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Summary Background There is no evidence that therapeutic drug monitoring is helpful in patients with inflammatory bowel disease patients in clinical remission with infliximab therapy. Methods Eighty consecutive inflammatory bowel disease patients in clinical remission on infliximab maintenance therapy were included and followed-up for at least one year. Infliximab trough level and antibody to infliximab concentration were measured prior to enrollment. At the time of enrollment, physicians in charge were free to alleviate infliximab therapy. Discrepancies between blind and therapeutic drug monitoring-based adjustments were assessed at the end of the follow-up period. Relapse-free survival was analyzed using univariate and multivariate analyses. Results The mean infliximab trough level was 3.1 μg/mL. Antibody to infliximab was found in 15 (19%) patients. At the end of the follow-up period, 18 (22.5%) patients experienced a relapse. The 3, 6, 9 and 12-month relapse-free rates were 98%, 87%, 86% and 80%, respectively. In our multivariate analysis, relapse-free survival was negatively associated with discrepancies between therapeutic drug monitoring-based and blind adjustments of infliximab therapy, absence of concomitant immunomodulator, the absence of mucosal healing, prior use of infliximab, infliximab therapy duration > 2 years and C-reactive protein levels > 5 mg/L at the time of enrollment. Conclusion In patients with inflammatory bowel disease in clinical remission on infliximab therapy, de-escalation of infliximab therapy should be considered based on therapeutic drug monitoring rather than according to symptoms and CRP.

Objectif La simulation haute-fidelite est de plus en plus utilisee comme outil de formation. L’objectif de notre etude etait d’evaluer l’interet de la simulation haute-fidelite comme aide aux medecins urgentistes pour mieux appliquer les procedures de soins.

International audience; MPhi and DC are key elements in the control of tissue homeostasis and response to insult. In this work, we demonstrate that MPhi and DC are the major producers of the phenylalanine catabolizing enzyme IL-4-induced gene 1 (IL4I1) under inflammatory conditions. IL4I1 was first described in B cells, which indeed can produce IL4I1 in vitro, although at much lower levels. In vivo, IL4I1 is highly expressed by MPhi and DC of Th1 granulomas (sarcoidosis, tuberculosis) but poorly detected in Th2 granulomas (schistosomiasis). In vitro, expression of the enzyme is induced in mononuclear phagocytes by various pro-inflammatory stimuli through the activation of the transcription factors NF-kappaB and/or STAT1. B cells also express IL4I1 in response to NF-kappaB-activating stimuli such as CD40L; however, in contrast to myeloid cells, B cells are insensitive to IFN-gamma but respond to stimulation of the IL-4/STAT6 axis. As we show that the expression of IL4I1 by a monocytic cell line inhibits T-cell proliferation and production of IFN-gamma and inflammatory cytokines, we propose that IL4I1 participates in the downregulation of Th1 inflammation in vivo.

International audience; We previously reported a strong IL4I1 gene expression in primary mediastinal B-cell lymphoma (PMBL) and recently identified the protein as a secreted L-phenylalanine oxidase, physiologically expressed by myeloid cells, which inhibits T-cell proliferation in vitro. Here, we analyzed the pattern of IL4I1 protein expression in 315 human lymphoid and non-lymphoid malignancies. Besides PMBL, IL4I1 expression in tumors was very frequent. IL4I1 was detected in tumor-associated macrophages from most of the tumors and in neoplastic cells from follicular lymphoma, classic and nodular lymphocyte predominant Hodgkin lymphomas and small lymphocytic lymphoma, three of which are germinal center derived. IL4I1-positive tumor cells were also detected in rare cases of solid cancers, mainly mesothelioma. The enzymatic activity paralleled protein expression, suggesting that IL4I1 is functional in vivo. Depending on the tumor type, IL4I1 may impact on different infiltrating lymphocyte populations with consequences on tumor evolution. In the particular case of follicular lymphoma cells, which are susceptible to antitumor cytotoxic T cells killing but depend on interactions with local T helper cells for survival, a high level of IL4I1 expression seems associated with the absence of bone marrow involvement and a better outcome. These findings plead for an evaluation of IL4I1 as a prognosis factor.

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I–binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2–restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT137–45–specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT137–45, have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.

We have recently described a new form of light chain deposition disease (LCDD) presenting as a severe cystic lung disorder requiring lung transplantation. There was no bone marrow plasma cell proliferation. Because of the absence of disease recurrence after bilateral lung transplantation and of serum-free light chain ratio normalization after the procedure, we hypothesized that monoclonal light chain synthesis occurred within the lung. The aim of this study was to look for the monoclonal B-cell component in 3 patients with cystic lung LCDD. Histologic examination of the explanted lungs showed diffuse nonamyloid κ light chain deposits associated with a mild lymphoid infiltrate composed of aggregates of small CD20+, CD5−, CD10− B lymphocytes reminiscent of bronchus-associated lymphoid tissue. Using polymerase chain reaction (PCR), we identified a dominant B-cell clone in the lung in the 3 studied patients. The clonal expansion of each patient shared an unmutated antigen receptor variable region sequence characterized by the use of IGHV4-34 and IGKV1 subgroups with heavy and light chain CDR3 sequences of more than 80% amino acid identity, a feature evocative of an antigen-driven process. Combined with clinical and biologic data, our results strongly argue for a new antigen-driven primary pulmonary lymphoproliferative disorder.

The interpretation of cellular infiltrate from renal transplant recipients with borderline (BL) changes is still a challenging problem. To analyze the immune phenotype of such infiltrate, we quantified the mRNA expression of Foxp3 and interleukinL-10 and granzyme B (GB) in 15 kidney biopsies with BL changes. Controls were patients presenting type IA acute rejection and nonrejecting patients. Only levels of GB mRNA correlated significantly with response to antirejection therapy. Levels of Foxp3 mRNA in BL changes were intermediate between type IA acute rejection and nonrejecting controls. To determine the balance of alloagressive to graft-protecting T cells, we quantified the Foxp3/GB ratio. BL changes T cells infiltrate expressed a significantly higher Foxp3/GB ratio than that in IA acute rejection. These results suggest that T cell infiltrate from BL change exhibit a tolerogenic rather than a cytotoxic phenotype.

Background Rituximab induces clinical response in advanced B-cell lymphoma and is efficient in removing circulating B-cell from peripheral blood. We therefore postulated that rituximab might be a useful in vivo purging agent before high-dose therapy in this setting. Patients and methods Fourteen patients with relapsed follicular, marginal zone and mantle cell lymphomas (11, two and one cases, respectively) and a PCR-detectable molecular marker were treated first with rituximab, then a mobilization chemotherapeutic regimen, followed by high-dose therapy with peripheral blood stem cell transplantation. PCR analyses were performed in peripheral blood before rituximab and during follow-up, and in harvest. Results Harvests were free of PCR-detectable molecular marker in nine of the 11 studied cases (82%). After high-dose therapy, clinical complete remission was obtained in 13 (93%) patients and molecular remission in 11 (79%). With a median follow-up of 3 years, the 14 transplanted patients were alive, 11 of them remaining in clinical complete remission and eight in molecular remission at last follow-up. Conclusion Rituximab treatment followed by high dose therapy appears to be effective in achieving complete clinical and molecular response. In vivo harvest purging is predictive of prolonged clinical and molecular remission.

