A randomized crossover study to evaluate Ro 115-1240, a selective alpha1A/1L-adrenoceptor partial agonist in women with stress urinary incontinence.

OBJECTIVES: To investigate the potential therapeutic benefits of the selective alpha1A/1l-adrenoceptor partial agonist Ro 115-1240 in women with mild-to-moderate stress urinary incontinence (SUI). PATIENTS AND METHODS: Thirty-seven women with mild-to-moderate SUI were enrolled in a randomized, placebo-controlled crossover study. Patients received 1.5 mg Ro 115-1240 twice daily or matching placebo for 2 or 4 weeks. Voiding diaries were used to record the number of SUI episodes, urge incontinence episodes and pads used. Sitting blood pressures and heart rate were recorded at each visit. RESULTS: Ro 115-1240 was associated with a significantly lower mean weekly number of SUI episodes than placebo (8.4 vs 6.0; P= 0.0079), a 28% relative improvement over placebo. There was also a significantly lower mean number of pads used and wet pads changed/week with Ro 115-1240 than with placebo (P = 0.0055 and 0.0066, respectively). The most frequently reported treatment-emergent adverse events were scalp tingling, headache, chills, piloerection, and pruritus. Generally these events were transient and mild to moderate. There was a slightly lower mean sitting heart rate with Ro 115-1240 than with placebo, but no difference in mean systolic or diastolic blood pressure between treatments. CONCLUSIONS: This study suggests that selective alpha1A/1l-adrenoceptor partial agonists have the potential to improve the symptoms of SUI with little or no cardiovascular effect. These results are encouraging and a randomized controlled trial of Ro 115-1240 in a larger population with SUI is warranted to substantiate these findings.

Cell proliferation and global methylation status changes in mouse liver after phenobarbital and/or choline-devoid, methionine-deficient diet administration.

Our laboratory is testing the hypothesis that hypomethylation of DNA [a decreased content of 5-methylcytosine (5MeC) compared with cytosine] facilitates aberrant oncogene expression involved in tumorigenesis, using a model system of mouse strains with differing susceptibilities to liver tumorigenesis. The B6C3F1 (C57BL/6 x C3H/He) mouse serves as the relatively susceptible strain and C57BL/6 serves as the relatively resistant strain. Phenobarbital (PB) and/or administration of a choline-devoid, methionine-deficient diet (CMD) were employed as non-genotoxic hepatocarcinogens. We have examined hepatocyte and nonhepatocyte proliferation in conjunction with an assessment of global methylation changes in liver DNA of B6C3F1 and C57BL/6 mice following these promoter treatments. Bromodeoxyuridine incorporation into DNA, used to measure cell proliferation indirectly, was visualized by immunohistochemistry and quantified by a Macintosh-based image analysis system. Increased hepatocyte proliferation was demonstrated following all three treatments. This increase was larger in C57BL/6 (the relatively resistant strain) as compared with B6C3F1. In contrast, global hypomethylation was evident to a larger extent in the B6C3F1 mouse, as compared with C57BL/6. PB led to hypomethylation (>20% decrease as compared with controls) at weeks 1, 2 and 4 in B6C3F1, but not in C57BL/6 at the same time points. CMD diet administration led to hypomethylation in both strains. At week 1, 21 and 9% decreases in global methylation status were observed in B6C3F1 and C57BL/6 respectively. Evaluation of these data suggests that the heightened sensitivity of the B6C3F1 mouse compared with the C57BL/6 is due, in part, to a decreased capacity for, or fidelity of, maintaining normal methylation status. The relatively resistant strain is better able to maintain the normal methylation status of DNA in the face of a higher level of cell proliferation.

Protein characterization and targeted disruption of Grg, a mouse gene related to the groucho transcript of the Drosophila Enhancer of split complex.

The Grg gene encodes a 197 amino acid protein homologous to the amino-terminal domain of the product of the groucho gene of the Drosophila Enhancer of split complex. Analysis with a polyclonal antisera specific for the Grg protein revealed that Grg is a 25 kd nuclear protein that can participate in specific protein-protein interactions. A null mutation of the Grg gene was constructed by gene targeting. Mice homozygous for this mutation completed embryogenesis and were born, but exhibited varying degrees of post-natal growth deficiency. No dosage-sensitive genetic interaction was detected between the Notch1 and Grg genes in mice heterozygous for a Notch1 mutant allele and homozygous for the Grg null mutation.

Characterization of E-selectin-deficient mice: demonstration of overlapping function of the endothelial selectins.

