Epidemiology of Plasmodium falciparum infection and drug resistance markers in Ota Area, Southwestern Nigeria.

Purpose: Effective routine monitoring and surveillance of parasite genes is a necessary strategy in the control of parasites' resistance to antimalarial drugs, according to the WHO's recommendation. This cross-sectional study therefore aimed at carrying out an epidemiological analysis on malaria incidence in Ado-Odo/Ota, Ogun State. Patients and methods: Blood and corresponding saliva samples were collected from 1,243 subjects of all ages and sex presenting with fever and a parasitemia level ≥2,000 between September 2016 and March 2018. Samples were collected from selected health facilities in the study area of Ogun state to establish the prevalence of falciparum malaria and determine resistance genes harbored by the parasites. The overall prevalence of falciparum malaria in the study site by microscopic examination was 45.86%. The highest incidence of 57.42% was recorded among male subjects. Point mutations of K76T and N86Y in the Pfcrt and pfmdr-1 genes, as well as non-synonymous mutations in Pfk13 genes, were screened for and sequenced for further analysis. Results: Pfcrt was detectable in 57.42% of blood and 51.02% of saliva samples, respectively. About 34.78% of the subjects that were confirmed microscopically harbored the Pfmdr-1 mutated gene while 26.67% of the saliva samples revealed Pfmdr-1. Epidemiological studies identified the presence of wild-type Pfk13 genes in 21.84% of blood and 44.44% of saliva samples correspondingly. For each of the genes evaluated, saliva portrayed great diagnostic performance when compared with blood. Conclusion: Findings from this study have established the prevalence of malaria and the resistance pattern of P. falciparum in the study area. The findings may help in formulating drug policies and suggest the use of saliva as a noninvasive point-of-care method of diagnosing malaria potentially deployable to rural endemic areas.

Therapeutic elastase inhibition by alpha-1-antitrypsin gene transfer limits neointima formation in normal rabbits.

PURPOSE: Alpha-1-antitrypsin (AAT) is the major circulating elastase inhibitor. Deficiency of elastase inhibition leads to emphysema and vascular abnormalities including accelerated neointima. Because recent evidence suggests that tissue AAT levels determine inhibitory function, the authors hypothesize that local tissue-based expression of AAT limits elastase activity sufficiently to guide arterial response to injury. MATERIALS AND METHODS: Rabbit common femoral arteries were injured by mechanical overdilation and treated with buffer, viral control, or an adenovirus expressing AAT (Ad/AAT). After 3 and 28 days, intima-to-media (I/M) ratios were evaluated. Additionally, early changes in elastase inhibition potential (3 d), extracellular elastin and collagen content (3 d), and local macrophage and neutrophil infiltration (7 d) were determined. RESULTS: Ad/AAT significantly decreased neointima formation after mechanical dilation injury after 28 days: buffer controls exhibited mean I/M ratios of 0.76 +/- 0.06, whereas viral controls reached 0.77 +/- 0.09; in contrast, Ad/AAT reduced I/M ratios to 0.44 +/- 0.06. Both early elastin and collagen content were preserved in the Ad/AAT group relative to controls. The Ad/AAT group also reversed the local inflammation that characterized viral controls. CONCLUSIONS: This strategy demonstrates that local increases in elastase inhibition potential promote a neointima-resistant small-caliber artery, which may offer new promise in management of patients undergoing angioplasty.

Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.

A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.

Adrenal teratoma in four domestic ferrets (Mustela putorius furo).

Teratomas are histologically complex neoplasms that are composed of structures derived from multiple germ cell layers (ectoderm, mesoderm, and endoderm). These neoplasms are uncommon in domestic animals and are usually found in the gonads. This paper describes teratomas of the adrenal gland in four domestic ferrets (Mustela putorius furo). Three of four of the neoplasms contained tissues from ectodermal, mesodermal, and endodermal germ cell layers; two of four contained rudimentary teeth. In one case, malignant epithelial cells had metastasized to local lymph nodes. Teratomas, although uncommon, should be included in the differential diagnosis for adrenal neoplasms in domestic ferrets.

