Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma.

Intratumoral heterogeneity (ITH) has increasingly being described for multiple cancers as the root cause of therapy resistance. Recent studies have started to explore the scope of ITH in glioblastoma (GBM), a highly aggressive and fatal form of brain tumor, to explain its inevitable therapy resistance and disease relapse. In this review, we detail the emerging data that explores the extensive genetic, cellular and functional ITH present in GBM. We discuss current experimental models of human GBM recurrence and suggest harnessing new technologies (CRISPR-Cas9 screening, CyTOF, cellular barcoding, single cell analysis) to delineate GBM ITH and identify treatment-refractory cell populations, thus opening new therapeutic windows. We will also explore why current therapeutics have failed in clinical trials and how ITH can inform us on developing empiric therapies for the treatment of recurrent GBM.

CD133+ brain tumor-initiating cells are dependent on STAT3 signaling to drive medulloblastoma recurrence.

Medulloblastoma (MB), the most common malignant paediatric brain tumor, is currently treated using a combination of surgery, craniospinal radiotherapy and chemotherapy. Owing to MB stem cells (MBSCs), a subset of MB patients remains untreatable despite standard therapy. CD133 is used to identify MBSCs although its functional role in tumorigenesis has yet to be determined. In this work, we showed enrichment of CD133 in Group 3 MB is associated with increased rate of metastasis and poor clinical outcome. The signal transducers and activators of transcription-3 (STAT3) pathway are selectively activated in CD133+ MBSCs and promote tumorigenesis through regulation of c-MYC, a key genetic driver of Group 3 MB. We screened compound libraries for STAT3 inhibitors and treatment with the selected STAT3 inhibitors resulted in tumor size reduction in vivo. We propose that inhibition of STAT3 signaling in MBSCs may represent a potential therapeutic strategy to treat patients with recurrent MB.

In vivo efficacy and pharmacological properties of a novel glycopeptide (YV4465) against vancomycin-intermediate Staphylococcus aureus.

Infections caused by vancomycin-intermediate Staphylococcus aureus (VISA) are associated with high rates of vancomycin treatment failure. The lipophilic vancomycin-carbohydrate conjugate YV4465 is a new glycopeptide antibiotic that is active against a variety of clinically relevant multidrug-resistant Gram-positive pathogens in vitro. YV4465 was 50- and 1000-fold more effective than vancomycin against VISA and vancomycin-resistant enterococci, respectively. This study evaluated the in vivo efficacy against VISA as well as the pharmacokinetics and toxicology of YV4465. A neutropenic mouse thigh infection model was used for the determination of efficacy and pharmacodynamic properties against VISA. YV4465 produced a dose-dependent reduction in VISA titres in thigh muscle; bacterial titres were reduced by up to ca. 2log(10)CFU/g from the pre-treatment titre at a dosage of 8 mg/kg. Single-dose pharmacokinetic studies demonstrated an increase in drug exposure to the animal following linear kinetics with a prolonged half-life (t(1/2)) compared with vancomycin. The peak plasma concentration (C(max)) following an intravenous dose of 12 mg/kg was 703 µg/mL. Acute toxicology studies revealed that YV4465 did not cause any significant alterations in biochemical parameters related to major organs such as the liver and kidneys at its pharmacodynamic endpoint (>ED(2-log kill)). These studies demonstrate that YV4465 has the potential to be developed as a next-generation glycopeptide antibiotic for the treatment of infections caused by VISA.

Vasorelaxation responses to insulin in laminar vessel rings from healthy, lean horses.

Hyperinsulinemia causes laminitis experimentally and is a risk factor for naturally occurring laminitis. The aim of this study was to investigate the effects of insulin on laminar vascular relaxation and to induce insulin-associated vascular dysfunction in vitro. Relaxation responses of isolated laminar arterial and venous rings to acetylcholine and insulin were evaluated. To alter vascular function in response to insulin, all vessel rings were incubated with insulin or vehicle, submaximally contracted, administered insulin again and relaxation responses recorded. Laminar arteries were also incubated with the mitogen-activated protein kinase (MAPK) inhibitor, PD-98059. Relaxation in response to acetylcholine was not different between arteries and veins, but veins relaxed less in response to insulin than arteries. In arteries incubated with insulin, the subsequent relaxation response to insulin was blunted. Veins had minimal relaxation to insulin regardless of incubation. Arteries incubated with PD-98059 relaxed more in response to insulin than arteries not exposed to PD-98059, indicating that MAPK plays a role in maintenance of basal tone in laminar arteries. A differing response of laminar veins and arteries to insulin-induced relaxation may be important in understanding the link between hyperinsulinemia and laminitis. In vitro induction of vascular dysfunction in response to insulin in laminar arteries may be useful for testing therapeutic interventions and for understanding the pathophysiology of laminitis.

