Longitudinal MRI study: the effects of azathioprine in MS patients refractory to interferon beta-1b.

An open-label study was performed to assess the effectiveness of oral azathioprine (AZA) on augmenting the response to interferon beta-1b (IFNbeta-1b) in patients with treatment-refractory relapsing-remitting MS. Six IFNbeta-1b-treated MS patients with continued disease activity were studied on IFNbeta-1b and AZA therapy for a median period of 15 months. A 69% reduction in the number of contrast-enhancing lesions was observed during the combination therapy (p = 0.002).

Interferon beta-1b and intravenous methylprednisolone promote lesion recovery in multiple sclerosis.

OBJECTIVE: To determine whether lesion evolution in relapsing-remitting multiple sclerosis (RRMS) patients is altered by treatment with interferonbeta1b (IFNbeta-1b) or by intravenous methylprednisolone (IVMP) as measured by magnetization transfer imaging. METHODS: Magnetization transfer ratios (MTR) of 225 contrast enhancing lesions (CEL), in four RRMS patients were serially determined for 12 months before and 12-18 months after contrast enhancement in a baseline vs treatment trial with IFNbeta-1b. During the baseline period, 185 new CEL were identified: 76 were treated with IVMP (1 g/day x 5 days) and designated steroid CEL (S-CEL); the remaining 109 were considered baseline lesions (BCEL). During IFNbeta-1b treatment, 40 CEL (IFN-CEL) were identified. After image co-registration, regions of interest (ROIs) defining new CEL were transferred to the MTR image set to determine the mean lesion MTR on each monthly exam. The lesion MTR was compared to MTR of normal appearing white matter (NAWM) on the same exam. RESULTS: As early as 12 months prior to enhancement, the MTR of CEL was reduced compared to NAWM (mean 9.43 +/- 3.2%; P<0.001). The further reduction in MTR (28% +/- 4.0) at the time of contrast enhancement was not significantly different for BCEL, S-CEL or IFN-CEL Following enhancement, lesion recovery for IFN-CEL (P=0.02) and S-CEL (P=0.002) was significantly higher than BCEL CONCLUSION: IFNbeta-1b and IVMP reduce tissue damage and promote lesion recovery in RRMS patients. The additional benefit of IVMP compared to IFNbeta-1b may be related to its inhibitory effect on demyelination.

Enhancing magnetic resonance imaging lesions and cerebral atrophy in patients with relapsing multiple sclerosis.

OBJECTIVE: To examine the relation between the frequency of enhancing magnetic resonance imaging lesions and their characteristics of enhancement and atrophy in patients with early relapsing multiple sclerosis. DESIGN: Analysis of number of enhancing lesions, ventricular volumes and diameters, and lesion characteristics on monthly magnetic resonance imaging scans during natural history follow-up. SETTING: A clinical research institution. PATIENTS: Sixteen patients with confirmed early relapsing multiple sclerosis. MAIN OUTCOME MEASURE: Cerebral atrophy as measured by ventricular enlargement. RESULTS: Numbers of enhancing lesions correlated well with an increase of ventricular size. This correlation was strongest for patients with a high proportion of concentric ring-enhancing lesions with central contrast pallor. CONCLUSIONS: Inflammatory events, especially those within lesions with associated blood-brain barrier breakdown, affect the ensuing loss of brain parenchyma. Patients with a high proportion of lesions with central contrast pallor, which is likely associated with more extensive tissue damage, have a higher rate of atrophic changes.

Increased activated human T cell lymphotropic virus type I (HTLV-I) Tax11-19-specific memory and effector CD8+ cells in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis: correlation with HTLV-I provirus load.