We report on the characteristics of 21 patients with hepatosplenic gammadelta T-cell lymphoma (HSgammadeltaTCL), an entity recognized since 1994 in the Revised European American Lymphoma (REAL) classification. Median age was 34 years. Patients had splenomegaly (n = 21), hepatomegaly (n = 15), and thrombocytopenia (n = 20). Histopathologic findings were homogeneous and showed the presence of medium-sized lymphoma cells within the sinusoids of splenic red pulp, liver, and bone marrow. Marrow involvement was usually mild but could be demonstrated by phenotyping in all patients. Cells were CD3+CD5-, expressed the gammadelta T-cell receptor, and had a nonactivated cytotoxic cell phenotype (TIA-1+, granzyme B-). Most patients were CD4-/CD8- (16 of 18); CD56+ (15 of 18), expressed the Vdelta1epitope (Vd1+/Vd2-/Vd3-) (9 of 12); and were negative for Epstein-Barr virus (EBV) (18 of 20). Isochromosome arm 7q was documented in 9 of 13 patients. Eight patients had previously undergone kidney transplantation or had a history of systemic lupus, Hodgkin disease, or malaria. Prognosis was poor; median survival time was 16 months, and all but 2 patients ultimately died despite consolidative or salvage high-dose therapy. In conclusion, HSgammadeltaTCL is a disease with distinctive clinical, histopathologic, and phenotypic characteristics. Bone marrow biopsy with combined phenotyping is sufficient for diagnosis, and splenectomy is therefore unwarranted. Current treatment modalities appear to be ineffective in most patients.

Anti-Hu syndrome is a paraneoplastic neurologic disease seemingly associated with an efficient antitumoral immune response against HuD protein expressed by both small cell lung cancer (SCLC) and neurons. Since anti-Hu antibodies are not pathogenic, and oligoclonal CD8(+) T cells infiltrate neoplastic and nervous tissues, we examined MHC class I-restricted immunogenicity of human HuD. Among 14 HuD-derived peptides potentially immunogenic in HLA-A*0201 restriction, 10 had actual in vitro binding capacity to the HLA molecule, 8 elicited specific cytotoxic T lymphocytes (CTLs) in a humanized murine model after peptidic vaccination, 2 also elicited specific CTLs in healthy humans, and 1 was naturally processed and presented to the immune system.

BACKGROUND: This study evaluated the ability of a modified cell separator (Cobe Spectra Apheresis) system to isolate monocytes (MOs) by elutriation. The evaluation was performed in two independent international laboratories. The capacity of collected MOs to differentiate into dendritic cells (DCs) was also assessed. STUDY DESIGN AND METHODS: MNCs from platelet apheresis residues were elutriated on a modified cell separator (Cobe Spectra Apheresis system) using a custom disposable set. Cells were separated according to their size and density. Recovery and purity of the collected cell product were evaluated by impedance counting and flow cytometry. DCs were differentiated in culture from the elutriated MOs and characterized by their surface markers and stimulatory capacity in a mixed WBC reaction assay. RESULTS: Six apheresis mononuclear cell products were used by each laboratory. The separation was achieved in less than 1 hour. Collected MOs had the potential to differentiate into DCs. CONCLUSION: The modified cell separator is an easy and fast device to obtain highly enriched MOs with a DC differentiation potential. The system is closed and employs a single-use disposable set and is more amenable to good tissue practice. This method could become a valuable tool for DC-based active immunotherapy.

Successful immunization requires that mature dendritic cells (mDCs) prime T cells in secondary lymphoid tissue (LT). Previously, the authors have shown that LT T cell activation has an increased costimulatory threshold for a proliferative response as compared with peripheral blood (PB) T cells. Therefore, to optimize mDC immunogenicity, DC maturation was studied using LT T cells as responders. While mDCs obtained with soluble CD40Ligand (sCD40L) or a sCD40L/IFNgamma combination similarly expressed the CD83 and CCR7 molecules on their membrane, only the latter secreted IL-12. sCD40L/IFNgamma mDCs, as compared with sCD40L mDCs, enhanced allogeneic LT T cell proliferation, LT CD4+ cell IFNgamma production and LT CD8+ cell cytotoxicity. Enhancement could be predominantly ascribed to IL-12 secreted by sCD40L/IFNgamma mDCs and to additional costimulatory signals as shown remarkably in the IFNgamma response when IL-12 was neutralized. Therefore, in addition to their membrane phenotype, mDCs to be used in immunization protocols should be assessed for IL-12 secretion as a surrogate marker for an optimum costimulatory potential.

Vaccine-based therapies are being developed for a variety of cancers and their efficacy will be determined by their ability to stimulate T cells in the secondary lymphoid tissue. We found that T cells isolated from human secondary lymphoid organs (LT-T), in contrast to peripheral blood T cells (PB-T) are hyporesponsive to cross-linked anti-CD3 mAb (CD3c) even in the presence of exogenous IL-2. Using mAb to trigger CD2 and CD28 co-stimulatory molecules, we found that such dual co-stimulation of LT-T induces profound and sustained responses including CD25 expression, IL-2 secretion and proliferation. Different levels of co-stimulation produced a hierarchical pattern of responses in LT-T, which correlated with the degree of CD3-TCR down-regulation. Mature antigen-presenting cells (APC) restored the capacity of LT-T to proliferate to stimulation of the CD3-TCR complex. Blocking studies demonstrated that optimal proliferation was critically dependent on co-stimulation via CD2 and CD28 engaged by their ligands on the APC. Therefore, LT-T have increased co-stimulatory requirements as compared to PB-T, i.e. multiple co-stimulatory signals coupled to CD3-TCR triggering. Furthermore, LT-T were found to be dependent on APC for survival, in contrast to PB-T. Clearly, LT-T do not behave in a comparable way to PB-T and in vitro experiments assessing novel cancer vaccines should therefore use LT-T as the most appropriate population of responder T cells.

It is now widely accepted that polymerase chain reaction (PCR) analysis of cutaneous T-cell clonality is of diagnostic value in cutaneous T-cell lymphomas (CTCLs) and most helpful in the diagnosis of mycosis fungoides (MF). However, the diagnostic and prognostic value of circulating clonal T cells remains unclear. We studied T-cell clonality in the peripheral blood (PB) and the cutaneous lesion, sampled at the same time, in 363 consecutively seen patients with a clinical suspicion of cutaneous lymphoma. Using a PCR technique providing a specific imprint of T-cell clones (PCRγ–denaturing gradient gel electrophoresis), we found that detection of identical circulating and cutaneous T-cell clones was associated with the diagnosis of CTCL (P

Hepatosplenic gammadelta T-cell lymphoma is a recently identified entity in which lymphoma cells bearing the gammadelta T-cell receptor (TCR) infiltrate the sinusoids of the liver and the sinuses of the splenic red pulp and bone marrow, without lymph node involvement. It is also characterized by a recurrent cytogenetic finding, isochromosome 7q (i7q10). The authors report a case of hepatosplenic lymphoma of alphabeta T-cell phenotype that shares the same clinical, histologic, and cytogenetic characteristics of the previously described hepatosplenic gammadelta T-cell lymphoma. Fluorescent in situ hybridization performed with chromosome 7 probes showed the typical pattern of isochromosome 7q. Genomic analysis of the TCR gamma locus failed to detect a clonal rearrangement. This unique case of hepatosplenic lymphoma of alphabeta T-cell phenotype supports the possibility that lymphoid populations of different alphabeta or gammadelta phenotype that share similar homing and presumably functional properties could give rise to lymphomas displaying similar clinical and pathologic findings.

