Biomedical subjects

M H Kaplan

Publications and source records attributed to M H Kaplan.

At least 37 records · Page 2Linked to original sources

PubMed1999

Cell-associated HIV-1 messenger RNA and DNA in T-helper cell and monocytes in asymptomatic HIV-1-infected subjects on HAART plus an inactivated HIV-1 immunogen.

OBJECTIVE: We examined the effect of an HIV-1-specific immune-based therapy on cell-associated HIV-1 DNA and RNA. DESIGN: Five HIV-1-infected subjects receiving HIV-1 immunogen plus HAART were compared with three HIV-1-infected subjects who received incomplete Freund's adjuvant (IFA) plus HAART. METHODS: Cell-associated HIV-1 RNA or DNA in lymphocytes and monocytes was determined using a dual immunophenotyping/in situ hybridization assay with or without in situ PCR amplification. RESULTS: Cell-associated HIV-1 RNA in CD4 cells correlated with plasma RNA overall. CD4, HIV-1 gag-pol messenger (m)RNA+ cells decreased in the immunogen plus HAART group compared with the IFA plus HAART group. Decreases in HIV-1 DNA+ CD4 cells were observed in the immunogen plus HAART compared with the IFA plus HAART group. Decreases in HIV-1 gag-pol mRNA+ monocytes were observed in the immunogen plus HAART group compared with the IFA plus HAART group. Consistent with the findings in CD4 cells, decreases in HIV-1 DNA+ monocytes were observed in the immunogen plus HAART group compared with the IFA plus HAART group. CONCLUSIONS: These preliminary observations support the rationale for examining the combination of immune-based therapies and antiretroviral drugs for effective HIV-1 control.

AIDS Vaccines↗

PubMed1999

Marking IL-4-producing cells by knock-in of the IL-4 gene.

IL-4 is a cytokine which can be expressed by a number of cell types including Th2 cells, mast cells and a population of CD4+ NK1.1+ NK T cells. Although phenotypic markers exist for identifying each of these cell types, there is at present no known cell surface marker common to all IL-4-producing cells. Using gene targeting in embryonic stem cells, we have modified the IL-4 locus by knock-in of a transmembrane domain to generate mice that express a membrane-bound form of IL-4 (mIL-4). Flow cytometry using an IL-4-specific mAb allowed the detection of IL-secreting Th2 cells, mast cells and NK T cells from mIL-4 mice. Furthermore, the analysis of immune responses in mIL-4 mice following immunization with anti-CD3 and anti-IgD has allowed us to identify distinct subpopulations of IL-4-producing NK T cells. Thus, the expression of IL-4 in a membrane-bound form provides a novel method for the identification and characterization of IL-4-producing cells.

Animals↗

PubMed1999

Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization.

The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

Alleles↗

PubMed1999

Comparison of levels of human immunodeficiency virus type 1 RNA in plasma as measured by the NucliSens nucleic acid sequence-based amplification and Quantiplex branched-DNA assays.

This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although the Quantiplex and NucliSens values were significantly different (P < 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays: r = 0.912, P < 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively.

DNA, Viral↗

PubMed1998

Decreased neonatal dietary fat absorption and T cell cytotoxicity in pancreatic lipase-related protein 2-deficient mice.

The pancreas secretes several different lipases. The most abundant is pancreatic triglyceride lipase (PTL). The pancreas also synthesizes two homologues of PTL, the pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Cytotoxic T-lymphocytes also express PLRP2 under certain conditions. We sought to determine the role of PLRP2 in fat absorption and in T-cell cytotoxicity by creating a PLRP2-deficient mouse. Adult PLRP2-deficient mice had normal fat absorption. In contrast, suckling PLRP2-deficient mice had fat malabsorption evidenced by increased fecal weight, increased fecal fats, and the presence of undigested and partially digested dietary triglycerides in the feces. As a result, the PLRP2-deficient pups had a decreased rate of weight gain. To assess T cell cytotoxicity, we immunized PLRP2-deficient mice with a mastocytoma cell line, P815, and determined the ability of splenocytes from the immunized mice to kill P815 cells in a 51Cr release assay. PLRP2-deficient cells had deficient killing activity in this assay, and PLRP2-deficient splenocytes released fewer fatty acid from the target cells than did control cells. Our results provide the first evidence of a physiological function for PLRP2. PLRP2 participates in T cell cytotoxicity, and PLRP2 performs a crucial role in the digestion of dietary fats in suckling animals.

Age Factors↗

PubMed1998

A signal transducer and activator of transcription (Stat)4-independent pathway for the development of T helper type 1 cells.

