Biomedical subjects

M T Philipp

Publications and source records attributed to M T Philipp.

At least 19 recordsLinked to original sources

PubMed2004

Pre-treatment and post-treatment assessment of the C(6) test in patients with persistent symptoms and a history of Lyme borreliosis.

It was recently reported that antibody to C(6), a peptide that reproduces an invariable region of the VlsE lipoprotein of Borrelia burgdorferi, declined in titer by a factor of four or more in a significant proportion of patients after successful antibiotic treatment of acute localized or disseminated Lyme borreliosis. The present study evaluated the C(6) test as a predictor of therapy outcome in a population of patients with post-treatment Lyme disease syndrome. The serum specimens tested were from patients with well-documented, previously treated Lyme borreliosis who had persistent musculoskeletal or neurocognitive symptoms. All of the patients had participated in a recent double-blind, placebo-controlled antibiotic trial in which serum samples were collected at baseline and 6 months thereafter, i.show $132#e. 3 months following treatment termination. In this patient population no correlation was found between a decline of C(6) antibody titer of any magnitude and treatment or clinical outcome. Antibodies to C(6) persisted in these patients with post-treatment Lyme disease syndrome following treatment, albeit at a markedly lower prevalence and titer than in untreated patients with acute disseminated Lyme disease. The results indicate that C(6) antibody cannot be used to assess treatment outcome or the presence of active infection in this population.

Anti-Bacterial Agents↗

PubMed2001

Antibody response to IR6, a conserved immunodominant region of the VlsE lipoprotein, wanes rapidly after antibiotic treatment of Borrelia burgdorferi infection in experimental animals and in humans.

Invariable region (IR)(6), an immunodominant conserved region of VlsE, the antigenic variation protein of Borrelia burgdorferi, is currently used for the serologic diagnosis of Lyme disease in humans and canines. A longitudinal assessment of anti-IR(6) antibody levels in B. burgdorferi-infected rhesus monkeys revealed that this level diminished sharply after antibiotic treatment (within 25 weeks). In contrast, antibody levels to P39 and to whole-cell antigen extracts of B. burgdorferi either remained unchanged or diminished less. A longitudinal analysis in dogs yielded similar results. In humans, the anti-IR(6) antibody titer diminished by a factor of > or =4 in successfully treated patients and by a factor of <4 in treatment-resistant patients. This result suggests that the quantification of anti-IR(6) antibody titer as a function of time should be investigated further as a test to assess response to Lyme disease therapy or to determine whether a B. burgdorferi infection has been eliminated.

Adult↗

PubMed2001

Borrelia burgdorferi stimulates in vitro a selective expansion of CD3-CD4-CD8- (triple negative) autoreactive cells in naive rhesus monkey lymphocytes.

Autoreactive cell lines were generated from cell suspensions of freshly isolated naive monkey lymph node (LN) cells and peripheral blood mononuclear cells by cocultivation with freeze-thawed Borrelia burgdorferi spirochetes (Bb/FT). These cells produced interleukin (IL)-6 when cocultured with autologous antigen-presenting cells (APCs) alone without Bb/FT. IL-6 production was not observed when control cell lines were stimulated in the same fashion. CD4+-enriched T cell populations obtained from the LN autoreactive cell line also produced IL-6 when cultivated with APCs alone. When these cells were cultivated further in the presence of APCs, a population of cells whose phenotype was CD56+/-CD4-CD8-CD3- was predominantly selected. These cells both proliferated and produced IL-6 when cocultured with APCs alone. The possible relevance of these cells to Lyme disease pathogenesis remains to be determined.

Animals↗

PubMed2001

CD19(+) cells produce IFN-gamma in mice infected with Borrelia burgdorferi.

