Doctor says you are cured, but you still feel the pain. Borrelia DNA persistence in Lyme disease

ArticleinMicrobes and Infection 19(9-10) · June 2017with 87 Reads
Cite this publication
Abstract
Lyme disease is a zoonosis caused by infection with Borrelia burgdorferi (Bb). A great amount of research has attempted to elucidate the mechanisms by which Bb causes inflammation and chronic symptomatology in some patients. Patients often seek unconventional treatments that lack scientific evidence, as medical care is unable to effectively explain and treat their illness. Bb-DNA can persist for long periods of time in some individuals, even after antibiotic therapy. Herein, scientific rationale is presented for a new therapeutic approach against remaining bacterial DNA, and/or increasing the ability of human macrophages to remove extracellular Bb DNA.

Do you want to read the rest of this article?

Request Full-text Paper PDF
  • Article
    Full-text available
    Post-infectious chronic fatigue syndrome is a public health problem. Etiologies and physiopathological mechanisms are unknown. Some viruses are known to be involved in post-infectious chronic fatigue syndrome, but the role of bacterial infection is still questioned, especially in cases of post-treatment Lyme disease syndrome where subjective symptoms are regularly attributed to the presence of the dormant bacterium without scientific evidence. However, the medical experience of recalcitrant infections, relapses, and reactivations questions the role of “dormant bacteria” in asymptomatic latent infections as well as in subjective symptoms. We summarized scientific literature data on post-bacterial infection chronic fatigue syndrome, the role of dormant bacteria in latent infections, and bacterial asymptomatic carriage. Subjective symptoms described in post-infectious chronic fatigue syndromes are still misunderstood and there is no evidence suggesting that such symptoms could be related to dormant bacterial infection or carriage of viable bacteria. Psychological trauma may be part of these subjective symptoms. Post-infectious chronic fatigue syndrome could nonetheless be due to unknown microorganisms. Antibiotic treatment is not required for latent infections, except for latent syphilis and latent tuberculosis infections to prevent, after the primary infection, progression to the secondary or tertiary stage of the disease.
  • Article
    Full-text available
    RESUMEN Esta es una revisión crítica y organizada de la información disponible y actualizada acerca de la enfermedad de Lyme y la infección por Borrelia en el Perú. Varios estudios de serología contra Borrelia burgdorferi, y de casos de enfermedad de Lyme han sido reportados en el Perú en las pasadas dos décadas. Nueva información sugiere la existencia de nuevas especies de Borrelia burgdorferi sensu lato en Sudamérica, y posiblemente en el Perú. Futuros estudios genéticos y microbiológicos en esta parte del continente, no sólo en casos con Western blot indeterminado, sino también en vectores y posibles reservorios, son necesarios para medir la extensión de estas nuevas especies de Borrelia y su implicancia clínica.
  • Article
    Host cells trigger signals for innate immune responses upon recognition of conserved structures in microbial pathogens. Nucleic acids, which are critical components for inheriting genetic information in all species including pathogens, are key structures sensed by the innate immune system. The corresponding receptors for foreign nucleic acids include members of Toll-like receptors, RIG-I-like receptors, and intracellular DNA sensors. While nucleic acid recognition by these receptors is required for host defense against the pathogen, there is a potential risk to the host of self-nucleic acids recognition, thus precipitating autoimmune and autoinflammatory diseases. In this review, we discuss the roles of nucleic acid-sensing receptors in guarding against pathogen invasion, discriminating between self and non-self, and contributing to autoimmunity and autoinflammatory diseases.
  • Article
    While many antimicrobial peptides (AMPs) disrupt bacterial membranes, some translocate into bacteria and interfere with intracellular processes. Buforin II and DesHDAP1 are thought to kill bacteria by interacting with nucleic acids. Here, molecular modeling and experimental measurements are used to show that neither nucleic acid binding peptide selectively binds DNA sequences. Simulations and experiments also show that changing lysines to arginines enhances DNA binding, suggesting that including additional guanidinium groups is a potential strategy to engineer more potent AMPs. Moreover, the lack of binding specificity may make it more difficult for bacteria to evolve resistance to these and other similar AMPs. This article is protected by copyright. All rights reserved.
  • Chapter
    The analysis of how antimicrobial peptides (AMPs) interact with bacterial membranes and intracellular targets is important for our understanding of how these molecules affect bacteria. Increased knowledge may aid the design of AMPs that work on their target bacterium without inducing bacterial resistance. Here, we describe different methods to investigate the mode of action of peptides against the Gram-positive bacterium Staphylococcus aureus. ATP leakage analysis can be used to evaluate the ability of AMPs to perturb bacteria. DNA-binding and SOS response induction can be analyzed to investigate intracellular targets.
  • Article
    Tissue factor pathway inhibitor 1 (TFPI-1) is a serine protease inhibitor that inhibits tissue factor (TF)-mediated coagulation. The C-terminal region of TFPI-1 could be cleaved off and proved to be antimicrobial against a broad-spectrum of microorganism. In a previous study, a C-terminal peptide, TC24 (with 24 amino acids), derived from tongue sole (Cynoglossus semilaevis) TFPI-1, was synthesized and found antibacterial against Micrococcus luteus. In the present study, the antibacterial spectrum and the action mode of TC24 was further examined, and its in vivo function was analyzed. Our results showed that TC24 also possesses bactericidal activity against Staphylococcus aureus and Vibrio vulnificus. During its interaction with the target bacterial cells, TC24 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA and total RNA. In vivo study showed that administration of tongue sole with TC24 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. These results indicated that TC24 is a novel antimicrobial peptide against bacterial and viral pathogens, and that the observed effect of TC24 on bacterial RNA adds new insights to the action mechanism of fish antimicrobial peptides. Moreover, TC24 may play an important role in fighting pathogenic infection in aquaculture.
  • Article
    A 45-year-old woman presented to the emergency department with 1 week of diffuse pruritic body rash, nausea, and high-grade fever. Her medical history was significant for multiple transient and ill-defined neurologic and gastrointestinal complaints for which she had sought numerous medical opinions without a satisfactory diagnosis. She sought care from a “Lyme-literate doctor,” a term used by certain physicians who prescribe prolonged antibiotic courses to treat chronic Lyme and other tick-borne diseases. Approximately 6 months prior to presentation, that physician diagnosed her with chronic Lyme disease and babesiosis based on results from a Lyme specialty laboratory that used nonvalidated serologic studies. Of note, she reported no preceding rash. Subsequently, she was treated with multiple antibiotic regimens without improvement. During the 3 months prior to admission, she received doxycycline, minocycline, and most recently, trimethoprim-sulfamethoxazole, which she took daily during the 5 weeks before her hospitalization.
  • Article
    Combinatorial library composed of rigid rod-like peptides with a triple-helical scaffold was constructed. The component peptides were designed to have various combinations of basic and neutral (or hydrophobic) amino acid residues based on collagen-like (Gly-Pro-Yaa)-repeating sequences, inspired from the basic and amphiphilic nature of naturally occurring antimicrobial peptides. Screening of the peptide pools resulted in identification of antimicrobial peptides. A structure-activity relationship study revealed that the position of Arg-cluster at N-terminus and cystine knots at C-terminus in the triple helix significantly contributed to the antimicrobial activity. The most potent peptide RO-A showed activity against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. In addition, Escherichia coli exposed to RO-A resulted in abnormal elongation of the cells. RO-A was also shown to have remarkable stability in human serum and low cytotoxicity to mammalian cells. This article is protected by copyright. All rights reserved.
