Apoptosis induced by death receptors.

Death receptors belong to the TNF receptor family and are characterised by an intracellular death domain that serves to recruit adapter proteins such as TRADD and FADD and cysteine proteases such as Caspase-8. Activation of Caspase-8 on the aggregated receptor leads to apoptosis. Triggering of death receptors is mediated through the binding of specific ligands of the TNF family, which are homotrimeric type-2 membrane proteins displaying three receptor binding sites. There are various means of modulating the activation of death receptors. The status of the ligand (membrane-bound vs. soluble) is critical in the activation of Fas and of TRAIL receptors. Cleavage of membrane-bound FasL to a soluble form (sFasL) does not affect its ability to bind to Fas but drastically decreases its cytotoxic activity. Conversely, cross-linking epitope-tagged sFasL with anti-tag antibodies to mimic membrane-bound ligand results in a 1000-fold increase in cytotoxicity. This suggests that more than three Fas molecules need to be aggregated to efficiently signal apoptosis. Death receptors can also be regulated by decoy receptors. The cytotoxic ligand TRAIL interacts with five receptors, only two of which (TRAIL-R1 and -R2) have a death domain. TRAIL-R3 is anchored to the membrane by a glycolipid and acts as a dominant negative inhibitor of TRAIL-mediated apoptosis when overexpressed on TRAIL-sensitive cells. Intracellular proteins interacting with the apoptotic pathway are potential modulators of death receptors. FLIP resembles Caspase-8 in structure but lacks protease activity. It interacts with both FADD and Caspase-8 to inhibits the apoptotic signal of death receptors and, at the same time, can activate other signalling pathways such as that leading to NF-kappa B activation.

Monoubiquitination and Activity of the Paracaspase MALT1 Requires Glutamate 549 in the Dimerization Interface.

The mucosa-associated lymphoid tissue protein-1 (MALT1, also known as paracaspase) is a protease whose activity is essential for the activation of lymphocytes and the growth of cells derived from human diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL). Crystallographic approaches have shown that MALT1 can form dimers via its protease domain, but why dimerization is relevant for the biological activity of MALT1 remains largely unknown. Using a molecular modeling approach, we predicted Glu 549 (E549) to be localized within the MALT1 dimer interface and thus potentially relevant. Experimental mutation of this residue into alanine (E549A) led to a complete impairment of MALT1 proteolytic activity. This correlated with an impaired capacity of the mutant to form dimers of the protease domain in vitro, and a reduced capacity to promote NF-κB activation and transcription of the growth-promoting cytokine interleukin-2 in antigen receptor-stimulated lymphocytes. Moreover, this mutant could not rescue the growth of ABC DLBCL cell lines upon MALT1 silencing. Interestingly, the MALT1 mutant E549A was unable to undergo monoubiquitination, which we identified previously as a critical step in MALT1 activation. Collectively, these findings suggest a model in which E549 at the dimerization interface is required for the formation of the enzymatically active, monoubiquitinated form of MALT1.

Resistance to nucleoside analog reverse transcriptase inhibitors mediated by human immunodeficiency virus type 1 p6 protein.

Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within the pol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6(gag)-p6(pol) region of HIV-1, placed immediately upstream of pol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6(Gag)), or by changes in activation of the viral protease (p6(Pol)). Duplication of the proline-rich p6(Gag) PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naïve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6(Pol)), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.

Degradation of ZAP-70 following antigenic stimulation in human T lymphocytes: role of calpain proteolytic pathway.

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.

Purification of recombinant proteins by chemical removal of the affinity tag.

The efficient removal of a N- or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and a Plasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag in Escherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.

Outcomes of patients on dual-boosted PI regimens: experience of the Swiss HIV cohort study.

