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Biomedical subjects

M S Kennedy

Publications and source records attributed to M S Kennedy.

At least 19 recordsLinked to original sources
PubMed1992

Metabolism of [14C]- and [32P]pyridoxal 5'-phosphate and [3H]pyridoxal administered intravenously to pigs and goats.

To gain more information about the kinetics of vitamin B-6 metabolism in vivo, the metabolism of tracer was examined after the simultaneous intravenous administration of [32P] and [14C]pyridoxal phosphate and [3H]pyridoxal in two 93-kg pigs and two 60-kg goats. In the pigs, [14C] removal was monophasic with T1/2 of 16 and 18 min and clearance of 165 and 248 mL/min. In the goats, [14C] removal was biphasic with T1/2 of 49 and 114 min for 0-30 min and 209 and 227 min for 0.5-6 h (clearance 20 and 17 mL/min). Uptake of pyridoxal phosphate by liver and resecretion into the plasma were too small to cause a detectable decrease in the [32P]:[14C] ratio. Pyridoxal removal from plasma was similar in both species, with a half-life of approximately 12 min from 0-30 min and approximately 50 min for 0.5-3 h. Clearance of [3H]pyridoxal in the four animals ranged from 412 to 2258 mL/min. Little [14C] entered the erythrocytes. The [3H] entered readily but was converted to pyridoxal phosphate faster in the pigs than in the goats. [14C] and [3H] were excreted as pyridoxic acid at the same rate. However, during the 54 h after injection the goats excreted approximately 60% of the [14C] doses in the urine compared with approximately 30% in the pigs. About 5-10% of the [14C] and [3H] doses were recovered in goat milk over 54 h. Pyridoxal kinase activity was higher in lactating mammary tissue than in liver, kidney or muscle of goats.

Animals↗
PubMed1991

Penetration of CD4 T cells by HIV-1. The CD4 receptor does not internalize with HIV, and CD4-related signal transduction events are not required for entry.

Receptor binding of HIV to the CD4 molecule is required for efficient infection of T cells, but the post-binding steps that result in penetration of HIV are not well understood. CD4 is induced to internalize upon T cell activation, and mAb to CD4 modify signal transduction and T cell activation as does HIV in some systems. It is not known whether HIV binding triggers CD4 endocytosis or whether signal transduction events are required for penetration. Selected inhibitors of signal transduction were evaluated for their effects on penetration using two assays that are dependent on penetration. After short term exposure to inhibitor and HIV, cells were analyzed for reverse-transcribed HIV DNA (DNA amplification assay), or productive infection is monitored (infectivity assay). Viral penetration was tested in the presence of H7 (protein kinase C inhibition), EGTA (extracellular Ca2+ chelation), cyclosporine A (inhibition of Ca2+/calmodulin-dependent activation), or pertussis toxin (inhibition of G protein function). All agents were used at concentrations that were inhibitory for their respective signal transduction pathways. None of the inhibitors affected viral penetration. We tracked the CD4 molecule with fluorescent probes that do not interfere with HIV binding in a system where CD4 T cells were saturated with HIV and the penetration event was relatively synchronized. Under conditions where detection of CD4 was more sensitive than the detection of HIV, HIV internalization was readily detected but CD4 internalization was not.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

HIV-1 binding to CD4 T cells does not induce a Ca2+ influx or lead to activation of protein kinases.