The differentiation of T helper (Th) cells is regulated by members of the signal transducer and activator of transcription (STAT) family of signaling molecules. We have generated mice lacking both Stat4 and Stat6 to examine the ability of Th cells to develop in the absence of these two transcription factors. Stat4, Stat6(-/-) lymphocytes fail to differentiate into interleukin (IL)-4-secreting Th2 cells. However, in contrast to Stat4(-/-) lymphocytes, T cells from Stat4, Stat6(-/-) mice produce significant amounts of interferon (IFN)-gamma when activated in vitro. Although Stat4, Stat6(-/-) lymphocytes produce less IFN-gamma than IL-12-stimulated control lymphocytes, equivalent numbers of IFN-gamma-secreting cells can be generated from cultures of Stat4, Stat6(-/-) lymphocytes activated under neutral conditions and control lymphocytes activated under Th1 cell-promoting conditions. Moreover, Stat4, Stat6(-/-) mice are able to mount an in vivo Th1 cell-mediated delayed-type hypersensitivity response. These results support a model of Th cell differentiation in which the generation of Th2 cells requires Stat6, whereas a Stat4-independent pathway exists for the development of Th1 cells.

Animals↗

PubMed1998

Detection and titration of human herpesvirus-8-specific antibodies in sera from blood donors, acquired immunodeficiency syndrome patients, and Kaposi's sarcoma patients using a whole virus enzyme-linked immunosorbent assay.

A human herpesvirus-8 (HHV-8) enzyme-linked immunosorbent assay (ELISA) with a whole virus lysate as antigen was developed and used to measure the seroprevalence rate and levels of IgG antibodies to HHV-8 in sera/plasma of various patient groups and blood donors. The virus antigen was prepared from the KS-1 cell line, which produces lytic virus, and therefore contains a broad array of viral proteins. Seroprevalence studies using this ELISA showed the following: 10 of 91 blood donors (11%) had an average HHV-8 antibody titer of 118; 67 of 72 (93%) classic Kaposi's sarcoma (KS) patients were positive with an average titer of 14,111; and 57 of 62 (92%) KS/human immunodeficiency virus (HIV) patients were positive with an average titer of 4,000. A study on a very limited number of serial serum samples from patients before and after diagnosis with KS showed highly elevated antibody titers to HHV-8 virus after KS lesions developed. Preliminary data show that 50% of the sera from HIV-1(+) homosexual patients contain IgG antibodies to HHV-8 suggesting that this population is at high risk for developing KS. Antibody results correlated well with the confirmatory immunofluorescent assays (IFA) using KS-1 cells as the substrate. This HHV-8 IgG antibody detection ELISA is sensitive and specific and does not cross-react with Epstein-Barr virus (EBV) or other human herpesviruses. The results of this HHV-8 antibody survey suggest that this rapid ELISA assay can be used to screen large numbers of sera to find those at risk for developing KS.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Th2 cells are required for the Schistosoma mansoni egg-induced granulomatous response.

The development of Schistosoma mansoni ova-induced granulomas is regulated by cytokines secreted by distinct Th cell subsets. To determine the importance of Th1 and Th2 cells in granuloma formation, we have studied the immune response to S. mansoni ova in Stat4- and Stat6-deficient mice, which lack Th1 and Th2 cells, respectively. Lymphocytes from both naive and infected Stat6-deficient mice produced minimal levels of Th2 cell cytokines and Ag-specific IgG1 and IgE, but showed enhanced production of IFN-gamma and Ag-specific IgG2a and IgG2b following schistosome egg injection. This shift away from a Th2 cell-mediated immune response was coupled with the development of pulmonary and hepatic granulomas that were greatly decreased in size compared with those in control littermates. Hepatic granulomas in Stat6-deficient mice were composed of predominantly mononuclear cells with very sparse appearance of eosinophils, and their diminished size was accompanied by decreased amounts of liver hydroxyproline content as a measure of collagen deposition. In contrast, lymphocytes from infected Stat4-deficient mice produced Th2 cell cytokines in amounts comparable to those produced by control littermates, but low levels of IFN-gamma. While infected Stat4-deficient mice developed pulmonary granulomas following schistosome egg injection that were modestly impaired in size, the granuloma size and amount of collagen deposition in the liver were equivalent to those seen in control littermates. These studies demonstrate that Th2 cells are required for the full development of the granulomas and tissue-destructive fibrotic pathology associated with the immune response to S. mansoni ova.

Animals↗

PubMed1998

Regulation of T helper cell differentiation by STAT molecules.

