Clinical significance of anti-RNP and anti-Sm autoantibodies as determined by immunoblotting and immunoprecipitation in sera from patients with connective tissue diseases.

Antibodies to Sm and RNP antigens have been detected by immunoblotting and immunoprecipitation of small nuclear ribonucleoproteins in 168 sera from patients with connective tissue diseases previously characterized by immunodiffusion. Anti-RNP and anti-Sm antibodies immunoprecipitated U1 and U1-U6 snRNA respectively. By immunoblotting anti-Sm reacted with B-B' and D polypeptides and we have distinguished two types of anti-RNP sera: 1) 'full spectrum' anti-RNP sera reacted with the 68 kD, A, C and B-B' polypeptides; 2) 'partially reactive' anti-RNP sera reacted with various combinations of these polypeptides but not the four of them. A strong specificity of anti-Sm antibodies for systemic lupus erythematosus (SLE) was found with all three methods but immunoblotting was more sensitive and detected anti-Sm in 76% of SLE sera. Sera containing a high titer of 'full spectrum' anti-RNP without anti-Sm activity were only detected in mixed connective tissue disease (MCTD) whereas anti-68 kD antibodies alone seemed to be less specific. This strong association between 'full spectrum' anti-RNP antibodies and MCTD supports the hypothesis that MCTD is a distinct clinical entity associated with a specific serologic marker.     

The B cell repertoire in rheumatoid arthritis. I. Frequency of EBV-inducible circulating precursors producing autoantibodies.

The frequency of B cell precursors producing antibodies against various autoantigens (Fc fragment of IgG, F(ab')2 fragment of IgG, type II collagen, cytoskeleton filaments and insulin) was determined in patients with rheumatoid arthritis (RA) using immortalization of peripheral blood B cells by the Epstein-Barr virus (EBV) and limiting dilution analysis. Equally large numbers of B cell precursors producing IgM-rheumatoid factors (RFs) were present in the peripheral blood of seronegative and seropositive RA patients and of controls. On average, 1 out of 15,000 B cells could be induced by EBV to secrete IgM-RFs, which represents 0.5-1% of the EBV-induced proliferating clones. By cloning or somatic hetero-hybridization of EBV cell lines derived from patients and controls, we obtained two types of monoclonal RFs: one polyreactive, reacting with Fc but also with the other autoantigens tested, and the other monoreactive, reacting with Fc only and that previously had only been found in the RA B cell repertoire. Moreover, patients and controls had similar numbers of circulating B cell precursors secreting IgM antibodies against other autoantigens that might be regarded as specific targets of RA (F(ab')2 fragment of IgG and type II collagen), and against cytoskeleton filaments that are targets of natural autoantibodies, increased in RA. The frequencies of EBV-induced B cells producing antibodies against all these autoantigens were of the same order of magnitude as the frequency of EBV-induced B cells producing RFs. The patients also possessed a similar number of precursors producing antibodies against insulin, an autoantigen irrelevant to the pathogenesis of the disease, taken as control. These data tend to demonstrate no abnormality in the autoantibody repertoire of B cells activable by EBV in RA, especially those secreting RFs. In vitro spontaneous RF secretion by circulating B cells was observed in seropositive RA patients but not in seronegative patients and in the controls tested. We enumerated the number of B cells spontaneously secreting RFs in seropositive RA patients and found that it correlated with the serum RF titer, but not with the number of RF-secreting B cells activated by EBV. The mean frequency values of B cells secreting RFs either spontaneously or after EBV infection were of the same order of magnitude, showing that the expanded population of in vivo-activated B cells was not (at least partially) infectable by EBV. This raised the possibility that EBV triggers a repertoire which may not reflect the status of B cells secreting autoantibodies in autoimmune diseases.     

Presence of circulating antibodies against gag-gene MuLV proteins in patients with autoimmune connective tissue disorders

An immunoblotting procedure using viral proteins from purified murine sarcoma virus or MSV-(MLV) has been developed to characterize antiviral antibodies in sera from patients with autoimmune connective tissue disorders. Fifty-eight sera with anti-Sm, anti-RNP, anti-SS-B (La), and other undefined specificities were found to react with several major viral polypeptide bands. Most of them corresponded to gag-gene-encoded products: pr65^gag, p40^gag, p30, p15, p12 and p10. Other bands with molecular weights averaging 90K, 60K, 45K, and 28K were recognized by a few sera. Immunological specificity of the reaction was assessed by reproducing the tests with IgG purified from sera and from corresponding F(ab′)2 fragments. Moreover, the specificity of the reaction with gag proteins was confirmed by repeating the tests with p30 and p15 prior purified by immunoprecipitation with anti-p30 and anti-p15 goat sera. Furthermore, the gag polypeptides were recognized by human sera by replacing MSV-(MLV) by three other murine retroviruses of different origin. An indirect confirmation of these results was obtained by applying this method to sera of MRL lpr/lpr mice which develop an autoimmune syndrome comparable to that of human systemic lupus erythematosus. In agreement with previously published results (C. Rordorf, C. Gambke, and J. Gordon (1983), J. Immunol. Methods59, 105–112), we found that anti-gag-gene antibodies were present in the sera of individual mice. Patterns of reactivity were found to vary with the age of the animals. No retroviral polypeptide was significantly detected in the great majority (80%) of sera from normal donors. However, 5 out of 25 sera showed faint bands although to a lesser extent than pathological sera. These five sera also reacted with HeLa cell purified HnRNPs, suggesting that their normal status should be reconsidered.     

