H1N1 hemagglutinin-specific HLA-DQ6-restricted CD4+ T cells can be readily detected in narcolepsy type 1 patients and healthy controls.

Following the 2009 H1N1 influenza pandemic, an increased risk of narcolepsy type 1 was observed. Homology between an H1N1 hemagglutinin and two hypocretin sequences has been reported. T cell reactivity to these peptides was assessed in 81 narcolepsy type 1 patients and 19 HLA-DQ6-matched healthy controls. HLA-DQ6-restricted H1N1 hemagglutinin-specific T cell responses were detected in 28.4% of patients and 15.8% of controls. Despite structural homology between HLA-DQ6-hypocretin and -H1N1 peptide complexes, T cell cross-reactivity was not detected. These results indicate that it is unlikely that cross-reactivity between H1N1 hemagglutinin and hypocretin peptides presented by HLA-DQ6 is involved in the development of narcolepsy.

IgG is elevated in obese white adipose tissue but does not induce glucose intolerance via Fcγ-receptor or complement.

BACKGROUND/OBJECTIVES: In obesity, B cells accumulate in white adipose tissue (WAT) and produce IgG, which may contribute to the development of glucose intolerance. IgG signals by binding to Fcγ receptors (FcγR) and by activating the complement system. The aim of our study was to investigate whether activation of FcγR and/or complement C3 mediates the development of high-fat diet-induced glucose intolerance. METHODS: We studied mice lacking all four FcγRs (FcγRI/II/III/IV-/-), only the inhibitory FcγRIIb (FcγRIIb-/-), only the central component of the complement system C3 (C3-/-), and mice lacking both FcγRs and C3 (FcγRI/II/III/IV/C3-/-). All mouse models and wild-type controls were fed a high-fat diet (HFD) for 15 weeks to induce obesity. Glucose metabolism was assessed and adipose tissue was characterized for inflammation and adipocyte functionality. RESULTS: In obese WAT of wild-type mice, B cells (+142%, P<0.01) and IgG (+128% P<0.01) were increased compared to lean WAT. Macrophages of FcγRI/II/III/IV-/-mice released lower levels of cytokines compared to wild-type mice upon IgG stimulation. Only C3-/- mice showed reduced HFD-induced weight gain as compared to controls (-18%, P<0.01). Surprisingly, FcγRI/II/III/IV-/- mice had deteriorated glucose tolerance (AUC +125%, P<0.001) despite reduced leukocyte number (-30%, P<0.05) in gonadal WAT (gWAT), whereas glucose tolerance and leukocytes within gWAT in the other models were unaffected compared to controls. Although IgG in gWAT was increased (+44 to +174%, P<0.05) in all mouse models lacking FcγRIIb, only FcγRI/II/III/IV/C3-/- mice exhibited appreciable alterations in immune cells in gWAT, for example, increased macrophages (+36%, P<0.001). CONCLUSIONS: Lack of FcγRs reduces the activity of macrophages upon IgG stimulation, but neither FcγR nor C3 deficiency protects against HFD-induced glucose intolerance or reduces adipose tissue inflammation. This indicates that if obesity-induced IgG contributes to the development of glucose intolerance, this is not mediated by FcγR or complement activation.

Macrophage-mediated gliadin degradation and concomitant IL-27 production drive IL-10- and IFN-γ-secreting Tr1-like-cell differentiation in a murine model for gluten tolerance.

