Rapid bedside rejuvenation of red blood cell with an autologous cell salvage device.

BACKGROUND AND OBJECTIVES: During storage, red blood cells (RBCs) undergo physicochemical changes which affect the quality, function, and in vivo survival of transfused packed RBCs (pRBC). Changes include decreased 2,3-diphosphoglycerate (2,3-DPG) levels, decreased ATP, changes in mechanical properties and oxidative injury. RBC rejuvenation is a method used to increase levels of 2,3-DPG and ATP in pRBCs. This process requires incubating the pRBCs with a rejuvenation solution and subsequent washing. Standard blood bank protocols using the COBE 2991 Cell Processor require several hours of preparation. The objective of this study was to verify if a bedside protocol for rejuvenating pRBC and washing with the Sorin Xtra autologous cell salvage system could be used. MATERIALS AND METHODS: Outdated pRBC units were obtained and rejuvenated in a model operating suite using a dry air incubator for 1 h at 37°C. Six units of pRBCs were pre-diluted with saline (1000 ml) and six units were not pre-diluted with saline. All units were washed with normal saline (1000 ml) using an apheresis-design cell salvage device in manual mode and wash volume set to 3000 ml. Samples were collected and analyzed for standard RBC quality parameters at baseline and post-wash. RESULTS: Total pRBC wash efficiency was 94% ± 12% at a final hematocrit of 67.7 ± 5.9% while maintaining post-wash hemolysis 0.24 ± 0.12 %. Pre-dilution prior to washing did not confer statistically significant differences in final RBC quality parameters with the notable exceptions of calculated hemolysis and supernatant potassium levels (P < 0.05). The washing process can be completed within 10 min. The post-wash RBC parameters are appropriate for immediate transfusion to patients.

Fluticasone propionate/formoterol for COPD management: a randomized controlled trial.

PURPOSE: To evaluate fluticasone propionate/formoterol (FP/FORM) in COPD. PATIENTS AND METHODS: COPD patients with forced expiratory volume in 1 s (FEV1) ≤50% predicted and ≥1 moderate/severe COPD exacerbation in the last 12 months were randomized to FP/FORM 500/20 or 250/10 µg bid, or formoterol (FORM) 12 µg bid for 52 weeks. The primary outcome was the annualized rate of moderate/severe COPD exacerbations. RESULTS: In total, 1,765 patients were randomized. There were fewer discontinuations with FP/FORM 500/20 µg (20.6%) and 250/10 µg (24.0%) compared with FORM (26.1%). None of the two FP/FORM doses reduced the moderate/severe exacerbation rate versus FORM (rate ratios [RR]: 0.93; P≤0.402). There was a trend toward a lower moderate/severe exacerbation rate with FP/FORM 500/20 µg versus FORM in patients with ≥2 exacerbations in the preceding year (RR: 0.79; P=0.084). Pre- and post-dose FEV1 and forced vital capacity were greater with FP/FORM 500/20 µg versus FORM (P≤0.039). There was a trend toward a lower EXAcerbations of Chronic pulmonary disease Tool (EXACT) exacerbation rate with FP/FORM 500/20 µg versus FORM (RR: 0.87; P=0.077). There were more St George's Respiratory Questionnaire for COPD (SGRQ-C) responders with FP/FORM 500/20 µg than FORM (odds ratios [OR] at weeks 6, 23 and 52 ≥1.28; P≤0.054). EXACT-respiratory symptoms total and breathlessness scores were lower with both FP/FORM 500/20 µg and 250/10 µg versus FORM (P≤0.066). Acute ß2-agonist-induced effects and 24-hour Holter findings were similar for all treatments. Mean 24-hour urinary cortisol was similarly reduced with both FP/FORM doses. Radiologically confirmed pneumonia was seen in 2.4%, 3.2% and 1.5% of FP/FORM 500/20 µg, FP/FORM 250/10 µg and FORM-treated patients, respectively. Adverse events were otherwise similar across treatment groups. CONCLUSION: FP/FORM did not reduce exacerbation rates versus FORM. Numerical benefits were observed with FP/FORM 500/20 µg versus FORM for secondary variables, including lung function, EXACT exacerbations, SGRQ-C and EXACT-respiratory symptoms total and breathlessness scores. Few efficacy differences were evident between FP/FORM 250/10 µg and FORM. Pneumonia was more frequent in FP/FORM-treated patients, although the absolute difference was low. Adverse events were otherwise similar between treatments.

