Eubacterium rectale Attenuates HSV-1 Induced Systemic Inflammation in Mice by Inhibiting CD83.

The purpose of this study was to determine whether administration of the microorganism Eubacterium rectale (E. rectale) could regulate dendritic cell (DC) activation and systemic inflammation in herpes simplex virus type 1-induced Behçet's disease (BD). E. rectale, butyrate-producing bacteria, was administered to BD mice. Peripheral blood leukocytes (PBL) and lymph node cells were isolated and analyzed by flow cytometry. 16S rRNA metagenomic analysis was performed in the feces of mice to determine the differences in the composition of the microbial population between normal and BD mice. Serum cytokine levels were measured by enzyme-linked immunosorbent assay. The frequency of DC activation marker CD83 positive cells was significantly increased in PBL of BD mice. Frequencies of CD83+ cells were also significantly increased in patients with active BD. 16S rRNA metagenomic analysis revealed different gut microbiota composition between normal and BD mice. The administration of E. rectale to BD mice reduced the frequency of CD83+ cells and significantly increased the frequency of NK1.1+ cells with the improvement of symptoms. The co-administration of colchicine and E. rectale also significantly reduced the frequency of CD83+ cells. Differences in gut microbiota were observed between normal mice and BD mice, and the administration of E. rectale downregulated the frequency of CD83, which was associated with BD deterioration. These data indicate that E. rectale could be a new therapeutic adjuvant for BD management.

Behçet's Disease-Do Microbiomes and Genetics Collaborate in Pathogenesis?

Behçet's disease (BD) is a multisystem autoinflammatory condition characterized by mucosal ulceration, breakdown of immune privilege sites and vasculitis. A genetic basis for BD has been described in genome-wide and validation studies. Similarly, dysbiosis of oral and gut microbiomes have been associated with BD. This review will describe links between genetic polymorphisms in genes encoding molecules involved in gut biology and changes seen in microbiome studies. A potential decrease in bacterial species producing short chain fatty acids linked to mutations in genes involved in their production suggests a potential therapy for BD.

Succinivibrionaceae is dominant family in fecal microbiota of Behçet's Syndrome patients with uveitis.

Behçet's Syndrome (BS) is a multisystem vasculitis with various clinical manifestations. Pathogenesis is unclear, but studies have shown genetic factors, innate immunity and autoinflammation to have an important role in the disease course. Diversity in the microbial community of gut microbiota may significantly contribute to the activation of the innate immune system. The clinical features of BS present themselves in clusters and each cluster may be a consequence of different disease mechanisms. For this reason we aimed to investigate the gut microbiota of BS patients with uveitis. In addition to healthy controls, we have aimed to compare the gut microbiota of BS with that of Familial Mediterranean Fever (FMF) and Crohn's Disease (CD) as both diseases have innate and autoinflammatory features in their pathogenesis. Seven patients with BS, 12 patients with FMF, 9 patients with CD and 16 healthy controls (HC) were included in the study. Total genomic DNAs were isolated from fecal samples of the patients. Partial 16S rRNA gene was sequenced using the PGM Ion Torrent (Thermo Fisher Scientific, Waltham, MA, USA) for microbiota analysis. Statistical analysis showed that significant differences were detected on the microbial community of four groups. Succinivibrionaceae is dominant and the signature family, whereas Bacteroides was absent in BS patients.

Modulation of gut microbiota through nutritional interventions in Behçet's syndrome patients (the MAMBA study): study protocol for a randomized controlled trial.

