Simultaneous determination of polyamines, N-acetylated polyamines and the polyamine analogues BE-3-3-3 and BE-4-4-4-4 by capillary gas chromatography with nitrogen-phosphorus detection

IgM paraproteins from patients with CANOMAD (chronic ataxic neuropathy, ophthalmoplegia, M-protein, agglutination, anti-disialosyl antibodies) react with NeuAc(alpha 2-8)NeuAc epitopes on a wide range of gangliosides including GQ1b, GT1a, GD1b and GD3. The tissue distribution of reactive antigens in human peripheral nerve has not been addressed in detail. In addition, the origin of these antibodies is unknown. Here we report that purified anti-disialosyl paraproteins from two affected patients bind a wide array of human peripheral nerve structures including dorsal root ganglia, dorsal and ventral root axons, femoral and oculomotor nerves. We also show that these paraproteins bind lipopolysaccharides of Campylobacter jejuni isolates from 3/3 cases of Miller Fisher syndrome, and to a less frequent extent, from cases of Guillain-Barré syndrome and enteritis controls. In conjunction with our previous studies, these data provide a possible causal link between the origin and pathogenic effects of anti-disialosyl antibodies in human paraproteinaemic neuropathy.     

Measurements of colour constancy by using a forced-choice matching technique

In order to compare the reactivity of glutamic acid decarboxylase (GAD) antibodies recognizing linear and conformational epitopes as islet cell cytoplasmic antibodies (ICA), monoclonal antibodies were generated. An ELISA displacement test using two biotinylated monoclonals recognizing a linear (M61/7E11) or a conformational GAD65 epitope (M65/6B12) was performed to identify epitope regions recognized by monoclonal GAD antibodies. The GAD binding by monoclonal GAD antibodies was tested by immunofluorescence on fixed and unfixed pancreatic sections of human, rat, and mouse, and by Dot-blot experiments. 16/23 (69.6%) of the monoclonals were specifically reactive with GAD65 and 7/23 (30.4%) were reactive with both GAD isoforms. 8/16 (50%) of monoclonal GAD65 antibodies recognized a linear GAD epitope located at the N-terminus (pattern 1). 5/16 (31.3%) displaced M65/6B12, indicating the recognition of a conformational GAD epitope (pattern 2). Monoclonals belonging to patterns 1 and 2 showed strong ICA binding. 3/16 (18.8%) of monoclonals specific for GAD65 with weak or no immunostaining of pancreatic islets (pattern 3) did not inhibit the binding of both biotinylated antibodies in the displacement test, indicating other epitope specificities. In conclusion, GAD antibodies recognizing both conformational and linear epitopes of the GAD65 molecule are involved in ICA binding with strong reactivity. Furthermore, results obtained with monoclonals of pattern 3 suggest the occurrence of GAD65 epitopes partly inaccessible on cryosections, which may result in an ICA-negative test of GAD65 autoantibody positive sera.     

Serologic responses of young colostrum fed dairy calves to antigens of Pasteurella haemolytica A1

The potential and limitations of early calfhood vaccination to induce active immunity to Pasteurella haemolytica A1 in conventional colostrum fed calves were investigated. Holstein dairy calves (n = 29) were vaccinated at 2 and 4 weeks of age, or at 6 and 8 weeks of age with a commercial culture supernatant vaccine (Presponse, Langford Inc., Guelph, Ont., Canada), or remained unvaccinated as controls. Serum antibody titres were measured using an indirect bacterial agglutination assay, a leukotoxin neutralization assay, and enzyme immunoassays for antibodies of the IgM, IgG1, and IgG2 isotypes binding purified capsular polysaccharide of P. haemolytica A1. Seroconversion (fourfold or greater increase in serum antibody titre) rates were compared using Fisher's exact test. The effects of passive antibody titres and age on response to vaccination were assessed by linear modelling. Vaccination at 2 and 4 weeks of age was associated with 40%, and 0% of calves seroconverting on the basis of agglutinating antibody titres, and leukotoxin neutralizing titres respectively, and 50%, 0%, and 0% seroconverting on the basis of IgM, IgG1 and IgG2 antibodies to capsular polysaccharide, respectively. Agglutinating antibody responses were not related to prevaccination antibody titres, or to age at vaccination. Higher responses (p = 0.08) to leukotoxin were observed in older calves (after taking differences in prevaccination titres into account). Statistical analyses of responses to capsular polysaccharide among calves with comparable prevaccination IgG1 antibody titres revealed significantly higher IgM, IgG1 and IgG2 responses in older calves. Rising titres of IgM antibodies in nonvaccinated calves after 5 weeks of age suggest natural exposure to P. haemolytica A1 or antigens which result in serologic cross-reactions as a means of priming immune responses.     

