[Polyclonal rabbit antithymocyte globulin (thymoglobulin): immunomodulatory effects and new aspects of its clinical application].

Antithymocyte immunoglobulins remain to be one of the most effective immunosuppressants used in transplantology and in the treatment of autoimmune diseases. The unique features of the mechanisms of individual antithymocyte globulin preparations should be borne in mind. Due to its polyclonal nature, thymoglobulin provides a wide spectrum of diverse immunomodulatory effects, which is the basis for its wide use in order to reduce the risk for graft rejection and a graft-versus-host reaction and to treat aplastic anemia.

Cytokine-producing activity of bone marrow erythrokaryocytes and its regulation under normal conditions.

We showed that inhibition of cytokine production by bone marrow adherent cells and their soluble products is a mechanism of regulation of cytokine-producing activity of bone marrow erythrokaryocytes under normal conditions.

Autologous bone marrow cells in the treatment of cirrhosis of the liver.

The data characterizing tolerance and efficiency of autologous bone marrow cells in the treatment of patients with cirrhosis of the liver are presented. Injection of autologous bone marrow cells was not associated with the development of adverse reactions. Cell therapy of patients with compensated cirrhosis arrested asthenic syndrome, reduced cytolysis, increased the level of serum albumin and platelet count. Ultrasonic examination revealed reduction of portal hypertension (the area of the spleen and the portal vein lumen decreased). In patients with decompensated cirrhosis, a positive response presenting as reduction of the disease severity (by 1.9 points) was observed in 48.6% cases. Positive shifts in these patients were associated with a decrease of ALT and AST levels, reduction of laboratory signs of cirrhosis, increase in platelet count, and reduction of the asthenic syndrome. Hence, therapy with autologous bone marrow cells is safe and, according to preliminary results, can be regarded as a new approach to the treatment of patients with cirrhosis of the liver.

Application of autologous bone marrow stem cells in the therapy of spinal cord injury patients.

We studied the safety and efficiency of transplantation of autologous bone marrow cells in complex therapy of patients with spinal cord injury in the late period of the disease. In control group patients, meningomyeloradiculolis was performed, while in the main group surgical treatment was supplemented by transplantation of autologous bone marrow cells. Transplantation of BM stem cells into the cyst cavity and intravenously was well tolerated, did not cause allergic or inflammatory reactions in the early and delayed periods after surgery, and did not induce the formation of ossification foci in the nervous tissue. Analysis of the neurological status by ASIA, Bartel, and Ashworth scales showed that in the main group the positive clinical dynamics was more often observed than in the control. The decrease in neurological deficit included improvement of sensory and motor activity and conducting sensory function. Thus, transplantation of autologous bone marrow cells can be a novel safe strategy for the treatment of patients in the late period after spinal trauma.

[Diagnosis and treatment of aquired aplastic anemia].

AIM: To analyse the results of diagnosis and treatment of patients with aquired aplastic anemia (AA) in one center. MATERIAL AND METHODS: All AA patients, diagnosed and treated in one clinic in 1998-2005, were included in the trial. In severe and very severe AA (SAA/VSAA) the patients (n = 19) received combined immunosuppressive therapy (IST) with antithymocytic globulin (ATG) and cyclosporin A (CsA), in non-severe AA (NSAA) the patients (n = 9) were given monotherapy with CsA. Allogenic transplantation of bone marrow (alloTBM) was made in 4 young patients with SAA/VSAA. RESULTS: The diagnosis of AA was established in 33 patients (19 males and 14 females): NSAA in 9, SAA in 19, VSAA in 5, idiopathic--in 26, posthepatic--in 5, associated with pregnancy--in 2 patients. Age median was 20 years (13-53). The clone of paroxysmal nocturnal hemoglobinuria (PNH) was identified in 7 of 33 patients (21%), antigen HLA-DRB1 *15 in 6 of 11 patients (55%). In median of 26-month follow-up 31 patients (94%) were alive. In IST, complete or partial remissions were obtained in 88% patients. Median of the interval to achievement of transfusion independence made up 2.5 months. All the patients after alloTBM are in complete remission, chronic extensive transplant against host reaction was observed in one case. CONCLUSION: Introduction of updated protocols provides long-term survival of more than 80% AA patients. To optimize treatment outcomes, it is necessary to include newly diagnosed AA patients into ongoing multicenter studies.

Autologous stem cell transplantation for progressive multiple sclerosis: update of the European Group for Blood and Marrow Transplantation autoimmune diseases working party database.

