Adjunctive therapy for Alzheimer’s Disease- Are you pro ...sites.utexas.edu/pharmacotherapy-rounds/files/2019/05/YOungE.pdf · administration of a probiotic mixture of Lactobacillus

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Adjunctive therapy for Alzheimer’s Disease- Are you pro probiotics?

Eric H. Young, Pharm.D.

Ph.D. Graduate Student in Pharmaceutical Sciences Division of Pharmacotherapy, The University of Texas at Austin

Pharmacotherapy Education & Research Center, UT Health San Antonio

Friday, May 24, 2019

Learning objectives: 1. Describe the epidemiology and clinical presentation of Alzheimer’s Disease2. Discuss the pathophysiology and the gut microbiome’s role in the development of Alzheimer’s

Disease3. Identify current treatment options and how probiotics can influence Alzheimer’s Disease

progression4. Explain the role probiotics have in adjunctive therapy for treatment of Alzheimer’s Disease

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Epidemiology and Clinical Presentation of Alzheimer’s Disease (AD) Epidemiology of AD

1. Sixth leading cause of death for all ages; fifth leading cause of death in patients 65 years of age and older1

2. Affects around 24 million people worldwide; 5.5 million in the United States2 a. 4.6 million cases a year

3. Highest prevalence in North America and Western Europe at age 60 years3 a. 6.4% of the population in North America; 5.4% in Western Europe

4. Almost 15-fold increase in dementia prevalence between ages of 60 and 85 years4 5. In the United States, most common in African-Americans and Hispanic populations5

a. Longitudinal study of 2,126 patients done in New York City showed standardized incidence rate for non-Hispanic black elders to be 4.2% per person-year; 3.8% in Caribbean Hispanics3

Clinical presentation6

1. Progressive memory and orientation loss 2. Characterized by β-amyloid and tau protein deposition 3. Neuroinflammation caused by activated microglia and reactive astrocytes 4. Other cognitive deficits

a. Impaired judgement b. Language disturbances

5. Other neuropsychiatric symptoms: a. Early progression symptoms like depression and anxiety b. Psychotic symptoms and behavioral symptoms seen with disease progression

6. Life expectancy averages four to eight years after diagnosis Risk factors3

1. Age 2. Family history 3. Genetics 4. Lifestyle 5. Other comorbidities

a. Cardiovascular disease b. Smoking c. Hypertension d. Type 2 diabetes e. Obesity f. Traumatic brain injury

Screening tools for dementia

Table 1. Screening tools for dementia7 Instrument Gold standard Cutoff for dementia (score less than or equal to ) MMSE8 DSM-IV diagnosis 23 TYM8 DSM-IV diagnosis 30 GPCOG9 DSM-IV diagnosis 10 (on total score)

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ACE-R8 DSM-IV diagnosis 73 MoCA10 Clinical diagnosis of AD 26

MMSE: Mini-Mental State Examination; TYM: Test Your Memory; GPCOG: General Practitioner assessment of Cognition; ACE-R: Addenbrookes Cognitive Assessment- Revised; MoCA: Montreal Cognitive Assessment Pathophysiology of AD General characteristics of AD pathophysiology

1. Aβ and tau accumulation 2. Neurotoxicity 3. Cognitive impairment

Pathophysiology

1. Amyloid plaque accumulation and inflammation a. Amyloid plaques are found in the extracellular lesions in the brain and cerebral

vasculature6 b. Amyloid plaque formation caused by Aβ overproduction/reduced clearance11 c. Astrocytes and microglia become activated due to Aβ accumulation- responsible for Aβ

clearance i. Prolonged microglia activation results in production of cytotoxic cytokines (IL-1,

IL-6, TNF-α), leading to enhanced neuroinflammation6 d. Oxidative stress and free radical release

i. Release of superoxide free radical species due to mitochondrial damage caused by Aβ accumulation6

e. Loss of calcium homeostasis i. Increased levels of calcium can stimulate Aβ aggregation and accumulation

2. Tau protein accumulation a. Tau hyperphosphorylation leads to neuronal cell death and intracellular neurofibrillary

changes12 b. Found in neurofibrillary tangles in the hippocampus and cerebral cortex6

3. Glutamate (Glu) and NMDA receptor activation a. Glu mediates neuronal plasticity, neural transmission, memory processes, and learning13 b. Increased glutamate production caused by microglia activation can lead to

“excitotoxicity,” which occurs due to chronic, moderate activation of NMDA receptors, leading to neurodegeneration6,13

