Background: The first visible signs of Crohns disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human alpha-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods: An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results: There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions: Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

Tuberculosis remains a big threat, with 1.6 million deaths in 2017, including 0.3 million deaths among patients with HIV. The risk of developing active disease increases considerably during an HIV coinfection. Alveolar macrophages are the first immune cells to encounter the causative agent Mycobacterium tuberculosis, but during the granuloma formation other cells are recruited in order to combat the bacteria. Here, we have investigated the effect of efferocytosis of apoptotic neutrophils by M. tuberculosis and HIV-coinfected macrophages in a human in vitro system. We found that the apo-ptotic neutrophils enhanced the control of M. tuberculosis in single and HIV-coinfected macrophages, and that this was dependent on myeloperoxidase (MPO) and reactive oxygen species in an autophagy-independent manner. We show that MPO remains active in the apoptotic neutrophils and can be harnessed by infected macrophages. In addition, MPO inhibition removed the suppression in M. tuberculosis growth caused by the apoptotic neutrophils. Antimycobacterial components from apoptotic neutrophils could thus increase the microbicidal activity of macrophages during an M. tuberculosis/HIV coinfection. This cooperation between innate immune cells could thereby be a way to compensate for the impaired adaptive immunity against M. tuberculosis seen during a concurrent HIV infection.

Background: We have previously shown an association of elevated coinhibitory molecule 2B4 expression with iNKT cells alterations in HIV disease. Herein, we show a comparative analysis of 2B4 expression on iNKT cells of HIV long-term nonprogressors (LTNPs) and progressors. Methods: Antiretroviral therapy-naive HIV-seropositive individuals (progressors, n = 16) and LTNPs (n = 10) were recruited for this study. We used multicolor flow cytometry on frozen peripheral blood mononuclear cells to determine iNKT subset frequencies, the levels of coinhibitory 2B4 expression, and intracellular interferon-gamma (IFN-gamma) production. CD1d tetramer was used to characterize iNKT cells. Results: We report significantly lower level of 2B4 expression on bulk LTNPs iNKT cells and on their CD4 subsets compared with HIV progressors. Furthermore, the iNKT cells from LTNPs produced higher amount of IFN-gamma than HIV progressors as detected by intracellular cytokine staining. Interestingly, the frequency of 2B4(+)iNKT cells of progressors but not LTNPs significantly correlates with CD4 T-cell count, HIV viral load, and IFN-gamma(+)production by iNKT cells. Conclusion: Our results suggest that in addition to suppressed HIV replication, diminished 2B4 expression and associated coinhibitory signaling, and substantial production of IFN-gamma could contribute to preserved iNKT cell phenotype in LTNPs.

Immunotherapies directly enhancing anti-tumor CD8(+) T cell responses have yielded measurable but limited success, highlighting the need for alternatives. Anti-tumor T cell responses critically depend on antigen presenting dendritic cells (DC), and enhancing mobilization, antigen loading and activation of these cells represent an attractive possibility to potentiate T cell based therapies. Here we show that expansion of DCs by Flt3L administration impacts in situ vaccination with oncolytic Newcastle Disease Virus (NDV). Mechanistically, NDV activates DCs and sensitizes them to dying tumor cells through upregulation of dead-cell receptors and synergizes with Flt3L to promote anti-tumor CD8(+) T cell cross-priming. In vivo, Flt3L-NDV in situ vaccination induces parallel amplification of virus- and tumor-specific T cells, including CD8(+) T cells reactive to newly-described neoepitopes, promoting long-term tumor control. Cross-presenting conventional Type 1 DCs are indispensable for the anti-tumor, but not anti-viral, T cell response, and type I IFN-dependent CD4(+) Th1 effector cells contribute to optimal anti-tumor immunity. These data demonstrate that mobilizing DCs to increase tumor antigen cross-presentation improves oncolytic virotherapy and that neoepitope-specific T cells can be induced without individualized, ex vivo manufactured vaccines. Strategies to advance T cell based immune therapies are mostly focusing on the improvement of CD8 T cell effector functions, such as cytotoxicity or recruitment to the tumor. Here authors show that by combining in situ vaccination with oncolytic Newcastle Disease Virus and Flt3L-driven dendritic cell expansion, the anti-tumor T cell response is amplified via increased antigen cross-presentation.

Background: Papillary thyroid carcinoma (PTC) is the most prevalent malignancy of the endocrine system. This study was aimed at evaluating the serum substance P (SP) levels, the tissue distribution of Neurokinin-1 receptors (NK1-R), and their possible diagnostic value in PTC. Method: The present case-control study included 31 healthy volunteers and 31 cases (age range: 25-64, 40.26 +/- 12.77), who were primarily diagnosed with PTC and were candidates for total thyroidectomy. Pre-operative serum level of SP was measured using a commercial ELISA kit. The tissue distribution of NK1-R was assessed immunohistochemically. Results: The serum level of SP in the patient group was higher than the healthy volunteers (P = 0.005). Besides, the expression of NK1-R was higher in tumoral tissues compared with their normal surroundings (P = 0.005). However, we observed no significant correlation between either SP level or NK1-R expression and the disease stage or lymph node involvement. Conclusion: SP level and NK1-R expression were upregulated in PTC patients, showing the involvement of SP/NK1R complex in PTC pathophysiology. Nonetheless, proposing SP/NK1R as a diagnostic factor requires further studies because we found no correlation between SP/NK1R and clinical stage or lymph node involvement.

Maturation of lymphoid cells is controlled by the action of stage and lineage-restricted transcription factors working in concert with the general transcription and chromatin remodeling machinery to regulate gene expression. To better understand this functional interplay, we used Biotin Identification in human embryonic kidney cells to identify proximity interaction partners for GATA3, TCF7 (TCF1), SPI1, HLF, IKZF1, PAX5, ID1, and ID2. The proximity interaction partners shared among the lineagerestricted transcription factors included ARID1a, a BRG1-associated factor complex component. CUT&RUN analysis revealed that ARID1a shared binding with TCF7 and GATA3 at a substantial number of putative regulatory elements in mouse T cell progenitors. In support of an important function for ARID1a in lymphocyte development, deletion of Aridla in early lymphoid progenitors in mice resulted in a pronounced developmental arrest in early T cell development with a reduction of CD4(+)CD8(+) cells and a 20-fold reduction in thymic cellularity. Exploring gene expression patterns in DN3 cells from Wt and Aridla-deficient mice suggested that the developmental block resided in the DN3a to DN3b transition, indicating a deficiency in beta-selection. Our work highlights the critical importance of functional interactions between stage and lineage-restricted factors and the basic transcription machinery during lymphocyte differentiation.

