Human phagosome processing of Mycobacterium tuberculosis antigens is modulated by interferon‐γ and interleukin‐10

Intracellular pathogens, such as Mycobacterium tuberculosis, reside in the phagosomes of macrophages where antigenic processing is initiated. Mycobacterial antigen–MHC class II complexes are formed within the phagosome and are then trafficked to the cell surface. Interferon‐γ (IFN‐γ) and interleukin‐10 (IL‐10) influence the outcome of M. tuberculosis infection; however, the role of these cytokines with regard to the formation of M. tuberculosis peptide–MHC‐II complexes remains unknown. We analysed the kinetics and subcellular localization of M. tuberculosis peptide–MHC‐II complexes in M. tuberculosis‐infected human monocyte‐derived macrophages (MDMs) using autologous M. tuberculosis‐specific CD4⁺ T cells. The MDMs were pre‐treated with either IFN‐γ or IL‐10 and infected with M. tuberculosis. Cells were mechanically homogenized, separated on Percoll density gradients and manually fractionated. The fractions were incubated with autologous M.  tuberculosis ‐specific CD4⁺ T cells. Our results demonstrated that in MDMs pre‐treated with IFN‐γ, M. tuberculosis peptide–MHC‐II complexes were detected early mainly in the phagosomal fractions, whereas in the absence of IFN‐γ, the complexes were detected in the endosomal fractions. In MDMs pre‐treated with IL‐10, the M. tuberculosis peptide–MHC‐II complexes were retained in the endosomal fractions, and these complexes were not detected in the plasma membrane fractions. The results of immunofluorescence microscopy demonstrated the presence of Ag85B associated with HLA‐DR at the cell surface only in the IFN‐γ‐treated MDMs, suggesting that IFN‐γ may accelerate M. tuberculosis antigen processing and presentation at the cell membrane, whereas IL‐10 favours the trafficking of Ag85B to vesicles that do not contain LAMP‐1. Therefore, IFN‐γ and IL‐10 play a role in the formation and trafficking of M. tuberculosis peptide–MHC‐II complexes.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

Tumour‐Infiltrating FoxP3+ and IL‐17‐Producing T Cells Affect the Progression and Prognosis of Gallbladder Carcinoma after Surgery

Tumour‐infiltrating lymphocytes (TILs) have been found to play crucial roles in a series of cancers. However, the impact of these cells on gallbladder carcinoma (GBC) remains poorly understood. In this study, we examined infiltrating FoxP3+, IL‐17+, CD4+ and CD8+ cells by immunohistochemistry in specimens of 104 patients with GBC and evaluated the association of these cells with clinicopathological features and prognosis. The number of FoxP3+ cells was increased in a stepwise manner from CC to GA and GBC (GA versus CC, P = 0.036; early GBC versus GA, P = 0.032; advanced versus early GBC, P = 0.025). Both intratumoral FoxP3+ and IL‐17+ cells correlated with nodal metastasis and TNM stage. Additionally, there were more infiltrating FoxP3+ cells in specimens with distant metastasis (P = 0.014). The group with high FoxP3+ cells showed poor overall survival (OS, P < 0.001) and disease‐free survival (DFS, P < 0.001), and high infiltration of IL‐17‐producing cells was also a predictor of poor OS (P = 0.024). Multivariate analysis revealed that the presence of intratumoral FoxP3+ cells was an independent prognostic indicator for poor DFS (P < 0.01). In summary, these findings indicate that FoxP3+ and IL‐17+ cells cooperatively facilitate pathogenesis and progression of GBC and show prognostic significance for OS or DFS.     