The penetration of CD4+ cells by human immunodeficiency virus (HIV) involves a high affinity interaction between the viral attachment protein, gp120, and the cellular receptor, CD4. The mechanism by which the virus penetrates the host cell subsequent to viral binding is unknown. We have investigated the possibility that HIV penetration induces changes in the metabolic state of the infected cell similar to those seen with the perturbation of CD4 cells by monoclonal antibodies (MAb) directed against the CD4 molecule, or with specific antigen-mediated activation. The activation of cellular protein kinases was examined. The basal level of activity was not altered in the presence of HIV. Kinase activity was markedly increased in cells stimulated with phytohemagglutinin (PHA), and was qualitatively and quantitatively changed by a brief exposure to the phorbol ester TPA (12-o-tetradecanoyl phorbol-13-acetate). The phosphorylation state of the CD4 molecule was examined by radioimmunoprecipitation and found to be unaltered by the binding of HIV under conditions in which TPA induced rapid CD4 phosphorylation. The activity of the CD4-associated protein tyrosine kinase p56lck was measured by in vitro assays of 32PO4 incorporation in CD4 immunoprecipitates from HIV-incubated cells. TPA incubation resulted in a rapid loss of CD4-associated p56lck activity, presumably due to dissociation of the enzyme from CD4. Concanavalin A stimulation resulted in a similar change but with a slower time course. However, no change in CD4-associated activity was detected in HIV-incubated cells. We found that Ca2+ influx was not induced by the binding of HIV to CD4+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

CD4 Antigens↗
PubMed1991

Analysis of synergism/antagonism between HIV-1 antibody-positive human sera and soluble CD4 in blocking HIV-1 binding and infectivity.

We tested human immunodeficiency virus type 1 (HIV-1) antibody-positive human sera and sCD4, alone and in combination, for synergistic, additive, or antagonistic effects on blocking of HIV binding and infectivity. Data were analyzed by an application of the median effect principle derived from the law of mass action. This allows the assessment of synergism/antagonism at any desired level of effect. Using three assays (whole virus binding to CD4 cells, neutralization of HIV infectivity, and binding of purified gp120 to solid-phase sCD4), we generally observed additive effects or slight synergism between antibody and sCD4 in inhibiting gp120-CD4 interaction. We used a fourth assay to measure the irreversible inactivation of HIV infectivity by sCD4, a property that can also be mediated by antibody but with considerably less potency than sCD4. The reduction in HIV infectivity mediated by mixtures of sCD4 and antibody was always equal to or greater than the arithmetic sum of the reductions by either agent alone. The relevant antiviral effects of sCD4 and anti-HIV sera may include reversible blockage of receptor binding, irreversible inactivation of HIV infectivity, and in the case of antibody, additional reactions that are independent of receptor binding. Although predictions concerning the in vivo situation are speculative, we find no evidence in vitro for antagonism between sCD4 and antibody with respect to the net effect of the two in blocking HIV binding and infectivity.

Binding, Competitive↗
PubMed1989

Structural features of CD4 required for binding to HIV.

A soluble form of the human CD4 glycoprotein (sCD4), the cellular receptor for human HIV, was treated with various physical, chemical, and enzymic regimens and tested over a range of concentrations for its capacity to inhibit the binding of HIV to CD4+ T cells. Reduction of disulfide bonds and alkylation in denaturing buffer (8 M urea) destroyed the inhibitory activity of sCD4, whereas reduction and alkylation in PBS had no effect. Derivatization or digestion of carbohydrate groups by periodate oxidation or by glycolytic enzyme digestion did not affect sCD4 inhibitory capacity. Digestion with trypsin or endoproteinase Glu-C destroyed activity. A limited digestion of sCD4 with endoproteinase Glu-C resulted in a mixture of fragments, however, and the mixture had inhibitory activity equivalent to that of intact sCD4. Within this mixture, a fragment of 23 kDa was identified that binds to HIV. Although sCD4 can be digested to yield fully active fragments, the requirement for intrachain disulfide bonding indicates that the minimum sized portion of CD4 that will retain full affinity for HIV will have to be formulated with a proper tertiary structure.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Serial determinations of HIV-1 titers in HIV-infected homosexual men: association of rising titers with CD4 T cell depletion and progression to AIDS.

