologically [8, 9]. Although PDM is generally seen in those who smoke heavily [9], some other studies also suggest inconsistent findings with this conclusion [10]. In addition, although obesity is considered as a significant risk factor for PDM [11], this conclusion is also just based on case reports, lacking epidemiological evidence. Literature also indicates conflicting information on whether age at first birth, parity, and duration of breastfeeding are potential risk factors for PDM [4, 12]. Moreover, while some studies claim that ethnicity is associated with PDM [13], these studies were limited to a few of geographical areas and races since all data are concluded from small scale studies. Furthermore, as we have known now, the data on the predisposing factors for PDM in Chinese patients is very rare too. Hindawi Mediators of Inflammation Volume 2017, Article ID 5309081, 9 pages https://doi.org/10.1155/2017/5309081
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Research ArticlePeriductal Mastitis An Inflammatory Disease Related toBacterial Infection and Consequent Immune Responses
Lu Liu12 Fei Zhou1 Pin Wang13 Lixiang Yu1 Zhongbing Ma1 Yuyang Li1
Dezong Gao1 Qiang Zhang1 Liang Li1 and Zhigang Yu1
1Department of Breast Surgery The Second Hospital of Shandong University Jinan Shandong 250033 China2School of Medicine Shandong University Jinan Shandong 250012 China3Department of Breast Surgery TheThird Peoplersquos Hospital of Chengdu Chengdu Sichuan 610031 China
Correspondence should be addressed to Liang Li liliang19772004sinacom and Zhigang Yu yzgmedmailcomcn
Received 26 August 2016 Revised 20 November 2016 Accepted 6 December 2016 Published 15 January 2017
Academic Editor Yona Keisari
Copyright copy 2017 Lu Liu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Periductal mastitis (PDM) is a prolonged inflammatory disease but the cause of PDM is poorly understood In the present casecontrol study 87 PDM and 87 healthy controls were enrolled and the results were evaluated to identify the significant risk factorsfor PDM To investigate the roles of bacterial infection and critical cytokines expression 16S rRNA gene sequencing and bacterialculturing were conducted We also measured the levels of interferon-120574 interleukin-12A and interleukin-17A by semiquantitativeimmunohistochemistry method In a multivariable logistic regression model we identified overweightobesity and late onset ofmenarche as independent risk factors for PDM In contrast age of first birth gt27 years had a protective effect With 16S rRNA genesequencing we confirmed bacterial infections were found in all PDM patients but none of the control patients was positive onthe gene expression of 16S rRNA Our results also demonstrated significant increases of the IFN-120574 and IL-12A expression in PDMbut there was no difference in IL-17A expression in these two groups Taken together this study suggests that reproductive factorsand overweightobesity are possible predisposing risk factors for PDM Bacterial infection and the increased expression of someproinflammatory cytokines are associated with the pathogenesis of this disease
1 Introduction
Periductal mastitis (PDM) is histopathologically definedas a chronic inflammation of the breast with dilation ofthe mammary duct plasma cell infiltration and abscessformation A variety of different terms including mammaryductal ectasia and plasma cellmastitis have been used for thiscondition [1 2] The use of different terms probably reflectsdifferent stages in the disease process In recent years themorbidity rate of PDM has risen rapidly [3] Moreover inseveral studies mammary ductal ectasia has been found to beresponsible for 5ndash25 of all symptomatic breast conditions[4 5] The clinical presentations of PDM are not specificand are easily confused with breast carcinoma in imagingmanifestations [6 7]
Thus far the etiology of PDM is still unknown Severalfactors have been associated with an increased risk for this
disease including smoking obesity and diabetes mellitusHowever all of these stated risks are identified based onsmall case series reports and have not been confirmedepidemiologically [8 9] Although PDM is generally seenin those who smoke heavily [9] some other studies alsosuggest inconsistent findings with this conclusion [10] Inaddition although obesity is considered as a significant riskfactor for PDM [11] this conclusion is also just based oncase reports lacking epidemiological evidence Literaturealso indicates conflicting information on whether age at firstbirth parity and duration of breastfeeding are potential riskfactors for PDM [4 12] Moreover while some studies claimthat ethnicity is associated with PDM [13] these studies werelimited to a few of geographical areas and races since all dataare concluded from small scale studies Furthermore as wehave known now the data on the predisposing factors forPDM in Chinese patients is very rare too
HindawiMediators of InflammationVolume 2017 Article ID 5309081 9 pageshttpsdoiorg10115520175309081
2 Mediators of Inflammation
Etiologically the exact role of bacterial infection in PDMhas been debated for many years Bacterial infection wasthought to be a possible etiological factor of PDM Howeverthe current overall data are not conclusive as some studiesalso suggest sterile lesions in PDM patients without invasionof any bacteria [14] Among the studies that demonstrated thepresence of bacterial infection both aerobes and anaerobeshave been found in the subsequent culturing or sequencing ofsamples from PDM patients including Pseudomonas aerug-inosa and Staphylococcus aureus [12] Although the diversityof bacterial strains which infected the lesions of PDM mightbe due to different detecting methods and culture mediaused in different individual study the overall results stillsuggest that a specific pathogenic bacterium may not existfor the infection of PDM [15 16] Therefore we hypothesizethat the main cause of this disease may be the consequentimmune response following bacterial infection instead of theinfection itself However there is little data available nowon the literature about the role of immune response in thisdisease It has been well-established that adaptive immuneresponses play very important roles in the development ofinflammatory diseases especially in those related to bacterialinfection Key players of the adaptive immune response are Thelper (Th) cells [17] Th cells have been divided into threemajor subsets based on their different cytokine expressionprofiles The Th1 subset is characterized by the productionof interferon-120574 (IFN-120574) and IL-12 while the major cytokinesfeatured in the Th2 subset are interleukin-4 (IL-4) and IL-10 respectively TheTh17 subset was described more recentlyand is simply defined by the production of IL-17 As reportedwhile Th1 subset controls intracellular bacterial infectionsthe Th2 subset controls parasitic infections and the Th17subset controls fungal and extracellular bacterial infectionsrespectively [18] Since PDM is a condition related to bacterialinfection it is reasonable to assume that Th1 and Th17 maymediate the possible immune responses following bacterialinfection in PDM
In the present study besides exploring and identifyingthe possible risk factors for PDM we also investigate therelationship between bacterial infection and PDM and thesignificance of the expression of consequent Th1 and Th17cytokines in this disease Our results may help to furtherelucidate the pathogenesis of PDM Furthermore since thereare very few well-designed investigations tending to explorethe risk factors and possible causes for PDM in Chinesepatients now our results here may lay an important groundfor the future epidemiological study on this issue in ChineseHan population
2 Materials and Methods
21 Study Population This was a retrospective nonmatchedcase control study Eighty-seven patients with PDM wererecruited from the Department of Breast Surgery at theSecond Hospital of Shandong University The study wasconducted from January 2011 to March 2015 The inclusioncriteria for patients with PDM were as follows (I) newlydiagnosed and histologically confirmed periductal mastitis(II) Han ethnic group and (III) no evidence or history of
cancer Eighty-seven healthy controls were collected fromthe Physical Examination Center of the Second Hospital ofShandong University from June 2015 to August 2015 Thecriteria for the control group were as follows (I) normalresults on breast physical examinations breast ultrasoundscans andor mammographic screening and without anypossible benign breast diseases (fibroadenoma PDM breastcyst intraductal papillomas etc) (II) Han ethnic groupand (III) no evidence or history of breast cancer All eligiblesubjects were women in reproductive age and willing toparticipate in the study voluntarily All procedures performedhere involving human participants were approved by the Sec-ond Hospital of Shandong University Research Committee
22 Pathological Diagnosis Vacuum-assisted incisional orexcisional biopsy was performed based on the clinical find-ings at the time of admission Pathological results werereviewed by two pathologists PDM patients were includedin this study only if they had confirmed pathologicalchanges All patients with other possible causes of mammaryinflammation were ruled out such as breast tuberculosisfat necrosis and granulomatous lobular mastitis as well asinflammation due to lactation or pregnancy
23 Data Collection Possible etiologic factors were writtendown at the time of admission including sociodemographicstatus smoking status alcohol consumption reproductivehistory and medical history In addition the followinginformation was also obtained patient age marital statusmenopause status height weight body mass index (BMI)active and passive smoking alcohol abuse heart and otherchronic disease history history of allergies history of benignbreast disease galactostasis age at menarche age of firstchild birth parity miscarriage history and duration ofbreastfeeding For individual patient clinicopathologic char-acteristics were also collected including major complaint onpresentation size of lesion and histology findings
24 Bacteria Detection For the specimen collection patientswho received any antibiotics before admission and had sinusor fistula or abscess incision drainage were ruled out from thestudy Finally 33 specimens from 31 patients including 21 pussamples and 12 tissue samples were collected in this studyNormal tissues adjacent to benign breast pathology (includ-ing fibroadenoma mastalgia and intraductal papilloma ofthe breast) were used as control specimens (119899 = 12) Breasttissue samples were collected under aseptic conditions at thetime of operation Tissues were divided into two sectionswith one section frozen immediately in liquid nitrogen andanother section sent to the laboratory for bacterial cultureFresh breast specimens were homogenized and incubatedonto Columbia blood agar (Babio Biotech Co Ltd JinanChina) and anaerobic blood culture bottles (Biotech CoLtd) The agar plates were incubated at 37∘C and humidifiedincubatorwas suppliedwith 10carbondioxideThe coloniesformed in the plates were picked and analyzed using aMicroScan Walkaway-96 system (Dade-Behring MicroScanSacramento CA) Gram staining and a stain for acid-fastbacilli were conducted for all specimens All samples were
Mediators of Inflammation 3
also analyzed by 16S rRNA gene sequencing Genomic DNAwas prepared using the bacterial DNAextraction kit (TiangenBiotech Co Ltd Beijing China) according to the manu-facturerrsquos recommendations The hypervariable regions V1ndashV3 of the 16S rRNA gene were PCR-amplified using uni-versal primers (V3F 51015840-CCAGACTCCTACGGGAGGCAG-31015840 V3R 51015840-CGTATTACCGCGGCTGCTG-31015840 27F 51015840-AGAGTT TGA TCMTGGCTC AG-31015840 519R 51015840-GWA TTA CCGCGG CKG CTG-31015840) (Sangon Biotech Co Ltd ShanghaiChina) [19] The 203 and 492 bp PCR products were purifiedafter agarose gel electrophoresis and sequences were deter-mined using the ABI PRISM 3730 DNA sequencer (PerkinElmer Inc Waltham MA USA) Nucleotide sequences wereanalyzed using the National Center for Biotechnology Infor-mation BLAST software (httpswwwncbinlmnihgov)
25 Immunohistochemistry For the patients who were testedfor bacterial infection tissue samples which are mentionedabove were also embedded in paraffin (119899 = 31) forimmunohistochemistry study of proinflammatory cytokinesNormal tissues adjacent to benign breast pathology (includ-ing fibroadenoma mastalgia and intraductal papilloma ofbreast) were used as control specimens (119899 = 22) Immuno-histochemistry was performed on 4 120583m thick sections Andthen the streptavidin-peroxidase-biotin (SP) immunohisto-chemical method was performed to detect the expression ofIFN-120574 IL-12A and IL-17A Briefly after deparaffinization andrehydration the human tissue sections were incubated in 3hydrogen peroxide in methanol to quench the endogenousperoxidase activity followed by incubation with normalgoat serum to block nonspecific binding Samples werethen incubated overnight at 4∘C with rabbit anti-IFN-120574(1 500 ab9657 Abcam Cambridge MA USA) anti-IL-12A(1 500 ab131039 Abcam) or anti-IL-17A (1 500 ab9565Abcam)The secondary antibody was from the SP reagent kit(PV9000 Zhongshan Biotech Co Ltd Beijing China) Thecolor reaction was performed with 331015840-Diaminobenzidine(Zhongshan Biotech Co Ltd) counterstained with hema-toxylin dehydrated treated with xylene and then mountedFor negative controls the antibodies were replaced withPBS To determine the ratio of positive cells for these threecytokines 5 fields (times400) were randomly examined per slideand scored by two pathologists who are blinded to thepatientsrsquo informationThe levels of IFN-120574 IL-12A and IL-17Aexpression were semiquantitatively expressed using a visualgrading system based on the extent of staining (percentage ofpositive cells graded on scale from 0 to 3 0 none 1 1ndash30 231ndash60 3 gt60) and the intensity of staining (graded on ascale of 0ndash3 0 none 1 weak staining 2 moderate staining 3strong staining) The combination of extent (E) and intensity(I) of staining was obtained by the product of E and I with EIvarying from 0 to 9 for each spot Low expression was definedas EI le 3 and others were considered high expression [20]
26 Statistical Analysis Statistical analysis was performedwith SPSS 170 software (SPSS Inc Chicago IL USA)The results were reported as counts (percentage) for thecategorical variables and mean plusmn standard deviation forthe continuous variables A two-sample Studentrsquos 119905-test was
Table 1 Demographic characteristics in case and control groups
Variable Cases (119899 = 87) Controls (119899 = 87) lowast119875
MarriageYes 82 (9425) 86 (9885) 0211No 5 (575) 1 (115)
MenopauseYes 6 (690) 0 (0) 0029No 81 (9310) 87 (100)
HypertensionYes 5 (575) 0 (0) 0059No 82 (9425) 87 (100)
Diabetes mellitusYes 5 (575) 0 (0) 0059No 82 (9425) 87 (100)
Heart diseasesYes 4 (460) 0 (0) 0121No 83 (9540) 87 (100)
Passive smokingYes 23 (4694) 16 (3556) 0263No 26 (5306) 29 (6444)
History of allergiesYes 9 (1034) 7 (805) 0670No 78 (8966) 80 (9195)
Note Data are number () of patientslowast119875 values were determined with the chi-square test
used to compare the difference between the means of thecontinuous variables Chi-square test (or Fisher exact test)was used to compare the differences between the proportionsif appropriate Risk factors for PDM were further assessedusing both univariate and multivariable (unconditional)logistic regression models The continuous variables wereevaluated according to the clinically relevant categories Thecategories for BMI were based on the criteria of beingoverweight and obese for Chinese individuals (overweight24ndash279 kgm2 obese ge28 kgm2) [21] The categories forother continuous variables were on the basis of references(menarche age le 14 years gt14 years times of parity le 1 gt1age at birth of first child le 27 years gt27 years duration ofbreastfeeding le 12 months gt12 months) [22 23] Odds ratio(OR) with 95 confidence interval (95 CI) was used toestimate the association between risk factors and exposureThe significance level was set to 119875 lt 005 (two-tailed)
3 Results
31 Study Population Characteristics To investigate the riskfactors for PDM 87 patients and 87 healthy control subjectswere enrolled in this study The characteristics of patientsand controls are summarized in Table 1 The median ageof patients at presentation was 34 (range 20ndash62) years andthe median age of controls was 34 (range 26ndash45) yearsThe median age did not differ significantly between the two
4 Mediators of Inflammation
Table 2 Clinical characteristics and presentations of patients withPDM
Characteristic ValueMean size of mass (cm) 391 plusmn 257
Affected sidesRight 34 (3908)Left 45 (5172)Bilateral 8 (920)
Quadrant(s)Upper-outer 20 (2299)Lower-outer 8 (920)Upper-inner 9 (1034)Lower-inner 9 (1034)Two or more quadrants 15 (1724)Subareolar region 26 (2989)
Clinical presentationMass without pain 63 (7241)Mass with pain 15 (1724)Erythroswelling 31 (3563)Nipple discharge 6 (690)Sinus 12 (1379)Nipple retraction 24 (2759)
Biopsy methodsIncisional or excisional biopsy 24 (2759)Vacuum-assisted biopsy 63 (7241)
Note Data are number () of patients unless otherwise indicated
groups (119875 = 0134) The majority of subjects in this studywere married There were no statistically significant differ-ences between these two groups in terms of for the history ofhypertension type 2 diabetes mellitus heart disease or anyhistory of allergy Neither alcohol abuse nor active smokingwas reported in the case and control groups There was notany form of breast cancer in these patients of PDM confirmedby pathologic examination
Most patients had unilateral breast disease with only 8patients exhibiting bilateral symptoms Breast mass with orwithout pain was the most common complaint The clinicalpresentations were summarized in Table 2 The medianduration between last pregnancy and onset of PDM was 5years (range 1ndash35 years) which implied that that reproductiveage might relate to PDM to some degree (Figure 1)
32 Univariate and Multivariate Analysis for the Risk Factorsfor PDM To examine the relationship between multiplefactors and PDM a univariate analysis was performed Theresults were shown in Table 3 An analysis of the full datasetindicated that overweightobesity (OR 306 95 CI 153ndash613 119875 = 0002) a late onset of menarche (OR 431 95CI 195ndash950 119875 lt 0001) having more than one child(OR 275 95 CI 118ndash641 119875 = 0019) history of benignbreast disease (OR 454 95 CI 107ndash1014 119875 = 0039)and nipple retraction (OR 3276 95 CI 432ndash24861 119875 =0001) were statistically significant risk factors for PDM Lateage at first birth (OR 022 95 CI 009ndash057 119875 = 0002)
0
5
10
15
20
25
30
20 25 30 35 40 45 50 55 60 62
Num
ber o
f cas
es
Age (year)
Age of onsetAge at birth of last child
Figure 1 The distribution of age of onset and age at birth of firstchildThemedian age of onset was 34 years while the median age atbirth of last child was 28 years It seemed that there was a correlationbetween these two items
had a protective effect for the disease When stratified at12 months duration of breastfeeding was not significantlyrelated to PDM
All factors in the univariate analysis with 119875 values lt 02were regarded as candidate predictors for a logistic regressionmodel and a backward variable selection process was used Inthe multivariable model (Table 4) overweightobesity (OR136 95CI 108ndash170119875 = 0008) and late onset ofmenarche(OR 241 95 CI 138ndash421 119875 = 0002) were independentpredictors of PDM In contrast late age at first birth had aprotective effect against PDM (OR 018 95 CI 003ndash098119875 = 0048)
33 Microorganism Infection in PDM To investigate the roleof bacterial infection in the development of PDM multiplemicroorganismdetectionmethodwas employed in this studyAcid-fast bacilli staining was negative in both case andcontrol groups However colony formation was found ononly one pus specimen plate in bacterial culturing studyand two distinct bacterial strains were further identified asBrevibacterium flavum and a rare Gram-positive bacteriumBacillus cereuswith 16S rRNAgene sequencingTherewas noanaerobic bacterial growth from any sample
Furthermore with sequencing of the 16S rRNA gene wedemonstrated that one or more species of bacteria could bedetected in all of the patient specimens but all the controltissues from the benign breast disease were negative on thistest In the case group a mixture of different bacterial infec-tion was most often observed (3871) followed by singlebacterial infection of Pseudomonas spp (2903) (Table 5)Enterococcus faecium Corynebacterium kroppenstedtii Bacil-lus firmus Sporosarcina and Staphylococcus aureus were alsofound in some samples This result validated the assumptionthat bacterial infection might be an etiological factor forPDM
34 Expression of Proinflammatory Cytokines in PDM Asbacteria were found in the lesions of PDM we thentest whether the critical proinflammatory cytokines from
Mediators of Inflammation 5
Table 3 Univariate analysis of selected risk factors for PDMlowast
Variable Cases (119899 = 87) Controls (119899 = 87) OR (95 CI) 119875
Age (years)lt35 48 (5517) 56 (6437) 10 (Ref) 0217ge35 39 (4483) 31 (3563) 147 (080ndash270)
BMI (kgm2)lt24 34 (3908) 65 (7471) 10 (Ref)
0002ge24 34 (3908) 20 (2299) 325 (163ndash648)Unknown 19 (2184) 2 (230)
Age at menarche (years)le14 57 (6552) 74 (8506) 10 (Ref)
0001gt14 30 (3448) 10 (1149) 390 (176ndash862)Unknown 0 (000) 3 (345)
Parityle1 64 (7356) 72 (8276) 10 (Ref)
0019gt1 22 (2529) 9 (1034) 275 (118ndash641)Unknown 1 (115) 6 (690)
MiscarriagesNo 23 (2644) 25 (2874) 10 (Ref)
0470Yes 25 (2874) 36 (4138) 076 (035ndash162)Unknown 39 (4482) 26 (2988)
Age at birth of first child (years)le27 34 (3908) 26 (2989) 10 (Ref)
0001gt27 8 (920) 29 (3333) 021 (008ndash054)Unknown 45 (5172) 32 (3678)
Duration of breastfeeding (months)le12 34 (3908) 42 (4828) 10 (Ref)
0688gt12 36 (4138) 39 (4483) 114 (060ndash216)Unknown 17 (1954) 6 (689)
History of benign breast diseaseYes 6 (690) 3 (345) 454 (107ndash1914)
0039No 37 (4253) 84 (9555) 10 (Ref)Unknown 44 (5057) 0 (000)
GalactostasisYes 23 (2644) 32 (3678) 086 (044ndash170)
0670No 40 (4598) 48 (5517) 10 (Ref)Unknown 24 (2758) 7 (805)
Nipple retractionYes 24 (2759) 1 (115) 3276 (432ndash24861) 0001No 63 (7241) 86 (9885) 10 (Ref)
Note Data are number () of patients BMI body mass index calculated as weight in kilograms divided by the square of height in meters OR odd ratio CIconfidence intervallowastUsing binary logistic regression
Table 4 Multivariate logistic regression analysis of the associations between various factors and PDM
Variable B SE Wald OR 95 CI 119875
Overweightobesity 031 012 700 136 108ndash170 0008Age at first birth minus170 086 392 018 003ndash098 0048Age at menarche 088 029 953 241 138ndash421 0002
6 Mediators of Inflammation
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 2 Representative illustrations of the expression of cytokines in PDM and normal breast tissues (a) Low-powermagnification of PDM(hematoxylin and eosin times40) (b) Low expression of IFN-120574 (IHC times200) (c) High expression of IFN-120574 (IHC times200) (d) Low expression ofIL-12A (IHC times100) (e) High expression of IFN-120574 (IHC times200) (f) Low expression of IL-17A (IHC times100) (g) High expression of IL-17A(IHC times200) (h) The expression of inflammatory cytokines in normal breast ductal epithelium and stromal cells (IHC times200)
Table 5 Multiple bacteria could be detected in PDM patients
Bacteria 119873 () (119899 = 31)Mixture of different bacteria 12 (3871)Pseudomonas
Pseudomonas aeruginosa 5 (1613)Uncultivated Pseudomonas 2 (645)Pseudomonas delhiensis 1 (323)Pseudomonas otitidis 1 (323)
Enterococcus faecium 2 (645)Corynebacterium kroppenstedtii 1 (323)Bacillus firmus 1 (323)Sporosarcina 1 (323)Staphylococcus aureus 1 (323)Uncultured bacterium clone 4 (1290)
following immune responses IFN-120574 IL-12A and IL-17Awere involved in the disease progressionwith an IHCmethod(Figure 2) After staining and taking photographs a scoringevaluation of the density for each cytokine in this IHCphotographs was performed and data were summarized inTable 6 As shown in Table 7 the expressions of IFN-120574 and IL-12A in stromal inflammatory cells of the breast were increasedin PDM compared to normal breast tissues However therewere no significant differences in the expression of IL-17Abetween these two groups
To investigate the cytokines expression profiles at differ-ent stages of PDM the expressions of IFN-120574 IL-12A and IL-17A were then analyzed by grouping with the presence orabsence of abscess Fifteen patients with abscess formationin their lesions and 16 patients without abscess formationwere included in this study As shown in the Figure 3higher expression levels of IFN-120574 and IL-17A were found
Table 6 The expressions of IFN-120574 IL-12A and IL-17A in PDMcompared with normal breast tissues
Score IFN-120574 IL-12A IL-17ACase Control Case Control Case Control
0 2 1 0 2 1 21 2 3 0 3 10 12 5 13 11 12 8 103 1 2 6 2 1 44 8 3 7 1 10 36 9 0 4 1 0 28 0 0 0 0 0 09 4 0 2 1 0 0Absent 0 0 1 0 1 0Samples were lost in the process of IHC
Table 7 The correlationship of IFN-120574 IL-12A and IL-17A expres-sion in PDM compared with normal breast tissues
Cytokines Case Control 119875lowast
Low expression of IFN-120574 10 19lt0001
High expression of IFN-120574 21 3Low expression of IL-12A 13 19 0022High expression of IL-12A 17 3Low expression of IL-17A 20 17 0404High expression of IL-17A 10 5lowast119875 values were determined with the chi-square test
Low expression was defined as the total score le 3 and others were definedas high expression
in stromal inflammatory cells from PDM patients withabscess formation than those without abscess formationThese results suggest that proinflammatory cytokines may
Mediators of Inflammation 7
0
5
10
15
Cases with abscess formationCases without abscess formation
Num
ber o
f cas
es
lowast lowast lowast
IFN
-120574(minus
)
IFN
-120574(+
)
IL-17
A(minus
)
IL-17
A(+
)
IL-12
A(minus
)
IL-12
A(+
)
