A selective antibiotic for Lyme disease.

Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

Azlocillin can be the potential drug candidate against drug-tolerant Borrelia burgdorferi sensu stricto JLB31.

Lyme disease is one of most common vector-borne diseases, reporting more than 300,000 cases annually in the United States. Treating Lyme disease during its initial stages with traditional tetracycline antibiotics is effective. However, 10-20% of patients treated with antibiotic therapy still shows prolonged symptoms of fatigue, musculoskeletal pain, and perceived cognitive impairment. When these symptoms persists for more than 6 months to years after completing conventional antibiotics treatment are called post-treatment Lyme disease syndrome (PTLDS). Though the exact reason for the prolongation of post treatment symptoms are not known, the growing evidence from recent studies suggests it might be due to the existence of drug-tolerant persisters. In order to identify effective drug molecules that kill drug-tolerant borrelia we have tested two antibiotics, azlocillin and cefotaxime that were identified by us earlier. The in vitro efficacy studies of azlocillin and cefotaxime on drug-tolerant persisters were done by semisolid plating method. The results obtained were compared with one of the currently prescribed antibiotic doxycycline. We found that azlocillin completely kills late log phase and 7-10 days old stationary phase B. burgdorferi. Our results also demonstrate that azlocillin and cefotaxime can effectively kill in vitro doxycycline-tolerant B. burgdorferi. Moreover, the combination drug treatment of azlocillin and cefotaxime effectively killed doxycycline-tolerant B. burgdorferi. Furthermore, when tested in vivo, azlocillin has shown good efficacy against B. burgdorferi in mice model. These seminal findings strongly suggests that azlocillin can be effective in treating B. burgdorferi sensu stricto JLB31 infection and furthermore in depth research is necessary to evaluate its potential use for Lyme disease therapy.

The Borrelia burgdorferi VlsE Lipoprotein Prevents Antibody Binding to an Arthritis-Related Surface Antigen.

Arp is an immunogenic protein of the Lyme disease spirochete Borrelia burgdorferi and contributes to joint inflammation during infection. Despite Arp eliciting a strong humoral response, antibodies fail to clear the infection. Given previous evidence of immune avoidance mediated by the antigenically variable lipoprotein of B. burgdorferi, VlsE, we use passive immunization assays to examine whether VlsE protects the pathogen from anti-Arp antibodies. The results show that spirochetes are only able to successfully infect passively immunized mice when VlsE is expressed. Subsequent immunofluorescence assays reveal that VlsE prevents binding of Arp-specific antibodies, thereby providing an explanation for the failure of Arp antisera to clear the infection. The results also show that the shielding effect of VlsE is not universal for all B. burgdorferi cell-surface antigens. The findings reported here represent a direct demonstration of VlsE-mediated protection of a specific B. burgdorferi surface antigen through a possible epitope-shielding mechanism.

Holistic Approach in Patients With Presumed Lyme Borreliosis Leads to Less Than 10% of Confirmation and More Than 80% of Antibiotic Failures.

BACKGROUND: There is no precise idea whether patients with chronic symptoms attributed to Lyme borreliosis (LB) have LB or another disease. METHODS: We evaluated patients consulting for a presumed LB with a holistic approach including presumptive treatment. We included symptomatic patients consulting for presumed LB. They were classified as confirmed LB when they met four criteria, and possible LB if three with a positive clinical response to presumptive treatment. RESULTS: Amongst the 301 patients, 275 (91%) were exposed to tick bites, and 165 (54%) were bitten by a tick. At presentation, 151 patients (50.1%) had already been treated with a median of one (1-22) course of antimicrobials, during 34 (28-730) days. Median number of symptoms was three (1-12) with a median duration of 16 (1-68) months. Median number of signs was zero (0-2). ELISA was positive in 84/295 (28.4%) for IgM and 86/295 (29.1%) for IgG, and immunoblot was positive in 21/191 (10.9%) for IgM and 50/191 (26.1 %) for IgG. Presumptive treatment after presentation failed in 46/88 patients (52%). Diagnosis of LB was confirmed in 29 patients (9.6%), and possible in 9 (2.9%). Of the 243 patients with non-LB diagnosis, diseases were psychological, musculoskeletal, neurological or other origin in 76 (31.2%), 48 (19.7%), 37 (15.2%) and 82 (33.7%) patients respectively. Patients with other diseases were significantly younger, having more symptoms, longest duration of symptoms, less clinical signs and less frequent LB positive serologies. CONCLUSIONS: Overdiagnosis and overtreatment of LB is worsening. Health authorities should investigate this phenomenon.

