Characterization of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidases from Borrelia burgdorferi: Antibiotic targets for Lyme disease.

BACKGROUND: Borrelia burgdorferi causes Lyme disease, the most common tick-borne illness in the United States. The Center for Disease Control and Prevention estimates that the occurrence of Lyme disease in the U.S. has now reached approximately 300,000 cases annually. Early stage Borrelia burgdorferi infections are generally treatable with oral antibiotics, but late stage disease is more difficult to treat and more likely to lead to post-treatment Lyme disease syndrome. METHODS: Here we examine three unique 5'-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidases (MTNs or MTANs, EC 3.2.2.9) responsible for salvage of adenine and methionine in B. burgdorferi and explore their potential as antibiotic targets to treat Lyme disease. Recombinant Borrelia MTNs were expressed and purified from E. coli. The enzymes were extensively characterized for activity, specificity, and inhibition using a UV spectrophotometric assay. In vitro antibiotic activities of MTN inhibitors were assessed using a bioluminescent BacTiter-Glo™ assay. RESULTS: The three Borrelia MTNs showed unique activities against the native substrates MTA, SAH, and 5'-deoxyadenosine. Analysis of substrate analogs revealed that specific activity rapidly dropped as the length of the 5'-alkylthio substitution increased. Non-hydrolysable nucleoside transition state analogs demonstrated sub-nanomolar enzyme inhibition constants. Lastly, two late stage transition state analogs exerted in vitro IC50 values of 0.3-0.4 µg/mL against cultured B. burgdorferi cells. CONCLUSION: B. burgdorferi is unusual in that it expresses three distinct MTNs (cytoplasmic, membrane bound, and secreted) that are effectively inactivated by nucleoside analogs. GENERAL SIGNIFICANCE: The Borrelia MTNs appear to be promising targets for developing new antibiotics to treat Lyme disease.

Lipid Profiles in Lyme Borreliosis: A Potential Role for Apheresis?

Dyslipidemia and dyslipoproteinemia are common causes of metabolic and cardiovascular diseases. On the other hand, intracellular bacteria, such as Borrelia burgdorferi, utilize host lipids to survive and disseminate within the host. Recent data suggest that elevated lipids are a contributing factor to the maintenance and severity of Lyme disease and its complications. Here we review and discuss the role of lipids in Borreliosis and report on a pilot trial to examine the potential roles of circulating lipids and lipoproteins in patients with Borrelia infection. In this analysis we assessed the clinical and lipid profiles of 519 patients (319 women, 200 men) with a proven history of Lyme disease, before and after an extracorporeal double membrane filtration. Lipid profiles pre- and post-apheresis were analyzed in conjunction with clinical symptoms and parameters of inflammation. Circulating cholesterol, triglycerides, LDL, LP(a), and other inflammatory lipids were significantly reduced after the apheresis, while symptoms of the disorder and bioindexes of inflammation such as CRP improved. Further studies should be initiated to investigate the possibly causal relation between Lyme disease and circulating lipids and to design appropriate therapeutic strategies.

Lysosomal ß-glucuronidase regulates Lyme and rheumatoid arthritis severity.

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most prevalent arthropod-borne illness in the United States and remains a clinical and social challenge. The spectrum of disease severity among infected patients suggests that host genetics contribute to pathogenic outcomes, particularly in patients who develop arthritis. Using a forward genetics approach, we identified the lysosomal enzyme ß-glucuronidase (GUSB), a member of a large family of coregulated lysosomal enzymes, as a key regulator of Lyme-associated arthritis severity. Severely arthritic C3H mice possessed a naturally occurring hypomorphic allele, Gusbh. C57BL/6 mice congenic for the C3H Gusb allele were prone to increased Lyme-associated arthritis severity. Radiation chimera experiments revealed that resident joint cells drive arthritis susceptibility. C3H mice expressing WT Gusb as a transgene were protected from severe Lyme arthritis. Importantly, the Gusbh allele also exacerbated disease in a serum transfer model of rheumatoid arthritis. A known GUSB function is the prevention of lysosomal accumulation of glycosaminoglycans (GAGs). Development of Lyme and rheumatoid arthritis in Gusbh-expressing mice was associated with heightened accumulation of GAGs in joint tissue. We propose that GUSB modulates arthritis pathogenesis by preventing accumulation of proinflammatory GAGs within inflamed joint tissue, a trait that may be shared by other lysosomal exoglycosidases.