To discern the T cell subtype associated with T cell differentiation, the expression of CD45RA and CD27 was measured from total CD8(high) cells and from human T cell lymphotropic virus type I (HTLV-I) Tax11-19 peptide-specific CD8(+) cells in peripheral blood lymphocytes of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Phenotypically defined memory and/or effector cells (CD45RA(-)CD27(+), CD45RA(+)CD27(-), and CD45RA(-)CD27(-)) were increased in HAM/TSP CD8(+) cells, compared with those of HTLV-I-seronegative healthy control subjects. The percentage of human leukocyte antigen (HLA)-DR-positive cells was also increased in CD8(+) cells of HAM/TSP, compared with those in HLA-DR(+)CD8(+) cells of healthy control subjects. HTLV-I provirus load correlated with the frequency of Tax11-19-specific CD8(+) cells. The high frequency of memory and/or effector type HTLV-I Tax11-19-specific CD8(+) cells suggests that continuous restimulation driven by HTLV-I antigens in vivo may be associated with the pathogenesis of HAM/TSP.

Increased lymphoproliferative response to human herpesvirus type 6A variant in multiple sclerosis patients.

Several reports have suggested an association of human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) based on immunohistochemical demonstration of HHV-6 antigens in inflammatory lesions, detection of increased HHV-6 specific serum antibody titers, and amplification of HHV-6 DNA from sera and cerebrospinal fluid of MS patients but not in controls. Characterization of the cellular immune response of MS patients to HHV-6 may further clarify the role of HHV-6 in MS and provide insight into the pathogenesis of this immune-mediated disease. We have compared lymphoproliferative responses to HHV-6A (U1102)-, HHV-6B (Z29)-, and HHV-7 (H7SB)-infected cell lysates in healthy controls and patients with MS. Most healthy controls (71%) proliferated to HHV-6B lysate, and fewer (33%) responded to the HHV-6A lysate. In contrast, 67% of MS patients had a lymphoproliferative response to HHV-6A, which is a significant increase in comparison with healthy controls. A similar frequency of lymphoproliferative response (78%) to HHV-6B was demonstrated in MS patients. Lymphoproliferation to HHV-7 lysate was demonstrated in 23% of healthy controls and 28% of MS patients. These results indicate that the lymphoproliferative response to the HHV-6A variant, which was recently reported to have greater neurotropism, is increased in MS patients.

HTLV-I/II seroindeterminate Western blot reactivity in a cohort of patients with neurological disease.

The human T-cell lymphotropic virus type I (HTLV-I) is associated with a chronic, progressive neurological disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis. Screening for HTLV-I involves the detection of virus-specific serum antibodies by EIA and confirmation by Western blot. HTLV-I/II seroindeterminate Western blot patterns have been described worldwide. However, the significance of this blot pattern is unclear. We identified 8 patients with neurological disease and an HTLV-I/II seroindeterminate Western blot pattern, none of whom demonstrated increased spontaneous proliferation and HTLV-I-specific cytotoxic T lymphocyte activity. However, HTLV-I tax sequence was amplified from the peripheral blood lymphocytes of 4 of them. These data suggest that patients with chronic progressive neurological disease and HTLV-I/II Western blot seroindeterminate reactivity may harbor either defective HTLV-I, novel retrovirus with partial homology to HTLV-I, or HTLV-I in low copy number.

Reduction in HTLV-I proviral load and spontaneous lymphoproliferation in HTLV-I-associated myelopathy/tropical spastic paraparesis patients treated with humanized anti-Tac.

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease that results from an interaction of retroviral infection and immune activation. In this study, five doses (1 mg/kg) of humanized anti-Tac antibody were administered to 9 HAM/TSP patients at weeks 0, 2, 6, 10, and 14. Preliminary immunological studies on HAM/TSP patients treated with humanized anti-Tac indicate that there is a selective down-regulation of activated T cells and a decrease in the HTLV-I viral load in peripheral blood lymphocytes, most likely through the selective removal of HTLV-I-infected, activated CD4+ lymphocytes.

Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients.

