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A Grossmann

Publications and source records attributed to A Grossmann.

At least 55 records · Page 3Linked to original sources
PubMed1991

Influence of aging on intracellular free calcium and proliferation of mouse T-cell subsets from various lymphoid organs.

The influence of aging on T-cell activation and proliferation was examined in lymphocytes derived from peripheral blood, spleen, and lymph nodes of WBB6F1 C57B1/6J x WB/Re) mice. Following activation with anti-CD3 monoclonal antibodies, the greatest age-related changes were seen in CD4+ cells derived from spleens of 27- to 30-month-old mice. These CD4+ lymphocytes showed reduced [Ca2+]i signaling and decreased proliferation in the presence of exogenous interleukin 2. CD8+ cells from spleens of old animals showed reduced [Ca2+]i but not altered proliferation. Both CD4+ and CD8+ cells derived from peripheral blood of old mice showed decreased peak [Ca2+]i, but no defect in cell proliferation. In contrast, age-related deficits in either [Ca2+]i or proliferation were not observed in CD4+ and CD8+ cells from lymph nodes. Additionally, the percentage of CD4+ cells was decreased in all lymphoid organs from old mice, while the percentage of CD8+ cells was similar in lymphoid organs of old and young mice. Old mice had a significant increase in expression of Pgp-1 in CD4+ cells from spleen and peripheral blood and CD8+ cells derived from lymph node. Our studies indicate that there are differential effects of aging in T lymphocytes derived from different lymphoid organs in mice. Among the cell sources and subsets examined, the age-related changes noted in CD4+ cells from mouse peripheral blood were the most similar to those previously observed in the corresponding peripheral blood lymphocyte subset in humans.

Aging↗
PubMed1991

Immunization with inactivated lactate dehydrogenase-elevating virus followed by virus infection favors the selection of hybridomas synthesizing antibodies cross-reacting with intermediate filaments and the LDV envelope protein VP3.

Autoantibodies against Golgi antigen and a tumor surface antigen (TSA) of aetiologically unrelated murine cell transformants that develop in immunocompetent mice as early as 6-7 days after treatment with live LDV do not appear after immunization with inactivated virus. However, combination of immunization with inactivated LDV and treatment with live LDV enhances the production of autoantibodies against determinants of intermediate filaments. The basis of the stimulation of this group of autoantibodies is at least in part due to antigenic mimicry between the envelope protein VP3 of LDV and determinants of intermediate filaments, since a panel of monoclonal antibodies cross-reacts with both.

Animals↗
PubMed1990

Proliferative capacity of human peripheral blood lymphocytes sorted on the basis of glutathione content.

Glutathione (GSH) is important in defense against oxygen free radical damage, in detoxification of xenobiotics, and in mitogenesis. The reducing conditions provided by low molecular weight thiols such as 2-mercaptoethanol (ME) have been shown to promote the growth of lymphocytes in culture. We wished to determine the effects of 2-ME on GSH content, and to determine to what extent GSH status affected lymphocyte proliferation. GSH content was quantitated in human peripheral blood lymphocytes (PBL) using a flow cytometric assay with monochlorobimane. This analysis was performed on PBL as well as on the CD4+ T-cell subset, as identified with fluorescent anti-CD4 monoclonal antibodies (mAb). Cells were viably sorted on the basis of their GSH content, and incubated for 3 days with mitogenic concentrations of PHA (for PBL) or anti-CD3 mAb (for CD4+ cells) in the presence of bromodeoxyuridine (BrdU). BrdU/Hoechst cell cycle analysis was then performed on these cells. High GSH sorted cells had a higher percentage of cells capable of entering the cell cycle than low GSH sorted cells. This data indicates that some of the heterogeneity in proliferative capacity within PBL in culture is directly or indirectly related to GSH content. Incubation of cells in 2-ME prevented the loss of GSH that occurs when cells are cultured. 2-ME improved the proliferative capacity of unsorted cells, and of cells sorted for high and low GSH. Acridine orange staining of anti-CD3 mAb stimulated cells sorted for high and low GSH indicated that an early event in cell activation was affected by GSH content.

