Biomedical subjects

D Levitt

Publications and source records attributed to D Levitt.

At least 73 records · Page 4Linked to original sources

PubMed1986

Selective infection of astrocytes by Chlamydia trachomatis in primary mixed neuron-glial cell cultures.

Both human biovars of Chlamydia trachomatis were able to productively infect primary cultures of fetal rat brain cells. Infected brain cells released bacteria that reinfected McCoy cells well as other cultured brain cells. The chlamydiae infected cultured astrocytes but were never observed to grow inside neurons, suggesting a selective susceptibility of specific brain cells to chlamydial infections.

Animals↗

PubMed1985

Effects of mycoplasma contamination on immunoglobulin biosynthesis by human B lymphoblastoid cell lines.

The synthesis and secretion of immunoglobulin M (IgM), as well as the relative ratio of membrane and secretory mu heavy chain (mu m and mu s, respectively), were evaluated in mycoplasma-contaminated B lymphoblastoid cell lines. The ratio of mu m to mu s was drastically lowered in infected cultures, and mu s chains could now combine with light chains. A 50 to 100% increase in IgM synthesis occurred in contaminated cultures, and small amounts of IgM were detectable in the culture media. These molecules possessed mu chains typical of secreted IgM. Reexpression of mu on the surface of B lymphoblastoid cells was substantially delayed in mycoplasma-contaminated cultures. Thus, mycoplasma contamination alters the synthesis and expression of a specific differentiated gene product (immunoglobulin) in B cell lines; such changes could significantly affect the interpretation of data on immunoglobulin synthesis by B cells with different phenotypes. This system may also provide a means of studying how mycoplasma infection alters specific gene expression in B cell lines.

Antibody Formation↗

PubMed1985

Binding, ingestion, and multiplication of Chlamydia trachomatis (L2 serovar) in human leukocyte cell lines.

We examined the ability of lymphoblastoid-myeloid cell lines to bind, ingest, and permit multiplication of Chlamydia trachomatis (L2 serovar). Four types of chlamydia-cell line interactions were observed: minimal bacterial binding; bacterial binding, followed by ingestion and high-level multiplication; bacterial binding, followed by ingestion but minimal multiplication; and bacterial binding, but minimal ingestion or replication. Our data demonstrate that at 37 degrees C in vitro the L2 serovar can both bind avidly to a cell without entering it and enter nonphagocytic cells but not grow.

Cell Line↗

PubMed1985

In vivo polyclonal stimulation of antibody secretion by two types of bacteria.

We previously proposed that one benefit of early polyclonal antibody secretion after bacterial infection might be the formation of antibodies protective against infection by a second pathogen. In order to analyse this possibility, the polyclonal and anti-phosphorylcholine (PC) plaque-forming cell (PFC) response induced by Staphylococcus aureus Cowan I (SA) was compared with the same responses stimulated by unencapsulated Streptococcus pneumonia strain R36a. S. aureus stimulated significant anti-PC PFCs, despite an inability to detect immunoassay-reactive PC on the surface of SA (present on R36a). The induction of these antibodies appeared very similar to the mechanism for producing polyclonal responses. R36a also stimulated both anti-PC and polyclonal PFCs, but by different means. In summary, our data suggest that each type of bacteria induces polyclonal and anti-PC responses through different mechanisms, and that the polyclonal antibody response stimulated by one group of bacteria can contain antibodies directed towards a second and different genus of bacteria.

Animals↗

PubMed1984

Sera used for complement-mediated cytolysis can alter B cell function in vitro.

Nonspecific effects of reagents used to selectively isolate lymphocyte subpopulations are most often analyzed according to their influence on cell viability and proliferation. While characterizing monoclonal anti-human B cell antibodies, we detected consistent changes in B cell function between individual donors, no matter which antibody was employed to deplete B cell subpopulations. The source of these alterations was traced to the commercial complement-containing sera used for cytolysis. Different lots of serum from 3 separate commercial vendors and 2 animal species (rabbit and guinea pig) routinely altered B lymphocyte differentiation after stimulation with either pokeweed mitogen or Staphylococcus aureus (Cowan I), while cell viability, lymphocyte subpopulation distribution, and proliferative responses were unaffected.

Animals↗

PubMed1984

Pneumococcal cell wall phosphorylcholine elicits polyclonal antibody secretion in mice.

Immunization of mice with phosphorylcholine (PC)-bearing Staphylococcus pneumoniae Type 2, strain 36a (R36a) results in both a PC-specific and a polyclonal increase in splenic plaque-forming cells. The polyclonal increase was observed in all strains tested, including those bearing an X-linked immune defect resulting in an undetectable anti-PC immune response. The magnitude of the polyclonal response is directly related to the amount of bacterial surface PC as detected by enzyme-linked immunosorbent assay. Congenitally athymic (nude) mice mount an anti-PC plaque-forming cell response after R36a immunization but fail to produce a significant polyclonal response. From our results it appears that PC on the cell wall of a bacterium acts both as a polyclonal activator and a specific antigen, stimulating each by different mechanisms.

