Comparison of the direct platelet immunofluorescence test (direct PIFT) with a modified direct monoclonal antibody-specific immobilization of platelet antigens (direct MAIPA) in detection of platelet-associated IgG

Glycoprotein (GP)-specific platelet-associated IgG (PA-IgG) may be demonstrable in autoimmune-mediated thrombocytopenia. We studied 159 consecutive patients with histories of thrombocytopenia by a modified direct monoclonal antibody-specific immobilization of platelet antigens (direct MAIPA) assay, which immobilizes GP IIb/ IIIa, GP Ib/IX and GP Ia/IIa simultaneously. This modification requires smaller quantities of platelets than standard measurements performed separately. PA-IgG was present in 84/159 (53%) patients, as shown by the direct platelet immunofluorescence test (PIFT) with flow cytometry as a reference. PA-IgG against GP IIb/IIIa and/or GP Ib/IX and/or GP Ia/IIa was noted in 46 patients (29%), of whom 93% (43/46)-were also PA-IgG positive. The amount of PA-IgG detected by PIFT correlated directly with that detected by direct MAIPA (r = 0.71; P < 0.001). Only three patients 12548 with negative direct PIFT had GP-specific PA-IgG. GPV-specific PA-IgG was detected in 13 (10%) of the 125 patients, in whom further studies could be performed. In the subgroup of patients with GP-specific PA-IgG, the median fluorescence intensities of direct PIFT were higher than in patients with no GP-specific PA-IgG (P < 0.001). Direct PIFT and direct MAIPA divided the patients into asymmetric subgroups. However, the relative roles of these tests in the diagnosis of autoimmune-mediated thrombocytopenia await further studies.     

Isolation and characterization of free form prostate specific antigen (f-PSA) in sera of men with prostate cancer

PURPOSE: The free uncomplexed form of prostate specific antigen (f-PSA) from prostate cancer sera was partially isolated and characterized because the molecular form of f-PSA in the serum is unknown. MATERIALS AND METHODS: 230 ml. of sera from 59 men with bone metastasis and individual PSA values of >2000 ng./mL were combined and centrifuged for 60 minutes at 30,000 RPM (4C). The sera were fractionated by gel filtration column chromatography (Sephacryl S-200, 2.5 cm. x 92 cm.). Free and complexed PSA in the eluted fractions were isolated by measuring immunoreactivity of PSA (Tosoh AIA-600 assay); f-PSA from 23 separate runs were combined, concentrated and re-chromatographed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to immobilize the isolated proteins onto a nitrocellulose membrane and a polyvinylidene difluoride (PVDF) membrane. Monoclonal antibody (F5) was used to probe PSA on nitrocellulose membrane and the PSA band was detected by Emission Chemoluminescence (ECL) kit. Amino terminal sequence analysis of the isolated f-PSA was performed with a gas-phase sequentor (Applied Biosyntens 4760 A) using the program designed by the manufacturer. RESULTS: 0.5 cc of f-PSA (27,000 ng./mL) was obtained from serums after rechromatography. SDS-PAGE showed one double band around 30 kDa; with ECL technique, one major band at 30-kDa was identified as PSA. The amino terminal sequence analysis of this band showed residue 1 through 9 and 146 through 152. CONCLUSIONS: In our preliminary experiment, the free form of serum PSA is partially isolated directly from human sera. Amino terminal sequence analysis has shown that serum f-PSA is not a pre-mature or zymogen form of PSA because serum f-PSA has a N-terminus identical to that of seminal fluid PSA. A nicked form of f-PSA is also found in these patient sera.     

Normal human epidermal keratinocytes express in vitro specific molecular forms of (pro)filaggrin recognized by rheumatoid arthritis-associated antifilaggrin autoantibodies

We describe a series of molecular dynamics simulations performed on a model of charged lipid bilayer (dipalmitoylphosphatidylserine) and water, in presence of sodium and lithium ions, with an atomic detail. The structure of the lipid membranes was strongly affected by the presence of lithium, as manifested by the observation of a transition from a disordered to a gel state. Concerning the mechanism of such a transition, it was associated to the dehydration that we detected in the lipid-water interface in the presence of lithium. This dehydration introduced an increase in the lipid-lipid interactions, and as a consequence, a diminution of the disorder of the membrane. When both types of ions are present in the aqueous phase, lithium shown a special affinity for the lipid membrane displacing almost all the sodium ions toward the middle of the water layer. As a result, we observed remarkable differences in the atom and electric field distributions across the lipid membrane. Concerning the diffusion and orientation of water molecules across the lipid-water interface, we also observed a strong dependency of the type ion. On the other hand, the mobility and the hydration shell of lithium and sodium ions are strongly perturbed by the presence of the charged lipid bilayer. The lipid layer was responsible for a dehydration of the ions compared to bulk water. This dehydration was compensated by an increase of coordination number of the ions with the lipid oxygens. Also, the residence times of water in the first hydration shell of lithium and sodium ions are perturbed by the presence of the lipid membrane.     

