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Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by variable impairment of cognitive function and interpersonal relationships. Furthermore, some individuals with ASD have gastrointestinal disorders that have been correlated with impairments in intestinal microbiota. Gut microbiota are important not only for intestinal health, but also for many other functions including food digestion, energy production, immune system regulation, and, according to current data, behavior. Disruption of the indigenous microbiota, microbial dysbiosis (imbalance between microorganisms present in the gut), overgrowth of potentially pathogenic microorganisms, a less diverse microbiome, or lower levels of beneficial bacteria in children with ASD can affect behavior. Metabolome analysis in children with ASD has identified perturbations in multiple metabolic pathways that might be associated with cognitive functions. Recent studies have shown that the intestinal microbiome provides environmental signals that can modify host response to stimuli by modifying the host epigenome, which affects DNA methylation, histone modification, and non-coding RNAs. The most studied microbiota-produced epigenetic modifiers are short-chain fatty acids, although other products of intestinal microbiota might also cause epigenetic modifications in the host's DNA. Here we review evidence suggesting that epigenetic alterations caused by modification of gene expression play an important role in understanding ASD.

Background Targeted therapies have improved survival and quality of life for patients with non-small-cell lung cancer with actionable driver mutations. However, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 gene (HER2, also known as ERBB2) exon 20 insertions (Ex20mut) are characterized by a poor response to currently approved tyrosine kinase inhibitors and immunotherapies. The underlying immune biology is not well understood. Materials and methods We carried out messenger RNA expression profiling of lung adenocarcinomas (ADCs) with ERBB2 (n = 19) and EGFR exon 20-insertion mutations (n = 13) and compared these to tumors with classical EGFR mutations (n = 40, affecting EGFR exons 18, 19 or 21) and EGFR/ERBB2 mutation-negative lung ADC (EGFR/ERBB2wt, n = 26) focusing on immunologically relevant transcripts. Tumor-infiltrating immune cells were estimated from gene expression profiles. Results Cytotoxic cells were significantly lower in EGFR-mutated tumors regardless of the affected exon, while Th1 cells were significantly lower in EGFR-Ex20mut compared to EGFR/ERBB2wt tumors. We assessed the differentially expressed genes of ERBB2-Ex20mut and EGFR-Ex20mut tumors compared to EGFR-Ex18/19/21mut and EGFR/ERBB2wt tumors. Of these, the genes GUSB, HDAC11, IFNGR2, PUM1, RASGRF1 and RBL2 were up-regulated, while a lower expression of CBLC, GBP1, GBP2, GBP4 and MYC was observed in all three comparison groups. The omnibus test revealed 185 significantly (FDR = 5%) differentially expressed genes and we found these four most significant gene expression changes in the study cohort: VHL and JAK1 were overexpressed in ERBB2-Ex20mut and EGFR-Ex20mut tumors compared to both EGFR-Ex18/19/21mut and EGFR/ERBB2wt tumors. RIPK1 and STK11IP showed the highest expression in ERBB2-Ex20mut tumors. Conclusions Targeted gene expression profiling is a promising tool to read out the characteristics of the tumor microenvironment from routine diagnostic lung cancer biopsies. Significant immune reactivity and specific immunosuppressive characteristics in ERBB2-Ex20mut and EGFR-Ex20mut lung ADC with at least some degree of immune infiltration support further clinical evaluation of immune-modulators as partners of immune checkpoint inhibitors in such tumors., Highlights • Gene expression profiling to characterize the tumor microenvironment is feasible using diagnostic lung cancer biopsies. • EGFR exon 20-mutated tumors show a higher expression of VHL and an immunologic ‘colder’ phenotype than EGFR/ERBB2wt tumors. • ERBB2 exon 20-mutated tumors show an overexpression of RIPK1 and STK11IP and a reduction of cytotoxic natural killer cells. • Drugs targeting these alterations are potential partners of checkpoint blockade in exon 20-mutated non-small-cell lung cancer.

