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Background In primary aldosteronism (PA), aldosterone is inappropriately released into the systemic circulation and binds to the mineralocorticoid receptor (MR) in the distal tubule and collecting duct of the kidney. As a result, there is sodium reabsorption and potassium wasting leading to extracellular volume expansion, hypertension, hypokalemia, and, ultimately, suppressed renin. For medication management of aldosterone-producing adenomas with MR antagonists, spironolactone or eplerenone, are recommended. Finerenone is a novel, selective, non-steroidal MR antagonist approved July 2021 to reduce the risk of kidney and heart complications in patients with diabetic kidney disease. Interestingly, finerenone is at least as potent as spironolactone, and more selective for the MR (at least 500-fold) than eplerenone (1). We present a case using finerenone for the treatment of PA. Clinical Case OA 62-year-old male with CKD Stage G4/A3, T2DM, obesity, refractory hypertension and persistent hypokalemia underwent two radiofrequency ablations of a left adrenal nodule for the management of PA. Because of biochemical evidence for persistent hyperaldosteronism, pharmacologic therapy was initiated with eplerenone, which was chosen over spironolactone based on tolerability profile. Other antihypertensives included labetalol and nifedipine. Eplerenone was gradually titrated to 150 mg twice daily along with potassium replacement, but PRA remained below goal at 0.35 ng/mL/h with average BP 164/97 mmHg lying, 169/92 mmHg sitting, and 139/90 mmHg standing. Other pertinent labs included average SCr 4.62 mg/dL and eGFR 15 mL/min/1.73m2. A joint decision was made to replace eplerenone with finerenone 10 mg once daily. Two weeks after starting finerenone, PRA increased to 0.59 ng/mL/h, and SCr, eGFR, and potassium were 4.79 mg/dL, 14 mL/min/1.73m2, and 4.2 mEq/L, respectively. BP was 165/92 mmHg lying, 154/92 mmHg sitting, and 118/82 mmHg standing. Finerenone was increased to 20 mg once daily with close follow-up monitoring revealing an average PRA 0.42 ng/mL/h, SCr 4.28 mg/dL, eGFR 16 mL/min/1.73m2, potassium 3.5 mEq/L, and average BP 163/90 mmHg lying, 164/94 mmHg sitting, and 149/84 mmHg standing. Finerenone was well-tolerated and no changes were made to potassium replacement during this time. Conclusion Current MR antagonists, spironolactone and eplerenone, have concerns with tolerability due to minimal selectivity and efficacy, respectively. A new generation of MR antagonists, such as finerenone, with high potency and selectivity may lead to practice changes for management of PA. Based on our patient, finerenone increased PRA and similarly controlled BP compared to eplerenone. Further research will be required to evaluate finerenone for PA as a safe, effective, and convenient alternative MR antagonist, specifically with evaluation of dose-dependent effects beyond 20 mg daily as currently described in the literature. Reference 1. Kolkhof P, Bärfacker L. 30 years of the mineralocorticoid receptor: mineralocorticoid receptor antagonists: 60 years of research and development. J Endocrinol. 2017;234(1): T125-T140. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Population isolates such as Finland provide benefits in genetic studies because the allelic spectrum of damaging alleles in any gene is often concentrated on a small number of low-frequency variants (0.1% ≤ minor allele frequency < 5%), which survived the founding bottleneck, as opposed to being distributed over a much larger number of ultra--rare variants. While this advantage is well-- established in Mendelian genetics, its value in common disease genetics has been less explored. FinnGen aims to study the genome and national health register data of 500,000 Finns, already reaching 224,737 genotyped and phenotyped participants. Given the relatively high median age of participants (63 years) and dominance of hospital-based recruitment, FinnGen is enriched for many disease endpoints often underrepresented in population-based studies (e.g., rarer immune-mediated diseases and late onset degenerative and ophthalmologic endpoints). We report here a genome-wide association study (GWAS) of 1,932 clinical endpoints defined from nationwide health registries. We identify genome--wide significant associations at 2,491 independent loci. Among these, finemapping implicates 148 putatively causal coding variants associated with 202 endpoints, 104 with low allele frequency (AFWe studied a benchmark set of 15 diseases that had previously been investigated in large genome-wide association studies. FinnGen discovery analyses were meta-analysed in Estonian and UK biobanks. We identify 30 novel associations, primarily low-frequency variants strongly enriched, in or specific to, the Finnish population and Uralic language family neighbors in Estonia and Russia.These findings demonstrate the power of bottlenecked populations to find unique entry points into the biology of common diseases through low-frequency, high impact variants. Such high impact variants have a potential to contribute to medical translation including drug discovery.

In the era of precision medicine, digital technologies and artificial intelligence, drug discovery and development face unprecedented opportunities for product and business model innovation, fundamentally changing the traditional approach of how drugs are discovered, developed and marketed. Critical to this transformation is the adoption of new technologies in the drug development process, catalyzing the transition from serendipity-driven to data-driven medicine. This paradigm shift comes with a need for both translation and precision, leading to a modern Translational Precision Medicine approach to drug discovery and development. Key components of Translational Precision Medicine are multi-omics profiling, digital biomarkers, model-based data integration, artificial intelligence, biomarker-guided trial designs and patient-centric companion diagnostics. In this review, we summarize and critically discuss the potential and challenges of Translational Precision Medicine from a cross-industry perspective.