Celiac disease is caused by inflammatory T-cell responses against the insoluble dietary protein gliadin. We have shown that, in humanized mice, oral tolerance to deamidated chymotrypsin-digested gliadin (CT-TG2-gliadin) is driven by tolerogenic interferon (IFN)-γ- and interleukin (IL)-10-secreting type 1 regulatory T-like cells (Tr1-like cells) generated in the spleen but not in the mesenteric lymph nodes. We aimed to uncover the mechanisms underlying gliadin-specific Tr1-like-cell differentiation and hypothesized that proteolytic gliadin degradation by splenic macrophages is a decisive step in this process. In vivo depletion of macrophages caused reduced differentiation of splenic IFN-γ- and IL-10-producing Tr1-like cells after CT-TG2-gliadin but not gliadin peptide feed. Splenic macrophages, rather than dendritic cells, constitutively expressed increased mRNA levels of the endopeptidase Cathepsin D; macrophage depletion significantly reduced splenic Cathepsin D expression in vivo and Cathepsin D efficiently degraded recombinant γ-gliadin in vitro. In response to CT-TG2-gliadin uptake, macrophages enhanced the expression of Il27p28, a cytokine that favored differentiation of gliadin-specific Tr1-like cells in vitro, and was previously reported to increase Cathepsin D activity. Conversely, IL-27 neutralization in vivo inhibited splenic IFN-γ- and IL-10-secreting Tr1-like-cell differentiation after CT-TG2-gliadin feed. Our data infer that endopeptidase mediated gliadin degradation by macrophages and concomitant IL-27 production drive differentiation of splenic gliadin-specific Tr1-like cells.

A biased view toward celiac disease.

Celiac disease is an autoimmune-like disorder that is triggered by dietary gluten and has a strong genetic association with the human leukocyte antigen locus, specifically, HLA-DQ2.5/DQ8. Here, Dahai-Koirala et al. apply ex vivo single-cell sequencing of TCRs from celiac disease patients, and show that biased T-cell receptor usage underpins the response to two gluten epitopes, which has implications for disease pathogenesis, diagnosis, and treatment.

Lactobacillus plantarum NCIMB8826 ameliorates inflammation of colon and skin in human APOC1 transgenic mice.

Genetic predisposition and environmental factors, including the gut microbiota, have been suggested as major factors in the development and progression of atopic dermatitis. Hyperlipidemic human APOC1(+/+) transgenic mice display many features of human atopic dermatitis, such as scaling, lichenification, excoriations, and pruritus, along with a disturbed skin barrier function. Cytokine analysis of serum shows an increase of various pro-inflammatory cytokines, including interleukin (IL)-12p40, IL-6, and IL-1α, but lower levels of interferon-γ. These mice also display aspects of colitis evident from macroscopic and histological abnormalities. Genome-wide transcriptome analysis of the intestine shows up-regulation of several genes associated with mast cells and eosinophils and this observation was confirmed by demonstrating increased numbers of IgE(+) and FcRε(+) mast cells in the colon and in the skin. Oral treatment with Lactobacillus plantarum NCIMB8826 resulted in decreased numbers of mast cells in the colon. Moreover, this L. plantarum strain ameliorated skin pathology, evident from improved skin barrier integrity, absence of skin thickening, and less excoriations. These results suggest that modulation of intestinal immune homeostasis contributes to the suppression of atopic dermatitis.

Randomised clinical study: Aspergillus niger-derived enzyme digests gluten in the stomach of healthy volunteers.

BACKGROUND: Aspergillus niger prolyl endoprotease (AN-PEP) efficiently degrades gluten molecules into non-immunogenic peptides in vitro. AIM: To assess the efficacy of AN-PEP on gluten degradation in a low and high calorie meal in healthy subjects. METHODS: In this randomised, double-blind, placebo-controlled, cross-over study 12 healthy volunteers attended to four test days. A liquid low or high calorie meal (4 g gluten) with AN-PEP or placebo was administered into the stomach. Via a triple-lumen catheter gastric and duodenal aspirates were sampled, and polyethylene glycol (PEG)-3350 was continuously infused. Acetaminophen in the meals tracked gastric emptying time. Gastric and duodenal samples were used to calculate 240-min area under the curve (AUC0-240 min ) of ?-gliadin concentrations. Absolute ?-gliadin AUC0-240 min was calculated using duodenal PEG-3350 concentrations. RESULTS: AN-PEP lowered α-gliadin concentration AUC0-240 min, compared to placebo, from low and high calorie meals in stomach (low: 35 vs. 389 µg × min/mL; high: 53 vs. 386 µg × min/mL; P < 0.001) and duodenum (low: 7 vs. 168 µg × min/mL; high: 4 vs. 32 µg × min/mL; P < 0.001) and absolute α-gliadin AUC0-240 min in the duodenum from low (2813 vs. 31 952 µg × min; P < 0.001) and high (2553 vs. 13 095 µg × min; P = 0.013) calorie meals. In the placebo group, the high compared to low calorie meal slowed gastric emptying and lowered the duodenal α-gliadin concentration AUC0-240 min (32 vs. 168 µg × min/mL; P = 0.001). CONCLUSIONS: AN-PEP significantly enhanced gluten digestion in the stomach of healthy volunteers. Increasing caloric density prolonged gastric residence time of the meal. Since AN-PEP already degraded most gluten from low calorie meals, no incremental effect was observed by increasing meal caloric density. ClinicalTrials.gov, Number: NCT01335503; www.trialregister.nl, Number: NTR2780.

