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Biomedical subjects

S E Malawista

Publications and source records attributed to S E Malawista.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of Borrelia burgdorferi-tick interactions in vivo by outer surface protein A antibody.

Borrelia burgdorferi outer surface protein (Osp) A is preferentially expressed by spirochetes in the Ixodes scapularis gut and facilitates pathogen-vector adherence in vitro. Here we examined B. burgdorferi-tick interactions in vivo by using Abs directed against OspA from each of the three major B. burgdorferi sensu lato genospecies: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. Abs directed against B. burgdorferi sensu stricto (isolate N40) destroy the spirochete and can protect mice from infection. In contrast, antisera raised against OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) bind to B. burgdorferi N40 but are not borreliacidal against the N40 isolate. Our present studies assess whether these selected OspA Abs interfere with B. burgdorferi-tick attachment in a murine model of Lyme disease with I. scapularis. We examined engorged ticks that had fed on B. burgdorferi N40-infected scid mice previously treated with OspA (N40, ACA-1, ZQ-1, or mAb C3.78) or control Abs. OspA-N40 antisera or mAb C3.78 destroyed B. burgdorferi N40 within the engorged ticks. In contrast, treatment of mice with OspA-ACA-1 and OspA-ZQ-1 antisera did not kill B. burgdorferi N40 within the ticks but did effectively interfere with B. burgdorferi-I. scapularis adherence, thereby preventing efficient colonization of the vector. These studies show that nonborreliacidal OspA Abs can inhibit B. burgdorferi attachment to the tick gut, highlighting the importance of OspA in spirochete-arthropod interactions in vivo.

Animals↗
PubMed2001

Murine Lyme disease: no evidence for active immune down-regulation in resolving or subclinical infection.

Macrophages in vitro rapidly ingest and kill Borrelia burgdorferi, yet some spirochetes in vivo may survive in the host and lead to complications of Lyme disease. One strategy for such survival may be the down-regulation of the immune system. To test this, we evaluated the degree of macrophage activation in a site of active disease-the heart-by examining cytokine expression in murine macrophages from control and B. burgdorferi-infected animals. Using double-label immunofluorescent staining in situ, we showed that infiltrating macrophages in infected hearts produce interleukin (IL)-1. By semiquantitative reverse transcription-polymerase chain reaction analysis, increased levels of mRNA were measured for the proinflammatory cytokines IL-1, tumor necrosis factor-alpha, and IL-12 during peak and resolving disease. No increases in the anti-inflammatory cytokines IL-10 and transforming growth factor-beta were detected. In an infected site without active disease--the peritoneal cavity--no increases in levels of proinflammatory or anti-inflammatory cytokines were detected in local macrophages. Thus, there is no evidence of pressure toward the down-regulation of inflammatory activity in the regions tested.

Animals↗
PubMed2001

Exploitation of interleukin-8-induced neutrophil chemotaxis by the agent of human granulocytic ehrlichiosis.

The agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium with a tropism for neutrophils; however, the mechanisms of bacterial dissemination are not yet understood. Interleukin-8 (IL-8) is a chemokine that induces neutrophil migration to sites of infection for host defense against pathogens. We now show that HGE bacteria, and the HGE-44 protein, induce IL-8 secretion in a promyelocytic (HL-60) cell line that has been differentiated along the neutrophil lineage with retinoic acid and in neutrophils. Infected HL-60 cells also demonstrate upregulation of CXCR2, an IL-8 receptor, but not CXCR1. Human neutrophils migrate towards Ehrlichia sp.-infected cells in a chemotaxis chamber assay, and this movement can be blocked with antibodies to IL-8. Finally, immunocompetent and severe combined immunodeficient mice administered CXCR2 antisera, and CXCR2(-/-) mice that lack the human IL-8 receptor homologue, are much less susceptible to granulocytic ehrlichiosis than are control animals. These results demonstrate that HGE bacteria induce IL-8 production by host cells and, paradoxically, appear to exploit this chemokine to enhance infection.

Animals↗
PubMed2000

Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes from a patient with leukocyte adhesion deficiency-1: normal displacement in close quarters via chimneying.

