Biomedical subjects

D Levitt

Publications and source records attributed to D Levitt.

At least 55 records · Page 3Linked to original sources

PubMed1988

Patterns of immunoenhancement and suppression induced by Chlamydia trachomatis in vivo and in vitro.

We investigated the cycle of immune enhancement and suppression seen in mice infected with Chlamydia trachomatis by using in vivo and in vitro model systems. BALB/c mice injected intravenously with chlamydia reveal a three- to seven-fold increase in numbers of plaque-forming cells producing antibodies against sheep red blood cells (SRBC), when immunized with SRBC 0 to 5 days after chlamydia infection. When mice are injected with SRBC 10 to 15 days after initial chlamydia infection, the specific anti-SRBC plaque-forming cell response is suppressed two- to three-fold. In vitro, low numbers (2 to 5 X 10(6) bacteria/ml) of chlamydia stimulate potent proliferative responses by B lymphocytes while high numbers (25 X 10(6) bacteria/ml) of bacteria generate strong, general T suppressor activity. This model has important implications for regulation of immune responses that arise at different times during chlamydial infections, as well as for the potential effectiveness of chlamydial vaccines.

Adjuvants, Immunologic↗

PubMed1988

Influence of dose and duration of infusion of interleukin-2 on toxicity and immunomodulation.

The purpose of this study was to investigate the effect of dose and duration of infusion of recombinant interleukin-2 (IL-2) on toxicity and immunomodulation. In a phase I/II study, IL-2 was administered intravenously (IV) daily for five consecutive days every other week for 4 weeks of treatment to 23 patients with progressive melanoma, renal, colon, or ovarian cancer by one of four regimens: groups I and II received 3 X 10(5) U/m2/d by two-hour or 24-hour infusion, respectively; groups III and IV received 3 X 10(6) U/m2/d by two-hour or 24-hour infusion, respectively. In a subsequent study, six patients (group V) received a single priming cycle of daily IL-2 for five days at 3 X 10(6) U/m2/d in divided 15-minute infusions every eight hours, before undergoing leukapheresis for lymphokine-activated killer (LAK) cell generation. Toxicity was mild with 3 X 10(5) U/m2/d, but severe chills and fever, moderate hypotension (not requiring IV pressors), and weight gain were observed with 3 X 10(6) U/m2/d. Toxicity was also related to the duration of infusion. In group IV (continuous infusion), fluid retention, weight gain, and azotemia were more frequent and severe than in groups III or V, in which the same total dose was administered by two-hour infusion or in three divided 15-minute infusions. IL-2 induced rebound lymphocytosis, which was directly dose-related and significantly higher in group IV (continuous infusion) than in groups III or V. Dramatic increases in the percentage and absolute number of cells expressing the IL-2 receptor were also most pronounced in group IV. With the higher dose of IL-2, LAK cells appeared in the circulation, and natural killer (NK) cytotoxicity was augmented. The results showed that the toxicity and immunomodulation by IL-2 are dose-dependent and are maximal by continuous infusion compared with two-hour or divided every eight hours infusions.

Adult↗

PubMed1988

Naturally occurring mouse antibodies against T-cell-secreted chondroitin sulphate proteoglycan.

Stimulated T lymphocytes and certain T-cell hybridomas secrete molecules capable of inducing B-lymphocyte proliferation and differentiation. It has been shown recently that one such B-cell stimulatory factor is associated with chondroitin sulphate proteoglycan (CSPG) and was designated T-cell proteoglycan fraction, or T-PGF. We report here that mouse spleen cells cultured at high densities or stimulated with lipopolysaccharide (LPS) at low cell densities secrete antibodies directed against T-PGF. Such antibodies react primarily with the CSPG component of T-PGF and can inhibit the induction of plaque-forming cells (PFC) by T-PGF. By fusing high-density cultures of unstimulated mouse spleen cells with the myeloma P3 x 63AG8.653, several anti-T-PGF (CSPG) hybridomas were derived that exhibited activities identical to anti-T-PGF (CSPG) obtained from high-density spleen cell culture supernatants. The role that these spontaneously secreted autoantibodies may play in immunoregulation is discussed.

Animals↗

PubMed1987

Development and regulation of chlamydia-responsive murine B lymphocytes.

