Single-cell immunophenotyping of the skin lesion erythema migrans identifies IgM memory B cells.

The skin lesion erythema migrans (EM) is an initial sign of the Ixodes tick-transmitted Borreliella spirochetal infection known as Lyme disease. T cells and innate immune cells have previously been shown to predominate the EM lesion and promote the reaction. Despite the established importance of B cells and antibodies in preventing infection, the role of B cells in the skin immune response to Borreliella is unknown. Here, we used single-cell RNA-Seq in conjunction with B cell receptor (BCR) sequencing to immunophenotype EM lesions and their associated B cells and BCR repertoires. We found that B cells were more abundant in EM in comparison with autologous uninvolved skin; many were clonally expanded and had circulating relatives. EM-associated B cells upregulated the expression of MHC class II genes and exhibited preferential IgM isotype usage. A subset also exhibited low levels of somatic hypermutation despite a gene expression profile consistent with memory B cells. Our study demonstrates that single-cell gene expression with paired BCR sequencing can be used to interrogate the sparse B cell populations in human skin and reveals that B cells in the skin infection site in early Lyme disease expressed a phenotype consistent with local antigen presentation and antibody production.

Serological testing for Lyme Borreliosis in general practice: A qualitative study among Dutch general practitioners.

Background: Concerns are raised about missed, delayed and inappropriate diagnosis of Lyme Borreliosis. Quantitative descriptive studies have demonstrated non-adherence to the guidelines for testing for Lyme Borreliosis.Objectives: To gain insight into the diagnostic practices that general practitioners apply for Lyme Borreliosis, their motives for ordering tests and how they act upon test results.Methods: A qualitative study among 16 general practitioners using semi-structured interviews and thematic content analysis.Results: Five themes were distinguished: (1) recognising localised Lyme Borreliosis and symptoms of disseminated disease, (2) use of the guideline, (3) serological testing in patients with clinically suspect Lyme Borreliosis, (4) serological testing without clinical suspicion of Lyme Borreliosis, and (5) dealing with the limited accuracy of the serological tests. Whereas the national guideline recommends using serological tests for diagnosing, general practitioners also use them for ruling out disseminated Lyme Borreliosis. Reasons for non-adherence to the guideline for testing were to reassure patients with non-specific symptoms or without symptoms who feared to have Lyme disease, confirmation of localised Lyme Borreliosis and routine work-up in patients with continuing unexplained symptoms. Some general practitioners referred all patients who tested positive to medical specialists, where others struggled with the explanation of the results.Conclusion: Both diagnosis and ruling out of disseminated Lyme Borreliosis can be difficult for general practitioners. General practitioners use serological tests to reassure patients and rule out Lyme Borreliosis, thereby deviating from the national guideline. Interpretation of test results in these cases can be difficult.

Placa roja en la mejilla con crecimiento progresivo: eritema migratorio / Progressively Expanding Red Plaque on the Cheek: Erythema Migrans

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Differences in Genotype, Clinical Features, and Inflammatory Potential of Borrelia burgdorferi sensu stricto Strains from Europe and the United States.

Borrelia burgdorferi sensu stricto isolates from patients with erythema migrans in Europe and the United States were compared by genotype, clinical features of infection, and inflammatory potential. Analysis of outer surface protein C and multilocus sequence typing showed that strains from these 2 regions represent distinct genotypes. Clinical features of infection with B. burgdorferi in Slovenia were similar to infection with B. afzelii or B. garinii, the other 2 Borrelia spp. that cause disease in Europe, whereas B. burgdorferi strains from the United States were associated with more severe disease. Moreover, B. burgdorferi strains from the United States induced peripheral blood mononuclear cells to secrete higher levels of cytokines and chemokines associated with innate and Th1-adaptive immune responses, whereas strains from Europe induced greater Th17-associated responses. Thus, strains of the same B. burgdorferi species from Europe and the United States represent distinct clonal lineages that vary in virulence and inflammatory potential.

Cutaneous Small/Medium CD4+ Pleomorphic T-Cell Lymphoma-Like Nodule in a Patient With Erythema Chronicum Migrans.

CD4+ small/medium pleomorphic T-cell lymphoma is a relatively rare subtype of cutaneous lymphoproliferative disorder with an indolent clinical behavior. The place of this condition among lymphomas is debatable. The authors describe a rare case of the direct association of CD4 small/medium pleomorphic T-cell lymphoma-like solitary nodule with Borrelia burgdorferi infection in a 5-year-old boy, discuss the reactive nature of this condition, and emphasize the importance of clinicopathological correlation.