Lymphocyte subset enumerations, antibody titers to specific proteins of human immunodeficiency virus (HIV), and measurement of infectious HIV titers in peripheral blood mononuclear cells were performed on serial blood specimens from 15 HIV-infected homosexual men with chronic lymphadenopathy syndrome (LAS); 6 of these men have subsequently progressed to AIDS (progressors), and 9 have remained clinically stable (nonprogressors). For the earliest samples studied, no test distinguished those who would progress to AIDS from those who have not. The two groups diverged significantly about 1 year before AIDS diagnosis in the progressor group. Virus titers rose in progressors but remained relatively stable in nonprogressors. CD4 T cells and the CD4 T cell subset, 4B4, declined more rapidly in progressors than in nonprogressors. HIV antibody titers tended to decline in progressors, but the differences were significant only for antibody and to the pol-encoded proteins, p51/65, and the gag-encoded polyprotein, p55. Before the onset of clinical AIDS, progressors are distinguished from nonprogressors by markedly different rates of CD4 cell depletion and virus replication, but the elements that control these dynamics remain to be defined.

AIDS-Related Complex↗
PubMed1988

B6 vitamer content of rat tissues measured by isotope tracer and chromatographic methods.

Feeding [14C]pyridoxine to growing rats for 146 days produced uniform labelling of the total vitamin B6 pool, thus permitting the radioactivity to be used as an absolute standard for evaluating the accuracy of vitamin B6 analyses. The results demonstrated that trichloroacetic acid extraction followed by cation exchange chromatography accurately measures the B6 vitamers. It is essential to homogenize tissues in a protein-denaturing agent in order to avoid shifts in the vitamer content, particularly in liver. In rats approximately 80% of the radioactivity was found in carcass and 8-9% each in liver and skin. Pyridoxamine phosphate equalled or exceeded the concentration of pyridoxal phosphate in heart, brain and kidney. The total vitamin B6 pool in weanling and adult rats averaged about 16 nmol/g body wt. Meta-phosphoric acid extraction followed by reverse phase chromatography gave good agreement with the cation exchange method in rat liver but with cat plasma yielded pyridoxal phosphate values below those of the cation exchange or enzymatic methods. The discrepancies encountered between different homogenization techniques and chromatographic methods emphasize the need for constant vigilance and continual verification of results by independent methods.

Animals↗
PubMed1988

Hemolytic disease of the newborn from anti-E. A case report.

Anti-E is rarely acknowledged as a cause of severe hemolytic disease of the newborn. A patient presented with a low titer (32) that increased following repeated amniocentesis, used to evaluate the fetal condition. The infant developed severe disease requiring multiple exchange transfusions but did well and was discharged in good condition.

Adult↗
PubMed1987

Monitoring of cyclosporine therapy with in vitro biological assays.

We examined the correlation between cyclosporine (CsA) levels and in vitro assays of immune function and hematopoiesis. Mixed lymphocyte reaction (MLR), mitogen responses, suppressor cell (SC), cell-mediated lympholysis (CML), and erythroid colony (EC) assays were studied in dogs, and in vitro megakaryocytopoiesis was studied in mice. Serum CsA concentrations were measured by radioimmunoassay. After oral or intramuscular CsA dosing, lymphocyte proliferation, as measured by MLR, inversely correlated with in vivo serum CsA concentration. MLR responses decreased rapidly, and nearly complete inhibition coincided with peak CsA levels. While CsA concentration-related suppression of lymphocyte stimulation was also observed in mitogen-stimulated cultures, results were less predictable and similar to results with in vitro CsA addition, and higher serum CsA levels were required to achieve comparable suppression. In vivo serum or in vitro CsA levels greater than 300 ng/ml completely inhibited the development of cytotoxic effector cells but had no measureable effect on the expression of suppressor cells in the same cultures. Furthermore, CsA serum also caused concentration-related inhibition of EC growth. The addition of human embryonic kidney-conditioned medium, however, abrogated CsA-related inhibition of EC growth, which suggested that CsA indirectly inhibited EC growth, presumably by interfering with CsA-sensitive accessory cells. This was supported by studies in an in vitro model of murine megakaryocytopoiesis. In normal conditioned medium, megakaryocyte colonies were unaffected by the presence of CsA. However, when cells were cultured in conditioned medium prepared in the presence of CsA, profound inhibition of megakaryocyte growth was observed. These studies show that biologic assays can be used reliably to measure concentration-related changes in immunosuppressive activity of CsA. Further clinical studies are needed to evaluate the usefulness of pharmacodynamic monitoring of CsA therapy.

