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Biomedical subjects

M Wachter

Publications and source records attributed to M Wachter.

18 recordsLinked to original sources
PubMed1992

Defective transport as a mechanism of acquired resistance to methotrexate in patients with acute lymphocytic leukemia.

Although the mechanisms of resistance to methotrexate (MTX) are known in experimental tumors made resistant to this drug, little information is available regarding acquired resistance to MTX in patients. A competitive displacement assay using the fluorescent lysine analogue of MTX, N-(4-amino-4-deoxy-N10-methylpteroyl)-N epsilon-(4'-fluorescein-thiocarbamyl)-L-lysine (PT430), was developed as a sensitive method of detection of transport resistance to MTX in cell lines, as well as in blast cells from patients with leukemia. Rapid uptake of PT430 at high concentrations (20 mumol/L) in leukemic blasts resulted in achievement of steady-state levels within 2 hours. Subsequent incubation with the folate antagonists, MTX and trimetrexate (TMTX), which differ in the mode of carrier transport, produced characteristic patterns of PT430 displacement. Flow cytometric analysis of the mean fluorescence intensity in the human CCRF-CEM T-cell lymphoblastic leukemia cell line and its MTX-resistant subline clearly identified the presence of transport deficiency in the resistant subline. Analysis of blasts from 17 patients with leukemia, nine with no prior chemotherapy and eight previously treated with chemotherapy, found evidence of MTX transport resistance in two of the four patients who were treated with MTX and considered to be clinically resistant to the drug. The finding that blast cells of some patients with leukemia considered clinically resistant to MTX is due to decreased MTX transport has important implications for clinical use of this drug and for new drug development.

Adolescent↗
PubMed1989

Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile.

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in Escherichia coli strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.

Bacterial Toxins↗
PubMed1988

Discordant maturation as the primary biological defect in chronic myelogenous leukemia.

Comparative studies of the in vitro growth characteristics of normal and chronic myelogenous leukemic (CML) progenitor cells have provided further evidence that discordant maturation is the primary biological defect in CML. The in vitro growth of total normal and CML granulocyte/macrophage colony forming unit (CFU-GM) populations were compared with early and intermediate (HLA-DR positive) CFU-GM derived from the same marrows. The absolute number of total CML CFU-GM exceeded the number generated by normal marrow through 7 days of culture due entirely to an excess of CML CFU-GM with limited proliferative capacity. Unlike normal colonies, relatively few of the leukemic colonies grew to a large size; the early and intermediate (HLA-DR positive) CML progenitors also exhibited limited proliferative capacity compared to normal. Highly enriched progenitor populations were prepared, and it was observed that the primitive (small) CML CFU-GM also had greatly reduced proliferative potential compared to primitive normal progenitors, but rather behaved similarly to normal mature (large) CFU-GM. Similarly, CML erythroid burst forming units were at a more advanced stage of maturation than normal erythroid burst forming units as evidenced by their reduced proliferative capacity, the observation that a reduced proportion required burst promoting activity to enable them to respond to erythropoietin and the observation that a larger fraction than normal could sustain a limited period of erythropoietin deprivation in the absence of burst promoting activity. Based on these findings and supporting evidence from our previous studies and those reported by other investigators, it is concluded that the dominance of the leukemic population is not due to unregulated proliferation but rather to discordant maturation resulting in expansion in the later maturational compartments which are not under strict regulatory control.

Bone Marrow↗
PubMed1987

Human bone marrow CFU-GM and BFU-E localized by light scatter cell sorting.

Flow cytometric separation was performed on the normal human bone marrow (BM) by using the low-angle (0 degrees) or high-angle (90 degrees) light scatter. Four distinct subpopulations of cells can be enriched from normal human BM and these fractions were subsequently evaluated for their morphological properties as well as their clonogenic capacity in various progenitor cell assays. Our results indicate that human erythroid and granulocyte-macrophage progenitor cells can be separated from BM low-density cells by cell sorting, and these cells show similar 0 degrees and 90 degrees light scatter properties to those observed with murine bone marrow studies. Flow cytometric analysis also suggests that the majority of sorted BFU-E and CFU-GM resides in the blast cell subset of human BM mononuclear cells.