It is now well appreciated that the cytokines interleukin (IL)-12 and IL-4 are important for the generation of Th1 and Th2 cells, respectively. Only recently, however, have the molecular mechanisms by which these cytokines affect Th cell differentiation begun to be defined. Previous work from our laboratory has demonstrated that members of the signal transducer and activator of transcription (STAT) gene family are critical for the differentiation of Th cell subsets. In particular, Stat4-deficient mice show an impairment in the generation of Th1 cells, whereas Stat6-deficient animals do not generate Th2 cells. We have now generated Stat4-Stat6 double-deficient mice to determine whether STAT-independent pathways exist for the development of Th cell subsets. It is surprising that Th1 but not Th2 cells can be generated from double-deficient mice in vitro and these animals are able to mount an in vivo Th1 cell-mediated immune response. These results suggest a model of Th cell differentiation in which Stat4 and Stat6 have different roles in the development of Th cell subsets.

Animals↗

PubMed1998

Application of a standardized cytomegalovirus antigenemia assay in the management of patients with AIDS.

One-hundred twenty AIDS patients, tested by an optimized cytomegalovirus antigenemia (CMV-Ag) assay, were followed over a 6-month period in an attempt to correlate viral (CMV) load with severe CMV organ-specific disease. Among patients available for follow-up, CMV organ-specific disease was present in seven of eight (88%) with pp65 antigen levels > or = 50 cells per 4 x 10(5) polymorphonuclear leukocytes. One-hundred six patients of 107 patients with levels < 50 pp65 reactive cells did not develop CMV organ-specific disease within our 6-month follow-up period. The CMV-Ag assay, as standardized in our clinical setting, served as a marker and predictor of CMV organ-specific disease in our AIDS patient population.

AIDS-Related Opportunistic Infections↗

PubMed1998

Membrane and soluble forms of Fas (CD95) and Fas ligand in peripheral blood mononuclear cells and in plasma from human immunodeficiency virus-infected persons.

The expression of membrane-bound Fas ligand (FasL) and Fas in lymphocytes and monocytes and levels of soluble forms of FasL (sFasL) and Fas (sFas) in plasma from human immunodeficiency virus (HIV)-positive and -negative subjects was evaluated. Surface FasL was detectable on monocytes, but poorly so on lymphocytes, even in the presence of KB8301, a metalloproteinase inhibitor. Unexpectedly, monocytes of HIV-positive subjects expressed less FasL than those of HIV-negative volunteers. sFasL levels in plasma of HIV-positive persons were elevated and correlated with levels in plasma and with HIV RNA burden. sFas levels in plasma of HIV-positive subjects were also elevated and correlated with Fas expression in apoptotic lymphocytes. Finally, culture-induced lymphocyte apoptosis of HIV-positive subjects was enhanced by anti-Fas agonistic antibody but was not inhibited by anti-FasL blocking antibodies. These results suggest that significant dysregulation of both Fas and FasL occurs in HIV infection and contributes to increased sensitivity of lymphocytes to apoptosis.

Apoptosis↗

PubMed1998

Stat proteins control lymphocyte proliferation by regulating p27Kip1 expression.

The proliferation of lymphocytes in response to cytokine stimulation is essential for a variety of immune responses. Recent studies with signal transducer and activator of transcription 6 (Stat6)-deficient mice have demonstrated that this protein is required for the normal proliferation of lymphocytes in response to interleukin-4 (IL-4). In this report, we show that the impaired IL-4-induced proliferative response of Stat6-deficient lymphocytes is not due to an inability to activate alternate signaling pathways, such as those involving insulin receptor substrates, or to a failure to upregulate IL-4 receptor levels. Cell cycle analysis showed that the percentage of Stat6-deficient lymphocytes that transit from the G1 to the S phase of the cell cycle following IL-4 stimulation is lower than that of control lymphocytes. Although the regulation of many genes involved in the control of cytokine-induced proliferation is normal in Stat6-deficient lymphocytes, protein levels of the cdk inhibitor p27Kip1 were found to be markedly dysregulated. p27Kip1 is expressed at significantly higher levels in Stat6-deficient lymphocytes than in control cells following IL-4 stimulation. The higher level of p27Kip1 expression seen in IL-4-stimulated Stat6-deficient lymphocytes correlates with decreased cdk2-associated kinase activity and is the result of the increased accumulation of protein rather than altered mRNA expression. Similarly, higher levels of p27Kip1 protein expression are also seen following IL-12 stimulation of Stat4-deficient lymphocytes than are seen following stimulation of control cells. These data suggest that Stat proteins may control the cytokine-induced proliferative response of activated T cells by regulating the expression of cell cycle inhibitors so that cyclin-cdk complexes may function to promote transition from the G1 to the S phase of the cell cycle.

