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BACKGROUND: Two-flow cytometry methods for quantification of degranulated basophil after allergen-specific activation were discussed. The methods are discerned by used membrane receptors--FcepsilonRI or IL-3Ralpha Our goal was to evaluate the diagnostic potential of the methods and to correlate them to allergen-specific IgE detection and skin prick test (SPT). METHODS: Patient's and control's groups were studied with Bulgarian grass pollen allergen B1 simultaneously by flow cytometry kits: Basotest and BD FastImmune test. Allergy diagnosis was based on clinical history and SPT. The determination of specific IgE was performed by ELISA--RIDASCREEN. RESULTS: There were no significant differences between the patient's results from Basotest and FastImmune (P>0.05). A significant correlation between values, analyzed by Basotest and by FastImmune was found (r=0.88). The sensitivity and specificity of the Basotest, FastImmune, specific IgE and SPT were 85%, 72%, 92% and 92% sensitivity and 100%, 92%, 100% and 85% specificity respectively. The efficiency was between 82% and 97%. There were a significant correlation between the specific IgE and flow cytometry tests: tau=0.92 (Basotest) and tau=0.71 (FastImmune) and a moderate significant correlation between the SPT and the in vitro tests: tau=0.26 (Basotest) and tau=0.31 (FastImmune). CONCLUSION: The successful use of the Bulgarian grass pollen allergen B1 and both flow cytometry tests was presented. These methods could be as specific tools for IgE-mediated diagnosis especially FastImmune in the case of low IgE receptor expression.

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In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.

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The levels of CD98 antigen expression were studied in 62 consecutive cases of adult acute leukemia including 24 acute lymphoblastic leukemia (ALL) and 38 acute myeloid leukemia (AML) using the monoclonal antibody CAF7 and flow cytometry. The mean follow-up was 13.5 months. The mean relative fluorescence intensity (MIF) of CAF7 varied between 6 and 83 channels (256 channels resolution). No correlation was established between CAF7 cell surface density and most of the predictive parameters such as age, sex, blood counts, immunophenotype, proliferative index (PI) or DNA index. Nevertheless expression of CAF7 correlated positively with survival duration (mean 210 vs 391 days, P = 0.048) and complete remission (CR) duration (mean 132 vs 361, days P = 0.032). The levels of CAF7 differed significantly between ALL and AML (P < 0.001), the ALL cases being all CAF7intermediate or CAF7high. In the AML group the low levels of CAF7 expression correlated with shorter CR duration (mean 132 vs 414 days, P = 0.017). The lack of correlation with other clinical and biological parameters suggested that CAF7 might have an independent prognostic significance in adult AML. Although PI was also positively related to survival duration (P = 0.02), it did not correlate with CR duration or the expression of CAF7. We suppose that the prognostic impact of CD98 is related to the control of cell growth and survival in which the molecule normally participates.

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We report here a patient who presented with pancytopenia, hypercellular bone marrow and three-lineage dysplasia associated with an increase of reticulin fibres. After a 5-month period, anaemia and thrombocytopenia progressed very rapidly and the white blood count increased showing 45% blasts with monocytoid morphology, but cytochemically undifferentiated in nature. The immunophenotype revealed an unusual expression of CD4, CD36 and HLA-DR in the absence of any other myeloid or lymphoid lineage-associated markers. The patient died unexpectedly during the course of chemotherapy. The occurrence of CD4, CD36 and HLA-DR on the blast cells cannot determine the lineage of differentiation with certainty but provides some evidence that the leukaemic cells were probably derived from a very early monocytic progenitor with maturation arrest. These cells had apparently complex interactions with pathologic megakaryocytes and cytokine production.

