Autophagy in Dendritic Cells and B Cells Is Critical for the Inflammatory State of TLR7-Mediated Autoimmunity.

Individuals suffering from autoimmune disorders possess a hyperactive cellular phenotype where tolerance to self-antigens is lost. Autophagy has been implicated in both the induction and prevention of autoimmunity, and modulators of this cellular recycling process hold high potential for the treatment of autoimmune diseases. In this study, we determine the effects of a loss of autophagy in dendritic cells (DCs), as well as both B cells and DCs, in a TLR7-mediated model of autoimmunity, similar to systemic lupus erythematosus, where both cell types are critical for disease. Although a loss of DC autophagy slowed disease, the combined loss of autophagy in both cell types resulted in a lethal sepsis-like environment, which included tissue inflammation and hyperproduction of inflammasome-associated cytokines. Ablation of B cell signaling reversed this phenotype, indicating that activation of these cells is an essential step in disease induction. Thus, autophagy plays a dichotomous role in this model of disease.

B cell autophagy mediates TLR7-dependent autoimmunity and inflammation.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in Tlr7 transgenic (Tg) mice with or without B cell-specific ablation of autophagy (Cd19-Cre Atg5(f/f)). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to Tlr7.1 Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in Tlr7.1 Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines.

γδT cells are prevalent in the proximal aorta and drive nascent atherosclerotic lesion progression and neutrophilia in hypercholesterolemic mice.

Unique innate immunity-linked γδT cells have been seen in early human artery lesions, but their role in lesion development has received little attention. Here we investigated whether γδT cells modulate atherogenesis in apolipoprotein E-deficient (ApoE KO) mice. We found that γδT cell numbers were markedly increased in the proximal aorta of ApoE-deficient vs. wild-type mice during early atherogenesis, particularly in the aortic root and arch, where they comprised most of the T cells and lesion progression is most rapid. γδT cells infiltrated intimal lesions in ApoE KO mice, but only the adventitia in wild-type mice, and were more prevalent than CD4+ T cells in early nascent lesions, as evaluated by en face confocal microscopy. These aortic γδT cells produced IL-17, but not IFN-γ, analyzed by ex vivo FACS. Furthermore, aortic arch lipid accumulation correlated strongly with abundance of IL-17-expressing splenic γδT cells in individual ApoE KO mice. To investigate the role of these γδT cells in early atherogenesis, we analyzed ApoE/γδT double knockout (DKO) compared to ApoE KO mice. We observed reduced early intimal lipid accumulation at sites of nascent lesion formation, both in chow-fed (by 40%) and Western diet-fed (by 44%) ApoE/γδT DKO mice. In addition, circulating neutrophils were drastically reduced in these DKO mice on Western diet, while expansion of inflammatory monocytes and splenic Th1 or Th17 lymphocytes was not affected. These data reveal, for the first time, a pathogenic role of γδT cells in early atherogenesis in ApoE KO mice, by mechanisms likely to involve their IL-17 production and induction of neutrophilia. Targeting γδT cells thus might offer therapeutic benefit in atherosclerosis or other inflammatory vascular diseases.

Human endogenous retrovirus-K18 superantigen expression and human herpesvirus-6 and human herpesvirus-7 viral loads in chronic fatigue patients.

