Functional Competence of Peritoneal Macrophages in Murine Lyme Borreliosis

In mice infected with the Lyme disease spirochete, Borrelia burgdorferi, spirochetes can be cultured from the uninflamed peritoneal cavity despite their proximity to resident macrophages. Here we have evaluated the phagocytic and killing capacity of peritoneal macrophages ex vivo from such mice, and compared them to cells from uninfected controls. We noted no significant differences in the numbers of cells recovered from lavage fluid nor in the proportion of lymphocytes in that fluid or their type or state of activation; B cells vs. T cells, CD4 vs. CD8, T cell receptor display (TCR vs. TCR), or B cell activation markers (CD24 vs. CD44). In addition, in studies conducted with coded samples, macrophages from infected animals were similar to controls in their ability (i) to bind and ingest spirochetes as shown by immunofluorescent kinetic studies imaged by confocal microscopy, (ii) to kill spirochetes as detected by acridine orange vital staining in live confocal microscopy, and (iii) to generate H₂O₂, a measure of their activation. Finally, we observed no difference in the ability of cells isolated from infected mice to kill the parasite, Toxoplasma gondii, a sensitive functional measure of the activation of killing capacity. Thus, despite the failure of immune clearance that results in persistent or chronic disease in vivo, there does not appear to be a global impairment of macrophage function in the infected host. It remains to be determined whether specific sites of inflammation, e.g., skin, joints, heart, are associated with a downregulation of phagocyte function locally.     

The Caspase 1 Inflammasome Is Not Required for Control of Murine Lyme Borreliosis

