Brigitte T. Huber

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

BackgroundIn 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.ResultsDNA 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.ConclusionsMouse cells contain upwards of 100 copies each of endogenous MLV DNA. Even much less than one cells 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.

Interferon-α-Induced Endogenous Superantigen: A Model Linking Environment and Autoimmunity

Abstract We earlier proposed that a human endogenous retroviral (HERV) superantigen (SAg) IDDMK 1,2 22 may cause type I diabetes by activating autoreactive T cells. Viral infections and induction of interferon-α (IFN-α) are tightly associated with the onset of autoimmunity. Here we establish a link between viral infections and IFN-α-regulated SAg expression of the polymorphic and defective HERV-K18 provirus. HERV-K18 has three alleles, IDDMK 1,2 22 and two full-length envelope genes, that all encode SAgs. Expression of HERV-K18 SAgs is inducible by IFN-α and this is sufficient to stimulate Vβ7 T cells to levels comparable to transfectants constitutively expressing HERV-K18 SAgs. Endogenous SAgs induced via IFN-α by viral infections is a novel mechanism through which environmental factors may cause disease in genetically susceptible individuals.

Vitamin E-Enhanced IL-2 Production in Old Mice: Naive But Not Memory T Cells Show Increased Cell Division Cycling and IL-2-Producing Capacity

Aging is associated with reduced T cell function, as demonstrated by decreased T cell proliferation and IL-2 production. These changes respond to supplemental vitamin E both in animals and humans, in part by the reduction of T cell suppressive PGE2, the production of which by macrophages is increased with age. To evaluate whether vitamin E has a direct PGE2-independent effect on T cell responses, T cells purified from the spleens of young and old mice were preincubated with vitamin E or vehicle control. Activation-induced cell division of T cells from old mice was lower than that by young, and the production of IL-2 following 48-h activation was less by T cells from old mice. There was an age-related decline in both the number of IL-2+ T cells and the amount of IL-2 produced per cell. Despite decreased IL-2 protein at 48 h, the expression of IL-2 mRNA at 6 h and IL-2 protein production at 6 and 16 h was greater by T cells from old mice compared with that of young. Age-related decline in cell division and IL-2 production at 48 h was only observed within the naive T cell subpopulation. Vitamin E increased both cell-dividing and IL-2-producing capacity of naive T cells from old mice, with no effect on memory T cells. These data indicate that naive T cells exhibit the greatest age-related defect and show for the first time that supplemental vitamin E has direct immunoenhancing effect on naive T cells from old mice.

Sequence, Purification, and Cloning of an Intracellular Serine Protease, Quiescent Cell Proline Dipeptidase

We recently observed that specific inhibitors of post-proline cleaving aminodipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell proline dipeptidase (QPP). QPP activity was purified from CD26− Jurkat T cells. The protein was identified by labeling with [3H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.

Molecular Mimicry in Lyme Arthritis Demonstrated at the Single Cell Level: LFA-1αL Is a Partial Agonist for Outer Surface Protein A-Reactive T Cells

Antibiotic treatment-resistant Lyme arthritis is a chronic inflammatory joint disease that follows infection with Borrelia burgdorferi (Bb). A marked Ab and T cell response to Bb outer surface protein A (OspA) often develops during prolonged episodes of arthritis. Furthermore, cross-reaction between the bacterial OspA and human LFA-1αL at the T cell level and the inability to detect Bb in the joint implicate an autoimmune mechanism. To analyze the nature of response to OspA and LFA-1αL, we used OspA-specific T cell hybrids from DR4 transgenic mice, as well as cloned human cells specific for OspA165–184, the immunodominant epitope, from five DRB1*0401+ patients, using OspA-MHC class II tetramers. Although OspA165–184 stimulated nearly all OspA-specific human T cell clones tested to proliferate and secrete IFN-γ and IL-13, LFA-1αL326–345 stimulated ∼10% of these clones to proliferate and a greater percentage to secrete IL-13. Assays with LFA- or OspA-DR4 monomers revealed that higher concentrations of LFA-DR4 were needed to stimulate dual-reactive T cell hybrids. Our analysis at the clonal level demonstrates that human LFA-1αL326–345 behaves as a partial agonist, perhaps playing a role in perpetuating symptoms of arthritis.

