In vivo microarterial freezing: experimental study

Quantitative techniques for in vivo and in vitro angiogenesis were developed using an image analyser. In the in vivo study, a Millipore chamber filled with mouse sarcoma 180 (S180) cells was transplanted subcutaneously to the dorsal side of a mouse, and the area of neovascularization induced by the tumour cells was quantified by image analysis. Images of vascular networks with poor contrast had their contrast improved by Laplacean transformation. The area of vascular network was 16.9 mm2 in the control group without tumour cells and 44.2 mm2 in the group with tumour cells, demonstrating a significant increase in neovascularized area by tumour cells. In the in vitro study, migration of vascular endothelial cells was induced with conditioned media of S180 cells. Image analysis was used to count automatically the nuclei of migrated endothelial cells, which were stained violet with Giemsa's solution. This automated measurement by image analyser is expected to save labour and time. Checkerboard analysis revealed that the endothelial cell migration induced by S180-conditioned medium was due to chemotaxis. The quantitation method using an automated image analyser is valuable in evaluating the induction of neovascularization by tumours and the effect of pharmacological agents on tumour angiogenesis in vivo and in vitro.     

In vivo gene transfer and expression in rat stomach by submucosal injection of plasmid DNA

We used electromyographic analysis to determine the muscle activity of the shoulder muscles during the lift-off test and during resisted internal rotation. The activity in the upper and lower subscapularis muscle during a lift-off test from the region of the midlumbar spine was approximately 70% of maximal voluntary contraction. This level was significantly higher than for all the other muscles tested (P < 0.05). The lift-off test with the hand placed in the region of the midlumbar spine resulted in one-third more electromyographic activity in the subscapularis muscle than when the test was modified and performed with the hand at the buttocks region. A resisted lift-off test resulted in higher activities in all the muscles, but only a small increase in the pectoralis major muscle. The pectoralis major muscle was significantly more active during resisted internal rotation with the arm in front of the body. Comparison of activity in the upper subscapularis with that in the lower subscapularis muscle showed no significant differences during any of the tests. This study documents the importance of the subscapularis muscle during the lift-off test and suggests that other potential internal rotators of the humerus have a limited role in maintaining internal rotation when the arm is placed behind the back.     

In vitro and in vivo antineoplastic effects of orthovanadate

A liquid chromatographic method for the determination of the macrolide antibiotics, roxithromycin and clarithromycin, in plasma is described. The method is fully automated, employing on-line solid-phase extraction for sample clean-up, using the Prospekt unit. Plasma samples, mixed with internal standard, were injected onto exchangeable CN cartridges. After washing, the compounds were eluted and transferred to a C18 analytical column for separation and electrochemical detection. Clarithromycin was used as internal standard when assaying roxithromycin and vice versa. The recovery of the solid-phase extraction method was 90% and higher, and the relative standard deviation was about 3%. The limit of quantitation was 0.5 mumol/l when 25 microliters of plasma was injected. Comparison with a liquid-liquid extraction method for sample clean-up showed good agreement.     

In vitro and in vivo binding of a CC-1065 analogue to human gene sequences: a polymerase-chain reaction study

In this paper we analyse the in vitro sequence selectivity of the CC-1065 analogue 2-[[5-[(1H-indol-2-yl]carbonyl)-1H-indol-2-yl] carbonyl]-7-methyl-1,2,8,8a-tetrahydrocyclopropa [c]-pyrrolo-[3,2-e]-indol-4-one (U-71184) employing the polymerase-chain reaction (PCR). In addition, we determined whether alteration of PCR by U-71184 is detected when DNA is isolated from cells cultured in the presence of this drug. As molecular model systems we employed the human estrogen receptor gene, the Ha-ras oncogene and the chromosome X-linked, (CGG)-rich fragile X mental retardation-1 gene. The first conclusion that can be drawn from the experiments reported in our paper is that U-71184 inhibits PCR in a sequence-dependent manner. A second conclusion of our experiments is that PCR performed on DNA from U-71184-treated cells is inhibited when the primers amplifying the estrogen receptor gene region are used. This approach might bring important information on both in vivo uptake of the drug by target cells and binding to DNA.     

