Sergey R. Konstantinov

Genomic ATG16L1 risk allele-restricted Paneth cell ER stress in quiescent Crohn's disease

Objective Although genome wide association studies have partly uncovered the genetic basis of Crohns disease (CD), it remains a challenge to link genetic polymorphisms to functional intestinal phenotypes. Paneth cells are specialised antimicrobial epithelial cells localised to the small-intestinal crypt-base. Here, we investigate whether genomic variations in ATG16L1 affect Paneth cell function. Design Genomic variation of ATG16L1 (T300A, rs2241880) was determined in DNA from 78 patients with CD and 12 healthy controls. Paraffin-embedded ileal biopsies from patients with genotype AA (n=17), GA (n=38) and patients with the GG allele (n=23) were stained for GRP78, phospho-EIF2α, lysozyme, cleaved-caspase 3, phosphohistone H3, phospho-IκB, p65, phospho-p38MAPK and PHLDA1. Microbial composition of biopsies was assessed by PCR. Disease phenotype was scored. Results In patients with quiescent disease but with an ATG16L1 risk allele, the endoplasmic reticulum (ER) stress markers GRP78 and pEIF2α were highly expressed in Paneth cells. Other CD risk gene variations did not correlate with Paneth cell ER stress. Functionally, patients with ER-stressed Paneth cells showed no changes in intestinal epithelial cells proliferation or apoptosis, Paneth cell or stem cell numbers, p65, phospho-IκB and phospho-p38 staining. However, a significantly increased presence of adherent-invasive Escherichia coli was observed in biopsies from patients with ER-stressed Paneth cells. Phenotypically, patients with GRP78 positive Paneth cells have relatively less colonic disease over ileal disease (−21%, p=0.04), more fistulas (+21%, p=0.05) and an increased need for intestinal surgery (+38%, p=0.002). Conclusions The ATG16L1 T300A polymorphism defines a specific subtype of patients with CD, characterised by Paneth cell ER stress even during quiescent disease. Paneth cell ER stress correlates with bacterial persistence, and is thus likely to modulate antimicrobial functionality of this cell type in patients with CD.

The Microbiome and Psoriatic Arthritis

Psoriatic arthritis is a chronic inflammatory joint disease, seen in combination with the chronic inflammatory skin disease psoriasis and belonging to the family of spondylarthritides (SpA). A link is recognized between psoriatic arthritis and inflammatory bowel disease (IBD). Environmental factors seem to induce inflammatory disease in individuals with underlying genetic susceptibility. The microbiome is a subject of increasing interest in the etiology of these inflammatory immune-mediated diseases. The intestinal microbiome is able to affect extra-intestinal distant sites, including the joints, through immunomodulation. At this point, evidence regarding a relationship between the microbiome and psoriatic arthritis is scarce. However, we hypothesize that common immune-mediated inflammatory pathways seen in the “skin–joint–gut axis” in psoriatic arthritis are induced or at least mediated by the microbiome. Th17 has a crucial function in this mechanism. Further establishment of this connection may lead to novel therapeutic approaches for psoriatic arthritis.

Functional genomic analyses of the gut microbiota for CRC screening

The evidence for a strong correlation between the gut microbiota and colorectal carcinogenesis is quickly gathering pace. This correlation raises important questions, such as whether analysis of the microbiota can be used for screening purposes, and whether targeted intervention can influence the risk of development and progression of neoplasia. The recovery of several pathobionts—such as members of the different bacterial phyla Proteobacteria, Bacteroidetes and Fusobacteria—from the tumour microenvironment of patients with colorectal cancer (CRC) now provides a link between specific microbial colonization and cancer. However, other intestinal bacteria belonging to another major intestinal phylum, Firmicutes, might be effective in the treatment of pathogenic inflammation related to CRC. Future approaches based on the analysis of the gut microbiota of patients with CRC combined with large human cohort studies might open up new possibilities for further prophylactic, screening and treatment strategies.

Similar Depletion of Protective Faecalibacterium prausnitzii in Psoriasis and Inflammatory Bowel Disease, but not in Hidradenitis Suppurativa