Alpha fucosidase and beta galactosidase in serum of a Lyme disease patients as a possible marker of accelerated senescence - a preliminary study.

Lyme disease (LD) is the most prevalent tick-borne disease in Europe. LD is caused by the spirochete Borrelia burgdorferi. LD is a chronic disease which can attack a number of organs: skin, heart, brain, joints. Chronic, low-grade inflammation involves general production of pro-inflammatory cytokines and inflammatory markers and is a typical feature of aging. So far, the best method of diagnosing LD is a time-consuming and expensive two-stage serological method. The aim of our study was to evaluate the activity of two lysosomal exoglycosidases: α-fucosidase (FUC) and ß-galactosidase (GAL) in the serum of patients with Lyme disease, as potential markers of LD. Due to the increasing number of patients with Lyme disease and a number of false results, new ways to diagnose this disease are still being sought. As elevated level of ß-galactosidase is a manifestation of residual lysosomal activity in senescent cells, the increase in its activity in serum during chronic Lyme disease might be a marker of a potentially accelerated senescence process. The study was performed on serum taken from cubital veins of 15 patients with Lyme disease and eight healthy subjects (control group). FUC and GAL activity was measured by the method of Chatterjee et al. as modified by Zwierz et al. In the serum of patients with Lyme disease, GAL activity significantly increased (p = 0.029), and the activity of FUC had a tendency to increase (p = 0.153), compared to the control group. A significant increase in GAL activity in the serum of patients with Lyme disease indicates an increased catabolism of glycoconjugates (glycoproteins, glycolipids, proteoglycans) and could be helpful in the diagnosis of Lyme disease, although this requires confirmation in a larger group of patients. As GAL is the most widely used assay for detection of senescent cells, an elevated level of ß-galactosidase might be a manifestation of accelerated senescence process in the course of Lyme disease.

HrpA, a DEAH-box RNA helicase, is involved in global gene regulation in the Lyme disease spirochete.

Spirochetes causing Lyme borreliosis are obligate parasites that can only be found in a tick vector or a vertebrate host. The ability to survive in these two disparate environments requires up and downregulation of specific genes by regulatory circuits that remain largely obscure. In this work on the Lyme spirochete, B. burgdorferi, we show that a disruption of the hrpA gene, which encodes a putative RNA helicase, results in a complete loss in the ability of the spirochetes to infect mice by needle inoculation. Studies of protein expression in culture by 2D gels revealed a change in the expression of 33 proteins in hrpA clones relative to the wild-type parent. Quantitative characterization of protein expression by iTRAQ analysis revealed a total of 187 differentially regulated proteins in an hrpA background: 90 downregulated and 97 upregulated. Forty-two of the 90 downregulated and 65 of the 97 upregulated proteins are not regulated under any conditions by the previously reported regulators in B. burgdorferi (bosR, rrp2, rpoN, rpoS or rrp1). Downregulated and upregulated proteins also fell into distinct functional categories. We conclude that HrpA is part of a new and distinct global regulatory pathway in B. burgdorferi gene expression. Because an HrpA orthologue is present in many bacteria, its participation in global regulation in B. burgdorferi may have relevance in other bacterial species where its function remains obscure. We believe this to be the first report of a role for an RNA helicase in a global regulatory pathway in bacteria. This finding is particularly timely with the recent growth of the field of RNA regulation of gene expression and the ability of RNA helicases to modulate RNA structure and function.

Lyme disease enolpyruvyl-UDP-GlcNAc synthase: fosfomycin-resistant MurA from Borrelia burgdorferi, a fosfomycin-sensitive mutant, and the catalytic role of the active site Asp.