Summary Background Patients with advanced gastric or gastro-oesophageal junction cancer that progresses on chemotherapy have poor outcomes. We compared pembrolizumab with paclitaxel in patients with advanced gastric or gastro-oesophageal junction cancer that progressed on first-line chemotherapy with a platinum and fluoropyrimidine. Methods This randomised, open-label, phase 3 study was done at 148 medical centres in 30 countries. Eligible patients were randomised (1:1) in blocks of four per stratum with an interactive voice-response and integrated web-response system to receive either pembrolizumab 200 mg every 3 weeks for up to 2 years or standard-dose paclitaxel. Primary endpoints were overall survival and progression-free survival in patients with a programmed cell death ligand 1 (PD-L1) combined positive score (CPS) of 1 or higher. Safety was assessed in all patients, irrespective of CPS. The significance threshold for overall survival was p=0·0135 (one-sided). This trial is registered at ClinicalTrials.gov, number NCT02370498. Findings Between June 4, 2015, and July 26, 2016, 592 patients were enrolled. Of the 395 patients who had a PD-L1 CPS of 1 or higher, 196 patients were assigned to receive pembrolizumab and 199 patients were assigned to receive paclitaxel. As of Oct 26, 2017, 326 patients in the population with CPS of 1 or higher had died (151 [77%] of 196 patients in the pembrolizumab group and 175 [88%] of 199 patients in the paclitaxel group). Median overall survival was 9·1 months (95% CI 6·2–10·7) with pembrolizumab and 8·3 months (7·6–9·0) with paclitaxel (hazard ratio [HR] 0·82, 95% CI 0·66–1·03; one-sided p=0·0421). Median progression-free survival was 1·5 months (95% CI 1·4–2·0) with pembrolizumab and 4·1 months (3·1–4·2) with paclitaxel (HR 1·27, 95% CI 1·03–1·57). In the total population, grade 3–5 treatment-related adverse events occurred in 42 (14%) of the 294 patients treated with pembrolizumab and 96 (35%) of the 276 patients treated with paclitaxel. Interpretation Pembrolizumab did not significantly improve overall survival compared with paclitaxel as second-line therapy for advanced gastric or gastro-oesophageal junction cancer with PD-L1 CPS of 1 or higher. Pembrolizumab had a better safety profile than paclitaxel. Additional trials of pembrolizumab in gastric and gastro-oesophageal cancer are ongoing. Funding Merck Sharp & Dohme, a subsidiary of Merck & Co.

We examined a large cohort of systemic lupus erythematosus (SLE) patients and found an unusual B cell subset highly expanded. These B cells displayed a unique phenotype and expressed antigens not present on other B cell populations, including high densities of CD11c, FcRL5, and the T-box transcription factor, T-bet, but unexpectedly, were CD40lo and CD24−. Although these CD11chi cells were largely negative for the memory B cell antigen CD27, their telomere length was consistent with memory, rather than naïve B cells. Notably, the increase of CD11chi B cells in SLE significantly correlated with disease severity scores as well as with other markers of disease activity including serum IL-21. While memory B cells characteristically express low densities of IL-21R, these memory CD11chi B cells were IL-21R bright. In culture, IL-21 was a potent driver of CD11c expression when B cells were co-stimulated through the B cell receptor and CD40. Lastly, CD11chi B cells in SLE significantly correlated with blood plasma cells as well as a distinct set of autoantibodies, and importantly, were efficiently driven to differentiate into plasma cells in response to activated T cells. These data indicate that among the many roles of IL-21 in regard to B cell activation and differentiation, this pleiotropic cytokine also regulates CD11chi B cells, and presumably contributes to autoimmunity through the differentiation of autoreactive CD11chi B cells in SLE.

Background Methotrexate (MTX) remains a cornerstone of treatment in multiple forms of inflammatory arthritis. AMPK is a highly conserved protein kinase present in all eukaryotic cells and is activated by an increasing intracellular ADP/ATP levels. Once activated, AMPK will promote ATP production by switching on catabolic and turning off anabolic pathways. We hypothesize that AMPK activation mediates a major portion of the anti-inflammatory effects of MTX. Methods We investigated the role of the anti-inflammatory effect of MTX via AMPK in human monocytes-derived macrophages (MDM) and mouse bone marrow-derived macrophages (BMDM) along with AICAR and A769662 (AMPK activators) and compound C (a selective AMPK inhibitor). AMPK phosphorylation and total AMPK were measured by Western blotting. Cells were then stimulated with LPS or TNF-α, and production of pro-inflammatory cytokines were measured in the supernatant. Results MTX induced AMPK activation with effects comparable to the AMPK activators (A769662, AICAR ) in hMDM and BMDM. MTX-induced AMPK activation was associated with a reduction in the production of IL-6, IL-1 β, and TNF-α in response to LPS and TNF stimulation. Compound C is able to partially reverse the effects of MTX. Conclusions MTX is able to induce AMPK activation in both MDM and BMDM, and suppress pro-inflammatory cytokines in a manner dependent on AMPK activity. These results have been confirmed genetically in macrophages deficient in AMPK subunits and in serum transfer arthritis. Our findings raise the possibility that some anti-inflammatory effects of MTX are mediated by AMPK, suggest that AMPK may be a target for the anti-inflammatory agents and a target for the development of new anti-inflammatory drugs.

