Multiple sclerosis patients with anti-lipid oligoclonal IgM show early favourable response to immunomodulatory treatment.

BACKGROUND AND PURPOSE: Interferon beta and Glatiramer acetate are safe immunomodulatory treatments (IT) for multiple sclerosis (MS), but not always effective. New drugs are available, although they show more side-effects and unknown long-term safety profile. Anti-lipid oligoclonal IgM bands (OCMB) distinguish MS patients with early aggressive course. We prospectively studied if IT are effective in these patients or if they are candidates for more aggressive drugs as first therapeutic option. METHODS: Seventy-five clinically isolated syndrome patients were studied. OCMB and conversion to MS were assessed. Patients suffering at least two demyelinating events within 3 years were considered eligible to start IT. RESULTS: Eighteen patients showed OCMB (M+) and 57 lacked them (M-). All M+ patients and only 25 M- patients were treated. The other 32 M- patients suffered less MS attacks than those required to initiate treatment. IT similarly reduced relapse rate in both treated groups (P < 0.0001) and reduced Expanded Disability Status Scale (EDSS) progression in M+ patients, whose EDSS score had significantly increased before treatment. EDSS did not change in M- patients during follow-up, regardless if they were treated or not. CONCLUSIONS: Oligoclonal IgM bands identify MS patients who are candidates for early immunomodulatory treatment as IT improves their initial aggressive disease course.

Influence of oligoclonal IgM specificity in multiple sclerosis disease course.

Oligoclonal IgM bands (OCMB) against myelin lipids predict an aggressive multiple sclerosis (MS) course. However, the clinical significance of OCMB without lipid specificity, present in other MS patients, remains unknown. We describe here a characterization of these antibodies and study their role in MS progression. Fifty-four MS patients showing CSF-restricted OCMB were included in this study at disease onset and followed-up during 61.1 +/- 2.7 months. The specificity of OCMB and the CSF B-cell profile were investigated. A second CSF IgM study was performed in a group of eight patients. Thirty-eight patients showed OCMB against myelin lipids (M+L+) and other sixteen had OCMB lacking this specificity (M+L-). The CD5+ B cell subpopulation, responsible for most persistent IgM responses, was considerably higher in M+L+ than in M+L- patients (3.3 +/- 0.6% versus 0.8 +/- 0.2, P = 0.009). In addition, M+L+ bands persisted during disease course, while M+L- disappeared during follow-up. M+L+ patients suffered more relapses (4.2 +/- 0.6 versus 1.6 +/- 0.3, P = 0.002) and reached higher disability (EDSS score of 2.2 +/- 0.2 versus 1.2 +/- 0.2, P = 0.02) than M+L- group. These data corroborate that anti-lipid OCMB associate with an aggressive MS course and show that OCMB that do not recognize myelin lipids represent a transient immune response related to a more benign disease course.

Clinically isolated syndromes: a new oligoclonal band test accurately predicts conversion to MS.

BACKGROUND: Patients with a clinically isolated demyelinating syndrome (CIS) are at risk of developing a second attack, thus converting into clinically definite multiple sclerosis (CDMS). Therefore, an accurate prognostic marker for that conversion might allow early treatment. Brain MRI and oligoclonal IgG band (OCGB) detection are the most frequent paraclinical tests used in MS diagnosis. A new OCGB test has shown high sensitivity and specificity in differential diagnosis of MS. OBJECTIVE: To evaluate the accuracy of the new OCGB method and of current MRI criteria (MRI-C) to predict conversion of CIS to CDMS. METHODS: Fifty-two patients with CIS were studied with OCGB detection and brain MRI, and followed up for 6 years. The sensitivity and specificity of both methods to predict conversion to CDMS were analyzed. RESULTS: OCGB detection showed a sensitivity of 91.4% and specificity of 94.1%. MRI-C had a sensitivity of 74.23% and specificity of 88.2%. The presence of either OCGB or MRI-C studied simultaneously showed a sensitivity of 97.1% and specificity of 88.2%. CONCLUSIONS: The presence of oligoclonal IgG bands is highly specific and sensitive for early prediction of conversion to multiple sclerosis. MRI criteria have a high specificity but less sensitivity. The simultaneous use of both tests shows high sensitivity and specificity in predicting clinically isolated demyelinating syndrome conversion to clinically definite multiple sclerosis.