The contribution of the inflammasome to the development of immune responses and disease during infection with the Lyme disease spirochete, Borrelia burgdorferi, is not well defined. Host defense against the spirochete is severely impaired in mice deficient in the adaptor molecule myeloid differentiation antigen 88 (MyD88), which is required not only for Toll-like receptor-mediated responses but also for the production of the proforms of interleukin 1β (IL-1β) and IL-18. These cytokines are released in active forms after cleavage by the inflammasome-associated enzyme caspase 1. To investigate the contribution of the inflammasome to host defense against B. burgdorferi, we examined Lyme borreliosis in mice deficient in either caspase 1 or apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), a molecule upstream of caspase 1 in the inflammasome signaling cascade. We found that caspase 1-deficient mice had a mild transient elevation in pathogen burden and a trend toward an increase in the prevalence of arthritis early in infection, but these differences resolved by day 14 postinfection. Caspase 1 deficiency had no effect on B. burgdorferi-induced humoral immunity, T-cell responses, or the abilities of macrophages to ingest and degrade spirochetes. The absence of the ASC protein had no effect on the control of the spirochete or the development of immune responses and disease. These findings reveal that the caspase 1 inflammasome is not critical to host defense against the extracellular pathogen Borrelia burgdorferi.Infection of humans with the Lyme disease spirochete, Borrelia burgdorferi, results in a characteristic pattern of skin lesions, arthritis, carditis, and neurologic abnormalities that reflect the immune response to the spirochete as it invades and disseminates in the mammalian host (7). In the murine model of Lyme borreliosis, spirochetes inoculated into the skin disseminate within days to infect all organ systems, but disease is primarily manifested in the joints and heart (4). Disease in the animal model is due largely to the innate immune response to spirochetes because histopathology reveals mainly neutrophils and macrophages within inflamed joints and hearts, respectively (5, 28, 36, 43), and occurs in the absence of adaptive (T- and B-cell-mediated) immunity (8, 28, 43).Recent studies have further defined the role of innate immunity in Lyme borreliosis. B. burgdorferi lipoproteins activate innate immune cells through the pattern recognition molecule Toll-like receptor 2 (TLR2), which is required for innate but not adaptive immune responses to the spirochete (2, 19, 49). Spirochete components also stimulate murine cells through TLR5 and TLR9 (44). The TLR cytosolic domains contain a Toll/interleukin 1 (IL-1) receptor domain (TIR) that interacts with myeloid differentiation antigen 88 (MyD88) and results in the activation of NF-κB and the production of proinflammatory cytokines, chemokines, and costimulatory molecules that are important for host defense (6, 12, 14). We and others have previously shown that B. burgdorferi-infected MyD88-deficient (MyD88^−/−) mice have significantly elevated pathogen burdens that persist through 90 days of infection despite the presence of high titers of anti-B. burgdorferi antibodies (9, 25). The elevated level of pathogen DNA in tissues was explained in part by our finding that MyD88^−/− peritoneal macrophages ingested spirochetes at the same rate as wild-type (WT) cells, but the kinetics of degradation was slower, with internalized spirochetes remaining in an elongated form for a longer period. Others have found that bone marrow-derived MyD88^−/− macrophages do not efficiently ingest spirochetes (44). The MyD88^−/− mice developed carditis and arthritis similar to the disease in WT mice analyzed at its peak (days 14 and 28) and during regression (day 45) (9, 25). Together, these results showed that MyD88-dependent signaling pathways are not required for B. burgdorferi-induced inflammation or disease regression but are necessary for efficient control of the pathogen burden by phagocytes. These studies did not distinguish whether interruption of MyD88-dependent TLR signaling pathways was solely responsible for the impaired control of the pathogen or whether other MyD88-dependent pathways also play a role.In addition to being a crucial signaling molecule for TLRs involved in B. burgdorferi recognition, MyD88 is required for IL-1 receptor (IL-1R)- and IL-18R-associated kinase signaling. TLR activation is a key inducer of the proforms of IL-1β and IL-18, and the secreted forms of these two cytokines require MyD88 for their receptors to mediate their effects (1, 34, 38). Behera et al. (6) have shown that IL-18 alone does not significantly contribute to host immunity in Lyme borreliosis because IL-18^−/− mice exhibit no defects in pathogen clearance or the development of disease. IL-1β, however, may play a role because human peripheral blood mononuclear cells secrete IL-1β after ingestion of live B. burgdorferi spirochetes (15). In support of this hypothesis, serum levels of IL-1β were reported to be elevated in Lyme disease patients, and the levels decreased significantly after doxycycline treatment (35). IL-1β mRNA levels in erythema migrans lesions were also shown to be elevated (31).To further delineate the role of MyD88-dependent signaling pathways in host defense against B. burgdorferi, we examined the course of Lyme borreliosis in mice deficient in either the intracellular cysteine protease caspase 1 or apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). Caspase 1 plays a key role in inflammatory responses by cleaving pro-IL-1β and pro-IL-18 into their active secreted forms (16, 22). These cytokines are matured in a large caspase 1-containing protein complex called the inflammasome (37). ASC, a component of the inflammasome, is required for eliciting the enzymatic activity of caspase 1. Caspase 1 contains an N-terminal caspase recruitment domain (CARD) shown to be involved in the assembly of protein platforms that promote proteolytic activation of recruited caspases in the context of apoptosis and inflammation (14). In addition to cleaving pro-IL-1 and pro-IL-18, caspase 1 is also involved in other proinflammatory pathways, including NF-κB signaling pathways associated with innate and adaptive immune responses (21, 23, 41). In contrast, ASC is essential only for the secretion of IL-1β/IL-18 but dispensable for caspase 1-mediated IL-6 and tumor necrosis factor alpha secretion and NF-κB and p38 activation (40). Thus, although both caspase 1^−/− mice and ASC^−/− mice have defects in the production of IL-1β/IL-18, caspase 1^−/− mice have additional defects in the activation of NF-κB.Several published reports have established that the inflammasome is important for immunity to intracellular bacteria and viruses, but much less is known about the contribution of the inflammasome to host defense against extracellular pathogens that elicit cytokines activated by caspase 1 (27, 29, 30, 32, 38, 42, 48). Thus, we sought to determine whether the inflammasome is also important during infection with the B. burgdorferi spirochete as representative of a subset of extracellular pathogens. We found that while B. burgdorferi can elicit IL-1β in a caspase 1-dependent fashion from mouse macrophages in vitro, the caspase 1-dependent inflammasome is not essential for the ultimate control of B. burgdorferi infection and disease.     

Aspects of Lyme Borreliosis in Europe

 Lyme borreliosis in Europe is somewhat different from that encountered in the USA. The aim of this review is to present historical, ecological, epidemiological, clinical, and therapeutic aspects associated with Lyme borreliosis in Europe. It becomes apparent that a considerable body of information about European Lyme borreliosis exists, which has been accumulated for more than 100 years.     