The many masks of cutaneous Lyme disease.

Early cutaneous Lyme disease, erythema migrans, may show different histopathologic patterns. The intent of this case series is to raise awareness of these findings to prevent misdiagnosis and keep this entity in the differential. Erythema migrans develops after a tick bite and subsequent infection with the spirochete, Borrelia burgdorferi. It most commonly manifests as a solitary, annular lesion with a bull's-eye appearance. Classic histopathologic findings include superficial and deep perivascular and interstitial lymphocytic infiltrates mixed with plasma cells and eosinophils. We identified and reviewed eight cases of early erythema migrans. Each patient had confirmed B. burgdorferi IgM seropositivity and IgG seronegativity. Histopathologic evaluation of these biopsies reveals a diversity of patterns. Seven of eight cases show sparse to mild perivascular and interstitial mixed infiltrate of variable amount of lymphocytes, eosinophils, neutrophils and plasma cells, with only one case showing a dense inflammatory infiltrate. Epidermal changes such as spongiosis and interface change are seen in some cases. Additionally, perineural lymphocytic infiltrate is seen in one case, periadnexal infiltrate in four cases and pigment incontinence in one case. Based on variable histopathologic findings, it is important to consider erythema migrans in the differential diagnosis for prompt diagnosis and treatment.

[Clinical and Laboratory Predictors for Forecasting the Outcomes of Ixodes Tick-Borne Borreliosis].

OBJECTIVE: Our aim was to identify the most informative clinical and laboratory predictors of chronicity of Ixodes tick-borne borreliosis in the acute phase of the disease based on the "optimal cut-off values" (COV) and the predicted probability of the outcomes. METHODS: A retrospective cohort controlled study was carried out. We used the technique of ROC-analysis to estimate the information content of the clinical and laboratory indicators in patients with Ixodes tick-borne borreliosis in the acute phase of the disease with erythemal (n =16), non-erythemal (n = 77) forms of Ixodes tick-borne borreliosis and co-infection with the tick-borne encephalitis (n = 68) for the prediction of the outcomes: recovery or chronization. RESULTS: A retrospective analysis of clinical and laboratory parameters recorded in the acute phase of the disease in 161 patients with chronic Ixodes tick-borne borreliosis. The calculations were performed for the informative clinical and laboratory prognostic predictors of the outcomes for the intervals above and below the COVvalues are defined probabilities of recovery or chronization of Ixodes tick-borne borreliosis. A general predictor of outcomes for all clinicalforms of the disease--the interleukin 8--was established: the probability of chronization after erythemal form is 100.0% at the level of its production over 107.89 pg/ml (AUC = 1.0), after non-erythemal form is 54.63 ± 0.23% at serum concentrations above 94.64 pg/ml (AUC = 0.770), after co-infection with the tick-borne encephalitis is 52.69 ± 0.27% at the level of interleukin 8 above 84.96 pg/ml (AUC = 0.780). CONCLUSION: The results of the study suggest the possibility of predicting the outcomes of infection in the acute phase, which allows to optimize the etiopathogenic therapy of the disease in a timely manner.

Testing patients with non-specific symptoms for antibodies against Borrelia burgdorferi sensu lato does not provide useful clinical information about their aetiology.

The aim of this study was to determine whether patients with antibodies against Borrelia burgdorferi sensu lato or who report a history of erythema migrans (EM) or tick bite are more likely to have non-specific symptoms such as musculoskeletal pain, fatigue, sensory disorder, and headache. The study group comprised 423 subjects with non-specific symptoms tested for antibodies against B. burgdorferi sensu lato between July 2012 and December 2014 because of suspicion of Lyme borreliosis (LB). Of these, 285 were females (67%) and 138 were males (33%); the median age was 53 years (range, 7-89 years). Patients with a confirmed diagnosis of LB and patients with a known underlying disease that could influence the development of the symptoms were excluded from the evaluation. Subjects were assigned to the seronegative group or to one of three seropositive groups, and the history of EM and tick bite was also recorded. Statistical analysis was performed with single chi-square tests of independence and multiple logistic regression models. No differences in the occurrence of non-specific symptoms were observed between patients grouped according to antibody status. A history of EM showed no significant effect on any of the non-specific symptoms. A history of tick bite was weakly correlated with joint pain and joint swelling (p <0.05). In conclusion, it is highly unlikely that the complaints are related to a previous infection with B. burgdorferi sensu lato. The results show that testing patients with non-specific symptoms for antibodies against B. burgdorferi sensu lato in the everyday clinical setting does not provide any useful information about their aetiology.

