The adverse effect of spasticity on 3-month poststroke outcome using a population-based model.

Several devices and medications have been used to address poststroke spasticity. Yet, spasticity's impact on outcomes remains controversial. Using data from a cohort of 460 ischemic stroke patients, we previously published a validated multivariable regression model for predicting 3-month modified Rankin Score (mRS) as an indicator of functional outcome. Here, we tested whether including spasticity improved model fit and estimated the effect spasticity had on the outcome. Spasticity was defined by a positive response to the question "Did you have spasticity following your stroke?" on direct interview at 3 months from stroke onset. Patients who had expired by 90 days (n = 30) or did not have spasticity data available (n = 102) were excluded. Spasticity affected the 3-month functional status (ß = 0.420, 95 CI = 0.194 to 0.645) after accounting for age, diabetes, leukoaraiosis, and retrospective NIHSS. Using spasticity as a covariable, the model's R (2) changed from 0.599 to 0.622. In our model, the presence of spasticity in the cohort was associated with a worsened 3-month mRS by an average of 0.4 after adjusting for known covariables. This significant adverse effect on functional outcomes adds predictive value beyond previously established factors.

Population-based study of wake-up strokes.

OBJECTIVE: Previous studies have estimated that wake-up strokes comprise 8%to 28% of all ischemic strokes, but these studies were either small or not population-based. We sought to establish the proportion and event rate of wake-up strokes in a large population-based study and to compare patients who awoke with stroke symptoms with those who were awake at time of onset. METHODS: First-time and recurrent ischemic strokes among residents of the Greater Cincinnati/Northern Kentucky region (population 1.3 million) in 2005 were identified using International Classification of Diseases-9 codes 430-436 and verified via study physician review. Ischemic strokes in patients aged 18 years and older presenting to an emergency department were included. Baseline characteristics were ascertained, along with discharge modified Rankin Scale scores and 90-day mortality. RESULTS: We identified 1,854 ischemic strokes presenting to an emergency department, of which 273 (14.3%) were wake-up strokes. There were no differences between wake-up strokes and all other strokes with regard to clinical features or outcomes except for minor differences in age and baseline retrospective NIH Stroke Scale score. The adjusted wake-up stroke event rate was 26.0/100,000. Of the wake-up strokes, at least 98 (35.9%) would have been eligible for thrombolysis if arrival time were not a factor. CONCLUSIONS: Within our population, approximately 14% of ischemic strokes presenting to an emergency department were wake-up strokes. Wake-up strokes cannot be distinguished from other strokes by clinical features or outcome. We estimate that approximately 58,000 patients with wake-up strokes presented to an emergency department in the United States in 2005.

Smoking and family history and risk of aneurysmal subarachnoid hemorrhage.

OBJECTIVE: Smoking and family history of aneurysmal subarachnoid hemorrhage (aSAH) are independent risk factors for aSAH. Using a population-based case-control study of hemorrhagic stroke, we hypothesized that having both a first-degree relative with a brain aneurysm or SAH (+FH) and current smoking interact to increase the risk of aSAH. METHODS: Cases of aneurysmal SAH were prospectively recruited from all 17 hospitals in the five-county region around the University of Cincinnati. Controls were identified by random digit dialing. Controls were matched to cases of aSAH by age (+/-5 years), race, and sex. Conditional multiple logistic regression was used to identify independent risk factors. For deviation from the additive model, the interaction constant ratio test was used. RESULTS: A total of 339 cases of aSAH were matched to 1,016 controls. Compared to current nonsmokers with no first-degree relatives with aSAH (-FH), the odds ratio (OR) for aSAH for current nonsmokers with +FH was 2.5 (95% confidence interval [CI] 0.9-6.9); for current smokers with -FH, OR = 3.1 (95% CI 2.2-4.4); and for current smokers with +FH, OR = 6.4 (95% CI 3.1-13. 2). The interaction constant ratio, which measured the deviation from the additive model, was significant: 2.19 (95% CI 0.80-5.99). The lower bound of the 95% CI >0.5 signifies a departure from the additive model. CONCLUSION: Evidence of a gene-environment interaction with smoking exists for aneurysmal subarachnoid hemorrhage. This finding is important to counseling family members and for screening of intracranial aneurysm (IA) as well as the design and interpretation of genetic epidemiology of IA studies.