Animals↗

PubMed1997

Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells.

A lymphocyte population that expresses surface markers found on T cells and natural killer (NK) cells secretes large amounts of interleukin-4 (IL-4) immediately after T cell receptor ligation. These NK-like T cells are thus thought to be important for the initiation of type 2 T helper cell (TH2) responses. CD1-deficient mice were found to lack this lymphocyte subset, but they could nevertheless mount a protypical TH2 response; after immunization with antibody to immunoglobulin D (IgD), CD1-deficient mice produced IgE. Thus, although dependent on CD1 for their development, IL-4-secreting NK-like T cells are not required for TH2 responses.

Animals↗

PubMed1997

Antiviral susceptibility testing-flow cytometric analysis (AST-FCA) for the detection of cytomegalovirus drug resistance.

Antiviral susceptibility testing-flow cytometric analysis (AST-FCA), an application of flow cytometry in conjunction with cell culture, was developed to identify susceptibility of cytomegalovirus (CMV) isolates to the antiviral drugs ganciclovir (GCV) and foscarnet (PFA). The viral isolates used in this study were obtained from peripheral blood of AIDS patients. Among GCV-susceptible strains, the mean 50% inhibitory concentration (IC50) using AST-FCA was 18 microns (SI50 = 1.4). Among GCV resistant strains, the mean IC50 was 47 microns (SI50 = 3.6). For PFA, the mean IC50 was 80 microns (SI50 = 1.0) for susceptible strains. IC50s for strains resistant to PFA, showed no significant reduction of infectivity at the highest drug concentration (i.e., 200 microns PFA) tested. Additional analyses confirmed the accuracy of AST-FCA by parallel testing using plaque reduction assay. AST-FCA is a novel, nonisotopic, and relatively simple to perform laboratory procedure for the identification of CMV drug-resistant strains.

Antiviral Agents↗

PubMed1997

Significance of leukocyte concentration in the performance of the quantitative cytomegalovirus (CMV) antigenemia assay.

BACKGROUND: The quantitative cytomegalovirus-antigenemia (CMV-Ag) assay is an important technology in the regimen of tests utilized in the care and management of acquired immunodeficiency syndrome (AIDS) and other immunocompromised patient groups. Performance of this assay is contingent upon the appropriate processing of the polymorphonuclear leukocyte (PMNL) compartment of the peripheral blood. However, a cell input standard in the performance of the CMV-Ag assay, has not been established. Interpretive differences between laboratories utilizing the CMV-Ag assay may reflect this lack of test uniformity. OBJECTIVES: To determine the effect of different PMNL concentrations on the quantitation of CMV in peripheral blood. The leukocyte concentration resulting in optimal rates of viral detection, will be compared with the shell vial assay-indirect immunofluorescent assay (SVA-IFA), and conventional tube culture (TC-CPE). STUDY DESIGN: A total of 74 freshly collected blood specimens were tested by the CMV-Ag assay, using cytospin preparations consisting of 2 x 10(5), 4 x 10(5) and in preliminary experiments, 8 x 10(5) PMNLs/slide. Data obtained from these studies were compared to SVA-IFA and TC-CPE. Viral load was monitored among 11 symptomatic patients through sequential testing of these patients at the start of ganciclovir (GCV), foscarnet (PFA), or combination drug therapy. RESULTS: Among 74 blood specimens tested by the CMV-Ag assay, cytospin preparations consisting of 4 x 10(5) compared with 2 x 10(5) PMNLs/slide, affected a mean positive cell increase of 215% (P = 0.03). PMNL slide preparations consisting 8 x 10(5) cells produced background levels which prevented accurate reading of slides. The CMV-Ag assay was more sensitive than the SVA-IFA, but equivalent to TC-CPE. Among 11 patients started on drug therapy, viral load was markedly reduced in 8 within 2-3 weeks; three patients (2 deceased within 3 weeks after receiving therapy), showed no decrease in viral load. One patient was identified as harboring a PFA resistant strain. CONCLUSIONS: A PMNL concentration of 4 x 10(5) cells facilitated the reading of CMV-Ag assay slide preparations. The modified CMV-Ag assay furthermore, is applicable in the monitoring of viral load for the tracking of susceptible or resistant CMV strains.

Antigens, Viral↗

PubMed1997

Effects of specimen collection, processing, and storage conditions on stability of human immunodeficiency virus type 1 RNA levels in plasma.

