Cytogenetic and array-CGH characterization of a complex de novo rearrangement involving duplication and deletion of 9p and clinical findings in a 4-month-old female.

Approximately 15 patients with partial trisomy 9p involving de novo duplications have been previously described. Here, we present clinical, cytogenetic, FISH and aCGH findings in a patient with a de novo complex rearrangement in the short arm of chromosome 9 involving an inverted duplication at 9p24-->p21.3 and a deletion at 9pter-->p24.2. FISH probes generated from BACs selected from the UCSC genome browser were utilized to verify this rearrangement. It is likely that some previously described duplications of 9p may also be products of complex chromosomal aberrations. This report in which FISH and aCGH were used to more comprehensively characterize the genomic rearrangement in a patient with clinical manifestations of 9p duplication syndrome underscores the importance of further characterizing cytogenetically detected rearrangements.

Urinary F2-isoprostanes are poor prognostic indicators for the development of bronchopulmonary dysplasia.

OBJECTIVE: Oxygen toxicity is thought to contribute to the development of bronchopulmonary dysplasia (BPD). Oxidant injury leads to formation of F(2)-isoprostanes (F(2)-IsoP). We hypothesized that urinary excretion of the stable metabolite of F(2)-IsoP, 8-iso-PGF(2alpha), would be higher in infants who develop BPD than those who did not. METHODS: Forty infants <30-weeks gestational age (GA) were enrolled, 24 infants with BPD and 16 without BPD. Urine specimens were collected weekly and stored at -80 degrees C until analyzed. Urinary 8-iso-PGF(2alpha) was measured by gas chromatography/mass spectrometry (GC-MS) and normalized to creatinine excretion. RESULTS: GA and birth weight (BW) were lower in infants who developed BPD than those who did not. Urinary 8-iso-PGF(2alpha) levels in the first or third weeks of age were not significantly different between the two groups. CONCLUSION: Urinary excretion of 8-iso-PGF(2alpha) in early postnatal life in preterm infants is not correlated with the development of BPD.

Mitochondrial glutathione and oxidative stress: implications for pulmonary oxygen toxicity in premature infants.

Administration of supplemental oxygen, despite being an important clinical therapy, can cause significant lung damage. Because they have underdeveloped lungs, prematurely born human infants frequently require supportive therapies that employ elevated oxygen concentrations, which put them at risk for developing pulmonary oxygen toxicity. This risk is made even greater by the immaturity of their cellular antioxidant defenses. Although the exact mechanisms of oxygen toxicity are still not fully defined, cellular damage is probably mediated by increased production of chemically reactive oxygen species (ROS) in the mitochondria. Cellular protection against ROS is provided by a variety of antioxidant molecules and enzymes, including the glutathione (GSH)-dependent antioxidant system. The GSH-dependent antioxidant enzyme system provides vital cellular protection against ROS, particularly hydrogen peroxide and certain organic hydroperoxides, under pathological and toxicological conditions, by using selenium-dependent and -independent peroxidases to reduce hydrogen peroxide or lipid peroxides to water or the respective alcohols, with the concurrent oxidation of GSH to glutathione disulfide (GSSG). In the mitochondria, limitations of GSH synthesis and transmembrane transport suggest that optimal functioning of the mitochondrial GSH system, and maintenance of adequate thiol-disulfide redox tone is essential to protect against the injurious effects of ROS. Manipulation of endogenous GSH concentrations can alter cellular responses to oxidant injury. Beneficial effects are evident when intracellular GSH concentrations are increased. In conditions that increase mitochondrial production of ROS, such as exposure to high concentrations of oxygen, therapies based on enhancing mitochondrial GSH concentrations could be highly beneficial.

Attenuation of hyperoxia-induced growth inhibition in H441 cells by gene transfer of mitochondrially targeted glutathione reductase.