4. Synaptic dysfunction a. Correlates with cognitive decline in AD6 b. Aβ accumulation can result in impaired synaptic plasticity6

i. Loss of homeostasis between long-term potentiation and long-term depression c. Impairment of nicotinic acetylcholine (ACh) receptor signaling and ACh release from

presynaptic terminal6 Contribution of the gut microbiota to AD development Gut-brain axis (GBA)

1. Communication occurs via neural, endocrinal, immunological, and humoral links14,15

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2. ENS also responsible for producing neurotransmitters and neuromodulators seen in AD14,16-17 a. Kynurenine- product of tryptophan breakdown that can result in immune system

activation and furthermore, increased inflammation16

Figure 1. Neurotransmitter and neuromodulator production by the GBA 14,16-17

b. ENS also shown to express amyloid precursor protein (APP)17 i. APP transgenic mice shown to develop accumulation of Aβ in enteric neurons,

leading to decreased enteric neuron abundance, dysmotility, and increased vulnerability to inflammation

Dysbiosis

1. Changes in bacterial composition seen in AD patients

Table 2. Microbiome changes in AD Study Species Overall microbiome

changes Relative abundance (Familial level)

Relative abundance (Genus level)

Harach et al.18 Mice ­ Bacteroidetes ¯ Firmicutes

Vogt et al.16 Humans ­ Bacteroidetes ­ Bacteroidaceae ­ Rikenellaceae

­ Bacteroides ­ Alistipes

¯ Firmicutes ¯ Ruminococcaceae ¯ Clostridiaceae ­ Gemellaceae

¯ Clostridium ­ Blautia ­ Gemella

¯ Actinobacteria ¯ Bifidobacteriaceae ¯ Bifidobacterium

a. Increased Bacteroidetes and decreased Firmicutes also commonly seen in other diseases that can contribute to AD

i. Insulin resistance and diabetes16 1. Decreased cerebral glucose metabolism 2. Increased amyloid deposition

ii. Bacteroides- gram-negative bacteria with an outer membrane made of LPS, which can increase inflammation16

Inflammatory responses

1. Increased intestinal permeability due to aging, which can lead to translocation of bacteria from gut to brain16

2. Bacterial amyloid production in the gut primes immune system to enhance immune response to endogenous β-amyloid production19

Kynurenine Glutamate

Acetylcholine

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a. Bacterial amyloids are similar in tertiary structure to endogenous Aβ b. Increased TLR-2, IL-6, TNF production

3. Bacterial lipopolysaccharide (LPS) production a. Injection of LPS into rats resulted in neuroinflammation and cognitive defects similar to

what is seen in AD20 b. Shown to activate toll-like receptor (TLR) 2 and 4 c. TLR-2 is also activated by Aβ and bacterial amyloids d. TLR-4 activation can result in promoting inflammatory response e. Promotes amyloid fibrillogenesis (neurotoxic)21 f. Can induce a more pathogenic β-pleated sheet conformation of prion amyloids21

Treatment of AD Goal: Delay progression of symptoms of neurocognitive and physical decline22

1. Mild to moderate AD: Acetylcholinesterase inhibitors (AChEIs)22,23 a. First-line agents for mild to moderate AD23 b. Reversibly binds and inactivates acetylcholinesterase, the enzyme responsible for

acetylcholine degradation23 2. Drugs23

a. Rivastigmine b. Galantamine c. Donepezil- only AChEI approved for use in all stages of AD22

3. Moderate to severe AD: N-methyl-D-aspartate (NMDA) receptor antagonists or combination with AChEI22

a. NMDA antagonists aid in preventing excitatory amino acid neurotoxicity without interfering with physiologic actions of glutamate22

4. Side effects23

Figure 2. Side effects of AD treatment4

Summary 1. AD is complex6 2. Alterations to the gut microbiome plays a role in the pathogenesis of AD 3. AD drugs have several side effects which can lead to low tolerability22,23 4. Limited pharmacotherapeutic options22 5. Current AD drugs show temporary cognitive and behavioral benefit22 6. AD drugs show no delay in disease progression22,23

Cholinesterase inhibitors

GI effects

Bradycardia

Fainting

Insomnia

Tremors

Weight loss

QT prolongation

NMDA receptor antagonists

Dizziness

Constipation

Headache

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Probiotics and AD Overview

1. What are probiotics? a. WHO definition: Live microorganisms which, when administered in adequate amounts,

confer a health benefit on the host24 b. Most commonly used bacteria in human nutrition belong to the following genera25

i. Lactobacillus (Firmicutes) ii. Bifidobacterium (Actinobacteria)

