Restoration of spatial working memory by genetic rescue of GluR-A-deficient mice

Gene-targeted mice lacking the AMPA receptor subunit GluR-A (also called GluR1 encoded by the gene Gria1,) have deficits in hippocampal CA3-CA1 long-term potentiation (LTP) and have profoundly impaired hippocampus-dependent spatial working memory (SWM) tasks, although their spatial reference memory remains normal. Here we show that forebrain-localized expression of GFP-tagged GluR-A subunits in GluR-A-deficient mice rescues SWM, paralleling its rescue of CA3-CA1 LTP. This provides powerful new evidence linking hippocampal GluR-A-dependent synaptic plasticity to rapid, flexible memory processing.     

CrELISA: a fast and robust enzyme-linked immunosorbent assay bypassing the need for purification of recombinant protein

A multitude of antigens has been recently identified by screening of cDNA expression libraries derived from human tumors with autologous sera. Using a phage autoantibody assay and small panels of sera derived from cancer patients or controls it has been shown that some of these antigens display cancer-associated autoantibody responses. The diagnostic and prognostic significance of these potentially cancer-related autoantibodies remains unclear until large-scale assays are developed and serological data are available for hundreds of cancer patients and controls. The major bottleneck for the development of large-scale assays are the cloning, expression and the purification of each of the respective antigens. Due to these limitations and despite the potential clinical relevance large-scale autoantibody tests are established for only a few of these tumor antigens. Here we describe an enzyme-linked immunosorbent assay, Crude lysate ELISA (CrELISA), suitable for antigens identified by expression screening based on crude lysates of antigen-expressing bacteria. This assay permits sensitive and specific autoantibody seroscreening without the need of laborious and time-consuming cloning, expression and purification of recombinant proteins. CrELISA is robust and provides a versatile high throughput procedure for the rapid evaluation of multiple antigens in large-scale serology.     

The knowledge and attitudes of orthodontic trainees towards orthodontic therapists: a national survey

Objective: To assess the knowledge and attitudes of orthodontic trainees towards orthodontic therapists (OTs) in the UK.

Design: Cross-sectional survey.

Setting: UK-based orthodontic trainees.

Materials and methods: An electronic survey was sent to all members of the Training Grades Group of the British Orthodontic Society assessing exposure to OTs and their knowledge regarding current supervision guidelines and scope of practice. Attitudes towards OTs were also explored.

Results: Seventy-six responses (response rate 57%) were returned. Nearly 90% of trainees had no formal training regarding OTs. A total of 15.5% were aware of the correct current supervision guidelines and there was large variation in the knowledge of OTs’ scope of practice. The majority of trainees were happy to supervise OTs, but only 22.4% felt prepared for this during training. In total, 63% of trainees felt that OTs could impact their own future job prospects.

Conclusions: Currently, there is minimal formal training provided to trainees regarding the role of OTs. This is reflected in the lack of knowledge regarding supervision guidelines and scope of practice. Overall, trainees felt OTs were positive for the workforce but were concerned regarding the impact of their own future employment.     

Characterization of Haemophilus influenzae type b fimbriae.

We confirmed that the fimbriae of Haemophilus influenzae type b conferred hemagglutinating activity (HA) towards human erythrocytes, and erythrocytes of certain other species. Most (17/25) cerebrospinal fluid isolates lacked detectable HA on direct testing, but selective enrichment for fimbriation (f+) indicated that 22 of 25 strains could produce these surface structures. HA was unchanged from pH 4.5 to 9.5 and was not inhibited by mannose or certain other simple sugars. The HA titer of a suspension of three f+ strains was slightly decreased at 50 degrees C; HA was lost by heating at 60 degrees C for 3 min. Growth on a variety of solid and liquid media and under differing degrees of oxygenation did not change the HA titer of a suspension of three f+ strains. Fimbriation was not lost on repeated subculture. Wild-type fimbriated strains, and those derived by transformation, did not contain detectable plasmid DNA. Transformation of a strain lacking fimbriae to f+ was associated with the appearance of an outer membrane protein of 24 kilodaltons. This protein was purified from one strain to homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by selective detergent solubilization and ammonium sulfate fractionation. Colonization capacity was equivalent with an isogenic untypable strain lacking or possessing fimbriae. Fimbriae of type b H. influenzae possess characteristics similar to those structures on other gram-negative bacteria; their role in cell physiology or pathogenesis of invasive disease is unknown.     

