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J A Dvorak

Publications and source records attributed to J A Dvorak.

At least 19 recordsLinked to original sources
PubMed1992

Merocyanine 540-sensitized photoinactivation of human erythrocytes parasitized by Plasmodium falciparum.

The purpose of this study was to evaluate the photosensitizing dye merocyanine 540 (MC540) as a means for extracorporeal purging of Plasmodium falciparum-infected erythrocytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoietic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine-sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC540-sensitized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood.

Animals↗
PubMed1990

Isolate-dependent differences in the oxidative metabolism of Trypanosoma cruzi epimastigotes.

Epimastigote cultures of the two cloned Trypanosoma cruzi stocks, CA-I/72 and HO-3/15, grown under identical conditions, differ both qualitatively and quantitatively in their cytochrome content. The CA-I/72 stock has a four-fold higher cytochrome b content (19.2 nM (mg protein)-1) than the HO-3/15 stock (4.9 nM (mg protein)-1). Cytochrome o is present at 29 nM (mg protein)-1 in the CA-I/72 stock but is below detectable limits in the HO-3/15 stock. There is no inter-stock difference in oxygen utilization (12-15 nM O2 min-1 (mg protein)-1) during exponential growth. However, stationary phase CA-I/72 epimastigotes utilize twice as much oxygen as HO-3/15 epimastigotes. Oxygen utilization by HO-3/15 epimastigotes incubated in Dulbecco's phosphate buffer solution (starvation conditions), was stimulated earlier and to higher levels by the addition of glucose than by CA-I/72 epimastigotes under identical conditions. Under starvation conditions and with the cytochrome chain partially inhibited by antimycin A,(anti-A) the addition addition of glucose also increased oxygen utilization by CA-I/72 epimastigotes. In contrast, anti-A did not influence glucose-stimulated oxygen utilization by HO-3/15 epimastigotes. Following partial inhibition with anti-A, salicylhydroxamic acid produced an additional 50% inhibition in oxygen utilization in both stocks irrespective of the growth phase of the organisms. These data indicate that marked intra-specific differences in oxidative metabolism exist within the T. cruzi population and that an alpha-glycerophosphate oxidase or similar salicylhydroxamic acid-inhibitable compound may be present in the organism.

Animals↗
PubMed1989

Modified Box-Cox transform for modulating the dynamic range of flow cytometry data.

We describe an algorithm, Vout = Integer ([2(12)-1/2(12 lambda)-1] V lambda in-1) + 1; lambda greater than 0 based upon Box-Cox transformations as an alternative to nonlinear electronic amplifiers to expand or compress high- or low-amplitude flow cytometer-derived signals. If the indexing parameter lambda less than 1, input channels in the high-amplitude input range are compressed in the output range as occurs when an electronic logarithmic amplifier is used. However, if lambda greater than 1, input channels in the low-amplitude input range are compressed in the output range as occurs when an electronic power amplifier is used. Our modified Box-Cox transform can be implemented either during data collection or off-line for the transformation of previously collected raw data. The transform is the equivalent of an infinite class of nonlinear amplifiers. As the transform is implemented in software, it does not suffer from many of the disadvantages of nonlinear electronic amplifiers.

Algorithms↗
PubMed1989

Quantitation of total DNA per cell in an exponentially growing population using the diphenylamine reaction and flow cytometry.

The diphenylamine assay used to estimate the absolute mass of DNA/cell as well as absolute differences in DNA content between cell populations is based upon the assumption that all of the cells are in the G0 or G1 phase of the DNA synthetic cycle. However, if cells are in exponential growth and synthesizing DNA, portions of the population will be in S or G2 phases and the diphenylamine assay will overestimate the total mass of DNA/cell. Conversely, flow cytometry (FCM) can estimate relative differences in total DNA/cell and the proportions of an exponentially growing population in G1, S, and G2 but cannot estimate absolute mass or differences in DNA/cell. In this report, we describe a methodology of combined diphenylamine and FCM assays of total DNA/cell which is applicable to any eukaryotic cell population. The method involves using the two assay methods concurrently and correcting the diphenylamine data for the FCM-derived distribution of the cells within the DNA synthetic cycle. The methodology was tested on single-cell-derived stocks of the obligate intracellular protozoan parasite Trypanosoma cruzi which displays marked but stable intraspecific heterogeneity.

Animals↗
PubMed1989

Leishmania mexicana mexicana: quantitative analysis of the intracellular cycle.