  • Article
    Full-text available
    Background The treatment of persistent symptoms attributed to Lyme disease remains controversial. We assessed whether longer-term antibiotic treatment of persistent symptoms attributed to Lyme disease leads to better outcomes than does shorter-term treatment. Methods In a randomized, double-blind, placebo-controlled trial conducted in Europe, we assigned patients with persistent symptoms attributed to Lyme disease — either related temporally to proven Lyme disease or accompanied by a positive IgG or IgM immunoblot assay for Borrelia burgdorferi — to receive a 12-week oral course of doxycycline, clarithromycin plus hydroxychloroquine, or placebo. All study groups received open-label intravenous ceftriaxone for 2 weeks before initiating the randomized regimen. The primary outcome measure was health-related quality of life, as assessed by the physical-component summary score of the RAND-36 Health Status Inventory (RAND SF-36) (range, 15 to 61, with higher scores indicating better quality of life), at the end of the treatment period at week 14, after the 2-week course of ceftriaxone and the 12-week course of the randomized study drug or placebo had been completed. Results Of the 281 patients who underwent randomization, 280 were included in the modified intention-to-treat analysis (86 patients in the doxycycline group, 96 in the clarithromycin–hydroxychloroquine group, and 98 in the placebo group). The SF-36 physical-component summary score did not differ significantly among the three study groups at the end of the treatment period, with mean scores of 35.0 (95% confidence interval [CI], 33.5 to 36.5) in the doxycycline group, 35.6 (95% CI, 34.2 to 37.1) in the clarithromycin–hydroxychloroquine group, and 34.8 (95% CI, 33.4 to 36.2) in the placebo group (P=0.69; a difference of 0.2 [95% CI, –2.4 to 2.8] in the doxycycline group vs. the placebo group and a difference of 0.9 [95% CI, –1.6 to 3.3] in the clarithromycin–hydroxychloroquine group vs. the placebo group); the score also did not differ significantly among the groups at subsequent study visits (P=0.35). In all study groups, the SF-36 physical-component summary score increased significantly from baseline to the end of the treatment period (P<0.001). The rates of adverse events were similar among the study groups. Four serious adverse events thought to be related to drug use occurred during the 2-week open-label ceftriaxone phase, and no serious drug-related adverse event occurred during the 12-week randomized phase. Conclusions In patients with persistent symptoms attributed to Lyme disease, longer-term antibiotic treatment did not have additional beneficial effects on health-related quality of life beyond those with shorter-term treatment. (Funded by the Netherlands Organization for Health Research and Development ZonMw; PLEASE ClinicalTrials.gov number, NCT01207739.)
  • Article
    Full-text available
    Borrelia burgdorferi is the tick-borne etiologic agent of Lyme disease. The spirochete must negotiate numerous barriers in order to establish a disseminated infection in a mammalian host. These barriers include migration from the feeding tick midgut to the salivary glands, deposition in skin, manipulation or evasion of the localized host immune response, adhesion to and extravasation through an endothelial barrier, hematogenous dissemination, and establishment of infection in distal tissue sites. Borrelia burgdorferi proteins that mediate many of these processes and the nature of the host response to infection are described.
  • Article
    Full-text available
    Lyme borreliosis is a zoonotic, tick-borne disease that infects humans worldwide. The disease is currently recognized as the most common vector-borne disease in Europe and North America. Disease is caused by several genospecies of the Borrelia burgdorferi sensu late complex. Humans are at high risk of infection in regions where highly competent reservoirs are the primary hosts for the subadult stages of the tick, in contrast to regions where less competent or refractory animals feed ticks. Human infections are also most frequently associated with spring and summer months when the nymph stage of the tick is active.
  • Article
    Lyme disease is the most common tick-borne illness in the United States and is also seen in areas of Europe and Asia. The growing deer and lxodes species tick populations in many areas underscore the importance of clinicians to properly recognize and treat the different stages of Lyme disease. Controversy regarding the cause and management of persistent symptoms following treatment of Lyme disease persists and is highlighted in this review.
  • Article
    Chronic Lyme disease is a poorly defined diagnosis that is usually given to patients with prolonged, unexplained symptoms or with alternative medical diagnoses. Data do not support the proposition that chronic, treatment-refractory infection with Borrelia burgdorferi is responsible for the many conditions that get labeled as chronic Lyme disease. Prolonged symptoms after successful treatment of Lyme disease are uncommon, but in rare cases may be severe. Prolonged courses of antibiotics neither prevent nor ameliorate these symptoms and are associated with considerable harm. Copyright © 2015 Elsevier Inc. All rights reserved.
  • Article
    The prognosis following appropriate antibiotic treatment of early or late Lyme disease is favorable but can be complicated by persistent symptoms of unknown cause termed posttreatment Lyme disease syndrome (PTLDS), characterized by fatigue, musculoskeletal pain, and cognitive complaints that persist for 6 months or longer after completion of antibiotic therapy. Risk factors include delayed diagnosis, increased severity of symptoms, and presence of neurologic symptoms at time of initial treatment. Two-tier serologic testing is neither sensitive nor specific for diagnosis of PTLDS because of variability in convalescent serologic responses after treatment of early Lyme disease. Optimal treatment of PTLDS awaits more precise understanding of the pathophysiologic mechanisms involved in this illness and future treatment trials. Copyright © 2015 Elsevier Inc. All rights reserved.
  • Article
    Lyme disease is the most common vector-borne illness in North America and Europe. The etiologic agent, Borrelia burgdorferi sensu lato, is transmitted to humans by certain species of Ixodes ticks, which are found widely in temperate regions of the Northern hemisphere. Clinical features are diverse, but death is rare. The risk of human infection is determined by the geographic distribution of vector tick species, ecologic factors that influence tick infection rates, and human behaviors that promote tick bite. Rates of infection are highest among children 5 to 15 years old and adults older than 50 years. Published by Elsevier Inc.
  • Article
    Full-text available
    Background: Animal studies suggest that Borrelia burgdorferi, the agent of Lyme disease, may persist after antibiotic therapy and can be detected by various means including xenodiagnosis using the natural tick vector (Ixodes scapularis). No convincing evidence exists for the persistence of viable spirochetes after recommended courses of antibiotic therapy in humans. We determined the safety of using I. scapularis larvae for the xenodiagnosis of B. burgdorferi infection in humans. Methods: Laboratory-reared larval I. scapularis ticks were placed on 36 subjects and allowed to feed to repletion. Ticks were tested for B. burgdorferi by polymerase chain reaction (PCR), culture, and/or isothermal amplification followed by PCR and electrospray ionization mass spectroscopy. In addition, attempts were made to infect immunodeficient mice by tick bite or inoculation of tick contents. Xenodiagnosis was repeated in 7 individuals. Results: Xenodiagnosis was well tolerated with no severe adverse events. The most common adverse event was mild itching at the tick attachment site. Xenodiagnosis was negative in 16 patients with posttreatment Lyme disease syndrome (PTLDS) and/or high C6 antibody levels and in 5 patients after completing antibiotic therapy for erythema migrans. Xenodiagnosis was positive for B. burgdorferi DNA in a patient with erythema migrans early during therapy and in a patient with PTLDS. There is insufficient evidence, however, to conclude that viable spirochetes were present in either patient. Conclusions: Xenodiagnosis using Ixodes scapularis larvae was safe and well tolerated. Further studies are needed to determine the sensitivity of xenodiagnosis in patients with Lyme disease and the significance of a positive result. Clinical Trials Registration NCT01143558.
