Sandhia Naik

Mutations in a Sar1 GTPase of COPII vesicles are associated with lipid absorption disorders.

Dietary fat is an important source of nutrition. Here we identify eight mutations in SARA2 that are associated with three severe disorders of fat malabsorption. The Sar1 family of proteins initiates the intracellular transport of proteins in COPII (coat protein)-coated vesicles. Our data suggest that chylomicrons, which vastly exceed the size of typical COPII vesicles, are selectively recruited by the COPII machinery for transport through the secretory pathways of the cell.

Absence of Toll-like receptor 4 explains endotoxin hyporesponsiveness in human intestinal epithelium

Background The Toll protein in Drosophila regulates dorsal ventral patterning during embryogenesis, and participates in antibacterial and antifungal host defense. Mammalian homologues are termed Toll-like receptors and, to date, nine have been cloned (TLR1–9) in humans. They are characterized by extracellular leucine-rich repeats and a cytoplasmic domain similar to the interleukin 1 receptor. Both TLR2 and TLR4 recognize various bacterial cell wall components including lipopolysaccharide (LPS). This results in the activation of the NF&kgr;B pathway. Peripheral blood mononuclear cells (PBMCs) express both TLR2 and TLR4. The authors hypothesized that the expression of TLR 2 and TLR4 in human intestinal epithelial cells differs from PBMCs because of the abundance of LPS in the intestinal lumen. Methods Epithelial cells were isolated from Caco-2 cells, fetal gut explants, and small bowel resection specimens using Hanks/ethylenediamine tetraacetic acid solution. PBMCs were used as positive controls. Ribonucleic acid (RNA) was isolated using the TRIzol method. Standard reverse transcription–polymerase chain reaction examined TLR2 and TLR4 messenger RNA (mRNA) expression. NF&kgr;B expression was determined using a luciferase reporter assay. Results TLR2 mRNA was highly expressed in PBMCs and was present in all human intestinal epithelial cells. TLR4 mRNA was detected only in PBMCs. TLR4 is not present in epithelium from children with inflammatory bowel disease. In Caco-2 cells, significant NF&kgr;B activation in response to LPS occurred only in the presence of TLR4 introduced by complementary deoxyribonucleic acid transfection. Conclusion Absence of TLR4 is associated with endotoxin hyporesponsiveness of intestinal epithelial cells. TLR4 is not directly involved in inflammation of the intestinal epithelium. Although TLR2 is normally present in the epithelial cell, it plays a limited role in inflammation. It may be activated during conditions in which bacterial cell wall concentrations within the intestine are pathologically high.

Preserved T-Cell Function in Children and Young Adults With Immune-Tolerant Chronic Hepatitis B

BACKGROUND & AIMS Chronic hepatitis B (CHB) infection acquired perinatally or in early childhood has been associated with a prolonged phase of immune tolerance from viral exposure into early adulthood. The immune-tolerant phase of the disease is characterized by high levels of hepatitis B virus (HBV) DNA and normal liver biochemistry, with minimal or no fibrosis. We investigated whether the age of patients with CHB affects their antiviral immunity and whether children and young adults have a veritable state of immunologic tolerance. METHODS We isolated T cells from different age groups of patients with CHB and used flow cytometric methods to measure production of effector and inflammatory cytokines (interferon, tumor necrosis factor, interleukin [IL]-17A, IL-22, and IL-8), T-helper (Th)2 cytokines (IL-10, IL-4), Th1 cytokines (IL-2 and IL-21), and the CC chemokine CCL3 (MIP-1). We also measured markers of T-cell exhaustion or inhibition (PD-1, LAG-3, TIM3, LAIR-1, and CTLA-4) and HBV-specific T cells. RESULTS Young patients with CHB have a Th1-cell cytokine profile and a partial profile of T-cell exhaustion. Direct quantification of the HBV-specific T-cell response showed that young patients with CHB have more HBV-specific T cells with the ability to proliferate and produce cytokines than adult patients with CHB. CONCLUSIONS HBV infection in younger patients is not associated with an immune profile of T-cell tolerance. On the contrary, children and young adults with chronic HBV infection have an HBV-specific immune profile that is less compromised than that observed in older patients.

Prevalence and management of anemia in children, adolescents, and adults with inflammatory bowel disease.