BACKGROUND: Chronic fatigue syndrome (CFS) is a complex, heterogeneous disease characterized by debilitating fatigue that is not improved with bed rest and worsens after physical activity or mental exertion. Despite extensive research into a cause of CFS, no definitive etiology has been determined; however, a large percentage of CFS patients note an acute infectious event that triggers their fatigue. METHODS: Blood and saliva were collected from 39 CFS cases and 9 healthy control subjects. Peripheral blood mononuclear cells (PBMCs) were tested for human endogenous retrovirus-K18 (HERV-K18) env transcripts using a TaqMan quantitative polymerase chain reaction (qPCR). In addition, viral copy number of human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7) were measured in both saliva and PBMCs using TaqMan qPCRs. Transcript levels and viral copy number were compared to patient CFS symptom severity. RESULTS: HERV-K18 env transcripts were not significantly different between healthy control subjects and CFS patients. Also, HERV-K18 env transcripts did not correlate with HHV-6 viral copy number or HHV-7 viral copy number in either PBMCs or saliva. HHV-6 viral copy number and HHV-7 viral copy number in both PBMCs and saliva were not significantly different between healthy control subjects and CFS patients. HERV-K18 env transcripts, HHV-6 viral copy number, and HHV-7 viral copy number did not correlate with CFS symptom severity. CONCLUSIONS: We fail to demonstrate a difference in HERV-K18 env transcripts, HHV-6 viral copy number, and HHV-7 viral copy number between CFS patients and healthy controls. Our data do not support the hypothesis of reactivation of HHV-6 or HHV-7 in CFS.

Th17 differentiation is the default program for DPP2-deficient T-cell differentiation.

Dipeptidyl peptidase 2 (DPP2) is an N-terminal dipeptidase, required for maintaining lymphocytes in a resting state. Mutant mice with T-cell-specific knock-down (kd) of DPP2 (lck-DPP2 kd) were generated and analyzed for their phenotype. Normal thymocyte development and a modest increase in the proportions of peripheral T cells were observed in these mice compared with littermate controls. Interestingly, the peripheral T cells were hyperactive upon TCR stimulation in vitro, although they did not express any activation markers. Furthermore, CD3-crosslinking in the naïve CD4(+) and CD8(+) T cells of lck-DPP2 kd mice resulted mainly in IL-17 production. Similarly, the mutant T cells secreted primarily IL-17 after in vivo priming and in vitro antigen-specific restimulation. These data suggest that IL-17 production is the default program for T-cell differentiation in the absence of DPP2. Thus, DPP2 seems to impose a threshold for quiescent T cells, preventing them from drifting into cell cycle.

Complement receptor 2 is expressed in neural progenitor cells and regulates adult hippocampal neurogenesis.

Injury and inflammation are potent regulators of adult neurogenesis. As the complement system forms a key immune pathway that may also exert critical functions in neural development and neurodegeneration, we asked whether complement receptors regulate neurogenesis. We discovered that complement receptor 2 (CR2), classically known as a coreceptor of the B-lymphocyte antigen receptor, is expressed in adult neural progenitor cells (NPCs) of the dentate gyrus. Two of its ligands, C3d and interferon-α (IFN-α), inhibited proliferation of wild-type NPCs but not NPCs derived from mice lacking Cr2 (Cr2(-/-)), indicating functional Cr2 expression. Young and old Cr2(-/-) mice exhibited prominent increases in basal neurogenesis compared with wild-type littermates, whereas intracerebral injection of C3d resulted in fewer proliferating neuroblasts in wild-type than in Cr2(-/-) mice. We conclude that Cr2 regulates hippocampal neurogenesis and propose that increased C3d and IFN-α production associated with brain injury or viral infections may inhibit neurogenesis.

Human complement receptor type 2 (CR2/CD21) transgenic mice provide an in vivo model to study immunoregulatory effects of receptor antagonists.

