Rotation thromboelastometry detects thrombocytopenia and hypofibrinogenaemia during orthotopic liver transplantation.

BACKGROUND: Orthotopic liver transplantation can be associated with haemorrhage, particularly in patients with severe liver dysfunction. We assessed the value of rotation thromboelastometry (ROTEM) to monitor coagulation in the operating theatre, its correlation with routine laboratory findings, and its ability to guide platelet (Plt) and fibrinogen (Fg) transfusion. METHODS: Twenty-three patients were included in this prospective observational study. Laboratory tests and ROTEM tests (EXTEM, INTEM, FIBTEM, and APTEM) were performed six times during the procedure. Correlations between laboratory findings and ROTEM parameters were sought. Thresholds for ROTEM parameters were determined with receiver-operating characteristic (ROC) curve analysis according to Plt count and Fg levels. RESULTS: Clot amplitude at 10 min (A10) of EXTEM was well correlated with Plt count and Fg levels (R(2)=0.46 and 0.52, respectively, P<0.0001). FIBTEM A10 was correlated with Fg (R(2)=0.55, P<0.0001). ROC analysis showed that EXTEM A10 with a threshold of 29 mm predicted thrombocytopenia with a sensitivity of 79% and a specificity of 60%, and a threshold of 26 mm predicted hypofibrinogenaemia with a sensitivity of 83% and a specificity of 75%. CONCLUSIONS: ROTEM is useful for the global assessment of coagulation in the operating theatre. EXTEM was the most informative for assessing the whole coagulation process and A10 showed value in guiding Plt and Fg transfusion.

ELISA in serodiagnosis of HCV infection.

A high level of anti-HCV is generally associated with viral replication and the number of recognized epitopes appears to be correlated with the viral charge. Nevertheless, the absence of detectable antibodies in about 60% of patients during the acute phase of the disease and in 10% of chronically infected (generally immunocompromised subjects) are heavy handicaps for HCV serology. Moreover, low levels of anti-HCV antibodies can persist after complete recovery, and HCV viremia does not appear to be associated with the presence of a special antibody specificity. The immunoblots presented as 'confirmatory test' always appear to be less sensitive than the screening tests and therefore are unable to discriminate between post-infection antibodies and false-positive reactions, as rare as they can be. In these cases, as in non-responder patients, PCR appears essential. The possible reasons of immune response limitations and the possible improvements of HCV serology are discussed.

Immunochemical structure of the hepatitis B surface antigen vaccine--I. Treatment of immobilized HBsAg by dissociation agents with or without enzymatic digestion and identification of polypeptides by protein blotting.

Since the immunosorbent techniques and the cycles of isopycnic and rate zonal velocity ultracentrifugations were shown to be unsuitable for the purification of hepatitis B surface antigen (HBsAg) particles from human sera because HBsAg was still largely contaminated by serum proteins, we applied a drastic dissociating treatment of HBsAg stabilized by adsorption on silica gel which appeared essential to remove extraneous components initially present in the HBsAg particles. Only albumin and sometimes IgG were recovered with the purified antigen. The polypeptide composition of our purified HBsAg preparations was analyzed by SDS-PAGE with subsequent transfer to a nitrocellulose sheet by blotting, incubation with 125I-anti-HBs and exposure to X-ray film. Samples from HBsAg-positive sera containing the hepatitis B virus e antigen (HBeAg) displayed three proteins: P 24.5 and GP 28 as major components and GP 36 as a minor component. Dimers of these polypeptides were also immunologically detected. When a supplementary step of trypsin or pepsin digestion was included in our purification procedure after adsorption to silica and acid dissociation of HBsAg, proteolytic cleavage fragments of HBsAg with mol. wts lower than 10,000 were obtained on SDS-PAGE after reduction. This finding shows that arginine and lysine residues inaccessible to tryptic digestion in the intact HBsAg lipoprotein particle were exposed to enzymatic hydrolysis by our treatment. However, HBsAg kept the antigenic and immunogenic properties of the native antigen. Therefore such a HBsAg preparation appeared as a new candidate for the vaccination against HBV and a useful material for the analysis of the HBs antigenic structure.

