Bernd Kitze

Pentoxifylline, a phosphodiesterase inhibitor, induces immune deviation in patients with multiple sclerosis

The outcome of immune responses can be predicted by the lymphokine production pattern of the participating cells. Cytokines of the T helper type 1 (Th1) cells mediate inflammatory responses and delayed-type hypersensitivity (DTH), whereas Th2-like T cells predominantly produce cytokines, which stimulate antibody production by B cells. Immunoregulatory therapy of autoimmune diseases with unknown antigens may be achieved by inhibiting the production of inflammatory cytokines and induction of protective cytokines of Th2-like T cells. To determine the immunoregulatory capacity of the phosphodiesterase inhibitor pentoxifylline (PTX), which is known to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), this drug was used in mitogen and antigen-stimulated lymphocyte cultures as well as in patients with multiple sclerosis. PTX significantly decreased TNF-alpha and interleukin-12 (IL-12), whereas it increased IL-4 and IL-10 production. In addition, PTX inhibited cell proliferation, which was associated with a marked reduction in CD25 (IL-2 receptor alpha-chain) and CD54 (intercellular adhesion molecule-1; ICAM-1) expression. Increasing doses of PTX significantly reduced TNF-alpha and IL-12 mRNA expression of blood mononuclear cells, but increased IL-4 and IL-10 expression in eight patients with relapsing-remitting multiple sclerosis. These results indicate that PTX modulates immune reactions favouring a Th2-like response and may therefore be useful for the treatment of autoimmune diseases with a dominant Th1-like T cell response.

Interferon-β-1 b decreased matrix metalloproteinase-9 serum levels in primary progressive multiple sclerosis

Abstract.Recent reports have shown that matrix-metalloproteinases (MMPs) facilitate T-cell migration into the CNS and play a role in disruption of the blood-brain-barrier and myelin break-down. An increase of MMP-9 serum levels predicts disease activity in relapsing remitting multiple sclerosis (RRMS). Interferon-β (IFN-β), which is an established treatment for RRMS, inhibits T-cell migration in vitro in parallel with the downregulation of MMP expression. Only limited data are available for primary progressive multiple sclerosis (PPMS) which differs in demographic and immunological aspects as well as in MRI criteria from RRMS. In this study, 19 patients with laboratory-supported definite PPMS were treated with 8 x 106 IU IFN-β1b (Betaferon®) subcutaneously every other day. Serum was collected before treatment and on months 1, 2, 3, 6 and 9 during treatment. Levels of MMP-9 and of its natural inhibitor known as tissue-inhibitor of matrix-metalloproteinase-1 (TIMP-1) were quantified by ELISA. In addition MMP-2 serum levels were determined by zymography. 19 healthy volunteers served as controls. Before treatment serum levels of MMP-9 were elevated in patients with PPMS compared with controls, whereas there was no difference in TIMP-1 serum levels. During treatment with IFN- β1b the concentration of MMP-9 in the serum of 18 out of 19 PPMS patients decreased,whereas serum levels of MMP-2 and TIMP-1 remained nearly unaffected. Our results demonstrate that the MMP-9 to TIMP-1 ratio in patients with PPMS is elevated in comparison with healthy controls. The suppression of MMP-9 by IFN-β1b indicates that this drug is immunomodulatory active in PPMS patients. Further studies are necessary to test if IFN-β exerts a beneficial effect in PPMS.

Immunoadsorption therapy in patients with multiple sclerosis with steroid-refractory optical neuritis

BackgroundIn multiple sclerosis relapses refractory to intravenous corticosteroid therapy, plasma exchange is recommended. Immunoadsorption (IA) is regarded as an alternative therapy, but its efficacy and putative mechanism of action still needs to be established.MethodsWe prospectively treated 11 patients with multiple sclerosis who had optical neuritis and fulfilled the indications for apheresis therapy (Trial registration DE/CA25/00007080-00). In total, five IA treatments were performed using tryptophan-IA. Clinical activity (visual acuity, Expanded Disability Status Scale, Incapacity Status Scale), laboratory values and visual evoked potentials were measured before, during and after IA, with a follow-up of six months. Moreover, proteomic analyses were performed to analyze column-bound proteins as well as corresponding changes in patients’ sera.ResultsAfter the third IA, we detected an improvement of vision in eight of eleven patients, whom we termed responders. Amongst these, the mean visual acuity improved from 0.15 ± 0.12 at baseline to 0.47 ± 0.32 after the third IA (P = 0.0252) up to 0.89 ± 0.15 (P < 0.0001) at day 180 ± 10 after IA. Soluble interleukin-2 receptor decreased in responders (P = 0.03), whereas in non-responders it did not. Proteomic analyses of proteins adsorbed to IA columns revealed that several significant immunological proteins as well as central nervous system protein fragments, including myelin basic protein, had been removed by IA.ConclusionsIA was effective in the treatment of corticosteroid-refractory optic neuritis. IA influenced the humoral immune response. Strikingly, however, we found strong evidence that demyelination products and immunological mediators were also cleared from plasma by IA.

