Profound lymphocyte depletion occurs in thymus, lymph nodes and spleens of normal and thymectomiz... more Profound lymphocyte depletion occurs in thymus, lymph nodes and spleens of normal and thymectomized mice early after a single high sublethal dose of cyclophosphamide. Among individual lymphocytes characterized by the different types of nucleolus, the "non-activable" lymphocytes with micronucleoli are affected most markedly by CY, whereas the proportion of the "active" lymphocytes is increased (up to 70% in the spleens). During the period of regeneration lymphocytes with micronucleoli increase rapidly, the increment thereof is very high on day 14 in the spleens of all mice, and still higher in the blood and lymph nodes of thymectomized animals. During the period of lymphocytopenia, in spite of the increased proportion of the "active" lymphocytes, the ability of residual cells of elicit GVH reactions and to incorporate 14C-uridine decreases. In contrast to changes in the early period, the recovery of the "active" lymphocytes and GVH reactivity, ...
Activation of mouse lymphocytes with Con A in the presence or absence of serum in the culture med... more Activation of mouse lymphocytes with Con A in the presence or absence of serum in the culture medium and the effect of two polymers, PVP and CS, on this stimulation was investigated. The presence of different sera in the culture medium resulted in a different degree of lymphocyte activation and the tested polymers affected it mildly, whereas in serum-free medium they had a strong inhibitory effect. This inhibition was not the outcome of a toxic effect nor of a reduced mitogen-binding capacity of Con A. In investigating the delayed agglutination of lymphocytes with small doses of Con A in vitro, it was found that PVP had a potentiating effect on agglutination in the presence of serum in the medium and an inhibitory effect in serum-free medium. The differences in the action of the tested polymers on stimulation and agglutination of cells in the presence and absence of serum in the medium in vitro suggest a pronounced effect of the sera on cultured cells. The results are discussed with...
The effect of colicins E3 and K on mouse lymphoid cells was studied. Low doses of colicin E3, whi... more The effect of colicins E3 and K on mouse lymphoid cells was studied. Low doses of colicin E3, which were not toxic, inhibited profoundly the Con A-induced activation of lymphocytes while the Con A binding capacity of the treated cells was retained. The inhibitory effect of colicin E3 was also found when the homing of 51Cr-labelled lymph node cells to the lymph nodes of syngeneic recipients was studied, while the rate of redistribution of H-2 antigens on the surface of the lymphocytes was not changed by colicin E3 or colicin K. The findings suggest that the cytoplasmic membrane is the site of the primary effect of colicin E3 on eukaryotic cells.
A microplate culture system has been standardized for blastic transformation of lymphocytes, usin... more A microplate culture system has been standardized for blastic transformation of lymphocytes, using various mitogens more or less specific to T lymphocytes (concanavalin A, phytohaemagglutinin, erythrogenic toxin) or B lymphocytes (lipopolysaccharides and their lipids A, pokeweed mitogen). The mitogen-stimulated lymphocyte activation was examined by the use of the nucleolar test determining the state of nucleolar RNA synthesis by morphologic criteria. In this introductory paper to a series of papers analyzing the specificity of action of the various chemical and biological immunosuppressive agents on the blastogenic responses of T and B lymphocytes, optimal concentrations of cells and blastogenic substances and other parameters were tested, and the kinetics of transformation was investigated in detail.
Sarcoma I cells from ascitic fluid of A/Ph mice were modified with iodoacetamide, diketene and gl... more Sarcoma I cells from ascitic fluid of A/Ph mice were modified with iodoacetamide, diketene and glutaraldehyde. Syngeneic and allogeneic mice were immunized with the modified cells and the effect of this immunization on the growth or rejection of the native sarcoma injected intramuscularly into the hind leg was ascertained. The antitumour effect of the modified cells was compared with that of the irradiated ones. Only cells treated with iodoacetamide gave a better antitumour effect than the unmodified cells irradiated with a minimal dose of gamma irradiation capable of stopping the proliferation of these cells.
