A mutation in X-linked inhibitor of apoptosis (G466X) leads to memory inflation of Epstein-Barr virus-specific T cells.

Mutations in the X-linked inhibitor of apoptosis (XIAP) gene have been associated with XLP-like disease, including recurrent Epstein-Barr virus (EBV)-related haemophagocytic lymphohystiocytosis (HLH), but the immunopathogenic bases of EBV-related disease in XIAP deficiency is unknown. We present the first analysis of EBV-specific T cell responses in functional XIAP deficiency. In a family of patients with a novel mutation in XIAP (G466X) leading to a late-truncated protein and varying clinical features, we identified gradual hypogammaglobulinaemia and large expansions of T cell subsets, including a prominent CD4(+) CD8(+) population. Extensive ex-vivo analyses showed that the expanded T cell subsets were dominated by EBV-specific cells with conserved cytotoxic, proliferative and interferon (IFN)-γ secretion capacity. The EBV load in blood fluctuated and was occasionally very high, indicating that the XIAP(G466X) mutation could impact upon EBV latency. XIAP deficiency may unravel a new immunopathogenic mechanism in EBV-associated disease.

Loss-of-function mutations within the IL-2 inducible kinase ITK in patients with EBV-associated lymphoproliferative diseases.

The purpose of this study was the appraisal of the clinical and functional consequences of germline mutations within the gene for the IL-2 inducible T-cell kinase, ITK. Among patients with Epstein-Barr virus-driven lymphoproliferative disorders (EBV-LPD), negative for mutations in SH2D1A and XIAP (n=46), we identified two patients with R29H or D500T,F501L,M503X mutations, respectively. Human wild-type (wt) ITK, but none of the mutants, was able to rescue defective calcium flux in murine Itk(-/-) T cells. Pulse-chase experiments showed that ITK mutations lead to varying reductions of protein half-life from 25 to 69% as compared with wt ITK (107 min). The pleckstrin homology domain of wt ITK binds most prominently to phosphatidylinositol monophosphates (PI(3)P, PI(4)P, PI(5)P) and to lesser extend to its double or triple phosphorylated derivates (PIP2, PIP3), interactions which were dramatically reduced in the patient with the ITK(R29H) mutant. ITK mutations are distributed over the entire protein and include missense, nonsense and indel mutations, reminiscent of the situation in its sister kinase in B cells, Bruton's tyrosine kinase.

An atypical case of X-linked lymphoproliferative disease revealed as a late cerebral lymphoma.

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency, partially characterized by a defect in cytotoxicity to Epstein-Barr virus. This viral infection is therefore often fatal in affected boys, whilst a variety of immune disorders or proliferative diseases may occur in surviving patients. We report an atypical case of a 41year-old male who presented with a primitive B-cell cerebral lymphoma, revealing an XLP. This presentation was unusual because of its late onset, the broad spectrum of the familial characteristics, its initial presentation as a cerebral lymphoma, and the occurrence of B-cell alymphocytosis associated with a-gamma-globulinemia.

Onset and duration of protective immunity against clinical disease and renal carriage in dogs provided by a bi-valent inactivated leptospirosis vaccine.

Protection against clinical disease and prevention of the renal carrier state remain the key objectives of vaccination against leptospirosis in the dog. In the present paper, groups of dogs were vaccinated twice with a commercial bacterin (EURICAN L) containing Leptospira interrogans serovars icterohaemorrhagiae and canicola and challenged with heterologous representatives of both serovars at 2 weeks (onset of immunity) or 14 months (duration of immunity) after the second vaccination. Control dogs were not vaccinated against leptospirosis and kept with the vaccinated dogs. The challenges, irrespective of the serovar, reliably produced clinical signs consistent with Leptospira infection in the control pups with up to 60% mortality. As expected clinical disease in the adult controls was less severe, but we were able to induce morbidity and mortality as well. Under these extreme challenge conditions, clinical signs in the vaccinated dogs were rare, and when observed, mild and transient in nature. Following experimental infection, 100% of the control pups and 83% of the adult controls became renal carriers. Despite the heavy challenges, none of the 18 vaccinated puppies (onset of immunity studies) and only 2 out of the 16 vaccinated adult dogs (duration of immunity studies) developed a renal carrier state. These results show that a primary course of two doses of EURICAN L provided quick onset and long-term protection against both clinical leptospirosis and the renal carrier stage. This vaccine should provide veterinarians with a powerful tool to prevent clinical disease in dogs and zoonotic transmission of leptospirosis to humans.