An ultrasensitive method for the detection of oligoclonal IgG bands.

We have developed an ultrasensitive isoelectrofocusing (IEF) method for the detection of oligoclonal (OGC) IgG bands in cerebrospinal fluid (CSF) and sera. In this procedure, double antibody and peroxidase immunodetection have been substituted by a single antibody labelled with alkaline phosphatase. Alkaline phosphatase immunodetection not only improves tenfold the sensitivity of the assay but also gives a sharper pattern resolution making the interpretation easier and increases the specificity of this technique. This preliminary report should be validated in a larger number of patients with and without multiple sclerosis (MS).

Tumoricidal activity of lauryl gallate towards chemically induced skin tumours in mice.

Lauryl gallate (antioxidant food additive E-312) prevents the formation of dimethylbenzanthracene-induced skin tumours in mice, and kills, selectively, tumoral cells on established tumours. This results in total remission, after topical application of the compound on the tumoral mass, without affecting the surrounding tissue.

Intrathecal IgM synthesis predicts the onset of new relapses and a worse disease course in MS.

BACKGROUND: The authors have recently described that intrathecal IgM synthesis (ITMS) correlates with a higher disability in patients with clinically definite MS (CDMS). OBJECTIVE: To follow-up a group of patients with MS in the initial stages of the disease to evaluate if the presence of ITMS correlates with a worse evolution. METHODS: Oligoclonal IgM bands were performed in 22 patients with MS with a mean of 1.14 months of evolution. Patients were followed for a period ranging from 6 to 36 months (mean, 21.4 months). During follow-up, time to conversion to CDMS, number of relapses, and changes in Expanded Disability Status Scale (EDSS) score were evaluated. RESULTS: Patients were divided into two groups according to the presence (Group 1, 10 patients) or absence (Group 2, 12 patients) of ITMS. No clinical differences were observed between the groups at inclusion in the study. During the follow-up, the probability of conversion to CDMS was greater in Group 1 (90% of the patients had converted to CDMS after 8 months of follow-up) than in Group 2 (51% of patients had converted to CDMS after 36 months of follow-up) (p = 0.0001). Patients from Group 1 had more relapses (mean, 2.0) than those from Group 2 (mean, 0.58) (p = 0.02). At the end of the study, patients from Group 1 had higher EDSS scores (mean, 1.70) than those from Group 2 (mean, 0.79) (p = 0.02). CONCLUSION: The presence of oligoclonal IgM bands in CSF can be a prognostic marker in the early phases of MS.

SHP-1 expression in peripheral T cells from patients with Sezary syndrome and in the T cell line HUT-78: implications in JAK3-mediated signaling.

SHP-1 is a key tyrosine phosphatase that acts as a negative regulator of signal transduction in lymphocytes, which has been found down-regulated in several T cell lines derived from human T cell malignancies. The standardization of a sensitive ELISA for the quantification of SHP-1 protein in peripheral T and B lymphocytes has enabled us to quantify the SHP-1 content of freshly isolated T cells from patients with Sezary syndrome and in the Sezary T cell line HUT-78. In all cases, a dramatic decrease in the content of this protein, when compared with the content in healthy volunteer controls, was observed. These results were corroborated when the expression of SHP-1 mRNA was analyzed. In order to study whether there was any correlation between SHP-1 protein expression and tyrosine phosphorylated state of JAK3, the state of phosphorylation of JAK3 was studied in the T cell line HUT-78, and found to be highly phosphorylated. These results suggest that SHP-1 might be involved in maintaining the IL-2R/JAK3 signaling pathway under control and point towards a role of SHP-1 in the pathogenesis of the disease.

Intrathecal IgM synthesis in neurologic diseases: relationship with disability in MS.