Warfarin use leads to larger intracerebral hematomas.

BACKGROUND: Among patients with intracerebral hemorrhage (ICH), warfarin use before onset leads to greater mortality. In a retrospective study, we sought to determine whether warfarin use is associated with larger initial hematoma volume, one determinant of mortality after ICH. METHODS: We identified all patients hospitalized with ICH in the Greater Cincinnati region from January through December 2005. ICH volumes were measured on the first available brain scan by using the abc/2 method. Univariable analyses and a multivariable generalized linear model were used to determine whether international normalized ratio (INR) influenced initial ICH volume after adjusting for other factors, including age, race, sex, antiplatelet use, hemorrhage location, and time from stroke onset to scan. RESULTS: There were 258 patients with ICH, including 51 patients taking warfarin. In univariable comparison, when INR was stratified, there was a trend toward a difference in hematoma volume by INR category (INR <1.2, 13.4 mL; INR 1.2-2.0, 9.3 mL; INR 2.1-3.0, 14.0 mL; INR >3.0, 33.2 mL; p = 0.10). In the model, compared with patients with INR <1.2, there was no difference in hematoma size for patients with INR 1.2-2.0 (p = 0.25) or INR 2.1-3.0 (p = 0.36), but patients with INR >3.0 had greater hematoma volume (p = 0.02). Other predictors of larger hematoma size were ICH location (lobar compared with deep cerebral, p = 0.02) and shorter time from stroke onset to scan (p < 0.001). CONCLUSION: Warfarin use was associated with larger initial intracerebral hemorrhage (ICH) volume, but this effect was only observed for INR values >3.0. Larger ICH volume among warfarin users likely accounts for part of the excess mortality in this group.

The increasing incidence of anticoagulant-associated intracerebral hemorrhage.

OBJECTIVE: To define temporal trends in the incidence of anticoagulant-associated intracerebral hemorrhage (AAICH) during the 1990s and relate them to rates of cardioembolic ischemic stroke. METHODS: We identified all patients hospitalized with first-ever intracerebral hemorrhage (ICH) in greater Cincinnati during 1988, from July 1993 through June 1994, and during 1999. AAICH was defined as ICH in patients receiving warfarin or heparin. Patients from the same region hospitalized with first-ever ischemic stroke of cardioembolic mechanism were identified during 1993/1994 and 1999. Incidence rates were calculated and adjusted to the 2000 US population. Estimates of warfarin distribution in the United States were obtained for the years 1988 through 2004. RESULTS: AAICH occurred in 9 of 184 ICH cases (5%) in 1988, 23 of 267 cases (9%) in 1993/1994, and 54 of 311 cases (17%) in 1999 (p < 0.001). The annual incidence of AAICH per 100,000 persons was 0.8 (95% CI 0.3 to 1.3) in 1988, 1.9 (1.1 to 2.7) in 1993/1994, and 4.4 (3.2 to 5.5) in 1999 (p < 0.001 for trend). Among persons aged > or =80, the AAICH rate increased from 2.5 (0 to 7.4) in 1988 to 45.9 (25.6 to 66.2) in 1999 (p < 0.001 for trend). Incidence rates of cardioembolic ischemic stroke were similar in 1993/1994 and 1999 (31.1 vs 30.4, p = 0.65). Warfarin distribution in the United States quadrupled on a per-capita basis between 1988 and 1999. CONCLUSIONS: The incidence of anticoagulant-associated intracerebral hemorrhage quintupled in our population during the 1990s. The majority of this change can be explained by increasing warfarin use. Anticoagulant-associated intracerebral hemorrhage now occurs at a frequency comparable to subarachnoid hemorrhage.

Long-term mortality after intracerebral hemorrhage.