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11-19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2-Ig complex to directly visualize HTLV-1 Tax11-19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8(+) lymphocytes specific for the HTLV-1 Tax11-19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11-19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11-19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11-19-specific CD8(+) T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-alpha and gamma-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11-19-specific CD8(+) T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

Virus-triggered acquired immunodeficiency by cytotoxic T-cell-dependent destruction of antigen-presenting cells and lymph follicle structure.

Virus-induced acquired immune suppression in mice infected with lymphocytic choriomeningitis virus is shown here to be caused by the CD8+-T-cell-dependent elimination of macrophages/antigen-presenting cells. Surprisingly, this is associated with severe destruction of the follicular organization of lymphoid organs, indicating a crucial role for dendritic cells and marginal zone macrophages in maintaining follicular structure. Once established, this immunopathology cannot be readily reversed by the elimination of CD8+ effector cells. Such a T-cell-mediated pathogenesis may play a pivotal role in acquired virus-induced immunosuppression and may represent one strategy by which virus escapes immune surveillance and establishes persistent infections in initially immunocompetent hosts.

Evidence for a role of IFN gamma in control of Listeria monocytogenes in T cell deficient mice.

The role of interferon (IFN) gamma in controlling chronic infections of Listeria monocytogenes (Listeria) was studied in athymic C57BL/6 nu/nu mice, and by treating thymectomized C57BL/6 +/+ mice with monoclonal rat CD4 and CD8-specific monoclonal antibodies (Mab). Mice treated with a combination of the two T cell subset antibodies were similar to athymic, nude mice in being able to control Listeria infection, keeping the titers below 3-5 log10 bacteria per organ, but they could not eliminate them completely. Treatment with antibodies to IFN gamma of nude or CD4+ + CD8+ - T cell-depleted mice suffering from chronic Listeria infection caused a marked increase of Listeria titers in liver and spleen. This result implies a role of IFN gamma in maintaining anti-Listeria resistance in mice lacking mature T cells.

Absence of effects of thymopentin on murine host resistance to Listeria monocytogenes.

The effects of various doses of Thymopentin, the synthetic 5 aminoacid fragment of thymopoietin injected on d -4 and d -1 on murine host resistance to Listeria monocytogenes were tested. The experiments did not reveal any regular dose-effect relationship; i.e. no or only marginal (more often small negative rather than positive) effects were observed on clearance and on resistance neither during the T cell independent first hour or the first 2 days of infection in euthymic or thymus deficient nude mice, nor during the T cell dependent phase after 2 days of infection in euthymic mice. Also survival of mice was not increased in a regular dose-effect relationship with dose ranges of Thymopentin from 0.3 ng-30 mg per 30 g mouse; furthermore, injections of 3 micrograms per 30 g mouse for varying time intervals from -3 days before up to 6 days after infection had no protective effect. Thus Thymopentin apparently does not induce measurable macrophage activation directly or cannot increase macrophage activation mediated by T cells in euthymic mice nor does it induce adequate T cell responses in nude mice to promote improved resistance to Listeria.

Mechanisms of macrophage-mediated tumor cell killing: a comparative analysis of the roles of reactive nitrogen intermediates and tumor necrosis factor.

The roles of tumor necrosis factor (TNF alpha) and reactive nitrogen intermediates (RNI) as effectors of macrophage-mediated tumor cell killing were investigated in a variety of tumor cell lines. Three TNF alpha-sensitive tumor targets were also susceptible to resting bone-marrow-derived mononuclear phagocytes (BMMP). This macrophage lytic activity was markedly diminished or even abolished by anti-TNF alpha, indicating that TNF alpha is the major effector of macrophage-mediated killing of these targets. The other 21 tumor cell lines examined were resistant to TNF alpha but, in their large majority, were more or less susceptible to killing by interferon gamma (IFN gamma)- and Corynebacterium parvum (CP)-activated BMMP. Among the various analogues of L-arginine used to assess the role of L-arginine-derived RNI as mediators of macrophage tumoricidal activity, NG-monomethyl-L-arginine (NMMA) was most efficient in suppressing RNI secretion by activated macrophages. In some macrophage tumor-cell combinations, NMMA inhibited both the generation of RNI and the expression of tumoricidal activity in a dose-dependent manner, suggesting a central role for RNI as effectors. In other combinations, NMMA in concentrations that abolished secretion of RNI either affected tumor-cell killing only after its induction by IFN gamma, or not at all. The findings not only support the thesis that macrophages posses various means of coping with tumor cells but also suggest that the mechanism becoming operative is determined predominantly by the pathway of macrophage activation and the properties of the tumor-cell type.