Reactive oxygen species (ROS) are implicated as agents of cellular damage in pulmonary oxygen toxicity. Glutathione (GSH) and GSH-dependent antioxidant enzymes protect against damage by ROS, and recycling of glutathione disulfide (GSSG) to GSH by glutathione reductase (GR) is essential for the optimum functioning of this system. Exposure to hyperoxia inhibits lung development in newborn animals and humans, and attenuates cell growth in proliferating cell cultures. Considerable evidence supports a role for ROS as growth-altering molecules. Previously, we have observed that gene transfer of GR to mitochondria in H441 cells, using a vector containing a mitochondrial leader sequence (LGR), protected these cells against t-BuOOH-induced cytotoxicity. The present studies tested the hypothesis that gene transfer of LGR would attenuate the cytostatic effects of hyperoxia exposure in H441 cells. H441 cells (0.9 x 10(6) cells/plate) transfected with adenovirus containing LGR or the complementary DNA (cDNA) for manganese superoxide dismutase in reverse orientation (DOS) as a control construct, and untransfected cells (CON) were maintained in 21% oxygen (normoxia) or 95% oxygen (hyperoxia) for 48 h, and cell growth was assessed by cell counts and by reduction of the tetrazolium dye 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan. Cells maintained in normoxia achieved normal growth (CON, 1.98; DOS, 1.91; LGR, 2.0 x 10(6) cells/plate). Hyperoxia inhibited cell growth and the reduction of MTT; however, cells transfected with LGR had greater mitochondrial GR activities (CON, 16+/-2; DOS, 19+/-3; LGR, 322+/-18 mU/mg of protein), sustained more normal growth patterns (CON, 1.25+/-0.12; DOS, 1.24 +/-0.21, LGR, 1.8+/-0.25 x 10(6) cells/plate), and had less inhibition of MTT reduction (CON, 29; DOS, 27; LGR, 16% inhibition, P<0.01) after exposure to hyperoxia for 48 h than was observed in cells transfected with DOS or in control cells not infected with virus. In addition, resistant cells had higher mitochondrial GSH levels and maintained mitochondrial GSH/GSSG ratios in hyperoxia, suggesting that maintaining mitochondrial GSH homeostasis determined critical aspects of cell division in these studies. The mechanisms for sustaining cell growth during hyperoxia in H441 cells with enhanced mitochondrial GR activities are unknown, but similar effects in infants exposed to supplemental oxygen could be highly beneficial.

Effects of intracoronary nicardipine, diltiazem and verapamil on coronary blood flow.

BACKGROUND: Intracoronary (IC) calcium channel blockers (CCB) such as diltiazem and verapamil are frequently utilized during percutaneous coronary interventions to maximize coronary blood flow. Their use, however, may be limited by systemic side effects such as hypotension and bradyarrhythmias. The vasoselective dihydropyridines, such as nicardipine, may be more effective at increasing coronary blood flow with fewer systemic side effects. This study compares the effects of nicardipine, diltiazem and verapamil on coronary blood flow, heart rate and blood pressure. METHODS: IC nicardipine (200 mcg), diltiazem (1 mg) and verapamil (200 mcg) were serially administered in a randomized, double-blinded fashion in minimally diseased (< 30% stenosis) left anterior descending or left circumflex arteries in nine patients. Epicardial coronary artery diameter (ECAD) was determined by quantitative coronary angiography and coronary blood flow velocity (CBFV) was measured by Doppler Flowire in each patient before and after each medication. RESULTS: Nicardipine significantly increased CBFV (p < 0.05) and had a longer duration of effect (p < 0.05), but had no difference in ECAD compared with diltiazem and verapamil. No differences were noted between CCB in changes in heart rate or mean arterial blood pressure. However, two patients had transient episodes of Type I second degree AV block after receiving diltiazem. CONCLUSION: When compared with diltiazem and verapamil, nicardipine appears to offer more potent and more prolonged vasodilatation with less risk of serious systemic side effects. Future studies are needed to assess the efficacy of IC nicardipine in patients with no-reflow.

Intravenous versus subcutaneous vitamin K1 in reversing excessive oral anticoagulation.

Our data suggest that compared with the subcutaneous route of administration, intravenous vitamin K1 results in a more prompt reduction in the international normalized ration. However, for most patients, subcutaneous vitamin K1 is an effective and safe alternative when used in conjunction with modification of subsequent warfarin dosing, because virtually all patients achieved a safe level of anticoagulation within 72 hours with this route of administration.

Gene transfer of mitochondrially targeted glutathione reductase protects H441 cells from t-butyl hydroperoxide-induced oxidant stresses.