We have examined characteristics of chlamydia-stimulated mouse B cells as well as cells that regulate polyclonal responses in vitro. B lymphocyte proliferation stimulated by chlamydia arises at a similar time as Escherichia coli lipopolysaccharide (LPS)-induced proliferative responses during ontogeny. In contrast, development of immunoglobulin (Ig)-secreting cells after chlamydia stimulation is delayed by several weeks relative to ontogeny of LPS-inducible plaque-forming cells (PFC). The lack of Ig secretion by immature B cells is not due to a deficiency of Lyb5+ B lymphocytes, since X-linked immunodeficient (xid) NBF1 mice that lack this B lymphocyte population respond well to chlamydia stimulation. Adherent cells are important for chlamydia-stimulated B lymphocyte differentiation, but are not as necessary for their proliferation. Neither adult adherent cells nor T cells can correct the inability of immature spleen cells to develop into Ig-secreting cells; spleen cells from 2-wk-old mice (i.e., immature B cells) will not suppress adult B lymphocyte responses to chlamydia. When B lymphocytes are separated according to their buoyant densities, chlamydia stimulates low density (activated) B cells to proliferate and differentiate better than high density (resting) cells. Proliferative responses to chlamydia arise earlier during ontogeny, do not require adherent cells, and can proceed to a relatively greater extent in resting B cell population (compared with activated B cells) than induction of Ig-secreting cells.

Animals↗

PubMed1987

Methanol fixation permits flow cytometric analysis of immunofluorescent stained intracellular antigens.

Fixation and immunofluorescent staining methods were developed for analyzing intracellular antigens with the cell flow cytometer. Fixing cell suspensions with 100% methanol provided best preservation of morphology, lowest fluorescent background staining and most intense specific immunofluorescence. Immunoglobulins present in B cell lines that were representative of different developmental stages could be distinguished quantitatively. Fluorescence histograms were compared with fluorescence microscope presentation of stained cells. Intracellular antigens that reacted with monoclonal antibodies could also be evaluated by flow cytometry. This method was utilized to assess plasmacyte development in mouse spleen cell cultures after stimulation with lipopolysaccharide.

Animals↗

PubMed1987

B-cell stimulation by T-cell-secreted proteoglycan.

We have further characterized a recently described B-cell stimulatory factor that contains chondroitin sulfate proteoglycan and 70- to 75-kDa protein, both of which are secreted by T cells and coisolate (T-cell proteoglycan fraction, T-PGF). Using T-PGF isolated from a T-cell hybridoma (T14), it was observed that the association between B-cell stimulatory activity and CSPG is stable and comigrates on Sephacryl S-200 columns eluted at high salt concentrations (1.5 M NaCl) and on CsCl gradients. The T-PGF stimulated larger numbers of low-density (activated) B cells, but better relative PFC formation occurred in high-density (resting) B-cell fractions. It is proposed that the B-cell stimulatory activity of T-PGF is, in fact, chondroitin sulfate proteoglycan.

B-Lymphocytes↗

PubMed1987

Blood conservation with membrane oxygenators and dipyridamole.

Cardiopulmonary bypass induces platelet activation and dysfunction, which result in platelet deposition and depletion. Reduced platelet numbers and abnormal platelet function may contribute to postoperative bleeding. A membrane oxygenator may preserve platelets and reduce bleeding more than a bubble oxygenator, and the antiplatelet agent dipyridamole may protect platelets intraoperatively and reduce bleeding postoperatively. A prospective randomized trial was performed in 44 patients undergoing elective coronary artery bypass grafting to assess the effects of the membrane oxygenator and dipyridamole on platelet counts, platelet activation products, and postoperative bleeding. Patients who were randomized to receive a bubble oxygenator and no dipyridamole had the lowest postoperative platelet counts, the greatest blood loss, and the most blood products transfused. Platelet counts were highest and blood loss was least in patients randomized to receive a membrane oxygenator and dipyridamole (p less than .05). A bubble oxygenator with dipyridamole and a membrane oxygenator without dipyridamole resulted in intermediate postoperative platelet counts and blood loss. Arterial thromboxane B2 and platelet factor 4 concentrations were elevated on cardiopulmonary bypass in all groups. Both the membrane oxygenator and dipyridamole were independently effective (by multivariate analysis) in preserving platelets. Optimal blood conservation was achieved with a membrane oxygenator and dipyridamole.