The present study describes a new culture protocol allowing the activation and proliferation of autologous tumor infiltrating T lymphocytes (TIL), and the generation of antitumor specific CTL in non-Hodgkin's lymphoma (NHL). Cells from eight patients with indolent NHL were used. We performed 3-week co-cultures of TIL with irradiated autologous malignant B cells in the presence of low doses of IL-1beta, IL-2 and IL-12. The proliferation, phenotype and cytotoxicity, and antitumor specificity of T cells recovered were studied. T-cell clonality was analyzed using TCRgamma gene rearrangement amplification by a multiplex PCR. Under these culture conditions, TIL proliferated, and the CD8+ T lymphocytes that were in a minority at the beginning of the culture increased dramatically in 6 out of 8 cases. In two cases, CD4+ T lymphocytes expanded. We showed that an oligoclonal selection of reactive T cells occurred in culture. Specific cytotoxicity developed against autologous malignant B cells in the 6 cases where there was an expansion of CD8+ T lymphocytes. Inhibition experiments performed with mAb directed against HLA class I and II molecules, CD4, CD8 and TCRgammadelta showed that the cytotoxic effector cells were CD8+ T lymphocytes probably expressing TCRalphabeta+. Cytokine secretion was analyzed in culture medium, and we detected significant levels of IFN-gamma, TNF-alpha, and IL-10 and no IL-4 (except in one case). Our results demonstrate that memory T cells from lymphoma patients can be amplified and differentiated into antitumor cytotoxic cells using a combination of the cytokines IL-1beta, IL-2, and IL-12 in association with non modified tumor cells.

Study objectives Non-Hodgkin's lymphomas (NHLs) are clonal proliferation of B or T lymphocytes. Assessment of clonality in lymphoid proliferations uses immunochemistry and, recently, molecular biology. The aim of our study is to assess the role of immunoglobulin gene rearrangement analysis on bronchoalveolar lymphocytes to aid in the diagnosis of B-cell pulmonary NHL. Patients and methods The study took place in a university hospital. There were seven consecutive patients with B-cell-type pulmonary lymphoma and nine control subjects. Gene rearrangement analysis using polymerase chain reaction (PCR) technique was performed on alveolar lymphocytes recovered by BAL. Results Analysis of the immunoglobulin heavy chain gene rearrangement showed a predominant clonal alveolar lymphocyte population in six of seven patients while all control subjects showed germline pattern. Conclusions Gene rearrangement analysis by PCR of alveolar lymphocytes would appear to be sensitive in patients with B-cell pulmonary NHL (six of seven patients) and specific (zero of nine in the control group). This simple test should be added only in the analysis of cells recovered by BAL in patients with suspected primary and secondary B-cell pulmonary NHL.

Background . The inflammatory response in 29 patients undergoing coronary artery bypass grafting using either roller or centrifugal (CFP) pumps was evaluated in a prospective study. Methods . Patients were randomized in roller pump (n = 15) and CFP (n = 14) groups. Terminal complement complex activation (SC5b-9) and neutrophil activation (elastase) were assessed during the operation. Cytokine production (tumor necrosis factor-α, interleukin-6, interleukin-8) and circulating adhesion molecules (soluble endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1) were assessed after the operation. Results . Release of SC5b-9 after stopping cardiopulmonary bypass and after protamine administration was higher in the CFP group ( p = 0.01 and p = 0.004). Elastase level was higher after stopping cardiopulmonary bypass using CFP ( p = 0.006). Multivariate analysis confirmed differences between roller pump and CFP groups in complement and neutrophil activation. After the operation, a significant production of cytokines was detected similarly in both groups, with peak values observed within the range of 4 to 6 hours after starting cardiopulmonary bypass. However, interleukin-8 levels were higher using CFP 2 hours after starting cardiopulmonary bypass ( p = 0.02). Plasma levels of adhesion molecules were similar in both groups within the investigation period. Conclusions . During the operation, CFP caused greater complement and neutrophil activation. After the operation, the inflammatory response was similar using either roller pump or CFP.

Previous reports have highlighted the disparity in biocompatibility of two differently engineered heparin coatings during the cardiopulmonary bypass (CPB) procedure. The aim of this prospective study was to evaluate the impact of the difference in haemocompatibility provided by either the Duraflo II equipment or the Carmeda equipment in the terminal inflammatory response observed after coronary artery surgery. Thirty patients were randomly allocated to two groups to be operated on using either Duraflo II equipment (group I) or Carmeda equipment (group 2) for extracorporeal circulation (ECC). Initial inflammatory response was assessed by terminal complement complex activation (SC5b-9). The late inflammatory response observed in the postoperative period was assessed by measuring cytokine production (tumour factor necrosis (TNFα), interleukin IL-6, interleukin IL-8) and circulating concentrations of adhesion molecules (ELAM-1, ICAM-1). The release of SC5b-9 after CPB and after protamine administration was lower in group 2 than in group 1 ( p = 0.0002 and p = 0.006, respectively). A significant production of cytokines was detected in both groups with peak values observed within the time range of 4-6 h after the start of CPB. However, no difference was observed between the groups except for the IL-8 level in group 2, which was lower 2 h after the start of CPB ( p = 0.01). Plasma levels of adhesion molecules were similar in both groups within the investigation period. Although the Carmeda equipment was more effective in reducing complement activation, the late inflammatory response was similar using either the Duraflo II or Carmeda equipment for extracorporeal circulation as reflected by the changes of cytokine and circulating adhesion molecule levels.

There is a dogma in tumor immunology that tumor-infiltrating lymphocytes (TIL) are defective based on their lack of antitumoral efficacy in vivo and on impaired response to in vitro functional tests. However, TIL have been compared usually with peripheral blood T lymphocytes, raising doubts on the conclusions drawn. Therefore, we compared TIL from B cell non-Hodgkin's lymphomas (NHL) with T cells from nonmalignant secondary lymphoid organs. NHL-TIL were unresponsive to activation by immobilized anti-CD3 mAb, although bypassing T cell receptor (TCR)/CD3 signaling led to proliferation. The poor proliferative responses of NHL-TIL could not be explained by quantitative defects in TCRzeta expression. NHL-TIL underwent marked spontaneous apoptosis in vitro with loss of approximately 50% of cells after 24 h of culture. This was associated with downregulation of the antiapoptotic Bcl-xL and Bcl-2 proteins, whereas viable NHL-TIL maintained their expression. IL-2, anti-CD3/IL-2, and manipulation of the Fas/Fas-ligand death pathway had no effect on NHL-TIL survival. Apoptosis was not due to increased cell cycling, as NHL-TIL were quiescent, nonproliferating cells. T cells from inflammatory, nonmalignant tissues gave similar functional results to NHL-TIL, suggesting the existence of factors common to the microenvironment of these diverse pathologies. Thus, the quiescent, anergic phenotype of NHL-TIL cannot be attributed solely to tumor factors, but rather is a feature of T cells from chronic inflammatory lesions.