In vitro and in vivo effects of retrovirus-mediated transfer of the connexin 43 gene in malignant gliomas: consequences for HSVtk/GCV anticancer gene therapy

OBJECTIVE: Patients with the deficit syndrome differ from other patients with schizophrenia relative to physiological correlates, course of illness, and response to treatment. Because of the abnormal seasonality of birth among persons with schizophrenia, the authors examined the relation between this risk factor and the deficit syndrome. METHOD: Findings in two clinical groups suggested an increase in summer births among deficit syndrome patients. The association between summer birth and the deficit syndrome was then examined in a catchment area study of first-admission patients with psychosis. RESULTS: In the catchment area sample, summer birth was also significantly associated with the deficit syndrome; negative symptoms broadly defined were not. CONCLUSIONS: These findings add to the increasing evidence that 1) patients with the deficit syndrome have a disease with an etiopathophysiology separate from that of other patients with what is now called schizophrenia and 2) the correlates of broadly defined negative symptoms are different from those for the deficit syndrome. The previously reported association between winter birth and schizophrenia appears to apply to nondeficit schizophrenia only.     

In vivo tissue specific modulation of rat insulin receptor gene expression in an experimental model of mineralocorticoid excess

Insulin receptor (IR) gene expression at the mRNA level was investigated in hindlimb skeletal muscle, epididymal adipose tissue and in the liver of rats exposed to prolonged in vivo administration of deoxycorticosterone acetate (DOCA). Following treatment, plasma insulin levels were reduced while glucose levels increased compared to values in control rats. DOCA-treated animals showed an increase in blood pressure and a reduction in body weight. This treatment also induced hypokalemia and decreased plasma protein levels. Sodium levels were unaffected. Moreover, no differences in DNA and protein content or in the indicator of cell size (protein/DNA) were observed in the skeletal muscle or adipose tissue of animals. In contrast, there was a clear increase in the protein and DNA contents of the liver with no change in the indicator of cell size. Northern blot assays revealed 2 major IR mRNA species of approximately 9.5 and 7.5 Kb in the 3 tissues from control animals. DOCA treatment induced no change in the levels of either RNA species in skeletal muscle. However, a decrease of approximately 22% was detected in the levels of both species in adipose tissue whereas the liver showed an increase of 64%. These results provide the first evidence for an in vivo tissue-specific modulation of IR mRNA levels under experimental conditions of mineralocorticoid excess.     

In vivo radiofrequency thermal balloon angioplasty of porcine coronary arteries: histologic effects and safety

The purpose of this study was to assess the safety and histologic effects of radiofrequency thermal balloon angioplasty in the coronary vasculature of normal pigs. Radiofrequency thermal balloon angioplasty was performed in 30 coronary arteries of 16 nonatherosclerotic pigs. Heated inflations were performed at either 50 degrees, 60 degrees, or 70 degrees C for 30 or 60 seconds, and were compared with five nonheated inflations in five additional arteries. All balloon inflations were performed at 2 atm pressure with a balloon/vessel diameter ratio of 1.2 to 1. Heart rate, arterial pressure, and left ventricular pressure were monitored continuously for each animal. A 12-lead ECG, coronary angiography, and two-dimensional transthoracic echocardiography were performed before and 1 hour after each balloon inflation. Each animal was subsequently put to death for postmortem cardiac examination. Heated inflations were well tolerated in 28 of the 30 arteries without significant adverse effects. During one inflation, ventricular fibrillation occurred because of prolonged ischemia from an occlusive guiding catheter. In another artery, a heated inflation resulted in a dissection with a transient decrease in distal coronary flow. Histologic examination revealed a significant increase in wall thinning and elastic fiber straightening with heating at 70 degrees C for both 30 and 60 seconds, and a significant increase in intracoronary thrombus with heating at 70 degrees C for 60 seconds. Depth of periarterial myocardial heat necrosis paralleled the increase in temperature, with an average depth of 166 microns at 50 degrees C, 312 microns at 60 degrees C, and 1031 microns at 70 degrees C. In vivo, radiofrequency coronary angioplasty can be performed relatively safely without significant electrical, hemodynamic, or ischemic changes beyond those seen with conventional nonthermal angioplasty. The extent of heat-induced vessel wall thinning, elastic tissue straightening, intracoronary thrombus formation, and periarterial myocardial necrosis are all related to balloon temperature or duration of heating.     