Lung cancer is still the leading cause of cancer death worldwide despite declining smoking prevalence in industrialised countries. Although lung cancer is highly associated with smoking status, a significant proportion of lung cancer cases develop in patients who never smoked, with an observable bias towards female never smokers. A better understanding of lung cancer heterogeneity and immune system involvement during tumour evolution and progression in never smokers is therefore highly warranted. We employed single nucleus transcriptomics of surgical lung adenocarcinoma (LADC) and normal lung tissue samples from patients with or without smoking history. Immune cells as well as fibroblasts and endothelial cells respond to tobacco smoke exposure by inducing a highly inflammatory state in normal lung tissue. In the presence of LADC, we identified differentially expressed transcriptional programmes in macrophages and cancer-associated fibroblasts, providing insight into how the niche favours tumour progression. Within tumours, we distinguished eight subpopulations of neoplastic cells in female smokers and never smokers. Through pseudotemporal ordering, we inferred a trajectory towards two differentiated tumour cell states implicated in cancer progression and invasiveness. A proliferating cell population sustaining tumour growth exhibits differential immune modulating signatures in both patient groups. Our results resolve cellular heterogeneity and immune interactions in LADC, with a special emphasis on female never smokers and implications for the design of therapeutic approaches.

OBJECTIVES Experimental studies have shown that vitamin D has an immunomodulatory effect on the innate and adaptive immune systems. Associations between vitamin D deficiency and development or progression of inflammatory bowel diseases (IBDs) are reported, but a cause-and-effect relationship between pretreatment 25 hydroxyvitamin D [25(OH)D] levels and response to anti-tumor necrosis factor-α (anti-TNF) therapy is not established. METHODS This retrospective study evaluated pediatric IBD patients who had 25(OH)D levels drawn within 3 months of initiating infliximab and/or adalimumab treatment. Demographic features, Paris classification, baseline 25(OH)D levels, disease activity, and laboratory results before and after 3 months of anti-TNF therapy were collected. The interaction between vitamin D insufficiency at induction and lack of response to anti-TNF therapy at 3 months was determined. RESULTS Of the 383 patients, 76 met inclusion criteria. Sixty-five patients (85.5%) had Crohn disease (CD) and 11 (14.5%) had ulcerative colitis. Seven patients had 25(OH)D levels obtained during both infliximab and adalimumab induction; hence 83 subjects were evaluated (infliximab: 70 patients, adalimumab: 13 patients). 25(OH)D

Introduction: Contemporary pediatric data on pouch outcomes are sparse, especially in the era of laparoscopic surgeries. We aimed to assess outcomes and predictors in children with ulcerative colitis/inflammatory bowel disease (IBD)-unclassified who underwent colectomy and ileal pouch-anal anastomosis. Methods: This was a multicenter retrospective cohort study from 17 IBD centers affiliated with the pediatric IBD Porto group of ESPGHAN. An electronic REDcap system was used to collate baseline characteristics, demographic, clinical, management and surgical data, short- and longterm outcomes, and to identify potential predictors of pouch outcome. Results: Of the 129 patients included, 86 (67%) developed pouchitis during follow-up of median 40 months (interquartile range 26–72), of whom 33 (26%) with chronic pouchitis. Patients operated on by surgeons performing

Inflammatory bowel diseases (IBD) are often treated with anti-tumor necrosis factor alpha (anti-TNFα) medications. Concomitant treatment of IBD with anti-TNFα agents and immunomodulators appears to be associated with an increased risk for lymphoma.Patients who developed lymphoma while on monotherapy with an anti-TNFα agent were identified at three centers. Institutional Review Board approval was obtained.Five adolescents and young adult patients with pediatric-onset IBD who were treated with infliximab (IFX) without exposure to thiopurines were subsequently diagnosed with lymphoma. Three of the five patients had bone involvement at presentation. Epstein-Barr virus was positive in 2 cases. Median time from diagnosis of IBD and exposure to IFX prior to diagnosis of lymphoma was 5 and 4.3 years, respectively.This case series reports long-term follow-up for young patients with IBD who were treated with IFX monotherapy and developed lymphoma. Three of the five patients had bone involvement. In general, the risk of lymphoma following exposure to anti-TNFα medications alone remains low, but the incidence of primary bone lymphomas in IBD has not been reported. Studies examining longer exposure times may be needed to determine the true lymphoma risk in patients treated with IFX monotherapy.

Gender dimorphism in autism spectrum disorders (ASD) is well known; however, the reasons for gender differences in autism are poorly understood. There are several hypotheses that might explain male prevalence in ASD including increased levels of androgens, "extreme male brain," and a combination of elevated levels of prenatal testosterone in conjunction with prenatal stress. In this review, differences in the gut microbiome and metabolome in humans and animals are described to explain gender differences in individuals with ASD, effects on behavior and social interactions and the impact of antibiotics, probiotics and fecal transplants. The bidirectional relationship between sex hormones and intestinal microbiota could also be relevant. Such interactions have been described in autoimmune diseases, but thus far are not implicated in ASD. We hypothesize that similar cross-talk exists in ASD between gut microbiota and sex hormones. Since intestinal microbiota may affect behavior, it is possible that prevalence of ASD in boys may be associated with more significant changes in the intestinal microbiome than in affected girls.