We have recently shown that production of IFN-gamma and IL-10, but not IL-4 is specifically induced in the lymph nodes of C3H/HeJ (disease susceptible) and C57BL/6J (disease resistant) mice 1 week after infection with Borrelia burgdorferi spirochetes. The present study was conducted to determine the phenotypes of ex vivo lymph node cells obtained from infected mice of both strains at this time point. The percentages of CD3(+), CD4(+), CD8(+), TCRalpha/beta (+) and TCRgamma/delta (+) cells decreased in both strains of mice compared to LN from naive mice. In contrast, there was a threefold increase in the proportion of CD19(+) cells. In view of this expansion of the B cell proportion, we examined the ability of purified CD19(+) cells and CD43(+) cells to produce both IL-10 and IFN-gamma when the cells were restimulated in vitro with B. burgdorferi freeze-thawed spirochetes. As expected, CD43(+) cells were able to produce both cytokines, but not IL-4. Surprisingly, CD19(+) (B) cells also were able to produce IFN-gamma in comparable amounts, in addition to IL-10. Intracellular staining of CD19(+) cells with anti-IFN-gamma antibody confirmed this finding. We discuss this novel phenomenon in terms of its possible underlying mechanisms and its relevance, both in the context of the immunology of Lyme disease and that of other infectious diseases.

Animals↗

PubMed2001

Analysis of Borrelia burgdorferi vlsE gene expression and recombination in the tick vector.

Expression and recombination of the antigenic variation vlsE gene of the Lyme disease spirochete Borrelia burgdorferi were analyzed in the tick vector. To assess vlsE expression, Ixodes scapularis nymphs infected with the B. burgdorferi strain B31 were fed on mice for 48 or 96 h or to repletion and then crushed and acetone fixed either immediately thereafter (ticks collected at the two earlier time points) or 4 days after repletion. Unfed nymphs also were examined. At all of the time points investigated, spirochetes were able to bind a rabbit antibody raised against the conserved invariable region 6 of VlsE, as assessed by indirect immunofluorescence, but not preimmune serum from the same rabbit. This same antibody also bound to B31 spirochetes cultivated in vitro. Intensity of fluorescence appeared highest in cultured spirochetes, followed by spirochetes present in unfed ticks. Only a dim fluorescent signal was observed on spirochetes at the 48 and 96 h time points and at day 4 postrepletion. Expression of vlsE in vitro was affected by a rise in pH from 7.0 to 8.0 at 34 degrees C. Hence, vlsE expression appears to be sensitive to environmental cues of the type found in the B. burgdorferi natural history. To assess vlsE recombination, nymphs were capillary fed the B. burgdorferi B31 clonal isolate 5A3. Ticks thus infected were either left to rest for 4 weeks (Group I) or fed to repletion on a mouse (Group II). The contents of each tick from both groups were cultured and 10 B. burgdorferi clones from the spirochetal isolate of each tick were obtained. The vlsE cassettes from several of these clones were amplified by PCR and sequenced. Regardless of whether the isolate was derived from Group I or Group II ticks, no changes were observed in the vlsE sequence. In contrast, vlsE cassettes amplified from B. burgdorferi clones derived from a mouse that was infected with B31-5A3 capillary-fed nymphs showed considerable recombination. It follows that vlsE recombination does not occur in the tick vector.

Animals↗

PubMed2001

C-terminal invariable domain of VlsE may not serve as target for protective immune response against Borrelia burgdorferi.

VlsE, the variable surface antigen of the Lyme disease spirochete, Borrelia burgdorferi, contains two invariable domains, at the amino and carboxyl termini, respectively, which collectively account for approximately one-half of the entire molecule's length and remain unchanged during antigenic variation. It is not known if these two invariable domains are exposed at the surface of either the antigen or the spirochete. If they are exposed at the spirochete's surface, they may elicit a protective immune response against B. burgdorferi and serve as vaccine candidates. In this study, a 51-mer synthetic peptide that reproduced the entire sequence of the C-terminal invariable domain of VlsE was conjugated to the carrier keyhole limpet hemocyanin and used to immunize mice. Generated mouse antibody was able to immunoprecipitate native VlsE extracted from cultured B. burgdorferi B31 spirochetes, indicating that the C-terminal invariable domain was exposed at the antigen's surface. However, this domain was inaccessible to antibody binding at the surface of cultured intact spirochetes, as demonstrated by both an immunofluorescence experiment and an in vitro killing assay. Mouse antibody to the C-terminal invariable domain was not able to confer protection against B. burgdorferi infection, indicating that this domain was unlikely exposed at the spirochete's surface in vivo. We concluded that the C-terminal invariable domain was exposed at the antigen's surface but not at the surface of either cultured or in vivo spirochetes and thus cannot elicit protection against B. burgdorferi infection.

