Search

Brain degenerative disorders such as Alzheimer’s disease (AD) can be exacerbated by aberrant metabolism. Supplementation with probiotic bacteria is emerging as a promising preventative strategy for both neurodegeneration and metabolic syndrome. In this study, we assess the impact of the Lab4b probiotic consortium on (i) cognitive and pathological markers of AD progression and (ii) metabolic status in 3xTg-AD mice subjected to metabolic challenge with a high fat diet. The group receiving the probiotic performed better in the novel object recognition test and displayed higher hippocampal neuronal spine density than the control group at the end of the 12 weeks intervention period. These changes were accompanied by differences in localised (brain) and systemic anti-inflammatory responses that favoured the Probiotic group together with the prevention of diet induced weight gain and hypercholesterolaemia and the modulation of liver function. Compositional differences between the faecal microbiotas of the study groups included a lower Firmicutes:Bacteroidetes ratio and less numbers of viable yeast in the Probiotic group compared to the Control. The results illustrate the potential of the Lab4b probiotic as a neuroprotective agent and encourage further studies with human participants.

Additional file 4: Figure S3. Heatmap of the differentially abundant genera amongst treatments in βgal mice. Median abundances were scaled according to row Z-score. Only genera with P < 0.5 are represented.

Additional file 3: Figure S2. Heatmap of the differentially abundant genera amongst treatments in TSHR-immunized mice. Median abundances were scaled according to row Z-score. Only genera with P < 0.5 are represented.

Additional file 1: Supplementary methods. Table S1. Characteristics of patients with sight-threatening GO recruited at the University Hospital Duisburg-Essen providing samples for hFMT production.

Background Graves’ disease (GD) is an autoimmune condition in which autoantibodies to the thyrotropin receptor (TSHR) cause hyperthyroidism. About 50% of GD patients also have Graves’ orbitopathy (GO), an intractable disease in which expansion of the orbital contents causes diplopia, proptosis and even blindness. Murine models of GD/GO, developed in different centres, demonstrated significant variation in gut microbiota composition which correlated with TSHR-induced disease heterogeneity. To investigate whether correlation indicates causation, we modified the gut microbiota to determine whether it has a role in thyroid autoimmunity. Female BALB/c mice were treated with either vancomycin, probiotic bacteria, human fecal material transfer (hFMT) from patients with severe GO or ddH2O from birth to immunization with TSHR-A subunit or beta-galactosidase (βgal; age ~ 6 weeks). Incidence and severity of GD (TSHR autoantibodies, thyroid histology, thyroxine level) and GO (orbital fat and muscle histology), lymphocyte phenotype, cytokine profile and gut microbiota were analysed at sacrifice (~ 22 weeks). Results In ddH2O-TSHR mice, 84% had pathological autoantibodies, 67% elevated thyroxine, 77% hyperplastic thyroids and 70% orbital pathology. Firmicutes were increased, and Bacteroidetes reduced relative to ddH2O-βgal; CCL5 was increased. The random forest algorithm at the genus level predicted vancomycin treatment with 100% accuracy but 74% and 70% for hFMT and probiotic, respectively. Vancomycin significantly reduced gut microbiota richness and diversity compared with all other groups; the incidence and severity of both GD and GO also decreased; reduced orbital pathology correlated positively with Akkermansia spp. whilst IL-4 levels increased. Mice receiving hFMT initially inherited their GO donors’ microbiota, and the severity of induced GD increased, as did the orbital brown adipose tissue volume in TSHR mice. Furthermore, genus Bacteroides, which is reduced in GD patients, was significantly increased by vancomycin but reduced in hFMT-treated mice. Probiotic treatment significantly increased CD25+ Treg cells in orbital draining lymph nodes but exacerbated induced autoimmune hyperthyroidism and GO. Conclusions These results strongly support a role for the gut microbiota in TSHR-induced disease. Whilst changes to the gut microbiota have a profound effect on quantifiable GD endocrine and immune factors, the impact on GO cellular changes is more nuanced. The findings have translational potential for novel, improved treatments.

Additional file 11: Table S2. Number of positive mice for each disease parameter. Numbers of TSHR-immunized mice positive for thyroid-stimulating antibodies (TSAbs), increased circulating thyroxine (T4), hyperplastic thyroid morphology, orbital brown fat (BAT) enlargement or muscle fiber atrophy are reported. The threshold for positivity was defined as upper 99% CI of the corresponding βgal groups. Table S3. Classification of total outcome of autoimmune hyperthyroidism and orbitopathy. Disease classification was done along the Z-Score values in Figure 8. The number of mice is given in %. Subclinical disease (Z-Score 0): These mice displayed clear signs of autoimmune hyperthyroidism and/or orbital pathology. Clinical disease is classified in mild and moderate/severe in accordance with the Z Score values as indicated (mild: Z Score 01; moderate/severe: Z-Score >1).