Animals↗
PubMed1987

Antibody response to human immunodeficiency virus in homosexual men. Relation of antibody specificity, titer, and isotype to clinical status, severity of immunodeficiency, and disease progression.

The titers and isotypes of antibodies to specific proteins of the human immunodeficiency virus were determined by Western blot analysis of sera from 107 homosexual men. Antibody titers were generally lower in sera from patients with the acquired immunodeficiency syndrome (AIDS) and in sera from men whose condition subsequently progressed to AIDS than in sera from men who had not progressed to AIDS. We found no evidence of isotypic prominence or restriction of the antibody response. In multivariate analysis, lower levels of CD4 helper cells were most highly associated with progression to AIDS. Lower antibody titers to the envelope protein gp110, the core protein p24, and the reverse transcriptase enzyme p51/65 were also predictive of progression to AIDS independent of their association with CD4 cell levels. These data suggest that differences in antibody levels are not simply a consequence of severe immunodeficiency but may be markers for control of infection.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Monitoring cyclosporin concentrations in marrow transplant recipients: comparison of two assay methods.

Renal dysfunction is the major dose-limiting toxicity associated with cyclosporin therapy. We have previously shown in patients undergoing allogeneic bone marrow transplantation that serum cyclosporin concentrations, as measured by radioimmunoassay (RIA), correlate significantly with the development of renal dysfunction. However, since the RIA measures both parent drug and metabolites, the relative role of each in the development of nephrotoxicity could not be determined. Therefore, we re-measured cyclosporin concentrations in the same serum samples by high-performance liquid chromatography (h.p.l.c.). Serum cyclosporin concentrations of less than 50, 50-100 and greater than 100 ng/ml, as measured by h.p.l.c., were considered equivalent to cyclosporin concentrations of less than 150, 150-250 and greater than 250 ng/ml, as measured by RIA. Contrary to results by RIA, cyclosporin concentrations measured by h.p.l.c. did not significantly correlate with renal dysfunction, which suggests that measurement of serum cyclosporin concentrations by h.p.l.c. provides no clinical advantage to RIA for monitoring cyclosporin concentrations to prevent renal dysfunction.

Adolescent↗
PubMed1987

Prevention of platelet alloimmunization in dogs with systemic cyclosporine and by UV-irradiation or cyclosporine-loading of donor platelets.

To study the immunosuppressive effects of three different treatments, 30 dogs received at weekly intervals eight platelet transfusions from a single random donor dog. The three experimental protocols were daily oral cyclosporine (Cs) treatment of recipients; in vitro ultraviolet (UV)-irradiation of donor platelets; and Cs-loading of donor platelets. All nine recipients of Cs, 11/12 (92%) recipients of UV-irradiated platelets, and 5/9 (56%) recipients of Cs-loaded donor platelets remained nonimmunized to repeated transfusions of donor platelets. In contrast, only 3 of 21 untreated controls (14%) were not alloimmunized by donor platelets. Moreover, 44% to 67% of the nonimmunized recipients remained tolerant to continued platelet transfusions from their original donor even after experimental therapy was discontinued. Forty-three percent to 100% of transfusions from secondary donors were also accepted without causing alloimmunization, suggesting that tolerance induced by prior treatment was not specific for the primary donor. However, survival of both the original and secondary donor platelets was reduced to about half the starting level, suggesting that some immune response to platelets had occurred. Also, recipients immunized by their original donor's platelets frequently developed refractoriness to platelets from other donors.