The macrophage activity marker sCD14 is increased in patients with multiple sclerosis and upregulated by interferon beta-1b

The soluble form of the CD14 molecule (sCD14), a macrophage activity marker, was measured in the plasma of 17 patients with primary progressive multiple sclerosis (PPMS) and 20 patients with relapsing remitting MS (RRMS). In patients with PPMS, sCD14 levels were determined before and after treatment with interferon beta (IFNB). In both PPMS and in RRMS, sCD14 levels were significantly elevated compared to healthy controls. In patients with PPMS, sCD14 levels increased significantly during the first 3 months of IFNB therapy, then slightly decreased, but still remained elevated compared with levels before therapy. Therefore, the elevated sCD14 levels may be a marker in evaluating biological response to IFNB therapy.

Diversity of intrathecal antibody synthesis against HTLV-I and its relation to HTLV-I associated myelopathy

The humoral immune response against human T-cell lymphotropic virus type I (HTLV-I) in the central nervous system (CNS) compartment and in the blood was investigated by enzyme immunoassay using 16 synthetic peptides corresponding to HTLV-I core and envelope sequences. We evaluated paired samples of cerebrospinal fluid and serum from HTLV-I seropositive Japanese patients, classified as follows: HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP;n = 39), patients with spinal cord disease ascribed to either HAM/TSP or to some concomitant, HTLV-I-unrelated disease (possible HAM/TSP;n = 6) or carriers without any clinical signs of HAM/TSP (n = 15). HTLV-I-peptide-specific intrathecal antibody synthesis was found in 79% of HAM/TSP patients, but only in 20% of carriers without HAM/TSP. The group of carriers without HAM/TSP showed local synthesis for some peptides (on average 0.3 peptides per patient). In most HAM/TSP patients, however, there was a diverse intrathecal immune response to several HTLV-I synthetic peptides (on average against 3.6 peptides per HAM/TSP patient), most frequently againstgag p19 100–130,env gp21 458–488, andenv gp46 175–199 and 288–317. The intrathecal antibody synthesis against several HTLV-I determinants may represent a pathogenic immune response in HAM/TSP and is possibly related to the infiltration of virus-infected T-cells in the spinal cord.

A soluble form of tumour necrosis factor receptor in cerebrospinal fluid and serum of HTLV-I-associated myelopathy and other neurological diseases

Paired samples of cerebrospinal fluid (CSF) and serum from 17 patients with human T-cell lymphotrophic virus I (HTLVI)-associated myelopathy, 5 patients with multiple sclerosis and 11 controls with non-inflammatory disorders (migraine, idiopathic epilepsy and myelopathy of unknown aetiology) were examined by enzyme-linked immunosorbent assay for the presence of the 60-kDa soluble form of tumour necrosis factor receptor (sTNF-R). The results were compared with blood-CSF barrier function, cell count and the intrathecal synthesis of HTLVI antibodies. No correlation could be demonstrated. High levels of sTNF-R were found in CSF of patients with HTLV-I-associated myelopathy and multiple sclerosis. In addition, intrathecal sTNF-R was also detected in the patients with non-inflammatory diseases, indicating that sTNF-R is definitively a normal constituent of CSF.