Short-Chain Fatty Acids Alter Metabolic and Virulence Attributes of Borrelia burgdorferi.

Borrelia burgdorferi responds to a variety of host-derived factors and appropriately alters its gene expression for adaptation under different host-specific conditions. We previously showed that various levels of acetate, a short-chain fatty acid (SCFA), altered the protein profile of B. burgdorferi In this study, we determined the effects of other physiologically relevant SCFAs in the regulation of metabolic/virulence-associated proteins using mutant borrelial strains. No apparent increase in the synthesis of outer surface protein C (OspC) was noted when a carbon storage regulator A (csrA of B. burgdorferi, or csrABb ) mutant (mt) was propagated within dialysis membrane chambers implanted within rat peritoneal cavity, while the parental wild type (wt; B31-A3 strain) and csrABb cis-complemented strain (ct) had increased OspC with a reciprocal reduction in OspA levels. Growth rates of wt, mt, ct, 7D (csrABb mutant lacking 7 amino acids at the C terminus), and 8S (csrABb with site-specific changes altering its RNA-binding properties) borrelial strains were similar in the presence of acetate. Increased levels of propionate and butyrate reduced the growth rates of all strains tested, with mt and 8S exhibiting profound growth deficits at higher concentrations of propionate. Transcriptional levels of rpoS and ospC were elevated on supplementation of SCFAs compared to those of untreated spirochetes. Immunoblot analysis revealed elevated levels of RpoS, OspC, and DbpA with increased levels of SCFAs. Physiological levels of SCFAs prevalent in select human and rodent fluids were synergistic with mammalian host temperature and pH to increase the levels of aforementioned proteins, which could impact the colonization of B. burgdorferi during the mammalian phase of infection.

Induction of Interleukin 10 by Borrelia burgdorferi Is Regulated by the Action of CD14-Dependent p38 Mitogen-Activated Protein Kinase and cAMP-Mediated Chromatin Remodeling.

Host genotype influences the severity of murine Lyme borreliosis, caused by the spirochetal bacterium Borrelia burgdorferi C57BL/6 (B6) mice develop mild Lyme arthritis, whereas C3H/HeN (C3H) mice develop severe Lyme arthritis. Differential expression of interleukin 10 (IL-10) has long been associated with mouse strain differences in Lyme pathogenesis; however, the underlying mechanism(s) of this genotype-specific IL-10 regulation remained elusive. Herein we reveal a cAMP-mediated mechanism of IL-10 regulation in B6 macrophages that is substantially diminished in C3H macrophages. Under cAMP and CD14-p38 mitogen-activated protein kinase (MAPK) signaling, B6 macrophages stimulated with B. burgdorferi produce increased amounts of IL-10 and decreased levels of arthritogenic cytokines, including tumor necrosis factor (TNF). cAMP relaxes chromatin, while p38 increases binding of the transcription factors signal transducer and activator of transcription 3 (STAT3) and specific protein 1 (SP1) to the IL-10 promoter, leading to increased IL-10 production in B6 bone marrow-derived monocytes (BMDMs). Conversely, macrophages derived from arthritis-susceptible C3H mice possess significantly less endogenous cAMP, produce less IL-10, and thus are ill equipped to mitigate the damaging consequences of B. burgdorferi-induced TNF. Intriguingly, an altered balance between anti-inflammatory and proinflammatory cytokines and CD14-dependent regulatory mechanisms also is operative in primary human peripheral blood-derived monocytes, providing potential insight into the clinical spectrum of human Lyme disease. In line with this notion, we have demonstrated that cAMP-enhancing drugs increase IL-10 production in myeloid cells, thus curtailing inflammation associated with murine Lyme borreliosis. Discovery of novel treatments or repurposing of FDA-approved cAMP-modulating medications may be a promising avenue for treatment of patients with adverse clinical outcomes, including certain post-Lyme complications, in whom dysregulated immune responses may play a role.

