Biomedical subjects

K R Alugupalli

Publications and source records attributed to K R Alugupalli.

10 recordsLinked to original sources

PubMed2001

Platelet activation by a relapsing fever spirochaete results in enhanced bacterium-platelet interaction via integrin alphaIIbbeta3 activation.

Borrelia hermsii, a spirochaete responsible for relapsing fever in humans, grows to high density in the bloodstream and causes thrombocytopenia. We show here that B. hermsii binds to human platelets. Extended culture in bacteriological medium resulted in both diminished infectivity in vivo and diminished platelet binding in vitro. Platelet binding was promoted by the platelet integrin alphaIIbbeta3: the bacterium bound to purified integrin alphaIIbbeta3, and bacterial binding to platelets was diminished by alphaIIbbeta3 antagonists or by a genetic defect in this integrin. Integrin alphaIIbbeta3 undergoes a conformational change upon platelet activation, and bacteria bound more efficiently to activated rather than resting platelets. Nevertheless, B. hermsii bound at detectable levels to preparations of resting platelets. The bacterium did not recognize a point mutant of alphaIIbbeta3 that cannot acquire an active conformation. Rather, B. hermsii was capable of triggering platelet and integrin alphaIIbbeta3 activation, as indicated by the expression of the platelet activation marker P-selectin and integrin alphaIIbbeta3 in its active conformation. The degree of platelet activation varied depending upon bacterial strain and growth conditions. Prostacyclin I2, an inhibitor of platelet activation, diminished bacterial attachment, indicating that activation enhanced bacterial binding. Thus, B. hermsii signals the host cell to activate a critical receptor for the bacterium, thereby promoting high-level bacterial attachment.

Animals↗

PubMed2001

Serial determinations of platelet counts in mice by flow cytometry.

Elucidation of the pathophysiological basis of platelet disorders in murine models requires a reliable method for the frequent determinations of platelet counts in individual mice. Here, we present a rapid, reproducible and accurate flow cytometric method for enumeration of platelets that involves fluorescent staining of platelets in whole blood with specific antibody and the addition of known numbers of fluorescent beads for standardization of the sample volume. Analysis of platelets obtained by tail bleeding indicated that this sampling procedure did not activate platelets, and that only five microliters of blood were required for platelet counting. Using this method, we followed platelet counts in mice infected with the relapsing fever spirochete Borrelia turicatae for 26 days, and found that this bacterium induces thrombocytopenia, a common manifestation of human relapsing fever. Therefore, this method can be used to follow the number and the activation state of circulating platelets from individual mice over extended periods of time and is applicable to a wide range of murine models of platelet disorders.

Animals↗

PubMed2000

Variable small protein (Vsp)-dependent and Vsp-independent pathways for glycosaminoglycan recognition by relapsing fever spirochaetes.

Tick-borne relapsing fever, caused by pathogenic Borrelia such as B. hermsii and B. turicatae, features recurrent episodes of bacteraemia, each of which is caused by a population of spirochaetes that expresses a different variable major protein. Relapsing fever is also associated with the infection of a variety of tissues, such as the central nervous system. In this study, we show that glycosaminoglycans (GAGs) mediate the attachment of relapsing fever spirochaetes to mammalian cells. B. hermsii strain DAH bound to immobilized heparin, and heparin and dermatan sulphate blocked bacterial binding to host cells. Bacterial binding was diminished by inhibition of host cell GAG synthesis or sulphation, or by the enzymatic removal of GAGs. GAGs mediated the attachment of relapsing fever spirochaetes to potentially relevant target cells, such as endothelial and glial cells. B. hermsii was able to attach to GAGs independently of variable major proteins, because strains expressing the variable major proteins Vsp33, Vlp7 or no variable major protein at all each recognized GAGs. Nevertheless, we found that a variable major protein of B. turicatae directly promoted GAG binding by this relapsing fever spirochaete. B. turicatae strain Oz1 serotype B, which expresses the variable major protein VspB, bound to GAGs more efficiently than did B. turicatae Oz1 serotype A, which expresses VspA. Recombinant VspB, but not VspA, bound to heparin and dermatan sulphate. Previous studies have shown that strain Oz1 serotype B grows to higher concentrations in the blood than does Oz1 serotype A. Thus, relapsing fever spirochaetes have the potential to express Vsp-dependent and Vsp-independent GAG-binding activities and, for one pair of highly related B. turicatae strains, differences in GAG binding correlate with differences in tissue tropism.

Animals↗

PubMed1997

Antibody response to Arcanobacterium haemolyticum infection in humans.

