Evaluation of Hemagglutinin Protein-Specific Immunoglobulin M for Diagnosis of Measles by an Enzyme-Linked Immunosorbent Assay Based on Recombinant Protein Produced in a High-Efficiency Mammalian Expression System

Recombinant hemagglutinin (H) of the measles virus (MV) expressed in a mammalian high-expression system based on the Semliki Forest virus replicon was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulin M (IgM) and IgG in patients with acute-phase measles. One hundred twelve serum specimens from 70 patients with measles were analyzed. Case definition was based on a commercial IgM ELISA that utilizes MV-infected cells (MV-ELISA) (Enzygnost; Behring Diagnostics); the clinical criteria of the Centers for Disease Control and Prevention (Atlanta, Ga.); and/or the increase in hemagglutinin test titers, neutralization test titers, and levels of MV-specific IgG whenever paired sera were available. The initial time courses of the IgM signal after the onset of rash are similar in the H- and MV-ELISAs. On days 0 to 19, both ELISAs detected IgM in 67 of 68 (98.5%) sera. Average maximal levels of IgM seem to persist, however, about 10 days longer in the MV-ELISA (up to day 25) than in the H-ELISA (day 15). From days 20 to 29 and 30 to 59, the H-ELISA detected only 64.3 (9 of 14) and 19.2% (5 of 26), respectively, of sera that were IgM positive by MV-ELISA. At least up to day 30, the performance of the H-ELISA seemed to be similar to that reported for commercial ELISAs based on whole MV. Our results demonstrate that MV H-specific IgM can be used to diagnose most measles cases from a single serum specimen collected within 19 days after the onset of rash and that the recombinant protein used in this study is suitable for this purpose.     

Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay

A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5'-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.     

Fate of the type II pneumocyte following tracheal occlusion in utero: a time-course study in fetal sheep

 Tracheal occlusion in utero has been shown to cause accelerated fetal lung growth and is now being considered as a therapeutic modality for pulmonary hypoplasia. We report the effects of tracheal ligation on the surfactant-producing type II pneumocyte population. Three groups of fetal lambs underwent tracheal ligation of 2 weeks’, 4 weeks’ and 6 weeks’ duration, respectively, and all were sacrificed at 136 days’ gestation (9 days pre-term). Nonoperated twins served as controls. The type II pneumocyte population was studied morphometrically using a combination of anti-surfactant protein B immunohistochemistry and computer-assisted stereologic morphometry at light and electron microscopic levels. Single-factor ANOVA was used for statistical analysis. Two weeks of tracheal ligation resulted in doubling of the total lung volume as a result of airspace distension and, to lesser extent, growth of the tissue compartment. With increasing duration of tracheal ligation, there was no additional lung growth. However, more prolonged tracheal occlusion was found to result in significant reduction of the surfactant system, as reflected in the marked decrease of total pneumocyte type II volume (3.14 cm³, 0.95 cm³, and 0.46 cm³, after 2, 4, and 6 weeks of ligation, compared with 5.96 cm³ for controls) and total pneumocyte type II number (13.9 × 10⁹, 3.8 × 10⁹, and 2.4 × 10⁹, compared with 53.2 × 10⁹ for controls). Ultrastructural analysis of the type II cells in obstructed lungs showed vacuolar degenerative changes that, after 6 weeks of ligation, were apparently irreversible. In utero tracheal ligation causes fetal lung hyperplasia, but results in reduction of and injury to the surfactant-producing cell population. Before tracheal occlusion can find widespread clinical application, its pathophysiology needs to be further elucidated. Received: 30 April 1997 / Accepted: 10 July 1997     

Autosomal genetics and Y-chromosome haplogroup L1b-M317 reveal Mount Lebanon Maronites as a persistently non-emigrating population