p53 autoantibodies in the sera, cyst and ascitic fluids of patients with ovarian cancer

LY231514 is a novel antifolate that principally inhibits thymidylate synthase, but with additional folate-dependent enzyme targets. A Phase I study of single-agent LY231514 administered as a daily i.v. infusion over 10 minutes for 5 days, repeated every 3 weeks, was conducted to evaluate the maximum tolerated dose, pharmacokinetic profile, and antitumor activity of the drug using this schedule. Thirty-eight patients with advanced malignancies that were refractory or not amenable to standard therapy were treated with a total of 116 courses of LY231514, escalating treatment doses through 10 dose levels, from 0.2-5.2 mg/m2/day. No objective clinical responses were observed, although minor antitumor activity not fulfilling the response criteria was seen in three patients. A maximum tolerated dose of 4.0 mg/m2/day was determined, with neutropenia as the predominant dose-limiting toxicity. Reversible disturbances of liver biochemistry, fulfilling the protocol definitions of dose-limiting toxicity, were also observed. Other toxicities included diarrhea, mucositis, skin rash, and fatigue. Pharmacokinetic studies were performed at all treatment levels. Analysis showed a linear relation between administered dose and both maximum plasma concentration (Cmax) and area under the plasma concentration/time curve. The drug was cleared with a day 1 total body clearance of 108.9 +/- 38.8 ml/min/m2, with plasma concentrations declining with a mean harmonic terminal half-life of 1.4 +/- 0.98 h. When given by this schedule, LY231514 is tolerable, and Phase II studies are in progress.     

Reactions of anti-immunoglobulin sera with synthetic T cell receptor peptides: implications for the three-dimensional structure and function of the TCR beta chain

The derived amino acid sequence of the human TCR beta chain shows considerable homology to lg lambda light chains in its variable (V) and constant (C) domains, and in its joining segment (J). We assessed the cross-reactivity between TCR beta chains and lg light chains by synthesizing a set of nested, overlapping 16-mer peptides that duplicated the sequence that corresponds to the continuous VDJC sequence of TCR beta chain and determining the capacity of rabbit antisera to human or murine lgs to react with these peptides. The reactivities we observed were consistent with homologies to lambda and kappa light chains. The strongest reactivity in ELISA binding and competitive inhibition was with a peptide that corresponds to the 'switch peptide' of light chains. The sequence is encoded by the C-terminal region of the J segment (Fr4) and the N-terminus of the C region. Other regions reactive with anti-light chain sera corresponded respectively to CDR1 and Fr3 segments of the V region, and a segment of the constant region predicted to loop out of the tight globular structure. The peptide immunochemical results, coupled with the identification of specific regions of sequence correspondence between TCR beta and the characterized lambda light chain Mcg, allowed us to develop a three-dimensional model of the beta chain consistent with its role in antigen recognition and response to superantigens.     

Selectivity of recognition of variable (V) regions of autoantibodies by intravenous immunoglobulin (IVIg)

In the present study, we demonstrate that intravenous immunoglobulin (IVIg) is capable of binding to variable (V) regions of anti-endothelial cell antibodies (AECA) of healthy donors and patients with systemic lupus erythematosus (SLE). Among V regions of AECAs, IVIg selectively recognized certain idiotypes expressed by the autoantibodies of a given individual, in the case of both natural and SLE-associated AECAs. These observations provide new and direct evidence that IVIg interacts idiotypically with V regions of autoantibodies and that the efficacy of such interaction depends on individual autoantibody specificity. Our findings may be relevant for the understanding of the mechanisms that control expression of natural autoantibody activity in serum and for that of the differences in response to IVIg therapy that are seen between patients with autoimmune disease.     

