Effects of faecal microbiota transplantation on the small intestinal mucosa in systemic sclerosis.

OBJECTIVES: In SSc, gastrointestinal tract (GIT) involvement is a major concern, with no disease-modifying and limited symptomatic therapies available. Faecal microbiota transplantation (FMT) represents a new therapeutic option for GIT-affliction in SSc, showing clinical promise in a recent controlled pilot trial. Here, we aim to investigate effects of FMT on duodenal biopsies collected from SSc patients by immunohistochemistry and transcriptome profiling. METHODS: We analysed duodenal biopsies obtained pre-intervention (week 0) and post-intervention (weeks 2 and 16) from nine SSc patients receiving an intestinal infusion of FMT (n = 5) or placebo (n = 4). The analysis included immunohistochemistry (IHC) with a selected immune function and fibrosis markers, and whole biopsy transcriptome profiling. RESULTS: In patients receiving FMT, the number of podoplanin- and CD64-expressing cells in the mucosa were lower at week 2 compared with baseline. This decline in podoplanin- (r = 0.94) and CD64-positive (r = 0.89) cells correlated with improved patient-reported lower GIT symptoms. Whole biopsy transcriptome profiling from week 2 showed significant enrichment of pathways critical for cellular and endoplasmic reticulum stress responses, microvillus and secretory vesicles, vascular and sodium-dependent transport, and circadian rhythm. At week 16, we found enrichment of pathways mandatory for binding activity of immunoglobulin receptors, T cell receptor complexes, and chemokine receptors, as well as response to zinc-ions. We found that 25 genes, including Matrix metalloproteinase-1 were upregulated at both week 2 and week 16. CONCLUSION: Combining selective IHC and unbiased gene expression analyses, this exploratory study highlights the potential for disease-relevant organ effects of FMT in SSc patients with GIT involvement. TRIAL REGISTRATION: ClinicalTrials.gov, http://clinicaltrials.gov, NCT03444220.

Deep learning for prediction of colorectal cancer outcome: a discovery and validation study.

BACKGROUND: Improved markers of prognosis are needed to stratify patients with early-stage colorectal cancer to refine selection of adjuvant therapy. The aim of the present study was to develop a biomarker of patient outcome after primary colorectal cancer resection by directly analysing scanned conventional haematoxylin and eosin stained sections using deep learning. METHODS: More than 12 000 000 image tiles from patients with a distinctly good or poor disease outcome from four cohorts were used to train a total of ten convolutional neural networks, purpose-built for classifying supersized heterogeneous images. A prognostic biomarker integrating the ten networks was determined using patients with a non-distinct outcome. The marker was tested on 920 patients with slides prepared in the UK, and then independently validated according to a predefined protocol in 1122 patients treated with single-agent capecitabine using slides prepared in Norway. All cohorts included only patients with resectable tumours, and a formalin-fixed, paraffin-embedded tumour tissue block available for analysis. The primary outcome was cancer-specific survival. FINDINGS: 828 patients from four cohorts had a distinct outcome and were used as a training cohort to obtain clear ground truth. 1645 patients had a non-distinct outcome and were used for tuning. The biomarker provided a hazard ratio for poor versus good prognosis of 3·84 (95% CI 2·72-5·43; p<0·0001) in the primary analysis of the validation cohort, and 3·04 (2·07-4·47; p<0·0001) after adjusting for established prognostic markers significant in univariable analyses of the same cohort, which were pN stage, pT stage, lymphatic invasion, and venous vascular invasion. INTERPRETATION: A clinically useful prognostic marker was developed using deep learning allied to digital scanning of conventional haematoxylin and eosin stained tumour tissue sections. The assay has been extensively evaluated in large, independent patient populations, correlates with and outperforms established molecular and morphological prognostic markers, and gives consistent results across tumour and nodal stage. The biomarker stratified stage II and III patients into sufficiently distinct prognostic groups that potentially could be used to guide selection of adjuvant treatment by avoiding therapy in very low risk groups and identifying patients who would benefit from more intensive treatment regimes. FUNDING: The Research Council of Norway.