Bone Marrow Cells↗
PubMed1987

Expression of two natural killer cell antigens, H-25 and H-366, by human immature myeloid cells and by erythroid and granulocytic/monocytic colony-forming units.

Two monoclonal antibodies (MoAbs), H-25 and H-366, shown previously to react with human peripheral blood large granular lymphocytes with natural killer (NK) cell activity and some peripheral blood monocytes, have now been shown to also react with a significant proportion of the myeloid and erythroid precursor cells in human bone marrow and peripheral blood. In FACS IV cell sorting and immune rosetting of bone marrow cells, the antigens recognized by H-25 and H-366 were found to be expressed on most blasts and promyelocytes but sequentially fewer of the more mature cells of the myeloid lineage. Both antigens were also found on most monocytes but only a minor proportion of lymphoid and nucleated red cells in the bone marrow. In vitro assays detecting hematopoietic colony-forming units revealed that these antigens are expressed by virtually all mature erythroid colony-forming units (day-7 CFU-E), and the majority of the more primitive erythroid burst forming units (day-14 BFU-E). H-25 but not H-366 was also found on a variable proportion of the day-7 and day-14 granulocytic/monocytic colony-forming units (CFU-GM) in the bone marrow. The same type of precursor cells are also found in the H-25 and H-366 positive cell populations isolated from peripheral blood. In preliminary testing of cells from acute leukemic patients, FACS analysis showed that both antigens are also expressed on leukemic cells from patients with T cell acute lymphocytic leukemia and with myeloid leukemias. These studies demonstrate that the H-25 and H-366 positive NK cells in the peripheral blood retain some of the cell surface properties of early hematopoietic precursor cells, thus providing further evidence supporting the bone marrow origin of NK cells.

Antibodies, Monoclonal↗
PubMed1986

Human gamma interferon enhances release from phytohemagglutinin-stimulated T4+ lymphocytes of activities that stimulate colony formation by granulocyte-macrophage, erythroid, and multipotential progenitor cells.

Human gamma interferon (HuIFN gamma) was evaluated for its effects on the release from human peripheral blood T lymphocytes (greater than 98% pure) stimulated by phytohemagglutinin (PHA) of activities that can stimulate granulocyte-macrophage (CFU-GM) colonies and clusters, erythroid (BFU-E) bursts, and mixed (CFU-GEMM) colonies. T lymphocytes did not release these activities in the absence of PHA with or without HuIFN gamma. In the presence of PHA, pure natural HuIFN gamma at concentrations of 0.1 to 100 U/mL significantly enhanced release of these colony-stimulating activities. Although enhanced release of granulocyte-macrophage colony-stimulating activities were noted when T lymphocytes were added to the conditioning medium in the presence of 0.1%, 0.5%, and 1% PHA, enhanced release of burst-promoting and mixed colony activities was seen only in the presence of 0.1% and 0.5% PHA. The enhanced release of colony-stimulating activities was not due to HuIFN gamma-suppression of the release from PHA-stimulated T lymphocytes of suppressor molecules. The enhancing effects of natural HuIFN gamma were neutralized with a monoclonal anti-natural HuIFN gamma, and recombinant HuIFN gamma mimicked the enhancing effects of the natural HuIFN gamma. This enhancing effect was noted only when HuIFN gamma was added with the T lymphocytes and PHA during the first 24 hours of incubation. T lymphocytes were separated into T4+, T8-, T8+, and T4- subsets (greater than 98% pure for the appropriate phenotypes) after incubation with OKT4- and OKT8- monoclonal antibodies and sorting on a fluorescence-activated cell sorter (FACS). All types of colony-stimulating activities were released from each population after stimulation with PHA, but enhanced release of these activities in the presence of HuIFN gamma was only detected with the T4+ or T8- subsets of lymphocytes. It cannot be concluded from these studies whether HuIFN gamma is enhancing the release of one or several types of colony-stimulating activities, but these studies suggest a role for HuIFN gamma and T4+ lymphocyte subsets in the regulation in vitro of the release of colony-stimulating activities.

