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M T Philipp

Publications and source records attributed to M T Philipp.

At least 37 records · Page 2Linked to original sources
PubMed1999

Killing of Borrelia burgdorferi by antibody elicited by OspA vaccine is inefficient in the absence of complement.

A Lyme disease vaccine, based on the Borrelia burgdorferi lipoprotein OspA, has recently undergone phase III trials in humans. The results of one of these trials indicate that vaccine efficacy positively correlates with anti-OspA antibody titer. Spirochete killing within the tick vector midgut, upon which vaccine efficacy appears to depend, may occur chiefly via a mechanism that involves antibody alone, as it has been reported that complement is degraded by tick saliva decomplementing factors. We compared the in vitro killing efficiencies of anti-OspA antibody elicited in rhesus monkeys by the OspA vaccine, in the presence and in the absence of monkey complement. Killing in the absence of complement was between 14 and 3,800 times less efficient than with complement present, depending on the spirochete strain. The relative inefficiency of the complement-independent killing mechanism by anti-OspA antibody may explain why OspA vaccine efficacy is critically dependent on antibody titer.

Animals↗
PubMed1999

Analysis of antibody response to invariable regions of VlsE, the variable surface antigen of Borrelia burgdorferi.

VlsE, the variable surface antigen of Borrelia burgdorferi, consists of two invariable domains at the amino and carboxyl termini and one central variable domain. The latter contains six invariable regions, IR(1) to IR(6), and six variable regions. In the present study, the antigenicity of all of the invariable regions in B. burgdorferi-infected monkeys, humans, and mice was assessed by peptide-based enzyme-linked immunosorbent assays. Only one invariable region, IR(6), was antigenic in all animals of the three host species. IR(2) and IR(4) were also antigenic in mice.

Amino Acid Sequence↗
PubMed1999

Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlsE.

VlsE, the variable surface antigen of Borrelia burgdorferi, contains an immunodominant conserved region named IR(6). In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C(6)) with the IR(6) sequence was explored. Sensitivity was assessed with serum samples (n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C(6) peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects.

Animals↗
PubMed1998

Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early disseminated and chronic phases of disease in the peripheral nervous system.

The histopathologic and immunohistochemical features of early and late neuroborreliosis of the peripheral nervous system were investigated in rhesus macaques infected with the JD1 strain of Borrelia burgdorferi. Infection was proven by culture or polymerase chain reaction analysis of skin biopsies and indirectly by Western blot analysis. Three months after infection, neuritis involving multiple nerves was the most consistent neurologic manifestation. Both macrophages and B lymphocytes but not T lymphocytes were present in the cellular infiltrates. Axonal structures surrounding infiltrates had changes consisting of demyelination and axonal phagocytosis. Some of the Schwann cells in lesions stained with anti-nitrotyrosine and anti-tumor necrosis factor-alpha antibodies. B. burgdorferi, or antigens thereof, were visualized immunohistochemically within macrophages. Forty-six months after infection, the most common changes were regenerative, whereas neuritis was infrequent. Aberrant axonal regeneration, irregularly sized myelinated fibers, and fibrosis were frequently observed. Possible mechanisms to explain the appearance and subsidence of Lyme neuritis are discussed.

Animals↗
PubMed1998

Differential expression of Borrelia burgdorferi proteins during growth in vitro.

In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165-1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of B. burgdorferi, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore, ospAB, bmpAB, groEL, and fla all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of ospC and bmpD indicated regulated expression of these genes. While bmpD exhibited a spike in mRNA expression in early stationary phase, ospC maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides P7.5 and P35 whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host.

Antigens, Bacterial↗
PubMed1998

Borrelia burgdorferi escape mutants that survive in the presence of antiserum to the OspA vaccine are killed when complement is also present.

As an initial attempt to investigate the possible role of outer surface protein A (OspA) escape mutants of Borrelia burgdorferi in decreasing the efficacy of the OspA vaccine, mutants of the HB19 strain of B. burgdorferi sensu stricto were selected in vitro from an uncloned, low-passage-number isolate. The antiserum used for selection was obtained from rhesus monkeys that had been given a vaccine of the same formulation and dose, and by the same route of administration, as that given to humans in several trials. All of the mutants selected in liquid medium and subsequently cloned twice in solid medium expressed a single abundant protein of 28 to 34 kDa instead of both OspA and OspB. Depending on the mutant, this protein reacted strongly, weakly, or not detectably with the anti-OspA antibody used for selection. Analysis of the ospAB locus of each of four representatives from these three groups of mutants by PCR with oligonucleotide primers that hybridize to flanking regions of the ospAB operon, and of the corresponding phenotype with monoclonal antibodies that bind to the amino or carboxyl terminus of the OspA or OspB polypeptide, indicated that in all cases a deletion within the operon had occurred. Spirochetes from the four mutant strains chosen for further analysis could be killed in antibody-dependent, complement-mediated killing assays with the selecting anti-OspA antibody, despite their resistance to killing with this antibody in the absence of complement. Complement-mediated killing occurred at an antibody concentration higher than that required to kill wild-type spirochetes. If anti-OspA antibody acts only within the tick, where complement is probably ineffective due to tick-derived decomplementing factors, then OspA escape mutants, if infectious, could seriously diminish the efficacy of OspA vaccines. On the other hand, if the killing of B. burgdorferi with anti-OspA antibody also takes place within the human host, then our results indicate that chimeric/deletion escape mutants will be killed as well.