Human CD4+ T lymphocytes recognize a highly conserved epitope of human T lymphotropic virus type 1 (HTLV‐1) env gp21 restricted by HLA DRB1*0101

HTLV‐1 causes two distinct human diseases, HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukaemia/lymphoma (ATL). Persistently infected individuals carry a risk of < 1% of developing either disease. These basic epidemiological data imply that virus–host interactions, especially immunogenetic factors, influence the outcome of infection. Several studies showed that the HLA class II DR1 DQ5 haplotype is over‐represented in HAM/TSP, but rare in ATL. Therefore, we selected four patients with HAM/TSP and one seronegative control who all carried the HLA DR1 DQ5 haplotype. We analysed the CD4+ T lymphocyte response against eight synthetic peptides of HTLV‐1 envelope (env) glycoprotein gp21, a crucial target antigen in HAM/TSP. The first of two immunodominant epitopes corresponded to a domain of the HTLV‐1 envelope protein which had previously been shown to be essential for HTLV‐1 envelope function. The second immunodominant epitope overlapped a highly conserved sequence of the retroviral transmembrane envelope protein. DR1 (DRB1*0101)‐restricted T lymphocytes were activated by the conserved peptide sequence in nanomolar concentrations. In contrast, this conserved sequence can also induce non‐specific, cAMP‐mediated immunosuppressive effects on T cells when added in micromolar concentrations to culture media, as shown by Haraguchi S, Good RA, James‐Yarish M, Cianciolo GJ, Day NK, Proc Natl Acad Sci USA 1995; 92:5568–71. Hence, HTLV‐1 env gp21 might exert either stimulating immunological or immunosuppressive effects in HTLV‐1‐infected individuals, depending on the level of its expression and the presence of HLA DRB1*0101.

The value of csf analysis for the differential diagnosis of HTLV-I associated myelopathy and multiple sclerosis

Cerebrospinal fluid (CSF) and serum of 17 patients with HAM/TSP (HTLV-I associated myelopathy/tropical spastic paraparesis), six with multiple sclerosis and six with idiopathic epilepsy (non inflammatory control) from Brazil were analysed for the presence of intrathecal synthesis of virus-specific antibodies against measles, rubella, varicella zoster virus and herpes simplex virus by enzyme-linked immunosorbent assay (ELISA). All HAM/TSP and multiple sclerosis cases had an intrathecal immune response (oligoclonal IgG). In HAM/TSP, only 1/17 case showed a polyspecific intrathecal immune response against measles and rubella virus. In multiple sclerosis, specific antibodies against measles and rubella (MRZ response) were observed in all patients but not in the control with idiopathic epilepsy. The diagnostic and theoretical relevance of mono- and polyspecific immune responses is discussed for these chronic neurological diseases.

Autoantibody synthesis in primary progressive multiple sclerosis patients treated with interferon beta-1b

We conducted an open-labeled clinical trial of interferon beta-1b (IFNB) treatment in 20 patients with primary progressive multiple sclerosis (PPMS) and longitudinally monitored autoantibodies against double-stranded DNA (dsDNA), thyroid peroxidase (TPO),myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), synapsin and S-100B. Before treatment, one patient had elevated TPO antibodies, four patients had elevated antibodies against S-100B, two patients against MOG or synapsin and one patient against MBP. In two patients we observed a continuous increase of dsDNA or TPO antibodies above the normal range. This rise paralleled IFNB treatment. In addition, 11 of 20 patients developed neutralizing antibodies against IFNB. There was no increase of autoantibodies directed against central nervous system antigens. Like patients with relapsing remitting or secondary progressive multiple sclerosis, PPMS patients may be at risk of an autoimmune response during IFNB treatment.

Preferential recognition of synthetic peptides from HTLV-I gp21 envelope protein by HLA-DRB1 alleles associated with HAM/TSP (HTLV-I-associated myelopathy/tropical spastic paraparesis)

To determine CD4+ T-cell epitopes of HTLV-I-envelope protein recognized by the HLA alleles associated with HAM/TSP, we established 20 CD4+ T-cell lines from peripheral blood mononuclear cells (PBMCs) of naive healthy donors using a panel of synthetic peptides spanning the entire length of HTLV-I-envelope proteins, gp46 and gp21. We quantitated the precursor frequencies of HTLV-1-envelope specific CD4+ T-cells and analyzed epitope specificity in the context of HLA alleles. The precursor frequencies ranged from 3.0 to 10.6 per 10(7) PBMCs in the naive healthy donors. The CD4+ T-cell epitopes of HTLV-I-envelope protein were clustered in amino acids 76 to 90, 136 to 160, 171 to 185 and 196 to 210 of gp46, and in amino acids 366 to 400 and 436 to 485 of gp21. The CD4+ T-cell epitopes of gp21 were preferentially recognized by HLA-DRB1 0101 and 1502 which were known to be associated with HAM/TSP. Thus, it was suggested that HTLV-I gp21 might contain the major CD4+ T-cell epitopes recognized by HLA-DRB1 alleles of HAM/TSP.