A novel protein C-factor VII chimera provides new insights into the structural requirements for cytoprotective protease-activated receptor 1 signaling.

Essentials The basis of cytoprotective protease-activated receptor 1 (PAR1) signaling is not fully understood. Activated protein C chimera (APCFVII-82 ) was used to identify requirements for PAR1 signaling. APCFVII-82 did not initiate PAR1 signaling, but conferred monocyte anti-inflammatory activity. APC-specific light chain residues are required for cytoprotective PAR1 signaling. SUMMARY: Background Activated protein C (APC) cell signaling is largely reliant upon its ability to mediate protease-activated receptor (PAR) 1 proteolysis when bound to the endothelial cell (EC) protein C (PC) receptor (EPCR). Furthermore, EPCR-bound PC modulates PAR1 signaling by thrombin to induce APC-like EC cytoprotection. Objective The molecular determinants of EPCR-dependent cytoprotective PAR1 signaling remain poorly defined. To address this, a PC-factor VII chimera (PCFVII-82 ) possessing FVII N-terminal domains and conserved EPCR binding was characterized. Methods Activated PC-FVII chimera (APCFVII-82 ) anticoagulant activity was measured with calibrated automated thrombography and activated FV degradation assays. APCFVII-82 signaling activity was characterized by the use of reporter assays of PAR1 proteolysis and EC barrier integrity. APCFVII-82 anti-inflammatory activity was assessed according to its inhibition of nuclear factor-κB (NF-κB) activation and cytokine secretion from monocytes. Results PCFVII-82 was activated normally by thrombin on ECs, but was unable to inhibit plasma thrombin generation. Surprisingly, APCFVII-82 did not mediate EPCR-dependent PAR1 proteolysis, confer PAR1-dependent protection of thrombin-induced EC barrier disruption, or limit PAR1-dependent attenuation of interleukin-6 release from lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, EPCR occupation by active site-blocked APCFVII-82 was, like FVII, unable to mimic EC barrier stabilization induced by PC upon PAR1 proteolysis by thrombin. APCFVII-82 did, however, diminish LPS-induced NF-κB activation and tumor necrosis factor-α release from monocytes in an apolipoprotein E receptor 2-dependent manner, with similar efficacy as wild-type APC. Conclusions These findings identify a novel role for APC light chain amino acid residues outside the EPCR-binding site in enabling cytoprotective PAR1 signaling.

Inhibition of major integrin αV ß3 reduces Staphylococcus aureus attachment to sheared human endothelial cells.

Essentials Staphylococcus aureus (S. aureus) binds and impairs function of vascular endothelial cells (EC). We investigated the molecular signals triggered by S. aureus adhesion to EC. Inhibition of the EC integrin αVß3 reduces S. aureus binding and rescues EC function. αVß3 blockade represents an attractive target to treat S. aureus bloodborne infections. SUMMARY: Background Vascular endothelial dysfunction with associated edema and organ failure is one of the hallmarks of sepsis. Although a large number of microorganisms can cause sepsis, Staphylococcus aureus (S. aureus) is one of the primary etiologic agents. Currently, there are no approved specific treatments for sepsis, and the initial management bundle is therefore focused on cardiorespiratory resuscitation and mitigation of the immediate threat of uncontrolled infection. The continuous emergence of antibiotic-resistant strains of bacteria necessitates the development of new therapeutic approaches for this disease. Objective To identify the molecular mechanisms leading to endothelial dysfunction as a result of S. aureus binding. METHODS: Binding of wild type and Clumping factor A (ClfA) deficient S. aureus Newman to the endothelium was measured in vitro and in the mesenteric circulation of C57Bl/6 mice. The effects of the αV ß3 blocker-cilengitide-on bacterial binding, endothelial VE-cadherin expression, apoptosis, proliferation and permeability were assessed. Results The major S. aureus cell wall protein ClfA bound to endothelial cell αV ß3 in the presence of fibrinogen. This interaction resulted in disturbances in barrier function mediated by VE-cadherin in endothelial cell monolayers, and ultimately cell death by apoptosis. With a low concentration of cilengitide, ClfA binding to αV ß3 was significantly inhibited both in vitro and in vivo. Moreover, preventing S. aureus from attaching to αV ß3 resulted in a significant reduction in endothelial dysfunction following infection. Conclusion Inhibition of S. aureus ClfA binding to endothelial cell αV ß3 by cilengitide prevents endothelial dysfunction.