High fat induces acute and chronic inflammation in the hypothalamus: effect of high-fat diet, palmitate and TNF-α on appetite-regulating NPY neurons.

BACKGROUND: Consumption of dietary fat is one of the key factors leading to obesity. High-fat diet (HFD)-induced obesity is characterized by induction of inflammation in the hypothalamus; however, the temporal regulation of proinflammatory markers and their impact on hypothalamic appetite-regulating neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons remains undefined. METHODS: Mice were injected with an acute lipid infusion for 24 h or fed a HFD over 8-20 weeks. Characterized mouse NPY/AgRP hypothalamic cell lines were used for in vitro experimentation. Immunohistochemistry in brain slices or quantitative real-time PCR in cell lines, was performed to determine changes in the expression of key inflammatory markers and neuropeptides. RESULTS: Hypothalamic inflammation, indicated by tumor necrosis factor (TNF)-α expression and astrocytosis in the arcuate nucleus, was evident following acute lipid infusion. HFD for 8 weeks suppressed TNF-α, while significantly increasing heat-shock protein 70 and ciliary neurotrophic factor, both neuroprotective components. HFD for 20 weeks induced TNF-α expression in NPY/AgRP neurons, suggesting a detrimental temporal regulatory mechanism. Using NPY/AgRP hypothalamic cell lines, we found that palmitate provoked a mixed inflammatory response on a panel of inflammatory and endoplasmic reticulum (ER) stress genes, whereas TNF-α significantly upregulated IκBα, nuclear factor (NF)-κB and interleukin-6 mRNA levels. Palmitate and TNF-α exposure predominantly induced NPY mRNA levels. Utilizing an I kappa B kinase ß (IKKß) inhibitor, we demonstrated that these effects potentially occur via the inflammatory IKKß/NF-κB pathway. CONCLUSIONS: These findings indicate that acute lipid and chronic HFD feeding in vivo, as well as acute palmitate and TNF-α exposure in vitro, induce markers of inflammation or ER stress in the hypothalamic appetite-stimulating NPY/AgRP neurons over time, which may contribute to a dramatic alteration in NPY/AgRP content or expression. Acute and chronic HFD feeding in vivo temporally regulates arcuate TNF-α expression with reactive astrocytosis, which suggests a time-dependent neurotrophic or neurotoxic role of lipids.

Estrogen receptor alpha mediates estrogen-inducible abnormalities in the developing penis.

Previously, we reported an association between estrogen receptor-alpha (ERalpha) upregulation and detrimental effects of neonatal diethylstilbestrol (DES) exposure in the rat penis. The objective of this study was to employ the ERalpha knockout (ERalphaKO) mouse model to test the hypothesis that ERalpha mediates DES effects in the developing penis. ERalphaKO and wild-type C57BL/6 mice received oil or DES at a dose of 0.2 microg/pup per day (0.1 mg/kg) on alternate days from postnatal days 2 to 12. Fertility was tested at 80-240 days of age and tissues were examined at 96-255 days of age. DES caused malformation of the os penis, significant reductions in penile length, diameter, and weight, accumulation of fat cells in the corpora cavernosa penis, and significant reductions in weight of the bulbospongiosus and levator ani muscles in wild-type mice. Conversely, ERalphaKO mice treated with DES developed none of the above abnormalities. While nine out of ten male mice sired pups in the wild-type/control group, none did in the wild-type/DES group. ERalphaKO mice, despite normal penile development, are inherently infertile. Both plasma and intratesticular testosterone levels were unaltered in the DES-treated wild-type or DES-treated ERalphaKO mice when compared with controls, although testosterone concentration was much higher in the ERalphaKO mice. Hence, the resistance of ERalphaKO mice to developing penile abnormalities provides unequivocal evidence of an obligatory role for ERalpha in mediating the harmful effects of neonatal DES exposure in the developing penis.

Estrogen-induced abnormal accumulation of fat cells in the rat penis and associated loss of fertility depends upon estrogen exposure during critical period of penile development.