Despite the great success of vaccines over two centuries, the conventional strategy is based on attenuated/altered microorganisms. However, this is not effective for all microbes and often fails to elicit a protective immune response, and sometimes poses unexpected safety risks. The expanding nano toolbox may overcome some of the roadblocks in vaccine development given the plethora of unique nanoparticle (NP)-based platforms that can successfully induce specific immune responses leading to exciting and novel solutions. Nanovaccines necessitate a thorough understanding of the immunostimulatory effect of these nanotools. We present a comprehensive description of strategies in which nanotools have been used to elicit an immune response and provide a perspective on how nanotechnology can lead to future personalized nanovaccines.

Epigenetics may play a central, yet unexplored, role in the profound changes that the maternal immune system undergoes during pregnancy and could be involved in the pregnancy-induced modulation of several autoimmune diseases. We investigated changes in the methylome in isolated circulating CD4(+) T-cells in non-pregnant and pregnant women, during the 1(st) and 2(nd) trimester, using the Illumina Infinium Human Methylation 450K array, and explored how these changes were related to autoimmune diseases that are known to be affected during pregnancy. Pregnancy was associated with several hundreds of methylation differences, particularly during the 2(nd) trimester. A network-based modular approach identified several genes, e.g., CD28, FYN, VAV1 and pathways related to T-cell signalling and activation, highlighting T-cell regulation as a central component of the observed methylation alterations. The identified pregnancy module was significantly enriched for disease-associated methylation changes related to multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. A negative correlation between pregnancy-associated methylation changes and disease-associated changes was found for multiple sclerosis and rheumatoid arthritis, diseases that are known to improve during pregnancy whereas a positive correlation was found for systemic lupus erythematosus, a disease that instead worsens during pregnancy. Thus, the directionality of the observed changes is in line with the previously observed effect of pregnancy on disease activity. Our systems medicine approach supports the importance of the methylome in immune regulation of T-cells during pregnancy. Our findings highlight the relevance of using pregnancy as a model for understanding and identifying disease-related mechanisms involved in the modulation of autoimmune diseases.

Background Collagenous colitis (CC) is an inflammatory bowel disease where chronic diarrhoea is the main symptom. Diagnostic markers distinguishing between CC and other causes of chronic diarrhoea remain elusive. This study explores neutrophil gelatinase-associated lipocalin (NGAL) and its mRNA lipocalin2 (LCN2) as histological and faecal disease markers in CC. Methods NGAL/LCN2 were studied in colonic biopsies from CC patients before and during budesonide treatment using RNA sequencing (n = 9/group), in situ hybridization (ISH) (n = 13-22/group) and immunohistochemistry (IHC) (n = 14-25/group). Faecal samples from CC (n = 3-28/group), irritable bowel syndrome diarrhoea (IBS-D) (n = 14) and healthy controls (HC) (n = 15) were assayed for NGAL and calprotectin. Results NGAL/LCN2 protein and mRNA expression were upregulated in active CC vs HC, and vs paired samples of treated CC in clinical remission. IHC and ISH localized increased NGAL/LCN2 mainly to epithelium of active CC, compared to almost absence in HC and treated CC. In contrast, calprotectin was solely expressed in immune cells. Despite great individual differences, faecal NGAL was significantly increased in active CC compared to HC, IBS-D and treated CC and had high test sensitivity. Faecal calprotectin levels were variably increased in active CC, but the values remained below usual clinical cut-offs. Conclusion NGAL/LCN2 is upregulated in the epithelium of active CC and reduced during budesonide-induced clinical remission to the level of HC and IBD-S. This was reflected in NGAL faecal concentrations. We propose NGAL as an IHC marker for disease activity in CC and a potential faecal biomarker discriminating CC from HC and IBS-D.

The SARS-CoV-2 virus is a newly emergent pathogen first identified in Wuhan, China, and responsible for the COVID-19 global pandemic. In this case report we describe a manifestation of non-bacterial thrombotic endocarditis with continuous peripheral embolization in a COVID-19-positive patient. The patient responded well to high-dose LMWH treatment with cessation of the embolic process.

The interplay of immune mediators is paramount to optimal host anti-viral immune responses, especially against chronic hepatitis B virus (HBV) infection. Here, we investigated the dynamic changes in host immune responses in chronic HBV-infected individuals with and without liver cirrhosis by examining the signatures of apoptosis and plasma levels of pro-inflammatory cytokines, chemokines, and cytotoxic proteins. A total of 40 chronic HBV patients with and without liver cirrhosis were studied for plasma levels of immune mediators, and signatures of apoptosis in peripheral blood mononuclear cells (PBMCs). The intracellular concentrations of reactive oxygen species (ROS) in patients with chronic HBV with liver cirrhosis was relatively higher as compared to chronic HBV patients. The onset of apoptosis was sustained due to ongoing liver inflammation in concert with plasma TNF-alpha and IL-6 levels. Plasma VEGF was upregulated among chronic HBV patients with liver cirrhosis, whereas CCL2, CCL5 and granzyme B levels were down-regulated. High levels of ROS, IL-6 and TNF-alpha correlated with ongoing inflammation among chronic HBV patients with liver cirrhosis, which likely attributed to the expression of biosignatures of apoptosis and activation in immune cells.

Antigen-specific immunotherapy is an appealing strategy to preserve beta-cell function in type 1 diabetes, although the approach has yet to meet its therapeutic endpoint. Direct administration of autoantigen into lymph nodes has emerged as an alternative administration route that can improve the efficacy of the treatment. In the first open-label clinical trial in humans, injection of aluminum-formulated glutamic acid decarboxylase (GAD-alum) into an inguinal lymph node led to the promising preservation of C-peptide in patients with recent-onset type 1 diabetes. The treatment induced a distinct immunomodulatory effect, but the response at the cell level has not been fully characterized. Here we used mass cytometry to profile the immune landscape in peripheral blood mononuclear cells from 12 participants of the study before and after 15 months of treatment. The immunomodulatory effect of the therapy included reduction of naive and unswitched memory B cells, increase in follicular helper T cells and expansion of PD-1+ CD69+ cells in both CD8+ and double negative T cells. In vitro stimulation with GAD(65) only affected effector CD8+ T cells in samples collected before the treatment. However, the recall response to antigen after 15 months included induction of CXCR3+ and CD11c+Tbet+ B cells, PD-1+ follicular helper T cells and exhausted-like CD8+ T cells. This study provides a deeper insight into the immunological changes associated with GAD-alum administration directly into the lymph nodes.