Amino Acid Sequence↗

PubMed2001

C-terminal invariable domain of VlsE is immunodominant but its antigenicity is scarcely conserved among strains of Lyme disease spirochetes.

VlsE, the variable surface antigen of Borrelia burgdorferi, contains two invariable domains located at the amino and carboxyl terminal ends, respectively, and a central variable domain. In this study, both immunogenicity and antigenic conservation of the C-terminal invariable domain were assessed. Mouse antiserum to a 51-mer synthetic peptide (Ct) which reproduced the entire sequence of the C-terminal invariable domain of VlsE from B. burgdorferi strain B31 was reacted on immunoblots with whole-cell lysates extracted from spirochetes of 12 strains from the B. burgdorferi sensu lato species complex. The antiserum recognized only VlsE from strain B31, indicating that epitopes of this domain differed among these strains. When Ct was used as enzyme-linked immunosorbent assay (ELISA) antigen, all of the seven monkeys and six mice that were infected with B31 spirochetes produced a strong antibody response to this peptide, indicating that the C-terminal invariable domain is immunodominant. None of 12 monkeys and only 11 of 26 mice that were infected with strains other than B31 produced a detectable anti-Ct response, indicating a limited antigenic conservation of this domain among these strains. Twenty-six of 33 dogs that were experimentally infected by tick inoculation were positive by the Ct ELISA, while only 5 of 18 serum samples from dogs clinically diagnosed with Lyme disease contained detectable anti-Ct antibody. Fifty-seven of 64 serum specimens that were collected from American patients with Lyme disease were positive by the Ct ELISA, while only 12 of 21 European samples contained detectable anti-Ct antibody. In contrast, antibody to the more conserved invariable region IR(6) of VlsE was present in all of these dog and human serum samples.

Amino Acid Sequence↗

PubMed2000

Antigenic conservation of an immunodominant invariable region of the VlsE lipoprotein among European pathogenic genospecies of Borrelia burgdorferi SL.

Lyme disease is caused by genetically divergent spirochetes, including 3 pathogenic genospecies: Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii. Serodiagnosis is complicated by this genetic diversity. A synthetic peptide (C(6)), based on the 26-mer invariable region (IR(6)) of the variable surface antigen of B. burgdorferi (VlsE), was used as ELISA antigen, to test serum samples collected from mice experimentally infected with the 3 genospecies and from European patients with Lyme disease. Regardless of the infecting strains, mice produced a strong antibody response to C(6), which indicates that IR(6) is antigenically conserved among the pathogenic genospecies. Twenty of 23 patients with culture-confirmed erythema migrans had a detectable antibody response to C(6). A sensitivity of 95.2% was achieved, with serum samples collected from patients with well-defined acrodermatitis chronica atrophicans. Fourteen of 20 patients with symptoms of late Lyme disease also had a positive anti-IR(6) ELISA. Thus, it is possible that C(6) may be used to serodiagnose Lyme disease universally.

Amino Acid Sequence↗

PubMed2000

Interleukin-10 modulates proinflammatory cytokines in the human monocytic cell line THP-1 stimulated with Borrelia burgdorferi lipoproteins.

We determined previously that lipoproteins of Borrelia burgdorferi stimulate inflammatory and anti-inflammatory cytokines (interleukin-10 [IL-10]) in monocytes. IL-10 could have an effect on innate and acquired immune responses to B. burgdorferi and influence the magnitude of the infectious inoculum and disease outcome. To understand the mechanism(s) of IL-10 action during early infection, when innate immunity expressed chiefly by skin macrophages is key, we investigated the effect of exogenous and endogenous IL-10 on the production of the macrophage-derived cytokines IL-6, IL-1beta, IL-12, and tumor necrosis factor alpha (TNF-alpha). We used the THP-1 human monocytic cell line and recombinant lipidated OspA (L-OspA) as the model target cell and stimulant, respectively. To determine the kinetics of cytokine production by THP-1 cells, we stimulated them with L-OspA and also with heat-killed B. burgdorferi cells (HBb) and lipopolysaccharide (LPS). Exogenous IL-10 dampened production of inflammatory cytokines, as elicited by lipoproteins. The inhibition of endogenous IL-10 function by anti-IL-10 antibody reduced the production of IL-12 and IL-6 but not that of IL-1beta and TNF-alpha. An inspection of the kinetics of cytokine production clarified this finding. TNF-alpha was produced prior to, and IL-beta was produced at the same time as, IL-10, whereas IL-6 and IL-12 were produced later. HBb, LPS, and L-OspA yielded similar kinetics of cytokine production. This result reinforces the notion that lipoproteins are the functional molecules in HBb and perhaps in vivo. It indicates also that signaling pathways utilized by LPS and lipoproteins may be extensively shared. The results are consistent with a major role played by IL-10 in controlling the initial phase of infection with this spirochete.