Human myeloma light chains with increased molecular weight: high frequency among lambda chains.

The discovery of a human myeloma protein comprising a kappa L-chain with an increased mol. wt of 30,000) (Bouvet et. al., 1980) prompted investigations on the incidence of such heavier L-chains among other human myeloma proteins. In 105 samples examined, 34 were found to have L-chains heavier than normal (23,000-24,000), ranging from 25,000 up to 31,000, and five of lighter mol. wt (21,000-22,000). These mol. wt abnormalities were detected by electrophoresis in sodium dodecyl sulfate 10% polyacrylamide gels (SDS-PAGE) after reduction with 2-mercaptoethanol. The mol. wt of three of the heavier kappa or lambda chains was also estimated by filtration through a Sephadex G100 column and by sedimentation equilibrium. All three methods indicated a mol. wt increase of about 15-25% as compared with the usual mol. wt. The distribution of the high mol. wt chains among all L-chains examined was found to be 11 out of 62 kappa chains (17.7%) and 23 out of 43 lambda chains (53%) (P less than 0.001). A preferential association of such L-chains with H-chains producing multiple bands in SDS-PAGE (P less than 0.01) and an association between multiple L-chain and multiple H-chain band (P less than 0.05) were also observed. In contrast, no abnormal L-chain was found in immunoglobulins from normal subjects. Spontaneous degradation of the normal H-chains sometimes yielded fragments of 30,000 mol. wt. These fragments were easily distinguishable from abnormal L-chains. The nature of extra mol. wt in heavy L-chains was investigated for the presence of carbohydrate moiety. Four large and three normal size L-chains were examined for amino-sugar and sialic acid content. A small amount (one residue per molecule) of amino-sugar was detected only in two normal and two heavy L-chains, whereas sialic acid was only found in the heaviest (27,000-30,000) L-chains (Lh) and in small percentage (one or two residues per molecule). Total sugar estimation in one Lh chain indicated a proportion not exceeding three or four residues per L-chain (mol. wt 1,000) and this is insufficient to explain the 15-25% (3,600-6,000) mol. wt increase. It is therefore possible that, at least in some heavy myeloma L-chains, an additional peptide is expressed. Whatever the nature of the increase it would be of interest to elucidate whether this is a marker of malignant process or of an intermediate step of normal Ig synthesis.

Demonstration of a firm association between hepatitis B surface antigen proteins bearing polymerized human albumin binding sites and core-specific determinants in serum hepatitis B viral particles.

Hepatitis B viral particles (HB-VP) were purified from sera of chronic hepatitis B surface antigen (HBsAg) positive carriers by consecutive isopycnic and rate-zonal sedimentation in sucrose gradients. Their immunological properties [HBsAg, hepatitis B core antigen (HBcAg) and hepatitis B e-antigen (HBeAg) activities] were examined by a radioimmunoassay based upon the classical "sandwich principle". A double antibody specificity radioimmunoassay (DAS-RIA) was then developed to determine whether envelope proteins (HBsAg) with binding activity for polymerized human serum albumin (pHSA-BA) were associated with core-specific antigenicities (HBc/HBeAg). An e-antigen activity cosedimenting with intact HB-VP (negative for HBcAg reactivity) was detected in association with HBsAg and receptors for pHSA. The presence of HBcAg-specific determinant(s) on HBeAg molecules was also indicated by DAS-RIA. So, we postulated that such hepatitis B virion (HBV) specific molecules are involved in immune complexes with anti-HBc as antibodies in sera of patients with chronic HBV infection. To define the significance of these molecular forms in HB-VP morphogenesis, we studied the effects of a mild treatment with a chaotropic salt, NaSCN, on HB-VP-rich fractions (DNA polymerase positive). A small mol. wt HBeAg derived from HB-VP by dissociating treatment was detected. We found that core-specific determinants (HBe/HBcAg) were bound to large surface proteins (HBsAg) with pHSA-BA and therefore probably contained the pre-S sequence. The selective release from HB-VP of such molecular forms, which could be a product of the major S-region transcript, suggests that they may be components of complete virions.