Dual-boosted protease inhibitors (DBPI) are an option for salvage therapy for HIV-1 resistant patients. Patients receiving a DBPI in the Swiss HIV Cohort Study between January1996 and March 2007 were studied. Outcomes of interest were viral suppression at 24 weeks. 295 patients (72.5%) were on DBPI for over 6 months. The median duration was 2.2 years. Of 287 patients who had HIV-RNA >400 copies/ml at the start of the regimen, 184 (64.1%) were ever suppressed while on DBPI and 156 (54.4%) were suppressed within 24 weeks. The median time to suppression was 101 days (95% confidence interval 90-125 days). The median number of past regimens was 6 (IQR, 3-8). The main reasons for discontinuing the regimen were patient's wish (48.3%), treatment failure (22.5%), and toxicity (15.8%). Acquisition of HIV through intravenous drug use and the use of lopinavir in combination with saquinavir or atazanavir were associated with an increased likelihood of suppression within 6 months. Patients on DBPI are heavily treatment experienced. Viral suppression within 6 months was achieved in more than half of the patients. There may be a place for DBPI regimens in settings where more expensive alternates are not available.

Colon-specific deletion of epithelial sodium channel causes sodium loss and aldosterone resistance.

Aldosterone promotes electrogenic sodium reabsorption through the amiloride-sensitive epithelial sodium channel (ENaC). Here, we investigated the importance of ENaC and its positive regulator channel-activating protease 1 (CAP1/Prss8) in colon. Mice lacking the αENaC subunit in colonic superficial cells (Scnn1a(KO)) were viable, without fetal or perinatal lethality. Control mice fed a regular or low-salt diet had a significantly higher amiloride-sensitive rectal potential difference (∆PDamil) than control mice fed a high-salt diet. In Scnn1a(KO) mice, however, this salt restriction-induced increase in ∆PDamil did not occur, and the circadian rhythm of ∆PDamil was blunted. Plasma and urinary sodium and potassium did not change with regular or high-salt diets or potassium loading in control or Scnn1a(KO) mice. However, Scnn1a(KO) mice fed a low-salt diet lost significant amounts of sodium in their feces and exhibited high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAP1/Prss8 in colonic superficial cells (Prss8(KO)) were viable, without fetal or perinatal lethality. Compared with controls, Prss8(KO) mice fed regular or low-salt diets exhibited significantly reduced ∆PDamil in the afternoon, but the circadian rhythm was maintained. Prss8(KO) mice fed a low-salt diet also exhibited sodium loss through feces and higher plasma aldosterone levels. Thus, we identified CAP1/Prss8 as an in vivo regulator of ENaC in colon. We conclude that, under salt restriction, activation of the renin-angiotensin-aldosterone system in the kidney compensated for the absence of ENaC in colonic surface epithelium, leading to colon-specific pseudohypoaldosteronism type 1 with mineralocorticoid resistance without evidence of impaired potassium balance.

Chicken BAFF--a highly conserved cytokine that mediates B cell survival.

Members of the tumor necrosis factor (TNF) family play key roles in the regulation of inflammation, immune responses and tissue homeostasis. Here we describe the identification of the chicken homologue of mammalian B cell activating factor of the TNF family (BAFF/BLyS). By searching a chicken EST database we identified two overlapping cDNA clones that code for the entire open reading frame of chicken BAFF (chBAFF), which contains a predicted transmembrane domain and a putative furin protease cleavage site like its mammalian counterparts. The amino acid identity between soluble chicken and human BAFF is 76%, considerably higher than for most other known cytokines. The chBAFF gene is most strongly expressed in the bursa of Fabricius. Soluble recombinant chBAFF produced by human 293T cells interacted with the mammalian cell-surface receptors TACI, BCMA and BAFF-R. It bound to chicken B cells, but not to other lymphocytes, and it promoted the survival of splenic chicken B cells in culture. Furthermore, bacterially expressed chBAFF induced the selective expansion of B cells in the spleen and cecal tonsils when administered to young chicks. Our results suggest that like its mammalian counterpart, chBAFF plays an important role in survival and/or proliferation of chicken B cells.