We found that transgenic (tg) mice stably expressing a bacterial artificial chromosome (BAC)-derived human complement receptor type 2 (CR2/CD21) gene demonstrate B cell specific hCR2 protein expression, normal B cell development and no changes in B cell subpopulations. To determine whether this BAC-encoded human CR2 (hCR2) can replace mouse CR2/CR1 in Cr2(-/-) mice and restore humoral immune responses to model foreign antigens (Ags), we generated hCR2(+/-)Cr2(-/-) tg mice and immunized them with sheep red blood cells (SRBC). We found that hCR2(+/-)Cr2(-/-) mice demonstrated anti-SRBC antibody (Ab) levels that were initially comparable to Cr2(-/-) mice after a single injection of the Ag, but then showed marked increases in anti-SRBC IgM and IgG1 levels after a second immunization. Identical results were found with a second model Ag, NP-Ficoll. To further confirm that this improvement in Ag-specific Ab production over Cr2(-/-) mice was indeed due to hCR2 expression, as well as to examine the effects of treating hCR2(+/-)Cr2(-/-) mice with an inhibitory anti-hCR2 monoclonal Ab (mAb) in vivo, we used mAb 171, an anti-hCR2 mAb that we have shown directly recognizes the C3d ligand binding site on hCR2. We first found that mAb 171 completely blocked hCR2-dependent co-activation of hCR2-tg B cells by anti-BCR/C3d complexes as measured in vitro by intracellular calcium influx. The i.p. injection of 1mg of mAb 171 was then found to induce for at least three weeks only partial loss of hCR2 surface expression, without modifying B and T cell numbers or the apparent activation status of the cells. Treatment of hCR2(+/-)Cr2(-/-) mice with mAb 171 also substantially suppressed the development of anti-SRBC and anti-NP Abs following immunization with Ags. The development of this model system should allow the study of the effects of manipulating hCR2 function in vivo with potential therapeutic compounds.

Failure to Detect XMRV-Specific Antibodies in the Plasma of CFS Patients Using Highly Sensitive Chemiluminescence Immunoassays.

In 2009, Lombardi et al. reported their startling finding that the gammaretrovirus xenotropic murine leukemia virus-related retrovirus (XMRV) is present in 67% of blood samples of patients suffering from chronic fatigue syndrome (CFS), as opposed to only 3.7% of samples from healthy individuals. However, we and others could not confirm these results, using a nested PCR assay. An alternative to this highly sensitive, but contamination-prone, technique is to measure the serological response to XMRV. Thus, we tested the plasma samples from our cohorts of CFS patients and healthy controls for the presence of XMRV-specific antibodies. Using two novel chemiluminescence immunoassays (CMIAs), we show that none of our samples have any XMRV-reactive antibodies. Taken together with our previous findings, we conclude that XMRV is not present in any human individual tested by us, regardless of CFS or healthy control.

Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences.

BACKGROUND: In 2006, a novel gammaretrovirus, XMRV (xenotropic murine leukemia virus-related virus), was discovered in some prostate tumors. A more recent study indicated that this infectious retrovirus can be detected in 67% of patients suffering from chronic fatigue syndrome (CFS), but only very few healthy controls (4%). However, several groups have published to date that they could not identify XMRV RNA or DNA sequences in other cohorts of CFS patients, while another group detected murine leukemia virus (MLV)-like sequences in 87% of such patients, but only 7% of healthy controls. Since there is a high degree of similarity between XMRV and abundant endogenous MLV proviruses, it is important to distinguish contaminating mouse sequences from true infections. RESULTS: DNA from the peripheral blood of 112 CFS patients and 36 healthy controls was tested for XMRV with two different PCR assays. A TaqMan qPCR assay specific for XMRV pol sequences was able to detect viral DNA from 2 XMRV-infected cells (~ 10-12 pg DNA) in up to 5 µg of human genomic DNA, but yielded negative results in the test of 600 ng genomic DNA from 100,000 peripheral blood cells of all samples tested. However, positive results were obtained with some of these samples, using a less specific nested PCR assay for a different XMRV sequence. DNA sequencing of the PCR products revealed a wide variety of virus-related sequences, some identical to those found in prostate cancer and CFS patients, others more closely related to known endogenous MLVs. However, all samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant intracisternal A-type particle (IAP) long terminal repeat and most were positive for murine mitochondrial cytochrome oxidase sequences. No contamination was observed in any of the negative control samples, containing those with no DNA template, which were included in each assay. CONCLUSIONS: Mouse cells contain upwards of 100 copies each of endogenous MLV DNA. Even much less than one cell's worth of DNA can yield a detectable product using highly sensitive PCR technology. It is, therefore, vital that contamination by mouse DNA be monitored with adequately sensitive assays in all samples tested.