Intestinal and multivisceral transplantation in patients with chronic intestinal failure.

BACKGROUND: Patients with chronic intestinal failure are treated primarily with parenteral nutrition, often for many years. If serious complications arise for intravenous nutritional therapy, it is possible to perform intestinal or multi-organ transplantation in selected patients. We have established a collaboration with Professor Michael Olausson at Sahlgrenska University Hospital in Gothenburg and Professor Andreas Tzakis at the Jackson Memorial Hospital in Miami, USA, to provide an option for Norwegian patients with chronic intestinal failure. MATERIAL AND METHOD: Retrospective long-term study of seven patients (five in Gothenburg and two in Miami) with chronic intestinal failure who underwent intestinal or multi-organ transplantation (ventricle, duodenum, pancreas and small intestine) in the period 2001-2009. At the same time, liver and kidney transplantations were performed on six and two patients, respectively. RESULTS: Four of seven patients are alive and have a good quality of life 24-120 months after the transplantation. The graft function is satisfactory, so that the patients' food intake is mainly oral. Three patients died following a serious infection one, ten and 24 months, respectively, after transplantation took place. INTERPRETATION: Intestinal and multi-organ transplantation is a demanding and expensive treatment. Life-long multi-disciplinary follow-up of the patients is necessary after the transplantation in order to ensure early diagnosis of rejection and infections. Collaboration with international centres has given Norwegian patients with chronic intestinal failure an option of transplantation with satisfactory long-term results.

AXL inhibition improves BRAF-targeted treatment in melanoma.

More than half of metastatic melanoma patients receiving standard therapy fail to achieve a long-term survival due to primary and/or acquired resistance. Tumor cell ability to switch from epithelial to a more aggressive mesenchymal phenotype, attributed with AXLhigh molecular profile in melanoma, has been recently linked to such event, limiting treatment efficacy. In the current study, we investigated the therapeutic potential of the AXL inhibitor (AXLi) BGB324 alone or in combination with the clinically relevant BRAF inhibitor (BRAFi) vemurafenib. Firstly, AXL was shown to be expressed in majority of melanoma lymph node metastases. When treated ex vivo, the largest reduction in cell viability was observed when the two drugs were combined. In addition, a therapeutic benefit of adding AXLi to the BRAF-targeted therapy was observed in pre-clinical AXLhigh melanoma models in vitro and in vivo. When searching for mechanistic insights, AXLi was found to potentiate BRAFi-induced apoptosis, stimulate ferroptosis and inhibit autophagy. Altogether, our findings propose AXLi as a promising treatment in combination with standard therapy to improve therapeutic outcome in metastatic melanoma.

Soluble AXL as a marker of disease progression and survival in melanoma.

Receptor tyrosine kinase AXL is a one-pass transmembrane protein upregulated in cancers and associated with lower survival and therapy resistance. AXL can be cleaved by the A Disintegrin and Metalloproteinases (ADAM)10 and ADAM17, yielding a soluble version of the protein. Elevated soluble AXL (sAXL) has been reported to be associated with disease progression in hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases. In the present work, we analyzed sAXL levels in blood from melanoma patients and showed that sAXL increases with disease progression. Additionally, increased sAXL levels were found correlated with shorter two-year survival in stage IV patients treated with ipilimumab. Furthermore, we showed that sAXL levels were related to the percentage of cells expressing AXL in resected melanoma lymph node metastases. This finding was verified in vitro, where sAXL levels in the cell media corresponded to AXL expression in the cells. AXL inhibition using the small-molecular inhibitor BGB324 reduced sAXL levels, while the cellular expression was elevated through increased protein stability. Our findings signify that quantification of sAXL blood levels is a simple and easily assessable method to determine cellular AXL levels and should be further evaluated for its use as a biomarker of disease progression and treatment response.

