ctDNA quantification improves estimation of outcomes in patients with high-grade osteosarcoma: a translational study from the OS2006 trial.

BACKGROUND: Osteosarcoma stratification relies on clinical parameters and histological response. We developed a new personalized stratification using less invasive circulating tumor DNA (ctDNA) quantification. PATIENTS AND METHODS: Plasma from patients homogeneously treated in the prospective protocol OS2006, at diagnosis, before surgery and end of treatment, were sequenced using low-passage whole-genome sequencing (lpWGS) for copy number alteration detection. We developed a prediction tool including ctDNA quantification and known clinical parameters to estimate patients' individual risk of event. RESULTS: ctDNA quantification at diagnosis (diagCPA) was evaluated for 183 patients of the protocol OS2006. diagCPA as a continuous variable was a major prognostic factor, independent of other clinical parameters, including metastatic status [diagCPA hazard ratio (HR) = 3.5, P = 0.002 and 3.51, P = 0.012, for progression-free survival (PFS) and overall survival (OS)]. At the time of surgery and until the end of treatment, diagCPA was also a major prognostic factor independent of histological response (diagCPA HR = 9.2, P < 0.001 and 11.6, P < 0.001, for PFS and OS). Therefore, the addition of diagCPA to metastatic status at diagnosis or poor histological response after surgery improved the prognostic stratification of patients with osteosarcoma. We developed the prediction tool PRONOS to generate individual risk estimations, showing great performance ctDNA quantification at the time of surgery and the end of treatment still required improvement to overcome the low sensitivity of lpWGS and to enable the follow-up of disease progression. CONCLUSIONS: The addition of ctDNA quantification to known risk factors improves the estimation of prognosis calculated by our prediction tool PRONOS. To confirm its value, an external validation in the Sarcoma 13 trial is underway.

Functional analysis of young patients with desmoid-type fibromatosis: Initial surveillance does not jeopardize long term quality of life.

BACKGROUND: With recent conservative strategies, prognosis of patients with desmoid-type fibromatosis (DTF) is about function preservation. We analyzed the long-term quality of life (QoL) of pediatric patients with DTF. METHODS: All French young patients (<21years) treated between 2005 and 2016 for a DTF in the EpSSG NRSTS-05 study were analyzed. A first wait-and-see strategy was recommended. Patients' QoL was analyzed with the internationally validated Child Health Questionnaire (CHQ). We focused on the relevant subscales scores: physical functioning (PF), role social limitations physical (RP), bodily pain (BP), general health perception (GH) and physical (PhS) and psychosocial (PsS) summary measures. RESULTS: Among the 81 patients, 52 families answered the CHQ (median delay since diagnosis = 6.2years; min2.2-max13.3 years). Median age at diagnosis was 11.5 years. Primary site: limbs (52%), head/neck (27%), or trunk (21%). Five year-Progression Free Survival was 39.1% (95%CI: 27.7-50.5%). As initial management for these 52 patients, 30 patients were first observed (57%), 13 had surgery (25%) and 9 received chemotherapy (18%). Total burden of therapy was exclusive surgery (9pts/18%), exclusive chemotherapy (18pts/35%), surgery + chemotherapy (13pts/25%), chemotherapy + radiotherapy (1 pt), surgery + chemotherapy + radiotherapy (1 pt), wait and see (10 pt). Regarding the parent forms, patients have significant lower PF (86.0vs.96.1; p = 0.03), RP (82.0vs.93.6; p = 0.04), GH (60vs.73; p < 0.005) and PhS (46.2 vs.53; p = 0.02) scores compared to healthy population. Comparison of QoL subscales scores according to initial strategy (wait-and-see vs.surgery/chemotherapy) did not reveal any difference (PF = 87.3vs.84.9; p = 0.80/RP = 83.4vs.78.7; p = 0.72/BP = 78.9vs.78.2; p = 0.95/GH = 59.7vs60; p = 0.97). Similar results were found using the children or adult forms. CONCLUSIONS: Initial wait-and-see strategy does not affect long term functional impairment.

Revisiting the initial diagnosis and blood staging of mycosis fungoides and Sézary syndrome with the KIR3DL2 marker.