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The humoral immune response against elastin and collagen was studied in parallel with the delayed type hypersensitivity (DTH) reaction to elastin and the percentage of lymphocyte subpopulations in peripheral blood in 20 patients with systemic sclerosis (SSc). An increase of anti-elastin antibodies of all subclasses was found with a significant prevalence of IgE and IgA antibodies. The profile of anti-collagen type I and type IV antibodies showed an increase of IgE isotypes. In 25% of the patients (5 out of 20) positive DTH reactions to elastin were observed as compared to the negative skin reactions in all control individuals. At the same time a significant hyporeactivity to common bacterial and mould antigens was found in 40% of the patients (versus 16% in the control group) which could be an explanation for the low incidence of positive anti-elastin DTH reaction. The DTH hyporeactivity in SSc cases was in contrast with the increased percentage of CD4 T cells (58.4 vs. 42.0) and increased CD4/CD8 ratio (2.5 vs. 1.5) in the peripheral blood of the patients. This finding together with the increased IgE antibodies to elastin and collagen type I and type IV might suggest a possible shift of the immune balance towards the Th2 type of immune response. This is in line with the increased CD8+CD57+ cells which correlated with the highest number of other parameters studied - disease duration, total skin score, IgE anti-elastin antibodies, IgG anti-collagen type I antibodies, CD4/CD8 ratio and CD19 B cells. The results of this study demonstrated the existence of both humoral and cell-mediated immune response against elastin in SSc patients. However, we could not define whether this was an essential part of pathogenetic mechanisms or a secondary phenomenon reflecting the extent of the damage of connective tissue.

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The expression pattern of A, B and H blood group antigens was evaluated by staining frozen sections with specific monoclonal antibodies developed by us and using the indirect immunoperoxidase method. The expression of blood group antigens was ubiquitously upregulated in the endothelial cells of fetal organs. In the process of their differentiation to endothelial naive embryonic mesenchymal cells expressed cytoplasmic ABH antigens. They were assumed as products of the activation of the respective genes. ABH antigen expression was considered as suggestive evidence for the assumption that blood group antigens could serve as early immunomorphologic markers of endothelial differentiation of mesenchymal cells, thus specifying the location of future blood vessels. Extending the conceptual framework of blood group antigens' significance we consider them as being possibly involved in the process of fetal morphogenesis.

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The levels of expression of the activation antigen CD98 were studied by mAB CAF7 in 51 newly diagnosed consecutive cases of childhood acute lymphoblastic leukemia aged from 1 to 13 years. The mean follow-up was 8 months. A wide range of CAF7 expression was observed, the highest mean fluorescence intensity exceeding the lowest by 20 times. No correlation was revealed between CAF7 cell surface density on the one hand and sex, age, WBC, platelet count, LDH level, FAB groups and immunophenotypes on the other. A positive association between the levels of CAF7 expression and the complete remission (CR) duration was observed. The group of CAF7(low) patients had a significantly shorter CR duration compared to the CAF7(intermediate) and CAF7(high) cases (P=0.0099). Half of the CAF7(low) patients did not respond to the induction therapy and failed to achieve remission. These correlations were clearly marked in common ALL (cALL), which was usually considered to have a favorable outcome. All CAF7(low) cALL cases had a significantly shorter CR duration (P=0.027). Thus CAF7 appears to provide additional information on the biological characteristics of childhood ALL and may have prognostic value regarding the response to therapy and remission duration.

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With regards to the geographical variation in acute lymphoblastic leukemia (ALL) distribution, we present data from the immunophenotyping of 171 newly diagnosed cases of childhood ALL in Bulgaria during a 4 year period (1990-1994). On the basis of 17 phenotypic markers the distribution of immunological subtypes was as follows: AUL 4%; Pro-B ALL 13%; common ALL 42%; Pre-B ALL 11%; B-ALL 1%; T-ALL 28% and unclassified 2%. Most of the cases were between 2 and 5 years of age. Common ALL was predominant (53%) in this age group. The male:female ratio was 1.7:1. The frequency of T-ALL (28%) was significantly higher (P < 0.01; t = 3.49) in comparison to that reported for the U.S.A. and West European countries (mean 13%). It was close to the frequency reported by some authors for France (20%), Greece (26%) and south Italy (28.1%). These countries and Bulgaria might form an environmental area with a moderate frequency of T-ALL.

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PROBLEM: To test the relative impact of epididymal versus ejaculated sperm in immunologic infertility. METHOD: Human antibody binding to epididymal and ejaculated spermatozoa was compared by flow cytometry (FCM) since it allows quantitative analysis of viable sperm while ignoring nonsperm cells. To select sera for FCM, GAT, TAT, and ELISA were applied on 145 sera from fertile men, idiopathically infertile and varicocele patients. RESULTS: All GAT/TAT-positive infertile patients, a representative group of varicocele patients and the fertile control, were assessed by FCM. Higher reactivity toward epididymal sperm revealed 18/22 sera while only four out of them bound to ejaculated sperm stronger than the control. All varicocele sera were positive against epididymal while negative against ejaculated spermatozoa. CONCLUSIONS: Epididymal sperm antigens may play a predominant role in some cases of immunologic infertility. Such patients might not be adequately diagnosed and respectively treated due to the limitations of diagnostic procedures applying only ejaculated spermatozoa.