Excessive fear is a hallmark of anxiety disorders, a major cause of disease burden worldwide. Substantial evidence supports a role of prefrontal cortex-amygdala circuits in the regulation of fear and anxiety, but the molecular mechanisms that regulate their activity remain poorly understood. Here, we show that downregulation of the histone methyltransferase PRDM2 in the dorsomedial prefrontal cortex enhances fear expression by modulating fear memory consolidation. We further show that Prdm2 knock-down (KD) in neurons that project from the dorsomedial prefrontal cortex to the basolateral amygdala (dmPFC-BLA) promotes increased fear expression. Prdm2 KD in the dmPFC-BLA circuit also resulted in increased expression of genes involved in synaptogenesis, suggesting that Prdm2 KD modulates consolidation of conditioned fear by modifying synaptic strength at dmPFC-BLA projection targets. Consistent with an enhanced synaptic efficacy, we found that dmPFC Prdm2 KD increased glutamatergic release probability in the BLA and increased the activity of BLA neurons in response to fear-associated cues. Together, our findings provide a new molecular mechanism for excessive fear responses, wherein PRDM2 modulates the dmPFC -BLA circuit through specific transcriptomic changes.

The use of X-chromosomal markers to resolve questions of relatedness has experienced a significant increase during the last years in forensic genetics. Perhaps primarily due to the emergence of commercial kits, but equally important due to an increased awareness of the utility of those markers. The X-chromosomal inheritance pattern entails that some cases, for instance paternal half-sisters, can potentially be resolved using a few X-chromosomal markers alone. For the statistical assessment in kinship cases it is of importance to have relevant population frequency data. In the present study 631 unrelated males from a Norwegian population sample are analyzed. The resulting haplotypes are compared to previously studied population samples and a deeper analysis of the linkage disequilibrium (LD) structure is conducted. We demonstrate that the power to detect LD will be low when few males, say below 300, are analyzed. We use entropy to describe the degree of LD between multiallelic loci and describe how this measure varies between different studied populations. Large population frequency databases have been recommended when using X-chromosomal markers, and we show that by combining reference da-tabases from genetically similar populations, more precise haplotype frequency estimates can be obtained for rare haplotypes which improves the statistical assessment of the weight of evidence. In addition, we promote the use of simulations to assess the utility of STR markers in contrast to standard forensic parameters. Specifically we perform extensive simulations on cases where X-chromosomal markers are important and illustrate how the results can be used to infer the information gained from these markers.

HIV transmission via genital and colorectal mucosa are the most common routes of dissemination. Here, we explored the effects of free and complement-opsonized HIV on colorectal tissue. Initially, there was higher antiviral responses in the free HIV compared to complementopsonized virus. The mucosal transcriptional response at 24 hr revealed the involvement of activated T cells, which was mirrored in cellular responses observed at 96 hr in isolated mucosal T cells. Further, HIV exposure led to skewing of T cell phenotypes predominantly to inflammatory CD4+ T cells, that is Th17 and Th1Th17 subsets. Of note, HIV exposure created an environment that altered the CD8+ T cell phenotype, for example expression of regulatory factors, especially when the virions were opsonized with complement factors. Our findings suggest that HIV-opsonization alters the activation and signaling pathways in the colorectal mucosa, which promotes viral establishment by creating an environment that stimulates mucosal T cell activation and inflammatory Th cells.

The American Zika virus (ZIKV) epidemic has highlighted the need to gain a better understanding of this emerging virus. The goal of this study was to describe the clinical symptoms, laboratory findings, and risk factors for symptomatic ZIKV infection in an area with ongoing transmission of other arboviral infections. We recruited patients at least 2 years of age seeking care at public health centers in Leon, Nicaragua, between January 2016 and August 2017, for fever, maculopapular rash, and/or nonsuppurative conjunctivitis with a duration of less than 1 week. A laboratory diagnosis of ZIKV was established using a combination of molecular and serological tests. Clinical and laboratory findings and potential risk factors were compared between participants with and without acute ZIKV infection. Fifty-eight (26%) of the 225 participants included in the analysis were found to have acute ZIKV infection. Pregnancy and reports of previous arboviral infection were associated with a higher risk of ZIKV infection. Rash, conjunctivitis, sore throat, and lower absolute neutrophil counts were associated with acute ZIKV infection. The clinical characteristics and risk factors identified were consistent with those identified by previous studies; however, we found sore throat to be a feature of ZIKV infection. We also found that neutrophil counts were lower in ZIKV-infected subjects. These clinical symptoms and laboratory datamay help clinicians suspect ZIKV infection during future outbreaks.

There are significant challenges to the development of a pediatric norovirus vaccine, mainly due to the antigenic diversity among strains infecting young children. Characterizing human norovirus serotypes and understanding norovirus immunity in naive children would provide key information for designing rational vaccine platforms. In this study, 26 Nicaraguan children experiencing their first norovirus acute gastroenteritis (AGE) episode during the first 18 months of life were investigated. We used a surrogate neutralization assay that measured antibodies blocking the binding of 13 different norovirus virus-like particles (VLPs) to histo-blood group antigens (HBGAs) in pre- and post-infection sera. To assess for asymptomatic norovirus infections, stools from asymptomatic children were collected monthly, screened for norovirus by RT-qPCR and genotyped by sequencing. Seroconversion of an HBGA-blocking antibody matched the infecting genotype in 25 (96%) of the 26 children. A subset of 13 (50%) and 4 (15%) of the 26 children experienced monotypic GII and GI seroconversion, respectively, strongly suggesting a type-specific response in naive children, and 9 (35%) showed multitypic seroconversion. The most frequent pairing in multitypic seroconversion (8/12) were GII.4 Sydney and GII.12 noroviruses, both co-circulating at the time. Blocking antibody titers to these two genotypes did not correlate with each other, suggesting multiple exposure rather than cross-reactivity between genotypes. In addition, GII titers remained consistent for at least 19 months post-infection, demonstrating durable immunity. In conclusion, the first natural norovirus gastroenteritis episodes in these young children were dominated by a limited number of genotypes and induced responses of antibodies blocking binding of norovirus VLPs in a genotype-specific manner, suggesting that an effective pediatric norovirus vaccine likely needs to be multivalent and include globally dominant genotypes. The duration of protection from natural infections provides optimism for pediatric norovirus vaccines administered early in life.

