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M S Klempner

Publications and source records attributed to M S Klempner.

At least 37 records · Page 2Linked to original sources
PubMed1997

Isolation, cloning, and expression of a 70-kilodalton plasminogen binding protein of Borrelia burgdorferi.

Surface receptors for plasminogen are expressed by many gram-positive and gram-negative bacteria and may play a role in the dissemination of organisms by binding plasminogen, which upon conversion to plasmin can digest extracellular matrix proteins. Two plasminogen binding proteins have been identified for Borrelia burgdorferi, outer surface protein A and a 70-kDa protein (BPBP). We purified BPBP by plasminogen affinity chromatography and obtained its amino acid sequence by Edman degradation of a tryptic digest. The gene coding for BPBP was isolated from a lambda-ZAP II genomic library with probes developed from sequenced portions of the protein. This gene was expressed in Escherichia coli; the recombinant product was seen by antibody raised against native BPBP and also bound 125I-labeled plasminogen. The experimentally derived amino acid sequences corresponded to the predicted sequence encoded by the BPBP gene. The deduced amino acid sequence for BPBP revealed significant similarity to p30, a 30-kDa protein of B. burgdorferi (54% identity and 65% similarity), to a 60-kDa protein in Borrelia coriaceae (66% identity and 80% similarity), to oligopeptide binding protein A of E. coli (34% identity and 57% similarity), and, more generally, to the periplasmic oligopeptide binding family of proteins.

Amino Acid Sequence↗
PubMed1996

Inhibition of Borrelia burgdorferi-bound fibrinolytic enzymes by alpha2-antiplasmin, PAI-1 and PAI-2.

Lyme disease is caused by Borrelia burgdorferi. Human plasminogen and urokinase-type plasminogen activator bind to the surface of the spirochete where plasmin is generated. We have suggested that bound urokinase and plasminogen are utilized by the organism to disseminate. We tested whether the physiological inhibitors of urokinase, plasminogen activator inhibitor-1 and -2 (PAI-1, PAI-2), could regulate the activity of spirochete-bound urokinase. The k(ass) of PAI-1 and PAI-2 for bound urokinase were 1.3 x 10(6) M(-1)s(-1) and 6.9 x 10(4)M(-1)s(-1), respectively, whereas the k(ass) for free urokinase were 7.2 x 10(6) M(-1)s(-1) and 5.3 x 10(5) M(-1)s(-1), respectively. Plasmin associated with the spirochete was not inhibited by alpha2-antiplasmin. These results suggest that PAI-1, PAI-2 and alpha2 antiplasmin would not be efficient regulators of fibrinolytic protease activity on the Borrelial surface and would not pose a barrier to utilization of these enzymes for dissemination in the human host.

Binding Sites↗
PubMed1996

Babesiosis in patients with AIDS: a chronic infection presenting as fever of unknown origin.

Babesiosis is a malaria-like, tick-transmitted zoonosis caused by protozoa of the family Piroplasmorida, which includes Babesia and Theileria species. In the United States, the infection is endemic in the Northeast and upper Midwest, although cases have recently been described in Northern California and Washington State. We report a case of babesiosis in a patient infected with HIV who presented with a prolonged fever of unknown origin; the patient had not undergone splenectomy. Parasitemia persisted despite initial clinical improvement after treatment with quinine and clindamycin. Babesiosis was controlled with a maintenance regimen consisting of clindamycin, doxycycline, and high-dose azithromycin, but the infection was not eradicated. Babesiosis should be considered in the differential diagnosis of HIV-infected patients with fevers and/or anemia in areas where the infection is endemic. HIV-infected patients who are severely immunosuppressed, even those without a history of splenectomy, may present with severe manifestations of babesiosis and develop a chronic infection, which may require therapy to prevent relapse of disease.

AIDS-Related Opportunistic Infections↗
PubMed1996

Binding of human urokinase type plasminogen activator and plasminogen to Borrelia species.

Borrelia burgdorferi binds human urokinase plasminogen activator (uPA), which cleaves plasminogen (Pgn) to plasmin. The ability of other Borrelia species to bind uPA, Pgn, or both was investigated. Borrelia coriacae, Borrelia garinii, Borrelia parkeri, Borrelia anserina, and Borrelia turicatae were compared with infectious and noninfectious B. burgdorferi isolates. All Borrelia species lacked endogenous proteases capable of digesting casein, but all species bound human uPA and Pgn, generating Pgn-dependent proteolytic activity. There were no significant differences in the amount of plasmin, Pgn, or uPA bound per spirochete of the different species. On unfixed borreliae, fluorochrome-conjugated uPA bound to all species. Early binding was at the terminus of B. burgdorferi, whereas diffuse binding was observed on B. coriacae, B. garinii, B. parkeri, and B. turicatae. These studies demonstrate that binding of human uPA and Pgn to borreliae occurs on multiple species with apparent differences in surface distribution.