Currently, there are 18 different religious communities living in Lebanon. While evolving primarily within Lebanon, these communities show a level of local isolation as demonstrated previously from their Y-haplogroup distributions. In order to trace the origins and migratory patterns that may have led to the genetic isolation and autosomal clustering in some of these communities we analyzed Y-chromosome STR and SNP sample data from 6327 individuals, in addition to whole genome autosomal sample data from 609 individuals, from Mount Lebanon and other surrounding communities. We observed Y chromosome L1b Levantine STR branching that occurred around 5000 years ago. Autosomal DNA analyses suggest that the North Lebanese Mountain Maronite community possesses an ancestral Fertile Crescent genetic component distinct from other populations in the region. We suggest that the Levantine L1b group split from the Caucasus ancestral group around 7300 years ago and migrated to the Levant. This event was distinct from the earlier expansions from the Caucasus region that contributed to the wider Levantine populations. Differential cultural adaption by populations from the North Lebanese Mountains are clearly aligned with the L1b haplotype STR haplogroup clusters, indicating pre-existing and persistent cultural barriers marked by the transmission of L1b lineages. Our findings highlight the value of uniparental haplogroups and STR haplotype data for elucidating biosocial events among these populations.Subject terms: Population genetics, Computational biology and bioinformatics     

Studies using a viral challenge and CD8 T cell depletions on the roles of cellular and humoral immunity in the control of an SHIV-89.6P challenge in DNA/MVA-vaccinated macaques

Here, we study immune responses in four DNA/MVA-vaccinated macaques following an SHIV-89.6P challenge and a subsequent CD8 cell depletion. Both post-challenge and post-depletion peaks of viremia contracted with the expansion, or re-emergence, of CD8 T cells. Post-depletion, CD8 cells expanded in the presence of higher levels of neutralizing Ab and CD4 help than post-challenge and had superior maturational characteristics as measured by expression of the anti-apoptotic protein Bcl-2, the IL-7 receptor CD127 and co-production of IFN-gamma and IL-2. Pre-challenge and pre-depletion titers of neutralizing Ab correlated inversely with peaks of viremia and directly with peaks for anti-viral CD4 cells. Thus, our results reveal CD8 cells playing a central role, and neutralizing Ab, a supporting role in SHIV-89.6P control. They also suggest a dynamic relationship between neutralizing Ab, antigen load and anti-viral CD4 cells in the maturation of high-quality anti-viral CD8 T cells.     

In vitro biosynthesis of plasma proteins under ischemic conditions of closed-circuit perfusion of healthy and intoxicated rabbit liver

We are elaborating on the kinetics and mechanisms of septic rabbit liver to de novo biosynthesize acute-phase response (APR) proteins under in vitro conditions of deepening ischemia in reference to their in vivo prevalence in serum and cerebrospinal fluids (CSF) collected at predetermined times. The significance of the data is interpreted as relevant to grafting cadaveric liver into end-stage liver diseased patients and APR-induced ischemic heart diseases (IHD). Hepatic APR was induced by CCl(4)-intubation, and the administration of cholera toxin (CT) or scorpion venom (SV), or both, to rabbits. Hepatic functional efficiency, in terms of biosynthesis of APR proteins in closed circuit perfusion of the isolated intoxicated liver with oxygenated saline or L-15 media paralleled the two-dimensional immunoelectrophoresis (2D-IEP) spectrum of APR serum proteins at time of liver isolation. We are suggesting: (a) in vitro biosynthesis of plasma proteins by isolated perfused liver is the result of in vivo decoded and retained APR inflammatory signals; and (b) decoded inflammatory signals are expressed not withstanding the perfusate's organic composition. Furthermore, 90 min of ischemic perfusion in saline or L-15 medium precipitated mitochondrial aberrations which resulted in further deterioration of de novo biosynthesis of APR plasma proteins. Regardless of the nature of the inflammatory stimuli, mitochondrial aberrations rendered the perfused organ a biologically inert tissue mass that was incapable of resuming biological function upon perfusion with oxygenated L-15 medium. This is most likely due to ischemia-induced irreversible hepatic necrosis. Thus, in vitro aberrations of mitochondrial function(s) critically limit the capability of the isolated liver to resume its organic function to sustain biosynthesis of de novo plasma proteins. Extrapolation of these results to the surgical management of end-stage liver diseases points to the importance of the status and the handling protocol(s) of the cadaver donor liver prior to successful grafting. We conclude that although histology of a cadaver liver may reveal well-preserved hepatic cellular organelles with at least minimal intra- and intercellular communication required for viable hepatic function, we deem it essential to further define acceptable minimal capabilities to de novo biosynthesize plasma proteins by a cadaver liver as a measure of its functional viability and suitability for transplantation. Ultimately, this measure may improve the success of liver transplants with minimal surgical and drug interventions.     

