Biomedical subjects

C R Brockelman

Publications and source records attributed to C R Brockelman.

At least 19 recordsLinked to original sources

PubMed1992

A specific DNA probe which identifies Babesia bovis in whole blood.

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.

Animals↗

PubMed1991

Development of an in vitro microtest to assess drug susceptibility of Babesia bovis and Babesia bigemina.

Continuous cultivation of the bovine hemoparasites Babesia bovis and Babesia bigemina was developed as an in vitro microtest to assess parasite susceptibility to babesicidal compounds. Reproducibility of parasite multiplication rates was independent of culture size, making it possible to use a microscale of 100 microliters for each test sample. Inhibitory concentrations (IC50s) of a commonly used babesicide, quinuronium sulfate, evaluated by this in vitro method were found to be 5 x 10(-8) g/ml for B. bovis and 2 x 10(-9) g/ml for B. bigemina.

Animals↗

PubMed1990

Finding and recognition of the bovine host by the cercariae of Schistosoma spindale.

The cercaria of Schistosoma spindale finds and identifies its bovine host with at least five behavioral phases. (1) Dispersal in and selection of midwater and water surface as the microhabitat are achieved by an intermittent swimming behavior with a weak geonegative but not photopositive orientation. (2) Attachments are stimulated by host-specific higher temperatures of the substrate but not by chemical host signals. (3) Remaining of the attached cercariae on the substrate is stimulated by host-specific higher temperatures of the substrate; chemical host signals have no effect. (4) The creeping of the cercariae is directed to the higher temperature in thermal gradients as weak as 0.07 degrees C/mm. Chemical gradients had no effect on the creeping direction. This behavior may enable the cercariae to migrate along hairs to the host's skin surface. (5) Penetrations are stimulated by the free fatty acid fraction of bovine skin-surface lipids. The characteristics of the stimulating fatty acids are the same as those identified for other schistosome species. Higher temperatures of the substrate alone do not stimulate penetrations. S. spindale cercariae do not use as many chemical host cues as stimuli for the identification of their host as do S. mansoni cercariae. S. spindale seems to be adapted to hairy hosts that are infected in shallow, muddy waters. The low host specificity of the cercarial host-finding behavior is compensated by an intimate parasite-snail intermediate host relationship, resulting in a high cercarial production of up to greater than 7,000 cercariae per snail per day.

Animals↗

PubMed1990

Opisthorchis viverrini: finding and recognition of the fish host by the cercariae.

The cercaria of Opisthorchis viverrini finds and recognizes its fish host by using at least four steps of behavioral patterns. (1) Dispersal and selection of plant-free water microhabitat are achieved by intermittent swimming behavior with positive phototactic orientation. (2) Attachment to the host is stimulated by water currents and a hydrophilic component of fish skin surface which has a molecular weight of more than 30,000. This component is sensitive to digestion with hyaluronidase and seems to be a glycosaminoglycan other than hyaluronic acid and chondroitin sulfate. (3) Remaining on the host's surface is induced by an unknown chemical component of fish skin surface mucus. (4) Penetration into the host is triggered by a hydrophilic component of fish skin surface of a molecular weight of more than 30,000. This host signal has a proteinaceus character as it is sensitive to proteinase digestion but not to hyaluronidase and glycosidases. The requirement of O. viverrini cercariae for complete glycosaminoglycans and proteins as signals in host identification may have the advantage that the numerous small molecules in mud and decaying materials in the water cannot interfere with host-finding.

Animals↗

PubMed1989

Comparative morphology of human and animal malaria parasites. I. Host-parasite interface.

Human and animal malaria parasites (Plasmodium falciparum, P. malariae, P. vivax, P. berghei, P. gallinaceum) were studied using special fixation and standardized methods, with special attention to their effects on host cells. Morphological alterations induced by the parasites in infected erythrocytes included knobs, invaginations, and caveola-vesicle complexes on the surface of the host cell and clefts, microvesicles, and small vesicles in the cytoplasm of the infected erythrocytes. For P. malariae, the ultrastructural study revealed invaginations with associated microvesicles, but knobs did not occur on the surface of infected erythrocytes. The development of invaginations and microvesicles in P. malariae-infected erythrocytes corresponded to the morphological alterations induced by P. vivax. A new hypothesis concerning the origin of Schüffner's dots is discussed.