Objectives: Insulin resistance (IR), which plays a fundamental role in the pathogenesis of metabolic syndrome and type 2 diabetes mellitus, is associated with serum levels of inflammatory markers and abdominal obesity. Whether the association of inflammation with IR is independent of obesity remains unresolved; moreover, whether this association varies with gender and urban living is not known. We therefore conducted a cross-sectional study to investigate the gender specific association between IR and high sensitive C-reactive protein (HsCRP), independent of abdominal obesity, in urban and rural communities in a South Asian population. Methods: We conducted a population-based cross-sectional survey in 8,080 South Indians above the age of 20 years in both urban and rural environments. The study assessed of waist circumference (WC), blood pressure (BP), socioeconomic status, family history, anxiety and stress levels, and fasting blood for plasma glucose, insulin, total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) and HsCRP. IR was calculated using the homeostasis model assessment (HOMA-IR). After the exclusion of individuals with diabetics, 6,309 subjects (mean age 42 years; 58% women) were eligible and constituted the study sample. Results: The HOMA-IR level (mean ± SD) was higher in women (1.98 ± 1.62), when compared to men (1.71 ± 1.38), and HsCRP levels were 4.00 ± 5.14 and 3.42 ± 7.22 mg/dl in women and men, respectively. There was stronger correlation of HsCRP with HOMA-IR in women (r= 0.371) when compared to men (r= 0.203). In a multiple regression analysis, after adjusting for WC and other co-variants such as, BP, TC, LDL, TG, socioeconomic status, physical activity and family history of diabetes, HsCRP continued to be a significant predictor only in women (p Conclusion: HsCRP is associated with IR independent of abdominal obesity in non-diabetic South Asian women, and at elevated HsCRP levels the association is stronger in urban women.

Human monoclonal antibodies (MAbs) offer potential advantages over murine MAbs for therapy because they are not likely to elicit immune responses and are expected to interact more efficiently with the human immune system to activate therapeutically useful functions. Traditional methods for obtaining human MAbs (i.e., immortalization of B cells by cell fusion or transformation) can result in low and unstable antibody secretion. Recently, methods have been devised for direct cloning of human variable region genes via polymerase chain reaction and phage combinatorial libraries. Both types of human MAb production can benefit from expression systems that support the stable, high-level antibody secretion required for therapeutic use. Using an existing human-derived hybridoma that secretes a human IgM antibody as a convenient source of antibody genes, we have demonstrated that cloned human antibody genes can be efficiently expressed in murine myeloma cells and that cell lines with properties suitable for large-scale economical production can be obtained. We were unable to detect any differences between the antibodies produced by the original hybridoma and the engineered cell line. In addition, we were able to express an IgG form of the antibody, showing that expression of a recombinant human antibody need not be limited to the original antibody class.

One of the main functions of the adaptive immune system is to mount specific responses to pathogens. These effector CD4 T helper cells, include Th1, Th2, Th9 and Th17 cells, produce distinct cytokines important in the eradication of different infectious pathogens--however excessive activation of these cells can be harmful to the host, contributing to immunopathology, including autoimmunity and asthma. ITK-deficient mice exhibit reduced responses in models of allergic asthma, a disease attributed to increased Th2 responses. Polymorphisms in the Itk promoter in humans have also been linked to increased asthma incidence. Th9 cells are a recently appreciated T helper cell population that produce IL-9, a cytokine implicated in allergic asthma, responses to microbial infections, inflammatory gut diseases and autoimmunity. However, the factors contributing to IL-9 regulation are still poorly understood. We have found that ITK-deficient CD4 cells completely fail to produce IL-9 in culture. This defect is one of the most profound phenotypes we have observed for Itk-/- cells to date, and is associate with reduced levels of IRF4 during Th9 differentiation. Furthermore, in preliminary experiments, we found decreased IL-9 and IL-13 expression in vivo using a papain hypersensitivity model. Our studies demonstrate important roles for Itk in Th9 differentiation, suggesting Itk as a therapeutic candidate for treatment of diseases involving Th9-mediated inflammation.