To define the optimal blood collection parameters for plasma human immunodeficiency virus type 1 (HIV-1) viral load testing, plasma HIV-1 RNA levels were quantitated with the NASBA HIV-1 RNA QT System from blood specimens that were collected, processed, and stored under a variety of conditions that might have affected HIV-1 RNA stability. We determined that when whole blood was processed within 2 h of specimen collection the levels of HIV-1 RNA detected in EDTA-, heparin-, and acid citrate dextrose (ACD)-anticoagulated plasma samples were comparable. The levels of HIV-1 RNA in serum specimens (mean = 4.126 log units) were significantly lower (P < 0.01) than the levels in corresponding plasma samples (mean = 4.501 log units). One cycle of freeze-thaw (-70 degrees C) did not significantly reduce the level of HIV-1 RNA detected in EDTA-, heparin-, or ACD-anticoagulated plasmas. The EDTA-anticoagulated plasmas showed the smallest decrease in HIV-1 RNA copies (0.050 log units). HIV-1 RNA levels decreased over a 6-month time period in serum as well as in EDTA-, ACD-, and heparin-anticoagulated plasmas stored at -70 degrees C. However, the only significant decreases were for serum (mean decrease = 0.317 log units) and heparin-anticoagulated samples (mean decrease = 0.384 log units). A comparison of the levels of HIV-1 RNA in cell-free plasma collected in VACUTAINER EDTA Plasma Preparation Tubes and in standard VACUTAINER EDTA tubes determined that HIV-1 RNA levels were stable for up to 30 h after collection when stored at either room temperature (mean standard deviation [SD] = +/- 0.101 log units) or at 4 degrees C (mean SD = +/- 0.102 log units) as cell-free plasma or as EDTA-anticoagulated whole blood (mean SD = +/- 0.109 log units). These data indicate that EDTA-anticoagulated plasma is the most suitable and stable matrix for HIV-1 RNA quantitation.

Anticoagulants↗

PubMed1997

Detection of the herpesvirus-like DNA sequences in matched specimens of semen and blood from patients with AIDS-related Kaposi's sarcoma by polymerase chain reaction in situ hybridization.

The DNA sequences of a novel human gamma-herpesvirus type 8 (HHV-8) have recently been detected in Kaposi's sarcoma (KS) lesions obtained from different populations in whom this neoplasm occurs, suggesting that this virus may be implicated in the etiology and/or pathogenesis of KS. To study the distribution and possible means of transmission of the putative viral agent, specimens of KS skin lesions, matched uninvolved skin, peripheral blood mononuclear cells (PB-MCs), and semen were collected from 12 HIV-positive homosexual men with acquired immune deficiency syndrome (AIDS)-related KS (AIDS-KS) and 2 human immunodeficiency virus (HIV)-negative homosexual men with KS. HHV-8 virus DNA was detected by polymerase chain reaction (PCR) studies in all 14 of these KS specimens and in 6 of 14 biopsies of normal-appearing skin distant from any KS lesions including 1 uninvolved skin specimen from an HIV-negative homosexual male with KS. In addition, 3 of 12 PBMC samples and 3 of 12 semen samples from the AIDS-KS patients were positive for HHV-8. The DNA sequences of HHV-8 were not detected in the matched semen and PBMC specimens obtained from 2 HIV-negative homosexual men with KS, 4 HIV-positive homosexual patients without KS, 2 HIV-seronegative healthy homosexual men, 5 HIV-positive heterosexual male intravenous drug users, or 5 healthy HIV-negative heterosexual donors. Using PCR in situ, positive signals for HHV-8 were demonstrated in the B lymphocyte subsets of PBMCs and/or in spermatozoa and mononuclear cells in the semen from some of the PCR-positive specimens from the AIDS-KS patients examined. These data show that HHV-8 is present in and could possibly be transmitted via semen and/or blood from some homosexual men with AIDS-KS.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Genomic organization of the murine CTL lipase gene.

Murine cytotoxic T-lymphocyte (CTL) lipase was originally identified as an IL-4-inducible gene in CD8-positive T cells. To further our understanding of both the function and the regulation of CTL lipase in T cells, we have cloned and characterized the murine gene. Two overlapping phage clones spanning 29 kb contain the entire CTL lipase gene. The exon structure is similar to those characterized for the human and canine pancreatic lipase-related protein 1 genes, with notable differences in the 5' end. Transcripts initiate from a site that matches a consensus for an initiator sequence. Potential cis-regulatory elements in the CTL lipase 5' regulatory region that would confer dual tissue specificity in exocrine pancreas and cytotoxic T lymphocytes are identified. The implications of this promoter organization are discussed.

Animals↗