BACKGROUND AND AIMSnPsoriasis and hidradenitis suppurativa [HS] co-occur more often with inflammatory bowel disease [IBD] than expected, due to shared pathogenic and genetic features. It is known that IBD patients harbour an altered intestinal microbiome characterised by a depletion of Faecalibacterium prausnitzii and increase of Escherichia coli. At present, it is unclear whether a similar intestinal microbiome trend can be identified in IBD-associated skin disorders. We therefore investigated the F. prausnitzii and E. coli abundance in psoriasis and HS, with and without concomitant IBD.nnnMETHODSnUsing quantitative polymerase chain reaction , we compared the F. prausnitzii and E. coli abundances in faecal samples from healthy controls [n = 33] with samples from patients with psoriasis [n = 29], IBD [n = 31], and concomitant IBD and psoriasis [n = 13]. Likewise, we analysed samples from patients with HS [n = 17], and concomitant IBD and HS [n = 17].nnnRESULTSnPsoriasis patients harboured a significantly lower abundance of F. prausnitzii in their stool than healthy controls [p < 0.001], which was similar to IBD patients. Together with the reduced F. prausnitzii levels, the psoriasis patients had a significantly higher abundance of E. coli [p < 0.001]. No significant difference in F. prausnitzii or E. coli abundance was found in HS. It was apparent that patients with concomitant IBD and associated skin disorder had the greatest decrease of F. prausnitzii and increase of E. coli.nnnCONCLUSIONSnThe study demonstrates, for the first time, an IBD-like decrease of F. prausnitzii together with an increase of E.coli in psoriasis, supporting the presence of a gut-microbiome-skin axis in psoriasis and IBD.

Do pregnancy-related changes in the microbiome stimulate innate immunity?

Pregnancy has a beneficial influence on the course of certain autoimmune diseases such as inflammatory bowel disease (IBD). It was recently reported that during pregnancy the microbiome undergoes profound changes that are associated with host physiological and immunological adaptations. Here we propose that microbiome remodeling during pregnancy is an active response of the mother, possibly to alter immune system status, and to facilitate metabolic and immunological adaptations that are needed for a successful pregnancy. Furthermore, these changes in the microbiome may ensure the transfer of specific traits into the neonatal gut. As the underlying mechanisms are not well understood, elucidating how pregnancy-related changes in the microbiome influence IBD would be of obvious value for designing rational therapy.

Human Fecal Microbiome–Based Biomarkers for Colorectal Cancer

Colorectal cancer may develop slowly over years from precursor lesions, and thus screening combined with early diagnosis is the key to disease prevention. Recent studies have elucidated specific traits in the gut microbiome associated with colorectal cancer and suggested that the microbiome may be useful in screening for colorectal cancer purposes but failed to provide protocols that can be applied in a practical situation. A recent study by Zackular and colleagues, presented on page 1112, provides an important way forward here in showing that specific analysis of multiple aspects of the microbiome composition in toto provides reliable detection of both precancerous and cancerous lesions. This important achievement when combined with other noninvasive techniques promises to provide highly effective tools for early colorectal cancer diagnosis and its prevention. Cancer Prev Res; 7(11); 1108–11. ©2014 AACR.

Fecal Microbiota Transfer May Increase Irritable Bowel Syndrome and Inflammatory Bowel Diseases–Associated Bacteria

