Oxidative inactivation of Actinobacillus actinomycetemcomitans leukotoxin by the neutrophil myeloperoxidase system.

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of inflammatory periodontal disease. We examined a potential mechanism for detoxification of this microbial product by the neutrophil myeloperoxidase system. Exposure to myeloperoxidase, H2O2, and a halide resulted in marked inactivation of leukotoxin, an effect which required each component of the myeloperoxidase system. Toxin inactivation was blocked by agents which inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The latter system, in fact, was more potent than reagent H2O2 in terms of the capacity to inactivate high concentrations of toxin. Toxin inactivation was complete within 1 to 2 min at 37 degrees C. These observations suggest a possible role for oxidative inactivation of leukotoxin by secretory products of neutrophils.     

The myocardial contractile responses to protamine sulfate and heparin

The administration of protamine sulfate for the reversal of heparin anticoagulation has been associated with adverse hemodynamic changes including hypotension and decreased cardiac output. The possible direct toxic effect of protamine on human right atrial trabeculae contracting isometrically in vitro was studied. Muscles were stimulated to contract at 1 Hz in Tyrode's solution (maintained at 34 degrees C, pH 7.4) into which protamine was continuously added. Following a polynomial regression analysis, a parabolic dose-response curve resulted. The equation was: y = 95.13 + 38.76x - 278.71x2 where y = relative developed force and x = concentration of protamine (milligrams per milliliters) (r = 0.82). The estimated concentration of protamine resulting in 50% developed force was 0.48 mg/ml. In a second series of experiments, protamine was added to the bath along with a neutralizing amount of heparin. This resulted in a limited reduction in the fall of relative developed force. Thus, protamine in high concentrations alone or in complex with heparin has a direct toxic effect on human myocardial muscle mechanics, and care is warranted in its clinical use.     

Replication and persistence of HPV DNA in cultured cells derived from laryngeal papillomas.

We have investigated the replication and persistence of human papillomavirus (HPV) type 6 and 11 DNA in cultured cells derived from laryngeal papillomas, with paradoxical findings. Measured by bromodeoxyuridine incorporation into heavy/light DNA separated on a cesium chloride gradient, viral DNA replicates in both primary and secondary cells. The ratio of the fraction of replicated viral to replicated cellular DNA was equal to or greater than 1 in all but one case and was closer to 2 in primary cells. Despite this efficient replication, HPV DNA is rapidly lost from the cells with passage. We propose that infected cells, or those with a high HPV copy number, show a selective decrease in plating efficiency compared to uninfected cells or those with a low copy number, which explains the loss of HPV DNA with repeated passage.     

Human myocardial responses to antibiotics: gentamicin, tobramycin and cephalothin

Although myocardial toxicity of certain antibiotics has been suggested by animal studies, their effects on human cardiac muscle has not been established. Accordingly, human right atrial trabeculae contracting isometrically in vitro were exposed to increasing doses of the antibiotics gentamicin, tobramycin and cephalothin. Contractile responses were measured and dose-response curves calculated by a polynomial regression analysis. All three antibiotics demonstrated a dose-related decrease in relative developed force although resting force remained constant. Concentrations of gentamicin, tobramycin and cephalothin that caused 50% depression of developed force were 1.97, 1.92 and 52.75 mg/mL, respectively. These doses were 200, 200 and 2600 times the accepted serum toxic concentrations for gentamicin, tobramycin and cephalothin, respectively. The depression caused by cephalothin is probably related to ionic changes within the bath solution. In conclusion, gentamicin and tobramycin have myocardial toxicity at high concentrations which should not occur clinically when used judiciously.     

Hemodynamics and survival of patients with portopulmonary hypertension.

It is not known whether patients with pulmonary arterial hypertension associated with portal hypertension (portopulmonary hypertension (PPHTN) have different disease characteristics from those of patients with other forms of pulmonary arterial hypertension. We performed a retrospective cohort study of patients with PPHTN and patients with pulmonary arterial hypertension that was idiopathic, familial, or associated with anorexigen use (IPAH) to determine whether hemodynamics or survival were different between these groups. We included consecutive patients who underwent initial pulmonary artery catheterization and vasodilator testing at our center between January 1997 and May 2001 and who were followed until January 2004. Patients with PPHTN (N = 13) had a higher cardiac index and lower pulmonary vascular resistance than patients with IPAH (N = 33) (P < or = 0.001). Right atrial pressure and pulmonary artery pressure were similar between the groups. Patients with PPHTN had a higher risk of death in multivariate analysis (hazard ratio: [HR] = 2.8, 95% CI 1.04-7.4; P = 0.04). These findings were not affected by adjustment for differences in laboratory values, hemodynamics, or therapy. In conclusion, patients with PPHTN have a higher risk of death than that of patients with IPAH, despite having a higher cardiac index and lower pulmonary vascular resistance. Future studies of the specific mechanisms of and therapy for pulmonary arterial hypertension should focus on the distinctions between the different forms of this disease.     