Identification of hepatitis B virus genome in faecal sample from wild living chimpanzee (Pan troglodytes troglodytes) in Gabon.

Non-invasive faecal sampling in the equatorial forest in Gabon allowed the first identification of the hepatitis B virus (HBV-Ch(RC170)) genome in samples collected from wild chimpanzees (Pan troglodytes troglodytes). The HBV-Ch(RCl70)sequence clustered with 100% bootstrap support with previous viral sequences obtained from Pan troglodytes subspecies. This is the first evidence of HBV infection in wild apes and confirms that the HBV-like strains thus far characterized in captive apes are directly related to those circulating in the wild.

Bidirectional negative regulation of human T and dendritic cells by CD47 and its cognate receptor signal-regulator protein-alpha: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation.

Proinflammatory molecules, including IFN-gamma and IL-12, play a crucial role in the elimination of causative agents. To allow healing, potent anti-inflammatory processes are required to down-regulate the inflammatory response. In this study, we first show that CD47/integrin-associated protein, a ubiquitous multispan transmembrane protein highly expressed on T cells, interacts with signal-regulator protein (SIRP)-alpha, an immunoreceptor tyrosine-based inhibition motif-containing molecule selectively expressed on myelomonocytic cells, and next demonstrate that this pair of molecules negatively regulates human T and dendritic cell (DC) function. CD47 ligation by CD47 mAb or L-SIRP-alpha transfectants inhibits IL-12R expression and down-regulates IL-12 responsiveness of activated CD4(+) and CD8(+) adult T cells without affecting their response to IL-2. Human CD47-Fc fusion protein binds SIRP-alpha expressed on immature DC and mature DC. SIRP-alpha engagement by CD47-Fc prevents the phenotypic and functional maturation of immature DC and still inhibits cytokine production by mature DC. Finally, in allogeneic MLR between mDC and naive T cells, CD47-Fc decreases IFN-gamma production after priming and impairs the development of a Th1 response. Therefore, CD47 on T cells and its cognate receptor SIRP-alpha on DC define a novel regulatory pathway that may be involved in the maintenance of homeostasis by preventing the escalation of the inflammatory immune response.

Regulation of SLAM-mediated signal transduction by SAP, the X-linked lymphoproliferative gene product.

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a short intracellular molecule that is mutated in humans with X-linked lymphoproliferative (XLP) disease. Although the exact role and mechanism of action of SAP are not known, it has the capacity to interact with the cytoplasmic region of SLAM and other related immune cell receptors. As SAP is composed almost exclusively of a Src homology 2 (SH2) domain, it has been proposed that it functions as a natural blocker of SH2 domain--mediated interactions. We report here that the SLAM receptor is capable of triggering a protein tyrosine phosphorylation signal in T cells via a mechanism that is strictly dependent on SAP expression. This signal involves the SH2 domain--containing inositol phosphatase (SHIP); the adaptor molecules Dok2, Dok1 and Shc; and Ras GTPase--activating protein RasGAP. SAP is essential for this pathway because it facilitates the selective recruitment and activation of the Src-related protein tyrosine kinase FynT. We also show that signaling via the SLAM-SAP pathway in an established T cell line can alter the profile of cytokine production during T cell activation. These findings identify a mechanism by which a putative adaptor molecule is required for receptor-mediated signaling events in the immune system. They also provide insights into the pathophysiology of a severe human lymphoproliferative disease.

Proximal protein tyrosine kinases in immunoreceptor signaling.

