Mucosal-associated invariant T cells from Clostridioides difficile-infected patients exhibit a distinct proinflammatory phenotype and enhanced cytotoxic activity.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells mainly found in the mucosa and peripheral blood. We have recently demonstrated that Clostridioides difficile activates MAIT cells in vitro. However, their role in the pathogenesis of C. difficile infection (CDI) in human patients remains elusive to date. In this study, we performed comprehensive immunophenotyping of MAIT cells derived from CDI patients and compared their phenotype to that of patients with inflammatory bowel diseases (IBD) and healthy controls. Our study revealed that blood MAIT cells from CDI patients exhibit an interleukin 17a (IL-17a)-dominated proinflammatory phenotype and an increased readiness to synthesize the proinflammatory cytokine interferon γ (IFN-γ) following in vitro re-stimulation. Moreover, the cytotoxic activity of MAIT cells, as measured by surface CD107a and intracellular granzyme B expression, was strongly increased in CDI. Multi epitope ligand cartography (MELC) analysis of intestinal biopsies from CDI patients revealed that MAIT cells exhibit an increased production of granzyme B and increased cytotoxicity compared to the control group. Together with previously published in vitro data from our group, our findings suggest that MAIT cells are functionally involved in the immune response against C. difficile and contribute to the pathogenesis of CDI.

Is the Routine Histopathologic Diagnosis of Ulcerative Colitis Based on Cross-cut Sections or on Well-oriented Sections?

BACKGROUND/AIM: For many years, it was empirically estimated that the majority of the routine colon biopsies in Swedish patients with ulcerative colitis (UC), exhibited cross-cut crypts. The aim of the present study was to assess the frequency of cross-cut crypts (CCC) and well-oriented crypts in routine colon biopsies in German patients with UC. PATIENTS AND METHODS: In total, 447 colon biopsies: 376 with UC and 71 controls were investigated. RESULTS: Out of 376 colon biopsies with UC, 73% exhibited ≥60% CCC. Out of the 237 biopsies showing ≥80% CCC, as many as 71% exhibited 100% CCC in individual biopsies. Similar percentages were found in control biopsies. CONCLUSION: The majority of the routine colon biopsies with UC, as well as control biopsies in German patients displayed CCC. Thus, an unnoticed, consequent, and systematic cutting technical hitch was introduced during the laboratory processing of colon biopsies. The reason(s) behind the similar histologic processing mode of colon biopsies at the two geographically disparate laboratories (Sweden and Germany) remains elusive. The cross-cutting mode influenced the narrative of biopsies in UC, inasmuch as some histological parameters listed among well-oriented colon sections were not present in sections displaying CCC.

Crypt-rings in tandem detected in infectious colitis, in IBD and in IBD-associated noninvasive neoplasia.

AIMS: Recently, eight novel histologic structures in colon mucosa with inflammation were described. Here, we assessed the frequency of one of them: crypt rings in tandem (CRT), in patients with infectious colitis (IC), IBD (ulcerative colitis; UC or Crohn colitis; CrC) and UC in remission (UCR). In addition, the frequency of dysplastic CRT (DCRT) in IBD-associated noninvasive neoplasia (IBDNIN) were also calculated. METHODS: Colon biopsies in 578 cases were reviewed: 42 cases with IC, 280 with IBD (180 UC and 100 CrC), 100 UCR and the remaining 156, IBDNIN. RESULTS: The proportions of CRT in IC was 16.7%, in IBD 14.3% %, in UCR 3%, and of DCRT in IBDNIN, 20%. No differences were recorded between the proportions of CRT in IC, UC and CrC. Conversely, the difference in CRT frequency between UC and UCR, and between CRT and DCRT were significant (P = 0.006, and p = 0.05, respectively). CONCLUSIONS: CRT evolved in IC and in IBD. The finding of CRT in IC strongly suggest that those characteristic crypts were shaped at the early stages of mucosal inflammation. CRT persisted in IBD with protracted inflammation but plummeted in UCR, that is when the mucosal inflammation waned. The proportion of DCRT was significantly higher than that of CRT. It is submitted that DCRT might had developed in IBDNIN using CRT as scaffolds. This is the first study in which a characteristic pathologic aberration of cryptogenesis was tracked in colon biopsies from patients with IBD and with IBD-associated neoplastic transformation.