Increased generation of reactive oxygen species (ROS) and low levels of antioxidants may cause morbidity in premature infants on supplemental oxygen. Glutathione (GSH)-dependent antioxidant systems protect against ROS, and regenerating GSH from GSH disulfide (GSSG) by the flavoenzyme GSH reductase (GR) is essential for the optimal function of this system. Previously, we have observed enhanced resistance to t-butyl hydroperoxide (t-BuOOH) in Chinese hamster ovary cells stably transfected with a vector (leader sequence GR [LGR]) for human GR cDNA that contained a functional synthetic mitochondrial targeting signal. The present studies were designed to investigate adenovirus-mediated gene transfer of LGR to H441 cells and resistance of such cells to t-BuOOH. Adenovirus-mediated transfection of H441 cells with LGR increased total GR activities more than 11-fold (mitochondria more than 10-fold and cytosolic more than 7-fold) and protected against t-BuOOH cytotoxicity, as indicated by lower fractional release of cellular lactate dehydrogenase (LDH) than was observed in wild-type untransfected cells (CON) or in cells transfected with a control gene (human manganese superoxide dismutase in the antisense orientation [DOS]) (*LGR 6.6 +/- 1.7; DOS 16 +/- 1.8; CON 16.6 +/- 0.7% LDH release). In addition, cells transfected with LGR retained higher GSH/GSSG ratios (*LGR 66 +/- 0.4; DOS 47 +/- 1; CON 52.6 +/- 2.3) and released less GSH + GSSG to the media in response to challenge with t-BuOOH (*LGR 0.05 +/- 0.01; DOS 0.08 +/- 0.01; CON 0.07 +/- 0.01 nmol/mg of protein) than did wild-type cells or cells transfected with a control vector, indicating an enhanced ability of the LGR cells to reduce GSSG formed in response to exposure to t-BuOOH. In conclusion, adenovirus-mediated gene transfer of LGR enhanced cellular GR activities and protected H441 cells from oxidant stresses.

Renal and hepatic nitrogen metabolism during NH4Cl ingestion in protein-deprived rats.

Three groups of rats were given either a standard protein diet, a protein-free diet, or a protein-free diet with the inclusion of 0.28 M NH4Cl in their drinking water, for 10 days. Body, liver and kidney masses were decreased similarly in the protein-free and protein-free NH4Cl groups. Ingestion of protein-free diet resulted in profound systemic acidosis in both groups, the simultaneous consumption of NH4Cl having no further effect. The activities of the urea-cycle enzymes carbamoyl-phosphate synthease, ornithine transcarbamoylase, arginosuccinate lyase and arginase were significantly reduced in the protein-free group, and the simultaneous ingestion of NH4Cl had no further effect. These results indicate that ammonium ingestion does not prevent the decrease in urea cycle enzyme activities during a period of dietary-protein deprivation. Renal phosphate-dependent glutaminase activity was unchanged in the protein-free group, but was significantly higher with simultaneous NH4Cl consumption, suggesting that the renal adaptation to acid ingestion is not compromised by a lack of dietary protein. Urinary ammonia excretion also increased in rats consuming protein-free diet and NH4Cl. Urinary urea excretion was greater in rats receiving protein-free diet and NH4Cl than in rats receiving protein-free diet only, at all time-points examined. These data demonstrate that urea synthesis is driven primarily by the need to dispose of protein-derived ammonia rather than bicarbonate.

Alterations in renal and hepatic nitrogen metabolism in rats during HCl ingestion.

The effect of prolonged metabolic acidosis on hepatic and renal enzymes associated with nitrogen metabolism was investigated. The rates of urinary ammonia and urea excretion were also determined. Administration of 9 mmol HCl daily for 8 days resulted in severe metabolic acidosis. The activity of the first two enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) and ornithine transcarbamoylase (OTC), was 30% greater in chronically acidotic rats than in pair-fed controls. There was also a fivefold increase in renal phosphate-dependent glutaminase (PDG) activity and an 18 to 24-fold increase in renal ammonia excretion. Urea excretion was not constant in the acidotic group, decreasing during the first 4 days and gradually returning to pair-fed control levels between the fourth and eighth day. The return to control levels of urinary urea excretion coincided with the plateau of urinary ammonia excretion that occurred by day 4 in the acidotic group. A similar pattern of urea nitrogen excretion has been observed in both NH4Cl and HCl acidosis, ie, an initial decrease in urea excretion followed by a gradual increase with time. These results suggest that hepatic urea synthesis does not play a significant role in long-term regulation of the acid-base balance in rats during chronic metabolic acidosis.

The effect of ammonium chloride on hepatic and renal metabolism in the rat.

The metabolic effects of an ammonium salt on the liver and kidney were investigated. Rats were allowed free access to a 0.28 M ammonium chloride (NH4Cl) solution for 7- and 8-day periods. Serum urea concentration was significantly increased after 8 days of NH4Cl ingestion. However the following hepatic urea cycle enzymes remained unchanged: CPS, OTC, ASS and ASL. The pattern of urinary urea excretion was variable. When the data for the 7-day period were pooled, there was no significant difference between the control and acidotic groups. However, when they were examined on a daily basis, acidosis significantly decreased urea excretion on day 2. Urea excretion then began to increase, reached the control value on day 4 and was significantly greater than the control value on day 7. Urinary ammonium excretion of the acidotic group was significantly increased on day 2 and continued to rise throughout the 7-day period. Renal phosphate-dependent glutaminase of the acidotic group was significantly increased on the eighth day. These data indicate that NH4Cl ingestion alters the pattern of urea excretion in a manner not previously demonstrated.