Immunoreceptor engagement results in the sequential activation of several classes of protein tyrosine kinases, including the Src and Syk/Zap-70 families. Recent progress has been made in our understanding of the regulation and function of these molecules. First, it was revealed that membrane compartmentation of protein tyrosine kinases may be essential for their proper biological function. Second, Src family kinases were found to act not only as positive regulators, but also as inhibitors of cell activation. Third, it was appreciated that Csk, a potent inhibitor of Src kinases, is regulated by an assortment of protein-protein interactions. Fourth, differences in the regulation of Syk and Zap-70 were observed, suggesting significant distinctions in the purpose of these two kinases in immunoreceptor signaling. And fifth, it was suggested that proximal kinases implicated in immunoreceptor-mediated signal transduction may be regulated by protein degradation via binding to c-Cbl, a ubiquitin ligase.

Dok-3, a novel adapter molecule involved in the negative regulation of immunoreceptor signaling.

Adapters are typically viewed as molecules coordinating the recruitment of positive effectors of cell signaling. Herein, we report the identification of Dok-3, a novel adapter molecule belonging to the Dok family. Our studies show that Dok-3 is highly expressed in several hemopoietic cell types, including B cells and macrophages. It undergoes rapid tyrosine phosphorylation in response to immunoreceptor-mediated cellular activation, seemingly as a result of the action of Src family kinases. This phosphorylation induces the binding of Dok-3 to at least two inhibitory molecules, the 5' inositol phosphatase SHIP and the protein tyrosine kinase Csk. We also demonstrate that augmented expression of wild-type Dok-3 in a B-cell line results in an inhibition of immunoreceptor-mediated nuclear factor of activated T-cells (NFAT) activation and cytokine release, while introduction of a Dok-3 mutant with impaired ability to associate with SHIP and Csk enhances B-cell responsiveness. Taken together, these results indicate that Dok-3 is an adapter involved in the recruitment of inhibitory molecules and that it may play a significant role in the negative regulation of immunoreceptor signaling in hemopoietic cells such as B cells and macrophages.

[Post-traumatic stress disorder. After the flood in Saguenay]. / L'état de stress post-traumatique. L'après-déluge au Saguenay.

OBJECTIVE: To measure the prevalence of posttraumatic stress disorder and emotional distress among victims of the Saguenay flood compared with those who were not affected by the flood. DESIGN: Cross-sectional study using a telephone survey of victims and a control group. SETTING: Chicoutimi, Que. PARTICIPANTS: Sixty-two adults in a flooded area and a control group of 79 volunteers chosen randomly from an adjacent area. MAIN OUTCOME MEASURES: Diagnostic criteria for posttraumatic stress disorder measured using the Post-traumatic Stress Disorder Reaction Index and high scores on the Self-Reporting Questionnaire on emotional distress. RESULTS: Socially and demographically, study group and control group were comparable. Prevalence of posttraumatic stress disorder in the study group was close to 20% (odds ratio [OR] 6.08; 95% confidence interval [CI] 1.63 to 22.64). Prevalence of emotional distress in the study group was 29% (OR 2.42; 95% CI 1.04 to 5.61). CONCLUSION: The Saguenay flood caused psychological distress that was measurable 4 months later. Health care professionals should be aware of the psychological effects of natural disasters.

Clnk, a novel SLP-76-related adaptor molecule expressed in cytokine-stimulated hemopoietic cells.

We have identified a novel Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76)-related molecule which we have termed Clnk (for cytokine-dependent hemopoietic cell linker). Unlike its relatives SLP-76 and B cell linker protein (Blnk), Clnk is not expressed uniformly within a given hemopoietic cell lineage. Even though it can be detected in several cell types, including T cells, natural killer cells, and mast cells, its expression seems to be strictly dependent on sustained exposure to cytokines such as interleukin (IL)-2 and IL-3. Strong support for the notion that Clnk is involved in immunoreceptor signaling was provided by the observation that it inducibly associated with at least one tyrosine-phosphorylated polypeptide (p92) in response to immunoreceptor stimulation. Moreover, transient expression of Clnk caused an increase in immunoreceptor-mediated signaling events in a T cell line. Taken together, these results show that Clnk is a novel member of the SLP-76 family selectively expressed in cytokine-stimulated hemopoietic cells. Furthermore, they suggest that Clnk may be involved in a cross-talk mechanism between cytokine receptor and immunoreceptor signaling.

