Effect of nonpharmacological interventions on nutrition status, complications and quality of life in head and neck cancer patients undergoing radiotherapy: A systematic review and meta-analysis.

PURPOSE: To evaluate the effect of nonpharmacological therapies on nutrition status, complications and quality of life in head and neck cancer patients and to provide a basis for clinical practice. METHODS: This systematic review was reported in accordance with the Preferred Reporting Items for Systematic Review and Meta-analysis statement. Ten databases were systematically searched for all available articles from construction to November 2023. Two researchers independently conducted literature screening, data extraction and quality evaluation. Cochrane Review Manager 5.3 was used for meta-analysis. RESULTS: Finally, 27 RCT studies including 2814 patients with head and neck cancer were included. Five categories of interventions were used: nutritional support, exercise, swallowing function training, psychological intervention and low-level laser therapy. Nonpharmacological interventions can improve body weight loss in patients with HNC at the end of treatment (MD: 1.66 kg; 95% CI: 0.80 to 2.51), and subgroup analysis showed that nutritional support, psychological intervention and low-level laser therapy were effective. Nonpharmacological interventions can also ameliorate decreases in BMI (MD: 0.71; 95% CI: 0.16 to 1.26) and reduce the incidence of malnutrition (RR: 0.76; 95% CI: 0.67 to 0.86), oral mucositis (RR: 0.54; 95% CI: 0.37 to 0.80) and gastrointestinal complications (RR: 0.61; 95% CI: 0.38 to 0.96) during radiotherapy; however, no significant differences were found in other complications and quality of life. CONCLUSION: Nonpharmacological interventions can improve the nutrition status of patients with head and neck cancer and reduce the incidence of severe oral mucositis and gastrointestinal complications during radiotherapy but have no significant impact on quality of life.

Optimal time to initiate early oral feeding in postoperative patients with upper gastrointestinal malignancy: A network meta-analysis.

BACKGROUND: With the development of enhanced recovery after surgery, early oral feeding is likely to become the preferred mode of nutrition after surgery for upper gastrointestinal tract malignancies. However, the optimal time to initiate early oral feeding remains unknown. OBJECTIVE: We aimed to compare the effects of different introduction times of early oral feeding in patients with upper gastrointestinal malignancies in terms of safety, tolerance, and effectiveness and to identify the optimal time for early oral feeding after surgery. METHODS: A random-effects meta-analysis was performed to identify evidence from relevant randomized controlled trials. Ten electronic databases were searched for randomized controlled trials from their earliest records to May 2023. Data were analyzed using the Stata 16.0 software. RESULTS: A total of 22 randomized controlled trials including 2510 patients and seven time points for oral feeding after surgery were considered. Regarding safety, oral feeding initiated on postoperative day 3 may be the safest (high-quality evidence) compared with other times. Regarding tolerance, oral feeding initiated on postoperative day 5 may be the most well-tolerated (moderate-quality evidence) compared with other times. Regarding effectiveness, oral feeding initiated on postoperative day 3 may be the most effective (moderate-quality evidence) compared with other times. CONCLUSIONS: Early oral feeding is safe, tolerable, and effective in postoperative patients with upper gastrointestinal malignancies. The optimal time to initiate early oral feeding after surgery was most likely postoperative day 3. The results of this meta-analysis provide evidence-based guidelines for clinical decision-making.

Structure Evolution and Properties Modification for Reaction-Bonded Silicon Carbide.

Complex structure reaction-bonded silicon carbide (RB-SiC) can be prepared by reactive melt infiltration (RMI) and digital light processing (DLP). However, the strength and modulus of RB-SiC prepared by DLP are not sufficient, due to its low solid content (around 40 vol.%), compared with the traditional fabrication techniques (solid content > 60 vol.%). With this understanding, a new method to improve the properties of RB-SiC was proposed, by the impregnation of composite precursor into the porous preform. The composite precursor was composed of phenolic (PF) resin and furfuryl alcohol (FA). PF and FA were pyrolyzed at 1850 °C to obtain amorphous carbon and graphite into the porous preform, respectively. The effects of multiphase carbon on the microstructure and performance of RB-SiC was studied. When the mass ratio of PF to FA was 1/4, the solid content of RB-SiC increased from 40 vol.% to 68.6 vol.%. The strength, bulk density and modulus were 323.12 MPa, 2.94 g/cm3 and 348.83 Gpa, respectively. This method demonstrated that the reaction process between liquid Si and carbon could be controlled by the introduction of multiphase carbon into the porous preforms, which has the potential to regulate the microstructure and properties of RB-SiC prepared by additive manufacturing or other forming methods.