The initial rolling interaction of leukocytes with the blood vessel wall during leukocyte trafficking has been postulated to rely on members of the selectin family of adhesion molecules. Two selectins, E-selectin and P-selectin, have been identified that are expressed on activated endothelial cells. Mice deficient in E-selectin expression have been produced in order to examine the role of this selectin in leukocyte trafficking. Mice homozygous for an E-selectin null mutation were viable and exhibited no obvious developmental alterations. E-selectin-deficient mice displayed no significant change in the trafficking of neutrophils in several models of inflammation. However, blocking both endothelial selectins by treatment of the E-selectin-deficient animals with an anti-murine P-selectin antibody, 5H1, significantly inhibited neutrophil emigration in two distinct models of inflammation. While neutrophil accumulation at early times during thioglycollate-induced peritonitis was dependent on P-selectin, neutrophil accumulation at later time points was blocked by 5H1 only in E-selectin-deficient mice but not in wild-type mice. Similarly, edema as well as leukocyte accumulation in a model of delayed-type hypersensitivity in the skin was almost completely prevented by blockade of P-selectin function with 5H1 in the E-selectin-deficient mice while the same treatment had no effect in wild-type mice. These data demonstrate that the majority of neutrophil migration in both models requires an endothelial selectin but that E-selectin and P-selectin are functionally redundant. These data have important implications in the use of selectin antagonists in the treatment of inflammatory disease.

Mice homozygous for a null mutation of activin beta B are viable and fertile.

We have made a null mutation in the mouse activin beta B gene by deleting the portion of the gene encoding the proteolytic cleavage site and the majority of the coding region for the mature processed protein. Mice homozygous for this mutation complete embryogenesis and are completely viable. Approximately 40% of the homozygous mutant animals are born with open eyes. Aside from the incompletely penetrant eye defects, histopathological analysis has not revealed any other abnormalities in homozygous mutant animals. Breeding tests have shown that both male and female homozygous mutant animals are fertile.

Enhanced antitumor efficacy in mice by combination treatment with interleukin-1 alpha and interferon-alpha.

The antitumor efficacy of recombinant murine interleukin-1 alpha (rMuIL-1 alpha) was evaluated either alone or in combination with recombinant human hybrid interferon alpha A/D (IFN-alpha A/D) against the murine B16 F10 malignant melanoma. Treatment of subcutaneous tumor-bearing mice intraperitoneally with rMuIL-1 alpha resulted in a dose-dependent inhibition of tumor growth with the greatest activity obtained with the maximum tolerated dose of rMuIL-1 alpha (10 micrograms per treatment). Augmented tumor inhibition comparable to that seen in mice treated with a high dose of rMuIL-1 alpha was observed in subcutaneous tumor-bearing mice injected with the combination of IFN-alpha A/D and a low dose of rMuIL-1 alpha. Similar inhibition of subcutaneous tumor growth was obtained in T-cell-deficient nude or natural killer cell-deficient beige mice. In contrast, treatment of mice bearing B16F10 experimental pulmonary metastases with rMuIL-1 alpha resulted in no decrease in the number of metastases, and rMuIL-1 alpha did not potentiate the antimetastatic activity of IFN-alpha A/D. A synergistic induction of IL-6 was induced in mice treated with the combination of rMuIL-1 alpha plus IFN-alpha A/D but the level of IL-6 induced was not correlated with inhibition of tumor growth because this elevation of IL-6 was not observed in tumor-bearing nude mice. No direct antiproliferative activity was demonstrable in vitro against B16 F10 cells with rMuIL-1 alpha, IL-6, or rMuIL-1 alpha plus IL-6, and addition of these cytokines did not enhance the antiproliferative activity of IFN-alpha A/D.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the methylation status and expression of the raf oncogene in phenobarbital-induced and spontaneous B6C3F1 mouse liver tumors.

The liver tumor-prone B6C3F1 mouse (C57Bl/6 female x C3H/He male), in conjunction with the more susceptible C3H/He paternal strain and the resistant C57BL/6 maternal strain, is an excellent model for studying the mechanisms involved in carcinogenesis. The study reported here indicated that the B6C3F1 mouse inherited a maternal raf allele containing a methylated site not present in the paternal allele. Seven days after partial hepatectomy or after administration of a promoting dose of phenobarbital (PB) for 14 d; raf in B6C3F1 mouse liver was hypomethylated. The additional methylated site in the allele inherited from C57BL/6 was not maintained. The methylation status of raf in the liver of the C57BL/6 mouse was not affected by PB treatment. This indicates that the B6C3F1 mouse is less capable of maintaining methylation of raf than the C57BL/6 strain is. In both PB-induced and spontaneous B6C3F1 liver tumors, raf was hypomethylated in a nonrandom fashion. The level of raf mRNA increased in seven of 10 PB-induced tumors but in only one of five spontaneous tumors, whereas the level of Ha-ras mRNA increased in nine of 10 PB-induced tumors and in four of five spontaneous tumors. The results of our investigation (a) support the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism involved in tumorigenesis, (b) support the notion that PB promotes liver tumors that develop along a pathway different from that leading to spontaneous tumors, and (c) indicate that differences in DNA methylation between C57BL/6 and B6C3F1 mice could, in part, account for the unusually high tendency of the latter strain to develop liver tumors.

