Biomedical subjects
Publications and source records attributed to J Tkaczuk.
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Human lymphocytes derived from the peripheral blood of a healthy woman were transfected with a plasmid carrying the simian virus 40 (SV40) large T antigen. The successfully transformed cells contained SV40 large T DNA and were negative for Epstein-Barr virus (EBV) and human T-cell leukaemia virus (HTLV)-1 genomes. The immortalized cell line was assigned to the T-lymphocyte lineage on the basis of morphological, immunological and cytochemical criteria. While the cells expressed CD1a and CD4 at the cell surface, the CD3 complex was solely intracytoplasmic. Immunoprecipitation studies indicated that these cells lacked T-cell receptor (TCR) alpha-chains but not beta-chains. They were negative for activation markers such as CD25, CD69 and major histocompatibility (MHC) class II molecules. In addition, the transformed cells exhibited a complete growth independency towards interleukin-2 (IL-2). However, after phorbol ester stimulation, CD25 and CD69 markers were expressed and IL-2 was secreted. This new human immortalized T-lymphocytic cell line, which is cell-surface TCR/CD3-negative, may be useful as an in vitro model for studying TCR/CD3 assembly, expression and signal transduction.
The mechanisms by which the bacterial root-canal infection leads to periapical bone destruction (cysts or granulomas) are not yet well understood. Previous works have shown elements of an active immune response in the lesions. In the present study, flow cytometry was used to improve the characterization of immune cells. Semiquantitative immunohistochemical analysis showed the presence of plasma cells, macrophages and B and T cells. The simultaneous use of several antibodies in flow cytometry allowed a more precise phenotype of the lymphocytes. The cysts displayed an abundance of B lymphocytes at the same time as a relative scarcity of CD8+ cells. CD4+ lymphocytes were the dominant lymphocyte population in most cases. A small number of gamma delta T lymphocytes and natural killer cells was found. These preliminary results show that flow cytometry may be used to characterize immune cells from inflamed tissue and opens the possibility for further functional studies.
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A new monoclonal antibody (MAb), CNA.42, was generated using the CEM T-cell line. It recognizes a 120-kd formalin-resistant glycosylated antigen that is mainly expressed by follicular dendritic reticulum cells (FDRCs). This antigen is also expressed by a few mononuclear cells in the paracortical area of reactive lymph nodes and by some cortical thymocytes. Two hundred and eighty-nine cases of hematopoietic tumors of various types were tested with this antibody. They showed either intact FDRC networks or FDRC networks dispersed among malignant cells. In follicular lymphomas, the follicular pattern was highlighted by CNA.42 MAb. Expanded FDRC networks were found in angioimmunoblastic T-cell lymphomas. Neoplastic cells were positive in 43.6% (24/55) of T-cell and 4.6% (6/129) of B-cell lymphomas. The highest percentage of cases with positive neoplastic cells was found in anaplastic large-cell lymphomas (62.5%; 15/24). In Hodgkin's disease, FDRC networks, sometimes encasing Hodgkin and Reed-Sternberg (HRS) cells, were found. HRS cells were also stained by this antibody in 23 (21.9%) of the 105 cases examined. A variety of normal nonlymphoid tissues and nonhematopoietic tumors, such as some neurogenic tumors, carcinoma, and occasional sarcomas, were found to be positive. Analysis of the reactivity of CNA.42 antibody with FDRCs of lymphoid tissue from different animal species showed similar reactivity to that observed in humans, suggesting widespread evolutionary conservation of the antigen recognized by this antibody. In daily diagnostic practice, CNA.42 MAb seems to be a suitable FDRC marker and possibly has an auxiliary role in recognizing T-cell lymphomas.
Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.