Autofluorescence spectroscopy augmented by multivariate analysis as a potential noninvasive tool for early diagnosis of oral cavity disorders.

OBJECTIVE: Oral leukoplakia is one of the common potentially malignant lesions encountered worldwide. We report the results of an in vivo clinical evaluation of autofluorescence (AF) spectroscopy for differential diagnosis of oral leukoplakia. Multivariate analysis of spectral data has been incorporated to improve the efficacy of the technique. The results of this noninvasive study are expected to provide potential for extending the technique to other disorders. MATERIALS AND METHODS: A total of 18 patients and 30 normal volunteers participated in this study. AF spectra were acquired from affected sites of patients and from right and left buccal mucosa of normal volunteers. Diagnostic performance was analyzed using spectral intensity ratio (SIR), and principal component analysis followed by linear discriminant analysis (PCA-LDA). RESULTS: AF spectra of leukoplakic patients showed characteristic emissions from flavin adenine dinucleotide (FAD) and porphyrin at 500 and 630 nm, respectively. But the emission from porphyrin is not very prominent in the case of healthy volunteers. Also, significant decrease in spectral intensity is observed for leukoplakia compared with normal volunteers in the unprocessed spectra. Method of SIR yielded 96% sensitivity and 100% specificity and an overall 100% for PCA-LDA respectively for efficient differentiation of the lesions. CONCLUSIONS: The result of this preliminary study shows that PCA-LDA or SIR applied to AF spectroscopy is a useful tool for the differential diagnosis of oral cavity disorders. This has been demonstrated in leukoplakia in a clinical setting, and it is expected that the technique can be extended to other oral cavity disorders as well.

Effect of potential therapeutic agents in reducing oxidative stress in pulmonary tissues of recurrent airway obstruction-affected and clinically healthy horses.

REASONS FOR PERFORMING STUDY: To determine and compare the reactive oxygen and nitrogen species (ROS and RNS) in pulmonary tissues of horses affected with recurrent airway obstruction (RAO) and clinically healthy horses, and to evaluate the effectiveness of potential therapeutic agents in reducing ROS and RNS in the tissues of these horses. OBJECTIVES: We hypothesised that RAO-affected horses would have high levels of reactive species and that the test agents would reduce them. The objectives were as follows: 1) to determine the level of ROS and RNS in pulmonary tissues (bronchial and arterial rings) of RAO-affected and clinically healthy horses; and 2) to determine the ability of pentoxifylline, pyrrolidine-dithiocarbamate and a combined use of endothelin A and B receptor antagonists (BQ123 and BQ788, respectively) in reducing reactive species. METHODS: Arterial and bronchial rings were collected from the diaphragmatic lung lobe of each horse immediately after euthanasia. The levels of ROS and RNS were measured in control tissues and those incubated with test agents, using an electron paramagnetic resonance instrument. RESULTS: The levels of ROS and RNS were significantly greater in arterial and bronchial tissues of RAO-affected than of clinically healthy horses. Pentoxifylline and endothelin antagonists reduced both ROS and RNS in tissues from RAO-affected horses. Basal levels of reactive species in clinically healthy horses were not affected by these agents. No difference in the level of reactive species was observed between arterial and bronchial tissues. CONCLUSIONS: Horses affected by RAO had higher ROS and RNS than clinically healthy horses. Pentoxifylline and endothelin antagonists effectively reduced ROS and RNS in pulmonary tissues of RAO-affected horses. POTENTIAL RELEVANCE: The study suggested a potential use for pentoxifylline and endothelin antagonists in treating RAO-affected horses. As endothelin is involved in physiological functions, therapeutic use of its antagonists is cautioned.

Sonic hedgehog regulates Bmi1 in human medulloblastoma brain tumor-initiating cells.

