Bronchial responsiveness to nonantigenic bronchoconstrictors in awake sheep

The experiments were designed to further characterize pulmonary responsiveness to nonantigenic aerosol bronchoconstrictors in unanesthetized sheep. The distribution of aerosol histamine responsiveness was described among 55 sheep. Within day reproducibility of aerosol histamine (n = 18) and carbachol (n = 8) responsiveness was studied and aerosol histamine and carbachol responsiveness were compared (n = 9). The effects of cyclooxygenase inhibition with meclofenamate (n = 7) and ibuprofen (n = 8) on pulmonary responsiveness to aerosol histamine was studied as was the effect of ibuprofen (n = 6) on pulmonary responsiveness to aerosol carbachol. A log normal unimodal distribution of pulmonary responsiveness to aerosol histamine was described. Within day pulmonary responsiveness to aerosol histamine was highly reproducible while pulmonary responsiveness to aerosol carbachol decreased slightly, but not significantly, on the second challenge. Pulmonary responsiveness to aerosol histamine correlated with pulmonary responsiveness to aerosol carbachol (r = 0.85, P less than 0.05). Meclofenamate did not significantly attenuate pulmonary responsiveness to aerosol histamine. Ibuprofen attenuated pulmonary responsiveness to aerosol histamine (P less than 0.05) but not to aerosol carbachol. These experiments supply basic information related to pulmonary responsiveness to nonantigenic bronchoconstrictors in awake sheep.     

Prolonged exposure of mice to He-O2 at high pressure: effects on seizure and anesthesia liability

Multiday exposures of CD-1 mice to He-O2 atmospheres at pressures from 30 to 100 atm result in marked increases of threshold pressures for type I high-pressure neurological syndrome seizures. The effect develops with a half time (t1/2) of 12 h and is reversible (t1/2 = 7 h). The maximum enhancement of Pc is attained at a conditioning pressure of 80 ATA. Pressure conditioning also results in suppression of the compression rate effect on Pc. Furthermore, reserpine blocks the increase in Pc during prolonged pressure exposure. The entire effect thus appears to be an extension in time of the monoaminergic compression rate effect on Pc. Pressure conditioning does not modify anesthesia tolerance, unlike N2 habituation which affects anesthesia threshold pressure as well as Pc. The results are compared with the effects of habituation to inert-gas narcotics and the implications of the data for an understanding of inert-gas high-pressure antagonism in intact animals are discussed.     

In vivo efficacy of monoclonal antibody — drug conjugates of three different subisotypes which bind the human tumor — associated antigen defined by the KS1/4 monoclonal antibody

Summary Monoclonal antibodies (mAbs) BW 625 and BW 704, of the IgG₃ isotype, bound to immunochemically indistinguishable epitopes on ganglioside II³(NeuAc)₂-GgOse₃-Cer. Despite this fact the mAbs showed a differential binding pattern on human glioma cell lines i.e. immunohistochemical data indicate that the detected epitopes are not identical. Furthermore, either mAb is able to mediate the antibody-dependent cellular cytotoxicity reaction (ADCC) and the human-complement-dependent cytotoxicity reaction (CDC) with epitope-expressing tumor cells. All cryopreserved tissue specimens from gliomas and neuroblastomas were immunohistochemically stained, whereas the other small round cell tumors of childhood, as well as melanomas and small-cell lung carcinomas, were essentially negative. Positive staining of normal cryopreserved tissues was restricted to amyelinic axons, Hassal's bodies and some connective tissue fibers in thymus and the tegumentary epithelium of skin. The high selectivity of mAb BW 704 for gliomas and neuroblastomas, the lack of cross-reactivity with major tissues and the strong ADCC and CDC potential argue for the use of mAb BW 704 in immunotherapy of neuroblastomas and gliomas.     

Acetaminophen Toxicity in Mice Lacking NADPH Oxidase Activity: Role of Peroxynitrite Formation and Mitochondrial Oxidant Stress

Previous data have indicated that activated macrophages may play a role in the mediation of acetaminophen toxicity. In the present study, we examined the significance of superoxide produced by macrophages by comparing the toxicity of acetaminophen in wild-type mice to mice deficient in gp91phox, a critical subunit of NADPH oxidase that is the primary source of phagocytic superoxide. Both groups of mice were dosed with 300?mg/kg of acetaminophen or saline and sacrificed at 1, 2, 4 or 24?h. Glutathione in total liver and in mitochondria was depleted by approximately 90% at 1?h in wild-type and knock out mice. No significant differences in toxicity (serum transaminase levels or histopathology) were observed between wild-type and mice deficient in gp91phox. Mitochondrial glutathione disulfide, as a percent of total glutathione, was determined as a measure of oxidant stress produced by increased superoxide, leading to hydrogen peroxide and/or peroxynitrite. The percent mitochondrial glutathione disulfide increased to approximately 60% at 1?h and 70% at 2?h in both groups of mice. Immunohistochemical staining for nitrotyrosine was present in vascular endothelial cells at 1?h in both groups of mice. Acetaminophen protein adducts were present in hepatocytes at 1?h in both wild-type and knock out animals. These data indicate that superoxide from activated macrophages is not critical to the development of acetaminophen toxicity and provide further support for the role of mitochondrial oxidant stress in acetaminophen toxicity.     

