Kavitha Narayan

MxA Gene Expression Analysis as an Interferon-β Bioactivity Measurement in Patients with Multiple Sclerosis and the Identification of Antibody-Mediated Decreased Bioactivity

AbstractBackground: Interferon-β (IFNβ) has proven to be an important advance in the therapy of multiple sclerosis (MS), but optimal markers for bioactivity have not been identified. To accurately measure bioactivity in MS patients treated with IFNβ, we developed and tested a real-time reverse transcriptase (RT)-PCR assay for gene expression of MxA, an IFNβ-induced gene in the peripheral blood of patients treated with IFNβ. Methods: We compared IFNβ-treated patients with MS to controls in expression of MxA relative to the house-keeping gene, GAPDH. 2′-5′oligoadenylate synthetase (OAS) gene expression was also tested by real-time RT-PCR on RNA from the same patient specimens. Anti-IFNβ antibody was measured by ELISA and a cytopathic effect assay. Results: Seven of 54 patients were found to have complete loss of bioactivity. MxA expression correlated well with OAS expression. All patients with lost bioactivity had high levels of binding antibodies or neutralizing antibodies. Conclusions: This is the first demonstration that a real-time RT-PCR assay can be used to monitor therapy with interferons. These data identify MxA mRNA as an excellent biomarker for INFβ action on the IFN receptor, and clarify the relationship between anti-IFNβ antibodies and bioactivity in patients with MS treated with IFNβ.

The nervous system as ectopic germinal center: CXCL13 and IgG in lyme neuroborreliosis.

Lyme neuroborreliosis (LNB) is a chronic infection in which B‐cell activation, plasma cell infiltration, and enhanced Ig production in infected tissue are prominent feature. However, little is known about how B cells and plasma cells invade and persist in target organs. To assess this issue, we developed real‐time PCR measurements of IgG and CXCL13 production. We used these RNA assays and specific enzyme‐linked immunosorbent assays for protein and demonstrated that human peripheral blood mononuclear cells (PBMCs), stimulated by Borrelia burgdorferi sonicate, produced CXCL13 and IgG. Magnetic separation of PBMC populations and flow cytometry showed that CXCL13 is produced by dendritic cells. We then measure the expression of CXCL13 and IgG in tissues and correlated the expression of these host genes with spirochetal load. We also measured expression of dbpA and BBK32, two spirochetal genes important in chronic infection. There was a strong correlation between host immune response gene expression (CXCL13 and IgG) and spirochetal load. Immunohistochemistry of infected nonhuman primates tissue confirmed that CXCL13 is expressed in the nervous system. We conclude that persistent production of CXCL13 and IgG within infected tissue, two characteristics of ectopic germinal centers, are definitive features of LNB. Ann Neurol 2005;57:813–823

Cardiac involvement in non-human primates infected with the Lyme disease spirochete Borrelia burgdorferi

To investigate cardiac involvement in the non-human primate (NHP) model of Lyme disease, we inoculated 39 adult Macaca mulatta with Borrelia burgdorferi sensu stricto strains N40 (BbN40) by needle (N=22, 14 immunocompetent (IC), seven permanently immunosuppressed (IS), and four transiently immunosuppressed (TISP)) or by tick-bite (N=4, all TISP) or strain 297 (Bb297) by needle (N=2 IS), or with B. garinii strains Pbi (N=4, 2 TISP and 2 IS), 793 (N=2, TISP) or Pli (N=2, TISP). Five uninfected NHPs were used as controls. Infection and inflammation was studied in the hearts and the aorta removed at necropsy 2–32 months after inoculation by (1) H&E and trichrome-staining; (2) immunohistochemistry and digital image analysis; (3) Western blot densitometry; and (4) TaqMan RT-PCR. All NHPs inoculated with BbN40 became infected and showed carditis at necropsy. The predominant cells were T cells, plasma cells, and macrophages. There was increased IgG and IgM in the heart independent of immunosuppression. The B-cell chemokine BLC was significantly increased in IS-NHPs. There was increased deposition of the complement membrane attack complex (MAC) in TISP and IS-NHPs. The spirochetal load was very high in all BbN40-inoculated IS-NHPs but minimal if any in IC or TISP NHPs. Double-immunostaining revealed that many spirochetes in the heart of BbN40-IS NHPs had MAC on their membranes. We conclude that carditis in NHPs infected with B. burgdorferi is frequent and can persist for years but is mild unless they are immunosupressed.

Multiplex analysis of expression of three IFNβ-induced genes in antibody-positive MS patients

Some interferon beta (IFNβ)–treated patients with multiple sclerosis develop antibody-mediated decreased bioactivity with resultant loss of therapeutic effect. The authors developed real-time multiplex reverse transcriptase PCR to measure expression of three IFNβ-inducible genes to directly assess IFNβ bioactivity in patients with neutralizing antibodies (NAbs). The three genes responded in tandem. Correlation of NAb level with bioactivity at low/moderate NAb levels was poor, indicating that for such patients, direct measurement of IFNβ bioactivity is most reliable.