The beta2 integrins are known to be important in the motile function of leukocytes in general and in the adhesive response to inflammatory stimuli in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the locomotion of human blood PMN from a patient with Leukocyte Adhesion Deficiency-1 (LAD), a disorder in which beta2 integrins on the cell surface are markedly deficient in number or function. In thin slide preparations such that the leukocytes were somewhat compressed between slide and cover slip, PMNLAD exhibited normal random locomotion and chemotaxis, apparently by using the opposing surfaces to generate the force for locomotion (chimneying). In thicker preparations, an adherence deficit was evident, but chemotaxis still occurred, even by PMNLAD anticoagulated in EDTA. Consistent with the paucity of beta2 integrins on the surface of the PMNLAD was their failure to aggregate in the presence of antibodies to beta2 integrins, even when they had been brought together by chemotaxis. We relate these findings to the reported independence from integrins of PMN in the lung vasculature in LAD, as well as in certain experimental conditions.

Antibodies, Monoclonal↗
PubMed2000

Resolution of Lyme arthritis, acute or prolonged: a new look.

I have presented a paradigm for the pathogenesis and resolution of Lyme arthritis founded upon the clinical facts that describe its natural history, and centered about a process of intermittent immune recognition of what may sometimes be an array of antigenic variants. The model applies both to acute and to prolonged (chronic) disease. If it proves to be correct, it represents a variation of the struggle between parasite and host that is probably not limited to Lyme disease.

Acute Disease↗
PubMed2000

Geographic clustering of an outer surface protein A mutant of Borrelia burgdorferi. Possible implications of multiple variants for Lyme disease persistence.

DNA sequences encoding full-length outer surface protein (Osp) A were amplified from four joint fluid samples over 4.5 months from a patient with chronic Lyme arthritis, with a variant from wild type only found in sample 3. Rather than a mutation in vivo, these findings suggested a mixed infection in which BORRELIA: containing the wild-type and mutant ospA were waxing and waning in the patient's joint. If so, we reasoned that the mutant should be present in the community. We therefore took the novel epitope resulting from the mutation, expressed as a fusion protein in Escherichia coli, and performed Western blots on 80 high-titred stored sera; however, all except that of our index patient were negative. We then collected 36 stored sera from patients with Lyme disease residing within 10 miles of where the index patient had lived. An additional two sera from this circumscribed area were positive (P = 0.038). These findings show that results from single samples can be misleading, and suggest that the OspAs expressed in force late in Lyme arthritis are the same ones introduced initially into the host. Moreover, they allow a speculative mechanism for disease persistence not previously considered, in which antigenically distinct B. burgdorferi variant proteins present themselves serially to the immune system.

Antigens, Surface↗
PubMed1999

Analysis of the PKC-gamma-related immunocrossreactive region of a novel leukocyte protein gamma-rp.

The newly discovered gamma-PKC-related-protein of human leukocytes (gamma-rp) crossreacts with a polyclonal antibody preparation originally designed to be specific for PKC-gamma (gammaMb-Ab). As this antibody is currently the only suitable probe for gamma-rp, we sought to characterize the binding of the two proteins. We determined that the gamma Mg-Ab does not recognize the native form of gamma-rp. However, with denaturing immunoblots of gamma-rp, we found that 1) the crossreactive gamma-rp epitope differs somewhat from that of classic rat brain PKC-gamma, but probably only to the degree of the rat/human PKC species difference; 2) the previously reported doublet bands of gamma-rp represent a single protein with cell-stimulus inducible modifications; 3) antibodies present in the gammaMg-Ab pool bind to two separate sites within the gamma-rp epitope; 4) access to one binding site is conformationally restricted, even after protein denaturation; 5) agonist-induced modification of gamma-rp does not significantly affect the total amount of gamma Mg-Ab that it can bind, but 6) does significantly affect the rate of antibody binding to one site. This investigation defines the appropriate experimental use of our antibody, and the significance of these findings for the future study and cloning of gamma-rp is discussed.

Amino Acid Sequence↗
PubMed1998

A vaccine consisting of recombinant Borrelia burgdorferi outer-surface protein A to prevent Lyme disease. Recombinant Outer-Surface Protein A Lyme Disease Vaccine Study Consortium.