Animals↗

PubMed1989

Effects of thalassaemic serum on the in vitro development of the malarial parasite Plasmodium falciparum.

Sera from thalassaemic individuals were experimentally tested for their suppressive effects on the in vitro development of asexual stages of Plasmodium falciparum. Cultures in sera collected from six patients who had classical haemoglobin H disease (alpha-thal1-alpha-thal2) and six patients who had haemoglobin H disease with haemoglobin (Hb) Constant Spring (alpha-thal1-CS) showed a significantly higher degree of inhibition of parasite multiplication than cultures of thalassaemic erythrocytes in sera from healthy individuals. Similarly, sera from 12 patients with beta-thalassaemia/haemoglobin E (beta-thal-HbE) diminished parasite development in vitro. Schizonts of P. falciparum cultured in erythrocytes from non-thalassaemic individuals containing normal Hb (alpha 2 beta 2) were also inhibited when thalassaemic serum was used in place of normal serum. The degree of inhibition was proportional to the concentration (vol/vol) of the thalassaemic serum.

Adult↗

PubMed1989

Development of in vitro microtest for the assessment of Plasmodium vivax sensitivity to chloroquine.

An in vitro microtest to assess P. vivax sensitivity to chloroquine has been developed using a medium mixture which contained RPMI, Waymouth (GIBCO) and 15% (vol/vol) human serum group AB. The rate of success was highest in samples which were not washed by centrifugation before culturing in microtest plates predosed with varying concentrations of chloroquine. Evaluation of the effective concentration of chloroquine using a program for probit analysis of log dose/response test proved superior to simply using the percentage of schizont maturation.

Adolescent↗

PubMed1989

Ultrastructural study on the interaction of beta-thalassemic-erythrocytes and Plasmodium falciparum.

beta-thalassemic erythrocytes and normal red blood cells were experimentally infected in vitro with Plasmodium falciparum cultured for 4 days and studied by means of transmission electron microscopy. Conspicuous alterations of the parasites appear in the advanced stage of schizogony leading to the fact that only a small amount of malaria pigment is formed, which in general is not crystalline, but always enclosed in extremely large vacuoles. Furthermore some of the developing merozoites reveal features of cellular degeneration, thus these merozoites lost their ability to invade new erythrocytes. Despite these findings alterations of the host cells are induced by the parasites, which, however, are comparable to those found in infected normal red blood cells, e.g. knobs appeared on the surface of the erythrocytes and membrane-bounded clefts became apparent in the cytoplasm of the host cell.

Animals↗

PubMed1989

Mefloquine sulfadoxine pyrimethamine (MSP) combination delays in vitro emergence of mefloquine resistance in multiple drug resistant Plasmodium falciparum.

Sulfadoxine-pyrimethamine-resistant and chloroquine resistant, but mefloquine sensitive (MIC 5 x 10(-9] isolates of Plasmodium falciparum were used to study the emergence of mefloquine resistance. The continuously cultured isolates which were exposed intermittantly to varying concentrations of mefloquine alone became insensitive to the drug at 1 x 10(-7) M after 12 months, whereas the culture line exposed to the combination of mefloquine, sulfadoxine and pyrimethamine became only slightly insensitive to mefloquine (MIC 2 x 10(-8]. Thus, the efficacy of mefloquine may be prolonged through use in combination with sulfadoxine-pyrimethamine.

Animals↗

PubMed1987

The influence of magnesium ion and ascorbic acid on the erythrocytic schizogony of Plasmodium vivax.