Modulation of Murine Lyme Borreliosis by Interruption of the B7/CD28 T-Cell Costimulatory Pathway

Recent studies have implicated cytokines associated with Th2 cells in the genetic resistance to murine Lyme borreliosis. Because the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th-cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to the Th2 cytokine profile and development of arthritis in BALB/c mice infected with Borrelia burgdorferi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin 4 (IL-4) and upregulation of gamma interferon (IFN-γ) responses by B. burgdorferi-specific T cells and by reduction of B. burgdorferi-specific immunoglobulin G. Despite the shift toward a Th1 cytokine pattern, which others have associated with disease susceptibility, the severity of arthritis was unchanged. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies or CTLA-4Ig enhanced IFN-γ production over that seen with CD86 blockade alone, yet augmentation of this Th1-associated cytokine did not enhance disease. These results demonstrate that IL-4 production by T cells in B. burgdorferi-infected BALB/c mice is dependent upon CD86/CD28 interaction and that this cytokine does not contribute significantly to host resistance to the development of arthritis. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-γ-producing T cells in B. burgdorferi infection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. These studies may provide insight into the role of the B7/CD28 pathway in other infectious and autoimmune diseases in which deviation of Th cell immune responses occurs and antigen is persistently present.Lyme disease is a multisystem illness due to infection with the tick-transmitted spirochete Borrelia burgdorferi. Experimental infection of laboratory mice with B. burgdorferi results in acute arthritis and carditis that reproducibly peak at 2 to 4 weeks of infection and then resolve within 3 months despite spirochete persistence (4). Studies using SCID mice, which lack functional T and B cells, have demonstrated that disease is due to the innate immunity of the host and can occur in the absence of specific immune responses (6, 28). The persistent and progressive nature of disease manifestations in SCID mice underscores the importance of T and B cells in initiating disease regression (5, 6, 28). Recent studies support the additional role of specific immunity in modulating disease severity via direct effects on spirochete burden through B. burgdorferi-specific antibodies (5) and indirectly through Th cell-associated cytokines that influence the activation of innate immune cells (14, 23). In particular, the dominance of Th1-type responses, which support macrophage activation, in patients with chronic Lyme arthritis has implicated this T-cell phenotype in the development and perpetuation of severe inflammatory disease (32, 37). Th1-type responses have also been observed during B. burgdorferi infection of C3H mice, a disease-susceptible strain, whereas Th2 responses, which promote B-cell functions, can be detected in BALB/c mice, a comparatively disease-resistant strain (14, 23). Despite the greater inflammatory response in C3H mice, their pathogen burden as assessed by quantitative PCR of spirochete DNA remains higher than that of disease-resistant mouse strains (36), suggesting that the recruitment of innate immune cells is appropriate yet ineffective at controlling infection (29).In addition to signals provided by T-cell antigen receptor engagement, the interaction of costimulatory molecules present on antigen-presenting cells (APCs) with their ligands on T cells is believed to be necessary for the initial priming of naive T cells. In particular, the B7/CD28 costimulatory pathway has been implicated in the differentiation of naive Th0 cells into Th1 and Th2 subsets (33). The mechanisms by which these molecules assist in the priming of the T-cell immune response are complex and poorly understood. Two members of the B7 family have been characterized, CD80 and CD86 (also known as B7-1 and B7-2, respectively), and differ not only in their binding properties to CD28 on T cells but also in the timing of their appearance on conventional APCs during the initiation of an immune response (11). CD86 appears earlier on the surface of mitogen-activated APCs and has a lower affinity for CD28 than does CD80. Once activated, T cells express CTLA-4, a second receptor to which both CD80 and CD86 bind with greater affinity than they bind CD28 (21). Interaction of CD80/CD86 with CTLA-4 can downregulate the T-cell immune response (35). Blockade of CD86 during the initiation of a T-cell response results in an immune response oriented toward a Th1 phenotype, whereas a similar blockade of CD80 does not consistently favor a Th2 phenotype (20). Experiments using mutant mice deficient in CD80 and/or CD86 reveal the important role of these molecules in sustaining a Th-cell phenotype and, in the case of CD86 expression, in the development of a Th2 response (20). Costimulation through the B7/CD28 pathway contributes to the expansion of autoimmune disease processes seen in experimental autoimmune encephalitis (17, 27), a predominantly Th1-associated disease, and autoimmune diabetes (19). Studies using a soluble recombinant form of CTLA-4 designated CTLA-4Ig have supported many of the observations made with anti-B7 antibodies (13, 19, 26).We have recently reported that the Th2 response of B. burgdorferi-infected BALB/c mice is preceded by a Th1 response and that the presence of interleukin 4 (IL-4) is associated with accelerated resolution of arthritis (12). A hind-foot inoculation route was used in that study so that T-cell responses could be examined in lymph nodes adjacent to joints afflicted with arthritis. We demonstrated that this route of inoculation induces moderately severe arthritis in BALB/c mice at day 14 of infection that undergoes more rapid regression than the arthritis seen in similarly infected C3H mice, in which IL-4 responses are not detectable. Previous studies have shown that treatment of mice with anti-IL-4 monoclonal antibody (MAb) exacerbates arthritis in BALB/c mice assessed at intervals corresponding to the plateau and resolution phases of disease, providing evidence that IL-4 modulates the severity of established arthritis (14, 23). The influence of Th2 cell effector functions on the development of arthritis remains unknown. In the current study, we have examined the effects of interruption of Th2 cell differentiation by B7/CD28 blockade with anti-CD80 and/or anti-CD86 MAb or CTLA-4Ig on the cytokine profiles and development of arthritis in BALB/c mice infected with B. burgdorferi.     