MurAs (enolpyruvyl-UDP-GlcNAc synthases) from pathogenic bacteria such as Borrelia burgdorferi (Lyme disease) and tuberculosis are fosfomycin resistant because an Asp-for-Cys substitution prevents them from being alkylated by this epoxide antibiotic. Previous attempts to characterize naturally Asp-containing MurAs have resulted in no protein or no activity. We have expressed and characterized His-tagged Lyme disease MurA (Bb_MurA(H6)). The protein was most soluble at high salt concentrations but maximally active around physiological ionic strength. The steady-state kinetic parameters at pH 7 were k(cat) = 1.07 ± 0.03 s(-1), K(M,PEP) = 89 ± 12 µM, and K(M,UDP-GlcNAc) = 45 ± 7 µM. Mutating the active site Asp to Cys, D116C, caused a 21-fold decrease in k(cat) and rendered the enzyme fosfomycin sensitive. The pH profile of k(cat) was bell-shaped and centered around pH 5.3 for Bb_MurA(H6), with pK(a1) = 3.8 ± 0.2 and pK(a2) = 7.4 ± 0.2. There was little change in pK(a1) with the D116C mutant, 3.5 ± 0.3, but pK(a2) shifted to >11. This demonstrated that the pK(a2) of 7.4 was due to D116, almost 3 pH units above an unperturbed carboxylate, and that it must be protonated for activity. This supports D116's proposed role as a general acid/base catalyst. As fosfomycin does not react with simple thiols, nor most protein thiols, the reactivity of D116C with fosfomycin, combined with the strongly perturbed pK(a2) for D116, strongly implies an unusual active site environment and a chemical role in catalysis for Asp/Cys. There is also good evidence for C115 having a role in product release. Both roles may be operative for both Asp- and Cys-containing MurAs.

CD14 signaling reciprocally controls collagen deposition and turnover to regulate the development of lyme arthritis.

CD14 is a glycosylphosphatidylinositol-anchored protein expressed primarily on myeloid cells (eg, neutrophils, macrophages, and dendritic cells). CD14(-/-) mice infected with Borrelia burgdorferi, the causative agent of Lyme disease, produce more proinflammatory cytokines and present with greater disease and bacterial burden in infected tissues. Recently, we uncovered a novel mechanism whereby CD14(-/-) macrophages mount a hyperinflammatory response, resulting from their inability to be tolerized by B. burgdorferi. Paradoxically, CD14 deficiency is associated with greater bacterial burden despite the presence of highly activated neutrophils and macrophages and elevated levels of cytokines with potent antimicrobial activities. Killing and clearance of Borrelia, especially in the joints, depend on the recruitment of neutrophils. Neutrophils can migrate in response to chemotactic gradients established through the action of gelatinases (eg, matrix metalloproteinase 9), which degrade collagen components of the extracellular matrix to generate tripeptide fragments of proline-glycine-proline. Using a mouse model of Lyme arthritis, we demonstrate that CD14 deficiency leads to decreased activation of matrix metalloproteinase 9, reduced degradation of collagen, and diminished recruitment of neutrophils. This reduction in neutrophil numbers is associated with greater numbers of Borrelia in infected tissues. Variation in the efficiency of neutrophil-mediated clearance of B. burgdorferi may underlie differences in the severity of Lyme arthritis observed in the patient population and suggests avenues for development of adjunctive therapy designed to augment host immunity.

5-Lipoxygenase-deficient mice infected with Borrelia burgdorferi develop persistent arthritis.

The enzyme 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid into the leukotrienes, which are critical regulators of inflammation and inflammatory diseases, such as asthma and arthritis. Although leukotrienes are present in the synovial fluid of Lyme disease patients, their role in the development of Lyme arthritis has not been determined. In the current study, we used a murine model of Lyme arthritis to investigate the role 5-LO products might have in the development of this inflammatory disease. After infection of Lyme arthritis-susceptible C3H/HeJ mice with Borrelia burgdorferi, mRNA expression of 5-LO and 5-LO-activating protein was induced in the joints, and the 5-LO product leukotriene B(4) was produced. Using C3H 5-LO-deficient mice, we demonstrated that 5-LO activity was not necessary for the induction of Lyme arthritis, but that its deficiency resulted in earlier joint swelling and an inability to resolve arthritis as demonstrated by sustained arthritis pathology through day 60 postinfection. Although production of anti-Borrelia IgG was decreased in 5-LO-deficient mice, bacterial clearance from the joints was unaffected. Phagocytosis of B. burgdorferi and efferocytosis of apoptotic neutrophils was defective in macrophages from 5-LO-deficient mice, and uptake of opsonized spirochetes by neutrophils was reduced. These results demonstrate that products of the 5-LO metabolic pathway are not required for the development of disease in all models of arthritis and that caution should be used when targeting 5-LO as therapy for inflammatory diseases.