Additional file 2: Figure S1. Supplementary results for the endpoint gut microbiota analysis. (A and B) Microbiota composition according to different anatomical samples in TSHR and βgal-immunized mice (samples/source: colon = 48, entire = 30, small = 51 from final timepoint and stool = 105 from baseline and mid-timepoint). (A) Alpha-diversity indices of the source of the microbiota sampled, Wilcoxon-Mann BH corrected test: ***P < 0.05. (B) NMDS of Bray-Curtis distances according to immunizations and sources at the endpoint. PERMANOVA between entire-colon samples P > 0.05. (C and D) Endpoint composition of the LGI microbiota amongst treatments in βgal-immunized mice. (n βgal mice/treatment at endpoint: control = 8, hFMT = 8, Lab4 = 10, vancomycin = 19). (C) Alpha diversity amongst treatments, Wilcoxon-Mann BH corrected test:***P < 0.001. (D to G) RandomForest of a model excluding vancomycin samples (n mice/treatment endpoint: control = 20, hFMT = 19, Lab4 = 20). (D) Confusion matrix for treatments w/o vancomycin samples. Diagonal boxes represent the number of samples correctly predicted. (E) Top-10 variables of treatment classification according to the Mean Decrease Gini, including the microbiota source as an effect related to figure E. (F) Confusion matrix for immunizations in a model w/o vancomycin samples. (n mice/immunization endpoint: TSHR = 33 and βgal = 26). (G) Top-10 variables of immunizations classification according to the Mean Decrease Gini, including the microbiota source and treatments as an effect related to figure G. Wilcoxon-Mann test with BH correction: ***P < 0.005; **P < 0.01; *P < 0.05.

Additional file 8: Figure S7. Mice eye signs and orbital tissues abnormalities analysed histologically. (A) Mice eye signs indicating orbital disease. Representative images of a ßgal mouse lacking pathological eye signs and of a TSHR-immunized mouse with acute signs of inflammation and/or proptosis. (B) UCP-1 (uncoupling protein -1) as a marker for brown fat tissue (BAT). Elevated portions of small vacuoled BAT were present in TSHR-immunized mice. Representative pictures of stainings are shown. (C, D) CD3 as a marker for T cells. Some CD3+ T cells (indicated by arrows) were detected in adipose tissues (C) and in muscle tissues (D) of βgal and TSHR-immunized mice. Immunohistochemistry of orbital tissues was carried out as described in detail before [40]

Federica Brosio,1 Giulia Masetti,1 Giulio Matteo,1 Armando Stefanati,2 Giovanni Gabutti2 1Postgraduate School of Hygiene and Preventive Medicine, Department of Medical Sciences, University of Ferrara, Ferrara, Italy; 2Department of Medical Sciences, University of Ferrara, Ferrara, Italy Abstract: Herpes zoster (HZ) is an acute vesicular dermatitis with a typical dermatomal distribution, caused by the varicella zoster virus (VZV), often preceded and accompanied by prodromal pain or pruritus. HZ may be related to several complications such as postherpetic neuralgia (PHN). The incidence and severity of the disease increase with aging, due to immunosenescence and in particular to the decline of the specific cell-mediated immunity (CMI). The impact of HZ in terms of morbidity and short- and long-term complications, the availability of suboptimal treatment options to date, and the high costs for the diagnostic and clinical-therapeutic management of patients have motivated the search for a new preventive approach through the development of a vaccine. The vaccine currently in use with live-attenuated virus (ZVL) has been shown to be effective in reducing the incidence of HZ, its impact, and the onset of PHN, although the efficacy is lower in older subjects and tends to decrease some years after immunization. A new adjuvanted recombinant subunit vaccine (HZ/su), containing the VZV glycoprotein E (gE) and the AS01B adjuvant system, is now a very promising alternative to ZVL; in several clinical studies, it showed a good safety profile and was able to elicit high immune humoral and cell-mediated responses, both maintained up to 9years. Furthermore, HZ/su vaccine was effective both in preventing HZ and in reducing the onset of PHN and other complications. HZ/su has been recommended and preferred over ZVL by the Advisory Committee on Immunization Practices (ACIP) for the prevention of HZ and its complications in immunocompetent adults aged ≥50years, even if already vaccinated with ZVL, through a two-dose schedule. HZ/su has been approved in Canada, USA, Europe, and Japan and is currently being approved in Australia. The aim of this review was to describe the epidemiological data, HZ and PHN risks and their impact on the social life and common life of infected people, and ZVL and HZ/su vaccine development including various clinical trials and efficacy, safety, and tolerability profiles. Keywords: herpes zoster, adjuvanted recombinant herpes zoster subunit vaccine, live-attenuated herpes zoster vaccine