Antigens, Surface↗

PubMed2000

Early induction of gamma interferon and interleukin-10 production in draining lymph nodes from mice infected with Borrelia burgdorferi.

Lymph node (LN) cells from C3H/HeJ mice (Lyme disease susceptible) infected for 1 week with Borrelia burgdorferi strain JD1 produced higher levels of gamma interferon (IFN-gamma) when stimulated in vitro with B. burgdorferi spirochetes than equivalent cells from B. burgdorferi-infected C57BL/6J mice (disease resistant). The interleukin-10 (IL-10) levels were comparable in the two strains, whereas the IL-4 levels were below detection limits. B. burgdorferi-stimulated LN cells from C57BL/6J mice produced significantly higher levels of IFN-gamma in the presence of neutralizing anti-IL-10 antibody than cells cultured with B. burgdorferi alone. No effect of IL-10 neutralization on IFN-gamma production by LN cells from C3H/HeJ mice was observed. Neutralizing antibody to IFN-gamma had no effect on the production of IL-10 by LN cells from C57BL/6J mice. A slight decrease in IL-10 production was detected in culture supernatants of equivalent cells from C3H/HeJ mice. The differential effect of IL-10 on IFN-gamma production in C57BL/6J and C3H/HeJ mice suggests that IL-10 is probably involved in the regulation of IFN-gamma production by LN cells during infection and may be at the root of the differential susceptibility to Lyme arthritis in these two strains of mice.

Animals↗

PubMed2000

Epitope mapping of the immunodominant invariable region of Borrelia burgdorferi VlsE in three host species.

VlsE, the variable surface antigen of Borrelia burgdorferi, contains a 26-amino-acid-long immunodominant invariable region, IR(6). In the present study, three overlapping 14-mer peptides reproducing the sequence of IR(6) were used as peptide-based enzyme-linked immunosorbent assay antigens to map this invariable region in infected monkeys, mice, and human Lyme disease patients. Antibodies of the two primate species appeared to recognize IR(6) as a single antigenic determinant, while mouse antibodies recognized multiple epitopes within this region.

Amino Acid Sequence↗

PubMed2000

Cryptic and exposed invariable regions of VlsE, the variable surface antigen of Borrelia burgdorferi sl.

Borrelia burgdorferi, the Lyme disease spirochete, possesses a surface protein, VlsE, which undergoes antigenic variation. VlsE contains two invariable domains and a variable one that includes six variable and six invariable regions (IRs). Five of the IRs are conserved among strains and genospecies of B. burgdorferi sensu lato. IR(6) is conserved, immunodominant, and exposed at the VlsE surface but not at the spirochete surface, as assessed in vitro. In the present study, the remaining conserved IRs (IR(2) to IR(5)) were investigated. Antisera to synthetic peptides based on each of the IR(2) to IR(5) sequences were produced in rabbits. Antipeptide antibody titers were similarly high in all antisera. Native VlsE was immunoprecipitable with antibodies to IR(2), IR(4), and IR(5) but not to IR(3), indicating that the first three sequences were exposed at the VlsE surface. However, negative surface immunofluorescence and in vitro antibody-mediated killing results indicated that none of the IRs were accessible to antibody at the spirochetal surface in vitro.

Animals↗

PubMed2000

DNA-Binding proteins possibly involved in regulation of the post-logarithmic-phase expression of lipoprotein P35 in Borrelia burgdorferi.