CD4 Antigens↗
PubMed1990

The effect of long-term caloric restriction on function of T-cell subsets in old mice.

The effect of caloric restriction (from weaning to old age) on CD3-stimulated CD4+ and CD8+ lymphocyte proliferation and calcium mobilization was examined. Young ad libitum (ad lib) fed, old ad lib fed, old calorically restricted, and old calorically restricted mice which were fed ad lib during the last 6 weeks of their life (restricted/refed) were compared in both BDF1 [(C57BL/6 x DBA/2)F1] and C57BL/6 mice. Proliferation of CD4+ cells was lower in old ad lib animals than in young animals; this difference was not seen in CD8+ cells. Those CD4+ cells which did proliferate in old ad lib animals underwent similar cell cycle progression as young cells. In calorically restricted and calorically restricted/refed animals, CD4+ cell proliferation was similar to the young animals, and CD8+ cells showed a higher proliferative capacity than cells from either young or old ad lib mice. Differences in proliferative capacity were not correlated with alterations in transmembrane signaling efficiency as peak [Ca2+]i was reduced in both T-cell subsets in all groups of old mice relative to young mice. Additionally, reduced [Ca2+]i was observed in the CD8+ subset for which there was no deficit in proliferation, and the enhanced proliferation seen in old restricted and old restricted/refed mice did not manifest as increased [Ca2+]i mobilization. The percentage of CD4+ cells from both mouse strains was reduced in all groups of old mice compared with young mice, while the percentage of CD8+ cells was generally similar in young and all groups of old mice. Our studies would suggest that lifelong caloric restriction of mice prevents the age-associated decrease in T-cell proliferative capacity but that the enhanced proliferation of these cells is not due to increased efficiency of transmembrane signaling.

Aging↗
PubMed1990

Aging-related deficiency in intracellular calcium response to anti-CD3 or concanavalin A in murine T-cell subsets.

To examine whether intracellular free calcium ([Ca2+]i) signals are reduced in murine T-lymphocyte subsets in aging, we used a flow cytometric assay with the dye indo-I and simultaneous immunofluorescence. Calcium response was reduced in both CD4+ and CD8+ subsets, after stimulation with concanavalin A or anti-CD3 monoclonal antibody over a broad range of concentrations. Experiments carried out in the presence of EGTA indicate that both calcium mobilization from internal stores and extracellular calcium influx were reduced. The reduced overall response was in part due to a decreased number of responding cells in aged animals. In contrast, the [Ca2+]i response produced by the crosslinking of Thy1 and Ly1 (CD5) surface molecules did not show significant differences between young and old mice, indicating that some signaling mechanisms are intact in aged animals. These data also suggest that there are substantial differences between humans and mice in age-related changes in signal transduction.

Aging↗
PubMed1990

Lactate dehydrogenase-elevating virus induces antibodies reactive with a surface antigen of aetiologically unrelated murine cell transformants.

Mice infected with lactate dehydrogenase-elevating virus (LDV) developed antibodies reactive with a tumour cell surface antigen (TSA) of Moloney sarcoma virus (Mo-MSV)-transformed mouse cells (Sac). We demonstrate that STU mice infected with LDV were protected against growth of syngeneic Sac tumour cells as early as 23 days post-infection (p.i.) and up to 5 months p.i. Nine LDV strains, including the neurovirulent LDV-C, elicited production of anti-TSA antibodies, which were restricted to the IgM isotype. Monoclonal anti-TSA antibodies were raised 4 days after infection of STU mice with LDV. When tested against several transformants of STU and BALB/c mouse origin they were found to react with Mo-MSV transformants (PV-TC-77, STU mouse origin; MSV85 C1 3, BALB/c mouse origin), methylcholanthrene-transformed MethA cells (BALB/c origin) and L929 cells. We suggest that the well known tumour growth inhibition by LDV is due to LDV-induced anti-TSA antibodies.