To screen for potential efficacy and assess the feasibility of intravenous (IV) doxycycline as a treatment for rheumatoid arthritis (RA).The study was a (stratified, block) randomized, double blind, 12 week, pilot trial of IV doxycycline 300 mg/day versus identical appearing IV placebo given over 2 h for 14 days. The primary comparison was to a hypothesized placebo rate of 20% as described by Paulus. If a total of 14 consecutive subjects receiving doxycycline treatment did not respond, it would be considered futile to proceed to a Phase III trial. We planned a placebo group of 14 subjects to verify the placebo response rate and estimate sample size required for a definitive Phase III trial, if such a trial was warranted based on the pilot study. American College of Rheumatology (ACR) RA response criteria were used. After 23 subjects entered, the study was closed due to recruitment difficulties.At baseline, mean (SD) tender joint count was 37 (11.9), swollen joint count 30 (9.6), morning stiffness 317 (319) min, and erythrocyte sedimentation rate 72 mm/h (27.5). Randomization resulted in 10 subjects receiving doxycycline and 13 receiving placebo. Treatment was stopped in 8 subjects: in 6, treatment was ineffective (one taking doxycycline, 5 placebo), and in 2, rashes occurred (one taking doxycycline, one placebo). Only one subject met ACR response criteria in the doxycycline group and none in the placebo group. Having no responders in the placebo group was consistent with placebo response rate of 20% or less. Several patients required peripherally inserted central catheters for venous access.The efficacy of IV doxycycline as a treatment for RA could not be ruled out. However, as the proportion of responders was small, it is unlikely that potential efficacy of IV doxycycline would outweigh potential disadvantages of IV administration.

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells through its receptor DR3 (Tnfrsf25). Currently known effects of TL1A on T cell differentiation or systemic immune responses cannot account for the resistance of Tnfrsf25-/- mice to diverse animal models of autoimmune and allergic inflammation. IL-9 is a cytokine that promotes inflammatory and allergic immune responses and is expressed by a distinct subset of T cells early after T cell activation or restimulation. We have found that TL1A potently promotes generation of T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. Unlike the TNF family member OX40 ligand, TL1A increases Th9 differentiation independently of STAT6 and the TNF-signaling adaptor TRAF6. Instead, TL1A affects Th9 differentiation through enhancing IL-2-dependent STAT5 activation and allowing increased binding to the IL-9 promoter. In two models of allergic asthma, we demonstrate that Tnfrsf25-/- mice have reduced disease severity accompanied by fewer IL-9 producing T cells in the lung. In addition to promoting Th9 differentiation, we find that TL1A enhances pathogenicity in Th9 transfer models of both uveitis and allergic asthma, where endogenous TL1A signaling is required for maximal pathology and IL-9 production at the site of inflammation. Taken together, these data identify TL1A as a novel cytokine that promotes Th9 differentiation and pathogenicity and as a possible therapeutic target in diseases dependent on IL-9.

Fas-Fas ligand (FasL) interactions are critical for apoptosis and T cell homeostasis, with deficiencies in either Fas or FasL leading to autoimmunity in both mice and humans. Fas can trigger apoptotic cell death directly through recruitment of a death-inducing signaling complex (DISC) consisting of FADD and Caspase-8. We have previously shown a correlation between lipid raft-associated Fas and sensitivity to apoptosis in CD4+ T cells, with the Fas-sensitive memory population having increased lipid raft receptor localization. Palmitoylation of human Fas at cysteine 199 (Cys-194 in mouse) is required for localization to lipid rafts. To investigate the role of palmitoylation in Fas signaling, we developed a transgenic mouse defective in palmitoylation of Fas receptor (Fas C194→V) backcrossed to the Fas-defective lpr/lpr mouse (FasC194Vlpr/lpr). These mice developed autoimmunity characteristic of lpr mice, but CD4-CD8- “Double-Negative” (DN) T cells did not accumulate. T cells expressing C194V mutant Fas were resistant to Fas- and TCR-induced cell death, indicating lipid raft localization is required for the apoptotic function of Fas, but not accumulation of DN T cells. High-resolution PALM imaging of cells expressing wild-type or C194V mutant Fas revealed receptor clustering into higher-order oligomers at the plasma membrane required palmitoylation. These findings identify palmitoylation as a key post-translational modification of Fas that controls the apoptotic signal.

The Tumour Necrosis Factor Superfamily (TNFSF) are an important source of costimulatory signals required for activation of T cells. The TNFSF is composed of 29 receptors and 18 ligands; one such pair is Death receptor 3 (DR3) and TNF-like ligand 1A (TL1A). Signalling via DR3 costimulates lymphocytes and polymorphysims in TL1A and DR3 have been associated with Crohn's Disease. In mice, signalling via DR3 has been shown to be required for maximal pathology in models of asthma and lupus and transgenic constitutive expression of TL1A results in an IL-13 and IL-17a driven intestinal pathology. Several TNFSF ligands, including TL1A, can be expressed on the membrane and cleaved to yield a soluble product. Some cytokines, such as TNF are active in soluble form whereas others, such as FasL, are not. The biological roles of these distinct forms of TL1A in vivo are not known. To investigate the biological function of membrane vs. soluble TL1A we used retrotransgenic technology to express cleavable vs. membrane bound TL1A throughout the murine hematopoietic system. Membrane restricted TL1A promoted a strong inflammatory pathology in both the ileum and lung. Full length TL1A expression yielded little membrane TL1A. These mice failed to develop pathology despite a greater than physiological level of TL1A in the serum. This work demonstrated that membrane restricted TL1A is a more potent ligand for DR3. We have generated membrane-restricted TL1A transgenic mice to investigate this further.