Bmi1 is a key stem cell regulatory gene implicated in the pathogenesis of many aggressive cancers, including medulloblastoma. Overexpression of Bmi1 promotes cell proliferation and is required for hedgehog (Hh) pathway-driven tumorigenesis. This study aimed to determine if Sonic hedgehog (Shh) modulates the key stem cell regulatory gene Bmi1 in childhood medulloblastoma brain tumor-initiating cells (BTICs). Although current literature suggests that there is a correlation between Shh pathway genes and Bmi1 expression, it is unclear whether there is indeed a direct regulatory mechanism. To address whether Shh induces expression of Bmi1, stem cell-enriched populations from medulloblastoma cell lines and primary samples were treated with Shh ligand and KAAD-cyclopamine (Shh antagonist). Our data indicate that Bmi1 expression positively correlates with increasing Shh ligand concentrations. Chromatin immunoprecipitation reveals that Gli1 preferentially binds to the Bmi1 promoter, and Bmi1 transcript levels are increased and decreased by Gli1 overexpression and downregulation, respectively. Knockdown experiments of Bmi1 in vitro and in vivo demonstrate that Hh signaling not only drives Bmi1 expression, but a feedback mechanism exists wherein downstream effectors of Bmi1 may, in turn, activate Hh pathway genes. These findings implicate Bmi1 and Hh as mutually indispensable pathways in medulloblastoma BTIC maintenance. Recent molecular characterization of medulloblastoma also reveals that Bmi1 is overexpressed across all subgroups of medulloblastoma, particularly in the most aggressive subtypes. Lastly, despite recent identification of BTIC markers, the molecular characterization of these cell populations remains unclear. In this work, we propose that the BTIC marker CD133 may segregate a cell population with a Hh-receptor phenotype, thus demonstrating a cell-cell interaction between the CD133+ Hh receptor cells and the CD133- Hh-secreting cells.

Insulin resistance in equine digital vessel rings: an in vitro model to study vascular dysfunction in equine laminitis.

REASONS FOR PERFORMING STUDY: One of the causes of equine laminitis is hyperinsulinaemia, which may be associated with endothelial dysfunction and insulin resistance of vessels. HYPOTHESIS AND OBJECTIVES: Insulin resistance can be induced in palmar digital vessels by continued exposure to insulin in vitro. The objective was to evaluate this in vitro model for future studies. METHODS: Palmar digital vessel segments were collected immediately after euthanasia from horses with normal insulin/glucose blood values. Four arterial and 4 venous rings (3 mm wide) were prepared and each ring mounted in a tissue bath, containing Tyrode's solution at 37°C, 2 g tension was applied and the rings allowed to equilibrate for 45 min. Of the 4 rings of each vessel type, one was used as a control. One each of the remaining 3 rings was used for incubation with insulin (to induce resistance), wortmannin (to block PI3-kinase) and PD-098059 (to block MAP-kinase), respectively, for 30 min. After the incubation period, the rings were contracted with phenylephrine. When the response reached a plateau, a single dose of insulin was added to the baths and the response of each ring monitored for 30 min. RESULTS: Insulin relaxed the control rings and those treated with PD 098059 but contracted those pretreated with insulin and wortmannin. Normal relaxation responses of the rings were converted to contractions by insulin resistance. Insulin resistance was confirmed by the qualitative response of insulin-incubated and wortmannin-incubated rings. CONCLUSIONS: This study demonstrated successful induction of insulin resistance in both arterial and venous rings. It also suggested that the MAP-kinase pathway plays a minor role in controlling vasomotor tone under normal physiological conditions. POTENTIAL RELEVANCE: The study suggests that the induction of insulin resistance in equine palmar digital vessel rings is reliable and provides a good in vitro model for studying the vascular insulin resistance which may occur in equine laminitis.

Plasma and pulmonary fluid endothelin in horses with seasonal recurrent airway obstruction.