Figure 3 Production of IFN-120574 IL-12A and IL-17A in cases withor without abscess formation (minus) low expression of cytokines(+) high expression of cytokines Fifteen patients had lesions withabscess formation while 16 patients did not have them More caseswith high expressions of IFN-120574 and IL-17A in stromal inflammatorycells could be found in patients with abscess formation lowast119875 gt 005
play an important role in the progression of the diseaseof PDM especially the IFN-120574 and IL-12A As mentionedabove these two cytokines are key factors involved in theTh1mediated adaptive immune responses following bacterialinfection
4 Discussion
Smoking obesity diabetes mellitus and reproductive factorshave been considered as significant risk factors for PDM inprevious studies [10 24] The results of the present studyconfirmed that overweightobesity and the late onset ofmenarche were independent risk factors for PDM whilelate age at first birth had a protective effect In additionour results also suggested that nipple retraction was relatedto PDM Bacterial infection could be found in all samplesof PDM patients by 16S rRNA gene sequencing but not inthe samples from the control subjects Among the infectedsamples in PDM patients a mixture of different bacterialstrains was the most common condition which could beseen and then followed by singlePseudomonas spp infectionIFN-120574 and IL-12A were upregulated in PDM compared tonormal breast tissues but the expression of IL-17A was notsignificantly different from control samples Moreover thereare increased expression pattern for both IFN-120574 and IL-17Ain PDM which suggested a following immune response afterbacterial infection may contribute the deterioration of PDM
The incidence of PDM is increasing rapidly during thepast decades Indeed in our previous investigation the detec-tion rate of mammary ductal ectasia was 024 (14761102)[25] However the etiology of PDM is unclear yet mostly dueto lack of conclusive evidences fromprevious study Although
some studies suggest smoking is a risk factor for PDM [11 12]smoking was not found to be closely related to PDM in thepresent study One of the plausible reasons might be the lowprevalence of smoking among Chinese women [26]
Obesity is related to low-grade chronic inflammationwhich can disrupt immune function [27 28] Furthermoreobesity can directly influence local mammary estrogen andinflammation [29] Gollapalli et al [8] reported that obesitywas a risk factor for breast abscess Bharat et al [13] found thathigher BMI values were also associated with nonpuerperalabscessThey also reported similar results in cases of granulo-matous lobular mastitis In PDM however they did not finda significant effect of obesity on patients when comparedwithhealth controls [30] In our study data demonstrated obesityis another risk factor for PDM Obesity may directly disruptthe local immune functioning of the breast and exacerbate thedevelopment of PDM
The relationship between reproductive factors and PDMis very interesting Inconsistent with the finding reportedpreviously from literature in which parity was not found tobe associated with the risk for duct ectasia [31] our resultssuggest that reproductive factors were related to PDM Wealso found late onset of menarche was a significant risk factorfor PDM while increased age at first birth was a protec-tive factor The changes of serum prolactin and lactationin patients may be responsible for the above associationThe exact mechanism underlying this relationship is stillunknown future investigation focusing on theses hormonesis warranted
The relationship between nipple retraction and PDM hasbeen well-documented [32] Nipple retraction could inducebreast deformation and influence the process of lactationFurthermore it also can obstruct drainage and lead toan accumulation of massive substances in ducts which istermed mastitis [33] In this study we confirmed that nippleretraction was a risk factor for PDM The prevalence rateof nipple retraction is approximately 326 according toprevious report [34] But over one quarter of PDM patientsexperienced nipple retraction in our study
Bacterial infection is considered to be another possibleetiology of PDM although it has been a long-standing con-troversial problem Al Benwan et al [15] found that S aureuswas the predominant pathogen followed by Bacteroides sppAnaerobic strep and P aeruginosaHowever the relationshipbetween bacteria and PDM was not confirmed in otherreport [31] Here we provided more evidence to confirm forthis assumption Inconsistent with previous report [35] wedid not find that either S aureus was the most commonbacterium in PDM or anaerobic bacteria was associated withthis disease based on our current data Interestingly thebacteria identified here were those microorganisms whichare often related to community-acquired infection includingPseudomonas spp and S aureus Collectively these findingsuggests that bacterial infection may only be acting as aninduction factor and the immune system which are follow-ing the bacterial infection might play a major role in thedevelopment of PDM However more study is needed for thefuture to determine the common route in which the bacterialinfection and the following immune response converge
8 Mediators of Inflammation
IFN-120574 and IL-12A are characteristic Th1 cytokines [36]Th1 cells are important for the eradication of invadingpathogens including bacteria parasites yeast and viruses[37] In this study the expressions of IFN-120574 and IL-12A wereupregulated in breast stromal inflammatory cells of PDMpatients Recently a lineage of CD4+ T cells producing IL-17A was described and accordingly named as Th17 cellsIL-17A has been shown to be important for host defenseagainst pathogens [38] However in our study there was nosignificant difference in IL-17A expression between the twogroups As IFN-120574 and IL-12A were detected in PDM tissuesamples there is a rationale to conclude that theTh1 immuneresponse plays a role in this diseaseThemechanism bywhichthese cytokines contribute to the course of disease is stillunknown and needs further investigation
The strengths of the present study include the following(a) a relatively large population of patients was included (b)the assessment of tissue was done by two histopathologistsexperienced in breast inflammation However our study alsohad several limitations First this was a nonmatched casecontrol study and there might be some uncontrolled biaseswhich may influence the credibility of the results Secondthere was an issue with missing data especially the dataof reproductive factors which may also lead to the bias ofresults Finally we have not investigated the mechanism bywhich IFN-120574 and IL-12A participated in the course of thedisease
5 Conclusions
The results of the present study indicated that over-weightobesity and later onset of menarche are independentrisk factors for PDM In contrast age of first birth gt27years is a protective factor for PDM The importance ofthese reproductive factors to the risk of PDM has notbeen previously reported and will provide important novelinformation to broaden the avenue of investigating theetiology of PDM in the future We also confirm that bacterialinfection with a mixture of different bacterial species is aclosely associated with the pathogenesis of PDM Moreoverproinflammatory cytokines IFN-120574 and IL-12A were alsoshown to be associated with PDM which indicates that theTh1 immune response may closely relate to this diseaseTherefore PDM could be an inflammatory disease related tobacterial infection and consequent immune responses withincreased cytokines As far as we know this is first reportabout the possible immune responses following bacterialinfection which is involved in development of PDM diseaseand more research is needed for the future to furtherinvestigate the pathogenesis of PDM on the mechanism bywhich bacterial infection and consequent immune responsesaffect the progression of the disease
Ethical Approval
All procedures performed here involving human participantswere in accordance with the ethical standards of the SecondHospital of Shandong University Research Committee and
with the 1964Helsinki Declaration its later amendments andcomparable ethical standards
Consent
Informed consent was obtained from all participants includ-ed in the study
Competing Interests
All authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Dr Zhigang Yu and Dr Liang Li have equal contribution tothis article
Acknowledgments
Theauthors thank the patients and staffs at theDepartment ofBreast Surgery of SecondHospital of ShandongUniversity fortheir collaboration and supportThis study was funded by theYouth Fund of the Second Hospital of Shandong University(Y2014010030) Fei Zhou has received research grants fromthe Second Hospital of Shandong University
References
[1] K Ramalingam A Srivastava S Vuthaluru A Dhar and RChaudhry ldquoDuct Ectasia and periductal mastitis in Indianwomenrdquo Indian Journal of Surgery vol 77 supplement 3 pp957ndash962 2015
[2] J M Dixon ldquoPeriductal mastitisduct ectasiardquoWorld Journal ofSurgery vol 13 no 6 pp 715ndash720 1989
[3] X Zhang Y Lin Q Sun and H Huang ldquoDermo-glandularflap for treatment of recurrent periductal mastitisrdquo Journal ofSurgical Research vol 193 no 2 pp 738ndash744 2015
[4] R M S Rahal R De Freitas-Junior and R R Paulinelli ldquoRiskfactors for duct ectasiardquo Breast Journal vol 11 no 4 pp 262ndash265 2005
[5] R Mansel D Webster and H Sweetland Hughes Mansel ampWebsterrsquos Benign Disorders and Diseases of the Breast (ThirdEdition) Saunders Philadelphia Pa USA 2009
[6] M H Lequin J van Spengler R van Pel C van Eijck and HvanOverhagen ldquoMammographic and sonographic spectrum ofnon-puerperal mastitisrdquo European Journal of Radiology vol 21no 2 pp 138ndash142 1995
[7] R Tomczak A Rieber H Zeitler N Rilinger R Kreienbergand H J Brambs ldquoThe value of MR-mammography at 15 teslain the differential diagnosis of non-puerperal mastitis andinflammatory breast carcinomardquo RoFo Fortschritte auf demGebiete der Rontgenstrahlen und der Nuklearmedizin vol 165no 2 pp 148ndash151 1996
[8] V Gollapalli J Liao A Dudakovic S L Sugg C E H Scott-Conner and R J Weigel ldquoRisk factors for development andrecurrence of primary breast abscessesrdquo Journal of the AmericanCollege of Surgeons vol 211 no 1 pp 41ndash48 2010
Mediators of Inflammation 9
[9] R Risager and N Bentzon ldquoSmoking and increased risk ofmastitisrdquo Ugeskrift for Laeger vol 172 no 33 pp 2218ndash22212010
[10] H N Oltean A S Soliman O S Omar et al ldquoRisk factors forchronic mastitis in Morocco and Egyptrdquo International Journalof Inflammation vol 2013 Article ID 184921 10 pages 2013
[11] P Schafer C Furrer and B Mermillod ldquoAn association ofcigarette smoking with recurrent subareolar breast abscessrdquoInternational Journal of Epidemiology vol 17 no 4 pp 810ndash8131988
[12] V Sakka ldquoNonpuerperal breast infection epidemiology andpredictors for recurrencesrdquo Clinical Microbiology and Infectionvol 18 p 237 2012
[13] A Bharat F Gao R L Aft W E Gillanders T J Eberlein andJ A Margenthaler ldquoPredictors of primary breast abscesses andrecurrencerdquoWorld Journal of Surgery vol 33 no 12 pp 2582ndash2586 2009
[14] N J Bundred J M J Dixon A B Lumsden et al ldquoAre thelesions of duct ectasia sterilerdquo British Journal of Surgery vol72 no 10 pp 844ndash845 1985
[15] K Al Benwan A Al Mulla and V O Rotimi ldquoA study of themicrobiology of breast abscess in a teaching hospital in KuwaitrdquoMedical Principles and Practice vol 20 no 5 pp 422ndash426 2011
[16] P-L Giacalone G Rathat S Fournet and C Rouleau ldquoSurgicaltreatment of recurring subareolar abscess using oncoplastictechniquesrdquo Journal of Visceral Surgery vol 147 no 6 pp e389ndashe394 2010
[17] A Geremia P Biancheri P Allan G R Corazza and ADi Sabatino ldquoInnate and adaptive immunity in inflammatorybowel diseaserdquo Autoimmunity Reviews vol 13 no 1 pp 3ndash102014
[18] P Miossec and J K Kolls ldquoTargeting IL-17 and TH17 cells inchronic inflammationrdquo Nature Reviews Drug Discovery vol 11no 10 pp 763ndash776 2012
[19] Y Khosravi Y Dieye B H Poh et al ldquoCulturable bacterialmicrobiota of the stomach of helicobacter pylori positive andnegative gastric disease patientsrdquo The Scientific World Journalvol 2014 Article ID 610421 10 pages 2014
[20] R Marechal P Demetter N Nagy et al ldquoHigh expressionof CXCR4 may predict poor survival in resected pancreaticadenocarcinomardquo British Journal of Cancer vol 100 no 9 pp1444ndash1451 2009
[21] M Yao Y Wu Q Fang L Sun T Li and H Qiao ldquoAssociationof ADIPOQ variants with type 2 diabetes mellitus susceptibilityin ethnic han Chinese from Northeast Chinardquo Journal ofDiabetes Investigation vol 7 no 6 pp 853ndash859 2016
[22] Q Zhang L-Y Liu FWang KMu and Z-G Yu ldquoThe changesin female physical and childbearing characteristics in china andpotential association with risk of breast cancerrdquo BMC PublicHealth vol 12 article 368 2012
[23] J Kotsopoulos J Lubinski L Salmena et al ldquoBreastfeedingand the risk of breast cancer in BRCA1 and BRCA2 mutationcarriersrdquo Breast Cancer Research vol 14 no 2 article R42 2012
[24] Y Dong J-J Yu Y Shibahara et al ldquoIntercellular adhesionmolecule 12 and E-selectin in plasma cell mastitis immuno-histochemical study of 35 casesrdquo Human Pathology vol 45 no3 pp 606ndash610 2014
[25] Y Y Li J J Du Q Liu and Z G Yu ldquoScreening for breastdiseases among 61102 women in Shandong Provincerdquo Journalof Shandong University vol 49 no 8 pp 157ndash160 2011
[26] Y-M Xu H-H Chen F Li et al ldquoPrevalence and correlatesof cigarette smoking among Chinese schizophrenia inpatientsreceiving antipsychotic mono-therapyrdquo PLOS ONE vol 9 no2 Article ID e88478 2014
[27] F M Wensveen S Valentic M Sestan T Turk Wensveen andB Polic ldquoThe ldquoBig Bangrdquo in obese fat events initiating obesity-induced adipose tissue inflammationrdquo European Journal ofImmunology vol 45 no 9 pp 2446ndash2456 2015
[28] Y B Shaik-Dasthagirisaheb A Kantarci and F C Gibson IIIldquoImmune response of macrophages from young and aged miceto the oral pathogenic bacterium Porphyromonas gingivalisrdquoImmunity amp Ageing vol 7 article 15 2010
[29] K A Brown ldquoImpact of obesity on mammary gland inflamma-tion and local estrogen productionrdquo Journal ofMammary GlandBiology and Neoplasia vol 19 no 2 pp 183ndash189 2014
[30] B Al-Khaffaf F Knox and N J Bundred ldquoIdiopathic granulo-matous mastitis a 25-year experiencerdquo Journal of the AmericanCollege of Surgeons vol 206 no 2 pp 269ndash273 2008
[31] R M S Rahal R F Junior C Reis F C Pimenta J C ANetto and R R Paulinelli ldquoPrevalence of bacteria in the nippledischarge of patients with duct ectasiardquo International Journal ofClinical Practice vol 59 no 9 pp 1045ndash1050 2005
[32] M N Hartley J Stewart and E A Benson ldquoSubareolardissection for duct ectasia and periareolar sepsisrdquo The BritishJournal of Surgery vol 78 no 10 pp 1187ndash1188 1991
[33] J Ming G Meng Q Yuan et al ldquoClinical characteristics andsurgical modality of plasma cell mastitis analysis of 91 casesrdquoThe American Surgeon vol 79 no 1 pp 54ndash60 2013
[34] H S Park C H Yoon and H J Kim ldquoThe prevalence ofcongenital inverted nipplerdquoAesthetic Plastic Surgery vol 23 no2 pp 144ndash146 1999
[35] F N L Versluijs-Ossewaarde R M H Roumen and R J AGoris ldquoSubareolar breast abscesses characteristics and resultsof surgical treatmentrdquoThe Breast Journal vol 11 no 3 pp 179ndash182 2005
[36] Y Luo H Yamada D P Evanoff and X Chen ldquoRole ofTh1-stimulating cytokines in bacillus Calmette-Guerin (BCG)-inducedmacrophage cytotoxicity against mouse bladder cancerMBT-2 cellsrdquo Clinical and Experimental Immunology vol 146no 1 pp 181ndash188 2006
[37] K J Rautajoki M K Kylaniemi S K Raghav K Rao and RLahesmaa ldquoAn insight into molecular mechanisms of human Thelper cell differentiationrdquoAnnals of Medicine vol 40 no 5 pp322ndash335 2008
[38] B Stockinger and M Veldhoen ldquoDifferentiation and functionof Th17 T cellsrdquo Current Opinion in Immunology vol 19 no 3pp 281ndash286 2007
Submit your manuscripts athttpswwwhindawicom
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Journal of
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Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
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Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Mediators of Inflammation
Etiologically the exact role of bacterial infection in PDMhas been debated for many years Bacterial infection wasthought to be a possible etiological factor of PDM Howeverthe current overall data are not conclusive as some studiesalso suggest sterile lesions in PDM patients without invasionof any bacteria [14] Among the studies that demonstrated thepresence of bacterial infection both aerobes and anaerobeshave been found in the subsequent culturing or sequencing ofsamples from PDM patients including Pseudomonas aerug-inosa and Staphylococcus aureus [12] Although the diversityof bacterial strains which infected the lesions of PDM mightbe due to different detecting methods and culture mediaused in different individual study the overall results stillsuggest that a specific pathogenic bacterium may not existfor the infection of PDM [15 16] Therefore we hypothesizethat the main cause of this disease may be the consequentimmune response following bacterial infection instead of theinfection itself However there is little data available nowon the literature about the role of immune response in thisdisease It has been well-established that adaptive immuneresponses play very important roles in the development ofinflammatory diseases especially in those related to bacterialinfection Key players of the adaptive immune response are Thelper (Th) cells [17] Th cells have been divided into threemajor subsets based on their different cytokine expressionprofiles The Th1 subset is characterized by the productionof interferon-120574 (IFN-120574) and IL-12 while the major cytokinesfeatured in the Th2 subset are interleukin-4 (IL-4) and IL-10 respectively TheTh17 subset was described more recentlyand is simply defined by the production of IL-17 As reportedwhile Th1 subset controls intracellular bacterial infectionsthe Th2 subset controls parasitic infections and the Th17subset controls fungal and extracellular bacterial infectionsrespectively [18] Since PDM is a condition related to bacterialinfection it is reasonable to assume that Th1 and Th17 maymediate the possible immune responses following bacterialinfection in PDM
In the present study besides exploring and identifyingthe possible risk factors for PDM we also investigate therelationship between bacterial infection and PDM and thesignificance of the expression of consequent Th1 and Th17cytokines in this disease Our results may help to furtherelucidate the pathogenesis of PDM Furthermore since thereare very few well-designed investigations tending to explorethe risk factors and possible causes for PDM in Chinesepatients now our results here may lay an important groundfor the future epidemiological study on this issue in ChineseHan population
2 Materials and Methods
21 Study Population This was a retrospective nonmatchedcase control study Eighty-seven patients with PDM wererecruited from the Department of Breast Surgery at theSecond Hospital of Shandong University The study wasconducted from January 2011 to March 2015 The inclusioncriteria for patients with PDM were as follows (I) newlydiagnosed and histologically confirmed periductal mastitis(II) Han ethnic group and (III) no evidence or history of
cancer Eighty-seven healthy controls were collected fromthe Physical Examination Center of the Second Hospital ofShandong University from June 2015 to August 2015 Thecriteria for the control group were as follows (I) normalresults on breast physical examinations breast ultrasoundscans andor mammographic screening and without anypossible benign breast diseases (fibroadenoma PDM breastcyst intraductal papillomas etc) (II) Han ethnic groupand (III) no evidence or history of breast cancer All eligiblesubjects were women in reproductive age and willing toparticipate in the study voluntarily All procedures performedhere involving human participants were approved by the Sec-ond Hospital of Shandong University Research Committee
22 Pathological Diagnosis Vacuum-assisted incisional orexcisional biopsy was performed based on the clinical find-ings at the time of admission Pathological results werereviewed by two pathologists PDM patients were includedin this study only if they had confirmed pathologicalchanges All patients with other possible causes of mammaryinflammation were ruled out such as breast tuberculosisfat necrosis and granulomatous lobular mastitis as well asinflammation due to lactation or pregnancy
23 Data Collection Possible etiologic factors were writtendown at the time of admission including sociodemographicstatus smoking status alcohol consumption reproductivehistory and medical history In addition the followinginformation was also obtained patient age marital statusmenopause status height weight body mass index (BMI)active and passive smoking alcohol abuse heart and otherchronic disease history history of allergies history of benignbreast disease galactostasis age at menarche age of firstchild birth parity miscarriage history and duration ofbreastfeeding For individual patient clinicopathologic char-acteristics were also collected including major complaint onpresentation size of lesion and histology findings
24 Bacteria Detection For the specimen collection patientswho received any antibiotics before admission and had sinusor fistula or abscess incision drainage were ruled out from thestudy Finally 33 specimens from 31 patients including 21 pussamples and 12 tissue samples were collected in this studyNormal tissues adjacent to benign breast pathology (includ-ing fibroadenoma mastalgia and intraductal papilloma ofthe breast) were used as control specimens (119899 = 12) Breasttissue samples were collected under aseptic conditions at thetime of operation Tissues were divided into two sectionswith one section frozen immediately in liquid nitrogen andanother section sent to the laboratory for bacterial cultureFresh breast specimens were homogenized and incubatedonto Columbia blood agar (Babio Biotech Co Ltd JinanChina) and anaerobic blood culture bottles (Biotech CoLtd) The agar plates were incubated at 37∘C and humidifiedincubatorwas suppliedwith 10carbondioxideThe coloniesformed in the plates were picked and analyzed using aMicroScan Walkaway-96 system (Dade-Behring MicroScanSacramento CA) Gram staining and a stain for acid-fastbacilli were conducted for all specimens All samples were
Mediators of Inflammation 3
also analyzed by 16S rRNA gene sequencing Genomic DNAwas prepared using the bacterial DNAextraction kit (TiangenBiotech Co Ltd Beijing China) according to the manu-facturerrsquos recommendations The hypervariable regions V1ndashV3 of the 16S rRNA gene were PCR-amplified using uni-versal primers (V3F 51015840-CCAGACTCCTACGGGAGGCAG-31015840 V3R 51015840-CGTATTACCGCGGCTGCTG-31015840 27F 51015840-AGAGTT TGA TCMTGGCTC AG-31015840 519R 51015840-GWA TTA CCGCGG CKG CTG-31015840) (Sangon Biotech Co Ltd ShanghaiChina) [19] The 203 and 492 bp PCR products were purifiedafter agarose gel electrophoresis and sequences were deter-mined using the ABI PRISM 3730 DNA sequencer (PerkinElmer Inc Waltham MA USA) Nucleotide sequences wereanalyzed using the National Center for Biotechnology Infor-mation BLAST software (httpswwwncbinlmnihgov)
25 Immunohistochemistry For the patients who were testedfor bacterial infection tissue samples which are mentionedabove were also embedded in paraffin (119899 = 31) forimmunohistochemistry study of proinflammatory cytokinesNormal tissues adjacent to benign breast pathology (includ-ing fibroadenoma mastalgia and intraductal papilloma ofbreast) were used as control specimens (119899 = 22) Immuno-histochemistry was performed on 4 120583m thick sections Andthen the streptavidin-peroxidase-biotin (SP) immunohisto-chemical method was performed to detect the expression ofIFN-120574 IL-12A and IL-17A Briefly after deparaffinization andrehydration the human tissue sections were incubated in 3hydrogen peroxide in methanol to quench the endogenousperoxidase activity followed by incubation with normalgoat serum to block nonspecific binding Samples werethen incubated overnight at 4∘C with rabbit anti-IFN-120574(1 500 ab9657 Abcam Cambridge MA USA) anti-IL-12A(1 500 ab131039 Abcam) or anti-IL-17A (1 500 ab9565Abcam)The secondary antibody was from the SP reagent kit(PV9000 Zhongshan Biotech Co Ltd Beijing China) Thecolor reaction was performed with 331015840-Diaminobenzidine(Zhongshan Biotech Co Ltd) counterstained with hema-toxylin dehydrated treated with xylene and then mountedFor negative controls the antibodies were replaced withPBS To determine the ratio of positive cells for these threecytokines 5 fields (times400) were randomly examined per slideand scored by two pathologists who are blinded to thepatientsrsquo informationThe levels of IFN-120574 IL-12A and IL-17Aexpression were semiquantitatively expressed using a visualgrading system based on the extent of staining (percentage ofpositive cells graded on scale from 0 to 3 0 none 1 1ndash30 231ndash60 3 gt60) and the intensity of staining (graded on ascale of 0ndash3 0 none 1 weak staining 2 moderate staining 3strong staining) The combination of extent (E) and intensity(I) of staining was obtained by the product of E and I with EIvarying from 0 to 9 for each spot Low expression was definedas EI le 3 and others were considered high expression [20]
26 Statistical Analysis Statistical analysis was performedwith SPSS 170 software (SPSS Inc Chicago IL USA)The results were reported as counts (percentage) for thecategorical variables and mean plusmn standard deviation forthe continuous variables A two-sample Studentrsquos 119905-test was
Table 1 Demographic characteristics in case and control groups
Variable Cases (119899 = 87) Controls (119899 = 87) lowast119875
MarriageYes 82 (9425) 86 (9885) 0211No 5 (575) 1 (115)
MenopauseYes 6 (690) 0 (0) 0029No 81 (9310) 87 (100)
HypertensionYes 5 (575) 0 (0) 0059No 82 (9425) 87 (100)
Diabetes mellitusYes 5 (575) 0 (0) 0059No 82 (9425) 87 (100)
Heart diseasesYes 4 (460) 0 (0) 0121No 83 (9540) 87 (100)
Passive smokingYes 23 (4694) 16 (3556) 0263No 26 (5306) 29 (6444)