Differential antagonism of platelet activating factor-induced neutrophilia and gastric hemorrhage in the rat.

Intravenous injection of platelet activating factor (PAF) in rats produced hypotension, neutrophilia, gastric congestion, and sloughing of the gastric epithelium. The congestion was quantified by measuring hemoglobin in the gastric mucosa. Other lesions were quantified by scores of gross pathology and histopathology. PAF-induced changes in neutrophil levels were prevented by pretreatment with the PAF-antagonist Ro24-4736, but not by the PAF-antagonist CV-3988. Both PAF antagonists reduced the hypotension, the amount of hemoglobin in the gastric mucosa, and the PAF-induced gastric pathology. These results suggest that PAF receptors involved in PAF-induced neutrophil mobilization respond differently from PAF receptors in the gastrointestinal and cardiovascular systems.

Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations.

The c-abl gene, originally identified as the cellular homolog of the transforming gene of the Abelson murine leukemia virus, encodes a protein-tyrosine kinase of unknown function that is expressed in all mammalian tissues. We have previously described the introduction of a mutation in the c-abl gene into the mouse germline via targeted gene disruption of embryonic stem cells. We now show that mice homozygous for this mutation are severely affected, displaying increased perinatal mortality, runtedness, and abnormal spleen, head, and eye development. We have examined components of the immune system and have found major reductions in B cell progenitors in the adult bone marrow, with less dramatic reductions in developing T cell compartments.

Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting.

The inviability of diploid androgenetic and parthenogenetic embryos suggests imprinting of paternal and maternal genes during germ cell development, and differential expression of loci depending on parental inheritance appears to be involved. To facilitate identification of imprinted genes, we have derived diploid androgenetic embryonic stem (ES) cell lines. In contrast to normal ES cells, they form tumors composed almost entirely of striated muscle when injected subcutaneously into adult mice. They also form chimeras following blastocyst injection, although many chimeras die at early postnatal stages. Surviving chimeras develop skeletal abnormalities, particularly in the rib cartilage. These results demonstrate that androgenetic ES cells are pluripotent and point to stage- and cell-specific expression of developmentally important imprinted genes.

Adjusting for confounded variables: pulmonary function and smoking in a special population.

Confounded variables present an obstacle to valid inference in many environmental and occupational studies. We describe a series of procedures that we used to address this problem in a study of pulmonary function and smoking. Subjects were drawn from the Multiple Risk Factor Intervention Trial (MRFIT), a prospective study of coronary heart disease. Confounding of smoking, hypertension, and hyperlipidemia was designed into the trial and was beyond the control of our ancillary study. We used statistical techniques to detect and characterize the pattern of confounding, identify important variables affecting pulmonary function, and perform appropriate adjustments for extraneous influences (i.e., other than smoking). Among the techniques we used were factor analysis, stepwise multiple regression, and bootstrap replication. Analysis of the adjusted pulmonary function measurements showed that they were satisfactorily standardized and free of artifact. Moreover, use of the adjusted values sharpened our statistical results concerning smoking, the ultimate object of the study. We contrast the use of external and internal standards and discuss methods for detecting, ruling out, or counteracting confounding.

Dietary seaweed (Laminaria) and mammary carcinogenesis in rats.

To test the potential in vivo antitumor effect of dietary seaweed, we induced mammary tumors in female Sprague-Dawley rats with the carcinogen 7,12-dimethylbenz(a)anthracene. Twenty-one-day-old rats (n = 108) were divided into two groups. Controls were fed a standard semipurified diet, and experimental rats received the control diet with 5% Laminaria, a brown seaweed, replacing 5% alphacel . At 55 days of age, each rat received 5 mg 7,12-dimethylbenz(a)anthracene intragastrically. Rats were palpated for mammary tumors and weighed weekly for 26 weeks. Complete autopsies were then done on all rats. The seaweed diet did not alter weight gain or weights of body organs at autopsy. Experimental rats had a significant delay in the time to tumor (p = 0.007); median time until tumor was 19 weeks in experimental rats and 11 weeks in control animals. Among mammary adenocarcinoma tumor-bearing animals, experimental rats had fewer adenocarcinomas/individual (p less than 0.05). There was also an overall 13% reduction in the number of experimental rats with histologically confirmed adenocarcinomas (76% among the control rats compared to 63% among the experimental rats). Components of Laminaria which might account for the observed difference in mammary tumor growth are varied and include the sulfated polysaccharide fucoidan . Rats in the top row of cages had a significant (p = 0.01) delay in time to tumor compared to rats in the lower four rows. In each row, the seaweed-fed rats had a longer time to tumor than did the control rats.