Hypomorphic mutation of the NF-kB essential modulator (NEMO) causes a spectrum of inflammatory disease and immunodeficiency. NEMO is critical in immunity, regulating the activation of transcription factors NF-kB and IRF3 to control inflammation and antiviral type I interferon. In an individual with persistent inflammatory disease, we identified a synonymous mutation in the gene encoding NEMO that enhances alternative splicing and production of a mutant protein (Δ-NEMO). Co-immunoprecipitation studies in patient T cells revealed impaired association of Δ-NEMO with TANK (TRAF family member-associated NF-kB activator) and, consequently, with the IRF3 kinase TANK-binding kinase 1 (TBK1). Concomitantly, patient fibroblasts displayed impaired production of IRF3-dependent type I and III interferon in response to poly(I:C) and permitted increased virus propagation. However, cells containing Δ-NEMO exhibited enhanced IRF3 nuclear translocation and binding to the IFNB1 promoter. Our results suggest that defective interferon production in this patient lies instead in impaired NF-kB activation, as poly(I:C)- and 5’pppRNA induced translocation of NF-kB in patient fibroblasts was reduced. This impairment in NF-kB was specific, as TNF-induced NF-kB activation and gene induction was normal. These data describe a novel mechanism of autoinflammatory disease due to a type I interferon deficiency. Thus, this represents a model of inflammatory diseases responsive to type I IFN therapy.

The role of the conformation and bioactivity of adsorbed vitronectin in enhancing osteoblast (the bone-forming cells) adhesion on nanophase (that is, grain sizes less than 100 nm) alumina was investigated in the present in vitro study. Results obtained from surface-enhanced Raman scattering (SERS) provided the first evidence of increased unfolding of vitronectin adsorbed on nanophase alumina. This conclusion was further supported by dose-dependent inhibition of cell adhesion on nanophase alumina pretreated with vitronectin following preincubation of osteoblasts with either Arginine-Glycine-Aspartic Acid-Serine (RGDS) or Lysine-Arginine-Serine-Arginine (KRSR) to block respective cell-membrane receptors. These events, namely enhanced unfolding of vitronectin that leads to exposure of bioactive epitopes (such as RGDS) in adsorbed vitronectin may explain the observed increased osteoblast adhesion on nanophase alumina.

The Fas death receptor mediates peripheral tolerance by apoptotic deletion of autoreactive T cells through Restimulation Induced Cell Death (RICD). Previously, we have shown that within a pool of T cells, effector memory were more sensitive to Fas-induced apoptosis than activated naïve CD4 T cells due to efficient DISC formation. Recently, we found that FasL induces an early MAPK response in the form of pJNK activation, preferentially in activated naïve, but not memory T cells. In apoptosis resistant FADD or Caspase-8 deficient Jurkat T cells, the pJNK response is exaggerated indicating that in the absence of a functional Fas apoptotic response, an alternate non-apoptotic pathway is elicited. Further, in HH cells, a CTCL resistant to Fas apoptosis, pJNK activation was upregulated when compared to HUT78, a highly apoptosis sensitive cell line. In an attempt to identify the upstream activators of Fas-induced pJNK, we studied the role of the kinase RIP1, a known component of the Fas DISC. Inhibition of RIPK1 decreased pJNK activation in Jurkat cells and RIP deficient Jurkat cells were unable to activate pJNK upon FasL stimulation. Immunoprecipitation studies also indicated a preferential recruitment of RIPK1 to the Fas DISC in apoptosis resistant T cells. These preliminary results indicate a role for Fas mediated non-apoptotic MAPK signaling in early activated naïve T cells that precedes its traditional apoptotic role in the elimination of effector memory cells.

The TNF-family cytokine TL1A costimulates T cells through its receptor DR3 and is required for diverse autoimmune disease models, but its role in innate lymphocyte biology has not been explored. Transgenic mice chronically expressing TL1A have increased activated T cells and develop small intestinal pathology characterized by high levels of IL-13, muscular and goblet cell hyperplasia, and infiltration with immune cells, all of which depend on DR3. Genetically eliminating T cells reduces T cell hyperactivation and gut infiltration with lymphocytes but not IL-13 production or mucosal hyperplasia. These data suggest that TL1A can induce another cell type to produce IL-13 and induce gastrointestinal pathology. Using a DsRED reporter mouse for IL-13, we find that the major IL-13 producing cells in TL1A transgenic mice lack T and B cell lineage markers and are phenotypically similar to ’type 2’ innate lymphocytes (ILC2) which expand and produce IL-13 in response to IL-25, IL-33 or parasitic infection. TL1A directly promotes IL-13 production by ILC2 ex-vivo. However, DR3 deficient mice mount a vigorous response to the intestinal nematode Nippostrongylus brasiliensis, and IL-13 producing innate lymphocytes can be expanded by IL-25 in the absence of DR3. Thus, TL1A can directly induce IL-13 production by innate lymphocytes through mechanisms distinct from parasitic infection. TL1A may be an attractive target for immunotherapy of diverse diseases associated with this cytokine.