6-Ketoprostaglandin F1 alpha↗

PubMed1987

Molecular basis of the cell-surface expression of immunoglobulin mu chain without light chain in human B lymphocytes.

Four distinct human B-lymphoid cell lines possess the ability to circumvent the mechanism regulating intracellular transport of immunoglobulin protein. These cells do not produce light chains, yet they express mu heavy chains on the cell surface at comparable levels to B-cell lines that produce native forms of both proteins. The mu-chain mRNA produced in all four cell lines was found to contain an identical deletion of most of the heavy-chain variable (VH) region (75% of the 3' portion), with no apparent alteration in constant (C) region structure. The truncated mu (mu*)-chain mRNA in these cells was created through the use of a cryptic splice donor site found within the human VH gene(s) utilized by these B-cell lines. The truncated mu chains exhibited a decreased ability to associate with the intracellular transport regulatory protein, heavy-chain binding protein (BiP). This result indicates that VH region structure, in addition to C mu 1 region structure, influences the formation of the BiP recognition site on the heavy chain. Furthermore, it suggests that the mechanism allowing for cell-surface expression of the mu* chains in the absence of light-chain pairing is the inability of BiP to bind to the mu* chains and hence prevent their intracellular transport. The high frequency with which the mu-only surface immunoglobulin positive phenotype is present in our collection of human B-cell lines and the isolation of one of the cell lines from a healthy individual also suggest that B cells of this type may represent a significant subpopulation among the normal human B-cell repertoire.

B-Lymphocytes↗

PubMed1987

Perfluorinated fatty acids alter merocyanine 540 dye binding to plasma membranes.

We have evaluated the effect of the perfluorinated fatty acids pentadecafluoro-n-octanoic acid (PFOA) and nonadecafluoro-n-decanoic acid (NDFDA) on the ability of a human B-lymphoblastoid cell line to bind the lipid-binding, membrane-impermeant, fluorescent dye merocyanine 540 (MC540). Subtoxic concentrations of perfluorinated fatty acids (0.9 mM PFOA; 0.5 mM NDFDA) greatly diminish binding of MC540 by normal plasma membranes, as determined by fluorescence flow cytometry. When perfluorinated fatty acids are added to cells at toxic or lethal concentrations (1.2 mM PFOA; 0.75 mM NDFDA), MC540 binding increases dramatically, with entrance of dye to internal membrane domains. Neither perfluorinated fatty acid molecule reduces the ability of surface immunoglobulin to migrate laterally and cap on cells. Our data suggest that perfluorinated fatty acids either interact directly with lipid binding sites for MC540, and thereby inhibit dye intercalation, or alter membrane lipid architecture and lipid packing to diminish MC540 binding. Both possibilities support a direct, physical, membrane-altering mechanism for perfluorinated fatty acid toxicity on mammalian cells.

Blood Physiological Phenomena↗

PubMed1987

Chlamydia trachomatis (L2 serovar) can be bound, ingested and destroyed by differentiated but not by undifferentiated human promyelocyte cell line HL-60.

As a model system for analysing interactions between chlamydiae and myeloid cells and their precursors, we have studied binding, ingestion and destruction of Chlamydia trachomatis (L2 serovar) by the human promyelocytic cell line HL-60. HL-60 cells were induced by phorbol myristate acetate (PMA) and dimethyl sulphoxide (DMSO) to differentiate along either the macrophage or the granulocyte pathway, respectively. Using an immunofluorescence assay and electron microscopy, we have shown that induced (differentiated) HL-60 cells, but not uninduced (undifferentiated) HL-60 or other cell lines treated with PMA or DMSO, exhibit increased binding, ingestion and elimination of C. trachomatis; these activities are associated with specific histochemical and antigenic markers of myeloid differentiation. These results suggest that myeloid cells acquire the ability to interact with and kill chlamydiae during cell development.

Cell Differentiation↗

PubMed1987

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii.

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.