BACKGROUND: The early diagnosis of Sézary syndrome (SS) is challenging. Loss of CD7 and CD26 expression on CD4+ T cells is the currently used criterion in the initial diagnosis and staging of patients with SS. OBJECTIVES: Our aim was to evaluate the respective value of CD26, CD7 and KIR3DL2 expression on CD4+ T cells and total lymphocytes at initial diagnosis of SS. METHODS: This prospective study included 254 patients with clinical features consistent with cutaneous T-cell lymphoma seen at our institution between March 2014 and February 2019. Peripheral blood analysis by flow cytometry was performed for each patient at the time of diagnosis and during follow-up. The diagnosis of SS was based on ISCL/EORTC criteria. RESULTS: The presence of KIR3DL2+ Sézary cells (SCs) ≥ 200 µL-1 correlated with the diagnosis of SS, with sensitivity of 88·6% and specificity of 96·3%. All 154 patients with either inflammatory skin disease or other haematological disease had KIR3DL2+ cells < 200 µL-1 , while eight of them had CD4+ CD26- T cells ≥ 1000 µL-1 . Of five patients with SS and lymphopenia, four had CD4+ CD7- T cells < 1000 µL-1 and three had CD4+ CD26- T cells < 1000 µL-1 . However, all of them had KIR3DL2+ CD4+ T cells ≥ 200 µL-1 . Among patients with available samples during evolution, all B1-staged patients with ≥ 200 µL-1 KIR3DL2+ SCs at diagnosis evolved to B2 stage within 7 months. CONCLUSIONS: KIR3DL2 expression on T cells is highly specific and helps the early diagnosis of SS, especially in those patients with lymphopenia. What's already known about this topic? In the ISCL/EORTC cutaneous T-cell lymphoma (CTCL) categorization of blood involvement (B0-B2), B2 is defined as a T-cell receptor clonal rearrangement in blood, associated with high blood-smear Sézary cell (SC) count. Flow cytometry was developed to circumvent interobserver variability of SC manual counts; however, it mostly relies on detection of cells lacking CD7 and/or CD26 expression. We previously reported the reliability of KIR3DL2 as the first positive SC marker. What does this study add? Based on our analysis of 254 patients, we propose that KIR3DL2 be added to the ISCL/EORTC criteria for initial diagnosis of Sézary syndrome (SS) and B2 staging. This marker improved sensitivity of SS B2-stage CTCL diagnosis with a specificity > 95%, especially for patients with lymphopenia. We found KIR3DL2 helped early diagnosis of SS and was more reliable than CD26 in assessing blood tumour burden during therapy. What is the translational message? SC quantification is the major means of staging at initial diagnosis and monitoring blood tumour burden in a clinical trials setting. We recommend using a threshold value of KIR3DL2+ SCs ≥ 200 µL-1 or KIR3DL2+ SCs/lymphocytes ≥ 10% in the diagnostic criteria of SS and propose a novel algorithm for CTCL B2 blood staging.

Identification of CD245 as myosin 18A, a receptor for surfactant A: A novel pathway for activating human NK lymphocytes.

CD245 is a human surface antigen expressed on peripheral blood lymphocytes, initially delineated by two monoclonal antibodies DY12 and DY35. Until now, CD245 molecular and functional characteristics remained largely unknown. We combined immunological and proteomic approaches and identified CD245 as the unconventional myosin 18A, a highly conserved motor enzyme reported as a receptor for the surfactant protein A (SP-A), that plays a critical role in cytoskeleton organization and Golgi budding. We report that the recruitment of CD245 strongly enhanced NK cell cytotoxicity. Further, we show that the enhancement of the NK lymphocytes killing ability toward CD137-ligand expressing target cells could result from the induction of CD137 expression following CD245 engagement. The SP-A receptor could therefore represent a novel and promising target in cancer immunotherapy.

[Managing late-effects after allogeneic stem cell transplantation in children: recommendations from the SFGM-TC]. / Prise en charge des séquelles après greffe de cellules souches hématopoïétiques chez l'enfant : recommandations de la SFGM-TC.

In this report, we address the issue of late-effects after allogeneic stem cell transplantation in children. In an effort to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapy (SFGM-TC) set up the fourth annual series of workshops which brought together practitioners from all member centers and took place in September 2013 in Lille.

Characteristics of endobronchial primitive tumors in children.

