[Human leukocyte differentiation antigens and CD classification]. / Lidské leukocytární antigeny z hlediska CD klasifikace.

The autor summarizes the information about human leukocyte antigens. Expression of membrane markers is included in accordance with CD (Cluster of Differentiation) classification. The CD molecules are used ubiquitously in human medical research, immunodiagnosis and treatment and also CD profiles are intended to be used by biologists, pathologists and clinicians generally. Profiles nearly 400 known leukocyte cell surface molecules are summarized in CD system (CD1-CD350). Most CD molecules are integral membrane proteins that have one or more pass through the plasma cell membranes. Knowing which regions of a protein are intracellular or extracellular is important in the selection of peptides for immunization, for the expression of domains of the protein, and to understand the interaction of the protein with other proteins. CD markers are great targets for diagnostic and research of different types of diseases as a potential treatment for a variety of tumors.

Lactosylceramide in lysosomal storage disorders: a comparative immunohistochemical and biochemical study.

Immunohistochemical studies of the presence of lactosylceramide (LacCer) in lysosomal storage disorders (LSDs) were done using anti-LacCer monoclonal antibody of the CDw 17 type (clone MG-2). No sign of an association between LacCer and the lysosomal system in normal cells was observed, except for histiocytes active in phagocytosis. A comparative study of a group of LSDs showed a general tendency for LacCer to increase in storage cells in Niemann-Pick disease type C (NPC), and types A and B, GM1 gangliosidosis, acid lipase deficiency, glycogen storage disease type II and mucopolysaccharidoses. LacCer accumulated in storage cells despite normal activity of relevant lysosomal degrading enzymes. The accumulation of LacCer displayed variability within storage cell populations, and was mostly expressed in neurons in NPC. An absence of the increase in LacCer in storage cells above control levels was seen in neuronal ceroid lipofuscinoses (neurons and cardiocytes) and in Fabry disease. Gaucher and Krabbe cells showed significantly lower levels, or even the absence, of LacCer compared with control macrophages. Results of immunohistochemistry were corroborated by semiquantitative lipid thin-layer chromatography (TLC). It is suggested that different associations of LacCer with the lysosomal storage process may reflect differences in glycosphingolipid turnover induced by the storage-compromised lysosomal/endosomal system.

Near-tetraploid poorly differentiated acute myeloid leukemia M0 diagnosed by short-term cultures with a phorbol ester TPA.

Leukemic blasts of two patients with acute leukemia exhibited similar characteristics. They were heterogeneous in size with a diameter of 14-30 microns in smears and unclassifiable by morphological, cytochemical, immunophenotypic and ultrastructural examinations. Cytogenetic examinations of both revealed a near-tetraploid karyotype. Blasts from both patients differentiated into macrophages in cultures with 10 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) which is a feature specific for myeloid blasts and the cases were thus classified as poorly differentiated acute myeloid leukemias (AML M0). Near-tetraploid poorly differentiated acute myeloid leukemias M0 seem to be a special category of AML in the morphologic, immunologic and cytogenetic (MIC) classification. The presence of very large blasts in the heterogeneous blast population in acute unclassified leukemias could be a morphological sign of near-tetraploid leukemias AML M0.

Characterization of platelet antigen for CD45RA monoclonal antibodies.

CD45RA monoclonal antibodies recognize the higher molecular weight isoforms (220 and 205 kDa) of leukocyte common antigen family (CD45), which are typically expressed on B cells and unstimulated T cells. We have found that there are at least three distinct CD45RA monoclonal antibodies which react with platelet 42 kDa (P42) intracellular protein antigen, which seems to be different from any to date described platelet proteins with similar molecular weight. This platelet antigen is a single chain protein, very likely not a glykoprotein, with isoelectrical point between 6.8 and 7.5. P42 does not seem to be a membrane protein and is not associated with platelet cytoskeleton. Results of immunofluorescence assay suggest that P42 may be redistributed to platelet surface after platelet aggregation.

Reaction of various human normal and leukemic cells and cells of different cell lines with rabbit antisera prepared against membrane fraction of B and T cell lines.

Serological analysis of human membrane antigens have been performed using rabbit xenosera against membrane fraction obtained from different cells (B cell lines: RAJI, RAMOS, DAUDI, UHKT-2: T cell line: MOLT-3, as well as normal pooled lymphocytes). The direct cytotoxicity testing demonstrated that xenogeneic rabbit B lymphoid antisera mediated a significant reaction in the complement-dependent cytotoxicity assay against an antigenic determinant on normal peripheral blood B lymphocytes but not against T lymphocytes and granulocytes. Moreover, these xenoantisera react with antigenic specificities present on the CLL leukocytes, on some AML myeloblasts, on the hairy cells, on majority of ALL and CML blasts, but not on eosinophilic leukocytes and on T lymphocytes from peripheral blood of patients with CLL, ALL, AML, and CML in remission. These xenoantisera react with antigenic specificities present on the cells of cultured lymphoid cell lines of B type, but not on cells of T cell lines.