Annexin A2 is a target of autoimmune T and B cell responses associated with synovial fibroblast proliferation in patients with antibiotic-refractory Lyme arthritis.

In this study, autoantibody responses to annexin A2 were found in 11-15% of 278 patients with Lyme disease, including in those with erythema migrans (EM), an early sign of the illness, and in those with antibiotic-responsive or antibiotic-refractory Lyme arthritis (LA), a late disease manifestation. In contrast, robust T cell reactivity to annexin A2 peptides was found only in patients with responsive or refractory LA. In LA patients, annexin A2 protein levels, which were higher in the refractory group, correlated with annexin A2 antibody levels in sera and synovial fluid. In addition, in patients with antibiotic-refractory LA who had anti-annexin A2 antibodies, synovial tissue had intense staining for annexin A2 protein, greater synovial fibroblast proliferation and more tissue fibrosis. Thus, a subset of LA patients had T and B cell responses to annexin A2, and in the refractory group, annexin A2 autoantibodies were associated with specific pathologic findings.

Spatiotemporal evolution of erythema migrans, the hallmark rash of Lyme disease.

To elucidate pathogen-host interactions during early Lyme disease, we developed a mathematical model that explains the spatiotemporal dynamics of the characteristic first sign of the disease, a large (≥5-cm diameter) rash, known as an erythema migrans. The model predicts that the bacterial replication and dissemination rates are the primary factors controlling the speed that the rash spreads, whereas the rate that active macrophages are cleared from the dermis is the principle determinant of rash morphology. In addition, the model supports the clinical observations that antibiotic treatment quickly clears spirochetes from the dermis and that the rash appearance is not indicative of the efficacy of the treatment. The quantitative agreement between our results and clinical data suggest that this model could be used to develop more efficient drug treatments and may form a basis for modeling pathogen-host interactions in other emerging infectious diseases.

Elevated levels of IL-23 in a subset of patients with post-lyme disease symptoms following erythema migrans.

BACKGROUND: The causes of post-Lyme disease symptoms are unclear. Herein, we investigated whether specific immune responses were correlated with such symptoms. METHODS: The levels of 23 cytokines and chemokines, representative of innate and adaptive immune responses, were assessed in sera from 86 antibiotic-treated European patients with erythema migrans, 45 with post-Lyme symptoms and 41 without symptoms, who were evaluated prior to treatment and 2, 6, and 12 months thereafter. RESULTS: At study entry, significant differences between groups were observed for the type 1 helper T cell (TH1)-associated chemokines CXCL9 and CXCL10, which were associated with negative Borrelia cultures, and the type 17 helper T cell (TH17)-associated cytokine interleukin 23 (IL-23), which was associated with positive cultures and the development of post-Lyme symptoms (P ≤ .02). Moreover, of the 41 patients with detectable IL-23 levels, 25 (61%) developed post-Lyme symptoms, and all 7 with IL-23 levels ≥ 230 ng/mL had such symptoms. Furthermore, antibody responses to the ECGF autoantigen were more common in patients with post-Lyme symptoms (P = .07) and were correlated directly with IL-23 levels (P = .02). Despite the presence of post-Lyme symptoms, all posttreatment culture results were negative, antiborrelial antibody responses declined, and there were no objective signs of disseminated disease, suggesting that spirochetal eradication had occurred with treatment in all patients. CONCLUSIONS: High TH1-associated responses correlated with more effective immune-mediated spirochetal killing, whereas high TH17-associated immune responses, often accompanied by autoantibodies, correlated with post-Lyme symptoms, providing a new paradigm for the study of postinfectious symptoms in a subset of patients with Lyme disease.

Decreased Th1-type inflammatory cytokine expression in the skin is associated with persisting symptoms after treatment of erythema migrans.