We previously reported that diethylstilbestrol (DES) or estradiol valerate (EV) exposure at a dose of 0.10-0.12 mg/kg, or higher, per day, on alternate days, from postnatal days 2-12, resulted in abnormal penis development and infertility (H. O. Goyal et al., 2005, J. Androl. 26, 32-43). The objective of this study was to identify a critical developmental period(s) during which EV exposure results in the observed penile abnormalities. Male pups received EV at a dose of 0.10-0.12 mg/kg on postnatal day(s) 1, 1-3, 4-6, 1-6, 7-12, 13-18, 19-24, or 25-30. Fertility was tested at 102-115 days of age and tissues were examined at 117-137 days. Both penile morphology and fertility were unaltered in rats treated with EV after 12 days of age. Conversely, except in rats treated on postnatal day 1 only, none of the males treated prior to 12 days of age sired pups, and all had abnormal penises, including varying degrees of abnormal accumulation of fat cells and loss of cavernous spaces and smooth muscle cells in the corpora cavernosa penis, which were maximal in the 1-6-day group. Also, the preputial sheath was partially released or its release was delayed, and the weight of the bulbospongiosus muscle was significantly reduced. Plasma testosterone (T) in the 1-6- and 4-6-day groups and intratesticular T in the 4-6-day group were significantly lower. The testosterone surge, characteristic of controls in the first week of life, was suppressed in the 1-3-day group. Estrogen receptor alpha mRNA expression was enhanced in the body of the penis in the 1-3-day group, but not in the 13-18-day group. Hence, EV exposure prior to 12 days of age (as short as 1-3 days postnatal), but not after 12 days of age, results in long-term abnormal penile morphology, characterized by abnormal accumulation of fat cells in the corpora cavernosa penis and, consequently, loss of fertility.

Exposure of neonatal male rats to estrogen induces abnormal morphology of the penis and loss of fertility.

The research objectives are to determine whether estrogen-induced infertility is associated with abnormal morphology of the penis and if morphological alterations can be reversed by testosterone (T). Male pups received diethylstilbestrol (DES) on alternate days from postnatal days 2 to 12. They received T or empty implants at 180 days, were tested for fertility at 188 days, and terminated at 200 days. While 5/7 control males sired pups, only 1/6 did in the DES group, and 0/8 in the DES plus T group. In addition to reductions in penile length and weight, the novel structural change induced by DES, and not reversed by T, was a replacement of cavernous spaces by fat cells in the penis body. Hence, T substitution for 8 days at adulthood did not reverse infertility in rats treated neonatally with DES and provided evidence that infertility probably resulted from absence of cavernous spaces and/or accumulation of fat cells in the penis body.

Abnormal morphology of the penis in male rats exposed neonatally to diethylstilbestrol is associated with altered profile of estrogen receptor-alpha protein, but not of androgen receptor protein: a developmental and immunocytochemical study.

Objectives of the study were to determine developmental changes in morphology and expression of androgen receptor (AR) and estrogen receptor (ER)alpha in the body of the rat penis exposed neonatally to diethylstilbestrol (DES). Male pups received DES at a dose of 10 microg per rat on alternate days from Postnatal Day 2 to Postnatal Day 12. Controls received olive oil vehicle only. Tissue samples were collected on Days 18 (prepuberty), 41 (puberty), and 120 (adult) of age. DES-induced abnormalities were evident at 18 days of age and included smaller, lighter, and thinner penis, loss of cavernous spaces and associated smooth muscle cells, and increased deposition of fat cells in the corpora cavernosa penis. Fat cells virtually filled the entire area of the corpora cavernosa at puberty and adulthood. Plasma testosterone (T) was reduced to an undetectable level, while LH was unaltered in all treated groups. AR-positive cells were ubiquitous and their profile (incidence and staining intensity) did not differ between control and treated rats of the respective age groups. Conversely, ERalpha-positive cells were limited to the stroma of corpus spongiosus in all age groups of both control and treated rats, but the expression in treated rats at 18 days was up-regulated in stromal cells of corpora cavernosa, coincident with the presence of morphological abnormalities. Hence, this study reports for the first time DES-induced developmental, morphological abnormalities in the body of the penis and suggests that these abnormalities may have resulted from decreased T and/or overexpression of ERalpha.

Molecular addition compounds. 18. Borane adducts with hydroxydialkyl sulfide borates for hydroboration. New, essentially odorless, water-soluble sulfide borane acceptors for hydroboration.