Primary endobronchial tumors are rare in children and they include a broad spectrum of lesions. The aim of this study was to determine the characteristic features, treatments and outcomes of these tumors. We report a retrospective analysis of all patients treated for endobronchial tumor in nine French hospitals between 1990 and 2010 and a comparison of the results with those reported in the medical literature. Twelve tumors were reported: five low grade muco epidermoid carcinomas, two inflammatory myofibroblastic tumors, two hemangiomas, one anaplastic large cell lymphoma, one carcinoid tumor, and one juvenile xanthogranuloma. The mean age of the patients was 7.5 ± 3.5 years. The most common sign revealing the disease was persistent atelectasis or recurrent pneumonia (eight cases). The other revealing signs were a persistent bronchospasm (three cases) and hemoptysis (one case). The clinical presentation, biology, serum tumor markers, and chest X-ray abnormalities were not specific to a particular histological diagnosis. Chest CT scan revealed the presence of an endobronchial tumor in 11 cases. Nine tumors could be diagnosed from a biopsy obtained by video endoscopy. Complete surgical resection was performed in seven patients. Bronchoscopic removal was performed in five cases and was successful in three. There were no deaths. Endobronchial tumors are rare in childhood and their histology is diverse. Chest CT scan and per-endoscopic endobronchial biopsies are required for diagnosis, when possible. Surgical or endoscopic treatment should be discussed by a multidisciplinary team. Despite the multiple etiologies, the prognosis of these tumors is good if diagnosis is early and if resection is complete. Long-term recurrences have been described, so long-term follow-up of these children is recommended.

[New developments in Sézary syndrome]. / Actualités sur le syndrome de Sézary.

Sézary syndrome (SS) represents 3% of cutaneous T-cell lymphomas (CTCL). It is an aggressive epidermotropic CTCL with a 5-year survival rate of 24%. According to EORTC (European organization for research and treatment of cancer), SS is defined by erythroderma, diffuse lymphadenopathy, atypical T lymphocytes (>1000/mm(3)), and the presence of a major blood, cutaneous and nodal T cell clone. A specific marker for atypical tumoral T lymphocytes known as Sézary cells was identified in 2001, namely KIR3DL2 (CD158k) receptor, which allows more specific diagnosis of SS; levels of this marker are highly correlated with the clinical course of the disease. In therapeutic terms, clinical trials are being conducted on new molecules that point towards an improved prognosis for this disease. We propose a review of Sézary syndrome, which is currently the subject of scientific papers concerning both physiopathology and therapeutics, with new prospects of targeted therapy.

Possible pathogenic role of the transmembrane isoform of CD160 NK lymphocyte receptor in paroxysmal nocturnal hemoglobinuria.

PIGA mutations in paroxysmal nocturnal hemoglobinuria (PNH) patients lead to a glycosylphosphatidylinositol (GPI)-linked membrane proteins expression deficiency. Herein, we report the constitutive expression of the transmembrane CD160 (CD160-TM) activating receptor on non PIGA-mutated PNH patients circulating NK cells. In healthy individuals, only the GPI-anchored isoform of CD160 receptors is expressed on the circulating NK lymphocytes, while the transmembrane isoform appears after ex vivo activation. Similarly to CD160-GPI, we identified CD160-TM as a receptor for the MHC class I molecules. We demonstrate that PNH patients NK lymphocytes spontaneously produce significant amounts of IFN-γ that is inhibited by anti-CD160-TM or anti-MHC class I mAbs. These results indicate that circulating NK cells from PNH patients exhibit a self-MHC class I molecule reactive effector function, which could be mediated through the recruitment of CD160-TM receptor. Our data provide new insights regarding the possible role of CD160-TM on PNH patients NK lymphocytes and in the pathogenesis of the disease.

[Recurrent and metastatic infantile fibrosarcoma: a case report]. / Fibrosarcome infantile récidivant et métastatique: à propos d'un cas.

Infantile fibrosarcoma is a rare malignant tumor that usually occurs during the 1st year of life. It accounts for approximately 5-10% of all sarcomas in infants younger than 1 year of age. It usually has indolent progression and metastatic spread is rare. We report the case of a patient who had infantile fibrosarcoma of the trunk. At birth, the baby presented a soft tissue mass of the scapulothoracic region. Histopathological examination after complete surgical resection at first suggested an angioma. Reanalysis of the histology after a metastatic relapse resulted in the diagnosis of infantile fibrosarcoma, which was confirmed by the presence of the specific translocation seen in infantile fibrosarcoma (ETV6/NTRK3). This patient's progression was uncommon because he developed 3 metastatic relapses. The treatment consisted of surgery, chemotherapy, and radiation therapy. The patient is alive with persistent complete remission. We discuss the diagnostic and therapeutic issues of infantile fibrosarcoma. There is a risk of erroneous diagnosis in newborn infants between benign angiomatous tumor and infantile fibrosarcoma. The fusion transcript ETV6-NTRK3 resulting from the specific chromosomal translocation t(12;15)(p13;q25) is now a useful diagnostic tool for infantile fibrosarcoma. Surgery with wide resection is the mainstay of treatment. However, infantile fibrosarcoma is a chemosensitive tumor. If initial surgery cannot be done without mutilation or is impossible, preoperative chemotherapy should be given. The role of radiation therapy is still debated.