Immunochemical characterization of anti-H monoclonal antibodies obtained from a mouse immunized with human saliva

Three IgM class anti-H monoclonal antibodies (1E3, 1E5 and 3H1) were obtained from a BALB/c mouse immunized with human O type saliva. These antibodies were found to agglutinate red cells from O group and A and B subgroups but not from Bombay and para-Bombay individuals whose H antigen was barely detected by anti-H reagents. The agglutination reactions of these antibodies were inhibited by H antigens from human tissues. It was also demonstrated that both 1E3 and 3H1 reacted with H disaccharide (Fuc alpha 1-->2Gal beta), H type 1 (Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta), H type 2 (Fuc alpha 1-->2Gal beta 1-->4GlcNAc beta), H type 3 (Fuc alpha 1-->2Gal beta 1-->3GalNAc alpha) and H type 4 (Fuc alpha 1-->2Gal beta 1-->3GalNAc beta) but not with Lea (Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc beta), Leb (Fuc alpha 1-->2Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc beta), X (Gal beta 1-->4[Fuc alpha-->3]GlcNAc beta) or Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta). On the other hand, 1E5 was found to react with H type 1, H type 2, Leb and Y. Because of the unique reactivities against various fucosyl linkages these monoclonal antibodies could be useful not only as anti-H reagents but also as reagents for the structural analysis of fucosylated glycoconjugates.     

Molecular basis for the interaction between human IgM and staphylococcal protein A

To examine the relationship between VH gene usage and reactivity of immunoglobulins, we cloned B cells from peripheral blood from adults and from human neonatal cord blood by EBV transformation. Nearly one-third of the B cell clones from both sources produced IgM reactive with staphylococcal protein A (SPA). None of such IgM reacted with other antigens, except for the crude extract of Staphylococcus aureus. All of 22 B cell clones producing IgM reactive with SPA expressed VH3 genes, while none of the control 15 clones secreting IgM nonreactive with SPA expressed VH3. The IgM proteins reactive with SPA could be clearly divided into two subjects based on the differential binding avidity to solid-phase SPA. Both kappa and lambda light chains were used in each subset of SPA-reactive IgM. Sequence analysis of the PCR products from seven VH3-IgM clones revealed that the VH3 genes were used in nearly germline configuration. The D and J gene usage was diverse. Comparison of amino acid sequences between antibodies with high and low avidity to SPA suggests that the differential avidity is related to amino acid sequence differences in the complementarity determining region 2 and framework region 3. The high frequencies of B cells committed to the production of SPA-reactive IgM in normal blood and the restricted use of VH3 heavy chain genes in nearly germline configuration in these cells support the notion that SPA behaves like a superantigen toward human B cells.     