The RIG-I-like receptors mediate antiviral signaling through NEMO-dependent activation of transcription factors NF-kB and IRF3, which are recognized for their roles in inflammation and the induction of Type I Interferon, respectively. We identified an individual with increased susceptibility to viral infection and persistent inflammatory disease. Sanger sequencing revealed a de novo synonymous mutation in the gene encoding NEMO that results in an in-frame splicing event and exclusive production of a shortened protein. To understand the role of NEMO in regulating the antiviral response, we stimulated patient and healthy control fibroblasts with RIG-I-like receptor ligand poly(I:C), which mimics viral RNA. Induction of IRF3 response genes IFNB1 and CXCL10 was reduced 35-fold and 500-fold (n=2), respectively, compared to healthy control, and patient cells were unable to control viral infection. In contrast, TNF induced the expression of proinflammatory genes TNFSF10 and IL17 7-fold and 4-fold higher in patient cells than in healthy control cells. Increased TNF induced gene expression was accompanied by increased p65 nuclear translocation indicative of increased canonical IKK activation. These results suggest that aberrant expression of an alternately spliced NEMO isoform constitutes a novel autoinflammatory disease characterized by unbalanced NF-kB and IRF3 mediated responses to viral infection.

Clearance of activated T cells by the Fas-FasL pathway is a critical mechanism of peripheral CD4 T cell tolerance. This process is preferentially higher in the effector subset of memory CD4+ T cells due to efficient recruitment and formation of the Fas death inducing signaling complex (DISC). In order to elucidate mechanisms of this heightened apoptosis sensitivity, we tested for differential DISC interacting proteins in Fas sensitive and Fas resistant CTCL cell lines using 2D-DIGE and Mass Spectrometry. We found several candidate proteins and invitro studies were done to confirm Fas receptor interactions through overexpression and Flourescence Energy Transfer (FRET) studies. One of these targets, Drebrin, is an actin binding protein previously found to play a role in T cell synapse formation by facilitating CXCR4 recruitment. Overexpression in 293 indicates that Drebrin interacts with Fas receptor. Further, endogenous drebrin preassociates with Fas receptor and remains associated with the Fas DISC even after Fas ligation only in Type I SKW cell line. Interestingly, Fas DISC associated Drebrin gets cleaved after Fas treatment and invitro caspase cleavage studies indicate that drebrin is a specific downstream target of caspase-8. Our studies propose Drebrin as a novel Fas interacting protein. Investigations are currently underway to study the mechanisms by which Drebrin modifies the Fas-DISC dynamics and the significance of the caspase-cleaved Drebrin in this process.

Autoreactive CD4+ T cells can be eliminated by the TNF superfamily member Fas through a process termed Restimulation Induced Cell Death (RICD). Previously, we have shown that effector memory CD4 T cells were the most Fas-induced apoptosis sensitive CD4 subset. This was due to efficient recruitment and activation of components of the death-inducing signaling complex (DISC) indicating a role for early signaling events in apoptosis regulation. To discover novel proteins recruited to the DISC, we used 2D-DIGE and Mass Spectrometry on Fas sensitive and resistant CTCL lines to identify differential binding partners in the Fas-DISC. We have identified a number of proteins including a SNARE family protein previously found to be involved in exocytosis. In transfection experiments, this protein interacted with Fas in a manner independent of the Fas death domain as shown by co-immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) between constructs fused to CFP and YFP. The endogenous protein preferentially interacted with Fas in cell lines and T cell subsets susceptible to Fas-induced cell death. These results suggest that that this protein is a novel component of the DISC and may regulate early events in Fas signaling, possibly through affecting receptor endocytosis or recycling to the plasma membrane. The mechanism by which this protein affects assembly and activation of the Fas DISC, and other proteins found in this proteomic screen are currently being investigated.

Mutations in the type 1 TNF receptor (TNFR1) cause the autoinflammatory disorder TNF receptor-associated periodic syndrome (TRAPS), a disease characterized by recurrent fevers with inflammation. TRAPS-associated mutations in TNFR1 cause misfolding, retention in the ER, and loss of function as a conventional receptor, but instead initiate an intracellular signaling cascade that results in hyper-responsiveness to innate stimuli. Using mice engineered to have TRAPS-associated mutations in TNFR1, we examined the contents of peritoneal lavage fluid after intraperitoneal LPS injection in order to identify the pathogenic cell type. TNFR1 mutant mice have increased monocyte/macrophage infiltrates when compared to wild-type mice, but a similar number of recruited neutrophils. In mixed marrow chimeras, wild-type and TNFR1 mutant neutrophils were recruited similarly into the peritoneum after LPS injection, suggesting that there is no cell-intrinsic predisposition of TNFR1 mutant neutrophils to accumulate at sites of inflammation. In support of this, neutrophils from TRAPS patients and healthy donors migrate similarly in response to CXCL8. In contrast, we found that murine peritoneal macrophages from TNFR1 mutant mice produce higher amounts of two potent chemokines, CXCL1 and CXCL2, in response to LPS in vitro. These results suggest that enhanced chemokine secretion by monocytes, rather than hyper-responsiveness of neutrophils to these stimuli, is critical to the pathogenesis of TRAPS.