The complete intracellular cycle of the Leishmania mexicana mexicana G. S. strain was quantified in human macrophages and in the mouse IC-21 macrophage line utilizing a culture system that allows the direct observation of individual intracellular parasites. A wide range of pre-replicative lag periods exists, implying that promastigotes may be in any phase of their DNA synthetic cycle when phagocytosed by the macrophage. Amastigotes replicated 2-3 times, after which the host cell died and liberated amastigotes that were taken up by other macrophages and continued to replicate. The mean amastigote population-doubling time in human macrophages (17.5 h) was not statistically different from promastigotes growing in axenic culture (16.4 h), but was nearly 2-fold less than amastigotes growing in mouse-derived IC-21 macrophages (33.7 h). These observations are markedly different from cover-glass culture assays of Leishmania-macrophage interactions and provide an unambiguous description of the intracellular cycle of Leishmania mexicana mexicana.

Animals↗
PubMed1988

Trypanosoma cruzi: elemental composition heterogeneity of cloned stocks.

Concentrates of the epimastigote stage of Trypanosoma cruzi stocks derived from single cell clones and cultured under identical conditions display a spectrum of 'colors' varying from dark brown to milk white. The color of the concentrate is reproducible for a parasite stock. An essential component of the culture medium for epimastigote growth is hemin, an iron-containing compound. Consequently, it seemed possible that the color spectrum of the epimastigote stocks reflected differences in the uptake, concentration or utilization of iron. This report describes the quantitative studies utilizing electron probe X-ray elemental mapping, energy dispersive X-ray microanalysis, and energy dispersive X-ray fluorescence spectrometry of the epimastigote stage of two T. cruzi stocks (CA-I/72 and HO-3/15) which display extreme color differences. Striking and statistically significant quantitative differences were found in the levels of Fe, Zn, and K between the two stocks. On the basis of atomic ratios, the CA-I/72 stock contains approximately two-fold more Fe per cell than the HO-3/15 stock. However, in the case of Zn the ratio is reversed; the HO-3/15 stock contains approximately two-fold more Zn per cell than the CA-I/72 stock. The marked inter-stock differences which exist in the levels of several elements could modulate the pathogenicity, survival, or adaptability of T. cruzi and, consequently, be important factors in our understanding of the complex problem of Chagas' disease.

Animals↗
PubMed1988

Nucleoside uptake in Trypanosoma cruzi: analysis of a mutant resistant to tubercidin.

Nucleoside salvage pathways are vital to the parasitic protozoan Trypanosoma cruzi, and have become important targets in the development of new chemotherapeutic agents against this organism. We produced a mutant T. cruzi clone with a defect in the uptake of the adenosine analogue tubercidin which allowed us to hypothesize that there are at least two distinct nucleoside transport pathways in this parasite. The mutant shows a marked defect in the uptake of tubercidin and thymidine, whereas the uptake of adenosine and inosine are normal. Inhibition and metabolic studies suggest that the defect is related to transport and that there are two transport processes relatively specific for purines and pyrimidines, respectively, although tubercidin is transported via the latter. This is similar to the reported dual nucleoside transport pathways in Leishmania donovani and may be a common system in the Trypanosomatidae. These transport processes are markedly different from those which have been described for mammalian cells and may play an important role in the design of strategies for the chemotherapy of human infection with these pathogenic parasites.

Adenosine↗
PubMed1988

Trypanosoma cruzi: cell biological behavior of epimastigote and amastigote forms in axenic culture.

A simple protocol to maintain Trypanosoma cruzi amastigote stocks indefinitely in axenic culture is described. The growth characteristics of amastigotes differ markedly from epimastigotes cultured under identical conditions. The amastigotes replicate for two generations, followed by a transformation to epimastigotes and resumption of growth. By changing the culture medium at the end of the second amastigote generation, transformation to epimastigotes is inhibited. Therefore, the protocol used to maintain amastigotes in culture is based upon changing the culture medium at preselected intervals. Flow cytometric analyses indicate that at the end of the exponential phase of growth the amastigote population consists of predominately G1 cells; changing the medium induces the amastigotes to begin a para-synchronous round of DNA synthesis without a pre-replicative lag phase. In contrast, when exponentially growing or stationary-phase epimastigotes are transferred to fresh culture medium, they grow asynchronously until reaching a limiting cell density. Amastigotes also differ from epimastigotes in being resistant to the lytic activity of human complement. These data demonstrate that marked differences in phenotypic expression exist between developmental stages of T. cruzi even when cultured under identical conditions.

Animals↗
PubMed1987

Comparative studies of the infection of Lewis rats with four Trypanosoma cruzi clones.