Liquid lung ventilation reduces neutrophil sequestration in a neonatal swine model of cardiopulmonary bypass.

OBJECTIVE: Liquid lung ventilation has been demonstrated to improve cardiorespiratory function after cardiopulmonary bypass. We hypothesized that liquid lung ventilation (LLV) would decrease the pulmonary inflammatory response after cardiopulmonary bypass (CPB). DESIGN: Prospective, randomized, experimental, controlled, nonblinded study. SETTING: Animal research laboratory at a university setting. SUBJECTS: A total of 24 neonatal piglets. INTERVENTIONS: After intubation with a cuffed endotracheal tube, swine were conventionally ventilated. After surgical cannulation, each piglet was placed on conventional nonpulsatile CPB and cooled to 18 degrees C (64.4 degrees F). Subsequently, the animals were exposed to 90 mins of low-flow CPB (35 mL/kg/min). Animals were rewarmed to 37 degrees C (98.6 degrees F), removed from CPB, and ventilated for 90 min. Ten animals received conventional gas ventilation only (control), seven received initiation of LLV before CPB (prevention), and seven received initiation of LLV during the rewarming phase of CPB (treatment). After the animals were killed, the lungs were removed en bloc. The left lobe was dissected and formalin-fixed at 20 cm H2O overnight, followed by paraffin embedding. Sections were taken from the paraffin-embedded lungs. Neutrophil accumulation and lung injury were assessed by histochemical staining with leukocyte esterase and morphometrics, respectively. One hundred microscopic images were digitized from each tissue sample for lung morphometrics, and neutrophil counts were obtained from every fifth image. MEASUREMENTS AND MAIN RESULTS: Lung tissue sections showed a significantly lower number of neutrophils per alveolar area in the prevention and treatment groups than in the control group (control 681 +/- 65, prevention 380 +/- 49, treatment 412 +/- 101 neutrophils per alveolar area [cells/mm2]; p <.05 for both prevention and treatment compared with control). There were no differences in lung injury as assessed with morphometrics or hemodynamic measurements between any of the three groups. CONCLUSIONS: The data suggest that LLV reduces the CPB-induced neutrophil sequestration in the pulmonary parenchyma independent of its effects on the circulatory physiology or evidence of early lung injury.

Enhancement of neointima formation with tissue-type plasminogen activator.

PURPOSE: Indirect evidence suggests that tissue plasminogen activator (tPA) either limits or does not alter restenosis. However, tPA enhances tumor invasiveness through matrix remodeling, and several elements of degraded matrix enhance smooth muscle cell mitogenesis. We use either local adenoviral-mediated overexpression of tPA or systemic infusion of recombinant tPA combined with mechanical overdilation of rabbit common femoral arteries to evaluate the impact of tPA on neointima formation. METHODS: Left common femoral arteries of New Zealand white rabbits were transfected in situ either with an adenoviral-construct-expressing tPA or a viral control (adenoviral-construct-expressing beta-galactosidase) or nonviral (buffer) control after balloon angioplasty injury. At 7 and 28 days, left common femoral artery segments were harvested (n = 4 for each group and time point). Vessel segments were examined for intimato-media ratio, smooth muscle cell proliferation, extracellular matrix, and inflammatory response. Thrombus formation was evaluated after 3 days (n = 3 for each group). In a second experiment, New Zealand white rabbits (n = 3 per group, per time point) underwent mechanical dilation followed by buffer treatment or systemic tPA infusion according to a widely clinically used accelerated infusion protocol. Treated artery segments were harvested after 7 or 28 days and processed for intima-to-media ratio determination and class-wide histochemical determination of collagenous extracellular matrix and collagen content. RESULTS: Both rate and degree of neointima formation increase dramatically with overexpression (250%-461% relative to controls at 7 and 28 days). Substantial early matrix degradation is observed in vessels treated with local overexpression of tPA, although no increases in local inflammation or in smooth muscle proliferation occur. Late enhancement of smooth muscle proliferation emerges, consistent with secondary impact of perturbed matrix components. Systemic infusion of tPA according to clinical protocols also results in early and late enhancement of neointima formation in this model (34%-52% relative to controls at at 7 and 28 days), with significant early collagenous matrix degradation. Systemic infusion, although significant, did not attain the degree of neointima formation present with overexpression. CONCLUSION: With some evidence of dose-dependence, tissue plasminogen activator enhances neointima formation after angioplasty in a rabbit model. Early matrix degradation precedes change in rates of proliferation and underlies this effect in spite of several antirestenotic actions including decreased thrombus and decreased macrophage recruitment in this model.