In neonatally thymectomized mice and in "B" mice obtained by adult thymectomy, lethal i... more In neonatally thymectomized mice and in "B" mice obtained by adult thymectomy, lethal irradiation and repopulation with foetal liver cells, blood lymphocytes were classified according to their nucleolar morphology as revealed by toluidine blue staining. A majority of the "active", i.e., RNA-synthesizing cells were found to be thymus-dependent. Direct evidence was obtained by testing the "active" lymphocytes for their sensitivity to anti- serum in vitro. During postnatal ontogenesis, the frequency distribution of individual nucleolar types of lymphocytes tends to reflect the developmental changes in the organ tested. The decline of "active" lymphocytes in the thymus has its counterpart in their increase in the lymph nodes and peripheral blood. In hydrocortisone-treated mice, the most affected lymphocytes were those from the thymus cortex -- both the prevalent population of cells with micronucleoli and the "active" lymphocytes -- where...
Proceedings of the National Academy of Sciences, 1983
Significant inhibition of spermatogenesis and appearance of antibodies against spermatogenic cell... more Significant inhibition of spermatogenesis and appearance of antibodies against spermatogenic cells identified by cytotoxicity and immunofluorescence reactions were observed in mice of inbred strains 129/Sv and BALB/c and in albino guinea pigs after syngeneic, allogeneic, and xenogeneic immunization with mouse F9 embryonic carcinoma cells and Freund's complete adjuvant. A similar syngeneic immunization with PYS-2 cells was ineffective. Appropriate absorption experiments confirmed the similarity between the antigens of F9 and spermatogenic cells and the absence of such a similarity with antigens of PYS-2 cells. These results support the hypothesis that the oncofetal F9 antigens represent spermatogenic differentiation antigens and thus play an essential role in spermatogenic cell differentiation.
Proceedings of the National Academy of Sciences, 1989
Most developmentally regulated epitopes identified on embryonal carcinoma cells and murine preimp... more Most developmentally regulated epitopes identified on embryonal carcinoma cells and murine preimplantation embryos are associated with a glycoprotein-bound large glycan called embryoglycan. To prepare monoclonal antibodies recognizing other, less immunogenic stage-specific embryonic epitopes, we used embryoglycan-negative embryonal carcinoma cells P19XT.1.1 as immunogen. One monoclonal antibody prepared by this strategy was found to react specifically with mouse embryonal carcinoma and embryo-derived stem cell lines. The target epitope, TEC-4, was found to be expressed on eggs and two-cell embryos but was undetectable on later stages of mouse embryos and adult mouse tissues. NaDodSO4/PAGE of immunoaffinity-isolated antigen revealed that TEC-4 epitope is associated with glycoproteins of apparent Mr 120,000 and 240,000. The epitope was resistant to oxidation by sodium periodate and to digestion by endoglycosidase F but was sensitive to treatment with protein-denaturing agents and prot...
Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates sign... more Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca2+ controlled by store-operated Ca2+ entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca2+. Changes in cytosolic Ca2+concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with kno...
Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein t... more Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FceRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FceRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FceRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, ...
The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated... more The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated mast cells. Sphingolipids are an essential player in the development and attenuation of this response. De novo synthesis of sphingolipids in mammalian cells is inhibited by the family of three ORMDL proteins (ORMDL1, 2, and 3). However, the cell and tissue-specific functions of ORMDL proteins in mast cell signaling are poorly understood. This study aimed to determine cross-talk of ORMDL2 and ORMDL3 proteins in IgE-mediated responses. To this end, we prepared mice with whole-body knockout (KO) of Ormdl2 and/or Ormdl3 genes and studied their role in mast cell-dependent activation events in vitro and in vivo. We found that the absence of ORMDL3 in bone marrow-derived mast cells (BMMCs) increased the levels of cellular sphingolipids. Such an increase was further raised by simultaneous ORMDL2 deficiency, which alone had no effect on sphingolipid levels. Cells with double ORMDL2 and ORMDL3 KO...