Effectiveness and Nephrotoxicity of Intravenous Polymyxin B in Carbapenem-Resistant Gram-Negative Bacterial Infections Among Chinese Children.

Background: No clinical study on the use of polymyxin B in Chinese children has been reported, thus making it difficult for pediatric clinicians to rationally select these drugs. Methods: A retrospective analysis of children treated with polymyxin B during hospitalization in a hospital from June 2019 to June 2021 was conducted to analyze its effectiveness and the incidence of acute kidney injury (AKI) during treatment with polymyxin B. Results: A total of 55 children were included in this study, and the results showed that the intravenous polymyxin B-based regimen had an effective rate of 52.7% in the treatment of Carbapenem-resistant Gram-negative bacterial (CR-GNB) infection in children. The results of the subgroup analysis showed that the course of treatment was longer in the favorable clinical response group than in the unfavorable outcome group (p = 0.027) and that electrolyte disturbances in children during the course of treatment could lead to unfavorable clinical outcomes (p = 0.042). The risk of incidence of AKI during treatment was 27.3%, and the all-cause mortality rate in the children on their discharge from the hospital was 7.3%. Conclusion: Polymyxin B can be used as a salvage therapy for CR-GNB infection in children when no other susceptible antibiotics are available, and the monitoring of kidney function should be strengthened.

Microstructure Evolution and Performance Improvement of Silicon Carbide Ceramics via Impregnation Method.

The high topological silicon carbide (SiC) ceramics can be prepared by stereolithography (SLA) combined with liquid silicon infiltration (LSI) techniques. This paper aims to enhance the performance of SiC ceramics prepared by SLA and LSI techniques via the cyclic impregnation/carbonization of the precursor of carbon source solution before LSI. The effects of impregnation/carbonization cycles on the microstructure and properties of C/SiC preform and sintered body were analyzed in detail. The results show that, with the increase of impregnation/carbonization cycles, the porosity in the C/SiC preform decreases obviously and the content of secondary SiC in the sintered body increases effectively. Especially, when the impregnation/carbonization cycle was performed twice, the sintered body had the optimal mechanical properties. The value of flexural strength, bulk density and elastic modulus were 258.63 ± 8.33 MPa, 2.95 ± 0.02 g/cm3 and 425.16 ± 14.15 GPa, respectively. In addition, the thermal dimensional stability of sintered body was also improved by this method. This method proves that SiC ceramics prepared by SLA combined with LSI have the potential of applications in space optical mirrors.

ARPC1B binds WASP to control actin polymerization and curtail tonic signaling in B cells.

Immune cells exhibit low-level, constitutive signaling at rest (tonic signaling). Such tonic signals are required for fundamental processes, including the survival of B lymphocytes, but when they are elevated by genetic or environmental causes, they can lead to autoimmunity. Events that control ongoing signal transduction are, therefore, tightly regulated by submembrane cytoskeletal polymers like F-actin. The actin-binding proteins that underpin the process, however, are poorly described. By investigating patients with ARPC1B deficiency, we report that ARPC1B-containing ARP2/3 complexes are stimulated by Wiskott Aldrich Syndrome protein (WASP) to nucleate the branched actin networks that control tonic signaling from the B cell receptor (BCR). Despite an upregulation of ARPC1A, ARPC1B-deficient cells were not capable of WASP-mediated nucleation by ARP2/3, and this caused the loss of WASP-dependent structures, including podosomes in macrophages and lamellipodia in B cells. In the B cell compartment, ARPC1B deficiency also led to weakening of the cortical F-actin cytoskeleton that normally curtails the diffusion of BCRs and ultimately resulted in increased tonic lipid signaling, oscillatory calcium release from the endoplasmic reticulum (ER), and phosphorylated Akt. These events contributed to skewing the threshold for B cell activation in response to microbial-associated molecular patterns (MAMPs). Thus, ARPC1B is critical for ARP2/3 complexes to control steady-state signaling of immune cells.