Thrombomodulin overexpression to limit neointima formation.

BACKGROUND-These studies were initiated to confirm that high-level thrombomodulin overexpression is sufficient to limit neointima formation after mechanical overdilation injury. METHODS AND RESULTS-An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on neointima formation 28 days after mechanical overdilation injury was evaluated. New Zealand White rabbit common femoral arteries were treated with buffer, viral control, or Adv/RSV-THM and subjected to mechanical overdilation injury. The treated vessels (n=4 per treatment) were harvested after 28 days and evaluated to determine intima-to-media (I/M) ratios. Additional experiments were performed to determine early (7-day) changes in extracellular elastin and collagen content; local macrophage, T-cell, and neutrophil infiltration; and local thrombus formation as potential contributors to the observed impact on 28-day neointima formation. The construct significantly decreased neointima formation after mechanical dilation injury in this model. By histological analysis, buffer controls exhibited mean I/M ratios of 0.76+/-0.06%, whereas viral controls reached 0.77+/-0.08%; in contrast, Adv/RSV-THM reduced I/M ratios to 0.47+/-0.06%. Local inflammatory infiltrate decreased in the Adv/RSV-THM group relative to controls, whereas matrix remained relatively preserved. Rates of early thrombus formation also decreased in Adv/RSV-THM animals. CONCLUSIONS-This construct thus offers a viable technique for promoting a locally neointima-resistant small-caliber artery via decreased thrombus bulk, normal matrix preservation, and decreased local inflammation without the inflammatory damage that has limited many other adenoviral applications.

Golgi targeting of the GLUT1 glucose transporter in lactating mouse mammary gland.

Lactose, the major carbohydrate of human milk, is synthesized in the Golgi from glucose and UDP-galactose. The lactating mammary gland is unique in its requirement for the transport of glucose into Golgi. Glucose transporter-1 (GLUT1) is the only isoform of the glucose transporter family expressed in mammary gland. In most cells, GLUT1 is localized to the plasma membrane and is responsible for basal glucose uptake; in no other cell type is GLUT1 a Golgi resident. To test the hypothesis that GLUT1 is targeted to Golgi during lactation, the amount and subcellular distribution of GLUT1 were examined in mouse mammary gland at different developmental stages. Methods including immunohistochemistry, immunofluorescence, subcellular fractionation, density gradient centrifugation, and Western blotting yielded consistent results. In virgins, GLUT1 expression was limited to plasma membrane of epithelial cells. In late pregnant mice, GLUT1 expression was increased with targeting primarily to basolateral plasma membrane but also with some intracellular signal. During lactation, GLUT expression was further increased, and targeting to Golgi, demonstrated by colocalization with the 110-kD coatomer-associated protein beta-COP, predominated. Removal of pups 18 d after delivery resulted in retargeting of GLUT1 from Golgi to plasma membrane and a decline in total cellular GLUT1 within 3 h. In mice undergoing natural weaning, GLUT1 expression declined. Changes in the amount and targeting of GLUT1 during mammary gland development are consistent with a key role for GLUT1 in supplying substrate for lactose synthesis and milk production.

Sex differences in diquat-induced hepatic necrosis and DNA fragmentation in Fischer 344 rats.