Absolute CD3+ CD158k+ lymphocyte count is reliable and more sensitive than cytomorphology to evaluate blood tumour burden in Sézary syndrome.

Background CD158k/KIR3DL2 is a specific marker for Sézary cells which can be used to diagnose Sézary syndrome (SS) in erythrodermic patients with abnormal circulating T cells. Objectives To evaluate the suitability of CD158k/KIR3DL2 for detecting and evaluating blood tumour load during the follow up of patients with SS. Methods The absolute CD3+ CD158k+ lymphocyte count was compared with the absolute count of cytomorphological Sézary cells and was correlated with clinical flares in a cohort of patients with SS. Twenty-five patients were included in the study and 48 blood samples were analysed. Results The absolute count of CD3+ CD158k+ cells strongly correlated with the absolute count of atypical circulating cells (r = 0.97, P < 10(-15)). The CD3+ CD158k+ lymphocyte cell count was in eight cases more sensitive than cytomorphology for detecting atypical circulating cells especially for small-sized tumour cells. The tumour burden evaluated by CD3+ CD158k+ immunostaining was significantly associated with clinical flare (P < 10(-4)). Conclusions CD3+ CD158k+ phenotyping is a reliable and objective test to monitor the blood tumour burden in patients with SS under systemic therapy.

Endostatin variations in childhood acute lymphoblastic leukaemia--comparison with basic fibroblast growth factor and vascular endothelial growth factor.

Angiogenic factors such as basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) were previously studied in childhood acute lymphoblastic leukaemia (ALL) but little is known concerning the anti-angiogenic response in ALL. At diagnosis, the plasma levels of the anti-angiogenic factor endostatin were significantly higher in 33 children with ALL than in controls (median values 17.7 and 7.6 ng/ml, respectively, p=0.0192) but no relationship was observed with plasma bFGF or VEGF levels. The highest levels were observed in patients with an hyperdiploïd karyotype. Expression of mRNA for collagen XVIII/endostatin in lymphoblasts was detected in 19/24 cases but protein secretion was found only in 14/28 supernatants of cultured lymphoblasts. No direct relationship appeared between secretion of endostatin by lymphoblasts and plasma levels. In addition, endostatin levels remained elevated in remission, suggesting that endostatin could have a stromal origin as well. No prognostic value of plasma endostatin could be assessed. In conclusion, the present data indicate that an anti-angiogenic response is observed in some ALL children, but its physiopathological importance remains to be established.

Protocadherin 15 (PCDH15): a new secreted isoform and a potential marker for NK/T cell lymphomas.

Natural killer cells are well known to play an important role in immune defense against tumor development and viral infections. To further characterize new functionally relevant structures in these cells, we studied a series of monoclonal antibodies that we have raised against the NK cell line YT. One of these antibodies previously described as AY19, recognizes a 85 kD surface glycoprotein. Here we report the identification of a new secreted isoform of protocadherin 15, PCDH15C, which represents a potential associated protein for p85. Importantly, whereas protocadherins are absent from the surface of normal hematopoietic cells, we describe, for the first time, that PCDH15 is expressed in cytotoxic tumor-derived T- and NK-cell lines as well as in biopsies of nasal NK/T-cell lymphomas.

Topotecan as a radiosensitizer in the treatment of children with malignant diffuse brainstem gliomas: results of a French Society of Paediatric Oncology Phase II Study.