BACKGROUND: Summer pasture-associated recurrent airway obstruction (SPA-RAO), a seasonal airway obstructive disease of horses, is characterized by clinical exacerbation after exposure to pasture during warm months of the year. Endothelin (ET)-1, potent bronchoconstrictor, mitogen, secretagogue, and proinflammatory mediator, has been implicated in the pathogenesis of asthma and equine heaves. HYPOTHESIS: Immunoreactive ET-1 concentrations increase during clinical exacerbation and return to basal values during periods of disease remission. ANIMALS: Twelve horses, 6 affected with SPA-RAO and 6 nonaffected. METHODS: Prospective, observational study. Bronchoalveolar lavage fluid (BALF), arterial and venous plasma samples, and clinical variables were obtained from affected horses during clinical exacerbation and remission. Samples and data of nonaffected horses were collected during the summer and winter on dates similar to affected horses. Immunoreactive ET-1 was determined using a commercial ELISA. RESULTS: The median and range ET-1 concentrations (pg/ml) in arterial (1.3, 0.7-1.8) and venous (1.3, 1.2-1.7) plasma and in BALF (0.3, 0.2-0.4), and pulmonary epithelial lining fluid (PELF) (25.5, 21-50) were greater in affected horses during clinical exacerbation compared with remission (P < .01). The concentrations of immunoreactive ET-1 were greater in affected horses during clinical exacerbation compared with nonaffected horses (P < .05). CONCLUSIONS AND CLINICAL IMPORTANCE: During clinical exacerbation of SPA-RAO, ET-1 is increased in circulation and pulmonary secretions. Intervention with ET receptor antagonists should provide further information on the role of ET-1 in SPA-RAO.

Inhibition of chemically induced carcinogenesis by drugs used in homeopathic medicine.

Homeopathy is considered as one modality for cancer therapy. However, there are only very few clinical reports on the activity of the drugs, as well as in experimental animals. Presently we have evaluated the inhibitory effects of potentized homeopathic preparations against N'-nitrosodiethylamine (NDEA) induced hepatocellular carcinoma in rats as well as 3-methylcholanthrene-induced sarcomas in mice. We have used Ruta, Hydrastis, Lycopodium and Thuja, which are commonly employed in homeopathy for treating cancer. Administration of NDEA in rats resulted in tumor induction in the liver and elevated marker enzymes such as gamma-glutamyl transpeptidase, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and alkaline phosphatase in the serum and in liver. Concomitant administration of homeopathic drugs retarded the tumor growth and significantly reduced the elevated marker enzymes level as revealed by morphological, biochemical and histopathological evaluation. Out of the four drugs studied, Ruta 200c showed maximum inhibition of liver tumor development. Ruta 200c and phosphorus 1M were found to reduce the incidence of 3-methylcholanthrene-induced sarcomas and also increase the life span of mice harboring the tumours. These studies demonstrate that homeopathic drugs, at ultra low doses, may be able to decrease tumor induction by carcinogen administration. At present we do not know the mechanisms of action of these drugs useful against carcinogenesis.

Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin.

In the present study, follicular fluids of estrous mares treated with saline solution (Control) or nitric oxide synthase (NOS) inhibitors were analyzed for nitric oxide (NO), estradiol-17beta (E2) and progesterone (P4) concentrations before and 36h after administration of human chorionic gonadotropin (hCG). Follicular fluids obtained before (0h) hCG administration from control mares had lower concentrations of NO than those obtained 36h after administration of hCG (58.3+/-17.8 micromol versus 340.4+/-57.7 micromol; P<0.05). A similar pattern was also noted for intrafollicular P4 in control mares, which had lower concentrations of intrafollicular P4 before hCG than 36h post-hCG administration (P<0.05). As expected, E2 concentrations of control follicles sampled before hCG administration were higher than those sampled 36h post-hCG administration (P<0.05). However, the E2 concentrations in follicles of mares treated with the NOS inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) did not decrease after hCG administration, unlike those in control mares (P>0.10). In addition, mares treated with NOS inhibitors had lower intrafollicular concentrations of NO and P4 than control mares, both before and after hCG administration (P<0.05). Increased intrafollicular concentrations of NO in control, hCG-stimulated mares provide evidence for the presence of an NO-generating system in the equine preovulatory follicle that is likely upregulated following administration of hCG.

Non-adrenergic non-cholinergic excitatory innervation in the airways: role of neurokinin-2 receptors.