Arcanobacterium haemolyticum causes pharyngitis, exanthema, and other infections. The evidence of the pathogenicity of A. haemolyticum depends on clinical descriptions of culture-positive patients and a comparison of carrier rates of patients with pharyngitis and healthy, matched controls. In this investigation, the antibody response of the host was studied for the first time, using SDS-PAGE and Western blot analyses. Paired acute and convalescent sera showed development of antibodies to A. haemolyticum in 7 of 8 patients. The antibodies reacted primarily with four distinct cell wall-associated proteins with estimated molecular masses of 80, 60, 50, and 30 kDa. Moreover, the reactivity of convalescent sera from 19 patients was compared with that of sera from 19 controls. Antibodies to A. haemolyticum were found in sera from 16 patients and 6 controls (P < .005); the antibody response of the patients was strong compared with that of the controls. These results indicate that A. haemolyticum infection induces an antibody response in the host.

Acute Disease↗

PubMed1997

Characterization of the lactoferrin-dependent inhibition of the adhesion of Actinobacillus actinomycetemcomitans, Prevotella intermedia and Prevotella nigrescens to fibroblasts and to a reconstituted basement membrane.

Lactoferrin was previously shown to inhibit the adhesion of A. actinomycetemcomitans, P. intermedia and P. nigrescens to human cells. Lactoferrin was also shown to competitively inhibit the binding of these bacteria to the basement membrane protein laminin. The present study aimed to determine the type of interactions inhibited by lactoferrin. Lactoferrin binds to fibroblast monolayers and Matrigel, a reconstituted basement membrane, through ionic interactions. The adhesion of A. actinomycetemcomitans to these substrata was mainly dependent on the ionic strength of the environment. P. intermedia and P. nigrescens also adhere to fibroblasts mainly by ionic interactions, while their adhesion to Matrigel seems to be mediated by specific mechanisms. Lectin-type interactions were not found to be involved in the binding of these bacteria to the substrata. Treatment of either A. actinomycetemcomitans or fibroblasts with lactoferrin decreased the adhesion in a dose-dependent manner, while lactoferrin treatment of Matrigel alone had no adhesion-counteracting effect. Adhesion of P. intermedia and P. nigrescens to Matrigel was not significantly affected by the ionic strength, but the presence of lactoferrin inhibited the adhesion. Lactoferrin bound to Matrigel, P. intermedia and P. nigrescens was rapidly released, while lactoferrin bound to A. actinomycetemcomitans and fibroblasts was retained. These findings indicate that lactoferrin-dependent inhibition of the adhesion of A. actinomycetemcomitans, P. intermedia and P. nigrescens to fibroblasts and Matrigel can involve binding of lactoferrin to both the bacteria and substrata. The decreased adhesion may be due to blocking of both specific adhesin-ligand as well as non-specific charge-dependent interactions.

Aggregatibacter actinomycetemcomitans↗

PubMed1996

Degradation of lactoferrin by periodontitis-associated bacteria.

The degradation of human lactoferrin by putative periodontopathogenic bacteria was examined. Fragments of lactoferrin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured by densitometry. The degradation of lactoferrin was more extensive by Porphyromonas gingivalis and Capnocytophaga sputigena, slow by Capnocytophaga ochracea, Actinobacillus actinomycetemcomitans and Prevotella intermedia, and very slow or absent by Prevotella nigrescens, Campylobacter rectus, Campylobacter sputorum, Fusobacterium nucleatum ssp. nucleatum, Capnocytophaga gingivalis, Bacteroides forsythus and Peptostreptococcus micros. All strains of P. gingivalis tested degraded lactoferrin. The degradation was sensitive to protease inhibitors, cystatin C and albumin. The degradation by C. sputigena was not affected by the protease inhibitors and the detected lactoferrin fragments exhibited electrophoretic mobilities similar to those ascribed to deglycosylated forms of lactoferrin. Furthermore a weak or absent reactivity of these fragments with sialic acid-specific lectin suggested that they are desialylated. The present data indicate that certain bacteria colonizing the periodontal pocket can degrade lactoferrin. The presence of other human proteins as specific inhibitors and/or as substrate competitors may counteract this degradation process.

Aggregatibacter actinomycetemcomitans↗

PubMed1996

Laminin binding to a heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans.

The interaction of Actinoabacillus actinomycetemcomitans with the basement membrane protein, laminin, was examined in a 125I-labeled protein-binding assay. The binding of laminin increased by lowering the pH. The ability to bind laminin was decreased in cells at the stationary phase of growth and by the presence of blood in the culture medium. Laminin binding to this bacterium was saturable, and the affinity constant was 4.6 nM. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot (ligand blot) analysis of cell-envelope and outer membrane of A. actinomycetemcomitans displayed a 125I-laminin-reactive protein band with a molecular weight of 29 k. The laminin-binding protein was the previously described outer membrane protein A of A. actinomycetemcomitans. It was identified by its heat-modifiable property, detergent-solubility profile and reactivity with outer membrane protein A-specific polyclonal antiserum. At acidic pH, 25I laminin bound to several cell-envelope components of A. actinomycetemcomitans, but at neutral pH, laminin bound only to the heat-modifiable protein. Despite the existence of the laminin-binding protein, cells grown in blood-containing media did not bind laminin. Several mammalian proteins interfered with laminin-bacterial interaction, including lactoferrin, which binds to the same bacterial protein that inhibited and displaced the laminin-bacterial interaction.