Asexual erythrocytic stages of Plasmodium vivax have been cultivated for one schizogonic cycle to investigate parasite requirements for metal ions and vitamins. Waymouth and RPMI 1640 (GIBCO) media were used in varying proportions resulting in varying concentrations of organic salts, vitamins, and growth factors. A 1:3 mixture gave the highest percentage (62.6%) of parasite development from the amoeboid forms to mature segmenters after 44 h of cultivation in a candle jar atmosphere at 38.5 degrees C. Nevertheless, the total parasite count was significantly higher (P less than 0.50) in the mixture which had a Waymouth: RPMI ratio of 1:2. Differentiation of schizonts to merozoites as well as parasite counts could be further enhanced by the addition of magnesium chloride to a final concentration of 1.8 mM magnesium ions. The minimal requirement for ascorbic acid which was studied in Science Mahidol (SCMI 612) medium appeared to vary among isolates. For example, all parasite population of four isolates tested declined proportionally with the decrease in concentration of ascorbic acid, the critical point being 3 micrograms/ml medium. However, two isolates used in this study could no longer differentiate to segmenters when the ascorbic acid concentration of the medium was less than 6 micrograms/ml.

Animals↗

PubMed1987

Thalassemic erythrocytes inhibit in vitro growth of Plasmodium falciparum.

Blood specimens from 100 thalassemic patients were screened in vitro for inhibitory effects on growth and multiplication of Plasmodium falciparum. The culture medium mixture designated REM consisted of 9 volumes of minimum essential medium (GIBCO Laboratories, Grand Island, N.Y.) and 1 volume of RPMI 1640 (GIBCO) supplemented with 10% heat-inactivated human serum. Parasite multiplication in erythrocytes containing normal hemoglobin cultured in RPMI or REM was similar. Significant reduction in parasite multiplication rates was observed in erythrocytes containing abnormal hemoglobin when these were cultured in REM. The degree of reduction in five types of thalassemic erythrocytes was in the following descending order: hemoglobin H disease with Hb Constant Spring, classical hemoglobin H disease, beta(0)-thalassemia-hemoglobin E in which blood harbored a high percentage of hemoglobin F-containing cells, beta (0)-thalassemia-hemoglobin E in which blood harbored few hemoglobin F-containing cells, and beta-thalassemia heterozygous variant.

Animals↗

PubMed1987

Optimal concentration of p-aminobenzoic acid and folic acid in the in vitro assay of antifolates against Plasmodium falciparum.

In vitro tests for Plasmodium falciparum sensitivity to pyrimethamine, sulfadoxine, and both drugs in combination were performed in four kinds of culture medium, each differing in p-amino benzoic acid (PABA) and folic acid concentrations. Results of the tests using pyrimethamine-sensitive and pyrimethamine-resistant isolates indicated that drug activity was reduced proportionally to the concentrations of these two growth factors in the medium. The optimal concentrations of PABA and folic acid for parasite growth and drug susceptibility, as evaluated by microscopic examination and by the extent of incorporation of radioactive 14C-pyrimethamine and 14C-sulfadoxine, were 10 ng/ml and 2 ng/ml, respectively. Depletion of PABA and folic acid from the medium had no effect on drug-resistant parasites but multiplication of drug-sensitive isolates was markedly reduced. Medium containing 0.5 ng/ml PABA and 10 ng/ml folic acid was the best for parasite growth regardless of the degree of drug sensitivity. Results obtained by using this medium agreed most closely with results from in vivo observations.

4-Aminobenzoic Acid↗

PubMed1987

Flunarizine and verapamil inhibit chloroquine-resistant Plasmodium falciparum growth in vitro.

Using pharmacological properties in relation to the biochemistry of P. falciparum, verapamil, flunarizine, and chlorpromazine which are calcium blockers were selected to test for their antimalarial activity against P. falciparum in vitro. Results revealed that the drugs inhibited parasite population growth in the following order of IC50: verapamil 1 X 10(-6) M, chlorpromazine 3.5 X 10(-6) M, and flunarizine 5 X 10(-6) M. These three calcium blockers have antimalarial effects on chloroquine resistant parasite (alone T9/94) but are less potent when compared with the efficacy of quinine or mefloquine in vitro.