Previously, we have shown that the transcription of p35, a lipoprotein gene of Borrelia burgdorferi, is upregulated or initiated during the post-logarithmic bacterial growth phase in vitro. To identify potential regulatory factors, we examined the formation of DNA-protein complexes by electromobility shift assay, using a 157-bp DNA fragment that spans the p35 promoter region and cell-free extracts of spirochetes harvested from both logarithmic and stationary growth phases. The binding properties of the extracts with the promoter region of the flaB gene, a constitutively expressed, growth-phase-independent gene, were also compared. The results from these experiments demonstrate that B. burgdorferi stationary-phase cell-free extracts have a growth-phase-dependent DNA binding protein that interacts specifically with the p35 promoter region. We show, in addition, that a segment from the 157-bp p35 promoter region which contains both a T-rich stretch and an inverted repeat is able to compete off the stationary-phase-specific complex when the segment is present in molar excess.

Antigens, Bacterial↗

PubMed2000

Characterization of a Borrelia burgdorferi VlsE invariable region useful in canine Lyme disease serodiagnosis by enzyme-linked immunosorbent assay.

Sera collected from dogs experimentally infected with Borrelia burgdorferi by tick inoculation were analyzed for an antibody response to each of the six invariable regions (IRs; i.e., IR(1) to IR(6)) of VlsE, the variable surface antigen of B. burgdorferi. Six synthetic peptides (C(1) to C(6)), which reproduced the six IR sequences were used as peptide-based, enzyme-linked immunosorbent assay (ELISA) antigens. Two IRs, IR(2) and IR(6), were found to be immunodominant. Studies with serially collected serum samples from experimentally infected dogs revealed that the antibody response to IR(6) appears earlier and is stronger than that to IR(2). Thus, the IR(6) sequence alone appeared to be sufficient for serodiagnosis. When C(6) alone was used as antigen, the peptide-based ELISA was positive in 7 of 23 dogs (30%) as early as 3 weeks postinfection. All dogs (n = 33) became strongly positive 1 or 2 weeks later, and this response persisted for the entire study, which lasted for 69 weeks. Of 55 sera submitted by veterinarians from dogs suspected of having Lyme disease, 19 were also positive by the C(6) ELISA, compared to 20 positives detected by immunoblot analysis using cultured B. burgdorferi lysates as antigen. The sensitivity of using C(2) and C(6) together for detecting specific antibody in both experimentally infected and clinically diagnosed dogs was not better than sensitivity with C(6) alone, confirming that C(6) suffices as a diagnostic probe. Moreover, the C(6) ELISA yielded 100% specificity with serum samples collected from 70 healthy dogs, 14 dogs with infections other than B. burgdorferi, and 15 animals vaccinated with either outer surface protein A, whole-spirochete vaccines, or the common puppy-vaccines. Therefore, this C(6) ELISA was both sensitive and specific for the serodiagnosis of canine Lyme disease and could be used with vaccinated dogs.

Amino Acid Sequence↗

PubMed2000

Transcriptional regulation in spirochetes.

Spirochetes belong to a widely diverse family of bacteria. Several species in this family can cause a variety of illnesses including syphilis and Lyme disease. Despite the fact that the complete genome sequence of two species, Borrelia burgdorferi and Treponema pallidum, have been deciphered, much remains to be understood about spirochetal gene regulation. In this review we focus on the environmental transitions that spirochetes undergo during their life cycles and the mechanisms of transcriptional regulation that might possibly mediate spirochetal adaptations to such changes.

Animals↗

PubMed1999

An immunodominant conserved region within the variable domain of VlsE, the variable surface antigen of Borrelia burgdorferi.