Histological features of early mycosis fungoides (MF) can simulate numerous inflammatory lesions and histological confirmation of MF is often delayed, compared with clinical diagnosis. Recently, using molecular techniques, the detection of a dominant T-lymphocyte clone has been reported in cutaneous lesions of MF. The aim of the present study was to determine the diagnostic value of a dominant T-lymphocyte clone as assessed by PCR-DGGE in early MF. Histopathological and molecular analyses were performed on cutaneous lesions from 104 patients clinically suspected as having MF. In this population, the positive predictive value of a PCR gamma(+) was 0.86. In addition, four of six patients whose lesions were PCR gamma(+) (detectable dominant T-cell clone) but not histologically MF progressed to MF within 2-48 months. In order to evaluate the relevance of PCR gamma-DGGE in MF follow-up, serial biopsies were performed in 24 patients. In 89 per cent of cases, the presence or absence of a PCR gamma(+) was constant during the course of the disease. When present, the DGGE imprint of PCR products was case-specific. These data demonstrate the diagnostic value in MF of T-lymphocyte clonality assessed by PCR gamma-DGGE on cutaneous lesions and show that the technique can be used in MF follow-up to evaluate residual disease with high specificity.

In order to precisely define human lymphocytes subsets, we used four color immunofluorescence analysis and flow cytometry. We report here the results of systematic studies performed on whole blood from 20 normal volunteers. This was performed using monoclonal antibodies recognizing 18 different CD molecules and analyzed here into 14 different 4-color immunofluorescence combinations. This showed that among T cells, the two major CD3+CD8+ and CD3+CD8- subsets differed greatly in the percentage of cells expressing CD26, CD27, CD28, CD38, CD45RA, CD45RO, CD57, S6F1, BY55, CD101 and their respective combination representing an array of subsets. Furthermore the minor NK subsets, i.e. CD2+CD3-CD8+, CD2-CD3-CD8+, CD2+CD3-CD8-, CD2-CD3-CD8- also differed in percentages of cells expressing CD16, CD56, CD57, S6F1, BY55 and their combinations.

The authors report four cases of sinonasal lymphomas with immunophenotypic (four cases) and genotypic (three cases) studies. These lymphomas are frequent in Oriental countries, but unusual in Western populations, particularly in France. Although they have been originally considered as T-cell lymphomas, their origin remains unclear, and several authors have recently suggested that they are a natural killer (NK)-cell proliferation. The tumor cells of the four cases reported here showed characteristics of NK cells. On cryostat sections, they were CD3 negative, but CD2 and CD56 positive. The cytoplasm of the tumor cells exhibited azurophilic granulations. Using polymerase chain reaction (PCR), no clonal rearrangement of the T-cell receptor γ gene was present. Furthermore, clinically, two presented a pharyngeal involvement, and two were also characterized by hepatosplenic involvement at the time of the diagnosis. Thus, the present cases provide additional evidence toward the NK-cell origin of these rare lymphomas. Many sinonasal lymphomas, including the present cases, are positive for Epstein-Barr virus (EBV) (LMP-1 antibody and EBER-messenger RNA probes). This may suggest an important role of EBV as a local factor in their pathogenesis.

Using Southern blotting for the diagnosis of clonality in peripheral T-cell lymphomas (PTCLs), analysis of the T-cell receptor (TCR) gamma gene rearrangement was shown to be more informative than that of the TCR beta gene rearrangement. In order to amplify every VJ gamma rearrangement, a polymerase chain reaction (PCR) procedure using newly designed GC-clamp primers has been developed. All primers can be mixed in a single multiplex PCR. PCR products are analysed by denaturing gradient gel electrophoresis (DGGE), providing tumour-specific imprints inasmuch as the procedure characterizes N sequence polymorphism at the VJ junctions. In a series of 30 PTCL cases, the PCR procedure demonstrated 27 cases to be clonally rearranged and failed in three cases. PCR was more accurate than Southern blotting, showing 47 rearranged gamma alleles, four of which were undetectable on the Southern blot. When lymphomas were studied at different sites and at relapse, the DGGE pattern remained unchanged. In PTCL, the proposed PCR is helpful for the diagnosis and staging of the disease and should improve the follow-up monitoring. The undetectability of clonal rearrangements in a few cases is discussed in the light of concepts of lymphomagenesis and T-cell differentiation.

Non-Hodgkin's lymphomas are divided into B- and T-cell neoplasms. The existence and the clinical relevance of lymphomas derived from the third lymphocyte lineage, ie, natural killer (NK) cells are still controversial. NK cells are lymphocytes that mediate cytotoxicity without prior sensitization. NK cells also have phenotypic and genotypic characteristics: they express the NK-related antigen CD56, T- cell markers such as CD2 and CD7, but do not express CD5 and T-cell receptor (TCR) proteins, and their TCR locus is not rearranged. Therefore, if NK cell lymphomas exist, they should express some T-cell markers, but not alpha beta or gamma delta TCR proteins. Such lymphomas are actually called TCR silent peripheral T cell lymphomas (PTCL). To detect and characterize NK cell lymphomas, we investigated the immunophenotype and immunogenotype of 35 patients with TCR silent PTCL. The first group included 16 patients with a lymphoma of CD5-CD56+ phenotype, which is identical to normal NK cells. These patients had either a nasal/nasopharyngeal lymphoma (11 cases) or a lymphoma with predominant non-nasal/non-nodal initial involvement (five cases). Eight of the nine cases for which immunogenotypic data were available lacked clonal rearrangement of the TCR gamma genes. Thus, these tumors are likely to be NK cell lymphomas. The second group of 15 cases had a CD5+ phenotype (14 were CD56-, and 1 was CD56+) and clonal rearrangement of TCR gamma genes, indicating that they were true PTCL with unproductive TCR rearrangement. The four remaining cases were CD5- CD56- lymphomas and disclosed either a clonal (two cases) or no clonal (two cases) rearrangements of the TCR gamma genes. Altogether these findings show that CD5-CD56+ so-called “TCR silent PTCL” bear the immunophenotype and immunogenotype of normal NK cells and display peculiar clinical features distinct from true PTCL.

Background and design Cutaneous pleomorphic small T-cell lymphoma is a rare, recently recognized lymphoma, different from mycosis fungoides and Sezary syndrome. Only a few cases have been reported and no treatment modalities have been defined. We reviewed the clinical, histologic, immunohistochemical, molecular biologic, and follow-up data of 11 primary cutaneous pleomorphic small T-cell lymphomas. Results The lesions presented as red purplish nodules, tumors, or plaques. The infiltrate consisted of small pleomorphic lymphoid cells without epidermotropism in nine patients and with a propensity to infiltrate the dermis and subcutaneous fat. Most cases were CD4+/CD8-. A T-cell clone was detected in the skin lesions of nine patients tested. The mean follow-up was 70.1 months and the median follow-up was 20 months. Ten patients are alive with three having persistent lesions. Interferon alfa-2a induced partial or complete remissions in five patients. Interferon alfa-2a combined with a regimen containing doxorubicin chlorhydrate induced a complete remission in a patient suffering a relapse after cyclophosphamide and interferon alone. Conclusions Cutaneous pleomorphic small T-cell lymphoma is a well-defined type of low-grade cutaneous lymphoma with favorable prognosis. Interferon and/or chemotherapy are the treatment of choice in patients with large tumor burden.

