Effects of Probiotics on Peripheral Immunity in Parkinson's Disease

Parkinson's Disease Clinical Trial

Official title:

Effects of Probiotics on Peripheral Immunity in Parkinson's Disease

Clinical Trial Details — Status: Enrolling by invitation

Administrative data

• NCT number: NCT05173701
• Other study ID #: PROB-PD
• Status: Enrolling by invitation
• Phase: N/A
• Start date: November 22, 2021
• Est. completion date: January 2023

Study information

• Verified date: June 2022
• Source: Università degli Studi dell'Insubria
• Contact: n/a
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial Summary

Parkinson's Disease (PD) is a common neurodegenerative disease, with no disease-modifying treatment available, therapy is therefore only symptomatic. The pathophysiology of the disease is still unclear, but inflammatory mechanisms are reported to play a prominent role. An involvement of peripheral adaptive immunity, with an imbalance in T cell subpopulations and in the expression of transcriptional factors (TF) in Cluster of Differentiation (CD) 4 positive T cells has been reported. An initial aggregation of α-synuclein (α-syn) in the gut with subsequent propagation along the vagus nerve to the brain has also been hypothesised. Interestingly, in an α-syn overexpressing murine model, the absence of gut microbiota prevented both microglia activation and motor impairment, pointing to a fundamental role of the microbiota in the development of PD. It has been shown that in Peripheral Blood Mononuclear Cells (PBMC) of PD patients, probiotics modulate the in vitro production of cytokines toward an anti-inflammatory profile. The investigators developed a clinical trial protocol for the evaluation of probiotics' effects on the peripheral immune system profile in Parkinson's Disease patients. ROS, Lymphocyte subpopulations, TF levels in PBMC will be assessed at baseline and after treatment with a mixture of probiotics in PD patients to assess immunomodulatory effects of said treatment. Motor and non-motor symptoms of PD will also be monitored through the trial period.

Description:

Introduction Parkinson's Disease (PD) is a common neurodegenerative disease, affecting up to 1-2 people in 1000 at any given time. Prevalence increases with age and is estimated at 1% in people over 65.

There is no available treatment to prevent PD onset or to delay its progression and therapy is focused on symptoms management. The administration of carbidopa/levodopa allows for control of motor symptoms, but it becomes less effective as the disease progresses and increasing daily doses causes more frequent and severe side effects.

The histopathologic hallmark of PD is the loss of dopaminergic neurons and accumulation of α-synuclein (α-syn) in surviving neurons, but the underlying pathophysiology is still unclear. Inflammatory mechanisms have been suggested to play a prominent role in the disease, with an imbalance between detrimental and protective immune functions, as well as neurotoxicity caused by reactive oxygen species (ROS).

Further evidence highlights the involvement of peripheral adaptive immunity in PD, reporting an imbalance in T cell subpopulations and in the expression of transcriptional factors in CD4+ T cells in PD patients.

In these patients, non-motor symptoms may precede the onset of a clinically established disease.

Etiopathogenesis of PD is still unknown, but seminal work by Braak et al. hypothesised an initial aggregation of α-syn in the gut with subsequent propagation along the vagus nerve to the brain, finally reaching the substantia nigra in the mesencephalon.

Interestingly, in an α-syn overexpressing murine model of PD, the absence of gut microbiota prevented both microglia activation and motor impairment, thus pointing to a fundamental role of the gut and microbiota in the pathogenesis and development of PD.

In a recent paper, Magistrelli et al. confirmed that probiotics may influence the peripheral immune system. Particularly, in peripheral blood mononuclear cells (PMBCs) of a cohort of PD patients, probiotics were able to modulate the production of cytokines toward an anti-inflammatory profile and to reduce the production of reactive oxygen species (ROS). The clinical effects of probiotics have been also explored in other pathological conditions. Tankou et al. administered VSL#3 in a cohort of 9 multiple sclerosis patients, whose peripheral immune system shifted toward an anti-inflammatory profile, with an inverse tendency after VSL#3 discontinuation. These results were also confirmed by the same group after administration of a mix of probiotics (four strains of Lactobacillus and three strains of Bifidobacterium).