Resistance to a non-immunogenic tumor, induced by Corynebacterium parvum or Listeria monocytogenes, is abrogated by anti-interferon gamma.

The complex processes that determine the outcome of the interaction of tumor and host were explored in the operationally simple and reproducible rat D-12 ascites tumor model. Animals exhibit weak spontaneous resistance against this tumor that is not augmented by repeated inoculation, by various routes, of irradiated syngeneic D-12 tumor cells, but considerably enhanced after local administration of heat-killed Corynebacterium parvum (CP) or Listeria monocytogenes (LM) organisms. Inoculation of conventional or monoclonal anti-rat IFN gamma antibodies into the same compartment did not affect spontaneous tumor resistance, but largely abrogated the tumor-protective effect triggered by CP or LM. Our findings support the concept that IFN gamma, produced by T cells in the course of the specific immune response raised against immunogenic micro-organisms, is able to enhance and to maintain local tumor resistance and thus to strengthen the capacity of the host to cope with a non-immunogenic tumor.

Treatment with anti-tumor necrosis factor alpha does not influence the immune pathological response against lymphocytic choriomeningitis virus.

The role of tumor necrosis factor alpha (TNF alpha) in the immunopathological events induced by infection with lymphocytic choriomeningitis (LCM) virus (LCMV) was assessed by treatment of C57Bl/6 mice with a sheep antibody to murine TNF alpha antiserum to strongly interfere with anti-Listeria host defense. However, despite its effectiveness in Listeria infections in vivo, antibody to TNF alpha used at 6 x 10(4) neutralizing units per day subcutaneously had no detectable influence on the kinetics of maturation of antiviral cytotoxic T-cell activity, inflammatory processes, or clearance of virus. First, onset and severity of LCMV-induced hepatitis, as assessed by cytotoxic T-cell activity, viral titers in the liver, serum liver enzyme values, and histology, were not detectably affected by antibody to TNF alpha. Second, incidence of lethal LCM disease after intracerebral infection and the kinetics of the primary footpad swelling reaction observed after local foot inoculation were not altered by anti-TNF alpha antibody treatment. From the data presented we conclude that TNF alpha as assayed by in vivo therapy with a polyclonal anti-TNF alpha antibody plays no detectable role in the host reaction against LCMV.

Role of tumor necrosis factor in Listeria resistance of nude mice.

The effects of sheep anti-murine recombinant tumor necrosis factor-alpha (TNF-alpha) on resistance to Listeria monocytogenes infection were studied in T cell-deficient nu/nu mice. The sheep anti-TNF-alpha antibody preparation was specific for TNF since it neutralized 300 U of recombinant murine TNF-alpha in vitro at a dilution of up to 1/1,000 but did not neutralize 32 U of interferon (IFN)-alpha, -beta or 32 U of IFN-gamma in vitro at a 1/20 dilution. When tested in vivo in sublethally Listeria-infected nu/nu or T cell-competent C57BL/6 or ICR mice, a single treatment of 0.2 ml anti-TNF-alpha given intraperitoneally on either day -1,0 or +1 resulted in the death of mice by day 5-7 due to the uncontrolled growth of Listeria; bacterial counts in spleen and liver were increased on days 3-5 by a factor of 10-1,000 in these organs. When examined histologically, organs from mice with the anti-TNF-alpha treatment contained more, and considerably bigger, lesions that exhibited central necrosis. The enhancing effect of anti-TNF-alpha on Listeria infection seemed greater early during Listeria infection on days 1-6 when compared to later phases of the infection around days 6-10. From the data presented we conclude that in addition to other lymphokines, such as IFN-gamma, TNF-alpha is of importance during the entire course of a Listeria infection in nu/nu mice.