BACKGROUND: Behçet's syndrome (BS) is a systemic inflammatory disorder of unknown etiology, and it is characterized by a wide range of potential clinical manifestations. Recent evidence suggests that the gut microbiota (GM) in BS has low biodiversity with a significant depletion in butyrate producers. The aim of the present project is to investigate whether a dietary intervention could ameliorate the clinical manifestations and modulate the GM of individuals with BS. METHODS: This is a randomized, open, cross-over study that involves 90 individuals with BS, who will be randomly assigned to one of three different diets for 3 months: a lacto-ovo-vegetarian diet (VD), a Mediterranean diet (MD), or a Mediterranean diet supplemented with butyrate (MD-Bt). The VD will contain inulin-resistant and resistant-starch-rich foods, eggs, and dairy in addition to plant-based food, but it will not contain meat, poultry, or fish. The MD will contain all food categories and will provide two portions per week of fish and three portions per week of fresh and processed meat. The MD-Bt will be similar to the MD but supplemented with 1.8 g/day of oral butyrate. The three different diets will be isocaloric and related to the participants' nutritional requirements. Anthropometric measurements, body composition, blood, and fecal samples will be obtained from each participant at the beginning and the end of each intervention phase. The primary outcomes will be represented by the change from baseline of the BS gastrointestinal and systemic symptoms. Changes from baseline in GM composition, short-chain fatty acid (SCFA) production, and the inflammatory and antioxidant profile will be considered as secondary outcomes. DISCUSSION: BS is a rare disease, and, actually, not all the available treatments are target therapies. A supportive treatment based on dietary and lifestyle issues, able to restore immune system homeostasis, could have a high impact on cost sustainability for the treatment of such a chronic and disabling inflammatory condition. TRIAL REGISTRATION: clinicaltrials.gov: NCT03962335. Registered on 21 May 2019.

Behçet's Disease Under Microbiotic Surveillance? A Combined Analysis of Two Cohorts of Behçet's Disease Patients.

Background: In Behçet's disease (BD), an auto-inflammatory vasculitis, an unbalanced gut microbiota can contribute to pro-inflammatory reactions. In separate studies, distinct pro- and anti-inflammatory bacteria associated with BD have been identified. Methods: To establish disease-associated determinants, we performed gut microbiome profiling in BD patients from the Netherlands (n = 19) and Italy (n = 13), matched healthy controls (HC) from the Netherlands (n = 17) and Italy (n = 15) and oral microbiome profiling in Dutch BD patients (n = 18) and HC (n = 15) by 16S rRNA gene sequencing. In addition, we used fecal IgA-SEQ analysis to identify specific IgA coated bacterial taxa in Dutch BD patients (n = 13) and HC (n = 8). Results: In BD stool samples alpha-diversity was conserved, whereas beta-diversity analysis showed no clustering based on disease, but a significant segregation by country of origin. Yet, a significant decrease of unclassified Barnesiellaceae and Lachnospira genera was associated with BD patients compared to HC. Subdivided by country, the Italian cohort displays a significant decrease of unclassified Barnesiellaceae and Lachnospira genera, in the Dutch cohort this decrease is only a trend. Increased IgA-coating of Bifidobacterium spp., Dorea spp. and Ruminococcus bromii species was found in stool from BD patients. Moreover, oral Dutch BD microbiome displayed increased abundance of Spirochaetaceae and Dethiosulfovibrionaceae families. Conclusions: BD patients show decreased fecal abundance of Barnesiellaceae and Lachnospira and increased oral abundance of Spirochaetaceae and Dethiosulfovibrionaceae. In addition, increased fecal IgA coating of Bifidobacterium, Ruminococcus bromii and Dorea may reflect retention of anti-inflammatory species and neutralization of pathosymbionts in BD, respectively. Additional studies are warranted to relate intestinal microbes with the significance of ethnicity, diet, medication and response with distinct pro- and inflammatory pathways in BD patients.

Is Helicobacter pylori associated with Behçet's syndrome? A meta-analysis.

BACKGROUND: Preceding studies have reported the relationship between Helicobacter pylori (H pylori) and Behçet's syndrome (BS), but there still exists controversy. In this study, we firstly conducted a meta-analysis to clarify the relationship of these two diseases. METHODS: Articles published until July 1, 2019, in the PubMed, MEDLINE, and Embase databases with restriction of English-language studies were searched and reviewed. According to the inclusion criteria, relevant statistical data were extracted and analyzed. RESULTS: Six articles were finally included. The result showed that BS individuals were 1.39 times more susceptible to H pylori infection (OR = 1.39, 95% CI = (1.03, 1.87)). In addition, it found that oral ulceration (OR = 27.98, 95% CI = (3.49, 224.49)), genital ulceration (OR = 3.15, 95% CI = (1.51, 6.56)), and cutaneous lesions (OR = 4.29, 95% CI = (2.14, 8.61)) were alleviated after H pylori eradication. Publication bias and sensitivity analysis showed no statistical heterogeneity. CONCLUSIONS: BS patients had higher rate of H pylori infection, and clinical symptoms including oral ulceration, genital ulceration, and cutaneous lesions can be improved after H pylori eradication. The results indicated that H pylori may be an etiological factor to BS.