Perfluorooctanoic acid inhibits the maturation rate of mouse oocytes cultured in vitro by triggering mitochondrial and DNA damage.

BACKGROUND: Perfluorooctanoic acid (PFOA) is widely used in the manufacture of household and industrial products. It has certain toxicity and leaves many residues in the environment. Numerous studies have shown that PFOA exhibits endocrine disrupting properties and immunotoxicity and induces developmental defects. However, there is very little information regarding its toxicity on oocytes. METHODS: We cultured denuded oocytes in maturation medium supplemented with 0, 300, or 500 PFOA during IVM and evaluated the maturation of oocytes from the aspects of ROS(DCFH-DA), mitochondria(MitoOrange and JC-1), DNA damage(P-H2AX), and cytoskeleton(ß-tubulin). RESULTS: Compared with the control group, the PFOA treatment group exhibited significantly reduced proportion of oocytes matutation. Furthermore, the DCFH-DA test showed that PFOA significantly increased reactive oxygen species (ROS) levels. PFOA disrupted mitochondrial distribution and decreased mitochondrial function as assessed using MitoOrange and JC-1. In addition, PFOA-treated oocytes exhibited a significantly higher percentage of P-H2AX, defective ß-tubulin, abnormal chromosome alignment, lower expression of the anti-apoptotic gene Bcl-2, and higher expression of the apoptotic genes caspase3 and Bax. CONCLUSION: In summary, PFOA could negatively and directly affect oocyte maturation in vitro and cause oxidative stress, mitochondrial function disruption, DNA damage, cytoskeleton damage, and apoptosis.

Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice.

Spleen tyrosine kinase (SYK) is a critical immune signaling molecule and therapeutic target. We identified damaging monoallelic SYK variants in six patients with immune deficiency, multi-organ inflammatory disease such as colitis, arthritis and dermatitis, and diffuse large B cell lymphomas. The SYK variants increased phosphorylation and enhanced downstream signaling, indicating gain of function. A knock-in (SYK-Ser544Tyr) mouse model of a patient variant (p.Ser550Tyr) recapitulated aspects of the human disease that could be partially treated with a SYK inhibitor or transplantation of bone marrow from wild-type mice. Our studies demonstrate that SYK gain-of-function variants result in a potentially treatable form of inflammatory disease.

Novel CARMIL2 loss-of-function variants are associated with pediatric inflammatory bowel disease.

CARMIL2 is required for CD28-mediated co-stimulation of NF-κB signaling in T cells and its deficiency has been associated with primary immunodeficiency and, recently, very early onset inflammatory bowel disease (IBD). Here we describe the identification of novel biallelic CARMIL2 variants in three patients presenting with pediatric-onset IBD and in one with autoimmune polyendocrine syndrome (APS). None manifested overt clinical signs of immunodeficiency before their diagnosis. The first patient presented with very early onset IBD. His brother was found homozygous for the same CARMIL2 null variant and diagnosed with APS. Two other IBD patients were found homozygous for a nonsense and a missense CARMIL2 variant, respectively, and they both experienced a complicated postoperative course marked by severe infections. Immunostaining of bowel biopsies showed reduced CARMIL2 expression in all the three patients with IBD. Western blot and immunofluorescence of transfected cells revealed an altered expression pattern of the missense variant. Our work expands the genotypic and phenotypic spectrum of CARMIL2 deficiency, which can present with either IBD or APS, aside from classic immunodeficiency manifestations. CARMIL2 should be included in the diagnostic work-up of patients with suspected monogenic IBD.

Analysis of differentially abundant proteins related to boar fertility in seminal plasma using iTRAQ-based quantitative proteomics.

