Comparison of Discrete Cardiovascular Fitness Groups on Plasma Catecholamine and Selected Behavioral Responses to Psychological Stress

Discrete cardiovascular fitness groups consisting of high-fit (n=10) and low-fit (n=9) men performed a well-learned vigilance task and their self-report, performance, and plasma catecholamine responses were compared. No significant differences were observed between the fitness groups on self-report or psychomotor performance responses to the vigilance task. However, the low-fit group took significantly longer than high-fit subjects to complete the first of three sets of anagrams administered immediately after the vigilance task. Plasma norepinephrine but not epinephrine response was greater in the low-fit group compared to their high-fit counterparts. The findings indicate that enhanced cardiovascular fitness may be characterized by an attenuated plasma norepinephrine response to a vigilance task with sustained cognitive performance subsequent to the task.     

Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

B7/BB-1 is a leucocyte differentiation antigen on human dendritic cells induced by activation.

Activation of a primary T-lymphocyte response requires additional signals apart from interaction of the T-cell receptor (TcR)/CD3 complex with major histocompatibility complex (MHC) antigens on the antigen-presenting cell. The CD28 antigen on T lymphocytes provides an important co-stimulatory signal to T lymphocytes and we therefore searched for the presence of its ligand, the B7/BB-1 antigen, on blood and tonsil dendritic cells (DC). Blood DC, prepared from peripheral blood mononuclear cells with a minimal period of in vitro culture, did not stain with the monoclonal antibody BB-1 using flow cytometry analysis. In contrast, tonsil DC stained weakly for B7/BB-1 compared to positive control cell lines. Polymerase chain reaction (PCR) was used to amplify a 605 base pair (bp) fragment from human B7/BB-1 mRNA and demonstrated significant amounts of B7/BB-1 mRNA in tonsil DC but no specific product was obtained from blood DC, confirming the surface-staining results. Weak expression of B7/BB-1 antigen was detected by immunofluorescence analysis following culture of blood DC with either interferon-gamma (IFN-gamma) or granulocyte-macrophage colony-stimulating factor (GM-CSF). These data support the concept that blood DC give rise to tissue and/or lymphoid DC, which acquire co-stimulatory ligands as a result of activation and/or differentiation.     

Dra/AfaE adhesin of uropathogenic Dr/Afa+ Escherichia coli mediates mortality in pregnant rats

Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa⁺ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE⁺ afaD and afaE afaD⁺ mutants. The clinical E. coli strain (afaE⁺ afaD⁺) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE⁺ afaD⁺ strain, followed by the group infected with the afaE⁺ afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE⁺ strains were the most invasive (afaE⁺ afaD strain > afaE⁺ afaD⁺ strain), while the afaE mutant strains (afaE afaD⁺ and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE⁺ E. coli strains in vitro.     

Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa

A multiresistant strain of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients attending clinics in Liverpool, United Kingdom. Suppression subtractive hybridization was used to identify sequences present in the Liverpool CF epidemic strain but absent from strain PAO1. Using dot blot and PCR amplification assays, the prevalence of such sequences among a panel of CF isolates was determined. Several sequences were found only in the Liverpool epidemic strain. Some sequences were present in the Liverpool epidemic strain and in a minority of other isolates, including sequences with homology to genes implicated in O6 serotype and siderophore production. The Liverpool epidemic strain and 81% of nonepidemic isolates contained a sequence identified as part of the PAGI-1 genomic island. Other strains implicated in epidemic spread, which were from Manchester, United Kingdom, and Melbourne, Australia, were also screened. None of the sequences identified was present in the Manchester strain. However, one of two Melbourne strains contained some of the sequences found in the Liverpool epidemic strain. All isolates implicated in epidemic spread and 76% of sporadic isolates contained the exoS gene. A sequence present in all isolates of the Liverpool epidemic strain was used to develop a diagnostic PCR test for identification of the strain from colonies or directly from sputum samples.     

Presence of type III secretion genes in Burkholderia pseudomallei correlates with Ara(-) phenotypes

Dot blot hybridization and PCR amplification of 14 Ara(+) and 8 Ara(-) Burkholderia pseudomallei strains showed that type III secretion (TTS) genes were present in all the Ara(-) strains but absent from all but one of the Ara(+) strains. The link between TTS genes and an Ara(-) phenotype suggests a role for TTS in virulence.     