Alterations in calcium antagonist receptors and calcium content in senescent brain and attenuation by nimodipine and nicardipine

Characteristics of L- and N-type calcium (Ca++) channel antagonist receptors in brains of senescence-accelerated prone mouse (SAMP8) showing age-related deterioration of learning and memory were examined by using (+)-[3H]PN 200-110 and [125I]omega-conotoxin GVIA as radioligands. There was a tendency toward consistent decrease in Bmax for both radioligands in seven brain regions of SAMP8 compared with the control mouse. The reduction in (+)-[3H]Pn 200-110 binding sites was statistically significant in the hippocampus, midbrain and pons/medulla oblongata, and that in [125I]omega-conotoxin binding sites was significant in the cerebral cortex, corpus striatum and pons/medulla oblongata. On the other hand, there was a marked elevation in Ca++ content in the brain of SAMP8. Chronic p.o. administration (0.3, 1 and 3 mg/kg/day for 3 weeks) of nimodipine and nicardipine to SAMP8 caused a significant increase in the Bmax values of (+)-[3H]PN 200-110 binding in the cerebral cortex and hippocampus. This may reflect up-regulation of brain Ca++ channel antagonist receptors as a result of the prolonged blockade by nimodipine and nicardipine. On the other hand, similar administration of amlodipine and nilvadipine failed to produce an enhancement of Bmax values of (+)-[3H]PN 200-110 binding, whereas both drugs at high doses evoked a significant increase in the apparent dissociation constant. Furthermore, the brain Ca++ content in SAMP8 was markedly reduced by chronic p.o. administration of Ca++ channel antagonists, and the decrease was equivalently observed for all of four 1,4-dihydropyridine antagonists in spite of the difference in the effect on brain receptors. In conclusion, the present study suggests that there is an altered Ca++ homeostasis in the SAMP8 brain that is effectively attenuated by chronic administration of nimodipine and nicardipine. Hence SAMP8 may be a suitable animal model for evaluating the therapeutic effects of Ca++ channel antagonists on neurological disorders associated with the aging brain.     

Arthroscopy of the upper ankle joint. List of indications from the literature--realistic expectations--complications

PROBLEM: Antiphospholipid antibodies (APLs) consist of very heterogenous autoantibodies. It has not been fully explored what kind of specificities are most relevant to recurrent pregnancy loss. Thus, we investigated the effects of specific APLs on recurrent aborters. METHOD: IgG and IgM antibodies against PE (treated with 1% acetic acid) and five negatively-charged phospholipids were measured by ELISA among 334 recurrent aborters without autoimmune disease. The relationships between APL specificities and subsequent pregnancy outcome were prospectively investigated in 38 recurrent aborters with positive APL who did not receive treatment with prednisolone and aspirin. Antibody levels exceeding the 99th percentile of 280 healthy women were considered positive. RESULTS: Positive IgG and/or IgM APLs were detected in 14%, IgG APLs in 12%, and IgG antibodies against PA, PG, PI, PS, CL and PE, respectively, in 9%, 7%, 7%, 7%, 8%, and 8%. In a prospective study of the 38 untreated patients, fetal loss recurred in 82% of the 33 IgG APL-positive patients, but in 40% of the five patients positive for only IgM APLs. The incidence of fetal loss in the next pregnancy of patients with IgG specific APL-positive against PE, PI, PS, or Cl was even higher at 90% and over, and fetal loss recurred in all of 21 patients with two or more IgG APL-positive against PE, PI, PS, or CL. CONCLUSION: These results suggest the possibility that two or more IgG APL-positive value against treated PE, PI, PS, or CL, may be more accurate as a predictive variable than that of only one IgG APL-positive in patients with recurrent pregnancy loss.     