Disease Expression of Lyme Borreliosis in Northeastern France

 Since very little is known about the clinical expression of Lyme borreliosis in Western Europe, a 3-year prospective study was conducted that included all patients seen for suspected Lyme borreliosis at the Strasbourg University Hospital in northeastern France. The diagnosis of Lyme borreliosis was made on the basis of the presence of erythema migrans or on the basis of another suggestive clinical manifestation and laboratory confirmation. A total of 132 patients, 70 women and 62 men, mean age 54 years, had Lyme borreliosis according to these criteria. Within this study group, 77% of the patients were regularly exposed to tick bites and 64% could remember one. Erythema migrans, the most frequent clinical manifestation, occurred in 60% of the patients and was the only sign of Lyme borreliosis in 40%. Lymphocytoma and acrodermatitis chronica atrophicans were rare (1 and 3 patients, respectively). Nervous system involvement (mainly radiculoneuropathy), the second most common clinical manifestation, was found in 40% of the patients and was the only sign of Lyme borreliosis in 22%. Musculoskeletal involvement was present in 26% of the patients and was an isolated finding in 14%. During the study period, no patient was diagnosed with Lyme carditis. There was serological evidence of Lyme borreliosis in 75% of the cases and direct evidence of borrelial infection in 10 (7.5%). The results show that the clinical expression of Lyme borreliosis in northeastern France is similar to that in other European countries but different from that in North America.     

Macrophage Polarization Drives Granuloma Outcome during Mycobacterium tuberculosis Infection

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), induces formation of granulomas, structures in which immune cells and bacteria colocalize. Macrophages are among the most abundant cell types in granulomas and have been shown to serve as both critical bactericidal cells and targets for M. tuberculosis infection and proliferation throughout the course of infection. Very little is known about how these processes are regulated, what controls macrophage microenvironment-specific polarization and plasticity, or why some granulomas control bacteria and others permit bacterial dissemination. We take a computational-biology approach to investigate mechanisms that drive macrophage polarization, function, and bacterial control in granulomas. We define a “macrophage polarization ratio” as a metric to understand how cytokine signaling translates into polarization of single macrophages in a granuloma, which in turn modulates cellular functions, including antimicrobial activity and cytokine production. Ultimately, we extend this macrophage ratio to the tissue scale and define a “granuloma polarization ratio” describing mean polarization measures for entire granulomas. Here we coupled experimental data from nonhuman primate TB granulomas to our computational model, and we predict two novel and testable hypotheses regarding macrophage profiles in TB outcomes. First, the temporal dynamics of granuloma polarization ratios are predictive of granuloma outcome. Second, stable necrotic granulomas with low CFU counts and limited inflammation are characterized by short NF-κB signal activation intervals. These results suggest that the dynamics of NF-κB signaling is a viable therapeutic target to promote M₁ polarization early during infection and to improve outcome.     