  • Article
    Full-text available
    One hundred twenty-four patients-53 with neuroborreliosis, 48 with erythema migrans, and 23 with Lyme arthritis-were tested in a prospective study for the presence of the DNA of Borrelia burgdorferi sensu lato in plasma, cerebrospinal fluid (CSF), urine, and synovial fluid by nested polymerase chain reaction (PCR). Specific DNA was detected using five amplification systems simultaneously: three targeted chromosomal genes encoding 16S rDNA, flagellin, and p66; and two plasmid sequences of OspA and OspC. Patients were examined clinically and by PCR before and after treatment and again after 3 and 6 months. Before treatment, the specific DNA was detected in 78 patients (62.9 %). Forty-one neuroborreliosis patients were DNA-positive (77.4 %), with CSF positivity in 26 patients, urine in 25, and plasma in 16. Twenty-six erythema migrans patients were DNA-positive (54.2 %), with plasma positivity in 18 cases and urine in 14. Eleven Lyme arthritis cases (47.8 %) were DNA positive (six in urine, five in plasma, and four in synovial fluid). The frequency of PCR positives was comparable in CSF and urine, and it was lower by approximately 50 % in plasma. Specific DNA was also found in a significant number of patients in later testing periods: 48 patients after treatment, 29 patients after 3 months, and 6 patients after 6 months. The prolonged PCR positivity was not explainable by persistent infection according to the clinical manifestations of the disease. Possible explanations of the problem are discussed.
  • Article
    Full-text available
    Phagocytosed Borrelia burgdorferi (Bb), the Lyme disease spirochete, induces a robust and complex innate immune response in human monocytes, in which TLR8 cooperates with TLR2 in the induction of NF-κB-mediated cytokine production, whereas TLR8 is solely responsible for transcription of IFN-β through IRF7. We now establish the role of Bb RNA in TLR8-mediated induction of IFN-β. First, using TLR2-transfected HEK.293 cells, which were unable to phagocytose intact Bb, we observed TLR2 activation by lipoprotein-rich borrelial lysates and TLR2 synthetic ligands but not in response to live spirochetes. Purified Bb RNA, but not borrelial DNA, triggered TLR8 activation. Neither of these 2 ligands induced activation of TLR7. Using purified human monocytes we then show that phagocytosed live Bb, as well as equivalent amounts of borrelial RNA delivered into the phagosome by polyethylenimine (PEI), induces transcription of IFN-β and secretion of TNF-α. The cytokine response to purified Bb RNA was markedly impaired in human monocytes naturally deficient in IRAK-4 and in cells with knockdown TLR8 expression by small interfering RNA. Using confocal microscopy we provide evidence that TLR8 colocalizes with internalized Bb RNA in both early (EEA1) and late endosomes (LAMP1). Live bacterial RNA staining indicates that spirochetal RNA does not transfer from the phagosome into the cytosol. Using fluorescent dextran particles we show that phagosomal integrity in Bb-infected monocytes is not affected. We demonstrate, for the first time, that Bb RNA is a TLR8 ligand in human monocytes and that transcription of IFN-β in response to the spirochete is induced from within the phagosomal vacuole through the TLR8-MyD88 pathway.
  • Article
    The persistence of dormant, noncultivable Borrelia burgdorferi after ceftriaxone treatment was examined. B. burgdorferi isolates were cultivated in Barbour-Stoenner-Kelly medium in the presence or absence of ceftriaxone, and cultures were monitored for up to 56 days. Viability of B. burgdorferi was assessed by subculture, growth, morphology, and pH (as a surrogate for metabolic activity). In addition, the presence of B. burgdorferi DNA and mRNA was assayed by PCR and by real-time reverse transcription (RT)-PCR, respectively. Spirochetes could not be successfully subcultured by day 3 after exposure to ceftriaxone. In cultures treated with ceftriaxone, the pH of the culture medium did not change through day 56, whereas it declined by at least 1 pH unit by 14 days in untreated cultures. These results suggest that B. burgdorferi viability is rapidly eliminated after antibiotic treatment. Nevertheless, DNA was detected by B. burgdorferi-specific PCR for up to 56 days in aliquots from both ceftriaxone-treated and untreated cultures. In addition, although ceftriaxone treatment resulted in a reduction in the quantities of transcript for ospC, ospA, flaB, and pfk, certain mRNAs could be detected through day 14. Transcript for all 4 genes was essentially undetectable after 28 days of treatment. Taken together, the results suggest that B. burgdorferi DNA and mRNA can be detected in samples long after spirochetes are no longer viable as assessed by classic microbiological parameters. PCR positivity in the absence of culture positivity following antibiotic treatment in animal and human studies should be interpreted with caution.
  • Article
    Lyme disease, which is caused by the tickborne spirochete Borrelia burgdorferi, is increasing in frequency; it is spreading to new geographic locations, and it is a particular problem in the northeastern United States.(1) Before the discovery of the causative agent, it was learned that natural infection, without antibiotic treatment, often occurred in stages.(2) The infection usually began with an expanding skin lesion called erythema migrans, sometimes followed weeks later by neurologic or cardiac involvement, and often followed months later by arthritis. Attacks of arthritis commonly recurred over a period of several years, and occasionally, erythema migrans reappeared faintly before episodes . . .
  • Article
    Lyme disease is caused by infection with several genospecies from the Borrelia burgdorferi sensu lato (s.l.) complex, and is transmitted by ixodid ticks. Human disease is an infrequent sequel to infection, which suggests that multiple factors underlie disease development. Several innate immune defects modulating disease development are observed in both natural and experimental infections, and significant heterogeneity exists between B. burgdorferi s.l. spirochaetes. These factors create a panel of presentations from asymptomatic carriage to overt and variable disease. In this short review we summarise the host immune responses associated with Lyme disease in humans, domestic species and laboratory mouse strains, and discuss B. burgdorferi s.l. pathogenicity. We also describe briefly the epidemiology of Lyme disease, and current options for the treatment and prevention.
  • Article
    Full-text available
    Localized elevation in type I IFN has been uniquely linked to the severe Lyme arthritis that develops in C3H mice infected with the spirochete Borrelia burgdorferi. In this study, the dynamic interactions that result in generation of these responses were further examined in C3H mice carrying the type I IFN receptor gene ablation, which effectively blocks all autocrine/paracrine signaling crucial to induction of downstream effectors. Reciprocal radiation chimeras between C3H and IFNAR1⁻/⁻ mice implicated both radiation-sensitive and radiation-resistant cells of the joint tissue in the proarthritic induction of type I IFN. Ex vivo analysis of cells from the naive joint revealed CD45⁺ cells residing in the tissue to be uniquely capable of initiating the type I IFN response to B. burgdorferi. Type I IFN responses were analyzed in real time by lineage sorting of cells from infected joint tissue. This demonstrated that myeloid cells, endothelial cells, and fibroblasts were responsible for propagating the robust IFN response, which peaked at day 7 postinfection and rapidly resolved. Endothelial cells and fibroblasts were the dominant sources of IFN signature transcripts in the joint tissue. Fibroblasts were also the major early source of chemokines associated with polymorphonuclear leukocyte and monocyte/macrophage infiltration, thus providing a focal point for arthritis development. These findings suggest joint-localized interactions among related and unrelated stromal, endothelial, and myeloid cell lineages that may be broadly applicable to understanding the pathogeneses of diseases associated with type I IFN signature, including systemic lupus erythematosus and some rheumatoid arthritides.
