Vav1 haploinsufficiency in a common variable immunodeficiency patient with defective T-cell function.

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by impaired antibody production, which is in many instances secondary to defective T cell function (T-CVID). We previously identified a subset of T-CVID patients characterized by defective expression of Vav1, a guanine nucleotide exchanger which couples the T-cell antigen receptor to reorganization of the actin cytoskeleton. Here we have addressed the possibility that an intrinsic defect in the Vav1 gene might underlie the reduction in Vav protein observed in T cells from these patients. We report the identification in one T-CVID patient of a heterozygous deletion in Vav1. The gene deletion, spanning exons 2-27, accounts for the reduction in Vav1 mRNA and protein in T cells from this patient. The disease-related pedigree of this patient suggests a de novo origin of the Vav1 deletion. The findings highlights Vav1 as an autosomal dominant disease gene associated with CVID with defective T-cell function.

Validation of a diagnostic score for the diagnosis of autoinflammatory diseases in adults.

Most autoinflammatory disorders typically come out in the pediatric population, although a limited number of patients may experience disease onset during adulthood. To date, a late disease onset has been described only in familial Mediterranean fever, caused by mutations in the MEFV gene, and in tumor necrosis factor receptor-associated periodic syndrome, caused by mutations in the TNFRSF1A gene. The relative rarity and lack of information on adult-onset autoinflammatory diseases make it likely that mutations will be found in an even smaller percentage of cases. With the aim of improving the genetic diagnosis in adults with suspected autoinflammatory disorders, we recently identified a set of variables related to the probability of detecting gene mutations in MEFV and TNFRSF1A and, in addition, we have also proposed a diagnostic score for identifying those patients at high risk of carrying mutations in these genes. In the present study we evaluated the preliminary score sensitivity and specificity on a wider number of patients in order to validate the goodness of fit of the model. Two hundred and nineteen consecutive patients with a clinical history of periodic fever attacks were screened for mutations in MEFV and TNFRSF1A genes; detailed information about family/personal history and clinical manifestations were also collected. For the validation of the score we considered data both from the 110 patients used to build the preliminary diagnostic score and from the additional 219 patients enrolled in the present study, for a total number of 329 patients. Early age at disease onset, positive family history for recurrent fever episodes, thoracic pain, abdominal pain and skin rash, which are the variables that had previously been shown to be significantly associated with a positive genetic test result (12), were used for validation. On univariate analysis the associations with a positive genetic test were: age at onset (odds ratio [OR] 0.43, p=0.003), positive family history for recurrent fever episodes (OR 5.81, p<0.001), thoracic pain (OR 3.17, p<0.001), abdominal pain (OR 3.80, p<0.001) and skin rash (OR 1.58, p=0.103). The diagnostic score was calculated using the linear combination of the estimated coefficients of the logistic multivariate model (cut-off equals to 0.24) revealing good sensitivity (0.778) and good specificity (0.718). In conclusion, our score may serve in the diagnostic evaluation of adult patients presenting with recurrent fever episodes suspected of having an autoinflammatory disorder, helping identify the few subjects among them who may be carriers of mutations in MEFV and TNFRSF1A genes.

Development and preliminary validation of a diagnostic score for identifying patients affected with adult-onset autoinflammatory disorders.

To date, the rate of detection of autoinflammatory gene mutations in patients suspected of having an autoinflammatory disorder is very low. However, most of these data refer to pediatric populations. The relative rarity and lack of information on adult-onset autoinflammatory diseases make it likely that mutations will be found in an even smaller percentage of cases. Our aim was to develop and validate a set of variables for predicting the risk that a given adult patient presenting with recurrent fever episodes carries mutations in the MEFV or TNFRSF1A genes, in order to increase the probability of obtaining positive results on genetic testing. One hundred and ten consecutive patients with a clinical history of periodic fever attacks were screened for mutations in the TNFRSF1A and the MEFV genes. The mean age at disease onset was 27.85 years. Detailed information about each patient?s family history, personal history, and clinical manifestations were retrospectively collected. A diagnostic score was constructed based on univariate and multivariate analysis in a randomly-selected dataset (training set; n=40). The score was validated on an independent set of the remaining patients (validation set; n=70). Age at onset (odds ratio 0.958, P =0.050), positive family history of recurrent fever episodes (OR 5.738, P = 0.006 ), thoracic pain (OR 7.390, P = 0.002), abdominal pain (OR 2.853, P = 0.038) and skin involvement (OR 8.241, P = 0.003) were independently correlated with a positive genetic test result. A diagnostic score was calculated using the linear combination of the estimated coefficients of the logistic model (cut off equal to 0.24) revealing high sensitivity (0.94), high specificity (0.94) and high accuracy (0.94). We have identified variables that appear to be strongly related to the probability of detecting gene mutations in MEF and TNFRSF1A in adults, thus improving the evaluation of patients with suspected autoinflammatory disorders.