Redox cycling metabolism of diquat catalyzes generation of reactive oxygen species, and diquat-induced acute hepatic necrosis in male Fischer 344 (F344) rats has been studied as a model of oxidant mechanisms of cell killing in vivo. At equal doses of diquat, female F344 rats sustained less hepatic damage than did male rats, as estimated by plasma alanine aminotransferase (ALT) activities after 6 h. Biliary efflux of glutathione disulfide (GSSG) was greater in male than in female rats at each dose of diquat, but even comparable rates of GSSG excretion were associated with less hepatic injury in female rats. Hepatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were similar in the two genders, and activities of glutathione reductase (GR) and glutathione S-transferase-alpha (GST-alpha) activities were higher in the male rats. Previous studies in male rats have implicated formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive "protein carbonyls" and related iron chelate-catalyzed redox reactions as mechanisms critical to diquat-induced acute cell death in vivo. However, diquat-treated female rats showed higher levels of DNPH-reactive proteins in livers and in bile than did males, both at identical doses of diquat and at doses that produced similar elevations in plasma ALT activities. In female rats, fragmentation of hepatic deoxyribonucleic acids (DNA) was increased by doses of diquat that did not increase plasma ALT activities, and increased fragmentation was observed prior to elevation of plasma ALT activities. In the present studies, hepatic necrosis was most closely associated with DNA fragmentation, although additional studies are needed to determine the mechanisms responsible for and the pathophysiological consequences of the fragmentation.

Molecular mechanisms of lipopolysaccharide induced ICAM-1 expression in A549 cells.

OBJECTIVE AND DESIGN: Lung intercellular adhesion molecule-1 (ICAM-1) expression is increased by LPS or hyperoxia on type II cells in vivo. The goals of the present study were to determine the mechanisms of ICAM-1 expression in a lung alveolar epithelial cell line (A549) exposed to lipopolysaccharide (LPS). MATERIALS: A549 cells, a transformed human cell line with characteristics of alveolar epithelial cells, were used. TREATMENT: Cells were exposed to LPS, TNF-alpha, IL-1beta, or media alone for up to 12 h. METHODS: Northern blot analyses were done to determine mRNA expression of ICAM-1 after exposures. Protein binding to NF-kappaB sequences were determined by gel mobility shift assays and super-shift analysis. RESULTS: ICAM-1 mRNA expression was induced in A549 cells with exposure to LPS for 1 to 4 h, and was diminished to baseline at 8 h, and the inductions were independent of TNF-alpha and IL-1beta expression. Nuclear protein extracts from LPS-exposed cells bound to a NF-kappaB sequence and the timing of increased binding correlated closely with ICAM-1 mRNA induction. Super-shift studies indicated that p65 was involved in the binding to the NF-kappaB sequence and p50 was not. CONCLUSION: LPS inducibility of ICAM-1 mRNA in A549 cells is independent of TNF- and IL-1 in A549 cells, and the similar time course of mRNA induction and NF-kappaB activation suggest the induction of ICAM-1 is mediated, in part, by NF-kappaB.

Dexamethasone enhances P-selectin mRNA expression in hyperoxic rat lungs.