Fas-FasL interactions play a critical role in TCR-induced apoptosis of activated CD4+ T cells. Fas deficiency prompts accumulation of CD44+ memory T cells and autoimmunity in mice, suggesting Fas plays a role in memory and/or autoreactive T cell apoptosis. We have recently shown that CD4+ effector memory cells were sensitive to Fas-induced cell death, and also a correlation between lipid raft-associated Fas and sensitivity to apoptosis. A critical cysteine residue near the transmembrane helix of Fas has been shown to be responsible for its association with lipid rafts. Whether the lipid raft association of Fas is regulated in different primary cell types is not known. We have compared the post-translational modification state and membrane localization of Fas receptor in purified naïve versus memory human CD4+ T cells. Memory T cells have increased lipid raft-associated Fas receptor versus naïve cells, which could explain the increased Fas sensitivity in the memory pool. Using a competitive inhibitor to palmitoylation, we found the sensitivity of effector memory T cells to Fas-induced apoptosis depended on Fas palmitoylation. Utilizing click chemistry to analyze the palmitoylation state of Fas showed increased receptor palmitoylation in cells sensitive to Fas-mediated apoptosis compared to resistant cells. These results suggest post-translational palmitoylation and lipid raft localization of Fas as a regulatory mechanism in Fas-induced apoptosis in memory T cells.

The death receptor Fas plays an essential role in TCR-induced apoptosis of activated CD4+ T cells, in a process termed restimulation-induced cell death (RICD). However, within a heterogeneous population of human CD4+ T cells, we have found that cells with an effector memory phenotype (CCR7-CD27-) are specifically programmed for sensitivity to Fas-induced apoptosis and RICD. Similary, mouse effector memory T cells (CD44hiCD62Llo) are the most sensitive to cells are very sensitive to RICD and direct killing by Fas receptor ligation. Fas resistant ‘central memory’ as well as Fas-sensitive ‘effector’ cells had similar levels of induced FasL, surface Fas, as well as pro-survival Bcl2/BCLxL levels. In ALPS patients with Fas mutations, resistance to Fas apoptosis was found predominantly in the effector memory subsets. Biochemical studies show a preponderance of lipid raft localized Fas and highly efficient formation of the death-inducing signaling complex (DISC) in memory and effector T cells. Thus sensitivity to TCR and Fas-mediated apoptoisis is mediated at the level of early signaling events in the Fas pathway. These observations may explain the specificity of the Fas/FasL system in T cell homeostasis toward chronically expressed and endogenous antigens to check autoreactivity and responses to chronic infections.

Fas-FasL interactions play a critical role in TCR-induced apoptosis of activated CD4+ T cells. However, naïve T cells activated through acute antigen stimulation expand and contract independently of the presence of the death receptor Fas. Fas deficiency prompts the accumulation of CD44+ memory phenotype T cells and autoimmunity in mice, suggesting Fas may play a critical role in memory and/or autoreactive T cell apoptosis. We have previously shown a correlation between lipid raft-associated Fas and sensitivity to apoptosis. Whether or not lipid raft microdomains influence Fas signaling in primary T cells, and whether lipid raft association of Fas is regulated in different primary cell types is unknown. Therefore, we have compared the sensitivity of purified naïve versus memory human CD4+ T cells to undergo Fas-mediated apoptosis. Effector memory phenotype cells lacking both the chemokine receptor CCR7 and the TNFSFR CD27 were the most sensitive to both anti-Fas antibody as well as FasL-induced cell death. Memory T cells have increased lipid raft-associated Fas receptor versus naïve cells, which could explain the increased Fas sensitivity in the memory pool. Using a competitive inhibitor to palmitoylation, we found sensitivity of effector memory T cells to Fas-induced apoptosis depended on Fas palmitoylation. These results suggest that effector memory cells are intrinsically more sensitive to Fas-induced apoptosis, possibly due to lipid raft localization of Fas.

Mutations in the type 1 TNF receptor (TNFR1) cause an autosomal-dominantly inherited autoinflammatory disorder called TNF receptor-associated periodic syndrome (TRAPS). The disease is characterized by recurrent fevers with abdominal pain, migratory rash and inflammation. Patients also develop serositis, including peritonitis, as a manifestation of disease flares. TRAPS-associated mutant receptors do not traffic to the cell surface and accumulate in the endoplasmic reticulum, which may lead to abnormal TNF-independent receptor signaling and sustained inflammation. We examined the contents of peritoneal lavage fluid after intraperitoneal injection of LPS in TNFR1 mutant mice. Interestingly, heterozygous TNFR1 mutant mice had increased neutrophil infiltrates into the peritoneal cavity after LPS injection. We have investigated whether the exaggerated infiltration of neutrophils in mice with TNFR1 mutations is due to an intrinsic neutrophil abnormality or the release of more neutrophil-attracting chemokines by macrophages. We have performed chemotaxis experiments using neutrophils isolated from TRAPS patients as well as TNFR1 mutant mice, and measured the migratory responses of neutrophils toward chemoattractants and macrophage-derived supernatants. These results will provide insights into whether the intracellularly retained mutant TNFR1 may function to enhance local neutrophil accumulation in response to inflammatory stimuli in a cell autonomous manner.