Animal fertility is one of the most important characteristics for the livestock breeding industry. Conventional semen analysis provides basic information on sperm quality, but the predictive value of such analysis with regard to fertility remains questionable. Therefore, it is important to determine and predict male fertility more accurately in the clinic. To identify seminal plasma proteins involved in fertility, isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography with tandem mass spectrometry (quantitative proteomic analysis) were used to identify differentially abundant proteins (DAPs) in seminal plasma between high- and low-reproductive-efficiency Landrace boars. A total of 141 DAPs were identified, of which 125 upregulated and 16 downregulated proteins were subjected to bioinformatics analysis. These DAPs were found to be mainly involved in proteolysis, ATP binding, and energy metabolism. We investigated the relevance of three DAPs-ceruloplasmin, carboxypeptidase E (CPE), and serpin family A member 12 (SERPINA12)-in an in vitro fertility assay. This assay revealed that the inhibition of these proteins with antibodies can reduce or increase the fertilization rate. These results indicate possible biomarkers for the selection of high-fertility boars and provide a theoretical basis for the use of protein biomarkers in the livestock breeding industry. SIGNIFICANCE: Our study identified differentially abundant proteins in the seminal plasma of high-reproductive-efficiency and low-reproductive-efficiency Landrace boars. These proteins may be used as biomarkers to screen out high-fertility boars. The study can provide not only a new method for improving the effects of artificial insemination and reproductive efficiency of boars but also an important reference for boar breeding. Meanwhile, because pigs and humans have similar physiological parameters and organ sizes, our findings can also serve as a reference for human reproduction research.

The E3 ubiquitin ligase UBR5 interacts with TTC7A and may be associated with very early onset inflammatory bowel disease.

Very early onset inflammatory bowel disease (VEOIBD) denotes children with onset of IBD before six years of age. A number of monogenic disorders are associated with VEOIBD including tetratricopeptide repeat domain 7A (TTC7A) deficiency. TTC7A-deficiency is characterized by apoptotic colitis in milder cases with severe intestinal atresia and immunodeficiency in cases with complete loss of protein. We used whole exome sequencing in a VEOIBD patient presenting with colitis characterized by colonic apoptosis and no identified known VEOIBD variants, to identify compound heterozygous deleterious variants in the Ubiquitin protein ligase E3 component N-recognin 5 (UBR5) gene. Functional studies demonstrated that UBR5 co-immunoprecipitates with the TTC7A and the UBR5 variants had reduced interaction between UBR5 and TTC7A. Together this implicates UBR5 in regulating TTC7A signaling in VEOIBD patients with apoptotic colitis.

Drug Screen Identifies Leflunomide for Treatment of Inflammatory Bowel Disease Caused by TTC7A Deficiency.

BACKGROUND & AIMS: Mutations in the tetratricopeptide repeat domain 7A gene (TTC7A) cause intestinal epithelial and immune defects. Patients can become immune deficient and develop apoptotic enterocolitis, multiple intestinal atresia, and recurrent intestinal stenosis. The intestinal disease in patients with TTC7A deficiency is severe and untreatable, and it recurs despite resection or allogeneic hematopoietic stem cell transplant. We screened drugs for those that prevent apoptosis of in cells with TTC7A deficiency and tested their effects in an animal model of the disease. METHODS: We developed a high-throughput screen to identify compounds approved by the US Food and Drug Administration that reduce activity of caspases 3 and 7 in TTC7A-knockout (TTC7A-KO) HAP1 (human haploid) cells and reduce the susceptibility to apoptosis. We validated the effects of identified agents in HeLa cells that stably express TTC7A with point mutations found in patients. Signaling pathways in cells were analyzed by immunoblots. We tested the effects of identified agents in zebrafish with disruption of ttc7a, which develop intestinal defects, and colonoids derived from biopsy samples of patients with and without mutations in TTC7A. We performed real-time imaging of intestinal peristalsis in zebrafish and histologic analyses of intestinal tissues from patients and zebrafish. Colonoids were analyzed by immunofluorescence and for ion transport. RESULTS: TTC7A-KO HAP1 cells have abnormal morphology and undergo apoptosis, due to increased levels of active caspases 3 and 7. We identified drugs that increased cell viability; leflunomide (used to treat patients with inflammatory conditions) reduced caspase 3 and 7 activity in cells by 96%. TTC7A-KO cells contained cleaved caspase 3 and had reduced levels of phosphorylated AKT and X-linked inhibitor of apoptosis (XIAP); incubation of these cells with leflunomide increased levels of phosphorylated AKT and XIAP and reduced levels of cleaved caspase 3. Administration of leflunomide to ttc7a-/- zebrafish increased gut motility, reduced intestinal tract narrowing, increased intestinal cell survival, increased sizes of intestinal luminal spaces, and restored villi and goblet cell morphology. Exposure of patient-derived colonoids to leflunomide increased cell survival, polarity, and transport function. CONCLUSIONS: In a drug screen, we identified leflunomide as an agent that reduces apoptosis and activates AKT signaling in TTC7A-KO cells. In zebrafish with disruption of ttc7a, leflunomide restores gut motility, reduces intestinal tract narrowing, and increases intestinal cell survival. This drug might be repurposed for treatment of TTC7A deficiency.