BACKGROUND: Despite the good prognosis of erythema migrans (EM), some patients have persisting symptoms of various character and duration post-treatment. Several factors may affect the clinical outcome of EM, e.g. the early interaction between Borrelia (B.) burgdorferi and the host immune response, the B. burgdorferi genotype, antibiotic treatment as well as other clinical circumstances. Our study was designed to determine whether early cytokine expression in the skin and in peripheral blood in patients with EM is associated with the clinical outcome. METHODS: A prospective follow-up study of 109 patients with EM was conducted at the Åland Islands, Finland. Symptoms were evaluated at 3, 6, 12 and 24 months post-treatment. Skin biopsies from the EM and healthy skin were immunohistochemically analysed for expression of interleukin (IL)-4, IL-10, IL-12p70 and interferon (IFN)-γ, as well as for B. burgdorferi DNA. Blood samples were analysed for B. burgdorferi antibodies, allergic predisposition and levels of systemic cytokines. FINDINGS: None of the patients developed late manifestations of Lyme borreliosis. However, at the 6-month follow-up, 7 of 88 patients reported persisting symptoms of diverse character. Compared to asymptomatic patients, these 7 patients showed decreased expression of the Th1-associated cytokine IFN-γ in the EM biopsies (p=0.003). B. afzelii DNA was found in 48%, B. garinii in 15% and B. burgdorferi sensu stricto in 1% of the EM biopsies, and species distribution was the same in patients with and without post-treatment symptoms. The two groups did not differ regarding baseline patient characteristics, B. burgdorferi antibodies, allergic predisposition or systemic cytokine levels. CONCLUSION: Patients with persisting symptoms following an EM show a decreased Th1-type inflammatory response in infected skin early during the infection, which might reflect a dysregulation of the early immune response. This finding supports the importance of an early, local Th1-type response for optimal resolution of LB.

Borrelia burgdorferi infection regulates CD1 expression in human cells and tissues via IL1-ß.

The appearance of group 1 CD1 proteins (CD1a, CD1b and CD1c) on maturing myeloid DC is a key event that converts myeloid DC to effective lipid APC. Here, we show that Borrelia burgdorferi, the causative agent of Lyme disease, triggers appearance of group 1 CD1 proteins at high density on the surface of human myeloid DC during infection. Within human skin, CD1b and CD1c expression was low or absent prior to infection, but increased significantly after experimental infections and in erythema migrans lesions from Lyme disease patients. The induction of CD1 was initiated by borrelial lipids acting through TLR-2 within minutes, but required 3 days for maximum effect. The delay in CD1 protein appearance involved a multi-step process whereby TLR-2 stimulated cells release soluble factors, which are sufficient to transfer the CD1-inducing effect in trans to other cells. Analysis of these soluble factors identified IL-1ß as a previously unknown pathway leading to group 1 CD1 protein function. This study establishes that upregulation of group 1 CD1 proteins is an early event in B. burgdorferi infection and suggests a stepwise mechanism whereby bacterial cell walls, TLR activation and cytokine release cause DC precursors to express group 1 CD1 proteins.

Early production of IL-22 but not IL-17 by peripheral blood mononuclear cells exposed to live Borrelia burgdorferi: the role of monocytes and interleukin-1.

If insufficiently treated, Lyme borreliosis can evolve into an inflammatory disorder affecting skin, joints, and the CNS. Early innate immunity may determine host responses targeting infection. Thus, we sought to characterize the immediate cytokine storm associated with exposure of PBMC to moderate levels of live Borrelia burgdorferi. Since Th17 cytokines are connected to host defense against extracellular bacteria, we focused on interleukin (IL)-17 and IL-22. Here, we report that, despite induction of inflammatory cytokines including IL-23, IL-17 remained barely detectable in response to B. burgdorferi. In contrast, T cell-dependent expression of IL-22 became evident within 10 h of exposure to the spirochetes. This dichotomy was unrelated to interferon-γ but to a large part dependent on caspase-1 and IL-1 bioactivity derived from monocytes. In fact, IL-1ß as a single stimulus induced IL-22 but not IL-17. Neutrophils display antibacterial activity against B. burgdorferi, particularly when opsonized by antibodies. Since neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection, protective and antibacterial properties of IL-22 may close this gap and serve essential functions in the initial phase of spirochete infection.

Antibody reactivity to Borrelia burgdorferi sensu stricto antigens in patients from the Brazilian Amazon region with skin diseases not related to Lyme disease.

In the present study, we report the occurrence of borreliosis in patients from the Brazilian Amazonic region. Nineteen (7.2%) out of 270 dermatological patients with different skin diseases (no one with clinical Lyme disease), tested positive by ELISA for Borrelia burgdorferi. Serum samples from 15 out of the 19 ELISA-positive patients were further evaluated by Western blot. Presence of Borrelia burgdorferi specific IgG was confirmed in eight (53.3%) out of the 15 patients. All eight patients with ELISA and Western blot positive reactions were treated with doxycycline, according to the Centers for Disease Control and Prevention guidelines. One of them had clinical manifestations of colagenosis and was sent to the Department of Internal Medicine for further investigation. Data presented here suggested that borreliosis "lato sensu" is in the Brazilian Amazon region.