Publications

BACKGROUND

The Pl(A2) polymorphism of GPIIIa has been associated with unstable coronary syndromes in some studies, but the association has remained debated. None of the previous studies have focused on families at high risk. Risk factors tend to cluster within kindreds with high prevalence of premature coronary heart disease (CHD). Therefore, a heightened prevalence of the Pl(A2) polymorphism among siblings of patients with CHD would support the hypothesis that Pl(A2) is linked, directly or indirectly, to CHD.

OBJECTIVES

To measure the prevalence of the Pl(A2) polymorphism among siblings of patients with CHD before the age of 60 years and to seek an association between the Pl(A2) polymorphism and established atherosclerotic and thrombogenic risk factors.

METHODS

From January 1994 to April 1996, we genotyped 116 asymptomatic siblings (60 Caucasians, 56 Afro-Caribbeans) of patients with CHD manifestations before the age of 60 years for the Pl(A) polymorphism (also called HPA-1). A control cohort was used for comparison, consisting of individuals that were matched for race and geographic area but were free of CHD (n = 268, 168 Caucasians and 100 Afro-Caribbeans). In addition, we have characterized the sibling cohort for other atherogenic and thrombogenic risk factors.

RESULTS

The prevalence of Pl(A2)-positive individuals (Pl(A2)[+], Pl(A1/A2) heterozygotes plus Pl(A2/A2) homozygotes) in the sibling cohort was high: 41.4%. When analyzed separately, the prevalence of Pl(A2)(+) siblings was 53.3% among Caucasians and 28.6% among Afro-Caribbeans. There was no association between Pl(A2) and other established atherogenic or thrombogenic risk factors. Interestingly, the clustering of other risk factors was lesser among Pl(A2)(+) siblings than their Pl(A1) counterparts.

CONCLUSIONS

This study supports the hypothesis that the prevalence of Pl(A2)(+) individuals is high in kindreds with premature CHD. Hence, like the established risk factors that tend to cluster in families with premature CHD and contribute strongly to the accelerated atherosclerotic process affecting these individuals, the Pl(A2) polymorphism of GPIIIa may represent an inherited risk that promotes the thromboembolic complications of CHD. That these asymptomatic Pl(A2)(+) siblings had overall less established risk factors than their Pl(A1) counterparts might represent an explanation for why they remained asymptomatic despite their Pl(A2) positivity.

Experimental and clinical studies strongly suggest that gelatinase A plays a central role in the evolution of glomerular injury and sclerosis. The sequences of the 5' flanking regions of the human and rat gelatinase A genes do not share similarities with other members of the matrix metalloproteinase gene family and are regulated in a distinctive manner. The human and rat gelatinase A genes include regions of significant homology (r2 human; RE-1 rat), which have been shown to act as potent cis-activators of transcription. The rat RE-1 sequence interacts specifically with the developmentally regulated transcription factors AP2 and YB-1, resulting in a synergistic activation of gelatinase A transcription. Although the human r2 sequence specifically interacts with AP2 (Mol Cell Biol 10: 6524-6532, 1990), there is no clear evidence for the presence of a canonical YB-1 binding site (Y-box) within this sequence. This study demonstrates, despite the absence of a canonical Y-box sequence in the r2 element, that YB-1 and AP2 specifically interact with r2, yielding synergistic transactivation of the human gelatinase A gene. It is concluded that the r2 element is the conserved functional analog of the RE-1 element, and that interactions of AP2 and YB-1 govern human gelatinase A gene expression.

The alphav integrins are likely to be an important group of molecules for regulating astrocyte behaviour within the central nervous system. Together with their ligand vitronectin, they are expressed by astrocytes in vivo and are further upregulated during neurological disease. Here we have characterised the expression of alphav integrins on primary astrocytes from both rat and mouse, and shown that they express just two members, alphavbeta5 and alphavbeta8. By using RGD peptides and function-blocking antibodies against the beta1 integrins and alphavbeta5, we find that both alphavbeta5 and alphavbeta8 can act as functional receptors for vitronectin. However, while alphavbeta5 is largely responsible for astrocyte adhesion to vitronectin this integrin appears to play no role in migration on vitronectin, with alphavbeta8 playing the dominant role in promoting migration on this substrate. beta1 integrins are not involved in mediating interactions between astrocytes and vitronectin. These results were confirmed in experiments with astrocytes derived from mice in which the beta5 gene had been deleted by homologous recombination. beta5 null astrocytes attached to vitronectin at a reduced rate, but showed no defect in migration on vitronectin relative to wild-type astrocytes. These data provide the first evidence that alphavbeta8 regulates migration and show that astrocyte alphavbeta5 and alphavbeta8 integrins have distinct functions.

PURPOSE

To validate the use of polymerase chain reaction (PCR)-amplified full-length cDNA as a substitute for mRNA in nucleic acid array and gene expression analysis.

METHODS

Total RNA was isolated from age-matched normal autopsy corneas and pseudophakic bullous keratopathy (PBK) corneas. Full-length cDNA was generated and PCR amplified using the Smart cDNA synthesis technology. Southern blot analysis of this cDNA was compared with Northern blot analysis of the RNA. Amplified cDNA was used to probe a commercial gene array. By immunohistochemistry, the expression pattern of several adhesion molecules represented on the array was assessed.