Over the last decade, hematopoietic stem cells transplantation (HSCT) has been increasingly used in the treatment of severe progressive autoimmune diseases. We report a retrospective survey of 183 multiple sclerosis (MS) patients, recorded in the database of the European Blood and Marrow Transplantation Group (EBMT). Transplant data were available from 178 patients who received an autologous graft. Overall, transplant related mortality (TRM) was 5.3% and was restricted to the period 1995-2000, with no further TRM reported since then. Busulphan-based regimens were significantly associated with TRM. Clinical status at the time of transplant and transplant techniques showed some correlations with toxicity. No toxic deaths were reported among the 53 patients treated with the BEAM (carmustine, etoposide, cytosine-arabinoside, melphalan)/antithymocyte globulin (ATG) regimen without graft manipulation, irrespective of their clinical condition at the time of the transplant. Improvement or stabilization of neurological conditions occurred in 63% of patients at a median follow-up of 41.7 months, and was not associated with the intensity of the conditioning regimen. In this large series, HSCT was shown as a promising procedure to slow down progression in a subset of patients affected by severe, progressive MS; the safety and feasibility of the procedure can be significantly improved by appropriate patient selection and choice of transplant regimen.

High-dose immunosuppression with autologous stem cell transplantation in severe refractory systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an immune-mediated disease that is responsive to suppression or modulation of the immune system. Patients with SLE who experience persistent multiorgan dysfunction, despite standard doses of intravenous cyclophosphamide (Cy), represent a subset of patients at high risk of early death. We investigated the efficacy and toxicity of high-dose immunosuppression and autologous hematopoietic stem cell transplantation (SCT) to treat such patients. Six patients (all female, age 15-29 years) with severe refractory SLE were enrolled in the clinic of our institution from 1998 to 2003. All patients were seriously ill, with SLE disease activity indices (SLEDAI) of 6-30, including two cases with central nervous system lupus, one case with lung vasculitis, and three cases with nephritis and nephrotic syndrome. All patients were registered in the European Group for Blood and Marrow Transplantation (EBMT)/European League Against Rheumatism (EULAR) database. Previous immunosuppression included pulse Cy intravenous, prednisolone (standard doses and pulse therapy), oral Cy and azathioprine, with little or no effect on disease progression. Autologous hemopoietic stem cells were collected from bone marrow (n = 4) or mobilized from peripheral blood with Cy and granulocyte colony-stimulating factor (G-CSF) (n = 2). Pre-transplant conditioning regimens included BEAM +/- ATG (n = 2), melphalan 140 mg/m2 + etoposid 1600 mg/m2 (n = 2) and Cy 200 mg/kg +/- ATG (n = 2). Median time to an absolute neutrophil count (ANC) greater than 0.5 x 10(9)/L and platelet count greater than 50 x 10(9)/L was 13 and 15 days, respectively. Three patients died on days 11, 22 and 63 due to transplant-related complications. The follow-up is now 60 and six months for two patients (complete remission), and 42 months for one other patient (partial response). All patients had experienced multiple and severe episodes of infections pre-SCT and long-term history of corticosteroid therapy (3-14 years). We conclude that achievement of prolonged, corticosteroid-free remissions is a reality. Judicious selection of patients earlier in disease or in remission, but with a high risk of relapse or further progression, will diminish transplantation-related mortality.

Altered expression of Pax-5 gene in human myeloma cells.

Recent phenotypic analysis of plasma cells showed that normal plasma cells do express the B-cell lineage-specific molecule CD19, but their malignant counterpart (myeloma cells) are CD19-. To clarify the meaning of loss of CD19 antigen on myeloma cells, we first compared the expression of CD19 and Pax-5 genes among B cells, normal plasma cells, myeloma cell lines, and primary myeloma cells, because the Pax-5 gene was reported to encode the transcriptional factor, B-cell-specific activating protein (BSAP), necessary for CD19 gene expression. Neither CD19 nor Pax-5 mRNA could be detected in those primary myeloma cells and cell lines, whereas normal plasma cells did express both CD19 and Pax-5 mRNA. Furthermore, we could confirm that BSAP-binding activity was not detected in the nuclear extract from CD19- myeloma cell line (KMS-5) but was detected in CD19+ B-cell line (Raji) by gel-shift assay. We further examined the expression of E2A and Id genes, because E2A and Id are considered to be positive and negative regulators in the expression of Pax-5 gene, respectively. However, no significant differences in the expression of these E2A and Id-2 genes could be observed between myeloma cells and normal plasma cells. Therefore, these data suggest that the altered expression of Pax-5, but not E2A or Id, is responsible for the loss of CD19 expression in human myeloma cells, although the underlying mechanism of the altered Pax-5 gene expression remains to be clarified.

Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells.