History of allergiesYes 9 (1034) 7 (805) 0670No 78 (8966) 80 (9195)
Note Data are number () of patientslowast119875 values were determined with the chi-square test
used to compare the difference between the means of thecontinuous variables Chi-square test (or Fisher exact test)was used to compare the differences between the proportionsif appropriate Risk factors for PDM were further assessedusing both univariate and multivariable (unconditional)logistic regression models The continuous variables wereevaluated according to the clinically relevant categories Thecategories for BMI were based on the criteria of beingoverweight and obese for Chinese individuals (overweight24ndash279 kgm2 obese ge28 kgm2) [21] The categories forother continuous variables were on the basis of references(menarche age le 14 years gt14 years times of parity le 1 gt1age at birth of first child le 27 years gt27 years duration ofbreastfeeding le 12 months gt12 months) [22 23] Odds ratio(OR) with 95 confidence interval (95 CI) was used toestimate the association between risk factors and exposureThe significance level was set to 119875 lt 005 (two-tailed)
3 Results
31 Study Population Characteristics To investigate the riskfactors for PDM 87 patients and 87 healthy control subjectswere enrolled in this study The characteristics of patientsand controls are summarized in Table 1 The median ageof patients at presentation was 34 (range 20ndash62) years andthe median age of controls was 34 (range 26ndash45) yearsThe median age did not differ significantly between the two
4 Mediators of Inflammation
Table 2 Clinical characteristics and presentations of patients withPDM
Characteristic ValueMean size of mass (cm) 391 plusmn 257
Affected sidesRight 34 (3908)Left 45 (5172)Bilateral 8 (920)
Quadrant(s)Upper-outer 20 (2299)Lower-outer 8 (920)Upper-inner 9 (1034)Lower-inner 9 (1034)Two or more quadrants 15 (1724)Subareolar region 26 (2989)
Clinical presentationMass without pain 63 (7241)Mass with pain 15 (1724)Erythroswelling 31 (3563)Nipple discharge 6 (690)Sinus 12 (1379)Nipple retraction 24 (2759)
Biopsy methodsIncisional or excisional biopsy 24 (2759)Vacuum-assisted biopsy 63 (7241)
Note Data are number () of patients unless otherwise indicated
groups (119875 = 0134) The majority of subjects in this studywere married There were no statistically significant differ-ences between these two groups in terms of for the history ofhypertension type 2 diabetes mellitus heart disease or anyhistory of allergy Neither alcohol abuse nor active smokingwas reported in the case and control groups There was notany form of breast cancer in these patients of PDM confirmedby pathologic examination
Most patients had unilateral breast disease with only 8patients exhibiting bilateral symptoms Breast mass with orwithout pain was the most common complaint The clinicalpresentations were summarized in Table 2 The medianduration between last pregnancy and onset of PDM was 5years (range 1ndash35 years) which implied that that reproductiveage might relate to PDM to some degree (Figure 1)
32 Univariate and Multivariate Analysis for the Risk Factorsfor PDM To examine the relationship between multiplefactors and PDM a univariate analysis was performed Theresults were shown in Table 3 An analysis of the full datasetindicated that overweightobesity (OR 306 95 CI 153ndash613 119875 = 0002) a late onset of menarche (OR 431 95CI 195ndash950 119875 lt 0001) having more than one child(OR 275 95 CI 118ndash641 119875 = 0019) history of benignbreast disease (OR 454 95 CI 107ndash1014 119875 = 0039)and nipple retraction (OR 3276 95 CI 432ndash24861 119875 =0001) were statistically significant risk factors for PDM Lateage at first birth (OR 022 95 CI 009ndash057 119875 = 0002)
0
5
10
15
20
25
30
20 25 30 35 40 45 50 55 60 62
Num
ber o
f cas
es
Age (year)
Age of onsetAge at birth of last child
Figure 1 The distribution of age of onset and age at birth of firstchildThemedian age of onset was 34 years while the median age atbirth of last child was 28 years It seemed that there was a correlationbetween these two items
had a protective effect for the disease When stratified at12 months duration of breastfeeding was not significantlyrelated to PDM
All factors in the univariate analysis with 119875 values lt 02were regarded as candidate predictors for a logistic regressionmodel and a backward variable selection process was used Inthe multivariable model (Table 4) overweightobesity (OR136 95CI 108ndash170119875 = 0008) and late onset ofmenarche(OR 241 95 CI 138ndash421 119875 = 0002) were independentpredictors of PDM In contrast late age at first birth had aprotective effect against PDM (OR 018 95 CI 003ndash098119875 = 0048)
33 Microorganism Infection in PDM To investigate the roleof bacterial infection in the development of PDM multiplemicroorganismdetectionmethodwas employed in this studyAcid-fast bacilli staining was negative in both case andcontrol groups However colony formation was found ononly one pus specimen plate in bacterial culturing studyand two distinct bacterial strains were further identified asBrevibacterium flavum and a rare Gram-positive bacteriumBacillus cereuswith 16S rRNAgene sequencingTherewas noanaerobic bacterial growth from any sample
Furthermore with sequencing of the 16S rRNA gene wedemonstrated that one or more species of bacteria could bedetected in all of the patient specimens but all the controltissues from the benign breast disease were negative on thistest In the case group a mixture of different bacterial infec-tion was most often observed (3871) followed by singlebacterial infection of Pseudomonas spp (2903) (Table 5)Enterococcus faecium Corynebacterium kroppenstedtii Bacil-lus firmus Sporosarcina and Staphylococcus aureus were alsofound in some samples This result validated the assumptionthat bacterial infection might be an etiological factor forPDM
34 Expression of Proinflammatory Cytokines in PDM Asbacteria were found in the lesions of PDM we thentest whether the critical proinflammatory cytokines from
Mediators of Inflammation 5
Table 3 Univariate analysis of selected risk factors for PDMlowast
Variable Cases (119899 = 87) Controls (119899 = 87) OR (95 CI) 119875
Age (years)lt35 48 (5517) 56 (6437) 10 (Ref) 0217ge35 39 (4483) 31 (3563) 147 (080ndash270)
BMI (kgm2)lt24 34 (3908) 65 (7471) 10 (Ref)
0002ge24 34 (3908) 20 (2299) 325 (163ndash648)Unknown 19 (2184) 2 (230)
Age at menarche (years)le14 57 (6552) 74 (8506) 10 (Ref)
0001gt14 30 (3448) 10 (1149) 390 (176ndash862)Unknown 0 (000) 3 (345)
Parityle1 64 (7356) 72 (8276) 10 (Ref)
0019gt1 22 (2529) 9 (1034) 275 (118ndash641)Unknown 1 (115) 6 (690)
MiscarriagesNo 23 (2644) 25 (2874) 10 (Ref)
0470Yes 25 (2874) 36 (4138) 076 (035ndash162)Unknown 39 (4482) 26 (2988)
Age at birth of first child (years)le27 34 (3908) 26 (2989) 10 (Ref)
0001gt27 8 (920) 29 (3333) 021 (008ndash054)Unknown 45 (5172) 32 (3678)
Duration of breastfeeding (months)le12 34 (3908) 42 (4828) 10 (Ref)
0688gt12 36 (4138) 39 (4483) 114 (060ndash216)Unknown 17 (1954) 6 (689)
History of benign breast diseaseYes 6 (690) 3 (345) 454 (107ndash1914)
0039No 37 (4253) 84 (9555) 10 (Ref)Unknown 44 (5057) 0 (000)
GalactostasisYes 23 (2644) 32 (3678) 086 (044ndash170)
0670No 40 (4598) 48 (5517) 10 (Ref)Unknown 24 (2758) 7 (805)
Nipple retractionYes 24 (2759) 1 (115) 3276 (432ndash24861) 0001No 63 (7241) 86 (9885) 10 (Ref)
Note Data are number () of patients BMI body mass index calculated as weight in kilograms divided by the square of height in meters OR odd ratio CIconfidence intervallowastUsing binary logistic regression
Table 4 Multivariate logistic regression analysis of the associations between various factors and PDM
Variable B SE Wald OR 95 CI 119875
Overweightobesity 031 012 700 136 108ndash170 0008Age at first birth minus170 086 392 018 003ndash098 0048Age at menarche 088 029 953 241 138ndash421 0002
6 Mediators of Inflammation
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 2 Representative illustrations of the expression of cytokines in PDM and normal breast tissues (a) Low-powermagnification of PDM(hematoxylin and eosin times40) (b) Low expression of IFN-120574 (IHC times200) (c) High expression of IFN-120574 (IHC times200) (d) Low expression ofIL-12A (IHC times100) (e) High expression of IFN-120574 (IHC times200) (f) Low expression of IL-17A (IHC times100) (g) High expression of IL-17A(IHC times200) (h) The expression of inflammatory cytokines in normal breast ductal epithelium and stromal cells (IHC times200)
Table 5 Multiple bacteria could be detected in PDM patients
Bacteria 119873 () (119899 = 31)Mixture of different bacteria 12 (3871)Pseudomonas
Pseudomonas aeruginosa 5 (1613)Uncultivated Pseudomonas 2 (645)Pseudomonas delhiensis 1 (323)Pseudomonas otitidis 1 (323)
Enterococcus faecium 2 (645)Corynebacterium kroppenstedtii 1 (323)Bacillus firmus 1 (323)Sporosarcina 1 (323)Staphylococcus aureus 1 (323)Uncultured bacterium clone 4 (1290)
following immune responses IFN-120574 IL-12A and IL-17Awere involved in the disease progressionwith an IHCmethod(Figure 2) After staining and taking photographs a scoringevaluation of the density for each cytokine in this IHCphotographs was performed and data were summarized inTable 6 As shown in Table 7 the expressions of IFN-120574 and IL-12A in stromal inflammatory cells of the breast were increasedin PDM compared to normal breast tissues However therewere no significant differences in the expression of IL-17Abetween these two groups
To investigate the cytokines expression profiles at differ-ent stages of PDM the expressions of IFN-120574 IL-12A and IL-17A were then analyzed by grouping with the presence orabsence of abscess Fifteen patients with abscess formationin their lesions and 16 patients without abscess formationwere included in this study As shown in the Figure 3higher expression levels of IFN-120574 and IL-17A were found
Table 6 The expressions of IFN-120574 IL-12A and IL-17A in PDMcompared with normal breast tissues
Score IFN-120574 IL-12A IL-17ACase Control Case Control Case Control
0 2 1 0 2 1 21 2 3 0 3 10 12 5 13 11 12 8 103 1 2 6 2 1 44 8 3 7 1 10 36 9 0 4 1 0 28 0 0 0 0 0 09 4 0 2 1 0 0Absent 0 0 1 0 1 0Samples were lost in the process of IHC
Table 7 The correlationship of IFN-120574 IL-12A and IL-17A expres-sion in PDM compared with normal breast tissues
Cytokines Case Control 119875lowast
Low expression of IFN-120574 10 19lt0001
High expression of IFN-120574 21 3Low expression of IL-12A 13 19 0022High expression of IL-12A 17 3Low expression of IL-17A 20 17 0404High expression of IL-17A 10 5lowast119875 values were determined with the chi-square test
Low expression was defined as the total score le 3 and others were definedas high expression
in stromal inflammatory cells from PDM patients withabscess formation than those without abscess formationThese results suggest that proinflammatory cytokines may
Mediators of Inflammation 7
0
5
10
15
Cases with abscess formationCases without abscess formation
Num
ber o
f cas
es
lowast lowast lowast
IFN
-120574(minus
)
IFN
-120574(+
)
IL-17
A(minus
)
IL-17
A(+
)
IL-12
A(minus
)
IL-12
A(+
)
Figure 3 Production of IFN-120574 IL-12A and IL-17A in cases withor without abscess formation (minus) low expression of cytokines(+) high expression of cytokines Fifteen patients had lesions withabscess formation while 16 patients did not have them More caseswith high expressions of IFN-120574 and IL-17A in stromal inflammatorycells could be found in patients with abscess formation lowast119875 gt 005
play an important role in the progression of the diseaseof PDM especially the IFN-120574 and IL-12A As mentionedabove these two cytokines are key factors involved in theTh1mediated adaptive immune responses following bacterialinfection
4 Discussion
Smoking obesity diabetes mellitus and reproductive factorshave been considered as significant risk factors for PDM inprevious studies [10 24] The results of the present studyconfirmed that overweightobesity and the late onset ofmenarche were independent risk factors for PDM whilelate age at first birth had a protective effect In additionour results also suggested that nipple retraction was relatedto PDM Bacterial infection could be found in all samplesof PDM patients by 16S rRNA gene sequencing but not inthe samples from the control subjects Among the infectedsamples in PDM patients a mixture of different bacterialstrains was the most common condition which could beseen and then followed by singlePseudomonas spp infectionIFN-120574 and IL-12A were upregulated in PDM compared tonormal breast tissues but the expression of IL-17A was notsignificantly different from control samples Moreover thereare increased expression pattern for both IFN-120574 and IL-17Ain PDM which suggested a following immune response afterbacterial infection may contribute the deterioration of PDM
The incidence of PDM is increasing rapidly during thepast decades Indeed in our previous investigation the detec-tion rate of mammary ductal ectasia was 024 (14761102)[25] However the etiology of PDM is unclear yet mostly dueto lack of conclusive evidences fromprevious study Although
some studies suggest smoking is a risk factor for PDM [11 12]smoking was not found to be closely related to PDM in thepresent study One of the plausible reasons might be the lowprevalence of smoking among Chinese women [26]
Obesity is related to low-grade chronic inflammationwhich can disrupt immune function [27 28] Furthermoreobesity can directly influence local mammary estrogen andinflammation [29] Gollapalli et al [8] reported that obesitywas a risk factor for breast abscess Bharat et al [13] found thathigher BMI values were also associated with nonpuerperalabscessThey also reported similar results in cases of granulo-matous lobular mastitis In PDM however they did not finda significant effect of obesity on patients when comparedwithhealth controls [30] In our study data demonstrated obesityis another risk factor for PDM Obesity may directly disruptthe local immune functioning of the breast and exacerbate thedevelopment of PDM
The relationship between reproductive factors and PDMis very interesting Inconsistent with the finding reportedpreviously from literature in which parity was not found tobe associated with the risk for duct ectasia [31] our resultssuggest that reproductive factors were related to PDM Wealso found late onset of menarche was a significant risk factorfor PDM while increased age at first birth was a protec-tive factor The changes of serum prolactin and lactationin patients may be responsible for the above associationThe exact mechanism underlying this relationship is stillunknown future investigation focusing on theses hormonesis warranted
The relationship between nipple retraction and PDM hasbeen well-documented [32] Nipple retraction could inducebreast deformation and influence the process of lactationFurthermore it also can obstruct drainage and lead toan accumulation of massive substances in ducts which istermed mastitis [33] In this study we confirmed that nippleretraction was a risk factor for PDM The prevalence rateof nipple retraction is approximately 326 according toprevious report [34] But over one quarter of PDM patientsexperienced nipple retraction in our study
Bacterial infection is considered to be another possibleetiology of PDM although it has been a long-standing con-troversial problem Al Benwan et al [15] found that S aureuswas the predominant pathogen followed by Bacteroides sppAnaerobic strep and P aeruginosaHowever the relationshipbetween bacteria and PDM was not confirmed in otherreport [31] Here we provided more evidence to confirm forthis assumption Inconsistent with previous report [35] wedid not find that either S aureus was the most commonbacterium in PDM or anaerobic bacteria was associated withthis disease based on our current data Interestingly thebacteria identified here were those microorganisms whichare often related to community-acquired infection includingPseudomonas spp and S aureus Collectively these findingsuggests that bacterial infection may only be acting as aninduction factor and the immune system which are follow-ing the bacterial infection might play a major role in thedevelopment of PDM However more study is needed for thefuture to determine the common route in which the bacterialinfection and the following immune response converge
8 Mediators of Inflammation
IFN-120574 and IL-12A are characteristic Th1 cytokines [36]Th1 cells are important for the eradication of invadingpathogens including bacteria parasites yeast and viruses[37] In this study the expressions of IFN-120574 and IL-12A wereupregulated in breast stromal inflammatory cells of PDMpatients Recently a lineage of CD4+ T cells producing IL-17A was described and accordingly named as Th17 cellsIL-17A has been shown to be important for host defenseagainst pathogens [38] However in our study there was nosignificant difference in IL-17A expression between the twogroups As IFN-120574 and IL-12A were detected in PDM tissuesamples there is a rationale to conclude that theTh1 immuneresponse plays a role in this diseaseThemechanism bywhichthese cytokines contribute to the course of disease is stillunknown and needs further investigation
The strengths of the present study include the following(a) a relatively large population of patients was included (b)the assessment of tissue was done by two histopathologistsexperienced in breast inflammation However our study alsohad several limitations First this was a nonmatched casecontrol study and there might be some uncontrolled biaseswhich may influence the credibility of the results Secondthere was an issue with missing data especially the dataof reproductive factors which may also lead to the bias ofresults Finally we have not investigated the mechanism bywhich IFN-120574 and IL-12A participated in the course of thedisease
5 Conclusions
The results of the present study indicated that over-weightobesity and later onset of menarche are independentrisk factors for PDM In contrast age of first birth gt27years is a protective factor for PDM The importance ofthese reproductive factors to the risk of PDM has notbeen previously reported and will provide important novelinformation to broaden the avenue of investigating theetiology of PDM in the future We also confirm that bacterialinfection with a mixture of different bacterial species is aclosely associated with the pathogenesis of PDM Moreoverproinflammatory cytokines IFN-120574 and IL-12A were alsoshown to be associated with PDM which indicates that theTh1 immune response may closely relate to this diseaseTherefore PDM could be an inflammatory disease related tobacterial infection and consequent immune responses withincreased cytokines As far as we know this is first reportabout the possible immune responses following bacterialinfection which is involved in development of PDM diseaseand more research is needed for the future to furtherinvestigate the pathogenesis of PDM on the mechanism bywhich bacterial infection and consequent immune responsesaffect the progression of the disease
Ethical Approval
All procedures performed here involving human participantswere in accordance with the ethical standards of the SecondHospital of Shandong University Research Committee and
with the 1964Helsinki Declaration its later amendments andcomparable ethical standards
Consent
Informed consent was obtained from all participants includ-ed in the study
Competing Interests
All authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Dr Zhigang Yu and Dr Liang Li have equal contribution tothis article
Acknowledgments
Theauthors thank the patients and staffs at theDepartment ofBreast Surgery of SecondHospital of ShandongUniversity fortheir collaboration and supportThis study was funded by theYouth Fund of the Second Hospital of Shandong University(Y2014010030) Fei Zhou has received research grants fromthe Second Hospital of Shandong University
References
[1] K Ramalingam A Srivastava S Vuthaluru A Dhar and RChaudhry ldquoDuct Ectasia and periductal mastitis in Indianwomenrdquo Indian Journal of Surgery vol 77 supplement 3 pp957ndash962 2015
[2] J M Dixon ldquoPeriductal mastitisduct ectasiardquoWorld Journal ofSurgery vol 13 no 6 pp 715ndash720 1989
[3] X Zhang Y Lin Q Sun and H Huang ldquoDermo-glandularflap for treatment of recurrent periductal mastitisrdquo Journal ofSurgical Research vol 193 no 2 pp 738ndash744 2015
[4] R M S Rahal R De Freitas-Junior and R R Paulinelli ldquoRiskfactors for duct ectasiardquo Breast Journal vol 11 no 4 pp 262ndash265 2005
[5] R Mansel D Webster and H Sweetland Hughes Mansel ampWebsterrsquos Benign Disorders and Diseases of the Breast (ThirdEdition) Saunders Philadelphia Pa USA 2009
[6] M H Lequin J van Spengler R van Pel C van Eijck and HvanOverhagen ldquoMammographic and sonographic spectrum ofnon-puerperal mastitisrdquo European Journal of Radiology vol 21no 2 pp 138ndash142 1995
[7] R Tomczak A Rieber H Zeitler N Rilinger R Kreienbergand H J Brambs ldquoThe value of MR-mammography at 15 teslain the differential diagnosis of non-puerperal mastitis andinflammatory breast carcinomardquo RoFo Fortschritte auf demGebiete der Rontgenstrahlen und der Nuklearmedizin vol 165no 2 pp 148ndash151 1996
[8] V Gollapalli J Liao A Dudakovic S L Sugg C E H Scott-Conner and R J Weigel ldquoRisk factors for development andrecurrence of primary breast abscessesrdquo Journal of the AmericanCollege of Surgeons vol 211 no 1 pp 41ndash48 2010
Mediators of Inflammation 9
[9] R Risager and N Bentzon ldquoSmoking and increased risk ofmastitisrdquo Ugeskrift for Laeger vol 172 no 33 pp 2218ndash22212010
[10] H N Oltean A S Soliman O S Omar et al ldquoRisk factors forchronic mastitis in Morocco and Egyptrdquo International Journalof Inflammation vol 2013 Article ID 184921 10 pages 2013
[11] P Schafer C Furrer and B Mermillod ldquoAn association ofcigarette smoking with recurrent subareolar breast abscessrdquoInternational Journal of Epidemiology vol 17 no 4 pp 810ndash8131988
[12] V Sakka ldquoNonpuerperal breast infection epidemiology andpredictors for recurrencesrdquo Clinical Microbiology and Infectionvol 18 p 237 2012
[13] A Bharat F Gao R L Aft W E Gillanders T J Eberlein andJ A Margenthaler ldquoPredictors of primary breast abscesses andrecurrencerdquoWorld Journal of Surgery vol 33 no 12 pp 2582ndash2586 2009
[14] N J Bundred J M J Dixon A B Lumsden et al ldquoAre thelesions of duct ectasia sterilerdquo British Journal of Surgery vol72 no 10 pp 844ndash845 1985
[15] K Al Benwan A Al Mulla and V O Rotimi ldquoA study of themicrobiology of breast abscess in a teaching hospital in KuwaitrdquoMedical Principles and Practice vol 20 no 5 pp 422ndash426 2011
[16] P-L Giacalone G Rathat S Fournet and C Rouleau ldquoSurgicaltreatment of recurring subareolar abscess using oncoplastictechniquesrdquo Journal of Visceral Surgery vol 147 no 6 pp e389ndashe394 2010
[17] A Geremia P Biancheri P Allan G R Corazza and ADi Sabatino ldquoInnate and adaptive immunity in inflammatorybowel diseaserdquo Autoimmunity Reviews vol 13 no 1 pp 3ndash102014
[18] P Miossec and J K Kolls ldquoTargeting IL-17 and TH17 cells inchronic inflammationrdquo Nature Reviews Drug Discovery vol 11no 10 pp 763ndash776 2012
[19] Y Khosravi Y Dieye B H Poh et al ldquoCulturable bacterialmicrobiota of the stomach of helicobacter pylori positive andnegative gastric disease patientsrdquo The Scientific World Journalvol 2014 Article ID 610421 10 pages 2014
[20] R Marechal P Demetter N Nagy et al ldquoHigh expressionof CXCR4 may predict poor survival in resected pancreaticadenocarcinomardquo British Journal of Cancer vol 100 no 9 pp1444ndash1451 2009
[21] M Yao Y Wu Q Fang L Sun T Li and H Qiao ldquoAssociationof ADIPOQ variants with type 2 diabetes mellitus susceptibilityin ethnic han Chinese from Northeast Chinardquo Journal ofDiabetes Investigation vol 7 no 6 pp 853ndash859 2016
[22] Q Zhang L-Y Liu FWang KMu and Z-G Yu ldquoThe changesin female physical and childbearing characteristics in china andpotential association with risk of breast cancerrdquo BMC PublicHealth vol 12 article 368 2012
[23] J Kotsopoulos J Lubinski L Salmena et al ldquoBreastfeedingand the risk of breast cancer in BRCA1 and BRCA2 mutationcarriersrdquo Breast Cancer Research vol 14 no 2 article R42 2012
[24] Y Dong J-J Yu Y Shibahara et al ldquoIntercellular adhesionmolecule 12 and E-selectin in plasma cell mastitis immuno-histochemical study of 35 casesrdquo Human Pathology vol 45 no3 pp 606ndash610 2014
[25] Y Y Li J J Du Q Liu and Z G Yu ldquoScreening for breastdiseases among 61102 women in Shandong Provincerdquo Journalof Shandong University vol 49 no 8 pp 157ndash160 2011
[26] Y-M Xu H-H Chen F Li et al ldquoPrevalence and correlatesof cigarette smoking among Chinese schizophrenia inpatientsreceiving antipsychotic mono-therapyrdquo PLOS ONE vol 9 no2 Article ID e88478 2014
[27] F M Wensveen S Valentic M Sestan T Turk Wensveen andB Polic ldquoThe ldquoBig Bangrdquo in obese fat events initiating obesity-induced adipose tissue inflammationrdquo European Journal ofImmunology vol 45 no 9 pp 2446ndash2456 2015
[28] Y B Shaik-Dasthagirisaheb A Kantarci and F C Gibson IIIldquoImmune response of macrophages from young and aged miceto the oral pathogenic bacterium Porphyromonas gingivalisrdquoImmunity amp Ageing vol 7 article 15 2010
[29] K A Brown ldquoImpact of obesity on mammary gland inflamma-tion and local estrogen productionrdquo Journal ofMammary GlandBiology and Neoplasia vol 19 no 2 pp 183ndash189 2014
[30] B Al-Khaffaf F Knox and N J Bundred ldquoIdiopathic granulo-matous mastitis a 25-year experiencerdquo Journal of the AmericanCollege of Surgeons vol 206 no 2 pp 269ndash273 2008
[31] R M S Rahal R F Junior C Reis F C Pimenta J C ANetto and R R Paulinelli ldquoPrevalence of bacteria in the nippledischarge of patients with duct ectasiardquo International Journal ofClinical Practice vol 59 no 9 pp 1045ndash1050 2005
[32] M N Hartley J Stewart and E A Benson ldquoSubareolardissection for duct ectasia and periareolar sepsisrdquo The BritishJournal of Surgery vol 78 no 10 pp 1187ndash1188 1991
[33] J Ming G Meng Q Yuan et al ldquoClinical characteristics andsurgical modality of plasma cell mastitis analysis of 91 casesrdquoThe American Surgeon vol 79 no 1 pp 54ndash60 2013