Adipocyte telomere length associates negatively with adipocyte size, whereas adipose tissue telomere length associates negatively with the extent of fibrosis in severely obese women.

Telomere length can be considered as a biological marker for cell proliferation and aging. Obesity is associated with adipocyte hypertrophy and proliferation as well as with shorter telomeres in adipose tissue. As adipose tissue is a mixture of different cell types and the cellular composition of adipose tissue changes with obesity, it is unclear what determines telomere length of whole adipose tissue. We aimed to investigate telomere length in whole adipose tissue and isolated adipocytes in relation to adiposity, adipocyte hypertrophy and adipose tissue inflammation and fibrosis. Telomere length was measured by real-time PCR in visceral adipose tissue, and isolated adipocytes of 21 obese women with a waist ranging from 110 to 147 cm and age from 31 to 61 years. Telomere length in adipocytes was shorter than in whole adipose tissue. Telomere length of adipocytes but not whole adipose tissue correlated negatively with waist and adipocyte size, which was still significant after correction for age. Telomere length of whole adipose tissue associated negatively with fibrosis as determined by collagen content. Thus, in extremely obese individuals, adipocyte telomere length is a marker of adiposity, whereas whole adipose tissue telomere length reflects the extent of fibrosis and may indicate adipose tissue dysfunction.

Feasibility of point-of-care creatinine testing in community pharmacy to monitor drug therapy in ambulatory elderly patients.

WHAT IS KNOWN AND OBJECTIVE: It is often necessary to adjust drug therapy if renal function is impaired in elderly patients taking drugs for diabetes and/or cardiovascular disease that are cleared by the kidneys. Although clinical guidelines recommend regular monitoring of renal function in these patients, in practice adherence to these recommendations varies from 28% to 75%. To determine whether drug dosing is appropriate, pharmacists need have up-to-date information about patients' renal function. In this study, the feasibility of point-of-care creatinine testing (POCCT) in a community pharmacy was evaluated as part of monitoring the drug therapy of ambulatory elderly patients. METHODS: Elderly patients on maintenance therapy with renally excreted drugs for diabetes or cardiovascular disease were eligible for POCCT. After informed consent was obtained, POCCT was performed by trained personnel. A pharmacist assessed the clinical relevance of electronically generated drug alerts based on the patient's calculated renal function and the Dutch guidelines for adjusting drug dosage in patients with chronic kidney disease. If appropriate, the patient's general practitioner (GP) was consulted and adjustments to treatment were communicated to the patient. The feasibility of POCCT was evaluated by means of questionnaires completed by patients and healthcare professionals (GPs and pharmacists). RESULTS: Of 338 potentially eligible patients, 149 (44%) whose renal function was not known were asked, by letter, to participate in the study. Of these individuals, 46 (31%) gave their informed consent and underwent POCCT. Response rates for completing the patient and professional questionnaires were 87% and 100%, respectively. More than half of the patients who underwent POCCT had mild-to-moderate renal impairment. On the basis of information provided by patients and healthcare professionals, POCCT would appear to be feasible in community pharmacies. WHAT IS NEW AND CONCLUSION: POCCT improves the management of drug therapy by community pharmacists and is feasible in daily practice.

Subtype-specific differences in the development of accessory mutations associated with high-level resistance to HIV-1 nucleoside reverse transcriptase inhibitors.