Immunochemical structure of the hepatitis B surface antigen vaccine--II. Analysis of antibody responses in human sera against the envelope proteins.

Antibody responses to the three envelope (env) proteins of hepatitis B viral particles (HB-VP): the S-encoded P25 polypeptide; the pre-S(2)- and S-encoded GP33/GP36 polypeptide; and the large entire env gene (pre-S + S) product, P39/GP42, were investigated using a Western immunoblotting assay (WIBA). HB-VP proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose by electroblotting were used as antigenic probes to determine the polypeptide specificity of these antibodies present in immune individuals. Antisera from human subjects either after a natural HBV infection or after active immunization with the hepatitis B vaccine licensed in France were selected on the basis of a positive serological RIA test for antibodies against hepatitis B surface antigen (HBsAg). In all studied cases, the lack of reactivity of the anti-HBs/P25 antibodies in blots from reduced SDS gels confirms that the S-related-determinants have a conformation sensitive to denaturing agents. In contrast, the anti-pre-S(2)/GP33-GP36 antibodies and the anti-pre-S(1)/P39-GP42 antibodies can be easily detected in WIBA, providing these antibodies recognize the disulfide-bond independent pre-S determinants on the denatured env proteins. However, antisera raised in guinea-pigs against individual HBsAg polypeptides contain antibodies reacting with denatured S-proteins, suggesting that the sequential S-determinants are lost during HBV morphogenesis. Antibody responses in HBV convalescing patients or vaccinated healthy donors are shown to be characterized by: an early transient polypeptide specific-antibody response to pre-S(2)-sequences (detected in WIBA); a persistent antibody response to conformation-dependent S-determinants (detected in RIA). This implies that effective long-term protection against HBV infection requires antibodies directed to native env proteins.

Antibodies to synthetic peptides from the pre-S1 and pre-S2 regions of one subtype of the hepatitis B virus (HBV) envelope protein recognize all HBV subtypes.

Immunodominant B and T cell epitopes have been demonstrated recently on the preS1 and PreS2 regions of the hepatitis B virus (HBV) envelope protein. Synthetic peptide analogs corresponding to the preS2 region elicit virus-neutralizing antibodies and protect chimpanzees against HBV infection. Antibodies raised by immunization with peptides derived from the preS1 sequence block the site involved in HBV attachment to cell receptors, and are expected to be virus-neutralizing. Results presented here show that antisera raised against synthetic peptide analogs carrying the immunodominant epitope of the preS1 and preS2 sequence, respectively, and corresponding to two HBV subtypes, adw2 and ayw, each recognized preS1 and preS2 specific epitopes on all serological subtypes of the HBV envelope protein. Thus, the sequence variability within the preS1 and preS2 regions does not represent an impediment to the development of synthetic peptide or genetically engineered hepatitis B preS immunogens for worldwide immunization.

Detection of antibodies to pre-S2 encoded epitopes of hepatitis B virus by monoclonal antibody-enzyme immunoassay.

An inhibition enzyme immunoassay (IEIA) for the detection of anti-pre-S2 antibody has been developed and used to evaluate anti-pre-S2 responses in the sera of patients recovering from acute type B hepatitis and in the sera of healthy recipients of HBV vaccine. In the assay, we used two monoclonal antibodies recognizing the nonoverlapping epitopes (pre-S2a and pre-S2b) of the pre-S2 protein of HBV envelope which compete with human anti-pre-S2 for the limited antibody-binding sites on recombinant HBsAg particles (pre-S and S gene product). Two variants of the method were assayed employing the reference pre-S2 antigen on the solid (IEIA-sp) or in the liquid phase (IEIA-lp). Two McAbs were used to detect antibodies reacting specifically with pre-S2a and pre-S2b epitopes of the pre-S2 sequence. Both variants gave similar results and were successfully used for the determination of anti-pre-s2 in sera. We demonstrated that during HBV infection as well as after vaccination against HBV both pre-S2 epitopes generate specific immune responses. Anti-pre-S2 were detected in 45.3% patients recovering from HBV infection and in 43.7% of healthy recipients of the HBV vaccine licensed in France. Anti-pre-S2a and anti-pre-S2b were detected in sera in dilutions up to 10(-5). IEIA may provide a specific and highly sensitive screening test for monitoring serum anti-pre-S2 levels during HBV infection and after immunization with HBV vaccine.