Background. Chikungunya infections range from subclinical infection to debilitating arthralgia and to chronic inflammatory rheumatism. Tumor necrosis factor (TNF) alpha, DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin), Toll-like receptor (TLR) 3, and blood groups have been directly or indirectly implicated in the susceptibility and pathogenesis of chikungunya. Methods. To test the hypothesis that polymorphisms in genes coding for these molecules determine clinical outcomes of chikungunya infection, a retrospective case-control study was performed in Leon, Nicaragua. The study included 132 case patients and 132 controls, matched for age, sex and neighborhood. Case patients had clinical symptoms of chikungunya, which was diagnosed by means of polymerase chain reaction. Controls were individuals not reporting abrupt presentation of clinical chikungunya-like symptoms. Polymorphisms were identified by TaqMan single-nucleotide polymorphism genotyping assays. Results. After adjustment for sociodemographic risk factors, chikungunya disease was associated with polymorphism in DC-SIGN and TLR3 genes (odds ratios, 5.2 and 3.3, respectively), and TNF-alpha with reduced persistent joint pain (0.24). Persistent joint pain was also associated with age, female sex and other comorbid conditions. Most interestingly, the Lewis-negative phenotype was strongly associated with both symptomatic chikungunya and immunoglobulin G seropositivity (odds ratios, 2.7, and 3.3, respectively). Conclusion. This study identified polymorphisms in DC-SIGN, TLR3, and TNF-alpha genes as well as Lewis-negative phenotype as risk factors for chikungunya infection and disease progression.

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, beta-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bd9 and Pygo-pus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of beta-catenins transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of beta-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of beta-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of beta-catenins transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by beta-catenins transcriptional activities upon stimulation with Wnt3a or during TGF-beta-induced EMT. Our results uncouple the signaling from the adhesion function of beta-catenin and underline the importance of Wnt/beta-catenin-dependent transcription in malignant tumor progression of breast cancer.

Introduction: celiac disease (CD) patients have a specific pattern of lymphocytic infiltrate in the duodenal mucosa. Flow cytometry is a complementary tool for the diagnosis of CD, which allows the quantification and characterization of intraepithelial lymphocytes (IELs) by what is commonly called a lymphogram. Here we describe our experience with this technique in the diagnosis of CD in adult patients. Methods: lymphograms from 157 patients performed in our center between 2009 and 2017 were retrospectively analyzed. Fourteen patients had a previous diagnosis of CD and followed a gluten-free diet (GFD), 21 had a new diagnosis of CD and the remaining were considered as non-celiac. The association of the lymphogram results (total IELs, CD3- lymphocytes and TcR gamma delta lymphocytes) with the CD diagnosis, compliance with the GFD, time since diagnosis and IgA anti-TG2 titer were determined. Results: the area under the ROC curve of TcR gamma delta lymphocytes for CD patients varied between 0.86 and 0.86. The percentage of TcR gamma delta lymphocytes in GFD-treated patients was lower; 12 (8.5) vs 20.5 (8.7), p = 0.0153. However, it remained high compared to non-CD; 12 (8.5) vs 6.7 (6), p = 0.135. The time since diagnosis and IgA anti-TG2 titer correlated with the lymphogram results. Helicobacter pylori infection and treatment with angiotensin receptor antagonist 2 (ARA2) were associated with differences in the lymphogram results in patients without CD. Conclusions: the duodenal lymphogram is a reliable complementary tool in adults for the diagnosis of CD. However, compliance and duration of the GFD and other factors may condition its diagnostic capacity.

Currently the only widely accepted corneal blindness treatment is human donor cornea transplantation. However, increasing shortage of donor corneas as well as high risk of rejection in some corneal diseases remain two major problems, which limit the success of corneal transplantation. Corneal neovascularization is considered as one of the main risk factors of graft failure. Different cell-free biosynthetic scaffolds fabricated from collagens or collagen-like peptides are being tested as donor cornea substitutes (DCS). Here, we report for the first-time composite biosynthetic DCS with integrated sustained release system of anti-VEGF drug, bevacizumab and their preliminary in vitro validation. We have tethered gold nanoparticles with bevacizumab and integrated into a collagen-based cell-free hydrogel scaffold. Developed grafts preserved good optical properties and were confirmed not toxic to human corneal epithelial cells. Bevacizumab has been shown to constantly releasing from the DCS up to 3 weeks and preserved its anti-angiogenic properties. These results provide background for further use of infused composite biosynthetic DCS with integrated nanosystem of bevacizumab sustained release in corneal disease accompanied by neovascularisation where conventional corneal transplantation might fail.

Norovirus has emerged as an important viral agent of acute pediatric gastroenteritis, with a growing genetic diversity reported in the last decades. Histo-blood group antigens (HBGAs) present on the surface of enterocytes are susceptibility factors for norovirus infection and differ between populations which could affects the epidemiology and evolution of these viruses. This study investigated the frequency, incidence and genetic diversity of noroviruses in a cohort of rotavirus A vaccinated children in association to the host HBGA (Secretor/Lewis) genetic susceptibility profile. Norovirus genogroups I and II (GI/GII) were screened by RT-qPCR in 569 stool samples from 132 children followed-up from birth to 11 months of age during 2014-2018. Noroviruses were identified in 21.2% of children enrolled in this study, with a norovirus detection rate of 5.6% (32/569), in 17.1% and 4.7% of acute diarrheic episodes (ADE) and non-ADE, respectively. The norovirus incidence was 5.8 infections per 100 child-months. Partial nucleotide sequencing characterized six different norovirus genotypes, with GII.4 Sydney 2012 being detected in 50% associated with three different polymerase genotypes (GII.31, GII.P16 and GII.P4 New Orleans 2009). FUT3 genotyping was yielded seven new mutations in this population. A significant association between symptomatic norovirus infection and secretor profile could be inferred.