Monoamine neurotransmitters as substrates for novel tick sulfotransferases, homology modeling, molecular docking, and enzyme kinetics.

Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the Northeastern United States. Current research focuses on elucidating biochemical pathways that may be disrupted to prevent pathogen transmission, thereby preventing disease. Genome screening reported transcripts coding for two putative sulfotransferases in whole tick extracts of the nymphal and larval stages. Sulfotransferases are known to sulfonate phenolic and alcoholic receptor agonists such as 17ß-estradiol, thereby inactivating the receptor ligands. We used bioinformatic approaches to predict substrates for Ixosc Sult 1 and Ixosc Sult 2 and tested the predictions with biochemical assays. Homology models of 3D protein structure were prepared, and visualization of the electrostatic surface of the ligand binding cavities showed regions of negative electrostatic charge. Molecular docking identified potential substrates including dopamine, R-octopamine and S-octopamine, which docked into Ixosc Sult 1 with favorable binding affinity and correct conformation for sulfonation. Dopamine, but not R- or S-octopamine, also docked into Ixosc Sult 2 in catalytic binding mode. The predictions were confirmed using cytosolic fractions of whole tick extracts. Dopamine was a good substrate (K(m) = 0.1-0.4 µM) for the native Ixodes scapularis sulfotransferases from larval and nymphal stages regardless of their fed/unfed status. Octopamine sulfonation was detected only after feeding when gene expression data suggests that Ixosc Sult 1 is present. Because dopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function(s) of Ixosc Sult 1 or 2 may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine.

Disordered lymphoid purine metabolism contributes to the pathogenesis of persistent Borrelia garinii infection in mice.

Extracellular ATP and adenosine are important regulators of immune responses; however, contribution of purinergic signaling to host defense during persistent microbial infections remains obscure. Lyme borreliosis is a common arthropod-borne infection caused by Borrelia burgdorferi sensu lato. In this study, we investigated whether lymphoid purinergic signaling contributes to the mechanisms by which borreliae species evade the immune system and trigger joint inflammation. Intracutaneous inoculation of Borrelia garinii to C3H/He mice induced symptomatic infection manifested in elevated levels of borrelia-specific IgG Abs, persistent spirochete dissemination into the tissues and joint swelling, as well as approximately 2- to 2.5-fold enlargement of draining lymph nodes with hyperplasia of B cell follicle area and L-selectin shedding from activated T lymphocytes. Purine catabolism was also activated in lymph nodes but not spleen and blood of infected C3H/He mice within the first 4 postinfection weeks, particularly manifested in transient upregulations of adenosine triphosphatase/ectonucleoside triphosphate diphosphohydrolase and ecto-5'-nucleotidase/CD73 on CD4(+)CD8(+) T lymphocytes and adenosine deaminase activity on B220(+) B lymphocytes. Compared with borrelia-susceptible C3H/He strain, lymphocytes from C57BL/6 mice displayed markedly enhanced adenosine-generating capability due to approximately three times higher ratio of ecto-5'-nucleotidase to adenosine deaminase. Borrelia-infected C57BL/6 mice efficiently eradicated the inoculated spirochetes at more chronic stage without any signs of arthritis. Strikingly, deletion of key adenosine-generating enzyme, ecto-5'-nucleotidase/CD73, was accompanied by significantly enhanced joint swelling in borrelia-infected CD73-deficient C57BL/6 mice. Collectively, these data suggest that insufficient basal adenosine level and/or pathogen-induced disordered lymphoid purine homeostasis may serve as important prerequisite for promotion of inflammatory responses and further host's commitment to persistence of bacterial infection and arthritis development.

Cyclooxygenase-1 orchestrates germinal center formation and antibody class-switch via regulation of IL-17.