BACKGROUND: The current Phase II study was conducted to evaluate the survival and toxicity observed in children with newly diagnosed brainstem gliomas who were treated with the daily radiotherapy with topotecan used as a radiosensitizer. METHODS: Eligible patients were those ages 3-18 years with previously untreated tumors arising in the pons diagnosed within the previous 6 months. Histologic confirmation was not mandatory provided that the clinical and magnetic resonance imaging findings were typical for a diffusely infiltrating brainstem lesion. Treatment was comprised of a 6-week course of topotecan administered intravenously at a dose of 0.4 mg/m(2)/day over 30 minutes within 1 hour before irradiation. Radiotherapy was comprised of a once-daily treatment of 1.8 grays (Gy) per fraction to a total dose of 54 Gy. RESULTS: Thirty-two patients were included in the current study between August 2000 and October 2002. All patients completed the combined treatment in accordance with the treatment design. Only partial responses were observed, occurring in 40% of the patients. The 9-month and 12-month survival rates were 34.4% +/- 8% and 25.5% +/- 8%, respectively. The median duration of survival for these 32 patients was 8.3 months. An intratumoral cystic/necrotic change was observed in five patients, with clinical impairment noted in two patients. One intratumoral hemorrhage occurred during radiotherapy, and was associated with transitory neurologic impairment. CONCLUSIONS: The findings of the current study regarding newly diagnosed brainstem glioma patients treated with topotecan given as a radiosensitizing agent did not reproduce the encouraging results obtained in preclinical studies. Therefore, the concomitant combination of topotecan and radiotherapy at this schedule and these doses cannot be recommended for the treatment of patients with brainstem gliomas.

[Cefepime-amikacin combination in febrile neutropenic children with malignant hemopathy or tumor]. / Association céfépime-amikacine chez les enfants neutropéniques fébriles atteints d'hémopathies ou de tumeurs malignes.

UNLABELLED: Our aim was to evaluate retrospectively the efficacy of a therapeutic strategy with a first line combination based on cefepime-amikacin in febrile neutropenic children treated with chemotherapy. PATIENTS AND METHODS: Sixty-five neutropenic febrile episodes in 43 children treated by the association cefepime-amikacin, were evaluated according to the clinical status, the depth and duration of neutropenia, the underlying disease and the initial treatment. RESULTS: Thirty-nine (60%) episodes were successfully treated by the association cefepime-amikacin. Among the 26 persisting febrile episodes, adjunction of vancomycin and amphotericin B was effective in 11 (76% of total rate success) and 5 (84% of total rate success) cases respectively. The efficacy of the first line antibiotherapy was not different as regards to the duration and the depth of neutropenia. Otherwise, febrile episodes after chemotherapy against solid tumours were rapidly controlled by the first and second line of the anti-microbial strategy. Children treated for haematological malignancies presented a lower response rate (P = 0.03). CONCLUSION: In febrile and neutropenic children treated with chemotherapy, the association cefepime-amikacin appeared to be a safe empirical treatment. In a neutropenic child, the immunodeficiency and possibly the clinical status should be the major factors of the infectious prognosis more than the duration of aplasia.

Structural and functional dissection of the cytoplasmic domain of the transmembrane adaptor protein SIT (SHP2-interacting transmembrane adaptor protein).

SIT (SHP2-interacting transmembrane adaptor protein) is a recently identified transmembrane adaptor protein, which is expressed in lymphocytes. Its structural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR-mediated recruitment of SH2 domain-containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly interacts with the SH2-containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine-based inhibition motif (ITIM). Moreover, SIT is capable to inhibit TCR-mediated signals proximal of activation of protein kinase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize the molecular interaction between SIT and intracellular effector molecules and to identify the protein(s) mediating its inhibitory function. We demonstrate that SIT not only interacts with SHP2 but also with the adaptor protein Grb2 via two consensus YxN motifs. However, mutation of both Grb2-binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine-based signaling motif Y(168) ASV completely abrogates the ability of SIT to inhibit T cell activation. Co-precipitation experiments revealed that the tyrosine kinase p50(csk) could represent the negative regulatory effector molecule that binds to this motif.

Increased expression of a novel early activation surface membrane receptor in cutaneous T cell lymphoma cells.

Using a newly generated monoclonal antibody we identified the 96 kDa transmembrane receptor SC5 expressed simultaneously on a human Sezary cell line and a minor T cell subset in normal individuals. SC5 antigen was detected mostly on CD45RO+ lymphocytes from both CD4+ and CD8+ subsets as well as on natural killer and B lineage cells. SC5 surface expression increased very early after polyclonal stimulation of CD3+ cells due to the transfer of intracellular SC5 molecules to the cell membrane. Engagement of SC5 receptor by its monoclonal antibody inhibited the anti-CD3-induced proliferation and cytokine secretion of peripheral blood T cells and cell clones, whereas SC5 monoclonal antibody did not affect the cytotoxic activity of CD8+ T cell clones. Extensive phenotypic analysis revealed that the percentage of SC5+ CD4+ circulating lymphocytes in Sezary syndrome patients was significantly increased in comparison with controls (p < 0.01) and correlated with the morphologically detected percentage of Sezary syndrome cells in peripheral blood (p < 0.001). In one patient we clearly demonstrated that the circulating malignant T cells coexpress SC5 molecules. Importantly, ligation of SC5 receptor in a cutaneous T cell lymphoma cell line profoundly inhibited the anti-CD3-induced proliferation. Consequently, the expression of SC5 receptor in the peripheral blood of Sezary syndrome patients may serve not only to detect the presence of circulating malignant CD4+ cells but also as a target for immunotherapy.