Recent studies have investigated whether the human histo-blood group antigen (HBGAs) could affect the effectiveness of the oral rotavirus vaccines, suggesting secretor positive individuals develop a more robust response. We investigated the Rotavirus A (RVA) shedding in association with the host susceptibility profile in children from a birth community-cohort in Rio de Janeiro, Brazil, from 2014 to 2018. A total of 132 children were followed-up between 0 to 11-month-old, stool samples were collected before/after the 1(st)/2(nd) RV1 vaccination doses and saliva samples were collected during the study. RVA shedding was screened by RT-qPCR and G/P genotypes determined by multiplex RT-PCR and/or Sanger nucleotide sequencing. The sequencing indicated an F167L amino acid change in the RV1 VP8* P[8] in 20.5% of shedding follow-ups and these mutant subpopulations were quantified by pyrosequencing. The HBGA/secretor status was determined and 80.3% of the children were secretors. Twenty-one FUT2 gene SNPs were identified and two new mutations were observed. The mutant F167L RV1 VP8* P[8] was detected significantly more in Le (a+b+) secretors (90.5%) compared to non-secretors and even to secretors Le (a-b+) (9.5%). The study highlights the probable association between RV1 shedding and HBGAs as a marker for evaluating vaccine strain host susceptibility.

Electro-stimulation is an effective way to manipulate the presentation of bio-factors at the materials interface. This study aimed to develop electrochemically-modified trabecular bone-like surfaces for manipulation of mesenchymal and hematopoietic cells. The electroactive surface was based on the conducting polymer polypyrrole for dynamic control of the presentation and mineralisation of chondrocyte-derived plasma membrane nanofragments (PMNFs) covalently immobilized on the surface. Electrochemical redox switching resulted in the PMNF-based formation of bone minerals with different morphologies, which further demonstrated to have distinct effects on the survival of mouse bone marrow-derived mesenchymal and hematopoietic cell populations cultured on the surface. This tunable electroactive surface could be a valuable tool for dynamically sensing and/or controlling stem cell functions in more suitable biomimetic microenvironments housing a stem cell niche.

Treatment with RNAi against HIV-1 transcripts efficiently inhibits viral replication but induces selection of escape mutants; therefore, the CCR5 coreceptor was suggested as an additional target. Blocking viral and host transcripts improved the antiviral effect. We have used short hairpin RNA (shRNA) targeting the human CCR5 (shCCR5) or the HIV-1 rev (shRev) transcripts to demonstrate distinctive properties of anti-CCR5 shRNA: shCCR5 induced more sustained protection than shRev; partial reduction in CCR5 expression substantially decreased HIV-1 infection, and shCCR5 performed better than shRev in the mixed shRNA-treated and untreated cultures. These observations indicate that CCR5 inhibitors should be conveniently included in HIV-1 gene silencing treatment schedules when only a certain cell fraction is protected to further reduce endogenous virus in a properly ART-treated HIV-1 infected individual.

Background and Aim: Increased frequencies of T regulatory (Treg) cells, key players in immune regulation, have been reported in inflammatory bowel diseases, including collagenous colitis (CC). However, traditional Treg identification techniques might have misinterpreted the frequencies of Treg cells in CC. Thus, we investigated the presence of genuine Treg cells in CC. Methods: Treg cells were analyzed in mucosal and peripheral blood samples of CC patients before and during treatment with the corticosteroid drug budesonide and in healthy controls. Samples were analyzed by flow cytometry by classifying CD3(+) CD4(+) cells as activated FoxP3(high)CD45RA. Treg cells, resting FoxP3(dim)CD45RA(+) Treg cells, and nonsuppressive FoxP3(dim)CD45RA-T helper cells. Traditional gating strategies that classified Treg cells as CD25(high)CD127(lo)(w), FoxP3(+)CD127(low), and CD4(+)CD25(+)FoxP3(+) were also used to facilitate comparison with previous studies. Results: Activated and resting Treg cell frequencies did not change in active CC mucosa or peripheral blood and were not affected by budesonide treatment. Instead, nonsuppressive FoxP3(dim)CD45RA-T helper cells were increased in active CC mucosa, and budesonide helped restore them to normal levels. In contrast, traditional Treg cell gating strategies resulted in increased Treg cell frequencies in active CC mucosa. No alterations were found in peripheral blood samples, independently of patient treatment or gating techniques. Conclusion: Previously reported increase of Treg cells is a result of incomplete Treg phenotyping, which included nonsuppressive FoxP3(dim)CD45RA - T helper cells. Because budesonide did not affect Treg percentage, its therapeutic effect in CC might involve alternative mechanisms.

IntroductionCollagenous colitis (CC) is an inflammatory bowel disease, which usually responds to budesonide treatment. Our aim was to study the immunological background of the disease. MethodsAnalyses of peripheral and mucosal MAIT (mucosa associated invariant T cells) and NK (natural killer) cells were performed with flow cytometry. Numbers of mucosal cells were calculated using immunohistochemistry. We studied the same patients with active untreated CC (au-CC) and again while in remission on budesonide treatment. Budesonide refractory patients and healthy controls were also included. The memory marker CD45R0 and activation marker CD154 and CD69 were used to further study the cells. Finally B cells, CD4(+) and CD8(+) T cells were also analysed. ResultsThe percentages of circulating CD56(dim)CD16(+) NK cells as well as MAIT cells (CD3(+)TCRVa7.2(+)CD161(+)) were decreased in au-CC compared to healthy controls. This difference was not seen in the mucosa; where we instead found increased numbers of mucosal CD4(+) T cells and CD8(+) T cells in au-CC. Mucosal immune cell numbers were not affected by budesonide treatment. In refractory CC we found increased mucosal numbers of MAIT cells, CD4(+) and CD8(+) T cells compared to au-CC. DiscussionPatients with active collagenous colitis have lower percentages of circulating MAIT and NK cells. However, there was no change of these cells in the colonic mucosa. Most mucosal cell populations were increased in budesonide refractory as compared to au-CC patients, particularly the number of MAIT cells. This may indicate that T cell targeting therapy could be an alternative in budesonide refractory CC.

The introduction of rotavirus A (RVA) vaccines has considerably reduced the RVA-associated mortality among children under 5 years of age worldwide. The ability of RVA to reassort gives rise to different combinations of surface proteins G (glycoprotein, VP7) and P (protease sensitive, VP4) RVA types infecting children. During the epidemiological surveillance of RVA in the Northwest Amazon region, an unusual rotavirus genotype G6P[8] was detected in feces of a 2-year-old child with acute gastroenteritis (AGE) that had been vaccinated with one dose of Rotarix(& REG;) (RV1). The G6P[8] sample had a DS-1-like constellation with a Wa-like VP3 gene mono-reassortment similar to equine-like G3P[8] that has been frequently detected in Brazil previously. The results presented here reinforce the evolutionary dynamics of RVA and the importance of constant molecular surveillance.