In the light of this evidence, the investigators designed a randomised controlled clinical trial whose primary objective is to test whether probiotics can influence the peripheral immune system in a cohort of PD patients. The aim of the protocol is to highlight changes in transcriptional factors messenger Ribonucleic Acid (mRNA) levels, lymphocytes subpopulations (Th1, Th2, Th17 and Treg), cytokine levels and ROS production.

Methods and analyses This explorative study is a randomized placebo-controlled double-blind study to evaluate the efficacy of probiotics in modulating the peripheral immune system in Parkinson's Disease subjects. Participants will be randomised in two comparable groups and treated with a mixture of probiotics or matching placebo once daily for three months. The primary objective of this study is to verify the effects of probiotics on the peripheral immune system. As exploratory outcomes, motor and non-motor symptoms of PD will be monitored, as well as cognitive function and quality of life over the three months treatment period via a selection of clinical rating scales.

Participant identification Subjects will be recruited among patients with scheduled routine follow-up visits at Azienda Ospedaliero-Universitaria Maggiore della Carità di Novara. Detailed information on comorbidities, current medical therapy and demographic data of these subjects will be readily available. The trial design will be briefly outlined and patients will be asked about their interest in participating. Willing subjects will attend a screening visit, written informed consent will be obtained before confirming eligibility.

Trial procedures

Clinical During enrolment visit, medical and neurological examination will assess the need for immediate variations in medical therapy for each participant. Within two weeks, any therapy modification will be completed and baseline visit will be scheduled.

During baseline visit (T0), physical and neurological examinations will be repeated to confirm the subject's conditions and persistence of inclusion criteria, a baseline blood withdrawal will be performed and all clinical evaluation scales will be completed to set baseline scores. Each participant, after the subscription of an appropriate informed consent, will be randomized and given a treatment box, containing single-dose sachets with 2,7g of powder of the allocated formulation. Patients will be instructed to take daily doses at home every morning before breakfast, mixing the content of one sachet in about 125 ml of fresh water or other cold, non-carbonated drink. Participants will be instructed to keep unused or empty packaging for recollection and compliance evaluation.

Follow-up visits will be scheduled at 4 weeks after T0 (T1) and at 12 weeks after T0 (T2). At T1, physical and neurological examination will be repeated and all clinical scales administered again. At T2, all assessments performed at T0 will be carried out again, including blood withdrawal.

At T0 and T2, withdrawal of 40 ml venous blood will be performed after a fasting night, between 8:00 and 10:00 am, in ethylenediaminetetraacetic acid-coated tubes (BD Vacutainer). Tubes will be coded and stored at room temperature until processing, within 24 hours. Complete blood count with differential analysis will be conducted on separate blood samples.

Laboratory methods

Cytokine measurement The possible influence of probiotic treatment on inflammatory profile will be evaluated by measuring at T0 and T2 the plasma levels of pro (e.g. Tumor Necrosis Factor (TNF)-α, Interferon (IFN)-γ) and anti-inflammatory cytokines (Interleukin (IL)-10, IL-4). Plasma aliquots from every sample will be separated and stored for cytokines assays. 2 mL of fresh blood will be centrifuged at 1400g for 10 minutes at room temperature and two plasma aliquots of 350 µL each will be stored in 1,5mL vials to assay cytokines levels.