Autoradiographic evidence that zona glomerulosa and capsular vessels of the human adrenal cortex are provided with different subtypes of adrenomedullin receptors

Frozen sections of normal adrenal glands, obtained from patients undergoing unilateral nephrectomy for kidney cancer, were labeled in vitro with human [125I]ADM(1-52). Autoradiography and quantitative densitometry showed the presence of abundant ADM(1-52) binding sites in both zona glomerulosa (ZG) and capsular vessels, which were displaced with about the same efficiency by cold ADM(1-52) and rat ADM(1-50). The selective calcitonin gene-related peptide type 1 (CGRPI) ligand CGRP(8-37) eliminated, although less efficiently than ADMs, [125I]ADM(1-52) binding in the ZG, but not in the capsular vessels. These findings suggest the existence of different receptor subtypes for ADM in the human adrenal cortex. The CGRP(8-37)-sensitive receptors located in the ZG may mediate the well-known inhibitory effect of ADM on aldosterone secretion, while the CGRP(8-37)-insensitive receptors present in the capsular vessel may be involved in the ADM-induced rise in adrenal blood flow.     

An analysis of the central pathway of vestibulo-sympathetic responses

The sera of 159 patients with monoclonal gammopathies were examined for the presence of anti-thyroglobulin (Tg) activity. An enzyme-linked immunosorbent assay was employed. Thirty-one (19.5%) sera were found to bind Tg. The activity against Tg was further confirmed by using purified immunoglobulins and employing competition assays. The anti-Tg antibodies were found in the sera of patients with IgG, IgM and IgA gammopathies. Anti-Tg antibodies were more frequent among patients with IgG gammopathy. Autoantibodies to Tg are found in patients with Hashimoto's thyroiditis, Graves' disease and occasionally in patients with thyroid carcinoma. Natural autoantibodies directed against human Tg have been detected, as well, in healthy subjects. None of the patients in the present study whose serum was found to contain high titers of anti-Tg human monoclonal antibodies had any clinical or biochemical evidence of thyroid disease. Our results of a high incidence of anti-Tg activity in the sera of patients with monoclonal gammopathies support previous reports of autoantibody properties characteristic of these immunoglobulins.     

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists

1. The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). 2. The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3. The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5-26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 microM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7-13.9 fold, whereas the affinity of DPCPX was unaffected. 4. The affinity of six xanthine-based adenosine receptor antagonists was 2.2-15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5. Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido ade nosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.     

Different anticoagulant and immunological properties of anti-prothrombin antibodies in patients with antiphospholipid antibodies

Lupus anticoagulant (LA) antibodies are acquired inhibitors of coagulation belonging-together with anticardiolipid (aCL) antibodies-to the family of antiphospholipid antibodies. Since LA antibodies affect coagulation reactions via recognition of the complex of lipid-bound prothrombin, they may be better named anti-prothrombin antibodies. We studied their immunological properties in the plasma of 59 patients with antiphospholipid antibodies by means of specific ELISA systems that allowed the characterization of the interaction of these antibodies with human prothrombin and anionic phospholipids. The mode of presentation of prothrombin was found to greatly influence the reactivity of anti-prothrombin antibodies. In fact, when plain polystyrene plates were used to immobilize prothrombin, virtually no binding was observed. Conversely, when prothrombin was coated on high-activated PVC ELISA plates, 34 samples (58%) contained antibodies that recognize human prothrombin in solid phase. In particular, IgG antibodies were found in 21 plasmas and IgM in 22; both IgG and IgM isotypes were present in 9 of these cases. A higher prevalence was observed in the ELISA for the detection of the antibodies directed at the calcium-mediated complex of phosphatidylserine (PS)-bound prothrombin: 53 samples (90%), preadsorbed with cardiolipin liposomes to remove aCL antibodies, showed the presence of IgG and/or IgM anti-prothrombin antibodies. When the results were analyzed according to the immunoglobulin isotypes, 44 (75%) and 39 (66%) samples were found to contain IgG and IgM anti-prothrombin antibodies, respectively. Both IgG and IgM were present in the plasma of 30 patients. Only half of these samples reacted also with PVC-bound prothrombin. Apparently, the higher rate of positivity of the ELISA for the detection of antibodies to the complex of PS-bound prothrombin was not due to differences in the amount of antigen available in the 2 systems, as judged by binding experiments performed with a rabbit polyclonal anti-human prothrombin antiserum. Finally, the anticoagulant properties of 14 total IgG preparations (12 of them contained anti-prothrombin antibodies positive in both ELISA systems, whereas the other 2 cases reacted either with PVC-bound prothrombin only or with PS-bound prothrombin only) were evaluated by diluted Russell's Viper Venom Time and by diluted activated Partial Thromboplastin Time. To rule out the beta 2-glycoprotein I (beta 2-GPI)-dependent anticoagulant effect of the aCL antibodies contained in the preparations, the coagulation tests were performed in beta 2-GPI deficient plasma. Six preparations failed to show anticoagulant activity in both assay systems, suggesting that 2 types of IgG anti-prothrombin antibodies exist, that differ with respect to their anticoagulant properties. These findings suggest that anti-prothrombin antibodies resemble aCL antibodies with respect to the behaviour in "in vitro" coagulation reactions and underline the wide heterogeneity of antiphospholipid antibodies.     