Dysplastic Crypts in Asymmetric Branching in Ulcerative Colitis: A Preliminary Report.

AIM: To report the detection of dysplastic crypts in asymmetric branching (DCAB) in biopsies from patients with ulcerative colitis (UC). PATIENTS AND METHODS: One hundred consecutive endoscopic biopsies from patients with UC undergoing surveillance were reviewed. RESULTS: Three biopsy/cases showed DCAB. The frequency of DCAB varied from two in one case, three in another case, and five in the remaining case. CONCLUSION: The final outcome of DCAB is to generate two or more dysplastic asymmetric offspring-crypts. Repeated DCAB offspring formation, together with new DCAB, would boost the pool of dysplastic crypts, resulting in an exponential expansion of the mucosal area occupied by dysplasia in UC.

Crypts in Asymmetric Branching in Patients With Ulcerative Colitis in Remission.

BACKGROUND/AIM: To report the frequency of crypts in asymmetric branching (CAB) in biopsies from all colorectal segments in patients with ulcerative colitis in remission (UCR). PATIENTS AND METHODS: Biopsies in 100 UC patients were investigated: 50 with UCR and 50 with ongoing long-lasting UC (LLU; i.e., controls). RESULTS: The frequency of CAB was significantly lower in UCR than in LLUC, both in the right colon and left colorectum. CAB frequency was not influenced by two important confounders: the age and sex of patients. CONCLUSION: CAB is a pathologic aberration of colorectal cryptogenesis evoked by chronic mucosal inflammation. When chronic inflammation waned in UCR, the production of CAB plummeted or ceased. Chronic inflammation and protracted disease-duration in LLUC increase the risk for colorectal dysplasia or carcinoma. Importantly, dysplastic CAB were recently detected in LLUC-associated dysplasia. Whether the abrogation of CAB is instrumental in reducing the neoplastic risk in UCR patients, deserves further investigation.

Crypts in Asymmetric Fission in Endoscopic Biopsies from German Patients With Inflammatory Bowel Disease.

BACKGROUND/AIM: We previously found in Swedish patients with inflammatory bowel disease (IBD), crypts in symmetric fission (CSF) and in asymmetric fission (CAF). This study aimed to examine CSF and CAF in a cohort of German patients with IBD. PATIENTS AND METHODS: H&E-sections from 106 IBD-patients [59 ulcerative colitis (UC) and 47 Crohn colitis (CCs)] were analysed. RESULTS: A total of 588 crypts in fission (CF) were found; 342 (58.2%) in UC and 246 (41.8%) in CCs. Out of the 505 CAFs found, 304 (60.2%) were recorded in UC, and 201(39.8%) in CCs (p=0.15272). CONCLUSION: Despite that German and Swedish populations reside in disparate geographical regions with different ecological milieus, the proportions of CAF and CSF were similar, thereby suggesting that CAF and CSF develop in IBD independently of the local environmental conditions in the two regions.

Crypts in Asymmetric Fission in Endoscopic Biopsies from Swedish Patients With Inflammatory Bowel Disease.

BACKGROUND/AIM: We previously found crypts in symmetric fission (CSF) and in asymmetric fission (CAF) in colectomy-specimens with ulcerative colitis. We now analyzed CSF and CAF (CSAF) in biopsies from 80 patients with inflammatory bowel disease (IBD) without dysplasia or carcinoma. PATIENTS AND METHODS: One unselected double-biopsy from affected endoscopic areas was investigated in the 80 cases. RESULTS: A total of 353 crypts in fission were found. The median number of CAF/biopsy was 3.7 and for CSF/biopsy, 0.7 (p<0.00001). CONCLUSION: CSAF often occur in unselected biopsies from patients with IBD. Whereas the increased frequency of CSF might mirror a compensatory mechanism of crypt production in areas occupied by inflammation, CAF reflects a pathological aberration of cryptogenesis, probably generated by somatic mutations. The biological significance of CAF in IBD without dysplasia or carcinoma, deserves to be further investigated.

