Quantitation of human immunodeficiency virus type 1 DNA by two PCR procedures coupled with enzyme-linked oligosorbent assay.

Two quantitative PCR methods with our nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format were developed for quantitation of human immunodeficiency virus type 1 (HIV-1). Quantitative competitive PCR (QC-PCR) was based on the coamplification of the wild-type nef region with a mimic competitive nef gene template carrying mutations in the capture region. Correlation of wild-type HIV-1 nef DNA to mimic template copy number permitted quantitation of HIV-1 copy numbers in the range of 20 to 2,000 copies per micrograms of DNA. Internally controlled PCR (IC-PCR) was based on coamplification of the nef region and the ras gene as an internal endogenous standard. Correlation to known amounts of HIV-1 DNA permitted quantitation by IC-PCR of HIV-1 copy numbers in the range of 10 to 2,000 copies per microgram of DNA. QC- and IC-PCR-ELOSA were performed on a panel of 53 seropositive patients and 12 seronegative controls. The methods showed similar coefficients of variation below 24%. Quantitations by QC- and IC-PCR-ELOSA were identical for 77% of patient samples. The copy level ranged between 443 +/- 156 and 21,453 +/- 13,511 copies per 10(5) CD4 cells for asymptomatic and AIDS patients, respectively. The simplicity and reliability of QC- and IC-PCR-ELOSA methods make them appropriate for routine laboratory use in the quantitation of viral and bacterial DNAs.     

Neutrophil-aggregating activity of monohydroxyeicosatetraenoic acids.

The following oxidative derivatives of arachidonic acid were prepared and assayed for their ability to aggregate cytochalasin-B-pretreated human neutrophils: 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids. The compounds were prepared by oxidation of arachidonic acid and purified by direct and reverse phase high performance liquid chromatography. Each lipid was racemic at the hydroxy residue and had a cistrans conjugated double bond adjacent to the hydroxy residue. Except for racemization, therefore, they were identical to hydroxyeicosatetraenoic acids generated by neutrophils exposed to diverse aggregating stimuli. In addition, 15-L-hydroxyeicosatetraenoic acid was prepared from soybean lipoxygenase. Of these 7 fatty acid preparations, only 5- and 12-hydroxyeicosatetraenoic acid aggregated the cells. Thus, the bioactions of these lipids are crucially dependent upon the position of the hydroxy residue. The 5- and 12-hydroxy derivatives were potent aggregating agents, inducing half-maximal responses at 200 and 40 nM, respectively. Their bioactions required extracellular calcium and magnesium. And the response to both fatty acids was effectively blocked by three inhibitors of cellular arachidonic acid metabolism: nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, and indomethacin. The 5- and 12- hydroxyeicosatetraenoic acids, therefore, may induce neutrophils to metabolize their endogenous arachidonate. Alternatively, the two hydroxy acids themselves may be further metabolized through pathways inhibited by arachidonate antimetabolites into a final mediator(s) of aggregate formation.     

Tissue-specific segregation of TCRgamma delta+ NKT cells according to phenotype TCR repertoire and activation status: parallels with TCR alphabeta+NKT cells

Whereas the majority of NKT cells in the mouse express an alpha beta TCR (NKTalpha beta cells), a small subset of NKT cells express a gamma delta TCR (NKTgamma delta). Here we have systematically analyzed the phenotype, TCR repertoire and activation status of NKTgamma delta cells in the thymus, liver, spleen and bone marrow of normal C57BL/6 mice. Our data indicate that NKTgamma delta cells segregate in a tissue-specific manner according to these parameters. While most NKTgamma delta cells in the thymus and liver have a recently activated CD62L(lo) phenotype and a TCR repertoire that is heavily biased to Vgamma1.1 and Vdelta6.3, the majority of NKTgamma delta cells in the spleen and bone marrow are CD62L(hi) and have a much less biased TCR repertoire. Moreover, expression of NK markers is high on NKTgamma delta cells in spleen and bone marrow but low in thymus and liver. Collectively our results reveal a tissue-specific segregation of NKTgamma delta cells that is strikingly similar to that recently described for CD1d-dependent and Cd1d-independent NKTalpha beta cells. We therefore propose that chronic TCR activation by tissue-specific endogenous ligands is a generic property of NKT cells of both the alpha beta and gamma delta lineages.     