Novel angiotensin II inhibitors in cardiovascular medicine.

Blockade of the renin-angiotensin-aldosterone cascade is now recognised as a very effective approach to treat hypertensive, heart failure and high cardiovascular risk patients and to retard the development of renal failure. The purpose of this review is to discuss the state of development of currently available drugs blocking the renin-angiotensin system, such as angiotensin converting enzyme (ACE) inhibitors, renin inhibitors and angiotensin II receptor antagonists, with a special emphasis on the results of the most recent trials conducted with AT(2) receptor antagonists in heart failure and Type 2 diabetes. In addition, the future perspectives of drugs with dual mechanisms of action, such as NEP/ACE inhibitors, also named vasopeptidase inhibitors, are presented.

Prevalence of comedications and effect of potential drug-drug interactions in the Swiss HIV Cohort Study.

BACKGROUND: Potential drug-drug interactions (PDDIs) might expand with new combination antiretroviral therapies (ART) and polypharmacy related to increasing age and comorbidities. We investigated the prevalence of comedications and PDDIs within a large HIV cohort, and their effect on ART efficacy and tolerability. METHODS: All medications were prospectively recorded in 1,497 ART-treated patients and screened for PDDIs using a customized version of the Liverpool drug interactions database. RESULTS: Overall, 68% (1,013/1,497) of patients had a comedication and 40% (599/1,497) had > or = 1 PDDI. Among patients with comedication, 2% (21/1,013) had red-flag interactions (contraindicated) and 59% (597/1,013) had orange-flag interactions (potential dose adjustment and/or close monitoring required). The latter involved mainly central nervous system drugs (49%), cardiovascular drugs (34%) and methadone (19%). In the multivariate analysis, factors associated with having a comedication were advanced age, female gender, obesity and HCV infection. Independent risk factors for PDDIs were regimens combining protease inhibitors and non-nucleoside reverse transcriptase inhibitors (odds ratio [OR] 3.06, 95% confidence interval [CI] 1.44-6.48), > or = 2 comedications (OR 1.89, 95% CI 1.32-2.70), current illicit drug use (OR 2.00, 95% CI 1.29-3.10) and patients with HCV infection (OR 1.74, 95% CI 1.19-2.56). Viral response was similar in patients with and without PDDIs (84.5% versus 86.4%; P=0.386). During follow-up, ART was modified in 134 patients with comedication regardless of the presence of PDDIs (P=0.524). CONCLUSIONS: PDDIs increase with complex ART and comorbidities. No adverse effect was noted on ART efficacy or tolerability; however, most PDDIs affected comedication but were manageable through dose adjustment or monitoring.

Pharmacokinetic and pharmacogenetic factors influencing plasma and cellular antiretroviral drug disposition