Role of etanercept in the treatment of tumor necrosis factor receptor-associated periodic syndrome: personal experience and review of the literature.

Tumor necrosis factor-alpha receptor (TNFR1)-associated periodic syndrome (TRAPS) is the most common autosomal-dominant autoinflammatory condition and is caused by mutations in the TNFRSF1A gene. TRAPS is characterized by recurrent attacks of fever typically lasting from 1 to 3 weeks; in addition to fever, common clinical features include mainly periorbital oedema, conjunctivitis, a migratory erythematous plaque simulating erysipela with underlying myalgia, and arthritis or arthralgia; serosal membrane inflammation is also possible. The identification of TNFRSF1A mutations as the genetic cause of TRAPS coincided with the wider use of biological agents in medicine and raised the possibility that blocking TNF could potentially represent the primary therapeutic goal in TRAPS, thus disclosing new treatment choices for this complex disease. In the past few years, isolated reports and case-series have been published suggesting that inhibition of TNF-alpha might represent a promising therapeutic approach in TRAPS. We present here our experience with etanercept in the treatment of patients affected with TRAPS, and we also add a review of the literature.

Idiopathic recurrent pericarditis refractory to colchicine treatment can reveal tumor necrosis factor receptor-associated periodic syndrome.

Recurrences develop in up to 20-50% of patients with acute pericarditis. Although different causes of recurrent pericarditis have been identified, the etiology remains obscure in most cases which are therefore labelled as idiopathic. Autoinflammatory syndromes include familial Mediterranean fever (FMF), due to mutations in the MEFV gene, and tumor necrosis factor receptor-associated periodic syndrome (TRAPS), due to mutations in the TNFRSF1A gene. Recurrent pericarditis is a common feature of both conditions, but it rarely occurs alone. Colchicine is the standard treatment for FMF, while patients with TRAPS do not respond to colchicine therapy, but are responsive to corticosteroids. Based on the proven efficacy of colchicine in preventing polyserositis in FMF, colchicine has been proposed for the treatment of recurrent pericarditis and is able to decrease the recurrence rate. Our aim was to investigate the possible involvement of TNFRSF1A mutations in a group of patients with idiopathic recurrent pericarditis who were refractory to colchicine treatment. Thirty consecutive patients (17 males, 13 females) diagnosed with idiopathic recurrent pericarditis, who were characterized by a poor response to colchicine treatment, were enrolled in the study. Mutations of the TNFRSF1A gene were searched for by amplifying, using polymerase chain reaction (PCR), genomic DNA, and direct sequencing. TNFRSF1A mutations were found in 4 of the 30 patients. None of these 4 patients had a family history of recurrent inflammatory syndromes or history of pericarditis. One of the 4 patients had a novel heterozygous deletion (DeltaY103-R104) and three patients carried a heterozygous low-penetrance R92Q mutation. Our data suggest that TRAPS should be kept in mind in the differential diagnosis of recurrent pericarditis, and mutation analysis of the TNFRSF1A gene should be considered, in addition to MEFV analysis, in patients of Mediterranean origin. A poor response to colchicine treatment and/or a steroid-dependence may be the clue to investigate TNFRSF1A mutations in patients with idiopathic recurrent pericarditis.

p66SHC promotes T cell apoptosis by inducing mitochondrial dysfunction and impaired Ca2+ homeostasis.

p66Shc, a redox enzyme that enhances reactive oxygen species (ROS) production by mitochondria, promotes T cell apoptosis. We have addressed the mechanisms regulating p66Shc-dependent apoptosis in T cells exposed to supraphysiological increases in [Ca2+]c. p66Shc expression resulted in profound mitochondrial dysfunction in response to the Ca2+ ionophore A23187, as revealed by dissipation of mitochondrial transmembrane potential, cytochrome c release and decreased ATP levels. p66Shc expression also caused a dramatic alteration in the cells' Ca2+-handling ability, which resulted in Ca2+ overload after A23187 treatment. The impairment in Ca2+ homeostasis was ROS dependent and caused by defective Ca2+ extrusion due at least in part to decreased plasma membrane ATPase (PMCA) expression. Both effects of p66Shc required Ca2+-dependent serine-36 phosphorylation. The mitochondrial effects of p66Shc were potentiated by but not strictly dependent on the rise in [Ca2+]c. Thus, Ca2+-dependent p66Shc phosphorylation causes both mitochondrial dysfunction and impaired Ca2+ homeostasis, which synergize in promoting T cell apoptosis.