Animals↗
PubMed1989

Reduced proliferation in T lymphocytes in aged humans is predominantly in the CD8+ subset, and is unrelated to defects in transmembrane signaling which are predominantly in the CD4+ subset.

Peripheral blood lymphocytes (PBL) from elderly donors have a reduced proliferative response to phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies (mAb) compared to those from young donors. To examine whether this is due to intrinsic deficiencies in proliferative potential of T-cell subsets, we compared the growth of unsorted PBL vs sorted CD4+ or CD8+ CD11- cells after anti-CD3 mAb or PHA stimulation. Unsorted PBL of elderly donors (greater than 65 years) showed a significant decrease in proliferation compared to young donors (20-30 years) when stimulated with anti-CD3 mAb or PHA. Sorted CD4+ and CD8+ cells were grown in culture in the absence of accessory cells under optimized growth conditions (CD28 mAb, interleukin 2 and beta-mercaptoethanol present). CD4+ cells from elderly donors showed no reduced growth after anti-CD3 mAb stimulation and only slightly decreased growth after stimulation with PHA. CD8+ CD11- cells from elderly donors, however, showed a 20-30% reduction in the proportion of cells proliferating in response to the mitogens and up to 40% reduction in the rate of cell-cycle progression of the responding cells. We examined whether this reduced proliferation is related to decreased efficiency of signal transduction by comparing this to the mobilization of intracellular free calcium ([Ca2+]i) and calcium channel activity after stimulation with anti-CD3 mAb or PHA. [Ca2+]i was measured in CD4 and CD8 subsets of young and elderly donors using a flow cytometric assay with the dye indo-1. Compared to cells from young donors, CD4+ cells from elderly donors showed a [Ca2+]i response which was up to 26% lower after stimulation with CD3 and 10% lower after stimulation with PHA. This appeared to be related to decreased calcium channel activity in elderly donors, rather than mobilization of intracellular Ca2+ stores. CD8+ cells from elderly donors, however, had a slightly, but significantly, greater [Ca2+]i response to CD3 mAb and PHA than did cells from young donors. Since the age-dependent defect in proliferation is mainly in CD8+ cells, but the [Ca2+]i decline is predominantly in the CD4+ subset, these results suggest that the reduced proliferation of T cells from older donors is not related to decreased efficiency of transmembrane signal transduction.

Adult↗
PubMed1989

Enhanced transmembrane signalling activity of monoclonal antibody heteroconjugates suggests molecular interactions between receptors on the T cell surface.

Signal transduction occurs through multiple receptors expressed on mature, resting T cells. In addition to the CD3-T cell receptor complex, the CD2, CD4, CD5, CD7, CD8 and CD28 receptors mobilize cytoplasmic calcium within minutes of binding with monoclonal antibodies and additional crosslinking occurs on the cell surface. As an approach to study the interactions between these receptors and their transduced signals, monoclonal antibodies to each of these receptors were covalently coupled as heteroconjugates and investigated for activity in cytoplasmic calcium mobilization using indo-1 and flow cytometry. Of a total of 35 conjugates studied, there were seven heteroconjugates that showed an increase in activity and these consisted of either certain conjugates of anti-CD3 or certain conjugates of anti-CD5. The CD3-CD2, CD3-CD4, CD3-CD6 and CD3-CD8 heteroconjugates each gained two to three orders of magnitude in titer in calcium mobilization compared to unconjugated CD3 or the CD3-CD3 conjugate. The increase in activity was not accompanied by an increase in binding titer, indicating that signal transduction occurred at lower levels of receptor occupancy. The increased activity was dependent in each case on the relevant second receptor, since unconjugated CD2, CD4, CD6 or CD8 MAb could block the activity of the corresponding heteroconjugate. Neither CD3-CD5, CD3-CD28 or CD3-CD3 conjugates gained activity, whereas CD3-CD7 heteroconjugates gained slightly in activity. The heteroconjugates with CD5 that acquired ability to mobilize calcium at low concns (less than 5 micrograms/ml) were CD5-CD4, CD5-CD8 and CD5-CD6. Their activity could be inhibited by either CD5 MAb or the second MAb of the heteroconjugate. The increased activity of CD3 or CD5 heteroconjugates was observed in the absence of extracellular calcium. Size exclusion chromatography of heteroconjugates demonstrated that 1:1 ratios were optimal, but larger conjugates were also active. These results suggest that certain receptors are capable for molecular interactions on the cell surface to form complexes with enhanced activity in signal transduction leading to calcium mobilization.