Microbiota stratification identifies disease-specific alterations in neuro-Behçet's disease and multiple sclerosis.

OBJECTIVES: Altered gut microbiota community dynamics are implicated in diverse human diseases including inflammatory disorders such as neuro-Behçet's disease (NBD) and multiple sclerosis (MS). Traditionally, microbiota communities are analysed uniformly across control and disease groups, but recent reports of subsample clustering indicate a potential need for analytical stratification. The objectives of this study are to analyse and compare faecal microbiota community signatures of ethno-geographical, age and gender matched adult healthy controls (HC), MS and NBD individuals. METHODS: Faecal microbiota community compositions in adult HC (n=14), NBD patients (n=13) and MS (n=13) were analysed by 16S rRNA gene sequencing and standard bioinformatics pipelines. Bipartite networks were then used to identify and re-analyse dominant compositional clusters in respective groups. RESULTS: We identified Prevotella and Bacteroides dominated subsample clusters in HC, MS, and NBD cohorts. Our study confirmed previous reports that Prevotella is a major dysbiotic target in these diseases. We demonstrate that subsample stratification is required to identify significant disease-associated microbiota community shifts with increased Clostridiales evident in Prevotella-stratified NBD and Bacteroides-stratified MS patients. CONCLUSIONS: Patient cohort stratification may be needed to facilitate identification of common microbiota community shifts for causation testing in disease.

Diagnostic value of the interferon-γ release assay for tuberculosis infection in patients with Behçet's disease.

BACKGROUND: To investigate the diagnostic value of the interferon-γ release assay (IGRA) for detecting tuberculosis (TB) infection in patients with Behçet's disease (BD). METHODS: We retrospective analyzed the data collected from 173 BD patients hospitalized between 2010 and 2015. Ninety-nine healthy volunteers were enrolled as a control group. IGRA was performed using T-SPOT.TB. The diagnosis of active TB (ATB) was based on clinical, radiological, microbiological, histopathological information and the response to anti-TB therapy. Latent TB (LTB) infection was defined as asymptomatic patients with positive T-SPOT.TB. RESULTS: TB infection was documented in 59 BD patients (34.1%). The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio of T-SPOT.TB for the diagnosis of ATB were 88.9%, 74.8%, 29.1%, 98.3%, 3.53 and 0.15, respectively. The receiver-operating-characteristic curve demonstrated that spot-forming cells (SFCs) of 70/106 PBMC was the optimal cutoff for diagnosing ATB, with an area under the curve of 0.891. Furthermore, the median SFCs in ATB group was significantly higher than those in LTB infection (466/106 PBMC vs. 68/106 PBMC, p = 0.007) or previous TB infection (466/106 PBMC vs. 96/106 PBMC, p = 0.018). A significant discrepancy between T-SPOT.TB and tuberculin skin test was noted (kappa coefficient = 0.391, p = 0.002). CONCLUSIONS: T-SPOT.TB, an IGRA, may assist in the diagnosis of ATB in BD patients, and the higher SFCs suggest ATB in BD patients.

Relative abundance of Megamonas hypermegale and Butyrivibrio species decreased in the intestine and its possible association with the T cell aberration by metabolite alteration in patients with Behcet's disease (210 characters).

OBJECTIVES: We have previously demonstrated that the phylum Actinobacteria, the family Lactobacillaceae, and the genus Bifidobacterium increased in relative abundance of gut microbiota in patients with Behcet's disease (BD). The phylum Firmicutes and the class Clostridia were predominant in the feces of normal individuals. The class Clostridia includes short-chain fatty acid-producing bacteria, important for the balance between regulatory T cells and helper T type 17 (Th17) cells. It is possible that the bacterial compositional alteration causes low intestinal short-chain fatty acid concentrations, leading to skewed immune functions in patients with BD. METHODS: To test the hypothesis, we examined species composition and gene functions from the 16S rRNA data by utilizing PICRUSt software. RESULTS: We have shown that relative abundance of Eggerthella lenta, Acidaminococcus species, Lactobacillus mucosae, Bifidobacterium bifidum, Lactobacillus iners, Streptococcus species, and Lactobacillus salivarius increased significantly in patients with BD. Relative abundance of Megamonas hypermegale, Butyrivibrio species, Streptococcus infantis, and Filifactor species increased significantly in normal individuals compared with BD patients. In the functional annotation analysis by PICRUSt, we found prevalent gene functions of the pentose phosphate pathway and the inosine monophosphate biosynthesis in patients with BD. The data suggested that BD gut microbes altered nucleic acid and fatty acid synthesis. CONCLUSIONS: These compositional and functional alterations of gut microbes may accompany unfavorable molecular exchanges between intestinal immunocompetent cells and gut microbes, and these interactions may have an association with the immune aberration in patients with BD.