Flow cytometric evaluation of immune phenotype In order to investigate possible changes in immune phenotype, the profile of both innate and adaptive immunity will be deepened by means of a cytofluorimetric evaluation according to the strategy described by Kustrimovic et al. in 2018 and with additional panels aimed specifically dedicated to innate immunity. The following cell subsets of the adaptive immune system will be assessed: CD4+ and CD8+ T naïve/memory cells, CD4+ T helper subsets and CD4+ regulatory T cells. Moreover, for innate immunity, monocytes and Natural Killer (NK) cells will be also assessed. Acquisition will be performed on a BD Fluorescence-activated cell sorting (FACS) Celesta flow cytometer (Becton Dickinson Italy, Milan, Italy) with BD FACS Diva software (version 8.0.1.1) and data will be analysed with FlowJo software (version 10.7.1)

Transcriptional factors mRNA evaluation According to previous studies by Kustrimovic and De Francesco, the ability of probiotic treatment to modify mRNA levels of the main transcriptional factors in CD4+ T lymphocytes will also be investigated. CD4+ positive cells will be obtained by PBMC, which will be isolated from whole blood using Ficoll-Paque Plus density gradient centrifugation. After resuspension, residual contaminating erythrocytes will be lysed by addition of 10 mL of lysis buffer ((g/L) NH4Cl 8.248, KHCO3 1.0, EDTA 0.0368). Cells will be washed twice in Purified Bovine Serum (PBS) by addition of 10 mL of PBS, then centrifuged at 300 g for 10 min at room temperature and resuspended in 10 mL of Roswell Park Memorial Institute medium (RPMI)/10% Fetal Bovine Serum (FBS). Manual cell count will then be performed to set CD4 separation reagents quantities.

Typical PBMC preparations will contain at least 80% lymphocytes. CD4+ T cells will then be isolated from PBMC by means of Dynabeads CD4 Positive Isolation kit. At least 50,000 separated CD4+ T cells will then be resuspended in PerfectPure RNA lysis buffer (5 Prime GmbH, Hamburg, Germany), and total RNA will be extracted by PerfectPure RNA Cell Kit™.

Reverse-transcription will be performed on resulting mRNA using a random primer and a high-capacity complementary DNA (cDNA) Real Time (RT) kit.

RT-Polymerase Chain Reactions (PCR) will be performed with 1 μM cDNA. Amplification of cDNA will allow for the analysis of mRNA levels of the transcription factor genes TBX21, STAT1, STAT3, STAT4, STAT6, RORC, GATA3, FOXP3 and NR4A2.

Treatment composition and rationale

Treatment:

Bifidobacterium animalis subsp. lactis BS01 ≥ 1 x 10^9 Colony Forming Units (CFU) Bifidobacterium longum 03 ≥ 1 x 10^9 CFU Bifidobacterium adolescentis BA02 ≥ 1 x 10^9 CFU Fructo-oligosaccharides FOS 2500 mg Maltodextrin q.s.

Placebo:

Maltodextrin q.s.

Each probiotics formulation contains Bifidobacterium animalis subsp. lactis (BS01), Bifidobacterium longum (BL03) and Bifidobacterium adolescentis (BA02). Fructo-oligosaccharides (FOS) is added as a prebiotical component and maltodextrin is used as a bulking agent.

The choice of probiotics for this study is based on solid literature data: in particular PBMC of PD patients treated with Bifidobacterium animalis subsp. Lactis (BS01) showed an increased production of anti-inflammatory cytokine IL-10. Said treatment also supported restoration of membrane integrity in a model of CacO2-cells.

All probiotics used are included in the "Qualified Presumption of Safety (QPS)-recommended biological agents intentionally added to food or feed as notified to European Food Safety Authority (EFSA) 12".

The placebo formulation is solely composed by maltodextrin.

Sample size calculation, data analysis The design of this study is explorative and equally randomised to treatment with probiotics or placebo. Due to the exploratory nature of the proposed study, a formal sample size calculation is not strictly required. Sample size for this study has thus been based mainly on previous results from another in vitro study, which yielded statistically significant results in a small cohort of 40 PD patients for all tested probiotic strains, with a global reduction in proinflammatory cytokines production and an increase in anti-inflammatory cytokines. In the present study, the expected sample will be doubled, with a total of 80 specimens to be collected both at T0 and T2.