The cyclooxygenase (COX) enzymes are known modulators of innate immune cell function; however, their contributions to adaptive immunity are relatively unknown. We investigated the roles of COX-1 and COX-2 in the humoral immune response to infection with the Lyme disease pathogen Borrelia burgdorferi. We report that in vitro, murine B cells constitutively expressed COX-1 and up-regulated expression of both COX-1 and COX-2 as well as their products PGE(2), PGF(2alpha), and thromboxane B(2) and their receptors following stimulation with B. burgdorferi or anti-CD40. In vitro inhibition of COX-1 and/or COX-2 in murine B cells resulted in decreased eicosanoid production and altered Ab production. Importantly, infection of mice lacking COX-1, but not COX-2, activity resulted in a defect in Ig class-switching and a lack of Borrelia-specific IgG production. This defect correlated with decreased germinal center formation and IL-6 and IL-17 production, and it could be partially recovered by restoration of IL-6, but fully recovered by IL-17. Furthermore, sera from COX-1 inhibitor-treated mice were dramatically less effective in killing B. burgdorferi, but borreliacidal activity was restored in COX-1 inhibitor-treated mice administered IL-17. We conclude that IL-17 plays a role in Ab production and Ig class-switching in response to infection and that COX-1 is a critical, previously unrecognized regulator of this response.

[Elevated transaminases - what to do if everything was done?]. / Erhöhte Transaminasen - wie weiter, wenn bereits alles gesucht wurde?

Transaminases, gamma-GT and alcalic phosphatase are classically termed as liver enzymes, however they can be found in almost every organ. Elevated levels of the transaminases ALAT (alanin-aminotransferase) and ASAT (aspartat-aminotransferase) are signs of disturbed permeability of the cells, in which these enzymes can be found. In contrast to ALAT, which is mainly liver-specific, the ASAT is found in other organs as well, e.g. heart and skeletal muscle. At a mild elevation of these enzymes a reevaluation is recommended, however if an elevation persists and is suspicious for a liver disease, a specific work up is necessary. In this manuscript, we discuss often overlooked problems and provide a diagnostic algorithm for the workup of elevated liver enzymes.

Activation of gamma delta T cells by Borrelia burgdorferi is indirect via a TLR- and caspase-dependent pathway.

Activation of the innate immune system typically precedes engagement of adaptive immunity. Cells at the interface between these two arms of the immune response are thus critical to provide full engagement of host defense. Among the innate T cells at this interface are gammadelta T cells. gammadelta T cells contribute to the defense from a variety of infectious organisms, yet little is understood regarding how they are activated. We have previously observed that human gammadelta T cells of the Vdelta1 subset accumulate in inflamed joints in Lyme arthritis and proliferate in response to stimulation with the causative spirochete, Borrelia burgdorferi. We now observe that murine gammadelta T cells are also activated by B. burgdorferi and that in both cases the activation is indirect via TLR stimulation on dendritic cells or monocytes. Furthermore, B. burgdorferi stimulation of monocytes via TLR, and secondary activation of gammadelta T cells, are both caspase-dependent.

[Activity of the caspase-3 in the culture of peripheral blood mononuclear cells stimulated with Borrelia burgdorferi antigens]. / Aktywnosc kaspazy 3 w hodowli komórek jednojadrzastych krwi obwodowej stymulowanych antygenami Borrelia burgdorferi.

OBJECTIVE: To investigate whether the decreased rate of apoptosis of peripheral blood mononuclear cells occurs in patients with chronic Lyme arthritis (LA), which could contribute to persistent inflammation in those patients. METHODS: PBMC from 7 patients with LA and 6 healthy persons (control--C) were incubated for 48 hours without stimulation (negative control) or under antigenic stimulation with Borrelia burgdorferi s.l. spirochetes: Borrelia afzelii VS46110 (B.a.), B. garinii 20047 (B.g.) or B. burgdorferi sensu stricte B-31 (B.ss.). Concentration of the apoptosis mediator active caspase-3, was measured by ELISA in the culture homogenate. RESULTS: Mean active caspase-3 concentration was higher in LA than in C in unstimulated culture. Increase of active caspase-3 concentration under antigenic stimulation occurred in C (significant for B.g.), but not in LA. CONCLUSIONS: 1) apoptosis rate of PBMC increases in presence of B. burgdorferi s.l., which may be a defense mechanism of this pathogen; 2) in late LA there is increased baseline susceptibility of PBMC to apoptosis, but exposition to B. burgdorferi s.l. seem not to exert further pro-apoptotic effect. This altered response to pro-apoptotic stimuli may be related to persistent inflammatory pathology in this group of patients.

