Lymphocyte proliferation assay in response to Borrelia burgdorferi in patients with Lyme arthritis: analysis of lymphocyte subsets

The objective of this study was to assess the validity of the lymphocyte proliferation assay in the diagnosis of Lyme arthritis (LA). We analyzed peripheral blood mononuclear cells of 11 patients with LA and 5 healthy controls for proliferative responses to live Borrelia burgdorferi by ³H-thymidine uptake. Before and after proliferation, the total number of cells was estimated and the relative amount of lymphocyte subsets was determined by FACS. Lymphoproliferative responses to B. burgdorferi in patients with LA were predominantly due to CD4+ cells and in a small part due to CD8+ cells and were antigen-dependent in comparison to healthy donors. Considering the number of cells before proliferation, responses of TCRγ/δ+ cells and CD56+ cells were higher than responses of TCRα/β+ and CD3+ cells in LA patients, but not in healthy donors. There were no demonstrable antigen-dependent CD19+ cell responses. Our results support the validity of lymphocyte proliferation assay in the diagnosis of LA. Received: 28 May 1997 / Accepted: 20 September 1997     

Diagnostic value of synovial fluid analysis in children with reactive arthritis

We report on three children with pauciarticular arthritis in whom the clinical picture and serology were compatible with both arthritis reactive to infection with Yersinia or Salmonella and with Lyme arthritis. Results of analysis of synovial fluid by polymerase chain reaction for enterobacterial or borrelial sequences were negative. Immunofluorescence with specific antibodies revealed the presence of amorphous enterobacterial antigens in synovial fluid cells. Since this staining did not reveal enterobacterial morphology, we infected synovial fluid cells of two children with juvenile rheumatoid arthritis in vitro with Yersinia or Salmonella. After 24 h typical rods were observed, but after about 1 week amorphous antigen similar to what had been found in the three patients was seen. In cases of reactive arthritis with ambiguous results of serological testing the diagnosis may be confirmed by demonstration of enterobacterial antigens in synovial fluid.     

Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia

CXCR6-GFP⁺ cells, which encompass 70% invariant natural killer T cells (iNKT cells), have been found primarily patrolling inside blood vessels in the liver. Although the iNKT cells fail to interact with live pathogens, they do respond to bacterial glycolipids presented by CD1d on liver macrophage that have caught the microbe. In contrast, in this study using dual laser multichannel spinning-disk intravital microscopy of joints, the CXCR6-GFP, which also made up 60–70% iNKT cells, were not found in the vasculature but rather closely apposed to and surrounding the outside of blood vessels, and to a lesser extent throughout the extravascular space. These iNKT cells also differed in behavior, responding rapidly and directly to joint-homing pathogens like Borrelia burgdorferi, which causes Lyme disease. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted dissemination attempts by these microbes into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway, an observation also made in vitro for iNKT cells from joint but not liver or spleen. These results suggest a novel, critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier and explains a large increase in pathogen burden of B. burgdorferi in the joint of iNKT cell-deficient mice, and perhaps the greater susceptibility of humans to this pathogen because of fewer iNKT cells in human joints.An essential component of homeostasis is the delivery of oxygen and nutrients to tissues via an extensive vascular network. This process, however, also creates a portal for pathogens to exploit the dissemination of invading bacteria. Intravascular immunity is an emerging concept that suggests that the host immune system remain vigilant and proactive within the vasculature, limiting or preventing pathogen dissemination (1). As such, it is not surprising that numerous cell types have been discovered patrolling the vasculature, including rolling neutrophils in places like skin, crawling monocytes (2), and invariant natural killer T cells (iNKT cells) (3) in skin, brain, muscle, and lung, and immobilized macrophages, including Kupffer cells and splenic macrophage in liver and spleen, respectively (4). Although macrophage have been demonstrated to directly catch, phagocytose, and destroy various pathogens, iNKT cells have to date been suggested to receive signals via antigen presentation and produce potent cytokines that can enhance immunity to infections (4–6), but their direct role in killing pathogens has not been reported. However, it is worth mentioning a recent publication suggesting that iNKT cells can produce granzyme B (7), a molecule known to be used by other immune cells to kill tumor cells as well as various fungi.The natural killer T cells, a subpopulation of T lymphocytes, express a T-cell receptor (TCR) with an invariant variable α-segment 14-joining α-segment 18 (Vα14-Jα18) TCR-α chain that is paired with a restricted subset of TCR Vβ chains in mice (Vα24-Jα18 or Vβ11 in humans) (5, 6). This highly restricted repertoire of TCRs expressed by iNKT cells allows them to recognize lipid antigens presented by CD1d (8, 9), a nonclassical MHC class I-like molecule. The most potent iNKT antigen identified was α-galactosylceramide (αGC) (10), and in vivo administration of this molecule leads to rapid stopping of patrolling iNKT cells with subsequent production of various cytokines, including IL-4 and IFN-γ (5, 6). More recently, several lipid antigens from pathogens have been reported, including α-galactosyl diacylglycerolipid from Borrelia burgdorferi, a spirochete responsible for human Lyme disease (8, 9, 11–13). Imaging of the intravascular immune response in vivo to αGC, which would exclusively bind CD1d, has shown a cessation of iNKT cell crawling and the production of cytokines. Unlike a specific CD1d ligand, an intact pathogen has many activating molecules, ranging from Toll-like receptor (TLR) ligands to complement to chemoattractants, and its direct interaction with iNKT cells is not fully elucidated.B. burgdorferi is arguably the prototype emerging pathogen, becoming a global public health concern estimated at as many as 300,000 patients a year in North America (www.cdc.gov/media/releases/2013/p0819-lyme-disease.html). Lyme disease is one of the most common vector-borne diseases and the number of infected patients is continuing to increase (14). A variety of symptoms have been identified, the most common late-stage manifestation being joint inflammation, known as Lyme arthritis (14). In most cases, treatment with antibiotics leads to resolution of symptoms, but in the absence of antibiotic therapy, intermittent or chronic synovial inflammation can occur (14, 15). Although iNKT cells have not been reported in joints of mice, mice lacking iNKT cells have a joint-specific 25-fold increase (PCR product) in pathogen burden in Lyme borreliosis (4, 13). Although it is well appreciated that iNKT cells receive signals from antigen-presenting cells to induce a systemic increase in IFN-γ, an important cytokine in the fight against infection, it is unclear why the absence of this response would favor localization of B. burgdorferi only in joints (4, 13). We hypothesized that the iNKT cells were also exerting their protective effects directly in the knee microvasculature. Indeed, we have identified a population of CXCR6-GFP⁺ cells, 60–70% of which were iNKT cells that resided in the tissues and preferentially surrounding the joint vasculature. These cells performed direct immune surveillance in joints that function as a cytotoxic barrier, killing pathogens via a granzyme-dependent mechanism. These cells appear to be less prevalent in human joints and may explain the greater susceptibility to Lyme borreliosis in these human tissues.     

Lyme disease: why the controversy?

Some Australians have become convinced of the existence of locally acquired Lyme disease (LD). The history of LD, since its recognition in the early 1970s, is reviewed as a model for investigative approaches to unknown syndromes. Australian Management Guidelines for LD include the requirement for diagnostic testing by National Association of Testing Authorities‐accredited laboratories using Therapeutic Goods Administration‐licensed tests, which result in the efficient diagnosis of LD in overseas travellers. Despite this, patients who have not left Australia pay many thousands of dollars for non‐specialist consultations and testing at overseas laboratories. Unproven long‐term therapy with multiple antibiotics has resulted in serious complications, including allergies, line sepsis, pancreatitis and pseudomembranous colitis. Studies have shown that LD vectors are not found in Australia, and Lyme Borrelia has not been found in Australian vectors, animals or patients with autochthonous illnesses. I propose that (i) A non‐controversial name for the chronic syndrome should be adopted, ‘Australian Multisystem Disorder’. (ii) Research funding should enable the development of a consensus case definition and studies of the epidemiology of this syndrome with laboratory investigations to identify an aetiology and surrogate markers of disease. Prospective, randomised treatment studies could then be undertaken using ethical protocols.     