Products25

Table 3. List of probiotics and strains Brand Name Bacterial Species Activia B. lactis DN-173 010, L. bulgaricus, L. laxtis, Streptococcus

thermophilus Culturelle L. rhamnosus GG Florastor Saccharomyces boulardii

AAD= Antibiotic associated diarrhea; CDAD= C. difficile associated diarrhea; IBS= irritable bowel syndrome See Appendix Table A for a more comprehensive list of probiotic products Adverse effects26

1. Abdominal cramping 2. Nausea 3. Fever 4. Soft stools 5. Flatulence 6. Taste disturbances

Rationale behind using probiotics in AD

1. Can help inhibit oxidative stress by reducing inflammation and increasing antioxidant enzymes27 a. May be able to improve cognitive behavior28

i. Study utilizing diabetic rats showed improved cognitive abilities after administration of a probiotic mixture of Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus fermentum

2. Microbes have been shown to activate vagal nerves, which interacts with neurons involved with learning and memory29

3. Certain bacteria are also known to stimulate production of acetylcholine in the hippocampus and cerebral cortex30

a. Lactobacillus plantarum shown to produce small amounts of acetylcholine

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Literature Review Preliminary studies

Table 4. Nimgampalle M and Kuna Y. Anti-Alzheimer Properties of Probiotic, Lactobacillus plantarum MTCC 1325 in Alzheimer’s Disease induced Albino Rats. J Clin Diagn Res. 2017; 11(8):KC01-KC0531 Objective: Assess anti-Alzheimer properties of acetylcholine-producing L. plantarum MTCC13235 against D-Galactose induced AD Design Interventional study on albino rats (wistar strain) from February 2015 to

June 2015 Intervention Male rats divided into four groups of six animals (n=48)

• Group 1 (Control): 1 mL/kg normal saline • Group 2 (AD-model): 120 mg/kg D-Galactose intraperitoneal

injection • Group 3 (Protective group- AD+L. plantarum (LP)): Received both

10mL/kg of D-Galactose and 12x108 CFU/mL of L. plantarum together for 60 days after the seventh week

• Group 4 (LP only): Received 12x108 CFU/mL of L. plantarum for 60 days after the seventh week)

AD induction period for 60 days prior to LP treatment

Outcomes • Changes in morphological features (e.g. general appearance, body weight, organ index)

• Evaluation of cognitive behavior o Used water maze experiment to assess cognitive function on

30th and 60th day of treatment • Assessment of gross behavioral activity on 30th and 60th day • Histopathological examination • Estimation of cholinergic system

Statistical analysis One-way ANOVA used to test significance of difference between groups, followed by Dunnet’s Multiple Range Test Statistically significant: p<0.05

Results Outcomes Morphological features

Outcome AD AD+LP Body weight at 30 days (g) 222.20 + 17.19 289.66 + 8.63 Body weight at 60 days (g) 195.32 + 7.59 301.26 + 17.42 Brain weight at 30 days (g) 1.63 + 0.20 2.03 + 0.10 Brain weight at 60 days (g) 1.67 + 0.19 1.89 + 0.09

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* = p<0.05 vs. control group; # = p<0.05 vs. AD model Mean escape latency was shorter in rats with AD+LP compared to all other groups

* = p<0.05 vs. control group; # = p<0.05 vs. AD model AD+LP showed improved activity compared to all other groups Histopathological examination

• Protective group (AD+LP) showed healthy neurons and hyperchromatic nuclear chromatin. At 30 days, showed some partially degenerated neurons, but at 60 days, showed recovery with healthy neurons with prominent nuclei (p<0.05)

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* = p<0.05 vs. control group; # = p<0.05 vs. AD model Acetylcholine content higher in AD+LP group compared to all other groups

* = p<0.05 vs. control group; # = p<0.05 vs. AD model Acetylcholinesterase content lower in AD+LP group compared to AD group

Author’s conclusions L. plantarum MTCC1325 has anti-Alzheimer properties against D-Galactose induced Alzheimer’s disease. Administration of LP in rats showed an increase in body weight, improved behavioral activity and restored histopathological abnormalities.

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Table 5. Leblhuber F, Steiner K, Schuetz B, et al. Probiotic Supplementation in Patients with Alzheimer’s Dementia- An Explorative Intervention Study. Curr Alzheimer Res. 2018; 15(12): 1106-1113.32 Objective: To analyze serum and fecal specimens of AD patients before and after probiotic supplementation.