The authors studied the intrathecal IgM synthesis (ITMS) in paired sera and CSF samples from 65 patients with MS, 28 with CNS infection, 40 with other neurologic diseases and eight control subjects. ITMS was found in 30 patients with MS and in 20 with CNS infection, but not in patients with other neurologic diseases or in control subjects. In infectious samples, the ITMS is likely a primary response. In MS group, it was associated with higher Expanded Disability Status Scale index (p = 0.017).

A sensitive and reproducible method for the detection of oligoclonal IgM bands.

We have developed a sensitive and specific isoelectrofocusing (IEF) method for the detection of oligoclonal (OGC) IgM in the cerebrospinal fluid (CSF) and sera. In this procedure, ampholytes, in the range pH 5-8, were used to improve the resolution, and serum was diluted in saline to avoid IgM precipitation. Samples were treated with 50 mM dithiothreitol (DTT) at pH 9.5 to reduce IgM and improve the migration of the protein. Using an anti-IgM labelled with alkaline phosphatase, the limit of detection was found to be 0.1 ng in 5 microl of sample.

Study of antiphospholipid antibodies in patients treated with antiepileptic drugs.

BACKGROUND: Antiphospholipid antibodies (lupus anticoagulant and anticardiolipin antibodies) are associated with a variety of clinical situations, including drug-intake, but their relationships with antiepileptic drugs have been scarcely investigated. OBJECTIVE: To determine the prevalence of antiphospholipid antibodies in patients treated with antiepileptic drugs and the associated risk of thrombotic events. PATIENTS AND METHODS: We performed the serologic study of thirty-six consecutively prospectively recruited epileptic patients treated with diverse antiepileptic drugs during 44.38 +/- 8.08 months (mean +/- SD) in which antiphospholipid antibodies were determined using cardiolipin and a mixture of phospholipid from rabbit brain as antigen for detection of cardiolipin and lupus anticoagulant by ELISA and in addition lupus anticoagulant was carried out also using coagulometric assays. A clinical evaluation was done in order to determine the presence of thrombotic events in the following five years. RESULTS: Antiphospholipid antibodies were detected in 43% of these patients, in most of them as anticardiolipin antibodies (IgM subtype). The patients did not present thrombotic events during the time of the study. CONCLUSION: Antiphospholipid antibodies are positive in a high proportion of these patients but thrombosis were not found during the study duration. This may be explained by the fact that the profile of aCL positivity not associated to positive LA observed in these patients does not confer a risk for thrombotic events.

Interleukin-6 dimers produced by endothelial cells inhibit apoptosis of B-chronic lymphocytic leukemia cells.

Tumoral lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) are long-lived cells in vivo, but they die rapidly by apoptosis in vitro. Here, it is reported that endothelial cells (ECs) inhibit the apoptosis of B-CLL cells, as determined by 4 different flow cytometric methods, and that this antiapoptotic effect is mediated mainly by soluble factor(s), as can be deduced from the following findings. First, EC-conditioned medium (ECCM) inhibited the apoptotic rate in B-CLL to approximately 50% of control. Second, the antiapoptotic effect mediated by EC/B-CLL cell contact was more apparent than real; using a fluorescence-based phagocytosis assay, it was demonstrated that this effect was due to the phagocytic capacity of ECs, which internalized apoptotic cells. Third, the protective effect of ECCM was associated neither with proliferation nor differentiation signals. Fourth, the survival factor was a dimeric form of IL-6 because anti-IL-6 antibodies completely neutralized the antiapoptotic effect mediated not only by the crude ECCM but also by the 45- to 55-kd active fractions obtained after gel filtration, which contained high levels of IL-6. These IL-6 dimers (IL-6(D)) were noncovalently associated. Sixth, human recombinant IL-6(D) (hrIL-6(D)) inhibited B-CLL apoptosis, whereas hrIL-6 monomers (hrIL-6(M)) did not. Binding and functional competition experiments showed not only that monomers and dimers had similar affinity for the IL-6R, but also that hrIL-6(M) inhibited the antiapoptotic activity of hrIL-6(D). These data suggest that IL-6(D) derived from ECs promote the survival of B-CLL cells.