In vivo effects of chicken interferon-gamma during infection with Eimeria

Newly hatched chickens are highly susceptible to infection by opportunistic pathogens during the first 1 or 2 weeks of life. The use of cytokines as therapeutic agents has been studied in animal models as well as in immunosuppressed patients. This approach has become more feasible in livestock animals, in particular poultry, with the recent cloning of cytokine genes and the development of new technologies, such as live delivery vectors. We have recently cloned the gene for chicken interferon-gamma (Ch-IFN-gamma). Poly-HIS-tagged recombinant Ch-IFN-gamma was expressed in Escherichia coli, was purified by Ni chromatography, and was found to be stable at 4 degrees C and an ambient temperature for at least several months and Several weeks, respectively. Ch-IFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis, induced nitrite secretion from chicken macrophages in vitro, and enhanced MHC class II expression on macrophages. Administration of recombinant Ch-IFN-gamma to chickens resulted in enhanced weight gain over a 12-day period. Furthermore, the therapeutic potential of Ch-IFN-gamma was assessed using a coccidial challenge model. Birds were treated with Ch-IFN-gamma or a diluent control and then infected with Eimeria acervulina. Infected birds treated with Ch-IFN-gamma showed improved weight gain relative to noninfected birds. The ability of Ch-IFN-gamma to enhance weight gain in the face of coccidial infection makes it an excellent candidate as a therapeutic agent.     

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.     

In vivo effects of the 5-HT3 antagonist alosetron on basal and cholera toxin-induced secretion in the human jejunum: a segmental perfusion study

BACKGROUND: 5-hydroxytryptamine type 3 receptor antagonists have been shown to reduce fluid and electrolyte secretion or promote absorption in experimental models of small intestinal secretion. The aim of this study was to compare the effects of a single dose (4 mg) of the 5-hydroxytryptamine type 3 receptor antagonist alosetron and placebo on jejunal fluid and electrolyte movement in humans under basal conditions (n = 7) and following cholera toxin-induced secretion (n = 5) in a randomized, double-blind, crossover design over two separate study periods. METHODS: One hour after oral alosetron or placebo, jejunal intubation was performed. A 30 cm segment of jejunum, isolated by two occluding balloons, was exposed to 15 microg of purified cholera toxin for 2 h prior to triple lumen perfusion with a plasma electrolyte solution containing [14C]-polyethylene glycol as a nonabsorbable volume marker. In the basal study, intestinal perfusion was performed without exposure to cholera toxin. Collection and analysis of the effluent from the jejunal segment allowed fluid and electrolyte movement to be calculated. RESULTS: Alosetron treatment increased basal jejunal fluid absorption (median 5.1 mL/cm/h [interquartile range 4.2 to 7.1] compared with placebo (3.8 [3.6 to 4.3] P = 0.028, two sided Wilcoxon matched pairs signed rank test)). However, following establishment of a secretory state by cholera toxin, alosetron failed to significantly promote absorption or reduce secretion (0.3 [-1.2 to 1.2] compared to placebo (-4.3 [-7.7 to -1.3] P = 0.14)). Adverse events during the study, which included anorexia, nausea, dizziness and loose bowel movements, were not considered to be clinically important. There were no clinically significant changes in laboratory parameters. CONCLUSION: The 5-hydroxytyptamine type 3 receptor antagonist, alosetron, increased basal fluid absorption in normal human small intestine but failed to have a beneficial effect in cholera toxin-induced secretion.     