OBJECTIVE: To characterize long-term mortality following intracerebral hemorrhage (ICH) in two large population-based cohorts assembled more than a decade apart. METHODS: All patients age > or = 18 hospitalized with nontraumatic ICH in the Greater Cincinnati/Northern Kentucky area were identified during 1988 (Cohort 1) and from May 1998 to July 2001 and August 2002 to April 2003 (Cohort 2). Mortality was tabulated using actuarial methods and compared with a log-rank test. RESULTS: There were 183 patients with ICH in Cohort 1 and 1,041 patients in Cohort 2. Patients in Cohort 1 were more likely to be white (p = 0.024) and undergo operation for their ICH (p = 0.002), whereas patients in Cohort 2 were more commonly on anticoagulants (p < 0.001). Among patients in Cohort 1, mortality at 7 days, 1 year, and 10 years was 31, 59, and 82%. Among patients in Cohort 2, mortality at 7 days and 1 year was 34 and 53%. Mortality rates did not differ between cohorts by log-rank test (p = 0.259). CONCLUSIONS: Intracerebral hemorrhage (ICH) mortality did not improve significantly between study periods. Operation for ICH became less frequent, whereas anticoagulant-associated ICH became more common.

o-Nitrotoluene-induced large intestinal tumors in B6C3F1 mice model human colon cancer in their molecular pathogenesis.

In the previous 500 2-year chemical bioassays within the National Toxicology Program, large intestinal tumors (cecal carcinomas) related to chemical exposure have not been observed in B6C3F1 mice. The recently completed o-nitrotoluene study provided the first cecal tumor response and an opportunity to evaluate the morphology and molecular profile of oncogenes and tumor suppressor genes that are relevant to humans. Morphologically, the carcinomas were gland-forming tumors lined by tall columnar epithelial cells that were positive for cytokeratin 20 and negative for cytokeratin 7. Using immunohistochemistry beta-catenin (encoded by Catnb) protein accumulation was detected in 80% (8/10) of the cecal carcinomas, while increased cyclin D1 and p53 protein expression was detected in 73% (8/11), respectively. There was no difference in adenomatous polyposis protein expression between normal colon and cecal carcinomas. All tumors examined exhibited mutations in exon 2 (corresponds to exon 3 in humans) in the Catnb gene. Mutations in p53 were identified in nine of 11 carcinomas, and all were in exon 7. Analysis of the K-ras gene revealed mutations in 82% (9/11) of carcinomas; all had specific G --> T transversions (Gly --> Val) at codons 10 or 12. The alterations in cancer genes and proteins found in the mouse large intestinal tumors included mutations that activate signal transduction pathways (K-ras and Catnb) and changes that disrupt the cell-cycle and bypass G(1) arrest (p53, cyclin D1). These alterations, which are hallmarks of human colon cancer, probably contributed to the pathogenesis of the large intestinal carcinomas in mice following o-nitrotoluene exposure.

Chemical-specific alterations in ras, p53, and beta-catenin genes in hemangiosarcomas from B6C3F1 mice exposed to o-nitrotoluene or riddelliine for 2 years.

The most prominent neoplastic lesions in mice in the 2-year studies of o-nitrotoluene and riddelliine were hemangiosarcomas. Fifteen o-nitrotoluene-induced hemangiosarcomas of the skeletal muscle, subcutaneous tissue, and mesentery; 12 riddelliine-induced hemangiosarcomas of the liver; and 15 spontaneous subcutaneous hemangiosarcomas were examined for genetic alterations in ras, p53, and beta-catenin genes. Mutations in at least one of these genes were identified in 13 of 15 (87%) of the o-nitrotoluene-induced hemangiosarcomas with missense mutations in p53 exons 5-8 detected in 11 of 15 (73%) of these neoplasms. Seven of 15 (47%) hemangiosarcomas from mice exposed to o-nitrotoluene had deletions at exon 2 splice sites or smaller deletions in the beta-catenin gene. K-ras mutation was detected in only 1 of the 15 (7%) o-nitrotoluene-induced hemangiosarcomas. In contrast to the o-nitrotoluene study, 7/12 (58%) riddelliine-induced hemangiosarcomas had K-ras codon 12 GTT mutations and, when screened by immunohistochemistry, 9/12 (75%) had strong staining for the p53 protein in malignant endothelial cells, the cells of origin of hemangiosarcomas. Riddelliine-induced hemangiosarcomas were negative for the beta-catenin protein. Spontaneous hemangiosarcomas from control mice lacked both p53 and beta-catenin protein expression and ras mutations. Our data indicated that p53 and beta-catenin mutations in the o-nitrotoluene-induced hemangiosarcomas and K-ras mutations and p53 protein expression in riddelliine-induced hemangiosarcomas most likely occurred as a result of the genotoxic effects of these chemicals. It also suggests that these mutations play a role in the pathogenesis of the respective hemangiosarcomas in B6C3F1(1) mice.