Lauryl gallate inhibits the activity of protein tyrosine kinase c-Src purified from human platelets.

The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu,Na,Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.

Mechanistic aspects of the induction of apoptosis by lauryl gallate in the murine B-cell lymphoma line Wehi 231.

The effect of lauryl gallate (antioxidant E-312) has been studied on the mouse B-cell lymphoma line Wehi 231. This compound is able to inhibit protein tyrosine kinases (PTKs) in whole cells and in crude extracts with a better efficiency than other well-known PTK inhibitors such as herbimycin or genistein. Initial events triggered upon the incubation of cells with lauryl gallate in phosphate-buffered saline (up to 1 h) include the inhibition of tyrosine phosphorylation, discharge of the mitochondrial transmembrane potential, and induction of mRNA for Bcl-2. Long-term cultures in complete medium supplemented with fetal calf serum (up to 24 h) in the presence of this compound exhibit clear apoptotic features such as increase in phosphatidylserine in the cell surface, decrease in the functionality of mitochondria, cytochrome c release to the cytosol, activation of caspases, hypodiploidy, and oligonucleosomal breakdown of DNA. Comparison between Wehi cells overexpressing Bcl-2 (Wehi-bcl-2) with Wehi-neo cells shows a delay in the manifestations of the apoptotic signs, indicating that Bcl-2 has a partial protective effect on the apoptosis induced by lauryl gallate. The proapoptotic effect of lauryl gallate is not dependent on DNA or protein synthesis, is not blocked by the chelation of calcium, and is not reverted by N-acetylcysteine.

The expression of integrins on activated T-cells in multiple sclerosis. Effect of intravenous methylprednisolone treatment.

We studied the effect of intravenous methylprednisolone (MP) on the expression of the integrins, LFA-1 and VLA-4, on activated blood T-lymphocytes in 17 patients with relapses of clinically definite relapsing-remitting MS. MP treatment did not induce changes in the expression of CD3, CD4, DR, LFA-1 or VLA-4 markers when measured in the total population of lymphocytes in MS patients in relation to treatment. Treatment influenced neither the LFA-1 nor VLA-4 positive cells within the CD3+ population. MP treatment clearly decreased the DR+ CD3+ cells (P < 0.01) and the percentage of DR+ CD3+ lymphocytes bearing VLA-4 (P < 0.01). However, this was not the case when we studied the percentage of lymphocytes which expressed LFA-1. Glucocorticoids did not influence the mean intensity of the expression of the two integrins quantified in either total or DR+ CD3+ lymphocytes. Although, further research seems warranted to investigate a possible effect of MP on lymphocyte integrin function, this work corroborates the idea that MP treatment may interfere with the mechanisms of T-cell migration into CNS, thus modulating the activity of multiple sclerosis.

Derivatives of gallic acid induce apoptosis in tumoral cell lines and inhibit lymphocyte proliferation.

The effect of gallic acid (3,4,5-trihydroxybenzoic acid) and its alkyl esters (methyl, propyl, octyl, and lauryl) has been studied on several tumoral and nontumoral cells. Three types of behavior have been observed; the first type is represented by the mouse B cell lymphoma Wehi 231 cell line in which death occurs according to the biochemical characteristics of classical apoptosis showing the DNA ladder fragmentation pattern. The second type is represented by the mouse fibroblast L929 cell line in which morphological characteristics such as cell shrinkage, chromatin condensation, and appearance of apoptotic bodies can be evidenced by microscopical observation. However, the typical DNA fragmentation is absent. Peripheral blood lymphocytes are representative of a third type of behavior. In a resting state they can withstand higher concentrations of these compounds. If the drug is washed, they proliferate normally upon the addition of the mitogen phytohemagglutinin (PHA). However, if the drug is added in the presence of PHA, a clear antiproliferative effect can be demonstrated. A special interest for these compounds stems from the fact that some of them are currently used as antioxidant food additives with the European Community codes E-310 (propylgallate), E-311 (octylgallate), and E-312 (laurylgallate).