Background: Children and adolescents with inflammatory bowel disease (IBD) are more likely to have Crohns disease (CD) than ulcerative colitis (UC) and their disease tends to be more extensive and severe than in adults. We hypothesized that the prevalence of anemia would therefore be greater in children and adolescents than in adults attending IBD outpatient clinics. Methods: Using the WHO age‐adjusted definitions of anemia we assessed the prevalence, severity, type, and response to treatment of anemia in patients attending pediatric, adolescent, and adult IBD clinics at our hospital. Results: The prevalence of anemia was 70% (41/59) in children, 42% (24/54) in adolescents, and 40% (49/124) in adults (P < 0.01). Overall, children (88% [36/41]) and adolescents (83% [20/24]) were more often iron‐deficient than adults (55% [27/49]) (P < 0.01). Multivariate logistic regression showed that both active disease (odds ratio [OR], 4.7 95% confidence interval [CI], 2.5, 8.8) and attending the pediatric clinic (OR 3.7; 95% CI, 1.6, 8.4) but not the adolescent clinic predicted iron deficiency anemia. Fewer iron‐deficient children (13% [5/36]) than adolescents (30% [6/20]) or adults (48% [13/27]) had been given oral iron (P < 0.05); none had received intravenous iron compared with 30% (6/20) adolescents and 41% (11/27) adults (P < 0.0001). Conclusions: Anemia is even more common in children than in older IBD patients. Oral iron was given to half of adolescents and adults but, despite similar tolerance and efficacy, only a quarter of children with iron‐deficient anemia. Reasons for the apparent underutilization of iron therapy include a perceived lack of benefit and concerns about side effects, including worsening of IBD activity. (Inflamm Bowel Dis 2012;)

Inhibition of NF-κB Signaling in Human Dendritic Cells by the Enteropathogenic Escherichia coli Effector Protein NleE

Intestinal dendritic cells (DCs) send processes between epithelial cells into the gut lumen to sample pathogens. Noninvasive enteropathogenic Escherichia coli (EPEC) colonize the gut using a type three secretion system (T3SS) to inject effector proteins into epithelial cells. We hypothesized that EPEC might also inject proteins into DC processes to dampen immune recognition. Using a T3SS-linked fluorescence resonance energy transfer-based system we show that EPEC injects effectors into in vitro grown human myeloid DCs. Injected cells emit a blue signal due to cleavage of the green fluorescence resonance energy transfer-based substrate CCF2/AM by β-lactamase. When cultured with a mutant EPEC unable to translocate effector proteins, myeloid DCs show rapid activation of NF-κB, secrete large amounts of proinflammatory cytokines and increase expression of CD80, CD83, and CD86, whereas wild-type EPEC barely elicits cytokine production and shuts off nuclear translocation of NF-κB p65. By deleting effector protein genes, we identified NleE as being critical for this effect. Expression of NleE in HeLa cells completely prevented nuclear p65 accumulation in response to IL1-β, and luciferase production in an NF-κB reporter cell line. DCs cocultured with wild-type EPEC or NleE-complemented strains were less potent at inducing MLR. EPEC was also able to inject effectors into DCs sending processes through model gut epithelium in a transwell system and into Peyer’s patch myeloid DCs. Thus, EPEC translocate effectors into human DCs to dampen the inflammatory response elicited by its own pathogen-associated molecular patterns.

HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant

BACKGROUND & AIMS Chronic infection with hepatitis B virus (HBV) progresses through different phases. The first, called the immune-tolerant phase, has been associated with a lack of disease activity. We examined HBV-DNA integration, clonal hepatocyte expansion, HBV antigen expression, and HBV-specific immune responses in patients in the immune-tolerant phase to assess whether this designation is appropriate or if there is evidence of disease activity. METHODS We studied HBV-DNA integration, clonal hepatocyte expansion, and expression of hepatitis B surface antigen and core antigen in liver tissues from 26 patients with chronic HBV infection (ages, 14-39 y); 9 patients were positive for hepatitis B e antigen (HBeAg) in the immune-tolerant phase and were matched for age with 10 HBeAg-positive patients with active disease and 7 HBeAg-negative patients with active disease. Peripheral blood samples were collected and HBV-specific T cells were quantified for each group. RESULTS Detection of HBV antigens differed among groups. However, unexpectedly high numbers of HBV-DNA integrations, randomly distributed among chromosomes, were detected in all groups. Clonal hepatocyte expansion in patients considered immune tolerant also was greater than expected, potentially in response to hepatocyte turnover mediated by HBV-specific T cells, which were detected in peripheral blood cells from patients in all phases of infection. CONCLUSIONS We measured HBV-specific T cells, HBV-DNA integration, and clonal hepatocyte expansion in different disease phases of young patients with chronic hepatitis B, with emphasis on the so-called immune-tolerant phase. A high level of HBV-DNA integration and clonal hepatocyte expansion in patients considered immune tolerant indicated that hepatocarcinogenesis could be underway-even in patients with early stage chronic HBV infection. Our findings do not support the concepts that this phase is devoid of markers of disease progression or that an immune response has not been initiated. We propose that this early phase be called a high-replication, low-inflammation stage. The timing of therapeutic interventions to minimize further genetic damage to the hepatocyte population should be reconsidered.

Optimal assessment of paediatric IBD with MRI and barium follow-through.

Objectives: The present UK criterion standard for assessing children with suspected inflammatory bowel disease (IBD) is upper endoscopy, ileocolonoscopy, and barium follow-through (BaFT). Significant doses of radiation, unpalatable contrast, and volume intolerance are involved with BaFT. Practice in investigating Crohn disease (CD) is changing with the increasing use of magnetic resonance imaging (MRI). The aim of the present study was to compare BaFT and a new abdominal MRI protocol in a paediatric IBD population. Methods: All consecutive patients with a new diagnosis of IBD or requiring reassessment from September 2008 to December 2010 were investigated with both abdominal MRI and BaFT in accordance with a specific local paediatric IBD protocol. The studies were reported by nonblinded radiologists with an interest in gastrointestinal imaging. The reports were compared in conjunction with case note review. Results: Eighty-seven patients underwent both BaFT and MRI abdomen. Thirty-one percent of patients had additional pathology on MRI, not seen on the BaFT. Sixty-seven percent of patients (n = 59) had an MRI finding equivalent to BaFT. Using histology as a criterion standard for detecting terminal ileal disease, BaFT had a sensitivity and specificity of 76% and 67%, and MRI had a sensitivity and specificity of 83% and 95%, respectively. Conclusions: This is the largest series of small bowel MRI in a paediatric population. MRI reports were at least equivalent to BaFT. MRI had higher sensitivity and, particularly, specificity in detecting terminal ileal pathology. These findings suggest that MRI should become the criterion standard investigation in children with IBD in centres with appropriate expertise, with zero radiation exposure being highly advantageous.

Factors associated with nonadherence to thiopurines in adolescent and adult patients with inflammatory bowel disease.

Objectives: We hypothesised that nonadherence to thiopurines is more common in adolescents than in adults with inflammatory bowel disease. Methods: We sought factors associated with thiopurine nonadherence defined by thiopurine metabolite levels. Results: Multivariate logistic regression confirmed that adolescents (odds ratio [OR] 4.6 [95% confidence interval [CI] 1.9–11.5]; P < 0.01) compared with adults, patients with Crohn disease (OR 3.3 [CI 1.1–10.5] P = 0.04) compared with ulcerative colitis, and patients living in more socially deprived areas (OR 1.03 [CI 1.0–1.1] P = 0.02) were more likely to be nonadherent to thiopurines. Conclusions: Adolescents are more frequently nonadherent than adults: prospective studies are required to determine the reasons for nonadherence in adolescents.

Mathematical modelling to restore circulating IGF-1 concentrations in children with Crohn's disease-induced growth failure: a pharmacokinetic study.