Data from previous in vitro study has been used to estimate orientational sample sizes for the in vitro effect of all tested probiotic strains and a sample size of 80 subjects allows for the determination of most tested cytokines variations with an expected power greater than 80%, setting the threshold for statistical significance at 0.05.

Statistical analysis on collected data will be performed after assessing normality of data distribution and using two-tailed Student's t test or Mann-Whitney test as appropriate for independent variables comparisons. Data correlations will be analysed through Pearson's or Spearman's correlation tests. ANOVA or the Kruskal-Wallis tests will be used for comparisons between more than two groups and paired t-test or Wilcoxon signed ranks test will be used when comparing paired groups (e.g. baseline vs end of treatment, treatment vs placebo). Thresholds for statistical significance will be set according to the specific test characteristics.

Recruitment information / eligibility

• Status: Enrolling by invitation
• Enrollment: 88
• Est. completion date: January 2023
• Est. primary completion date: September 2022
• Accepts healthy volunteers: No
• Gender: All
• Age group: N/A and older

Eligibility

Inclusion Criteria:

• a diagnosis of Parkinson's Disease;

• a disease duration between 2 and 5 years at baseline

Exclusion Criteria:

• past or concomitant autoimmune disease

• previous or ongoing immune-modulating or immunosuppressive therapy

• inflammatory bowel diseases, colorectal diseases or past major abdominal or pelvic surgery

• antibiotics therapy up to three months before enrolment

• usage of tube feeding

• known or suspected allergy to any component of the treatment or placebo mixtures

• known and established cognitive decline or any comorbidity preventing reliable completion of trial assessments

• motor fluctuations

Related Conditions & MeSH terms

• Parkinson Disease
• Parkinson's Disease

Intervention

Dietary Supplement:
• Probiotics
Selected probiotics mixture, in powder form, will be administered once daily for three months in the morning.
• Placebo
A placebo (2,7g maltodextrin) will be administered once daily for three months in the morning.

Locations

Country	Name	City	State
Italy	Azienda Ospedaliero-Universitaria "Maggiore della Carità"	Novara

Sponsors (3)

Lead Sponsor	Collaborator
Franca Marino	Università degli Studi del Piemonte Orientale "Amedeo Avogadro", Università degli Studi dell'Insubria

Country where clinical trial is conducted

Italy, 

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* Note: There are 33 references in all — Click here to view all references