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The expression of a novel B-cell-associated carbohydrate epitope (1D8) was studied by means of flow cytometry in 153 well defined cases of leukemias and lymphomas and 19 cases of lymphadenopathy used as controls. The 1D8 epitope was detected preferentially in proliferations of mature B-lymphocytes (11/15 CD20+ acute lymphoblastic leukemia (ALL), 14/16 chronic lymphocytic leukemia (CLL), 4/7 mantle cell non-Hodgkins lymphoma (NHL), 3/8 follicle cell NHL. However its expression did not appear lineage- or differentiation stage-restricted. Intensive expression on in vivo and in vitro-activated lymphocytes as well as in some high grade malignancies indicated a relationship to the functional state of cells. Bearing in mind the enhanced detection of 1D8 upon desialylation, the epitope might be involved in the regulation of adhesion/migration potential of normal leukocytes and their malignant counterparts.

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A novel murine monoclonal antibody (MAb) 1D8 was produced after immunization with the nylon wool-adherent fraction of human peripheral blood mononuclear cells (PBMNC). Its reactivity pattern was studied on a panel of hemopoetic normal cells, cell lines and malignancies. Mab 1D8 detects a lymphocyte-specific surface antigen expressed on a major subpopulation of mature B lymphocytes as well as on a minor T-cell subset. Activation-related changes in the expression of 1D8 molecule were observed on both B and T lymphocytes. As compared with the pattern of known activation-associated antigens, 1D8 seems to play a role in the early stages of lymphocyte activation. The antigen could not be immunoprecipitated by the conventional methods for glycoproteins.

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A new murine hybridoma, CAF7, is raised using as an antigen the T-leukemic cell line CEM. It produces a monoclonal antibody (mAb) specific for an activation antigen on human lymphocytes. CAF7 stains monocytes, very weakly resting lymphocytes, and granulocytes, part of the thrombocytes, but not erythrocytes. After activation with phytohemagglutinin (PHA), CAF7 expression on peripheral lymphocytes rises as early as 5 hr post stimulation and 72-hr blasts express it at a considerable level. The cell lines Jurkat, CEM, MOLT4, Raji, Reh, K562, and MONOMAC6 are positive for CAF7. CAF7 immunoprecipitated an antigen, which when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions migrated as a single band with a molecular weight of 120-140 kD. Under reducing conditions, it appears as two bands at 90-100 and 40 kD. The pattern of expression and the biochemical characteristics of CAF7 do not match any of the clusters defined in the Leukocyte Typing Workshops, but do resemble that of a previously described antigen, 4F2.

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The human ABH blood-group antigen expression of cytokeratin, S-100 protein, and the melanoma-associated antigen was studied in 12 cases of pigmented nevi. The biotin-streptavidin immunostaining system was applied. ABH antigens were found both in the endothelial cells and in the germinative epidermal layer. The flat overlaying epithelium, single melanocytes and melanoblasts were positive for cytokeratin. Some basilar cells and the giant multinucleate cells in the Spitz nevi stained for S-100 protein. The melanoma-associated antigen was visualized in the upper epidermal layers as well as in some melanocytes. The present article discusses the specificity of the antigens studied as well as some immunologic traits of potential malignant development of Spitz nevi.

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The parallel expression of ABH blood-group antigens and of the carcinoembryonic antigen was examined applying the biotin-streptavidin immunostaining system. Monoclonal antibodies to the ABH antigens newly produced by us and a polyclonal antibody to the carcinoembryonic antigen (Ortho) were used as primary antibodies. Human tumours derived from six different organs were studied. ABH antigens showed a heterogeneous expression. They were entirely missing in some neoplastic tissues and found in single or in clustered tumour cells in others. The staining for the carcinoembryonic antigen revealed stronger intensity and covered large malignant areas. The possible functions of ABH blood-group antigens as tumour-associated structures are discussed. A number of tumour-associated antigens have been identified which may serve to improve the early diagnostics of tumour processes in man. Recently, the blood-group antigens (BGA) from the ABO/H system have attracted the attention of many investigators who regard them as differentiation antigens and as tumour-associated structures. Oncogenesis is accompanied by a block in BGA biosynthesis as a result of an altered ontogenetical programme. This process leads to: a) deletion of BGA-structures in malignant cells; b) neoantigen expression (onco-developmental markers) and c) appearance of BGA-incompatible antigens. The aim of the present investigation is to examine the coexpression of A, B and H BGA and of the carcinoembryonic antigen (CEA) in malignant human tissues. Using the monoclonal antibodies (MAbs) to ABH-BGA newly produced in our laboratory, some insufficiently explored organs like the mammary gland and especially its metastases were also tested.