Colony-Forming Units Assay↗
PubMed1986

Enhancement of the proliferation of human marrow erythroid (BFU-E) progenitor cells by prostaglandin E requires the participation of OKT8-positive T lymphocytes and is associated with the density expression of major histocompatibility complex class II antigens on BFU-E.

The relationship between major histocompatibility complex class II antigens (MHC class II, eg, HLA-DR, Ia), T lymphocytes, and the enhancement of erythroid colony formation from BFU-E by prostaglandin E was analyzed using normal bone marrow cells. In primary methylcellulose culture, the addition of prostaglandin E1 (PGE1) to unseparated buffy coat, low-density, or nonadherent low-density (NAL) marrow cells resulted in an enhancement of the total number of erythroid (BFU-E) colonies observed. Treatment of bone marrow cells with a monoclonal antihuman MHC class II antibody plus complement (C') resulted in a reduction of the total number of colonies by approximately 50% and abrogation of the enhancing effect of PGE1. Analysis of accessory cell requirements by depletion of both adherent cells and sheep erythrocyte rosetting lymphocytes (E+ cells) and reconstitution using C' or anti-MHC class II antibody plus C'-treated T cell-depleted NAL (NALT-) marrow cells and E+ cell populations treated with C' or anti-MHC class II antibody plus C' demonstrated a requirement for MHC class II antigen-T cells, but not adherent cells, and a requirement for MHC class II antigen + BFU-E in order to observe the enhancing effect of PGE1 on erythroid colony formation. Positive selection of BFU-E in NALT- bone marrow expressing differing density distributions of MHC class II antigens was accomplished with monoclonal anti-MHC class II antibodies and sorting with a fluorescence-activated cell sorter (FACS). Addition of E+ cells to the different populations of MHC class II antigen+ NALT- cells demonstrated that the PGE-enhancing effects on erythroid colony formation were directly related to increasing density distributions of MHC class II antigens on BFU-E. Colony formation by BFU-E expressing a low density distribution of MHC class II antigens or having no detectable MHC class II antigens, as determined by FACS analysis, was not enhanced by PGE1 in the presence of MHC class II antigen-positive or -negative T cells.

Alprostadil↗
PubMed1985

Functional implications of oligomerization of simian virus 40 large T antigen during lytic virus infection.

The formation of oligomers of simian virus 40 (SV40) large T antigen in SV40-infected and -transformed monkey cells was analyzed by sucrose density gradient centrifugation. The overall distribution of total T antigen during lytic infection showed mainly low-molecular-weight forms (monomers and dimers) in the early phase (10 h postinfection) and an increase in the number of oligomers in the late phase of the lytic cycle (36 h postinfection), indicating an accumulation of these final products. In contrast, studying the conversion of newly synthesized T antigen into oligomers by appropriate pulse-chase radiolabeling of infected cells revealed that this processing decelerates considerably during the late phase of infection. This mechanism can be reaccelerated by blocking DNA replication with aphidicolin. Since none of these results could be obtained by using synchronized SV40-transformed monkey cells (COS-1), these observations are compatible with the idea that the process of T antigen oligomerization may be involved in viral, but not in cellular, DNA synthesis.

Antigens, Viral, Tumor↗
PubMed1985

Incidence of involvement of the B and T lymphocyte lineages in chronic myelogenous leukemia.