By using two-color phenotypic analysis with fluorescein isothiocyanate-anti-CD38 and phycoerythrin-anti-CD19 antibodies, we found that pre-B cells (CD38+CD19+) signifcantly decreased depending on the number of plasma cells (CD38++CD19+) in the bone marrow (BM) in the cases with BM plasmacytosis, such as myelomas and even polyclonal gammopathy. To clarify how plasma cells suppress survival of pre-B cells, we examined the effect of plasma cells on the survival of pre-B cells with or without BM-derived stromal cells in vitro. Pre-B cells alone rapidly entered apoptosis, but interleukin-7 (IL-7), a BM stromal cell line (KM-102), or culture supernatants of KM-102 cells could support pre-B cell survival. On the other hand, inhibitory factors such as transforming growth factor-beta1 (TGF-beta1) and macrophage inflammatory protein-1beta (MIP-1beta) could suppress survival of pre-B cells even in the presence of IL-7. Plasma cells alone could not suppress survival of pre-B cells in the presence of IL-7, but coculture of plasma cells with KM-102 cells or primary BM stromal cells induced apoptosis of pre-B cells. Supernatants of coculture with KM-102 and myeloma cell lines (KMS-5) also could suppress survival of pre-B cells. Furthermore, we examined the expression of IL-7, TGF-beta1, and MIP-1beta mRNA in KM-102 cells and primary stromal cells cocultured with myeloma cell lines (KMS-5). In these cells, IL-7 mRNA was downregulated, but the expression of TGF-beta1 and MIP-1beta mRNA was augmented. Therefore, these results suggest that BM-derived stromal cells attached to plasma (myeloma) cells were modulated to secrete lesser levels of supporting factor (IL-7) and higher levels of inhibitory factors (TGF-beta1 and MIP-1beta) for pre-B cell survival, which could explain why the increased number of plasma (myeloma) cells induced suppression of pre-B cells in the BM. This phenomenon may represent a feedback loop between pre-B cells and plasma cells via BM stromal cells in the BM.

Identification of early plasma cells in peripheral blood and their clinical significance.

In the peripheral blood (PB) we detected so-called early plasma cells that might already be committed to entering the bone marrow (BM). By two-colour staining with FITC-anti-CD38 antibody, their intensity (CD38++) of expression of CD38 antigen was between that of germinal centre (GC) B cells (low expression (CD38+)) and that of BM plasma cells (high expression (CD38++)), and their phenotype was CD38++ CD19+ CD10- CD20- CD21+ CD24- CD39+ CD5- VLA-4+ VLA-5- MPC-1- without expression of surface membrane IgM (SmIgM). Morphological and immunological examination of the sorted cells confirmed that they were plasmacytoid cells with expression of cytoplasmic IgG (cIgG). Variations of these early plasma cells were examined in various diseases. In active systemic lupus erythematosus, bacterial septicaemia and liver cirrhosis, early plasma cell levels were significantly increased in PB, and after subsidence of such inflammation (inactive states) these cells returned to normal levels. In contrast, normal early plasma cells were significantly suppressed in myelomas, whilst normal or slightly increased numbers of early plasma cells was found in benign monoclonal gammopathy (BMG). In addition, the number of normal early plasma cells returned to a normal level in myeloma cases with complete responses. Therefore, early plasma cells were identified phenotypically, and an increase and decrease in these cells in PB may reflect mobilization and suppression, respectively, of activated B cells into BM plasma cells.

High proportions of VLA-5- immature myeloma cells correlated well with poor response to treatment in multiple myeloma.

Using two-colour phenotypic analysis with anti-CD38 antibody, human myeloma cells can be classified into VLA-5- immature and VLA-5+ mature cells. We examined the relationship between variations of these subpopulations and clinical responses during treatment in multiple myeloma (MM). 39 patients with MM were treated with combined chemotherapy. First estimation of clinical responses after induction therapy showed that early clinical responses were correlated with the percentage of immature myeloma cells present after induction therapy (P < 0.01), not at diagnosis. After three courses of cyclic maintenance therapy, immature myeloma cells significantly decreased in proportion along with a decrease in total myeloma cells in maintained or more responsive cases (P < 0.01). On the other hand, immature myeloma cells were still found in high proportions in nonresponsive cases with no change (NC) or minor response (MR) (P < 0.01). Furthermore, in relapsing cases from partial response (PR) or progressive disease (PD) from nonresponsive cases, immature myeloma cells increased markedly. Therefore these results show that high proportions of VLA-5- immature myeloma cells remaining after induction therapy and during maintenance therapy correlate well with a declining clinical course of MM during maintenance therapy.

[Effect of B-cell suppressor factor and erythroblast suppressor factor on the colony-forming capacity of hematopoietic stem cells]. / Vliianie B-kletochnogo supressornogo faktora i supressornogo faktora éritroblastov na kolonieobrazuiushchuiu sposobnost' gemopoéticheskikh kletok-predshestvennikov.