OBJECTIVE AND DESIGN: To test the hypothesis that glucocorticoid administration would diminish the lung expression of P-selectin mRNA in hyperoxia-exposed rats. ANIMALS: Adult male Sprague-Dawley rats were divided into 6 separate groups containing 10 to 13 animals per group. TREATMENT: Rats were dosed with 1 mg/kg of dexamethasone or vehicle only, ip. Immediately after dosing, animals were placed in > 95 % oxygen. Some animals were maintained in room air and are presented as 0 h of exposure to hyperoxia. Another group of animals was dosed with 10 mg/kg lipopolysaccharide (LPS) ip immediately after dosing with either dexamethasone or vehicle as above. METHODS: At 24 or 48 h, lung samples were obtained, and lung weight to body weight ratios calculated. In the LPS studies, samples were obtained 4 h after LPS dosing. In a subset of animals, lung sections were hybridized for P-selectin mRNA. All data except for hybridizations were analyzed with three-way ANOVA, with subsequent post-hoc testing. P-selectin hybridizations were quantified by counting the number of positive vessels per high-powered field, and subsequently analyzed by unpaired Student's t-test. Immunohistochemical analyses for P-selectin expression were also performed to determine whether changes in P-selectin mRNA were associated with differences in protein expression. All data are expressed as means +/- SEM. RESULTS: Rats dosed with dexamethasone had higher lung/body weight ratios after 24 and 48 h of exposure to hyperoxia than did similarly exposed rats dosed only with vehicle (at 48 h, 0.87 +/- 0.04 versus 0.65 +/- 0.06, respectively, P < 0.05). The higher ratios in hyperoxic animals dosed with dexamethasone were associated with much higher levels of lung expression for P-selectin mRNA than was observed in similarly exposed rats dosed with vehicle alone (at 48 h, 3.93 +/- 1.02, versus 0.20 +/- 0.06, respectively, P < 0.01). In contrast dexamethasone dosing lead to lower lung P-selectin mRNA expression in animals exposed to LPS (1.23 +/- 1.08 in dexamethasone dosed animals versus 6.80 +/- 0.92 in vehicle only dosed animals). Consistent with the mRNA data, P-selectin immunoreactivity increased as a function of hyperoxia-exposure time in animals dosed with dexamethasone, while immunoreactivity decreased as a function of hyperoxia-exposure time in animals dosed with vehicle only. CONCLUSIONS: Increased P-selectin mRNA combined with increased P-selectin protein expression in animals exposed to hyperoxia and dosed with dexamethasone suggests that enhanced expression of P-selectin may contribute to the greater lung injury and inflammation caused by hyperoxia in rats treated with dexamethasone.

[Not Available]. / Die Höhe des Liquorflusses über dem Aquädukt MR-Liquorflussquantifizierung an 107 Patienten ohne klinische und bildmorphologische Hinweise auf eine Liquorzirkulationsstörung : MR-Liquorflussquantifizierung an 107 Patienten ohne klinische und bildmorphologische Hinweise auf eine Liquorzirkulationsstörung.

With the 2D-phase-contrast technique the volume flow of the CSF via the cerebral aqueduct can be quantified by MRI-means. In this study the stroke volume of CSF via the aqueduct per cardiac cycle (SVcc) is used to measure the extent of the volume flow. Normative values for the SVcc are not yet defined, however, they are indispensable for the clinical utility of this non-invasive method.The aim of the presented investigation is to evaluate, if the interthalamic width of the third ventricle is useful as a reference system for the extent of the SVcc via the aqueduct and if a normal CSF-flow can be defined.Hundred and seven patients (56 female, 51 male; age distribution 8 to 89 years) without clinical or imaging findings of a CSF-flow disturbance were examined on a standard 0.5 T MRI-scanner (Gyroscan, Philips). The measurements of the SVcc via the aqueduct were performed in a single slice perpendicular to the aqueduct in the level of its median third with a retrospective cardial-gated quantitative 2D-phase-contrast sequence. The interthalamic width of the third ventricle was measured in a transversal slice (bicommissural orientation, standard T1-weighted spin-echo sequence) in the level of the upper margin of the tectorial membrane.In 83 patients with a normal heart rate and without any further abnormalities in their imaging studies the SVcc is essentially dependent (r = 0.822) on the interthalamic width of the third ventricle (between 1 and 16 mm). Eleven patients with either a subcortical atrophy without leucencephalopathy, megacisterna magna, Dandy-Walker variant or bradycardia showed a significant increase of the SVcc (p < 0.05). On the other hand a significant decrease of the SVcc (p < 0.05) is seen in 13 patients with either tachycardia, Arnold-Chiari Type-1 malformation, relative aqueductal stenosis and/or severe periventricular leucencephalopathy. These results are in good agreement with the current conceptions on the physiology of the CSF-flow. As the above mentioned criterias of influence have ho pathological significance concerning a CSF-flow disturbance requiring therapy, we used the linear regression with y = B1*× +b0 (b1 = 22.2 ± 2.9; b0 = 43,5 ± 21.1) in all 107 patients to evaluate the extent of the SVcc (y) versus the interthalamic width of the third ventricle (x).This correlation offers the possibility to differentiate a hyperdynamic (above +3 standard error SE), a hypodynamic (below -3 SE) and a normodynamic (between ± 3 SE) CSF-flow via the cerebral aqueduct for the first time. Additional imaging findings and the heart rate must find their influence in the evaluation.