1 The hypothesis that the non-adrenergic, non-cholinergic excitatory (NANC-e) innervation is involved in the induction of asthma and that antagonists of NANC-e neurotransmitter could reduce bronchoconstriction during asthma was tested. 2 The first objective was to identify the neurotransmitter(s) of NANC-e innervation from a group of selected putative neurotransmitters. The second objective was to use the antagonist of the identified neurotransmitter(s) to determine its effectiveness against bronchoconstriction to ovalbumin (OVA) in sensitized guinea-pigs. 3 Neurotransmitter identification was performed using the "tracheal pouch"', a surgical preparation established for demonstrating NANC innervation, in anaesthetized guinea-pig airways. A segment of trachea was cannulated and clamped at one end and the other end was connected to a pressure transducer. The stump of the trachea was connected to a ventilator to keep the blood gas values within the normal range. The vagus nerve and the sympathetic nerves were isolated bilaterally and cut. The left carotid artery was cannulated to monitor blood pressure and for sampling blood for blood gas analysis. The jugular vein was cannulated for administration of test agents. 4 Both NANC-e and NANC-i (inhibitory) control responses of airways were obtained by bilateral vagal stimulation after complete autonomic blockade with atropine, propranolol and prazosin. The relaxation of the tracheal pouch was indicative of the NANC-i response and the increase in insufflation pressure of the ventilated peripheral airways was due to NANC-e stimulation. 5 The involvement of the putative neurotransmitters such as neurokinin-A (NK-A), histamine, serotonin and endothelin (ET) was investigated by using the respective antagonists, MEN-10376, pyrilamine maleate, cyproheptadine hydrochloride, and two ET receptor antagonists (BQ-123 and IRL-1038), respectively. The antagonists were administered at the dose rate of 4 mg kg-1 i.v. which was determined from preliminary studies by testing against the respective agonists. 6 MEN-10376 (neurokinin-2 receptor antagonist) significantly inhibited the insufflation pressure (peripheral airway pressure) increase caused by NANC-e stimulation. MEN-10376 also inhibited the fall in blood pressure caused by bilateral vagal stimulation. The 5-HT antagonist, cyproheptadine, significantly enhanced the NANC-e response. 7 After identifying the NANC-e neurotransmitter as NK-A, the effectiveness of its antagonist, MEN-10376, was evaluated for its ability to attenuate the increase in insufflation pressure (bronchoconstriction) induced in guinea-pigs sensitized by OVA. Guinea-pigs were sensitized to OVA (200 mg i.p.) and 10 days later prepared for the determination of tracheal pouch and insufflation responses to 100 microg of OVA administered i.v. (challenge dose). This caused an increase in insufflation pressure in the presence of adrenergic and cholinergic blockade, which was significantly attenuated by MEN-10376. 8 These studies indicated that neurokinin-2 receptors were involved in the vagally mediated efferent neurotransmission of NANC-e and that NANC-e plays a role in allergen-induced bronchoconstriction.

Intestinal bioavailability and biotransformation of 3-hydroxybenzo(a)pyrene in an isolated perfused preparation from channel catfish, Ictalurus punctatus.

The intestinal bioavailability and biotransformation of 3-hydroxybenzo(a)pyrene, a major metabolite of benzo(a)pyrene in many animal species, was investigated in an in situ isolated intestinal preparation from the channel catfish, and in vitro with preparations of catfish intestine and blood. 3-Hydroxybenzo(a)pyrene was a good substrate for adenosine 3'-phosphate 5'-phosphosulfate (PAPS)-sulfotransferase and UDP-glucuronosyltransferase in cytosol or microsomes prepared from intestinal mucosa. The benzo(a)pyrene-3-glucuronide and 3-sulfate conjugates were only very slowly hydrolyzed by intestinal beta-glucuronidase and sulfatase. The K(m) values for PAPS-sulfotransferase and UDP-glucuronosyltransferase were 0.4 and 1 microM, respectively, and V(max) were 1.61 +/- 1.08 nmol benzo(a)pyrene-3-sulfate/min/mg of cytosolic protein and 1.08 +/- 0.54 nmol benzo(a)pyrene-3-glucuronide/min/mg of microsomal protein. Hydrolytic enzyme activities were three orders of magnitude slower. In the in situ intestinal preparation, [(3)H]3-hydroxybenzo(a)pyrene was readily metabolized to the glucuronide and sulfate conjugates. After 1 h of incubation of 2 or 20 microM [(3)H]3-hydroxybenzo(a)pyrene in the in situ preparation, the luminal contents contained 3-hydroxybenzo(a)pyrene, benzo(a)pyrene-3,6-dione, benzo(a)pyrene-3-sulfate, and benzo(a)pyrene-3-glucuronide. Mucosal samples contained these components, as well as some unextractable material. The blood contained mainly benzo(a)pyrene-3-sulfate and an as yet unidentified metabolite of 3-hydroxybenzo(a)pyrene bound to hemoglobin. Some, but not all, blood samples contained small amounts of 3-hydroxybenzo(a)pyrene, benzo(a)pyrene-3-glucuronide, and benzo(a)pyrene-3,6-dione. These studies demonstrate the rapid phase 2 conjugation of a phenolic benzo(a)pyrene metabolite in intestinal mucosa, and the transfer of the phase 2 sulfate and glucuronide conjugates to blood.