MEN1 Tumor Suppressor Gene is Required for Long-term Memory Formation in an Aversive Operant Conditioning Model of Lymnaea stagnalis.

Activity-dependent transcription factors critically coordinate the gene expression program underlying memory formation. The tumor suppressor gene, MEN1, encodes a ubiquitously expressed transcription regulator required for synaptogenesis and synaptic plasticity in invertebrate and vertebrate central neurons. In this study, we investigated the role of MEN1 in long-term memory (LTM) formation in an aversive operant conditioning paradigm in the freshwater pond snail Lymnaea stagnalis (L. stagnalis). We demonstrated that LTM formation is associated with an increased expression of MEN1 coinciding with an up-regulation of creb1 gene expression. In vivo knockdown of MEN1 prevented LTM formation and conditioning-induced changes in neuronal activity in the identified pacemaker neuron RPeD1. Our findings suggest the involvement of a new pathway in LTM consolidation that requires MEN1-mediated gene regulation.

Variants in TRIM22 That Affect NOD2 Signaling Are Associated With Very-Early-Onset Inflammatory Bowel Disease.

BACKGROUND & AIMS: Severe forms of inflammatory bowel disease (IBD) that develop in very young children can be caused by variants in a single gene. We performed whole-exome sequence (WES) analysis to identify genetic factors that might cause granulomatous colitis and severe perianal disease, with recurrent bacterial and viral infections, in an infant of consanguineous parents. METHODS: We performed targeted WES analysis of DNA collected from the patient and her parents. We validated our findings by a similar analysis of DNA from 150 patients with very-early-onset IBD not associated with known genetic factors analyzed in Toronto, Oxford, and Munich. We compared gene expression signatures in inflamed vs noninflamed intestinal and rectal tissues collected from patients with treatment-resistant Crohn's disease who participated in a trial of ustekinumab. We performed functional studies of identified variants in primary cells from patients and cell culture. RESULTS: We identified a homozygous variant in the tripartite motif containing 22 gene (TRIM22) of the patient, as well as in 2 patients with a disease similar phenotype. Functional studies showed that the variant disrupted the ability of TRIM22 to regulate nucleotide binding oligomerization domain containing 2 (NOD2)-dependent activation of interferon-beta signaling and nuclear factor-κB. Computational studies demonstrated a correlation between the TRIM22-NOD2 network and signaling pathways and genetic factors associated very early onset and adult-onset IBD. TRIM22 is also associated with antiviral and mycobacterial effectors and markers of inflammation, such as fecal calprotectin, C-reactive protein, and Crohn's disease activity index scores. CONCLUSIONS: In WES and targeted exome sequence analyses of an infant with severe IBD characterized by granulomatous colitis and severe perianal disease, we identified a homozygous variant of TRIM22 that affects the ability of its product to regulate NOD2. Combined computational and functional studies showed that the TRIM22-NOD2 network regulates antiviral and antibacterial signaling pathways that contribute to inflammation. Further study of this network could lead to new disease markers and therapeutic targets for patients with very early and adult-onset IBD.

Defects in NADPH Oxidase Genes NOX1 and DUOX2 in Very Early Onset Inflammatory Bowel Disease.