Objectives Children with Crohns disease grow poorly, and inflammation depresses the response of insulin-like growth factor-1 (IGF-1) to growth hormone. Correcting the inflammation normalises growth velocity; however, removing inflammation cannot be achieved in all children. Our lack of understanding of IGF-1 kinetics has hampered its use, particularly as high IGF-1 concentrations over long periods may predispose to colon cancer. We hypothesised that mathematical modelling of IGF-1 would define dosing regimes that return IGF-1 concentrations into the normal range, without reaching values that risk cancer. Design Pharmacokinetic intervention study. Setting Tertiary paediatric gastroenterology unit. Participants 8 children (M:F; 4:4) entered the study. All completed: 5 South Asian British; 2 White British; 1 African British. Inclusion criteria: Children over 10 years with active Crohns disease (C reactive protein >10 mg/l or erythrocyte sedimentation rate >25 mm/h) and height velocity <–2 SD score. Exclusion criteria: closed epiphyses; corticosteroids within 3 months; neoplasia or known hypersensitivity to recombinant human IGF-1 (rhIGF-1). Interventions Subcutaneous rhIGF-1 (120 μg/kg) per dose over two admissions: the first as a single dose and the second as twice daily doses over 5 days. Primary outcome Significant increase in circulating IGF-1. Secondary outcomes Incidence of side effects of IGF-1. A mathematical model of circulating IGF-1 (Ac) was developed to include parameters of endogenous synthesis (Ksyn); exogenous uptake (Ka) from the subcutaneous dose (As): and IGF-1 clearance: where dAc/dt=Ksyn − Kout×Ac+Ka×As. Results Subcutaneous IGF-1 increased concentrations, which were maintained on twice daily doses. In covariate analysis, disease activity reduced Ksyn (p<0.001). Optimal dosing was derived from least squares regression fitted to a dataset of 384 Crohns patients, with model parameters assigned by simulation. Conclusions By using age, weight and disease activity scaling in IGF-1 dosing, over 95% of children will have normalised IGF-1 concentrations below +2.5 SDs of the normal population mean, a level not associated with cancer risk.

Subcutaneous rhIGF-1 Significantly Increases Circulating IGF-1 Concentrations in Children With Crohn's Disease Induced Growth Failure

Introduction Faltering growth is a complication of paediatric Crohns disease, affecting up to a third of patients. There is no recognised treatment targeted at improving linear growth. These children have low circulating insulin-like growth factor-1 (IGF-1), a hormone essential for linear growth and the primary mediator of growth hormone (GH) action. Low IGF-1 concentrations occur despite normal GH response to stimulation testing – the children thus having a functional GH insensitivity. Injections with recombinant human IGF-1 (rhIGF-1) have been reported to improve growth in animal models of colitis and in children with genetic GH insensitivity syndrome using doses of 80–120 μg/kg bd. rhIGF-I therapy has never previously been used in children with Crohns disease. We hypothesised that subcutaneous injections of rhIGF-1 would significantly increase circulating IGF-1 concentrations in children with Crohns disease induced growth failure, and that twice daily injections would maintain these concentrations. Methods Eight children with active Crohns disease and growth failure were recruited for an open-label pharmacokinetics study of rhIGF-1 (Increlex). A subcutaneous injection of rhIGF-1 (dose 120 μg/kg) was given, and levels were measured over 24 h. Children were also studied over a second period (5 days) of repeated doses. Blood sugar levels were monitored as hypoglycaemia is a potential adverse effect. Protein losing enteropathy was measured by stool α1-antitrypsin and related to IGF-1 levels attained. Results The median age(range) of the children was 12 years(10–14). 4 were female, 4 male, mean (SD) Paediatric Crohns Disease Activity Index (PCDAI) was 31.25(14.08). All the children had poor growth (mean growth velocity standard deviation score(SDS) −3.34 (SD 1.13)). All the subjects completed the study. The rhIGF-1 was well tolerated, with only one patient having an (asymptomatic) hypoglycaemic episode. All patients except for one had low baseline IGF-1 levels (mean SDS -1.78 (SD 1.37)) and all showed an increase in 3-h circulating IGF-1 levels following administration of rhIGF-1 (mean SDS 2.70 (SD 3.06)). This increase was significant(p = 0.007). IGF-1 levels were maintained above 0.0 SDS by twice daily injections. This occurred without any change in PCDAI over the 5-day trial period (p = 0.77) Protein-losing enteropathy did not inhibit this response. Conclusion Subcutaneous administration of rhIGF-1 significantly increased circulating concentrations of IGF-1 in children with Crohns disease-related growth retardation. These results support the initiation of trials to assess the impact of long-term rhIGF replacement therapy on linear growth.