ASSOCIATION OF ANTIBIOTIC TREATMENT-RESISTANT LYME ARTHRITIS WITH T CELL RESPONSES TO DOMINANT EPITOPES OF OUTER SURFACE PROTEIN A OF BORRELIA BURGDORFERI

OBJECTIVE To explore further the association of antibiotic treatment-resistant Lyme arthritis and T cell reactivity with outer surface protein A (OspA) of Borrelia burgdorferi, including the identification of T cell epitopes associated with this treatment-resistant course. METHODS The responses of peripheral blood and, if available, synovial fluid lymphocytes to B burgdorferi proteins, fragments, and synthetic peptides, as determined by proliferation assay and interferon-gamma production, were compared in 16 patients with treatment-responsive and 16 with treatment-resistant Lyme arthritis. RESULTS The maximum severity of joint swelling correlated directly with the response to OspA. Moreover, the only significant difference between patients with treatment-resistant and treatment-responsive arthritis was in reactivity with N-terminal and C-terminal fragments of OspA, OspA1 (amino acids [aa] 16-106), and OspA3 (aa 168-273). Epitope mapping showed that 14 of the 16 patients with treatment-resistant arthritis had responses to OspA peptides (usually 4 or 5 epitopes), whereas only 5 of the 16 patients with treatment-responsive arthritis had reactivity with these peptides (usually 1 or 2 epitopes) (P = 0.003). Patients with HLA-DRB1 alleles associated with treatment-resistant arthritis were more likely to react with peptide 15 (aa 154-173) and, to a lesser degree, with peptide 21 (aa 214-233) than patients with other alleles, whereas the responses to other epitopes were similar in both groups. CONCLUSION The maximum severity of joint swelling and the duration of Lyme arthritis after antibiotic treatment are associated with T cell responses to specific epitopes of OspA.

Epstein-Barr Virus Latent Membrane Protein LMP-2A Is Sufficient for Transactivation of the Human Endogenous Retrovirus HERV-K18 Superantigen

ABSTRACT Superantigens are microbial proteins that strongly stimulate T cells. We described previously that the Epstein-Barr virus (EBV) transactivates a superantigen encoded by the human endogenous retrovirus, HERV-K18. We now report that the transactivation is dependent upon the EBV latent cycle proteins. Moreover, LMP-2A is sufficient for induction of HERV-K18 superantigen activity.

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 Endogenous Retrovirus-K18 env as a risk factor in multiple sclerosis

Background The human endogenous retrovirus (HERV)-K18 Env is an Epstein-Barr virus (EBV)-associated superantigen. Given the evidence for a role of EBV in the etiology of multiple sclerosis (MS), HERV-K18 Env is a plausible candidate for association with MS. Objective To assess whether variation in HERV-K18 Env is a risk factor for MS. Methods We developed a single nucleotide polymorphism-based genotyping method to determine the distribution of the three alleles of HERV-K18 env. We then conducted a nested case-control study including 207 MS cases and 403 matched controls. Analyses were replicated in an independent series of 909 MS cases and 339 controls. Results Overall, there was a significant association between HERV-K18 env genotype and MS risk (χ2 P = 0.03). As compared with K18.2/K18.2 individuals, risk of MS was three fold higher among K18.3/K18.3 individuals (P = 0.03). An increase in MS risk among carriers of the K18.3 allele was also observed in the replication study, but did not reach statistical significance. In pooled analyses, K18.3/K18.3 individuals had a significantly increased risk of MS (relative risks [RR] comparing K18.3/K18.3 vs K18.2/K18.2 = 2.7; 95% confidence interval: 1.1–6.4). Conclusion Variation in EBV-associated superantigen HERV-K18 Env could influence the genetic susceptibility to MS.

Cutting Edge: Epstein-Barr Virus Transactivates the HERV-K18 Superantigen by Docking to the Human Complement Receptor 2 (CD21) on Primary B Cells

EBV, a ubiquitous human herpesvirus, is the causative agent of infectious mononucleosis and is associated with many carcinomas. We have previously shown that the EBV latent genes LMP-1 and LMP-2A (for latent membrane proteins 1 and 2A), transactivate a human endogenous retrovirus (HERV), HERV-K18, in infected B lymphocytes. The envelope (Env) protein of HERV-K18 encodes a superantigen that strongly stimulates a large number of T cells. In this study we report that HERV-K18 env is transactivated even earlier in the infection process, before the establishment of latency; namely, we found that EBV, through its interaction with its cellular receptor CD21, induces the HERV-K18 env gene in resting B lymphocytes. This transactivation is direct and immediate, as up-regulation of transcripts can be detected within 30 min after EBV exposure. Thus, EBV binding to human CD21 on resting B cells triggers the expression of an endogenous superantigen. The biological significance of this superantigen expression for the EBV life cycle is discussed.