RESULTS

The cDNA produced by the Smart cDNA system gave results very similar to those of northern blot analysis when examined for beta2-microglobulin, Rab geranylgeranyl transferase, and tenascin-C. This cDNA obtained from normal or PBK corneas was labeled and used to probe a 588 gene array (Clontech). Among other differences, beta6 integrin was detected only with the PBK probe, beta-catenin was markedly elevated in PBK, and beta4 integrin appeared to be reduced in PBK. Immunohistochemical patterns of these proteins were consistent with the hybridization signals on the gene array.

CONCLUSIONS

Smart cDNA synthesis and nucleic acid arrays were combined and validated for the first time to identify differential gene expression in normal and diseased corneas. These techniques require very little RNA such as that equivalent to a half of a single cornea, which is useful when the amount of tissue is limiting. Altered expression of adhesive proteins beta6 integrin and beta-catenin may be related to the formation of epithelial bullae and microcystic changes in PBK patients.

PURPOSE

Many occupational factors can cause asthma or reactivate preexisting disease. We carried out a critical review and synthesis of the available literature to estimate the proportion of adult asthma that is attributable to workplace factors.

METHODS

We reviewed published citations from 1966 through May 1999 as well as recent abstracts of studies providing risk estimates for asthma among various occupations. We extracted published attributable risk estimates, derived others from published data, and extrapolated estimates from the incidence rates of occupational asthma. We used a semiquantitative score to rank studies based on their characteristics.

RESULTS

We obtained 43 attributable risk estimates from 19 different countries: 23 were published estimates, 8 were derived from published data, and 12 were extrapolated from incidence data. The median value for the attributable risk of occupationally associated asthma was 9%(25th to 75th interquartile range: 5% to 19%). The derived estimates (median attributable risk = 25%) were significantly greater than published values (median = 9%, P = 0.002), whereas the extrapolated estimates were significantly lower (median = 5%, P = 0.04). The 12 highest scored studies based on their characteristics yielded a median risk estimate of 15%.

CONCLUSION

Occupational factors are associated with about 1 in 10 cases of adult asthma, including new onset disease and reactivation of preexisting asthma.

Work disability due to respiratory disease, especially asthma, is common and costly among working age adults. The goal of this analysis was to characterize the risk factors for such disability. We analyzed data from the Swedish part of the European Community Respiratory Health Survey (ECRHS), a random population-based sample of adults age 20 to 44, enriched with symptomatic subjects at increased likelihood of having asthma. We analyzed structured interview data available for 2,065 subjects and further analyzed methacholine challenge and skin prick test data for 1,562 of these. We defined respiratory work disability as reported job change or work loss due to breathing affected by a job. We used binary generalized linear modeling with a log link to estimate disability risk. Eighty-four subjects (4%) reported such work disability. This increased to 13% among those with asthma (45 of 350 subjects). Adjusting for covariates, occupations at high risk for asthma were associated with disability (prevalence ratio [PR] 1.8; 95% confidence interval [CI] 1.1 to 3.0), as was self-reported regular exposure to environmental tobacco smoke (ETS) at work (PR 1.8; 95% CI 1.1 to 3.1) and self- reported job exposure to vapors, gases, dust, or fumes (VGDF) (PR 4.3; 95% CI 2.2 to 8.6). Workplace ETS exposure was also associated with methacholine challenge-positive asthma reported to be symptomatic at work among male subjects (PR 4. 2; 95% CI 1.8 to 9.8), whereas high asthma-risk occupations were associated with this outcome among female subjects (PR 2.7; 95% CI 1. 05 to 7.1). Respiratory work disability, defined as breathing-related job change due to work loss, was associated with workplace exposures themselves, even after taking into account other covariates. Better control of workplace exposures, including workplace ETS, may reduce work disability caused by respiratory conditions, especially adult asthma.

Repeated treatment of female rats with the synthetic estrogen ethynylestradiol (EE(2)) increases the formation of the cyclosporine A (CyA) metabolites AM1c and AM9 by 3-fold, whereas the formation of AM1 and AM4N is not significantly enhanced. The formation of all four CyA metabolites was inhibited by greater than 80% by the CYP3A-selective substrate midazolam or polyclonal anti-rat CYP3A IgGs in liver microsomes of untreated and EE(2)-induced rats. In contrast, anti-rat CYP2C6 IgGs had little effect, indicating the involvement of a CYP3A but not 2C6 in this EE(2)-stimulated CyA metabolism. Semiquantitative reverse-transcriptase polymerase chain reaction was used to determine the mRNA content for four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in livers of control and EE(2)-treated female rats. EE(2) selectively induced CYP3A9 by 3.3-fold whereas the expression of CYP3A18 and CYP3A23 was slightly decreased; neither CYP3A2 mRNA nor CYP3A1 mRNA was detectable in these EE(2)-treated livers. To determine whether rat liver microsomal CYP3A9 was indeed responsible for the EE(2)-stimulated CyA metabolism, a recombinant CYP3A9 was heterologously expressed in Escherichia coli. When functionally reconstituted, this enzyme was active in metabolizing CyA preferentially to its AM9 and AM1c metabolites as compared with CYP3A4. These findings thus support the notion that the increased CyA-metabolizing capacity of EE(2)-treated female rat liver microsomes is due to the induction of the CYP3A9 enzyme.