Separation of ECHO virus type 6 antigens by centrifugation in caesium chloride density gradients

Summary ECHO virus 6 particles separated by caesium chloride density gradient centrifugation were studied by electron microscopy. Particles in fractions of a density of 1.33 g/cm³, carrying peak infectivity and N antigenicity, appeared in the electron microscope as complete virions. The diameter was found to be 25 m. Particles in fractions of a density of 1.30 g/cm³, carrying H antigenicity, appeared as more or less empty capsids. This was true for the naturally occurring H antigen as well as for H antigen obtained by heat treatment.     

Isolation of a peptide inhibitor of human rhinovirus

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.     

Human circulating specific antibody-forming cells after systemic and mucosal immunizations: differential homing commitments and cell surface differentiation markers

Circulating spontaneous antibody-secreting cells (ASC) induced by mucosal and systemic immunizations in human volunteers have been characterized with respect to differentiation stage and homing commitments. Irrespective of the immunization route, the large majority of ASC co-expressed CD19 and HLA-DR, which are normally lost during the transition of plasmablasts to plasmocytes, as well as CD38, a marker of activated B cell blasts, expressed also by plasmocytes. However, these cells expressed neither CD28, a molecule acquired by plasmocytes, nor CD22 and CD37, which are lost during the transition of plasmablasts to plasmocytes. Therefore, the large majority of ASC found in peripheral blood after oral and parenteral immunizations are terminally differentiated B cells, but not fully differentiated plasmocytes. As a whole, the mucosally derived ASC population seemed to be more homogenously differentiated. CD25 was detected on few ASC, whereas ASC expressing CD71 were more numerous, especially among systemically derived ASC. Almost all ASC expressed the adhesion molecules CD44 and α4-integrins, irrespective of immunization route. However, virtually all systemically derived ASC expressed L-selectin, recognizing the peripheral lymph node addressin, whereas only a minority of mucosally induced blood ASC expressed L-selectin. These studies are the first to demonstrate in humans that circulating precursors of mucosal B cell immunoblasts utilize organ-specific recognition mechanisms distinct from those of corresponding systemic B cells and appear to be more advanced in the B lineage maturation pathway. Specialization of receptor expression could explain both the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from non-mucosal immune mechanisms in humans.     

Four new adenosine deaminase mutations,altering a zinc-binding histidine,two conserved alanines,and a 5′ splice site

Three new missense mutations (H15D, A83D, and A179D) and a new splicing defect (573 + 1G→A) in the 5′ splice site of intron 5 were among six mutant adenosine deaminase (ADA) alleles found in three unrelated patients with severe combined immunodeficiency disease, the most common phenotype associated with ADA deficiency. When expressed in vitro, the H15D, A83D, and A179D proteins lacked detectable ADA activity. The splicing defect caused skipping of exon 5, resulting in premature termination of translation and a reduced level of mRNA. H15D is the first naturally occurring mutation of a residue that coordinates directly with the enzyme-associated zinc ion. Molecular modeling based on the atomic coordinates of murine ADA suggests that the D15 mutation would create a cavity or gap between the zinc ion and the side chain carboxylate of D15. This could alter the ability of zinc to activate a water molecule postulated to play a role in the catalytic mechanism. A83 and A179 are not directly involved in the active site, but are conserved residues located respectively in a helix 4 and β strand 4 of the α/β barrel. Replacement of these small hydrophobic Ala residues with the charged, more bulky Asp side chain may distort ADA structure and affect enzyme stability or folding.© 1995 wiley-Liss, Inc.     

Autonomic Nervous System Function and Essential Hypertension: Individual Response Specificity With and Without Beta-Adrenergic Blockade

The aim of the present research was to study individual response specificity in 22 male patients having essential hypertension (HT) and to compare these patients with age-matched male normotensive controls (NT). Four stimuli, letter identification, mental arithmetic, cold pressor and isometric exercise, were administered while recordings were made of: systolic and diastolic blood pressures, heart rate, respiration, forearm and hand blood flows, and skin conductance level and fluctuations. After each session urine samples were collected and epinephrine and norepinephrine levels were analyzed. Twelve subjects in the HT group were given beta-adrenergic blocking agents and retested 1 to 21 months (X?= 12 months) after the first session. Each response was standardized, using NT as the reference group. Intraclass correlations were computed to evaluate whether HT males reacted with a more consistent hierarchy of responses than did NT. Intraclass correlations were significantly higher among the patients than in the control group, regardless of whether the blood pressure response was included or excluded in the computation of the intraclass correlations. Thus, we conclude that male HT patients show more individual response specificity than NT controls. Beta-adrenergic receptor antagonists reduced levels of cardiovascular activity and attenuated reactivity but did not affect amount of specificity. Thus, intraclass correlations provide unique and useful information, since they are not related to blood pressure reactivity or to urinary catecholamine levels, nor affected by beta-adrenergic blockade.     