Immunophenotypic significance of the "lymphoid" CD38 antigen in myeloid blood malignancies.

In 72 patients with blood malignancies (leukemias), the expression and distribution of the "B-lineage" antigen CD38, was analyzed, individually and in combination with CD19, CD10, HLA-DR, CD13, CD14, CD33, CDw65, CD2 and CD7. The expression of CD38 on the surface of leukemic cells was totally different from its expression on normal hematopoietic cells. Its positivity in myeloid malignancies was as follows: In patients with acute myeloid leukemia in 21/28 cases-75% (probability of expression 0.68 +/- 0.2, p < 0.05) and in patients with chronic myeloid leukemia in 4/6 cases-66%. In lymphoproliferative malignancies the CD38 antigen was expressed as follows: In patients with acute non-T lymphoblastic leukemia in 12/16 cases-75% (probability of expression 0.7 +/- 0.2, p < 0.05) and in patients with chronic B lymphocytic leukemia in 6/8 cases-75%. CD38 was also found positive in patients with acute mixed lineage leukemia.

Lack of expression of adhesion molecules on leukemic cells: possible pathogenetic factor in blood malignancies.

In 68 patients with different leukemias the expression of the following adhesion molecules was examined: CD11a, CD18, CD54, CD44, CD58, and CD59. While in normal individuals all these molecules are broadly expressed on leukocytes, in patients with leukemias the following deviations were observed: (a) at least one of the examined molecules was missing in 64/68 cases (94%); in 12/68 cases (18%) both molecules LFA-1 and ICAM-1 were missing, in 37/68 (54%) either LFA-1 or ICAM-1, and in 15/68 cases (22%) adhesion molecules other than LFA-1 or ICAM-1 were missing; (b) the expression of CD11a/CE18, CD58, CD59 on leukemic cells was heterogeneous, without any clear correlation to the subclass of leukemia; (c) in the majority of cases, CD54 (45/68; 66%) and CD44 (36/68; 53%) were missing, however showing a tendency of expression on leukemic cells with more mature immunophenotype.

Aberrant markers expression in T- and B-lymphoid and myeloid leukemia cells of different differentiation stages.

The aim of the study was to ascertain if in T acute lymphoblastic leukemia (T-ALL), B acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML) of different differentiation stages the coexistence of aberrant markers correlate with the degree of leukemic blasts maturation. We evaluated the results of surface and intracellular markers in 42 T-ALL, 86 B-ALL and 71 AML cases. A large panel of monoclonal antibodies (MoAbs) against T-cell, B-cell, myeloid cell and non-lineage specific structures has been used. Patients had dual-color flow cytometric immunophenotyping performed by FACStar flow cytometer. The correct immunological diagnosis of followed new cases before any treatment has been performed and simultaneously the presence of atypical/aberrant phenotypes has been studied and correlated with leukemia cells differentiation stage. A great deal of T-ALL and AML, in opposite to B-ALL cases, revealed a high proportion of atypical phenotypes (55, 75 and 36%, respectively), which are absent in nonleukemic cells. We found out that these atypical phenotypes were present in T-ALL, AML (not clearly in B-ALL) through all differentiation stages and so we obtained an evidence that they might represent an abnormal/atypical rather than an immature phenotype, as it was postulated till now by several authors.

Expression of cytokine receptors on different myeloid leukemic cells.

We have studied the expression of cytokine receptors CD25 (IL-2Ra,55kD), CD116 (hGM-CSRF,145kD), CD117 (CSFR,145kD), CD120a (TNFR,55kD), CD120b (TNFR,75kD), CD121a (IL-1R, type I, 80kD), CD123 (IL-3R), CD124 (IL-4R, 140kD), CD126 (IL-6R, 80kD), CDw127 (IL-7R, 75kD), CDw128 (IL-8R), CD130 (gp130 subunit), CD131 (common beta), CD134 (OX40) and also CD95 (Fas antigen) on the myeloid leukemic cells. Cells from peripheral blood or bone marrow of 30 patients with disorders in myeloid lineage included mostly acute myeloid leukemias (with high leukocyte count and percentage of blasts) were analyzed for the expression of surface membrane molecules by indirect immunofluorescence method evaluated by flow cytometry. The findings indicate that some monoclonal antibodies have a reactivity against cytokine receptors of pathological cells in individual cases, but with very variable qualitative and quantitative (number copies/cell) expression (preliminary results). The leukemic cells demonstrate unique cytokine receptor profiles, which reveal the great diversity of immunophenotypes within the main functional characterization of blood malignancies. The immunophenotype heterogeneity of leukemic cells has proved to be much greater than to match with existing classification criteria. This fact could raise the necessity of further evaluation and specification of cytokine markers of the myeloid acute leukemias. On the other hand, detection of cytokine receptors on the leukemia cells is important for cytokine therapy.