Targeting of vascular adhesion protein-1 by positron emission tomography visualizes sites of inflammation in Borrelia burgdorferi-infected mice.

BACKGROUND: In the present study, we sought to evaluate the feasibility of targeting vascular adhesion protein-1 (VAP-1) by positron emission tomography (PET) for the longitudinal quantitative assessment of Borrelia burgdorferi infection-induced inflammation in mice. METHODS: Mice with B. burgdorferi infection-induced arthritis were studied. During a 7-week follow-up period, the progression of arthritis was monitored weekly with 68Ga-DOTA-Siglec-9 PET/computed tomography (CT) and measurement of tibiotarsal joint swellings. A subgroup of infected mice was treated with ceftriaxone. Finally, histopathological assessment of joint inflammation was performed and VAP-1 expression in joints were determined. RESULTS: Explicit joint swelling and 68Ga-DOTA-Siglec-9 uptake could be demonstrated in the affected joints from B. burgdorferi-infected mice. By contrast, no obvious accumulation of 68Ga-DOTA-Siglec-9 was detected in joints of uninfected mice. The maximum swelling and highest uptake in the affected joints were observed 4 weeks after the infection. 68Ga-DOTA-Siglec-9 uptake in joints correlated with joint swelling (P < 0.0001) and histopathological scoring of inflammation (P = 0.020). Despite short-term antibiotic treatment, the arthritis persisted, and the PET signal remained as high as in nontreated mice. Immunohistochemistry revealed strong-to-moderate expression of VAP-1 in the synovium of B. burgdorferi-infected mice, while only weak expression of VAP-1 was detected in uninfected mice. CONCLUSIONS: The present study showed that 68Ga-DOTA-Siglec-9 can detect B. burgdorferi infection-induced arthritis in mice. Furthermore, longitudinal PET/CT imaging allowed monitoring of arthritis development over time.

Nonpyrogenic Molecular Adjuvants Based on norAbu-Muramyldipeptide and norAbu-Glucosaminyl Muramyldipeptide: Synthesis, Molecular Mechanisms of Action, and Biological Activities in Vitro and in Vivo.

Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).

CD4+ cell-derived interleukin-17 in a model of dysregulated, Borrelia-induced arthritis.

Lyme borreliosis, which is caused in the United States by the spirochete Borrelia burgdorferi, may manifest as different arrays of signs, symptoms and severities between infected individuals. Recent studies have indicated that particularly severe forms of Lyme borreliosis in humans are associated with an increased Th17 response. Here, we hypothesized that a murine model combining the dysregulated immune response of an environment lacking interleukin-10 (IL-10) with a robust T-cell-driven inflammatory response would reflect arthritis associated with the production of IL-17 by CD4+ cells. We demonstrate that IL-10 regulates the production of IL-17 by Borrelia-primed CD4+ cells early after interaction with Lyme spirochetes in vitro and that infection of Borrelia-primed mice with B. burgdorferi leads to significant production of IL-17 that contributes to the development of severe arthritis. These results extend our previous findings by demonstrating that a dysregulated adaptive immune response to Lyme spirochetes can contribute to severe, Th17-associated arthritis. These findings may lead to therapeutic measures for individuals with particularly severe symptoms of Lyme borreliosis.

Arthritis is developed in Borrelia-primed and -infected mice deficient of interleukin-17.

Interleukin-17 (IL-17) has been shown to participate in the development of Lyme arthritis in experimental mice. For example, neutralization of IL-17 with antibodies inhibits induction of arthritis in Borrelia-primed and -infected C57BL/6 wild-type mice. We hypothesized that mice lacking IL-17 would fail to develop Borrelia-induced arthritis. IL-17-deficient and wild-type C57BL/6 mice were primed with heat-inactivated Borrelia and then infected with viable spirochetes 3 weeks later. No swelling or major histopathological changes of the hind paws were detected in IL-17-deficient or wild-type mice that were primed with Borrelia or infected with viable spirochetes. By contrast, IL-17-deficient and wild-type mice that were primed and subsequently infected with heterologous Borrelia developed severe swelling and histopathological changes of the hind paws. In addition, Borrelia-primed and -infected IL-17-deficient mice exhibited elevated gamma-interferon (IFN-γ) levels in sera and increased frequencies of IFN-γ-expressing lymphocytes in popliteal lymph nodes compared to Borrelia-primed and -infected wild-type mice. These results demonstrate that IL-17 is not required for development of severe pathology in response to infection with Borrelia burgdorferi, but may contribute to disease through an interaction with IFN-γ.