Quantitative and qualitative differences in parasitaemia levels and histopathological characteristics occurred in Lewis rats infected with four Trypanosoma cruzi clones. Rats infected with clone Sylvio-X10/7 sustained high parasitaemias and died during the acute phase whereas rats infected with clones Sylvio-X10/4, Miranda/78 or Miranda/80 did not. Myocardial fibrosis was more extensive in rats chronically infected with clone X10/4 than in rats infected with the 2 Miranda clones. Myocardial fibrosis developed more rapidly in rats than has been reported in mice. These characteristics are advantageous in developing animal models of human chagasic myocardiopathy.

Animals↗
PubMed1987

Temperature modulation of growth rates and glucosephosphate isomerase isozyme activity in Trypanosoma cruzi.

In an attempt to understand the conditions which might influence the geographical distribution of Trypanosoma cruzi isozyme profiles (zymodemes), the thermal response of different glucosephosphate isomerase (GPI) phenotypes was studied. T. cruzi clones with single- and triple-banded GPI phenotype showed a similar response to temperature with respect to both growth rates and GPI kinetic parameters. However, the relative activity of each GPI isozyme was dependent on parasite incubation temperature. In view of the similar kinetic properties of the isozymes, enzyme regulation is not a consequence of an adaptative response to thermal conditions and the suggestion of a phenotype distribution determined selectively by temperature is not supported by the present study.

Animals↗
PubMed1987

Trypanosoma cruzi: analysis of the population dynamics of heterogeneous mixtures.

Utilizing the previously reported inter-clonal differences in total DNA/organism, flow cytometry was used to analyze the population dynamics of Trypanosoma cruzi clone mixtures growing in liquid medium or vertebrate cells. The growth of clone mixtures in liquid medium can be described by unique parameters reflecting exponential growth rate (r), stationary phase population density (1/k), and the interaction between the clones (h). The relative numbers of each clone in the population change rapidly with time and the results are in quantitative agreement with mathematical models of competitive population growth. The relationship between the parameters for T. cruzi is such that, in general, there is no dynamic equilibrium with coexistence of clones with different growth rates; under all culture protocols, the faster growing clone will prevail. A computer simulation of the vertebrate cell cycle of T. cruzi suggests that clone mixtures grow relatively independently; the basic attributes of the model were substantiated experimentally. Although wide fluctuations in the proportion of each clone released occurred, the faster growing clone again predominated. Finally, these results underline the importance of working with well-defined clones in the laboratory to avoid inconsistencies and paradoxical results and stress the importance of the rapid isolation of single cell clones from clinical specimens when studying the relationship of the parasite to human disease.

Animals↗
PubMed1987

Flow cytometric analysis of the DNA synthetic cycle of Candida species.

The total DNA per cell and DNA synthetic cycle phases were determined by flow cytometry in five Candida isolates including three species: Candida albicans 208R1, Candida tropicalis ATCC 750, and Candida parapsilosis 970, 3138, and ATCC 22019. The cells were prepared for flow cytometry by fixation in Carnoy fixative followed by staining with mithramycin. Marked but stable and reproducible inter- and intraspecific differences in total DNA per cell of stationary-phase cultures were found which did not correlate directly to diphenylamine estimates of the same parameter. This discrepancy was resolved by mathematically converting flow cytometry data into diphenylamine data. The reason for the discrepancy was found in studies of the DNA synthetic cycle of these yeasts: a large but isolate-specific variable proportion of the population is arrested in the S and G2-M phases after the culture passes from exponential to stationary phase. Histograms of exponential-growth-phase Candida isolates demonstrate that the majority of the population is in the G2-M phase of the DNA synthetic cycle. The DNA content of the C. tropicalis and C. parapsilosis isolates studied is as high as or higher than that of C. albicans. Extranuclear fluorescent particles were observed in the C. tropicalis isolate. No equivalent particles could be detected in the other four Candida isolates. The nature of the particles is unknown.

Candida↗
PubMed1987

Studies of Trypanosoma cruzi clones in inbred mice. III. Histopathological and electrocardiographical responses to chronic infection.

Histopathological and electrocardiographical (ECG) changes occur in the heart of C3H/HeN and C57BL/6 mice infected for 1 year with Trypanosoma cruzi clones Sylvio-X10/4 (X10/4), Miranda/78 (M/78), or Miranda/80 (M/80). Heart parasitism and a variable degree of inflammation occurred following infection with clones X10/4 or M/78 but not with M/80. Clone X10/4 caused more extensive myocardial inflammation and fibrosis than clone M/78. Myocardial fibrosis was more extensive in C3H than in C57 mice infected with clone X10/4. The normal ECG pattern of C3H mice is distinctly different from C57 mice. The PR intervals of mice infected with clone X10/4 greater than M/78 greater than M/80 approximately equal to controls. ECG abnormalities occurred more frequently in mice infected with clone X10/4 than in controls or mice infected with either M/78 or M/80 regardless of strain or sex and were generally more severe in C57 than in C3H infected with X10/4. First degree atrioventricular block occurred more frequently in C3H mice infected with clone X10/4 or M/78 and C57 mice infected with X10/4 than in all other groups. Complete atrioventricular dissociation occurred frequently in C57 mice infected with X10/4 and rarely in other mice. These results demonstrate that the myocardial response of mice to T. cruzi infection, both histological and electrophysiological, is modulated by both the mouse strain and the parasite isolate used.