Antigens, Bacterial↗
PubMed1998

Borrelia burgdorferi stimulates the production of interleukin-10 in peripheral blood mononuclear cells from uninfected humans and rhesus monkeys.

Heat-killed Borrelia burgdorferi spirochetes stimulate in vitro production of interleukin-10 (IL-10) at both mRNA and protein levels in peripheral blood mononuclear cells (PBMC) of uninfected rhesus monkeys. A concomitant down-modulation of IL-2 gene transcription was observed. Neither IL-4 nor gamma interferon gene expression was ostensibly affected by B. burgdorferi spirochetes. These phenomena were observed regardless of whether the stimulating spirochetes belonged to the B. burgdorferi sensu stricto, Borrelia afzelii, or Borrelia garinii genospecies, the three main species that cause Lyme disease. B. burgdorferi also induced production of IL-10 in uninfected human PBMC, indicating that this effect might play a role in human Lyme disease. Purified lipidated outer surface protein A (OspA), but not its unlipidated form, induced the production of high levels of IL-10 in uninfected human PBMC. Thus, the lipid moiety is essential in the induction of IL-10 in these PBMC. B. burgdorferi M297, a mutant strain that lacks the plasmid that encodes OspA and OspB, also induced IL-10 gene transcription in PBMC, indicating that this phenomenon is not causally linked exclusively to OspA and its lipid moiety. These results demonstrate that B. burgdorferi can stimulate the production of an antiinflammatory, immunosuppressive cytokine in naive cells and suggest that IL-10 may play a role both in avoidance by the spirochete of deleterious immune responses and in limiting the inflammation that the spirochete is able to induce.

Animals↗
PubMed1997

Mononeuropathy multiplex in rhesus monkeys with chronic Lyme disease.

Peripheral neuropathy is a recognized but poorly understood manifestation of Lyme disease. We performed serial electrophysiological studies on 8 rhesus monkeys chronically infected with the JD1 strain of Borrelia burgdorferi and compared the results with those of similar studies on 10 uninfected control monkeys. Four infected and 2 uninfected animals underwent sural nerve biopsy. Five of the infected and 1 of the uninfected animals also had postmortem neuropathological examinations. Altogether, 5 of the infected monkeys demonstrated primarily axonal-loss-variety multifocal neuropathies. Only one nerve lesion exhibited findings compatible with demyelination. Pathologically, peripheral nerve specimens showed multifocal axonal degeneration and regeneration and occasional perivascular inflammatory cellular infiltrates without vessel wall necrosis. Free spirochetal structures were not seen, but several macrophages exhibited positive immunostaining with a highly specific anti-B. burgdorferi, 7.5-kd lipoprotein monoclonal antibody. In the infected animals, serial analysis of serum antibodies to B. burgdorferi showed increasing numbers of IgG specificities and new IgM specificities, suggesting persistent infection. Thus, peripheral neuropathy in the form of a mononeuropathy multiplex develops frequently in rhesus monkeys chronically infected with B. burgdorferi. The pathogenesis of these nerve lesions is not yet known, but our studies suggest an immune-mediated process perhaps driven by persistent infection with B. burgdorferi.

Animals↗
PubMed1997

The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey.

The efficacy of an outer surface protein A (OspA) vaccine in three different formulations was investigated in the rhesus monkey. The challenge infection was administered using Ixodes scapularis ticks that were infected with the B31 strain of Borrelia burgdorferi. Protection was assessed against both infection and disease, by a variety of procedures. Some of the animals were radically immune suppressed, as an attempt to reveal any putative low level infection in the vaccinated animals. The significant difference found between the spirochaetal infection rates of ticks that had fed on vaccinated vs. control monkeys, lack of seroconversion in the vaccinated animals, and the absence of spirochaetal DNA in the skin of vaccinated animals in the weeks following the challenge, indicate that vaccinated monkeys were protected against tick challenge. The post-mortem immunohistochemical and polymerase chain reaction analyses, however, suggest that these monkeys may have undergone a low-level infection that was transient.

Animals↗
PubMed1997

Lyme neuroborreliosis in the rhesus monkey.