BACKGROUND: Lyme disease is a multisystem inflammatory disease caused by infection with the tick-borne spirochete Borrelia burgdorferi and is the most common vector-borne infection in the United States. We assessed the efficacy of a recombinant vaccine consisting of outer-surface protein A (OspA) without adjuvant in subjects at risk for Lyme disease. METHODS: For this double-blind trial, 10,305 subjects 18 years of age or older were recruited at 14 sites in areas of the United States where Lyme disease was endemic; the subjects were randomly assigned to receive either placebo (5149 subjects) or 30 microg of OspA vaccine (5156 subjects). The first two injections were administered 1 month apart, and 7515 subjects also received a booster dose at 12 months. The subjects were observed for two seasons during which the risk of transmission of Lyme disease was high. The primary end point was the number of new clinically and serologically confirmed cases of Lyme disease. RESULTS: The efficacy of the vaccine was 68 percent in the first year of the study in the entire population and 92 percent in the second year among the 3745 subjects who received the third injection. The vaccine was well tolerated. There was a higher incidence of mild, self-limited local and systemic reactions in the vaccine group, but only during the seven days after vaccination. There was no significant increase in the frequency of arthritis or neurologic events in vaccine recipients. CONCLUSIONS: In this study, OspA vaccine was safe and effective in the prevention of Lyme disease.

Adolescent↗
PubMed1998

An apparently novel protein of human leukocytes, reactive with an antibody to protein kinase C-gamma, is rapidly modified upon cell activation: initial characterization in neutrophils and their cytoplasts.

On immunoblots of human neutrophil cytoplasts (U-CYT), a previously undescribed 97 kDa protein was revealed by intense and selective reaction with an antibody that was initially raised to recognize PKC-gamma. Denoted "gamma-rp" for gamma-related protein, this acidic cytosolic protein somewhat resembled the classic forms of PKC in several biochemical respects. Appearing as a doublet on low-percentage SDS-PAGE gels, both its mobility and staining pattern were rapidly altered by treatment of U-CYT with either phorbol ester or chemotactic peptide. Whole neutrophil gamma-rp was detectable only after TCA precipitation of intact cells. It was also detectable in human platelets, lymphocytes, and neutrophil-like differentiated HL60 cells, but not in fibroblasts, erythrocytes, monocytes, or monocyte-like differentiated HL60 cells. Our data suggest that gamma-rp merits further study as a potential participant in cellular activation, and as a possible structural or functional relative of PKC.

Adolescent↗
PubMed1998

[Functions of neutrophil motility of human blood: locomotion and chemotaxis in simplified systems].

Polymorphonuclear leukocytes (PMN, granulocytes) employ their plasma membranes and subjacent microfilament-rich peripheral cytoplasm for such motile functions as adherence and spreading, random locomotion, chemotaxis (directed locomotion), and phagocytosis. All of these functions are preserved in certain anucleate, granule-poor, cytoplasmic fragments (cytoplasts) derived from PMN. Thus, the sensing, transduncing, and effector capacities involved in these functions remain integrated without control from nuclei or from the other cellular organelles left behind when the cytoplast forms. More recently, we have begun to examine in intact PMN the role of divalent cations, which have been thought to be essential for motile function of leukocytes in general, and for the function of critical adhesion molecules in particular. In slide preparations under direct microscopic observation, EDTA (10 mM; to chelate divalent cations) did not impair either random locomotion or chemotaxis, nor did specific antibodies to beta-2 (CD 18) integrins or to other PMN integrins. Motile behavior appeared to benefit from the close approximation of slide and coverslip ("chimneying"). Thus, in "close quarters", PMN can generate the force for locomotion even when adhesion molecules are lacking or disabled. We relate these findings to the reported independence from integrins of PMN in certain experimental and diseases states.

Cell Movement↗
PubMed1997

Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes (PMN) in the presence of EDTA: PMN in close quarters require neither leukocyte integrins nor external divalent cations.

Divalent cations are thought essential for motile function of leukocytes in general, and for the function of critical adhesion molecules in particular. In the current study, under direct microscopic observation with concomitant time-lapse video recording, we examined the effects of 10 mM EDTA on locomotion of human blood polymorphonuclear leukocytes (PMN). In very thin slide preparations, EDTA did not impair either random locomotion or chemotaxis; motile behavior appeared to benefit from the close approximation of slide and coverslip ("chimneying"). In preparations twice as thick, PMN in EDTA first exhibited active deformability with little or no displacement, then rounded up and became motionless. However, on creation of a chemotactic gradient, the same cells were able to orient and make their way to the target, often, however, losing momentarily their purchase on the substrate. In either of these preparations without EDTA, specific antibodies to beta2 integrins did not prevent random locomotion or chemotaxis, even when we added antibodies to beta1 and alphavbeta3 integrins and to integrin-associated protein, and none of these antibodies added anything to the effects of EDTA. In the more turbulent environment of even more media, effects of anti-beta2 integrins became evident: PMN still could locomote but adhered to substrate largely by their uropods and by uropod-associated filaments. We relate these findings to the reported independence from integrins of PMN in certain experimental and disease states. Moreover, we suggest that PMN locomotion in close quarters is not only integrin-independent, but independent of external divalent cations as well.