Peptide-Based OspC Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Lyme Borreliosis

Sera from 210 patients with Lyme borreliosis (LB) were studied by an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (pepC10) comprising the C-terminal 10-amino-acid residues of OspC of Borrelia burgdorferi. We found that 36.3 and 45.0% of the serum samples from patients with erythema migrans (EM) and neuroborreliosis (NB), respectively, displayed immunoglobulin M (IgM) anti-pepC10 reactivities, while these samples rarely (≤8%) displayed IgG antibody reactivities. Sera from patients with acrodermatitis chronica atrophicans did not contain anti-pepC10 antibodies. The diagnostic performance of this newly developed peptide ELISA was compared with those of an ELISA based on the full-length recombinant OspC protein (rOspC) and a commercially available ELISA based on the B. burgdorferi flagellum (Fla). The sensitivity of the IgM pepC10 ELISA was slightly lower (P < 0.04) than that of the rOspC ELISA for EM patients (36.3 versus 43.8%), while there was no difference for NB patients (45.0 versus 48.0%). However, the optical density values obtained by the pepC10 ELISA were generally higher than those obtained by the rOspC ELISA, leading to a significantly better quantitative discrimination between seropositive patients with NB and controls (P < 0.008). The specificity of the pepC10 ELISA was similar to those of the rOspC ELISA and the Fla ELISA for relevant controls including patients with syphilis and mononucleosis. Although the overall diagnostic sensitivity of the Fla ELISA was superior, 8.8 and 12.0% of the EM and NB patients, respectively, were antibody positive only by the pepC10 ELISA. Thus, use of a diagnostic test for LB based on the detection of IgM antibodies to pepC10 and Fla has increased sensitivity for the diagnosis of early LB.     

Decorin-Binding Protein of Borrelia burgdorferi Is Encoded within a Two-Gene Operon and Is Protective in the Murine Model of Lyme Borreliosis

Isolated outer membranes of Borrelia burgdorferi were used in immunoblotting experiments with sera from immune mice to identify new putative Lyme disease vaccine candidates. One immunoreactive polypeptide migrated on polyacrylamide gels just proximal to outer surface protein C and comigrated with [³H]palmitate-labeled polypeptides. A degenerate oligonucleotide primer based upon internal amino acid sequence information was used to detect the corresponding gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 24-amino-acid putative signal peptide terminated by LLISC, a probable consensus sequence for lipoprotein modification, and a mature protein of 163 amino acids. Immunoblots of a recombinant fusion protein corresponding to this ORF supported the idea that the encoded protein was a previously reported decorin-binding protein (DBP) of B. burgdorferi N40 (B. P. Guo, S. J. Norris, L. C. Rosenberg, and M. Höök, Infect. Immun. 63:3467–3472, 1995). However, further DNA sequencing revealed the presence of a second ORF, designated ORF-1, whose termination codon was 119 bp upstream of the dbp gene. ORF-1 also encoded a putative lipoprotein with a mature length of 167 amino acids. Northern blots, Southern blots, and primer extension analyses indicated that ORF-1 and dbp comprised a two-gene operon located on the 49-kb linear plasmid. Both proteins, which were 40% identical and 56% similar, partitioned into Triton X-114 detergent extracts of B. burgdorferi isolated outer membranes. Mice infected with B. burgdorferi produced high titers of antibodies against the ORF-1-encoded protein and DBP during both early and later stages of chronic infection. Both DBP and the ORF-1-encoded protein were sensitive to proteinase K treatment of intact borreliae, suggesting that they were surface exposed. In active immunization experiments, 78% of mice immunized with recombinant DBP were immune to challenge. While it is not clear whether the two lipoproteins encoded by the ORF-1-dbp operon have analogous decorin-binding functions in vivo, the combined studies implicate DBP as a new candidate for a human Lyme disease vaccine.     