B cell lymphoproliferative disorders arising in organ transplant recipients (B cell posttransplant lymphoproliferative disorders [PTLD]) are generally associated with EBV. In previous reports, B cell PTLD were shown to express the full pattern of EBV latent genes, as in vitro-established lymphoblastoid cell lines. Although viral linear DNA was detected in 40% of lymphoproliferative disorders from immunocompromised hosts, immunophenotypic studies failed to detect late EBV replicative antigens. The aim of this study was to investigate the relationship of EBV latent gene expression in B cell PTLD to morphology, clonality, and immunophenotype, and to examine the replicative state of EBV in malignant cells. For this purpose, 9 cases of EBV-related B cell PTLD were analyzed. Immunoglobulin gene rearrangements were detected by Southern blot analysis. The presence of EBV was assessed by Southern blot and by in situ hybridization. B cell differentiation antigens, adhesion and activation molecules, and EBV latent and replicative gene expression were studied using immunohistochemistry techniques. We demonstrated that EBV-related B cell PTLD exhibited varying patterns of latent viral gene expression. Higher levels of adhesion molecules were detected in latent membrane protein 1 (LMP1) or LMP1 plus EBV nuclear antigen 2 (EBNA2)-positive tumors than in LMP1 and EBNA2-negative tumors. In contrast, there was no relationship between CD21 and CD23 expression and latent EBV phenotype. Activation of the EBV replicative cycle was highlighted by BamHI Z left frame 1 expression in 5 of 9 cases. Less frequent expression of late viral proteins suggested that the initiation of the EBV lytic cycle might not always lead to virions production.

Summary. Clinical studies in hairy cell leukaemia (HCL) have linked the frequent occurrence of infections due to intracellular pathogens and a profound monocytopenia. More recently, dendritic cells (DC), a subset of which are related to monocytes, were shown to be the professional antigen-presenting cells which stimulate the adaptive immune response. Using membrane markers and flow cytometry, we determined in peripheral blood whether various DC subsets and monocytes were impaired in HCL. Lymphoid and myeloid DC were virtually absent in five HCL patients with active disease. After treatment, both DC and monocytes recovered slowly. The decrease in DC suggests that defective antigen presentation could affect susceptibility to intracellular pathogens in HCL.

International audience; The L-phenylalanine oxidase IL4I1 inhibits T-cell proliferation in vitro through H(2) O(2) production, and is highly expressed in tumor-associated macrophages. IL4I1 is also detected by immunohistochemistry in neoplastic cells from several B-cell lymphomas and some non-lymphoid tumors. To evaluate IL4I1's effect on tumor growth, we developed a mouse melanoma model constitutively coexpressing IL4I1 and the GP33 epitope. After GP33 vaccination, tumors developed more frequently in mice injected with IL4I1-expressing cells in comparison with mice receiving control cells. Tumor escape was preceded by a rapid diminution of IFN-γ-producing cytotoxic antitumor CD8(+) T cells. Moreover, tumor incidence was already increased when only 20% of the injected cells expressed IL4I1. The minimal IL4I1 activities leading to tumor escape were close to those detected in human melanoma and mesothelioma. Thus, we demonstrate the immunosuppressive functions of IL4I1 in vivo and suggest that IL4I1 facilitates human tumor growth by inhibiting the CD8(+) antitumor T-cell response.

Intravenous (i.v.) infusions of Ig concentrates are an effective but expensive treatment for patients with autoimmune thrombocytopenic purpura (AITP). The optimal treatment protocol and the long-term results are uncertain, and the precise mechanism by which the platelet count increases is poorly understood. Twenty adult patients with chronic AITP were enrolled in a prospective study to compare the respective efficacy of two high-dose IVIgG induction regimens (1 g v 2 g/kg body weight) and the long-term effect of six 1 g/kg body weight i.v. IgG reinfusions. An initial response was observed in all 18 evaluable patients: the platelet count increased to a mean value of 251 x 10(9)/L (range 72 to 836 x 10(9)/L) and the mean pretreatment platelet count was multiplied by 14.6. No difference in efficiency was observed between the two i.v. IgG dosages. The degree of the platelet count increment correlated in both groups with the increase in the clearance of antibody-coated red blood cells, measured by an isotopic method, but not with the serum IgG elevation. Treatment was considered to have failed in 11 patients, 90 days after the last i.v. IgG reinfusion (D90), because the platelet counts were comparable with pretreatment values. In contrast, a complete response was observed at D90 in five patients (mean platelet count: 184 x 10(9)/L; range: 150 to 250 x 10(9)/L) and a partial response at D90 was obtained in the remaining two patients (platelet counts: 70 and 104 x 10(9)/L). Five of the 7 responders at D90 kept a platelet count above 50 x 10(9)/L during the entire follow-up period (mean 33 months; range: 5 to 66) with no further treatment; unfortunately, no clinical or biologic criteria were found to be predictive of the long-term response. This study shows that an i.v. IgG infusion regimen of 1 g/kg body weight could safely replace the classical 2 g/kg body weight dosage, at least in patients who do not have life-threatening thrombocytopenia. Moreover, repeated i.v. IgG reinfusion could be an alternative for AITP patients in whom splenectomy is contraindicated.

purpose: An increased risk of high-grade non-Hodgkin's lymphoma is observed in patients who are seropositive for human immunodeficiency virus (HIV). Treatment of such patients is complicated by their underlying acquired immunodeficiency syndrome (AIDS). Intensive strategies such as those used in non-HIV-related lymphoma may be poorly tolerated. However, patients without severe AIDS may derive significant benefits from such an approach. In a prospective multicenter study, treatment outcomes were assessed in 141 cases of HIV-seropositive lymphomas submitted to aggressive chemotherapy. patients and methods: Adult patients with lymphoma with a performance status less than 3 and no active opportunistic infection were consecutively treated with three cycles of doxorubicin 75 mg/m2, cyclophosphamide 1,200 mg/m2, vindesine 2 mg/m2 for 2 days, bleomycin 10 mg for 2 days, and prednisolone 60 mg/m2 for 5 days (ACVB). This treatment was followed by a consolidation phase of high-dose methotrexate plus leucovorin, ifosfamide, etoposide, asparaginase, and cytarabine (LNH84). Central nervous system prophylaxis with intrathecal methotrexate was routinely used. Zidovudine maintenance therapy was started after chemotherapy. Ninety-three patients had high-grade lymphomas (59 Burkitt's type) and 48 had intermediate-grade lymphomas. Disseminated stage III-IV was present in 86 patients, meningeal involvement in 29, and bone marrow infiltration in 30; 62 patients had more than 2 extranodal localizations. Lactate dehydrogenase levels were above the normal value in 95 cases. The median CD4-positive lymphocyte count was 227 X 106/L. results: Eighty-nine patients (63%) achieved complete remission (CR) and 19 (13%) partial remission, whereas 13 did not respond and 20 (14%) died during the course of ACVB, 8 of them from progressive disease. With a median follow-up of 28 months, median survival and disease-free survival were 9 and 16 months, respectively. Median survival for nonresponders was 5 months; 23 patients died of opportunistic infections while in persistent CR. In multivariate analysis, four factors were strongly associated with shorter survival: (1) CD4 count less than 100 × 106/L, (2) performance status greater than 1, (3) immunoblastic lymphoma, and (4) prior AIDS. In the absence of all risk factors, the probability of survival at 2 years was 50%. conclusion: In a selected group of HIV-related lymphomas, intensive chemotherapy with LNH84 is feasible and yields a high CR rate. Survival is short due to death from HIV-related infections; however, in a subgroup of patients without adverse prognostic factors, long-term remission was observed.

