Citations

Abstract Anti-neutrophil cytoplasmic Abs (cANCAs) against conformational epitopes of proteinase 3 (PR3) are regarded as an important pathogenic marker in Wegener’s granulomatosis (WG). Although the three-dimensional structure of PR3 is known, binding sites of mAbs and cANCAs have not been mapped to date. Competitive binding and biosensor experiments suggested the existence of four nonoverlapping areas on the PR3 surface. In this paper, we present an approach to identify these discontinuous surface regions that cannot be mimicked by linear peptides. The very few surface substitutions found in closely related PR3 homologs from primates, which were further varied by the construction of functional human-gibbon hybrids, resulted in the differential loss of three Ab binding sites, two of which were mapped to the N-terminal β-barrel and one to the linker segment connecting the N- and C-terminal barrels of PR3. The sera from WG patients differed in their binding to gibbon PR3 and the gibbon-human PR3 hybrid, and could be divided into two groups with similar or significantly reduced binding to gibbon PR3. Binding of almost all sera to PR3–α1-protease inhibitor (α1–PI) complexes was even more reduced and often absent, indicating that major antigenic determinants overlap with the active site surface on PR3 that associates with α1-PI. Similarly, the mouse mAbs CLB12.8 and 6A6 also did not react with gibbon PR3 and PR3–α1-PI complexes. Our data strongly suggest that cANCAs from WG patients at least in part recognize similar surface structures as do mouse mAbs and compete with the binding of α1-PI to PR3.

ABSTRACT Campylobacter jejuni and Salmonella , Shigella , and Yersinia species (along with Shiga toxin-producing Escherichia coli ) are the most common causes of acute bacterial diarrheal disease in the United States. Current detection techniques are time-consuming, limiting usefulness for patient care. We developed and validated a panel of rapid PCR assays for the detection and identification of C. jejuni , C. coli , Salmonella , and Yersinia species and Shigella and enteroinvasive E. coli in stool samples. A total of 392 archived stool specimens, previously cultured for enteric pathogens, were evaluated by PCR. Overall, 104 stool specimens had been culture positive ( C. jejuni / coli [ n = 51], Salmonella species [ n = 42], Shigella species [ n = 6], and Yersinia species [ n = 5]). Compared to culture, the overall sensitivity and specificity of PCR detection of these organisms were 92 and 98% (96/104 and 283/288), respectively, from fresh or Cary Blair stool ( P = 0.41); 87 and 98% (41/47 and 242/246), respectively, from fresh stool ( P = 0.53); and 96 and 98% (55/57 and 41/42), respectively, from Cary Blair stool ( P = 0.56). For individual genera, PCR was as sensitive as the culture method, with the exception of Salmonella culture using selenite enrichment for which PCR was less sensitive than culture from fresh, but not Cary Blair ( P = 0.03 and 1.00, respectively) stools. This PCR assay panel for the rapid diagnosis of acute infectious bacterial diarrheal pathogens has a sensitivity and specificity equivalent to that of culture for stools in Cary Blair transport medium. Paired with reflexive culture of stools testing positive, this should provide an improvement in care for patients with acute infectious diarrheal disease.

Abstract The alterations in metabolism and cerebral blood flow that occur following transient focal ischemia were studied in rabbits anesthetized with halothane and subjected to transient occlusion of an M2 segment of the middle cerebral artery (MCA). The parameters measured included intracellular brain pH and focal cortical blood flow (fCBF)—assessed by the umbelliferone technique—electrocorticograms, and cortical microcirculatory changes. A gradient of ischemia developed in the cortex between the patent and occluded vessels. Cortical sites with moderate and severely diminished flow were examined as a function of time before and after occlusion. Mean preocclusion fCBF was 50.8 ± 2.1 ml/100 g/min, and brain pH was 6.99 ± 0.04. Following occlusion, fCBF fell to 14.6 ± 2.3 ml/100 g/min, with an intracellular pH of 6.53 ± 0.03 in sites of severe ischemia in the territory of the occluded vessel. Sites between the patent and occluded branches revealed moderate changes in fCBF and intracellular pH of 26.7 ± 3.6 ml/100 g/min and 6.74 ± 0.03 ml/100 g/min, respectively. Sites adjacent to the patent M2 branch remained similar to baseline. Pretreatment intravenously with nimodipine, a dihydropyridine class of Ca2+ channel antagonist, improved flow in the territory of the occluded segment of the middle cerebral artery to 30.6 ± 2.2 ml/100 g/min, while maintaining the brain pH at 6.83 ± 0.03. Similarly, at sites intermediate between the patent and occluded M2 segments of the MCA, cortical blood flow remained at 53.5 ± 4.0 ml/100 g/min, and the pH at 6.95 ± 0.04, in 10 animals pretreated with nimodopine. This effect was apparently achieved by blocking the secondary, ischemia-induced vasoconstriction known to occur in areas of focal incomplete ischemia. The diphenylalkylamine class Ca2+ channel blockers was examined using verapamil. This was found to be without significant effect. The vehicle carrying nimodopine was also without effect. To authenticate further the reliability of MCA occlusion, a separate group of animals was examined with cautery occlusion of the M2 segment of the MCA. Nimodopine had an effect in this group of animals identical to that caused by occlusion of the vessel with a clip. Visual inspection of the cortex in control and treated animals revealed a marked difference in the development of cortical pallor and microcirculatory changes. We conclude that nimodopine protects the conducting vessels against the secondary vasoconstriction known to occur in areas of focal incomplete ischemia, and thus increases collateral flow. This agent may have the potential for brain protection in areas of incomplete focal ischemia from occlusion of a single major vessel.