Outcome

Type	Measure	Description	Time frame	Safety issue
Other	Changes in Unified Parkinson's Disease Rating Scale (UPDRS) scores	Scores for the UPDRS will be collected for all participants during scheduled visits.; Minimum score is 0 and maximum score is 199, with higher scores representing worse outcomes.	Baseline, 6 weeks, 12 weeks
Other	Changes in Hoehn and Yahr's (H&Y) assessment scale	Scores for the H&Y assessment will be collected for all participants during scheduled visits.; H&Y scale includes stages from 0 to 5. Intermediate stages 1.5 and 2.5 are widely used. A higher stage represents worse clinical conditions.	Baseline, 6 weeks, 12 weeks
Other	Changes in Non-Motor Symptoms Scale in Parkinson's disease (NMSS) scores	Scores for the NMSS will be collected for all participants during scheduled visits.; The NMSS is divided into 9 different domains and 30 single questions on non-motor symptoms severity and frequency. Severity is reported from 0 (None) to 3 (Severe), frequency is can range from 1 (Rarely) to 4 (Very Frequent). Each question yields a Frequency x Severity value and total minimum score is 0, while maximum is 360. Higher scores represent more frequent and more severe non-motor symptoms.	Baseline, 6 weeks, 12 weeks
Other	Changes in Beck's Depression Inventory Scale (BDI-II) scores	Scores for BDI-II will be collected for all participants during scheduled visits.; BDI-II contains 21 questions, each answer being scored on a scale value of 0 to 3. Higher total scores indicate more severe depressive symptoms.; Minimum score is 0 and maximum score is 63.	Baseline, 6 weeks, 12 weeks
Other	Changes in Zung's Self Rating Anxiety Scale (SAS) scores	Scores for SAS will be collected for all participants during scheduled visits. This scale consists of 20 statements for which a frequency self-assessment is requested.; Minimum score is 20, maximum score is 80, with higher scores representing worse anxiety symptoms.	Baseline, 6 weeks, 12 weeks
Other	Changes in Composite Autonomic Symptoms Scale 31 (COMPASS-31) scores	Scores for COMPASS-31 will be collected for all participants during scheduled visits.; COMPASS-31 total scores can range between 0 to 100, with higher values representing more severe symptoms.	Baseline, 6 weeks, 12 weeks
Other	Changes in Montreal Cognitive Assessment (MOCA) scores	Scores for MOCA will be collected for all participants during scheduled visits. Total scores for MOCA range between 0 and 30, with lower scores representing worse cognitive performance.	Baseline, 6 weeks, 12 weeks
Other	Changes in the Patient Assessment of Constipation - Quality Of Life (PAC-QOL) questionnaire	Scores for PAC-QOL will be collected for all participants during scheduled visits.; The range of possible scores on this questionnaire is 0 to 112, with higher scores indicative of a greater burden of constipation on quality of life.	Baseline, 6 weeks, 12 weeks
Other	Changes in reported Bristol Stool Form Chart assessment	Bristol Stool Form Chart will be used upon scheduled visits to evaluate stool form changes.; This data is qualitative and values are not representative per se of better or worse outcomes or symptoms.	Baseline, 6 weeks, 12 weeks
Other	Changes in Constipation Assessment Scale (CAS) scores	Scores for CAS will be collected for all participants during scheduled visits. CAS scores range from 0 to 16 and are calculated on 8 items rated from 0 (no problem) to 2 (severe problem). Higher scores represent worse constipation symptoms.	Baseline, 6 weeks, 12 weeks
Other	Changes in Wexner Constipation Scoring System (WCSS) scores	Scores for WCSS will be collected for all participants during scheduled visits. Possible values for the WCSS range from 0 to 30, with higher scores representing worse constipation symptoms.	Baseline, 6 weeks, 12 weeks
Primary	Changes in plasma IFN-? level	IFN-? level will be assessed in plasma samples via ELISA assay.	Baseline, 12 weeks
Primary	Changes in plasma TNF-a level	TNF-a level will be assessed in plasma samples via ELISA assay.	Baseline, 12 weeks
Primary	Changes in plasma IL-4 level	IL-4 level will be assessed in plasma samples via ELISA assay.	Baseline, 12 weeks
Primary	Changes in plasma IL-17A level	IL-17A level will be assessed in plasma samples via ELISA assay.	Baseline, 12 weeks
