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Biomedical subjects

C H King

Publications and source records attributed to C H King.

At least 37 records · Page 2Linked to original sources
PubMed1996

Intracellular growth and cytotoxicity of Mycobacterium haemophilum in a human epithelial cell line (Hec-1-B).

We developed an in vitro model to study the temperature-regulated cytotoxicity and intracellular growth of Mycobacterium haemophilum in cultured human epithelial and endothelial cells. M. haemophilum associated with human epithelial and endothelial cells at similar rates when incubated at 33 and 37 degrees C, but only the epithelial cell line supported the multiplication of this organism. M. haemophilum grew equally well with epithelial cells at both temperatures. The aminoglycoside antibiotic amikacin was used to study the intracellular growth of M. haemophilum in the epithelial cells at 33 and 37 degrees C. Although an approximately equal number of bacteria were found within cells after 2 days of incubation at both temperatures, intracellular replication of M. haemophilum was 1,000-fold greater at 33 than at 37 degrees C. This intracellular multiplication was associated with destruction of the monolayers at 33 but not at 37 degrees C, and only culture filtrates from infected monolayers incubated at 33 degrees C were cytotoxic to fresh epithelial cell monolayers. This strain of M. haemophilum also produced contact-dependent hemolysis of sheep erythrocytes, demonstrating the possible presence of a cytolysin. These studies suggest that M. haemophilum has a preference for growth with cultured human epithelial cells. In addition, intracellular growth is best at 33 degrees C in epithelial cells, and this correlated with cytotoxicity at this temperature. This phenotype may be caused by induction of a soluble cytotoxic component, possibly a hemolytic cytolysin.

Colony Count, Microbial↗
PubMed1996

Comparison of in vitro models for the study of Mycobacterium tuberculosis invasion and intracellular replication.

We recently evaluated several tissue culture model systems for the study of invasion and intracellular multiplication of Mycobacterium tuberculosis. These model systems include a human alveolar pneumocyte epithelial cell line, a murine macrophage cell line (J774), and fresh human peripheral blood-derived macrophages. Our data indicated that the initial level of association of M. tuberculosis with human alveolar pneumocyte cells (2%) was less than that observed with fresh human peripheral blood macrophages (9%) or J774 murine macrophages (13%) within 6 h of the addition of the bacteria. M. tuberculosis replicated in association with the pneumocyte cells by more than 55-fold by day 7 postinfection. In contrast, total bacteria] growth in the J774 cells and human macrophages was considerably less, with increases of only fourfold and threefold, respectively, over the same 7-day period. Amikacin, an aminoglycoside antimicrobial agent, was added to inhibit the growth of extracellular bacteria after the initial 6-h infection period. Decreases in viable counts were observed in all three cell cultures within the first 3 days after infection. However, unlike the case with either macrophage culture, intracellular bacterial CFU obtained from the infected pneumocytes increased by fourfold by day 7 after the addition of amikacin. These data indicate that M. tuberculosis infects and multiplies intracellularly in human lung epithelial cells and that these cells may be an alternative in vitro model for the study of intracellular multiplication of M. tuberculosis in the human lung.

Animals↗
PubMed1996

Dynamics and control of Schistosoma haematobium transmission in Kenya: an overview of the Msambweni Project.

Ecologic modeling of Schistosoma transmission in endemic communities has suggested that antiparasite therapy targeted at the most heavily infected segment of the human population (i.e., school-age children) should have a significant impact on local parasite transmission. Our 1984-1991 experience with age-targeted therapy in the Msambweni area of Kenya has shown an overall decrease in area transmission within 1-2 years following initiation of annual treatment of school-age groups. Snail monitoring confirmed a continuing but variable reduction of vector infection rates. However, subgroup analysis showed significant differences in transmission suppression between more developed coastal villages with piped-water kiosks and villages with only limited access to safe water supplies. Villages without piped water were marked by higher initial prevalences of S. haematobium infection, greater prevalence among adults, longer and more frequent contact with high-risk water sources, and persistently high transmission despite compliance with parasitologic screening or drug therapy. We conclude that targeted therapy had a significant impact on S. haematobium transmission in some areas, but that more extensive or more prolonged coverage is necessary to reduce the rate of new infection in high-risk villages. Defining field-use algorithms, based on decision analysis of economic and ecologic parameters, should provide effective guidelines for selective versus mass treatment in expanded control areas.

Adolescent↗
PubMed1996

Gender differences in growth of school-aged children with schistosomiasis and geohelminth infection.