Development of a life events/icon calendar questionnaire to ascertain occupational histories and other characteristics of migrant farmworkers.

BACKGROUND: Specialized methods are necessary to collect data from migrant farmworkers for epidemiologic research. METHODS: We developed a questionnaire that collected lifetime occupational histories and other lifestyle risk factors via a life events/icon calendar, and administered the questionnaire to a convenience sample of 162 migrant farmworkers in nine areas of the U.S. RESULTS: The average duration of the interviews was about 1 h 30 min, with an average of 45 min for the work history section. The occupational histories covered a median of 27.6 years per person for men and 20.8 years per person for women. The median number of years spent in farm jobs was 11.3 for men and 5.8 for women. The median number of farm jobs (crop/task combination) per person was 59 among men and 27 among women. Many farmworkers performed the same crop/task combinations at multiple times throughout their lives, yielding a median of 13 unique farm jobs and 8 unique crops among men and 7 jobs and 5 crops among women. CONCLUSIONS: The project demonstrated that it is feasible to collect detailed work histories and other risk factor data from farmworkers, documented the complexity of work histories encountered among farmworkers, and yielded recommendations for refining a questionnaire that will facilitate future epidemiologic research on farmworkers.

A model for predicting the frequency of high pesticide exposure events in the Agricultural Health Study.

The frequency of self-reported high pesticide exposure events (HPEE) has been recorded in the NCI/EPA/NIEHS Agricultural Health Study (AHS). Fourteen percent (14%) of the enrolled applicators responding reported "an incident or experience while using any pesticide which caused an unusually high exposure." These data show, as expected, that the probability of a report of an HPEE increases with the cumulative number of days of pesticide application reported by the applicator. We have developed a three-parameter model that predicts the risk odds ratio (OR) of an HPEE as a function of the number of days that pesticides are applied. These events are costly in terms of resulting health-care visits, lost time from work, and potential risk for cancer and other chronic diseases. We propose that failure to carefully follow all the pesticide manufacturer's label requirements, inexperience, and random events (i.e., breaking hose) are the three factors responsible for the events observed. Special precautions for new or infrequent users of pesticides are indicated.

Characteristics of pesticide use in a pesticide applicator cohort: the Agricultural Health Study.

Data on recent and historic pesticide use, pesticide application methods, and farm characteristics were collected from 35,879 restricted-use pesticide applicators in the first 2 years of the Agricultural Health Study, a prospective study of a large cohort of private and commercial licensed pesticide applicators that is being conducted in Iowa and North Carolina. (In Iowa, applicators are actually "certified," while in North Carolina they are "licensed"; for ease of reference the term license will be used for both states in this paper.) Commercial applicators (studied in Iowa only) apply pesticides more days per year than private applicators in either state. When the types of pesticides being used by different groups are compared using the Spearman coefficient of determination (r2), we find that Iowa private and Iowa commercial applicators tend to use the same type of pesticides (r2=0.88). White and nonwhite private applicators tended to use the same type of pesticides (North Carolina r2=0.89), as did male and female private applicators (Iowa r2=0.85 and North Carolina r2=0.84). There was less similarity (r2=0. 50) between the types of pesticides being used by Iowa and North Carolina private applicators. A greater portion of Iowa private applicators use personal protective equipment than do North Carolina private applicators, and pesticide application methods varied by state. This heterogeneity in potential exposures to pesticides between states should be useful for subsequent epidemiologic analyses using internal comparison groups.

Characteristics of persons who self-reported a high pesticide exposure event in the Agricultural Health Study.

Characteristics of persons who report high pesticide exposure events (HPEE) were studied in a large cohort of licensed pesticide applicators from Iowa and North Carolina who enrolled in the Agricultural Health Study between December 1993 and December 1995. Fourteen percent reported having "an incident or experience while using any pesticide which caused an unusually high personal exposure. " After taking into account total number of applications made and education, females (OR=0.76), applicators from NC (OR=0.65), and privately licensed applicators (OR=0.65) were less likely to have reported an HPEE. Work practices more common among both private and commercial applicators with an HPEE included delay in changing clothing or washing after pesticide application, mixing pesticide application clothing with the family wash, washing up inside the house after application, applying pesticides within 50 yards of their well, and storing pesticides in the home. Job characteristics more common among those with an HPEE included self-repair of application equipment and first pesticide use more than 10 years in the past. These job characteristics explained much of the difference in reported HPEE between males and females, but not between IA and NC subjects or between commercial or private applicators.