  • Article
    Full-text available
    An enigmatic feature of Lyme disease is the slow resolution of musculoskeletal symptoms that can continue after treatment, with some patients developing an inflammatory arthritis that becomes refractory to antibiotic therapy. Using intravital microscopy and the mouse model of Lyme borreliosis, we observed that Borrelia burgdorferi antigens, but not infectious spirochetes, can remain adjacent to cartilage for extended periods after antibiotic treatment. B. burgdorferi was not recovered by culture or xenodiagnosis with ticks after antibiotic treatment of WT mice and all but one of the immunodeficient mice with heightened pathogen burden due to impaired TLR responsiveness. Amorphous GFP+ deposits were visualized by intravital microscopy in the entheses of antibiotic-treated mice infected with GFP-expressing spirochetes and on the ear cartilage surface in sites where immunofluorescence staining detected spirochete antigens. Naive mice were not infected by tissue transplants from antibiotic-treated mice even though transplants contained spirochete DNA. Tissue homogenates from antibiotic-treated mice induced IgG reactive with B. burgdorferi antigens after immunization of naive mice and stimulated TNF-α production from macrophages in vitro. This is the first direct demonstration that inflammatory B. burgdorferi components can persist near cartilaginous tissue after treatment for Lyme disease. We propose that these deposits could contribute to the development of antibiotic-refractory Lyme arthritis.
  • Article
    Full-text available
    Recent studies in patients with dilated cardiomyopathy (DCM) have detected the genome of Borrelia burgdorferi sensu lato (BBSL) in endomyocardial biopsy (EMB) specimens using a qualitative polymerase chain reaction (PCR), suggesting a causal link between Lyme disease and DCM in areas in which Lyme disease is endemic. We aimed to study this relationship using a comprehensive molecular analysis detecting BBSL in EMB samples. We performed a comprehensive histopathological, immunohistochemical, ultrastructural, and molecular analysis targeting cardiotropic viruses and BBSL in EMB specimens of 41 individuals with recent-onset DCM and 15 controls with end-stage coronary artery disease. Specifically, quantitative PCR and electron microscopy of EMB specimens were employed. In addition, autoantibodies and manifestation of autoimmune diseases were evaluated in both groups. Individuals with recent-onset DCM presented more frequently with myocardial BBSL persistence as compared with the control group (24% vs. 0%, P = 0.035). In contrast, the prevalence of parvovirus B19 and cytomegalovirus was similar in both groups. Sequence analysis of borrelial DNA revealed the following genospecies: Borrelia burgdorferi sensu stricto in three patients (30%), Borrelia afzelii in two patients (20%), and Borrelia garinii in four patients (40%), the results being inconclusive in one case. BBSL-positive DCM patients had a higher prevalence of organ-specific autoimmune diseases in comparison with the remaining DCM patients (50% vs. 16%, P = 0.030). Myocardial persistence of BBSL may be involved in the pathophysiology of DCM in individuals living in areas in which Lyme disease is endemic.
  • Article
    Full-text available
    Signaling by innate immune receptors initiates and orchestrates the overall immune responses to infection. Macrophage receptors recognizing pathogens can be broadly grouped into surface receptors and receptors restricted to intracellular compartments, such as phagosomes and the cytoplasm. There is an expectation that ingestion and degradation of microorganisms by phagocytes contributes to activation of intracellular innate receptors, although direct demonstrations of this are rare, and many model ligands are studied in soluble form, outside of their microbial context. By comparing a wild-type strain of Staphylococcus aureus and a lysozyme-sensitive mutant, we have been able directly to address the role of degradation of live bacteria by mouse macrophages in determining the overall innate cellular inflammatory response. Our investigations revealed a biphasic response to S. aureus that consisted of an initial signal resulting from the engagement of surface TLR2, followed by a later, second wave on inflammatory gene induction. This second wave of inflammatory signaling was dependent on and correlated with the timing of bacterial degradation in phagosomes. We found that TLR2 signaling followed by TLR2/TLR9 signaling enhanced sensitivity to small numbers of bacteria. We further found that treating wild-type bacteria with the peptidoglycan synthesis-inhibiting antibiotic vancomycin made S. aureus more susceptible to degradation and resulted in increased inflammatory responses, similar to those observed for mutant degradation-sensitive bacteria.
  • Article
    Full-text available
    A collection of molecular sensors has been defined by studies in the last decade that can recognize a diverse array of pathogens and initiate protective immune and inflammatory responses. However, if the molecular signatures recognized are shared by both foreign and self-molecules, as is the case of nucleic acids, then the responses initiated by these sensors may have deleterious consequences. Notably, this adverse occurrence may be of primary importance in autoimmune disease pathogenesis. In this case, microbe-induced damage or mishandled physiologic processes could lead to the generation of microparticles containing self-nucleic acids. These particles may inappropriately gain access to the cytosol or endolysosomes and, hence, engage resident RNA and DNA sensors. Evidence, as reviewed here, strongly indicates that these sensors are primary contributors to autoimmune disease pathogenesis, spearheading efforts toward development of novel therapeutics for these disorders.
  • Article
    Full-text available
    Human β-defensin 3 (hBD3) is a cationic antimicrobial peptide with potent bactericidal activity in vitro. HBD3 is produced in response to pathogen challenge and can modulate immune responses. The amplified recognition of self-DNA by human plasmacytoid dendritic cells has been previously reported, but we show here that hBD3 preferentially enhances the response to bacterial DNA in mouse Flt-3 induced dendritic cells (FLDCs) and in human peripheral blood mononuclear cells. We show the effect is mediated through TLR9 and although hBD3 significantly increases the cellular uptake of both E. coli and self-DNA in mouse FLDCs, only the response to bacterial DNA is enhanced. Liposome transfection also increases uptake of bacterial DNA and amplifies the TLR9-dependent response. In contrast to hBD3, lipofection of self-DNA enhances inflammatory signalling, but the response is predominantly TLR9-independent. Together, these data show that hBD3 has a role in the innate immune-mediated response to pathogen DNA, increasing inflammatory signalling and promoting activation of the adaptive immune system via antigen presenting cells including dendritic cells. Therefore, our data identify an additional immunomodulatory role for this copy-number variable defensin, of relevance to host defence against infection and indicate a potential for the inclusion of HBD3 in pathogen DNA-based vaccines. This article is protected by copyright. All rights reserved.
  • Article
    Full-text available
    Bacterial lipoproteins and CpG-DNA are ligands for Toll-Like-Receptors (TLR) 2 and 9 respectively. Both classes of molecules were reported to induce experimental arthritis in rodents following direct intra-articular injection. Here we studied: 1) whether arthritis induction by Outer surface (Lipo)protein A (OspA) (B.burgdorferi) involved the TLR-2 as well as the TLR-4 or the CD-14 receptors in addition, and 2) re-examined the arthritogenic potential of CpG-DNA motifs in mice. Following intra-articular injection of the test substances [20µg recombinant, lipidated OspA; 1nM(6µg) to 10nM(60µg) synthetic CpG-DNA], inflammation was monitored by 99Tc scintigraphy (ratio left/right knee joint uptake > 1.1 indicates inflammation) and by histology. Lipoprotein OspA induced severe, acute arthritis in TLR-2+/+ w.t. but not in TLR-2-/- mice (p<0.01). There were no significant differences in the severity of arthritis induced in TLR-4+/+ w.t. and TLR-4-/- mutant mice, or between CD14+/+ w.t. and CD14-/- mice. CpG-DNA (1or 10 nM) did not cause notable inflammation in C57BL/6 mice; 99Tc ratios were < 1.0 and histology showed only minimal changes. Induction of arthritis by the OspA lipoprotein of B.burgdorferi involves the TLR-2 receptor, no evidence for additional participation of TLR-4 or CD14 receptors was found. Intra-articular injection of CpG-DNA did not produce manifest joint injury in mice, at variance with previous reports.