AIM To investigate the accuracy of fungal dysbiosis in mucosa and stool for predicting the diagnosis of Crohn’s disease (CD). METHODS Children were prospectively enrolled in two medical centers: one university hospital and one private gastroenterology clinic in the city of Riyadh, Kingdom of Saudi Arabia. The children with confirmed diagnosis of CD by standard guidelines were considered cases, and the others were considered non-inflammatory bowel disease controls. Mucosal and stool samples were sequenced utilizing Illumina MiSeq chemistry following the manufacturer’s protocols, and abundance and diversity of fungal taxa in mucosa and stool were analyzed. Sparse logistic regression was used to predict the diagnosis of CD. The accuracy of the classifier was tested by computing the receiver operating characteristic curves with 5-fold stratified cross-validation under 100 permutations of the training data partition and the mean area under the curve (AUC) was calculated. RESULTS All the children were Saudi nationals. There were 15 children with CD and 20 controls. The mean age was 13.9 (range: 6.7-17.8) years for CD children and 13.9 (3.25-18.6) years for controls, and 10/15 (67%) of the CD and 13/20 (65%) of the control subjects were boys. CD locations at diagnosis were ileal (L1) in 4 and colonic (L3) in 11 children, while CD behavior was non-stricturing and non-penetrating (B1) in 12 and stricturing (B2) in 3 children. The mean AUC for the fungal dysbiosis classifier was significantly higher in stools (AUC = 0.85 ± 0.057) than in mucosa (AUC = 0.71 ± 0.067) (P < 0.001). Most fungal species were significantly more depleted in stools than mucosal samples, except for Saccharomyces cerevisiae and S. bayanus, which were significantly more abundant. Diversity was significantly more reduced in stools than in mucosa. CONCLUSION We found high AUC of fungal dysbiosis in fecal samples of children with CD, suggesting high accuracy in predicting diagnosis of CD.

There is a male predominance in autism, with a male/female ratio of 4:1 and an even higher ratio (11:1) in individuals with high functioning autism. The reasons for gender differences in ASD are unknown. Genetic and environmental factors have been implicated, but no definitive evidence exists to explain male predominance. In this review, evidence is presented to support a hypothesis that the intestinal microbiota and metabolome play a role in gender dimorphism in children with autism. Metabolic products may affect not only gastrointestinal (GI) tract and the central nervous system, but also behavior, supporting communication between GI tract and central nervous system. Furthermore, mood and anxiety may affect intestinal function, indicating bidirectional flow in the gut-brain axis. Several hormone-based hypotheses are discussed to explain the prevalence of autism in males. Observations in animal models and studies in humans on the intestinal microbiome and metabolome are reviewed to support the proposed gender dimorphism hypothesis. We hypothesize that the intestinal microbiome is a contributing factor to the prevalence of ASD in boys either directly, through microbial metabolites and/or epigenetic factors capable of regulating host gene expression through DNA methylation and/or histone modification.

BACKGROUND C-reactive protein (CRP) is a serum marker that is used to measure disease activity in Crohn's disease (CD). However, a subset of CD patients have normal CRP during flares. In rheumatoid arthritis and lupus, genetic variants can restrict CRP elevations during flares. This study sought to determine if common CRP genetic variants affect CRP values during active CD. METHODS Subjects with CD who participated in the Partners HealthCare BioBank were genotyped for 5 common CRP genetic variants (rs2794520, rs3122012, rs3093077, rs2808635, and rs1800947). Medical records were reviewed to determine disease activity and the highest CRP value during active CD. CRP values during active infection or malignancy at the time of the test were excluded. CRP values were compared by genotype using the Mann-Whitney test. RESULTS The study included 199 subjects with active CD (21 to 86 years of age). Subjects with the rs2794520 TT genotype had a lower CRP than subjects with the CC genotype (58.3 mg/L vs 28.4 mg/L, P = 0.008). Subjects with the rs1800947 CG genotype had a lower CRP than those with the CC genotype (54.3 mg/L vs 22.4 mg/L, P < 0.0001); 41.6% of TT subjects had a normal CRP compared with 24.1% of CT subjects and 16.5% of CC subjects (P = 0.041). CONCLUSIONS This study demonstrates that rs2794520 and rs1800947 are associated with a restriction of CRP elevations during active CD. While CRP is typically a reliable biomarker in CD, there is a subset of CD patients with a genetically determined restriction of CRP in whom other disease markers should be utilized.