Animals↗

PubMed1984

Phosphorylcholine on isologous red blood cells induces polyclonal but not anti-phosphorylcholine plaque-forming cells in mice.

It has been demonstrated in the preceding report (Bach, M. A., Beckmann, E. and Levitt, D., Eur. J. Immunol. 1984. 14: 589) that phosphorylcholine (PC) on the bacterium Streptococcus pneumoniae R36a stimulated polyclonal as well as anti-PC plaque-forming cells (PFC) in mouse spleen in vivo. In this study, red blood cells from BALB/c mice (MRBC) were either conjugated with PC, 2,4,6-trinitrophenyl (TNP) or treated with phospholipase A2 (PLA2) to expose PC on the cell membrane (determined by hemagglutination with the anti-PC myeloma HOPC8). When BALB/c mice were immunized i.v. with the conjugated or enzyme-treated MRBC, a significant polyclonal antibody response occurred (p less than 0.05) using PC-MRBC or PLA2-treated MRBC, but not with TNP-MRBC or sham-treated MRBC. No anti-PC or anti-MRBC immunoglobulin-secreting cells developed after immunization. Repeated immunization with PC-MRBC resulted in similar levels of protein A PFC after each immunization but no anti-PC, anti-MRBC or anti-PC-MRBC PFC. Thus, PC on R36a or isologous RBC stimulated increased numbers of splenic plaque-forming cells. In the case of R36a, 10-25% of these PFC produced antibodies directed towards PC. In contrast, PC-MRBC or PLA2-treated MRBC, failed to evoke any anti-PC antibody responses.

Animals↗

PubMed1984

Epstein-Barr virus-induced lymphoblastoid cell lines derived from the peripheral blood of patients with X-linked agammaglobulinemia can secrete IgM.

We have established lymphoblastoid cell lines (LCL) from the peripheral blood of three individuals with X-linked agammaglobulinemia as well as three of their immunodeficient first-degree relatives. Lines could be induced with Epstein-Barr virus only when T lymphocytes were depleted from total mononuclear leukocytes. The LCLs derived from XLA patients expressed characteristics of IgM-secreting plasmacytes, including intense cytoplasmic fluorescence after staining with anti-mu, easily detectable amounts of IgM in culture supernatants, and radiolabeled IgM with both heavy and light chains in culture media and cell lysates. The cell lines induced from blood of the first-degree relatives of these patients were more varied. They often exhibited multiple isotypes for both heavy and light chains in different cells or expressed a B-lymphocyte phenotype (easily detectable surface Ig but no Ig secretion). These studies suggest that B-cell precursors are present in peripheral blood of individuals with classical XLA . Differentiation of such cells to mature cells of the B lineage can be accomplished using Epstein-Barr virus after removal of T cells.

Adult↗

PubMed1984

In vitro plaque-forming cell responses induced by Streptococcus pneumoniae in humans.

When peripheral blood lymphocytes (PBL) were stimulated in vitro with the rough form of type 2 Streptococcus pneumoniae R36a, the resulting plaque-forming cells (PFC) did not produce antibodies directed against phosphorylcholine, a major antigenic determinant of the cell wall C-polysaccharide. Instead, R36a stimulated polyclonal PFC in PBL and splenic lymphocytes. We compared the polyclonal responses stimulated by R36a with those induced by two well-characterized polyclonal activators (PA), Staphylococcus aureus Cowan I and pokeweed mitogen (PWM). We found that R36a was a poor mitogen for PBL, whereas the other two PA were potent mitogens; that the predominant isotype produced in response to all three PA was IgM; that adherent cells strongly inhibited the polyclonal PFC response to both R36a and Staph. aureus but not PWM; and that T cells were necessary for induction of polyclonal antibody-secreting cells by all three stimuli.

Adult↗

PubMed1984

Chlamydia trachomatis stimulates human peripheral blood B lymphocytes to proliferate and secrete polyclonal immunoglobulins in vitro.