Spontaneous activity of single neurons in the olfactory bulb of the rainbow trout (Salmo gairdneri) and its modulation by olfactory stimulation with amino acids

Summary Spontaneous action potentials recorded from 126 neurons in the olfactory bulb of Salmo gairdneri show a higher tendency towards bursting patterns of activity than those shown in previous reports about other fish. Granule cells and periglomerular cells are more likely to fire in bursts than mitral cells. Natural chemical stimulation of the olfactory mucosa with amino acid solutions produced a unique pattern of excitatory and inhibitory responses across all units. Chi-square values were calculated for stimulatory effectiveness between forty-five pairs of odours. L-serine and L-alanine consistently showed a high degree of similarity with several other odours. The converse was true for GABA and L-histidine, although this pair had a high chi-square value when mutually compared. Enantiomeric pairs of amino acids were often found to have opposite stimulatory effects on bulbar units. These results are discussed in relation to the possible properties and configurations of odorant receptor sites for amino acids in the fish olfactory mucosa.     

Changes in peritoneal myeloid populations and their proinflammatory cytokine expression during infection with Listeria monocytogenes are altered in the absence of gamma/delta T cells

Evidence that gamma/delta T cells play a broad, immunoregulatory role has been accumulating steadily. We show here that myeloid cells are disregulated after peritoneal infection with Listeria monocytogenes in mice lacking gamma/delta T cells. Inflammatory populations of neutrophils and monocytes recruited to the site of infection remained longer. Intracellular cytokine analysis showed that frequencies of myeloid cells producing interleukin-12 and tumor necrosis factor alpha were higher and remained elevated longer after infection in mice genetically deficient in gamma/delta T cells. In vivo dye-tracking studies indicated that the majority of inflammatory monocytes differentiated into resident tissue macrophages in situ. In vitro experiments confirmed that monocytes harvested from mice lacking gamma/delta T cells were defective in their maturation process. This evidence suggests that gamma/delta T cells promote differentiation in the monocyte/macrophage lineage. These cells are important for bactericidal activity, inflammatory cytokine production, clearance of inflammatory neutrophils, and ultimately, antigen presentation to T cells. Regulation of monocyte/macrophage differentiation may underlie a broad segment of the phenotypic alterations that have been reported in mice lacking gamma/delta T cells.     

Difference in allelic expression of the CLCN1 gene and the possible influence on the myotonia congenita phenotype

Mutations in the CLCN1 gene, encoding a muscle-specific chloride channel, can cause either recessive or dominant myotonia congenita (MC). The recessive form, Becker's myotonia, is believed to be caused by two loss-of-function mutations, whereas the dominant form, Thomsen's myotonia, is assumed to be a consequence of a dominant-negative effect. However, a subset of CLCN1 mutations can cause both recessive and dominant MC. We have identified two recessive and two dominant MC families segregating the common R894X mutation. Real-time quantitative RT-PCR did not reveal any obvious association between the total CLCN1 mRNA level in muscle and the mode of inheritance, but the dominant family with the most severe phenotype expressed twice the expected amount of the R894X mRNA allele. Variation in allelic expression has not previously been described for CLCN1, and our finding suggests that allelic variation may be an important modifier of disease progression in myotonia congenita.     

Routine mortality monitoring for detecting mass murder in UK general practice: test of effectiveness using modelling.

BACKGROUND: The Shipman Inquiry recommended mortality rate monitoring if it could be 'shown to be workable' in detecting a future mass murderer in general practice. AIM: To examine the effectiveness of cumulative sum (CUSUM) charts, cross-sectional Shewhart charts, and exponentially-weighted, moving-average control charts in mortality monitoring at practice level. DESIGN OF STUDY: Analysis of Scottish routine general practice data combined with estimation of control chart effectiveness in detecting a 'murderer' in a simulated dataset. METHOD: Practice stability was calculated from routine data to determine feasible lengths of monitoring. A simulated dataset of 405,000 'patients' was created, registered with 75 'practices' whose underlying mortality rates varied with the same distribution as case-mix-adjusted mortality in all Scottish practices. The sensitivity of each chart to detect five and 10 excess deaths was examined in repeated simulations. The sensitivity of control charts to excess deaths in simulated data, and the number of alarm signals when control charts were applied to routine data were estimated. RESULTS: Practice instability limited the length of monitoring and modelling was consequently restricted to a 3-year period. Monitoring mortality over 3 years, CUSUM charts were most sensitive but only reliably achieved >50% successful detection for 10 excess deaths per year and generated multiple false alarms (>15%). CONCLUSION: At best, mortality monitoring can act as a backstop to detect a particularly prolific serial killer when other means of detection have failed. Policy should focus on changes likely to improve detection of individual murders, such as reform of death certification and the coroner system.