Although there are several animal models of Lyme disease, only the rhesus monkey model exhibits all of the key manifestations of the disease. After infection with Borrelia burgdorferi, rhesus monkeys develop signs of early localized, early disseminated, and chronic Lyme disease. Specific features include erythema migrans, uveitis, myocarditis, arthritis, and disease of the peripheral and central nervous system. One of the unique features of the rhesus monkey model is the development of Lyme neuroborreliosis. Peripheral nervous system (PNS) involvement is usually in the form of a mononeuropathy multiplex with primarily axonal-loss features. Evidence of central nervous system (CNS) disease has included CSF pleocytosis, meningeal inflammation, spinal cord lesions, and polymerase chain reaction (PCR) data consistent with chronic CNS infection. The pathogenesis of Lyme neuroborreliosis is not well understood, but it is likely to involve complex interactions between B. burgdorferi and host immune mechanisms.

Animals↗
PubMed1997

Cell-density-dependent expression of Borrelia burgdorferi lipoproteins in vitro.

Previously, we had identified non-OspA-OspB surface proteins of Borrelia burgdorferi that are targeted by the antibody-dependent complement-mediated killing mechanism. Here we demonstrate by Western blotting that one of these proteins, P35, is upregulated at the onset of stationary phase in vitro. Northern analysis revealed that the upregulation of P35 is at the level of transcription. In addition, the expression of an open reading frame (ORF) located downstream of the p35 gene was found to be regulated in the same fashion as that of P35. This ORF encodes a 7.5-kDa lipoprotein. The transcriptional start sites for both of these genes were determined, to aid in the identification of the putative promoter regions. Additional sequencing of the 5' flanking region of the p35 gene revealed a region of dyad symmetry 52 bp upstream of the transcription start site. Southern analysis demonstrated that the expression of these genes was not due to a cell-density-dependent rearrangement in the genome of B. burgdorferi. These findings provide an in vitro model for studying mechanisms of gene regulation in B. burgdorferi.

Amino Acid Sequence↗
PubMed1996

Borrelia burgdorferi possesses a collagenolytic activity.

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi, an invasive spirochete. B. burgdorferi has a predilection for collagenous tissue and one major clinical manifestation of the disease is arthritis. We have identified a collagenolytic activity in B. burgdorferi detergent lysates using iodinated gelatin as well as iodinated pepsinized human collagen types II and IV as protein substrates. In addition, we describe several proteolytic activities in B. burgdorferi with molecular masses greater than 200 kDa on sodium dodecyl sulfate polyacrylamide gels containing copolymerized gelatin. We propose that the collagenolytic activity of B. burgdorferi has a role in invasion, in the pathogenesis of Lyme arthritis, and perhaps also in other manifestations of Lyme borreliosis.

Borrelia burgdorferi Group↗
PubMed1996

Dirofilaria immitis: ultrastructural localization, molecular characterization, and analysis of the expression of p27, a small heat shock protein homolog of nematodes.

A cDNA fragment encoding the complete coding region of a 27-kDa protein (p27) of Dirofilaria immitis was cloned. Antibody to the recombinant p27 bound to hypodermal tissues of third (L3) and fourth stage larvae (L4) of D. immitis and to both the hypodermis and the cuticle of L3s of Onchocerca volvulus, as visualized by immunoelectronmicroscopy. The deduced amino acid sequence of the central and C-terminal regions of p27 (amino acids S83 to H222) is 18-36% identical to members of the sHsp/alpha-crystallin family of proteins. The homologous region is thought to be responsible for the molecular chaperone activity of members of this family. The p27 cDNA does not encode a hydrophobic signal peptide. At least two homologous yet distinct p27 genes were identified in the D. immitis genome by Southern hybridization using the p27 cDNA as a probe. The p27 transcript was 0.9 kb in length on Northern blots. The expression of p27 in L3s of D. immitis was neither upregulated by heat shock (43 degrees C) nor by incubation at the physiologic temperature of 37 degrees C. Pulse-labeling experiments of both D. immitis and Brugia malayi L3s during the L3-L4 molt in vitro showed that synthesis of p27 is also not upregulated during this developmental phase. However, p27 is expressed constitutively throughout the D. immitis L3-L4 molt and therefore by both larval stages. In addition, both female and male adult worms of this species express p27 constitutively. P27, or an allomorph thereof, was detected in each of nine species representing four nematode superfamilies, thus indicating that this molecule is ubiquitous within the phylum Nematoda. In view of the hypodermal localization of p27, its constitutive expression, and its retention among nematodes, the function of this protein in essential housekeeping roles such as that of molecular chaperone during the molting process is discussed.

Amino Acid Sequence↗
PubMed1996

Host DNA can interfere with detection of Borrelia burgdorferi in skin biopsy specimens by PCR.