Antineuronal antibodies in acute cerebellar ataxia following Epstein-Barr virus infection

A 29-year-old man developed acute cerebellar ataxia following Epstein-Barr virus infection. Serum IgG and IgM antibodies reacted with both nuclear and cytoplasmic elements of neurons. Western blot revealed IgG binding to the 34- and 29-kd bands and IgM binding to the 44-, 37-, and 29-kd bands. The IgM reactivity gradually reduced. There was no identifiable neoplasm and the ataxia gradually improved. These findings suggest a role for autoimmune mechanisms in the pathogenesis of acute cerebellar ataxia.     

Decrease of hormone binding capacity of estrogen receptor by calcium

We have addressed the question as to whether calcium may modify the [3H]estradiol ([3H]E2) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues. Ca++ (0.1-10 mM) always produced an important loss of [3H]E2 binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [125I]Z-17 alpha-(2-iodovinyl)-11 beta-chloromethyl estradiol-17 beta (an compound with very high selectivity for ER) as well as [125I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca(++)-induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [3H]E2 binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca++ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca++ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.     

Murine immune responses to Neisseria meningitidis group C capsular polysaccharide and a thymus-dependent toxoid conjugate vaccine

The polysaccharide (PS) capsules of many pathogenic bacteria are poor immunogens in infants and young children as a result of the delayed response to PS antigens during ontogeny. The development of polysaccharide-protein conjugate vaccines for Haemophilus influenzae type b, which have proven to be efficacious in this age group, has led to active development by a number of investigators of conjugate vaccines for other diseases. We describe here the response of several mouse strains to the capsular PS of Neisseria meningitidis group C (MCPS) conjugated to tetanus toxoid (MCPS-TT) and the same response in BALB/c mice as a model of the immune consequences of conjugate vaccine immunization. The use of a conjugate vaccine results in a shift in the isotype elicited in response to the MCPS, from immunoglobulin M (IgM) and IgG3 to primarily IgG1. A response to MCPS-TT is seen even among mouse strains which respond poorly to MCPS itself, emphasizing the importance of a strain survey when choosing a mouse model for a vaccine. The marked increase in IgG1 antibody titer was accompanied by a large increase in bactericidal activity of sera from these animals. Animals primed with the conjugate vaccine demonstrated a booster response after secondary immunization with either the MCPS or the conjugate. The ability to produce a boosted IgG1 anti-MCPS response to the MCPS can be transferred to adoptive recipients by B cells alone from mice primed with MCPS-TT but not mice primed with MCPS alone. These data indicate that in BALB/c mice a single immunization with MCPS-TT is sufficient to induce a shift to IgG1 and generate a memory B-cell population that does not require T cells for boosting.     