Animals↗
PubMed1989

Autoimmunity induced by lactate dehydrogenase-elevating virus: monoclonal autoantibodies against Golgi antigens and other subcellular elements.

Immunocompetent mice infected with LDVAGIA developed autoantibodies against Golgi antigen as early as 6-7 days postinfection preceding anti-viral antibodies for nearly a week. The anti-Golgi antibody titres peaked between two and three weeks postinfection independent of the applied virus dose. Already one week postinfection anti-Golgi autoantibodies were prominent in IgG subclasses IgG2a and b. Spleen cells from these mice were fused with myeloma cells and the culture fluids were screened by indirect immunofluorescence for antibodies reactive with the Golgi antigen of normal cultured cells. A panel of cloned stable antibody-producing hybridomas has been obtained. Some antibodies showed broad cross-species reactivity, recognizing similar antigenic determinants in the Golgi region of mouse, rat and other mammalian cells and also in avian and piscine cells, whereas others recognized determinants in this cell compartment only in mammalian cells and one recognized Golgi antigen only in particular murine tumor cells. From 19 Golgi antibody producing hybridomas 3 secreted IgM-antibodies, 10 synthesized autoantibodies of the subclass IgG2a and 6 of IgG2b. Applied in immunoelectron microscopy mABs decorated the Golgi organelle. A considerable amount of LDVAGIA-induced hybridomas produced antibodies against conserved antigens associated with the cytoskeleton, mitochondria, and the nucleus, LDV-induced monoclonal anti-Golgi antibodies decorated the Golgi area in LDV- and mock-infected macrophages. However, cytoplasmic fluorescence characteristic for LDV-infected cells was not observed indicating that the anti-Golgi autoantibodies do not cross-react with the virus.

Animals↗
PubMed1988

Signal transduction through CD4 receptors: stimulatory vs. inhibitory activity is regulated by CD4 proximity to the CD3/T cell receptor.

The binding of antibody to the CD4 molecule inhibits mobilization of cytoplasmic free calcium ([Ca2+]i) in response to CD3 cross-linking on resting T cells. Similarly, when CD3 and CD4 are independently and simultaneously cross-linked, calcium mobilization is inhibited when compared to that induced by cross-linking CD3 alone. In contrast, when anti-CD4 and anti-CD3 are cross-linked together, calcium mobilization is substantially higher than from CD3 cross-linking alone. A heteroconjugate consisting of covalently bound CD3 and CD4 monoclonal antibodies (mAb) retains the ability to mobilize [Ca2+]i in CD4 cells at protein concentrations approximately two orders of magnitude lower than the free CD3 mAb, and the activity of the heteroconjugate is inhibitable by free CD4 mAb. The CD3/CD4 heteroconjugate also shows significantly greater activity in stimulation of inositol phosphate IP1, IP2 and IP3 synthesis in T cells than the CD3 mAb alone, and again the activity is inhibited by free CD4 mAb. The activity of the CD3/CD4 heteroconjugate is not simply due to oligomerization, since CD3/CD3 or CD4/CD4 homoconjugates or homoconjugate mixtures did not show increased activity. Other heteroconjugates (CD3/CD5 and CD3/CD28) were not different than the CD3/CD3 homoconjugate in their ability to increase [Ca2+]i. Purified CD4 T cells that do not respond to CD3 mAb in solution do respond to the CD3/CD4 heteroconjugate in solution by proliferating in the presence of a CD28 mAb, with a significant fraction of CD4 cells entering the second cycle within the first three days of stimulation. The CD3/CD4 heteroconjugate co-modulates the CD3 and CD4 receptors, indicating that the heteroconjugate is not simply anchoring the T cell receptor to the T cell surface like anti-CD3 on a solid surface. These results suggest that CD4 plays an active role in signal transduction when brought into close physical proximity to the CD3/T cell receptor complex during major histocompatibility complex class II-restricted antigen presentation.