In vivo effects of remoxipride and aromatic ring metabolites in the rat

The in vivo effects of remoxipride, in relation to some of its identified metabolites, were investigated in adult male Sprague-Dawley rats. The methods used included: (1) estimation of the in vivo rate of brain monoamine synthesis by measuring the accumulation of dihydroxyphenylalanine and 5-hydroxytryptophan after decarboxylase inhibition; (2) observations of spontaneous locomotor activity in a photocell-equipped open-field arena ( approximately 0. 5 m2); (3) treadmill locomotion ( approximately 4 m min-1); (4) inclined grid (60 degrees ) catalepsy test; (5) d-amphetamine-induced (1.0 mg kg-1) hyperlocomotion;(6) quinpirole-induced (0.4 mg kg-1) hypothermia. By use of one or more of these tests, the findings with remoxipride were as follows: First, remoxipride had a late onset of action (up to 3 h). Second, potency and efficacy depended on exposure to hepatic metabolism. Thus, intraperitoneal administration was more effective than the subcutaneous route, whereas virtually all biological effects were lost on intracerebroventricular administration. The ED50 values (micromol kg-1, neostriatal dihydroxyphenylalanine accumulation) for remoxipride and a range of its phenolic aromatic ring metabolites were: remoxipride (approximately 20), NCQ-344 (approximately 0.01), FLA-797 (approximately 0.1), FLA-908 (approximately 2.2), NCQ-436 (approximately 25) and NCQ-469 (approximately 30). Considering remoxipride as a nonclozapine atypical antipsychotic drug, together with the fact that remoxipride behaves as a prodrug in the laboratory studies above, further characterization of the pharmacodynamic profile of its metabolites remains a challenge.     

In vitro and in vivo effects of phenolic antioxidants against cisplatin-induced nephrotoxicity

We have investigated the effect of phenolic antioxidants on cisplatin-induced cytotoxicity in vero (African Green Monkey Kidney) cells and in rat renal cortical slices in vitro, and on cisplatin-induced nephrotoxicity in rats in vivo. Incubation of cisplatin with vero cells resulted in time- and concentration-dependent cytotoxicity, as characterized by decreased tryphan blue exclusion (TBE) and increased release of lactate dehydrogenase (LDH) into the medium. Cisplatin also caused reduction of glutathione (GSH) in a concentration-dependent manner. In the rat renal cortical slices model, incubation of cisplatin for 120 min caused an increase in malondialdehyde (MDA), a decrease in GSH and inhibited p-aminohippurate (PAH) uptake in a concentration-dependent manner. Among phenolic antioxidants, isoeugenol (IG) was found to be more active against cisplatin-induced cytotoxicity in vero cells as well as in rat renal cortical slices than eugenol (EG) and dehydrozingerone (DZ). However none of the test compounds were able to arrest the reduction of the GSH content induced by cisplatin in either the vero cells or the renal cortical slice model. Administration of cisplatin (3 mg/kg) i.p. to rats resulted in significant reduction of body weight, and elevation of blood urea nitrogen (BUN) and serum creatinine. Treatment with IG 10 mg/kg i.p. 1 h before cisplatin resulted in partial but significant protection against the cisplatin-induced reduction of body weight, and elevation of BUN and serum creatinine, the protection being 34, 46, and 62%, respectively. EG and DZ (10 mg/kg, i.p.) were found to be inactive in vivo. Because IG is a potent free radical scavenger and protects against cisplatin-induced toxicitiy, the present results have many clinical implications in chemotherapy and thus warrants further investigation.     

In vivo study on the immunomodulating effects of OM-85 BV on survival, inflammatory cell recruitment and bacterial clearance in Klebsiella pneumonia

Oral administration of the bacterial extract OM-85 BV has been shown to prime alveolar macrophages (AM) in such a way that they secrete significantly more nitric oxide, tumor necrosis factor-alpha and interleukin-1beta upon in vitro stimulation with lipopolysaccharide (LPS). As increased cytokine secretion by AM may account for the therapeutic effect of OM-85 BV in respiratory tract infections, we studied the effect of orally administered OM-85 BV on the outcome of Klebsiella pneumoniae-induced pneumonia. Mice received a daily oral dose of OM-85 BV (350 mg/kg body weight) for 5 days and were intratracheally infected with 333, 1000 or 3333 CFU K. pneumoniae on day 8. It was shown that OM-85 BV pretreatment of mice has no effect on bacterial clearance, neutrophil recruitment and survival in this acute respiratory tract infection. Also, OM-85 BV treatment had no protective effect in a recurrent infection with K. pneumoniae. It is concluded that AM activation by oral treatment with OM-85 BV is not sufficient to play a protective role in respiratory tract infection with K. pneumoniae.