Correlation between testicular mast cell count and spermatogenic epithelium in non‐obstructive azoospermia

Although there is emerging evidence that mast cells are involved in infertility, their exact role has not been elucidated clearly. Here we carried out a retrospective case–control study to find out whether there is a correlation between mast cell (MC) count and proliferation (Ki67 index) of the spermatogenic epithelium as well as of the Sertoli cells (vimentin‐positive) in non‐obstructive azoospermia (NOA). We assessed MCs, Ki67 and vimentin expression in Sertoli cells in testicular biopsies of germ cell aplasia (GCA, n = 14) and maturation arrest (MA, n = 14) vs. normal spermatogenesis (n = 14) cases. There was a significant decrease in the spermatogonial Ki67 index (1.25 ± 0.91, 4.21 ± 1.81 vs. 39.57 ± 3.92) and Johnsen score (2.48 ± 0.65, 4.89 ± 1.05 vs. 9.75 ± 0.30) as well as a significant increase (P < 0.001) in MC count (29.00 ± 4.11, 7.57 ± 1.95 vs. 3.00 ± 1.30) in seminiferous tubules of infertile cases with GCA and MA vs. controls. On the other hand, the percentage of vimentin‐expressing Sertoli cells was significantly decreased (P < 0.001) in biopsies of cases with MA (35.50 ± 15.62) compared to those of cases with GCA and controls (72.64 ± 10.67 and 98.57 ± 1.45 respectively). Additionally, a significant negative correlation was detected between MC count and Ki67 index as well as Johnsen score in the MA group which became more significant in the GCA group. The significant increase in MC count in the GCA group and to a lesser extent in the MA group indicates their possible role in NOA particularly at the spermatogonial proliferation level and this is supported by the significant negative correlation with the Ki67 index.     

Genetic association of interleukin‐2, interleukin‐4, interleukin‐6, transforming growth factor‐β, tumour necrosis factor‐α and blood concentrations of calcineurin inhibitors in Turkish renal transplant patients

Cytokines are essential for the control of the immune response as most of the immunosuppressive drugs target cytokine production or their action. The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are immunosuppressive drugs widely used after renal transplantation to prevent allograft rejection. They are characterized by large interindividual variability in their pharmacokinetics; therefore, monitoring their blood concentrations is important to predict their optimal dosage following transplantation. Calcineurin inhibitors inhibit the phosphatase activity of calcineurin, thereby suppressing the production of other cytokines such as transforming growth factor (TGF‐β), tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐6, IL‐2, and IL‐4. The aim of this study was to investigate the relationship between polymorphisms of cytokines and blood concentrations of CNIs in renal transplant patients. The study included 53 CsA‐treated renal transplant patients and 37 tacrolimus‐treated renal transplant patients. Cytokine polymorphisms were analysed using polymerase chain reaction (PCR) sequence‐specific primers with the cytokine CTS‐PCR‐sequence‐specific primers Tray Kit; University of Heidelberg. Blood concentrations of CNIs were determined with Cloned Enzyme Donor Immunoassay (CEDIA) method. Patients with TC genotype of TGF‐β at codon 10 had lower CsA blood concentrations than the TT and CC genotypes (P = 0.005) at 1 month in CsA treatment group. The ratio of blood concentration/dose of CsA for patients with TGF‐β1‐codon 10 TC genotype was lower than for patients with TT, CC genotypes, and the dose given to these patients was higher in the first month (P = 0.046). The ratio of blood concentration/dose of CsA for patients with IL‐2‐330 GG genotype was higher than for patients with GT, TT genotypes, and the dose given to these patients was lower at first month and sixth months (P = 0.043, P = 0.035 respectively). The tacrolimus blood concentrations were significantly higher in patients with the genotype GG of IL‐2‐330 (P = 0.012) at the third month. Patients who had the TC genotype TGF‐β codon 10 had lower CsA blood concentrations and this group had higher acute rejection (P = 0.033). These results suggest that the genotyping for TGF‐β‐codon 10, IL‐2‐330 and IL‐6‐174 polymorphisms may help individualized immunosuppressive dosage regiments.     