[34] H S Park C H Yoon and H J Kim ldquoThe prevalence ofcongenital inverted nipplerdquoAesthetic Plastic Surgery vol 23 no2 pp 144ndash146 1999
[35] F N L Versluijs-Ossewaarde R M H Roumen and R J AGoris ldquoSubareolar breast abscesses characteristics and resultsof surgical treatmentrdquoThe Breast Journal vol 11 no 3 pp 179ndash182 2005
[36] Y Luo H Yamada D P Evanoff and X Chen ldquoRole ofTh1-stimulating cytokines in bacillus Calmette-Guerin (BCG)-inducedmacrophage cytotoxicity against mouse bladder cancerMBT-2 cellsrdquo Clinical and Experimental Immunology vol 146no 1 pp 181ndash188 2006
[37] K J Rautajoki M K Kylaniemi S K Raghav K Rao and RLahesmaa ldquoAn insight into molecular mechanisms of human Thelper cell differentiationrdquoAnnals of Medicine vol 40 no 5 pp322ndash335 2008
[38] B Stockinger and M Veldhoen ldquoDifferentiation and functionof Th17 T cellsrdquo Current Opinion in Immunology vol 19 no 3pp 281ndash286 2007
Submit your manuscripts athttpswwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Mediators of Inflammation 3
also analyzed by 16S rRNA gene sequencing Genomic DNAwas prepared using the bacterial DNAextraction kit (TiangenBiotech Co Ltd Beijing China) according to the manu-facturerrsquos recommendations The hypervariable regions V1ndashV3 of the 16S rRNA gene were PCR-amplified using uni-versal primers (V3F 51015840-CCAGACTCCTACGGGAGGCAG-31015840 V3R 51015840-CGTATTACCGCGGCTGCTG-31015840 27F 51015840-AGAGTT TGA TCMTGGCTC AG-31015840 519R 51015840-GWA TTA CCGCGG CKG CTG-31015840) (Sangon Biotech Co Ltd ShanghaiChina) [19] The 203 and 492 bp PCR products were purifiedafter agarose gel electrophoresis and sequences were deter-mined using the ABI PRISM 3730 DNA sequencer (PerkinElmer Inc Waltham MA USA) Nucleotide sequences wereanalyzed using the National Center for Biotechnology Infor-mation BLAST software (httpswwwncbinlmnihgov)
25 Immunohistochemistry For the patients who were testedfor bacterial infection tissue samples which are mentionedabove were also embedded in paraffin (119899 = 31) forimmunohistochemistry study of proinflammatory cytokinesNormal tissues adjacent to benign breast pathology (includ-ing fibroadenoma mastalgia and intraductal papilloma ofbreast) were used as control specimens (119899 = 22) Immuno-histochemistry was performed on 4 120583m thick sections Andthen the streptavidin-peroxidase-biotin (SP) immunohisto-chemical method was performed to detect the expression ofIFN-120574 IL-12A and IL-17A Briefly after deparaffinization andrehydration the human tissue sections were incubated in 3hydrogen peroxide in methanol to quench the endogenousperoxidase activity followed by incubation with normalgoat serum to block nonspecific binding Samples werethen incubated overnight at 4∘C with rabbit anti-IFN-120574(1 500 ab9657 Abcam Cambridge MA USA) anti-IL-12A(1 500 ab131039 Abcam) or anti-IL-17A (1 500 ab9565Abcam)The secondary antibody was from the SP reagent kit(PV9000 Zhongshan Biotech Co Ltd Beijing China) Thecolor reaction was performed with 331015840-Diaminobenzidine(Zhongshan Biotech Co Ltd) counterstained with hema-toxylin dehydrated treated with xylene and then mountedFor negative controls the antibodies were replaced withPBS To determine the ratio of positive cells for these threecytokines 5 fields (times400) were randomly examined per slideand scored by two pathologists who are blinded to thepatientsrsquo informationThe levels of IFN-120574 IL-12A and IL-17Aexpression were semiquantitatively expressed using a visualgrading system based on the extent of staining (percentage ofpositive cells graded on scale from 0 to 3 0 none 1 1ndash30 231ndash60 3 gt60) and the intensity of staining (graded on ascale of 0ndash3 0 none 1 weak staining 2 moderate staining 3strong staining) The combination of extent (E) and intensity(I) of staining was obtained by the product of E and I with EIvarying from 0 to 9 for each spot Low expression was definedas EI le 3 and others were considered high expression [20]
26 Statistical Analysis Statistical analysis was performedwith SPSS 170 software (SPSS Inc Chicago IL USA)The results were reported as counts (percentage) for thecategorical variables and mean plusmn standard deviation forthe continuous variables A two-sample Studentrsquos 119905-test was
Table 1 Demographic characteristics in case and control groups
Variable Cases (119899 = 87) Controls (119899 = 87) lowast119875
MarriageYes 82 (9425) 86 (9885) 0211No 5 (575) 1 (115)
MenopauseYes 6 (690) 0 (0) 0029No 81 (9310) 87 (100)
HypertensionYes 5 (575) 0 (0) 0059No 82 (9425) 87 (100)
Diabetes mellitusYes 5 (575) 0 (0) 0059No 82 (9425) 87 (100)
Heart diseasesYes 4 (460) 0 (0) 0121No 83 (9540) 87 (100)
Passive smokingYes 23 (4694) 16 (3556) 0263No 26 (5306) 29 (6444)
History of allergiesYes 9 (1034) 7 (805) 0670No 78 (8966) 80 (9195)
Note Data are number () of patientslowast119875 values were determined with the chi-square test
used to compare the difference between the means of thecontinuous variables Chi-square test (or Fisher exact test)was used to compare the differences between the proportionsif appropriate Risk factors for PDM were further assessedusing both univariate and multivariable (unconditional)logistic regression models The continuous variables wereevaluated according to the clinically relevant categories Thecategories for BMI were based on the criteria of beingoverweight and obese for Chinese individuals (overweight24ndash279 kgm2 obese ge28 kgm2) [21] The categories forother continuous variables were on the basis of references(menarche age le 14 years gt14 years times of parity le 1 gt1age at birth of first child le 27 years gt27 years duration ofbreastfeeding le 12 months gt12 months) [22 23] Odds ratio(OR) with 95 confidence interval (95 CI) was used toestimate the association between risk factors and exposureThe significance level was set to 119875 lt 005 (two-tailed)
3 Results
31 Study Population Characteristics To investigate the riskfactors for PDM 87 patients and 87 healthy control subjectswere enrolled in this study The characteristics of patientsand controls are summarized in Table 1 The median ageof patients at presentation was 34 (range 20ndash62) years andthe median age of controls was 34 (range 26ndash45) yearsThe median age did not differ significantly between the two
4 Mediators of Inflammation
Table 2 Clinical characteristics and presentations of patients withPDM
Characteristic ValueMean size of mass (cm) 391 plusmn 257
Affected sidesRight 34 (3908)Left 45 (5172)Bilateral 8 (920)
Quadrant(s)Upper-outer 20 (2299)Lower-outer 8 (920)Upper-inner 9 (1034)Lower-inner 9 (1034)Two or more quadrants 15 (1724)Subareolar region 26 (2989)
Clinical presentationMass without pain 63 (7241)Mass with pain 15 (1724)Erythroswelling 31 (3563)Nipple discharge 6 (690)Sinus 12 (1379)Nipple retraction 24 (2759)
Biopsy methodsIncisional or excisional biopsy 24 (2759)Vacuum-assisted biopsy 63 (7241)
Note Data are number () of patients unless otherwise indicated
groups (119875 = 0134) The majority of subjects in this studywere married There were no statistically significant differ-ences between these two groups in terms of for the history ofhypertension type 2 diabetes mellitus heart disease or anyhistory of allergy Neither alcohol abuse nor active smokingwas reported in the case and control groups There was notany form of breast cancer in these patients of PDM confirmedby pathologic examination
Most patients had unilateral breast disease with only 8patients exhibiting bilateral symptoms Breast mass with orwithout pain was the most common complaint The clinicalpresentations were summarized in Table 2 The medianduration between last pregnancy and onset of PDM was 5years (range 1ndash35 years) which implied that that reproductiveage might relate to PDM to some degree (Figure 1)
32 Univariate and Multivariate Analysis for the Risk Factorsfor PDM To examine the relationship between multiplefactors and PDM a univariate analysis was performed Theresults were shown in Table 3 An analysis of the full datasetindicated that overweightobesity (OR 306 95 CI 153ndash613 119875 = 0002) a late onset of menarche (OR 431 95CI 195ndash950 119875 lt 0001) having more than one child(OR 275 95 CI 118ndash641 119875 = 0019) history of benignbreast disease (OR 454 95 CI 107ndash1014 119875 = 0039)and nipple retraction (OR 3276 95 CI 432ndash24861 119875 =0001) were statistically significant risk factors for PDM Lateage at first birth (OR 022 95 CI 009ndash057 119875 = 0002)
0
5
10
15
20
25
30
20 25 30 35 40 45 50 55 60 62
Num
ber o
f cas
es
Age (year)
Age of onsetAge at birth of last child
Figure 1 The distribution of age of onset and age at birth of firstchildThemedian age of onset was 34 years while the median age atbirth of last child was 28 years It seemed that there was a correlationbetween these two items
had a protective effect for the disease When stratified at12 months duration of breastfeeding was not significantlyrelated to PDM
All factors in the univariate analysis with 119875 values lt 02were regarded as candidate predictors for a logistic regressionmodel and a backward variable selection process was used Inthe multivariable model (Table 4) overweightobesity (OR136 95CI 108ndash170119875 = 0008) and late onset ofmenarche(OR 241 95 CI 138ndash421 119875 = 0002) were independentpredictors of PDM In contrast late age at first birth had aprotective effect against PDM (OR 018 95 CI 003ndash098119875 = 0048)
33 Microorganism Infection in PDM To investigate the roleof bacterial infection in the development of PDM multiplemicroorganismdetectionmethodwas employed in this studyAcid-fast bacilli staining was negative in both case andcontrol groups However colony formation was found ononly one pus specimen plate in bacterial culturing studyand two distinct bacterial strains were further identified asBrevibacterium flavum and a rare Gram-positive bacteriumBacillus cereuswith 16S rRNAgene sequencingTherewas noanaerobic bacterial growth from any sample
Furthermore with sequencing of the 16S rRNA gene wedemonstrated that one or more species of bacteria could bedetected in all of the patient specimens but all the controltissues from the benign breast disease were negative on thistest In the case group a mixture of different bacterial infec-tion was most often observed (3871) followed by singlebacterial infection of Pseudomonas spp (2903) (Table 5)Enterococcus faecium Corynebacterium kroppenstedtii Bacil-lus firmus Sporosarcina and Staphylococcus aureus were alsofound in some samples This result validated the assumptionthat bacterial infection might be an etiological factor forPDM
34 Expression of Proinflammatory Cytokines in PDM Asbacteria were found in the lesions of PDM we thentest whether the critical proinflammatory cytokines from
Mediators of Inflammation 5
Table 3 Univariate analysis of selected risk factors for PDMlowast
Variable Cases (119899 = 87) Controls (119899 = 87) OR (95 CI) 119875
Age (years)lt35 48 (5517) 56 (6437) 10 (Ref) 0217ge35 39 (4483) 31 (3563) 147 (080ndash270)
BMI (kgm2)lt24 34 (3908) 65 (7471) 10 (Ref)
0002ge24 34 (3908) 20 (2299) 325 (163ndash648)Unknown 19 (2184) 2 (230)
Age at menarche (years)le14 57 (6552) 74 (8506) 10 (Ref)
0001gt14 30 (3448) 10 (1149) 390 (176ndash862)Unknown 0 (000) 3 (345)
Parityle1 64 (7356) 72 (8276) 10 (Ref)
0019gt1 22 (2529) 9 (1034) 275 (118ndash641)Unknown 1 (115) 6 (690)
MiscarriagesNo 23 (2644) 25 (2874) 10 (Ref)
0470Yes 25 (2874) 36 (4138) 076 (035ndash162)Unknown 39 (4482) 26 (2988)
Age at birth of first child (years)le27 34 (3908) 26 (2989) 10 (Ref)
0001gt27 8 (920) 29 (3333) 021 (008ndash054)Unknown 45 (5172) 32 (3678)
Duration of breastfeeding (months)le12 34 (3908) 42 (4828) 10 (Ref)
0688gt12 36 (4138) 39 (4483) 114 (060ndash216)Unknown 17 (1954) 6 (689)
History of benign breast diseaseYes 6 (690) 3 (345) 454 (107ndash1914)
0039No 37 (4253) 84 (9555) 10 (Ref)Unknown 44 (5057) 0 (000)
GalactostasisYes 23 (2644) 32 (3678) 086 (044ndash170)
0670No 40 (4598) 48 (5517) 10 (Ref)Unknown 24 (2758) 7 (805)
Nipple retractionYes 24 (2759) 1 (115) 3276 (432ndash24861) 0001No 63 (7241) 86 (9885) 10 (Ref)
Note Data are number () of patients BMI body mass index calculated as weight in kilograms divided by the square of height in meters OR odd ratio CIconfidence intervallowastUsing binary logistic regression
Table 4 Multivariate logistic regression analysis of the associations between various factors and PDM
Variable B SE Wald OR 95 CI 119875
Overweightobesity 031 012 700 136 108ndash170 0008Age at first birth minus170 086 392 018 003ndash098 0048Age at menarche 088 029 953 241 138ndash421 0002
6 Mediators of Inflammation
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 2 Representative illustrations of the expression of cytokines in PDM and normal breast tissues (a) Low-powermagnification of PDM(hematoxylin and eosin times40) (b) Low expression of IFN-120574 (IHC times200) (c) High expression of IFN-120574 (IHC times200) (d) Low expression ofIL-12A (IHC times100) (e) High expression of IFN-120574 (IHC times200) (f) Low expression of IL-17A (IHC times100) (g) High expression of IL-17A(IHC times200) (h) The expression of inflammatory cytokines in normal breast ductal epithelium and stromal cells (IHC times200)
Table 5 Multiple bacteria could be detected in PDM patients
Bacteria 119873 () (119899 = 31)Mixture of different bacteria 12 (3871)Pseudomonas
Pseudomonas aeruginosa 5 (1613)Uncultivated Pseudomonas 2 (645)Pseudomonas delhiensis 1 (323)Pseudomonas otitidis 1 (323)
Enterococcus faecium 2 (645)Corynebacterium kroppenstedtii 1 (323)Bacillus firmus 1 (323)Sporosarcina 1 (323)Staphylococcus aureus 1 (323)Uncultured bacterium clone 4 (1290)
following immune responses IFN-120574 IL-12A and IL-17Awere involved in the disease progressionwith an IHCmethod(Figure 2) After staining and taking photographs a scoringevaluation of the density for each cytokine in this IHCphotographs was performed and data were summarized inTable 6 As shown in Table 7 the expressions of IFN-120574 and IL-12A in stromal inflammatory cells of the breast were increasedin PDM compared to normal breast tissues However therewere no significant differences in the expression of IL-17Abetween these two groups
To investigate the cytokines expression profiles at differ-ent stages of PDM the expressions of IFN-120574 IL-12A and IL-17A were then analyzed by grouping with the presence orabsence of abscess Fifteen patients with abscess formationin their lesions and 16 patients without abscess formationwere included in this study As shown in the Figure 3higher expression levels of IFN-120574 and IL-17A were found
Table 6 The expressions of IFN-120574 IL-12A and IL-17A in PDMcompared with normal breast tissues
Score IFN-120574 IL-12A IL-17ACase Control Case Control Case Control
0 2 1 0 2 1 21 2 3 0 3 10 12 5 13 11 12 8 103 1 2 6 2 1 44 8 3 7 1 10 36 9 0 4 1 0 28 0 0 0 0 0 09 4 0 2 1 0 0Absent 0 0 1 0 1 0Samples were lost in the process of IHC
Table 7 The correlationship of IFN-120574 IL-12A and IL-17A expres-sion in PDM compared with normal breast tissues
Cytokines Case Control 119875lowast
Low expression of IFN-120574 10 19lt0001
High expression of IFN-120574 21 3Low expression of IL-12A 13 19 0022High expression of IL-12A 17 3Low expression of IL-17A 20 17 0404High expression of IL-17A 10 5lowast119875 values were determined with the chi-square test
Low expression was defined as the total score le 3 and others were definedas high expression
in stromal inflammatory cells from PDM patients withabscess formation than those without abscess formationThese results suggest that proinflammatory cytokines may
Mediators of Inflammation 7
0
5
10
15
Cases with abscess formationCases without abscess formation
Num
ber o
f cas
es
lowast lowast lowast
IFN
-120574(minus
)
IFN
-120574(+
)
IL-17
A(minus
)
IL-17
A(+
)
IL-12
A(minus
)
IL-12
A(+
)
Figure 3 Production of IFN-120574 IL-12A and IL-17A in cases withor without abscess formation (minus) low expression of cytokines(+) high expression of cytokines Fifteen patients had lesions withabscess formation while 16 patients did not have them More caseswith high expressions of IFN-120574 and IL-17A in stromal inflammatorycells could be found in patients with abscess formation lowast119875 gt 005
play an important role in the progression of the diseaseof PDM especially the IFN-120574 and IL-12A As mentionedabove these two cytokines are key factors involved in theTh1mediated adaptive immune responses following bacterialinfection
4 Discussion
Smoking obesity diabetes mellitus and reproductive factorshave been considered as significant risk factors for PDM inprevious studies [10 24] The results of the present studyconfirmed that overweightobesity and the late onset ofmenarche were independent risk factors for PDM whilelate age at first birth had a protective effect In additionour results also suggested that nipple retraction was relatedto PDM Bacterial infection could be found in all samplesof PDM patients by 16S rRNA gene sequencing but not inthe samples from the control subjects Among the infectedsamples in PDM patients a mixture of different bacterialstrains was the most common condition which could beseen and then followed by singlePseudomonas spp infectionIFN-120574 and IL-12A were upregulated in PDM compared tonormal breast tissues but the expression of IL-17A was notsignificantly different from control samples Moreover thereare increased expression pattern for both IFN-120574 and IL-17Ain PDM which suggested a following immune response afterbacterial infection may contribute the deterioration of PDM
The incidence of PDM is increasing rapidly during thepast decades Indeed in our previous investigation the detec-tion rate of mammary ductal ectasia was 024 (14761102)[25] However the etiology of PDM is unclear yet mostly dueto lack of conclusive evidences fromprevious study Although
some studies suggest smoking is a risk factor for PDM [11 12]smoking was not found to be closely related to PDM in thepresent study One of the plausible reasons might be the lowprevalence of smoking among Chinese women [26]
Obesity is related to low-grade chronic inflammationwhich can disrupt immune function [27 28] Furthermoreobesity can directly influence local mammary estrogen andinflammation [29] Gollapalli et al [8] reported that obesitywas a risk factor for breast abscess Bharat et al [13] found thathigher BMI values were also associated with nonpuerperalabscessThey also reported similar results in cases of granulo-matous lobular mastitis In PDM however they did not finda significant effect of obesity on patients when comparedwithhealth controls [30] In our study data demonstrated obesityis another risk factor for PDM Obesity may directly disruptthe local immune functioning of the breast and exacerbate thedevelopment of PDM
The relationship between reproductive factors and PDMis very interesting Inconsistent with the finding reportedpreviously from literature in which parity was not found tobe associated with the risk for duct ectasia [31] our resultssuggest that reproductive factors were related to PDM Wealso found late onset of menarche was a significant risk factorfor PDM while increased age at first birth was a protec-tive factor The changes of serum prolactin and lactationin patients may be responsible for the above associationThe exact mechanism underlying this relationship is stillunknown future investigation focusing on theses hormonesis warranted
The relationship between nipple retraction and PDM hasbeen well-documented [32] Nipple retraction could inducebreast deformation and influence the process of lactationFurthermore it also can obstruct drainage and lead toan accumulation of massive substances in ducts which istermed mastitis [33] In this study we confirmed that nippleretraction was a risk factor for PDM The prevalence rateof nipple retraction is approximately 326 according toprevious report [34] But over one quarter of PDM patientsexperienced nipple retraction in our study
Bacterial infection is considered to be another possibleetiology of PDM although it has been a long-standing con-troversial problem Al Benwan et al [15] found that S aureuswas the predominant pathogen followed by Bacteroides sppAnaerobic strep and P aeruginosaHowever the relationshipbetween bacteria and PDM was not confirmed in otherreport [31] Here we provided more evidence to confirm forthis assumption Inconsistent with previous report [35] wedid not find that either S aureus was the most commonbacterium in PDM or anaerobic bacteria was associated withthis disease based on our current data Interestingly thebacteria identified here were those microorganisms whichare often related to community-acquired infection includingPseudomonas spp and S aureus Collectively these findingsuggests that bacterial infection may only be acting as aninduction factor and the immune system which are follow-ing the bacterial infection might play a major role in thedevelopment of PDM However more study is needed for thefuture to determine the common route in which the bacterialinfection and the following immune response converge
8 Mediators of Inflammation
IFN-120574 and IL-12A are characteristic Th1 cytokines [36]Th1 cells are important for the eradication of invadingpathogens including bacteria parasites yeast and viruses[37] In this study the expressions of IFN-120574 and IL-12A wereupregulated in breast stromal inflammatory cells of PDMpatients Recently a lineage of CD4+ T cells producing IL-17A was described and accordingly named as Th17 cellsIL-17A has been shown to be important for host defenseagainst pathogens [38] However in our study there was nosignificant difference in IL-17A expression between the twogroups As IFN-120574 and IL-12A were detected in PDM tissuesamples there is a rationale to conclude that theTh1 immuneresponse plays a role in this diseaseThemechanism bywhichthese cytokines contribute to the course of disease is stillunknown and needs further investigation
The strengths of the present study include the following(a) a relatively large population of patients was included (b)the assessment of tissue was done by two histopathologistsexperienced in breast inflammation However our study alsohad several limitations First this was a nonmatched casecontrol study and there might be some uncontrolled biaseswhich may influence the credibility of the results Secondthere was an issue with missing data especially the dataof reproductive factors which may also lead to the bias ofresults Finally we have not investigated the mechanism bywhich IFN-120574 and IL-12A participated in the course of thedisease
5 Conclusions
The results of the present study indicated that over-weightobesity and later onset of menarche are independentrisk factors for PDM In contrast age of first birth gt27years is a protective factor for PDM The importance ofthese reproductive factors to the risk of PDM has notbeen previously reported and will provide important novelinformation to broaden the avenue of investigating theetiology of PDM in the future We also confirm that bacterialinfection with a mixture of different bacterial species is aclosely associated with the pathogenesis of PDM Moreoverproinflammatory cytokines IFN-120574 and IL-12A were alsoshown to be associated with PDM which indicates that theTh1 immune response may closely relate to this diseaseTherefore PDM could be an inflammatory disease related tobacterial infection and consequent immune responses withincreased cytokines As far as we know this is first reportabout the possible immune responses following bacterialinfection which is involved in development of PDM diseaseand more research is needed for the future to furtherinvestigate the pathogenesis of PDM on the mechanism bywhich bacterial infection and consequent immune responsesaffect the progression of the disease
Ethical Approval
All procedures performed here involving human participantswere in accordance with the ethical standards of the SecondHospital of Shandong University Research Committee and
with the 1964Helsinki Declaration its later amendments andcomparable ethical standards
Consent
Informed consent was obtained from all participants includ-ed in the study
Competing Interests
All authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Dr Zhigang Yu and Dr Liang Li have equal contribution tothis article
Acknowledgments
Theauthors thank the patients and staffs at theDepartment ofBreast Surgery of SecondHospital of ShandongUniversity fortheir collaboration and supportThis study was funded by theYouth Fund of the Second Hospital of Shandong University(Y2014010030) Fei Zhou has received research grants fromthe Second Hospital of Shandong University
References
[1] K Ramalingam A Srivastava S Vuthaluru A Dhar and RChaudhry ldquoDuct Ectasia and periductal mastitis in Indianwomenrdquo Indian Journal of Surgery vol 77 supplement 3 pp957ndash962 2015
[2] J M Dixon ldquoPeriductal mastitisduct ectasiardquoWorld Journal ofSurgery vol 13 no 6 pp 715ndash720 1989
[3] X Zhang Y Lin Q Sun and H Huang ldquoDermo-glandularflap for treatment of recurrent periductal mastitisrdquo Journal ofSurgical Research vol 193 no 2 pp 738ndash744 2015
[4] R M S Rahal R De Freitas-Junior and R R Paulinelli ldquoRiskfactors for duct ectasiardquo Breast Journal vol 11 no 4 pp 262ndash265 2005
[5] R Mansel D Webster and H Sweetland Hughes Mansel ampWebsterrsquos Benign Disorders and Diseases of the Breast (ThirdEdition) Saunders Philadelphia Pa USA 2009
[6] M H Lequin J van Spengler R van Pel C van Eijck and HvanOverhagen ldquoMammographic and sonographic spectrum ofnon-puerperal mastitisrdquo European Journal of Radiology vol 21no 2 pp 138ndash142 1995
[7] R Tomczak A Rieber H Zeitler N Rilinger R Kreienbergand H J Brambs ldquoThe value of MR-mammography at 15 teslain the differential diagnosis of non-puerperal mastitis andinflammatory breast carcinomardquo RoFo Fortschritte auf demGebiete der Rontgenstrahlen und der Nuklearmedizin vol 165no 2 pp 148ndash151 1996
[8] V Gollapalli J Liao A Dudakovic S L Sugg C E H Scott-Conner and R J Weigel ldquoRisk factors for development andrecurrence of primary breast abscessesrdquo Journal of the AmericanCollege of Surgeons vol 211 no 1 pp 41ndash48 2010
Mediators of Inflammation 9
[9] R Risager and N Bentzon ldquoSmoking and increased risk ofmastitisrdquo Ugeskrift for Laeger vol 172 no 33 pp 2218ndash22212010