Sixteen renal transplant (RT) patients (10 men, 6 women, aged 49 +/- 10 years) with chronic hepatitis C received alpha interferon (IFN alpha) therapy (Intron A, Schering Plough) at a dose of 3 x 10(6) units s.c. 3 times a week, scheduled for 24 consecutive weeks. At the beginning of the study all had a stable renal function since at least 12 months (mean serum creatinine -SCr- 121 +/- 38 mmol/l). Fourteen patients were receiving cyclosporin A (CsA) either alone (1) or in combination with steroids and/or azathioprine -AZA- (double therapy: 8; triple therapy: 5); two patients were on conventional therapy. The mean daily doses of CsA were 2.6 mg/kd i.e. a mean whole blood trough level of 104 ng/ml. Six patients experienced renal failure either acute (5) or subacute (1) within 7 to 24 weeks after the start of IFN alpha therapy. Their mean SCr increased from 105 +/- 31 mmol/l to 207 +/- 63 mmol/l (p = 0.02) with de-novo proteinuria in one case (1 g/d) and an increase in pre-existing proteinuria in 2; 3 remained without proteinuria. The histological study showed in all cases a diffuse interstitial edema associated with dilatation of peritubular capillaries; mild inflammatory infiltrates were present in only 3 cases; mild glomerular lesions were not always found (glomerular ischemia, mesangial hypertrophy). There was no vascular lesions IFN alpha was withdrawn in these 6 patients, associated with methylprednisolone pulses in 5 cases. Renal function improved in two cases, stabilized in one and progressed to end stage renal failure in 3 within 4 to 12 months. Four patients had iterative renal biopsies showing in all cases diffuse interstitial fibrosis. This subgroup of patients did not statistically differ at the start of the study from those who did not develop renal failure according to baseline immunosuppression, HLA matching, total peripheral blood lymphocyte (PBL) count. PBL subtypes. INF alpha therapy was associated with acute or subacute renal failure in 37% of patients. The most prominent histological finding was a diffuse interstitial edema of rapid onset, without signs of cellular or vascular rejection. Thus we do not recommend to use IFN alpha therapy in RT patients with chronic hepatitis C, until the mechanisms of the subsequent renal failure be more understood.
A novel antigen detected by the CBF.78 monoclonal antibody (MoAb) is strongly expressed on cortical thymocytes and weakly expressed on resting peripheral T lymphocytes. Expression of the antigen is increased on phytohemagglutinin (PHA)- and anti-CD3-activated T lymphocytes and on Epstein-Barr virus-transformed B lymphocytes. The CBF.78 immunoprecipitated a protein of 116 kD from resting and PHA-activated peripheral blood mononuclear cells. CBF.78 MoAb did not inhibit T-cell proliferation induced by anti-CD3 antibody. This MoAb was effective for immunostaining on paraffin sections after microwave-oven heating of tissue sections. Among malignant lymphomas, the antigen recognized by CBF.78 MoAb was found to be mainly expressed by T-cell lymphomas (49+ of 74), particularly those of high-grade malignancy (31+ of 36), whereas only occasional B-cell lymphomas (4+ of 107) expressed the antigen. A distinctive pattern of reactivity was shown by 108 cases of anaplastic large cell lymphomas. Strong positivity for CBF.78 antibody was observed in 86+ of 108 cases, irrespective of B, T, or null phenotype. This multicenter study suggests that CBF.78 MoAb could be of diagnostic value in differentiating Hodgkin's-like anaplastic large cell lymphomas from cases of Hodgkin's disease rich in neoplastic cells. Only a few cases of Hodgkin's disease (13+ of 126) showed rare Reed-Sternberg cells that stained, In these few cases, staining was weak to moderate and confined to cytoplasm. CBF.78 MoAb was nonreactive with all nonhematopoietic neoplasms examined (0+ of 48). Further studies should delineate the function of this new antigen and its clinical utility.
Adrenal medullary chromaffin cells produce high levels of endogenous opioid peptides. Recent data suggest that transplantation injected locally into the spinal subarachnoid space reduced intractable malignant pain. In order to determine the feasibility, the efficacy and the risks of using adrenal medullary tissue for control of irreducible pain, we have developed a transplantation protocol on cancer pain patients selected when they required chronic intrathecal injection of morphine and progressively increasing doses to maintain the level of analgesic effects. At the present time, our clinical trial involves 8 patients. We report here our initial results (mean follow-up: 5 months). The various data collected before and after the intrathecal administration of chromaffin cells included: 1) Pain evaluation over time, with concomitant narcotic intake, 2) CSF sampling through an implanted access port to determine the following biological parameters: biochemical assay for opioid peptides, cell count and phenotyping of lymphocytes, 3) peripheral blood samples for lymphocyte typing. The results confirm the efficacy of adrenal medullary transplantation into spinal CSF for controlling irreducible cancer pain. Complementary intrathecal and oral morphine were totally stopped in 2 cases and stabilized in 5 others. It seems essential to have an important volume of grafted tissue to achieve analgesia with high levels of metenkephalin in CSF. A progressive decrease in metenkephalin release was observed from 2 to 4 months after the transplantation. Two patients with a long-term follow-up (8 and 12 months) needed another intrathecal chromaffin cell graft.