A modified gel filtration technique producing an unusual exclusion volume of IgM: a simple way of preparing monoclonal IgM.

The vast majority of monoclonal IgM proteins is eluted just before the total volume of the column when filtered through G200 Sephadex or S200 Sephacryl gels equilibrated in a 0.005 M phosphate buffer but eluted with 0.05 M phosphate buffer containing 1.7 M NaCl. This unusual behaviour in low-ionic buffer is probably due to the poor solubility of IgM in diluted buffers. It allows a 1-step purification procedure under mild conditions and is suitable for both large and small scale preparations.

Interest of immunomodulation as a mean to improve the preparation of polyclonal and monoclonal antibody reagents.

Immunomodulation by monoclonal antibodies (mAbs) was investigated in mice in order to improve the preparation of antibody reagents. Three different types of representative immunogens were chosen: a human soluble protein (secretory immunoglobulin A, SIgA), a bacterial polysaccharide from E. coli K1 and an envelope protein from the hepatitis B virus. These Ag are all of importance for diagnosis and exhibit different levels of immunogenicity. Antibody-mediated enhancement was observed against restricted and defined regions of each immunogen i.e.: the Fab epitopes of SIgA, the preS1 domain of the HBV envelope and associated cell wall components of the capsular PS. The epitopes which were enhanced appeared to be different from those recognized by the modulating mAb. Negative modulations were also observed. Moreover, new epitopes seemed to be generated. In both cases the level and direction of the modulation were irrespective of isotypy and affinity of the mAbs. Interestingly the positive modulatory effect was found to be correlated with an in vitro assay based on the binding of immune complex to antigen-presenting cells.

An indirect immunofluorescence staining procedure for detection of human Fc gamma receptors on streptococci.

Fc gamma receptors on streptococci are usually revealed by hemagglutinating techniques (IgG coated red blood cells) or uptake of radiolabeled IgG. The results obtained with these methods are not always satisfactory. For this reason, we developed a technique involving indirect immunofluorescence staining. Bacterial smears were treated with human Fc gamma fragment and their binding to streptococcal Fc gamma receptors was revealed by a fluorescent F(ab')2 fragment of anti-human Fc gamma sheep antibodies purified on an IgG immunosorbent. These purified sheep F(ab')2 fragments did not contain any IgG nor Fc gamma as shown by SDS polyacrylamide gel electrophoresis. Under these conditions indirect immunofluorescence staining was a highly specific and sensitive method of detecting Fc gamma receptors on streptococci. Distribution of Fc gamma receptors was studied in 237 streptococcal strains of human origin belonging to groups A, B, C, D and G; these receptors were also looked for in 21 strains of alpha-hemolytic streptococci which did not possess the group carbohydrate and 12 strains of pneumococci. Fc gamma receptors were found only in group A, C and G streptococci, but all strains of these groups did not possess Fc gamma receptors.

Human secretory component. II. Easy detection of abnormal amounts of combined secretory component in human sera.

A simple method, allowing easy detection of abnormally increased sIgA levels is described. It consists in quantitation of combined SC by gel double diffusion, using appropriate anti-SC immune sera. The technical conditions, locating the threshold of sensitivity of precipitation at about 25 microgram/ml, a value higher than that found in normal sera, were established. Comparison with other classical methods (SRID, ELISA and IHA) emphasizes the validity and simplicity of the technique which has shown convenient whenever a large number of sera have to be tested.