In vitro pharmacologic effect of two endothelin-1 antagonists on equine colonic arteries and veins.

OBJECTIVE: To evaluate the effectiveness of 2 potential endothelin (ET)-1 antagonists in blocking the contractile responses of equine colonic vessels to increasing concentrations of ET-1. SAMPLE POPULATION: Mesenteric vessels from 6 clinically healthy horses. PROCEDURE: Colonic vessels (arterial and venous rings) were placed in organ baths with oxygenated Tyrode solution at 37 C. Each was attached to a force transducer interfaced with a polygraph, and 2 g of tension was applied and equilibrated for 45 minutes. Then, B-1 (PD 142893) and B-2 (PD 145065) ET-1 antagonists were tested. One ring from each vessel type was used as a control for determining concentration-response relationships of ET-1 (10(-10) to 10(-6)M). Three rings of each vessel type were incubated with 3 concentrations of each antagonist (10(-7), 10(-6), and 10(-5) M) for 30 minutes before ET induced contractions were determined. The maximum contractile response and pA2 values were determined. RESULTS: Vessels contracted in a concentration-dependent manner to ET-1. Arteries responded slowly but reached greater contractions. Veins responded immediately with sustained contractions. Both antagonists inhibited contractions in a concentration-dependent manner with significant differences at 10(-6) and 10(-5)M for arteries and 10(-5) M for veins. Complete blockade of contractions was observed with B-2 (10(-5)M). The pA2 values for B-1 were 8.26 and 6.82 for arteries and veins, respectively, whereas they were 8.25 and 7.21 for B-2. CONCLUSION AND CLINICAL RELEVANCE: Both antagonists effectively blocked ET-1-induced contractions of equine colonic vessels. Because B-2 is water soluble and caused complete blockade at 10(-5) M, it appears to be the preferred antagonist.

Comparative responses of bronchial rings to mediators of airway hyperreactivity in healthy horses and those affected with summer pasture-associated obstructive pulmonary disease.

OBJECTIVE: To compare responses of bronchial rings obtained from healthy horses and horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) to selected mediators of airway hyperreactivity in vitro. SAMPLE POPULATION: Bronchial rings from 6 healthy horses and 6 horses affected with SPAOPD. PROCEDURE: Bronchial rings obtained from each group of horses were mounted in organ baths and attached to force transducers interfaced with a polygraph. After applying 2g of tension, each ring was allowed to equilibrate for 45 minutes in Tyrode's solution at 37 C. Cumulative concentration-response relationships to graded concentrations of selected mediators (10(-8) to 10(-4) M) were determined and analyzed for significance at each concentration. RESULTS: Acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 induced concentration-dependent contractile responses in bronchial rings. Prostaglandin F2alpha induced weak and inconsistent contractile responses. The other 2 agents, norepinephrine and substance P, did not induce concentration-dependent responses. Considering the overall group-drug effect, acetylcholine, histamine, 5-hydroxytryptamine, and leukotriene D4 were effective in inducing consistent concentration-dependent contractile responses in both groups. Only 5-hydroxytryptamine and histamine induced significant responses in contractility between groups. The response of bronchial rings from horses with SPAOPD to 5-hydroxytryptamine was significantly greater than those from control horses, whereas the response to histamine was significantly lower. Significant responses were evident at concentrations ranging from 10(-6) to 10(-4) M for both drugs. CONCLUSIONS AND CLINICAL RELEVANCE: Because the airways of horses with SPAOPD had increased responsiveness to 5-hydroxytryptamine in vitro, treatment modalities using 5-hydroxytryptamine antagonists should be investigated to address this phenomenon.

In vitro responses of equine colonic arterial and venous rings to adenosine triphosphate.