Antibodies, Monoclonal↗
PubMed1997

Chemotaxis by human neutrophils and their cytokineplasts treated with inhibitors of nitric oxide synthase: no suppression of orientation or trajectory.

Inhibitors of nitric oxide (NO) synthase are reported to inhibit both the adherence of polymorphonuclear leukocytes (PMN) to substrate and chemotaxis (directed locomotion) of PMN as determined in Boyden chamber assays. In the current study, we examined both human blood PMN and granule-poor motile cytoplasts derived from them (cytokineplasts, CKP), under direct microscopic observation with concomitant time-lapse video recording, for their ability to respond chemotactically to an erythrocyte destroyed by laser microirradiation. In this system we can observe directly and continuously the orientation and trajectory of PMN before, during, and after establishment of a chemotactic gradient. For both PMN and CKP we employed three different inhibitors of NO synthase (N(omega)-methyl-L-arginine, N-iminoethyl-L-ornithine, and diphenyleneiodonium) in at least twice the concentrations employed to inhibit chemotaxis of PMN in Boyden chambers or killing of bacteria in CKP. Although small differences in adhesion might not have been appreciated, treated PMN and CKP were each indistinguishable from untreated controls in their ability to orient in a newly created chemotactic gradient and in their trajectories toward the chemotactic target.

Arginine↗
PubMed1996

Direct demonstration of antigenic substitution of Borrelia burgdorferi ex vivo: exploration of the paradox of the early immune response to outer surface proteins A and C in Lyme disease.

The outer surface proteins (Osps) of Borrelia burgdorferi, the etiologic agent of Lyme disease, are principle targets of protective immune responses against this organism. Whereas most North American strains of B. burgdorferi in culture express an abundant amount of Osp A, antibodies to this protein are either absent or only weakly detected in the sera of naturally infected patients or experimentally infected mice. In contrast, Osp C, which has variable expression on cultured organisms; elicits an early, strong humoral response. To examine this paradox, we have studied the in vivo adaptation of a cloned population of B. burgdorferi strain N40 during the early course of experimental murine borreliosis. As in human disease, antibodies to Osp A were only weakly present in the early immune repertoire after murine inoculation with low dose (10(3)) spirochetes. In contrast, antibodies to Osp C were prominent, even though on cultured spirochetes Osp C mRNA and protein expression could not be detected by reverse transcription polymerase chain reaction (RT-PCR) or indirect immunofluorescence, respectively. These observations led us to investigate the expression of Osp A and Osp C in vivo. By direct fluorescent staining of uncultured spirochetes ex vivo and by PCR amplification of spirochetal mRNA, we show that Osp C is indeed expressed by some spirochetes after infection in the mouse. Spirochetes expressing Osp A could also be detected within the first 2 wk of infection, but not at 30 d. Osp A mRNA, although present at day 14 of infection, could not be amplified by RT-PCR at day 30, suggesting that the expression of this Osp is transient. This further implies that the late burst in Osp A antibodies in both mice and humans may be anamnestic. These results indicate that either Osp C is upregulated on spirochetes after infection, or Osp C-expressing spirochetes expand preferentially over those expressing Osp A during infection. These results have important implications for vaccine design and offer one explanation for the failure of Osp A antibodies to eradicate spirochetes from the infected host.

Adaptation, Biological↗
PubMed1996

Paradoxical effects of colchicine on the activation of human neutrophilis by chemotactic factors and inflammatory microcrystal.

Neutrophil activation by chemotactic factors and by inflammatory microcrystals is accompanied by increases in protein tyrosine phosphorylation and by the activation of the NADPH oxidase. The addition of colchicine inhibited both responses induced by triclinic monosodium urate or calcium pyrophosphate crystals. On the other hand, colchicine enhanced the tyrosine phosphorylation of specific protein in neutrophils stimulated by chemotactic factor and augmented the production of superoxide anions induced by these same agonists. The effects of colchicine were shared by other anti-microtubule agents (nocodazole and vinblastine) but not by its inactive analogue beta-lumicolchicine, trimethylcolchicinic acid, indomethacin, or phenylbutazone. Furthermore, the (enhancing as well as inhibitory) effects of colchicine on tyrosine phosphorylation and superoxide anion production were reversed by taxol. Finally, in human cytoplasts colchicine again inhibited microcrystal-stimulated tyrosine phosphorylation but did not change chemotactic factor-stimulated phosphorylation. These data strongly support the hypothesis that microtubule-related mechanisms are involved in the modulation of the tyrosine phosphorylation response in human neutrophils, and suggest that a relationship may exist between the augmentation of tyrosine phosphorylation and of the stimulation of the NADPH oxidase induced by chemotactic factors.