Borrelia↗
PubMed1996

Phase I trial of interleukin 2 in combination with the soluble tumor necrosis factor receptor p75 IgG chimera.

Our purpose was to determine the effective biological dose and/or maximum tolerated dose of recombinant human tumor necrosis factor receptor:IgG chimera (rhuTNFR:Fc; Immunex, Seattle, WA) in combination with interleukin 2 (IL-2) with regard to reduction in IL-2 toxicity and modulation of biological effects of high-dose IL-2 administration. Twenty-four patients with metastatic cancer were treated with escalating doses of rhuTNFR:Fc at 1, 1, 5, 10, and 20 mg/m2 i.v. on days 1 and 15 (dose levels 1-5) or 10, 20, and 30 mg/m2 days 1 and 15 plus 50% dose on days 3, 5, 17, and 19 (dose levels 6-8) prior to IL-2 at doses of 300,000 IU/kg (dose level 1) and 600,000 IU/kg (dose levels 2-8) i.v. every 8 h on days 1-5 and 15-19. The t1/2 of rhuTNFR in patients receiving IL-2 was 72 h. The median number of IL-2 doses was 24, and central nervous system, skin, and cardiac arrhythmias were the major dose-limiting toxicities. TNF bioactivity was inhibited, and the polymorphonuclear leukocyte chemotactic defect normally seen with IL-2 was not observed. Increases in C-reactive protein, IL-6, IL-8, and IL-1 receptor antagonist levels were partially suppressed relative to historical controls, whereas peripheral blood mononuclear cell phenotypes, urinary nitrate, endothelial adhesion molecule expression in skin biopsies, and cellular infiltrates in tumor biopsies were consistent with findings in patients treated with IL-2 alone. Four patients developed thyroid dysfunction. There were five responses: two complete responses (both melanoma) and three partial responses (response rate, 21%). rhuTNFR:Fc may modulate the toxicity and some of the biological effects of IL-2 while preserving antitumor activity. Dose level 6 (10 mg/m2 on days 1 and 15, and 5 mg/m2 on days 3, 5, 17, and 19) has been chosen for a randomized, double-blind, placebo-controlled trial of IL-2 with and without rhuTNFR:Fc.

Adult↗
PubMed1995

Binding of human plasminogen and urokinase-type plasminogen activator to the Lyme disease spirochete, Borrelia burgdorferi.

Many bacteria that spread in the skin produce enzymes that digest extracellular matrix components. Borrelia burgdorferi spreads from a skin inoculation site to form the characteristic erythema migrans skin lesion. It was determined that B. burgdorferi does not produce collagenase, elastase, hyaluronidase, or other enzymes that digest extracellular matrix components. However, B. burgdorferi bound human plasmin, plasminogen (Pgn), and urokinase-type plasminogen activator (uPA). When spirochetes were sequentially incubated with Pgn and uPA, bioactive plasmin was generated on the surface of B. burgdorferi. B. burgdorferi did not produce an endogenous Pgn activator. Fluorochrome-conjugated uPA and Pgn colocalized to the terminus of the spirochete. In a mouse model, uPA-treated B. burgdorferi were more infectious than control spirochetes. Binding of host uPA and Pgn to form a bioactive extracellular matrix protease on B. burgdorferi represents a mechanism that could facilitate dissemination and localization of spirochetes to sites of vascular injury.

Animals↗
PubMed1995

Binding of human plasminogen to Borrelia burgdorferi.

We studied the binding of plasminogen to Borrelia burgdorferi, a spirochete which causes Lyme disease and produces no endogenous proteases which digest extracellular matrix proteins. Using 125I-labeled plasminogen, we demonstrated that B. burgdorferi bound human plasminogen and that this binding was inhibitable with unlabeled plasminogen. 125I-labeled plasminogen binding by B. burgdorferi was also inhibited by the lysine analog epsilon-aminocaproic acid. There was no significant difference in the binding of Glu- or Lys-plasminogen to B. burgdorferi. Binding of plasminogen was similar in low-passage (infectious) and high-passage (noninfectious) isolates of B. burgdorferi. Plasminogen bound to the surface of B. burgdorferi could be converted into plasmin by a human urokinase-type plasminogen activator. 125I-labeled plasminogen ligand blots of borrelial membrane proteins demonstrated two prominent binding proteins at approximately 70 and approximately 30 kDa. By Western blot (immunoblot), the 30-kDa protein was found to be outer surface protein A (Osp A) of B. burgdorferi. 125I-labeled plasminogen binding to both the 70-kDa protein and Osp A was inhibited by approximately 90% with a 1,000-fold excess of unlabeled plasminogen. By scanning densitometry, the 70-kDa band bound > 10 time more 125I-labeled plasminogen than did Osp A. An Osp A-deficient mutant of B. burgdorferi and wild-type B. burgdorferi bound equal amounts of 125I-labeled plasminogen. Ligand blots of membrane proteins from an Osp A-deficient mutant showed association of 125I-labeled plasminogen at only the 70-kDa protein. Two-dimensional gel electrophoresis showed that the 70-kDa protein had a pI of approximately 5.3, clearly separable from Osp A. The association of host plasmin(ogen) with borrelial surface proteins provides a mechanism by which B. burgdorferi can digest extracellular matrix and disseminate.