Impaired generation of anti-viral cytotoxicity against lymphocytic choriomeningitis and vaccinia virus in mice treated with CD4-specific monoclonal antibody.

The role of CD4+ helper T cells in induction of anti-viral cytotoxic T-cell response was investigated by treating normal and thymectomized C57B1/6 mice with CD4-specific monoclonal antibodies (MoAb). In CD4-specific MoAb-treated mice infected with Vaccinia or lymphocytic choriomeningitis virus (LCMV), cytotoxic T-cell activity was 5-15 times lower than in normal controls when measured in a 51Cr release assay and computed as lytic units 6 and 8 days respectively after virus inoculation. This difference in the levels of effector T-cell activities did not reflect slower kinetics of cytotoxic T-cell induction in antibody-treated versus control mice, since it was also obvious at 8 days after infection for Vaccinia virus and 10 and 12 days after inoculation with LCMV. CD4-specific MoAb-induced inhibition of cytotoxic T-cell responses in vivo was seen up to 150 days after treatment in thymectomized mice. However, no significant suppressive effect of the same antibody treatment on T-cell cytotoxicity could be observed in animals treated on day 3 or later after infection with Vaccinia virus. Injection of CD4-depleted mice with recombinant interleukin 2 (rIL-2) partially corrected the impaired virus-specific cytotoxic T-cell response, suggesting that IL-2 supply may be limiting in mice lacking T helper cells.

Latent infections in spinal ganglia with thymidine kinase-deficient herpes simplex virus.

A herpes simplex virus type 1 variant [C239(TK-)] harboring a deletion in the thymidine kinase (TK) gene was assessed for capacity to establish latent infections. Outbred Swiss Webster mice were inoculated on both hind footpads, and numbers of neurons expressing latency-associated transcript and amounts of viral DNA in latently infected lumbosacral spinal ganglia were scored. C239(TK-) established levels of latent infection that were only slightly lower than those found for either a TK rescued variant of this agent or the parent wild-type KOS. However, in contrast to the TK+ viruses, C239(TK-) could not be reactivated when spinal ganglia were cultured in vitro. The results presented show that expression of the viral TK gene plays no major role in establishment of the latent state but that it functions during reactivation of latent virus from explanted ganglia maintained in vitro.

Effects of treatment with IL-2 receptor specific monoclonal antibody in mice. Inhibition of cytotoxic T cell responses but not of T help.

Contribution of IL-2R-bearing activated lymphocytes to antiviral host defense was investigated in C57BL/6 mice by treatment in vivo with IL-2R-specific mAb PC61. When treated on days 0 and 1 with respect to infection with either vaccinia virus, lymphocytic choriomeningitis (LCM) virus (LCMV) or vesicular stomatitis virus, 6-day immune mice had low numbers of CD8+ T cells that were reduced to about 10% of the values found for infected but otherwise untreated controls. In contrast, the number of CD4+ T cells was within normal ranges. Correspondingly, induction of strictly T help-dependent antiviral neutralizing IgG antibody titers remained unaffected by the mAb treatment, whereas generation of antiviral cytotoxic T cell activity was abrogated. Anti-IL-2R treatment of thymectomized mice 14 and 15 days after infection prevented generation of secondary antiviral cytotoxic T cells in restimulation cultures in vitro initiated 24 days later. Treatment with IL-2R-specific mAb was comparable to treatment with CD8-specific mAb in preventing mice to eliminate virus. Because of the involvement of antiviral cytotoxic T cells in disease manifestations, treatment with IL-2R-specific mAb protected mice from lethal LCM after intracerebral infection with LCMV and inhibited the footpad swelling reaction caused by local infection with the same virus.