Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

Autoimmune diseases in myelodysplastic syndrome favors patients survival: A case control study and literature review

Background

We conducted a monocentric retrospective study of patients with myelodysplastic syndromes (MDS) and autoimmune or inflammatory disorders (AIMs) and a literature review. We analyzed the association with subgroups of the WHO 2016 MDS classification and patient's survival in a case control study. Risk factors associated with survival were analyzed by uni- and multivariate analysis.

In vitro controllability of the MagScrew total artificial heart system

The purpose of this study was to evaluate the in vitro responses to preload and afterload of our total artificial heart (TAH), the MagScrew TAH. The TAH consists of two blood pumps and a control logic, developed at the Cleveland Clinic, OH, and the MagScrew actuator and its electronic control system, developed by Foster-Miller Technologies, Inc., Albany, NY. Tests were performed on a mock circulatory loop, using water as a test fluid. Preload sensitivity of the Mag-Screw TAH demonstrated a Frank-Starling response to preload in automatic mode. A peak flow of 10 L/min was obtained, with a left atrial pressure of 13 mm Hg. The relationship between right atrial pressure and left atrial pressure was well balanced when tested with a left bronchial shunt flow of 5% and a range of pulmonary artery and aortic pressures. With respect to afterload response, the left pump showed a relatively low sensitivity, which allowed the pump to maintain perfusion over a wide range of aortic pressures. The right pump, on the other hand, was much more sensitive to pulmonary artery pressure, which provided a measure of protection against pulmonary congestion. The very effective physiologic response of the MagScrew TAH is believed to result from employment of a left master, alternating ejection control logic, high inherent sensitivity of the blood pumps to atrial pressure, a lower effective stroke volume for the right pump, and a scaling of right side motor ejection voltage to 80% of that used for the left side ejection.     

Human gammadelta T cells express a higher TCR/CD3 complex density than alphabeta T cells

The aim of our study was to compare CD3 expression on gammadelta T cells and alphabeta T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human gammadelta T cells constitutively express approximately twofold more of the TCR/CD3 complex than alphabeta T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of gammadelta T cells compared to alphabeta T cells. These clinical laboratory results confirm the fundamental data described elsewhere. gammadelta T cells deserve further clinical investigations to understand their precise role in human immunity.     

Detection of extended broad-spectrum beta-lactamases inEnterobacteriaceae in four French hospitals

In 210 strains ofEnterobacteriaceae which were isolated in four hospitals and which showed reduced susceptibility to cefotaxime, high synergy was demonstrated between amoxicillin (20µg) + clavulanate (10µg) and cefotaxime (30µg) using a simple double-disk test. Isoelectric focusing on gel and specific iodometric detection using ceftriaxone identified four extended broad-spectrum-lactamases (isoelectric points 7.6, 6.3, 7.0 and 5.9) produced by the strains.     

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Role of double-stranded RNA and Npro of classical swine fever virus in the activation of monocyte-derived dendritic cells

Classical swine fever virus (CSFV) is a noncytopathogenic (ncp) positive-sense RNA virus that replicates in myeloid cells including macrophages and dendritic cells (DC). The virus does not induce type I interferon (IFN-alpha/beta), which in macrophages has been related to the presence of the viral Npro gene. In the present work, the role of viral double-stranded (ds)RNA and Npro in the virus-host cell interaction has been analyzed. Higher levels of detectable dsRNA were produced by a genetically engineered cytopathogenic (cp) CSFV compared with ncp CSFV, and cp CSFV induced IFN-alpha/beta in PK-15 cells. With DC, there was only a small difference in the levels of dsRNA between the cp and ncp viruses, and no IFN-alpha/beta was produced. However, the cp virus induced a higher degree of DC maturation, in terms of CD80/86 and MHC II expression. Npro deletion mutants induced an increase in DC maturation and IFN-alpha/beta production-for both ncp and cp viruses-despite reduced replication efficiency in the DC. Deletion of Npro did not influence dsRNA levels, indicating that the interference was downstream of dsRNA turnover regulation. In conclusion, the capacity of CSFV to replicate in myeloid DC, and prevent IFN-alpha/beta induction and DC maturation, requires both regulated dsRNA levels and the presence of viral Npro.     

Influences of hormone replacement therapy on postmenopausal women's health perceptions

OBJECTIVE: To assess the beliefs of climacteric women regarding their health, menopause, and hormone replacement therapy (HRT). DESIGN: Medical students asked to interview 526 healthy women, ranging from 40 to 64 years of age, between January and February of 2002. Of that number, 26 (4.9%) declined to participate in the interview. Thus, 500 women were interviewed about their beliefs and perceptions regarding their quality of life and health risks, as well as their opinions on menopause and HRT. RESULTS: The mean age of the sample was 53.3 +/- 6.2 years; 83.4% were postmenopausal, and 18.8% were HRT users. Of the women interviewed, 38.6% believed that their health was good. Although 78.8% thought that cancer is the main cause of death, 64% of them considered themselves to be at high risk for cardiovascular disease and osteoporosis. Most (64%) believed that menopause deteriorates the quality of life and that it increases cardiovascular risk (52.4%) and osteoporosis (72.0%). The HRT users perceived that they had better health status (48.9% v 36.2%, P < 0.02) and smaller cardiovascular risk (54.3% v 66.3%, P < 0.04) than did the nonusers; however, they ignored the preventive effect of estrogens in osteoporosis. CONCLUSIONS: Women believe that menopause deteriorates their health. The HRT users perceived themselves to be healthier and to have a smaller risk for cardiovascular disease.     

Vasopressin-induced taurine efflux from rat pituicytes: a potential negative feedback for hormone secretion

Previous work on the whole neurohypophysis has shown that hypotonic conditions increase release of taurine from neurohypophysial astrocytes (pituicytes). The present work confirms that taurine is present in cultured pituicytes, and that its specific release increases in response to a hypotonic shock. We next show that vasopressin (VP) and oxytocin (OT) also specifically release taurine from pituicytes. With an EC₅₀ of ∼2 n m , VP is much more potent than OT, and the effects of both hormones are blocked by SR 49059, a V_1a receptor antagonist. This pharmacological profile matches the one for VP- and OT-evoked calcium signals in pituicytes, consistent with the fact that VP-induced taurine efflux is blocked by BAPTA-AM. However, BAPTA-AM also blocks the taurine efflux induced by a 270 mosmol l⁻¹ challenge, which per se does not evoke any calcium signal, suggesting a permissive role for calcium in this case. Nevertheless, the fact that structurally unrelated calcium-mobilizing agents and ionomycin are able to induce taurine efflux suggests that calcium may also play a signalling role in this event. It is widely accepted that in hypotonic conditions taurine exits cells through anionic channels. Antagonism by the chloride channel inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) suggests the same pathway for VP-induced taurine efflux, which is also blocked in hypertonic conditions (330 mosmol l⁻¹). Moreover, it is likely that the osmosensitivity of the taurine channel is up-regulated by calcium. These results, together with our in situ experiments showing stimulation of taurine release by endogenous VP, strengthen the concept of a glial control of neurohormone output.