Germ Cells Are Not Required to Establish the Female Pathway in Mouse Fetal Gonads

The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells.     

Preparation and stability of sixteen murine tissues and organs for flow cytometric cell cycle analysis

Three different technical protocols were used to prepare samples for flow cytometric (FCM) analysis. Each protocol developed worked best for only certain organs. Protocol I involved mincing small pieces of fresh tissue in the propidium iodide (PI) staining solution and filtering through packed glass wool. The organs that were prepared by protocol I were: submandibular gland, urinary bladder, liver, thymus, bone marrow, spleen, lung, kidney and testis. Protocol II involved exposure of the organ to 0.5% acetic acid for 48 h prior to mincing in the PI. The organs that were prepared by protocol II were: uterus, rectum, colon, ileum, and heart. Protocol III utilized an exposure to 0.5% acetic acid, pepsinization, and then staining with PI. The tissues that were prepared by protocol III were the epithelium of the anterior surface of the cornea and the epithelium of the surface of the tongue. A total of 16 different organs and tissues were successfully prepared. For each organ, averaged DNA histograms were analyzed by nonparametric and parametric programs and the results (phase fractions) are presented in tabular form. Several of the organs used came from animals exposed to 1.0 mg/kg vincristine (VC) for 5-6 h to test the capability of the different protocols to detect the enlargement of the G2 + M compartment by the accumulation of VC-arrested mitotic figures. The stability of the many different sample preparations was tested by comparing averaged DNA histograms obtained on the day of sample preparation to averaged DNA histograms of the same set of samples after storage at 4 degrees C, with or without fixation in 10% phosphate-buffered formalin, for days to weeks. After staining with propidium iodide, fixation of the sample with a final concentration of 2-3% phosphate-buffered formalin, was the procedure adopted to assure sample stability. The demonstration of sample stability permits sample preparation to occur at one site followed by transport of the samples to the FCM laboratory at another geographical location. The major findings of this work were a) technical protocols were developed which resulted in acceptable nuclear suspensions for FCM from 16 different murine organs or tissues, b) the stability of these samples can be assured by fixing the PI stained nuclear suspension with formalin, and c) each different protocol was capable of detecting and preserving at least some of the mitotic figures arrested and collected by vincristine.     

NADPH oxidase-derived oxidant stress is critical for neutrophil cytotoxicity during endotoxemia

Neutrophils can cause liver injury during endotoxemia through generation of reactive oxygen species. However, the enzymatic source of the oxidant stress and the nature of the oxidants generated remain unclear. Therefore, we investigated the involvement of NADPH oxidase in the pathophysiology by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) in the galactosamine/endotoxin (700 mg/kg Gal:100 microg/kg ET) model of liver injury. In addition, we measured chlorotyrosine as indicator for hypochlorous acid formation by myeloperoxidase. Gal/ET treatment of male C3HeB/FeJ mice resulted in sinusoidal neutrophil accumulation and parenchymal cell apoptosis (14 +/- 3% of cells) at 6 h. At 7 h, 35% of neutrophils had transmigrated. The number of apoptotic cells increased to 25 +/- 2%, and the overall number of dead cells was 48 +/- 3%; many of them showed the characteristic morphology of necrosis. Hepatocytes, which colocalized with extravasated neutrophils, stained positive for chlorotyrosine and 4-hydroxynonenal (4-HNE) protein adducts. In contrast, animals pretreated with DPI (2.5 mg/kg) were protected against liver injury at 7 h (necrosis = 20 +/- 2%). These livers showed little chlorotyrosine or 4-HNE staining, but apoptosis and neutrophil accumulation and extravasation remained unaffected. However, DPI-treated animals showed serious liver injury at 9 h due to sustained apoptosis. The results indicate that NADPH oxidase is responsible for the neutrophil-derived oxidant stress, which includes formation of hypochlorous acid by myeloperoxidase. Thus NADPH oxidase could be a promising therapeutic target to prevent neutrophil-mediated liver injury. However, the long-term benefit of this approach needs to be investigated in models relevant for human liver disease.