[10] H N Oltean A S Soliman O S Omar et al ldquoRisk factors forchronic mastitis in Morocco and Egyptrdquo International Journalof Inflammation vol 2013 Article ID 184921 10 pages 2013
[11] P Schafer C Furrer and B Mermillod ldquoAn association ofcigarette smoking with recurrent subareolar breast abscessrdquoInternational Journal of Epidemiology vol 17 no 4 pp 810ndash8131988
[12] V Sakka ldquoNonpuerperal breast infection epidemiology andpredictors for recurrencesrdquo Clinical Microbiology and Infectionvol 18 p 237 2012
[13] A Bharat F Gao R L Aft W E Gillanders T J Eberlein andJ A Margenthaler ldquoPredictors of primary breast abscesses andrecurrencerdquoWorld Journal of Surgery vol 33 no 12 pp 2582ndash2586 2009
[14] N J Bundred J M J Dixon A B Lumsden et al ldquoAre thelesions of duct ectasia sterilerdquo British Journal of Surgery vol72 no 10 pp 844ndash845 1985
[15] K Al Benwan A Al Mulla and V O Rotimi ldquoA study of themicrobiology of breast abscess in a teaching hospital in KuwaitrdquoMedical Principles and Practice vol 20 no 5 pp 422ndash426 2011
[16] P-L Giacalone G Rathat S Fournet and C Rouleau ldquoSurgicaltreatment of recurring subareolar abscess using oncoplastictechniquesrdquo Journal of Visceral Surgery vol 147 no 6 pp e389ndashe394 2010
[17] A Geremia P Biancheri P Allan G R Corazza and ADi Sabatino ldquoInnate and adaptive immunity in inflammatorybowel diseaserdquo Autoimmunity Reviews vol 13 no 1 pp 3ndash102014
[18] P Miossec and J K Kolls ldquoTargeting IL-17 and TH17 cells inchronic inflammationrdquo Nature Reviews Drug Discovery vol 11no 10 pp 763ndash776 2012
[19] Y Khosravi Y Dieye B H Poh et al ldquoCulturable bacterialmicrobiota of the stomach of helicobacter pylori positive andnegative gastric disease patientsrdquo The Scientific World Journalvol 2014 Article ID 610421 10 pages 2014
[20] R Marechal P Demetter N Nagy et al ldquoHigh expressionof CXCR4 may predict poor survival in resected pancreaticadenocarcinomardquo British Journal of Cancer vol 100 no 9 pp1444ndash1451 2009
[21] M Yao Y Wu Q Fang L Sun T Li and H Qiao ldquoAssociationof ADIPOQ variants with type 2 diabetes mellitus susceptibilityin ethnic han Chinese from Northeast Chinardquo Journal ofDiabetes Investigation vol 7 no 6 pp 853ndash859 2016
[22] Q Zhang L-Y Liu FWang KMu and Z-G Yu ldquoThe changesin female physical and childbearing characteristics in china andpotential association with risk of breast cancerrdquo BMC PublicHealth vol 12 article 368 2012
[23] J Kotsopoulos J Lubinski L Salmena et al ldquoBreastfeedingand the risk of breast cancer in BRCA1 and BRCA2 mutationcarriersrdquo Breast Cancer Research vol 14 no 2 article R42 2012
[24] Y Dong J-J Yu Y Shibahara et al ldquoIntercellular adhesionmolecule 12 and E-selectin in plasma cell mastitis immuno-histochemical study of 35 casesrdquo Human Pathology vol 45 no3 pp 606ndash610 2014
[25] Y Y Li J J Du Q Liu and Z G Yu ldquoScreening for breastdiseases among 61102 women in Shandong Provincerdquo Journalof Shandong University vol 49 no 8 pp 157ndash160 2011
[26] Y-M Xu H-H Chen F Li et al ldquoPrevalence and correlatesof cigarette smoking among Chinese schizophrenia inpatientsreceiving antipsychotic mono-therapyrdquo PLOS ONE vol 9 no2 Article ID e88478 2014
[27] F M Wensveen S Valentic M Sestan T Turk Wensveen andB Polic ldquoThe ldquoBig Bangrdquo in obese fat events initiating obesity-induced adipose tissue inflammationrdquo European Journal ofImmunology vol 45 no 9 pp 2446ndash2456 2015
[28] Y B Shaik-Dasthagirisaheb A Kantarci and F C Gibson IIIldquoImmune response of macrophages from young and aged miceto the oral pathogenic bacterium Porphyromonas gingivalisrdquoImmunity amp Ageing vol 7 article 15 2010
[29] K A Brown ldquoImpact of obesity on mammary gland inflamma-tion and local estrogen productionrdquo Journal ofMammary GlandBiology and Neoplasia vol 19 no 2 pp 183ndash189 2014
[30] B Al-Khaffaf F Knox and N J Bundred ldquoIdiopathic granulo-matous mastitis a 25-year experiencerdquo Journal of the AmericanCollege of Surgeons vol 206 no 2 pp 269ndash273 2008
[31] R M S Rahal R F Junior C Reis F C Pimenta J C ANetto and R R Paulinelli ldquoPrevalence of bacteria in the nippledischarge of patients with duct ectasiardquo International Journal ofClinical Practice vol 59 no 9 pp 1045ndash1050 2005
[32] M N Hartley J Stewart and E A Benson ldquoSubareolardissection for duct ectasia and periareolar sepsisrdquo The BritishJournal of Surgery vol 78 no 10 pp 1187ndash1188 1991
[33] J Ming G Meng Q Yuan et al ldquoClinical characteristics andsurgical modality of plasma cell mastitis analysis of 91 casesrdquoThe American Surgeon vol 79 no 1 pp 54ndash60 2013
[34] H S Park C H Yoon and H J Kim ldquoThe prevalence ofcongenital inverted nipplerdquoAesthetic Plastic Surgery vol 23 no2 pp 144ndash146 1999
[35] F N L Versluijs-Ossewaarde R M H Roumen and R J AGoris ldquoSubareolar breast abscesses characteristics and resultsof surgical treatmentrdquoThe Breast Journal vol 11 no 3 pp 179ndash182 2005
[36] Y Luo H Yamada D P Evanoff and X Chen ldquoRole ofTh1-stimulating cytokines in bacillus Calmette-Guerin (BCG)-inducedmacrophage cytotoxicity against mouse bladder cancerMBT-2 cellsrdquo Clinical and Experimental Immunology vol 146no 1 pp 181ndash188 2006
[37] K J Rautajoki M K Kylaniemi S K Raghav K Rao and RLahesmaa ldquoAn insight into molecular mechanisms of human Thelper cell differentiationrdquoAnnals of Medicine vol 40 no 5 pp322ndash335 2008
[38] B Stockinger and M Veldhoen ldquoDifferentiation and functionof Th17 T cellsrdquo Current Opinion in Immunology vol 19 no 3pp 281ndash286 2007
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Mediators of Inflammation
Table 2 Clinical characteristics and presentations of patients withPDM
Characteristic ValueMean size of mass (cm) 391 plusmn 257
Affected sidesRight 34 (3908)Left 45 (5172)Bilateral 8 (920)
Quadrant(s)Upper-outer 20 (2299)Lower-outer 8 (920)Upper-inner 9 (1034)Lower-inner 9 (1034)Two or more quadrants 15 (1724)Subareolar region 26 (2989)
Clinical presentationMass without pain 63 (7241)Mass with pain 15 (1724)Erythroswelling 31 (3563)Nipple discharge 6 (690)Sinus 12 (1379)Nipple retraction 24 (2759)
Biopsy methodsIncisional or excisional biopsy 24 (2759)Vacuum-assisted biopsy 63 (7241)
Note Data are number () of patients unless otherwise indicated
groups (119875 = 0134) The majority of subjects in this studywere married There were no statistically significant differ-ences between these two groups in terms of for the history ofhypertension type 2 diabetes mellitus heart disease or anyhistory of allergy Neither alcohol abuse nor active smokingwas reported in the case and control groups There was notany form of breast cancer in these patients of PDM confirmedby pathologic examination
Most patients had unilateral breast disease with only 8patients exhibiting bilateral symptoms Breast mass with orwithout pain was the most common complaint The clinicalpresentations were summarized in Table 2 The medianduration between last pregnancy and onset of PDM was 5years (range 1ndash35 years) which implied that that reproductiveage might relate to PDM to some degree (Figure 1)
32 Univariate and Multivariate Analysis for the Risk Factorsfor PDM To examine the relationship between multiplefactors and PDM a univariate analysis was performed Theresults were shown in Table 3 An analysis of the full datasetindicated that overweightobesity (OR 306 95 CI 153ndash613 119875 = 0002) a late onset of menarche (OR 431 95CI 195ndash950 119875 lt 0001) having more than one child(OR 275 95 CI 118ndash641 119875 = 0019) history of benignbreast disease (OR 454 95 CI 107ndash1014 119875 = 0039)and nipple retraction (OR 3276 95 CI 432ndash24861 119875 =0001) were statistically significant risk factors for PDM Lateage at first birth (OR 022 95 CI 009ndash057 119875 = 0002)
0
5
10
15
20
25
30
20 25 30 35 40 45 50 55 60 62
Num
ber o
f cas
es
Age (year)
Age of onsetAge at birth of last child
Figure 1 The distribution of age of onset and age at birth of firstchildThemedian age of onset was 34 years while the median age atbirth of last child was 28 years It seemed that there was a correlationbetween these two items
had a protective effect for the disease When stratified at12 months duration of breastfeeding was not significantlyrelated to PDM
All factors in the univariate analysis with 119875 values lt 02were regarded as candidate predictors for a logistic regressionmodel and a backward variable selection process was used Inthe multivariable model (Table 4) overweightobesity (OR136 95CI 108ndash170119875 = 0008) and late onset ofmenarche(OR 241 95 CI 138ndash421 119875 = 0002) were independentpredictors of PDM In contrast late age at first birth had aprotective effect against PDM (OR 018 95 CI 003ndash098119875 = 0048)
33 Microorganism Infection in PDM To investigate the roleof bacterial infection in the development of PDM multiplemicroorganismdetectionmethodwas employed in this studyAcid-fast bacilli staining was negative in both case andcontrol groups However colony formation was found ononly one pus specimen plate in bacterial culturing studyand two distinct bacterial strains were further identified asBrevibacterium flavum and a rare Gram-positive bacteriumBacillus cereuswith 16S rRNAgene sequencingTherewas noanaerobic bacterial growth from any sample
Furthermore with sequencing of the 16S rRNA gene wedemonstrated that one or more species of bacteria could bedetected in all of the patient specimens but all the controltissues from the benign breast disease were negative on thistest In the case group a mixture of different bacterial infec-tion was most often observed (3871) followed by singlebacterial infection of Pseudomonas spp (2903) (Table 5)Enterococcus faecium Corynebacterium kroppenstedtii Bacil-lus firmus Sporosarcina and Staphylococcus aureus were alsofound in some samples This result validated the assumptionthat bacterial infection might be an etiological factor forPDM
34 Expression of Proinflammatory Cytokines in PDM Asbacteria were found in the lesions of PDM we thentest whether the critical proinflammatory cytokines from
Mediators of Inflammation 5
Table 3 Univariate analysis of selected risk factors for PDMlowast
Variable Cases (119899 = 87) Controls (119899 = 87) OR (95 CI) 119875
Age (years)lt35 48 (5517) 56 (6437) 10 (Ref) 0217ge35 39 (4483) 31 (3563) 147 (080ndash270)
BMI (kgm2)lt24 34 (3908) 65 (7471) 10 (Ref)
0002ge24 34 (3908) 20 (2299) 325 (163ndash648)Unknown 19 (2184) 2 (230)
Age at menarche (years)le14 57 (6552) 74 (8506) 10 (Ref)
0001gt14 30 (3448) 10 (1149) 390 (176ndash862)Unknown 0 (000) 3 (345)
Parityle1 64 (7356) 72 (8276) 10 (Ref)
0019gt1 22 (2529) 9 (1034) 275 (118ndash641)Unknown 1 (115) 6 (690)
MiscarriagesNo 23 (2644) 25 (2874) 10 (Ref)
0470Yes 25 (2874) 36 (4138) 076 (035ndash162)Unknown 39 (4482) 26 (2988)
Age at birth of first child (years)le27 34 (3908) 26 (2989) 10 (Ref)
0001gt27 8 (920) 29 (3333) 021 (008ndash054)Unknown 45 (5172) 32 (3678)
Duration of breastfeeding (months)le12 34 (3908) 42 (4828) 10 (Ref)
0688gt12 36 (4138) 39 (4483) 114 (060ndash216)Unknown 17 (1954) 6 (689)
History of benign breast diseaseYes 6 (690) 3 (345) 454 (107ndash1914)
0039No 37 (4253) 84 (9555) 10 (Ref)Unknown 44 (5057) 0 (000)
GalactostasisYes 23 (2644) 32 (3678) 086 (044ndash170)
0670No 40 (4598) 48 (5517) 10 (Ref)Unknown 24 (2758) 7 (805)
Nipple retractionYes 24 (2759) 1 (115) 3276 (432ndash24861) 0001No 63 (7241) 86 (9885) 10 (Ref)
Note Data are number () of patients BMI body mass index calculated as weight in kilograms divided by the square of height in meters OR odd ratio CIconfidence intervallowastUsing binary logistic regression
Table 4 Multivariate logistic regression analysis of the associations between various factors and PDM
Variable B SE Wald OR 95 CI 119875
Overweightobesity 031 012 700 136 108ndash170 0008Age at first birth minus170 086 392 018 003ndash098 0048Age at menarche 088 029 953 241 138ndash421 0002
6 Mediators of Inflammation
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 2 Representative illustrations of the expression of cytokines in PDM and normal breast tissues (a) Low-powermagnification of PDM(hematoxylin and eosin times40) (b) Low expression of IFN-120574 (IHC times200) (c) High expression of IFN-120574 (IHC times200) (d) Low expression ofIL-12A (IHC times100) (e) High expression of IFN-120574 (IHC times200) (f) Low expression of IL-17A (IHC times100) (g) High expression of IL-17A(IHC times200) (h) The expression of inflammatory cytokines in normal breast ductal epithelium and stromal cells (IHC times200)
Table 5 Multiple bacteria could be detected in PDM patients
Bacteria 119873 () (119899 = 31)Mixture of different bacteria 12 (3871)Pseudomonas
Pseudomonas aeruginosa 5 (1613)Uncultivated Pseudomonas 2 (645)Pseudomonas delhiensis 1 (323)Pseudomonas otitidis 1 (323)
Enterococcus faecium 2 (645)Corynebacterium kroppenstedtii 1 (323)Bacillus firmus 1 (323)Sporosarcina 1 (323)Staphylococcus aureus 1 (323)Uncultured bacterium clone 4 (1290)
following immune responses IFN-120574 IL-12A and IL-17Awere involved in the disease progressionwith an IHCmethod(Figure 2) After staining and taking photographs a scoringevaluation of the density for each cytokine in this IHCphotographs was performed and data were summarized inTable 6 As shown in Table 7 the expressions of IFN-120574 and IL-12A in stromal inflammatory cells of the breast were increasedin PDM compared to normal breast tissues However therewere no significant differences in the expression of IL-17Abetween these two groups
To investigate the cytokines expression profiles at differ-ent stages of PDM the expressions of IFN-120574 IL-12A and IL-17A were then analyzed by grouping with the presence orabsence of abscess Fifteen patients with abscess formationin their lesions and 16 patients without abscess formationwere included in this study As shown in the Figure 3higher expression levels of IFN-120574 and IL-17A were found
Table 6 The expressions of IFN-120574 IL-12A and IL-17A in PDMcompared with normal breast tissues
Score IFN-120574 IL-12A IL-17ACase Control Case Control Case Control
0 2 1 0 2 1 21 2 3 0 3 10 12 5 13 11 12 8 103 1 2 6 2 1 44 8 3 7 1 10 36 9 0 4 1 0 28 0 0 0 0 0 09 4 0 2 1 0 0Absent 0 0 1 0 1 0Samples were lost in the process of IHC
Table 7 The correlationship of IFN-120574 IL-12A and IL-17A expres-sion in PDM compared with normal breast tissues
Cytokines Case Control 119875lowast
Low expression of IFN-120574 10 19lt0001
High expression of IFN-120574 21 3Low expression of IL-12A 13 19 0022High expression of IL-12A 17 3Low expression of IL-17A 20 17 0404High expression of IL-17A 10 5lowast119875 values were determined with the chi-square test
Low expression was defined as the total score le 3 and others were definedas high expression
in stromal inflammatory cells from PDM patients withabscess formation than those without abscess formationThese results suggest that proinflammatory cytokines may
Mediators of Inflammation 7
0
5
10
15
Cases with abscess formationCases without abscess formation
Num
ber o
f cas
es
lowast lowast lowast
IFN
-120574(minus
)
IFN
-120574(+
)
IL-17
A(minus
)
IL-17
A(+
)
IL-12
A(minus
)
IL-12
A(+
)
Figure 3 Production of IFN-120574 IL-12A and IL-17A in cases withor without abscess formation (minus) low expression of cytokines(+) high expression of cytokines Fifteen patients had lesions withabscess formation while 16 patients did not have them More caseswith high expressions of IFN-120574 and IL-17A in stromal inflammatorycells could be found in patients with abscess formation lowast119875 gt 005
play an important role in the progression of the diseaseof PDM especially the IFN-120574 and IL-12A As mentionedabove these two cytokines are key factors involved in theTh1mediated adaptive immune responses following bacterialinfection
4 Discussion
Smoking obesity diabetes mellitus and reproductive factorshave been considered as significant risk factors for PDM inprevious studies [10 24] The results of the present studyconfirmed that overweightobesity and the late onset ofmenarche were independent risk factors for PDM whilelate age at first birth had a protective effect In additionour results also suggested that nipple retraction was relatedto PDM Bacterial infection could be found in all samplesof PDM patients by 16S rRNA gene sequencing but not inthe samples from the control subjects Among the infectedsamples in PDM patients a mixture of different bacterialstrains was the most common condition which could beseen and then followed by singlePseudomonas spp infectionIFN-120574 and IL-12A were upregulated in PDM compared tonormal breast tissues but the expression of IL-17A was notsignificantly different from control samples Moreover thereare increased expression pattern for both IFN-120574 and IL-17Ain PDM which suggested a following immune response afterbacterial infection may contribute the deterioration of PDM
The incidence of PDM is increasing rapidly during thepast decades Indeed in our previous investigation the detec-tion rate of mammary ductal ectasia was 024 (14761102)[25] However the etiology of PDM is unclear yet mostly dueto lack of conclusive evidences fromprevious study Although
some studies suggest smoking is a risk factor for PDM [11 12]smoking was not found to be closely related to PDM in thepresent study One of the plausible reasons might be the lowprevalence of smoking among Chinese women [26]
Obesity is related to low-grade chronic inflammationwhich can disrupt immune function [27 28] Furthermoreobesity can directly influence local mammary estrogen andinflammation [29] Gollapalli et al [8] reported that obesitywas a risk factor for breast abscess Bharat et al [13] found thathigher BMI values were also associated with nonpuerperalabscessThey also reported similar results in cases of granulo-matous lobular mastitis In PDM however they did not finda significant effect of obesity on patients when comparedwithhealth controls [30] In our study data demonstrated obesityis another risk factor for PDM Obesity may directly disruptthe local immune functioning of the breast and exacerbate thedevelopment of PDM
The relationship between reproductive factors and PDMis very interesting Inconsistent with the finding reportedpreviously from literature in which parity was not found tobe associated with the risk for duct ectasia [31] our resultssuggest that reproductive factors were related to PDM Wealso found late onset of menarche was a significant risk factorfor PDM while increased age at first birth was a protec-tive factor The changes of serum prolactin and lactationin patients may be responsible for the above associationThe exact mechanism underlying this relationship is stillunknown future investigation focusing on theses hormonesis warranted
The relationship between nipple retraction and PDM hasbeen well-documented [32] Nipple retraction could inducebreast deformation and influence the process of lactationFurthermore it also can obstruct drainage and lead toan accumulation of massive substances in ducts which istermed mastitis [33] In this study we confirmed that nippleretraction was a risk factor for PDM The prevalence rateof nipple retraction is approximately 326 according toprevious report [34] But over one quarter of PDM patientsexperienced nipple retraction in our study
Bacterial infection is considered to be another possibleetiology of PDM although it has been a long-standing con-troversial problem Al Benwan et al [15] found that S aureuswas the predominant pathogen followed by Bacteroides sppAnaerobic strep and P aeruginosaHowever the relationshipbetween bacteria and PDM was not confirmed in otherreport [31] Here we provided more evidence to confirm forthis assumption Inconsistent with previous report [35] wedid not find that either S aureus was the most commonbacterium in PDM or anaerobic bacteria was associated withthis disease based on our current data Interestingly thebacteria identified here were those microorganisms whichare often related to community-acquired infection includingPseudomonas spp and S aureus Collectively these findingsuggests that bacterial infection may only be acting as aninduction factor and the immune system which are follow-ing the bacterial infection might play a major role in thedevelopment of PDM However more study is needed for thefuture to determine the common route in which the bacterialinfection and the following immune response converge
8 Mediators of Inflammation
IFN-120574 and IL-12A are characteristic Th1 cytokines [36]Th1 cells are important for the eradication of invadingpathogens including bacteria parasites yeast and viruses[37] In this study the expressions of IFN-120574 and IL-12A wereupregulated in breast stromal inflammatory cells of PDMpatients Recently a lineage of CD4+ T cells producing IL-17A was described and accordingly named as Th17 cellsIL-17A has been shown to be important for host defenseagainst pathogens [38] However in our study there was nosignificant difference in IL-17A expression between the twogroups As IFN-120574 and IL-12A were detected in PDM tissuesamples there is a rationale to conclude that theTh1 immuneresponse plays a role in this diseaseThemechanism bywhichthese cytokines contribute to the course of disease is stillunknown and needs further investigation
The strengths of the present study include the following(a) a relatively large population of patients was included (b)the assessment of tissue was done by two histopathologistsexperienced in breast inflammation However our study alsohad several limitations First this was a nonmatched casecontrol study and there might be some uncontrolled biaseswhich may influence the credibility of the results Secondthere was an issue with missing data especially the dataof reproductive factors which may also lead to the bias ofresults Finally we have not investigated the mechanism bywhich IFN-120574 and IL-12A participated in the course of thedisease
5 Conclusions
The results of the present study indicated that over-weightobesity and later onset of menarche are independentrisk factors for PDM In contrast age of first birth gt27years is a protective factor for PDM The importance ofthese reproductive factors to the risk of PDM has notbeen previously reported and will provide important novelinformation to broaden the avenue of investigating theetiology of PDM in the future We also confirm that bacterialinfection with a mixture of different bacterial species is aclosely associated with the pathogenesis of PDM Moreoverproinflammatory cytokines IFN-120574 and IL-12A were alsoshown to be associated with PDM which indicates that theTh1 immune response may closely relate to this diseaseTherefore PDM could be an inflammatory disease related tobacterial infection and consequent immune responses withincreased cytokines As far as we know this is first reportabout the possible immune responses following bacterialinfection which is involved in development of PDM diseaseand more research is needed for the future to furtherinvestigate the pathogenesis of PDM on the mechanism bywhich bacterial infection and consequent immune responsesaffect the progression of the disease
Ethical Approval
All procedures performed here involving human participantswere in accordance with the ethical standards of the SecondHospital of Shandong University Research Committee and
with the 1964Helsinki Declaration its later amendments andcomparable ethical standards
Consent
Informed consent was obtained from all participants includ-ed in the study
Competing Interests
All authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Dr Zhigang Yu and Dr Liang Li have equal contribution tothis article
Acknowledgments
Theauthors thank the patients and staffs at theDepartment ofBreast Surgery of SecondHospital of ShandongUniversity fortheir collaboration and supportThis study was funded by theYouth Fund of the Second Hospital of Shandong University(Y2014010030) Fei Zhou has received research grants fromthe Second Hospital of Shandong University
References
[1] K Ramalingam A Srivastava S Vuthaluru A Dhar and RChaudhry ldquoDuct Ectasia and periductal mastitis in Indianwomenrdquo Indian Journal of Surgery vol 77 supplement 3 pp957ndash962 2015
[2] J M Dixon ldquoPeriductal mastitisduct ectasiardquoWorld Journal ofSurgery vol 13 no 6 pp 715ndash720 1989
[3] X Zhang Y Lin Q Sun and H Huang ldquoDermo-glandularflap for treatment of recurrent periductal mastitisrdquo Journal ofSurgical Research vol 193 no 2 pp 738ndash744 2015
[4] R M S Rahal R De Freitas-Junior and R R Paulinelli ldquoRiskfactors for duct ectasiardquo Breast Journal vol 11 no 4 pp 262ndash265 2005
[5] R Mansel D Webster and H Sweetland Hughes Mansel ampWebsterrsquos Benign Disorders and Diseases of the Breast (ThirdEdition) Saunders Philadelphia Pa USA 2009
[6] M H Lequin J van Spengler R van Pel C van Eijck and HvanOverhagen ldquoMammographic and sonographic spectrum ofnon-puerperal mastitisrdquo European Journal of Radiology vol 21no 2 pp 138ndash142 1995
[7] R Tomczak A Rieber H Zeitler N Rilinger R Kreienbergand H J Brambs ldquoThe value of MR-mammography at 15 teslain the differential diagnosis of non-puerperal mastitis andinflammatory breast carcinomardquo RoFo Fortschritte auf demGebiete der Rontgenstrahlen und der Nuklearmedizin vol 165no 2 pp 148ndash151 1996
[8] V Gollapalli J Liao A Dudakovic S L Sugg C E H Scott-Conner and R J Weigel ldquoRisk factors for development andrecurrence of primary breast abscessesrdquo Journal of the AmericanCollege of Surgeons vol 211 no 1 pp 41ndash48 2010
Mediators of Inflammation 9
[9] R Risager and N Bentzon ldquoSmoking and increased risk ofmastitisrdquo Ugeskrift for Laeger vol 172 no 33 pp 2218ndash22212010
[10] H N Oltean A S Soliman O S Omar et al ldquoRisk factors forchronic mastitis in Morocco and Egyptrdquo International Journalof Inflammation vol 2013 Article ID 184921 10 pages 2013
[11] P Schafer C Furrer and B Mermillod ldquoAn association ofcigarette smoking with recurrent subareolar breast abscessrdquoInternational Journal of Epidemiology vol 17 no 4 pp 810ndash8131988
[12] V Sakka ldquoNonpuerperal breast infection epidemiology andpredictors for recurrencesrdquo Clinical Microbiology and Infectionvol 18 p 237 2012