Antibodies, Monoclonal↗
PubMed1987

Lactate dehydrogenase-elevating virus induces anti-Golgi apparatus antibodies.

A recently described autoantibody-inducing agent in mice was further characterized. Tentatively designated AGIA (anti-Golgi apparatus-inducing agent), this agent has previously been shown to cause antibody production against Golgi apparatus (GA) antigen of cells from different vertebrate species as well as against tumour surface antigen of the Moloney murine sarcoma virus-non-producer transformant Sac. It was shown to possess the properties characteristic of lactate dehydrogenase-elevating virus (LDV). It induced elevation of lactate dehydrogenase levels in the blood, persistent lifelong viraemia in mice and serum titres of up to 10(11) infectious doses (ID50) per ml in the acute phase of infection. Its replication in vitro was limited to subpopulations of murine peritoneal macrophages. Electron microscopy of AGIA-infected macrophages and of serum of infected mice revealed virus-like particles with a morphology resembling LDV. The buoyant density of AGIA was approximately 1.14 g/ml. Both the enzyme-elevating activity and the autoantibody-inducing activity were shown to belong to LDV. Infection of STU mice with two established strains of LDV (LDVROW and LDVPLA) was also found to induce both autoantibody groups. In both cases, after infection with AGIA as well as after infection with the two known LDV isolates, anti-Sac cell antibodies occurred at comparable titres. However, anti-GA antibody titres were rather low after infection with LDVROW and LDVPLA compared with AGIA infection. Serological cross-reactivity was demonstrated between AGIA-, LDVROW- and LDVPLA-infected macrophages. AGIA induced anti-GA antibodies in all six mouse strains tested (STU, DBA/2, BALB/c, C3H/He, NMRI, C57BL/6); however, anti-Sac cell antibodies did not develop in C57BL/6 mice.

Animals↗
PubMed1986

Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry.

Measurement of intracellular ionized calcium concentrations ([Ca2+]i) has been indispensable in elucidating the central role of [Ca2+]i as a trigger of cellular responses to activating stimuli. Such studies have employed the dye quin2, which has not been readily adapted to analysis of individual small cells. We show here that the calcium response of large numbers of single cells can be analyzed with the use of flow cytometry and the recently described dye, indo-1. Such analyses demonstrate for the first time the heterogeneous nature of the [Ca2+]i response to mitogenic stimuli within populations of peripheral blood lymphocytes (PBL). By simultaneous quantitation of one- or two-color surface immunofluorescence labels, some of this heterogeneity of [Ca2+]i response in PBL is shown to be related to cellular immunophenotype. Almost all T cells responded to anti-CD3 antibody; however, the response is greater among CD4+ than CD8+ cells, and within the CD4+ population the rate of response to stimulation by antibody to CD3 differed between subpopulations defined by expression of the common leukocyte marker p220. In contrast, not all T cells responded to phytohemagglutinin (PHA), even at very high doses. As with anti-CD3, after stimulation with PHA, CD4+ cells showed a larger proportion of responding cells than did CD8+ cells. In separate experiments, indo-1 was found not to impair reproductive viability of PBL, thereby providing the potential for analysis of functional activity after the separation of cells by sorting on the basis of the [Ca2+]i response to stimuli. Mixing experiments indicated that a response of a subpopulation representing as little as 0.3% of total cells could be readily detected. Thus, the flow cytometric assay with indo-1 is the first technique that allows the quantitative analysis of response differences of small subpopulations of cells and intercellular variation in [Ca2+]i.