Cortico-spinal inhibition reflects time but not event preparation: neural mechanisms of preparation dissociated by transcranial magnetic stimulation.

Changes in cortico-spinal excitability related to time and event preparation were investigated by transcranial magnetic stimulation (TMS) of the motor cortex during the foreperiod of a movement-precuing task. Subjects performed a four alternative choice reaction time (RT) task involving a button-press with the index or middle finger (FI) of the left or right hand. Advance information about the to-be-signaled response was provided by a precue, which preceded the response signal by a 1 s foreperiod. The precue either indicated the hand (right or left) or FI (index or middle) with which the response would be executed or was uninformative. TMS was delivered to the left or right cortical hand area at one of five possible times during the foreperiod: -1000, -500, -333, -166 or 0 ms prior to the response signal. Surface EMG activity from a prime mover involved in flexion of the response FIs (Flexor digitorum superficialis) was used to measure the magnitude of the motor evoked potential (MEP) elicited by TMS. Cortico-spinal excitability--as assessed by the magnitude of the MEP evoked in the target muscle contralateral to the stimulated hemisphere--progressively decreased during the foreperiod. The identity of the precued responses, however, had no effect on MEP magnitude. These results suggest that preparation to respond at a particular time inhibited excitability of the cortico-spinal tract, while advance preparation to perform specific responses affected more central structures only.

Interactions of CD45-associated protein with the antigen receptor signaling machinery in T-lymphocytes.

CD45 is a transmembrane protein tyrosine phosphatase playing an essential role during T-cell activation. This function relates to the ability of CD45 to regulate p56(lck), a cytoplasmic protein tyrosine kinase necessary for T-cell antigen receptor (TCR) signaling. Previous studies have demonstrated that CD45 is constitutively associated in T-lymphocytes with a transmembrane molecule termed CD45-AP (or lymphocyte phosphatase-associated phosphoprotein). Even though the exact role of this polypeptide is unclear, recent analyses of mice lacking CD45-AP have indicated that its expression is also required for optimal T-cell activation. Herein, we wished to understand better the function of CD45-AP. The results of our studies showed that in T-cells, CD45-AP is part of a multimolecular complex that includes not only CD45, but also TCR, the CD4 and CD8 coreceptors, and p56(lck). The association of CD45-AP with TCR, CD4, and CD8 seemed to occur via the shared ability of these molecules to bind CD45. However, binding of CD45-AP to p56(lck) could take place in the absence of other lymphoid-specific components, suggesting that it can be direct. Structure-function analyses demonstrated that such an interaction was mediated by an acidic segment in the cytoplasmic region of CD45-AP and by the kinase domain of p56(lck). Interestingly, the ability of CD45-AP to interact with Lck in the absence of other lymphoid-specific molecules was proportional to the degree of catalytic activation of p56(lck). Together, these findings suggest that CD45-AP is an adaptor molecule involved in orchestrating interactions among components of the antigen receptor signaling machinery. Moreover, they raise the possibility that one of the functions of CD45-AP is to recognize activated Lck molecules and bring them into the vicinity of CD45.

High expression of inhibitory receptor SHPS-1 and its association with protein-tyrosine phosphatase SHP-1 in macrophages.