Hotspot for deletions in the CGG repeat region of FMR1 in fragile X patients

The fragile X syndrome is the most frequent cause of inheritedmental retardation. The molecular mechanism of the disorderis based on the expansion of a CGG repeat in the 5' UTR of theFMR1 gene In the majority of fragile X patients. The instabilityof this CGG repeat containing region is not restricted to theCGG repeat Itself but expands to the flanking region as well.We describe four unrelated fragile X patients that are mosaicfor both a full mutation and a small deletion in the CGG repeatcontaining region. Sequence analysis of the regions surroundingthe deletions showed that both the (CGG)_n repeat and some flankingsequences were missing in all four patients. The 5' breakpointsof the deletions were found to be located between 75–53bp proximal to the CGG repeat. This suggests the presence ofa hot spot region for deletions in the CGG repeat region ofthe FMR1 gene and emphasizes the instability of this regionIn the presence of an expanded CGG repeat.     

Functional mapping of the human globus pallidus: contrasting effect of stimulation in the internal and external pallidum in Parkinson's disease

Our objective was to elaborate a functional map of the globus pallidus by correlating the intrapallidal localization of quadripolar electrodes implanted in parkinsonian patients with the clinical effect of the stimulation of each contact. Five patients with L-DOPA-responsive Parkinson's disease presenting severe motor fluctuations and L-DOPA-induced dyskinesias were treated by continuous bilateral high-frequency stimulation of the globus pallidus. The effects of stimulation on parkinsonian disability were tested through each of the four stimulating contacts of each electrode. The anatomical localization of each of the stimulating contacts was determined by confronting the pre- and post-operative magnetic resonance imaging with the anatomical atlas of Schaltenbrand and Wharen.(34) The registration procedure comprised digitization of the atlas, the use of deformation tools to fit atlas sections with magnetic resonance imaging sections, and three-dimensional reconstruction of both the atlas and the magnetic resonance imaging sections. Analysis of the 32 stimulating contacts tested did not reveal a somatotopic organization in the pallidal region investigated but demonstrated that high-frequency stimulation had contrasting effects depending on whether it was applied to the external or the internal pallidum. Akinesia was improved by stimulation of the external pallidum but worsened by stimulation of the internal pallidum. In contrast, parkinsonian rigidity was improved by stimulation of either part of the pallidum. The areas in the internal pallidum where stimulation worsened akinesia were those in which stimulation reduced or suppressed L-DOPA-induced dyskinesias. Conversely, stimulation applied to the external pallidum induced dyskinesias. The fact that rigidity was improved by stimulation of the internal and external pallidum suggests that the neuronal bases of parkinsonian rigidity are different from those of akinesia and dyskinesias. The effect on akinesia and dyskinesias is in agreement with the current model of basal ganglia circuitry(10) if high-frequency stimulation activates rather than inhibits pallidal neurons, a possibility which is very likely since there are marked anatomical, biochemical and electrophysiological differences between the globus pallidus and the subthalamic nucleus.This study demonstrates that high-frequency stimulation of the globus pallidus in parkinsonian patients has contrasting effects depending on whether it is applied to the external or the internal part of this nucleus. The effect on akinesia and dyskinesias suggests that stimulation activates pallidal neurons, a result which challenges the generally accepted concept that high-frequency stimulation inactivates neurons in the region stimulated.