Dynamic Changes of Microglia/Macrophage M1 and M2 Polarization in Theiler's Murine Encephalomyelitis

Microglia and macrophages play a central role for demyelination in Theiler's murine encephalomyelitis (TME) virus infection, a commonly used infectious model for chronic‐progressive multiple sclerosis. In order to determine the dynamic changes of microglia/macrophage polarization in TME, the spinal cord of Swiss Jim Lambert (SJL) mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and demyelinating leukomyelitis were confirmed by immunohistochemistry and histology. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase indicative of incomplete remyelination. A total of 59 genes out of 151 M1‐ and M2‐related genes were differentially expressed in TME virus‐infected mice over the study period. The onset of virus‐induced demyelination was associated with a dominating M1 polarization, while mounting M2 polarization of macrophages/microglia together with sustained prominent M1‐related gene expression was present during the chronic‐progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32⁺ M1‐, arginase‐1⁺ M2‐ and Ym1⁺ M2‐type cells associated with progressive demyelination. The present study provides a comprehensive database of M1‐/M2‐related gene expression involved in the initiation and progression of demyelination supporting the hypothesis that perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME.     

Lyme Borreliosis in Human Patients in Florida and Georgia,USA

The aim of this study was to determine the cause of illness in several human patients residing in Florida and Georgia, USA, with suspected Lyme disease based upon EM-like skin lesions and/or symptoms consistent with early localized or late disseminated Lyme borreliosis. Using polymerase chain reaction (PCR) assays developed specifically for Lyme group Borrelia spp., followed by DNA sequencing for confirmation, we identified Borrelia burgdorferi sensu lato DNA in samples of blood and skin and also in lone star ticks (Amblyomma americanum) removed from several patients who either live in or were exposed to ticks in Florida or Georgia. This is the first report to present combined PCR and DNA sequence evidence of infection with Lyme Borrelia spp. in human patients in the southern U.S., and to demonstrate that several B. burgdorferi sensu lato species may be associated with Lyme disease-like signs and symptoms in southern states. Based on the findings of this study, we suggest that human Lyme borreliosis occurs in Florida and Georgia, and that some cases of Lyme-like illness referred to as southern tick associated rash illness (STARI) in the southern U.S. may be attributable to previously undetected B. burgdorferi sensu lato infections.     

Comparison of Immunodot and Western Blot Assays for Diagnosing Lyme Borreliosis

Two commercially available serologic tests for use in diagnosing Lyme borreliosis were evaluated by using a test panel comprised of sera from patients diagnosed with Lyme borreliosis, non-Lyme disease controls, and healthy subjects. The test methods examined were a Western blot assay and an immunodot assay. The study was initiated to determine how the immunodot assay, which contains purified and recombinant proteins to those borrelial antigens recommended for immunoglobulin M (IgM) detection in the Dearborn criteria, would compare with the Western blot assay as a confirmatory method for serologic diagnosis of Lyme borreliosis. Results obtained showed that the two test methods performed comparably for detecting IgG antibodies. For IgM antibody detection, the immunodot and Western blot assays had similar sensitivities; however, the immunodot assay was more specific and had greater positive predictive value than the Western blot assay. The results obtained indicate that the immunodot assay performs as well as or better than the Western blot assay for diagnosing Lyme borreliosis. Furthermore, because it uses a limited panel (n = 5) of antigens, the immunodot is easier to read and interpret than standard Western blots.     

Critical Evaluation of Urine-Based PCR Assay for Diagnosis of Lyme Borreliosis

Many approaches were made in recent years to establish urine PCR as a diagnostic tool for Lyme borreliosis, but results are contradictory. In the present study, a standardized protocol spiking urine from healthy donors with a defined amount of whole Borrelia or Borrelia DNA was established. The development of a nested real-time PCR targeting ospA enabled a highly sensitive and quantitative analysis of these samples. We show the following. (i) Storage of spiked urine samples for up to 6 months at −20°C had no negative effect on spike recovery. (ii) Centrifugation of 10 ml of urine at 40,000 × g for 30 min resulted in a concentration of both spikes, i.e., whole Borrelia and DNA. (iii) The inhibition of DNA spike recovery in 48% (11 of 23 samples) of urine samples tested could be attributed to nuclease activity. This was abrogated by alkalizing the urine or by working with the samples on ice. Despite optimized conditions, analysis of urine samples of 12 patients with erythema migrans, the clinical stage considered to be associated with the highest bacterial load, revealed a positive result in only one sample. All 12 samples were negative by an alternative PCR targeting flagellin. The results of our study support doubts that urine is a suitable material for diagnosis of Lyme borreliosis.     