Estimating recharge distribution by incorporating runoff from mountainous areas in an alluvial basin in the Great Basin region of the southwestern United States.

A method is described to estimate the distribution of ground water recharge within hydrographic basins in the Great Basin region of the southwestern United States on the basis of estimated runoff from high mountainous areas and subsequent infiltration in alluvial fans surrounding the intermontane basins. The procedure involves a combination of Geographic Information System (GIS) analysis, empirical surface-runoff modeling, and water-balance calculations. The method addresses the need to develop and incorporate constraints on the distribution of recharge in regional-scale ground water flow modeling of arid and semiarid environments. The conceptual approach and methodology were developed for Crescent Valley, Nevada. However, the concept and method are generally applicable to any region where excess precipitation in upland areas is conveyed to lower elevations before it infiltrates to recharge the ground water system. Application of the procedure to a ground water flow model of Crescent Valley appears both qualitatively and quantitatively to result in a more accurate representation of actual recharge conditions than might otherwise have been prescribed.

The role of oxidative stress in indium phosphide-induced lung carcinogenesis in rats.

Indium phosphide (IP), widely used in the microelectronics industry, was tested for potential carcinogenicity. Sixty male and 60 female Fischer 344 rats were exposed by aerosol for 6 h/day, 5 days/week, for 21 weeks (0.1 or 0.3 mg/m(3); stop exposure groups) or 105 weeks (0 or 0.03 mg/m(3) groups) with interim groups (10 animals/group/sex) evaluated at 3 months. After 3-month exposure, severe pulmonary inflammation with numerous infiltrating macrophages and alveolar proteinosis appeared. After 2 years, dose-dependent high incidences of alveolar/bronchiolar adenomas and carcinomas occurred in both sexes; four cases of squamous cell carcinomas appeared in males (0.3 mg/m(3)), and a variety of non-neoplastic lung lesions, including simple and atypical hyperplasia, chronic active inflammation, and squamous cyst, occurred in both sexes. To investigate whether inflammation-related oxidative stress functioned in the pathogenesis of IP-related pulmonary lesions, we stained lungs of control and high-dose animals immunohistochemically for four markers indicative of oxidative stress: inducible nitric oxide synthase (i-NOS), cyclooxygenase-2 (COX-2), glutathione-S-transferase Pi (GST-Pi), and 8-hydroxydeoxyguanosine (8-OHdG). Paraffin-embedded samples from the 3-month and 2-year control and treated females were used. i-NOS and COX-2 were highly expressed in inflammatory foci after 3 months; at 2 years, all four markers were expressed in non-neoplastic and neoplastic lesions. Most i-NOS staining, mainly in macrophages, occurred in chronic inflammatory and atypical hyperplastic lesions. GST-Pi and 8-OHdG expression occurred in cells of carcinoma epithelium, atypical hyperplasia, and squamous cysts. These findings suggest that IP inhalation causes pulmonary inflammation associated with oxidative stress, resulting in progression to atypical hyperplasia and neoplasia.

Unique renal tubule changes induced in rats and mice by the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D) and WY-14643.