Light or moderate intensity infection with Schistosoma mansoni may contribute to growth deficits. We report on the effects of treatment for S. mansoni on growth and development in Brazilian schoolchildren. Anthropometric measurements were taken from 539 S. mansoni-infected children and their age- and sex-matched egg-negative controls between the ages of 7 and 15 years. The children as a whole exhibited chronic malnutrition, with growth retardation in height evident in 21% of the population. Infected children, however, were significantly smaller in height, weight, mid upper arm circumference (UAC), tricep skinfold (TSF), and subscapular skinfold (SSF) measurements than control children (P < 0.05). These differences were due primarily to a greater disparity between infected and egg-negative girls in height (P < 0.01), weight (P = 0.01), UAC (P = 0.O2), and TSF (P < 0.01). Nevertheless, girls demonstrated a better level of development and nutrition compared with boys. While infected boys were shorter and weighed less than controls, these differences were not significant. Growth and development in girls was negatively correlated with intensity of infection. Coinfection with S. mansoni and Trichuris appeared to act synergistically in the development of malnutrition.

Adolescent↗
PubMed1995

Molecular biology, virulence, and pathogenicity of mycobacteria.

The diseases resulting from infections with Mycobacterium species are important sources of morbidity and mortality throughout the world today, with particularly devastating effects in tropical and developing countries. Almost 2 billion people have been infected with Mycobacterium tuberculosis, the causative agent of tuberculosis, and approximately 3 million people die each year from this disease. Tuberculosis also has re-emerged as an important public health problem in the United States, and this resurgence has been accompanied by an increased incidence of tuberculosis resistant to the standardly used anti-tuberculosis drugs. Researchers' ability to investigate the molecular basis of the pathogenicity and drug resistance of the mycobacteria has been hampered by a lack of appropriate experimental tools. However, during the past 5 years, tremendous progress has been made in the development of the molecular biology of mycobacteria, and molecular tools are now available for detailed analysis of their genetics and for elucidation of the molecular mechanisms of their pathogenicity. The development of these tools is briefly reviewed, and the uses of the tools to investigate drug resistance in Mycobacterium tuberculosis, to identify mycobacterial virulence factors, and to explore intracellular survival strategies are described.

DNA, Bacterial↗
PubMed1993

Expression of contact-dependent cytolytic activity by Mycobacterium tuberculosis and isolation of the genomic locus that encodes the activity.

We investigated the presence of cytolytic activity in the virulent H37Rv and attenuated H37Ra strains of Mycobacterium tuberculosis and in the vaccine strain Mycobacterium bovis BCG. The virulent strain H37Rv expressed > 3-fold more contact-dependent cytolytic activity than the attenuated strain H37Ra, and the vaccine strain M. bovis BCG did not produce cytolytic activity. We also isolated an approximately 3.2-kbp fragment of the M. tuberculosis H37Rv genome that was capable of inducing this contact-dependent hemolytic activity in a nonhemolytic strain of Escherichia coli.

Animals↗
PubMed1992

Age-targeted chemotherapy for control of urinary schistosomiasis in endemic populations.

Severity of urinary tract morbidity increases with intensity and duration of Schistosoma haematobium infection. We assessed the ability of yearly drug therapy to control infection intensity and reduce S. haematobium-associated disease in children 5-21 years old in an endemic area of Kenya. In year 1, therapy resulted in reduced prevalence (66% to 22%, P < 0.001) and intensity of S. haematobium infection (20 to 2 eggs/10 mL urine), with corresponding reductions in the prevalence of hematuria (52% to 19%, P < 0.001). There was not, however, a significant first-year effect on prevalence of urinary tract abnormalities detected by ultrasound. Repeat therapy in years 2 and 3 resulted in significant regression of hydronephrosis and bladder abnormalities (41% to 6% prevalence, P < 0.01), and further reductions in proteinuria. Repeat age-targeted therapy was associated with decreased prevalence of infection among young children (< 5 yr) entering into the targeted age group. Two years after discontinuation of therapy, intensity of S. haematobium infection and ultrasound abnormalities remained suppressed, but hematuria prevalence began to increase (to 33% in 1989). Reinstitution of annual therapy in 1989 and 1990 reversed this trend. We conclude that annual oral therapy provides an effective strategy for control of morbidity due to S. haematobium on a population basis, both through regression of disease in treated individuals, and prevention of infection in untreated subjects.

Adolescent↗
PubMed1991

Protease priming of neutrophil superoxide production. Effects on membrane lipid order and lateral mobility.