Rare Deleterious PARD3 Variants in the aPKC‐Binding Region are Implicated in the Pathogenesis of Human Cranial Neural Tube Defects Via Disrupting Apical Tight Junction Formation

Increasing evidence that mutation of planar cell polarity (PCP) genes contributes to human cranial neural tube defect (NTD) susceptibility prompted us to hypothesize that rare variants of genes in the core apical–basal polarity (ABP) pathway are risk factors for cranial NTDs. In this study, we screened for rare genomic variation of PARD3 in 138 cranial NTD cases and 274 controls. Overall, the rare deleterious variants of PARD3 were significantly associated with increased risk for cranial NTDs (11/138 vs.7/274, P < 0.05, OR = 3.3). These NTD‐specific variants were significantly enriched in the aPKC‐binding region (6/138 vs. 0/274, P < 0.01). The East Asian cohort in the ExAC database and another Chinese normal cohort further supported this association. Over‐expression analysis in HEK293T and MDCK cells confirmed abnormal aPKC binding or interaction for two PARD3 variants (p.P913Q and p.D783G), resulting in defective tight junction formation via disrupted aPKC binding. Functional analysis in human neural progenitor cells and chick embryos revealed that PARD3 knockdown gave rise to abnormal cell polarity and compromised the polarization process of neuroepithelial tissue. Our studies suggest that rare deleterious variants of PARD3 in the aPKC‐binding region contribute to human cranial NTDs, possibly by disrupting apical tight junction formation and subsequent polarization process of the neuroepithelium.     

Interferon‐γ enhances both the anti‐bacterial and the pro‐inflammatory response of human mast cells to Staphylococcus aureus

Human mast cells (huMCs) are involved in both innate and adaptive immune responses where they release mediators including amines, reactive oxygen species (ROS), eicosanoids and cytokines. We have reported that interferon‐γ (IFN‐γ) enhances FcγR‐dependent ROS production. The aim of this study was to extend these observations by investigating the effect of IFN‐γ on the biological responses of huMCs to Staphylococcus aureus. We found that exposure of huMCs to S. aureus generated intracellular and extracellular ROS, which were enhanced in the presence of IFN‐γ. IFN‐γ also promoted bacteria killing, β‐hexosaminidase release and eicosanoid production. Interferon‐γ similarly increased expression of mRNAs encoding CCL1 to CCL4, granulocyte–macrophage colony‐stimulating factor (GM‐CSF), tumour necrosis factor‐α and CXCL8 in S. aureus‐stimulated huMCs. The ability of IFN‐γ to increase CXCL8 and GM‐CSF protein levels was confirmed by ELISA. Fibronectin or a β₁ integrin blocking antibody completely abrogated IFN‐γ‐dependent S. aureus binding and reduced S. aureus‐dependent CXCL8 secretion. These data demonstrate that IFN‐γ primes huMCs for enhanced anti‐bacterial and pro‐inflammatory responses to S. aureus, partially mediated by β₁ integrin.     

Up‐regulation of autophagy in small intestine Paneth cells in response to total‐body γ‐irradiation

Macroautophagy (mAG) is a lysosomal mechanism of degradation of cell self‐constituents damaged due to variety of stress factors, including ionizing irradiation. Activation of mAG requires expression of mAG protein Atg8 (LC3) and conversion of its form I (LC3‐I) to form II (LC3‐II), mediated by redox‐sensitive Atg4 protease. We have demonstrated upregulation of this pathway in the innate host defense Paneth cells of the small intestine (SI) due to ionizing irradiation and correlation of this effect with induction of pro‐oxidant inducible nitric oxide synthase (iNOS). CD2F1 mice were exposed to 9.25 Gy γ‐ionizing irradiation. Small intestinal specimens were collected during 7 days after ionizing irradiation. Assessment of ionizing irradiation‐associated alterations in small intestinal crypt and villus cells and activation of the mAG pathway was conducted using microscopical and biochemical techniques. Analysis of iNOS protein and the associated formation of nitrites and lipid peroxidation products was performed using immunoblotting and biochemical analysis, and revealed increases in iNOS protein, nitrate levels and oxidative stress at day 1 following ionizing irradiation. Increase in immunoreactivity of LC3 protein in the crypt cells was observed at day 7 following ionizing irradiation. This effect predominantly occurred in the CD15‐positive Paneth cells and was associated with accumulation of LC3‐II isoform. The formation of autophagosomes in Paneth cells was confirmed by transmission electron microscopy (TEM). Up‐regulation of LC3 pathway in the irradiated SI was accompanied by a decreased protein–protein interaction between LC3 and chaperone heat shock protein 70. A high‐level of LC3‐immunoreactivity in vacuole‐shaped structures was spatially co‐localized with immunoreactivity of 3‐nitro‐tyrosine. The observed effects were diminished in iNOS knockout B6.129P2‐NOS2^tm1Lau/J mice subjected to the same treatments. We postulate that the observed up‐regulation of mAG in the irradiated small intestine is at least in part mediated by the iNOS signalling mechanism. Published in 2009 by John Wiley & Sons, Ltd.     

