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.

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.

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).

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.

Use of the ELISA to detect and quantify histocompatibility antigens and their subunits.

A solid phase enzyme immunoassay (ELISA) has been developed for the detection and quantification of human histocompatibility antigens and their subunits. The assay involves the binding to a microELISA plate of a mouse monoclonal antibody reacting with a common antigenic determinant to all HLA (A, B, C) antigens. The standard conditions for the assay and the curves obtained for the quantification of total HLA, free beta 2m, and free heavy chain subunit (alpha) present in a biological sample are described and the sensitivity and potential uses of the method are discussed.

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.

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).

Intrathecal synthesis of soluble class I antigens in multiple sclerosis.

We have studied the intrathecal synthesis of soluble class I antigens (sHLA), reflected by the index IH = (CSF sHLA/serum sHLA)/(CSF albumin/serum albumin), in multiple sclerosis (MS). IH was increased in patients in relapse, but normal in patients in remission; these findings show that there is a high lymphocyte activation within the central nervous system in patients with clinically active MS.

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).

Intrathecal synthesis of soluble class I antigens (sHLA) in patients with HIV infection and tuberculous meningitis.

sHLA are soluble class I antigens produced by lymphocytes on early activation. We have studied the sHLA index IH = (CSF sHLA/serum sHLA)/(CSF albumin/serum albumin), which reflects the intrathecal synthesis (ITS) of sHLA in 23 intravenous drug abusers with central nervous system (CNS) HIV infection. Their mean IH value was increased and directly correlated with ITS of IgG against HIV when the total group of patients was studied; however, 8 of them, who suffered from concomitant tuberculous meningitis, had a decreased IH. The relationship between this index, blood-brain barrier (BBB) function, and HIV and tuberculous infection was also studied. We consider IH an index of lymphocyte activation within the CNS. Its decrease in patients with CNS HIV infection may reflect the presence of a meningeal opportunistic infection due to Mycobacterium tuberculosis.

Quantification of soluble serum HLA class I antigens in healthy volunteers and AIDS patients.

A solid-phase enzyme immunoassay (ELISA) has been used to quantify human soluble Class I histocompatibility antigens in serum samples from voluntary blood donors and AIDS patients. Statistical analysis of the results showed significantly raised levels (p less than 0.01) of free HLA Class I in sera from AIDS patients (2.95 +/- 1.80 micrograms/ml) when compared with the blood donors (1.06 +/- 0.6 micrograms/ml). The assay is specific, reproducible and easy to perform. Potential uses of this determination are discussed.

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.

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.

Introducting of the 3-deoxy group in 3,6-dideoxyhexoses: a novel cofactor function for pyridoxamine-5'-phosphate.

A cofactor needed for the reduction of CDP-4-keto-6-deoxy-D-glucose to CDP-4-keto-3,6-dideoxy D-glucose has been detected and purified. Its spectra in neutral, acid, and basic solutions, its behavior on thin-layer chromatography, and other properties led to its identification as pyridoxamine-5'-phosphate. Moreover, authentic pyridoxamine-5'-phosphate substituted for the purified cofactor with about the same specific activity. Other vitamin B(6) derivatives did not stimulate the reaction, and inhibition was observed with some of them.

Studies on the quaternary structure of class I major histocompatibility complex antigens. Effect of different agents on the interaction between subunits.

The effect of temperature, urea, guanidine HCl, ionic and nonionic detergents, organic solvents, chaotropic salts, pH, and divalent cations has been investigated on purified human histocompatibility antigens solubilized by papain (HLApap) or solubilized by sodium cholate (HLAchol). HLApap and HLAchol are fairly stable proteins to agents acting predominantly on hydrogen bonds (temperature, urea) or hydrophobic forces (ionic and nonionic detergents). However, agents which affect ionic interactions (pH, salts, divalent cations) dissociate the molecules into subunits. A single binding site for beta 2-microglobulin with an affinity constant of 1.0 X 10(7) M-1 was found for the alpha chain of HLAchol. The dissociated subunits can be separated by affinity chromatography on Sepharose-rabbit IgG anti-human beta 2-microglobulin and reassociate in vitro when incubated under the appropriate conditions. The results point toward an important role of ionic interactions between subunits in the stabilization of the quaternary structure of HLA.

Purification and properties of membrane and cytosolic phosphatidylinositol-specific phospholipases C from human spleen.

Two phosphatidylinositol-specific phospholipases C (PI-PLC) have been purified from human spleen. PI-PLCm represents the main activity detected in the membrane, while PI-PLCc is the main activity present in the cytoplasm. PI-PLCm can be resolved into two peaks of activity of high Mr (60,000-70,000) and low Mr (16,000-18,000). High salt concentration ((NH4)2SO4, 2M) dissociates the high Mr form yielding the low molecular form and increasing the specific activity. The same effect of dissociation and potentiation of the activity is observed when membranes solubilized by n-octyl glucoside are subjected to the high voltage conditions of an isoelectric focusing run. The purified Pi-PLCm has a Mr of about 18,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration and a basic pI (9.0-9.2). Purified PI-PLCc has a Mr of 57,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration) and a slightly acid pI (6.2). Other characteristics of both enzymes, such as cations dependence, substrate specificity, optimum pH, and kinetic parameters, are also discussed.

Description of an enzyme-linked immunosorbent assay for the detection of protein tyrosine kinase.

A solid immunoassay for the detection of protein tyrosine kinases has been developed. It is based on the binding of the synthetic polypeptide poly(Glu.Na,Tyr) 4:1 to microELISA wells, where the phosphorylation reaction takes place in the presence of ATP and enzyme. The phosphorylated tyrosine residues produced in the reaction are finally detected, in the same well, by means of an ELISA using monoclonal antiphosphotyrosine antibody, peroxidase-labeled goat anti-mouse IgG antibody, and substrate. The amount of protein tyrosine kinase activity present in the sample is proportional to the color at 492 nm developed in each well.

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.