Dipeptidyl peptidase 2 apoptosis assay determines the B-cell activation stage and predicts prognosis in chronic lymphocytic leukemia.

OBJECTIVE: Dipeptidyl peptidase 2 (DPP2/DPP7) is a regulator of quiescence as inhibition of DPP2 results in apoptosis of resting, but not activated lymphocytes. The purpose of the present study was to investigate the prognostic value of DPP2 inhibition and the role of DPP2 in cell cycle in chronic lymphocytic leukemia (CLL). MATERIALS AND METHODS: We screened 152 peripheral blood samples from patients with CLL in an apoptosis assay with AX8819, a DPP2-specific inhibitor. The apoptotic response was correlated with B-cell receptor signaling and cell cycle and molecular prognostic factors. RESULTS: We categorized CLL into two prognostic subgroups. Inhibition of DPP2 induced apoptosis in 60% of CLL, while 40% were resistant to apoptosis. Resistance to apoptosis correlated with unmutated IgV(H) and increased ZAP-70 expression and was associated with unfavorable clinical outcomes. Sensitive CLL B cells expressed high p27, low c-Myc protein levels and decreased Syk phosphorylation, indicative of a resting phenotype. DPP2 inhibition in those cells resulted in apoptosis accompanied by enhanced phosphorylation of Syk, degradation of p27 and p130, and upregulation of c-Myc, indicative of activation and inappropriate cell cycle entry. Resistant CLL demonstrated baseline low p27 and high c-Myc protein levels and increased pSyk, indicative of an activated phenotype. Inhibition of heat shock protein 90 in this subset of CLL partially reversed apoptosis resistance. CONCLUSIONS: The DPP2 apoptosis assay provides a reliable prognostic factor in CLL. CLL B cells sensitive to DPP2 inhibition are in true G(0), while resistant CLL B-cells are partially activated. DPP2 inhibition alone or with concomitant inhibition of heat shock protein 90 warrants investigation as a therapeutic modality in CLL.

Infectious arthritis and immune dysregulation: lessons from Lyme disease.

PURPOSE OF REVIEW: Borrelia burgdorferi colonization of the joints induces an inflammatory response, which in some individuals progresses to chronic arthritis. In this review, we discuss novel pathways that are implicated in disease development by modulating host defenses to B. burgdorferi infection. RECENT FINDINGS: The use of transgenic mice and gene expression analyses has revealed novel pathways involved in pathogenesis of Lyme disease. It is now clear that B. burgdorferi exploits an array of salivary gland proteins of the tick to evade immune responses in the mammalian host. The spirochete also modulates its surface protein profile upon infection and induces anti-inflammatory cytokines, favoring survival of the pathogen. The host defense involves toll-like receptors (TLRs), such as TLR2 and others, in B. burgdorferi recognition. To further dissect the genetic predisposition to treatment-refractory Lyme arthritis, HLA-DR transgenic mice have been used. SUMMARY: The cause and pathogenesis of Lyme arthritis are complex. Elucidating the mechanisms that govern this chronic inflammatory response will provide direct insights into other infectious arthritides and the development of novel therapeutic approaches against B. burgdorferi infection.

HLA-DR alleles determine responsiveness to Borrelia burgdorferi antigens in a mouse model of self-perpetuating arthritis.