Peroxisome proliferators are non-mutagenic carcinogens in the liver of rodents, acting both as initiators and promoters. The National Toxicology Program (NTP) conducted a study of several peroxisome proliferators (PPs), including Wyeth (WY)-14643 as a prototypical PP and 2,4-dichlorophenoxyacetic acid (2,4-D) as a weak PP, in Sprague-Dawley rats. B6C3F1 mice, and Syrian hamsters. In the kidney, an unusual change was observed in the outer stripe of the outer medulla, especially in rats treated with 2,4-D or WY-14643. This change was characterized by foci of tubules that were partially or completely lined by basophilic epithelial cells with decreased cytoplasm and high nuclear density. Changes typical of chronic nephropathy such as interstitial fibrosis or basement membrane thickening were not associated with these foci. Results of immunohistochemical staining for catalase and cytochrome P-450 4A in the kidney indicated increased staining intensity in renal tubular epithelial cells primarily in the region where the affected tubules were observed: however, the altered cells were negative for both immunohistochemical markers. Ultrastructurally, affected cells had long brush borders typical of the P3 tubule segment. The most distinguishing ultrastructural change was a decreased amount of electronlucent cytoplasm that contained few differentiated organelles and, in particular, a prominent reduced volume and number of mitochondria; changes in peroxisomes were not apparent. In addition to the lesion in rats, mice treated with the highest dose of 2,4-D, but not WY-14643, manifested similar renal tubular changes as seen by light microscopy. Neither chemical induced renal tubular lesions in hamsters. Hepatocellular changes characteristic of PPs were present in all 3 species treated with WY-14643, but not 2,4-D. These results indicate that the rat is the species most sensitive to the nephrotoxic effects of PPs and there is a site specificity to this toxicity related to areas of PP-related enzyme induction. Although 2,4-D is considered a weak PP for the liver, it was the most effective at inducing renal lesions, indicating that the toxic potency of various PPs will depend on the target organ.

Effects of iodine on inducible nitric oxide synthase and cyclooxygenase-2 expression in sulfur mustard-induced skin.

In a previous study we demonstrated the protective effect of topical iodine as postexposure treatment for sulfur mustard (SM) application. The iodine treatment results in significantly reduced inflammation and necrosis and increased epidermal hyperplasia. The expression and localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in paraffin-embedded skin samples from that study were evaluated in the present investigation. We compared the immunoreactivity of iNOS and COX-2 using five samples from each of the following four test sites: untreated control sites, SM-exposed sites, sites treated with iodine mixture 15 min after SM exposure, and sites treated with iodine 30 min after SM exposure. All animals were killed 2 days after irritant exposure. iNOS immunoreactivity was present only in skin sites exposed to SM without iodine treatment. The ulcerated skin was covered with a relatively thick band of exudate composed of iNOS-immunostained polymorphonuclear cells and macrophages. In untreated skin, COX-2 immunostaining was limited to the thin suprabasal epidermal layer. In SM-exposed skin, induction of COX-2 was noted in inflammatory cells located close to the site of epidermal injury. In skin sites treated with iodine 15 or 30 min after SM exposure, the regenerating hyperplastic epithelium showed moderate cytoplasmic staining localized to the epithelium overlying the basal layer. This pattern of staining was also present in the nearby dermal fibroblasts. Thus, in contrast to the skin samples exposed to SM without iodine treatment, the epidermal layer expressing immunohistochemical positivity for COX-2 was thicker and corresponded to the epidermal hyperplasia noted in samples treated with iodine. It is well documented that prostaglandins (PGs) promote epidermal proliferation, thereby contributing to the repair of injured skin. That the induction of the COX-2 shown in our study may also play a role in the healing process is indicated by the present evidence. The results suggest that nitric oxide radicals (NO*) are involved in mediating the damage induced by the SM and that iodine-related reduction in acute epidermal inflammation is associated with reduced iNOS expression.

Glutathione S-transferase pi expression in forestomach carcinogenesis process induced by gavage-administered 2,4-hexadienal in the F344 rat.