One of the mechanisms of peripheral tolerance in CD4 T cells, mediated by the TNF superfamily member Fas/FasL, occurs through a process termed Restimulation Induced Cell Death. Previously, we showed that naïve and memory activated CD4 T cell subsets vary in their response to Fas apoptosis, with the effector memory subsets having the highest Fas sensitivity. Since this phenotype was maintained stably in activated cell cultures, genetic or epigenetic regulation is likely. To examine the role of miRNAs in regulating the phenotype of effector memory (Em) T cells, we performed a miRNA array via Solexa sequencing to profile miRNAs differentially expressed in CD4 T cell subsets. One of these, miR181a, was upregulated in naïve and Cm, but down modulated in memory and Em cells. This downregulation correlated with the upregulation of its target genes including DUSP6. Mir181a is required for positive selection of T cells in the thymus, however, its function in peripheral TCR signaling is unknown. To probe the functional consequences of differential miR181a expression in peripheral T cell subsets we utilized a siRNA knockdown and lentiviral overexpression approach. Thus far we have not observed overt differences in Fas sensitivity, however, we have seen skewing of cytokine responses in miR181a knockdown, including changes in IL-17 and IFN-g expression following TCR triggering. These results suggest a role for miR-181a in regulating certain aspects of the effector memory T cell phenotype.

DR3 is a TNF-receptor specifically expressed on lymphocytes which can costimulate lymphocyte activation. Its ligand, TL1A, can be produced by at least 3 different cell subsets: endothelial cells, lymphocytes and DC. TL1A and DR3 have been reported to be upregulated in biopsy samples and in animal models of IBD, providing circumstantial evidence of the involvement of this TNF ligand-receptor pair in IBD. We have generated TL1A transgenic mice that constitutively express TL1A by either T-cells or DC. Both of these transgenic lines develop spontaneous IBD. Constitutive expression of TL1A by T cells leads to a severe pathology found primarily in the ileum and duodenum in all founders, from an early age. Consistent with the role of TL1A as a T cell costimulator, the T cell population had an activated phenotype and increased effector subsets. Surprisingly, despite the inflammation, we observed increased FoxP3+ cells. Interestingly, IL-13 and to a lesser extent IL-17 are the cytokine mRNA that are the most elevated in the gut. In contrast, constitutive expression of TL1A by DC leads to a milder pathology that is more restricted to the ileum, and is detectable in older mice. Ectopic expression of TL1A appears to be able to overcome T-cell self-tolerance and drive IBD with no other overt stimuli. These findings suggest that TL1A may be an interesting target for therapy.

Laws committing dangerous sexual offenders indefinitely are legitimate only insofar as "dangerousness" can be predicted and treated. The court has relied on the clinician to identify and measure the crucial variables in violent behavior and assign a probability of occurrence. Yet most critical studies have concluded that not only has predictive accuracy not been demonstrated but also that such accuracy cannot be achieved. We argue that any conclusion at this time is premature. There are data indicating the soundness of clinical as opposed to statistical prediction of dangerous ness. Both approaches show high false positive error rates, but the im provement in accuracy of clinical studies over statistical studies cannot be denied. In examining the success of treatment, the authors present data indi cating that a program of psychological rehabilitation can have a profound effect on the majority of dangerous sexual offenders. The authors conclude that the indefinite commitment of dangerous offenders is legitimate and appropriate, but the policy carries with it heavy responsibilities. Enforcing such an approach means that the clinician must play an important role in his relationship to the court.

On presente l'observation d'une jeune femme apres une mastectomie radicale et une greffe de peau pour demontrer qu'une greffe de peau peut assurer une couverture suffisante pour reconstruire le sein avec un implant de silicone

A study that explored children's understanding of the body and illness was conducted with 23 thalassemic outpatients and 27 healthy controls. The findings were supportive of a developmental progression in children's understanding that is reflective of their underlying cognitive maturity. Behavioral compliance was related to psychiatric adjustment. There was a trend toward poorer psychiatric adjustment in children with impaired understanding of their illness. These findings hold implications for both educational and psychiatric intervention with this group. Optimal health care of chronically ill children requires knowledge of the developmental course and interplay of children's cognition and their familial and personal adjustment.

International audience; The self-activation of initiator caspases is dependent on their oligomerization driven by interaction with the death fold domains (DFD) of adaptor proteins. Here, we show that the E2 protein of human papillomavirus type 18 triggers apoptosis by assembling cytoplasmic filaments together with caspase 8, in which its efficient self-activation occurs. The E2 protein binds directly to the death effector domains (DED) of caspase 8 through non-DFD interaction. This interaction is independent of FADD, but it can cooperate with FADD homotypic binding to caspase 8 to induce its oligomerization; hence cell death, while it is antagonized by competitive binding of MC159 FLICE inhibitory protein. The amino-terminal domain of E2 contains a 27 amino-acid a-helix, which is necessary and sufficient to induce caspase oligomerization and cell death. Our results provide evidence for adaptor-independent oligomerization of caspase 8, mediated by non-DFD direct interactions with the HPV18 E2 protein, thus deciphering a new pathway for caspase 8 activation.