Adult↗
PubMed1996

Microbial killing by human neutrophil cytokineplasts: similar suppressive effects of reversible and irreversible inhibitors of nitric oxide synthase.

Employing anucleate, granule-poor, motile fragments from human blood neutrophils (cytokineplasts; CKP), we previously provided evidence for a new staphylococcal killing pathway for human neutrophils involving reactive nitrogen intermediates: the NO synthase inhibitor N(omega)-monomethyl-L-arginine (NMMA), an analogue of L-arginine (L-Arg), substantially decreased the killing capacity of CKP for Staphylococcus aureus (Staph), an effect reversible by excess L-Arg but not D-Arg. We have extended these findings to two irreversible NO synthase inhibitors: the first, N-iminoethyl-L-ornithine (L-NIO), is an L-Arg analogue; the other, diphenyleneiodonium (DPI), is not. After 60 min of incubation with bacteria, despite having taken up somewhat fewer staphylococci than did controls, cytoplasts treated with NO synthase inhibitors had many more live, CKP-associated bacteria: for NMMA, 6.9 times more (40.0% of the inoculum vs. 5.8%; n = 8, P = 0.003); for L-NIO, 3.6 times more (25.5 vs. 7%; n = 4, P = 0.004); for DPI, 5.8 times more (37.4 vs. 6.4%; n = 7, P = 0.002). Results were similar after only 20 min of incubation. In two experiments in which the Gram-negative bacterium, Serratia marcescens, was employed instead of Staph, the results were again similar. In contrast, killing of either bacterium by intact neutrophils (PMN) was not inhibited by NMMA, by L-NIO, or by DPI, a failure most likely attributable to their granule content. The irreversible inhibitors of NO synthase will be especially useful in analyzing particular effects on CKP employed in multicellular systems.

Arginine↗
PubMed1995

An ospA frame shift, identified from DNA in Lyme arthritis synovial fluid, results in an outer surface protein A that does not bind protective antibodies.

Passive immunization with murine or human Abs to outer surface protein A (OspA) can protect mice against Borrelia burgdorferi, but OspA Abs elicited during natural infection in mice or humans are unable to clear the spirochete from the infected host. To examine Ab binding by OspA during the course of human infection, we amplified the operon encoding full-length ospA and ospB from synovial fluids of a patient with chronic Lyme arthritis, the first such recoveries from human material, at four separate time points over 4.5 mo, and expressed OspA in Escherichia coli. OspA mAbs that passively protected mice from infection did not bind one of the expressed OspAs, because of a deletion in ospA that resulted in a frame shift and premature stop codon near the carboxyl terminus. However, expressed OspA from a later synovial fluid sample did not contain this deletion. Thus, although altered forms of OspA, which potentially can influence host immune effectiveness, do occur in the human host, they cannot be the only factors responsible for microbial persistence.

Adolescent↗
PubMed1995

Cytoplasts generate oxidants but require added neutrophil granule constituents for fungicidal activity against Candida albicans hyphae.

Killing of Candida albicans hyphae requires oxidant generation by neutrophils (PMNL), but it is uncertain whether hyphal killing is mediated by PMNL oxidants alone or requires contributions by granule constituents. This was assessed using U-cytoplasts (U-CYT), anucleate PMNL fragments depleted of cytoplasmic granules but retaining motility and respiratory burst activity. Granule-depleted U-CYT killed Staphylococcus aureus, but hyphae remained viable despite targeted generation of putatively fungicidal oxidants by avidly adherent U-CYT. Hyphal killing occurred by combining U-CYT with sublethal concentrations of purified PMNL granule extracts approximating those present in equivalent numbers of intact PMNL. Contributions of granule constituents were not entirely attributable to purified granule constituents with known antimicrobial activity (lactoferrin, cathepsin G, myeloperoxidase, and human neutrophil peptide defensins 1-3) individually or combined. Thus, oxidant generation by intact PMNL may be necessary but not always sufficient to mediate hyphal killing without complementary nonoxidative mechanisms.

Candida albicans↗