Abnormal tumor necrosis factor receptor I cell surface expression and NF‐κB activation in tumor necrosis factor receptor–associated periodic syndrome

Objective

Tumor necrosis factor receptor–associated periodic syndrome (TRAPS) is an autosomal‐dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene. The cellular mechanisms by which mutations in this gene trigger inflammation are currently unclear. Because NF‐κB is the major intracellular signaling component inducing secretion of proinflammatory cytokines, we sought to determine whether differences in the clinical phenotype of patients with TRAPS may be attributable to variable effects of TNFRSF1A mutations on TNFRI expression, localization, or NF‐κB activity.

Methods

Peripheral blood mononuclear cells were obtained from patients (following informed consent), and cellular nuclear and cytosolic fractions were generated by subcellular fractionation. Localization of IκBα and NF‐κB was determined by Western blotting of the resultant fractions. NF‐κB subunit activity was determined by enzyme‐linked immunosorbent assay analysis and confirmed by electrophoretic mobility shift assay. Subcellular localization of TNFRI was determined by immunofluorescence confocal microscopy or by immunoblotting following affinity isolation of plasma membrane by subcellular fractionation.

Results

Cells from patients with the fully penetrant C73R mutation had marked activation of the proinflammatory p65 subunit of NF‐κB. In contrast, cells from patients with the low‐penetrant R92Q mutation displayed high levels of DNA binding by the p50 subunit, an interaction previously linked to repression of inflammation. Interestingly, although cells from patients with the C73R mutation have no TNFRI shedding defect, there was nonetheless an unusually high concentration of functional TNFRI at the plasma membrane.

Conclusion

High levels of TNFRI at the cell surface in patients with the C73R mutation hypersensitizes cells to stimulation by TNF, leading to increased NF‐κB p65 subunit activation and an exaggerated proinflammatory response.

     

Antiproliferative effects of cannabinoid agonists on deep infiltrating endometriosis

Deep infiltrating endometriosis (DIE) is characterized by chronic pain, hyperproliferation of endometriotic cells and fibrosis. Since cannabinoids are endowed with antiproliferative and antifibrotic properties, in addition to their psychogenic and analgesic effects, cannabinoid agonists have been evaluated in DIE both in vitro and in vivo. The in vitro effects of the cannabinoid agonist WIN 55212-2 were evaluated on primary endometriotic and endometrial stromal and epithelial cell lines extracted from patients with or without DIE. Cell proliferation was determined by thymidine incorporation and production of reactive oxygen species by spectrofluorometry. ERK and Akt pathways were studied by immunoblotting. Immunoblotting of α-smooth muscle actin was studied as evidence of myofibroblastic transformation. The in vivo effects of WIN 55212-2 were evaluated on Nude mice implanted with human deep infiltrating endometriotic nodules. The in vitro treatment of stromal endometriotic cells by WIN 55212-2 decreased cell proliferation, reactive oxygen species production, and α-smooth muscle actin expression. The decrease in cell proliferation induced by WIN 55212-2 was not associated with a decrease in ERK activation, but was associated with the inhibition of Akt activation. WIN 55212-2 abrogated the growth of endometriotic tissue implanted in Nude mice. Cannabinoid agonists exert anti-proliferative effects on stromal endometriotic cells linked to the inhibition of the Akt pathway. These beneficial effects of cannabinoid agonists on DIE have been confirmed in vivo.     

ALCOHOL AND BLOOD PRESSURE IN A WORKING POPULATION

1. The association between alcohol consumption and blood pressure was studied in 491 Government employees. The men, aged 21–45 years, volunteered to complete a health questionnaire and submitted to standardized measurements of blood pressure, heart rate and body size. 2. Average weekly alcohol consumption correlated with systolic pressure (R = 0.18, P<0.001) but not with diastolic pressure. Systolic pressure increased progressively with increasing alcohol consumption with no obvious threshold effect. An effect of alcohol was seen independent of age, obesity (Quetelet's index) or cigarette smoking. 3. Results indicate that alcohol ranks close to obesity as a preventable cause of essential hypertension in the community.     