Antibodies, Monoclonal↗
PubMed1985

The frequency of mutants in human fibroblasts UV-irradiated at various times during S-phase suggests that genes for thioguanine- and diphtheria toxin-resistance are replicated early.

Human cells deficient in rate of excision repair of DNA damage induced by UV-radiation, i.e., xeroderma pigmentosum (XP) cells, are much more sensitive to the mutagenic effect of UV than are cells from normal persons. The lower frequency of mutants in the latter cells has been attributed to the fact that, unlike XP cells, they excise most of the potentially mutagenic lesions before these can be converted into mutations. If semi-conservative DNA synthesis on a template still containing unexcised lesions is responsible for introducing mutations and if replication of the gene of interest, e.g., hypoxanthine (guanine)phosphoribosyltransferase (HPRT) for thioguanine resistance or the elongation factor 2 (EF-2) for diphtheria toxin resistance, occurs at a particular time during S-phase, it should be possible to shorten the time available for such repair by synchronizing cells and irradiating them just as the gene is to be replicated. The predicted result would be a much higher frequency of mutants at one part in the S-phase than at other times. To test this, cells were synchronized using the alpha-polymerase inhibitor aphidicolin, which blocks cells at the G1/S border. Autoradiography, cytofluorimetry, and incorporation of tritiated thymidine studies showed that DNA synthesis started immediately after release from aphidicolin and was completed in 8-10 h. Cells irradiated with 6 J/m2 at various times post-release were assayed for survival and mutations. The frequency of thioguanine- or diphtheria toxin-resistant cells in the population was highest in cells irradiated during the first fifth of the S-phase, i.e., 0-1.5 h post-release. It was significantly lower in cells irradiated at later times. In contrast, UV-induced cytotoxicity showed no significant time dependence during S-phase. These data suggest that the HPRT and EF-2 genes are replicated early in S-phase.

Aphidicolin↗
PubMed1984

The amino-acid sequence of bovine glutathione peroxidase.

The amino-acid sequence of the seleno-enzyme glutathione peroxidase from bovine erythrocytes was completely determined. Fragmentation of the carboxymethylated protein comprised cleavages with trypsin, with endoproteinase Lys-C, and with cyanogen bromide in 70% formic acid. The resulting peptides were separated by reversed-phase high-performance chromatography or by gel filtration. For sequence determination automated solid or liquid phase techniques of Edman degradation were used. The proper alignment of fragments was experimentally proven in all but one instance. In this case, consistent indirect evidence was provided. The monomer of glutathione peroxidase was shown to consist of 198 amino acids representing a molecular mass ob about 21 900 Da. The active site selenocysteine was localized at position 45. In addition, four cysteine residues were found at positions 74, 91, 111, and 152. The N-terminal part of the sequence obtained revealed a pronounced homology with a partial sequence of the rat liver enzyme. Moreover, tentative sequence data predicted from X-ray crystallographic analysis of bovine glutathione peroxidase were found to agree in about 80% of the residues with the sequence presented. Differences between the predicted and the experimentally determined sequence are discussed.

Amino Acid Sequence↗
PubMed1983

Non-reactivity of the selenoenzyme glutathione peroxidase with enzymatically hydroperoxidized phospholipids.

Selenium-containing glutathione peroxidase (EC 1.11.1.9) was purified 6000-fold from bovine red blood cells to apparent homogeneity. Lipoxygenase (EC 1.13.11.12) was enriched 20-fold from soybean acetone powder. Linoleic acid was peroxidized with lipoxygenase and then used as a substrate in the glutathione peroxidase reaction. Analogous experiments were conducted with synthetic 1,2-dilinoleoyl-L-alpha-glycerophosphocholine and with natural bovine heart cardiolipin. The peroxidized phospholipids were reactive with glutathione peroxidase only after enzymatic attack by phospholipase A2 (EC 3.1.1.4). This result implies that the membrane-protective function of glutathione peroxidase includes preceeding phospholipase action and excludes a direct interaction of this enzyme with membrane-bound lipid hydroperoxides.

Animals↗