Background : No immunohistologic techniques are currently available to demonstrate clon-ality of T-cell lymphomas. Monoclonal antibodies to the variable region of the T-cell receptor (TCR) have been produced that identify minor populations of normal peripheral blood T lymphocytes. Objective : We investigated the expression of TCR V-region genes in cutaneous lymphomas to determine whether immunostaining with these antibodies may be a simple method to detect clonal T-cell proliferations and help to distinguish benign lymphoid infiltrates from malignant lymphoma. Methods : Cutaneous samples were obtained from 18 cutaneous T-cell lymphomas (14 mycosis fungoides, 1 Sezary syndrome, 2 pleomorphic T-cell lymphoma, and 1 large cell anaplastic lymphoma) and 8 benign lymphoid infiltrates. Frozen sections were incubated with monoclonal antibodies and stained by the alkaline phosphatase-antialkaline phosphatase technique. Staining was performed with a panel of 7 anti-TCR V-region antibodies, 6 T-cell markers, 1 anti- β chain antibody, and 1 anti- δ chain antibody. Results : Clonality could be demonstrated in 2 of 18 cutaneous lymphomas. We observed the strictly intraepidermal localization of clonal proliferation in one case of early-stage mycosis fungoides. Conclusion : Anti-TCR V-region antibodies may identify a strictly epidermotropic clone in early mycosis fungoides. However, the panel of antibodies currently available stains only a minority of cutaneous T-cell lymphomas. The usefulness of these antibodies as a clonotypic marker needs to be reevaluated when a larger panel of antibodies becomes available.

Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation. J Clin Invest

SUMMARY The CD3-T cell receptor (TcR) complex is central to the immune response. Upon binding by specific ligands, internalized CD3-TcR molecules increase, and either T cell response or unresponsiveness may ensue depending on the triggering conditions. Using semi-solid agar culture, we have shown previously that quiescent CD4 but not CDS lymphocytes generate clonal colonies under phyto-haemagglutinin stimulation. Here we have demonstrated that the agar induces selective CD3-TcR modulation in the CD8 and not in the CD4 subset. CDS lymphocytes preactivated in liquid culture and recultured in agar with exogenous recombinant interleukin-2 generate colonies with a modulated CD3-TcR surface expression. The peptides composing the CD3-TcR complex are synthesized in CD8 colonies as well as in CD4; however, the CD3 gamma chain is phosphorylated at a higher level in CD8 colonies. A component of the agar polymer, absent in agarose, appears to be the ligand that induces differential CD3-TcR modulation in the CD8 subset. In contrast to agar culture, CD8 colonies can be derived from quiescent CD8 lymphocytes in agarose. These CD8 colonies express unmodulated CD-TcR. CD3-TcR modulation with anti-CD3 monoclonal antibody prior to culturing in agarose inhibits the colony formation. We conclude that given triggering conditions can result in both CD3-TcR modulation and inhibition of the proliferative response selectively in the CD8 lymphocyte subset and not in the CD4.

[F-18]-fluoro-2-deoxy-D: -glucose positron emission tomography (FDG-PET) is a non-invasive imaging technique which has recently been validated for the assessment of therapy response in patients with aggressive non-Hodgkin's lymphoma. Our objective was to determine its value for the evaluation of immunotherapy efficacy in immunocompetent Balb/c mice injected with the A20 syngeneic B lymphoma cell line. The high level of in vitro FDG uptake by A20 cells validated the model for further imaging studies. When injected intravenously, the tumour developed as nodular lesions mostly in liver and spleen, thus mimicking the natural course of an aggressive human lymphoma. FDG-PET provided three-dimensionnal images of tumour extension including non-palpable lesions, in good correlation with ex vivo macroscopic examination. When mice were pre-immunized with an A20 cell lysate in adjuvant before tumour challenge, their significantly longer survival, compared to control mice, were associated with a lower incidence of lymphoma visualized by PET at different time points. Estimation of tumour growth and metabolism using the calculated tumour volumes and maximum standardized uptake values, respectively, also demonstrated delayed lymphoma development and lower activity in the vaccinated mice. Thus, FDG-PET is a sensitive tool relevant for early detection and follow-up of internal tumours, allowing discrimination between treated and non-treated small animal cohorts without invasive intervention.

Lymphoma-derived immunoglobulin idiotype (Id) is a tumour-specific antigen used for antitumour vaccination in follicular lymphoma (FL). However, FL Ids are subject to hypermutation and subclones may escape antitumour cytotoxic T-cell response. To investigate the intraclonal epitope diversity, we sequenced the FL heavy chain gene (consensus Id gene) and subclones of 24 patients. The derived polypeptide sequences were analysed by bioinformatics for human leucocyte antigen (HLA)-A0201-restricted epitope prediction. Epitopes were classified according to BIMAS and SYFPEITHI scores. Surprisingly in these highly mutated polypeptides, the epitopes concentrated in short hotspots in the conserved framework regions (FRs), both in HLA-A0201(+) and HLA-A0201(-) patients. Similar hotspots have been observed by others in chronic lymphocytic leukaemia Ids which in contrast to FL have low mutation frequency. FR3 amino acids 78-88 displayed the best-score epitopes in Ids containing a VH3-family segment, the most represented in FL Ids. Such VH3-FR3(78-88) epitopes were previously demonstrated as immunogenic. Modifications of the epitope pattern in subclones of HLA-A0201(+) patients were generally absent from high-score peptides, including VH3-FR3(78-88) epitopes (83% unmodified). Therefore, no tendency for loss of HLA-A0201-restricted epitopes was evidenced and, given their limited intraclonal diversity, VH3-FR3(78-88) epitopes may provide a useful target for the induction of cytotoxic response in Id-vaccinated FL patients.