Figure S1. Temporal stability of fecal microbiota and cage effect of the immunizations. Weighted Unifrac distances of mice fecal microbial communities represented over the time course of the experiment according to the immunization (A) or the cage (B). Permutational MANOVA of weighted Unifrac distances according to timepoint, immunizations, caging, and their interactions (time × cage; time × immunization; immunization × cage) as described in Additional file 2. The time had a significant effect on the stability of the fecal microbiota (P = 0.001), in particular between the baseline (T0) and the last timepoint (T4, P = 0.003); and between the T1 and T4 (P = 0.009). The interaction between time and immunization was significant (P = 0.007). Cage was also significant, in particular the interaction cage × timepoint (P = 0.001) and cage × immunization (P = 0.002). Significant differences within the same immunization group cage has been observed (TSHR group in C4 and C5, P = 0.01). (PDF 152 kb)

Figure S1. Bifidobacterium counts derived from the 28F-combo primers in the TSHR-immunized mice in Center 1 (n = 5) and Center 2 (n = 10). ANOVA with Tukey’s HSD post hoc analysis (95% confidence interval), P value = 0.003 generated with STAMP. (PDF 21 kb)

Supplementary methods. (DOCX 121 kb)

The rumen microbiome is fundamental for the productivity and health of dairy cattle and diet is known to influence the rumen microbiota composition. In this study, grape-pomace, a natural source of polyphenols, and copper sulfate were provided as feed supplementation in 15 Holstein-Friesian calves, including 5 controls. After 75 days of supplementation, genomic DNA was extracted from the rumen liquor and prepared for 16S rRNA-gene sequencing to characterize the composition of the rumen microbiota. From this, the rumen metagenome was predicted to obtain the associated gene functions and metabolic pathways in a cost-effective manner. Results showed that feed supplementations did alter the rumen microbiome of calves. Copper and grape-pomace increased the diversity of the rumen microbiota: the Shannon's and Fisher's alpha indices were significantly different across groups (p-values 0.045 and 0.039), and Bray-Curtis distances could separate grape-pomace calves from the other two groups. Differentially abundant taxa were identified: in particular, an uncultured Bacteroidales UCG-001 genus and OTUs from genus Sarcina were the most differentially abundant in pomace-supplemented calves compared to controls (p-values 0.003 and 0.0002, respectively). Enriched taxonomies such as Ruminiclostridium and Eubacterium sp., whose functions are related to degradation of the grape- pomace constituents (e.g. flavonoids or xyloglucan) have been described (p-values 0.027/0.028 and 0.040/0.022 in Pomace vs Copper and Controls, respectively). The most abundant predicted metagenomic genes belonged to the arginine and proline metabolism and the two- component (sensor/ responder) regulatory system, which were increased in the supplemented groups. Interestingly, the lipopolysaccharide biosynthetic pathway was decreased in the two supplemented groups, possibly as a result of antimicrobial effects. Methanogenic taxa also responded to the feed supplementation, and methane metabolism in the rumen was the second most different pathway (up-regulated by feed supplementations) between experimental groups.

Figure S1. NMDS plot based on the weighted Unifrac distances of Center2 immune and control mice including TSHR-immunized mice from Center 1. TSHR-immunized mice from Center 1 were more similar to TSHR-immunized mice from Center 2 (P = 0.2) than to the βgal (P = 0.024) than the untreated (P = 0.04). (PDF 28 kb)

Influenza immunization coverage rates remains far below the optimal value recommended by World Health Organization, even in groups considered more at risk, as elderly. A possible explanation of lacking compliance to vaccine uptake can rely on deprivation. A local deprivation index, specifically developed, was proposed for the classification of the residents in Ferrara’s municipality in order to evaluate the characteristics of over 65 people accepting/refusing the influenza immunization (2010-2015). The variables involved in the Ferrara’s deprivation index were primarily related to demographic aspects like age, being widow/widower, education, composition of the family and characteristics of the house. In groups with higher deprivation, elderly were more frequent (53.4% in very deprived). Influenza immunization coverage rates were unsatisfactory in all categories of deprivation. A decreasing trend in coverage rates with decreasing deprivation was observed with statistical significance, in general population and in males but not in females. In addition to factors composing the local deprivation index, being separated, living in a family of three members and working as independent contractor were features preventing from the immunization in very deprived elderly., Journal of Preventive Medicine and Hygiene, Vol 59, No 4s2 (2018): Influenza vaccination coverage in the elderly and socio-economic inequalities in Italy. How local deprivation indices can help to improve the prevention of influenza in specific population sub-groups. - Suppl.2

Figure S1. Schematic representation of the GO immunization protocol and sample collection. Table S1. Summary of disease characteristics induced in mice in Center 1 and Center 2 using TSHR expression plasmid illustrating the heterogeneity of response. Table S2. Quarterly Health Screen Reports on viral, bacterial, mycoplasma and parasite screen in both centers. Table S3. Composition of the commercial chows provided ad libitum in Center 1 and Center 2. (DOCX 106 kb)