Methods Design Prospective interventional study Population Study included patients with an ICD-10 code for AD Intervention All patients received 28 days of multispecies probiotic

• L. acidophilus • L. casei • L. lactis • L. paracasei • L. plantarum • L. salivarius • B. lactis • B. bifidum

Fecal samples collected before and after supplementation Measurements • Quantification of microbiota via assays (Immundiagnostik)

• DNA extraction via MagNA Pure LC DNA Isolation Kit • Quantitative polymerase chain reaction (qPCR) via MutaPLATE qPCR

Assays • Fecal inflammation markers measured through enzyme-linked

immunosorbent assays Outcomes Changes in biomarkers of immune activation

Changes in fecal bacterial composition Statistical analysis Spearman rank correlation analysis performed to test for associations between

variables Significant differences: p<0.05

Results Baseline characteristics

Characteristic (n=18) Value Age (years) 76.7 + 9.1 MMSE 17.9 + 7.9 Clock drawing test 4.1 + 2.8 Kyn/Trp (mcgmol/mmol) 38.6 + 15.1 Neopterin (nmol/L) 10.0 + 5.2 C-reactive protein (mg/L) 1.6 + 2.3

Outcomes Biomarker/Bacterial strain (Serum)

Before 28 days (n=15)

After 28 days (n=15)

P-value

Neopterin (nmol/L) 9.8 + 4.9 12.8 + 10.1 >0.05 Kynurenine (mcmol/L) 1.82 + 0.29 2.06 + 0.42 <0.05

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• Fecal S100A12 correlated with fecal α1-antitrypsin (rs= 0.789; p<0.001)

and inversely with CDT (rs= -0.674; p<0.01) • Clostridium cluster I inverse correlation with zonulin (rs= -0.557; p<0.05) • Faecalibacterium prausnitzii correlated with Akkermansia muciniphila

(rs=0.619; p<0.01) • BDNF correlated with vitamin D concentrations (rs=0.767; p<0.001) and

inversely with nitrite levels (rs=-0.575; p<0.05)

Kynurenine/tryptophan ratio (mcmol/L)

38.2 + 13.8 39.4 + 10.5 >0.05

Biomarker/Bacterial strain (Fecal)

Before 28 days (n=18)

After 28 days (n=18)

P-value

α-antitrypsin (mg/g) 37.9 + 23.7 44.7 + 35.8 >0.05

Calprotectin (mg/L) 84.7 + 71 119 + 131 >0.05 Zonulin (mcg/L) 93.1 + 56.3 66.6 + 54.2 0.1 S100A12 3.5 + 6.0 N/A >0.05 Faecalibacterium prausnitzii (RNA copy/g feces, log10)

8.25 + 1.47 9.04 + 1.43 <0.001

Author’s conclusions

Probiotic supplementation had a significant impact on serum kynurenine levels, thus influencing the activation of immunologic processes.

Reviewer’s Conclusions Strengths Limitations

• One of first studies to compare changes of the microbiome before and after probiotic supplementation

• Not randomized nor blinded • No control group; difficult to determine

whether probiotics increase/decrease AD progression

• Small patient size Reviewer’s conclusion

Probiotics show promising results in altering the microbiome and certain biomarkers, but study has little clinical implication.

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Clinical question: Can the use of probiotics in patients with AD provide symptom relief and/or reduce disease progression?

Table 6. Akbari E, Asemi Z, Kakhaki RD, et al. Effect of probiotic supplementation on cognitive function and metabolic status in Alzheimer’s disease: A Randomized, Double-Blind and Controlled Trial. Front Aging Neurosci. 2016; 8:25633 Objective: To assess whether reinforcement of intestinal microbiota through probiotic supplementation helps improve cognitive and metabolic disorders in AD patients.

Methods Design 12-week randomized, double-blind, and controlled clinical trial conducted in

Iran Registration Iranian Website for Registration of Clinical Trials (IRCT): IRCT201511305623N60 Population Inclusion criteria Exclusion criteria • Patients with AD diagnosed

using NINDS-ADRDA criteria and revised criteria from National Institute on Aging-Alzheimer’s Association

• Age between 60 and 95 years

• Metabolic disorders • Chronic infections • Clinically relevant disorders • Consumption of probiotic

supplements at least six weeks before study

• Consumption of any forms of probiotics (e.g. yogurt, fermented foods)

Intervention Patients were matched for disease severity based on gender, BMI, and age and then randomly divided into two groups Dietary intakes recorded at baseline, 3rd, 6th, 9th weeks, and at the end of the trial Patients received either:

• 200mL/day of probiotic milk containing strains of L. acidophilus, L. casei, B. bifidum, and L. fermentum (2x109 CFU/g) for 12 weeks

• Milk containing no probiotics Outcomes Primary outcome: Measurements from Mini-Mental State Examination (MMSE)

Secondary outcome: Biomarkers of oxidative stress and metabolic profiles Statistical analysis Analyses utilized intention-to-treat population

Student’s t-test used to detect differences in anthropometric measures and macro/micronutrient intakes Pearson Chi-square test used to compare categorical variables Sample size calculated to be 25 people (α=0.05, β=0.2)

Results Baseline characteristics

Characteristic Control (n=30) Probiotic (n=30) P-value Age (years) 82.00 + 1.69 77.67 + 2.62 0.13

Weight at baseline (kg) 56.63 + 2.21 59.03 + 1.99 0.42 Weight at end-of-trial (kg)

56.80 + 2.17 59.50 + 1.98 0.36

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Outcomes

MDA, malondialdehyde; HOMA-IR, homeostasis model of assessment-estimated insulin resistance; HOMA-B, homeostasis model of assessment-estimated B-cell function; QUICKI, quantitative insulin sensitivity check index See Appendix Table A for detailed results

Outcome Control (n=30) Probiotic (n=30) P-value Change from baseline to end-of-trial

Change from baseline to end-of-trial

MMSE score -0.47 +1.90 <0.0001 MDA (mcmol/L) +0.06 -1.00 <0.0001 hs-CRP (mcg/mL) +2.05 -1.17 <0.0001 HOMA-IR +0.65 +0.30 0.002 HOMA-B +12.82 -5.30 0.002 QUICKI -0.02 -0.01 0.006 Triglycerides (mg/dL) -2.58 -25.27 0.003 VLDL (mg/dL) -0.51 -5.05 0.003

Author’s conclusions

Consumption of probiotics for 12 weeks had positive effects on the MMSE as well as several biomarkers of insulin metabolism but did not have any significant impact on biomarkers of stress and inflammation.

Reviewer’s conclusions Strengths Limitations

• Double-blind randomized controlled trial

• Accounted for effects on multiple biochemical markers (e.g. insulin, metabolic profile)

• Pharmacological treatment for AD in these patients were unknown

• Trial only lasted for 12 weeks; long term effects of probiotic supplementation unknown

• Diet was not tightly controlled in study, may confound results of macro/micronutrient intake

Overall conclusions Study shows promising results that probiotics can help with improving cognitive ability in AD patients but utilizing MMSE as its sole primary outcome warrants future testing to confirm probiotics’ place in AD therapy.

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Table 7. Agahi A, Hamidi GA, Daneshvar R, et al. Does Severity of Alzheimer’s disease contribute to its responsiveness to modifying gut microbiota? A double-blind clinical trial. Front Neurol. 2018; 9:66234 Objective: To test whether a mixture of probiotics can affect cognitive status as well as observing how serum concentration of inflammatory and oxidative biomarkers respond to oral bacteriotherapy.

Methods Design 12-week randomized, double-blind, and controlled clinical trial conducted in

Iran Registration IRCT 2017061534549N1 Population Inclusion criteria Exclusion criteria • Patients with AD diagnosed

using NINDS-ADRDA criteria and revised criteria from National Institute on Aging-Alzheimer’s Association

• Age between 60 and 95 years

• Metabolic disorders • Chronic infections • Clinically relevant disorders • Consumption of probiotic

supplements at least six weeks before study

• Consumption of any forms of probiotics (e.g. yogurt, fermented foods)

Intervention Patients were matched for disease severity based on gender, BMI, and age and then randomly divided into two groups Dietary intakes recorded at baseline, 3rd, 6th, 9th weeks, and at the end of the trial

• Patients received either: o Capsules containing L. fermentum, L. plantarum, B. lactis and

capsules containing L. acidophilus, B. bifidum, B. longum § Received either capsule every other day

o Placebo capsules with 500mg maltodextrine Outcomes Primary outcome: Results from Test Your Memory (TYM)

Secondary outcome: Biomarkers of oxidative stress and inflammation Statistical analysis Unpaired student’s t-test used to detect differences in anthropometric

measures Probiotic supplementation on TYM test and biomarkers determined by one-way analysis of variance (ANOVA), followed by Tukey’s post test Data reported as mean + standard error of the mean (SEM) Significant differences calculated at p<0.05 Sample size calculated to be 25 people (α=0.05, β=0.2)