Extracellular cysteine protease produced by Streptococcus pyogenes participates in the pathogenesis of invasive skin infection and dissemination in mice.

The role of an extracellular cysteine protease encoded by the speB gene in group A Streptococcus (GAS) skin infection was studied with a mouse model. Mice were injected subcutaneously with a wild-type GAS serotype M3 strain or a cysteine protease-inactivated isogenic derivative grown to stationary phase. The mortality rate of mice injected with the M3 speB mutant strain was significantly decreased (P < 0.0008) compared to that of animals injected with the wild-type parental organism. The abscesses formed in animals infected with the cysteine protease mutant strain were significantly smaller (P < 0.0001) than those caused by the wild-type organism and slowly regressed over 3 to 4 weeks. In striking contrast, infection with the wild-type GAS isolate generated necrotic lesions, and in some animals the GAS disseminated widely from the injection site and produced extensive cutaneous damage. All of these animals developed bacteremia and died. GAS dissemination was accompanied by severe tissue and blood vessel necrosis. Cysteine protease expression in the infected tissue was identified by immunogold electron microscopy. These data demonstrate that cysteine protease expression contributes to soft tissue pathology, including necrosis, and is required for efficient systemic dissemination of the organism from the initial site of skin inoculation.

Local overexpression of thrombomodulin for in vivo prevention of arterial thrombosis in a rabbit model.

-Endothelial thrombomodulin plays a critical role in hemostasis by binding thrombin and subsequently converting protein C to its active form, a powerful anticoagulant. Thrombomodulin thus represents a central mechanism by which patency is maintained in normal vessels. However, thrombomodulin expression decreases in perturbed endothelial cells, predisposing to thrombotic occlusion. An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on in vivo thrombus formation was subsequently examined in a stasis/injury model of arterial thrombosis. The construct prevented arterial thrombosis formation in all animals, while viral and nonviral controls typically developed occluding thrombi. By histological analysis, nonviral controls exhibited intravascular thrombus occluding a mean of 70.52+/-3.72% of available lumen, while viral controls reached 86. 85+/-2.82% thrombotic occlusion; in contrast, Adv/RSV-THM reduced thrombosis to 28.61+/-3.31% of lumen in cross section. No significant intima-to-media ratio was observed in the thrombomodulin group relative to controls. Local infiltration of granulocytes and macrophages significantly decreased in the Adv/RSV-THM group relative to controls, while neutrophilic infiltration increased in viral controls relative to nonviral controls. This construct thus offers a viable technique for promoting a locally thromboresistant small-caliber artery, without the inflammatory damage that has limited many other adenoviral applications.

Neuronal nitric oxide synthase is expressed in rat vascular smooth muscle cells: activation by angiotensin II in hypertension.

The nitric oxide synthase (NOS) inhibitor nitro-L-arginine augmented the contractions to angiotensin (Ang) II in carotid artery rings without endothelium from spontaneously hypertensive rats (SHR) but not normotensive Wistar-Kyoto rats, suggesting the possibility of nonendothelial NOS activity in SHR arteries. In SHR artery without endothelium, the potentiation of Ang II contraction by nitro-L-arginine was prevented by L-arginine, but not by D-arginine, and was observed also in the presence of oxyhemoglobin, monomethyl-L-arginine, and 7-nitroindazole, but not in the presence of aminoguanidine. In further support of NOS activation by Ang II in nonendothelial cells, Ang II but not acetylcholine stimulated cGMP levels by 2-fold in SHR arteries without endothelium; nitro-L-arginine decreased both basal and Ang II-stimulated cGMP levels. When NOS activity in SHR arteries was measured, no calcium-independent L-citrulline formation was detectable, while up to 47% of the total calcium-dependent NOS activity was present in nonendothelial cells. Expression of neuronal NOS was revealed in the media of SHR arteries by immunohistochemistry, Western blot, and reverse transcriptase-polymerase chain reaction. Expression of this NOS isoform was greater in SHR than in Wistar-Kyoto rat preparations. Finally, endothelial NOS was observed in the endothelium, but no detectable levels of inducible NOS were found in these tissues. These results demonstrate the expression of neuronal NOS in rat vascular smooth muscle cells and its activation on stimulation by Ang II in spontaneously hypertensive, but not normotensive, animals.