Animals↗
PubMed1986

A biochemical comparison of glucosephosphate isomerase isozymes from Trypanosoma cruzi.

The glucosephosphate isomerase (D-glucose-6-phosphate Ketol-isomerase, EC 5.3.1.9) isozymes of Trypanosoma cruzi were characterized with respect to their native and subunit molecular size, isoelectric point and in vitro thermostability. The molecular weight data are consistent with a dimeric enzyme structure. The apparent native and subunit size homogeneity and differences in pI values imply that the electrophoretic mobility differences of isozymes in native gels are determined by their molecular charge. Minor differences in peptide maps indicate the existence of some heterogeneity in the primary structure of the isozymes. The stability of triple-banded glucosephosphate isomerase electrophoretic profiles was confirmed, supporting the view that these phenotypes represent non-interconvertible enzyme species.

Animals↗
PubMed1986

Trypanosoma cruzi: constancy of clone pathogenicity for inbred mice during long-term in vitro maintenance.

The reproducibility of infection of C3H/He mice with T. cruzi clones Sylvio-X10/4 and Sylvio-X10/7 maintained in the laboratory for 946 and 496 days respectively was assayed. Clone X10/7 from 15 different in vitro passages consistently induced an acute lethal infection (94.3% mortality) and constant survival time (Mean = 24.6 d.p.i.). Female mice survived significantly longer than males and mice older than 30 days at the time of infection survived significantly longer than younger mice. The mortality of mice infected with clone X10/4 from 23 different in vitro passages was lower (5.1%) and their survival longer (Mean = 42.7 d.p.i.) than mice infected with clone X10/7. High anti-T. cruzi IgG titres were detected in the plasma of all mice killed after 30 d.p.i. Histologically, the heart, skeletal muscles and/or large intestine contained intracellular parasites in 59% of the mice; parasites were found in mice of all groups tested. The hearts of all mice were comparably inflammed regardless of parasite passage number or duration of infection. These data demonstrate that the presentation and course of infection of inbred mice with two T. cruzi clones is not changed by either the duration or protocol used for in vitro maintenance; the reported loss or change in virulence of T. cruzi strains during long-term in vitro maintenance did not occur. Consequently, T. cruzi clones and inbred mice provide a dependable and reproducible means to study host-parasite interactions.

Animals↗
PubMed1986

A histopathological analysis of the course of myocarditis in C3H/He mice infected with Trypanosoma cruzi clone Sylvio-X10/4.

The histopathology of hearts of C3H/He (MTV-) mice infected for 2 to 440 days with Trypanosoma cruzi clone Sylvio-X10/4 was examined. Inflammation was present in most hearts at 14 days post-infection (d.p.i.) and persisted thereafter. Myocardial fibrosis, which was first seen 45 d.p.i. and was more marked after 200 d.p.i., developed at a time when few parasites were present. Fibrosis predominated in subepicardial and subendocardial myocardium, areas of most severe inflammation. Parasites were restricted to myocardial fibres and were found in all mice infected for 14 to 30 days and, in fewer numbers, in 54% of mice infected for more than 30 days. T. cruzi-induced myocarditis in mice was not a self-resolving disease but a dynamic, active process occurring continuously over a long time following infection. This clone-derived T. cruzi stock produced a myocarditis in inbred mice similar to that seen in human chronic Chagas' disease.

Animals↗
PubMed1986

Identification and analysis of epimastigote surface and metabolic proteins in Trypanosoma cruzi.

Metabolic and surface membrane proteins of four epimastigote-stage Trypanosoma cruzi clones were analyzed by one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No major inter-clonal differences were observed in the metabolic protein patterns, indicating that these proteins are highly conserved. However, marked quantitative and qualitative differences were observed in surface-labeled protein patterns following both one and two-dimensional electrophoretic analyses. Inter and intra-clonal differences in antigenic properties also were demonstrated by immunoprecipitation of the surface proteins with sera from animals immunized or infected with various T. cruzi stocks. Thus, a wide spectrum of both phenotypic and antigenic diversity exists in T. cruzi which may be relevant to problems of the diagnosis and immunotherapy of Chagas' disease.

Animals↗