Specificities of anti-neutrophil autoantibodies in patients with rheumatoid arthritis (RA)

Phosphatidylcholine-phospholipase D has been proposed to play a key role in the transduction of the proliferative responses of a wide range of mitogens and growth factors. We now report that the antigen receptors on T lymphocytes derived from human tonsillar or murine splenic preparations are coupled to phosphatidylcholine (PtdCho)-phospholipase D (PLD) activation following stimulation of these T cells with anti-CD3 antibodies. However, since we also demonstrate that the antigen receptors on murine thymocytes are coupled to PtdCho-PLD activation, we propose that it is unlikely that this PLD pathway plays a central role in the transduction of T-cell proliferative responses, but rather, may be involved in either driving cells into cycle or maintaining cell cycle progression, processes required both for proliferation and activation-induced cell death. Whilst the molecular mechanisms underlying T-cell receptor (TCR)-coupling to PtdCho-PLD activation in these cells have not been fully defined, kinetics studies and experiments using pharmacological inhibitors of protein tyrosine phosphatases (PTPases) and reconstituting CD3-coupled PtdCho-PLD activity in streptolysin-O permeabilized cells, suggest that the TCR/CD3 complex, under optimal conditions of activation, may be predominantly coupled to PtdCho-PLD activation downstream of tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma), phosphatidylinositol (PtdIns)P2 hydrolysis, calcium mobilization and protein kinase C (PKC) activation.     

钢 淬透性的末端淬火试验方法（Jominy试验）标准探讨

根据试验室实践，对“钢淬透性的末端淬火试验方法GB／T225—2006”标准进行了分析和探讨，分析了新国标中存在的一些问题，同时还提出了一些建议。     

The persistence of leptospiral agglutinins titers in human sera diagnosed by the microscopic agglutination test

Human serum albumin (HSA) is used in large amounts as an excipient in many biopharmaceutical formulation to prevent loss of the active ingredient through adsorption and/or degradation. Traditionally, iso-electric focusing has been used to demonstrate charge heterogeneity in HSA preparations. In an effort to develop new methods for the analysis of formulation components, a capillary zone electrophoresis method was developed for the analysis of HSA. Under initial separation conditions using untreated silica capillaries and 20 mM sodium phosphate, pH 6.0 as electrophoretic buffer, HSA migrated as a single peak. Addition of 1,4-diaminobutane allowed separation of several components which could be further resolved by varying the buffer pH. Optimal separation conditions were attained at 5 mM 1,4-diaminobutane and pH 8.5. The reproducibility of the separation conditions was verified by using capillaries from a different manufacturer. A comparative analysis of HSA preparations from different manufacturers provided evidence that the method may be used to qualitatively differentiate individual preparations. The analysis of rhEPO formulations, composed largely of HSA, showed levels of heterogeneity comparable to that of HSA preparations. Electrospray ionisation mass spectrometry (ESA-MS) was used as an independent method to confirm the heterogeneous nature of HSA.     

Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb-IIIa in a patient with an unusual platelet membrane glycoprotein composition

The subject (E.B.) is a 63-year-old woman with autoimmune thrombocytopenic purpura (AITP) who was first examined some 6 years ago with symptoms of epistaxis and gum bleeding, severe thrombocytopenia, and large platelets. Her serum tested positively with control platelets in the MAIPA assay performed using monoclonal antibodies (MoAb) to glycoprotein (GP) IIIa (XIIF9, Y2/51), yet was negative in the presence of MoAbs to GP IIb (SZ 22) or to the GP IIb-IIIa complex (AP2, P2). The patient's platelets failed to aggregate with all agonists tested except for ristocetin. IgG isolated from the patient's serum inhibited ADP-induced aggregation of control platelets. Unexpectedly, flow cytometry showed an altered expression of membrane glycoproteins on the patient's platelets. Levels of GP Ib-IX were much higher than previously located by us in platelets. In contrast, the expression of GP IIb-IIIa was about half that seen with control subjects. When Western blotting was performed, a striking finding was a strong band of 250 kDa recognized by a series of MoAbs to GP Ib alpha in addition to the band in the normal position of GP Ib alpha. Finally, ADP-stimulated (E.B.) platelets failed to express activation-dependent epitopes on GP IIb-IIIa as recognized by PAC-1, AP6, or F26 and additionally gave a reduced P-selectin expression after thrombin addition. In conclusion, we present a novel patient with a severely perturbed platelet function where an altered membrane GP profile is associated with the presence of an autoantibody recognizing a complex-dependent determinant on GP IIb-IIIa and inhibitory of platelet aggregation.     