Bacterial Proteins↗
PubMed1993

Invasion of human skin fibroblasts by the Lyme disease spirochete, Borrelia burgdorferi.

The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell surface by treatment with ceftriaxone (1 microgram/mL) for 5 days. Despite the absence of visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear region within human fibroblasts by laser scanning confocal microscopy. Intracellular spirochetes specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent laser scanning confocal microscopy. These observations suggest that B. burgdorferi can adhere to, penetrate, and invade human fibroblasts in organisms that remain viable.

Bacterial Adhesion↗
PubMed1992

Fibroblasts protect the Lyme disease spirochete, Borrelia burgdorferi, from ceftriaxone in vitro.

The Lyme disease spirochete, Borrelia burgdorferi, can be recovered long after initial infection, even from antibiotic-treated patients, indicating that it resists eradication by host defense mechanisms and antibiotics. Since B. burgdorferi first infects skin, the possible protective effect of skin fibroblasts from an antibiotic commonly used to treat Lyme disease, ceftriaxone, was examined. Human foreskin fibroblasts protected B. burgdorferi from the lethal action of a 2-day exposure to ceftriaxone at 1 microgram/mL, 10-20 x MBC. In the absence of fibroblasts, organisms did not survive. Spirochetes were not protected from ceftriaxone by glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity. Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone. Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective effect. Thus, several eukaryotic cell types provide the Lyme disease spirochete with a protective environment contributing to its long-term survival.

Adenocarcinoma↗
PubMed1991

Neutrophil chemotactic factors in synovial fluids of patients with Lyme disease.

We examined synovial fluid samples from 14 patients with Lyme arthritis for the presence of neutrophil chemotactic factors. Thirteen of the synovial fluids stimulated chemotaxis of normal human neutrophils. The chemotactic activity was heat-sensitive and was not inhibited by antibody to C5a or antibody to interleukin-8, or by a competitive inhibitor of the chemotactic peptide f-Met-Leu-Phe. A culture supernatant of Borrelia burgdorferi also contained neutrophil chemoattractants. Chromatography demonstrated that the chemoattractants in the synovial fluid samples were different from those in the B burgdorferi culture supernatant. One of the major chemotactic factors in Lyme disease synovial fluid had a calculated molecular weight of 13,900. We conclude that a novel, host-derived chemoattractant is present in the synovial fluid of patients with Lyme disease.

Chemotactic Factors↗
PubMed1991

Infectivity of Borrelia burgdorferi correlates with resistance to elimination by phagocytic cells.

The Lyme disease spirochete, Borrelia burgdorferi, causes a disseminated infection in vivo, implying resistance to clearance by phagocytic cells. Because B. burgdorferi loses its infectivity after in vitro cultivation, the relationships between serial passaging of the organism in vitro, its susceptibility to elimination by phagocytes, and its infectivity were examined. When three different high-passage strains were incubated for 4 h at 37 degrees C with peripheral blood mononuclear cells, macrophages, or polymorphonuclear neutrophils, 45%-67% of the organisms were eliminated. In contrast, two low-passage strains were resistant to elimination by phagocytes, and only 5%-6% of the organisms were removed after 4 h. All five strains equally stimulated the neutrophil oxidative burst, indicating that evasion of phagocytes was not a result of avoidance of recognition by these cells. The two low-passage strains were infective when injected into mice, whereas the three high-passage ones were not. These observations indicate that infectivity of the Lyme disease spirochete correlates with resistance to elimination by phagocytic cells.

Animals↗
PubMed1990

Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone: prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects.