Phagocyte superoxide (O2-) response is primed by a variety of physiologic compounds including the neutrophil secretory proteases cathepsin G and elastase. To study whether protease priming of neutrophil O2- response is related to changes in membrane physical state, we examined enzyme effects on the order and lateral mobility of lipid probes in intact neutrophil membranes. Exposure to cathepsin G (5 micrograms/ml) or elastase (10 micrograms/ml) caused a significant decrease in fluorescence anisotropy of the probe trimethylammonium diphenylhexatriene in neutrophil plasma membranes (0.279 to 0.256 for cathepsin G, 0.274 to 0.256 for elastase, p less than 0.02 for both), indicating a decrease in phospholipid chain order in the surface membrane bilayer. Cathepsin G and elastase also caused significant increases in membrane lipid lateral mobility as measured by excimer formation of the fluorescent probe 1-pyrenedecanoic acid (for cathepsin G, a 107% increase, and for elastase, a 44% increase in excimer/monomer fluorescence ratio, p less than 0.001). Enzyme effects on membrane structure were dependent on intact proteolytic activity, and were cell specific; the proteases had no effect on lipid order or lateral mobility in liposomes. In corollary studies, the possible association between the physical state of the polymorphonuclear leukocyte membrane and O2- generation was analyzed with the membrane modifying compounds, linoleic acid, ethanol, and cholesterol. Cell exposure to linoleic acid (1 microM) caused a significant decrease in lipid order and an increase in lipid lateral mobility along with increased O2- production to N-formyl-Met-Leu-Phe (fMLP) (191%) and phorbol myristate acetate (PMA) (39%), p less than 0.02 for each. 3 mM ethanol also augmented O2- response to fMLP (31%) and PMA (48%) and caused a significant decrease in lipid order, but did not affect lipid lateral mobility. Treatment with cholesteryl hemisuccinate (100 micrograms/ml) resulted in increased lipid order and decreased lipid lateral mobility, as well as decreased neutrophil superoxide response to fMLP (-61%, p less than 0.001) and PMA (-50%, p less than 0.02). We then examined whether modulation of membrane physical state may explain the mechanism of action of a known priming agent by studying the effects of low concentrations of a diacylglycerol. Cells treated with 10 microM 1-oleoyl-2-acetyl-sn-glycerol had a greater than 8-fold increase in superoxide response to fMLP (p less than 0.001) while demonstrating a significant decrease in lipid order (0.289 to 0.281, p less than 0.01) and a 50% increase in lipid lateral mobility (p less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1991

Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase.

Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.

Antibodies, Monoclonal↗
PubMed1991

Cytokine- and LPS-induced synthesis of interleukin-8 from human mesangial cells.

The cytokine neutrophil-activating peptide-1/interleukin-8 (NAP/IL-8) activates neutrophils (PMN) and elicits selective diapedesis of PMN into the extracellular space. The glomerular mesangial cell (MC) is a specialized pericyte that controls glomerular filtration and synthesizes and responds to a variety of cytokines. Because of its location within the glomerulus, the MC is in a pivotal position to orchestrate events underlying immune injury. Since immune-injured glomeruli have been shown to produce NAP/IL-8 activity in vitro, we assessed whether lipopolysaccharide (LPS)- or cytokine-activated MC might be a source of this activity. Pure human MC, devoid of monocyte/macrophage and fibroblast contamination, were grown by explant from collagenase-treated glomeruli. Human recombinant interleukin-1 alpha (IL-1 alpha, 20 ng/ml), IL-1 beta (50 ng/ml), tumor necrosis factor alpha (TNF, 100 ng/ml) and lipopolysaccharide (LPS, 10 micrograms/ml) stimulated release of a neutrophil chemotactic factor from cultured MC. Both concentrated (fivefold) and unconcentrated MC supernatants stimulated directed neutrophil migration under agarose at a level similar to that of the bacterial chemotactic factor, FMLP. In contrast, unstimulated MC-conditioned media and IL-1 alpha, IL-1 beta. TNF and LPS in medium alone did not directly induce PMN migration. Molecular sizing studies using sequential membrane ultrafiltration identified significant TNF-stimulated, MC-derived chemotactic activity in the 3000 to 10000 kD fraction. An anti-NAP/IL-8 monoclonal antibody, 46E5, significantly inhibited PMN chemotaxis stimulated by TNF-stimulated, MC-conditioned media in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1991

Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells.

A cloned and axenically cultured strain of Hartmannella vermiformis was used as a model to study intracellular multiplication of Legionella pneumophila in amoebae. The growth of L. pneumophilia in both H. vermiformis and a human monocyte-like cell line (U937) was investigated with cytoskeletal and metabolic inhibitors. L. pneumophila replicated only intracellularly in these cellular models, and electron microscopy showed ultrastructural similarities in the initial phase of multiplication. Treatment of amoebae with an inhibitor of microfilament-dependent phagocytosis (cytochalasin D, 0.5 or 1.0 micrograms/ml) did not inhibit intracellular growth of L. pneumophila; however, intracellular multiplication was inhibited by treatment of U937 monocytes with the same concentrations of cytochalasin D. Methylamine (10 to 100 mM), an inhibitor of adsorptive pinocytosis, inhibited the replication of L. pneumophila in amoebae in a dose-dependent manner. All doses of methylamine tested (10 to 50 mM) inhibited growth of L. pneumophila in U937 monocytes. Cytochalasin D and methylamine had no effect on the multiplication of L. pneumophila in culture medium or on the viability of amoebae or U937 monocytes. Intracellular replication of L. pneumophila in H. vermiformis may be accomplished by a cytochalasin D-independent mechanism, such as adsorptive pinocytosis. In contrast, both cytochalasin D- and methylamine-sensitive mechanisms may be essential for the intracellular multiplication of L. pneumophila in U937 monocytes.

Analysis of Variance↗