Dear Sir: With a great interest we have read the article by Vrieze et al,1 entitled “Transfer of Intestinal Microbiota From Lean Donors Increases Insulin Sensitivity in Individuals With Metabolic Syndrome.” The demonstrated potential of the fecal microbiota transplantation (FMT) to improve insulin sensitivity in humans has resulted in a number of enthusiastic responses, including ours. However, we would like to emphasize that the interpretation of the results of the study may not be always as straightforward as presented in the article. We feel the need to outline below several important findings in the aforementioned article regarding the data on the changes in the human microbiota after FMT. As presented in Figure 2 and in Supplementary Tables 3 and 4, but not discussed in the paper, the allogenic FMT has led to a significantly increased number of bacteria such as Alcaligens faecalis et rel. in the small intestinal iopsy samples of the patients involved. The abundance f Dorea formicigenerans et rel, Clostridium sphenoides et rel, C exile et rel, and Ruminococcus lactaris et rel has also been significantly elevated in the fecal samples after the allogenic FMT. It is notable that bacteria identified as Alcaligens spp are often found inside the lymphoid tissue and that Alcaligenes-specific systemic immune responses have recently been associated with Crohn’s disease.2 Furtherore, an earlier study has identified D formicigenerans et rel, C sphenoides et rel, C nexile et rel, and R lactaris et rel as being associated with irritable bowel syndrome.3 In addition, 2 independent studies have showed that D formicigenerans is enriched on the mucosa of patients with ulcerative colitis and that some antibiotics applied to treat ulcerative colitis could reduce the colonic population of D formicigenerans.4,5 Furthermore, the donors’ feces have not been creened for the abundance of these bacterial species beore the FMT. Although data on post-FMT irritable bowel yndrome, ulcerative colitis, or Crohn’s disease are lackng, adverse effects after FMT have been occasionally reorted. For instance, there were 2 patients who died in a tudy involving 18 patients undergoing FMT to treat ecurrent C difficile colitis. FMT and specifically the use of nasogasric tube, which has also been applied in the study of Vrieze et al1 have explicitly not been excluded as a contributing risk factor to the death of one of the patients as a result of peritonitis.6 Thus, we feel that the data vailable are certainly not all consistent with safety of the rocedure. We advise application of a rapid diagnostic test ased on polymerase chain reaction detection within the ime FMT frame of 6 hours,1 because rapid, 1-hour polymerase chain reaction-based diagnostics are now available in a number of centers. Such tests will not compromise the freshness of the samples, but will reduce the FMTassociated risk and assist the clinicians to make an evidence-based decision that is essential immediately before the transfer. We therefore advise on the need for the FMT protocol adjustments regarding these issues when the procedure is applied to the treatment of the metabolic syndrome and beyond. We feel that with such fairly simple modifications, the safety of the existing FMT protocol can be substantially enhanced. Furthermore, specific attention needs to be given to the recipients’ microbiota and its compositional changes after FMT with regard to the quantitative changes in the intestinal bacteria associated with irritable bowel syndrome, and other inflammatory and infectious diseases in the gastrointestinal tract. This may reduce the risks associated with the outgrowth of potentially harmful bacteria in anatomically different parts of the intestine after FMT. We would like to further comment on the putative mechanism underlying the increased insulin sensitivity and the suggested key role for butyrate and butyrateproducing bacteria in there.1 FMT has not significantly changed the concentration of butyrate detected in the fecal samples of the allogenic FMT group where the improved insulin sensitivity has been observed. Furthermore, the number of the butyrate-producing bacteria has not been significant between the 2 groups. Therefore, we think that there may have been another mechanism in place related to the abundance of specific strains in the small intestine, which could explain the observed increased insulin sensitivity in the allogenic FMT group. Escherichia coli et rel were significantly increased in the small intestinal biopsies of individuals with metabolic syndrome from the autologous group, but decreased in the allogenic FMT group. A rise in the number of E coli and related Proteobacteria species has been associated with greater adiposity and insulin insensitivity, as recently reported in women in the third trimester of pregnancy.7 Therefore, we hypothesize that the reduction in the abundance of E coli in the small intestinal mucosa may have ccounted for the improved insulin sensitivity in the alogenic FTM group. We feel that future experiments need o be performed in this direction to prove or reject the ypothesis. Perhaps a technical detail in the paper is the presentaion of the results in Figure 1. Different from the main ext, there are no data on the autologous gut microbiota nfusion regarding both peripheral and hepatic insulin ensitivity. In conclusion, the critical discussion on the data and n the FMT protocol presented in the paper with a speific focus on the putative intestinal inflammatory disases that may arise after the FMT is timely. It will repond to the high interest in FMT by patients, esearchers, and clinicians that this study has evoked.

Diet, microbiome, and colorectal cancer

The scientific interests in the colorectal cancer (CRC) associated microbiome have increased significantly in the past decade. Mechanistically, several members of the human microbiome and products thereof have been implicated as inductors of the pathogenic inflammation related to CRC. Conversely, the activities of the human intestinal microbial community influenced by specific diet might confer a protective effect against the CRC risks and progression. As the microbiome is both a key contributor and one of the tools to prevent CRC, the current review gives a summary of the CRC-associated microbiome and the dietary strategies relevant to CRC. As more evidences become available, new microbiome-based treatments and specific diets may emerge to reduce the CRC risk and improve CRC patients quality of life.

Bacterial invasion of the pancreas revealed after analyses of the pancreatic cyst fluids

The involvement of bacterial translocation (BT) in the promotion of carcinogenesis has gained a considerable attention in the last years. At this point however BT has not been studied in the context of pancreatic cystic lesions and their development into pancreatic ductal adenocarcinoma. The aim of the study was to analyze if bacteria are present in pancreatic cyst fluid (PCF) collected from patients with suspected pancreatic cysts. Total DNA was isolated from sixty nine PCF. The occurrence of bacteria in PCF was analyzed using bacterial 16S rRNA gene-specific PCR-based method followed by sequence identification and quantitative PCR assay tuned up to different pathogenic and commensal human bacteria. Forty-seven out of sixty-nine samples (68%) were found positive for harboring bacterial 16S rRNA gene. Follow up sequencing analyses of the PCR products revealed that bacterial species related to Fusobacterium spp., Bacteroides spp., and Bacillus spp. were predominating the PCF samples. The results suggest that specific bacteria can translocate to the pancreas and become detectable in the PCF.