Antagonistic Interplay between MicroRNA-155 and IL-10 during Lyme Carditis and Arthritis.

MicroRNA-155 has been shown to play a role in immune activation and inflammation, and is suppressed by IL-10, an important anti-inflammatory cytokine. The established involvement of IL-10 in the murine model of Borrelia burgdorferi-induced Lyme arthritis and carditis allowed us to assess the interplay between IL-10 and miR-155 in vivo. As reported previously, Mir155 was highly upregulated in joints from infected severely arthritic B6 Il10-/- mice, but not in mildly arthritic B6 mice. In infected hearts, Mir155 was upregulated in both strains, suggesting a role of miR-155 in Lyme carditis. Using B. burgdorferi-infected B6, Mir155-/-, Il10-/-, and Mir155-/- Il10-/- double-knockout (DKO) mice, we found that anti-inflammatory IL-10 and pro-inflammatory miR-155 have opposite and somewhat compensatory effects on myeloid cell activity, cytokine production, and antibody response. Both IL-10 and miR-155 were required for suppression of Lyme carditis. Infected Mir155-/- mice developed moderate/severe carditis, had higher B. burgdorferi numbers, and had reduced Th1 cytokine expression in hearts. In contrast, while Il10-/- and DKO mice also developed severe carditis, hearts had reduced bacterial numbers and elevated Th1 and innate cytokine expression. Surprisingly, miR-155 had little effect on Lyme arthritis. These results show that antagonistic interplay between IL-10 and miR-155 is required to balance host defense and immune activation in vivo, and this balance is particularly important for suppression of Lyme carditis. These results also highlight tissue-specific differences in Lyme arthritis and carditis pathogenesis, and reveal the importance of IL-10-mediated regulation of miR-155 in maintaining healthy immunity.

The role of infectious agents, antibiotics, and antiviral therapy in the treatment of extranodal marginal zone lymphoma and other low-grade lymphomas.

OPINION STATEMENT: There is strong evidence to corroborate the association with Helicobacter pylori (Hp) to gastric extranodal marginal zone lymphoma (ENMZL) and hepatitis C virus (HCV) to splenic/nodal marginal zone lymphoma. Koch's postulates generally hold for these two associations and eradication of the infectious agent is well supported. Hp eradication (HPE) is recommended as front-line therapy for early stage gastric ENMZL regardless of Hp status. Complete response (CR) rate for Hp-negative patients is not as high as for Hp-positive patients; however, the benign nature of HPE and high rates of salvage allow this strategy to be safe while sparing some Hp-negative patients from systemic therapy or radiation. Similarly for HCV-seropositive patients, treatment with antivirals should be strongly considered as first-line for those who do not require immediate cytoreductive therapy or at some point even after completing chemoimmunotherapy. The controversy regarding the role for antibiotics is greatest for primary ocular adnexal lymphoma (POAL). Considering the low incidence of Chlamydia psittaci (Cp) infection with OAL and the challenges to reliably identifying Cp, we typically do not consider doxycycline in POAL treatment. Involved-field radiotherapy (IFRT) remains the treatment of choice for most with unilateral POAL. However, if reliable detection of Cp is available and Cp is identified, patients with unilateral low tumor stage POAL who do not require immediate radiotherapy could be considered for doxycycline as front-line treatment. Other infectious associations to indolent lymphomas have been made, including Borrelia borgdorferi (Bb) in cutaneous lymphoma and Campylobacter in immunoproliferative small intestinal disease (IPSID), but these associations are not as strong and primary treatment targeting the infectious agents is not recommended.

Using new non-invasive quick method to detect Borrelia Burgdorferi (B.B.) infection from specific parts of the heart in "seemingly normal" ECGs, and from the ECGs of Atrial Fibrillation (AF), a majority of AF ECGs are found to have: 1) Significant B.B. infection, 2) Markedly increased ANP, 3) Increased Cardiac Troponin I & 4) Markedly reduced Taurine. These 4 factors were mainly localized at infected areas of the SA node area, R-&L-Atria & pulmonary veins at the L-atrium.