  • Article
    To determine the burden and viability of Borrelia burgdorferi in the skin and joints of patients with Lyme disease. Standard and quantitative polymerase chain reaction (PCR) techniques were used to detect B burgdorferi DNA in skin samples from 90 patients with erythema migrans (EM) and in synovial fluid (SF) from 63 patients with Lyme arthritis (LA) and in synovial tissue from 9 patients. Quantitative PCR determinations of B burgdorferi DNA, messenger RNA (mRNA), and ribosomal RNA (rRNA) were made in 10 skin samples from EM patients and 11 SF samples from LA patients. Skin lesions in most patients with EM had positive PCR results for B burgdorferi DNA. In the majority of patients with LA, a late disease manifestation, PCR results in pretreatment SF samples were positive. In patients with antibiotic-refractory arthritis, positive PCR results persisted for as long as 11 months, but positive results in samples taken during the postantibiotic period did not correlate with relapse or with the subsequent duration of arthritis, and at synovectomy, all results of PCR of synovial tissue were negative. B burgdorferi mRNA, a marker of spirochetal viability, was detected in 8 of 10 skin samples from EM patients, but in none of 11 SF samples from LA patients, even when obtained prior to antibiotic administration. Moreover, the median ratio of spirochetal rRNA to DNA, a measure of ribosomal activity, was 160 in the 10 EM skin samples, but only 0.15 in the 3 LA SF samples with positive results. B burgdorferi in the skin lesions of EM patients were active and viable, whereas those in the SF of LA patients were moribund or dead at any time point. Thus, detection of B burgdorferi DNA in SF is not a reliable test of active joint infection in Lyme disease.
  • Article
    Full-text available
    Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are 2 similar diseases characterized by a cytokine storm, overwhelming inflammation, multiorgan dysfunction, and death. Animal models of HLH suggest that disease is driven by IFN-γ produced by CD8⁺ lymphocytes stimulated by persistent antigen exposure. In these models and patients with "primary" HLH, the antigen persists due to genetic defects, resulting in ineffective cytotoxic responses by CD8⁺ T cells and poor pathogen clearance. However, infectious triggers are often not identified in patients with MAS, and some patients with HLH or MAS lack defects in cytotoxic T cell killing. Herein, we show that repeated stimulation of TLR9 produced an HLH/MAS-like syndrome on a normal genetic background, without exogenous antigen. Like previous HLH models, TLR9-induced MAS was IFN-γ dependent; however, unlike other models, disease did not require lymphocytes. We further showed that IL-10 played a protective role in this model and that blocking IL-10 signaling led to the development of hemophagocytosis. IL-10 may therefore be an important target for the development of effective therapeutics for MAS. Our data provide insight into MAS-like syndromes in patients with inflammatory diseases in which there is chronic innate immune activation but no genetic defects in cytotoxic cell function.
  • Article
    Full-text available
    Phagocytosed Borrelia burgdorferi (Bb) induces inflammatory signals that differ both quantitatively and qualitatively from those generated by spirochetal lipoproteins interacting with Toll-like receptor (TLR) 1/2 on the surface of human monocytes. Of particular significance, and in contrast to lipoproteins, internalized spirochetes induce transcription of IFN-β. Using inhibitory immunoregulatory DNA sequences (IRSs) specific to TLR7, TLR8, and TLR9, we show that the TLR8 inhibitor IRS957 significantly diminishes production of TNF-α, IL-6, and IL-10 and completely abrogates transcription of IFN-β in Bb-stimulated monocytes. We demonstrate that live Bb induces transcription of TLR2 and TLR8, whereas IRS957 interferes with their transcriptional regulation. Using confocal and epifluorescence microscopy, we show that baseline TLR expression in unstimulated monocytes is greater for TLR2 than for TLR8, whereas expression of both TLRs increases significantly upon stimulation with live spirochetes. By confocal microscopy, we show that TLR2 colocalization with Bb coincides with binding, uptake, and formation of the phagosomal vacuole, whereas recruitment of both TLR2 and TLR8 overlaps with degradation of the spirochete. We provide evidence that IFN regulatory factor (IRF) 7 is translocated into the nucleus of Bb-infected monocytes, suggesting its activation through phosphorylation. Taken together, these findings indicate that the phagosome is an efficient platform for the recognition of diverse ligands; in the case of Bb, phagosomal signaling involves a cooperative interaction between TLR2 and TLR8 in pro- and antiinflammatory cytokine responses, whereas TLR8 is solely responsible for IRF7-mediated induction of IFN-β.
  • Article
    Full-text available
    TLRs play an essential role in the induction of immune responses by detecting conserved molecular products of microorganisms. However, the function of TLR8 is largely unknown. In the current study, we investigated the role of TLR8 signaling in immunity in mice. We found that Tlr8(-/-) DCs overexpressed TLR7, were hyperresponsive to various TLR7 ligands, and showed stronger and faster NF-κB activation upon stimulation with the TLR7 ligand R848. Tlr8(-/-) mice showed splenomegaly, defective development of marginal zone (MZ) and B1 B cells, and increased serum levels of IgM and IgG2a. Furthermore, Tlr8(-/-) mice exhibited increased serum levels of autoantibodies against small nuclear ribonucleoproteins, ribonucleoprotein, and dsDNA and developed glomerulonephritis, whereas neither Tlr7(-/-) nor Tlr8(-/-)Tlr7(-/-) mice showed any of the phenotypes observed in Tlr8(-/-) mice. These data provide evidence for a pivotal role for mouse TLR8 in the regulation of mouse TLR7 expression and prevention of spontaneous autoimmunity.
  • Article
    Yrjänäinen H, Hytönen J, Hartiala P, Oksi J, Viljanen MK. Persistence of borrelial DNA in the joints of Borrelia burgdorferi-infected mice after ceftriaxone treatment. APMIS 2010; 118: 665–73. We have earlier shown that Borrelia burgdorferi-infected and ceftriaxone-treated mice have viable spirochetes in their body, since immunosuppressive treatment allows B. burgdorferi to be detected by culture. However, the niche of the persisting spirochetes remained unknown. In the present study, we analyzed the tissues of B. burgdorferi-infected and ceftriaxone-treated mice by culture and PCR to reveal the foci of persisting spirochetes. C3H/HeN mice were infected via intradermal needle injection with B. burgdorferi s.s. N40. The mice were treated as follows: (i) short (5 days) and (ii) long (18 days) course of ceftriaxone at 2 weeks of infection and killed after either 10 or 30 weeks, or (iii) the mice received ceftriaxone for 5 days at 18 weeks of infection and were killed 21 weeks after the treatment. All samples of ceftriaxone-treated mice were culture negative, whereas all untreated controls were culture positive. Importantly, B. burgdorferi DNA was detected in the joints of 30–100% of the treated mice. In conclusion, these results combined with earlier results suggest that the joint or a tissue adjacent to the joint is the niche of persisting B. burgdorferi in ceftriaxone-treated mice.