Whether methicillin-resistant Staphylococcus aureus (MRSA) constitutes per se an independent risk factor for morbidity and mortality after surgery as compared with methicillin-sensitive Staphylococcus aureus (MSSA) remains a subject of debate. The aim of this study was to assess whether innate defenses against MRSA and MSSA strains are similarly impaired after cardiac surgery. Both intracellular (isolated neutrophil functions) and extracellular (plasma) defenses of 12 patients undergoing scheduled cardiac surgery were evaluated preoperatively (day 0) and postoperatively (day 3) against two MSSA strains with a low level of catalase secretion and two MRSA strains with a high level of catalase secretion, inasmuch as SA killing by neutrophils relies on oxygen-dependent mechanisms. After surgery, an increase in plasma concentration of IL-10, an anti-inflammatory cytokine able to inhibit reactive oxygen species secretion and bactericidal activity of neutrophils, was evidenced. Despite the fact that univariate analysis suggested a specific impairment of neutrophil functions against MRSA strains, two-way repeated-measures ANOVA failed to demonstrate that the effect of S. aureus phenotype was significant. On the other hand, an increase in type-II secretory phospholipase A2 activity, a circulating enzyme involved in SA lysis, was evidenced and was associated with an enhancement of extracellular defenses (bactericidal activity of plasma) against MRSA. Overall, cardiac surgery and S. aureus phenotype had a significant effect on plasma bactericidal activity. Cardiac surgery was characterized by enhanced antibacterial defenses of plasma, whereas neutrophil killing properties were reduced. The overall effect of S. aureus phenotype on neutrophil functions did not seem significant.

International audience; Therapeutic vaccination aims at a strong stimulation of antigen-specific CD8(+) T-cells, so that they differentiate into effectors active in vivo against antigenic targets. Two adenovirus vectors ( Ad) encoding two HLA-A*0201-restricted HIV epitope sequences (pol 476 and pol 589) were constructed. The Ad differ by the presence or absence of a ubiquitin monomer sequence (AdUb(+) and AdUb(-)). The effect of transgene product ubiquitination was analyzed on (1) in vivo, the immunization of Ad vaccinated HLA-A* 0201 humanized HHD mice and ( 2) in vitro, the presentation of the transgene encoded peptides by transduced human dendritic cells ( DC). In vivo, we found that immunization of humanized HHD mice with AdUb(+) elicited a transgene product-specific ;interferon (INF)-gamma CD8(+) T-cell response detectable by enzyme-linked immunospot (ELISPOT), whereas the AdUb(-) construction did not. Antigen-specific cytotoxic T lymphocytes (CTL) were also generated in HHD mice immunized with AdUb(+) and not with AdUb(-). In vitro, using human AdUb(+)-transduced DC, a sizeable expansion of pol 476 and pol 589 tetramer positive CD8(+) T cells as well as CD8(+) CTL were obtained in healthy donors. Compared to AdUb(-)-transduced DC, AdUb(+)-transduced DC triggered a higher number of pol 476-specific IFN-gamma-secreting CD8(+) T cells. In agreement, AdUb(+) transduced DC, used as target in a Cr-51-release assay, were more efficiently lysed by peptide-specific CTL than AdUb(-)-transduced DC. In conclusion, the addition of an ubiquitin sequence to the adenoviral transgene, used as an antigen source, resulted in both in vivo enhanced CD8(+) T-cell immunogenicity in HHD mice and in vitro increased HLA class I-restricted presentation of encoded peptides by human DC.

Toxoplasma gondii encysts in skeletal muscle. Although only rarely found at muscle biopsy, this parasite has previously been regarded as a possible cause of polymyositis. We report a case of biopsy-proven toxoplasmic myositis in a non–HIV-infected patient that led to recognition of idiopathic CD4 lymphocytopenia (ICL), a rare condition typically associated with opportunistic infections. Interestingly, the CD25+ subset that corresponds to the CD4+ regulatory T cells controlling autoimmune processes was lacking. Steroid and antiprotozoal therapy led to recovery. Muscle Nerve 27: 761–765, 2003

The molecular markers that distinguish primary mediastinal large B-cell lymphoma (PMBL) from nonmediastinal diffuse large B-cell lymphomas (NM-DLBLs) remain to be identified. Using cDNA representational difference analysis to compare PMBL and NM-DLBL transcripts, we isolated a cDNA fragment homologous to the mouse B-cell interleukin 4 (IL-4)–inducible gene FIG1(interleukin 4–induced gene 1) transcript. The human FIG1mRNA encodes a 567 amino acid protein that comprises a signal peptide and a large flavin-binding amino oxidase domain, and shares significant homology with secreted apoptosis-inducing L-amino acid oxidases. Northern blot studies showed that FIG1 mRNA expression is mainly restricted to lymphoid tissues. It is expressed at low levels in thymus, spleen, tonsils, and reactive lymph nodes, and is highly up-regulated in IL-4+CD40–activated tonsillar B cells. Interestingly, in human B-cell lines, FIG1 mRNA expression appeared restricted to the PMBL-derived MedB-1 and Karpas 1106 cell lines. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR), we demonstrated that all but one PMBL (16/17) displayed high FIG1 mRNA levels, whereas most NM-DLBLs (12/18) and all low-grade B-cell lymphomas tested (8/8) exhibited low FIG1 mRNA levels. The difference between PMBLs and NM-DLBLs was statistically significant (Fisher test;P = .0003). Southern blot studies did not show rearrangement of the FIG1 gene. FIG1 gene expression might be due to a constitutive activation of a cytokine signaling pathway in PMBL.

T-cell clones of unknown significance (TCUS), assessed by monoclonal or oligoclonal T-cell patterns in PCR-DGGE, were detected in blood of 7/9 patients with anti-Hu syndrome. Clonal patterns were also detected in 2/2 neoplastic lymph nodes, and in 2/2 inflamed dorsal root ganglia from three patients. Only some T-cell clones found in target tissues were also detected in blood or non-target tissues, and likely corresponded to TCUS. In one patient, an identical T-cell clone was found in both neoplastic lymph node tissue and dorsal root ganglia, but not in blood. Dorsal root-infiltrating lymphocytes were cytotoxic CD8(+) TIA-1(+) T-cells. They were often found in close contact to sensory neurons, most of which expressed MHC-1. Taken together, these data support a direct effector role of cytotoxic CD8(+) T-cells, the same clones being likely operative in sensory neuron damage and immune-mediated tumor growth control.

The therapeutic use of dendritic cells (DC) in antigen-specific anti-tumor vaccines, requires sufficient numbers of functional DC, the preparation of which should comply with the code of Good Manufacturing Practice. In addition, the expression of tumor specific antigen should be possible in these DC. As a preclinical step, the method reported here was developed in healthy volunteers. Monocytes (Mo) were isolated by leukapheresis from 12 donors, purified by elutriation and then cultured for 6 days in sealed bags in AIM-V serum free medium with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-13 (IL-13). Between 6×10 8 and 1×10 9 immature DC (iDC) could be differentiated from one leukapheresis. Cells displayed a characteristic iDC phenotype (CD1a + , CD14 − , CD80 + , CD86 + , HLA DR + , CD83 − ), and had potent allogeneic and antigen dependent autologous T cell-stimulatory capacity. Moreover, iDC could be further differentiated into mature DC by CD40 ligation as assessed by CD83 expression and the upregulation of HLA-DR and costimulatory molecules. After infection with a recombinant adenovirus encoding for beta-galactosidase (βGal), 50% to 80% of iDC expressed βGal without toxicity. Adenovirus infection increased the expression of both costimulatory molecules and CD83, and also increased allogeneic stimulatory capacity. Thus, the method developed here allows us to use large numbers of functional iDC as will be required for therapeutic uses in man. These DC can express a transgenic protein.

