Monoclonal antibodies to bovine serum albumin: affinity and specificity determinations

A panel of 12 monoclonal antibodies (MAb) to bovine serum albumin (BSA) was developed and characterized as to their physiochemical and immunological properties. Affinity constants of the MAb varied over a wide range from 10(5) to 10(8) M-1. MAb were assembled into several groups of non- or minimally interacting antibodies by analysis of competitive binding experiments, and BSA domain and subdomain specificities of the MAb were assigned by analysis of results of MAb binding to purified BSA fragments. Further fine specificity delineation was accomplished by examination of cross-reactivity patterns to several mammalian albumins. The data suggest that some of the low affinity MAb recognize sites on different portions of the BSA molecule, indicating that similar epitopes exist on different domains of the BSA molecule.     

Negative regulation of FcepsilonRI signaling by FcgammaRII costimulation in human blood basophils

BACKGROUND: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor FcepsilonRI of rodent mast cells is decreased by cross-linking these receptors to the low-affinity IgG receptor FcgammaRII. The inhibition is thought to involve the tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the FcgammaRIIB cytoplasmic tail, creating binding sites for SH2-containing protein (Src homology domain containing protein tyrosine phosphatase 1 and 2 [SHP-1, SHP-2]) and/or lipid (SH2 domain-containing polyphosphatidyl-inositol 5-phosphatase) phosphatases that oppose activating signals from the costimulated antigen receptors. OBJECTIVE: In human basophils and mast cells FcepsilonRI signaling generates mediators and cytokines responsible for allergic inflammation. We proposed to determine whether FcepsilonRI signaling is inhibited by FcgammaRII costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. METHODS: FcgammaR expression on human basophils was examined using flow cytometry and RT-PCR analysis. FcgammaRII/FcepsilonRI costimulation was typically accomplished by priming cells with anti-dinitrophenol (DNP) IgE and anti-DNP IgG and stimulating with DNP-BSA. Phosphatases were identified by Western blotting, and their partitioning between membrane and cytosol was determined by cell fractionation. Biotinylated synthetic peptides and phosphopeptides corresponding to the FcgammaRIIB ITIM sequence were used for adsorption assays. RESULTS: We report that peripheral blood basophils express FcgammaRII (in both the ITIM-containing FcgammaRIIB and the immunoreceptor tyrosine-based activation motif-containing FcgammaRIIA forms) and that costimulating FcgammaRII and FcepsilonRI inhibits basophil FcepsilonRI-mediated histamine release, IL-4 production, and Ca(2+) mobilization. The inhibition of basophil FcepsilonRI signaling by FcgammaRII/FcepsilonRI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation. Human basophils express all 3 SH2-containing phosphatases. CONCLUSIONS: Evidence that FcgammaRII/FcepsilonRI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylated FcgammaRIIB ITIM sequence specifically recruit SHP-1 from basophil lysates particularly implicates this protein phosphatase in the negative regulation of FcepsilonRI signaling by costimulated FcgammaRII.     

Partial androgen insensitivity with phenotypic variation caused by androgen receptor mutations that disrupt activation function 2 and the NH(2)- and carboxyl-terminal interaction

Partial androgen insensitivity with sex phenotype variation in two unrelated families was associated with missense mutations in the androgen receptor (AR) gene that disrupted the AR NH(2)-terminal/carboxy terminal interaction. Each mutation caused a single amino acid change within the region of the ligand-binding domain that forms activation function 2 (AF2). In one family, the mutation I737T was in alpha helix 4 and in the other F725L was between helices 3 and 4. Neither mutation altered androgen binding as determined by assays of mutant AR in the patient's cultured genital skin fibroblasts or of recombinant mutant receptors transfected into COS cells. In transient cotransfection assays in CV1 cells, transactivation with the AR mutants at low concentrations of DHT was reduced several fold compared with wild-type AR but increased at higher concentrations. Defects in NH(2)-terminal/carboxy terminal interactions were identified in mammalian two hybrid assays. In similar assays, there was reduced binding of the p160 coactivators TIF2/SRC2 and SRC1 to the mutant AR ligand binding domains (LBD). In the family with AR I737T, sex phenotype varied from severely defective masculinization in the proband to a maternal great uncle whose only manifestation of AIS was severe gynecomastia. He was fertile and passed the mutation to two daughters. The proband of the F725L family was also incompletely masculinized but was raised as a male while his half-sibling by a different father was affected more severely and reared as a female. These studies indicate that the function of an AR AF2 mutant in male development can vary greatly depending on the genetic background.     