On the high affinity binding site for [3H]-1,3-dipropyl-8-cyclopentylxanthine in frog brain membranes

1. Radioligand binding properties of the adenosine receptor ligands, [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), and [3H]-R-phenylisopropyladenosine ([3H]-R-PIA) were investigated in frog brain membranes. 2. The specific binding of the adenosine antagonist, [3H]-DPCPX to frog brain membranes showed one binding site with Kd and Bmax values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 microM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [3H]-DPCPX (40 nM) binding to frog brain membranes. 3. [3H]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the Kd value of [3H]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. Bmax values were not significantly (P > 0.05) modified (0.261 +/- 0.018 pmol mg-1 protein, with Mg2+, and 0.266 +/- 0.026 pmol mg-1 protein, in presence of GTP) by the presence of Mg2+ or GTP. 4. The specific binding of [3H]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 microM) and increased to 123 +/- 4% by Mg2+ (8 mM). [3H]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with Kd and Bmax values of 0.9 nM and 0.229 +/- 0.008 pmol mg-1 of protein, respectively. 5. The concentration-inhibition curves of adenosine agonists and antagonists versus [3H]-DPCPX binding showed the following order of potencies: CPA> R-PIA~ NECA> S-PIA> > CGS 21680, for the agonists, and XAC ~-DPCPX> > XCC> PACPX, for the antagonists.6. The present results suggest that the adenosine binding site in the frog brain membranes is G-protein coupled, but that the antagonist affinities and the pharmacological profile is different from the Al or A2 adenosine receptors.     

CT findings in neonatal hypothermia

PROBLEM: Antiphospholipid antibodies (APAs) are important in the etiology of reproductive failure. Studies have shown that binding proteins are necessary for the detection of APAs. One of these, beta 2-glycoprotein, has been shown to be necessary for detection of anticardiolipin antibodies. It is felt that some APAs may be directed to the binding protein itself, or to a combination of the binding protein and phospholipid. METHOD OF STUDY: In this study, a comparison of APAs vs. anti beta 2-glycoprotein antibodies was performed on the sera of 123 women younger than 40 years of age with a history of reproductive failure. Antibodies to six phospholipid epitopes, cardiolipin, phosphatidyle-thanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and phosphatidylserine, were measured. RESULTS: Of the 123 women tested, 33 had one or more positive immunoglobulin (Ig)G antibodies to phospholipids, of which 9 were to cardiolipin. However, only 1 of 123 women had IgG antibodies to beta 2-glycoprotein and she was APA negative. Thirty-eight of 123 women had one or more IgM antibodies to phospholipids, with none directed to cardiolipin IgM. In contrast, only 8 of the 123 women had IgM antibodies to beta 2-glycoprotein. Five of the eight patients had IgM APA; 4 of 5 had IgM antibodies to PE, 1 to PI. CONCLUSIONS: There is no correlation between beta 2-glycoprotein antibodies and APA status in this population. To date, our most sensitive test for detecting phospholipid autoimmune-mediated in vitro fertilization failure still appears to be the ELISA assay for APA.     

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

In order to identify the individual contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1Pg] ([D139-->N]r-Pg), [K4Pg] ([D413-->N]r-Pg), and [K5Pg] ([D515--N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu1]Pg and, to a lesser extent, the [K5Pg] mutant, [D518-->N/Glu1]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C50) occurred at 3.3 mM for wtr-[Glu1]Pg and at 0.7 mM for [D518-->N/Glu1]r-Pg. Subsequent inhibition by EACA occurred with a C50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during the activation. Similar initial activation rates of both [D139-->N]r-Pg and [D413N]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C50 for this process that was similar to wtr-[Glu1]Pg and [D518-->N/Glu1]r-Pg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative assessment of IgM antibodies towards an immunodominant B-cell epitope within the preS2 domain of HBV in the natural course and during combined prednisone/interferon alpha 2b treatment of chronic hepatitis B virus infection