Acholeplasma laidlawii↗

PubMed1986

Both species of chlamydia and two biovars of Chlamydia trachomatis stimulate mouse B lymphocytes.

We have investigated the ability of both species of chlamydiae (C. trachomatis and C. psittaci), two major biovars of C. trachomatis (lymphogranuloma venereum and trachoma), and the two developmental forms of chlamydia (reticulate and elementary bodies) to stimulate murine spleen lymphocytes. All of these forms of the bacteria induce potent proliferation and differentiation to plaque-forming cells by B lymphocytes in vitro. Chlamydiae induce a broad antibody response, suggesting that stimulation is polyclonal in nature. Although all chlamydiae possess a lipopolysaccharide (LPS) genus-specific molecule similar to LPS found on Re mutant enterobacteria, polyclonal B cell stimulation is likely caused by molecules other than LPS, since i) polymyxin B failed to inhibit chlamydia-induced immunostimulation and ii) C3H/HeJ mice (LPS nonresponders) produced normal numbers of PFC after culture with chlamydia (but not LPS). Thus, a cross-species moiety that is not LPS is responsible for polyclonal stimulation by chlamydia. Because these bacteria can exist in latent forms in an animal, and all forms are immunostimulatory, the question of whether these bacteria can alter immune responses if released during other infections or immunizations has been raised.

Animals↗

PubMed1986

Separation of lymphocyte subpopulations using biotin-avidin erythrocyte rosettes.

The major method for isolating murine lymphocyte subpopulations involves negative selection using antibody plus complement-mediated cytolysis. We have developed an efficient rosette method for enriching murine B and T cells using biotin-conjugated antibodies and avidin-coated sheep erythrocytes. Rosetted and non-rosetted subpopulations are separated rapidly on Percoll cushions. In systems employing rabbit anti-mouse immunoglobulin or monoclonal rat anti-mouse Thy-1.2 conjugated to biotin, positively-selected cells are greater than 90% pure while negatively-depleted populations possess less than 2% contamination with unwanted cells. Recoveries from starting spleen cell populations range between 50 and 75%. This method provides an easily performed alternative for obtaining positively and negatively selected cell populations and can be used with any biotin-conjugated antibody protein.

Animals↗

PubMed1986

Binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) analyzed by flow cytometry.

We have developed a method for quantitatively assessing binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) in several mammalian cell lines using fluorescence staining and flow cytometry. Cells were incubated with chlamydia at 4 degrees C to monitor binding; ingestion was determined by raising the temperature to 37 degrees C for 1-4 h and removing extracellular bacteria with pronase. Growth of bacteria was measured by assessing brightly stained intracellular inclusions. Fixation with methanol prior to fluorescent staining provided the most intense specific staining with minimal background, as well as preserving cell morphology. Our data reveal relatively slow ingestion of L2 by McCoy fibroblasts (maximum ingestion by 4 h) and a sizeable population of McCoy cells (30-40% of total cells) that ingest L2 but do not permit its growth under certain infectious conditions. It was possible to correlate specific histogram patterns on the flow cytometer with fluorescent microscope observations. This system provides a means of analyzing quantitative interactions between chlamydia and individual host cells.

Animals↗

PubMed1986

Stimulation of mouse B cells by a factor that coisolates with T-cell proteoglycan.

We have isolated a factor that copurifies with chondroitin sulfate proteoglycan secreted by mouse splenocytes and some murine T-cell hybridomas. This factor will stimulate proliferation and plaque-forming cell differentiation of B lymphocytes from mouse spleens, even after T cells have been depleted (less than 2% Thy 1.2-bearing cells). Adherent macrophages enhance the activity of this factor, but their function can be replaced in macrophage- and T-cell-depleted populations by small concentrations of a protein mitogen from Salmonella typhimurium. The stimulatory fraction contains chondroitin sulfate, a major protein which has a molecular weight of 74,000 and a minor moiety at 50,000. Stimulatory activity of this material is destroyed by (i) boiling, (ii) mild alkali treatment, and (iii) protease digestion. It is unaffected by RNase and chondroitinase treatments, suggesting that the factor is a protein. Our data define a new B-cell stimulatory substance(s) and suggest that it may be associated with chondroitin sulfate proteoglycan secreted by immune cells.