2,4-Hexadienal (2,4-Hx), an unsaturated aldehyde formed by in vivo and in vitro peroxidation of unsaturated lipid induced, in National Toxicology Program (NTP) gavage studies of F344 rats, forestomach hyperplasia in 13-week and 2-year exposures and squamous papilloma and carcinoma in 2-year studies. Hyperplasia was characterized by thickening of all layers of epithelium with particularly prominent proliferation of the basal cells. The present investigation describes the nature and potential significance of glutathione-S-transferase-Pi (GST-Pi) immunoexpression of normal forestomach epithelium, compared to that of 2,4-Hx-related basal cell hyperplasia and squamous cell papilloma and carcinoma. Paraffin-embedded forestomachs from these NTP studies were used to investigate possible correlations between the carcinogenic process and expression of GST-Pi, a physiological metabolic barrier and an inducible phase II detoxifying enzyme suggested to decrease the responsiveness of reactive oxygen species (ROS) and organic electrophilic compounds. The amount of immunopositive staining was graded on a scale of 0 (no staining) to 4 (marked staining). The simple basal epithelium of control rats showed strong immunopositivity. In cases of basal cell hyperplasia from the 13-week and 2-year studies, these cells usually expressed strong immunopositivity for GST-Pi (grade 3 to 4). In the 2-year treated animals only, occasional focal reduction (grade 0 to 2) in immunoreactivity for GST-Pi was noted. In papillomas and squamous cell carcinomas, a wide range of GST-Pi expression was observed, perhaps indicating irregularities in its induction or change in the phenotype of these cells compared to normal or hyperplastic ones. Reduced expression of GST-Pi by the foci of basal cell hyperplasia and in tumor cells may suggest changes in cellular protection from oxidative or electrophilic DNA damage; these changes may result in genetic alterations and be the precursor to clonal expansion.

Proteolytic cleavage of the Fe-S subunit hinge region of Rhodobacter capsulatus bc(1) complex: effects of inhibitors and mutations.

The three-dimensional structure of the mitochondrial bc(1) complex reveals that the extrinsic domain of the Fe-S subunit, which carries the redox-active [2Fe2S] cluster, is attached to its transmembrane anchor domain by a short flexible hinge sequence (amino acids D43 to S49 in Rhodobacter capsulatus). In various structures, this extrinsic domain is located in different positions, and the conformation of the hinge region is different. In addition, proteolysis of this region has been observed previously in a bc(1) complex mutant of R. capsulatus [Saribas, A. S., Valkova-Valchanova, M. B., Tokito, M., Zhang, Z., Berry E. A., and Daldal, F. (1998) Biochemistry 37, 8105-8114]. Thus, possible correlations between proteolysis, conformation of the hinge region, and position of the extrinsic domain of the Fe-S subunit within the bc(1) complex were sought. In this work, we show that thermolysin, or an endogenous activity present in R. capsulatus, cleaves the hinge region of the Fe-S subunit between its amino acid residues A46-M47 or D43-V44, respectively, to yield a protease resistant fragment with a M(r) of approximately 18 kDa. The cleavage was affected significantly by ubihydroquinone oxidation (Q(o)) and ubiquinone reduction (Q(i)) site inhibitors and by specific mutations located in the bc(1) complex. In particular, using either purified or detergent dispersed chromatophore-embedded R. capsulatus bc(1) complex, we demonstrated that while stigmatellin blocked the cleavage, myxothiazol hardly affected it, and antimycin A greatly enhanced it. Moreover, mutations in various regions of the Fe-S subunit and cyt b subunit changed drastically proteolysis patterns, indicating that the structure of the hinge region of the Fe-S subunit was modified in these mutants. The overall findings establish that protease accessibility of the Fe-S subunit of the bc(1) complex is a useful biochemical assay for probing the conformation of its hinge region and for monitoring indirectly the position of its extrinsic [2Fe2S] cluster domain within the Q(o) pocket.

Effects of apocynin and natural antioxidant from spinach on inducible nitric oxide synthase and cyclooxygenase-2 induction in lipopolysaccharide-induced hepatic injury in rat.