Sapovirus is an important etiological agent of acute gastroenteritis (AGE), mainly in children under 5 years old living in lower-income communities. Eighteen identified sapovirus genotypes have been observed to infect humans. The aim of this study was to identify sapovirus genotypes circulating in the Amazon region. Twenty-eight samples were successfully genotyped using partial sequencing of the capsid gene. The genotypes identified were GI.1 (n = 3), GI.2 (n = 7), GII.1 (n = 1), GII.2 (n = 1), GII.3 (n = 5), GII.5 (n = 1), and GIV.1 (n = 10). The GIV genotype was the most detected genotype (35.7%, 10/28). The phylogenetic analysis identified sapovirus genotypes that had no similarity with other strains reported from Brazil, indicating that these genotypes may have entered the Amazon region via intense tourism in the Amazon rainforest. No association between histo-blood group antigen expression and sapovirus infection was observed.

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::Nras(Q61K); Cdkn2a(-/-) melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism. Melanoma cells can switch between proliferative and invasive phenotypes. Here the authors show that the embryonic stem cell factor Sall4 is a negative regulator of melanoma phenotype switching where its loss leads to the acquisition of an invasive phenotype, due to derepression of invasiveness genes.

Mouse embryonic stem cells (mESCs) can bemaintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confersm ESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a beta-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

Background: Efficient healthcare based on prognostic variables in hospitalised patients with COVID-19 could reduce the risk of complications and death. Recently, soluble urokinase Plasminogen Activator Receptor (suPAR) was shown to predict respiratory failure, kidney injury, and clinical outcome in patients with SARS-CoV-2 infection. The aim of this study was to investigate the value of suPAR as a prognostic tool, in comparison with other variables, regarding disease severity and length of hospital stay in patients with COVID-19.Patients and Methods: Individuals hospitalised with COVID-19 (40 males, 20 females; median age 57.5 years) with a median symptom duration of 10 days and matched, healthy controls (n = 30) were included. Admission levels of suPAR were measured in serum by enzyme-linked immunosorbent assay. Blood cell counts, C-reactive protein (CRP) levels, lactate dehydrogenase (LDH), plasma creatinine and estimated glomerular filtration rates were analysed and oxygen demand, level of care and length of hospitalisation recorded.Results: Patients had significantly higher suPAR levels compared to controls (P &lt; 0.001). Levels were higher in severely/critically (median 6.6 ng/mL) compared with moderately ill patients (median 5.0 ng/mL; P = 0.002). In addition, suPAR levels correlated with length of hospitalisation (rho = 0.35; P = 0.006). Besides suPAR, LDH, CRP, neutrophil count, neutrophil-to-monocyte and neutrophil-to-lymphocyte ratio, body mass index and chronic renal failure were discriminators of COVID-19 severity and/or predictors of length of hospitalisation.Conclusion: Admission levels of suPAR were higher in patients who developed severe/critical COVID-19 and associated with length of hospital stay. In addition, we showed that suPAR functioned as an independent predictor of COVID-19 disease severity.

Objectives Type I interferons (IFNs) are central and reflective of disease activity in systemic lupus erythematosus (SLE). However, IFN-alpha levels are notoriously difficult to measure and the type I IFN gene signature (IGS) is not yet available in clinical routine. This study evaluates galectin-9 and an array of chemokines/cytokines in their potential as surrogate markers of type I IFN and/or SLE disease activity. Methods Healthy controls and well-characterized Swedish SLE patients from two cross-sectional cohorts (n=181; n=59) were included, and a subgroup (n=21) was longitudinally followed. Chemokine/cytokine responses in immune complex triggered IFN-alpha activity was studied in healthy donor peripheral blood mononuclear cells (PBMC). Levels of chemokines/cytokines and galectin-9 were measured by immunoassays. Gene expression was quantified by qPCR. Results The IGS was significantly (p&lt;0.01) correlated with galectin-9 (rho=0.54) and CXCL10 (rho=0.37) levels whereas serum IFN-alpha correlated with galectin-9 (rho=0.36), CXCL10 (rho=0.39), CCL19 (rho=0.26) and CCL2 (rho=0.19). The strongest correlation was observed between galectin-9 and TNF (rho=0.56). IFN-alpha and disease activity (SLEDAI-2K) were correlated (rho=0.20) at cross-sectional analysis, but no significant associations were found between SLEDAI-2K and galectin-9 or chemokines. Several inflammatory mediators increased at disease exacerbation although CCL19, CXCL11, CXCL10, IL-10 and IL-1 receptor antagonist were most pronounced. Immune complex-stimulation of PBMC increased the production of CCL2, CXCL8 and TNF. Conclusion Galectin-9 and CXCL10 were associated with type I IFN in SLE but correlated stronger with TNF. None of the investigated biomarkers showed a convincing association with disease activity, although CXCL10 and CCL19 performed best in this regard.

The intestinal epithelium limits host-luminal interactions and maintains gut homeostasis. Breakdown of the epithelial barrier and villous atrophy are hallmarks of coeliac disease. Besides the well characterized immune-mediated epithelial damage induced in coeliac mucosa, constitutional changes and early gluten direct effects disturb intestinal epithelial cells. The subsequent modifications in key epithelial signaling pathways leads to outnumbered immature epithelial cells that, in turn, facilitate epithelial dysfunction, promote crypt hyperplasia, and increase intestinal permeability. Consequently, underlying immune cells have a greater access to gluten, which boosts the proinflammatory immune response against gluten and positively feedback the epithelial damage loop. Gluten-free diet is an indispensable treatment for coeliac disease patients, but additional therapies are under development, including those that reinforce intestinal epithelial healing. In this chapter, we provide an overview of intestinal epithelial cell disturbances that develop during gluten intake in coeliac disease mucosa.

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Intraepithelial immune response can be studied by flow cytometry. The proportion of CD3(+) cells differs between Crohns disease and ulcerative colitis.

BACKGROUND AND AIMS: Diarrhoea is a common, debilitating symptom of gastrointestinal disorders. Pathomechanisms probably involve defects in trans-epithelial water transport, but the role of aquaporin [AQP] family water channels in diarrhoea-predominant diseases is unknown. We investigated the involvement of AQPs in the pathobiology of collagenous colitis [CC], which features chronic, watery diarrhoea despite overtly normal intestinal epithelial cells [IECs].