Comparison of the fertilizing capability of spermatozoa from ejaculates, epididymal aspirates and testicular biopsies using intracytoplasmic sperm injection

A prospective study was carried out to compare the fertilizing capability and pregnancy outcome following intracytoplasmic sperm injection (ICSI) using spermatozoa obtained from ejaculates, or surgically from epididymis or seminiferous tubules. A total of 77 ICSI cycles (one per patient) was included. In all, 28 patients had severe oligoasthenoteratozoospermia, 19 patients had obstructive azoospermia and 30 patients had non-obstructive azoospermia. The main outcome measures were fertilization rate per injected metaphase II oocyte and the clinical pregnancy rate per embryo transferred back to the female recipients. In patients with severe oligoasthenoteratozoospermia, the fertilization and pregnancy rates were 79 and 25 %. In patients with obstructive azoospermia, for whom epididymal spermatozoa were used, these were 75 and 28%, and in the non-obstructive group for which testicular spermatozoa were used for injection, they were 69 and 21% respectively. These rates were not significantly different in the three groups (P = 0.85 and P = 0.14 respectively), suggesting that spermatozoa from the ejaculates and epididymal or testicular biopsies are able to fertilize equally by using ICSI. Live birth per embryo transfer was significantly reduced in patients with non-obstructive azoospermia compared to the other two groups. The high abortion rate (50%) in the group in which testicular spermatozoa were used raises doubts about the developmental competence of such embryos.      

Important role of interleukin‐3 in the early phase of collagen‐induced arthritis

Objective

Activation of basophils contributes to memory immune responses and results in exacerbation of collagen‐induced arthritis (CIA). We undertook the present study to analyze the production and biologic effects of interleukin‐3 (IL‐3), a strong activator of basophils, in CIA.

Methods

Arthritis was induced by immunization with type II collagen. Mice were treated with blocking monoclonal antibodies against IL‐3 or with recombinant IL‐3. Clinical scoring, histologic analysis, fluorescence‐activated cell sorter analysis, enzyme‐linked immunosorbent assay, and cell culturing were performed to assess disease activity and IL‐3 production.

Results

IL‐3 was produced in large quantities by collagen‐specific CD4+ T cells in the spleen and was present in the synovial tissue during onset of arthritis, but was down‐regulated in paws with severe inflammation. Blockade of IL‐3 during the time of arthritis onset resulted in profound improvement of the disease, with reductions in synovial leukocyte and cytokine levels, peripheral blood basophil levels, and anticollagen antibody titers. Blockade of IL‐3 during the late phase of arthritis had no beneficial effect. Administration of recombinant IL‐3 during onset of arthritis induced a marked exacerbation of the disease, with increased peripheral blood basophil and plasma IL‐6 levels and increased titers of anticollagen antibody. In studies of the regulation of IL‐3 expression in CD4+ T cells, IL‐6 and IL‐4 suppressed the release of IL‐3 by activated CD4+ T cells, whereas lipopolysaccharide and CpG DNA up‐regulated IL‐3 secretion in activated CD4+ T cells by acting on costimulatory cells.

Conclusion

Taken together, the present results demonstrate for the first time that IL‐3 has an important role in the early phase of CIA.

     

Platelet turnover and kinetics in immune thrombocytopenic purpura: results with autologous 111In-labeled platelets and homologous 51Cr- labeled platelets differ

Mean platelet survival and turnover were simultaneously determined with autologous 111In-labeled platelets (111In-AP) and homologous 51Cr- labeled platelets (51Cr-HP) in ten patients with chronic immune thrombocytopenic purpura (ITP). In vivo redistribution of the 111In-AP was quantitated with a scintillation camera and computer-assisted image analysis. The patients were divided into two groups: those with splenic platelet sequestration (spleen-liver 111In activity ratio greater than 1.4), and those with diffuse sequestration in the reticuloendothelial system. The latter patients had more severe ITP reflected by pronounced thrombocytopenia, decreased platelet turnover, and prominent early hepatic platelet sequestration. Mean platelet life span estimated with 51Cr-HP was consistently shorter than that of 111In-AP. Platelet turnover determined with 51Cr-HP was thus over-estimated. The difference in results with the two isotope labels was apparently due to greater in vivo elution of 51Cr. Although the limitations of the techniques should be taken into account, these findings indicate that platelet turnover is not always normal or increased in ITP, but is low in severe disease. We suggest that this may be ascribed to damage to megakaryocytes by antiplatelet antibody. The physical characteristics in 111In clearly make this radionuclide superior to 51Cr for the study of platelet kinetics in ITP.