The immunoreactivity of inducible nitric oxide synthase, and cyclooxygenase-2 was compared among groups of male Wistar rats comprising those injected with lipopolysaccharide following pretreatment with either natural antioxidant from spinach or the antioxidant apocynin, with lipopolysaccharide without pretreatment with antioxidants, with each of the two antioxidants alone, and untreated controls. The grade of staining of both inducible nitric oxide synthase and cyclooxygenase-2 increased with the severity of the inflammatory reaction in the lipopolysaccharide-treated animals, compared to the antioxidant-treated groups. Interpretation of the results of the immunostained tissues indicated that pretreatment with either antioxidant significantly (P<0.05) attenuated the lipopolysaccharide-stimulated inducible nitric oxide synthase induction. Analysis of the cycloxygenase-2-stained liver samples indicated that the pretreatment with the natural antioxidant NAO significantly (P<0.05) attenuated lipopolysaccharide-stimulated cycloxygenase-2 induction; whereas, in animals pretreated with apocynin, there was a trend of reduction in the cyclooxygenase-2 expression, but not statistically significant (P>0.05). The negative nitrotyrosine immunoreactivity of the lipopolysaccharide-related hepatic lesions may indicate that there was relatively low interaction between superoxide anions and nitric oxide to form peroxynitrite or that the expression levels of the nitrotyrosine were below the limit of detection. In all treatment groups a positive correlation (P<0.05, r=0.86) found between the inducible nitric oxide synthase and cyclooxygenase-2 scores suggests a strong relationship between these two parameters. The results indicate the possible therapeutic efficacy of NAO and apocynin in the prevention of liver damage related to clinical endotoxemia known to be associated with oxidative stress.

An unusual human IgM antibody with a protruding HCDR3 and high avidity for its peptide ligands.

The crystal structure of the Fv molecule from a human monoclonal IgM cryoglobulin (Mez) was determined at 2.6 A resolution. Amino acid sequences of framework regions (FR) of the Mez light (L) and heavy (H) chain variable domains (VL and VH) are highly similar to their counterparts in another human Fv (Pot) previously subjected to X-ray analysis in our laboratory. As expected, the three-dimensional (3-D) structures of FR are quite similar in the two proteins, as are four of the six complementarity-determining regions (CDRs): CDRs 1 and 2 for both L and H chains. Absence of Pro 95L from the LCDR3 loop in Mez VL (relative to Pot LCDR3) results in compression of this loop and creates more space in the VL-VH interface. In the two IgMs, HCDR3 conformations differ significantly from all previously defined conformations for these loops. Pot has a 12-residue HCDR3 that collapses to fill all available space in the VL-VH domain interface, resulting in the formation of a relatively flat platform for antigen binding. In Mez, the HCDR3 is two residues longer and is comprehensively different. A semi-rigid ascending segment dominated by a Pro-Pro-Tyr sequence protrudes out into solvent. The descending portion has the sequence Gly-Trp-Gly-Gly-Gly, which promotes high local flexibility. This segment folds across the VL-VH domain interface to interact with residues in LCDR3. These features partition the Mez active site into two compartments, a large cavity between VL and VH and a smaller cavity lined entirely by constituents of the three heavy chain CDRs. Such an unusual topographical feature indicates why the Mez IgM does not bind to the Fc portion of intact human IgG antibodies in immunoassays yet interacts with high avidity with many Fc-derived octapeptides. The cavities are expected to be the repositories for the Fc-derived peptides, while the semi-rigid protrusion of the Mez HCDR3 prevents the close approach of another macromolecule (e.g. intact IgG) to the active site.

Mutations of ras protooncogenes and p53 tumor suppressor gene in cardiac hemangiosarcomas from B6C3F1 mice exposed to 1,3-butadiene for 2 years.

1,3-Butadiene is a multisite carcinogen in rodents. Incidences of cardiac hemangiosarcomas were significantly increased in male and female B6C3F1 mice that inhaled 1,3-butadiene (BD) for 2 years. Eleven BD-induced cardiac hemangiosarcomas were examined for genetic alterations in ras protooncogenes and in the p53 tumor suppressor gene. Nine of 11 (82%) BD-induced hemangiosarcomas had K-ras mutations and 5 of 11 (46%) had H-ras mutations. All of the K-ras mutations were G-->C transversions (GGC-->CGC) at codon 13; this pattern is consistent with reported results in BD-induced lung neoplasms and lymphomas. Both K-ras codon 13 CGC mutations and H-ras codon 61 CGA mutations were detected in 5 of 9 (56%) hemangiosarcomas. The 11 hemangiosarcomas stained positive for p53 protein by immunohistochemistry and were analyzed for p53 mutations using cycle sequencing of polymerase chain reaction (PCR) amplified DNA isolated from paraffin-embedded sections. Mutations in exons 5 to 8 of the p53 gene were identified in 5 of 11 (46%) hemangiosarcomas, and all of these were from the 200- or 625-ppm exposure groups that also had K-ras codon 13 CGC mutations. Our data indicate that K-ras, H-ras, and p53 mutations in these hemangiosarcomas most likely occurred as a result of the genotoxic effects of BD and that these mutations may play a role in the pathogenesis of BD-induced cardiac hemangiosarcomas in the B6C3F1 mouse.