OBJECTIVES: To identify accessory mutations associated with high-level resistance to reverse transcriptase (RT) inhibitors in HIV-1 subtypes B and C. METHODS: Changes relative to the wild-type for codons 1-400 of RT were analysed from treatment-experienced patients infected with subtypes B (5464 patients) and C (1920 patients). Positions associated with the accumulation of mutations conferring resistance to thymidine analogues and to non-nucleoside RT inhibitors (NNRTIs) were identified. A subtype-specific single-replication cycle drug susceptibility assay was used to determine whether some of the mutations affected drug susceptibility or viral infectivity. RESULTS: In subtype B, mutations at 31 and 26 positions were associated with the accumulation of thymidine analogue mutations (TAMs) and NNRTI mutations, respectively; in subtype C, 18 and 13 positions were identified, respectively. Amino acid changes at the following positions were differentially associated with (i) the accumulation of 0-4+ TAMs in subtypes B and C (away from consensus): 43 (27.0% B versus 2.5% C); 118 (36.4% B versus 16.2% C); 135 (12.5% B versus 28.0% C); and 326 (2.6% towards consensus in B versus 7.6% away in C) and (ii) the accumulation of 0-3+ NNRTI mutations (away from consensus): 43 (10.2% B versus 0.5% C); and 68 (5.2% B versus 10.3% C). Codon changes K43E, E44D and V118I were found to have no effect on susceptibility to three NRTIs with or without TAMs in either subtype; however, some accessory mutations had subtype-specific effects on viral infectivity. CONCLUSIONS: Differences between subtypes B and C were observed in the development and effect of accessory mutations associated with high-level resistance to RT inhibitors.

KIR-ligand mismatches are associated with reduced long-term graft survival in HLA-compatible kidney transplantation.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system with the ability to detect HLA class I disparities via killer-cell immunoglobulin-like receptors (KIR). To test whether such KIR-ligand mismatches contribute to the rejection of human solid allografts, we did a retrospective cohort study of 397 HLA-DR-compatible kidney transplantations and determined the KIR and HLA genotypes of recipients and the HLA genotypes of donors. In transplantations compatible for HLA-A, HLA-B and HLA-DR (n = 137), in which a role for T cells and HLA antibodies in rejection was minimized, KIR-ligand mismatches were associated with an approximately 25% reduction in 10-year death-censored graft survival (p = 0.043). This effect was comparable to the effect of classical HLA-A and HLA-B incompatibility, and in HLA-A,-B-incompatible transplantations (n = 260) no significant additional effect of KIR-ligand mismatches was observed. Multivariate Cox regression analysis confirmed the effect of KIR-ligand mismatching as an independent risk factor in HLA-A,-B,-DR-compatible transplantations (hazard ratio 2.29, range 1.03-5.10, p = 0.043). This finding constitutes the first indication that alloreactive NK cells may thwart the success of HLA-compatible kidney transplantations, and suggests that suppression of NK-cell activity can improve the survival of such kidney grafts.

Efficient degradation of gluten by a prolyl endoprotease in a gastrointestinal model: implications for coeliac disease.

BACKGROUND: Coeliac disease is caused by an immune response to gluten. As gluten proteins are proline rich they are resistant to enzymatic digestion in the gastrointestinal tract, a property that probably contributes to the immunogenic nature of gluten. AIMS: This study determined the efficiency of gluten degradation by a post-proline cutting enzyme, Aspergillus niger prolyl endoprotease (AN-PEP), in a dynamic system that closely mimics the human gastrointestinal tract (TIM system). METHODS: Two experiments were performed. In the first, a slice of bread was processed in the TIM system with and without co-administration of AN-PEP. In the second, a standard fast food menu was used. Samples of the digesting meals were taken from the stomach, duodenum, jejunum and ileum compartments at time zero until 4 hours after the start of the experiment. In these samples the levels of immunogenic peptides from gliadins and glutenins were assessed by monoclonal antibody-based competition assays, Western blot analysis and proliferation T-cell assays. RESULTS: AN-PEP accelerated the degradation of gluten in the stomach compartment to such an extent that hardly any gluten reached the duodenum compartment. CONCLUSION: AN-PEP is capable of accelerating the degradation of gluten in a gastrointestinal system that closely mimics in-vivo digestion. This implies that the co-administration of AN-PEP with a gluten-containing meal might eliminate gluten toxicity, thus offering patients the possibility of abandoning (occasionally) their strict gluten-free diet.