Profound lymphocyte depletion occurs in thymus, lymph nodes and spleens of normal and thymectomiz... more Profound lymphocyte depletion occurs in thymus, lymph nodes and spleens of normal and thymectomized mice early after a single high sublethal dose of cyclophosphamide. Among individual lymphocytes characterized by the different types of nucleolus, the "non-activable" lymphocytes with micronucleoli are affected most markedly by CY, whereas the proportion of the "active" lymphocytes is increased (up to 70% in the spleens). During the period of regeneration lymphocytes with micronucleoli increase rapidly, the increment thereof is very high on day 14 in the spleens of all mice, and still higher in the blood and lymph nodes of thymectomized animals. During the period of lymphocytopenia, in spite of the increased proportion of the "active" lymphocytes, the ability of residual cells of elicit GVH reactions and to incorporate 14C-uridine decreases. In contrast to changes in the early period, the recovery of the "active" lymphocytes and GVH reactivity, ...
Activation of mouse lymphocytes with Con A in the presence or absence of serum in the culture med... more Activation of mouse lymphocytes with Con A in the presence or absence of serum in the culture medium and the effect of two polymers, PVP and CS, on this stimulation was investigated. The presence of different sera in the culture medium resulted in a different degree of lymphocyte activation and the tested polymers affected it mildly, whereas in serum-free medium they had a strong inhibitory effect. This inhibition was not the outcome of a toxic effect nor of a reduced mitogen-binding capacity of Con A. In investigating the delayed agglutination of lymphocytes with small doses of Con A in vitro, it was found that PVP had a potentiating effect on agglutination in the presence of serum in the medium and an inhibitory effect in serum-free medium. The differences in the action of the tested polymers on stimulation and agglutination of cells in the presence and absence of serum in the medium in vitro suggest a pronounced effect of the sera on cultured cells. The results are discussed with...
The effect of colicins E3 and K on mouse lymphoid cells was studied. Low doses of colicin E3, whi... more The effect of colicins E3 and K on mouse lymphoid cells was studied. Low doses of colicin E3, which were not toxic, inhibited profoundly the Con A-induced activation of lymphocytes while the Con A binding capacity of the treated cells was retained. The inhibitory effect of colicin E3 was also found when the homing of 51Cr-labelled lymph node cells to the lymph nodes of syngeneic recipients was studied, while the rate of redistribution of H-2 antigens on the surface of the lymphocytes was not changed by colicin E3 or colicin K. The findings suggest that the cytoplasmic membrane is the site of the primary effect of colicin E3 on eukaryotic cells.
A microplate culture system has been standardized for blastic transformation of lymphocytes, usin... more A microplate culture system has been standardized for blastic transformation of lymphocytes, using various mitogens more or less specific to T lymphocytes (concanavalin A, phytohaemagglutinin, erythrogenic toxin) or B lymphocytes (lipopolysaccharides and their lipids A, pokeweed mitogen). The mitogen-stimulated lymphocyte activation was examined by the use of the nucleolar test determining the state of nucleolar RNA synthesis by morphologic criteria. In this introductory paper to a series of papers analyzing the specificity of action of the various chemical and biological immunosuppressive agents on the blastogenic responses of T and B lymphocytes, optimal concentrations of cells and blastogenic substances and other parameters were tested, and the kinetics of transformation was investigated in detail.
Sarcoma I cells from ascitic fluid of A/Ph mice were modified with iodoacetamide, diketene and gl... more Sarcoma I cells from ascitic fluid of A/Ph mice were modified with iodoacetamide, diketene and glutaraldehyde. Syngeneic and allogeneic mice were immunized with the modified cells and the effect of this immunization on the growth or rejection of the native sarcoma injected intramuscularly into the hind leg was ascertained. The antitumour effect of the modified cells was compared with that of the irradiated ones. Only cells treated with iodoacetamide gave a better antitumour effect than the unmodified cells irradiated with a minimal dose of gamma irradiation capable of stopping the proliferation of these cells.