OBJECTIVE: Arthritis is a prominent manifestation of Lyme disease, which is caused by infection with Borrelia burgdorferi (Bb). Chronic Lyme arthritis persisting even after antibiotic treatment is linked to HLA-DRB1*0401 (DR4) and related alleles. In contrast, patients whose Lyme arthritis resolves within 3 months postinfection show an increased frequency of HLA-DRB1*1101 (DR11). The aim of this study was to analyze the underlying mechanism by which HLA-DR alleles confer genetic susceptibility or resistance to antibiotic-refractory Lyme arthritis. METHODS: We generated DR11-transgenic (DR11-Tg) mice on a murine MHCII-/- background and compared their immune response to Bb antigens with the response of DR4-Tg mice after immunization with Bb outer surface protein A (OspA) or infection with live Bb. RESULTS: T cells from OspA-immunized and Bb-infected DR11-Tg mice had defective production of interferon-gamma as compared with those from DR4-Tg mice. In contrast, DR11-Tg mice developed higher titers of anti-OspA and anti-Bb antibodies, respectively, than did DR4-Tg mice. Consistent with this observation, we found that the Bb-infected DR11-Tg mice had a decreased spirochetal burden as compared with the DR4-Tg mice, as measured by quantitative polymerase chain reaction. CONCLUSION: This study provides direct evidence that in the presence of HLA-DR11, the immune response against Bb antigens is directed toward a protective antibody response. In contrast, an inflammatory Th1 response is induced in the presence of DR4. These observations offer an explanation for the differential genetic susceptibility of DR4+ and DR11+ individuals to the development of chronic Lyme arthritis and, eventually, the progression to antibiotic-refractory Lyme arthritis.

Neurogenin 3-specific dipeptidyl peptidase-2 deficiency causes impaired glucose tolerance, insulin resistance, and visceral obesity.

The control of glucose metabolism is a complex process, and dysregulation at any level can cause impaired glucose tolerance and insulin resistance. These two defects are well-known characteristics associated with obesity and onset of type 2 diabetes. Here we introduce the N-terminal dipeptidase, DPP2, as a novel regulator of the glucose metabolism. We generated mice with a neurogenin 3 (NGN3)-specific DPP2 knockdown (kd) to explore a possible role of DPP2 in maintaining metabolic homeostasis. These mice spontaneously developed hyperinsulinemia, glucose intolerance, and insulin resistance by 4 months of age. In addition, we observed an increase in food intake in DPP2 kd mice, which was associated with a significant increase in adipose tissue mass and enhanced liver steatosis but no difference in body weight. In accordance with these findings, the mutant mice had a higher rate of respiratory exchange than the control littermates. This phenotype was exacerbated with age and when challenged with a high-fat diet. We report, for the first time, that DPP2 enzyme activity is essential for preventing hyperinsulinemia and maintaining glucose homeostasis. Interestingly, the phenotype of NGN3-DPP2 kd mice is opposite that of DPP4 knockout mice with regard to glucose metabolism, namely the former have normal glucagon-like peptide 1 levels but present with glucose intolerance, whereas the latter have increased glucagon-like peptide 1, which is accompanied by augmented glucose tolerance.

HHV-6A infection induces expression of HERV-K18-encoded superantigen.

BACKGROUND: The human endogenous retrovirus K-18 (HERV-K18) encodes a superantigen that causes deregulation of the immune system. This provirus is transcriptionally silent, but can be induced by Epstein-Barr virus (EBV) infection and IFN-alpha treatment. OBJECTIVES: Since the herpesvirus EBV induces HERV-K18 expression in human B cells, it was of interest to determine if other herpesviruses would have similar HERV-K18 transactivation properties. Human herpesvirus (HHV)-6A, a neurotropic virus associated with multiple sclerosis, was a logical candidate for these studies. STUDY DESIGN: HSB2 cells (HHV-6-negative control), HSB2-ML cells (containing latent HHV-6A genome) and HSB2/HHV-6A cells (HSB-2 cells productively infected with HHV-6A) were compared for their level of HERV-K18 transcription, using quantitative RT-PCR. RESULTS: Latently infected HSB2-ML cells showed a significant increase in HERV-K18 RNA compared to the control cells. HERV-K18 expression was even greater in HSB2 cells productively infected with HHV-6A for 78h. CONCLUSION: These results imply that HHV-6A, either in latent form or during acute infection, directly transactivates HERV-K18. This HERV-K18 induction may be mediated through IFN-alpha that is produced by the HHV-6A-infected cells. The functional implications of superantigen expression are discussed.