  • Article
    Full-text available
    The human cathelicidin LL-37 has broad-spectrum antimicrobial activity. It also participates at the interface of innate and adaptive immunity by chemoattracting immune effector cells, modulating the production of a variety of inflammatory mediators by different cell types, and regulating the differentiation of monocytes into dendritic cells. In this study, we investigated the effects of LL-37 on the differentiation of human monocytes into anti-inflammatory macrophages (MPhi-2; driven by M-CSF) versus proinflammatory macrophages (MPhi-1; driven by GM-CSF) as well as on fully differentiated MPhi-1 and MPhi-2. Results revealed that monocytes cultured with M-CSF in the presence of LL-37 resulted in macrophages displaying a proinflammatory signature, namely, low expression of CD163 and little IL-10 and profound IL-12p40 production on LPS stimulation. The effects of LL-37 on M-CSF-driven macrophage differentiation were dose- and time-dependent with maximal effects observed at 10 microg/ml when the peptide was present from the start of the cultures. The peptide enhanced the GM-CSF-driven macrophage differentiation. Exposure of fully differentiated MPhi-2 to LL-37 for 6 d resulted in macrophages that produced less IL-10 and more IL-12p40 on LPS stimulation than control MPhi-2. In contrast, LL-37 had no effect on fully differentiated MPhi-1. Peptide mapping using a set of 16 overlapping 22-mer peptides covering the complete LL-37 sequence revealed that the C-terminal portion of LL-37 is responsible for directing macrophage differentiation. Our results furthermore indicate that the effects of LL-37 on macrophage differentiation required internalization of the peptide. Together, we conclude that LL-37 directs macrophage differentiation toward macrophages with a proinflammatory signature.
  • The objective of this study was to investigate the level of Toll-like receptors (TLRs) 7, 8, and 9 in peripheral mononuclear blood cells (PMBCs) from patients with primary Sjögren's syndrome (pSS) and whether TLR7 and 9 exist in the parotid glands of pSS patients. TLR7, 8, and 9 mRNA levels in PMBC from 37 pSS patients and 24 controls were determined using real-time polymerase chain reaction. TLR7- and TLR9-positive cells in parotid glands from 20 pSS patients and 10 controls were observed using immunofluorescence and immunohistochemistry, respectively. Statistical analysis was performed by Student t test. TLR7 and TLR9 mRNA levels were 2.17- and 2.33-fold higher in pSS patients than control subjects, respectively (P < .05), whereas TLR8 levels were not. TLR7- and TLR9-positive cells of the pSS group were localized in the epithelial islands, lymphocytes, and ductal epithelial cells of the parotid glands and were more abundant than the TLR7- and TLR9-positive cells that were only localized to the ductal epithelial cells of parotid glands (P < .05). TLR7 and TLR9 mRNA levels are up-regulated in the PMBC of pSS patients, and TLR7- and TLR9-positive cells exist in the epithelial islands, lymphocytes, and ductal epithelial cells of the parotid glands of individuals affected by pSS, but are limited to the ductal epithelial cells of controls.
  • Article
    Full-text available
    The human lactoferrin-derived peptide hLF1-11 displays antimicrobial activities in vitro and is effective against infections with antibiotic-resistant bacteria and fluconazole-resistant Candida albicans in animals. However, the mechanisms underlying these activities remain largely unclear. Since hLF1-11 is ineffective in vitro at physiological salt concentrations, we suggested modulation of the immune system as an additional mechanism of action of the peptide. We investigated whether hLF1-11 affects human monocyte-macrophage differentiation and determined the antimicrobial activities of the resulting macrophages. Monocytes were cultured for 7 days with GM-CSF in the presence of hLF1-11, control peptide, or saline for various intervals. At day 6, the cells were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or heat-killed C. albicans for 24 h. Thereafter, the levels of cytokines in the culture supernatants, the expression of pathogen recognition receptors, and the antimicrobial activities of these macrophages were determined. The results showed that a short exposure of monocytes to hLF1-11 during GM-CSF-driven differentiation is sufficient to direct differentiation of monocytes toward a macrophage subset characterized by both pro- and anti-inflammatory cytokine production and increased responsiveness to microbial structures. Moreover, these macrophages are highly effective against C. albicans and Staphylococcus aureus. In conclusion, hLF1-11 directs GM-CSF-driven differentiation of monocytes toward macrophages with enhanced effector functions.
  • Article
    Since their initial use in the 1980s, IFNs have become an essential component of the therapy for many diseases such as hepatitis and multiple sclerosis. Although they have been extremely useful in conditions that pose therapeutic challenges, complications associated with their use have been widely reported including emerging reports of several autoimmune diseases. Many of these reports have shed light on the pathogenesis of autoimmune disorders and helped to highlight not only the critical role of type I IFNs in defense against viral infections but also the pivotal role they occupy in the interface between innate and adaptive immunity. Many patients with autoimmune disease have increased responsiveness to type I IFNs (alpha/beta), and therapy with these cytokines has induced or unmasked autoimmune disease in many additional patients. The objective of this paper is to discuss the role of type I IFNs in autoimmunity. The literature regarding type I IFNs and autoimmunity was reviewed using the Medline database from 1950 to 2009. Search terms included 'interferon alpha' and 'autoimmune disease' and 'interferon beta' and 'autoimmune disease'. Case reports, case series, reviews and prospective studies were included in the analysis. In the literature a variety of autoimmune disorders have reportedly been induced by the use of type I IFNs, being used, although these are primarily in the form of case reports and case series. Nevertheless, there is a growing body of molecular evidence to support the clinical association. The role of IFNs in the induction of autoimmunity is complex with interplay of many genetic and environmental factors that influence the balance between normal and aberrant immune responsiveness, ultimately leading to the observed clinical manifestations.
  • Article
    The mechanisms that determine bacterial shape are in many ways poorly understood. A prime example is the Lyme disease spirochete, Borrelia burgdorferi (B. burgdorferi), which mechanically couples its motility organelles, helical flagella, to its rod-shaped cell body, producing a striking flat-wave morphology. A mathematical model is developed here that accounts for the elastic coupling of the flagella to the cell cylinder and shows that the flat-wave morphology is in fact a natural consequence of the geometrical and material properties of the components. Observations of purified periplasmic flagella show two flagellar conformations. The mathematical model suggests that the larger waveform flagellum is the more relevant for determining the shape of B. burgdorferi. Optical trapping experiments were used to measure directly the mechanical properties of these spirochetes. These results imply relative stiffnesses of the two components, which confirm the predictions of the model and show that the morphology of B. burgdorferi is completely determined by the elastic properties of the flagella and cell body. This approach is applicable to a variety of other structures in which the shape of the composite system is markedly different from that of the individual components, such as coiled-coil domains in proteins and the eukaryotic axoneme.