Some mAbs recognizing polymorphic epitopes of HLA-DR molecules exhibit striking differences of reactivity with the same HLA-DR molecules expressed by different cell types. In this study, we investigated the basis for the differential reactivity of the polymorphic anti-DR mAb OHA TM901 with HLA-DR9 molecules expressed by human PBLs or LCLs. By immunoprecipitation experiments we showed that OHA TM901 recognizes a subset of HLA-DR9 molecules from LCLs. This subset corresponds to HLA-DR9 molecules containing immature-type oligosaccharides. The absence of OHA TM901 reactivity with HLA-DR9 PBLs, as revealed by cytofluorometry analysis, suggests that this subset is either not expressed or expressed at a very low level on PBLs. These results indicate that overexpression of HLA-DR molecules in immortalized LCLs could lead to cell-surface expression of underglycosylated forms which are generally not found on the cell surface of PBLs.
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We report characterization of CD45 isoforms expressed by CD8+ lymphocytes in peripheral blood and in the graft of 40 kidney transplanted patients who underwent kidney biopsy on the basis of clinical signs suggesting rejection. Standard histological examination of the biopsy fragments and three-color cytofluorimetric analysis of lymphocytes extracted from the same fragments by mechanical and enzymatic treatment were performed simultaneously and compared to the peripheral blood lymphocytes. In 14/40 biopsies where lymphocyte extraction succeeded, the predominant subset was CD8 (CD4/CD8 mean ratio was 0.53). Almost all CD8+ cells were activated: among these CD8+ cells, 55 percent were HLA-DR+, and 68 percent CD45RO+, i.e. of a memory cell type with cytotoxic activity. This situation resembles the in vitro observation made during mitogenic stimulation of lymphocytes by phytohemagglutinin, OKT3 or CML ("culture mixte lymphocytaire"). Beside their evident interest for the diagnosis, these data could be useful for our understanding of the physiopathology of the rejection crisis.
Production of monoclonal antibodies directed against polymorphic epitopes of HLA class II molecules using whole human cells as immunogen has often proved ineffective, because most of the antibodies produced are directed against non-MHC human cell surface molecules. One approach to overcome this problem is the use of transfected mouse L cells expressing a single HLA class II allele as immunogen. By immunizing C3H mice with DR103-transfected L cells, we obtained 3 mAb, OHA TM901, OHA TM902, and OHA TM903, that recognize different polymorphic epitopes of the HLA-DR molecule. The molecular specificities of the 3 mAb were determined on a large panel of B-lymphoblastoid cell lines (B-LCL), peripheral blood cells and HLA class II transfectants from the XIth International Histocompatibility Workshop. Interestingly, the 3 polymorphic mAb detect new HLA-DR epitopes shared by several specificities: OHA TM901 reacts with DR1 (DR101, DR103), DR9 (DR901) and DR10 (DR1001) molecules; OHA TM902 recognizes the same molecules but also DR8 (DR801, 802, 803); OHA TM903 reacts with all DR types except DR3 (DR301, 302), DR7 (DR701, 702) and DR52. Surprisingly, OHA TM901 reacts with DR9 transfectants and B-LCL but not with DR9 peripheral blood lymphocytes. Biochemical analyses indicate that the 3 mAb immunoprecipitate HLA-DR products and react in western blots with DR alpha/beta-dimer but not with free alpha- or beta-chains. This study shows that transfected L cells are very useful tools for the production and the fine characterization of mAb recognizing polymorphic epitopes of HLA class II molecules.