[13] A Bharat F Gao R L Aft W E Gillanders T J Eberlein andJ A Margenthaler ldquoPredictors of primary breast abscesses andrecurrencerdquoWorld Journal of Surgery vol 33 no 12 pp 2582ndash2586 2009
[14] N J Bundred J M J Dixon A B Lumsden et al ldquoAre thelesions of duct ectasia sterilerdquo British Journal of Surgery vol72 no 10 pp 844ndash845 1985
[15] K Al Benwan A Al Mulla and V O Rotimi ldquoA study of themicrobiology of breast abscess in a teaching hospital in KuwaitrdquoMedical Principles and Practice vol 20 no 5 pp 422ndash426 2011
[16] P-L Giacalone G Rathat S Fournet and C Rouleau ldquoSurgicaltreatment of recurring subareolar abscess using oncoplastictechniquesrdquo Journal of Visceral Surgery vol 147 no 6 pp e389ndashe394 2010
[17] A Geremia P Biancheri P Allan G R Corazza and ADi Sabatino ldquoInnate and adaptive immunity in inflammatorybowel diseaserdquo Autoimmunity Reviews vol 13 no 1 pp 3ndash102014
[18] P Miossec and J K Kolls ldquoTargeting IL-17 and TH17 cells inchronic inflammationrdquo Nature Reviews Drug Discovery vol 11no 10 pp 763ndash776 2012
[19] Y Khosravi Y Dieye B H Poh et al ldquoCulturable bacterialmicrobiota of the stomach of helicobacter pylori positive andnegative gastric disease patientsrdquo The Scientific World Journalvol 2014 Article ID 610421 10 pages 2014
[20] R Marechal P Demetter N Nagy et al ldquoHigh expressionof CXCR4 may predict poor survival in resected pancreaticadenocarcinomardquo British Journal of Cancer vol 100 no 9 pp1444ndash1451 2009
[21] M Yao Y Wu Q Fang L Sun T Li and H Qiao ldquoAssociationof ADIPOQ variants with type 2 diabetes mellitus susceptibilityin ethnic han Chinese from Northeast Chinardquo Journal ofDiabetes Investigation vol 7 no 6 pp 853ndash859 2016
[22] Q Zhang L-Y Liu FWang KMu and Z-G Yu ldquoThe changesin female physical and childbearing characteristics in china andpotential association with risk of breast cancerrdquo BMC PublicHealth vol 12 article 368 2012
[23] J Kotsopoulos J Lubinski L Salmena et al ldquoBreastfeedingand the risk of breast cancer in BRCA1 and BRCA2 mutationcarriersrdquo Breast Cancer Research vol 14 no 2 article R42 2012
[24] Y Dong J-J Yu Y Shibahara et al ldquoIntercellular adhesionmolecule 12 and E-selectin in plasma cell mastitis immuno-histochemical study of 35 casesrdquo Human Pathology vol 45 no3 pp 606ndash610 2014
[25] Y Y Li J J Du Q Liu and Z G Yu ldquoScreening for breastdiseases among 61102 women in Shandong Provincerdquo Journalof Shandong University vol 49 no 8 pp 157ndash160 2011
[26] Y-M Xu H-H Chen F Li et al ldquoPrevalence and correlatesof cigarette smoking among Chinese schizophrenia inpatientsreceiving antipsychotic mono-therapyrdquo PLOS ONE vol 9 no2 Article ID e88478 2014
[27] F M Wensveen S Valentic M Sestan T Turk Wensveen andB Polic ldquoThe ldquoBig Bangrdquo in obese fat events initiating obesity-induced adipose tissue inflammationrdquo European Journal ofImmunology vol 45 no 9 pp 2446ndash2456 2015
[28] Y B Shaik-Dasthagirisaheb A Kantarci and F C Gibson IIIldquoImmune response of macrophages from young and aged miceto the oral pathogenic bacterium Porphyromonas gingivalisrdquoImmunity amp Ageing vol 7 article 15 2010
[29] K A Brown ldquoImpact of obesity on mammary gland inflamma-tion and local estrogen productionrdquo Journal ofMammary GlandBiology and Neoplasia vol 19 no 2 pp 183ndash189 2014
[30] B Al-Khaffaf F Knox and N J Bundred ldquoIdiopathic granulo-matous mastitis a 25-year experiencerdquo Journal of the AmericanCollege of Surgeons vol 206 no 2 pp 269ndash273 2008
[31] R M S Rahal R F Junior C Reis F C Pimenta J C ANetto and R R Paulinelli ldquoPrevalence of bacteria in the nippledischarge of patients with duct ectasiardquo International Journal ofClinical Practice vol 59 no 9 pp 1045ndash1050 2005
[32] M N Hartley J Stewart and E A Benson ldquoSubareolardissection for duct ectasia and periareolar sepsisrdquo The BritishJournal of Surgery vol 78 no 10 pp 1187ndash1188 1991
[33] J Ming G Meng Q Yuan et al ldquoClinical characteristics andsurgical modality of plasma cell mastitis analysis of 91 casesrdquoThe American Surgeon vol 79 no 1 pp 54ndash60 2013
[34] H S Park C H Yoon and H J Kim ldquoThe prevalence ofcongenital inverted nipplerdquoAesthetic Plastic Surgery vol 23 no2 pp 144ndash146 1999
[35] F N L Versluijs-Ossewaarde R M H Roumen and R J AGoris ldquoSubareolar breast abscesses characteristics and resultsof surgical treatmentrdquoThe Breast Journal vol 11 no 3 pp 179ndash182 2005
[36] Y Luo H Yamada D P Evanoff and X Chen ldquoRole ofTh1-stimulating cytokines in bacillus Calmette-Guerin (BCG)-inducedmacrophage cytotoxicity against mouse bladder cancerMBT-2 cellsrdquo Clinical and Experimental Immunology vol 146no 1 pp 181ndash188 2006
[37] K J Rautajoki M K Kylaniemi S K Raghav K Rao and RLahesmaa ldquoAn insight into molecular mechanisms of human Thelper cell differentiationrdquoAnnals of Medicine vol 40 no 5 pp322ndash335 2008
[38] B Stockinger and M Veldhoen ldquoDifferentiation and functionof Th17 T cellsrdquo Current Opinion in Immunology vol 19 no 3pp 281ndash286 2007
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Mediators of Inflammation 5
Table 3 Univariate analysis of selected risk factors for PDMlowast
Variable Cases (119899 = 87) Controls (119899 = 87) OR (95 CI) 119875
Age (years)lt35 48 (5517) 56 (6437) 10 (Ref) 0217ge35 39 (4483) 31 (3563) 147 (080ndash270)
BMI (kgm2)lt24 34 (3908) 65 (7471) 10 (Ref)
0002ge24 34 (3908) 20 (2299) 325 (163ndash648)Unknown 19 (2184) 2 (230)
Age at menarche (years)le14 57 (6552) 74 (8506) 10 (Ref)
0001gt14 30 (3448) 10 (1149) 390 (176ndash862)Unknown 0 (000) 3 (345)
Parityle1 64 (7356) 72 (8276) 10 (Ref)
0019gt1 22 (2529) 9 (1034) 275 (118ndash641)Unknown 1 (115) 6 (690)
MiscarriagesNo 23 (2644) 25 (2874) 10 (Ref)
0470Yes 25 (2874) 36 (4138) 076 (035ndash162)Unknown 39 (4482) 26 (2988)
Age at birth of first child (years)le27 34 (3908) 26 (2989) 10 (Ref)
0001gt27 8 (920) 29 (3333) 021 (008ndash054)Unknown 45 (5172) 32 (3678)
Duration of breastfeeding (months)le12 34 (3908) 42 (4828) 10 (Ref)
0688gt12 36 (4138) 39 (4483) 114 (060ndash216)Unknown 17 (1954) 6 (689)
History of benign breast diseaseYes 6 (690) 3 (345) 454 (107ndash1914)
0039No 37 (4253) 84 (9555) 10 (Ref)Unknown 44 (5057) 0 (000)
GalactostasisYes 23 (2644) 32 (3678) 086 (044ndash170)
0670No 40 (4598) 48 (5517) 10 (Ref)Unknown 24 (2758) 7 (805)
Nipple retractionYes 24 (2759) 1 (115) 3276 (432ndash24861) 0001No 63 (7241) 86 (9885) 10 (Ref)
Note Data are number () of patients BMI body mass index calculated as weight in kilograms divided by the square of height in meters OR odd ratio CIconfidence intervallowastUsing binary logistic regression
Table 4 Multivariate logistic regression analysis of the associations between various factors and PDM
Variable B SE Wald OR 95 CI 119875
Overweightobesity 031 012 700 136 108ndash170 0008Age at first birth minus170 086 392 018 003ndash098 0048Age at menarche 088 029 953 241 138ndash421 0002
6 Mediators of Inflammation
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 2 Representative illustrations of the expression of cytokines in PDM and normal breast tissues (a) Low-powermagnification of PDM(hematoxylin and eosin times40) (b) Low expression of IFN-120574 (IHC times200) (c) High expression of IFN-120574 (IHC times200) (d) Low expression ofIL-12A (IHC times100) (e) High expression of IFN-120574 (IHC times200) (f) Low expression of IL-17A (IHC times100) (g) High expression of IL-17A(IHC times200) (h) The expression of inflammatory cytokines in normal breast ductal epithelium and stromal cells (IHC times200)
Table 5 Multiple bacteria could be detected in PDM patients
Bacteria 119873 () (119899 = 31)Mixture of different bacteria 12 (3871)Pseudomonas
Pseudomonas aeruginosa 5 (1613)Uncultivated Pseudomonas 2 (645)Pseudomonas delhiensis 1 (323)Pseudomonas otitidis 1 (323)
Enterococcus faecium 2 (645)Corynebacterium kroppenstedtii 1 (323)Bacillus firmus 1 (323)Sporosarcina 1 (323)Staphylococcus aureus 1 (323)Uncultured bacterium clone 4 (1290)
following immune responses IFN-120574 IL-12A and IL-17Awere involved in the disease progressionwith an IHCmethod(Figure 2) After staining and taking photographs a scoringevaluation of the density for each cytokine in this IHCphotographs was performed and data were summarized inTable 6 As shown in Table 7 the expressions of IFN-120574 and IL-12A in stromal inflammatory cells of the breast were increasedin PDM compared to normal breast tissues However therewere no significant differences in the expression of IL-17Abetween these two groups
To investigate the cytokines expression profiles at differ-ent stages of PDM the expressions of IFN-120574 IL-12A and IL-17A were then analyzed by grouping with the presence orabsence of abscess Fifteen patients with abscess formationin their lesions and 16 patients without abscess formationwere included in this study As shown in the Figure 3higher expression levels of IFN-120574 and IL-17A were found
Table 6 The expressions of IFN-120574 IL-12A and IL-17A in PDMcompared with normal breast tissues
Score IFN-120574 IL-12A IL-17ACase Control Case Control Case Control
0 2 1 0 2 1 21 2 3 0 3 10 12 5 13 11 12 8 103 1 2 6 2 1 44 8 3 7 1 10 36 9 0 4 1 0 28 0 0 0 0 0 09 4 0 2 1 0 0Absent 0 0 1 0 1 0Samples were lost in the process of IHC
Table 7 The correlationship of IFN-120574 IL-12A and IL-17A expres-sion in PDM compared with normal breast tissues
Cytokines Case Control 119875lowast
Low expression of IFN-120574 10 19lt0001
High expression of IFN-120574 21 3Low expression of IL-12A 13 19 0022High expression of IL-12A 17 3Low expression of IL-17A 20 17 0404High expression of IL-17A 10 5lowast119875 values were determined with the chi-square test
Low expression was defined as the total score le 3 and others were definedas high expression
in stromal inflammatory cells from PDM patients withabscess formation than those without abscess formationThese results suggest that proinflammatory cytokines may
Mediators of Inflammation 7
0
5
10
15
Cases with abscess formationCases without abscess formation
Num
ber o
f cas
es
lowast lowast lowast
IFN
-120574(minus
)
IFN
-120574(+
)
IL-17
A(minus
)
IL-17
A(+
)
IL-12
A(minus
)
IL-12
A(+
)
Figure 3 Production of IFN-120574 IL-12A and IL-17A in cases withor without abscess formation (minus) low expression of cytokines(+) high expression of cytokines Fifteen patients had lesions withabscess formation while 16 patients did not have them More caseswith high expressions of IFN-120574 and IL-17A in stromal inflammatorycells could be found in patients with abscess formation lowast119875 gt 005
play an important role in the progression of the diseaseof PDM especially the IFN-120574 and IL-12A As mentionedabove these two cytokines are key factors involved in theTh1mediated adaptive immune responses following bacterialinfection
4 Discussion
Smoking obesity diabetes mellitus and reproductive factorshave been considered as significant risk factors for PDM inprevious studies [10 24] The results of the present studyconfirmed that overweightobesity and the late onset ofmenarche were independent risk factors for PDM whilelate age at first birth had a protective effect In additionour results also suggested that nipple retraction was relatedto PDM Bacterial infection could be found in all samplesof PDM patients by 16S rRNA gene sequencing but not inthe samples from the control subjects Among the infectedsamples in PDM patients a mixture of different bacterialstrains was the most common condition which could beseen and then followed by singlePseudomonas spp infectionIFN-120574 and IL-12A were upregulated in PDM compared tonormal breast tissues but the expression of IL-17A was notsignificantly different from control samples Moreover thereare increased expression pattern for both IFN-120574 and IL-17Ain PDM which suggested a following immune response afterbacterial infection may contribute the deterioration of PDM
The incidence of PDM is increasing rapidly during thepast decades Indeed in our previous investigation the detec-tion rate of mammary ductal ectasia was 024 (14761102)[25] However the etiology of PDM is unclear yet mostly dueto lack of conclusive evidences fromprevious study Although
some studies suggest smoking is a risk factor for PDM [11 12]smoking was not found to be closely related to PDM in thepresent study One of the plausible reasons might be the lowprevalence of smoking among Chinese women [26]
Obesity is related to low-grade chronic inflammationwhich can disrupt immune function [27 28] Furthermoreobesity can directly influence local mammary estrogen andinflammation [29] Gollapalli et al [8] reported that obesitywas a risk factor for breast abscess Bharat et al [13] found thathigher BMI values were also associated with nonpuerperalabscessThey also reported similar results in cases of granulo-matous lobular mastitis In PDM however they did not finda significant effect of obesity on patients when comparedwithhealth controls [30] In our study data demonstrated obesityis another risk factor for PDM Obesity may directly disruptthe local immune functioning of the breast and exacerbate thedevelopment of PDM
The relationship between reproductive factors and PDMis very interesting Inconsistent with the finding reportedpreviously from literature in which parity was not found tobe associated with the risk for duct ectasia [31] our resultssuggest that reproductive factors were related to PDM Wealso found late onset of menarche was a significant risk factorfor PDM while increased age at first birth was a protec-tive factor The changes of serum prolactin and lactationin patients may be responsible for the above associationThe exact mechanism underlying this relationship is stillunknown future investigation focusing on theses hormonesis warranted
The relationship between nipple retraction and PDM hasbeen well-documented [32] Nipple retraction could inducebreast deformation and influence the process of lactationFurthermore it also can obstruct drainage and lead toan accumulation of massive substances in ducts which istermed mastitis [33] In this study we confirmed that nippleretraction was a risk factor for PDM The prevalence rateof nipple retraction is approximately 326 according toprevious report [34] But over one quarter of PDM patientsexperienced nipple retraction in our study
Bacterial infection is considered to be another possibleetiology of PDM although it has been a long-standing con-troversial problem Al Benwan et al [15] found that S aureuswas the predominant pathogen followed by Bacteroides sppAnaerobic strep and P aeruginosaHowever the relationshipbetween bacteria and PDM was not confirmed in otherreport [31] Here we provided more evidence to confirm forthis assumption Inconsistent with previous report [35] wedid not find that either S aureus was the most commonbacterium in PDM or anaerobic bacteria was associated withthis disease based on our current data Interestingly thebacteria identified here were those microorganisms whichare often related to community-acquired infection includingPseudomonas spp and S aureus Collectively these findingsuggests that bacterial infection may only be acting as aninduction factor and the immune system which are follow-ing the bacterial infection might play a major role in thedevelopment of PDM However more study is needed for thefuture to determine the common route in which the bacterialinfection and the following immune response converge
8 Mediators of Inflammation
IFN-120574 and IL-12A are characteristic Th1 cytokines [36]Th1 cells are important for the eradication of invadingpathogens including bacteria parasites yeast and viruses[37] In this study the expressions of IFN-120574 and IL-12A wereupregulated in breast stromal inflammatory cells of PDMpatients Recently a lineage of CD4+ T cells producing IL-17A was described and accordingly named as Th17 cellsIL-17A has been shown to be important for host defenseagainst pathogens [38] However in our study there was nosignificant difference in IL-17A expression between the twogroups As IFN-120574 and IL-12A were detected in PDM tissuesamples there is a rationale to conclude that theTh1 immuneresponse plays a role in this diseaseThemechanism bywhichthese cytokines contribute to the course of disease is stillunknown and needs further investigation
The strengths of the present study include the following(a) a relatively large population of patients was included (b)the assessment of tissue was done by two histopathologistsexperienced in breast inflammation However our study alsohad several limitations First this was a nonmatched casecontrol study and there might be some uncontrolled biaseswhich may influence the credibility of the results Secondthere was an issue with missing data especially the dataof reproductive factors which may also lead to the bias ofresults Finally we have not investigated the mechanism bywhich IFN-120574 and IL-12A participated in the course of thedisease
5 Conclusions
The results of the present study indicated that over-weightobesity and later onset of menarche are independentrisk factors for PDM In contrast age of first birth gt27years is a protective factor for PDM The importance ofthese reproductive factors to the risk of PDM has notbeen previously reported and will provide important novelinformation to broaden the avenue of investigating theetiology of PDM in the future We also confirm that bacterialinfection with a mixture of different bacterial species is aclosely associated with the pathogenesis of PDM Moreoverproinflammatory cytokines IFN-120574 and IL-12A were alsoshown to be associated with PDM which indicates that theTh1 immune response may closely relate to this diseaseTherefore PDM could be an inflammatory disease related tobacterial infection and consequent immune responses withincreased cytokines As far as we know this is first reportabout the possible immune responses following bacterialinfection which is involved in development of PDM diseaseand more research is needed for the future to furtherinvestigate the pathogenesis of PDM on the mechanism bywhich bacterial infection and consequent immune responsesaffect the progression of the disease
Ethical Approval
All procedures performed here involving human participantswere in accordance with the ethical standards of the SecondHospital of Shandong University Research Committee and
with the 1964Helsinki Declaration its later amendments andcomparable ethical standards
Consent
Informed consent was obtained from all participants includ-ed in the study
Competing Interests
All authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Dr Zhigang Yu and Dr Liang Li have equal contribution tothis article
Acknowledgments
Theauthors thank the patients and staffs at theDepartment ofBreast Surgery of SecondHospital of ShandongUniversity fortheir collaboration and supportThis study was funded by theYouth Fund of the Second Hospital of Shandong University(Y2014010030) Fei Zhou has received research grants fromthe Second Hospital of Shandong University
References
[1] K Ramalingam A Srivastava S Vuthaluru A Dhar and RChaudhry ldquoDuct Ectasia and periductal mastitis in Indianwomenrdquo Indian Journal of Surgery vol 77 supplement 3 pp957ndash962 2015
[2] J M Dixon ldquoPeriductal mastitisduct ectasiardquoWorld Journal ofSurgery vol 13 no 6 pp 715ndash720 1989
[3] X Zhang Y Lin Q Sun and H Huang ldquoDermo-glandularflap for treatment of recurrent periductal mastitisrdquo Journal ofSurgical Research vol 193 no 2 pp 738ndash744 2015
[4] R M S Rahal R De Freitas-Junior and R R Paulinelli ldquoRiskfactors for duct ectasiardquo Breast Journal vol 11 no 4 pp 262ndash265 2005
[5] R Mansel D Webster and H Sweetland Hughes Mansel ampWebsterrsquos Benign Disorders and Diseases of the Breast (ThirdEdition) Saunders Philadelphia Pa USA 2009
[6] M H Lequin J van Spengler R van Pel C van Eijck and HvanOverhagen ldquoMammographic and sonographic spectrum ofnon-puerperal mastitisrdquo European Journal of Radiology vol 21no 2 pp 138ndash142 1995
[7] R Tomczak A Rieber H Zeitler N Rilinger R Kreienbergand H J Brambs ldquoThe value of MR-mammography at 15 teslain the differential diagnosis of non-puerperal mastitis andinflammatory breast carcinomardquo RoFo Fortschritte auf demGebiete der Rontgenstrahlen und der Nuklearmedizin vol 165no 2 pp 148ndash151 1996
[8] V Gollapalli J Liao A Dudakovic S L Sugg C E H Scott-Conner and R J Weigel ldquoRisk factors for development andrecurrence of primary breast abscessesrdquo Journal of the AmericanCollege of Surgeons vol 211 no 1 pp 41ndash48 2010
Mediators of Inflammation 9
[9] R Risager and N Bentzon ldquoSmoking and increased risk ofmastitisrdquo Ugeskrift for Laeger vol 172 no 33 pp 2218ndash22212010
[10] H N Oltean A S Soliman O S Omar et al ldquoRisk factors forchronic mastitis in Morocco and Egyptrdquo International Journalof Inflammation vol 2013 Article ID 184921 10 pages 2013
[11] P Schafer C Furrer and B Mermillod ldquoAn association ofcigarette smoking with recurrent subareolar breast abscessrdquoInternational Journal of Epidemiology vol 17 no 4 pp 810ndash8131988
[12] V Sakka ldquoNonpuerperal breast infection epidemiology andpredictors for recurrencesrdquo Clinical Microbiology and Infectionvol 18 p 237 2012
[13] A Bharat F Gao R L Aft W E Gillanders T J Eberlein andJ A Margenthaler ldquoPredictors of primary breast abscesses andrecurrencerdquoWorld Journal of Surgery vol 33 no 12 pp 2582ndash2586 2009
[14] N J Bundred J M J Dixon A B Lumsden et al ldquoAre thelesions of duct ectasia sterilerdquo British Journal of Surgery vol72 no 10 pp 844ndash845 1985
[15] K Al Benwan A Al Mulla and V O Rotimi ldquoA study of themicrobiology of breast abscess in a teaching hospital in KuwaitrdquoMedical Principles and Practice vol 20 no 5 pp 422ndash426 2011
[16] P-L Giacalone G Rathat S Fournet and C Rouleau ldquoSurgicaltreatment of recurring subareolar abscess using oncoplastictechniquesrdquo Journal of Visceral Surgery vol 147 no 6 pp e389ndashe394 2010
[17] A Geremia P Biancheri P Allan G R Corazza and ADi Sabatino ldquoInnate and adaptive immunity in inflammatorybowel diseaserdquo Autoimmunity Reviews vol 13 no 1 pp 3ndash102014
[18] P Miossec and J K Kolls ldquoTargeting IL-17 and TH17 cells inchronic inflammationrdquo Nature Reviews Drug Discovery vol 11no 10 pp 763ndash776 2012
[19] Y Khosravi Y Dieye B H Poh et al ldquoCulturable bacterialmicrobiota of the stomach of helicobacter pylori positive andnegative gastric disease patientsrdquo The Scientific World Journalvol 2014 Article ID 610421 10 pages 2014
[20] R Marechal P Demetter N Nagy et al ldquoHigh expressionof CXCR4 may predict poor survival in resected pancreaticadenocarcinomardquo British Journal of Cancer vol 100 no 9 pp1444ndash1451 2009
[21] M Yao Y Wu Q Fang L Sun T Li and H Qiao ldquoAssociationof ADIPOQ variants with type 2 diabetes mellitus susceptibilityin ethnic han Chinese from Northeast Chinardquo Journal ofDiabetes Investigation vol 7 no 6 pp 853ndash859 2016
[22] Q Zhang L-Y Liu FWang KMu and Z-G Yu ldquoThe changesin female physical and childbearing characteristics in china andpotential association with risk of breast cancerrdquo BMC PublicHealth vol 12 article 368 2012
[23] J Kotsopoulos J Lubinski L Salmena et al ldquoBreastfeedingand the risk of breast cancer in BRCA1 and BRCA2 mutationcarriersrdquo Breast Cancer Research vol 14 no 2 article R42 2012
[24] Y Dong J-J Yu Y Shibahara et al ldquoIntercellular adhesionmolecule 12 and E-selectin in plasma cell mastitis immuno-histochemical study of 35 casesrdquo Human Pathology vol 45 no3 pp 606ndash610 2014
[25] Y Y Li J J Du Q Liu and Z G Yu ldquoScreening for breastdiseases among 61102 women in Shandong Provincerdquo Journalof Shandong University vol 49 no 8 pp 157ndash160 2011
[26] Y-M Xu H-H Chen F Li et al ldquoPrevalence and correlatesof cigarette smoking among Chinese schizophrenia inpatientsreceiving antipsychotic mono-therapyrdquo PLOS ONE vol 9 no2 Article ID e88478 2014
[27] F M Wensveen S Valentic M Sestan T Turk Wensveen andB Polic ldquoThe ldquoBig Bangrdquo in obese fat events initiating obesity-induced adipose tissue inflammationrdquo European Journal ofImmunology vol 45 no 9 pp 2446ndash2456 2015
[28] Y B Shaik-Dasthagirisaheb A Kantarci and F C Gibson IIIldquoImmune response of macrophages from young and aged miceto the oral pathogenic bacterium Porphyromonas gingivalisrdquoImmunity amp Ageing vol 7 article 15 2010
[29] K A Brown ldquoImpact of obesity on mammary gland inflamma-tion and local estrogen productionrdquo Journal ofMammary GlandBiology and Neoplasia vol 19 no 2 pp 183ndash189 2014
[30] B Al-Khaffaf F Knox and N J Bundred ldquoIdiopathic granulo-matous mastitis a 25-year experiencerdquo Journal of the AmericanCollege of Surgeons vol 206 no 2 pp 269ndash273 2008
[31] R M S Rahal R F Junior C Reis F C Pimenta J C ANetto and R R Paulinelli ldquoPrevalence of bacteria in the nippledischarge of patients with duct ectasiardquo International Journal ofClinical Practice vol 59 no 9 pp 1045ndash1050 2005
[32] M N Hartley J Stewart and E A Benson ldquoSubareolardissection for duct ectasia and periareolar sepsisrdquo The BritishJournal of Surgery vol 78 no 10 pp 1187ndash1188 1991
[33] J Ming G Meng Q Yuan et al ldquoClinical characteristics andsurgical modality of plasma cell mastitis analysis of 91 casesrdquoThe American Surgeon vol 79 no 1 pp 54ndash60 2013
[34] H S Park C H Yoon and H J Kim ldquoThe prevalence ofcongenital inverted nipplerdquoAesthetic Plastic Surgery vol 23 no2 pp 144ndash146 1999
[35] F N L Versluijs-Ossewaarde R M H Roumen and R J AGoris ldquoSubareolar breast abscesses characteristics and resultsof surgical treatmentrdquoThe Breast Journal vol 11 no 3 pp 179ndash182 2005
[36] Y Luo H Yamada D P Evanoff and X Chen ldquoRole ofTh1-stimulating cytokines in bacillus Calmette-Guerin (BCG)-inducedmacrophage cytotoxicity against mouse bladder cancerMBT-2 cellsrdquo Clinical and Experimental Immunology vol 146no 1 pp 181ndash188 2006
[37] K J Rautajoki M K Kylaniemi S K Raghav K Rao and RLahesmaa ldquoAn insight into molecular mechanisms of human Thelper cell differentiationrdquoAnnals of Medicine vol 40 no 5 pp322ndash335 2008
[38] B Stockinger and M Veldhoen ldquoDifferentiation and functionof Th17 T cellsrdquo Current Opinion in Immunology vol 19 no 3pp 281ndash286 2007
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Mediators of Inflammation