Aggregatibacter actinomycetemcomitans↗

PubMed1995

Lactoferrin interaction with Actinobacillus actinomycetemcomitans.

The interaction of lactoferrin with Actinobacillus actinomycetemcomitans was examined in a 125I-labeled protein binding assay. The binding of human and bovine lactoferrins reached maximum within 1 h. Lactoferrin binding to the bacterium was pH-dependent and reversible. Scatchard analysis indicated the existence of two different types of binding sites on the bacterium, one with a high affinity constant k alpha approximately 8.8 x 10(-7) M) and the other with a low one (k alpha approximately 1.8 x 10(-6) M). Bacteria in the exponential phase of growth showed higher binding than cells in the stationary phase. Bacteria grown in medium containing serum and/or lysed erythrocytes bound lactoferrin to a lesser extent. Heat-inactivated serum, lysed erythrocytes and other proteins such as mucin and laminin inhibited lactoferrin binding to A. actinomycetemcomitans in a competitive binding assay. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis and Western blot analysis of the cell envelope as well as the outer membrane of A. actinomycetemcomitans revealed lactoferrin-reactive protein bands at 29 kDa and 16.5 kDa. The 29-kDa band displayed a heat-modifiable lactoferrin-reactive form with a molecular weight of 34 kDa. Neither proteinase K-treated cell envelope nor lipopolysaccharide of this bacterium showed reactivity with lactoferrin. These data suggests a specific interaction of lactoferrin with outer membrane proteins of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗

PubMed1995

Inhibitory effect of lactoferrin on the adhesion of Actinobacillus actinomycetemcomitans and Prevotella intermedia to fibroblasts and epithelial cells.

Adhesion of the periodontitis-associated bacteria Actinobacillus actinomycetemcomitans and Prevotella intermedia to monolayers of fibroblasts, HEp-2, KB and HeLa cells was quantified with radiolabeled bacteria. Bacterial adhesion was also examined microscopically with Giemsa-stained non-radioactive preparations. The degree of bacterial adherence was dependent on the growth phase of the bacteria. Strains at the exponential phase adhered to a greater extent than those at the stationary phase of growth. Both human and bovine lactoferrins competitively inhibited the adhesion of A. actinomycetemcomitans and P. intermedia to all tested cell monolayers. The inhibitory effect was dose-dependent in the concentration range 0.5-2500 micrograms/ml and not related to the bacterial growth phase. In the presence of lactoferrin, decreased association of bacteria with the cell monolayers was also found by microscopic examination of the preparations. The present findings indicate that lactoferrin may prevent the establishment of bacteria in periodontal tissues through adhesion-counteracting mechanisms in addition to its bacteriostatic and bactericidal properties.

Aggregatibacter actinomycetemcomitans↗

PubMed1994

Effect of lactoferrin on interaction of Prevotella intermedia with plasma and subepithelial matrix proteins.

A lactoferrin-binding protein with an estimated molecular mass of 57 kDa was identified in the cell envelope of Prevotella intermedia by gel electrophoresis and Western-blot analysis. Peroxidase-labeled bovine lactoferrin and human lactoferrin showed similar specific binding to this protein. Whole cells of P. intermedia were also examined for interactions with 5 125I-labeled plasma and subepithelial matrix proteins. A high degree of binding was found with fibronectin, collagen type I and type IV and laminin, whereas a moderate interaction was detected with fibrinogen. The ability of bovine lactoferrin to affect the interactions of the above proteins with P. intermedia was examined. In the presence of unlabeled bovine lactoferrin, a dose-dependent inhibition of binding was observed with all 5 proteins tested. Unlabeled bovine lactoferrin also dissociated the bacterial complexes with these proteins. The complexes with laminin or collagen type I were more effectively dissociated than fibronectin or fibrinogen, whereas the interaction with collagen type IV was affected to a lesser extent. A strain-dependent variation in the effect of bovine lactoferrin was observed. These data establish the presence of a specific lactoferrin-binding protein in the cell envelope of P. intermedia. The ability of lactoferrin to inhibit the binding of some plasma and subepithelial matrix proteins to P. intermedia could be a protective mechanism against the establishment of this pathogen in the periodontal pocket.

Animals↗