105Changes in Nitric Oxide Levels After Deep Dermal injury in the Female,Red Duroc Pig (FRDP)

Introduction : Hypertrophic scar is a devastating sequel to burns and other tangential skin injuries. It follows deep dermal injuries and does not occur after superficial injuries. Nitric oxide (NO) plays many important roles in wound healing from inflammation to scar remodeling. Studies have shown that expression of nitric oxide synthase and nitric oxide production are decreased in human hypertrophic scar. However little is known about NO involvement in the early stages of hypertrophic scarring, because of the lack of an animal model. It was recently reported that the female red Duroc pig (FRDP) makes thick scar, which is similar to human hypertrophic scar. We hypothesized that NO production in wounds on the female, red Duroc pig is similar to that of human hypertrophic scar and that NO involvement in deep wounds is different from that in superficial wounds. Methods : Superficial (0.015” to 0.030”) and deep (0.045” to 0.060”) wounds were created on the backs of four FRDPs. Biopsies were collected at weeks 1.5, 4, 8 and 21 post wounding including samples of uninjured skin. Nitric oxide levels were measured with the Griess reaction assay and normalized with tissue protein level. Results : Superficial wounds healed with an invisible scar whereas the deep wounds healed with scar resembling mild hypertrophic scar. The thickness of the scars from the deep wounds was significantly greater than uninjured skin and healed superficial wounds (p < 0.01). NO levels were increased at 1.5 weeks in deep wounds compared to superficial wounds and uninjured skin (p < 0.05). At 8 weeks, NO levels in deep wounds had returned to the level of uninjured tissue and superficial wounds. By 21 weeks, NO levels had decreased significantly when compared to superficial wounds (p < 0.01). There were no differences in NO levels between uninjured skin and superficial wounds at any time point (p > 0.05). Conclusions : NO production is similar in late, deep wounds on the female, red Duroc pig to that reported in the literature for human hypertrophic scar further validating this animal model. NO production is quite different after deep wounds as compared to superficial wounds in the FRDP. Early elevation in nitric oxide production might account for excessive inflammation in deep wounds that become thick scars in the FRDP. Nitric oxide regulators and effects at early stages of scar formation should be elucidated further and the FRDP appears to be a useful model.     

Interaction of Inflammatory Cells and Oral Microorganisms VII. In Vitro Polymorphonuclear Responses to Viable Bacteria and to Subcellular Components of Avirulent and Virulent Strains of Actinomyces viscosus

Both virulent (V) and avirulent (AV) strains of Actinomyces viscosus T14 are capable of colonizing the oral cavity of gnotobiotic rats, but only T14-V causes destructive periodontal disease. The basis for this difference in in vivo pathogenicity has not been adequately defined. In the present study we compared the capacities of T14-AV and T14-V to provoke in vitro extracellular release of lysosomal constituents from human polymorphonuclear leukocytes (PMNs). In serum-free cultures, viable T14-V but not T14-AV stimulated discharge of PMN lysosomes. The release response was correlated with PMN phagocytic activity; thus, PMNs readily ingested T14-V but not T14-AV. To explain these differences in PMN-bacteria interactions, subcellular fractions of T14-AV or T14-V were incubated with PMNs. A crude, insoluble sonic extract derived from T14-V caused PMN lysosome release, but a similar fraction from T14-AV was inactive. However, following extensive washing and treatment with deoxyribonuclease or sodium dodecyl sulfate, cell wall fractions of T14-AV stimulated lysosome release. These procedures apparently removed an extracellular polysaccharide slime which is synthesized by T14-AV but not by T14-V. There was a significant reduction in the capacities of viable T14-V or cell wall fractions of T14-V or T14-AV to provoke PMN lysosome release when these agents were preincubated with a slime material isolated from T14-AV. This inhibitory influence of slime was overcome by the addition of fresh or heated (56°C, 30 min) serum to the PMN-bacteria cultures. The data suggest a relationship between the abilities of the avirulent and virulent strains of A. viscosus T14 to act as periodontal pathogens in vivo and to serve as stimuli for PMN lysosome release in vitro.