Evolutionary comparison of laboratory cardiac and infectious parameters during septic shock: about an original case

A massive release of troponin Ic and CKMB was described in a patient during septic shock. According to experimental animal models previously described, this release of biological markers by myocardial tissue could be due to an inflammatory process of myocardial tissue during septic shock without myocardial infarction in non cardiac critically ill patients.     

Antiphospholipid antibodies and the outcome of pregnancy after the first in-vitro fertilization and embryo transfer cycle

Increased antiphospholipid antibody prevalence has been demonstrated by a number of recent studies in in-vitro fertilization (IVF) patients but the potential effects of antiphospholipid antibodies on the different components of the reproductive process and the consideration of whether to test IVF patients for antiphospholipid antibodies are controversial. The present study was undertaken to investigate the possible association between the presence of circulating antiphospholipid antibodies (namely the lupus anticoagulant and anticardiolipin antibodies), among a series of 21 consecutive IVF patients having a clinical spontaneous abortion after their first embryo transfer. As a control group (n=42), the nearest IVF cycle resulting in an ongoing pregnancy before and after each miscarried IVF cycle (i.e. the closest cycles in temporal relationship to the index cycle) was used. One patient (4.8%) in the study group and two women (4.8%) among controls were seropositive for antiphospholipid antibodies. These low and similar seropositivity rates found in the two groups studied lead us to conclude that antiphospholipid antibodies testing in IVF patients should be considered only in those women having repeated failures of implantation/clinical abortion after embryo transfer but not in an infertile general population reaching an IVF programme.      

The effectiveness of a treatment protocol for male lower urinary tract symptoms in general practice: a practical randomised controlled trial

BACKGROUND: Randomised controlled trials have shown the efficacy of several treatment modalities for lower urinary tract symptoms (LUTS) in selected populations. The effectiveness in daily practice has hardly been investigated, especially in primary care and is dependent on choices between all possible treatment options and best investigated in a comprehensive study, including all treatment modalities (watchful waiting, alpha-blockers, 5-alpha-reductase inhibitors, and surgery). AIM: Assessment of the effectiveness of a comprehensive treatment protocol for LUTS in primary care. DESIGN OF STUDY: Randomised controlled trial. SETTING: Fourteen general practices in the Netherlands. METHOD: Intervention: treatment protocol based on a formalised expert opinion. Control condition: usual care. Study population: 208 subjects with moderate to severe LUTS (IPSS > or =8, median = 13). OUTCOME MEASURES: symptom severity (IPSS [International Prostate Symptom Score]), bother score (Dan-PSS [Danish Prostate Symptom Score]), and maximum urinary flow (Q(max)); incidence of acute urinary retention and urinary tract infections. RESULTS: In the intervention group markedly more subjects used an alpha-blocker at end of follow-up than in the usual care group (24% versus 6%). No significant differences were found between intervention and control group in IPSS, Q(max) or Dan-PSS. CONCLUSION: alpha-blockers and watchful waiting are the most frequent treatment modalities for LUTS in primary care. Our study showed no evidence that a protocol using well-defined indications for all possible treatment modalities based on a formalised expert opinion procedure has added value. Based on our results, we cannot recommend a broadening of the indication for alpha-blockers, which, however, seems to be the current trend.     

Safety of blood products and B19 parvovirus]

More than 25 years after the discovery of the parvovirus B19 (B19), the issue of the safety of blood components and the screening of this virus in blood donations is still debated. Although more often transmitted by respiratory route, B19 may also be transmitted by transfusion of blood components. This risk of exposure has been estimated to a frequency ranging from 1/625 to 1/50,000, according to the sensitivity of the detection methods and to seasonal epidemiologic circumstances. Usually, B19 is responsible for benign pathologies. However, such an infection can have a serious clinical outcome in three categories of susceptible recipients: (i) patients with shortened red cell survival (thalassemia major, sickle cell disease, other hemolytic diseases); (ii) immunocompromised patients (previously exposed to B19 or not) (iii) and pregnant women (not previously exposed the B19), with a risk of hydrops fetalis or of intrauterine death. Selected blood components, not collected during the short but highly viremic pre-seroconversion phase, could be reserved for these three groups of at-risk recipients. The screening of such viremic donations could be performed with nucleic acid testing (NAT), but an alternate strategy could be the selection of B19 immunised donors far from the primo-infection (positive for B19 IgG and negative for B19 IgM, or only positive for IgG at two controls distant of several months). However, the existence of persistently B19-infected individuals carrying B19 DNA despite the presence of specific IgG (estimated at 1% of blood donors) could constitute a potential threat for transfused immunocompromised recipients. The screening of such donors, which could be performed through a very highly sensitive NAT, would be justified only if the infectivity of such blood donations is demonstrated. If not, a screening of blood donors positive for B19 IgG would be a sufficient preventive measure.