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 2 Representative illustrations of the expression of cytokines in PDM and normal breast tissues (a) Low-powermagnification of PDM(hematoxylin and eosin times40) (b) Low expression of IFN-120574 (IHC times200) (c) High expression of IFN-120574 (IHC times200) (d) Low expression ofIL-12A (IHC times100) (e) High expression of IFN-120574 (IHC times200) (f) Low expression of IL-17A (IHC times100) (g) High expression of IL-17A(IHC times200) (h) The expression of inflammatory cytokines in normal breast ductal epithelium and stromal cells (IHC times200)
Table 5 Multiple bacteria could be detected in PDM patients
Bacteria 119873 () (119899 = 31)Mixture of different bacteria 12 (3871)Pseudomonas
Pseudomonas aeruginosa 5 (1613)Uncultivated Pseudomonas 2 (645)Pseudomonas delhiensis 1 (323)Pseudomonas otitidis 1 (323)
Enterococcus faecium 2 (645)Corynebacterium kroppenstedtii 1 (323)Bacillus firmus 1 (323)Sporosarcina 1 (323)Staphylococcus aureus 1 (323)Uncultured bacterium clone 4 (1290)
following immune responses IFN-120574 IL-12A and IL-17Awere involved in the disease progressionwith an IHCmethod(Figure 2) After staining and taking photographs a scoringevaluation of the density for each cytokine in this IHCphotographs was performed and data were summarized inTable 6 As shown in Table 7 the expressions of IFN-120574 and IL-12A in stromal inflammatory cells of the breast were increasedin PDM compared to normal breast tissues However therewere no significant differences in the expression of IL-17Abetween these two groups
To investigate the cytokines expression profiles at differ-ent stages of PDM the expressions of IFN-120574 IL-12A and IL-17A were then analyzed by grouping with the presence orabsence of abscess Fifteen patients with abscess formationin their lesions and 16 patients without abscess formationwere included in this study As shown in the Figure 3higher expression levels of IFN-120574 and IL-17A were found
Table 6 The expressions of IFN-120574 IL-12A and IL-17A in PDMcompared with normal breast tissues
Score IFN-120574 IL-12A IL-17ACase Control Case Control Case Control
0 2 1 0 2 1 21 2 3 0 3 10 12 5 13 11 12 8 103 1 2 6 2 1 44 8 3 7 1 10 36 9 0 4 1 0 28 0 0 0 0 0 09 4 0 2 1 0 0Absent 0 0 1 0 1 0Samples were lost in the process of IHC
Table 7 The correlationship of IFN-120574 IL-12A and IL-17A expres-sion in PDM compared with normal breast tissues
Cytokines Case Control 119875lowast
Low expression of IFN-120574 10 19lt0001
High expression of IFN-120574 21 3Low expression of IL-12A 13 19 0022High expression of IL-12A 17 3Low expression of IL-17A 20 17 0404High expression of IL-17A 10 5lowast119875 values were determined with the chi-square test
Low expression was defined as the total score le 3 and others were definedas high expression
in stromal inflammatory cells from PDM patients withabscess formation than those without abscess formationThese results suggest that proinflammatory cytokines may
Mediators of Inflammation 7
0
5
10
15
Cases with abscess formationCases without abscess formation
Num
ber o
f cas
es
lowast lowast lowast
IFN
-120574(minus
)
IFN
-120574(+
)
IL-17
A(minus
)
IL-17
A(+
)
IL-12
A(minus
)
IL-12
A(+
)
Figure 3 Production of IFN-120574 IL-12A and IL-17A in cases withor without abscess formation (minus) low expression of cytokines(+) high expression of cytokines Fifteen patients had lesions withabscess formation while 16 patients did not have them More caseswith high expressions of IFN-120574 and IL-17A in stromal inflammatorycells could be found in patients with abscess formation lowast119875 gt 005
play an important role in the progression of the diseaseof PDM especially the IFN-120574 and IL-12A As mentionedabove these two cytokines are key factors involved in theTh1mediated adaptive immune responses following bacterialinfection
4 Discussion
Smoking obesity diabetes mellitus and reproductive factorshave been considered as significant risk factors for PDM inprevious studies [10 24] The results of the present studyconfirmed that overweightobesity and the late onset ofmenarche were independent risk factors for PDM whilelate age at first birth had a protective effect In additionour results also suggested that nipple retraction was relatedto PDM Bacterial infection could be found in all samplesof PDM patients by 16S rRNA gene sequencing but not inthe samples from the control subjects Among the infectedsamples in PDM patients a mixture of different bacterialstrains was the most common condition which could beseen and then followed by singlePseudomonas spp infectionIFN-120574 and IL-12A were upregulated in PDM compared tonormal breast tissues but the expression of IL-17A was notsignificantly different from control samples Moreover thereare increased expression pattern for both IFN-120574 and IL-17Ain PDM which suggested a following immune response afterbacterial infection may contribute the deterioration of PDM
The incidence of PDM is increasing rapidly during thepast decades Indeed in our previous investigation the detec-tion rate of mammary ductal ectasia was 024 (14761102)[25] However the etiology of PDM is unclear yet mostly dueto lack of conclusive evidences fromprevious study Although
some studies suggest smoking is a risk factor for PDM [11 12]smoking was not found to be closely related to PDM in thepresent study One of the plausible reasons might be the lowprevalence of smoking among Chinese women [26]
Obesity is related to low-grade chronic inflammationwhich can disrupt immune function [27 28] Furthermoreobesity can directly influence local mammary estrogen andinflammation [29] Gollapalli et al [8] reported that obesitywas a risk factor for breast abscess Bharat et al [13] found thathigher BMI values were also associated with nonpuerperalabscessThey also reported similar results in cases of granulo-matous lobular mastitis In PDM however they did not finda significant effect of obesity on patients when comparedwithhealth controls [30] In our study data demonstrated obesityis another risk factor for PDM Obesity may directly disruptthe local immune functioning of the breast and exacerbate thedevelopment of PDM
The relationship between reproductive factors and PDMis very interesting Inconsistent with the finding reportedpreviously from literature in which parity was not found tobe associated with the risk for duct ectasia [31] our resultssuggest that reproductive factors were related to PDM Wealso found late onset of menarche was a significant risk factorfor PDM while increased age at first birth was a protec-tive factor The changes of serum prolactin and lactationin patients may be responsible for the above associationThe exact mechanism underlying this relationship is stillunknown future investigation focusing on theses hormonesis warranted
The relationship between nipple retraction and PDM hasbeen well-documented [32] Nipple retraction could inducebreast deformation and influence the process of lactationFurthermore it also can obstruct drainage and lead toan accumulation of massive substances in ducts which istermed mastitis [33] In this study we confirmed that nippleretraction was a risk factor for PDM The prevalence rateof nipple retraction is approximately 326 according toprevious report [34] But over one quarter of PDM patientsexperienced nipple retraction in our study
Bacterial infection is considered to be another possibleetiology of PDM although it has been a long-standing con-troversial problem Al Benwan et al [15] found that S aureuswas the predominant pathogen followed by Bacteroides sppAnaerobic strep and P aeruginosaHowever the relationshipbetween bacteria and PDM was not confirmed in otherreport [31] Here we provided more evidence to confirm forthis assumption Inconsistent with previous report [35] wedid not find that either S aureus was the most commonbacterium in PDM or anaerobic bacteria was associated withthis disease based on our current data Interestingly thebacteria identified here were those microorganisms whichare often related to community-acquired infection includingPseudomonas spp and S aureus Collectively these findingsuggests that bacterial infection may only be acting as aninduction factor and the immune system which are follow-ing the bacterial infection might play a major role in thedevelopment of PDM However more study is needed for thefuture to determine the common route in which the bacterialinfection and the following immune response converge
8 Mediators of Inflammation
IFN-120574 and IL-12A are characteristic Th1 cytokines [36]Th1 cells are important for the eradication of invadingpathogens including bacteria parasites yeast and viruses[37] In this study the expressions of IFN-120574 and IL-12A wereupregulated in breast stromal inflammatory cells of PDMpatients Recently a lineage of CD4+ T cells producing IL-17A was described and accordingly named as Th17 cellsIL-17A has been shown to be important for host defenseagainst pathogens [38] However in our study there was nosignificant difference in IL-17A expression between the twogroups As IFN-120574 and IL-12A were detected in PDM tissuesamples there is a rationale to conclude that theTh1 immuneresponse plays a role in this diseaseThemechanism bywhichthese cytokines contribute to the course of disease is stillunknown and needs further investigation
The strengths of the present study include the following(a) a relatively large population of patients was included (b)the assessment of tissue was done by two histopathologistsexperienced in breast inflammation However our study alsohad several limitations First this was a nonmatched casecontrol study and there might be some uncontrolled biaseswhich may influence the credibility of the results Secondthere was an issue with missing data especially the dataof reproductive factors which may also lead to the bias ofresults Finally we have not investigated the mechanism bywhich IFN-120574 and IL-12A participated in the course of thedisease
5 Conclusions
The results of the present study indicated that over-weightobesity and later onset of menarche are independentrisk factors for PDM In contrast age of first birth gt27years is a protective factor for PDM The importance ofthese reproductive factors to the risk of PDM has notbeen previously reported and will provide important novelinformation to broaden the avenue of investigating theetiology of PDM in the future We also confirm that bacterialinfection with a mixture of different bacterial species is aclosely associated with the pathogenesis of PDM Moreoverproinflammatory cytokines IFN-120574 and IL-12A were alsoshown to be associated with PDM which indicates that theTh1 immune response may closely relate to this diseaseTherefore PDM could be an inflammatory disease related tobacterial infection and consequent immune responses withincreased cytokines As far as we know this is first reportabout the possible immune responses following bacterialinfection which is involved in development of PDM diseaseand more research is needed for the future to furtherinvestigate the pathogenesis of PDM on the mechanism bywhich bacterial infection and consequent immune responsesaffect the progression of the disease
Ethical Approval
All procedures performed here involving human participantswere in accordance with the ethical standards of the SecondHospital of Shandong University Research Committee and
with the 1964Helsinki Declaration its later amendments andcomparable ethical standards
Consent
Informed consent was obtained from all participants includ-ed in the study
Competing Interests
All authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Dr Zhigang Yu and Dr Liang Li have equal contribution tothis article
Acknowledgments
Theauthors thank the patients and staffs at theDepartment ofBreast Surgery of SecondHospital of ShandongUniversity fortheir collaboration and supportThis study was funded by theYouth Fund of the Second Hospital of Shandong University(Y2014010030) Fei Zhou has received research grants fromthe Second Hospital of Shandong University
References
[1] K Ramalingam A Srivastava S Vuthaluru A Dhar and RChaudhry ldquoDuct Ectasia and periductal mastitis in Indianwomenrdquo Indian Journal of Surgery vol 77 supplement 3 pp957ndash962 2015
[2] J M Dixon ldquoPeriductal mastitisduct ectasiardquoWorld Journal ofSurgery vol 13 no 6 pp 715ndash720 1989
[3] X Zhang Y Lin Q Sun and H Huang ldquoDermo-glandularflap for treatment of recurrent periductal mastitisrdquo Journal ofSurgical Research vol 193 no 2 pp 738ndash744 2015
[4] R M S Rahal R De Freitas-Junior and R R Paulinelli ldquoRiskfactors for duct ectasiardquo Breast Journal vol 11 no 4 pp 262ndash265 2005
[5] R Mansel D Webster and H Sweetland Hughes Mansel ampWebsterrsquos Benign Disorders and Diseases of the Breast (ThirdEdition) Saunders Philadelphia Pa USA 2009
[6] M H Lequin J van Spengler R van Pel C van Eijck and HvanOverhagen ldquoMammographic and sonographic spectrum ofnon-puerperal mastitisrdquo European Journal of Radiology vol 21no 2 pp 138ndash142 1995
[7] R Tomczak A Rieber H Zeitler N Rilinger R Kreienbergand H J Brambs ldquoThe value of MR-mammography at 15 teslain the differential diagnosis of non-puerperal mastitis andinflammatory breast carcinomardquo RoFo Fortschritte auf demGebiete der Rontgenstrahlen und der Nuklearmedizin vol 165no 2 pp 148ndash151 1996
[8] V Gollapalli J Liao A Dudakovic S L Sugg C E H Scott-Conner and R J Weigel ldquoRisk factors for development andrecurrence of primary breast abscessesrdquo Journal of the AmericanCollege of Surgeons vol 211 no 1 pp 41ndash48 2010
Mediators of Inflammation 9
[9] R Risager and N Bentzon ldquoSmoking and increased risk ofmastitisrdquo Ugeskrift for Laeger vol 172 no 33 pp 2218ndash22212010
[10] H N Oltean A S Soliman O S Omar et al ldquoRisk factors forchronic mastitis in Morocco and Egyptrdquo International Journalof Inflammation vol 2013 Article ID 184921 10 pages 2013
[11] P Schafer C Furrer and B Mermillod ldquoAn association ofcigarette smoking with recurrent subareolar breast abscessrdquoInternational Journal of Epidemiology vol 17 no 4 pp 810ndash8131988
[12] V Sakka ldquoNonpuerperal breast infection epidemiology andpredictors for recurrencesrdquo Clinical Microbiology and Infectionvol 18 p 237 2012
[13] A Bharat F Gao R L Aft W E Gillanders T J Eberlein andJ A Margenthaler ldquoPredictors of primary breast abscesses andrecurrencerdquoWorld Journal of Surgery vol 33 no 12 pp 2582ndash2586 2009
[14] N J Bundred J M J Dixon A B Lumsden et al ldquoAre thelesions of duct ectasia sterilerdquo British Journal of Surgery vol72 no 10 pp 844ndash845 1985
[15] K Al Benwan A Al Mulla and V O Rotimi ldquoA study of themicrobiology of breast abscess in a teaching hospital in KuwaitrdquoMedical Principles and Practice vol 20 no 5 pp 422ndash426 2011
[16] P-L Giacalone G Rathat S Fournet and C Rouleau ldquoSurgicaltreatment of recurring subareolar abscess using oncoplastictechniquesrdquo Journal of Visceral Surgery vol 147 no 6 pp e389ndashe394 2010
[17] A Geremia P Biancheri P Allan G R Corazza and ADi Sabatino ldquoInnate and adaptive immunity in inflammatorybowel diseaserdquo Autoimmunity Reviews vol 13 no 1 pp 3ndash102014
[18] P Miossec and J K Kolls ldquoTargeting IL-17 and TH17 cells inchronic inflammationrdquo Nature Reviews Drug Discovery vol 11no 10 pp 763ndash776 2012
[19] Y Khosravi Y Dieye B H Poh et al ldquoCulturable bacterialmicrobiota of the stomach of helicobacter pylori positive andnegative gastric disease patientsrdquo The Scientific World Journalvol 2014 Article ID 610421 10 pages 2014
[20] R Marechal P Demetter N Nagy et al ldquoHigh expressionof CXCR4 may predict poor survival in resected pancreaticadenocarcinomardquo British Journal of Cancer vol 100 no 9 pp1444ndash1451 2009
[21] M Yao Y Wu Q Fang L Sun T Li and H Qiao ldquoAssociationof ADIPOQ variants with type 2 diabetes mellitus susceptibilityin ethnic han Chinese from Northeast Chinardquo Journal ofDiabetes Investigation vol 7 no 6 pp 853ndash859 2016
[22] Q Zhang L-Y Liu FWang KMu and Z-G Yu ldquoThe changesin female physical and childbearing characteristics in china andpotential association with risk of breast cancerrdquo BMC PublicHealth vol 12 article 368 2012
[23] J Kotsopoulos J Lubinski L Salmena et al ldquoBreastfeedingand the risk of breast cancer in BRCA1 and BRCA2 mutationcarriersrdquo Breast Cancer Research vol 14 no 2 article R42 2012
[24] Y Dong J-J Yu Y Shibahara et al ldquoIntercellular adhesionmolecule 12 and E-selectin in plasma cell mastitis immuno-histochemical study of 35 casesrdquo Human Pathology vol 45 no3 pp 606ndash610 2014
[25] Y Y Li J J Du Q Liu and Z G Yu ldquoScreening for breastdiseases among 61102 women in Shandong Provincerdquo Journalof Shandong University vol 49 no 8 pp 157ndash160 2011
[26] Y-M Xu H-H Chen F Li et al ldquoPrevalence and correlatesof cigarette smoking among Chinese schizophrenia inpatientsreceiving antipsychotic mono-therapyrdquo PLOS ONE vol 9 no2 Article ID e88478 2014
[27] F M Wensveen S Valentic M Sestan T Turk Wensveen andB Polic ldquoThe ldquoBig Bangrdquo in obese fat events initiating obesity-induced adipose tissue inflammationrdquo European Journal ofImmunology vol 45 no 9 pp 2446ndash2456 2015
[28] Y B Shaik-Dasthagirisaheb A Kantarci and F C Gibson IIIldquoImmune response of macrophages from young and aged miceto the oral pathogenic bacterium Porphyromonas gingivalisrdquoImmunity amp Ageing vol 7 article 15 2010
[29] K A Brown ldquoImpact of obesity on mammary gland inflamma-tion and local estrogen productionrdquo Journal ofMammary GlandBiology and Neoplasia vol 19 no 2 pp 183ndash189 2014
[30] B Al-Khaffaf F Knox and N J Bundred ldquoIdiopathic granulo-matous mastitis a 25-year experiencerdquo Journal of the AmericanCollege of Surgeons vol 206 no 2 pp 269ndash273 2008
[31] R M S Rahal R F Junior C Reis F C Pimenta J C ANetto and R R Paulinelli ldquoPrevalence of bacteria in the nippledischarge of patients with duct ectasiardquo International Journal ofClinical Practice vol 59 no 9 pp 1045ndash1050 2005
[32] M N Hartley J Stewart and E A Benson ldquoSubareolardissection for duct ectasia and periareolar sepsisrdquo The BritishJournal of Surgery vol 78 no 10 pp 1187ndash1188 1991
[33] J Ming G Meng Q Yuan et al ldquoClinical characteristics andsurgical modality of plasma cell mastitis analysis of 91 casesrdquoThe American Surgeon vol 79 no 1 pp 54ndash60 2013
[34] H S Park C H Yoon and H J Kim ldquoThe prevalence ofcongenital inverted nipplerdquoAesthetic Plastic Surgery vol 23 no2 pp 144ndash146 1999
[35] F N L Versluijs-Ossewaarde R M H Roumen and R J AGoris ldquoSubareolar breast abscesses characteristics and resultsof surgical treatmentrdquoThe Breast Journal vol 11 no 3 pp 179ndash182 2005
[36] Y Luo H Yamada D P Evanoff and X Chen ldquoRole ofTh1-stimulating cytokines in bacillus Calmette-Guerin (BCG)-inducedmacrophage cytotoxicity against mouse bladder cancerMBT-2 cellsrdquo Clinical and Experimental Immunology vol 146no 1 pp 181ndash188 2006
[37] K J Rautajoki M K Kylaniemi S K Raghav K Rao and RLahesmaa ldquoAn insight into molecular mechanisms of human Thelper cell differentiationrdquoAnnals of Medicine vol 40 no 5 pp322ndash335 2008
[38] B Stockinger and M Veldhoen ldquoDifferentiation and functionof Th17 T cellsrdquo Current Opinion in Immunology vol 19 no 3pp 281ndash286 2007
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Mediators of Inflammation 7
0
5
10
15
Cases with abscess formationCases without abscess formation
Num
ber o
f cas
es
lowast lowast lowast
IFN
-120574(minus
)
IFN
-120574(+
)
IL-17
A(minus
)
IL-17
A(+
)
IL-12
A(minus
)
IL-12
A(+
)
Figure 3 Production of IFN-120574 IL-12A and IL-17A in cases withor without abscess formation (minus) low expression of cytokines(+) high expression of cytokines Fifteen patients had lesions withabscess formation while 16 patients did not have them More caseswith high expressions of IFN-120574 and IL-17A in stromal inflammatorycells could be found in patients with abscess formation lowast119875 gt 005
play an important role in the progression of the diseaseof PDM especially the IFN-120574 and IL-12A As mentionedabove these two cytokines are key factors involved in theTh1mediated adaptive immune responses following bacterialinfection
4 Discussion
Smoking obesity diabetes mellitus and reproductive factorshave been considered as significant risk factors for PDM inprevious studies [10 24] The results of the present studyconfirmed that overweightobesity and the late onset ofmenarche were independent risk factors for PDM whilelate age at first birth had a protective effect In additionour results also suggested that nipple retraction was relatedto PDM Bacterial infection could be found in all samplesof PDM patients by 16S rRNA gene sequencing but not inthe samples from the control subjects Among the infectedsamples in PDM patients a mixture of different bacterialstrains was the most common condition which could beseen and then followed by singlePseudomonas spp infectionIFN-120574 and IL-12A were upregulated in PDM compared tonormal breast tissues but the expression of IL-17A was notsignificantly different from control samples Moreover thereare increased expression pattern for both IFN-120574 and IL-17Ain PDM which suggested a following immune response afterbacterial infection may contribute the deterioration of PDM
The incidence of PDM is increasing rapidly during thepast decades Indeed in our previous investigation the detec-tion rate of mammary ductal ectasia was 024 (14761102)[25] However the etiology of PDM is unclear yet mostly dueto lack of conclusive evidences fromprevious study Although
some studies suggest smoking is a risk factor for PDM [11 12]smoking was not found to be closely related to PDM in thepresent study One of the plausible reasons might be the lowprevalence of smoking among Chinese women [26]
Obesity is related to low-grade chronic inflammationwhich can disrupt immune function [27 28] Furthermoreobesity can directly influence local mammary estrogen andinflammation [29] Gollapalli et al [8] reported that obesitywas a risk factor for breast abscess Bharat et al [13] found thathigher BMI values were also associated with nonpuerperalabscessThey also reported similar results in cases of granulo-matous lobular mastitis In PDM however they did not finda significant effect of obesity on patients when comparedwithhealth controls [30] In our study data demonstrated obesityis another risk factor for PDM Obesity may directly disruptthe local immune functioning of the breast and exacerbate thedevelopment of PDM
The relationship between reproductive factors and PDMis very interesting Inconsistent with the finding reportedpreviously from literature in which parity was not found tobe associated with the risk for duct ectasia [31] our resultssuggest that reproductive factors were related to PDM Wealso found late onset of menarche was a significant risk factorfor PDM while increased age at first birth was a protec-tive factor The changes of serum prolactin and lactationin patients may be responsible for the above associationThe exact mechanism underlying this relationship is stillunknown future investigation focusing on theses hormonesis warranted
The relationship between nipple retraction and PDM hasbeen well-documented [32] Nipple retraction could inducebreast deformation and influence the process of lactationFurthermore it also can obstruct drainage and lead toan accumulation of massive substances in ducts which istermed mastitis [33] In this study we confirmed that nippleretraction was a risk factor for PDM The prevalence rateof nipple retraction is approximately 326 according toprevious report [34] But over one quarter of PDM patientsexperienced nipple retraction in our study
Bacterial infection is considered to be another possibleetiology of PDM although it has been a long-standing con-troversial problem Al Benwan et al [15] found that S aureuswas the predominant pathogen followed by Bacteroides sppAnaerobic strep and P aeruginosaHowever the relationshipbetween bacteria and PDM was not confirmed in otherreport [31] Here we provided more evidence to confirm forthis assumption Inconsistent with previous report [35] wedid not find that either S aureus was the most commonbacterium in PDM or anaerobic bacteria was associated withthis disease based on our current data Interestingly thebacteria identified here were those microorganisms whichare often related to community-acquired infection includingPseudomonas spp and S aureus Collectively these findingsuggests that bacterial infection may only be acting as aninduction factor and the immune system which are follow-ing the bacterial infection might play a major role in thedevelopment of PDM However more study is needed for thefuture to determine the common route in which the bacterialinfection and the following immune response converge
8 Mediators of Inflammation
IFN-120574 and IL-12A are characteristic Th1 cytokines [36]Th1 cells are important for the eradication of invadingpathogens including bacteria parasites yeast and viruses[37] In this study the expressions of IFN-120574 and IL-12A wereupregulated in breast stromal inflammatory cells of PDMpatients Recently a lineage of CD4+ T cells producing IL-17A was described and accordingly named as Th17 cellsIL-17A has been shown to be important for host defenseagainst pathogens [38] However in our study there was nosignificant difference in IL-17A expression between the twogroups As IFN-120574 and IL-12A were detected in PDM tissuesamples there is a rationale to conclude that theTh1 immuneresponse plays a role in this diseaseThemechanism bywhichthese cytokines contribute to the course of disease is stillunknown and needs further investigation
The strengths of the present study include the following(a) a relatively large population of patients was included (b)the assessment of tissue was done by two histopathologistsexperienced in breast inflammation However our study alsohad several limitations First this was a nonmatched casecontrol study and there might be some uncontrolled biaseswhich may influence the credibility of the results Secondthere was an issue with missing data especially the dataof reproductive factors which may also lead to the bias ofresults Finally we have not investigated the mechanism bywhich IFN-120574 and IL-12A participated in the course of thedisease