Primary	Changes in plasma IL-10 level	IL-10 level will be assessed in plasma samples via ELISA assay.	Baseline, 12 weeks
Primary	Changes in plasma Transforming Growth Factor (TGF)-ß level	TGF-ß level will be assessed in plasma samples via ELISA assay.	Baseline, 12 weeks
Primary	Changes in ROS production capacity	ROS production will be evaluated by the superoxide dismutase-sensitive cytochrome C reduction assay and results of these assays will be expressed as nmol of reduced cytochrome C / 10^6 cells / 30 min.	Baseline, 12 weeks
Secondary	Changes in Naive-Memory lymphocytes subpopulations	Lymphocytes subpopulations will be assessed through flow cytometry with panel Naive-Mem + CD45V500 (CD45/CD3/CD4/CD8/CD45RA/CCR7).; The following subpopulations will be assessed as percentage of parent population: CD3+ lymphocytes (% of ly CD45+), CD4+ T lymphocytes (% of CD3+) CD4+ T Naive (% of CD4+), CD4+ T Central Memory (CM) (% of CD4+), CD4+ T Effector Memory (EM), (% of CD4+), CD4+ T Effector Memory cells Re-expressing CD45RA (EMRA) (% of CD4+).	Baseline, 12 weeks
Secondary	Changes in T helper (Th) lymphocytes subpopulations	Lymphocytes subpopulations will be assessed through flow cytometry with panel Th+CD3+CD45V500 (CD45/CD3/CD4/CXCR3/CCR4/CCR6).; The following subpopulations will be assessed as percentage of parent population: CD3+ lymphocytes (% of lyCD45+), CD4+ T lymphocytes (% of CD3+), Th1 (% of CD4+), Th1-Th17 (% of CD4+), Th2 (% of CD4+), Th17 (% of CD4+).	Baseline, 12 weeks
Secondary	Changes in Regulatory T cells (Treg) lymphocytes subpopulations	Lymphocytes subpopulations will be assessed through flow cytometry with panel TREG+CD3+CD45V500 (CD45/CD3/CD4/CD25/CD127/CD45RA).; The following subpopulations will be assessed as percentage of parent population: CD3+ lymphocytes (% Ly CD45+), CD4+ T lymphocytes (% of CD3+), CD4+ Treg (% of CD4+), Naïve Treg (% of Treg), Active Treg (% of Treg).	Baseline, 12 weeks
Secondary	Changes in Monocyte subpopulations	Monocytes subpopulations will be assessed through flow cytometry with Monocyte Panel (CD45/HLA-DR/CD14/CD16).; The following subpopulations will be assessed as percentage of parent population: Classical Monocytes (% of all HLA-DR+ Monocytes), Intermediate Monocytes (% of all HLA-DR+ Monocytes), Non-Classical Monocytes (% of all HLA-DR+ Monocytes).; The three assessed subsets will also be characterised by their Median Fluorescence Intensity (MFI).	Baseline, 12 weeks
Secondary	Changes in NK subpopulations	NK cells subpopulations will be assessed through flow cytometry with NK-NKT Cell Panel (CD45/CD3/CD56/CD16/CD57/CD14/CD19).; The following subpopulations will be assessed as percentage of parent population: NK cells (% of Ly CD45+), CD56dim/CD16bright (% of NK), CD56bright/CD16dim (% of NK), CD57- (% of CD56dim/CD16bright), CD57+ (% of CD56dim/CD16bright), CD57- (% of CD56bright/CD16dim), CD57+ (% of CD56bright/CD16dim).	Baseline, 12 weeks
Secondary	Changes in CD4+ T cells TBX21 mRNA levels	mRNA levels of the transcription factor gene TBX21 will be assessed through RT-PCR	Baseline, 12 weeks
Secondary	Changes in CD4+ T cells STAT1 mRNA levels	mRNA levels of the transcription factor gene STAT1 will be assessed through RT-PCR	Baseline, 12 weeks
Secondary	Changes in CD4+ T cells STAT3 mRNA levels	mRNA levels of the transcription factor gene STAT3 will be assessed through RT-PCR	Baseline, 12 weeks
Secondary	Changes in CD4+ T cells STAT4 mRNA levels	mRNA levels of the transcription factor gene STAT4 will be assessed through RT-PCR	Baseline, 12 weeks
Secondary	Changes in CD4+ T cells STAT6 mRNA levels	mRNA levels of the transcription factor gene STAT6 will be assessed through RT-PCR	Baseline, 12 weeks
Secondary	Changes in CD4+ T cells RORC mRNA levels	mRNA levels of the transcription factor gene RORC will be assessed through RT-PCR	Baseline, 12 weeks
Secondary	Changes in CD4+ T cells GATA3 mRNA levels	mRNA levels of the transcription factor gene GATA3 will be assessed through RT-PCR	Baseline, 12 weeks
Secondary	Changes in CD4+ T cells FOXP3 mRNA levels	mRNA levels of the transcription factor gene FOXP3 will be assessed through RT-PCR	Baseline, 12 weeks
Secondary	Changes in CD4+ T cells NR4A2 mRNA levels	mRNA levels of the transcription factor gene NR4A2 will be assessed through RT-PCR	Baseline, 12 weeks