Animals↗

PubMed1985

Observation on complete schizogony of Plasmodium vivax in vitro.

Attempts to grow Plasmodium vivax in vitro were made on 43 isolates in three different culture media. Complete schizogony occurred in the new medium SCMI 612 in which 34 out of 43 isolates produced merozoites. The RPMI 1640 and Waymouth media suitable for the cultivation of P. falciparum were also used with markedly less success. Results of the experiments indicate differences in nutritional requirements between the two species of Plasmodium.

Animals↗

PubMed1985

In vitro sensitivity of Plasmodium falciparum to sulfadoxine and pyrimethamine.

An in vitro microtechnique of Rieckmann et al., (1978) modified by Yisunsri and Rieckmann (1980) using 3 media; Waymouth, Waymouth plus 10% human serum, and RPMI was assessed to determine the sensitivity of P. falciparum to sulfadoxine, pyrimethamine and its combination. The study confirmed the synergism between sulfadoxine and pyrimethamine. There was no interaction between media and drug tested. MIC1 and MIC2 of sulfadoxine in different media showed significant difference (p less than 0.001). No significant difference was observed in MIC1 and MIC2 of pyrimethamine in the three media used (p greater than 0.05). For sulfadoxine-pyrimethamine combination, MIC1 and MIC2 in Waymouth alone and plus 10% human serum showed no significance (p greater than 0.05) while in RPMI showed positive correlation (p less than 0.001). MIC1 might be more applicable for clinical evaluation than MIC2. At present Waymouth medium with 5% patient serum, is considered to be the most suitable for testing sensitivity of malarial parasites.

Culture Media↗

PubMed1984

Mefloquine-pyrimethamine-sulfadoxine combination delays emergence of mefloquine resistant Plasmodium falciparum in continuous culture.

The efficacy of mefloquine against Plasmodium falciparum in continuous culture was studied. The development of mefloquine resistance in P. falciparum was significantly inhibited by a combination of mefloquine, pyrimethamine and sulfadoxine. By contrast, more resistant variants were selected in continuous culture without drug pressure or with pyrimethamine-sulfadoxine pressure. The most mefloquine resistant variants were selected by step-wise increases in mefloquine pressure.

Animals↗

PubMed1983

Plasmodium falciparum: variations in p-aminobenzoic acid requirements as related to sulfadoxine sensitivity.

Plasmodium falciparum, Thai strain FCM-5S sensitive to sulfadoxine, was grown continuously for 1 year in a medium with a low concentration of p-aminobenzoic acid (PABA) without the emergence of a sulfadoxine-resistant line. The minimum PABA-requirement for growth of sulfadoxine-sensitive Thai strains in vitro was found to be 25.0 ng/ml, whereas resistant Burmese and Gambian strains completed their schizogony in the absence of PABA. Cultivation in dialyzed and nondialyzed human serum suggested that the sulfadoxine-resistant parasite might use another serum factor in place of PABA for folate biosynthesis, thus overcoming the effect of sulfadoxine, a PABA antagonist. The significantly lower rate of [14C]sulfadoxine incorporation into erythrocytes infected with sulfadoxine-resistant strains strongly supports this suggestion.

4-Aminobenzoic Acid↗

PubMed1983

Continuous cultivation and improved drug responsiveness of Plasmodium falciparum in p-aminobenzoic acid-deficient medium.

Long term cultivation of three culture lines of Plasmodium falciparum was achieved in Waymouth and RPMI media, both supplemented with 10% heat-inactivated human serum. Observations on parasite multiplication over a 6-mo period showed no difference in the rate of parasite population increase. Growth of asexual erythrocytic stages in Waymouth medium was as good as that in RPMI when measured by the rate of glucose utilization and extent of 3H-isoleucine and 3H-methionine incorporation. Responsiveness of the parasites to pyrimethamine and sulfadoxine was demonstrated to increase when tested in Waymouth medium because it contained no p-aminobenzoic acid to compete with the drugs.

4-Aminobenzoic Acid↗