Low incidence of enterocolitis and colonic mucosal inflammation in Norwegian patients with Hirschsprung's disease.

BACKGROUND: Hirschsprung's disease (HD) may be associated with inflammation in the colon. Further, the etiology of Hirschsprung-associated enterocolitis (HEC) is unclear. To learn more about these features, we examined our cohort of HD patients during a period of 6 years for inflammation in their colonic mucosa as well as for signs of HEC. MATERIALS AND METHODS: Rectal suction biopsies and operative full thickness aganglionic and ganglionic colonic specimens from 36 patients were examined. Signs of inflammation were recorded in hematoxylin/eosin/saffron (HES)-stained sections and with fluorescence conjugated polyclonal antibodies to IgA and IgG applied on serial sections. The suction biopsies were also evaluated for the presence of mucus inspissation and crypt dilatation. Clinical signs of HEC were recorded from medical files of the same 36 patients. RESULTS: HES-staining revealed that seven patients had inflammation in the suction biopsies; these patients were significantly older than the patients without inflammation. Slight mucus inspissation was identified in suction biopsies of five out of 33 patients, but crypt abscesses or ulcerations were not found in any specimens. Virtually all very young patients (<3 months) had slight crypt dilatation. We identified inflammation in resected colonic segments from 17 out of 36 patients. Thirteen of these 17 had a diverting colostomy, and only one out of 14 patients with colostomy had no inflammation. Inflammatory changes were similar in ganglionic and aganglionic bowel. By immunofluorescence (IF) staining, inflammation was found in resected colonic segments from five patients. Four of these had a colostomy. HEC was diagnosed in three patients, and inflammation detected in resected specimens from only one of these three. CONCLUSIONS: We have not been able to identify particular characteristics in the colonic or rectal mucosa that are linked to development of HEC. Inflammation in the resected specimen was mainly found in patients with a diverting colostomy, and then in both ganglionic and aganglionic colon.

A Three-dimensional Ex Vivo Viability Assay Reveals a Strong Correlation Between Response to Targeted Inhibitors and Mutation Status in Melanoma Lymph Node Metastases.

Although clinical management of melanoma has changed considerably in recent years, intrinsic treatment resistance remains a severe problem and strategies to design personal treatment regimens are highly warranted. We have applied a three-dimensional (3D) ex vivo drug efficacy assay, exposing disaggregated cells from 38 freshly harvested melanoma lymph node metastases and 21 patient derived xenografts (PDXs) to clinical relevant drugs for 7 days, and examined its potential to evaluate therapy response. A strong association between Vemurafenib response and BRAF mutation status was achieved (P < .0001), while enhanced viability was seen in some NRAS mutated tumors. BRAF and NRAS mutated tumors responded comparably to the MEK inhibitor Cobimetinib. Based on the ex vivo results, two tumors diagnosed as BRAF wild-type by routine pathology examinations had to be re-evaluated; one was subsequently found to have a complex V600E mutation, the other a double BRAF mutation (V600E/K601 N). No BRAF inhibitor resistance mechanisms were identified, but PIK3CA and NF1 mutations were identified in two highly responsive tumors. Concordance between ex vivo drug responses using tissue from PDXs and corresponding patient tumors demonstrate that PDX models represent an indefinite source of tumor material that may allow ex vivo evaluation of numerous drugs and combinations, as well as studies of underlying molecular mechanisms. In conclusion, we have established a rapid and low cost ex vivo drug efficacy assay applicable on tumor tissue from patient biopsies. The 3D/spheroid format, limiting the influence from normal adjacent cells and allowing assessment of drug sensitivity to numerous drugs in one week, confirms its potential as a supplement to guide clinical decision, in particular in identifying non-responding patients.

CD38 is a marker of human lacteals.