Seronegative Lyme arthritis caused by Borrelia garinii

A case of a female patient suffering from Lyme arthritis (LA) without elevated antibody levels to Borrelia burgdorferi sensu lato is reported. Seronegative Lyme arthritis was diagnosed based on the classic clinical manifestations and DNA-detected Borrelia garinii in blood and synovial fluid of the patient, after all other possible causes of the disease had been ruled out. The disease was resistant to the first treatment with antibacterial agents. Six months after the therapy, arthritis still persisted and DNA of Borrelia garinii was repeatedly detected in the synovial fluid and the tissue of the patient. At the same time, antigens or parts of spirochaetes were detected by electron microscopy in the synovial fluid, the tissue and the blood of the patient. The patient was then repeatedly treated by antibiotics and synovectomy has been performed. Received: 28 August 2001 / Accepted: 1 January 2002     

Repeated detection ofBorrelia burgdorferi DNA in synovial fluid of a child with Lyme arthritis

Summary In the pathogenesis of late Lyme borreliosis the relative importance of the causative organism,Borrelia burgdorferi, and the immune response of the host, including autoimmune phenomena, is not yet known. We describe a 7-year-old boy with Lyme arthritis from whom two synovial fluid samples were obtained 5 months apart and up to 17 months after the first appearance of arthritis. Both synovial fluid specimens were shown to contain borrelial DNA by nested polymerase chain reaction for the amplification of portions of the genes for flagellin and OspA. Thus,Borrelia burgdorferi may persist within the joint even during late stages of the disease.     

Interstitial granulomatous dermatitis with plaques and arthritis

Interstitial granulomatous dermatitis and arthritis (IGDA) is a rare disease entity with female predominance. The case of a 53-year-old woman with erythemas, plaques and nodules associated with polyarthritis is presented. She was treated with cyclosporin A, with improvement of the joint affliction and complete clearance of skin lesions. The differential diagnosis of IGDA is discussed briefly.     

Lyme arthritis in Belgium. Report of three cases

Summary Three cases of lyme arthritis are reported. The clinical manifestations of lyme disease and the literature are discussed.     

Diagnosis not to be missed: Lyme carditis,rare but reversible cause of complete atrioventricular block

Lyme carditis is a known cause of atrioventricular block and in most cases, atrioventricular block is reversible with appropriate antibiotic treatment. The diagnosis can be challenging if the disease is either not suspected, or if the initial cutaneous manifestation of erythema migrans is missed. It is important to diagnose Lyme carditis as the cause of complete heart block if unnecessary pacemaker implantation is to be avoided. We present a 43-year-old male who presented with complete heart block and also illsustained ventricular tachycardia due to Lyme carditis that reversed completely with antibiotic therapy.     

Seroprevalencia de enfermedad de Lyme en el suroccidente de Asturias

IntroductionTo correctly interpret the serological markers of Lyme disease, it is very important to determine the region's infection rate. The aim of this study was to ascertain the prevalence of specific antibodies against Borrelia burgdorferi in a rural district in northern Spain.MethodsThe presence of IgG antibodies against B. burgdorferi was determined by qualitative enzyme immunoassay in the serum of 1,432 people divided into 3 groups: 316 blood donors, 432 individuals who attended the hospital without infection and 684 for whom Lyme serology testing was specifically requested as part of a differential diagnosis. In the latter group, the presence or absence of an occupational risk factor was recorded.ResultsAntibodies against B. burgdorferi were detected in 189 individuals (13.2%): 16 (5.1%) in the blood donors group, 62 (14.4%) in subjects who attended hospital without infection and 111 (16.2%) in subjects in whom a differential diagnosis of Lyme disease was requested (p < 0.0001). In subjects with an occupational risk factor, the prevalence was 23.5%, peaking at 45.8% in men over 65 years.ConclusionOur study showed a high prevalence of antibodies against B. burgdorferi and higher than that seen in other areas with similar characteristics in Spain. However, our results are similar to those published from other European regions. The prevalence in the blood donors group was lower than that observed in the other groups. Older age, the male gender and occupational risks were associated with a higher prevalence of Lyme disease.     