Enamel structure and composition in the tricho-dento-osseous syndrome

Tricho-dento-osseous syndrome (TDO) is an autosomal dominant disorder characterized by curly hair, hypoplastic enamel, taurodontism, and dense bone. The purpose of this investigation was to characterize the enamel defects in a TDO population in North Carolina. Twelve TDO teeth and 12 normal teeth were examined. The enamel thickness was decreased in all TDO teeth ranging from having no enamel to about 60% the thickness of normal teeth. Half of the TDO teeth had primarily prismless enamel while the remainder had at least occasional areas of prismatic enamel. TDO enamel crystallites appeared similar to normal crystallites with TEM. The mineral per volume of TDO enamel (n = 9) (68.5%) was significantly less, on average, compared with normal enamel (n = 8) (84.5). The genetic mutation responsible for the TDO phenotype results in alteration of a developmental pathway(s) common to hair, teeth and bone. This further illustrates that these embryologically diverse tissues share common developmental controls at the molecular level.     

Differences in sensitivity to melphalan between con A-activated and nonactivated human T-cell subsets

In vitro treatment with melphalan (L-PAM, L-phenylalanine mustard), 2 micrograms/2 X 10(6) cells, significantly decreased the total number of E-rosette-positive (E+) T lymphocytes from peripheral blood (PBL) of healthy human donors as well as those of the OKT4 (precursor suppressor/helper/inducer T cells) and OKT17 populations (suppressor cells within the OKT4 subset). The OKT8 population (cytotoxic/mature suppressor cells) was not affected by a similar L-PAM treatment. The sensitivity of concanavalin A (Con A)-activated E+ T-cell populations to subsequent L-PAM treatment in vitro was different from that of Con A-untreated T cells: Thus, L-PAM treatment did not affect the expression of OKT3 and OKT4 antigens, increased the percentage of OKT17 cells, and inhibited the expression of OKT8 antigen. Depletion of OKT8 from Con A-activated E+ T cells (OKT4+-OKT8(-)-OKT17+) did not affect their suppressive activity on PHA stimulation in L-PAM-treated as well as untreated cells. Further depletion of OKT17+ cells from the OKT4+-OKT8(-)-OKT17+ subset (OKT4+-OKT8(-)-OKT17-) abolished the suppressive effect on PHA stimulation. Suppressive activity of the OKT4+-OKT8(-)-OKT17- subset was again evident after treatment of this population with L-PAM. The results obtained indicate that the sensitivity to L-PAM treatment of various T-cell phenotypes is changed by Con A activation and that after depletion of specific T suppressor cells L-PAM seems affect the immunoregulatory circuit within the Con A-activated OKT4 subset.     

Autoantibodies to vascular smooth muscle are pathogenic for vasculitis

We have previously shown that microvascular smooth muscle activates CD4+ T lymphocytes in sterile co-culture, presents antigen, and produces inflammatory cytokines. Adoptive transfer of lymphocytes co-cultured with syngeneic smooth muscle cells to healthy recipient mice results in vasculitic lesions predominantly in postcapillary venules. The present study assessed the pathogenic role of immunoglobulin and B cells in a murine model of vasculitis. Here, we show that transferred B cells, including plasmablast cells, accumulated, persisted, and proliferated in lung and secondary lymphoid organs of recipient mice. The induction of vasculitis was accompanied by production of IgM and IgG2a autoantibodies specific for vascular smooth muscle intracellular antigens. Circulating immunoglobulin had a pathogenic role in this vasculitis model, because the disease could be induced by transfer of serum from vasculitic mice to untreated animals but not by transfer of serum depleted of anti-smooth muscle autoantibodies. Additionally, the pathogenic mechanisms triggered by the transfer of vasculitogenic serum were dependent on T lymphocytes because both wild-type and B cell-deficient mice developed the disease after serum transfer, whereas RAG2-deficient mice did not. Thus, immunoglobulin and cell-mediated pathways work in concert to produce vasculitis in this model.