OBJECTIVE: To evaluate the in vitro effects of adenosine tryphosphate (ATP) on vasomotor tone of equine colonic vasculature. SAMPLE POPULATION: Arteries and veins from the left ventral colon of 14 mixed-breed horses euthanatized for reasons unrelated to cardiovascular or gastrointestinal tract disease. PROCEDURES: Endothelium-intact and -denuded arterial and venous rings were precontracted with 10(-7) and 1.8 x 10(-8) M endothelin-1, respectively. In 1 trial, endothelium-intact rings were also incubated with 10(-4) M N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) production. Adenosine triphosphate (10(-8) to 10(-3) M) was added in a noncumulative manner, and relaxation percentage versus time curves were generated. Areas under the curves (ie, percentage of relaxation time) were calculated. RESULTS: Relaxation response of arterial and venous rings to ATP was dose-dependent. Percentage of relaxation time in response to 10(-4) and 10(-3) MATP was significantly greater, compared with that for rings not treated with ATP Removal of endothelium attenuated but did not eliminate the relaxation response. Addition of L-NAME did not attenuate the relaxation response in arteries. At higher concentrations, the vascular response to ATP was biphasic. CONCLUSIONS AND CLINICAL RELEVANCE: ATP applied to equine colonic arterial and venous rings with and without intact endothelium induced a biphasic response characterized by transient contraction followed by slow, substantial, and sustained relaxation. This ATP-induced response is possibly mediated by a mechanism other than NO. Adenosine triphosphate may be a useful treatment to modulate colonic vasomotor tone in horses with strangulating volvulus of the ascending colon.

Effect of micelle fatty acid composition and 3,4,3', 4'-tetrachlorobiphenyl (TCB) exposure on intestinal [(14)C]-TCB bioavailability and biotransformation in channel catfish in situ preparations.

Polychlorinated biphenyls are transferred in the diet along aquatic food chains. This study investigated the effect of dietary micelle composition and 3,4,3',4'-tetrachlorobiphenyl (TCB) exposure upon the subsequent systemic bioavailability and intestinal metabolism of [(14)C]-TCB in a catfish in situ intestinal preparation. Initial in vitro experiments examined the solubility of [(14)C]-TCB in micelles of varying fatty acid composition. Micelles composed of single fatty acids demonstrated greater [(14)C]-TCB solubility with those fatty acids of longer chain length. Similarly, micelles of the long-chain fatty acid, linoleic acid, solubilized more [(14)C]-TCB than mixed micelles formulated from equal amounts of myristic (14:0), palmitic (16:0), stearic (18:0), or linoleic (18:2) acids. Systemic bioavailability of [(14)C]-TCB (60 microM) from an in situ perfused intestinal preparation was 2.2-fold greater when delivered to the intestine in linoleic acid micelles as compared to the mixed micelle preparation. Catfish exposed in vivo to either 0.5 or 5.0 mg TCB/kg feed for 10 days resulted in a 45 to 47% decrease in the subsequent systemic bioavailability of [(14)C]-TCB in the in situ intestinal preparation. Total intestinal cytochrome P450 content was not significantly affected by TCB preexposure. Immunodetectable CYP1A was found only in the 5.0 mg TCB/kg diet treatment. Corresponding intestinal aryl hydrocarbon hydroxylase (AHH) activities were 2.46 +/- 1.16, 2.43 +/- 1.58, and 11.35 +/- 10.25 pmol/min/mg protein for the control, 0.5, and 5 mg TCB/kg diet groups, respectively. [(14)C]-TCB in the in situ preparation was metabolized to only a small degree upon a single pass through the intestinal mucosa of the catfish. High variability and low rates of metabolism precluded the association of the magnitude of metabolism with dietary TCB pretreatment. Analysis of tissue sample extracts demonstrated 4 minor peaks, 3 of which were tentatively identified by co-elution with standards as 2-OH-3,4,3',4'-TCB, 4-OH-3,5,3',4'-TCB, and 5-OH-3, 4,3',4'-TCB. A fourth remains unidentified. Histological changes in the intestine such as thinning of the submucosa and increased numbers of goblet cells were evident at the 5.0 mg TCB/kg diet dose. These results suggest that TCB intestinal bioavailability may be linked to micelle composition as well as TCB exposure history. Furthermore, single pass intestinal metabolism appears to be a minor contributor to the biotransformational modification of dietary TCB.