Increased soluble serum HLA class I antigens in patients with lymphoma.

sHLA are soluble forms of class I histocompatibility antigens detected in human serum and cerebrospinal fluid. These molecules are secreted by B and T lymphocytes and the secretion increases dramatically upon mitogenic activation of these cells. sHLA was quantified by an ELISA sandwich method in sera from healthy blood donors, and from patients with Non Hodgkin's Lymphoma (NHL) and Hodgkin's Disease (HD) both at diagnosis and at remission. Pretreatment sHLA serum levels in NHL and in HD were compared with the values found in controls. sHLA levels are increased in patients with NHL (low grade: 6.68 +/- 1.80; high grade: 2.65 +/- 0.53 microg/ml X +/- S.E.) and HD (6.44 +/- 0.98) at diagnosis and in relapses when compared with controls (0.89 +/- 0.08). This increment is statistically significant (low grade NHL: p = 0.0038; high grade NHL: p = 0.0049 and HD: p = 0.0005 versus control group). No statistical differences between titers of sHLA after complete remission and sHLA in the control group were found. The high levels of sHLA detected in patients with lymphoma are mainly due to low molecular weight HLA molecules (55 KD) (60-75% of the HLA present in serum in the control group and 75-100% in serum of patients with lymphoma).

Methimazole has no dose-related effect on the serum concentrations of soluble class I major histocompatibility complex antigens, soluble interleukin-2 receptor, and beta 2-microglobulin in patients with Graves' disease.

Soluble class I major histocompatibility antigens (sHLA), beta 2-microglobulin (beta 2-M), and soluble interleukin-2 receptor (sIL-2R), are secreted by B and T lymphocytes upon activation, and have been used as markers of immune activation in several diseases. Thirty-two Graves' disease patients were randomly assigned to three methimazole (MMI) regimens of treatment: (1) low-dose, starting with 45 mg/day, and lowering the dose thereafter to maintain normal serum thyroid hormones; (2) MMI 60 mg/day + levothyroxine, and (3) MMI 30 mg/day + levothyroxine. Serum sHLA, beta 2-M, sIL-2R, TSH receptor antibodies (TSH-R Ab), T3, and free T4 (fT4) were measured at diagnosis and at weeks 4, 12, and 24 (end of treatment). Patients were followed-up after treatment for at least 24 weeks (24 to 89). At diagnosis, serum levels of sIL-2R, beta 2-M, sHLA, and TSH-R Ab were elevated. Serum sIL-2R, beta 2-M, sHLA, and TSH-R Ab decreased with treatment. No effect of the varying MMI regimens on these parameters was observed. Soluble IL-2R correlated positively with T3, fT4, beta 2-M, sHLA, and TSH-R Ab. Statistically significant, but weak, correlations (r < 0.35) were observed between beta 2-M, sHLA, and TSH-R Ab, between beta 2-M, T3, and fT4, and between TSH-R Ab and T3. Recurrence rates were not associated either with the MMI regimen or any of the parameters studied, with the exception of elevated initial TSH-R Ab levels. Serum sHLA, beta 2-M, and sIL-2R are increased in untreated Graves' disease, and decrease during treatment. No MMI dose-related differences were observed in these parameters, and in the recurrence rate. Unfortunately, sHLA, beta 2-M, and sIL-2R were not useful predictors of prolonged remission after MMI treatment.

Thioesterase and protein deacylase activities of porcine pancreatic phospholipase A2.

The thioesterase activity of porcine pancreatic phospholipase A2 has been investigated with non-phospholipid substrates. The acyl-CoA hydrolase activity towards acyl-CoA derivatives is specific for long chain fatty acids (14 C, 16 C) but is unable to hydrolyze short chain acyl-CoA compounds (below 8 C). The same enzyme also shows protein deacylase activity liberating [3H]palmitic acid from [3H]palmitoyl-acyl carrier protein.