Results Baseline characteristics

Characteristic Control (n=23) Probiotic (n=25)

P-value

Age (years) 80.57 + 1.79 79.70 + 1.72 0.36

Weight at baseline (kg) 60.63 + 1.26 60.12 + 1.12 0.42 Weight at end-of-trial (kg)

60.58 + 2.36 60.32 + 1.42 0.48

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Outcomes

See Appendix Tables B and C for detailed results

Outcome Control (n=23) Probiotic (n=25) P-value Change between pre- and post-treatment

Change between pre- and post-treatment

Change in TYM score +3.12 +2.78 0.82 Inflammatory markers IL-10 (pg/mL) +0.17 -0.14 >0.05 IL-6 (pg/mL) -0.21 +1.09 >0.05 TNF-α (pg/mL) +1.50 +0.33 >0.05

Author’s conclusions Probiotic use had little effect on biochemical markers of inflammation. Benefits from probiotic supplementation may be influenced by disease severity.

Reviewer’s conclusions Strengths Limitations

• Double-blind randomized controlled trial • Included biomarkers of inflammation (in

contrast to previous study) • TYM for cognitive impairment provided a

more complete understanding of a patient’s cognitive status

• Pharmacological treatment for AD in these patients were unknown

• TYM is not indicated for patients with severe AD

• Trial only lasted for 12 weeks; long term effects of probiotic supplementation unknown

• Uneven distribution of AD severity in patient population

• Diet was not tightly controlled in study, may confound results of macro/micronutrient intake

• Not all markers of inflammation that are cognitive related were measured

Overall conclusions Probiotic supplementation is not beneficial in patients with severe AD. However, this study shows promise in confirming probiotic’s role in helping those with mild to moderate AD.

Conclusions and recommendations

1. AD is commonly seen in patients over the age of 60 years 2. AD is a complex disease with numerous factors playing a role in its pathogenesis, including

dysbiosis of the gut microbiome 3. Studies have promising results in showing that probiotics potentially can improve cognitive

function in AD patients, particularly in patients with mild to moderate AD 4. Given the limitations outlined in the previous studies, I would be cautious in starting an AD

patient with probiotics until we see a larger clinical trial with longer safety outcomes is performed.

Areas of future exploration:

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1. Strain-specific probiotic interventions 2. Long-term probiotic supplementation in AD patients 3. Potential benefits of fecal microbiota transplantation in at-risk patients and those with mild to

moderate AD

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Appendix Table A. List of probiotics with strain, clinical condition, effectiveness, and dose

Brand Name

Bacterial Species Clinical condition Effectiveness Dose (bacteria count/dosing)

Activia B. lactis DN-173 010, L. bulgaricus, L. laxtis, Streptococcus thermophilus

IBS C 4 oz/once daily

Align Bifidobacterium infantis 35624

IBS B 1 billion/once daily

BioGaia L. reuteri protectis SD2112 Infectious diarrhea treatment IBS

A C

100 million/once daily

Bio-K+ L. acidophilus CL1285, L. casei LBC80R

AAD prevention CDAD prevention

N/A 50 billion/twice daily

Culturelle L. rhamnosus GG AAD prevention Infectious diarrhea treatment/prevention CDAD prevention Crohn’s disease IBS

A A/B B/C C B/C*

10 billion/once daily

Danactive L. casei DN-114001 AAD prevention Infectious diarrhea prevention CDAD prevention

A 3.1 oz (10 billion)/cup

Florastor Saccharomyces Boulardii AAD prevention Infectious diarrhea treatment/prevention CDAD prevention Crohn’s disease

A A/B B/C C

250mg/twice daily

Mutaflor E. coli NISSLE 1917 UC induction/maintenance

B/A 100mg/twice daily

VSL*3 Streptococcus thermophilus, B. breve, B. longum, B. infantis, L. acidophilus, L. planatrum, L. paracasei, L. delbreuckii/bulgaricus

IBS UC induction/maintenance Pouchitis

B/C B/A A

122.5 billion (capsule) 450 billion (satchet)

IBS=irritable bowel syndrome; AAD=antibiotic-associated diarrhea; CDAD=C. difficile associated diarrhea;UC=ulcerative colitis

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Table B. Mean values of behavioral test and biomarker measurements in probiotic and control groups

Table C. Comparison of the change percent of the biochemical factors between the control and probiotic groups.

Table D. Pre- and post-treatment cognitive scores and biochemical values in the CON and PRO groups

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