Genome-Wide Network Analysis Reveals the Global Properties of IFN-β Immediate Transcriptional Effects in Humans

IFN-β effectively controls clinical exacerbations and magnetic resonance imaging activity in most multiple sclerosis patients. However, its mechanism of action has not been yet fully elucidated. In this study we used DNA microarrays to analyze the longitudinal transcriptional profile of blood cells within a week of IFN-β administration. Using differential expression and gene ontology analyses we found evidence of a general decrease in the cellular activity of T lymphocytes resembling the endogenous antiviral response of IFNs. In contrast, most of the differentially expressed genes (DEGs) from untreated individuals were involved in cellular physiological processes. We then used mutual information (MI) to build networks of coregulated genes in both treated and untreated individuals. Interestingly, the connectivity distribution (k) of networks generated with high MI values displayed scale-free properties. Conversely, the observed k for networks generated with suboptimal MI values approximated a Poisson distribution, suggesting that MI captures biologically relevant interactions. Gene networks from individuals treated with IFN-β revealed a tight core of immune- and apoptosis-related genes associated with higher values of MI. In contrast, networks obtained from untreated individuals primarily reflected cellular housekeeping functions. Finally, we trained a neural network to reverse engineer the directionality of the main interactions observed at the biological process level. This is the first study that incorporates network analysis to investigate gene regulation in response to a therapeutic drug in humans. Implications of this method in the creation of personalized models of response to therapy are discussed.

Spinal cord involvement in the nonhuman primate model of Lyme disease

Lyme borreliosis is a multisystemic disease caused by infection with various genospecies of the spirochete Borrelia burgdorferi. The organs most often affected are the skin, joints, the heart, and the central and peripheral nervous systems. Multiple neurological complications can occur, including aseptic meningitis, encephalopathy, facial nerve palsy, radiculitis, myelitis, and peripheral neuropathy. To investigate spinal cord involvement in the nonhuman primate (NHP) model of Lyme borreliosis, we inoculated 25 adult Macaca mulatta with B. burgdorferi sensu strictu strains N40 by needle (N=9) or by tick (N=4) or 297 by needle (N=2), or with B. burgdorferi genospecies garinii strains Pbi (N=4), 793 (N=2), or Pli (N=4) by needle. Immunosuppression either transiently (TISP) or permanently (IS) was used to facilitate establishment of infection. Tissues and fluids were collected at necropsy 7–24 weeks later. Hematoxylin and eosin staining was used to study inflammation, and immunohistochemistry and digital image analysis to measure inflammation and localize spirochetes. The spirochetal load and C1q expression were measured by TaqMan RT-PCR. The results showed meningoradiculitis developed in only one of the 25 NHPs examined, TISP NHP 321 inoculated with B. garinii strain Pbi. Inflammation was localized to nerve roots, dorsal root ganglia, and leptomeninges but rarely to the spinal cord parenchyma itself. T cells and plasma cells were the predominant inflammatory cells. Significantly increased amounts of IgG, IgM, and C1q were found in inflamed spinal cord. Taqman RT-PCR found spirochetes in the spinal cord only in IS-NHPs, mostly in nerve roots and ganglia rather than in the cord parenchyma. C1q mRNA expression was significantly increased in inflamed spinal cord. This is the first comprehensive study of spinal cord involvement in Lyme borreliosis.

Intrathecal antibody production in a mouse model of Lyme neuroborreliosis

Intrathecal antibody (ITAb) production is a common feature of neurological diseases, yet very little is known about its mechanisms. Because ITAb is prominent in human Lyme neuroborreliosis (LNB), in the present study we established a mouse model of LNB to study ITAb production. We injected different strains of Borrelia burgdorferi into a variety of mouse strains by the intracerebral (i.c.) route to develop the model. Spirochetal infection and ITAb production were identified by complementary methods. This study demonstrates that the mouse model of LNB can be utilized to test hypotheses related to the mechanisms of ITAb production.

Management of neutralizing antibodies against beta-IFN in beta-IFN-treated multiple sclerosis patients

JO N 2854 from the onset of therapy can lower the incidence of NAbs [10]. However, ideally, only MS patients at risk for developing NAbs should be subjected to prophylactic corticosteroid treatment. Therefore, identification of patients at risk for the development of Nabs is an important goal in our ability to prevent ADB. From all specimens analyzed at the Neurology Clinical Laboratory at UMDNJ-New Jersey Medical School for anti-beta-IFN antibodies for one year, we identified 24 NAbs-positive patients, in whom at least two samples were tested within the first year of therapy, and who stayed on the same beta-IFN preparation for at least the first 2 years of treatment. BAbs, NAbs, and bioactivity were measured as previously described [6, 7]. A total of 132 serum samples were obtained from the 24 patients within the first two years, an average of 5.5 samples per patient, of which 75 were NAb-positive (57 %). All 24 became NAb-positive in the first year of therapy; the predominant development of NAbs within the first year has been previously reported [11]. Moderate or high levels of BAbs in the first year, i.e. levels above 15U in the absence of NAbs, were strongly predictive of the subsequent development of neutralizing antibodies. In 11 patients who met this criterion, termed “at-risk” patients, measurable NAb developed and in 7/11 patients this occurred 2–6 months after initiation of therapy. Only 2 patients who met the “at-risk” criteria and were subsequently retested did not develop NAbs. Thus, in our data set, the risk of becoming NAb-positive, using the above “at-risk” criterion, was 11/13 or 85 %. Five MS patients were treated for NAb positivity with IV methylprednisolone with an induction protocol of 5 days of 1 g/d, followed Andrew R. Pachner Jennifer Brady Israel Steiner Kavitha Narayan