以急性脑膜炎为首发症状的莱姆病：附一例报告

为加强对莱姆病的认识，报道１例以急性脑膜炎为首发症状、经血清学检测（ＥＬＩＳＡ及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ两种方法）证实为神经莱姆病的诊治经过。表明北京地区有莱姆病存在。如发现淋巴细胞增多性脑膜炎而原因不明时，神经莱姆病应作为鉴别诊断之一，及时送检血清及脑脊液莱姆病的抗体检测将有助于诊断。     

Enfermedad de Lyme

Lyme disease (LD) is a worldwide-distributed multisystemic process caused by Borrelia burgdorferi sensu lato (s.l.) and transmitted by hard ticks. In fact, it is the most common tick-borne infectious disease in the northern hemisphere. In Spain it is transmitted by Ixodes ricinus ticks and Borrelia garinii is the genoespecies of B. burgdorferi s.l. mostly involved in our area. LD is known as “the last great imitator” due to the broad clinical spectrum that may cause. Except in the case of erythema migrans (pathognomonic feature of the disease), the remaining clinical manifestations should be confirmed using microbiological tests.This review is intended to provide readers a current vision of the etiology, epidemiology, clinical manifestations, laboratory diagnosis and treatment of Lyme disease in our environment. Controversial aspects arising from the use of non-validated microbiological tests that are being used without scientific rigor are highlighted.     

Frequencies of Borrelia burgdorferi-reactive T lymphocytes in Lyme arthritis

Summary Using a limiting dilution system, frequencies of Borrelia burgdorferi-reactive T cells were determined in the blood and synovial fluid of four patients with chronic Lyme arthritis (LA), one patient with acrodermatitis chronica atrophicans (ACA), two patients with other inflammatory joint diseases, and two healthy individuals. B. burgdorferi-reactive precursor T cells ranged from 1/750 to 1/8 220 in case of LA and ACA patients and from 1/820 to 1/31 400 in case of controls. In vivo activated B. burgdorferi-reactive T cells were almost absent in control subjects. With one exception, they were detected in LA patients at frequencies ranging from 1/1 300 to 1/15 400. Interestingly, even after successful antibiotic therapy of LA patients, similar frequencies of in vivo activated B. burgdorferi-reactive T cells were observed in the peripheral blood, provided that low cell concentrations were used for culture. At higher cell numbers, the fraction of B. burgdorferi-reactive T cells apparently dropped, suggesting regulatory phenomena.     

Diagnosis and Treatment of Lyme Carditis: JACC Review Topic of the Week

The incidence of Lyme disease, a tick-borne bacterial infection, is dramatically increasing in North America. The diagnosis of Lyme carditis (LC), an early disseminated manifestation of Lyme disease, has important implications for patient management and preventing further extracutaneous complications. High-degree atrioventricular block is the most common presentation of LC, and usually resolves with antibiotic therapy. A systematic approach to the diagnosis of LC in patients with high-degree atrioventricular block will facilitate the identification of this usually transient condition, thus preventing unnecessary implantation of permanent pacemakers in otherwise healthy young individuals.     

Is It Possible to Make a Correct Diagnosis of Lyme Disease on Symptoms Alone? Review of Key Issues and Public Health Implications

There is much confusion and misinformation about the diagnosis of Lyme disease, as well as its treatment. This review explains why one cannot make a correct diagnosis of Lyme disease based on symptoms alone. It also provides evidence to support the validity of two-tier testing for the laboratory diagnosis of Lyme disease. The public health consequences of failing to consider these issues are discussed.