Animals↗
PubMed1986

Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T4) molecule: conformation dependence, epitope mapping, antibody inhibition, and potential for idiotypic mimicry.

Human immunodeficiency virus (HIV), the retrovirus that causes the acquired immunodeficiency syndrome, is cytopathic for CD4+ T cells and binds to these cells via a complex of the 110,000 m.w. viral-envelope glycoprotein, gp110, and the CD4 molecule. We treated virus with several physical, chemical, and enzymic agents to determine their effect on the capacity of HIV to bind to the CD4+ T cell line, CEM. Reduction and alkylation (but not alkylation alone) and trypsin digestion (but not glycolytic enzyme digestions) of HIV destroyed its capacity to bind. If the tertiary protein structure conferred by disulfide bonding is not disrupted, the tertiary and secondary conformations dependent on noncovalent forces appear to be thermodynamically favored, because treatment with denaturants such as sodium dodecyl sulfate, 8 M urea, alcohol, or heat (56 degrees C or 65 degrees C for 30 min) followed by removal of the denaturants did not affect binding. Irreversible denaturation and loss of binding occurred after heating at 100 degrees C for 10 min. HIV binding to CD4+ T cells was inhibited either by murine monoclonal antibodies to the CD4 molecule or by human polyclonal or murine monoclonal antibodies to the gp110 molecule. On the basis of results of binding inhibition obtained with a panel of alpha-CD4 monoclonal antibodies, the receptor site for virus on the CD4 molecule was mapped to the amino-terminal portion of the molecule. Four candidate alpha-CD4 monoclonal antibodies that were potent inhibitors of virus binding (OKT4A, OKT4D, OKT4F, and Leu-3a) were examined for the possibility that their binding sites (idiotopes) might share structural and conformational similarity with the CD4-binding site on gp110. Polyclonal human or rabbit anti-HIV sera (that reacted with gp110 and inhibited virus binding) did not react with or inhibit the binding of these four alpha-CD4 monoclonal antibodies. Conversely, rabbit anti-idiotypic sera raised against each of the four candidate CD4 monoclonal antibodies did not react with virus or inhibit virus binding to CD4+ T cells. Further search or different approaches may yet yield an idiotype that is a structural and conformational "internal image" of the CD4-binding site of virus.

Antibodies, Monoclonal↗
PubMed1986

Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule.

Human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) is tropic for human T cells with the helper-inducer phenotype, as defined by reactivity with monoclonal antibodies specific for the T4 molecule. Treatment of T4+ T cells with monoclonal antibodies to T4 antigen blocks HTLV-III/LAV binding, syncytia formation, and infectivity. Thus, it has been inferred that the T4 molecule itself is a virus receptor. In the present studies, the surfaces of T4+ T cells were labeled radioactively, and then the cells were exposed to virus. After the cells were lysed, HTLV-III/LAV antibodies were found to precipitate a surface protein with a molecular weight of 58,000 (58K). By blocking and absorption experiments, this 58K protein was identified as the T4 molecule. No cell-surface structures other than the T4 molecule were involved in the antibody-antigen complex formation. Two monoclonal antibodies, each reactive with a separate epitope of the T4 molecule, were tested for their binding capacities in the presence of HTLV-III/LAV. When HTLV-III/LAV was bound to T4+ T cells, the virus blocked the binding of one of the monoclonal antibodies, T4A (OKT4A), but not of the other, T4 (OKT4). When HTLV-III/LAV was internally radiolabeled and bound to T4+ T cells which were then lysed, a viral glycoprotein of 110K (gp110) coprecipitated with the T4 molecule. The binding of gp110 to the T4 molecule may thus be a major factor in HTLV-III/LAV tropism and may prove useful in developing therapeutic or preventive measures for the acquired immune deficiency syndrome.

Acquired Immunodeficiency Syndrome↗