Patients with total hip arthroplasty were screened for the presence of proinflammatory cytokines in the systemic circulation. Only increased levels of interleukin-6 were detected in patients having had total hip arthroplasty more than 10 years ago. These increased levels of interleukin-6 were associated with a decrease in bone mineral density associated with polyethylene wear and with radiologic osteolysis in some patients. These abnormalities were not found in control subjects without total hip arthroplasty or in patients who had a prosthesis in place for less than 6 years. The elevation in interleukin-6 levels found in patients with the oldest prostheses could constitute a marker for periprosthetic osteolysis.

Positive selection of CD34+ cells in autologous grafts, designed to deplete tumour cells, also results in T-cell depletion. To assess the reconstitution of the different lymphocyte subsets and of the T-cell repertoire diversity following autologous transplantation of selected CD34+ peripheral blood stem cells (PBSC), we analysed sequential blood samples in eight patients autografted for advanced B-cell non-Hodgkin's lymphoma in a phase I-II pilot study. Although natural killer cell recovery was rapid, T- and B-cell recovery was delayed with a median of 110/microliters CD4+, 175/microliters CD8+ T cells and 45/microliters B cells at 12 months post-transplant. The naive CD45RA+ T-cell compartment was profoundly deficient up to 12 months for both CD4+ and CD8+ subsets. A transient expansion of memory CD8+CD45RO+ T cells consisting of an increased percentage of CD57+CD28- cells occurred within the first 3 months post-transplant, but the memory CD4+CD45RO+ T cells remained far below the normal value. The CD8+CD28+ T-cell subset did not recover. Using multiplex PCR analysis of the T-cell receptor gamma locus, we found that the repertoire diversity improved at 12 months after being poor and oligoclonal during the first 3 months post-transplant. As shown by monoplex PCRgamma analysis of every VJ combination, despite T-cell depletion of the graft, mature T cells were carried over with the selected CD34+ PBSC and contributed to the T-cell recovery after transplantation.

Background The differential diagnosis of cutaneous lymphoid hyperplasia and B-cell lymphoma may be difficult. Whether the detection of clonal immunoglobulin gene rearrangement in the cutaneous lesion is predictive of a malignant outcome remains controversial. We therefore studied cases of cutaneous lymphoid hyperplasia by polymerase chain reaction analysis. Design Retrospective study of patients seen between 1988 and 1996. Setting Two dermatology university departments. Patients Twenty-four patients with cutaneous lymphoid hyperplasias were included according to clinical, histopathological, and immunophenotypic criteria. Main Outcome Measures Clinical, histopathological, and laboratory findings. Results There were 13 men and 11 women (mean age, 49 years) who presented with erythematous or violaceous papules or nodules. The lesions were unique in 13 cases and multiple in 11 cases. All patients had immunochemical evidence of a mixed T- and B-cell infiltrate with polytypic B cells. Polyclonality was demonstrated in 23 patients, whereas a dominant B-cell clone was detected in 1 patient. No lymphoma developed during the follow-up (median, 4 years). In the same period, we studied 53 cases of B-cell lymphomas. Thirty-five (66%) of the 53 cases had a detectable clonal immunoglobulin gene rearrangement. Conclusions In the majority of our cases, polyclonality demonstrated by polymerase chain reaction analysis was in accordance with the diagnosis of cutaneous lymphoid hyperplasia. In 1 of the 24 patients, the presence of a B-cell clone could be evidenced. This fact did not modify the treatment as there were no histological or immunophenotypic signs suggestive of a lymphoma.

Objective: To determine whether idiopathic sensory neuropathies could be associated with circulating dominant T-cell clones, a T-cell equivalent to monoclonal gammopathy of unknown significance.Background: A number of predominantly sensory neuropathies remain of unknown etiology. Circulating dominant T-cell clones may be observed in the elderly, in autoimmune disorders, and in chronic viral infections.Methods: Twenty patients with chronic sensory or predominantly sensory neuropathies considered idiopathic after intensive investigation were evaluated for the presence of dominant T-cell clones in blood using PCR amplification of the variable region of the T-cell receptor γ-chain gene. They were classified as chronic idiopathic axonal polyneuropathy (CIAP) or sensory neuronopathy, i.e., chronic idiopathic ataxic neuropathy (CIAN), according to clinical and electrophysiologic criteria.Results: Occurrence of clonal expansions of T cells was strikingly high in patients with idiopathic sensory neuropathies (16/20, 80%), with a similar proportion in CIAP (12/15, 80%) and CIAN (4/5, 80%), as compared with elderly normal controls (2/10, 20%), elderly controls with degenerative neurologic diseases(2/10, 20%), and elderly patients with idiopathic chronic inflammatory demyelinating polyneuropathy (2/10, 20%) (all p < 0.005).Conclusion: Both CIAN and CIAP are associated with dominant T-cell clones of unknown significance that cannot simply be attributed to the age of patients. Relevance of T-cell clones to the pathogenesis of idiopathic sensory neuropathies remains to be determined.

BACKGROUND Acute graft-versus-host disease (aGVHD) is still one of the main causes of morbidity and mortality after allogeneic bone marrow transplantation. Attempts to avoid GVHD are associated with an increased risk of relapse, probably because the graft-versus-leukemia effect is also abrogated. It was recently suggested that a high frequency of host-specific donor helper T cell precursors (HTLp) might be predictive of significant aGVHD (grade > or = II). METHODS We retrospectively studied the frequency of HTLp by means of simplified limiting-dilution analysis to determine its predictive value for aGVHD and relapse. Pre-bone marrow transplantation, host-specific donor HLTp frequencies were analyzed in 32 patients who had received marrow from HLA-identical siblings for hematological malignancies, in terms of aGVHD and relapse. RESULTS HTLp frequencies were significantly higher in patients who had aGVHD > or = grade II (n=14) than in those without aGVHD (n=18) (P=0.007). Patients who relapsed (n=13) had significantly lower HTLp frequencies than those who did not relapse (n=19) (P

A distinct group of cutaneous lymphomas has been described on the basis of CD30 antigen expression by at least 75% of the tumoral cells. When confined to the skin, these CD30 positive cutaneous lymphomas seem to be associated with a better prognosis than CD30 negative counterparts and spontaneous regression may even occur. We observed a spontaneously regressive evolution in 6 out of 9 CD30 positive primary cutaneous large cell-lymphomas diagnosed during a 5-year period. Clinicopathological data of regressive cases were analysed. The mean age of patients was 56.5 years. They were 3 males and 3 females. Skin lesions were solitary nodule or plaque measuring from 1.5 cm to 11 cm in diameter. Histologically, the lesions were classified as pleomorphic, medium and large cell (5 cases) or large cell anaplastic lymphoma (1 case) according to the updated Kiel classification. Delay for spontaneous regression varied from 1 to 6 months. Three of the 6 patients had cutaneous relapses, followed by a spontaneous regression. All patients remained disease-free with an overall median follow-up of 30 months. Histologically, some distinctive signs such as epidermal pseudo-epitheliomatous hyperplasia, epidermotropism, edema, dermal vascular hyperplasia, seemed to be more frequently associated with spontaneously regressive evolution.