Lyme disease is found in a majority of people we tested. Once Borrelia Burgdorferi (B.B.) spirochete enters human body, it not only causes pain by infecting joints, but it also often enters the brain and the heart. Infection of brain can be quickly detected from the pupil and infection of the heart by ECGs non-invasively. By evaluating recorded ECGs of atrial fibrillation (AF), using U.S. patented non-invasive highly sensitive electromagnetic field (EMF) resonance phenomenon between 2 identical molecules or between a molecule and its antibody, we examined 25 different AF patients' ECGs and found the majority of them suffer from various degrees of B.B. spirochete infection in SA node areas, also in the right & left atria, and pulmonary vein near and around its junction at left atrium & lesser degrees of infection at the AV node & His Bundle. When B.B. infection reaches over 224-600ng or higher at these areas, AF often appears in the majority of all AF analyzed. In order to develop AF, the 4 abnormal factors must be present simultaneously: 1) B.B. infection must be increased to 224-600ng or higher, 2) Atrial Natriuretic Peptide (ANP) must be markedly reduced from normal value of less than 4ng to over 100-400ng, 3) A significant increase of Cardiac Troponin I from normal value of less than 3ng to over 12ng and 4) Taurine must also be markedly reduced from normal value of 4-6ng to 0.25ng. These 4 changes were mainly found only at infected sites of the SA node area, both atria and between the end of the T wave & the beginning of the SA node area, which corresponds to U waves at recorded ECG. Origin of the U wave is mainly due to abnormal electrical potential of pulmonary vein at L-atrium. If all 4 factors do not occur at the infection site, no AF will develop. In seemingly normal ECGs, if using this method, one can detect invisible B.B. infection in early stages. Long before AF appears, AF can be prevented by improved treatment with Amoxicillin 500ng 3 times/day + Taurine 175mg x 3 times/day, with or without EPA 180 mg & DHA 120 mg, to avoid serious current limitations in the use of Doxycycline 100 mg 2 times/day, for 4 weeks.

Murine Borrelia arthritis is highly dependent on ASC and caspase-1, but independent of NLRP3.

INTRODUCTION: The protein platform called the NOD-like-receptor -family member (NLRP)-3 inflammasome needs to be activated to process intracellular caspase-1. Active caspase-1 is able to cleave pro-Interleukin (IL)-1ß, resulting in bioactive IL-1ß. IL-1ß is a potent proinflammatory cytokine, and thought to play a key role in the pathogenesis of Lyme arthritis, a common manifestation of Borrelia burgdorferi infection. The precise pathways through which B. burgdorferi recognition leads to inflammasome activation and processing of IL-1ß in Lyme arthritis has not been elucidated. In the present study, we investigated the contribution of several pattern recognition receptors and inflammasome components in a novel murine model of Lyme arthritis. METHODS: Lyme arthritis was elicited by live B. burgdorferi, injected intra-articularly in knee joints of mice. To identify the relevant pathway components, the model was applied to wild-type, NLRP3-/-, ASC-/-, caspase-1-/-, NOD1-/-, NOD2-/-, and RICK-/- mice. As a control, TLR2-/-, Myd88-/- and IL-1R-/- mice were used. Peritoneal macrophages and bone marrow-derived macrophages were used for in vitro cytokine production and inflammasome activation studies. Joint inflammation was analyzed in synovial specimens and whole knee joints. Mann-Whitney U tests were used to detect statistical differences. RESULTS: We demonstrate that ASC/caspase-1-driven IL-1ß is crucial for induction of B. burgdorferi-induced murine Lyme arthritis. In addition, we show that B. burgdorferi-induced murine Lyme arthritis is less dependent on NOD1/NOD2/RICK pathways while the TLR2-MyD88 pathway is crucial. CONCLUSIONS: Murine Lyme arthritis is strongly dependent on IL-1 production, and B. burgdorferi induces inflammasome-mediated caspase-1 activation. Next to that, murine Lyme arthritis is ASC- and caspase-1-dependent, but NLRP3, NOD1, NOD2, and RICK independent. Also, caspase-1 activation by B. burgdorferi is dependent on TLR2 and MyD88. Based on present results indicating that IL-1 is one of the major mediators in Lyme arthritis, there is a rationale to propose that neutralizing IL-1 activity may also have beneficial effects in chronic Lyme arthritis.