BACKGROUND & AIMS: Defects in intestinal innate defense systems predispose patients to inflammatory bowel disease (IBD). Reactive oxygen species (ROS) generated by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases in the mucosal barrier maintain gut homeostasis and defend against pathogenic attack. We hypothesized that molecular genetic defects in intestinal NADPH oxidases might be present in children with IBD. METHODS: After targeted exome sequencing of epithelial NADPH oxidases NOX1 and DUOX2 on 209 children with very early onset inflammatory bowel disease (VEOIBD), the identified mutations were validated using Sanger Sequencing. A structural analysis of NOX1 and DUOX2 variants was performed by homology in silico modeling. The functional characterization included ROS generation in model cell lines and in in vivo transduced murine crypts, protein expression, intracellular localization, and cell-based infection studies with the enteric pathogens Campylobacter jejuni and enteropathogenic Escherichia coli. RESULTS: We identified missense mutations in NOX1 (c.988G>A, p.Pro330Ser; c.967G>A, p.Asp360Asn) and DUOX2 (c.4474G>A, p.Arg1211Cys; c.3631C>T, p.Arg1492Cys) in 5 of 209 VEOIBD patients. The NOX1 p.Asp360Asn variant was replicated in a male Ashkenazi Jewish ulcerative colitis cohort. All NOX1 and DUOX2 variants showed reduced ROS production compared with wild-type enzymes. Despite appropriate cellular localization and comparable pathogen-stimulated translocation of altered oxidases, cells harboring NOX1 or DUOX2 variants had defective host resistance to infection with C. jejuni. CONCLUSIONS: This study identifies the first inactivating missense variants in NOX1 and DUOX2 associated with VEOIBD. Defective ROS production from intestinal epithelial cells constitutes a risk factor for developing VEOIBD.

Mutations in Plasmalemma Vesicle Associated Protein Result in Sieving Protein-Losing Enteropathy Characterized by Hypoproteinemia, Hypoalbuminemia, and Hypertriglyceridemia.

BACKGROUND & AIMS METHODS: Severe intestinal diseases observed in very young children are often the result of monogenic defects. We used whole exome sequencing (WES) to examine the genetic cause in a patient with a distinct severe form of protein losing enteropathy (PLE) characterized by hypoproteinemia, hypoalbuminemia, and hypertriglyceridemia. METHODS: WES was performed at the Centre for Applied Genomics, Hospital for Sick Children, Toronto, Canada. Exome library preparation was performed using the Ion Torrent AmpliSeq RDY Exome Kit. Functional studies were carried out based on the identified mutation. RESULTS: Using whole exome sequencing we identified a homozygous nonsense mutation (1072C>T; p.Arg358*) in the PLVAP (plasmalemma vesicle associated protein) gene in an infant from consanguineous parents who died at five months of age of severe protein losing enteropathy. Functional studies determined that the mutated PLVAP mRNA and protein were not expressed in the patient biopsy tissues, presumably secondary to nonsense-mediated mRNA decay. Pathological analysis showed that the loss of PLVAP resulted in disruption of endothelial fenestrated diaphragms. CONCLUSIONS: PLVAP p.Arg358* mutation resulted in loss of PLVAP expression with subsequent deletion of the diaphragms of endothelial fenestrae leading to plasma protein extravasation, protein-losing enteropathy and ultimately death.

Variants in nicotinamide adenine dinucleotide phosphate oxidase complex components determine susceptibility to very early onset inflammatory bowel disease.

BACKGROUND & AIMS: The colitis observed in patients with very early onset inflammatory bowel disease (VEOIBD; defined as onset of disease at younger than 6 years of age) often resembles that of chronic granulomatous disease (CGD) in extent and features of colonic inflammation observed by endoscopy and histology. CGD is a severe immunodeficiency caused by defects in the genes that encode components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. We investigated whether variants in genes that encode NADPH oxidase components affect susceptibility to VEOIBD using independent approaches. METHODS: We performed targeted exome sequencing of genes that encode components of NADPH oxidases (cytochrome b light chain and encodes p22(phox) protein; cytochrome b-245 or NADPH oxidase 2, and encodes Nox2 or gp91(phox); neutrophil cytosol factor 1 and encodes p47 (phox) protein; neutrophil cytosol factor 2 and encodes p67 (phox) protein; neutrophil cytosol factor 4 and encodes p40 (phox) protein; and Ras-related C3 botulinum toxin substrate 1 and 2) in 122 patients with VEOIBD diagnosed at The Hospital for Sick Children, University of Toronto, from 1994 through 2012. Gene variants were validated in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients with VEOIBD. In a second approach, we examined Tag single nucleotide polymorphisms in a subset of patients with VEOIBD in which the NOX2 NADPH oxidase genes sequence had been previously analyzed. We then looked for single nucleotide polymorphisms associated with the disease in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients. We analyzed the functional effects of variants associated with VEOIBD. RESULTS: Targeted exome sequencing and Tag single nucleotide polymorphism genotyping identified 11 variants associated with VEOIBD; the majority of patients were heterozygous for these variants. Expression of these variants in cells either reduced oxidative burst or altered interactions among proteins in the NADPH oxidase complex. Variants in the noncoding regulatory and splicing elements resulted in reduced levels of proteins, or expression of altered forms of the proteins, in blood cells from VEOIBD patients. CONCLUSIONS: We found that VEOIBD patients carry heterozygous functional hypomorphic variants in components of the NOX2 NADPH oxidase complex. These do not cause overt immunodeficiency, but instead determine susceptibility to VEOIBD. Specific approaches might be developed to treat individual patients based on their genetic variant.