Peripheral blood specimens were obtained from 22 patients with Philadelphia chromosome (Ph1) positive chronic myelogenous leukemia (CML) (16 in chronic phase, 2 in an accelerated phase, and 4 in blast crisis). Studies were performed to determine the frequency of the presence of the Ph1 chromosome in cells of lymphoid lineages. Rosetted (E+) lymphocytes (T lymphocytes) from nine patients in chronic phase and one patient in blast crisis were stimulated with T cell growth factor interleukin 2 (IL-2) and/or phytohemagglutinin (PHA). All ten patients had sufficient T lymphocyte metaphases for analysis and of a total of 461 metaphases examined, only one contained the Ph1 chromosome. Nucleated cells of density less than 1.077 g/mL were infected with Epstein-Barr virus (EBV). Following infection, cell lines were established from individual colonies attached to egg albumin-coated Lab-Tek slide chambers (clonal cell lines) or from suspension culture in 96-well tissue culture cluster dishes (nonclonal cell lines). Cell surface and intracellular marker analysis confirmed the B lymphocyte phenotype of all the cell lines examined. B lymphoblastoid cell lines were established from 16 of the 22 patients. All lines from 12 patients were Ph1-negative. From two chronic phase patients, both Ph1-positive and Ph1-negative lines were established. From one patient in an accelerated phase, only Ph1-positive lines were established. From another patient in blast crisis (of myeloblastic phenotype), only Ph1-positive lines were established initially; however, five months later, after the patient had been treated with mitoxantrone, only Ph1-negative lines were derived from this patient. Based on these results, it appears that most B cells and mature T cells in most CML patients are Ph1-negative, but that about 25% of patients have predominantly Ph1-positive B cells or a mixture of Ph1-positive and Ph1-negative B cells that are capable of growing as established cell lines after transformation with EBV.

Adolescent↗
PubMed1985

Regulation of human peripheral blood erythroid burst-forming unit growth by T lymphocytes and T lymphocyte subpopulations defined by OKT4 and OKT8 monoclonal antibodies.

To reexamine the influence that T lymphocytes have on the regulation of human peripheral blood burst-forming unit (BFU-E) proliferation in the absence of a statistically significant number of monocytes, very low numbers (3 to 10 X 10(3)/mL) of a null cell fraction highly enriched for BFU-E were cultured alone and in the presence of 5 X 10(5) sheep erythrocyte-purified, autologous T lymphocytes in a methylcellulose culture system containing erythropoietin. T lymphocytes consistently enhanced the growth of BFU-E from the null cell fraction, as reflected in both their number and size. Irradiation of T lymphocytes prior to coculture with null cells markedly reduced this enhancement, strongly suggesting that T lymphocytes synthesize erythroid burst-promoting factors (BPA). To determine whether there were functional differences between the two major T lymphocyte populations as defined by OKT4 (T helper/inducer) and OKT8 (T suppressor/cytotoxic) murine monoclonal antibodies to stimulate the growth of BFU-E, both T cell subpopulations were isolated by negative (panning) or positive (fluorescence-activated cell sorting) selection and cocultured with null cells. No statistically significant differences emerged between unseparated, OKT4+ and OKT8+ T lymphocytes in their ability to stimulate the growth of BFU-E. Thus, these studies provide further evidence that T lymphocytes are a major population of BPA-producing cells and further that OKT4+ and OKT8+ T lymphocytes equally elaborate these factors.

Animals↗
PubMed1985

Release of granulocyte-macrophage colony stimulating factors from major histocompatibility complex class II antigen-positive monocytes is enhanced by human gamma interferon.