METHODS: We assessed the expression of all AQP family members in mucosal samples of CC patients before and during treatment with the corticosteroid drug budesonide, steroid-refractory CC patients and healthy controls. Samples were analysed by genome-wide mRNA sequencing [RNA-seq] and quantitative real-time PCR [qPCR]. In some patients, we performed tissue microdissection followed by RNA-seq to explore the IEC-specific CC transcriptome. We determined changes in the protein levels of the lead candidates in IEC by confocal microscopy. Finally, we investigated the regulation of AQP expression by corticosteroids in model cell lines.

RESULTS: Using qPCR and RNA-seq, we identified loss of AQP8 expression as a hallmark of active CC, which was reverted by budesonide treatment in steroid-responsive but not refractory patients. Consistently, decreased AQP8 mRNA and protein levels were observed in IECs of patients with active CC, and steroid drugs increased AQP8 expression in model IECs. Moreover, low APQ8 expression was strongly associated with higher stool frequency in CC patients.

CONCLUSION: Down-regulation of epithelial AQP8 may impair water resorption in active CC, resulting in watery diarrhoea. Our results suggest that AQP8 is a potential drug target for the treatment of diarrhoeal disorders.

BACKGROUND AND AIMS: The pathophysiology of the inflammatory bowel disease collagenous colitis (CC) is poorly described. Our aim was to use RNA sequencing of mucosal samples from patients with active CC, CC in remission, refractory CC, ulcerative colitis (UC), and controls to gain insight into CC pathophysiology, identify genetic signatures linked to CC, and uncover potentially druggable disease pathways.

METHODS: We performed whole transcriptome sequencing of CC samples from patients before and during treatment with the corticosteroid drug budesonide, CC steroid-refractory patients, UC patients, and healthy controls (n=9-13). Bulk mucosa and laser-captured microdissected intestinal epithelial cell (IEC) gene expression were analyzed by gene-set enrichment and gene-set variation analyses to identify significant pathways and cells, respectively, altered in CC. Leading genes and cells were validated using reverse transcription quantitative PCR and/or immunohistochemistry.

RESULTS: We identified an activation of the adaptive immune response to bacteria and viruses in active CC that could be mediated by dendritic cells. Moreover, IECs display hyperproliferation and increased antigen presentation in active CC. Further analysis revealed that genes related to the immune response (DUOX2, PLA2G2A, CXCL9), DNA transcription (CTR9), protein processing (JOSD1, URI1) and ion transport (SLC9A3) remained dysregulated even after budesonide-induced remission. Budesonide-refractory CC patients fail to restore normal gene expression, and displayed a transcriptomic profile close to UC.

CONCLUSIONS: Our study confirmed the implication of innate and adaptive immune responses in CC, governed by IECs and dendritic cells, respectively; and identified ongoing epithelial damage. Refractory CC could share pathomechanisms with UC.

Purpose The diagnosis microscopic colitis (MC) consisting of collagenous colitis (CC) and lymphocytic colitis (LC) relies on histological assessment of mucosal biopsies from the colon. The optimal biopsy strategy for reliable diagnosis of MC is controversial. The aim of this study was to evaluate the distribution of histopathological features of MC throughout the colon. Methods Mucosal biopsies from multiple colonic segments of patients with MC who participated in one of the three prospective European multicenter trials were analyzed. Histological slides were stained with hematoxylin-and-eosin, a connective tissue stain, and CD3 in selected cases. Results In total, 255 patients were included, 199 and 56 patients with CC and LC, respectively. Both groups exhibited a gradient with more pronounced inflammation in the lamina propria in the proximal colon compared with the distal colon. Similarly, the thickness of the subepithelial collagenous band in CC showed a gradient with higher values in the proximal colon. The mean number of intraepithelial lymphocytes was &gt; 20 in all colonic segments in patients within both subgroups. Biopsies from 86 to 94% of individual segments were diagnostic, rectum excluded. Biopsies from non-diagnostic segments often showed features of another subgroup of MC. Conclusion Conclusively, although the severity of the histological changes in MC differed in the colonic mucosa, the minimum criteria required for the diagnosis were present in the random biopsies from the majority of segments. Thus, our findings show MC to be a pancolitis, rectum excluded, questioning previously proclaimed patchiness throughout the colon.

Background Perinatal childhood exposures, including probiotic supplementation, may affect epigenetic modifications and impact on immune maturation and allergy development. The aim of this study was to assess the effects of pre- and postnatal Lactobacillus reuteri supplementation on DNA methylation in relation to immune maturation and allergy development. Methods DNA methylation patterns were investigated for allergy-related T helper subsets using a locus-specific method and at a genome-wide scale using the Illumina 450K array. From a randomised, double-blind, placebo-controlled allergy prevention trial with pre- and postnatal probiotic supplementation, CD4+ T helper cells were obtained at birth (from cord blood), and 12 and 24 months of age (total (placebo/probiotics); locus-specific method: CB = 32 (17/15), 12 months = 24 (9/15), 24 months = 35 (15/20); Illumina: CB = 19 (10/9), 12 months = 10 (6/4), 24 months = 19(11/8)). Results Comparing probiotics to placebo, the greatest genome-wide differential DNA methylation was observed at birth, where the majority of sites were hypomethylated, indicating transcriptional accessibility in the probiotic group. Bioinformatic analyses, including network analyses, revealed a module containing 91 genes, enriched for immune-related pathways such as chemotaxis, PI3K-Akt, MAPK and TGF-beta signalling. A majority of the module genes were associated with atopic manifestations (OR = 1.43, P = 2.4 x 10(-6)), and a classifier built on this model could predict allergy development (AUC = 0.78, P = 3.0 x 10(e-3)). Pathways such as IFN-gamma signalling and T-cell activation were more hypermethylated at birth compared with later in life in both intervention groups over time, in line with DNA methylation patterns in the IFNG locus obtained by the locus-specific methodology. Conclusion Maternal L. reuteri supplementation during pregnancy alters DNA methylation patterns in CD4+ T cells towards enhanced immune activation at birth, which may affect immune maturation and allergy development.