[Usefulness of examinations of serum levels of matrix metalloproteinases 1, MMP-3, MMP-9, tissue inhibitor of metalloproteinases 1, hyaluronic acid and antibodies against cyclic citrullinated peptide in Lyme arthritis, rheumatoid arthritis and patients with arthritic complaints]. / Przydatnosc badania wybranych metaloproteinaz (MMP-3, MMP-9) i ich inhibitora TIMP-1, a takze kwasu hialuronowego (HA) i przeciwcial anty-CCP w róznicowaniu Lyme arthritis, rzs i grupy osób z dolegliwosciami bólowymi kostno-stawowymi, bez zakazenia Borrelia burgdorferi--doniesienie wstepne.

Lyme disease is a multisystem disease that can affect skin, nervous system, heart and joints. Lyme arthritis can develope in about 60% of "not treated" Lyme disease patients, 10% of patients may develope chronic arthritis. Lyme arhritis symptoms (especially chronic arthritis) is similar to rheumatoid arthritis. The purpose of this study was to establish the usefulness of examinations of serum levels of matrix metalloproteinases MMP-3, MMP-9, tissue inhibitor of metalloproteinases 1 (TIMP-1), hialuronic acid (HA) and antibodies against cyclic citrullinated peptide (anti-CCP) in Lyme arthritis, rheumatoid arthritis (RA) and patients with arthritic complaints. Plasma levels of MMP-3, HA and anti-CCP were significantly higher in RA group than in Lyme arthritis group and patients with arthritic complaints. There were no significant differences in serum levels of MMP-3, MMP-9, TIMP-1, HA, anti-CCP between Lyme arthritis patients and patients with arthritic complaints and these parameters are not usefull in differential diagnoses of Lyme arthritis.

CD14 mediates cross talk between mononuclear cells and fibroblasts for upregulation of matrix metalloproteinase 9 by Borrelia burgdorferi.

Lyme disease is an infection caused by a tick-borne spirochete, Borrelia burgdorferi. Matrix metalloproteinase 9 (MMP-9) was selectively upregulated in the erythema migrans skin lesions of patients with acute Lyme disease. In this study, the mechanism of upregulation of MMP-9 was investigated in vitro and in vivo. The concentrations of MMP-9 and soluble CD14 were markedly elevated in serum from patients with acute Lyme disease and were also upregulated in U937 cells by B. burgdorferi in a time- and concentration-dependent manner. MMP-9 mRNA was expressed at baseline in fibroblasts in the presence or absence of B. burgdorferi. However, when fibroblasts were incubated with supernatants from U937 cells with B. burgdorferi or recombinant CD14, the expression of MMP-9 was significantly increased. This effect was completely abolished by the anti-CD14 antibody. These data suggest that the upregulation of MMP-9 by B. burgdorferi involves the CD14 pathway in infiltrating inflammatory cells. Fibroblasts could be recruited to amplify local production of MMP-9 by acquiring CD14 from macrophages.

Role of novel protein kinase C isoforms in Lyme arthritis.

Inflammation caused by Borrelia burgdorferi infection occurs as a result of induction of pro-inflammatory cytokines from activation of multiple signalling pathways. It has previously been shown that mitogen-activated protein kinase (MAPK) and Janus kinase/signal transducer and activator of transcription signalling pathways are activated by B. burgdorferi in cultured human chondrocytes. Protein kinase C (PKC) signalling pathways are potential candidates that may control these downstream signalling pathways. Here we show that B. burgdorferi infection leads to phosphorylation and activation of novel PKC isoforms (PKC delta, epsilon, eta and theta) in a time-dependent manner. A specific inhibitor of novel PKC isoforms blocked the induction of pro-inflammatory molecules in response to B. burgdorferi infection as did transient transfection of novel PKC dominant-negative plasmids into chondrocytes. B. burgdorferi-induced p38 MAPK phosphorylation was also significantly inhibited by an inhibitor of novel PKC isoforms, suggesting that PKC activation occurs upstream of p38 activation. In vivo, administration of an inhibitor of classical and novel PKC isoforms to C3H/HeN mice infected with B. burgdorferi resulted in significantly reduced ankle inflammation and swelling. In conclusion, these data suggest that novel PKC isoforms are specifically activated by B. burgdorferi infection and this can contribute to the regulation of inflammation in vitro and in vivo.