Atopic dermatitis is associated with a low-producer transforming growth factor beta(1) cytokine genotype

BACKGROUND: Atopic dermatitis (AD) is associated with hyperresponsiveness of lymphocytes to allergens. In acute AD only T(H)2-type lymphocytes are activated, whereas in more chronic forms of AD, the activity of both T(H)1- and T(H)2-type lymphocytes increases. IL-10 and transforming growth factor beta(1) (TGF-beta(1)) are immunosuppressive cytokines that inhibit the activity of both T(H) cell types in human subjects. OBJECTIVE: The aim of this study was to determine whether children with moderately severe chronic AD had IL10 or TGFB1 genotypes known to be associated with low cytokine production. METHODS: Using amplification refractory mutation screening PCR, we examined TGFB1 and IL10 gene polymorphisms, which are known to affect cytokine production, in 68 children with moderately severe AD and in 50 nonatopic children. RESULTS: The odds ratio of children with AD having a low TGFB1 producer genotype was 4.8 (95% CI, 2.4--9.7) compared with the control subjects (P <.0001). There were no differences in the frequency of IL10 gene polymorphisms between groups. CONCLUSION: TGFB1 genotype may partly explain the strong genetic predisposition to AD.     

SAP mediates specific cytotoxic T-cell functions in X-linked lymphoproliferative disease

Cytotoxic T cells (CTLs) and natural killer cells play a major role in the immune response to Epstein-Barr virus (EBV) infection. In X-linked lymphoproliferative (XLP) disease, a severe immunodeficiency, immunodysregulatory phenomena are observed following EBV infection, suggesting that defects exist in these effector populations. The gene defective in XLP is SAP (signaling lymphocytic activation molecule [SLAM]-associated protein), an adaptor protein that mediates signals through SLAM and other immunoglobulin superfamily receptors including 2B4. We generated EBV-specific T-cell lines from controls and XLP patients and examined CTL function in response to different stimuli. We show that XLP patients can generate EBV-T-cell lines that are phenotypically similar to those from controls. XLP patient EBV-T-cell lines showed a significant decrease in interferon-gamma (IFN-gamma) production in response to 2B4 and autologous EBV-transformed lymphoblastoid cell line (LCL) stimulation but not in response to SLAM. Furthermore, XLP EBV-T-cell lines demonstrated markedly decreased cytotoxic activity against autologous LCLs. By retroviral gene transfer of the SAP gene into XLP EBV-T-cell lines, we show reconstitution of IFN-gamma production and of cytotoxic activity confirming SAP-dependent defects. These studies demonstrate that in XLP the lack of SAP affects specific signaling pathways resulting in severe disruption of CTL function.     

Mycobacterium vaccae reduces scratching behavior but not the rash in NC mice with eczema: a randomized, blinded, placebo-controlled trial

In a randomized, double-blinded, placebo-controlled trial, we previously showed that intra-dermal administration of a killed Mycobacterium vaccae suspension to school-aged children with atopic dermatitis ameliorates their disease. We wished to test the hypothesis that M. vaccae may also prevent the development of eczema. As it was not possible to do this in children, we studied the NC/Nga eczema mouse model. Thirty NC/Nga mice were randomized into a blinded, placebo-controlled trial where they received either 0.1 or 0.01 mg of M. vaccae (SRP299) or placebo given subcutaneously at 1 and 8 wk of age. Clinical eczema scores, as well as scratching frequency using a digital videotape system were assessed during the 26-wk study. Digital scratch scores correlated with clinical severity (p=0.001). Although there were no significant differences in age of onset or severity of the rash between the three study arms, mice injected with 0.1 mg but not 0.01 mg of SPR299 had significantly lower peak scratch frequencies than controls (Hazard ratio 0.2; 95% confidence interval 0.1-0.7; p=0.01). We conclude that in this NC/Nga mouse model, SRP299 did have a beneficial effect in reducing pruritis, a major clinical symptom of eczema, although it does not prevent the rash from developing.