Infectious Chlamydia trachomatis (LGV strain), obligate intracellular bacteria, stimulated human peripheral blood lymphocytes to proliferate and secrete immunoglobulins in vitro. In contrast, mock-infected preparations were unable to induce similar responses in peripheral blood lymphocytes. Although levels of immunoglobulin secreted into the media of LGV-stimulated cultures were greater than 10 micrograms/ml, we estimated that less than 1% of these molecules were directed against the bacteria itself, suggesting polyclonal antibody production. Since stimulation with Formalin-killed bacteria resulted in comparable numbers of plaque-forming cells (PFC) as infectious particles, we concluded that the polyclonal immunoglobulin response was not dependent on the in vitro chlamydial infectious process. The polyclonal PFC response induced by LGV was highly sensitive to monocyte inhibition. Although LGV induced proliferation of predominantly B cells, the numbers of generated PFC was increased by the addition of autologous T cells. Neither lymphocyte proliferation nor PFC responses of normal human volunteers correlated significantly with the presence or titer of antichlamydial antibodies in their sera.

Antibodies, Bacterial↗

PubMed1984

Phosphorylcholine on Streptococcus pneumoniae R36a is responsible for in vitro polyclonal antibody secretion by human peripheral blood lymphocytes.

We have shown that Pc on the C-polysaccharide of Streptococcus pneumoniae R36a is responsible for polyclonal PFC responses induced in vitro by this bacterium in humans. R36a grown in media containing EA instead of CL, and therefore having phosphorylethanolamine instead of Pc in their C-polysaccharide, were unable to induce substantial PFC responses. When EA-substituted bacteria were chemically conjugated with Pc, their ability to induce polyclonal PFC was restored. Specific removal of Pc from the surface of the bacteria by the use PLC also resulted in abrogation of the polyclonal antibody response. These data are consistent with our hypothesis that polyclonal activation resulting from R36a stimulation may be mediated by a recently described Pc-binding receptor that is distributed on the surface of a subpopulation of B lymphocytes in humans and mice.

Adult↗

PubMed1984

Analysis of surface mu-chain expression in human lymphoblastoid cell lines that do not produce light chains.

It has been suggested that light chains (LC) are necessary for the surface expression of mu heavy chains. Fluorescent antibody screening of 42 human lymphoblastoid cell lines transformed in our laboratory, however, disclosed four lines that expressed surface mu-chains without LC. The biosynthesis, glycosylation, and turnover of mu-chains in these cell lines was compared to mu-chain production in cell lines synthesizing both heavy chains and LC. LC production could not be detected in the mu +LC- cell lines by either surface or biosynthetic labeling. The mu-chains expressed on the surface of the LC- cells appeared as disulfide-linked dimers and migrated slightly faster on SDS-polyacrylamide gels (70 Kd) than did mu-chains from IgM monomers (H2 L2) (78 Kd) after reduction. Biosynthetic labeling in the presence of tunicamycin demonstrated that the smaller size of free mu heavy chains was due to incomplete glycosylation of these molecules and not to amino acid deletions. The mu-chains produced by mu + LC- cells lines were degraded faster than mu-chains from LC+ cell lines, but their rate of transit to the cell surface was identical in both cell types. Thus, although LC are necessary for the formation of an intact antigen-binding site, they are not involved in the synthesis or expression of membrane mu-chains.

Acetylglucosaminidase↗

PubMed1983

Excessive numbers and activity of peripheral blood B cells in infants with Chlamydia trachomatis pneumonia.

The peripheral blood lymphocytes of seven infants who had lower respiratory infections caused by Chlamydia trachomatis (chlamydial pneumonia) were studied for abnormalities that may be related to the hyperimmunoglobulinemia characteristic of this infection. Both proportions and numbers of B cells and plasma cells were strikingly elevated in these infants, as indicted by the percentage of peripheral blood mononuclear cells (PBMC) that reacted with fluorochrome-labeled antibodies to human immunoglobulins. Cells expressing IgM and IgD on their surface, and cells possessing IgM and IgG in their cytoplasm were especially increased above levels found in normal adults, infants, and a group of infants with other infections. Cells from infected infants secreted exceptionally large amounts of IgM, IgG, and IgA when cultured in the absence of added mitogens. These data suggest that chlamydial pneumonia induces substantial B-cell activation during a period of development when antibody responses are normally difficult to stimulate.

Antibodies, Monoclonal↗

PubMed1983

Induction of chondroitin sulfate proteoglycan synthesis and secretion in lymphocytes and monocytes.

The ability of mononuclear leukocytes to synthesize and secrete proteoglycans was evaluated. Using radiolabeling with H2 35SO4, it is shown that peripheral blood mononuclear cells (PBMC) and their major subpopulations (B cells, T cells, and monocytes), as well as mouse spleen cells, all secreted easily detectable proteoglycan. After 24-h labeling periods, 90% of macromolecular 35S could be detected in culture media. This material was primarily (greater than 95%) chondroitin-4-sulfate proteoglycan (CSPG). Production and secretion of CSPG could be stimulated more than 200% in PBMC and 300% in T cell populations by high concentrations of concanavalin A and phorbol 12-myristate-13-acetate; lipopolysaccharide induced a small (twofold) but reproducible increase in CSPG secretion by adherent mononuclear leukocytes. The CSPG secreted by PBMC was relatively small in size compared to chondrocyte CSPG (130,000 daltons vs. 2-4 million daltons) but possessed similar sizes of glycosaminoglycan chains and greater solubility in low ionic strength solutions. This sulfated polyanion, which was produced endogenously by leukocytes and was actively secreted, might function as a co-mediator or "second messenger" in certain immune responses.