Derivatives of benzo(c)fluorene. XXIV. Cytostatic effect of benfluron on human leukemic cells in vitro.

Benfluoron (BF), a new cytostatic drug, synthesized at the Institute of Pharmacy and Biochemistry in Prague, was tested for its cytostatic and cytotoxic effect. The concentrations of BF ranging from 0.1-2 micrograms/ml had a significant cytostatic effect on nine stabilized human leukemic cell lines. This effect was demonstrated by cell counts, cell viability and 3H-thymidine incorporation. The BF concentrations of 2 micrograms/ml and higher were considerably cytostatic causing the cell death and cell degradation. The effect of BF on the cell growth being irreversible could not be eliminated by washing the cells and recultivating them in fresh medium. The BF concentrations halving the total number of viable cells (EC50 value) induced a higher cytotoxicity in lymphoblastoid cell lines then in myeloid ones. BF did not influence the binding of monoclonal antibodies with membrane markers of different cell subpopulations. Prospective application of BF as a cytostatic and immunosuppressive drug is discussed.

Phenotypic heterogeneity and aberrant markers expression in T-cell leukemia.

For exact determination of lineage assessment there is a need of surface membrane and intracellular (cytoplasmic and nuclear) immunophenotyping performed by flow cytometry. We evaluated in detail the results of surface and intracellular immunophenotyping of 34 T-ALL cases. The great heterogeneity of T-cell differentiation markers has been observed which did not allow relevant subclassification of T-ALL according to the existing subclassification schemes and the proposed three-stage model of physiological T-cell differentiation. Therefore, a simplified classification based on the CD3 marker expression either on cell membrane or in cytoplasm has been created with allocation of T-ALL into two main phenotypic groups. From 34 in detail examined T-ALL cases a great deal-27 (79%) belonged to an immature phenotype (Stage I) and only 7 (21%) expressed more mature phenotype (Stage II). Simultaneously the presence of atypical/aberrant T-cell phenotypes has been studied. We showed that in T-ALL it was possible to specify some cases with leukemia-associated phenotype with coexistence of atypical markers which are absent in nonleukemic cells. In a majority of cases early B-lineage marker (CD10) and in a smaller proportion of them non-lineage associated marker (CD34) were observed. Myeloid marker CD13 was observed in one case of the immature T-ALL, together with CD10 and CD34. As these atypical markers were present through all differentiation stages of T-ALL we obtained a strong evidence that they might represent an abnormal rather than an immature phenotype. The prognostic significance of T-ALL subtypes and aberrant markers coexpression have been discussed. Simultaneously it was shown that quantitative immunofluorescence could provide an additional important diagnostic marker also in T-ALL cases.

Expression of CDw12 and CD17 cell surface antigens on leukemic cells from patients with blood malignancies.

In 67 cases of newly diagnosed blood malignancies, NonT-ALL, T-ALL, AMLL, AML, CML, CLL, HCL, PLL, MDS, B splenic lymphoma, AUL, as well as in 9 cell lines (U937, HEL, Jurkat, HL60, UHKT2, KG1, Raji, K562, REH), we have analysed the expression and distribution of 2 relatively incompletely studied antigenic markers from the CD nomenclature: CDw12 and CD17, individually and in combination with well characterized ones. We present our data for the usefulness of these molecules in immunodiagnosis of leukemias and lymphomas.

Partial purification and separation of H-2 antigens by free-flow preparative electrophoresis.

The membrane fraction of murine spleen cells was solubilized by autolysis in 0.25% potassium cholate and 3M potassium chloride, and the solubilized membrane components were fractionated by ultrafiltration and preparative free-flow electrophoresis. This procedure yielded partially purified fractions with enriched specific antigenic activities. The H-2Ka and H-2Da antigens were found to possess distinctly different electrophoretic mobilities.

[Standardization of ICO-G2 monoclonal antibodies against X-hapten]. / Standartizatsiia monoklonal'nykh antitel IKO-G2 protiv X-gaptena.

Results obtained from comparative analysis of ICO-G2 monoclonal antibody with some standard CD 15 monoclonal antibodies (FMC-11, TG-1, G-2, B-3-4) and with MG-1, MG-3, MG-5, MG-6 monoclonal antibodies (Prague) performed by flow cytometry on FACS 440 instrument (Becton Dickinson) indicate that all monoclonal antibodies have a similar reactivity and may identify the same X-haptene structure (also known as Lex) included into the differentiation cluster as CD15.