Evaluation of Fifteen Commercially Available Serological Tests for Diagnosis of Lyme Borreliosis

 The performance of 11 commercially available enzyme immunoassays (EIA) and four Western blot (WB) tests for the detection of IgM and IgG antibodies against Borrelia burgdorferi were compared. A total of 229 serum specimens were used: 26 from patients with early Lyme borreliosis, 13 from patients with late Lyme borreliosis, 62 from healthy controls and 128 from patients with disorders clinically mimicking Lyme borreliosis and/or known to cause cross-reactivity in Lyme borreliosis serological tests (patient control group). In specimens from patients with early Lyme borreliosis, the sensitivity of the individual tests ranged from 35 to 81% for detection of IgM. In late Lyme borreliosis, sensitivity of the tests ranged from 46 to 92%. In healthy controls the specificity of the tests ranged from 89 to 100% and from 82 to 97% for IgM and IgG tests, respectively. In the patient control group, specificity of the tests ranged from 75 to 90% for IgM and from 84 to 100% for IgG tests. The Behring (Germany) and Genzyme Virotech (Germany) IgM EIA tests showed the best performance in detecting early Lyme borreliosis. For the detection of late Lyme borreliosis, the Dako (Denmark) IgG test was the best despite its low sensitivity. The maximum sensitivity of Western blotting for detecting IgM in patients with early Lyme borreliosis and IgG in patients with late Lyme borreliosis was 50 and 46%, respectively. The use of an EIA-WB two-test protocol improved the specificity and positive predictive values of the EIA results but caused a significant loss in sensitivity. Patients with Epstein-Barr virus or cytomegalovirus infection who had a positive reaction in the IgM EIA could not be discriminated from patients with early Lyme borreliosis with the help of Western blotting. Hence, positive and negative predictive values in combination with sensitivity and specificity values indicated that the exclusion of these infections was more relevant than the confirmation of a positive IgM EIA with Western blot.     

Detection of Borreliacidal Antibodies in Lyme Borreliosis Patient Sera Containing Antimicrobial Agents

The borreliacidal-antibody test has been used for the serological detection and confirmation of Lyme borreliosis. However, the presence of antimicrobial agents in serum can confound the accurate detection of borreliacidal antibodies. In this study, we developed a Bacillus subtilis agar diffusion bioassay to detect small concentrations of antimicrobial agents in serum. We also used XAD-16, a nonionic polymeric resin, to adsorb and remove high concentrations of amoxicillin, cefotaxime, ceftriaxone, cefuroxime, doxycycline, and erythromycin without significantly affecting even small concentrations of immunoglobulin M (IgM) or IgG borreliacidal antibodies. High concentrations of penicillin could also be removed by adding 1 U of penicillinase without significantly influencing the levels of borreliacidal antibodies. These simple procedures greatly enhance the clinical utility of the borreliacidal-antibody test.     

Cellular Metabolism and Macrophage Functional Polarization

Macrophages are a functionally heterogeneous cell population that is mainly shaped by a variety of microenvironmental stimuli. Interferon γ (IFN-γ), interleukin-1β (IL-1β), and lipopolysaccharide (LPS) induce a classical activation of macrophages (M1), whereas IL-4 and IL-13 induce an alternative activation program in macrophages (M2). Reprogramming of intracellular metabolisms is required for the proper polarization and functions of activated macrophages. Similar to the Warburg effect observed in tumor cells, M1 macrophages increase glucose consumption and lactate release and decreased oxygen consumption rate. In comparison, M2 macrophages mainly employ oxidative glucose metabolism pathways. In addition, fatty acids, vitamins, and iron metabolisms are also related to macrophage polarization. However, detailed metabolic pathways involved in macrophages have remained elusive. Understanding the bidirectional interactions between cellular metabolism and macrophage functions in physiological and pathological situations and the regulatory pathways involved may offer novel therapies for macrophage-associated diseases.