Airway fibrosis in rats induced by vanadium pentoxide.

Vanadium pentoxide (V(2)O(5)) is a cause of occupational asthma and bronchitis. We previously reported that intratracheal instillation of rats with V(2)O(5) causes fibrosis of the lung parenchyma (J. C. Bonner, P. M. Lindroos, A. B. Rice, C. R. Moomaw, and D. L. Morgan. Am. J. Physiol. Lung Cell. Mol. Physiol. 274: L72-L80, 1998). In this report, we show that intratracheal instillation of V(2)O(5) induces airway remodeling similar to that observed in individuals with asthma. These changes include airway smooth muscle cell thickening, mucous cell metaplasia, and airway fibrosis. The transient appearance of peribronchiolar myofibroblasts, which were desmin and vimentin positive, coincided with a twofold increase in the thickness of the airway smooth muscle layer at day 6 after instillation and preceded the development of airway fibrosis by day 15. The number of nuclear profiles within the smooth muscle layer also increased twofold after V(2)O(5) instillation, suggesting that hyperplasia accounted for thickening of the smooth muscle layer. The majority of cells incorporating bromodeoxyuridine at day 3 were located in the connective tissue surrounding the airway smooth muscle wall that was positive for vimentin and desmin. These data suggest that myofibroblasts are the principal proliferating cell type that contributes to the progression of airway fibrosis after V(2)O(5) injury.

The 21- and 23-kD forms of TCR zeta are generated by specific ITAM phosphorylations.

The T cell receptor (TCR) zeta subunit contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that translate effective extracellular ligand binding into intracellular signals by becoming phosphorylated into 21- and 23-kD forms. We report here that the 21-kD form of TCR zeta is generated by phosphorylation of the tyrosines in the second and third ITAMs, whereas the 23-kD form is formed by the additional phosphorylation of the membrane-proximal ITAM tyrosines. The stable formation of the 21- and 23-kD species requires the binding of the tandem SH2 domains of ZAP-70. We also report that TCR-mediated signaling processes can proceed independently of either the 21- or 23-kD species of TCR zeta.

Specific inhibitors of platelet-derived growth factor or epidermal growth factor receptor tyrosine kinase reduce pulmonary fibrosis in rats.

The proliferation of myofibroblasts is a central feature of pulmonary fibrosis. In this study we have used tyrosine kinase inhibitors of the tyrphostin class to specifically block autophosphorylation of the platelet-derived growth factor receptor (PDGF-R) or epidermal growth factor receptor (EGF-R). AG1296 specifically inhibited autophosphorylation of PDGF-R and blocked PDGF-stimulated [3H]thymidine uptake by rat lung myofibroblasts in vitro. AG1478 was demonstrated as a selective blocker of EGF-R autophosphorylation and inhibited EGF-stimulated DNA synthesis in vitro. In a rat model of pulmonary fibrosis caused by intratracheal instillation of vanadium pentoxide (V2O5), intraperitoneal delivery of 50 mg/kg AG1296 or AG1478 in dimethylsulfoxide 1 hour before V2O5 instillation and again 2 days after instillation reduced the number of epithelial and mesenchymal cells incorporating bromodeoxyuridine (Brdu) by approximately 50% at 3 and 6 days after instillation. V2O5 instillation increased lung hydroxyproline fivefold 15 days after instillation, and AG1296 was more than 90% effective in preventing the increase in hydroxyproline, whereas AG1478 caused a 50% to 60% decrease in V2O5-stimulated hydroxyproline accumulation. These data provide evidence that PDGF and EGF receptor ligands are potent mitogens for collagen-producing mesenchymal cells during pulmonary fibrogenesis, and targeting tyrosine kinase receptors could offer a strategy for the treatment of fibrotic lung diseases.