A metagenomic study of the gut microbiome in Behcet's disease.

BACKGROUND: Behcet's disease (BD) is a recalcitrant, multisystemic inflammatory disease that can lead to irreversible blindness. Microbial agents have been considered to contribute to the pathogenesis of this disease, but the underlying mechanisms remain unclear. In this study, we investigated the association of gut microbiome composition with BD as well as its possible roles in the development of this disease. METHODS: Fecal and saliva samples were collected from 32 active BD patients and 74 healthy controls. DNA extracted from fecal samples was subjected to metagenomic analysis, whereas DNA extracted from saliva samples was subjected to 16S rRNA gene sequencing analysis. The results were used to compare the composition and biological function of the microbiome between patients and healthy controls. Lastly, transplantation of pooled fecal samples from active BD patients into B10RIII mice undergoing experimental autoimmune uveitis (EAU) was performed to determine the causal relationship between the gut microbiome and BD. RESULTS: Fecal samples from active BD patients were shown to be enriched in Bilophila spp., a sulfate-reducing bacteria (SRB) and several opportunistic pathogens (e.g., Parabacteroides spp. and Paraprevotella spp.) along with a lower level of butyrate-producing bacteria (BPB) Clostridium spp. and methanogens (Methanoculleus spp. Methanomethylophilus spp.). Analysis of microbial functions revealed that capsular polysaccharide transport system, oxidation-reduction process, type III, and type IV secretion systems were also increased in active BD patients. Network analysis showed that the BD-enriched SRB and opportunistic pathogens were positively correlated with each other, but they were negatively associated with the BPB and methanogens. Animal experiments revealed that fecal microbiota transplantation with feces from BD patients significantly exacerbated EAU activity and increased the production of inflammatory cytokines including IL-17 and IFN-γ. CONCLUSIONS: Our findings revealed that BD is associated with considerable gut microbiome changes, which is corroborated by a mouse study of fecal microbiota transplants. A model explaining the association of the gut microbiome composition with BD pathogenesis is proposed.

Rediscover the clinical value of small intestinal bacterial overgrowth in patients with intestinal Behçet's disease.

BACKGROUND AND AIMS: Many patients with intestinal Behçet's disease (BD) still suffer from gastrointestinal symptoms despite the disease being in endoscopic or radiological remission. Previous studies report that small intestinal bacterial overgrowth (SIBO) can be associated with inflammatory bowel disease. However, there have been no reports about SIBO in patients with intestinal BD. We sought to identify the frequency of SIBO in patients with inactive intestinal BD by hydrogen breath test (HBT) and to investigate the efficacy of rifaximin as a treatment for SIBO. METHODS: Twenty-five patients with intestinal BD who had gastrointestinal symptoms even in endoscopic or radiological remission status were enrolled between January 2012 and January 2016. The patients filled out a questionnaire regarding their subjective gastrointestinal symptoms and took an HBT. Patients with positive HBT results were recommended to take 800-mg rifaximin daily for 14 days. RESULTS: Nine patients (9/25, 36%) had a positive HBT test. Eight (8/9, 88.9%) were women, and their mean age was 48.7 years. The most common symptom was abdominal distension (8/9, 88.9%), followed by abdominal discomfort (6/9, 66.7%). Rifaximin was prescribed to the nine patients with positive HBT, but two patients refused to take the medication. Four weeks later, six of the seven patients taking rifaximin (85.7%) reported symptom improvement, and none of them reported adverse events. CONCLUSIONS: More than one-third of the patients with inactive intestinal BD who had gastrointestinal symptoms were accompanied by SIBO using HBT. Rifaximin might be an effective and safe drug to treat these patients.