A critical study of the use of staphylococci containing protein A for separation of IgG and IgM antibodies.

This study was to determine the best conditions for using staphylococci bearing protein A to separate IgG from IgM. The validity of the technique was evaluated for detection of IgM with antimicrobial activity and for typing monoclonal IgM. The results indicate that separation of IgG and IgM is not entirely satisfactory in normal sera and worse in hyperglobulinemic sera. The detection and titration of IgM antimicrobial antibodies (rubella and hepatitis B core (HBc) specific IgM) was unreliable because IgG was only partially absorbed by staphylococcal cells, while a significant portion of IgM was bound. The use of higher concentrations of staphylococci did not improve the results because the more IgG was absorbed, the more IgM was also bound. It is shown that with anti-HBc specific IgM the risk of misinterpretation is very high with a sensitive radioimmunoassay technique allowing detection of trace amounts of nonabsorbed IgG. In contrast staphylococcal protein A proved useful in typing monoclonal IgM.

A monoclonal antibody enzyme immunoassay for the detection of epitopes encoded by the pre-S2 region of the hepatitis B virus genome.

Pre-S2-coded sequences of the hepatitis B virus (HBV) represent important serological markers of HBV infection and elicit the antibodies essential for recovery from type B hepatitis. Monoclonal antibodies (McAbs) directed against two non-overlapping epitopes (pre-S2a and pre-S2b) of the pre-S2 protein of HBV were used to develop an enzyme immunosorbent assay (EIA). The assay was based on the solid-phase sandwich principle in which two different epitope-specific antibodies were used as immunadsorbents and as enzyme-labelled probes. The assay sensitivity was in the pg range and permitted precise quantitation of the pre-S2 sequences in sera. Using the 'site-specific' monoclonal assay we demonstrated that pre-S2a and pre-S2b epitopes are expressed on HBsAg particles of both ay and ad subtypes. The assay is the most sensitive currently available method for the detection of pre-S2 epitopes and may be used for routine immunodiagnosis of hepatitis B.

Both Fc alpha domains of human IgA are involved in in vitro interaction between secretory component and dimeric IgA.

The sites of interaction between 125I labelled human secretory component (SC) and dimeric IgA were located by studying the inhibitory effect of various antibodies to IgA. Several Fab' fragments were isolated from three sera of hyperimmunized rabbits. The specificity of these different antibody preparations, as determined by a RIA inhibition test or by ELISA, showed that two were directed against both domains of Fc alpha, two against C alpha 2, two against C alpha 3 and one against Fd alpha. A monoclonal antibody against C alpha 3 was also used. The results indicate that both the C alpha 2 and C alpha 3 domains are equally and independently involved in the interaction between SC and dimeric IgA.

Human secretory component. IV: Antigenic regions involved in in vitro binding to dimeric IgA.

The aim of this report was to identify the region(s) of the secretory component (SC) molecule involved in in vitro binding to dimeric IgA. Inhibition of the SC binding was tested by Fab' antibody fragments directed against the accessible (A) and inaccessible (I) regions of SC. Antibodies directed against the main 38.5-kDa trypsin fragment of SC, and antibodies from two immune sera with a wide anti-SC spectrum, were also used. The specificity and activity of the five antibody preparations were established by double-diffusion in gel and by their SIgA combining capacity. Inhibition curves were established by RIA using constant amounts of 125I SC, dimeric IgA and increasing quantities of the various Fab' antibodies. These results indicated involvement of a larger part than the I region of the SC molecule in combination with dimeric IgA, perhaps including a second (minor?) site of binding on the accessible parts in addition to the major region located on I.

IgM reassociation in the absence of J-chain.