The role of alkaline phosphatase in intracellular lipid accumulation in the human hepatocarcinoma cell line,HepG2

Inhibition of tissue non-specific alkaline phosphatase (TNALP) decreases intracellular lipid accumulation in human preadipocytes and the murine preadipocyte cell line, 3T3-L1. Therefore, the current study was performed to determine if TNALP is required for intracellular lipid deposition in the human hepatocyte cell line, HepG2. Intracellular lipid accumulation, TNALP activity and peroxisome proliferator activated receptor (PPAR) γ gene expression were measured in HepG2 and 3T3-L1 cells in the presence and absence of the TNALP inhibitors levamisole and histidine. Sub-cellular TNALP activity was localized using cytochemical analysis. Both PPARγ gene expression and TNALP activity increased during intracellular lipid accumulation in HepG2 and 3T3-L1 cells. Inhibition of TNALP blocked intracellular lipid accumulation but did not alter expression of the PPARγ gene. In HepG2 cells, TNALP co-localized with adipophilin on the lipid droplet membrane. These data suggest a role for TNALP in lipid droplet formation, possibly downstream from PPARγ, within HepG2 and 3T3-L1 cells.     

Regulatory T‐cells in acute dengue viral infection

Although regulatory T‐cells (T_regs) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of T_regs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4⁺ CD25⁺T‐cells (FoxP3⁺ cells). Dengue virus (DENV) viral loads were measured by quantitative real‐time polymerase chain reaction (PCR) and DENV‐specific T‐cell responses were measured by ex‐vivo interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV‐NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T‐cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte‐antigen 4 (CTLA‐4) and Fas. While the frequency of FoxP3⁺ cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3⁺ cells did not correlate with either ex‐vivo IFN‐γ DENV‐NS3‐, NS5‐ or NS1‐specific T‐cell responses. FoxP3⁺ cells of patients with acute dengue were predominantly CD45RA⁺ FoxP3^low, followed by CD45RA‐FoxP3^low, with only a small proportion of FoxP3⁺ cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T‐cells, with only CCR4 only being expressed at high levels in the effector T_reg population. Therefore, although FoxP3⁺ cells are expanded in acute dengue, they predominantly consist of naive T_regs, with poor suppressive capacity.     

PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation

Phosphatidylinositol‐3‐kinase gamma (PI3Kγ) is a leukocyte‐specific lipid kinase with signaling function downstream of G protein‐coupled receptors to regulate cell trafficking, but its role in T cells remains unclear. To investigate the requirement of PI3Kγ kinase activity in T‐cell function, we studied T cells from PI3Kγ kinase‐dead knock‐in (PI3Kγ^KD/KD) mice expressing the kinase‐inactive PI3Kγ protein. We show that CD4⁺ and CD8⁺ T cells from PI3Kγ^KD/KD mice exhibit impaired TCR/CD28‐mediated activation that could not be rescued by exogenous IL‐2. The defects in proliferation and cytokine production were also evident in naïve and memory T cells. Analysis of signaling events in activated PI3Kγ^KD/KD T cells revealed a reduction in phosphorylation of protein kinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycle progression. Furthermore, PI3Kγ^KD/KD CD4⁺ T cells displayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells. PI3Kγ^KD/KD mice also exhibited an impaired response to immunization and a reduced delayed‐type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation, as well as for mounting an efficient T cell‐mediated immune response. The results suggest that PI3Kγ kinase inhibitors could be beneficial in reducing the undesirable immune response in autoimmune diseases.     