The Microbiome in Connective Tissue Diseases and Vasculitides: An Updated Narrative Review.

OBJECTIVE: To provide a narrative review of the most recent data concerning the involvement of the microbiome in the pathogenesis of connective tissue diseases (CTDs) and vasculitides. METHODS: The PubMed database was searched for articles using combinations of words or terms that included systemic lupus erythematosus, systemic sclerosis, autoimmune myositis, Sjögren's syndrome, undifferentiated and mixed CTD, vasculitis, microbiota, microbiome, and dysbiosis. Papers from the reference lists of the articles and book chapters were reviewed, and relevant publications were identified. Abstracts and articles written in languages other than English were excluded. RESULTS: We found some evidence that dysbiosis participates in the pathogenesis of systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, and Behçet's disease, but there are still few data concerning the role of dysbiosis in other CTDs or vasculitides. CONCLUSIONS: Numerous studies suggest that alterations in human microbiota may be involved in the pathogenesis of inflammatory arthritides as a result of the aberrant activation of the innate and adaptive immune responses. Only a few studies have explored the involvement of dysbiosis in other CTDs or vasculitides, and further research is needed.

Sequencing of 16S rRNA reveals a distinct salivary microbiome signature in Behçet's disease.

Behçet's disease (BD) is characterized by recurrent oro-genital ulcers, mucocutaneous lesions, and serious organ involvement. We investigated the salivary microbiome in BD using high-throughput sequencing of the 16S rRNA V4 region. Stimulated saliva samples were collected from 31 BD patients and 15 healthy controls, and in 9 BD patients, a second saliva sample was collected following dental and periodontal treatment. Sequence analysis identified a total of 908 operational taxonomic units (OTUs) present across all samples. Patients had a microbial community structure that is significantly less diverse than healthy controls. The most overabundant species in BD was Haemophilus parainfluenzae, while the most depleted included Alloprevotella rava and species in the genus Leptotrichia. Periodontal treatment improved oral health indices in BD but had no short-term effect on bacterial community structure. Neither the BD-associated genetic risk locus within the HLA-B/MICA region nor being on immunosuppressive medications explained the differences between patients and controls.

Bifidobacteria Abundance-Featured Gut Microbiota Compositional Change in Patients with Behcet's Disease.

Gut microbiota compositional alteration may have an association with immune dysfunction in patients with Behcet's disease (BD). We conducted a fecal metagenomic analysis of BD patients. We analyzed fecal microbiota obtained from 12 patients with BD and 12 normal individuals by sequencing of 16S ribosomal RNA gene. We compared the relative abundance of bacterial taxa. Direct comparison of the relative abundance of bacterial taxa demonstrated that the genera Bifidobacterium and Eggerthella increased significantly and the genera Megamonas and Prevotella decreased significantly in BD patients compared with normal individuals. A linear discriminant analysis of bacterial taxa showed that the phylum Actinobacteria, including Bifidobacterium, and the family Lactobacillaceae exhibited larger positive effect sizes than other bacteria in patients with BD. The phylum Firmicutes and the class Clostridia had large effect sizes in normal individuals. There was no significant difference in annotated species numbers (as numbers of operational taxonomic unit; OTU) and bacterial diversity of each sample (alpha diversity) between BD patients and normal individuals. We next assigned each sample to a position using three axes by principal coordinates analysis of the OTU table. The two groups had a significant distance as beta diversity in the 3-axis space. Fecal sIgA concentrations increased significantly in BD patients but did not correlate with any bacterial taxonomic abundance. These data suggest that the compositional changes of gut microbes may be one type of dysbiosis (unfavorable microbiota alteration) in patients with BD. The dysbiosis may have an association with the pathophysiology of BD.

Usefulness of Measuring Serum Procalcitonin Levels in Patients with Inflammatory Bowel Disease.