Mutations in tetratricopeptide repeat domain 7A result in a severe form of very early onset inflammatory bowel disease.

BACKGROUND & AIMS: Very early onset inflammatory bowel diseases (VEOIBD), including infant disorders, are a diverse group of diseases found in children younger than 6 years of age. They have been associated with several gene variants. Our aim was to identify the genes that cause VEOIBD. METHODS: We performed whole exome sequencing of DNA from 1 infant with severe enterocolitis and her parents. Candidate gene mutations were validated in 40 pediatric patients and functional studies were carried out using intestinal samples and human intestinal cell lines. RESULTS: We identified compound heterozygote mutations in the Tetratricopeptide repeat domain 7 (TTC7A) gene in an infant from non-consanguineous parents with severe exfoliative apoptotic enterocolitis; we also detected TTC7A mutations in 2 unrelated families, each with 2 affected siblings. TTC7A interacts with EFR3 homolog B to regulate phosphatidylinositol 4-kinase at the plasma membrane. Functional studies demonstrated that TTC7A is expressed in human enterocytes. The mutations we identified in TTC7A result in either mislocalization or reduced expression of TTC7A. Phosphatidylinositol 4-kinase was found to co-immunoprecipitate with TTC7A; the identified TTC7A mutations reduced this binding. Knockdown of TTC7A in human intestinal-like cell lines reduced their adhesion, increased apoptosis, and decreased production of phosphatidylinositol 4-phosphate. CONCLUSIONS: In a genetic analysis, we identified loss of function mutations in TTC7A in 5 infants with VEOIBD. Functional studies demonstrated that the mutations cause defects in enterocytes and T cells that lead to severe apoptotic enterocolitis. Defects in the phosphatidylinositol 4-kinase-TTC7A-EFR3 homolog B pathway are involved in the pathogenesis of VEOIBD.

Higher activity of the inducible nitric oxide synthase contributes to very early onset inflammatory bowel disease.

OBJECTIVES: The NOS2 gene encodes for the inducible nitric oxide synthase (iNOS), responsible for nitric oxide (NO) production, which contributes to antimicrobial and antipathogenic activities. Higher levels of both iNOS and NO-induced damage have been observed in inflammatory bowel disease (IBD) patients. NOS2 may have a role in a specific subset of IBD patients with severe and/or extensive colitis. Therefore, the aim of this study is to examine the role of NOS2 in such a subset, very early onset IBD (VEO-IBD). METHODS: Seventeen tag single nucleotide polymorphisms (SNPs) in the NOS2 gene were successfully genotyped in VEO-IBD patients. Genetic associations were replicated in an independent VEO-IBD cohort. Functional analysis for iNOS activity was performed on the most significantly associated functional variant. RESULTS: The NOS2 rs2297518 SNP was found to be associated in VEO-IBD in two independent cohorts. Upon combined analysis, a coding variant (S608L) showed the strongest association with VEO-IBD (Pcombined=1.13 × 10(-6), OR (odds ratio)=3.398 (95% CI (confidence interval) 2.02-5.717)) as well as associations with VEO-Crohn's disease and VEO-ulcerative colitis (UC). This variant also showed an association with UC diagnosed between 11 and 17 years of age but not with adult-onset IBD (>17 years). B-cell lymphoblastoid cell lines genotyped for the risk variant as well as Henle-407 cells transfected with a plasmid construct with the risk variant showed higher NO production. Colonic biopsies of VEO-IBD patients showed higher immunohistochemical staining of nitrotyrosine, indicating more nitrosative stress and tissue damage. CONCLUSIONS: These studies suggest the importance of iNOS in genetic susceptibility to younger IBD presentation due to higher NO production.