Objectives Large cell neuroendocrine carcinoma of the lung (LCNEC) is associated with an unfavorable prognosis and only few patients are eligible for surgery. In most patients, chemotherapy is recommended alone or in addition to resection. Novel immunotherapies blocking the PD-L1 pathway have been introduced into therapeutic regimens for NSCLC with great success. In order to evaluate a possible efficacy of an anti-PD-L1 therapy, we analyzed the frequency of PD-L1 expression in LCNEC. Material and methods We retrospectively reviewed data from 76 patients with LCNEC treated in our institution between 1998 and 2010. The expression of PD-L1 was examined on the tumor cells and the tumor surrounding tissue by immunohistochemistry. An expression of >1% was considered as positive. Statistical analysis was performed to determine significant predictors for survival. Results 56 of 76 patients with LCNEC were treated with a potentially therapeutic surgical approach. Tumor-specific survival (TSS) of the entire cohort was 29% at five years. 17 patients (22.3%) had PD-L1 positive tumors and 12 of these had no additional PD-L1 expression in the adjacent immune cell infiltrate. Tumor-flanking immune cells were found PD-L1 positive 28 patients; 16 of these had no additional expression on the tumor cells. The most considerable difference in survival was found when comparing patients with isolated PD-L1 expression on tumor cells and PD-L1 negative immune cell infiltrate to their counterpart (positive immune-cell infiltrate and PD-L1 negative tumor cell surface; 5-year TSS: 0% vs. 60%; p Conclusion PD-L1 expression in LCNEC was associated with poorer survival whereas PD-L1 expression in the tumor microenvironment seemed to have a beneficial effect. Therapeutic approaches have to be evaluated in future.

Background The advanced lung cancer inflammation index [ALI: body mass index × serum albumin/neutrophil-to-lymphocyte ratio (NLR)] reflects systemic host inflammation, and is easily reproducible. We hypothesized that ALI could assist guidance of non-small-cell lung cancer (NSCLC) treatment with immune checkpoint inhibitors (ICIs). Patients and methods This retrospective study included 672 stage IV NSCLC patients treated with programmed death-ligand 1 (PD-L1) inhibitors alone or in combination with chemotherapy in 25 centers in Greece and Germany, and a control cohort of 444 stage IV NSCLC patients treated with platinum-based chemotherapy without subsequent targeted or immunotherapy drugs. The association of clinical outcomes with biomarkers was analyzed with Cox regression models, including cross-validation by calculation of the Harrell's C-index. Results High ALI values (>18) were significantly associated with longer overall survival (OS) for patients receiving ICI monotherapy [hazard ratio (HR) = 0.402, P < 0.0001, n = 460], but not chemo-immunotherapy (HR = 0.624, P = 0.111, n = 212). Similar positive correlations for ALI were observed for objective response rate (36% versus 24%, P = 0.008) and time-on-treatment (HR = 0.52, P < 0.001), in case of ICI monotherapy only. In the control cohort of chemotherapy, the association between ALI and OS was weaker (HR = 0.694, P = 0.0002), and showed a significant interaction with the type of treatment (ICI monotherapy versus chemotherapy, P < 0.0001) upon combined analysis of the two cohorts. In multivariate analysis, ALI had a stronger predictive effect than NLR, PD-L1 tumor proportion score, lung immune prognostic index, and EPSILoN scores. Among patients with PD-L1 tumor proportion score ≥50% receiving first-line ICI monotherapy, a high ALI score >18 identified a subset with longer OS and time-on-treatment (median 35 and 16 months, respectively), similar to these under chemo-immunotherapy. Conclusions The ALI score is a powerful prognostic and predictive biomarker for patients with advanced NSCLC treated with PD-L1 inhibitors alone, but not in combination with chemotherapy. Its association with outcomes appears to be stronger than that of other widely used parameters. For PD-L1-high patients, an ALI score >18 could assist the selection of cases that do not need addition of chemotherapy., Highlights • ALI is prognostic and predictive for patients with advanced NSCLC treated with immunotherapy monotherapy, but not chemo-immunotherapy. • Its association with outcomes is stronger than that of other parameters (PD-L1 TPS, NLR, lung immune prognostic index, EPSILoN). • For PD-L1-high patients, an ALI score >18 could assist the selection of cases that do not need addition of chemotherapy.