A direct binding enzyme-linked immunosorbent assay (ELISA) was established for quantitative determination of serum IgM antibodies towards a synthetic peptide corresponding to a selected segment (14-21) of the preS2-gene product containing an immunodominant linear B-cell epitope. The prevalence of IgM anti-preS2 (14-21) antibody titers > 1,000 for hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B virus (HBV) infection was 38% (22/58) and 10% (2/21) for HBeAg-negative subjects (P < 0.005). IgM anti-preS2 (14-21) reactivity was detected during the clinical course of chronic HBV infection and IgM anti-peptide antibody titers declined and disappeared before spontaneous HBe/anti-HBe seroconversion. Recombinant interferon (IFN)-alpha 2b with an antecedent short course of corticosteroids was administered to eight Chinese patients with chronic HBV infection. The IgM anti-preS2 (14-21) reactivity was monitored consecutively during treatment and patients were followed for more than 1 year. A close association between the presence of pretreatment IgM anti-preS2 (14-21) in serum and the capacity to respond favorably to the combined prednisone/IFN-alpha 2b therapy was detected. The IgM anti-preS2 (14-21) titers decreased during treatment with subsequent loss of detectable antibodies 8-16 weeks after the initiation of therapy. This decrease was concomitant with an alanine aminotransferase (ALT) augmentation preceding the disappearance of HBV-DNA and anti-HBe seroconversion. Long-term remission was not observed in treated patients who lacked detectable levels of pretreatment IgM anti-preS2 (14-21) in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Of four murine, anti-Shigella dysenteriae type 1 O-polysaccharide antibodies, three employ V-genes that differ extensively from those of the fourth

Three murine, monoclonal antibodies, IgM 5286 F2, IgM 5297 C1, and IgG 5338 H4 were generated against Shigella dysenteriae type 1 O-specific polysaccharide (O-SP)-conjugate. They are specific for the O-SP, which is a poly-[alpha-L-rhamnopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-al pha-D-galactopyranosyl-(1-->3)-2-deoxy-2-amino-N-acetyl-alpha-D-glucopyr anosyl]. The VH and VL genes of these antibodies were cloned and their sequences determined. They showed 93% homology, but were quite different to the primary sequence of IgM 3707 E9, of the same O-SP-specificity, previously reported. The fine-specificities of both IgG 5338 H4 and IgM 3707 E9 were for the same disaccharide moiety in the O-SP, while IgMs 5286 F2 and 5297 C1 showed fine-specificity for the entire repeating unit of the O-SP. Therefore, divergent sequences can confer upon antibodies similar-, or even identical-carbohydrate-epitope fine-specificity. In addition, close primary sequence-homology does not preclude differences in antibody fine-specificity.     

Immunologic epitope, gene, and immunity involved in pneumococcal glycoconjugate

Pneumococcal infection persists as a major cause of pneumonia, otitis media, and meningitis in infants. Children less than 2 years of age show the highest incidence of pneumococcal diseases. Production of monoclonal antibody (MAb) to polysaccharide (PS) and binding characteristics to PS epitopes were studied. Removal of the O-acetyl group from 9V PS by alkali hydrolysis resulted in a decreased binding with rabbit 9V antiserum (AS). However, the binding reaction with 9V MAb was less affected by the loss of O-acetyl content. Type 9V IgG MAb provided passive protection and enhanced the opsonophagocytic activity of polymorphonuclear (PMN) leukocytes to kill type 9V pneumococci. The pathogenecity of pneumococci is attributed to various virulence factors distributed on the cell surface, including capsular polysaccharide and protein antigens, for example, pneumolysin, autolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesion (PsaA), and hemin binding protein. Some of these protein antigens may be used as a component to combine with pneumococcal PS vaccine or as a carrier of conjugate vaccine. Clinical trials of pneumococcal conjugate vaccines showed that covalent linkage of capsular PS to protein carriers improved the immunogenicity of the PS. Development of glycoconjugate vaccine for selected pneumococcal types will help solve the problem of poor immunogenecity of PS vaccine in young children used for prevention of pneumococcal infection.