N-terminal disulfide-bridging of Borrelia outer surface protein C increases its diagnostic and vaccine potentials.

OspC is the main target for IgM in early-stage Lyme disease. As such it is employed as its native or recombinant form in routine immunoassays for the determination of Borrelia-specific antibodies. However, recombinant OspC has so far not shown the antigenicity of the native protein. The latter contains an intrinsic signal sequence and an adjacent cysteine residue, the attachment site of the lipid membrane anchor which has been discussed to have an adjuvant effect on the immune reaction. In expression experiments, we have found a recombinant variant, an OspC covalently homodimerized via an N-terminal disulfide bridge, that shows a remarkably enhanced antigenicity without lipid attachment. Three such OspCs derived from different Borrelia strains were subsequently expressed in E. coli and purified under non-reducing conditions. In non-reducing SDS-PAGE, OspC(Δ1-18) exhibited a 48-kDa band of dimeric OspC. When incubated with IgM-OspC-positive human sera, the reaction at 48kDa was always stronger than at 24kDa of monomeric OspC(Δ1-18, C19G). A lineblot with OspC(Δ1-18) also showed a higher diagnostic accuracy than that obtained with OspC(Δ1-18, C19G) based on a higher affinity of IgM for the dimeric form. When used for the immunization of mice, dimeric OspC(Δ1-18) induced consistent high-titre antibodies against OspC, whereas OspC(Δ1-18, C19G) failed to provoke significant titres in some animals. We conclude that the disulfide-bridging of 2 OspC molecules via their N termini forms a complex that is more suitable for the determination of IgM-OspC and is a promising candidate for a monovalent vaccine.

Ineffectiveness of tigecycline against persistent Borrelia burgdorferi.

The effectiveness of a new first-in-class antibiotic, tigecycline (glycylcycline), was evaluated during the early dissemination (1 week), early immune (3 weeks), or late persistent (4 months) phases of Borrelia burgdorferi infection in C3H mice. Mice were treated with high or low doses of tigecycline, saline (negative-effect controls), or a previously published regimen of ceftriaxone (positive-effect controls). Infection status was assessed at 3 months after treatment by culture, quantitative ospA real-time PCR, and subcutaneous transplantation of joint and heart tissue into SCID mice. Tissues from all saline-treated mice were culture and ospA PCR positive, tissues from all antibiotic-treated mice were culture negative, and some of the tissues from most of the mice treated with antibiotics were ospA PCR positive, although the DNA marker load was markedly decreased compared to that in saline-treated mice. Antibiotic treatment during the early stage of infection appeared to be more effective than treatment that began during later stages of infection. The viability of noncultivable spirochetes in antibiotic-treated mice (demonstrable by PCR) was confirmed by transplantation of tissue allografts from treated mice into SCID mice, with dissemination of spirochetal DNA to multiple recipient tissues, and by xenodiagnosis, including acquisition by ticks, transmission by ticks to SCID mice, and survival through molting into nymphs and then into adults. Furthermore, PCR-positive heart base tissue from antibiotic-treated mice revealed RNA transcription of several B. burgdorferi genes. These results extended previous studies with ceftriaxone, indicating that antibiotic treatment is unable to clear persisting spirochetes, which remain viable and infectious, but are nondividing or slowly dividing.

Adenoviral delivery of interleukin-10 fails to attenuate experimental Lyme disease.

Production of interleukin-10 (IL-10) by C57BL/6 mice following infection with Borrelia burgdorferi has been proposed as a mechanism whereby resistance to the development of experimental Lyme arthritis is maintained. In the current study, we sought to determine the role of IL-10 during infection of arthritis- and carditis-susceptible C3H mice. Infection of C3H IL-10(-/-) mice led to increased joint swelling and arthritis severity scores over those of wild-type C3H mice. Measurement of B. burgdorferi numbers in joints or disseminated tissues indicated a more efficient clearance of spirochetes in the absence of IL-10, similar to that reported in C57BL/6 IL-10(-/-) mice. However, in contrast to previous in vitro work, infection of C3H IL-10(-/-) mice led to decreased in vivo expression of the cytokines KC, IL-1beta, IL-4, and IL-12p70 in the infected joints. Finally, adenoviral expression of IL-10 in the infected joints of C3H mice was unable to modulate the development of severe Lyme arthritis and had no effect on spirochete clearance or Borrelia-specific antibody production. Development of Lyme carditis appeared to be independent of modulation by IL-10. These results suggest that IL-10 limits the development of joint inflammation in both arthritis-resistant and -susceptible mouse strains infected with B. burgdorferi and that increased IL-10 production cannot rescue genetic susceptibility to development of pathology in this model.