CD38 is a type II transmembrane glycoprotein involved in signaling and adhesion which is expressed mainly by immature hematopoietic cells and activated lymphoid cells. Central lymphatic channels of human small intestinal villi, the so-called lacteals, were coincidentally found to express CD38. Gastric and large intestinal mucosae, pancreas, liver, lung, nasal mucosa, kidney, thymus, palatine tonsil, Peyer's patches, appendix, and mesenteric lymph nodes, and rodent intestinal mucosa were subsequently examined for lymphatic expression of CD38. Cryosections prepared from biopsy or surgical resection specimens were immunostained with four different antibodies to CD38 combined with antibodies to von Willebrand factor and CD31 to differentiate lymphatics from blood vessels, or with antibody to lysosomal protein. Sections were evaluated by ordinary and confocal immunofluorescence microscopy. Jejunal cryosections were subjected to in situ hybridization for CD38. All CD38 antibodies decorated human lacteals, and some of these were positive for CD38 mRNA. Lymphatics draining Peyer's patches and appendix as well as afferent lymphatics of mesenteric lymph nodes expressed CD38 weakly. CD38 was not detected on lymphatics in other organs or in rodent lacteals. We propose that CD38 is a novel marker of human small intestinal lymphatic vessels.

[Coeliac disease--new clinical findings and diagnostic tools]. / Cøliaki--nye kliniske erkjennelser og diagnostiske hjelpemidler.

Coeliac disease is a disorder characterised by intolerance to wheat gluten and related proteins from rye and barley, ingestion of which leads to a harmful immune activation in the small intestinal mucosa. The symptoms are often vague and do not suggest an intestinal disorder. The diagnosis is often easy once the question is raised; it requires a combination of serological tests and small intestinal biopsies. Coeliac disease can also exist with a normal serology. The trend in many countries is to require less pronounced mucosal abnormalities than previously for the diagnosis.

Regional induction of adhesion molecules and chemokine receptors explains disparate homing of human B cells to systemic and mucosal effector sites: dispersion from tonsils.

Ethical constraints restrict direct tracking of immune-cell migration throughout the human body in vivo. We, therefore, used deletion of the immunoglobulin M (IgM) heavy-chain constant-gene (Cmu) segment as a marker to provide a dispersal signature of an effector B-cell subset (IgD(+)IgM(-)CD38+) induced selectively in human tonsils. By DNA analysis, the Cmu deletion identified dissemination of such blasts and their plasma-cell progeny to peripheral blood, lymph nodes, and bone marrow, as well as to mucosae and glands of the upper airways. Also the endocervix was often positive, while the small intestine was mainly negative, as could be expected from the identified homing-molecule profile of the marker cells, with relatively low levels of integrin alpha4beta7 and CC chemokine receptor 9 (CCR9). Of further importance for vaccine design, the circulating cells expressed abundantly CD62L (L-selectin) and CCR7, which provided a mechanism for integration of respiratory and systemic immunity. Most mucosal vaccines are at present administered perorally, and our results suggested that the nasal route is no alternative for vaccination against rotavirus or other small-intestinal infections in humans. However, immunization of nasopharynx-associated lymphoid tissue clearly appears preferable to target respiratory pathogens and may to some extent also protect against infections of the female genital tract.

Effect of elemental diet on mucosal immunopathology and clinical symptoms in type 1 refractory celiac disease.