Hemopoietic precursor cloning in healthy donors and patients with lymphoproliferative disorders and its regulation in donors by two novel suppressive factors (BSF and ErSF) are investigated. It is shown that the count of erythroid precursors is increased in peripheral blood of patients with non-Hodgkin's lymphoma in contrast to the count of granulocyte-macrophage precursors. BSF inhibits entirely granulocyte-macrophage, macrophage and erythrocyte colony growth. In contrast, ErSF abrogates selectively erythroid precursor cell proliferation and differentiation. The role of the above factor in normal hemopoiesis inhibition mechanism during lymphoproliferative diseases are discussed.

[Quantitative and functional characteristics of T-lymphocytes and monocytes in lymphosarcoma patients at various stages of the neoplastic process]. / Kolichestvennaia i funktsional'naia kharakteristika T-limfotsitov i monotsitov u bol'nykh limfosarkomoi na raznykh stadiiakh opukholevogo protsessa.

IKO monoclonal antibodies were used to study peripheral blood mononuclear types in 92 patients with various histomorphological types of diffuse lymphosarcoma at various stages of tumorous process. Absolute counts of CD7 and CD5 cells (T cells) were found reducing as was CD4 cell (T helpers) level as the disease progressed. CD8 cell (T suppressors) count reliably increased only in the phase of lymphoblastic lymphosarcoma leukemic degeneration. A group of patients with stage IV lymphoblastic lymphosarcoma was detected with drastically increased counts of mononuclears carrying mature T cell markers. Clinical course of the disease in these patients was characterized by the highest malignancy degree and metastatic involvement of the central nervous system. Examination of peripheral blood monocyte/macrophage ratio in the same patient population (n = 26) revealed reduced Fc receptor expression, EA phagocytosis, and increased levels of circulating immune complexes, these shifts augmenting with the tumor progress. The results may be valuable for prediction of lymphosarcoma course and for immunocorrection.

[Structure and function of erythrocytes in lymphomas]. / Struktura i funktsiia éritrotsitov pri limfomakh.

Significant changes have been recorded in the concentration of sulfhydryl groups, histidine, lipoproteins, catalase activity, saponin resistance, and kinetics of chemiluminescent responses of red blood cells in lymphoma patients. Lymphosarcoma is characterized by changes in the structure and function of red blood cells at the early stage of the process, whereas in lymphogranulomatosis changes are observed with the disease progressing, when pronounced signs of tumor intoxication are noted and anemia is present. In lymphosarcoma patients an increase of peripheral blood mononuclears is recorded which expresses the erythroid differentiating antigens with the use of monoclonal antibodies against glycophorin A (ZAE-3) and human erythroblast antigen AG-EB (HAE-9). In lymphogranulomatosis patients it was not detected.

[Expression of erythroid differentiation antigens on mononuclear cells of peripheral blood in neoplastic progression of non-Hodgkin's malignant lymphoma]. / Ekspressiia éritroidnykh differentsirovochnykh antigenov na mono- nuklearakh perifericheskoi krovi pri opukholevoi progressii nekhodzhkinskikh zlokachestvennykh limfom.

High level expression of erythroid differentiation antigens on peripheral blood mononuclear cells associated with increased BFUe number has been identified in a malignant lymphoma patient. A possible pathogenetic role of such disturbance in leukemogenesis has been considered.

[The expression of lymphopoietic differentiation antigens on the membrane of the peripheral blood mononuclear cells in lymphosarcoma tumor progression]. / Izuchenie ékspressii differentsirovochnykh antigenov limfopoéza na membrane mononuklearov perifericheskoi krovi pri opukholevoi progressii limfosarkom.

Monoclonal antibodies were used in the indirect immunofluorescence test to assay the antigenic features of lymphoid cells in patients with lymphosarcoma. Some patients with progression of lymphoblastic lymphoma showed an increased content of T lymphocyte expressing early activation antigens, antigens of early hemopoietic progenitors, and CALLA-antigens.

[Suppressive effect of the blast cells of AKR strain mice on antibody formation in vivo and lymphocyte proliferation in vitro]. / Supressivnyi éffekt blastnykh kletok myshei linii AKR na antiteloobrazovanie in vivo i proliferatsiiu limfotsitov in vitro.

Blast cells obtained from the "erythropoietic spleen" of FG-stimulated young mice and cells accumulating in the spleens of preleukemic AKR mice have a marked suppressive effect on spontaneous and mitogen-induced proliferation of young mouse splenocytes in vitro and suppress the development of humoral immune response in immunized recipients during syngeneic transfer in vivo. Some disturbances in erythron system in preleukemic AKR mice manifested in the accumulation of immature erythroid precursors which are suppressors of immunocompetent lymphocytes are suggested to be a pathogenetic link in the development of leukemia.