It is demonstrated that a diagnostic PCR for Borrelia burgdorferi can be inhibited in the presence of more than 500 ng of host (monkey skin) DNA. The inhibitor is the host DNA itself. An acceptable value for analytical sensitivity can be obtained by diluting the skin-B. burgdorferi proteinase K lysate to a level below the inhibitory concentration of the host DNA. Dilution of the lysate may obviate the need for further DNA purification.

Animals↗
PubMed1995

Chronic lyme disease in the rhesus monkey.

BACKGROUND: We have previously reported the clinical, pathologic, and immunologic features of "early" Borrelia burgdorferi infection in rhesus monkeys (3). We have now evaluated these features during the chronic phase of Lyme disease in this animal model. EXPERIMENTAL DESIGN: Clinical signs, and pathologic changes at the gross and microscopic levels, were investigated 6 months post-infection in several organ systems of five rhesus macaques (Macaca mulatta), which were infected with Borrelia burgdorferi by allowing infected Ixodes scapularis nymphal ticks to feed on them. A sixth animal was used as an uninfected control. Borrelia antigens recognized by serum antibody were identified longitudinally by Western blot analysis, and C1q-binding immune complexes were quantified. Localization of the spirochete in the tissues was achieved by immunohistochemistry and in vitro culture. The species of spirocheta cultured was confirmed by the polymerase chain reaction. RESULTS: Chronic arthritis was observed in five out of five animals. The knee and elbow joints were the most consistently affected. Articular cartilage necrosis and/or degenerative arthropathy were the most severe joint structural changes. Synovial cell hyperplasia and a mononuclear/lymphocyte infiltrate were commonly seen. Nerve lesions were also observed, including nerve sheath fibrosis and focal demyelinization of the spinal cord. Peripheral neuropathy was observed in five out of five animals and could be correlated in the most severely affected monkey with the presence of higher levels of circulating immune complexes. Differences in disease severity did not correlate with differences in the antigens recognized on Western blot analysis. CONCLUSIONS: B. burgdorferi infection in rhesus macaques mirrors several aspects of both the early and chronic phases of the disease in humans. This animal model will facilitate the study of the pathogenesis of Lyme arthritis and neuroborreliosis.

Animals↗
PubMed1994

Brugia malayi: the diagnostic potential of recombinant excretory/secretory antigens.

The diagnostic potential of recombinant E/S antigens of the lymphatic filaria Brugia malayi was investigated by Western blot. A cDNA expression library was constructed using B. malayi male adult worm mRNA, and E/S recombinants were identified with a rabbit antiserum raised against E/S products collected in vitro from B. malayi male and female adult worms. Two of these recombinants, Bm12 and Bm14L, were studied after subcloning the cDNA inserts in an Escherichia coli plasmid expression and purification vector, obtaining the inserts' nucleotide sequence, and purifying the expressed proteins. By homology of their deduced amino acid sequence with that of previously identified proteins, Bm12 was identified as the B. malayi gp 15/400 antigen, and Bm14 as a member of the hsp90 family of heat shock proteins. The antigenic cross-reactivity of the purified recombinant proteins was assessed with 28 serum samples from patients infected with Ascaris, Trichuris, or hookworm, and also with a few samples from patients with onchocerciasis and loiasis. For Bm12, the specificity for all of the intestinal helminthiasis together was 75%. Bm14L, on the other hand, cross-reacted with all of the ascariasis serum samples with which it was tested. Presence of antibodies cross-reactive with B. malayi was confirmed in all of these serum samples by examining their antibody reactivity with Western blots of extracts of whole B. malayi adult worms. A semiquantitative (+ or -) assessment of the sensitivity of Bm12 for antibody detection was performed using 6 serum samples from patients with chronic filariasis and 24 samples from patients with microfilaremia. All of these serum samples contained anti-Bm12 antibody (sensitivity of 100%). Finally, the ability of Bm12 to detect antibody before the onset of patency was established with a longitudinal collection of serum samples obtained from 2 African green vervets (Cercopithecus aethiops) and 3 rhesus macaques (Macaca mulatta), all of which were infected with B. malayi. Anti-Bm12 antibodies were detectable in all animals between 4 and 11 weeks before patency.

Amino Acid Sequence↗
PubMed1994

Animal models of Lyme disease: pathogenesis and immunoprophylaxis.

Valuable insights into the pathogenesis and immunoprophylaxis of Lyme disease are beginning to emerge from studies in animal models. This review highlights two animal models: the mouse, which has allowed us to investigate the role of both the immune response and spirochete phenotype in determining the outcome of the disease; and the Rhesus monkey, which manifests signs of nerve involvement, in addition to showing erythema migrans and arthritis.

Animals↗