BACKGROUND/AIMS: The relationships between serum procalcitonin, inflammatory bowel disease (IBD) and intestinal Behçet's disease (BD) have not been completely determined. We aimed to evaluate the usefulness of measuring serum procalcitonin levels to assess disease activity and infection stage in patients with IBD and intestinal BD. METHODS: We retrospectively analyzed clinical data from 129 patients with IBD and intestinal BD for whom serum procalcitonin and C-reactive protein (CRP) levels were measured between January 2006 and February 2013. RESULTS: The median serum procalcitonin levels in the IBD and intestinal BD with septic shock or sepsis (n=8), with localized infection (n=76), and without infection (n=45) were 3.46 ng/mL (range, 0.17 to 63.66 ng/mL), 0.22 ng/mL (range, 0.05 to 140.18 ng/mL), and 0.07 ng/mL (range, 0.00 to 31.50 ng/mL), respectively (p=0.001). The serum CRP levels in the IBD and intestinal BD patients did not differ according to the infection stage. Variations in serum procalcitonin levels were not observed in the IBD and intestinal BD patients with different disease activities. CONCLUSIONS: Serum procalcitonin levels may not be affected by IBD and intestinal BD activity itself, although they may be affected by concomitant infection. Serum procalcitonin measurements could be more useful than CRP in determining the infection stage that reflects the severity of infection in IBD and intestinal BD patients.

Behçet's syndrome patients exhibit specific microbiome signature.

BACKGROUND AND AIMS: Behçet syndrome is a systemic inflammatory condition characterized by muco-cutaneous and ocular manifestations, with central nervous system, vascular and/or gastro-intestinal involvement. The association of microbiota with Behçet syndrome has not been shown yet. Our work was aimed to compare the gut microbiota structure and the profiles of short-chain fatty acids production in Behçet syndrome patients and healthy control relatives. METHODS: Here, we compared the fecal microbiota of 22 patients with Behçet syndrome and that of 16 healthy co-habiting controls, sharing the same diet and lifestyle by pyrosequencing of the V3-V4 hypervariable regions of the 16 rDNA gene and biochemical analyses. RESULTS: Our analyses showed significant differences in gut microbiota between Behçet patients and healthy cohabitants. In particular we found that Behçet's patients were significantly depleted in the genera Roseburia and Subdoligranulum. Roseburia showed a relative abundance value of 10.45±6.01% in healthy relatives and 4.97±5.09% in Behçet's patients, and Subdoligranulum, which reached a relative abundance of 3.28±2.20% in healthy controls, was only at 1.93±1.75% of abundance in Behçet's patients. Here we report, for the first time, that a peculiar dysbiosis of the gut microbiota is present in patients with Behçet syndrome and this corresponds to specific changes in microbiome profile. A significant decrease of butyrate production (P=0.0033) in Behçet's patients was demonstrated. Butyrate is able to promote differentiation of T-regulatory cells, and consequently the results obtained prompt us to speculate that a defect of butyrate production might lead to both reduced T-reg responses and activation of immuno-pathological T-effector responses. CONCLUSIONS: Altogether, our results indicate that both a peculiar dysbiosis of the gut microbiota and a significant decrease of butyrate production are present in patients with Behçet syndrome.

Behçet's syndrome and micro-organisms.

Behçet's syndrome (BS) is a multi-systemic vasculitis of unknown aetiology. More than one mechanism seems to be operative in the pathogenesis of Behçet's syndrome, including genetic and environmental factors, causing different manifestations of the syndrome. There are several clues to the role of environmental factors and especially micro-organisms in the pathogenesis. These include clinical findings such as a decrease in the frequency of a positive pathergy reaction with surgical cleaning of the skin before the procedure, the acne-arthritis association carrying similar features to acne-associated reactive arthritis, a higher rate of tonsillectomy, cold sores, late birth order, higher number of siblings, history of travel to countries with a high incidence of BS and earlier age at first sexual intercourse. Moreover, basic research on both viruses and bacteria suggests that micro-organisms may be playing a role, possibly through heat shock proteins and T-cell hypersensitivity.

Serum anti-Saccharomyces cerevisiae antibodies in Greek patients with Behcet's disease.

We tested 59 Greek patients with Behcet's Disease (BD) for serum anti-Saccharomyces cerevisiae antibodies. No increase of these antibodies was detected in the cases compared to 55 healthy unrelated blood donors from the same population. This finding is in contrast with the correlation between Saccharomyces cerevisiae antibodies and BD as reported in other populations. It seems that environmental factors may contribute to disease expression in different populations, producing different effects according to the individual's genetic predisposition. Saccharomyces cerevisiae antibodies do not seem to be of any significance in the Greek population.