Dexamethasone enhancement of hyperoxic lung inflammation in rats independent of adhesion molecule expression.

Infants and adults on oxygen often are treated with glucocorticoids in an attempt to reduce lung inflammatory injury. However, glucocorticoids hasten the development of hyperoxic lung injury in some animal models. The purpose of this study was to test the hypothesis that dexamethasone alters the lung inflammatory responses to hyperoxia exposure. We studied male Sprague-Dawley rats, placing them in >95% oxygen immediately after administration of 0, 0.1, 1, or 10 mg/kg of dexamethasone. At 0, 24, or 48 hr of exposure to hyperoxia, extravascular lung water contents were measured, and lung inflammatory responses were assessed by lung myeloperoxidase activities, lung neutrophil counts, and lung expression of E-Selectin and intercellular adhesions molecule-1 (ICAM-1). Dexamethasone, independent of exposure to hyperoxia, led to marked increases in lung neutrophil counts, without increases in lung myeloperoxidase activities or increases in the expression of the adhesion molecules. Hyperoxia exposure also enhanced lung neutrophil accumulation, and extravascular lung water increased earlier in animals exposed to hyperoxia and dexamethasone than in those exposed to hyperoxia alone. In conclusion, the increase in lung neutrophils in dexamethasone-treated rats without enhanced expression of E-Selectin or intracellular adhesions molecule-1 suggests that dexamethasone leads to lung neutrophil accumulation by its effect on neutrophils. The more rapid development of hyperoxic lung injury associated with earlier lung neutrophil accumulation suggests that dexamethasone-induced lung neutrophil sequestration primes the lung for the development of hyperoxic lung injury and supports further the conclusion that lung inflammation contributes significantly to the development of hyperoxic lung injury.

Increased soluble E-Selectin is associated with lung inflammation, and lung injury in hyperoxia-exposed rats.

The pulmonary damage caused by prolonged exposure to high oxygen concentrations is accompanied by lung inflammation, which may contribute to the expression of hyperoxic lung injury. In turn, adhesion molecules are crucial for initiating inflammatory responses. The goal of the present study was to investigate the association of contents of soluble adhesion molecules in plasma or alveolar fluids of hyperoxic rats with lung expression of adhesion molecules, lung inflammation and lung injury. We exposed adult Sprague-Dawley rats to > 95% oxygen for up to 60 h and measured the contents of intercellular adhesion molecule-I (ICAM-I) and E-Selectin in plasma and lung tissue expression of the same molecules, and we assessed lung myeloperoxidase (MPO) activties and lung water contents as indices of lung inflammation and injury, respectively. We also assessed ICAM-I content in lavage samples, because ICAM-I may be shed from the alveolar epithelium. Lung water was elevated at 60 h of hyperoxia-exposure, and this effect was preceded by increases in lung MPO activities. Lung ICAM-I expression was more than doubled at 48 h, although soluble ICAM-I contents were not elevated in plasma or lavage. Soluble E-Selectin was increased by more than 50% at 24 h of hyperoxia-exposure, while lung expressions of E-Selectin were not increased until 48 h. The sequence of the events observed in the present studies suggests that E-Selectin contributes to lung inflammation in hyperoxia and the acceleration of lung injury immediately following the inflammatory response suggests a pivotal role for inflammation in this injury.