The human fetal γ-globin gene is repressed in the adult stage through complex regulatory mechanisms involving transcription factors and epigenetic modifiers. Reversing γ-globin repression, or maintaining its expression by manipulating regulatory mechanisms, has become a major clinical goal in the treatment of β-hemoglobinopathies. Here, we identify the orphan nuclear receptor Coup-TFII (NR2F2/ARP-1) as an embryonic/fetal stage activator of γ-globin expression. We show that Coup-TFII is expressed in early erythropoiesis of yolk sac origin, together with embryonic/fetal globins. When overexpressed in adult cells (including peripheral blood cells from human healthy donors and β⁰39 thalassemic patients) Coup-TFII activates the embryonic/fetal globins genes, overcoming the repression imposed by the adult erythroid environment. Conversely, the knock-out of Coup-TFII increases the β/γ+β globin ratio. Molecular analysis indicates that Coup-TFII binds in vivo to the β-locus and contributes to its conformation. Overall, our data identify Coup-TFII as a specific activator of the γ-globin gene.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Semen changes the gene expression in endometrial and oviductal tissues modulating important processes for reproduction. We tested the hypothesis that mating and/or sperm-free seminal plasma deposition in the reproductive tract affect the expression of genes associated with sperm-lining epithelium interactions, ovulation, and pre-implantation effects (nerve growth factor, NGF; α/β hydrolase domain-containing protein 2, ABHD2; C-terminal tensin-like protein, CTEN or TNS4; and versican, VCAN) in the period 10-72 h post-mating. In Experiment 1, does (n = 9) were treated with gonadotropin-releasing hormone (GnRH) (control), GnRH-stimulated, and vaginally infused with sperm-free seminal plasma (SP-AI), or GnRH-stimulated and naturally mated (NM). In Experiment 2, does (n = 15) were GnRH-stimulated and naturally mated. Samples were retrieved from the internal reproductive tracts (cervix-to-infundibulum) 20 h post-treatment (Experiment 1) or sequentially collected at 10, 24, 36, 68, or 72 h post-mating (Experiment 2, 3 does/period). All samples were processed for gene expression analysis by quantitative PCR. Data showed an upregulation of endometrial CTEN and NGF by NM, but not by SP-AI. The findings suggest that the NGF gene affects the reproductive tract of the doe during ovulation and beyond, influencing the maternal environment during early embryonic development.

Simple Summary In mammals, the expression of regulatory genes is modified by the interaction between semen and the female reproductive tract. This study intends to unveil how mating or insemination with sperm-free seminal plasma, as well as the presence of preimplantation embryos, affects inflammation and angiogenesis in different segments of the reproductive tract of female rabbits. Gene expression of anti-inflammatory cytokines and angiogenesis mediators was analyzed in segmented tracts (cervix to infundibulum) in response to mating and sperm-free seminal plasma infusion. Moreover, the gene expression at different times post-mating was also analyzed. Results showed that gene expression changes were mainly localized in the uterus in the natural mating group, describing a clear temporal variation, while limited to the oviduct in the sperm-free seminal plasma group. These changes suggest an early response in the uterus and late modulation in the oviduct, distinctly demonstrating that semen and seminal plasma, through their interaction with the female reproductive tract, can differentially modulate the expression of anti-inflammatory and angiogenesis mediators. The maternal environment modulates immune responses to facilitate embryo development and ensure pregnancy. Unraveling this modulation could improve the livestock breeding systems. Here it is hypothesized that the exposure of the female rabbit reproductive tract to semen, as well as to early embryos, modulates inflammation and angiogenesis among different tissue segments. qPCR analysis of the gene expression changes of the anti-inflammatory interleukin-10 (IL10) and transforming growth factor beta family (TGF beta 1-3) and the angiogenesis mediator vascular endothelial growth factor (VEGF-A) were examined in response to mating or insemination with sperm-free seminal plasma (SP). Reproductive tract segment (cervix to infundibulum) samples were obtained in Experiment 1, 20 h after gonadotropin-releasing hormone (GnRH) stimulation (control), natural mating (NM) or vaginal infusion with sperm-free SP (SP-AI). Additionally, segmented samples were also obtained at 10, 24, 36, 68 or 72 h after GnRH-stimulation and natural mating (Experiment 2). The results of gene expression, analyzed by quantitative PCR, showed that NM effects were mainly localized in the uterine tissues, depicting clear temporal variation, while SP-AI effects were restricted to the oviduct. Changes in anti-inflammatory and angiogenesis mediators indicate an early response in the uterus and a late modulation in the oviduct either induced by semen or preimplantation embryos. This knowledge could be used in the implementation of physiological strategies in breeding systems to face the new challenges on rabbit productivity and sustainability.

Recent technical developments and early clinical examples support that precision medicine has potential to provide novel diagnostic and therapeutic solutions for patients with complex diseases, who are not responding to existing therapies. Those solutions will require integration of genomic data with routine clinical, imaging, sensor, biobank and registry data. Moreover, user-friendly tools for informed decision support for both patients and clinicians will be needed. While this will entail huge technical, ethical, societal and regulatory challenges, it may contribute to transforming and improving health care towards becoming predictive, preventive, personalised and participatory (4P-medicine).

An amendment to this paper has been published and can be accessed via the original article.

Background Psychological stress and increased permeability are implicated as contributing factors in the initiation and worsening of gastrointestinal diseases. A link between stress and intestinal permeability has been shown in animal models as well as in human small intestine, but stress effects on the human colorectal mucosal barrier has not been reported. Objective To investigate the potential effects of acute psychological stress on colorectal mucosal barrier function and to explore stress-induced molecular events in the rectal mucosa under healthy conditions. Methods Endoscopic biopsies were taken from the rectosigmoid region of healthy volunteers, who had been subjected to dichotomous listening stress and after a control session, respectively. Paracellular and transcellular permeability were assessed in modified Ussing chambers. RNA expression (microarray technology confirmed by quantitative real-time polymerase chain reaction) and biological pathway analysis were used to investigate the local mucosal response to acute stress. Results Dichotomous listening stress induced a subjective and objective stress response, and significantly increased paracellular but not transcellular permeability. We also identified a stress-induced reduction in RNA expression of genes related to immune cell activation and maturation (CR2, CD20, TCLA1, BANK1, CD22, FDCSP), signaling molecules of homing of immune cells to the gut (chemokines: CCL21, CXCL13, and CCL19, and receptors: CCR7, CXCR5), and innate immunity (DUOX2). Eight of the 10 top down-regulated genes are directly involved in B cell activation, signaling and migration. The systemic stress response correlated positively with paracellular permeability and negatively with DUOX2 expression. Conclusion Dichotomous listening stress increases paracellular permeability and modulates immune cell activity in the rectal mucosa. Further studies are warranted to identify the primary mechanisms of stress-mediated reduction of mucosal defensive activity and barrier dysfunction, and their potential implications for gastrointestinal disorders.