Human monoclonal IgM pentamers with different biophysical properties (euglobulin, pseudoglobulin or cryoglobulin) were reduced and reassociated in the absence of J-chain. Reassembly occurred for 50-82% of the monomers. The reassociated molecules consisted of covalent oligomers and pentamers. The deficiency of J-chain (estimated to be less than 0.17% of normal) was shown by alkaline-urea overloaded gel electrophoresis followed by silver staining. The addition of exogenous J-chain, from polymeric IgA or IgM, did not significantly modify the reassembly ratio. Thus J-chain does not seem to be an absolute requirement for IgM polymerization.

Cervicovaginal anti-HIV antibodies in index women from HIV-discordant, exclusively heterosexual, couples.

Cervicovaginal IgA and IgG anti-gp160 antibodies were evaluated in cervicovaginal secretions from twelve HIV-discordant heterosexual couples, matched with twelve HIV-concordant heterosexual couples, at similar stage of HIV disease. The mean reciprocal end-point titers of cervicovaginal IgA or IgG to gp160 were similar in cases and in controls. These observations suggest that cervicovaginal antibodies to HIV do not appear as biological indicators sufficiently relevant to explain a possible reduced infectivity of the female index case in HIV-discordant couples, by comparison with HIV-concordant couples.

Local synthesis of IgG antibodies to HIV within the female and male genital tracts during asymptomatic and pre-AIDS stages of HIV infection.

Paired sera and cervicovaginal secretions or seminal fluids, obtained from HIV-1-infected, clinically asymptomatic women (n = 41) and men (n = 12), were investigated in order to test the hypothesis of a local synthesis of IgG to HIV in the female and male reproductive tracts. Anti-gp41 + p24 IgG was evaluated by an IgG immunocapture assay, and anti-gp160 IgG by an indirect ELISA. Estimation of anti-HIV IgG-specific activities was carried out after ponderal determination of total IgG and evaluation of anti-HIV IgG activity. IgG to gp41 + p24, as well as IgG to gp160, were specifically detected in all sera, cervicovaginal secretions, and seminal fluid samples from all tested HIV-1-infected subjects. The mean specific activities of IgG to gp41 + p24 in cervicovaginal secretions and in seminal fluids were about 33-fold (in women) and 16-fold (in men) that of the corresponding sera; similarly, the mean specific activities of IgG to gp160 in genital secretions were about 17-fold (in women) and 10-fold (in men) that of the corresponding sera. IgGs to HIV are constantly detected in genital secretions from HIV-1-infected subjects, and appear to be largely synthesized in situ within the genital tract of both genders.

High-level ability of secretory IgA to block HIV type 1 transcytosis: contrasting secretory IgA and IgG responses to glycoprotein 160.

The IgG and secretory IgA (S-IgA) responses to the HIV-1 envelope (gp160 antigen) were analyzed in the colostrum (Col) and in the cervicovaginal fluid (CVF) of HIV-l-infected women. We show IgG antibodies (Abs) to the recombinant gp160 to be predominant as compared with the corresponding S-IgA isotype. The low level of the S-IgA response cannot be related to a general disturbance of the mucosal-associated Iymphoid tissue (MALT) because the level of a current Ab to a caries-associated antigen from Streptococcus sobrinus was in the normal range in these secretions. The major subclass of IgA to gp160 was of the alpha1 isotype both in Col and in CVF. However, the specific activities of S-IgA1 and S-IgA2 were different when expressed as the ratio of the anti-gp160 related to total Ig of each subclass. Indeed, the specific activity of the S-IgA2 was predominant over S-IgA1 in the Col, whereas the reciprocal results were found in CVF, showing a subcompartmentalization of these secretions. The ability of S-IgA and IgG to block one of the pathways involved in the HIV-1 penetration across mucosa, i.e., transcytosis through epithelial cells, was evaluated using a functional in vitro assay. Both S-IgA and IgG Abs impaired virus transcytosis, irrespective of the level of antigp160 specific activities. However, specific S-IgA was more efficient than IgG. These features suggest that mucosal specific S-IgA to HIV-1 could be relevant in decreasing infectivity of HIV-1 in corporal fluids.