The removal of a transdural pedicle screw placed for thoracolumbar spine fracture.

STUDY DESIGN: This report describes the method of removal of a pedicle screw that had been misplaced through the thecal sac and the cauda equina instead of its proper location within the pedicle. OBJECTIVES: A patient who previously had undergone placement of pedicle screws and Roy-Camille plates for fixation of L1 burst fracture presented to the authors with neurologic deficits and a cerebrospinal fluid leak. Computed tomography myelogram and surgical findings demonstrated misplacement of three of the screws, one of which was placed through the dura with resulting impingement of nerve roots within the thecal sac. SUMMARY OF BACKGROUND DATA: Removal of the screws was required for neural decompression and resolution of the cerebrospinal fluid leak. METHODS: The patient underwent laminectomy to expose the screw and primary durotomy to include the entry point of the screw. This technique allowed safe removal under direct vision, with direct protection of the nerve roots. A subsequent anterior fusion procedure was performed. RESULTS: The patient regained full neurologic function, and his cerebrospinal fluid leak ceased. At 2-year follow-up evaluation, he describes only mild, occasional back pain. CONCLUSIONS: Penetration of the dura and injury to the neural elements can occur when pedicle screw instrumentation is used. Removal of the screw under these circumstances may cause additional neurologic injury. Durotomy and direct visualization of the neural elements in this case was a valuable adjunct to safe removal of the screw.

Delayed-type hypersensitivity skin testing predicts progression to AIDS in HIV-infected patients.

OBJECTIVE: To evaluate the prognostic significance of cutaneous delayed-type hypersensitivity (DTH) skin testing in persons infected with HIV. DESIGN: Cohort study. SETTING: United States Air Force (USAF) Medical Center. PATIENTS: Consecutive sample of 889 HIV-infected USAF personnel or dependents undergoing their first staging evaluation from 1985 through August 1990 in the USAF HIV Natural History Study. MEASUREMENTS: All patients were evaluated with DTH skin testing including purified protein derivative and four control skin test antigens: mumps, candida, tetanus toxoid, and trichophyton. In addition, all patients underwent CD4+ T-cell surface marker determinations. The relation between DTH skin test response at first evaluation and progression to Walter Reed stage 6 (presence of an AIDS-defining opportunistic infection) was evaluated using Kaplan-Meier survival analysis. RESULTS: Patients with more than 400 CD4+ T cells/mm3 are more likely than those having fewer than 400 CD4+ T cells per mm3 to respond to at least one (94% compared with 67%, P < 0.001) or at least two (86% compared with 45%, P < 0.001) DTH skin tests. Mean CD4 counts are lower for anergic compared with nonanergic patients and for patients responding to a single control skin test compared with those responding to two or more skin tests (P < 0.05). The DTH skin test response at first evaluation was also found to predict progression to AIDS; the relative risk at 5 years of follow-up was 2.5 (95% CI, 1.2 to 5.2) for anergy compared with a single positive skin test and 3.0 (CI, 1.4 to 6.2) for a single compared with two or more skin test responses. The DTH skin test response at first evaluation was a predictor of progression (P < 0.001) when controlling for initial CD4 count and Walter Reed stage in a Cox proportional hazards regression analysis. CONCLUSIONS: The DTH skin test response, a functional measure of cellular immunity, is an independent predictor of progression to AIDS in persons with HIV.

Anticoccidial effect of monensin against Eimeria mitis and Eimeria dispersa.

Four chick and five poult trials were conducted in order to investigate the anticoccidial efficacy of monensin against Eimeria mitis in chickens and Eimeria dispersa in turkeys. The chicks were fed a basal diet with either 0 or 100 ppm of monensin. The poults were fed a basal diet with either 0 or 60 ppm of monensin. Two days after the initiation of each experiment, the chicks and poults were crop-intubated with oocysts of E. mitis and E. dispersa, respectively. A group was also included that was not infected and not medicated. Growth and feed intake were recorded. At 6 or 7 days postinoculation, the birds were killed by cervical dislocation and were scored for the incidence and severity of intestinal abnormalities. The Eimeria mitis infection reduced (P less than .01) gain and the feed:gain ratio, compared with uninfected birds; the E. dispersa infection only reduced (P less than .05) gain. Although well-defined, discrete lesions were not observed, marked intestinal abnormalities were noted in birds infected with either E. mitis or E. dispersa. Also, the infected, unmedicated birds had increased (P less than .01) intestinal scores compared with uninfected birds or those with infection but treated with monensin. Monensin eliminated the reduction in gain and feed efficiency seen in the infected birds. The infected birds fed monensin had intestinal scores, gain, and feed:gain ratios similar (P greater than .10) to those for the birds that were not infected and that did not receive medication.