Abstract: The improvement in antiretroviral drug therapy has transformed HIV infection into a chronic disease. However, treatment failure and drug toxicity are frequent. Inadequate response to treatment is clearly multifactorial and, therefore, dosage individualisation based on demographic factors, genetic markers and measurement of cellular and plasma drug level may enhance both drug efficacy and tolerability. At present, antiretroviral drugs levels are monitored in plasma, whereas only drugs penetrating into cells are able to exert an antiviral activity, suggesting that cellular drug determination may more confidently reflect drug exposure at the site of pharmacological action. The overall objective of this thesis is to provide a better understanding of the Pharmacokinetic and pharmacogenetic factors influencing the plasma and cellular disposition of antiretroviral drugs. To that endeavour, analytical methods for the measurements of plasma and cellular drug levels have been developed and validated using liquid chromatography methods coupled with ultraviolet and tandem mass spectrometry detection, respectively. Correlations between plasma and cellular exposures were assessed during observational and experimental studies. Cytochrome (CYP) 2B6, efflux transporters (ABCB1, ABCC1, ABCC2 and ABCG2) and orosomucoid (ORM) polymorphisms were determined and were related to plasma and cellular exposures, as well as toxicity of antiretroviral drugs. A Pharmacokinetic population model was developed to characterise inter- and intra-patient variability of atazanavir pharmacokinetics, and to identify covariates influencing drug disposition. In that context, a Pharmacokinetic interaction study between atazanavir and lopinavir, both boosted with ritonavir, has beén conducted to assess the safety and pharmacokinetics of this boosted double-protease inhibitors regimen. Well to moderately-correlated cellular and plasma drug levels are .observed or protease inhibitors, whereas for efavirenz and nevirapine these correlations are weak. Cellular exposure, and CYP2B6 genotype (516G>T) are predictors of efavirenz neuropsychological toxicity. Nevirapine plasma exposure is also influenced by CYPZB6 polymorphism. Nelfinavir cellular exposure appears to be significantly associated only with ABCB1 genotype (3435C>T and intron 26 + 80T>C). Indinavir and lopinavir clearance and lopinavir cellular/plasma exposure ratio are influenced by the concentration of the variant S of ORM, suggesting-a specific binding of these drugs to this variant. Nelfinavir and efavirenz are not influenced by ORM concentration and phenotype. The Pharmacokinetic parameters of atazanavir are adequately described by our population model. The atazanavir-lopinavir interaction study indicates no influence on plasma and cellular atazanavir pharmacokinetics, while limited decrease in lopinavir concentrations was observed after atazanavir addition. The residual variability unexplained by the considered variables suggests that other covariates either uncontrolled at present or remaining to be identified, such as genetic and environmental factors influence antiretroviral drug pharmacokinetics, with substantial impact on treatment efficacy and tolerability. In that context, a comprehensive approach taking into account drug pharmacokinetics and patient genetic background is expected to contribute to increase treatment success, and to reduce the occurrence of adverse drug reactions by stratifying patients in an individualised antiretroviral therapy approach. Résumé Facteurs pharmacocinétiques et pharmacogénétiques influençant l'exposition plasmatique et cellulaire des antirétroviraux Les progrès de la thérapie antirétrovirale ont transformé l'infection par le VIH d'une affection mortelle à une maladie chronique. En dépit de ce succès, l'échec thérapeutique et la toxicité médicamenteuse restent fréquents. Une réponse inadéquate au traitement est clairement multifactorielle et une individualisation de la posologie des médicaments qui se baserait sur les facteurs démographiques et génétiques des patients et sur les taux sanguins des médicaments pourrait améliorer à la fois l'efficacité et la tolérance de la thérapie. Par ailleurs, seules les concentrations plasmatiques sont actuellement considérées pour le suivi thérapeutique des médicaments, alors que les taux cellulaires pourraient mieux refléter l'activité de ses médicaments qui agissent au niveau intracellulaire. L'objectif global de cette thèse était de mieux comprendre les facteurs pharmacocinétiques et pharmacocénétiques influençant l'exposition plasmatique et cellulaire des médicaments antirétroviraux. A cet effet, des méthodes pour quantifier les concentrations plasmatiques et cellulaires des antirétroviraux ont été développées et validées en utilisant la chromatographie liquide couplée à la détection ultraviolette et la spectrométrie de masse en tandem, respectivement. La corrélation entre l'exposition cellulaire et plasmatique de ces médicaments a été étudiée lors d'études observationnelles et expérimentales. Les polymorphismes du cytochrome (CYP) 