Clinicopathologic implications of TNFAIP3/A20 deletions in extranodal NK/T‐cell lymphoma

The A20/Tumor necrosis factor‐alpha‐induced protein 3 (A20/TNFAIP3) is a negative regulator of NF‐κB signaling. We analyzed the clinicopathologic implications of A20 deletions in extranodal NK/T‐cell lymphoma (NKTL). Fluorescence in situ hybridization analysis of the A20 gene was performed using archived formalin‐fixed tissues in 49 cases of NKTL. Among the 49 NKTL patients (median age, 48 y [10‐79]), stage I‐II (75% [36/48]) and upper aerodigestive tract (UAT)‐origin (84% [41/49]) were predominant. All A20 deletions were monoallelic and found in cases with UAT‐origin, accounting for 18% (9/49) of all NKTLs and 22% (9/41) of UAT‐origin. In univariate analysis, overall survival (OS) and progression‐free survival (PFS) were associated with stage, international prognostic index (IPI), B symptoms and number of extranodal sites, and OS with performance status and non‐UAT‐origin, but none with A20 deletion. In multivariate analysis, IPI predicted OS (P = .008 [HR = 23.4]) and PFS (P = .005 [HR = 34.0]). Risk was divided by B symptoms (P = .001 [OS]; P = .034 [PFS]) in low IPI subset (n = 36), and by A20 deletion (P = .029 [PFS]) in high IPI subset (n = 13). These results suggest a clinicopathologic implication of A20 in progression of NKTL.     

Phenotypic characterization of regulatory T cells from antiretroviral‐naive HIV‐1‐infected people

Regulatory T (Treg) cells play a key role in dampening excessive immune activation. However, antiretroviral therapy (ART) ‐naive HIV‐1 infection maintains the immune system in a sustained state of activation that could alter both Treg cell surface markers and functions. As Treg cell surface markers are directly linked to their functions the overall objective of this study was to determine how ART‐naive HIV infection affects the phenotypic properties of Treg cells. Our data showed that in ART‐naive HIV‐1 infection, Treg cells are dominated by effector (CD45RA⁺ CD27⁻ CCR7⁻ CD62L⁻) and effector memory (CD45RA⁻ CD27⁻ CCR7⁻ CD62L⁻) cells. In contrast Treg cells from HIV‐negative individuals were mainly naive (CD45RA⁺ CD27⁺ CCR7⁺ CD62L⁺) and central memory (CD45RA⁻ CD27⁺ CCR7⁺ CD62L⁺) cells. Whereas effector and effector memory Treg cells showed enhanced expression of CD39 (P < 0·05), CD73 (P < 0·001), HLA‐DR and CD38 (P < 0·001); naive and central memory Treg cells showed a significant reduction in the expression of these markers. Overall Treg cell frequencies within total CD4⁺ T cells correlated positively with plasmatic HIV‐1 viral load. As increased viral load is associated with augmented CD4⁺ T‐cell destruction; this could suggest a resistance of peripheral Treg cells to HIV‐1 destruction. Hence the modulation of Treg cell phenotype and frequencies could be considered in designing immunotherapeutic strategies targeting immune system restoration during HIV‐1 infection.     