Control of Borrelia burgdorferi-specific CD4+-T-cell effector function by interleukin-12- and T-cell receptor-induced p38 mitogen-activated protein kinase activity.

Infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in a Th1 response and proinflammatory cytokine production. Mice deficient for MKK3, an upstream activator of p38 mitogen-activated protein (MAP) kinase, develop a lower Th1 response and exhibit an impaired ability to produce proinflammatory cytokines upon infection with the spirochete. We investigated the contribution of p38 MAP kinase activity in gamma interferon (IFN-gamma) production in CD4+ T cells in response to specific antigen through T-cell receptor (TCR)- and interleukin-12 (IL-12)-mediated signals. The specific inhibition of p38 MAP kinase in T cells and the administration of a pharmacological inhibitor of the kinase during the course of infection with the spirochete resulted in reduced levels of IFN-gamma in the sera of infected mice. Our results also demonstrate that although p38 MAP kinase activity is not required for the differentiation of B. burgdorferi-specific CD4+ T cells, the production of IFN-gamma by Th1 effector cells is regulated by the kinase. Both TCR engagement and IL-12 induced the production of the Th1 cytokine through the activation of the p38 MAP kinase pathway. Thus, the inhibition of this pathway in vitro resulted in decreased levels of IFN-gamma during restimulation of B. burgdorferi-specific T cells in response to anti-CD3 and IL-12 stimulation. These results clarify the specific contribution of the p38 MAP kinase in the overall immune response to the spirochete and its role in the effector function of B. burgdorferi-specific T cells.

Activity of lysosomal exoglycosidases in the serum of patients with chronic Lyme arthritis.

To examine the role of lysosomal exoglycosidases in the pathogenesis of Lyme arthritis we studied a group of 18 patients aged 18-72 (mean: 46 yr) diagnosed with chronic arthritis in the course of borreliosis. The control group was composed of 20 healthy volunteers (health service employees) aged 25-65 (mean: 45 yr) with no detectable serum anti-Borrelia burgdorferi antibodies. We found that N-acetyl-beta-d-hexosaminidase (HEX) was significantly increased and beta-galactosidase and alpha-mannosidase also showed an increase in Lyme arthritis patients compared to healthy persons and normalised after treatment with doxycycline. Our results suggest that HEX is a sensitive enzymatic marker of Lyme arthritis and it may be used to monitor the course of the disease and the efficiency of treatment.

Bb2Bb3 regulation of murine Lyme arthritis is distinct from Ncf1 and independent of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase.

Several quantitative trait loci regulating murine Lyme arthritis severity have been mapped, including a highly significant linkage found on chromosome 5, termed Bb2Bb3. Within this region, the Ncf1 gene of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been identified as a major regulator of arthritis severity in rodent models of rheumatoid arthritis, an effect attributed to protective properties of reactive oxygen species. To assess the role of Ncf1 in Lyme arthritis, we introgressed Bb2Bb3 from severely arthritic C3H/He mice onto mildly arthritic C57BL/6 mice. This increased Lyme arthritis severity, whereas the reciprocal transfer conferred protection from disease. A single nucleotide polymorphism was identified in the Ncf1 gene that did not influence the protein sequence or expression of Ncf1. Although polymorphonuclear leukocytes from C57BL/6 mice generated a greater oxidative burst than polymorphonuclear leukocytes from C3H/He mice, studies with the Bb2Bb3 congenic mice demonstrated this difference was not linked to Ncf1 alleles. Furthermore, Lyme arthritis severity was not altered in mice lacking either the Ncf1 or Gp91phox subunits of the NADPH oxidase complex. Together, these results argue that Ncf1 is not a candidate gene for regulation of Lyme arthritis and reveal Lyme arthritis to be independent of NADPH oxidase activity, distinguishing it from other models of rheumatoid arthritis.