Clinico-biological markers for the prognosis of status epilepticus in adults

Journal of Neurology - Prediction of mortality, functional outcome and recovery after status epilepticus (SE) is a challenge. Biological and clinical markers have been proposed to reflect the brain...     

Neuron Specific Enolase,S100-beta protein and progranulin as diagnostic biomarkers of status epilepticus

Journal of Neurology - Status epilepticus (SE) is a life-threatening prolonged epileptic seizure. A rapid diagnosis is fundamental to initiate antiepileptic treatment and to prevent the development...     

Cerebrospinal fluid and blood biomarkers of status epilepticus

Status epilepticus is a condition resulting either from the failure of the mechanisms responsible for seizure termination or from the initiation of mechanisms that lead to abnormally prolonged seizures and require urgent administration of antiepileptic drugs. Refractory status epilepticus requires anesthetics drugs and may lead to brain injury with molecular and cellular alterations (eg, inflammation, and neuronal and astroglial injury) that could induce neurologic sequels and further development of epilepsy. Outcome scores based on demographic, clinical, and electroencephalography (EEG) condition are available, allowing prediction of the risk of mortality, but the severity of brain injury in survivors is poorly evaluated. New biomarkers are needed to predict with higher accuracy the outcome of patients admitted with status in an intensive care unit. Here, we summarize the findings of studies from patients and animal models of status epilepticus. Specific protein markers can be detected in the cerebrospinal fluid and the blood. One of the first described markers of neuronal death is the neuron-specific enolase. Gliosis resulting from inflammatory responses after status can be detected through the increase of S100-beta, or some cytokines, like the High Mobility Group Box 1. Other proteins, like progranulin may reflect the neuroprotective mechanisms resulting from the brain adaptation to excitotoxicity. These new biomarkers aim to prospectively identify the severity and development of disability, and subsequent epilepsy of patients with status. We discuss the advantages and disadvantages of each biomarker, by evaluating their brain specificity, stability in the fluids, and sensitivity to external interferences, such as hemolysis. Finally, we emphasize the need for further development and validation of such biomarkers in order to better assess patients with severe status epilepticus.     

Hepatitis B virus Dane particles bind to human plasma apolipoprotein H

Human apolipoprotein H (apo H) was found to bind specifically to hepatitis B surface antigen (HBsAg) from hepatitis B virus (HBV)-infected individuals. We used recombinant HBsAg proteins to analyze HBV domains recognized by apo H. We showed that the myristylated pre-S1 domain of HBsAg strongly interacted with apo H. This binding involved phospholipid components of the HBV envelope because their removal by detergent prevented apo H-HBsAg interaction. The opposite effects of iron and zinc metal ions on binding suggest that the oxidation of phospholipids also affects apo H-HBsAg interaction. After fractionation of viral particles on a sucrose gradient, and their addition to microtiter plates coated with apo H or anti-HBsAg, we observed that the maximal anti-HBsAg capture activity corresponded to a sucrose concentration of 36%, whereas the maximal apo H capture activity corresponded to a concentration of 39%. Electron microscopy and polymerase chain reaction (PCR) Southern blot studies of these fractions showed that the fraction with maximal apo H binding predominantly contained full Dane particles. Finally, we studied apo H-HBsAg binding relative to the presence of hepatitis B virus markers and observed that apo H binding activity for HBsAg was higher in sera from patients in the active virus replication phase.     

Oxidation and biotinylation of beta 2 glycoprotein I glycan chains induce an increase in its affinity for anionic phospholipids similar to that obtained by the addition of anti-beta 2 glycoprotein I or anti-cardiolipin antibodies

Binding of beta 2 glycoprotein I (β₂GPI) to apoptotic cells plays a key role in the opsonization of apoptotic bodies and the formation of antiphospholipids antibodies. Here, we describe the binding of β₂GPI to apoptotic cells using β₂GPI labelled with biotin-hydrazide (β₂GPI-bh) after oxidation of its glycan chains. Flow cytometry analyses and confocal microscopy showed that β₂GPI-bh, contrary to native β₂GPI, bound to apoptotic cells, either permeable or non-permeable to propidium iodide (PI), as did annexin-V–FITC. But, in the absence of divalent ions, β₂GPI-bh, contrary to annexin V, was still able to bind to apoptotic cells. Binding equilibrium studies, performed on solid-state anionic phospholipids (AnPL), revealed that β₂GPI-bh had a greater apparent affinity for AnPL than native β₂GPI. In presence of the anti-β₂GPI mAb 8C3, the ability of native β₂GPI to bind to AnPL was increased and binding to apoptotic PI⁺ and PI⁻ CEM cells was observed whereas binding of β₂GPI-bh was barely affected by the addition of 8C3. However, the 8C3-enhanced ability of native β₂GPI to bind to AnPL was still weaker than that of β₂GPI-bh. It is not clear why the oxidation and biotinylation of glycan chains of β₂GPI increases its affinity for AnPL, but it seems that if such oxidative process occurs naturally, it could participate in enhancing antiphospholipid formation.