Length polymorphism in the Period 3 gene is associated with sleepiness and maladaptive circadian phase in night‐shift workers

The objective of the current study was to determine if night‐shift workers carrying the five‐repeat variant of the Period 3 gene show elevated levels of nocturnal sleepiness and earlier circadian phase compared with homozygotes for the four‐repeat allele. Twenty‐four permanent night‐shift workers were randomly selected from a larger study. Participants took part in an observational laboratory protocol including an overnight multiple sleep latency test and half‐hourly saliva collection for calculation of dim‐light melatonin onset. Period 3^–/5 shift workers had significantly lower multiple sleep latency test during overnight work hours compared with Period 3^4/4 workers (3.52 ± 23.44 min versus 10.39 ± 6.41 min, P = 0.003). We observed no significant difference in sleepiness during early morning hours following acute sleep deprivation. Long‐allele carriers indicated significantly higher sleepiness on the Epworth Sleepiness Scale administered at 17:00 hours (12.08 ± 2.55 versus 8.00 ± 1.94, P < 0.001). We observed a significantly earlier melatonin onset in Period 3^–/5 individuals compared with Period 3^4/4 shift workers (20:44 ± 6:37 versus 02:46 ± 4:58, P = 0.021). Regression analysis suggests that Period 3 genotype independently predicts sleepiness even after controlling for variations in circadian phase, but we were unable to link Period 3 to circadian phase when controlling for sleepiness. Period 3^–/5 shift workers showed both subjective and objective sleepiness in the pathological range, while their Period 3^4/4 counterparts showed sleepiness within normal limits. Period 3^–/5 night workers also show a mean circadian phase 6 h earlier (i.e. less adapted) than Period 3^4/4 workers. Because Period 3^–/5 workers have maladaptive circadian phase as well as pathological levels of sleepiness, they may be at greater risk for occupational and automotive accidents. We interpret these findings as a call for future research on the role of Period 3 in sleepiness and circadian phase, especially as they relate to night work.     

Glycogen synthase kinase‐3β ablation limits pancreatitis‐induced acinar‐to‐ductal metaplasia

Acinar‐to‐ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre‐malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase‐3beta (GSK‐3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK‐3β promotes TGF‐α‐induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK‐3β attenuates caerulein‐induced ADM formation and PanIN progression in Kras^G12D transgenic mice. Furthermore, we demonstrate that GSK‐3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas‐driven cell proliferation. Mechanistically, we show that GSK‐3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK‐3β participates in early pancreatitis‐induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Suppression of the allogeneic response by the anti‐allergy drug N‐(3,4‐dimethoxycinnamonyl) anthranilic acid results from T‐cell cycle arrest

Previously we have shown that indoleamine 2,3‐dioxygenase (IDO) and the tryptophan metabolite, 3‐hydroxykynurenine (3HK) can prolong corneal allograft survival. IDO modulates the immune response by depletion of the essential amino acid tryptophan by breakdown to kynurenines, which themselves act directly on T lymphocytes. The tryptophan metabolite analogue N‐(3,4‐dimethoxycinnamonyl) anthranilic acid (DAA, ‘Tranilast’) shares the anthranilic acid core with 3HK. Systemic administration of DAA to mice receiving a fully MHC‐mismatched allograft of cornea or skin resulted in significant delay in rejection (median survival of controls 12 days, 13 days for cornea and skin grafts, respectively, and of treated mice 24 days (P < 0·0001) and 17 days (P < 0·03), respectively). We provide evidence that DAA‐induced suppression of the allogeneic response, in contrast to that induced by tryptophan metabolites, was a result of cell cycle arrest rather than T‐cell death. Cell cycle arrest was mediated by up‐regulation of the cell cycle‐specific inhibitors p21 and p15, and associated with a significant reduction in interleukin‐2 production, allowing us to characterize a novel mechanism for DAA‐induced T‐cell anergy. Currently licensed as an anti‐allergy drug, the oral bioavailability and safe therapeutic profile of DAA make it a candidate for the prevention of rejection of transplanted cornea and other tissues.     