2B6, ainsi que des transporteurs d'efflux (ABCB1, ABCC1, ABCC2 et ABCG2) et de l'orosomucoïde (ORM) ont été déterminés et corrélés avec l'exposition plasmatique et cellulaire des antirétroviraux, ainsi qu'à leur toxicité. Un modèle de pharmacocinétique de population a été établi afin de caractériser la variabilité inter- et intra-individuelle de l'atazanavir, et d'identifier les covariables pouvant influencer le devenir de ce médicament. Dans ce contexte, une étude d'interaction entre l'atazanavir et le lopinavir a été effectuée afin de déterminer la sécurité et le profil pharmacocinétique de ce régime thérapeutique. Des corrélations modérées à bonnes ont été observées entre les taux cellulaires et plasmatiques des inhibiteurs de protéase, alors que pour l'efavirenz et la névirapine ces corrélations sont faibles. L'exposition cellulaire, ainsi que le génotype du CYP2B6 (516G>T) sont des indices de la toxicité neuropsychologique de l'efavirenz. L'exposition plasmatique de la névirapine est également influencée par le polymorphisme du CYPZB6. L'exposition cellulaire du nelfinavir est significativement associée au génotype du ABCB1 (3435C>T et intron 26 + 80T>C). La clairance de l'indinavir et du lopinavir, ainsi que le rapport entre exposition cellulaire et plasmatique du lopinavir sont influencés par la concentration du variant S de l'ORM, suggérant une liaison spécifique de ces médicaments à ce variant. La clairance du nelfinavir et de l'efavirenz n'est pas influencée ni par la concentration ni par le phénotype de l'ORM. Les paramètres pharmacocinétiques de l'atazanavir ont été décrits de façon adéquate par le modèle de population proposé. De plus, le lopinavir n'influence pas les concentrations plasmatiques et cellulaires de l'atazanavir; alors que celui-ci conduit à une baisse limitée des taux de lopinavir. L'importante variabilité pharmacocinétique des antirétroviraux suggère que d'autres facteurs génétiques et environnementaux -qui restent encore à découvrir- influencent également leur disponibilité. Dans un proche futur, une prise en charge qui tienne. compte de la pharmacocinétique des médicaments et des caractéristiques génétiques du patient devrait permettre d'individualiser le traitement, contribuant certainement à une amélioration de la réponse thérapeutique et à une diminution de la toxicité. Résumé grand public Facteurs pharmacocinétiques et pharmacogénétiques influençant l'exposition plasmatique et cellulaire des antirétroviraux Les progrès effectués dans le traitement de l'infection par le virus de l'immunodéficience humaine acquise (VIH), ont permis de transformer une maladie avec un pronostic sombre, en une maladie chronique traitable avec des médicaments de plus en plus efficaces. Malgré ce succès, de nombreux patients ne répondent pas de façon optimale à leur traitement et/ou souffrent d'effets indésirables médicamenteux entraînant fréquemment une modification de leur thérapie. Actuellement, le suivi de la réponse au traitement s'effectue par la mesure chez les patients de la quantité de virus et du nombre des cellules immunitaires dans le sang, ainsi que par la concentration sanguine des médicaments administrés. Cependant, comme le virus se réplique à l'intérieur de la cellule, la mesure des concentrations médicamenteuses au niveau intracellulaire pourrait mieux refléter l'activité pharmacologique au site d'action. De plus, il a été possible de mettre en évidence la grande variabilité des concentrations plasmatiques de médicaments chez des patients prenant pourtant la même dose de médicament. Comme cette variabilité est notamment due à des facteurs génétiques qui sont susceptibles d'influencer la réponse au traitement antirétroviral, des analyses génétiques ont été également effectuées chez ces patients. Cette thèse a eu pour objectif de mieux comprendre les facteurs pharmacologiques et génétiques influençant l'activité et la toxicité des médicaments antirétroviraux afin de réduire la variabilité de la réponse thérapeutique. A cet effet, une méthode de dosage permettant la quantification des médicaments anti-HIV au niveau intracellulaire a été développée. Par ailleurs, nos études ont également porté .sur les variations génétiques influençant la quantité et l'activité des protéines impliquées dans le métabolisme et dans le transport des médicaments antirétroviraux. Enfin, les conséquences de ces variations sur la réponse clinique et la toxicité du traitement ont été évaluées. Nos études ont mis en évidence des associations significatives entre les variations génétiques considérées et la concentration sanguine, cellulaire et la toxicité de quelques médicaments antirétroviraux. La complémentarité des connaissances pharmacologiques, génétiques et virales pourrait aboutir à une stratégie globale permettant d'individualiser le traitement et la dose administrée, en fonction des caractéristiques propres de chaque patient. Cette approche pourrait contribuer à une optimisation du traitement antirétroviral dans la perspective d'une meilleure- efficacité thérapeutique à long terme et d'une diminution des effets indésirables rencontrés.

