Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells

The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.     

Iduronate-2-sulfatase gene mutations in 16 patients with mucopolysaccharidosis type II (Hunter syndrome)

Mutations of the iduronate-2-sulfatase gene were identifiedin 16 patients with mucopolysaccharidosis type II (Hunter syndrome).Together with another 10 cases reported by us earlier it emergesthat about 20% of the patients have deletions of the whole geneor other major structural alterations. One, two or three basepair deletions are found in about 23% of the cases while theremaining about 57% carry point mutations predicting amlno acidreplacement, premature termination of translation, or aberrantsplicing. Molecular analysis of mRNA in splice site mutantsshowed that these latter defects frequently resulted in useof cryptic splice sites in exons or introns. 62% of the smalldeletions and point mutations have occurred in 3 of the 9 iduronate-2-sulfatasegene exons. Knowledge of the primary genetic defect allows fastand reliable carrier detection and prenatal diagnosis as wellas insight into the relationship between genotype and phenotype.     

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.      

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we describe the problems and solutions of setting up the recombinant SARS-CoV S-based ELISA for antibody detection. The SARS-CoV S-based immunoglobulin M (IgM) and IgG ELISAs were evaluated and compared with the corresponding N-based ELISA for serodiagnosis of SARS-CoV pneumonia, using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS-CoV pneumonia patients in Hong Kong. Results obtained by the recombinant S (rS)-based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers, followed by addition of different regeneration buffer, identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection. The specificities of the S-based ELISA for IgG and IgM detection were 98.6% and 93.9%, with corresponding sensitivities of 58.9% and 74.7%, respectively. The sensitivity of the rN IgG ELISA (94.7%) is significantly higher than that of the rS IgG ELISA (P < 0.001), whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA (55.2%) (P < 0.01). An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS-CoV pneumonia.     

Implementing a Transition Program from Paediatric to Adult Services in Phenylketonuria: Results after Two Years of Follow-Up with an Adult Team

We aimed to report the implementation of a phenylketonuria (PKU) transition program and study the effects of follow-up with an adult team on metabolic control, adherence, and loss of follow-up. Fifty-five PKU patients were analysed in the study periods (SP): 2 years before (SP1) and after the beginning of adult care (SP2). Retrospective data on metabolic control and number of clinic appointments were collected for each SP, and protein intakes were analysed. In SP2, three patients (6%) were lost to follow-up. There was a small but statistically significant increase in median number of annual blood spots from SP1 to SP2: 11 (7–15) vs. 14 (7–20); p = 0.002. Mean ± SD of median blood Phe remained stable (525 ± 248 µmol/L vs. 552 ± 225 µmol/L; p = 0.100); median % of blood Phe < 480 µmol/L decreased (51 (4–96)% vs. 37 (5–85)%; p = 0.041) and median number of clinic appointments increased from SP1 to SP2: (5 (4–6) vs. 11 (8–13); p < 0.001). No significant differences were found regarding any parameter of protein intake. Our results suggest that the implementation of an adult service was successful as impact on metabolic control was limited and attendance remained high. Continuous dietetic care likely contributed to these results by keeping patients in follow-up and committed to treatment.     

Social Inequalities in Changes in Diet in Adolescents during Confinement Due to COVID-19 in Spain: The DESKcohort Project

Adolescence is a critical period in the consolidation of healthy lifestyles that can last into adulthood. To analyze changes in food consumption and eating behaviors in high-school adolescents during the first confinement, a cross-sectional study was conducted at the end of confinement in Spain. Changes in the frequency or quantity of consumption of different types of food and food-related behaviors were analyzed. Socioeconomic and health-related variables were also considered. To determine whether dietary changes were related to socioeconomic position (SEP), Poisson regression models with robust variance were estimated. Overall, there were some changes towards a healthier diet such as an increase in fruit consumption (38.9%) and a decrease in the consumption of soft drinks (49.8%), sweets and pastries (39.3%), and convenience foods (49.2%). Some changes, however, were related to less healthy behaviors, such as a more irregular pattern of meal distribution (39.9%) or an increase in snacking between meals (56.4%). Changes towards less healthy eating were also related to students’ SEP. The risk of worsening the diet was found to be 21% higher in adolescents from a more disadvantaged SEP. Future public policies could be adapted to avoid increasing nutritional and health inequalities.     

Documento de consenso de la Sociedad Española de Anestesiología y Reanimación (SEDAR), Sociedad Española de Medicina Interna (SEMI) y Sociedad Española de Medicina de Urgencias y Emergencias (SEMES) para la definición de competencias mínimas de ecografía en Cuidados Intensivos y Urgencias y la obtención del Diploma acreditativo

The use of ultrasound as a clinical diagnostic tool and guide of bedside procedures has become an indispensable examination in the acute critically ill patient. The training of professionals in minimum skills of knowledge, management and indications of use of ultrasound required to be defined by the Scientific Societies. The Intensive Care Ultrasound Working Group of the Spanish Society of Anesthesiology and Resuscitation (SEDAR), of the Spanish Society of Internal Medicine (SEMI) and the Spanish Society of Emergency Medicine (SEMES) has developed this consensus document in which the recommended training program and the minimum competencies to be achieved with regard to the use of Ultrasound in Intensive Care, Anesthesia and Emergency medicine are defined.This document defines the training program and the skills to acquire in order to achieve the diploma in lung, abdominal and vascular ultrasound. This document can serve as a guide to define the skills to be acquired in the training programs of residents (MIRs) of specialists working in intensive care, anesthesia, and emergency medicine.     

The Role of Community Health Workers Within the Continuum of Services for HIV,Viral Hepatitis,and Other STIs Amongst Men Who Have Sex with Men in Europe

Journal of Community Health - Little is known about Community Health Workers (CHWs) who work in non-clinical settings to provide sexual health support around HIV, viral hepatitis, and other...     

Role of purinergic system in tracheobronchial reactivity of healthy and bronchopathic subjects

We have investigated the effect of inhaled adenosine on bronchomotor tone in 16 healthy and 24 allergic and non-allergic bronchopathic subjects. We determined the inhaled adenosine dose-response curves after no treatment and after treatment with aminophylline (240 mg in 10 min), reproterol (90 mcg in 2 min) and salbutamol (100 mcg in 2 min) administered intravenously 15 min before adenosine and reproterol (500 mcg) and salbutamol (200 mcg) administered by inhalation from a metered aerosol 30 min before adenosine on separate days. Without prior treatment, inhaled adenosine caused bronchoconstriction in both groups of subjects (normal, and atopic and non-atopic asthmatic ones), but the peripheral airways revealed only a moderate change in normal subjects. Aminophylline caused a greater inhibition of adenosine bronchoconstriction than did reproterol. These results suggest that inhaled adenosine bronchoconstriction involved purinergic transmission and that it is mediated via a P1/Ri (A-1/R1) receptor. Aminophylline is a potent antagonist at purinoceptor level. Reproterol inhibits bronchoconstriction by a mechanism independent of its effect on beta-adrenergic receptors. Because salbutamol, i.e. a beta 2-agonist bronchodilator, did not inhibit adenosine-induced bronchoconstriction (or very nearly so), our results support the view that reproterol antagonizes P1/Ri (A1/R1) tracheobronchial receptors.