Dipeptidyl peptidase 2 is an essential survival factor in the regulation of cell quiescence.

Most cells in the body are in a resting state and undergo cell cycle progression only upon growth factor stimulation or activation. while much research on proliferation and activation has been performed, very little about signals that maintain quiescent cells in G(0) is known, preventing cell cycle entry or apoptosis. In this study, the pathways of apoptosis induction in quiescent peripheral blood cells and fibroblasts mediated by inhibition or downregulation of Dipeptidyl Peptidase 2 (DPP2) have been explored. A decrease in DPP2 activity was found to cause resting cells to exit from G(0), accompanied by a decrease in p130, p27(Kip1) and p21(Cip1) protein levels. In addition, DPP2-inhibited or downregulated cells exhibit an increase in early G(1)/S progressors, with increases in the levels of retinoblastoma (pRb), p107 and cyclin D proteins. Furthermore, decrease of DPP2 activity leads to an increase in c-Myc and a decrease in Bcl-2, two events that have been associated with apoptosis induction. This apoptosis by DPP2 downregulation is prevented in p53(-/-) cells or by ectopic expression of proteins that suppress p53 or c-Myc activity. Thus, DPP2 is essential for maintaining lymphocytes and fibroblasts in G(0), and its inhibition results in apoptosis mediated by induction of c-Myc and p53.

Vitamin E reverses impaired linker for activation of T cells activation in T cells from aged C57BL/6 mice.

Supplemental vitamin E alleviates age-related defects in interleukin (IL)-2 production, T cell proliferation, and immune synapse formation. Here, we evaluated the effect of in vitro supplementation with 46 mumol/L of vitamin E on T cell receptor-proximal signaling events of CD4(+) T cells from young (4-6 mo) and old (22-26 mo) C57BL mice. Aged murine CD4(+) T cells stimulated via CD3 and CD28, tyrosine 191 of the adaptor protein Linker for Activation of T cells (LAT), was hypo-phosphorylated. Supplementation with vitamin E eliminated this difference in the tyrosine phosphorylation of LAT. By using a flow cytometric assay, the age-related differences in the activation-induced phosphorylation of LAT were observed in both naïve and memory T cell subsets. In addition, supplementation with vitamin E eliminates the age-related differences in LAT phosphorylation in both T cell subsets. Neither age nor vitamin E supplementation altered the fraction of LAT entering the membrane compartment. Furthermore, neither age nor vitamin E influenced the phosphorylation of Lck and Zap70, indicating that associated changes in LAT phosphorylation were not caused by alterations in activation states of the upstream kinases Lck and Zap70.

HLA type and immune response to Borrelia burgdorferi outer surface protein a in people in whom arthritis developed after Lyme disease vaccination.

OBJECTIVE: To investigate whether persons with treatment-resistant Lyme arthritis-associated HLA alleles might develop arthritis as a result of an autoimmune reaction triggered by Borrelia burgdorferi outer surface protein A (OspA), the Lyme disease vaccine antigen. METHODS: Persons in whom inflammatory arthritis had developed after Lyme disease vaccine (cases) were compared with 3 control groups: 1) inflammatory arthritis but not Lyme disease vaccine (arthritis controls), 2) Lyme disease vaccine but not inflammatory arthritis (vaccine controls), and 3) neither Lyme disease vaccine nor inflammatory arthritis (normal controls). HLA-DRB1 allele typing, Western blotting for Lyme antigen, and T cell reactivity testing were performed. RESULTS: Twenty-seven cases were matched with 162 controls (54 in each control group). Odds ratios (ORs) for the presence of 1 or 2 treatment-resistant Lyme arthritis alleles were 0.8 (95% confidence interval [95% CI] 0.3-2.1), 1.6 (95% CI 0.5-4.4), and 1.75 (95% CI 0.6-5.3) in cases versus arthritis controls, vaccine controls, and normal controls, respectively. There were no significant differences in the frequency of DRB1 alleles. T cell response to OspA was similar between cases and vaccine controls, as measured using the stimulation index (OR 1.6 [95% CI 0.5-5.1]) or change in uptake of tritiated thymidine (counts per minute) (OR 0.7 [95% CI 0.2-2.3]), but cases were less likely to have IgG antibodies to OspA (OR 0.3 [95% CI 0.1-0.8]). Cases were sampled closer to the time of vaccination (median 3.59 years versus 5.48 years), and fewer cases had received 3 doses of vaccine (37% versus 93%). CONCLUSION: Treatment-resistant Lyme arthritis alleles were not found more commonly in persons who developed arthritis after Lyme disease vaccination, and immune responses to OspA were not significantly more common in arthritis cases. These results suggest that Lyme disease vaccine is not a major factor in the development of arthritis in these cases.