Popliteal pterygium syndrome: a clinical study of three families and report of linkage to the Van der Woude syndrome locus on 1q32

Popliteal pterygium syndrome (PPS) is a rare autosomal dominant disorder, thought to occur with an incidence of approximately 1 in 300 000 live births. The main clinical manifestations are popliteal webbing, cleft lip, cleft palate, lower lip pits, syndactyly, and genital and nail anomalies. This report describes the clinical features in two families with PPS and one isolated case, showing the range of anomalies found both within and between the families. PPS has some features in common with Van der Woude syndrome (VWS), also inherited as an autosomal dominant condition, with cleft lip/palate and, more distinctively, lower lip pits. Although the gene for VWS has not yet been identified, it has been localised to within 1.6 cM in the region 1q32-41. To determine whether PPS and VWS represent allelic forms of the same gene, three families were genotyped for markers flanking and within the critical region. A multipoint lod score of 2.7 was obtained, with no evidence of recombination, supporting the hypothesis that these two disorders are allelic.     

Causal dimensions of college students' perceptions of physical symptoms

According to attribution theory, controllability, locus, and stability are important dimensions underlying causal explanations. The extent to which these theoretical dimensions underlie lay explanations for physical symptoms is unclear. Accordingly, in this study, attributes relevant to the lay public were empirically derived using a multidimensional scaling (MDS) procedure. Undergraduates (N=194) provided similarity judgments for 18 potential causes of physical discomfort. The MDS analysis yielded a three-dimensional solution. The first dimension captured the distinction between physical and nonphysical causes. The second dimension distinguished either variable versus stable causes or those that are controllable versus uncontrollable by health care professionals. The third dimension differentiated causes under low versus high personal control. These findings empirically confirm the theoretically proposed dimensions of personal control and stability and suggest the utility of considering the physical/nonphysical and controllability by health care professional distinctions in future work on attributions in the health domain.     

Activation of the endothelium by IL-1 alpha and glucocorticoids results in major increase of complement C3 and factor B production and generation of C3a.

Constitutive secretion of complement C3 and factor B by the endothelial cell (EC) is lowered by therapeutic concentrations of glucocorticoids such as hydrocortisone or dexamethasone, whereas regulatory protein factor H production is increased by these hormones. In contrast, the proinflammatory cytokine IL-1 alpha has a stimulatory effect on C3 and factor B secretion by the endothelium and an inhibitory effect on factor H secretion. In this study, we examined the combined effect of IL-1 alpha and glucocorticoids on C3 and factor B expression by the endothelial cell. When dexamethasone or hydrocortisone were added to IL-1 alpha, significant potentialization of IL-1 alpha-induced stimulation of C3 and factor B production was observed, occurring at various concentrations of either stimuli. Dose-response experiments indicate that, in vitro, optimal concentrations are in the range of 10(-7) to 10(-5) M for dexamethasone and 50-200 U for IL-1 alpha. In contrast, dexamethasone counteracts, in an additive way, the inhibitory effect of IL-1 alpha on regulatory complement protein factor H production by EC. Such a potentialization between glucocorticoids and IL-1 alpha was not observed for another marker of endothelial activation, IL-1 alpha-induced stimulation of coagulation tissue factor expression. The association of glucocorticoids and IL-1 alpha therefore appears to be a specific and major stimulus for the secretion of complement C3 and factor B, two acute-phase proteins, by the endothelium. As a result of the in vitro endothelium stimulation by glucocorticoids and IL-1 alpha, C3a is generated in the vicinity of the endothelial cell. This study further suggests that complement activation, with its deleterious consequences, may result from the stimulation of endothelium in situations where high levels of IL-1 alpha and endogenous glucocorticoids coexist, such as in septic shock.     

Expression analysis of chick Wnt and frizzled genes and selected inhibitors in early chick patterning.

Wnt signaling is an important component in patterning the early embryo and specifically the neural plate. Studies in Xenopus, mouse, and zebrafish have shown that signaling by members of the Wnt family of secreted signaling factors, their Frizzled receptors and several inhibitors (sFRP1, sFRP2, sFRP3/Frzb1, Crescent/Frzb2, Dkk1, and Cerberus) are involved. However, very little is known about the expression of genes in the Wnt signaling pathway during early anterior neural patterning in chick. We have performed an expression analysis at neural plate stages of several Wnts, Frizzled genes, and Wnt signaling pathway inhibitors using in situ hybridization. The gene expression patterns of these markers are extremely dynamic. We have identified two candidate molecules for anterior patterning of the neural plate, Wnt1 and Wnt8b, which are expressed in the rostral ectoderm at these stages. Further functional studies on the roles of these markers are underway.