New automatic test for the study of platelet response to variations in osmotic pressure: application in the evaluation of the viability of platelet concentrates

Studying the osmotic resistance or swelling of platelets has often been suggested as a global test to assess the viability of those cells. A number of authors have also analysed the behaviour of platelets in hypotonic media by a variety of complementary methods (cell count, morphology, determinations of substances released, photometric measurement of aggregation induced by aggregating agents, etc). Most studies are currently based on the so-called "osmotic shock response" test, which measures according to time the light transmitted through platelet-rich plasma (PRP) after dilution in distilled water. In this study, the authors describe a new automated and reproducible test using slow dialysis to assess platelet osmotic resistance. The "Fragilimeter", a device initially described by the authors to characterise RBC fragility, has been adapted to the study of platelet osmotic behaviour. The variations in light transmission through a platelet suspension according to NaCl concentration are linked to the change in cellular volume and lysis and characterise the viability of the cells. The results obtained with normal platelets revealed the good reproducibility of the technique. The osmotic resistance is evaluated for two parameters: anticoagulant (citrate, EDTA) and cellular concentration. The test was applied to quality control of stored platelet concentrates for transfusion, prepared with different cell separators.     

Quantitation of Salmonella O-antibodies in human sera by enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) has been applied to the detection of antibodies against Salmonella O-antigens in human sera. Phenol-water extracted lipopolysaccharides (LPS) from serogroups A (O-antigens 2, 12), B (4, 5, 12) and D (9, 12) were used as antigens. When compared to the tube agglutination method according to Widal employing sera from patients with verified or suspected typhoid--or paratyphoid fever and from healthy controls it was found that ELISA (i) correlated significantly with the Widal reaction, (ii) was up to 100-fold more sensitive, and (iii) showed a greater reproducibility.     

A comparative study of skin tests and determination of the serum level of specific IgE immunoglobulins by means of the radioallergosorbent test in subjects with atopy

On 73 patients suspected for atopic diseases, 258 skin tests and radioallergosorbent tests (RAST) for specific IgE in serum were carried out. The results of both tests were compared in connection with the clinical picture. A good qualitative positive correlation was found, especially for grass pollen and house dust. False positive results with RAST cannot be excluded. The RAST seems to be a valuable adjunct to extensive allergologic diagnostic investigation, particularly in cases where difficulties can be expected in the performance or the interpretation of skin tests.     

Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di-tert-butyl-4-methylphenol (BHT)

Several alkylphenols (e.g., 2,6-di-tert-butyl-4-methylphenol, BHT) form reactive quinone methide intermediates (e.g., 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, BHT-QM) upon oxidation by cellular enzymes. In order to pursue the role of protein alkylation in alkylphenol toxicity, we used an immunochemical approach to identify protein targets alkylated by BHT. Synthetic BHT-N-acetylcysteine (BHT-NAC) was coupled to keyhole limpet hemocyanin and used as an antigen from which polyclonal antibodies were raised in New Zealand white rabbits. Rabbit serum contained an antibody which was highly specific for BHT-NAC, as determined by competitive ELISA. The BHT antibody was used as a probe to look for the presence of BHT-protein adducts in in vitro incubations with rat liver microsomes or tissue slices and also in vivo in liver tissue from male Sprague-Dawley rats exposed to BHT. Western blotting of protein gels revealed BHT-dependent protein alkylation over a wide molecular weight range. Prominent recurrent bands were observed at approximately 34.5, 52, 64.5, 74, and 97 kDa. Detection of adducts was inhibited in microsomal incubations by cytochrome P450 inhibitors, deuterated BHT, and the omission of NADPH. Similar protein alkylation patterns were observed in rat liver microsomes exposed to synthetically prepared BHT-QM as in the enzyme-mediated incubations. In rats gavaged with up to 1000 mg/kg BHT, the amount of protein alkylation observed was maximal at 24 h postdosing and was dose-dependent. Two alkylated proteins were isolated and identified by N-terminal sequencing: a mitochondrial beta-oxidation enzyme, enoyl-CoA hydratase, and a plasma membrane/cytoskeletal linker protein from the ezrin/moesin/radixin family.     

IgG levels in the sera of melanoma patients (author''s transl)

397 sera from 185 melanoma patients have been tested. We classified our subjects into three groups, according to the stage of disease. An alteration of the level of IgG 4 sub-class was found and related to the extension of the disease. The percentage of abnormalities was more frequent in stage II and III (55 p. 100 and 53 p. 100) than in stage I (19 p. 100). High titers of IgG 4 subclass were essentially detected in advanced disease. The biological significance is discussed.     

Anti-cytoplasmic autoantibodies reactive with epithelial cells of the salivary gland in sera from patients with Sj?gren''s syndrome: their disease- and organ-specificities

The current emphasis on outcomes management and outcomes research projects is stimulating interest in the psychometric properties of computerized clinical data bases among nurses and other health care providers. The knowledge, behavior, and status subscales of the Omaha System's Problem Rating Scale for Outcomes (PRSO) were evaluated for interrater reliability and content validity. The authors describe the methods used and the results of that research.