High concentrations of tumor necrosis factor (TNF) alpha have been detected in the plasma of patients undergoing immunotherapy with interleukin 2 (IL-2), suggesting that this cytokine may play a role in the fever and shocklike state induced by the administration of high-dose IL-2. Dexamethasone has been shown to inhibit the synthesis of TNF by monocytes activated in vitro by endotoxin. To determine if dexamethasone can exert a similar suppressive effect on IL-2-induced TNF synthesis in vivo, the concentration of TNF alpha was measured in plasma samples serially obtained (a) from cancer patients participating in a phase I dose escalation clinical trial with high-dose IL-2 administered in conjunction with dexamethasone (IL-2/Dex) and (b) from patients participating in concurrent studies with IL-2 alone. In contrast to the high plasma levels of TNF alpha detected in patients receiving IL-2 alone, TNF levels in most of the IL-2/Dex patients remained below the threshold of detectability of our TNF radioimmunoassay. The concurrent administration of dexamethasone also prevented the IL-2-induced increase in serum levels of C-reactive protein, a hepatic acute phase reactant whose synthesis is regulated by proinflammatory cytokines such as TNF. The steroid-treated patients also failed to develop the neutrophil chemotactic defect characteristic of IL-2 recipients. The concomitant administration of dexamethasone increased the maximum tolerated dose of IL-2 approximately threefold and markedly reduced the hypotension and organ dysfunction ordinarily observed in these patients. These results demonstrate that dexamethasone inhibits the release of TNF into the circulation of patients undergoing immunotherapy with IL-2. They further suggest that the altered spectrum and reduced severity of IL-2 side effects observed in patients receiving dexamethasone may be attributable in part to the suppressive effect of steroids on IL-2-induced TNF synthesis.

C-Reactive Protein↗
PubMed1990

An acquired chemotactic defect in neutrophils from patients receiving interleukin-2 immunotherapy.

Bacterial sepsis is a frequent complication in patients with cancer who are receiving high doses of interleukin-2. We evaluated the function of neutrophils from such patients to determine whether there was any abnormality in this form of host defense. Before interleukin-2 therapy, neutrophils from 31 patients with metastatic cancer were normal in assays of random migration and chemotaxis. Superoxide production, phagocytosis, secretion of granule proteins, and bactericidal activity were also normal. Neutrophils from the patients near the end of the first course of interleukin-2 had severely impaired chemotaxis in response to a formylated peptide stimulus (mean [+/- SEM], 49.6 +/- 7.4 percent of base line; P less than 0.001). The detect in chemotaxis improved 5 to 10 days after patients completed the first course of interleukin-2 therapy but recurred toward the end of the second course of such therapy (35.3 +/- 6.9 percent of base line; P less than 0.001). The chemotactic response to a second stimulus (zymosan-activated serum) was also abnormal, but random migration, superoxide production, bactericidal activity, and the secretion of neutrophil granule constituents remained normal or increased throughout treatment with interleukin-2. We conclude that patients who receive interleukin-2 immunotherapy acquire an acute, profound, and reversible defect in neutrophil chemotaxis that may contribute to the high morbidity resulting from bacterial infections in these patients.

Blood Bactericidal Activity↗
PubMed1990

Yersinia pestis pH 6 antigen: genetic, biochemical, and virulence characterization of a protein involved in the pathogenesis of bubonic plague.

We studied a protein antigen, designated pH 6 Ag, that has the same regulation of expression as the previously described Yersinia pestis pH 6 Ag. Monospecific antiserum to this antigen recognized several proteins, ranging from 15 to over 75 kilodaltons (kDa), which were strongly expressed when Y. pestis was cultivated at 37 degrees C and pH 6 but were expressed weakly, if at all, at 37 degrees C and pH 8 and at 26 degrees C. The antigen appeared to be composed of aggregates of a 15-kDa subunit. Escherichia coli minicell analysis and Western blotting (immunoblotting) of minicell extracts containing the cloned pH 6 Ag locus revealed that a 1.7-kilobase-pair (kb) region of Y. pestis chromosomal DNA produced 16- and 15-kDa immunoreactive proteins. We used transposon mutagenesis of the pH 6 Ag-coding region to demonstrate that the 16- and 15-kDa polypeptides were produced by the same cistron. The pH 6 Ag structural gene, psaA, was located within a 0.5-kb region of DNA. A Tn10lacZ transposon insertion 1.2 kb upstream of the psaA locus but outside the psaA transcriptional unit caused decreased expression of pH 6 Ag in both E. coli and Y. pestis and defined the psaE locus necessary for maximum pH 6 Ag expression. This locus itself was not regulated by temperature or pH. However, psaA remained responsive to both of these environmental signals in a Y. pestis psaE mutant. Mutation of either psaE or psaA resulted in at least a 100-fold reduction in the intravenous 50% lethal dose of Y. pestis in mice. Accordingly, pH 6 Ag is involved in the pathogenesis of bubonic plague.

Animals↗