The potential of peroxisome proliferator-activated receptor gamma (PPARgamma) ligands in the treatment of hematological malignancies.

PPARgamma has emerged as a key regulator of cell growth and survival, whose activity is modulated by a number of synthetic and natural ligands. Here we shall review the activities of PPARgamma ligands in the control of immune cell proliferation, differentiation and apoptosis and their potential therapeutic applications to hematological malignancies.

A role for protein kinase C activity in interleukin-1 (IL-1) induction of IL-2 gene expression but not in IL-1 signal transduction.

Interleukin-1 (IL-1) is known to synergize with phorbol esters in the induction of interleukin-2 (IL-2) expression in T-lymphoid leukemia cells and proliferation of mouse thymocytes. We used a plasmid construct containing the bacterial gene for chloramphenicol acetyltransferase under the control of the human IL-2 promoter to study the nature of this synergism in the murine thymoma cell line EL4. Although IL-1 induction of the IL-2 promoter in these cells required costimulus with phorbol myristate acetate, the signal induced by IL-1 was qualitatively different. We provide evidence to support the hypothesis that the phorbol ester signal is mediated by protein kinase C, and we show that the IL-1 signal is not. That IL-1 and phorbol myristate acetate represent different stimuli was shown by their response to protein kinase C inhibitors, capacity to synergize with increased intracellular free calcium, and requirement for protein synthesis. In addition we show that pretreatment with IL-1 can prime EL4 cells to subsequent activation by concentrations of phorbol esters not normally sufficient to induce IL-2 expression. Pretreated cells remained primed for at least 40 h after removal of the IL-1. Neither phorbol myristate acetate nor a calcium ionophore was capable of preactivating EL4 cells.

Human gastric epithelium produces IL-4 and IL-4delta2 isoform only upon Helicobacter pylori infection.

Recent evidence suggests that interleukin-4 (IL-4) is related to mucosal tolerance by which an injurious immune response is prevented, suppressed or shifted to a non-injurious response. We investigated the expression of IL-4 and its splice variant isoform IL-4delta2 in gastric epithelial cells of healthy subjects and gastritis patients infected with Helicobacter pylori (H. pylori) with or without the cag pathogenicity island (cag-PAI). IL-4 and IL-4delta2 mRNAs were evaluated in microdissected gastric epithelium and in AGS cell lines co-cultured with H. pylori B128 or SS1 strains. IL-4 mRNA was consistently detected in microdissected gastric epithelial cells from healthy subjects. The IL-4 mRNA expression was low in H. pylori?infected patients, and markedly reduced in cag-PAI-positive ones. IL-4delta2 mRNA was expressed on gastric epithelium of H. pylori-infected patients, but not in healthy subjects. The IL-4delta2 expression was lower in cag-PAI-positive than in cag-PAI-negative H. pylori infected patients. AGS cells also produced IL-4 mRNA upon SS1 strain stimulation, whereas IL-4delta2 mRNA expression was detected in AGS co-cultured with either SS1 or B128 strains. An inverse correlation was documented between IL-4 and IL-4delta2 mRNA expression by microdissected gastric epithelial cells and the score of gastritis. IL-4, but not IL-4delta2, is expressed by gastric epithelium of healthy subjects, whereas IL-4delta2 and lesser IL-4 mRNA are detectable in the gastric epithelium of H. pylori-infected patients. Data suggest that gastric epithelial cells might regulate the balance between tolerance and immune response by the fine tuning of IL-4 and IL-4delta2 expression.

Inhibition of CD4/p56lck signaling by a dominant negative mutant of the Shc adaptor protein.

T-cell antigen receptor stimulation results in phosphorylation of the SH2 containing Shc proteins and recruitment of the Grb2/mSos complex suggesting that Shc proteins are involved in transducing T-cell activating signals to Ras. We have measured the effects of the isolated Shc-SH2 domain and the dominant negative RasN17 protein on activation of the T-cell specific transcription factor NF-AT. The isolated Shc-SH2 domain was designed to compete with endogenous Shc binding to upstream tyrosine phosphorylated proteins and to interfere with coupling to regulators of Ras activation. We have demonstrated that both the Shc-SH2 domain and the RasN17 protein significantly inhibited NF-AT activation by the CD4 coreceptor and the CD4 associated tyrosine kinase p56lck. In contrast, only the RasN17 protein reduced NF-AT activation by the TCR/CD3 complex. Furthermore, tyrosine kinase activity and p56lck protein were found in complexes immunoprecipitated with Shc specific antisera after CD4 triggering but not after CD3 triggering. These results indicate that both CD4 and CD3 signal to Ras and that this signaling is mediated by independent pathways of activation of the Shc adaptor protein.