Neutrophil sequestration and pulmonary dysfunction in a canine model of open heart surgery with cardiopulmonary bypass. Evidence for a CD18-dependent mechanism.

BACKGROUND: Previous studies documented an inflammatory reaction to cardiopulmonary bypass with neutrophil (PMN) sequestration in the lungs, contributing to microvascular injury and postoperative pulmonary dysfunction. This study explored the hypothesis that the beta 2 integrin CD18, a leukocyte adhesion molecule, mediates this response. METHODS AND RESULTS: Fifteen adult, mixed-breed dogs underwent 90 minutes of cardiopulmonary bypass with 3 hours of subsequent recovery. Seven additional dogs were treated before cardiopulmonary bypass with a 1-mg/kg IV bolus of R15.7 IgG, a monoclonal antibody to CD18. Both groups were compared with 5 sham bypass control dogs. Bypassed dogs demonstrated an increased number of PMNs sequestered in the lungs 3 hours after bypass compared with sham bypass control dogs (1466 +/- 75 versus 516 +/- 43 PMN/mm2 alveolar surface area, mean +/- SEM, P < .001). Also, when PMNs from bypass dogs were compared with those from sham dogs, they produced more H2O2 (305 +/- 45 versus 144 +/- 48 amol H2O2 per phagocyte per 20 minutes, P < .05). Bypass dogs had significantly decreased arterial oxygenation 3 hours after the procedure compared with shams (457 +/- 20 versus 246 +/- 49 mm Hg, P < .05), and they had a significantly increased lung wet-to-dry weight ratio (5.38 +/- 0.14 versus 4.54 +/- 0.15, P = .003), demonstrating a significant increase in lung water. R15.7 markedly attenuated pulmonary PMN accumulation in bypass dogs (412 +/- 73 PMN/mm2, P < .001) and significantly inhibited PMN production of H2O2 (146 +/- 18 amol H2O2 per phagocyte per 20 minutes, P < .05) Bypass dogs pretreated with R15.7 also had improved oxygenation (445 +/- 28 mm Hg, P < .05) and tended to have less lung water accumulation after bypass (4.99 +/- 0.20). CONCLUSIONS: Pulmonary dysfunction after cardiopulmonary bypass is caused, at least in part, by a neutrophil-mediated, CD18-dependent mechanism.

Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice.

The avian skeletal alpha-actin gene was used as a template for construction of a myogenic expression vector that was utilized to direct expression of a human IGF-I cDNA in cultured muscle cells and in striated muscle of transgenic mice. The proximal promoter region, together with the first intron and 1.8 kilobases of 3'-noncoding flanking sequence of the avian skeletal alpha-actin gene directed high level expression of human insulin-like growth factor I (IGF-I) in stably transfected C2C12 myoblasts and transgenic mice. Expression of the actin/IGF-I hybrid gene in C2C12 muscle cells increased levels of myogenic basic helix-loop-helix factor and contractile protein mRNAs and enhanced myotube formation. Expression of the actin/IGF-I hybrid gene in mice elevated IGF-I concentrations in skeletal muscle 47-fold resulting in myofiber hypertrophy. IGF-I concentrations in serum and body weight were not increased by transgene expression, suggesting that the effects of transgene expression were localized. These results indicate that sustained overexpression of IGF-I in skeletal muscle elicits myofiber hypertrophy and provides the basis for manipulation of muscle physiology utilizing skeletal alpha-actin-based vectors.

Disruption of the adenosine deaminase gene causes hepatocellular impairment and perinatal lethality in mice.

We have generated mice with a null mutation at the Ada locus, which encodes the purine catabolic enzyme adenosine deaminase (ADA, EC 3.5.4.4). ADA-deficient fetuses exhibited hepatocellular impairment and died perinatally. Their lymphoid tissues were not largely affected. Accumulation of ADA substrates was detectable in ADA-deficient conceptuses as early as 12.5 days postcoitum, dramatically increasing during late in utero development, and is the likely cause of liver damage and fetal death. The results presented here demonstrate that ADA is important for the homeostatic maintenance of purines in mice.