  • Article
    Borrelial specific DNA was examined in a group of 62 patients with different forms of Lyme borreliosis (LB) (32 patients suffered from neuroborreliosis, 19 manifested erythema migrans, and 11 joint involvement). Nested-PCR system with five newly derived primers was used in parallel. The study was organized prospectively, the presence of DNA was tested for plasma, CSF, joint fluid and urine before treatment, and plasma, joint fluid and urine were examined after treatment. Before therapy, 36 patients (58.1%) were DNA positive on the whole; 21 positive patients (65.6%) were found in the group of neuroborreliosis, 8 (42.1%) showed signs of skin involvement, and 7 (63.6%) were positive in arthritis. After treatment, 11 patients (36.7%) were positive in neuroborreliosis, 3 (17.6%) in skin form, and 6 (54.5%) in joint form of LB. Among 97 positive amplifications the most frequent target was found in primer corresponding with 16S rDNA (50 samples, 51.5%). Lower but very similar results were obtained with primers for OspA (18 positive amplifications; 18.6%), OspC (13 positive amplifications; 13.4%), and flagellin (13 positive amplifications; 13.4%). There were 11 patients in whom only DNA and no specific antibodies were found. Specific DNA was found in all clinical groups of LB with similar sensitivity. Examination of the borrelial DNA in urine displayed the same sensitivity as in CSF and had a two times higher sensitivity than in plasma.
  • Article
    Fifteen patients with acute exacerbation of rheumatoid arthritis each received a 1 g bolus intravenous injection of ceftriaxone. Serum and synovial fluid was sampled at intervals between 1 h and 24 h later and assayed for ceftriaxone. Synovial fluid leucocyte counts and albumin content were measured concomitantly. Detectable levels of ceftriaxone were found in synovial fluid and serum 24 h after injection. Synovial fluid ceftriaxone concentration ranged between 66% and 100% of the concomitant serum levels. No correlation was observed between synovial fluid ceftriaxone concentration and synovial fluid leucocyte count and albumin concentration.
  • Article
    Concentrations of ceftriaxone in serum and intervertebral disc tissue were determined with high-pressure liquid chromatography in forty-five patients after a single intravenous loading dose of 1000 milligrams given at different intervals before an operation on the spine. The mean serum concentrations in this study corresponded well with reported values. The mean tissue concentrations were 5.6 micrograms per gram (95 per cent confidence interval, 3.6 to 6.8 micrograms per gram) one to less than two hours after administration of the antibiotic, 6.4 micrograms per gram (95 per cent confidence interval, 2.8 to 10.0 micrograms per gram) two to less than four hours, and 3.6 micrograms per gram (95 per cent confidence interval, 0.6 to 6.6 micrograms per gram) at fourteen to less than sixteen hours. These drug concentrations exceed the minimum inhibitory concentration that was effective against 90 per cent of the bacteria for methicillin-sensitive Staphylococcus aureus; for Streptococcus pyogenes, agalactiae, viridans, pneumoniae, and bovis; and for community-acquired Enterobacteriaceae. The average serum-to-tissue ratio was 191:1 at less than one-half hour and 13:1 at less than one and a half hours. The lower values of the 95 per cent confidence intervals for the concentration of the antibiotic exceeded the minimum inhibitory concentrations in the disc tissue against most susceptible bacteria during the period between one and a half and four hours, but a larger bolus would be needed to maintain this level for a longer period (such as in a longer operation) and as prophylaxis against methicillin-sensitive Staphylococcus aureus and coagulase-negative staphylococci.
  • Article
    Full-text available
    A nested PCR was developed for the detection of Borrelia burgdorferi-specific DNA in the urine of patients with erythema migrans. The target for the nested PCR was a specific region of the flagellin gene; the detection limit was less than five organisms of B. burgdorferi including all three species B. burgdorferi sensu stricto, B. afzelii, and B. garinii. A prospective, controlled, blinded study was performed with 26 patients with erythema migrans to evaluate the nested PCR method with clinical samples. B. burgdorferi-specific DNA could be detected in urine specimens from 22 of 24 patients with erythema migrans (sensitivity, 91.61%). Immediately after therapy, 11 of 19 patients still yielded positive results (58%). Eight weeks after therapy, 2 of 16 patients (13%) were positive by PCR of urine, and 20 weeks after treatment none of seven investigated urine samples was reactive. Essential for the sensitivity that was obtained was the development of a simple DNA extraction procedure. The results of the study indicate that the described method is highly sensitive and allows for the effective control of the efficacy of antibiotic therapy in patients with early Lyme borreliosis.
  • Article
    Unmethylated CpG motifs are often found in bacterial DNA, and exert immunostimulatory effects on hematopoietic cells. Bacteria produce severe joint inflammation in septic and reactive arthritides; bacterial DNA may be involved in this process. We injected bacterial DNA originating from Escherichia coli and Staphylococcus aureus and oligonucleotides containing CpG directly into the knee joints of mice of different strains. Arthritis was seen by histopathology within 2 hours and lasted for at least 14 days. Unmethylated CpG motifs were responsible for this induction of arthritis, as oligonucleotides containing these motifs produced the arthritis. The arthritis was characterized by an influx of monocytic, Mac-1+ cells and by a lack of T lymphocytes. Depletion of monocytes resulted in abrogation of the synovial inflammation. Tumor necrosis factor (TNF)-alpha, a cytokine produced by cells of the monocyte/macrophage lineage, is an important mediator of this disease, as expression of mRNA for TNF-alpha was evident in the inflamed joints, and the CpG-mediated inflammation was abrogated in mice genetically unable to produce this cytokine. These findings demonstrate that bacterial DNA containing unmethylated CpG motifs induces arthritis, and indicate an important pathogenic role for bacterial DNA in septic arthritis.
  • Article
    Full-text available
    Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that affects over one million people in the United States. SLE is characterized by the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. It is thought that the resulting immune complexes accumulate in vessel walls, glomeruli and joints and cause a hypersensitivity reaction type III, which manifests as glomerulonephritis, arthritis and general vasculitis. The aetiology of SLE is unknown, but several studies suggest that increased liberation or disturbed clearance of nuclear DNA-protein complexes after cell death may initiate and propagate the disease. Consequently, Dnase1, which is the major nuclease present in serum, urine and secreta, may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and thus for the prevention of SLE (refs 7-11). To test this hypothesis, we have generated Dnase1-deficient mice by gene targeting. We report here that these animals show the classical symptoms of SLE, namely the presence of ANA, the deposition of immune complexes in glomeruli and full-blown glomerulonephritis in a Dnase1-dose-dependent manner. Moreover, in agreement with earlier reports, we found Dnase1 activities in serum to be lower in SLE patients than in normal subjects. Our findings suggest that lack or reduction of Dnase1 is a critical factor in the initiation of human SLE.
  • Article
    To understand the mechanisms of arthritis triggered by CpG-containing oligonucleotides (ODN). Following the induction of CpG ODN-triggered arthritis in mice, we analyzed the impact of depletion of immune cells, including neutrophils, natural killer (NK) cells, and monocyte/macrophages, on the arthritis, as well as the impact in SCID mice lacking T and B cells. In addition, tumor necrosis factor alpha (TNFalpha) knockout mice were studied, and intraarticular administration of p65 antisense to nuclear factor kappaB (NF-kappaB) was used to examine effects in CpG ODN-triggered arthritis. Cytokine messenger RNA expression in synovial tissue was evaluated by in situ hybridization. The presence of macrophages was mandatory for the mediation of arthritis triggered by CpG ODN, whereas the absence of neutrophils, NK cells, T cells, and B cells was of minor importance in this regard. The proinflammatory cytokines TNFalpha, interleukin-1beta, and interleukin-12, which originate from macrophages, were frequently found in the inflamed joints, and TNFalpha was confirmed to be an important mediator in the development of arthritis, since the incidence and severity of joint inflammation were markedly reduced in TNFalpha knockout mice. NF-kappaB exerted an important regulatory role in the development of CpG ODN-mediated arthritis, since local administration of antisense to the p65 subunit of NF-kappaB diminished the incidence of inflammation by 50%. Macrophages and their products play an important role in the development of arthritis triggered by bacterial DNA containing CpG motifs.