The aminoterminal phosphotyrosine binding domain of Shc associates with ZAP-70 and mediates TCR dependent gene activation.

T-cell antigen receptor stimulation results in recruitment to the zeta chain and phosphorylation both of the syk family protein tyrosine kinase ZAP-70 and of the Shc adaptor protein, which transduces activating signals to Ras. Both ZAP-70 and Ras are required for T-cell activation. We have investigated the functional link between these two molecules in TCR signaling. She was found to associate with ZAP-70 in response to TCR triggering. This association was dependent on the presence of the aminoterminal phosphotyrosine binding (PTB) domain of She. The analysis of She binding to a potential PTB domain binding site on ZAP-70 confirmed the interaction of the She PTB domain with ZAP-70 and identified the ZAP-70 phosphotyrosine residue involved in this interaction. To test the role of the She PTB domain in transducing TCR derived signals we measured the effects of the isolated She PTB domain on the activation of the T-cell specific transcription factor NF-AT. The isolated She PTB domain was designed to compete non productively with endogenous She for binding to up-stream tyrosine phosphorylated proteins and thus interfere with coupling to regulators of Ras activation. A significant inhibition of NF-AT activation by TCR triggering was observed, showing a functional involvement of She in TCR signaling through its PTB domain and suggesting an important role for She association with ZAP-70.

Constitutive activation of the Ras/MAP kinase pathway and enhanced TCR signaling by targeting the Shc adaptor to membrane rafts.

The Shc adaptor is responsible for coupling receptor tyrosine kinases and tyrosine kinase-associated receptors to the Ras/MAP kinase pathway. Shc is believed to be regulated by a change in subcellular localization from the cytosol to the plasma membrane, where it recruits Grb-2/Sos complexes and hence permits juxtaposition of the guanine nucleotide exchange factor Sos to Ras, resulting in GDP/GTP exchange and Ras activation. Shc has been recently shown to inducibly colocalize in detergent-resistant membrane rafts together with the activated TCR and associated signaling molecules. To understand whether Shc localization in membrane rafts is sufficient to regulate Shc function, we constructed a Shc chimera containing the Ras membrane localization motif at the C-terminus. We show that membrane targeted Shc was constitutively localized in the plasma membrane of T-cells, and was mostly compartmentalized in lipid rafts. Membrane targeted Shc was phosphorylated on tyrosine residues and bound Grb-2/Sos in the absence of TCR engagement. Furthermore, expression of membrane targeted Shc resulted in constitutive downstream signaling, including Erk2 activation and enhancement of TCR dependent activation of the TCR responsive transcription factor NF-AT. Hence localization of Shc in membrane rafts is sufficient for Shc to acquire a signaling competent state. Interestingly, a membrane targeted Shc mutant lacking both Grb-2 binding sites was not only incapable of signaling in the absence of TCR triggering, but transdominantly inhibited endogenous Shc, supporting a non redundant role for Shc in the activation of the Ras/MAP kinase pathway in T-cells.

The BCR signalosome: where cell fate is decided.

B cell antigen receptor (BCR) engagement results in the assembly of a multimolecular complex at the cytosolic side of the plasma membrane, known as signalosome. Here we briefly review the current knowledge on the molecules which participate in the BCR signalosome and on the response modulators which control the final signal output by enhancing or dampening BCR signaling.

Restoration of thymic development in an Lck(-/-) thymoma overexpressing ZAP-70.

Thymic development is strictly controlled by Src and Syk family protein tyrosine kinases. The major players in this process are Lck and ZAP-70, which regulate critical differentiation steps of thymopoiesis. Notwithstanding the critical role of Lck and ZAP-70 in thymocyte development as compared to the related kinases Fyn and Syk, a partial functional redundancy between members of the same family of protein tyrosine kinases has emerged from studies on genetically manipulated mouse models. Furthermore, a close functional interplay between Lck and ZAP-70 in intracellular signaling has been shown to occur in thymocytes. Here we present the characterization of a thymoma from an Lck(-/-) mouse, where the block in thymocyte development is overcome and the transition between the CD4(-)CD8(-) and CD4(+)CD8(+) stages is fully restored. Determination of the expression levels of Fyn, ZAP-70 and Syk in thymocytes form the Lck(-/-) thymoma revealed high levels of ZAP-70 overexpression and recovery of a specific subset of phosphoproteins as compared to Lck(-/-) thymocytes. Hence ZAP-70 overexpression in thymocytes is associated with recovery from the developmental arrest caused by the absence of Lck, suggesting a role for ZAP-70 downstream of Lck in the maturation of CD4(+)CD8(+) thymocytes.