Human gamma interferon (HuIFN gamma) was assessed for its capacity to enhance release of granulocyte-macrophage colony stimulating factors (GM-CSF) from human peripheral blood monocytes. Natural HuIFN gamma (2 X 10(7) NIH reference units per milligram) at concentrations as low as 0.01 U/mL to 10 U/mL reproducibly enhanced release of GM-CSF. This enhancement was detected when T lymphocytes were depleted from monocyte preparations and when T lymphocytes and monocytes were depleted from populations of human bone marrow cells stimulated by monocyte-conditioned media to form colonies and clusters. T lymphocytes alone or in the presence of HuIFN gamma did not release GM-CSF. The enhancing activity of HuIFN gamma was removed by preincubating HuIFN gamma with neutralizing concentrations of monoclonal anti-HuIFN gamma, and recombinant HuIFN gamma mimicked the effects of natural HuIFN gamma, suggesting that the effects were due to HuIFN gamma itself. HuIFN gamma suppression of the release of inhibitory activity from monocytes was ruled out as a reason for the noted enhancing activity of HuIFN gamma. The enhancing activity of HuIFN gamma was confined to the MHC class II antigen-positive population of monocytes. Removal of these cells with monoclonal antibody plus complement (C') ablated the enhancing activity, high concentrations of certain monoclonal antibodies in the absence of C' blocked the enhancing activity and, when monocytes were sorted into MHC class II antigen-positive and -negative cells by fluorescence-activated cell sorting, it was only the positive cell fraction that responded to the enhancing activity of HuIFN gamma.

Cells, Cultured↗
PubMed1983

On the specificity of sialidase. Synthesis and properties of N5-acetyl-beta-D-neuraminoylpeptides - AcNeu-Gly-OH, AcNeu-Glu-OH, AcNeu-Phe-OH - and the corresponding alpha-ketosides.

By means of the mixed anhydride procedure the benzyl alpha-ketoside of N5-acetyl-D-neuraminic acid was linked to L-glycine, L-glutamic acid and L-phenylalanine. Hydrogenolytic cleavage of the benzyl group resulted in the corresponding free N5-acetyl-beta-D-neuraminoylpeptides. This new class of compounds is no substrate for Vibrio cholerae sialidase. The enzyme does not split the benzyl alpha-ketosides of N5-acetyl-D-neuraminoylpeptides nor is its activity inhibited by these compounds. The results strongly support the assumption that in sialidase substrates the carboxy group must be located close to the ketosidic oxygen. N-(N5-acetyl-beta-D-neuraminoyl)-L-phenylalanine was readily hydrolysed by carboxypeptidase A from bovine pancreas.

Animals↗
PubMed1979

Measurement of myocardial PCO2 with a microelectrode: its relation to coronary sinus PCO2.

A micro-PCO2 electrode, with dimensions of 1 x 10 mm, and a 63% response time of 14 s was inserted into the left ventricular myocardium of the pentobarbital-anesthetized dog. Continuous recordings were made of myocardial PCO2 (PmCO2), arterial PCO2 (PaCO2), and coronary sinus PCO2 (CSPCO2) during variation of respiratory rate. PmCO2 and CSPCO2 were compared at varying coronary flow. PmCO2 was similar to and closely followed changes in CSPCO2. The difference between PmCO2 and CSPCO2 was -0.52 +/- 3.63 (SD) mmHg, and PmCO2 exceeded PaCO2 by 20.69 +/- 5.12 mmHg. After coronary occlusion, PmCO2, rose promptly, but CSPCO2 was only slightly elevated until the occlusion was released, when a CO2 efflux into the coronary sinus occurred. It is concluded that the electrode measures extracellular PCO2 and that extracellular and myocardial PCO2 are essentially equal. PmCO2 rises rapidly following coronary occlusion.

Animals↗
PubMed1978

Relative effect of CO2 on canine coronary vascular resistance.

We determined the effect of alterations in coronary arterial PCO2 on coronary vascular resistance (CVR) at a constant coronary sinus (CS) PO2 and the effect of coronary arterial PO2 variation on CVR at a constant CS PCO2. A linear but opposing effect on CVR was found for both gases. The sensitivity of CVR to O2 change, represented as CVR/CS PO2, was approximately twice that of the ratio CVR/CS PCO2. (0.0852 +/- 0.006 vs. -0.0362 +/- 0.005). The entire range of CVR variation obtainable through CO2 variation was as great as that resulting from O2 variation. During randomized variation of O2 and CO2, CVR can be mathematically related in a multiple linear expression to CS PO2 and CS PCO2.

Animals↗