  • Article
    Background It is controversial whether prolonged antibiotic treatment is effective for patients in whom symptoms persist after the recommended antibiotic treatment for acute Lyme disease. Methods We conducted two randomized trials: one in 78 patients who were seropositive for IgG antibodies to Borrelia burgdorferi at the time of enrollment and the other in 51 patients who were seronegative. The patients received either intravenous ceftriaxone, 2 g daily for 30 days, followed by oral doxycycline, 200 mg daily for 60 days, or matching intravenous and oral placebos. Each patient had well-documented, previously treated Lyme disease but had persistent musculoskeletal pain, neurocognitive symptoms, or dysesthesia, often associated with fatigue. The primary outcome measures were improvement on the physical- and mental-health–component summary scales of the Medical Outcomes Study 36-Item Short-Form General Health Survey (SF-36) — a scale measuring the health-related quality of life — on day 180 of the study. Results After a planned interim analysis, the data and safety monitoring board recommended that the studies be discontinued because data from the first 107 patients indicated that it was highly unlikely that a significant difference in treatment efficacy between the groups would be observed with the planned full enrollment of 260 patients. Base-line assessments documented severe impairment in the patients' health-related quality of life. In intention-to-treat analyses, there were no significant differences in the outcomes with prolonged antibiotic treatment as compared with placebo among either the seropositive or the seronegative patients. Conclusions There is considerable impairment of health-related quality of life among patients with persistent symptoms despite previous antibiotic treatment for acute Lyme disease. However, in these two trials, treatment with intravenous and oral antibiotics for 90 days did not improve symptoms more than placebo.
  • Article
    Full-text available
    Systemic lupus erythematosus (SLE) is a highly prevalent human autoimmune diseases that causes progressive glomerulonephritis, arthritis and an erythematoid rash. Mice deficient in deoxyribonuclease I (Dnase1) develop an SLE-like syndrome. Here we describe two patients with a heterozygous nonsense mutation in exon 2 of DNASE1, decreased DNASE1 activity and an extremely high immunoglobulin G titer against nucleosomal antigens. These data are consistent with the hypothesis that a direct connection exists between low activity of DNASE1 and progression of human SLE.
  • Article
    Autoimmune diseases remain one of the mysteries that perplex immunologists. What makes the immune system, which has evolved to protect an organism from foreign invaders, turn on the organism itself? A popular answer to this question involves the lymphoid network's primordial function: autoimmunity is a by-product of the immune response to microbial infection. For decades there have been tantalizing associations between infectious agents and autoimmunity: beta-hemolytic streptococci and rheumatic fever; B3 Coxsackieviruses and myocarditis; Trypanosoma cruzi and Chagas' disease; diverse viruses and multiple sclerosis; Borrelia burgdorfii and Lyme arthritis; and B4 Coxsackievirus, cytomegalovirus or rubella and type 1 diabetes, to name the most frequently cited examples. In addition, animal models have provided direct evidence that infection with a particular microbe can incite a particular autoimmune disease. Nonetheless, many of the associations appear less than convincing and, even for those that seem to be on solid footing, there is no real understanding of the underlying mechanism(s).
  • Article
    To determine whether post Lyme syndrome (PLS) is antibiotic responsive. The authors conducted a single-center randomized double-masked placebo-controlled trial on 55 patients with Lyme disease with persistent severe fatigue at least 6 or more months after antibiotic therapy. Patients were randomly assigned to receive 28 days of IV ceftriaxone or placebo. The primary clinical outcomes were improvement in fatigue, defined by a change of 0.7 points or more on an 11-item fatigue questionnaire, and improvement in cognitive function (mental speed), defined by a change of 25% or more on a test of reaction time. The primary laboratory outcome was an experimental measure of CSF infection, outer surface protein A (OspA). Outcome data were collected at the 6-month visit. Patients assigned to ceftriaxone showed improvement in disabling fatigue compared to the placebo group (rate ratio, 3.5; 95% CI, 1.50 to 8.03; p = 0.001). No beneficial treatment effect was observed for cognitive function or the laboratory measure of persistent infection. Four patients, three of whom were on placebo, had adverse events associated with treatment, which required hospitalization. Ceftriaxone therapy in patients with PLS with severe fatigue was associated with an improvement in fatigue but not with cognitive function or an experimental laboratory measure of infection in this study. Because fatigue (a nonspecific symptom) was the only outcome that improved and because treatment was associated with adverse events, this study does not support the use of additional antibiotic therapy with parenteral ceftriaxone in post-treatment, persistently fatigued patients with PLS.
  • Article
    Xenodiagnosis by ticks was used to determine whether spirochetes persist in mice after 1 month of antibiotic therapy for vectorborne Borrelia burgdorferi infection. Immunofluorescence and polymerase chain reaction (PCR) were used to show that spirochetes could be found in Ixodes scapularis ticks feeding on 4 of 10 antibiotic-treated mice up to 3 months after therapy. These spirochetes could not be transmitted to naive mice, and some lacked genes on plasmids correlating with infectivity. By 6 months, antibiotic-treated mice no longer tested positive by xenodiagnosis, and cortisone immunosuppression did not alter this result. Nine months after treatment, low levels of spirochete DNA could be detected by real-time PCR in a subset of antibiotic-treated mice. In contrast to sham-treated mice, antibiotic-treated mice did not have culture or histopathologic evidence of persistent infection. These results provide evidence that noninfectious spirochetes can persist for a limited duration after antibiotics but are not associated with disease in mice
  • Article
    The spirochete Borrelia burgdorferi causes acute inflammation in mice that resolves with the development of pathogen-specific adaptive immunity. B. burgdorferi lipoproteins activate innate immune cells via Toll-like receptor 2 (TLR2), but TLR2-deficient mice are not resistant to B. burgdorferi-induced disease, suggesting the involvement of other TLRs or non-TLR mechanisms in the induction of acute inflammation. For this study, we used mice that were deficient in the intracellular adapter molecule myeloid differentiation antigen 88 (MyD88), which is required for all TLR-induced inflammatory responses, to determine whether the interruption of this pathway would alter B. burgdorferi-induced disease. Infected MyD88−/− mice developed carditis and arthritis, similar to the disease in wild-type (WT) mice analyzed at its peak (days 14 and 28) and during regression (day 45). MyD88−/− macrophages produced tumor necrosis factor alpha only when spirochetes were opsonized, suggesting a role for B. burgdorferi-specific antibody in disease expression. MyD88−/− mice produced stronger pathogen-specific Th2-dependent immunoglobulin G1 (IgG1) responses than did WT mice, and their IgM titers remained significantly elevated through 90 days of infection. Despite specific antibodies, the pathogen burden was 250-fold higher in MyD88−/− mice than in WT mice 45 days after infection; by 90 days of infection, the pathogen burden had diminished substantially in MyD88−/− mice, but it was still elevated compared to that in WT mice. The elevated pathogen burden may be explained in part by the finding that MyD88−/− peritoneal macrophages could ingest spirochetes but degraded them more slowly than WT macrophages. Our results show that MyD88-dependent signaling pathways are not required for B. burgdorferi-induced inflammation but are necessary for the efficient control of the pathogen burden by phagocytes.