Health-Related Quality of Life in patients with hepatitis C in double and triple therapy

Abstract OBJECTIVE Comparing Health-Related Quality of Life (HRQoL) scores in patients with chronic hepatitis C undergoing double and triple antiviral therapy and analyzing possible factors related to HRQoL. METHOD HRQoL was assessed using the Short Form 36 and Chronic Liver Disease Questionnaire, which were applied at baseline and at weeks 4, 12 and 16 of treatment to 32 patients divided into two groups: double therapy with pegylated interferon (IFN-PEG) and ribavirin, and triple therapy with PEG-IFN, ribavirin and telaprevir. RESULTS The reduction of HRQoL was greater in patients receiving triple therapy compared to those treated with two drugs, the most critical time is at 12 weeks in both groups. After removal of telaprevir, the triple therapy group significantly improved their HRQoL scores. Anxiety and depression before treatment, employment status and race are significantly related to diminished HRQoL. CONCLUSION Patients undergoing double and triple therapy have diminished HRQoL indexes, but the addition of telaprevir chooses a more significant decrease.

The role of LEKTI in fate determination of keratinocyte stem cells

SPINK5 (serine protease inhibitor Kazal-type 5) encodes the putative proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor). In skin, LEKTI expression is restricted to the stratum granulosum of the epidermis and the inner root sheath of hair follicles. Mutations that create premature termination codons in SPINK5 have been reported as the cause of Netherton syndrome (NS), a human autosomal recessive disorder characterized by congenital ichthyosis with defective cornification, a specific hair shaft defect known as trichorrexis invaginata or 'bamboo hair', and severe atopic manifestations, including atopic dermatitis and hayfever. Althought recombinant human LEKTI inhibits a battery of serine proteases including plasmin, trypsin, subtilisin A, cathepsin G, and elastase, the precise role of LEKTI in the physiopathology of NS remains unclear. Spink5−/− mice display a NS-like phenotype. Surprisingly, a psoriasis-like hyperplasia, basement membrane breakdown followed by evagination of spindle-shaped epidermal cells into the dermal compartment, and the presence of numerous sweat gland-like structures were also observed when the skin of Spink5−/− newborn mice, which die at birth, was transplanted onto the back of nude mice. Collectively, these observations suggest that LEKTI may play a role on cell proliferation and stem cell fate. Our current work aims at elucidating the mechanisms by which LEKTI impact these biological processes. Using keratinocyte stem cells obtained from NS patients, we have identified LEKTI as a regulator node in several signaling pathways involved in stem cell behavior.

Regulation of inflammasome activity.

Summary Interleukin-1beta (IL-1beta) is a potent inflammatory cytokine, which is implicated in acute and chronic inflammatory disorders. The activity of IL-1beta is regulated by the proteolytic cleavage of its inactive precursor resulting in the mature, bioactive form of the cytokine. Cleavage of the IL-1beta precursor is performed by the cysteine protease caspase-1, which is activated within protein complexes termed 'inflammasomes'. To date, four distinct inflammasomes have been described, based on different core receptors capable of initiating complex formation. Both the host and invading pathogens need to control IL-1beta production and this can be achieved by regulating inflammasome activity. However, we have, as yet, little understanding of the mechanisms of this regulation. In particular the negative feedbacks, which are critical for the host to limit collateral damage of the inflammatory response, remain largely unexplored. Recent exciting findings in this field have given us an insight into the potential of this research area in terms of opening up new therapeutic avenues for inflammatory disorders.

Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A.

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.