Lesion Load May Predict Long-Term Cognitive Dysfunction in Multiple Sclerosis Patients

Background

Magnetic Resonance Imaging (MRI) techniques provided evidences into the understanding of cognitive impairment (CIm) in Multiple Sclerosis (MS).

Objectives

To investigate the role of white matter (WM) and gray matter (GM) in predicting long-term CIm in a cohort of MS patients.

Methods

303 out of 597 patients participating in a previous multicenter clinical-MRI study were enrolled (49.4% were lost at follow-up). The following MRI parameters, expressed as fraction (f) of intracranial volume, were evaluated: cerebrospinal fluid (CSF-f), WM-f, GM-f and abnormal WM (AWM-f), a measure of lesion load. Nine years later, cognitive status was assessed in 241 patients using the Symbol Digit Modalities Test (SDMT), the Semantically Related Word List Test (SRWL), the Modified Card Sorting Test (MCST), and the Paced Auditory Serial Addition Test (PASAT). In particular, being SRWL a memory test, both immediate recall and delayed recall were evaluated. MCST scoring was calculated based on the number of categories, number of perseverative and non-perseverative errors.

Results

AWM-f was predictive of an impaired performance 9 years ahead in SDMT (OR 1.49, CI 1.12–1.97 p = 0.006), PASAT (OR 1.43, CI 1.14–1.80 p = 0.002), SRWL-immediate recall (OR 1.72 CI 1.35–2.20 p<0.001), SRWL-delayed recall (OR 1.61 CI 1.28–2.03 p<0.001), MCST-category (OR 1.52, CI 1.2–1.9 p<0.001), MCST-perseverative error(OR 1.51 CI 1.2–1.9 p = 0.001), MCST-non perseverative error (OR 1.26 CI 1.02–1.55 p = 0.032).

Conclusion

In our large MS cohort, focal WM damage appeared to be the most relevant predictor of the long-term cognitive outcome.     

Altered helper T lymphocyte function associated with chronic hepatitis B virus infection and its role in response to therapeutic vaccination in humans

Theradigm-hepatitis B virus (HBV) is an experimental lipopeptide vaccine designed to stimulate induction of HBV-specific CTL responses in HLA-A2 individuals. Previous studies had demonstrated high immunogenicity in healthy volunteers, but comparatively weak CTL responses in chronically infected HBV patients. Herein, we examined helper T lymphocyte (HTL) responses in chronically infected patients. Despite normal proliferation and IL-2 secretion, IL-12 and IFN-gamma secretion in vitro in response to the vaccine was reduced compared with healthy volunteers. A similar pattern of cytokine secretion was observed following mitogen stimulation, suggesting a general altered balance of Th1/Th2 responses. Further analysis indicated that HTL recall responses to whole tetanus toxoid protein were reduced in chronically infected subjects, and reduced responsiveness correlated with the outcome of Theradigm-HBV immunization. Finally, experiments in HBV transgenic mice indicated that the nonnatural Pan DR HTL epitope, PADRE, is capable of inducing high levels of IFN-gamma secretion and that its inclusion in a lipopeptide incorporating an immunodominant Ld-restricted CTL epitope resulted in breaking tolerance at the CTL level. Overall, our results demonstrate an alteration in the quality of HTL responses induced in chronically infected HBV patients and suggest that use of a potent HTL epitope may be important to overcome CTL tolerance against specific HBV Ags.     

Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice

In this study we evaluated the ability of activated intrahepatic APCs to inhibit hepatitis B virus (HBV) replication in transgenic mice. Intrahepatic APCs were activated by administration of an anti-CD40 agonistic mAb (alphaCD40). We showed that a single i.v. injection of alphaCD40 was sufficient to inhibit HBV replication noncytopathically by a process associated with the recruitment of dendritic cells, macrophages, T cells, and NK cells into the liver and the induction of inflammatory cytokines. The antiviral effect depended on the production of IL-12 and TNF-alpha by activated APCs; however, it was mediated primarily by IFN-gamma produced by NK cells, and possibly T cells, that were activated by IL-12. Collectively, these results suggest that activated APCs can directly produce antiviral cytokines (IL-12, TNF-alpha) and trigger the production of other cytokines (i.e., IFN-gamma) by other cells (e.g., NK cells and T cells) that do not express CD40. These results provide insight into a hitherto unsuspected antiviral function of intrahepatic APCs, and they suggest that therapeutic activation of APCs may represent a new strategy for the treatment of chronic HBV infection.     

Ongoing murine T1 or T2 immune responses to the hepatitis B surface antigen are excluded from the liver that expresses transgene-encoded hepatitis B surface antigen

Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver.     

Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines

We have previously shown that hepatitis B virus (HBV) replication is inhibited noncytopathically in the livers of transgenic mice following injection of HBV-specific cytotoxic T lymphocytes (CTLs) or infection with unrelated hepatotropic viruses, including lymphocytic choriomeningitis virus (LCMV) and adenovirus. These effects are mediated by gamma interferon (IFNgamma), tumor necrosis factor alpha (TNFalpha), and IFNalpha/beta. In the present study, we crossed HBV transgenic mice with mice genetically deficient for IFNgamma (IFNgammaKO), the TNFalpha receptor (TNFalphaRKO), or the IFNalpha/beta receptor (IFNalpha/betaRKO) in order to determine the relative contribution of each cytokine to the antiviral effects observed in each of these systems. Interestingly, we showed that HBV replicates in unmanipulated IFNgammaKO and IFNalpha/betaRKO mice at levels higher than those observed in control mice, implying that baseline levels of these cytokines control HBV replication in the absence of inflammation. We also showed that IFNgamma mediates most of the antiviral effect of the CTLs while IFNalpha/beta is primarily responsible for the early inhibitory effect of LCMV and adenovirus on HBV replication. In addition, we showed that the hepatic induction of IFNalpha/beta observed after injection of poly(I. C) is sufficient to inhibit HBV replication and that a similar antiviral effect is achieved by systemic administration of very high doses of IFNalpha. We also compared the relative sensitivity of LCMV and adenovirus to control by IFNgamma, TNFalpha, or IFNalpha/beta in these animals. Importantly, IFNalpha/betaRKO mice, and to a lesser extent IFNgammaKO mice, showed higher hepatic levels of LCMV RNA and adenovirus DNA and RNA than control mice, underscoring the importance of both interferons in controlling these other viral infections as well.     

Intracellular hepatitis B virus nucleocapsids survive cytotoxic T-lymphocyte-induced apoptosis

Following antigen recognition, hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) induce a necroinflammatory liver disease in HBV-transgenic mice. An early event in this process is CTL-dependent activation of apoptosis in a small fraction of HBV-positive hepatocytes. Here we show that cytoplasmic HBV nucleocapsids and their cargo of replicative DNA intermediates survive CTL-induced apoptosis of hepatocytes in vitro. These results suggest that destruction of infected cells per se is not sufficient to destroy the replicating HBV genome in infected tissue and that other events in addition to this process are required for viral clearance to occur.     

Targeting murine immune responses to selected T cell- or antibody-defined determinants of the hepatitis B surface antigen by plasmid DNA vaccines encoding chimeric antigen

By exchanging sequences from the middle-surface (MS) and small-surface (S) Ag of hepatitis B virus (HBV) with corresponding sequences of the MS Ag of woodchuck hepatitis virus, we constructed chimeric MS variants. Using these constructs as DNA vaccines in mice, we selectively primed highly specific (non-cross-reactive) Ab responses to pre-S2 of the HBV MS Ag and the "a" determinant of the HBV S Ag, as well as L(d)- or K(b)-restricted CTL responses to HBV S epitopes. In transgenic mice that constitutively express large amounts of HBV surface Ag in the liver we could successfully suppress serum antigenemia (but not Ag production in the liver) by adoptive transfer of anti-pre-S2 or anti-"a" immunity but not CTL immunity. DNA vaccines greatly facilitate construction of chimeric fusion Ags that efficiently prime specific, high-affinity Ab and CTL responses. Such vaccines, in which sequences of an Ag of interest are exchanged between different but related viruses, are interesting tools for focusing humoral or cellular immunity on selected antigenic determinants and elucidating their biological role.     

Immunogenicity and tolerogenicity of hepatitis B virus structural and nonstructural proteins: implications for immunotherapy of persistent viral infections

Persistent hepatitis B virus (HBV) infection is characterized by a weak and narrowly focused CD8+ T-cell response to HBV that is thought to reflect the induction of central and/or peripheral tolerance to HBV proteins in neonatal and adult onset infections, respectively. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may lead to viral clearance in chronically infected individuals. The present study was performed to compare the relative immunogenicities and tolerogenicities of HBV structural (envelope [ENV]) and nonstructural (polymerase [POL]) proteins at the CD8+ cytotoxic T lymphocyte (CTL) level in transgenic mice that replicate HBV in the liver and secrete infectious virus into the blood, thus representing an excellent model of persistent HBV infection. Interestingly, the mice were tolerant to the ENV but not to the POL proteins at the CTL level. Furthermore, the POL-specific CTLs had no impact on HBV replication or liver function in vivo, even though they were readily induced and reached the liver after DNA immunization, reflecting their relatively low avidity and the low level at which the POL protein is expressed by the hepatocyte. Collectively, these results suggest that the factors that make POL less tolerogenic also make POL-specific CTLs relatively inefficient effector cells when they reach the target organ. Immunotherapeutic strategies to control HBV infection by inducing virus-specific CTL responses in chronically infected subjects should be evaluated in light of this observation.     

Interleukin-18 inhibits hepatitis B virus replication in the livers of transgenic mice

Interleukin-18 (IL-18) produced by activated antigen-presenting cells stimulates natural killer (NK) cells, natural killer T (NKT) cells, and T cells to secrete gamma interferon (IFN-gamma). In this study, injection of a single 10- micro g dose of recombinant murine IL-18 rapidly, reversibly, and noncytopathically inhibited hepatitis B virus (HBV) replication in the livers of HBV transgenic mice. Furthermore, HBV replication was inhibited by as little as 1 micro g of IL-18 injected repetitively, and also by a single 0.1- micro g dose of IL-18 injected together with 1 ng of IL-12, neither of which inhibited HBV replication individually, demonstrating synergy between these cytokines in this system. The antiviral effect of IL-18 was mediated by its ability to activate resident intrahepatic NK cells and NKT cells to produce IFN-gamma and by its ability to induce IFN-alpha/beta production in the liver. These results suggest that IL-18 has the potential to contribute to the control of HBV replication during self-limited infection and that it may have therapeutic value for the treatment of patients with chronic hepatitis.