[Hematopoiesis from the aspect of CD antigen expression]. / Hematopoéza z hlediska exprese CD antigenu.

The author summarized the expression of membrane markers (CD antigens) on haematopoietic cells. The proliferation and differentiation of cells in individual lineages from initial haematopoietic stem cell and their myeloid and lymphoid precursors is evaluated.

[The Th1/Th2 paradigm. The role of the Th1 and Th2 lymphocyte subpopulations in the regulation of immune processes]. / Th1/Th2 paradigma. Role Th1 a Th2 lymfocytárních subpopulací v regulaci imunitních deju.

A short report on the interaction of Th1 and Th2 lymphocyte subpopulations in the regulation of immune processes is reviewed. Th1 and Th2 subsets have been characterized on the basis of cytokines they secrete and the immune functions they mediate and also on the basis of cytokine and chemokine receptor expression. The clinical and diagnostic influence of immune processes in pathological stages is also mentioned.

[Progression of multiple myeloma during treatment with recombinant G-CSF and absence of G-CSF and IL-6 cell surface receptors on malignant cells]. / Progrese mnohocetného myelomu pri lécbe rekombinantním G-CSF, s neprítomností receptoru pro G-CSF a IL-6 na povrchu maligních bunek.

Recombinant human hematopoietic growth factors are widely used in the treatment of multiple myeloma (MM) especially due to the increasing role of autologous blood stem cell transplantation (ABSCT). We report a patient with MM in whom rapid extramedullary progression of disease was observed during stem cell mobilization with G-CSF. In 56-year-old man with relapsing IgG lambda MM myeloablative therapy with ABSCT was planned 2 years after diagnosis. G-CSF is increasing doses was used for mobilization. Ten days after the start of G-CSF therapy 2 extramedullary (subcutaneous) myeloma infiltrates appeared. For the second mobilization high dose cyclophosphamide and VP-16 with subsequent G-CSF was used. During the time of chemotherapy tumour infiltrates disappeared, however, after one week of G-CSF treatment rapid progression of disease with the formation of multiple extramedullary infiltrates occurred and the patient died in June 1996. Small pieces of subcutaneous tumour infiltrates were removed at autopsy and immediately frozen in liquid nitrogen. Using the panel of specific antibodies the expression of cytokine receptors (IL-1, 2, 3, 6, 7, 8, 10, SCF, gp130, G-CSF, GM-CSF, EPO) and Fas, Pgp and 24-34 kD multidrug resistance-associated protein were examined. However, no expression of cytokine receptors on tumor cells was found. On the contrary, high positivity of surface MDR associated proteins was observed.

[Immunohistochemical detection of cytokine receptors on cryostat tissue sections]. / Imunohistochemická detekce cytokinových receptoru na kryostatových tkánových rezech.

We have studied tissue expression of the cytokine receptors using a high sensitivity biotin-streptavidin system on cryostat sections. We used a panel of monoclonal antibodies from the 6th International Workshop on Human Leukocyte Differentiation Antigens, namely CD25 (IL-2R alpha), CD95 (FAS antigen), CD116 (GM CSFR), CD117 (SCFR), CD120 alpha (TNFR I), CD120b (TNFR II), CD121a (IL-1R I), CDw123 (IL-3R), CD124 (IL-4R), CD126 (IL-6R), CD127 (IL-7R), CDw128 (IL-8R), CD130 (gpl130), CD131 (IL-3R), CD132 (IL-2R gamma), CD134 (OC-40), CD135 (FLT3/FLK2). Examined tissues (lymph nodes and spleens) were obtained from 12 patients with folicular non-Hodgkin's lymphoma, periferal T non-Hodgkin's lymphoma, B lymphoma, myeloma, Hodgkin's disease, two cases of T cell rich B-lymphoma, autoimmune haemolytic anemia and two cases of rudimentary trombocytopenic purpura. Our results indicate that immunohistological technology using native tissues on cryostat sections, monoclonal antibodies and the visualisation with biotin-streptavidin is a particularly suitable supplementary staining procedure for detection of the cytokine receptors in tissues.

[Presence of CD markers on hematopoietic cells]. / Výskyt CD znaku na hematopoetických bunkách.

The author summarizes the expression of membrane markers of haematopoietic cells. The proliferation and differentiation of cells in individual lineages as well as immunophenotype mozaik of membrane molecules included into CD nomenclature is evaluated.

[The CD nomenclature of leukocyte antigens]. / CD nomenklatura leukocytárních antigenu.

The author gives the basic information about human leucocyte antigens of haematopoietic cells. At present, the membrane markers (antigens) included into CD nomenclature are gradually detected, classified and ranged, many of them being well defined as to genetic determination, chemical structure and function.