5 Conclusions
The results of the present study indicated that over-weightobesity and later onset of menarche are independentrisk factors for PDM In contrast age of first birth gt27years is a protective factor for PDM The importance ofthese reproductive factors to the risk of PDM has notbeen previously reported and will provide important novelinformation to broaden the avenue of investigating theetiology of PDM in the future We also confirm that bacterialinfection with a mixture of different bacterial species is aclosely associated with the pathogenesis of PDM Moreoverproinflammatory cytokines IFN-120574 and IL-12A were alsoshown to be associated with PDM which indicates that theTh1 immune response may closely relate to this diseaseTherefore PDM could be an inflammatory disease related tobacterial infection and consequent immune responses withincreased cytokines As far as we know this is first reportabout the possible immune responses following bacterialinfection which is involved in development of PDM diseaseand more research is needed for the future to furtherinvestigate the pathogenesis of PDM on the mechanism bywhich bacterial infection and consequent immune responsesaffect the progression of the disease
Ethical Approval
All procedures performed here involving human participantswere in accordance with the ethical standards of the SecondHospital of Shandong University Research Committee and
with the 1964Helsinki Declaration its later amendments andcomparable ethical standards
Consent
Informed consent was obtained from all participants includ-ed in the study
Competing Interests
All authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Dr Zhigang Yu and Dr Liang Li have equal contribution tothis article
Acknowledgments
Theauthors thank the patients and staffs at theDepartment ofBreast Surgery of SecondHospital of ShandongUniversity fortheir collaboration and supportThis study was funded by theYouth Fund of the Second Hospital of Shandong University(Y2014010030) Fei Zhou has received research grants fromthe Second Hospital of Shandong University
References
[1] K Ramalingam A Srivastava S Vuthaluru A Dhar and RChaudhry ldquoDuct Ectasia and periductal mastitis in Indianwomenrdquo Indian Journal of Surgery vol 77 supplement 3 pp957ndash962 2015
[2] J M Dixon ldquoPeriductal mastitisduct ectasiardquoWorld Journal ofSurgery vol 13 no 6 pp 715ndash720 1989
[3] X Zhang Y Lin Q Sun and H Huang ldquoDermo-glandularflap for treatment of recurrent periductal mastitisrdquo Journal ofSurgical Research vol 193 no 2 pp 738ndash744 2015
[4] R M S Rahal R De Freitas-Junior and R R Paulinelli ldquoRiskfactors for duct ectasiardquo Breast Journal vol 11 no 4 pp 262ndash265 2005
[5] R Mansel D Webster and H Sweetland Hughes Mansel ampWebsterrsquos Benign Disorders and Diseases of the Breast (ThirdEdition) Saunders Philadelphia Pa USA 2009
[6] M H Lequin J van Spengler R van Pel C van Eijck and HvanOverhagen ldquoMammographic and sonographic spectrum ofnon-puerperal mastitisrdquo European Journal of Radiology vol 21no 2 pp 138ndash142 1995
[7] R Tomczak A Rieber H Zeitler N Rilinger R Kreienbergand H J Brambs ldquoThe value of MR-mammography at 15 teslain the differential diagnosis of non-puerperal mastitis andinflammatory breast carcinomardquo RoFo Fortschritte auf demGebiete der Rontgenstrahlen und der Nuklearmedizin vol 165no 2 pp 148ndash151 1996
[8] V Gollapalli J Liao A Dudakovic S L Sugg C E H Scott-Conner and R J Weigel ldquoRisk factors for development andrecurrence of primary breast abscessesrdquo Journal of the AmericanCollege of Surgeons vol 211 no 1 pp 41ndash48 2010
Mediators of Inflammation 9
[9] R Risager and N Bentzon ldquoSmoking and increased risk ofmastitisrdquo Ugeskrift for Laeger vol 172 no 33 pp 2218ndash22212010
[10] H N Oltean A S Soliman O S Omar et al ldquoRisk factors forchronic mastitis in Morocco and Egyptrdquo International Journalof Inflammation vol 2013 Article ID 184921 10 pages 2013
[11] P Schafer C Furrer and B Mermillod ldquoAn association ofcigarette smoking with recurrent subareolar breast abscessrdquoInternational Journal of Epidemiology vol 17 no 4 pp 810ndash8131988
[12] V Sakka ldquoNonpuerperal breast infection epidemiology andpredictors for recurrencesrdquo Clinical Microbiology and Infectionvol 18 p 237 2012
[13] A Bharat F Gao R L Aft W E Gillanders T J Eberlein andJ A Margenthaler ldquoPredictors of primary breast abscesses andrecurrencerdquoWorld Journal of Surgery vol 33 no 12 pp 2582ndash2586 2009
[14] N J Bundred J M J Dixon A B Lumsden et al ldquoAre thelesions of duct ectasia sterilerdquo British Journal of Surgery vol72 no 10 pp 844ndash845 1985
[15] K Al Benwan A Al Mulla and V O Rotimi ldquoA study of themicrobiology of breast abscess in a teaching hospital in KuwaitrdquoMedical Principles and Practice vol 20 no 5 pp 422ndash426 2011
[16] P-L Giacalone G Rathat S Fournet and C Rouleau ldquoSurgicaltreatment of recurring subareolar abscess using oncoplastictechniquesrdquo Journal of Visceral Surgery vol 147 no 6 pp e389ndashe394 2010
[17] A Geremia P Biancheri P Allan G R Corazza and ADi Sabatino ldquoInnate and adaptive immunity in inflammatorybowel diseaserdquo Autoimmunity Reviews vol 13 no 1 pp 3ndash102014
[18] P Miossec and J K Kolls ldquoTargeting IL-17 and TH17 cells inchronic inflammationrdquo Nature Reviews Drug Discovery vol 11no 10 pp 763ndash776 2012
[19] Y Khosravi Y Dieye B H Poh et al ldquoCulturable bacterialmicrobiota of the stomach of helicobacter pylori positive andnegative gastric disease patientsrdquo The Scientific World Journalvol 2014 Article ID 610421 10 pages 2014
[20] R Marechal P Demetter N Nagy et al ldquoHigh expressionof CXCR4 may predict poor survival in resected pancreaticadenocarcinomardquo British Journal of Cancer vol 100 no 9 pp1444ndash1451 2009
[21] M Yao Y Wu Q Fang L Sun T Li and H Qiao ldquoAssociationof ADIPOQ variants with type 2 diabetes mellitus susceptibilityin ethnic han Chinese from Northeast Chinardquo Journal ofDiabetes Investigation vol 7 no 6 pp 853ndash859 2016
[22] Q Zhang L-Y Liu FWang KMu and Z-G Yu ldquoThe changesin female physical and childbearing characteristics in china andpotential association with risk of breast cancerrdquo BMC PublicHealth vol 12 article 368 2012
[23] J Kotsopoulos J Lubinski L Salmena et al ldquoBreastfeedingand the risk of breast cancer in BRCA1 and BRCA2 mutationcarriersrdquo Breast Cancer Research vol 14 no 2 article R42 2012
[24] Y Dong J-J Yu Y Shibahara et al ldquoIntercellular adhesionmolecule 12 and E-selectin in plasma cell mastitis immuno-histochemical study of 35 casesrdquo Human Pathology vol 45 no3 pp 606ndash610 2014
[25] Y Y Li J J Du Q Liu and Z G Yu ldquoScreening for breastdiseases among 61102 women in Shandong Provincerdquo Journalof Shandong University vol 49 no 8 pp 157ndash160 2011
[26] Y-M Xu H-H Chen F Li et al ldquoPrevalence and correlatesof cigarette smoking among Chinese schizophrenia inpatientsreceiving antipsychotic mono-therapyrdquo PLOS ONE vol 9 no2 Article ID e88478 2014
[27] F M Wensveen S Valentic M Sestan T Turk Wensveen andB Polic ldquoThe ldquoBig Bangrdquo in obese fat events initiating obesity-induced adipose tissue inflammationrdquo European Journal ofImmunology vol 45 no 9 pp 2446ndash2456 2015
[28] Y B Shaik-Dasthagirisaheb A Kantarci and F C Gibson IIIldquoImmune response of macrophages from young and aged miceto the oral pathogenic bacterium Porphyromonas gingivalisrdquoImmunity amp Ageing vol 7 article 15 2010
[29] K A Brown ldquoImpact of obesity on mammary gland inflamma-tion and local estrogen productionrdquo Journal ofMammary GlandBiology and Neoplasia vol 19 no 2 pp 183ndash189 2014
[30] B Al-Khaffaf F Knox and N J Bundred ldquoIdiopathic granulo-matous mastitis a 25-year experiencerdquo Journal of the AmericanCollege of Surgeons vol 206 no 2 pp 269ndash273 2008
[31] R M S Rahal R F Junior C Reis F C Pimenta J C ANetto and R R Paulinelli ldquoPrevalence of bacteria in the nippledischarge of patients with duct ectasiardquo International Journal ofClinical Practice vol 59 no 9 pp 1045ndash1050 2005
[32] M N Hartley J Stewart and E A Benson ldquoSubareolardissection for duct ectasia and periareolar sepsisrdquo The BritishJournal of Surgery vol 78 no 10 pp 1187ndash1188 1991
[33] J Ming G Meng Q Yuan et al ldquoClinical characteristics andsurgical modality of plasma cell mastitis analysis of 91 casesrdquoThe American Surgeon vol 79 no 1 pp 54ndash60 2013
[34] H S Park C H Yoon and H J Kim ldquoThe prevalence ofcongenital inverted nipplerdquoAesthetic Plastic Surgery vol 23 no2 pp 144ndash146 1999
[35] F N L Versluijs-Ossewaarde R M H Roumen and R J AGoris ldquoSubareolar breast abscesses characteristics and resultsof surgical treatmentrdquoThe Breast Journal vol 11 no 3 pp 179ndash182 2005
[36] Y Luo H Yamada D P Evanoff and X Chen ldquoRole ofTh1-stimulating cytokines in bacillus Calmette-Guerin (BCG)-inducedmacrophage cytotoxicity against mouse bladder cancerMBT-2 cellsrdquo Clinical and Experimental Immunology vol 146no 1 pp 181ndash188 2006
[37] K J Rautajoki M K Kylaniemi S K Raghav K Rao and RLahesmaa ldquoAn insight into molecular mechanisms of human Thelper cell differentiationrdquoAnnals of Medicine vol 40 no 5 pp322ndash335 2008
[38] B Stockinger and M Veldhoen ldquoDifferentiation and functionof Th17 T cellsrdquo Current Opinion in Immunology vol 19 no 3pp 281ndash286 2007
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Mediators of Inflammation
IFN-120574 and IL-12A are characteristic Th1 cytokines [36]Th1 cells are important for the eradication of invadingpathogens including bacteria parasites yeast and viruses[37] In this study the expressions of IFN-120574 and IL-12A wereupregulated in breast stromal inflammatory cells of PDMpatients Recently a lineage of CD4+ T cells producing IL-17A was described and accordingly named as Th17 cellsIL-17A has been shown to be important for host defenseagainst pathogens [38] However in our study there was nosignificant difference in IL-17A expression between the twogroups As IFN-120574 and IL-12A were detected in PDM tissuesamples there is a rationale to conclude that theTh1 immuneresponse plays a role in this diseaseThemechanism bywhichthese cytokines contribute to the course of disease is stillunknown and needs further investigation
The strengths of the present study include the following(a) a relatively large population of patients was included (b)the assessment of tissue was done by two histopathologistsexperienced in breast inflammation However our study alsohad several limitations First this was a nonmatched casecontrol study and there might be some uncontrolled biaseswhich may influence the credibility of the results Secondthere was an issue with missing data especially the dataof reproductive factors which may also lead to the bias ofresults Finally we have not investigated the mechanism bywhich IFN-120574 and IL-12A participated in the course of thedisease
5 Conclusions
The results of the present study indicated that over-weightobesity and later onset of menarche are independentrisk factors for PDM In contrast age of first birth gt27years is a protective factor for PDM The importance ofthese reproductive factors to the risk of PDM has notbeen previously reported and will provide important novelinformation to broaden the avenue of investigating theetiology of PDM in the future We also confirm that bacterialinfection with a mixture of different bacterial species is aclosely associated with the pathogenesis of PDM Moreoverproinflammatory cytokines IFN-120574 and IL-12A were alsoshown to be associated with PDM which indicates that theTh1 immune response may closely relate to this diseaseTherefore PDM could be an inflammatory disease related tobacterial infection and consequent immune responses withincreased cytokines As far as we know this is first reportabout the possible immune responses following bacterialinfection which is involved in development of PDM diseaseand more research is needed for the future to furtherinvestigate the pathogenesis of PDM on the mechanism bywhich bacterial infection and consequent immune responsesaffect the progression of the disease
Ethical Approval
All procedures performed here involving human participantswere in accordance with the ethical standards of the SecondHospital of Shandong University Research Committee and
with the 1964Helsinki Declaration its later amendments andcomparable ethical standards
Consent
Informed consent was obtained from all participants includ-ed in the study
Competing Interests
All authors declare that there are no competing interestsregarding the publication of this paper
Authorsrsquo Contributions
Dr Zhigang Yu and Dr Liang Li have equal contribution tothis article
Acknowledgments
Theauthors thank the patients and staffs at theDepartment ofBreast Surgery of SecondHospital of ShandongUniversity fortheir collaboration and supportThis study was funded by theYouth Fund of the Second Hospital of Shandong University(Y2014010030) Fei Zhou has received research grants fromthe Second Hospital of Shandong University
References
[1] K Ramalingam A Srivastava S Vuthaluru A Dhar and RChaudhry ldquoDuct Ectasia and periductal mastitis in Indianwomenrdquo Indian Journal of Surgery vol 77 supplement 3 pp957ndash962 2015
[2] J M Dixon ldquoPeriductal mastitisduct ectasiardquoWorld Journal ofSurgery vol 13 no 6 pp 715ndash720 1989
[3] X Zhang Y Lin Q Sun and H Huang ldquoDermo-glandularflap for treatment of recurrent periductal mastitisrdquo Journal ofSurgical Research vol 193 no 2 pp 738ndash744 2015
[4] R M S Rahal R De Freitas-Junior and R R Paulinelli ldquoRiskfactors for duct ectasiardquo Breast Journal vol 11 no 4 pp 262ndash265 2005
[5] R Mansel D Webster and H Sweetland Hughes Mansel ampWebsterrsquos Benign Disorders and Diseases of the Breast (ThirdEdition) Saunders Philadelphia Pa USA 2009
[6] M H Lequin J van Spengler R van Pel C van Eijck and HvanOverhagen ldquoMammographic and sonographic spectrum ofnon-puerperal mastitisrdquo European Journal of Radiology vol 21no 2 pp 138ndash142 1995
[7] R Tomczak A Rieber H Zeitler N Rilinger R Kreienbergand H J Brambs ldquoThe value of MR-mammography at 15 teslain the differential diagnosis of non-puerperal mastitis andinflammatory breast carcinomardquo RoFo Fortschritte auf demGebiete der Rontgenstrahlen und der Nuklearmedizin vol 165no 2 pp 148ndash151 1996
[8] V Gollapalli J Liao A Dudakovic S L Sugg C E H Scott-Conner and R J Weigel ldquoRisk factors for development andrecurrence of primary breast abscessesrdquo Journal of the AmericanCollege of Surgeons vol 211 no 1 pp 41ndash48 2010
Mediators of Inflammation 9
[9] R Risager and N Bentzon ldquoSmoking and increased risk ofmastitisrdquo Ugeskrift for Laeger vol 172 no 33 pp 2218ndash22212010
[10] H N Oltean A S Soliman O S Omar et al ldquoRisk factors forchronic mastitis in Morocco and Egyptrdquo International Journalof Inflammation vol 2013 Article ID 184921 10 pages 2013
[11] P Schafer C Furrer and B Mermillod ldquoAn association ofcigarette smoking with recurrent subareolar breast abscessrdquoInternational Journal of Epidemiology vol 17 no 4 pp 810ndash8131988
[12] V Sakka ldquoNonpuerperal breast infection epidemiology andpredictors for recurrencesrdquo Clinical Microbiology and Infectionvol 18 p 237 2012
[13] A Bharat F Gao R L Aft W E Gillanders T J Eberlein andJ A Margenthaler ldquoPredictors of primary breast abscesses andrecurrencerdquoWorld Journal of Surgery vol 33 no 12 pp 2582ndash2586 2009
[14] N J Bundred J M J Dixon A B Lumsden et al ldquoAre thelesions of duct ectasia sterilerdquo British Journal of Surgery vol72 no 10 pp 844ndash845 1985
[15] K Al Benwan A Al Mulla and V O Rotimi ldquoA study of themicrobiology of breast abscess in a teaching hospital in KuwaitrdquoMedical Principles and Practice vol 20 no 5 pp 422ndash426 2011
[16] P-L Giacalone G Rathat S Fournet and C Rouleau ldquoSurgicaltreatment of recurring subareolar abscess using oncoplastictechniquesrdquo Journal of Visceral Surgery vol 147 no 6 pp e389ndashe394 2010
[17] A Geremia P Biancheri P Allan G R Corazza and ADi Sabatino ldquoInnate and adaptive immunity in inflammatorybowel diseaserdquo Autoimmunity Reviews vol 13 no 1 pp 3ndash102014
[18] P Miossec and J K Kolls ldquoTargeting IL-17 and TH17 cells inchronic inflammationrdquo Nature Reviews Drug Discovery vol 11no 10 pp 763ndash776 2012
[19] Y Khosravi Y Dieye B H Poh et al ldquoCulturable bacterialmicrobiota of the stomach of helicobacter pylori positive andnegative gastric disease patientsrdquo The Scientific World Journalvol 2014 Article ID 610421 10 pages 2014
[20] R Marechal P Demetter N Nagy et al ldquoHigh expressionof CXCR4 may predict poor survival in resected pancreaticadenocarcinomardquo British Journal of Cancer vol 100 no 9 pp1444ndash1451 2009
[21] M Yao Y Wu Q Fang L Sun T Li and H Qiao ldquoAssociationof ADIPOQ variants with type 2 diabetes mellitus susceptibilityin ethnic han Chinese from Northeast Chinardquo Journal ofDiabetes Investigation vol 7 no 6 pp 853ndash859 2016
[22] Q Zhang L-Y Liu FWang KMu and Z-G Yu ldquoThe changesin female physical and childbearing characteristics in china andpotential association with risk of breast cancerrdquo BMC PublicHealth vol 12 article 368 2012
[23] J Kotsopoulos J Lubinski L Salmena et al ldquoBreastfeedingand the risk of breast cancer in BRCA1 and BRCA2 mutationcarriersrdquo Breast Cancer Research vol 14 no 2 article R42 2012
[24] Y Dong J-J Yu Y Shibahara et al ldquoIntercellular adhesionmolecule 12 and E-selectin in plasma cell mastitis immuno-histochemical study of 35 casesrdquo Human Pathology vol 45 no3 pp 606ndash610 2014
[25] Y Y Li J J Du Q Liu and Z G Yu ldquoScreening for breastdiseases among 61102 women in Shandong Provincerdquo Journalof Shandong University vol 49 no 8 pp 157ndash160 2011
[26] Y-M Xu H-H Chen F Li et al ldquoPrevalence and correlatesof cigarette smoking among Chinese schizophrenia inpatientsreceiving antipsychotic mono-therapyrdquo PLOS ONE vol 9 no2 Article ID e88478 2014
[27] F M Wensveen S Valentic M Sestan T Turk Wensveen andB Polic ldquoThe ldquoBig Bangrdquo in obese fat events initiating obesity-induced adipose tissue inflammationrdquo European Journal ofImmunology vol 45 no 9 pp 2446ndash2456 2015
[28] Y B Shaik-Dasthagirisaheb A Kantarci and F C Gibson IIIldquoImmune response of macrophages from young and aged miceto the oral pathogenic bacterium Porphyromonas gingivalisrdquoImmunity amp Ageing vol 7 article 15 2010
[29] K A Brown ldquoImpact of obesity on mammary gland inflamma-tion and local estrogen productionrdquo Journal ofMammary GlandBiology and Neoplasia vol 19 no 2 pp 183ndash189 2014
[30] B Al-Khaffaf F Knox and N J Bundred ldquoIdiopathic granulo-matous mastitis a 25-year experiencerdquo Journal of the AmericanCollege of Surgeons vol 206 no 2 pp 269ndash273 2008
[31] R M S Rahal R F Junior C Reis F C Pimenta J C ANetto and R R Paulinelli ldquoPrevalence of bacteria in the nippledischarge of patients with duct ectasiardquo International Journal ofClinical Practice vol 59 no 9 pp 1045ndash1050 2005
[32] M N Hartley J Stewart and E A Benson ldquoSubareolardissection for duct ectasia and periareolar sepsisrdquo The BritishJournal of Surgery vol 78 no 10 pp 1187ndash1188 1991
[33] J Ming G Meng Q Yuan et al ldquoClinical characteristics andsurgical modality of plasma cell mastitis analysis of 91 casesrdquoThe American Surgeon vol 79 no 1 pp 54ndash60 2013
[34] H S Park C H Yoon and H J Kim ldquoThe prevalence ofcongenital inverted nipplerdquoAesthetic Plastic Surgery vol 23 no2 pp 144ndash146 1999
[35] F N L Versluijs-Ossewaarde R M H Roumen and R J AGoris ldquoSubareolar breast abscesses characteristics and resultsof surgical treatmentrdquoThe Breast Journal vol 11 no 3 pp 179ndash182 2005
[36] Y Luo H Yamada D P Evanoff and X Chen ldquoRole ofTh1-stimulating cytokines in bacillus Calmette-Guerin (BCG)-inducedmacrophage cytotoxicity against mouse bladder cancerMBT-2 cellsrdquo Clinical and Experimental Immunology vol 146no 1 pp 181ndash188 2006
[37] K J Rautajoki M K Kylaniemi S K Raghav K Rao and RLahesmaa ldquoAn insight into molecular mechanisms of human Thelper cell differentiationrdquoAnnals of Medicine vol 40 no 5 pp322ndash335 2008
[38] B Stockinger and M Veldhoen ldquoDifferentiation and functionof Th17 T cellsrdquo Current Opinion in Immunology vol 19 no 3pp 281ndash286 2007
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Mediators of Inflammation 9
[9] R Risager and N Bentzon ldquoSmoking and increased risk ofmastitisrdquo Ugeskrift for Laeger vol 172 no 33 pp 2218ndash22212010
[10] H N Oltean A S Soliman O S Omar et al ldquoRisk factors forchronic mastitis in Morocco and Egyptrdquo International Journalof Inflammation vol 2013 Article ID 184921 10 pages 2013
[11] P Schafer C Furrer and B Mermillod ldquoAn association ofcigarette smoking with recurrent subareolar breast abscessrdquoInternational Journal of Epidemiology vol 17 no 4 pp 810ndash8131988
[12] V Sakka ldquoNonpuerperal breast infection epidemiology andpredictors for recurrencesrdquo Clinical Microbiology and Infectionvol 18 p 237 2012
[13] A Bharat F Gao R L Aft W E Gillanders T J Eberlein andJ A Margenthaler ldquoPredictors of primary breast abscesses andrecurrencerdquoWorld Journal of Surgery vol 33 no 12 pp 2582ndash2586 2009
[14] N J Bundred J M J Dixon A B Lumsden et al ldquoAre thelesions of duct ectasia sterilerdquo British Journal of Surgery vol72 no 10 pp 844ndash845 1985
[15] K Al Benwan A Al Mulla and V O Rotimi ldquoA study of themicrobiology of breast abscess in a teaching hospital in KuwaitrdquoMedical Principles and Practice vol 20 no 5 pp 422ndash426 2011
[16] P-L Giacalone G Rathat S Fournet and C Rouleau ldquoSurgicaltreatment of recurring subareolar abscess using oncoplastictechniquesrdquo Journal of Visceral Surgery vol 147 no 6 pp e389ndashe394 2010
[17] A Geremia P Biancheri P Allan G R Corazza and ADi Sabatino ldquoInnate and adaptive immunity in inflammatorybowel diseaserdquo Autoimmunity Reviews vol 13 no 1 pp 3ndash102014
[18] P Miossec and J K Kolls ldquoTargeting IL-17 and TH17 cells inchronic inflammationrdquo Nature Reviews Drug Discovery vol 11no 10 pp 763ndash776 2012
[19] Y Khosravi Y Dieye B H Poh et al ldquoCulturable bacterialmicrobiota of the stomach of helicobacter pylori positive andnegative gastric disease patientsrdquo The Scientific World Journalvol 2014 Article ID 610421 10 pages 2014
[20] R Marechal P Demetter N Nagy et al ldquoHigh expressionof CXCR4 may predict poor survival in resected pancreaticadenocarcinomardquo British Journal of Cancer vol 100 no 9 pp1444ndash1451 2009
[21] M Yao Y Wu Q Fang L Sun T Li and H Qiao ldquoAssociationof ADIPOQ variants with type 2 diabetes mellitus susceptibilityin ethnic han Chinese from Northeast Chinardquo Journal ofDiabetes Investigation vol 7 no 6 pp 853ndash859 2016
[22] Q Zhang L-Y Liu FWang KMu and Z-G Yu ldquoThe changesin female physical and childbearing characteristics in china andpotential association with risk of breast cancerrdquo BMC PublicHealth vol 12 article 368 2012
[23] J Kotsopoulos J Lubinski L Salmena et al ldquoBreastfeedingand the risk of breast cancer in BRCA1 and BRCA2 mutationcarriersrdquo Breast Cancer Research vol 14 no 2 article R42 2012
[24] Y Dong J-J Yu Y Shibahara et al ldquoIntercellular adhesionmolecule 12 and E-selectin in plasma cell mastitis immuno-histochemical study of 35 casesrdquo Human Pathology vol 45 no3 pp 606ndash610 2014
[25] Y Y Li J J Du Q Liu and Z G Yu ldquoScreening for breastdiseases among 61102 women in Shandong Provincerdquo Journalof Shandong University vol 49 no 8 pp 157ndash160 2011
[26] Y-M Xu H-H Chen F Li et al ldquoPrevalence and correlatesof cigarette smoking among Chinese schizophrenia inpatientsreceiving antipsychotic mono-therapyrdquo PLOS ONE vol 9 no2 Article ID e88478 2014
[27] F M Wensveen S Valentic M Sestan T Turk Wensveen andB Polic ldquoThe ldquoBig Bangrdquo in obese fat events initiating obesity-induced adipose tissue inflammationrdquo European Journal ofImmunology vol 45 no 9 pp 2446ndash2456 2015
[28] Y B Shaik-Dasthagirisaheb A Kantarci and F C Gibson IIIldquoImmune response of macrophages from young and aged miceto the oral pathogenic bacterium Porphyromonas gingivalisrdquoImmunity amp Ageing vol 7 article 15 2010
[29] K A Brown ldquoImpact of obesity on mammary gland inflamma-tion and local estrogen productionrdquo Journal ofMammary GlandBiology and Neoplasia vol 19 no 2 pp 183ndash189 2014
[30] B Al-Khaffaf F Knox and N J Bundred ldquoIdiopathic granulo-matous mastitis a 25-year experiencerdquo Journal of the AmericanCollege of Surgeons vol 206 no 2 pp 269ndash273 2008
[31] R M S Rahal R F Junior C Reis F C Pimenta J C ANetto and R R Paulinelli ldquoPrevalence of bacteria in the nippledischarge of patients with duct ectasiardquo International Journal ofClinical Practice vol 59 no 9 pp 1045ndash1050 2005
[32] M N Hartley J Stewart and E A Benson ldquoSubareolardissection for duct ectasia and periareolar sepsisrdquo The BritishJournal of Surgery vol 78 no 10 pp 1187ndash1188 1991
[33] J Ming G Meng Q Yuan et al ldquoClinical characteristics andsurgical modality of plasma cell mastitis analysis of 91 casesrdquoThe American Surgeon vol 79 no 1 pp 54ndash60 2013
[34] H S Park C H Yoon and H J Kim ldquoThe prevalence ofcongenital inverted nipplerdquoAesthetic Plastic Surgery vol 23 no2 pp 144ndash146 1999
[35] F N L Versluijs-Ossewaarde R M H Roumen and R J AGoris ldquoSubareolar breast abscesses characteristics and resultsof surgical treatmentrdquoThe Breast Journal vol 11 no 3 pp 179ndash182 2005
[36] Y Luo H Yamada D P Evanoff and X Chen ldquoRole ofTh1-stimulating cytokines in bacillus Calmette-Guerin (BCG)-inducedmacrophage cytotoxicity against mouse bladder cancerMBT-2 cellsrdquo Clinical and Experimental Immunology vol 146no 1 pp 181ndash188 2006
[37] K J Rautajoki M K Kylaniemi S K Raghav K Rao and RLahesmaa ldquoAn insight into molecular mechanisms of human Thelper cell differentiationrdquoAnnals of Medicine vol 40 no 5 pp322ndash335 2008
[38] B Stockinger and M Veldhoen ldquoDifferentiation and functionof Th17 T cellsrdquo Current Opinion in Immunology vol 19 no 3pp 281ndash286 2007
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
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