Gene activating and proapoptotic potential are independent properties of different CD4 epitopes.

CD4 engagement triggers an early signaling cascade which initiates late events such as transcription factor activation. The outcome of CD4 engagement is T-cell commitment to alternative, dramatically different fates, such as activation and apoptosis. We have tested a panel of anti-CD4 mAbs specific for different CD4 epitopes, as well as HIV-1 gp120, for the capacity to activate crucial early events such as enhancement of p56(lck) kinase activity and Shc phosphorylation. The same CD4 epitopes were characterized for their capacity both to deliver a gene activating signal and to program T-cells to activation dependent death. No correlation could be found between capacity of specific CD4 epitopes to deliver a gene activating signal and capacity to prime T-cells to apoptosis, suggesting that gene activating and proapoptotic potential are independent functions of CD4 epitopes. Furthermore, while triggering of the calcium pathway appears critical in NF-AT activation, optimal p56(lck) activation and Shc phosphorylation might be required for initiation of the apoptotic pathway.

The small GTPase Rab29 is a common regulator of immune synapse assembly and ciliogenesis.

Accumulating evidence underscores the T-cell immune synapse (IS) as a site of intense vesicular trafficking, on which productive signaling and cell activation crucially depend. Although the T-cell antigen receptor (TCR) is known to exploit recycling to accumulate to the IS, the specific pathway that controls this process remains to be elucidated. Here we demonstrate that the small GTPase Rab29 is centrally implicated in TCR trafficking and IS assembly. Rab29 colocalized and interacted with Rab8, Rab11 and IFT20, a component of the intraflagellar transport system that regulates ciliogenesis and participates in TCR recycling in the non-ciliated T cell, as assessed by co-immunoprecipitation and immunofluorescence analysis. Rab29 depletion resulted in the inability of TCRs to undergo recycling to the IS, thereby compromizing IS assembly. Under these conditions, recycling TCRs accumulated in Rab11(+) endosomes that failed to polarize to the IS due to defective Rab29-dependent recruitment of the dynein microtubule motor. Remarkably, Rab29 participates in a similar pathway in ciliated cells to promote primary cilium growth and ciliary localization of Smoothened. These results provide a function for Rab29 as a regulator of receptor recycling and identify this GTPase as a shared participant in IS and primary cilium assembly.

Sequences of two cDNA clones from the medfly Ceratitis capitata encoding antibacterial peptides of the cecropin family.

Using a back translated oligodeoxyribonucleotide probe, encoding a conserved motif in insect antibacterial peptides, we have isolated two cDNA clones from the medfly, Ceratitis capitata. Sequence determination shows that the cDNAs encode two closely related peptides which are members of the cecropin family.

Calcium dependent activation of the NF-AT transcription factor by p59fyn.

A reporter gene under the control of a T-cell antigen receptor element was activated in Jurkat cells by antigen receptor triggering or by a combination of phorbol myristate acetate, which activates protein kinase C, and a calcium ionophore. Both these signals were necessary for expression of the reporter gene. When co-transfected with a construct capable of overexpressing the tyrosine kinase p59fyn, the reporter gene was activated by PMA alone. Thus p59fyn could replace the calcium ionophore but not activation of protein kinase C. The activation by p59fyn plus PMA was blocked by EGTA and by the immunosuppressant drug cyclosporin A.

Intracellular single chain Fv antibody inhibits Ras activity in T-cell antigen receptor stimulated Jurkat cells.

A mammalian expression vector directing the synthesis of a cytoplasmic single chain Fv version of the Y13-259 anti-Ras antibody was constructed and co-transfected into the human lymphoid cell line Jurkat together with a reporter construct containing the bacterial gene for chloramphenicol acetyl transferase under the transcriptional control of several copies of the binding site for the transcription factor NF-AT. The Ras specific antibody interferes with NF-AT activation upon direct activation of the T-cell antigen receptor, whereas activation by direct protein kinase C stimulation is less sensitive to the anti-Ras antibody. Furthermore, the observed inhibition is dependent on the ratio of antibody to reporter plasmid utilized in the transfection experiments.