Borrelia burgdorferi vlsE antigenic variation is not mediated by RecA.

RecA is a key protein linking genetic recombination to DNA replication and repair in bacteria. Previous functional characterization of Borrelia burgdorferi RecA indicated that the protein is mainly involved in genetic recombination rather than DNA repair. Genetic recombination may play a role in B. burgdorferi persistence by generation of antigenic variation. We report here the isolation of a recA null mutant in an infectious B. burgdorferi strain. Comparison of the in vitro growth characteristics of the mutant with those of the wild-type strain under various conditions showed no significant differences. While the RecA mutant was moderately more sensitive to UV irradiation and mitomycin C than the wild-type strain, the lack of RecA abolished allelic exchange in the mutant. Absence of RecA did not affect the ability of the mutant to infect mice. However, the RecA mutant was attenuated for joint infection in competitive-infection assays with the wild-type strain. vlsE sequence variation in mice was observed in both wild-type and RecA mutant spirochetes, indicating that the mechanism of antigenic variation is not homologous genetic recombination.

Borrelia-associated crystalline keratopathy with intracorneal detection of Borrelia garinii by electron microscopy and polymerase chain reaction.

PURPOSE: First report of a patient with Borrelia-associated crystalline keratopathy with intracorneal evidence of Borrelia garinii by polymerase chain reaction (PCR) and electron microscopy (EM). METHODS: Report of a 67-year-old patient with medical history of recurrent iridocyclitis and arthritis presented with a bilateral, progressive, asymmetric crystalline keratopathy, which was particularly pronounced in the peripheral temporal superior cornea. After penetrating keratoplasty, crystalline keratopathy with stromal haziness recurred. Corneal regrafting was performed. The corneal specimen from the penetrating keratoplasty was examined by light and EM as well as by PCR. RESULTS: In the explanted corneal graft, as well as retrospectively in the corneal specimen from the first keratoplasty, spirochetelike bodies and fragments were detected by light and EM. Borrelia burgdorferi sensu lato DNA was demonstrated by broad-range (16S rDNA) PCR. A more precise identification as Borrelia garinii serotype 5 was possible by analyses of the flaB and ospA gene sequences. Borrelia-specific serological tests showed borderline titers in immunofluorescence and weak reaction in immunoblot, respectively. CONCLUSIONS: This case illustrates that borreliae must be considered as a cause of crystalline keratopathy; Borrelia-specific serological tests can be false negative; explanted cornea specimens of etiologically unclear crystalline keratopathy should be analyzed by EM or PCR for detection of pathogens; and prolonged antibiotic treatment might be effective to prevent progression or recurrence of the disease.

Persistence of Borrelia burgdorferi following antibiotic treatment in mice.

The effectiveness of antibiotic treatment was examined in a mouse model of Lyme borreliosis. Mice were treated with ceftriaxone or saline solution for 1 month, commencing during the early (3 weeks) or chronic (4 months) stages of infection with Borrelia burgdorferi. Tissues from mice were tested for infection by culture, PCR, xenodiagnosis, and transplantation of allografts at 1 and 3 months after completion of treatment. In addition, tissues were examined for the presence of spirochetes by immunohistochemistry. In contrast to saline solution-treated mice, mice treated with antibiotic were consistently culture negative, but tissues from some of the mice remained PCR positive, and spirochetes could be visualized in collagen-rich tissues. Furthermore, when some of the antibiotic-treated mice were fed on by Ixodes scapularis ticks (xenodiagnosis), spirochetes were acquired by the ticks, as determined based upon PCR results, and ticks from those cohorts transmitted spirochetes to naïve SCID mice, which became PCR positive but culture negative. Results indicated that following antibiotic treatment, mice remained infected with nondividing but infectious spirochetes, particularly when antibiotic treatment was commenced during the chronic stage of infection.