[Severe cutaneous Streptococcus pyogenes infections in the child: results of a multicenter survey]. / Infections cutanées sévères à Streptococcus pyogenes chez l'enfant: résultats d'une enquête multicentrique.

UNLABELLED: To assess pediatric cases of severe cutaneous infections due to Streptococcus pyogenes. Since the beginning of 1980, the incidence of cellulitis and necrotizing fasciitis due to S. pyogenes has increased in adults. Serotyping of obtained isolates are in most cases M1, M3 or M5 protein. PATIENTS AND METHOD: A retrospective (1990-2000) survey was carried out in pediatric hospital centers. RESULTS: Three cases of necrotizing fasciitis and 15 of cellulitis were observed. In 30% of the cases, vancella lesions were associated; in the other cases, minor wounds were the site of the infection. Bacteriologic diagnosis was made by local samples in 14 cases; blood cultures were positive in four cases. In 11 cases, initial intravenous treatment consisted of third generation cephalosporin, in six cases of penicillin M or G and in one case of fusidic acid. In the second time, penicillin M was perfused in the majority of the cases. Mean duration of intravenous antibiotics perfusion was 15 days. There were no sequelae or death in this survey. CONCLUSIONS: Despite this study had limited epidemiological characteristics, it confirms that these two infections are rare. The frequency is probably underestimated, due to the difficulty in performing a diagnosis. The major site of infection was the varicella lesion. These two infections are so similar that it is frequent to mistake one infection for the other. Nonsteroidal anti-inflammatory drugs and site of infections did not influence prognosis. The treatment of cellulitis is penicillinotherapy whereas in necrotizing fasciitis early major surgery is often correlated with the rate of survival.

The Yersinia tyrosine phosphatase YopH targets a novel adhesion-regulated signalling complex in macrophages.

The Yersinia protein tyrosine phosphatase (PTP) YopH is translocated into eukaryotic cells by a type III secretion system that requires bacterial-host cell contact. YopH is composed of two modular effector domains: a substrate-binding domain located in the N-terminal region (residues 1-130) and a PTP catalytic domain located in the C-terminal region (residues 206-468). Previous studies have shown that YopH selectively targets tyrosine-phosphorylated proteins of approximate molecular weight 120 kDa (p120) and 55 kDa (p55) in murine macrophages. It has been demonstrated that p120 actually represents two tyrosine-phosphorylated target proteins, Cas and Fyb. We used the substrate-binding domain of YopH to affinity purify tyrosine-phosphorylated target proteins from lysates of J774A.1 macrophages. Protein microsequencing identified p55 as murine SKAP-HOM. Direct interaction between SKAP-HOM and a catalytically inactive form of YopH was demonstrated in vitro and in macrophages. In addition, we obtained evidence that SKAP-HOM is tyrosine phosphorylated in response to macrophage cell adhesion and that it forms a signalling complex with Fyb. We suggest that dephosphorylation of SKAP-HOM and Fyb by YopH allows yersiniae to interfere with a novel adhesion-regulated signal transduction pathway in macrophages.

Coupling the TCR to downstream signalling pathways: the role of cytoplasmic and transmembrane adaptor proteins.

Engagement of the T-cell receptor (TCR) complex initiates a cascade of intracellular events ultimately leading to T-cell proliferation and differentiation. One of the first detectable consequences of TCR triggering is the activation of cytoplasmic protein kinases which, through phosphorylation of specific substrates, couple the TCR to downstream signalling cascades. Although it is well established that activation of the Ras- and the calcium-dependent calcineurin pathway is required for the achievement of T-cell activation, the precise mechanism as to how the TCR is connected to these intracellular effector molecules is still unclear. Major progress has been made in this regard with the molecular characterization of novel cytoplasmic and transmembrane molecules called adaptor proteins which integrate TCR-mediated signals at the intracellular level thus allowing fine tuning of T-cell responses.