Factors associated with self-reported, pesticide-related visits to health care providers in the agricultural health study.

To investigate factors associated with pesticide-related visits to health care providers (i.e., doctor or hospital visits), responses to self-administered questionnaires received from 35,879 licensed restricted-use pesticide applicators participating in the Agricultural Health Study were analyzed. (In Iowa, applicators are actually certified, whereas in North Carolina they are licensed; for ease of reference, the term license will be used for both states in this paper.) The cohort reported a total of more than 10.9 million pesticide-application days. These applications were associated with one or more pesticide-related health care visits by 2,214 applicators (7.0% of the applicator cohort for whom health care visit data were available). The odds of a pesticide-related health care visit were increased for commercial applicators compared to private applicators [odds ratio (OR = 1.77; 95% confidence interval (CI), 1.52-2.06) and for applicators who used insecticides 70 times or more in their lifetime compared to those who used insecticides less frequently (OR = 1.43; CI, 1.26-1.63). After adjusting for the number of applications in a logistic regression model, significantly higher odds of health care visits were observed among North Carolina applicators compared to Iowa applicators (OR = 1.35; CI, 1.17-1.52), among applicators who mixed their own pesticides (OR = 1.65; CI, 1. 22-2.23), and among applicators who personally repaired their pesticide application equipment at least once per year (OR = 1.12; CI, 1.06-1.25). Significantly lower odds were found among female versus male applicators (OR = 0.68; CI, 0.46-0.99) and among applicators who graduated from high school versus those who did not (OR = 0.82; CI, 0.71-0.94 for high school graduates and OR = 0.79; CI, 0.68-0.91 for those with at least some college). Several methods of pesticide application to crops, seed, or stored grain were also associated with significantly elevated odds ratios of health care visits. These observations suggest that several steps can be taken to reduce the number of health care visits resulting from occupational exposure to pesticides. The implications of this pattern of pesticide-related health care visits may have etiologic implications for cancer and other chronic diseases.

The Agricultural Health Study.

The Agricultural Health Study, a large prospective cohort study has been initiated in North Carolina and Iowa. The objectives of this study are to: 1) identify and quantify cancer risks among men, women, whites, and minorities associated with direct exposure to pesticides and other agricultural agents; 2) evaluate noncancer health risks including neurotoxicity reproductive effects, immunologic effects, nonmalignant respiratory disease, kidney disease, and growth and development among children; 3) evaluate disease risks among spouses and children of farmers that may arise from direct contact with pesticides and agricultural chemicals used in the home lawns and gardens, and from indirect contact, such as spray drift, laundering work clothes, or contaminated food or water; 4) assess current and past occupational and nonoccupational agricultural exposures using periodic interviews and environmental and biologic monitoring; 5) study the relationship between agricultural exposures, biomarkers of exposure, biologic effect, and genetic susceptibility factors relevant to carcinogenesis; and 6) identify and quantify cancer and other disease risks associated with lifestyle factors such as diet, cooking practices, physical activity, smoking and alcohol consumption, and hair dye use. In the first year of a 3-year enrollment period, 26,235 people have been enrolled in the study, including 19,776 registered pesticide applicators and 6,459 spouses of registered farmer applicators. It is estimated that when the total cohort is assembled in 1997 it will include approximately 75,000 adult study subjects. Farmers, the largest group of registered pesticide applicators comprise 77% of the target population enrolled in the study. This experience compares favorably with enrollment rates of previous prospective studies.

An esthetic approach to vertical root extrusion in a patient with an anterior open bite: report of case.

A previous attempt to locate the root canal system in the maxillary left central incisor with a bur created a perforating root defect at the level of the alveolar crest. The patient received root canal therapy. Next, vertical root extrusion was chosen to expose sound tooth structure apical to the defect so that a crown could be constructed. With an anterior open bite, the natural crown was left intact and it was possible to position the anchor bar in an incisal and lingual position, maintaining esthetics for the patient during both the extrusive and stabilization phases of treatment.