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The present study assesses the influence of the immunophenotyping procedure, the type of monoclonal antibody (MAb) and flow cytometer used, upon the reproducibility of the results of estimation of the normal values for CD3, CD4, and CD8 positive lymphocyte subpopulations. A hundred healthy adults were immunophenotyped by direct immunofluorescence with commercially available (OKT3-FITC, OKT4-PE and OKT8-PE), and locally prepared (HL3-FITC, HL4-PE and HL8-PE)MAb. The samples were analysed on FACStar flow cytometer. The maximal values of the analytical (CD3-0.96, CD4-1.05, CD8-1.02), the individual (CD3-2.33, CD4-2.88, CD8-2.86), and the population (CD3-8.7, CD4-8.8, CD8-7.5) variation prove that the precision of immunophenotyping procedure has no effect on the variation of the values of the CD3+, CD4+ and CD8+ subpopulations. The values of all three markers showed Gaussian type of distribution. Regression analysis proved that the values of these subpopulations determined by MAb from two sources were highly correlated (r > 0.9) but the paired t-test gave statistically significant difference in the values of CD8 (1.23%, P > 0.001). Mean, SD and 95% range of the sizes of the positive subsets determined by OKT3, OKT4 and OKT8 showed that the results were very close to the results from a multicenter study of adult Caucasians. The values we find fall within the cited critical range for these markers; so we can conclude that if good care is taken for the precision of the procedure, MAb source and type of flow cytometer do not influence the values of the assayed subsets.

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Human blood group antigens are genetically determined structures of undefined nature found not only in red blood cells but also in various tissues. Monoclonal antibodies against A and B human blood group antigens (B7H4 and D1E8) were obtained. Their tissue compatibility was tested in 11 different normal human organs taken from people with A1 and B blood group phenotypes. The antigen-antibody reaction was visualized using the indirect immunoperoxidase technique. The results of the study showed that the antibodies B7H4 and D1E8 react specifically with the tissue blood group antigens in healthy subjects. The findings obtained in most of the tested organs were in accord with those described by other researchers. Differences were found only in the stomach, urothelium, the lungs and the thyroid gland. A possible explanation of the results is the difference in the epitopes recognized by the monoclonal antibodies used by us and the blood group antibodies applied by other researchers. It is suggested that the monoclonal antibodies against the human blood group antigens A and B obtained by us could be used to establish suspected modification of ABH-antigens in the process of oncogenesis.

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Blood plasma, cell mass, white blood cells (WBC) and erythrocytes were investigated for their relation to cord formation of M. tuberculosis, cultivated in blood according to Pryce's method. It was found that cord formation was related to blood cell and followed the zone of sedimentation of WBC. In lysed blood cord formation could be revealed on the whole glass surface, but disappeared if the lysed blood was filtered through Seitz filter - evidences which were accepted as indications that cord formation was dependent on WBC. However, destroying WBC in lysed blood by freezing and thawing or eliminating them by centrifugation did not disturb cord formation and addition of WBC to simple media did not result in cord formation. It was concluded that cord formation was called forth by lysed erythrocytes and this was confirmed through adding erythrocytes stromata to simple media, where cord formation appeared. The practical value of the technique applied and the eventual role of erythrocyte stromata's lipids in cord formation are discussed.

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Mouse bone marrow cells are cultivated in a liquid culture system in the presence of fibroblast conditioned medium. Under these conditions, proliferation of macrophage and granulocyte precursor cells is induced. Cells of a 5-day-old culture are shown to act as cytotoxic effector cells against tumor targets such as P815, E14, YAC and L5178Y. The effector cell is of macrophage origin since it is susceptible to treatment with the alloantiserum Mph-1.2 plus complement. The kinetics of the reaction resembles the kinetics for killer (K) lymphocyte lysis. In contrast to bone marrow cells, peritoneal macrophages do not show cytotoxic activity against antibody-coated tumor targets although they are susceptible to activation to cytotoxicity by lymphokines. The possible relationship of bone marrow effector cells and K lymphocytes is discussed.

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