SHPS-1 (or SIRP) is a member of the immunoglobulin (Ig) superfamily abundantly expressed in neurons and other cell types. Within its cytoplasmic domain, it possesses at least two immunoreceptor tyrosine-based inhibitory motifs, which are targets for tyrosine phosphorylation and mediate the recruitment of SHP-2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase. Since other immunoreceptor tyrosine-based inhibitory motifs-containing receptors have critical roles in the negative regulation of hemopoietic cell functions, we wanted to examine the expression of SHPS-1 in cells of hematological lineages. By analyzing a panel of hemopoietic cell lines, evidence was provided that SHPS-1 is abundantly expressed in macrophages and, to a lesser extent, in myeloid cells. No expression was detected in T-cell or B-cell lines. Expression of SHPS-1 could also be documented in normal ex vivo peritoneal macrophages. Further studies showed that SHPS-1 was an efficient tyrosine phosphorylation substrate in macrophages. However, unlike in non-hemopoietic cells, tyrosine-phosphorylated SHPS-1 in macrophages associated primarily with SHP-1 and not SHP-2. Finally, our analyses allowed us to identify several isoforms of SHPS-1 in mouse cells. In part, this heterogeneity was due to differential glycosylation of SHPS-1. Additionally, it was caused by the production of at least two distinct shps-1 transcripts, coding for SHPS-1 polypeptides having different numbers of Ig-like domains in the extracellular region. Taken together, these findings indicate that SHPS-1 is likely to play a significant role in macrophages, at least partially as a consequence of its capacity to recruit SHP-1.

A unique insert in the linker domain of Syk is necessary for its function in immunoreceptor signalling.

Accumulating data indicate that the 'linker' region of Syk, which lies between its tandem Src homology 2 (SH2) domains and kinase region, provides a critical function for the biological activity of Syk. This importance has been ascribed to the presence of tyrosine phosphorylation sites capable of mediating the recruitment of cellular effectors. We and others previously identified an alternatively spliced variant of Syk, termed SykB, which lacks a 23 amino acid sequence in the linker domain. As this 'linker insert' is also not present in the closely related enzyme Zap-70, it seems plausible that Syk possesses this unique sequence for functional reasons. To understand its role better, we have compared the abilities of Syk and SykB to participate in immunoreceptor-triggered signal transduction. The results of our experiments revealed that, unlike Syk, SykB was inefficient at coupling stimulation of FcepsilonRI on basophils or the antigen receptor on T cells to the early and late events of cellular activation. Further studies showed that the functional defect in SykB was not caused by the absence of crucial tyrosine phosphorylation sites, or by a reduced intrinsic kinase activity. Rather, it correlated with the reduced ability of SykB to bind phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) in vitro and in vivo. In combination, these results demonstrated that the unique insert in the linker domain of Syk is crucial for its capacity to participate in immunoreceptor signalling. Furthermore, they provided evidence that the linker region can regulate the ability of Syk to bind ITAMs, thus identifying a novel function for this domain.

The Syk protein tyrosine kinase is essential for Fcgamma receptor signaling in macrophages and neutrophils.

The cytoplasmic protein tyrosine kinase Syk has two amino-terminal SH2 domains that engage phosphorylated immunoreceptor tyrosine-based activation motifs in the signaling subunits of immunoreceptors. Syk, in conjunction with Src family kinases, has been implicated in immunoreceptor signaling in both lymphoid and myeloid cells. We have investigated the role of Syk in Fcgamma receptor (FcgammaR)-dependent and -independent responses in bone marrow-derived macrophages and neutrophils by using mouse radiation chimeras reconstituted with fetal liver cells from Syk-/- embryos. Chimeric mice developed an abdominal hemorrhage starting 2 to 3 months after transplantation that was ultimately lethal. Syk-deficient neutrophils derived from the bone marrow were incapable of generating reactive oxygen intermediates in response to FcgammaR engagement but responded normally to tetradecanoyl phorbol acetate stimulation. Syk-deficient macrophages were defective in phagocytosis induced by FcgammaR but showed normal phagocytosis in response to complement. The tyrosine phosphorylation of multiple cellular polypeptides, including the FcgammaR gamma chain, as well as Erk2 activation, was compromised in Syk-/- macrophages after FcgammaR stimulation. In contrast, the induction of nitric oxide synthase in macrophages stimulated with lipopolysaccharide and gamma interferon was not dependent on Syk. Surprisingly, Syk-deficient macrophages were impaired in the ability to survive or proliferate on plastic petri dishes. Taken together, these results suggest that Syk has specific physiological roles in signaling from FcgammaRs in neutrophils and macrophages and raise the possibility that in vivo, Syk is involved in signaling events other than those mediated by immunoreceptors.