Identification of Glyceraldehyde‐3‐Phosphate and Alcohol Dehydrogenases as Autoantigens in Doberman Hepatitis

An autoimmune background is suspected for Doberman hepatitis (DH). It is based on the finding of mononuclear cell infiltrates in the liver, strong female bias, association to the homozygous risk factor dog leucocyte antigen (DLA) allele DRB1*00601 and aberrant major histocompatibility complex (MHC) class II expression on hepatocytes that correlates with the degree of inflammation in the liver. The aim of this study was to search for autoantibodies against liver‐related antigens associated with DH. Twenty‐five Dobermans with subclinical DH (SDH), 13 that clinically manifest DH (CDH) and 17 healthy controls were studied. Immunoblotting analysis detected specific antibodies in the DH sera. By mass spectrometry the targets were identified as liver‐related enzymes glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and alcohol dehydrogenase (ADH). Using ELISA, anti‐GAPDH IgG was detected in 36% (9/25) of SDH dogs and 69.2% (9/13) of the CDH dogs compared to healthy controls (0/17) (P < 0.0005). Anti‐ADH IgG was detected in 72% (18/25) of SDH dogs and 76.9% (10/13) of CDH dogs and only in one (1/17) control (P < 0.0005). The finding of novel autoantigens, GAPDH and ADH strengthen the hypothesis that DH is an autoimmune disease of the liver. These findings suggest that DH could be diagnosed by screening for autoantibodies against the defined antigens.     

Increasing sleep duration to lower beat‐to‐beat blood pressure: a pilot study

Strong evidence has accumulated over the last several years, showing that low sleep quantity and/or quality plays an important role in the elevation of blood pressure. We hypothesized that increasing sleep duration serves as an effective behavioral strategy to reduce blood pressure in prehypertension or type 1 hypertension. Twenty‐two participants with prehypertension or stage 1 hypertension, and habitual sleep durations of 7 h or less, participated in a 6‐week intervention study. Subjects were randomized to a sleep extension group (48 ± 12 years, N = 13) aiming to increase bedtime by 1 h daily over a 6‐week intervention period, or to a sleep maintenance group (47 ± 12 years, N = 9) aiming to maintain habitual bedtimes. Both groups received sleep hygiene instructions. Beat‐to‐beat blood pressure was monitored over 24 h, and 24‐h urine and a fasting blood sample were collected pre‐ and post‐intervention. Subjects in the sleep extension group increased their actigraphy‐assessed daily sleep duration by 35 ± 9 min, while subjects in the sleep maintenance condition increased slightly by 4 ± 9 min (P = 0.03 for group effect). Systolic and diastolic beat‐to‐beat blood pressure averaged across the 24‐h recording period significantly decreased from pre‐ to post‐intervention visit in the sleep extension group by 14 ± 3 and 8 ± 3 mmHg, respectively (P < 0.05). Though the reduction of 7 ± 5 and 3 ± 4 mmHg in the sleep maintenance group was not significant, it did not differ from the blood pressure reduction in the sleep extension group (P = 0.15 for interaction effect). These changes were not paralleled by pre‐ to post‐intervention changes in inflammatory or sympatho‐adrenal markers, nor by changes in caloric intake. While these preliminary findings have to be interpreted with caution due to the small sample size, they encourage future investigations to test whether behavioral interventions designed to increase sleep duration serve as an effective strategy in the treatment of hypertension.     

Efficacy and safety of the SQ‐standardized grass allergy immunotherapy tablet in mono‐ and polysensitized subjects

The efficacy of single‐allergen‐specific immunotherapy in polysensitized subjects is a matter of debate. We therefore performed a post hoc analysis of pooled data from six randomized, double‐blind, placebo‐controlled trials (N = 1871) comparing the efficacy and safety of the SQ‐standardized grass allergy immunotherapy tablet (AIT), Grazax (Phleum pratense 75 000 SQ‐T/2800 BAU, ALK, Denmark), in mono‐ and polysensitized subjects. A statistically significant reduction in the mean total combined symptom/medication score (TCS) of 27% was demonstrated in actively treated subjects compared with placebo (P < 0.0001). This was not dependent on sensitization status (P = 0.5772), suggesting a similar treatment effect in mono‐ and polysensitized subjects (i.e. reductions of the TCSs of 28% and 26%, respectively, both P < 0.0001). Finally, a comparable and favourable safety profile of grass AIT was demonstrated in the two subgroups. Thus, no difference in efficacy and safety of single‐allergen grass AIT was observed between mono‐ and polysensitized subjects.