In neonatally thymectomized mice and in "B" mice obtained by adult thymectomy, lethal i... more In neonatally thymectomized mice and in "B" mice obtained by adult thymectomy, lethal irradiation and repopulation with foetal liver cells, blood lymphocytes were classified according to their nucleolar morphology as revealed by toluidine blue staining. A majority of the "active", i.e., RNA-synthesizing cells were found to be thymus-dependent. Direct evidence was obtained by testing the "active" lymphocytes for their sensitivity to anti- serum in vitro. During postnatal ontogenesis, the frequency distribution of individual nucleolar types of lymphocytes tends to reflect the developmental changes in the organ tested. The decline of "active" lymphocytes in the thymus has its counterpart in their increase in the lymph nodes and peripheral blood. In hydrocortisone-treated mice, the most affected lymphocytes were those from the thymus cortex -- both the prevalent population of cells with micronucleoli and the "active" lymphocytes -- where...
Proceedings of the National Academy of Sciences, 1983
Significant inhibition of spermatogenesis and appearance of antibodies against spermatogenic cell... more Significant inhibition of spermatogenesis and appearance of antibodies against spermatogenic cells identified by cytotoxicity and immunofluorescence reactions were observed in mice of inbred strains 129/Sv and BALB/c and in albino guinea pigs after syngeneic, allogeneic, and xenogeneic immunization with mouse F9 embryonic carcinoma cells and Freund's complete adjuvant. A similar syngeneic immunization with PYS-2 cells was ineffective. Appropriate absorption experiments confirmed the similarity between the antigens of F9 and spermatogenic cells and the absence of such a similarity with antigens of PYS-2 cells. These results support the hypothesis that the oncofetal F9 antigens represent spermatogenic differentiation antigens and thus play an essential role in spermatogenic cell differentiation.
Proceedings of the National Academy of Sciences, 1989
Most developmentally regulated epitopes identified on embryonal carcinoma cells and murine preimp... more Most developmentally regulated epitopes identified on embryonal carcinoma cells and murine preimplantation embryos are associated with a glycoprotein-bound large glycan called embryoglycan. To prepare monoclonal antibodies recognizing other, less immunogenic stage-specific embryonic epitopes, we used embryoglycan-negative embryonal carcinoma cells P19XT.1.1 as immunogen. One monoclonal antibody prepared by this strategy was found to react specifically with mouse embryonal carcinoma and embryo-derived stem cell lines. The target epitope, TEC-4, was found to be expressed on eggs and two-cell embryos but was undetectable on later stages of mouse embryos and adult mouse tissues. NaDodSO4/PAGE of immunoaffinity-isolated antigen revealed that TEC-4 epitope is associated with glycoproteins of apparent Mr 120,000 and 240,000. The epitope was resistant to oxidation by sodium periodate and to digestion by endoglycosidase F but was sensitive to treatment with protein-denaturing agents and prot...
Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates sign... more Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca2+ controlled by store-operated Ca2+ entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca2+. Changes in cytosolic Ca2+concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with kno...
Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein t... more Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FceRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FceRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FceRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, ...
The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated... more The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated mast cells. Sphingolipids are an essential player in the development and attenuation of this response. De novo synthesis of sphingolipids in mammalian cells is inhibited by the family of three ORMDL proteins (ORMDL1, 2, and 3). However, the cell and tissue-specific functions of ORMDL proteins in mast cell signaling are poorly understood. This study aimed to determine cross-talk of ORMDL2 and ORMDL3 proteins in IgE-mediated responses. To this end, we prepared mice with whole-body knockout (KO) of Ormdl2 and/or Ormdl3 genes and studied their role in mast cell-dependent activation events in vitro and in vivo. We found that the absence of ORMDL3 in bone marrow-derived mast cells (BMMCs) increased the levels of cellular sphingolipids. Such an increase was further raised by simultaneous ORMDL2 deficiency, which alone had no effect on sphingolipid levels. Cells with double ORMDL2 and ORMDL3 KO...
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