Globularia alypum L. Modulates Inflammatory Markers in Human Colon and Shows a Potential Antioxidant Role in Myeloid Leukemic Cells.

Globularia alypum (GA), a plant of the Globulariacea family, has long been used as a traditional cure for inflammatory and metabolic illnesses. In addition to various in vitro model studies, the current work focuses on the antioxidant and anti-inflammatory properties of GA in human colon biopsies. The phenol components in GA aqueous extract (GAAE) were identified by Liquid Chromatography-Electrospray Ionization Mass Spectrometry. The antioxidant ability of GAAE was tested in vitro utilizing chemiluminescence and flow cytometry using fluorescent yeasts n conjunction with PLB-985-human myeloid leukemia cells. Experiments on human colon biopsies after a biopsy challenge with Escherichia coli-lipopolysaccharides aimed to see if GAAE had an anti-inflammatory impact on human colon inflammation. Western blotting was used to assess the expression of several inflammatory markers. According to the findings, GAAE had a significant influence on hydrogen peroxide and cellular reactive oxygen species. GAAE inhibited the activities of cyclooxygenase 2 and nuclear factor B in inflamed biopsies, indicating anti-inflammatory action. The present study is the first to show that GA has a beneficial effect on human colon inflammation, thanks to its significant antioxidant activity in vitro. According to these preliminary data, GA may be utilized to treat a range of human inflammatory illnesses.

Micro ATR-FTIR spectroscopic imaging of colon biopsies with a large area Ge crystal.

A new large-area germanium ATR crystal is utilised with an FTIR microscope to improve the acquired images of de-paraffinized colon biopsy sections, without recourse to a synchrotron source. The large crystal (⌀ = 28 mm) offers significant improvements compared to slide-on small germanium crystal (⌀ = 3.5 mm); for example, it facilitates more uniform distribution of higher signal intensity within the field of view and more rapid acquisition time. Mapping of a larger sample area up to ca. 350 × 350 µm2 with this new set-up, coupled with imaging using an FPA detector, is demonstrated for the first time on biological specimens. The performance of k-means clustering algorithm applied to classify the different anatomical structures of the colon biopsies is greatly improved with mapping. Comparison of H&E stained adjacent tissue sections with false-colour k-means images strongly support differentiation of five distinct regions within tissues. The efficiency of the methodology to categorise colon tissues at various stages of malignancy is analysed via multivariate chemometrics. The second derivative spectra extracted from the crypt region of the colon were subjected to Partial Least Squares classification. Good separation between data in clusters occurs when projecting spectra on a PLS score plot on a plane made by the first 3 principal components. Important spectral biomarkers for colon malignancy classification were identified to exist mostly in the fingerprint region of the FTIR spectrum based on the chemometrics analysis.

The Proteome of Ulcerative Colitis in Colon Biopsies from Adults - Optimized Sample Preparation and Comparison with Healthy Controls.

PURPOSE: The purpose of the study was to optimize the sample preparation and to further use an improved sample preparation to identify proteome differences between inflamed ulcerative colitis tissue from untreated adults and healthy controls. EXPERIMENTAL DESIGN: To optimize the sample preparation, we studied the effect of adding different detergents to a urea containing lysis buffer for a Lys-C/trypsin tandem digestion. With the optimized method, we prepared clinical samples from six ulcerative colitis patients and six healthy controls and analysed them by LC-MS/MS. We examined the acquired data to identify differences between the states. RESULTS: We improved the protein extraction and protein identification number by utilizing a urea and sodium deoxycholate containing buffer. Comparing ulcerative colitis and healthy tissue, we found 168 of 2366 identified proteins differently abundant. Inflammatory proteins are higher abundant in ulcerative colitis, proteins related to anion-transport and mucus production are lower abundant. A high proportion of S100 proteins is differently abundant, notably with both up-regulated and down-regulated proteins. CONCLUSION AND CLINICAL RELEVANCE: The optimized sample preparation method will improve future proteomic studies on colon mucosa. The observed protein abundance changes and their enrichment in various groups improve our understanding of ulcerative colitis on protein level.