An improved RT-PCR method for the detection of killer-cell immunoglobulin-like receptor (KIR) transcripts.

Killer cell immunoglobulin-like receptors (KIRs) are expressed on human natural killer (NK) cells and a proportion of T cells. As the specificity of these NK and T cells is, at least in part, determined by the combination of KIRs they express, it is important to be able to determine the KIR expression pattern of NK and T cell clones to understand their function. However, for most KIR genes, specific reagents to detect expression are currently either unavailable or sensitive to allelic variations. In this study, a reverse transcriptase-polymerase chain reaction (RT-PCR) that uses new primer sets for the gene-specific detection of KIR transcripts is presented and validated. The key advantage of this RT-PCR method over previously published ones is that it was designed to detect transcripts of all confirmed allelic variants of the KIR genes, while remaining gene-specific.

The impact of single amino acid substitutions in CD3gamma on the CD3epsilongamma interaction and T-cell receptor-CD3 complex formation.

The human T-cell receptor-CD3 complex consists of at least eight polypeptide chains; CD3gamma- and delta-dimers associate with the disulfide linked alphabeta- and zetazeta-dimers to form a functional receptor complex. The exact structure of this complex is still unknown. We now have examined the interaction between CD3gamma and CD3 in human T-cells. For this purpose, we have generated site-directed mutants of CD3gamma that were introduced in human T-cells defective in CD3gamma expression. Cell-surface and intracellular expression of the introduced CD3gamma chains was determined, as was the association with CD3delta, CD3, and the T-cell receptor. Although the introduction of wild type CD3gamma and CD3gamma (78Y-F) fully restored T-cell receptor assembly and expression, the introduction of CD3gamma (82C-S), CD3gamma (85C-S), and CD3gamma (76Q-E) all resulted in an impaired association between CD3gamma and CD3 and a lack of cell-surface expressed CD3gamma. Finally, the introduction of CD3gamma (76Q-L) and CD3gamma (78Y-A) restored the expression of TCR-CD3deltagammazeta2 complexes, although the association between CD3gamma and CD3 was impaired. These results indicate that several amino acids in CD3gamma are essential for an optimal association between CD3gamma and CD3 and the assembly of a cell-surface expressed TCR-CD3deltagammazeta2 complex.

A novel and sensitive method for the detection of T cell stimulatory epitopes of alpha/beta- and gamma-gliadin.

BACKGROUND: It is now generally accepted that coeliac disease (CD) is caused by inflammatory T cell responses to gluten peptides bound to HLA-DQ2 or -DQ8 molecules. There is overwhelming evidence that CD patients can mount T cell responses to peptides found in both alpha-gliadin and gamma-gliadin molecules. Assays that would detect the presence or absence of such peptides in food would thus be accurate indicators of safety for consumption by CD patients. AIMS: The development of a sensitive method to detect T cell stimulatory epitopes of alpha-gliadin and gamma-gliadin molecules in food products. METHODS: Monoclonal antibodies (mAb) were raised against peptides encoding the T cell stimulatory epitopes of alpha-gliadin (amino acids (aa) 59-71) and aa gamma-gliadin (aa 142-153 and aa 147-159). These mAb competition assays were developed that quantitatively detect T cell stimulatory epitopes present on both intact proteins and peptides of sizes recognisable by CD4(+) T cells. RESULTS: With the mAb based competition assays, T cell epitopes were detected in pepsin/trypsin digests of wheat proteins and ethanol extracts of various food products, with detection levels lower than those reached with gluten specific T cells. Moreover, the presence of T cell stimulatory epitopes was also detected in preparations of barley, rye, and triticale, other cereals known to be toxic for CD patients. CONCLUSIONS: A new antibody based method has been developed, detecting the presence of T cell stimulatory gluten peptides. This can be used to further ensure the safety of food consumed by CD patients.

Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells.

Hairy cell leukemia (HCL) is a chronic mature B-cell leukemia characterized by malignant B cells that have typical hairy protrusions. To characterize possible HCL-associated tumor antigens, we generated an HCL-specific and HLA class II (DPw4)-restricted proliferative CD4+ T-cell clone. To identify the target antigen of these T cells, we constructed a synthetic peptide library dedicated to bind HLA DPw4, and identified a mimicry epitope recognized by the T-cell clone. With this epitope, the recognition motif of the T-cell clone was deduced and a peptide of human synaptojanin 2 (Syn 2) was identified that stimulated the HCL-reactive T-cell clone. Both Northern and Western blot analyses showed that Syn 2 expression was increased in HCL samples compared to other B cells. Besides, the Syn 2-expressing cell line AML193, with the introduced restrictive HLA-DPw4 molecules, was recognized by the HCL-specific T-cell clone. These results indicate that Syn 2 is a target of autoreactive HCL-specific T cells. Since Syn 2 is a phosphatidylinositol 4,5-biphosphatase involved in cell growth and rearrangement of actin filaments, the increased Syn 2 expression may correlate with the disease etiology or the characteristic morphologic alterations caused by the disease.

An immunodominant DQ8 restricted gliadin peptide activates small intestinal immune response in in vitro cultured mucosa from HLA-DQ8 positive but not HLA-DQ8 negative coeliac patients.

BACKGROUND: Studies on intestinal T cell clones from the mucosa of patients with coeliac disease have led to the identification of immunogenic gliadin epitopes. One is HLA-DQ8 restricted, its recognition by T cells being increased by introduction of negatively charged residues operated by tissue transglutaminase. AIM: To test HLA-DQ8 restricted epitope in both native (QYPSGQGSFQPSQQNPQA) and deamidated (QYPSGEGSFQPSQENPQA) forms in an organ culture system of treated coeliac mucosa from HLA-DQ8 positive and HLA-DQ8 negative patients. PATIENTS AND METHODS: Jejunal biopsies obtained from 10 patients with coeliac disease (six HLA-DQ8 positive and four HLA-DQ8 negative) were cultured in vitro with a peptic-tryptic digest (PT) of gliadin, or with the native (peptide A) or deamidated (peptide B) peptide. Intraepithelial CD3(+) and lamina propria total CD25(+) and CD3(+)CD25(+) cells were counted, lamina propria intercellular adhesion molecule 1 (ICAM-1) expression was evaluated, as well as that of Fas molecules on epithelial cells. RESULTS: In HLA-DQ8 positive, but not in HLA-DQ8 negative, coeliacs the density of intraepithelial CD3(+) cells, lamina propria total CD25(+), and CD3(+)CD25(+) cells, as well as expression of ICAM-1 and Fas molecules were significantly increased in biopsies cultured with PT, peptide A, or peptide B compared with biopsies cultured in medium alone. CONCLUSION: These data show that the DQ8 restricted gliadin peptide is immunogenic only in the intestinal mucosa of HLA-DQ8 positive coeliac patients in both native and deamidated forms.

T cell responses modulated through interaction between CD8alphaalpha and the nonclassical MHC class I molecule, TL.

The thymus leukemia antigen (TL) is a nonclassical class I molecule, expressed abundantly on intestinal epithelial cells. We show that, in contrast to other major histocompatibility complex (MHC) class I molecules that bind CD8alphabeta, TL preferentially binds the homotypic form of CD8alpha (CD8alphaalpha). Thus, TL tetramers react specifically to CD8alphaalpha-expressing cells, including most intestinal intraepithelial lymphocytes. Compared with CD8alphabeta, which recognizes the same MHC as the T cell receptor (TCR) and thus acts as a TCR coreceptor, high-affinity binding of CD8alphaalpha to TL modifies responses mediated by TCR recognition of antigen presented by distinct MHC molecules. These findings define a novel mechanism of lymphocyte regulation through CD8alphaalpha and MHC class I.