Biologically distinct conformations of Bcl-x can be resolved using 2D isoelectric focusing.

Bcl-x, a potent regulator of cellular decisions of life and death, has multiple survival-enhancing activities that rely on distinct protein regions. Evidence suggests that depending on the local environment and the binding of protein or peptide partners, Bcl-x can take on several conformations that expose different protein regions. However, biological occurrence of conformational forms has been very difficult to study, because structure determination techniques use large quantities of protein, purified under conditions that change Bcl-x conformation. We show here that standard 2D isoelectric focusing techniques can be used to distinguish conformationally distinct forms of Bcl-x in cell lysates. Conformational isoelectric forms were manipulated through the use of detergents and buffers of differing pH. Our data indicate that post-translational modifications are not needed for or associated with conformational changes, distinguishing the dominant isoelectric forms of Bcl-x. We found that Bcl-x conformational isoelectric forms have preferred subcellular localization patterns. Moreover, conformational forms are differently regulated in certain locations during cytokine starvation of IL-3-dependent cells. Therefore, we provide evidence that 2DIEF can be used to view biologically distinct conformational differences in Bcl-x on minute quantities of unpurified protein from cells or lysates.

EBV LMP-2A employs a novel mechanism to transactivate the HERV-K18 superantigen through its ITAM.

EBV encodes latent membrane protein (LMP)-2A that functions as a homologue of the activated BCR. We have previously shown that LMP-2A transactivates a human endogenous retrovirus, HERV-K18, in infected B-lymphocytes. The Env protein of HERV-K18 encodes a superantigen that strongly stimulates a large number of T cells. To delineate the mechanism through which LMP-2A transactivates HERV-K18 env, we tested a panel of tyrosine mutants of LMP-2A in a murine B lymphoma that stably harbors HERV-K18. Our analysis revealed that the immunoreceptor tyrosine-based activation motif (ITAM) of LMP-2A is important for HERV-K18 env transactivation. ITAM contains 2 tyrosines that initiate signaling cascades when both residues are phosphorylated. However, in our study, single-tyrosine mutants of ITAM still retained full induction of HERV-K18 env. After truncating 25 kb of genomic sequence downstream of HERV-K18, LMP-2A failed to transactivate HERV-K18 env. Thus, an LMP-2A-inducible element is located downstream of HERV-K18.

Emergence of chronic Lyme arthritis: putting the breaks on CD28 costimulation.

Lyme disease is a debilitating infection that is caused upon a bite of Borrelia burgdorferi (Bb)-infected ticks. One of the most prominent clinical manifestations is the development of chronic Lyme arthritis. Months after Bb infection, approximately 60% of untreated Lyme patients experience intermittent arthritic attacks that may last for years. The use of the CD28(-/-) mouse in Bb infection has helped to shed light into the mechanisms that govern this inflammatory process, which seems to be tightly regulated. In this current review, the effect of immunoregulation, as well as CD28 deficiency in the development of chronic Lyme arthritis is discussed.