Antigenic variation is an effective strategy evolved by pathogenic microbes to avoid immune destruction. Variable Ags such as the variable major protein of Borrelia hermsii, the variant surface glycoprotein of African trypanosomes, and the pilin of Neisseria gonorrhoeae include an immunodominant variable domain and one or more invariable domains that are not antigenic. Short, nonantigenic, invariable regions also may be present within the variable domain. VlsE (variable major protein-like sequence, expressed), the variable surface Ag of Borrelia burgdorferi, the Lyme disease spirochete, also contains both variable and invariable domains. In addition, interspersed within the VlsE variable domain there are six invariable regions (IR1-6) that together amount to half of this portion's primary structure. We show here that these IRs are conserved among strains and genospecies of the B. burgdorferi sensu lato complex. Surprisingly, unlike the invariable regions of variable major protein, variant surface glycoprotein, and pilin, which are not antigenic in natural infections, the most conserved of the IRs, IR6, is immunodominant in Lyme disease patients and in monkeys infected with B. burgdorferi. IR6 is exposed on the surface of VlsE, as assessed by immunoprecipitation experiments, but is inaccessible to Ab on the spirochete's outer membrane, as demonstrated by immunofluorescence and in vitro killing assays. VlsE thus significantly departs from the antigenic variation paradigm, whereby immunodominance is only manifest in variable portions. We submit that IR6 may act as a decoy epitope(s) and contribute to divert the Ab response from other, perhaps protective regions of VlsE.

Amino Acid Sequence↗

PubMed1999

Clinical outcome of a protocol to produce immunosuppression in rhesus monkeys (Macaca mulatta): application to infectious disease and gene therapy studies.

Induced immunosuppression is required for a number of studies using rhesus monkeys (Macaca mulatta). This report describes the clinical outcome and safety of a dose-finding experiment that determined doses of cyclophosphamide and prednisone that could be used to induce a state of immunosuppression in rhesus monkeys. After determining the optimum dose of immunosuppressive agents, the protocol was then used on animals participating in infectious disease and gene therapy studies. Splenectomy was performed in some animals to increase the severity of immunosuppression. The onset, duration, and severity of lymphopenia and leukopenia were consistent in all animals. In most animals, physical examination findings and clinical serum chemistry profiles demonstrated only transient abnormalities. With proper clinical monitoring, combination treatment with cyclophosphamide and prednisone is an effective and safe method for inducing immunosuppression in rhesus monkeys.

Animals↗

PubMed1999

Induction of pro- and anti-inflammatory cytokines by Borrelia burgdorferi lipoproteins in monocytes is mediated by CD14.

We previously showed that heat-killed Borrelia burgdorferi spirochetes and lipidated outer surface protein A (L-OspA) stimulated the in vitro production of interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC) from uninfected humans and rhesus monkeys (G. Giambartolomei et al., Infect. Immun. 66:2691-2697, 1998). Here we demonstrate that uninfected human peripheral blood monocytes, but not B or T cells, are the cells that transcribe the IL-10 cytokine gene in response to heat-killed B. burgdorferi. B. burgdorferi similarly induced an upregulation of the IL-1beta and IL-6 cytokine genes in monocytes and the production of IL-10 and IL-6 in culture supernatants of the human monocytic cell line THP-1. Purified L-OspA (but not unlipidated OspA [U-OspA] or U-OspC) also stimulated the production of both cytokines in THP-1 cells in a dose-dependent fashion, suggesting that acylation of the OspA protein molecule is required for the production of both anti- and pro-inflammatory cytokines in naive monocytes. A lipohexapeptide that contained the tripalmitoyl-modified cysteine motif (Pam3Cys-Hex) of B. burgdorferi lipoproteins but with an arbitrary peptide sequence had the same effect. Monoclonal antibodies (MAbs) MY4 and 60bca, both of which bind to CD14 and are known to block lipopolysaccharide (LPS)-mediated cytokine production, were able to block L-OspA-mediated IL-10 and IL-6 cytokine production. In contrast, MAb 26ic, which also binds to CD14 but does not block LPS function, failed to inhibit L-OspA-mediated cytokine production. These data suggest that activation of monocytes and production of both anti- and pro-inflammatory cytokines induced by lipoproteins proceeds via the CD14 receptor. LPS binding protein was not required for OspA-induced cytokine production. Our results demonstrate that pro- and anti-inflammatory cytokines induced by B. burgdorferi lipoproteins in PBMC are produced by monocytes and that lipoprotein and LPS signaling pathways share at least the initial signaling event that involves the CD14 receptor.

Acute-Phase Proteins↗