Animals↗

PubMed1986

Toxicity of perfluorinated fatty acids for human and murine B cell lines.

The toxicity of two perfluorinated fatty acids, penta decafluoro-n-octanoic acid (PFOA) and nonadecafluoro-n-decanoic acid (NDFDA), on three mammalian B cell lines was evaluated. Cells were exposed to the perfluorinated molecules for either 24 or 48 hr under a variety of culture conditions. Immunoglobulin secretion and surface membrane expression were unaffected by both PFOA and NDFDA at sublethal concentrations. Lethal effects of PFOA and NDFDA are diminished by either lowering culture temperature (37 to 20 degrees C) or including fetal bovine serum or human serum albumin in media. At lethal concentrations, both PFOA and NDFDA possess detergent activity since they can release IgM in soluble form from a cell line that does not secrete immunoglobulins, and brief exposure (15 min) to 1-2 mM of both perfluorinated fatty acids results in solubilization of F4 cells equivalent to the anionic detergent deoxycholic acid. Our data suggest that at subtoxic concentrations, neither PFOA nor NDFDA alters expression or secretion of a differentiated gene product (IgM). At lethal levels, both chemicals cause increased solubilization of proteins from lymphoblastoid cell lines.

Animals↗

PubMed1986

Chlamydia trachomatis (L2 serovar) binds to distinct subpopulations of human peripheral blood leukocytes.

We have previously shown that infants with pneumonitis caused by Chlamydia trachomatis, an obligate intracellular bacterium, possess increased percentages of B lymphocytes but not T lymphocytes in their peripheral blood. It was then demonstrated that chlamydiae induce proliferation in vitro of human peripheral blood B lymphocytes and, in the presence of T cells, differentiation of B cells to immunoglobulin-secreting cells. In this study, we show that C. trachomatis (L2 serovar) binds preferentially to 50% of human B lymphocytes from peripheral blood but only to a small percentage, if any, of T cells. Both monocytes and granulocytes bind and ingest chlamydiae. Despite chlamydial binding to B cells and ingestion by monocytes, no uptake by B cells and limited growth (fewer than 0.5% inclusion-containing cells) in monocytes occur. There is a dramatic decrease in the percentage of cells associated with the bacteria after culture. These results are the first demonstration of binding of C. trachomatis (L2 serovar) to lymphocytes and represent a direct step toward correlating physical interactions between bacteria and lymphocytes with specific immunostimulatory activities in vitro.

Adhesiveness↗

PubMed1986

Potential of human polymorphonuclear leukocytes to synthesize and secrete sulfated proteoglycans.

To analyze the function of proteoglycans (PG) in different types of leukocytes, both the relative amounts and specific types of proteoglycans produced by cultured human peripheral blood polymorphonuclear leukocytes (PMN) were were determined and compared to mononuclear leukocytes (PBMC). Media from 3-day cultured PMN contained significantly less (less than 10%) 35SO2-4-labeled PG than media from PBMC cultures. Incorporation of 35SO2-4 into cell-associated material was comparable for both types of white blood cells. In contrast to PBMC, PMN could not increase their synthesis or secretion of PG after exposure to concanavalin A or phorbol-12-myristate-13-acetate. Various inducers of leukocyte chemotaxis also failed to enhance PG production by PMNs. Release of prelabeled PG from PMNs could be induced by exposure to either opsonized or unopsonized zymosan (yeast) as well as the bacteria S. aureus, suggesting that particle ingestion may be accompanied by PG exocytosis. Both chondroitinase ABC and AC digested greater than 90% of PMN 35S-labeled material in media and 75% in cell lysates; HNO3 treatment removed less than 5% of N-linked 35SO4 from radiolabeled media and 25% from cells. Treatment with 0.5 N NaOH released shortened glycosaminoglycan chains from 35S-labeled PMN cell lysates. beta-D-xylosides did not stimulate an increase in polysaccharide chain production by cultured PMNs. These data suggest that PMNs can produce chondroitin 4-sulfate PG whose synthesis is not affected by treatments that alter PMN functions; in contrast to PBMCs, PMNs will actively release these molecules when exposed to micro-organisms that stimulate phagocytosis.

Cells, Cultured↗