Correlation of lung macrophage age and surface antigen in the hamster.

A monoclonal antibody specific for a surface antigen found on hamster lung macrophages has been produced. Macrophages obtained from LSH Syrian golden hamsters by pulmonary lavage have varying amounts of this antigen on their surface. We compared the age of alveolar macrophages (using 3H-thymidine) with the amount of surface antigen. Lung macrophages were obtained by repeated saline lavage at 1, 3, 5, and 10 days after 3H-thymidine injection. Monoclonal antibody was then reacted with these cells followed by fluorescein isothiocyanate-conjugated protein A. Cell size and fluorescence were analyzed by flow cytometry. A wide range of fluorescent intensity was observed; the cells were sorted into four subpopulations (SPs). SP1 had the lowest fluorescence per cell, and SP4 had the highest. The sorted cells were placed on glass slides, and autoradiographs were made. The percentage of labeled macrophages in each SP was determined. At 1 day after thymidine injection, cells with a paucity of antigen (SP1) were the most highly labeled; 12.5% of SP1 macrophages were labeled, but only 1.4 and 1.1% of SP3 and SP4 were labeled, respectively. The labeling was relatively even in all four SPs at 3 days, but at 5 days the labeling of cells in SP2 and SP3 was highest. By day 10, labeled macrophages had large amounts of surface antigen and were in SP3 and SP4. These findings suggest that pulmonary macrophages that have recently synthesized DNA lack surface antigen. As time passes, cells mature and more antigen is acquired. The amount of surface antigen reflects cell age and provides a useful tool to isolate and study macrophage SPs.

Effects of exercise on particle deposition in Syrian golden hamsters.

The effects of exercise and its associated increase in ventilation on the deposition of inhaled particles were investigated. Both total retention and patterns of distribution of a 99mTc sulfur colloid aerosol (activity median aerodynamic diameter, 0.38 micron; geometric standard deviation, 1.35) were measured in male Syrian golden hamsters. Animals were either anesthetized, resting, or exercising on a treadmill during a 15-min aerosol exposure. Each hamster's oxygen consumption (VO2) was continuously monitored during the exposure; immediately after, the animal was killed. The lungs were excised, inflated, and dried in a microwave oven. The rigid lungs were sliced and dissected in a predetermined way so that retention at specific locations could be compared. The radioactivity and weight of 40 pieces from each of 12 hamsters were measured. The uniformity of deposition was described by an evenness index (EI) for each piece: EI = (cpm/g)piece/(cpm/g) whole lung. With theoretical uniformity of retention, all EI values should be 1.0. During aerosol exposure, the exercising group had a VO2 of 5.0 +/- 0.6 (SD) ml STPD/min/100 g, which was 2 times the resting group (2.5 +/- 0.4) and 4 times the anesthetized group (1.2 +/- 0.2). The total retention of particles in the lungs increased in a parabolic manner as a function of VO2; the exercising animals had a retention 6 times greater than the anesthetized animals. The increased retention in running hamsters may reflect either increased ventilation alone or increased collection efficiency. Each animal's activity level also affected local distribution of particles in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Malignant mesothelioma in urban dogs.

Clinical and postmortem materials from six dogs with a diagnosis of malignant mesothelioma were studied retrospectively. The dogs were urban pets with clinical signs of malignant effusions. Two mesotheliomas were pleural, one pericardial, and one peritoneal. Both pleura and pericardium were involved in one dog, and the pleura and peritoneum in another. On gross examination at necropsy, diffuse granular or velvety plaques covering mesothelial surfaces were found in all dogs; firm discrete pleural nodules also were present in two dogs. Neither distant metastases nor areas of deep lung invasion were found. The tumors varied histologically, but the most common type was epithelial with a papillary pattern. Ultrastructurally, the neoplastic cells had prominent surface microvilli, numerous desmosomes, and tonofilaments. Lung tissue from these dogs and from control dogs was evaluated for the presence of ferruginous bodies. Asbestos bodies were found in three of five dogs with mesotheliomas but rarely were found in control dogs. As a group, the mesothelioma cases had significantly more asbestos bodies and total ferruginous bodies than controls. The clinical and morphologic appearance of canine mesothelioma is similar to human mesothelioma and also may be associated with exposure to airborne fibers.