Abstract A simple and reproducible method of quantitating the amount of hematoporphyrin derivative by fluorescence microscopy in biopsies of tumors from patients undergoing photoradiation therapy is presented, substantiated by laboratory studies of experimental central nervous system tumors and normal rat tissues. The literature regarding the quantitation of hematoporphyrin is reviewed, and implications of this study for the future application of photoradiation therapy and the design of laboratory studies are discussed.

Abstract Between 1935 and 1974, 3598 episodes of head trauma among Olmsted County, Minnesota, residents resulted in 1097 skull fractures. Of these, 53% were simple, 16% were depressed, 12% were compound, and 19% were basilar. The age- and sex-adjusted incidence of skull fractures was 44.3 per 100,000 person-years overall, was somewhat greater in the urban than in the rural areas of Olmsted County, and was relatively stable for the final 30 years of the study. Age-specific incidence rates were highest for the very young, and simple linear fractures were the predominant type of skull fracture in this age group and among the elderly. The male:female ratio of incidence rates varied from 2.1:1 to 4.5:1 depending on fracture type. Motor vehicle accidents accounted for 38% of the skull fractures and were a particularly important cause among young males. Falls accounted for 37% of the skull fractures and were the major cause of fractures in the elderly and pediatric age groups. The results of this population-based study may be helpful in formulating recommendations for the evaluation and management of head-injured patients.

Purpose Up to 75% of women experience hot flashes, which can negatively impact quality of life. As hot flash physiology is not definitively understood, it cannot be assumed that effective agents represent class effects. Therefore, there is a continued need for rigorous evaluation to identify effective nonhormonal options for hot flash relief. Methods A randomized, double-blind trial evaluated citalopram at target doses of 10, 20, or 30 mg/d versus placebo for 6 weeks. Postmenopausal women with at least 14 bothersome hot flashes per week recorded hot flashes for 7 days before starting treatment and were then titrated to their target doses. The primary end point was the change from baseline to 6 weeks in hot flash score. Results Two hundred fifty-four women were randomly assigned onto this study. Data for hot flash scores and frequencies showed significant improvement in hot flashes with citalopram over placebo, with no significant differences among doses. Reductions in mean hot flash scores were 2.0 (23%), 7.0 (49%), 7.7 (50%), and 10.7 (55%) for placebo and 10, 20, and 30 mg of citalopram, respectively (P ≤ .002). Improvement in secondary outcomes, such as the Hot Flash Related Daily Interference Scale, was statistically superior in the 20-mg arm. Citalopram was well-tolerated, with no significant negative adverse effects. Conclusion Citalopram is an effective, well-tolerated agent in managing hot flashes. There does not appear to be a significant dose response above 10 mg/d, but broader helpful effects of the agent appear to be more evident at 20 mg/d.

Abstract Human blood eosinophils exposed ex vivo to hematopoietic cytokines (e.g., IL-5 or GM-CSF) subsequently display enhanced responsiveness to numerous chemoattractants, such as chemokines, platelet-activating factor, or FMLP, through a process known as priming. Airway eosinophils, obtained by bronchoalveolar lavage after segmental Ag challenge, also exhibit enhanced responsiveness to selected chemoattractants, suggesting that they are primed during cell trafficking from the blood to the airway. Earlier work has shown that chemoattractants stimulate greater activation of ERK1 and ERK2 following IL-5 priming in vitro, thus revealing that ERK1/ERK2 activity can be a molecular readout of priming under these circumstances. Because few studies have examined the intracellular mechanisms regulating priming as it relates to human airway eosinophils, we evaluated the responsiveness of blood and airway eosinophils to chemoattractants (FMLP, platelet-activating factor, CCL11, CCL5, CXCL8) with respect to degranulation, adherence to fibronectin, or Ras–ERK signaling cascade activation. When compared with blood eosinophils, airway eosinophils exhibited greater FMLP-stimulated eosinophil-derived neurotoxin release as well as augmented FMLP- and CCL11-stimulated adherence to fibronectin. In airway eosinophils, FMLP, CCL11, and CCL5 stimulated greater activation of Ras or ERK1/ERK2 when compared with baseline. Ras activation by FMLP in blood eosinophils was also enhanced following IL-5 priming. These studies are consistent with a model of in vivo priming of eosinophils by IL-5 or related cytokines following allergen challenge, and further demonstrate the key role of priming in the chemoattractant-stimulated responses of eosinophils. These data also demonstrate the importance of the Ras–ERK signaling pathway in the regulation of eosinophil responses to chemoattractants in the airway. Human airway eosinophils respond to several chemoattractants with increased activation of the Ras–ERK cascade, eosinophil-derived neurotoxin release, and adherence to fibronectin relative to blood eosinophils.