Contribution of nitric oxide synthases 1, 2, and 3 to airway hyperresponsiveness and inflammation in a murine model of asthma.

Asthma is a chronic disease characterized by increased airway responsiveness and airway inflammation. The functional role of nitric oxide (NO) and the various nitric oxide synthase (NOS) isoforms in human asthma is controversial. To investigate the role of NO in an established model of allergic asthma, mice with targeted deletions of the three known isoforms of NOS (NOS1, 2, and 3) were studied. Although the inducible (NOS2) isoform was significantly upregulated in the lungs of ovalbumin (OVA)-sensitized and -challenged (OVA/OVA) wild-type (WT) mice and was undetectable in similarly treated NOS2-deficient mice, airway responsiveness was not significantly different between these groups. OVA/OVA endothelial (NOS3)-deficient mice were significantly more responsive to methacholine challenge compared with similarly treated NOS1 and NOS1&3-deficient mice. Airway responsiveness in OVA/OVA neuronal (NOS1)-deficient and neuronal/endothelial (NOS1&3) double-deficient mice was significantly less than that observed in similarly treated NOS2 and WT groups. These findings demonstrate an important function for the nNOS isoform in controlling the inducibility of airway hyperresponsiveness in this model of allergic asthma.

Outcome of 350 implanted chest ports placed by interventional radiologists.

PURPOSE: To determine the outcome of implanted chest ports placed by interventional radiologists. MATERIALS AND METHODS: Between June 1993 and July 1996, a single institution placed 350 implanted chest ports in 346 patients by means of the subclavian vein approach. The medical records of these patients were reviewed to determine the outcome of the ports. Ports were implanted for chemotherapy (n = 341), blood transfusion (n = 7), or antibiotics (n = 2). RESULTS: Immediate complications were seven (2%) pneumothoraces and one (0.3%) hematoma. Four (1.1%) of the pneumothoraces necessitated hospital admission and treatment with a chest tube. The remaining three were managed on an outpatient basis. One was successfully treated in the interventional suite by catheter suction. Two pneumothoraces were observed and resolved spontaneously. Mean time of patient follow-up was 260 days (range, 22-929 days). Total time of follow-up was 91,000 catheter days. Delayed complications were 10 cases of thrombosis (2.9% or 0.11 per 1,000 catheter days) of the subclavian vein, four infections (1.1% or 0.04 per 1,000 catheter days), four catheter coiling or tip malpositions (1.1% or 0.04 per 1,000 catheter days), three catheter occlusions (0.9% or 0.03 per 1,000 catheter days), and one catheter leak (0.3% or 0.01 per 1,000 catheter days). Six (1.7%) ports had to be removed as a result of a delayed complication. CONCLUSION: Chest port implantation by interventional radiologists within the radiology department is a successful and safe procedure with complication rates equivalent to, or lower than, those reported in surgical placement series.

Utilization pattern and efficacy of nonsurgical techniques to establish drainage for high biliary obstruction.

PURPOSE: To review the frequency and success of percutaneous and endoscopic techniques in the relief of high biliary obstruction. MATERIALS AND METHODS: A search of the radiologic achieves was performed identifying 70 patients with cholangiographic demonstration of high biliary obstruction defined as proximal to the distal third of the extrahepatic bile duct. Record review determined the frequency and success rates of percutaneous and endoscopic techniques in providing biliary decompression for obstructive jaundice. RESULTS: Endoscopic retrograde cholangiopancreatography was performed in 35 of 70 patients, providing initial endoscopic biliary decompression (EBD) in six patients (two subsequently required percutaneous intervention). Percutaneous biliary drainage (PBD) was attempted in 60 of 70 patients, providing initial decompression in 55 patients. PBD provided decompression after failed endoscopic biliary drainage in 18 of 26 patients. Endoscopic drainage was never attempted after failed percutaneous drainage. Overall EBD success was 23% and overall PBD success was 95%. The complication rate attributed to EBD was 26%; that attributed to PBD was 25%. For those patients who underwent attempts at both EBD and PBD, the complication rate was 16%. CONCLUSION: At an institution with well-developed gastrointestinal medical services and interventional radiologic services, PBD was more successful in providing initial biliary decompression than endoscopic techniques for high biliary obstruction.