BACKGROUND & AIMS: Patients with celiac disease (CD) who do not improve or exhibit villous atrophy on a gluten-free diet may have type 1 refractory CD (RCD) with a polyclonal mucosal T-cell infiltrate, or type 2 RCD with a monoclonal infiltrate, also termed cryptic T-cell lymphoma. Both conditions are difficult to treat. Here we describe the effects of a nonimmunogenic elemental diet on clinical symptoms and mucosal immunopathology in type 1 RCD. METHODS: Ten CD patients on a strict gluten-free diet were diagnosed with type 1 RCD after extensive clinical evaluation in a tertiary referral hospital. A 4-week amino-acid-based liquid elemental diet regimen was given with no other treatment, except in 1 patient who also received methotrexate. Duodenal biopsy specimens were obtained before and after treatment for histologic assessment, immunophenotyping of intraepithelial lymphocytes, T-cell receptor clonality, mucosal interleukin (IL)-15 expression, flow-cytometric analysis of interferon (IFN)-gamma-secreting T cells, and whole biopsy specimen IFN-gamma messenger RNA determination. RESULTS: Nine patients completed the treatment; however, 1 patient did not tolerate the diet. Histologic improvement and reduced epithelial IL-15 were seen in 8 patients, whereas IFN-gamma-secreting mucosal T cells and IFN-gamma messenger RNA levels decreased in 4 and 7 patients, respectively. Clinical improvement was noted in 6 patients, with 1 patient showing normalization of hypoalbuminemia. Three patients could discontinue their total parenteral nutrition. CONCLUSIONS: Persistent mucosal IFN-gamma and IL-15 production often occurs in type 1 RCD despite conventional treatment. Elemental diet is a therapeutic option that can provide long-term immunopathologic and clinical improvement of this difficult condition.

IgVH gene analysis suggests that peritoneal B cells do not contribute to the gut immune system in man.

The contribution of peritoneal B cells to the intestinal lamina propria plasma cell population is well documented in mice, but unknown in humans. We have analyzed immunoglobulin (Ig) genes of human peritoneal B cells, because such genes show distinctive characteristics in mucosal B cells, particularly highly mutated variable regions. Here, we report the characteristics of variable region genes used by IgM, IgA and IgG in peritoneal cells. We focused on the properties of IgV(H)4-34 to allow comparisons of like-with-like between different isotypes and cells from different immune compartments. We observed that the IgM genes were mostly unmutated, and that the mutated subset had less mutations than would be expected in a mucosal B cell population. Likewise, the IgV(H)4-34 genes used by IgA and IgG from peritoneal B cells had significantly lower numbers of mutations than observed in the mucosal counterparts. Other trends observed, while not reaching statistical significance, followed the trend of peripheral B cells. The peritoneal B cell population had more IgA1 than IgA2 sequences, and there was no dominance of J(H)4 in the IgA from peritoneum or spleen, in contrast to the mucosal sequences. Overall, this study suggested that human peritoneal B cell are either peripheral or mixed in origin; they are unlikely to represent an inductive compartment for the mucosal B cell system.

Microbial colonization drives lymphocyte accumulation and differentiation in the follicle-associated epithelium of Peyer's patches.

Peyer's patches (PPs) are lined by follicle-associated epithelium (FAE) with Ag-transporting M cells. To investigate the spatial relationships of B cells, T cells, and dendritic cells (DCs) in PPs during microbial colonization, their in situ redistribution was examined in germfree (GF) rats exposed to a conventional pathogen-free microflora (conventionalized, CV). Although occasional B and T cells occurred in the FAE of GF rats, it contained mainly immature DCs (CD4(+)CD86(-)), whereas mature DCs (CD86(high)) were seen in the interfollicular zones even under GF conditions. In CV rats, DCs had disappeared from the FAE, which instead contained clusters by B and T cells associated with induction of putative M cell pockets. CD86 was seen neither in the FAE nor in the follicles under GF conditions, but it became apparent on intraepithelial B cells 5 wk after colonization. The level of CD86 on these B cells was comparable to that on germinal center B cells, although the B cell follicles did not show direct contact with the M cell areas. B cells in the follicular mantles acquired Bcl-2 after 12 wk in CV rats, whereas B cells in the FAE did not express Bcl-2 at a substantial level throughout the experimental period. The cellular redistribution patterns and phenotypic characteristics observed after colonization suggested that immature DCs, but not B cells, are involved in Ag presentation during primary immune responses against intestinal bacteria. However, the spatial cellular relationships sequentially being established among DCs, B cells, and T cells in PPs, are most likely important for the induction of post-germinal center B cells subsequently residing within the M cell pockets.