Acyl-CoA:diacylglycerol acyltransferase (DGAT)1 and DGAT2 catalyze triglyceride (TG) biosynthesis in humans. Biallelic loss-of-function mutations in human DGAT1 result in severe congenital diarrhea and protein-losing enteropathy. Additionally, pharmacologic inhibition of DGAT1 led to dose-related diarrhea in human clinical trials. Here we identify a previously unknown DGAT1 mutation in identical twins of South Asian descent. These male patients developed watery diarrhea shortly after birth, with protein-losing enteropathy and failure to thrive. Exome sequencing revealed a homozygous recessive mutation in DGAT1, c.314T>C, p.L105P. We show here that the p.L105P DGAT1 enzyme produced from the mutant allele is less abundant, resulting in partial loss of TG synthesis activity and decreased formation of lipid droplets in patient-derived primary dermal fibroblasts. Thus, in contrast with complete loss-of-function alleles of DGAT1, the p.L105P missense allele partially reduces TG synthesis activity and causes a less severe clinical phenotype. Our findings add to the growing recognition of DGAT1 deficiency as a cause of congenital diarrhea with protein-losing enteropathy and indicate that DGAT1 mutations result in a spectrum of diseases.

There is evidence that symptoms of maldigestion or malabsorption in autistic individuals are related to changes in the indigenous microbiota. Analysis of colonic bacteria has revealed microbial dysbiosis in children with autism; however, characteristics of the duodenal microbiome are not well described. In the present study the microbiome of the duodenal mucosa of subjects with autism was evaluated for dysbiosis, bacteria overgrowth, and microbiota associated with carbohydrate digestion. The relationship between the duodenal microbiome and disaccharidase activity was analyzed in biopsies from 21 autistic subjects and 19 children without autism.Microbiota composition was determined by 16S ribosomal RNA gene sequencing, and disaccharidase activity via biochemical assays.Although subjects with autism had a higher frequency of constipation (P 0.005), there was no difference in disaccharidase activity between groups. In addition, no differences in microbiome diversity (species richness and evenness) were observed. Bacteria belonging to the genus Burkholderia were more abundant in subjects with autism, whereas members of the genus Neisseria were less abundant. At the species level, a relative decrease in abundance of 2 Bacteroides species and Escherichia coli was found in autistic individuals. There was a positive correlation between the abundance of Clostridium species, and disaccharidase activity, in autistic individuals.There are a variety of changes at the genus and species level in duodenal microbiota in children with autism that could be influenced by carbohydrate malabsorption. These observations could be affected by variations in individual diets, but also may represent a more pervasive dysbiosis that results in metabolites that affect the behavior of autistic children.

Background and Aims: Although increasing evidence suggests a role for fungi in inflammatory bowel disease [IBD], data are scarce and mostly from adults. Our aim was to define the characteristics of fungal microbiota in newly diagnosed treatment-naïve children with Crohn’s disease [CD]. Methods: The children referred for colonoscopy were prospectively enrolled in the study at King Khalid University Hospital, King Saud University, and Al Mofarreh Polyclinics in Riyadh. Tissue and stool samples were collected and frozen till sequencing analysis. The children with confirmed CD diagnosis were designated as cases and the others as non- IBD controls; 78 samples were collected from 35 children [15 CD and 20 controls]. Statistical analysis was performed to investigate CD associations and diversity. Results: CD-associated fungi varied with the level of phylogenetic tree. There was no significant difference in abundance between normal and inflamed mucosa. Significantly abundant CD-associated taxa included Psathyrellaceae [p = 0.01], Cortinariaceae [p = 0.04], Psathyrella [p = 0.003], and Gymnopilus [p = 0.03]. Monilinia was significantly depleted [p = 0.03], whereas other depleted taxa, although not statistically significant, included Leotiomycetes [p = 0.06], Helotiales [p = 0.08], and Sclerotiniaceae [p = 0.07]. There was no significant difference in fungal diversity between CD and controls. Conclusions: We report highly significant fungal dysbiosis in newly diagnosed treatment-naïve CD children. Depleted and more abundant taxa suggest anti-inflammatory and pro-inflamatory potentials, respectively. Further studies with larger sample size and including functional analysis are needed to clarify the significance of the fungal community in the pathogenesis of CD.