Animals↗

PubMed1983

Endothelial albumin permeability measured with a new technique in perfused rabbit lung.

This study describes two methods of measuring the albumin permeability-surface area product (PS) of the pulmonary microvascular endothelium in isolated, perfused rabbit lungs. Both involve direct sampling of lung tissue to measure how much labeled albumin has entered the extravascular space during a known exposure period. In one, the traditional "multiple-sample" method, extravascular albumin is measured for several different exposure times to determine the kinetics of transcapillary albumin movement, and PS is calculated from that data. We found single-exponential kinetics, with a half time of 77 min and a PS of 2.28 X 10(-3) cm3 . min-1 . g blood-free wet lung-1. The second, "single-sample" method, is a new modification that involves exposing the tissue to labeled albumin for only 3 min, followed by a 3-min vascular washout. Tissue is then analyzed for extravascular albumin and PS calculated assuming the interstitial albumin concentration is essentially zero. The PS obtained was 2.44 X 10(-3) cm3 . min-1 . g blood-free wet lung-1, not significantly different from the value obtained using the more accepted method. Possible sources of error in the new method are discussed, and we conclude that it is an easy, quick, reliable way of accurately measuring macromolecular PS in the lung and presumably other organs as well.

Animals↗

PubMed1983

Hyper IgM immunodeficiency. A primary dysfunction of B lymphocyte isotype switching.

Immunological evaluations (lymphocyte markers, B cell differentiation, T cell function) were performed on peripheral blood mononuclear cells from four individuals with hyper IgM immunodeficiency. Number, proportion, and proliferation of T lymphocytes and T lymphocyte subpopulations were relatively normal in affected individuals. The percentage and number of B cells expressing surface IgM and IgD were either normal or elevated in both blood and lymph nodes. However, surface IgG- and IgA-bearing B lymphocytes were completely absent. In vitro stimulation of blood lymphocytes with both T cell-dependent and T-cell independent polyclonal B cell activators resulted in normal numbers of IgM plasma cells and IgM secretion in cultures, but failed to induce any IgG- or IgA-producing cells. This failure of isotype switching was intrinsic to the B cell population and did not involve aberrant T cell help or suppression. Therefore, individuals with this disorder possess an intrinsic B cell dysfunction that is not related to abnormal T cell regulation.

Adolescent↗

PubMed1983

The skin test antigen stimulated killer (STAK) cell mediating NK like CMC is OKM1 positive and OKT3 negative.

Recently we demonstrated that candida antigen stimulated natural killer cell like cell-mediated cytolysis (NK like CMC) in peripheral blood mononuclear cells (PBMNC) isolated from normal individuals (Tartof et al., 1980). Utilizing monoclonal antibodies directed against human mononuclear cell subpopulations in conjunction with a fluorescence activated cell sorter (FACS) we determined that, similar to the previously described NK cell, the skin test antigen stimulated killer (STAK) cell is a larger OKM1 positive, OKT3 negative cell. We obtained similar results using two different skin test antigens. Thus, stimulation of NK like CMC in PBMNC by skin test antigens probably represents activation of NK or NK like cells.

Antibodies, Monoclonal↗

PubMed1983

Binding of phosphorylcholine by non-immunoglobulin molecules on mouse B cells.

Phosphorylcholine (PC), a molecule found in the cell wall of most serotypes of pneumococcus, has been used extensively as a probe for the study of network interactions during immune responses. The frequency of B lymphocytes capable of interacting with PC has not been directly examined. We used immunofluorescence to study the binding of PC and monoclonal anti-TEPC15 anti-idiotopic antibodies to murine lymphocytes. In addition to identifying PC-specific Ig molecules, PC was bound by a non-Ig molecule on the surface of a relatively large subset of B cells; this non-Ig marker shared an idiotypic determinant with the PC-binding myeloma protein HOPC8 (H8). PC-bearing R36a pneumococci bind to a similar subset of lymphocytes. This binding is inhibited specifically by PC coupled to bovine serum albumin and also by a monoclonal anti-H8 antibody. We suggest that bacterial interaction with B cells through non-Ig molecules capable of binding a dominant antigen like PC may possess functional significance, possibly during the events that lead to antibody induction by these microorganisms.

Animals↗