Hydroxydialkyl sulfides of general formula RS(CH2CH2)nH (R = Et, t-Bu, i-Am; n = 1-3) and thiodiethanol monomethyl ether (9) have been synthesized and used as borane carriers. The compounds 3 and 6 (R = Et, n = 2, 3), 7 (R = t-Bu, n = 3), and 9 are completely miscible with water and exhibit only very mild odor. The sulfides were transformed into the corresponding borates by treatment either with boric acid or with diborane. The borates complex 3 mol of borane per 1 mol of borate to give highly reactive, stable, liquid adducts, hydroborating 1-octene in 15 min at room temperature. The adducts derived from water soluble sulfides 3 and 9, selected for the hydroboration of more hindered olefins, reacted readily with (-)-beta-pinene, 1-methylcyclohexene, and 2,3-dimethyl-2-butene. The carrier borates liberated from the adducts during hydroboration are readily hydrolyzed to give 3 and 9, which can be washed off with water from trialkylboranes or oxidation products. Consequently, hydroxydialkyl sulfides 3 and 9 are the first completely water-soluble sulfide borane carriers that can be washed off in the workup of hydroboration products. The adducts derived from 3 and 9 are new, highly promising reagents suitable for large scale hydroborations and reductions.

Comparative assessment of the effect of aflatoxin B1 on hepatic dysfunction in some mammalian and avian species.

1. Aflatoxin B1 (1.5 mg/kg body weight, i.p.) was administered to rats, mice, quail and chickens to examine the comparative effect on hepatic microsomal drug-metabolizing enzymes, cytosolic glutathione S-transferase and serum enzymes. 2. Administration of aflatoxin B1 to rats resulted in a significant decrease in microsomal cytochrome P-450, NADPH-cytochrome c reductase, activities of aminopyrine N-demethylase, aniline hydroxylase, cytosolic glutathione S-transferase and liver glutathione content. However, no significant changes in these parameters were seen in mice. 3. Quail showed a significant decrease in the content of cytochrome P-450 and the activities of aminopyrine N-demethylase, aniline hydroxylase and cytosolic glutathione S-transferase. A similar treatment did not affect these biotransformation enzymes in chickens. 4. The activities of serum enzymes, sorbitol dehydrogenase, alanine aminotransferase and aspartate aminotransferase were increased significantly in rats and quail. Mice exhibited a significant increase in the activities of sorbitol dehydrogenase and aspartate aminotransferase, while chickens showed a significant increase only in alanine aminotransferase.

Lack of in vitro and in vitro effects of fenbendazole on phase I and phase II biotransformation enzymes in rats, mice and chickens.

Intraperitoneal administration of 10 mg fenbendazole/kg bw daily for 5 d caused no significant alterations in the activities of hepatic microsomal drug-metabolizing enzymes viz aminopyrine N-demethylase, aniline hydroxylase and cytosolic glutathione S-transferase in rats, mice and chickens. Similarly no significant difference in the amount of microsomal cytochrome P-450 and NADPH-cytochrome c reductase was found between control and treated animals. In vitro incubation of fenbendazole with rat, mouse and chicken microsomes suggests that the drug neither binds to microsomal protein cytochrome P-450 nor inhibits the activities of aminopyrine N-demethylase and aniline hydroxylase. Similarly in vitro addition of fenbendazole to cytosolic glutathione S-transferase from the above species did not alter the activity of this enzyme. The results indicate that fenbendazole does not alter the activity of hepatic microsomal monooxygenase system significantly in rats, mice and chickens at a dosage level of 10 mg/kg body weight. In vitro studies also indicate that fenbendazole does not interact with the hepatic microsomal monooxygenase system, indicating it is not a substrate for cytochrome P-450-dependent monooxygenase system.

Comparison of the effects of piperine administered intragastrically and intraperitoneally on the liver and liver mixed-function oxidases in rats.

Piperine, a major pungent constituent of black and red peppers, was administered to rats intragastrically and intraperitoneally to study whether it alters the activities of hepatic mixed-function oxidases (MFO) and serum enzymes as specific markers of hepatotoxicity. An intragastric dose of 100 mg/kg of piperine to adult, male Sprague-Dawley rats caused an increase in hepatic microsomal cytochrome P-450 and cytochrome b5, NADPH-cytochrome c reductase, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h following treatment. On the other hand, a 10 mg/kg dose given i.p. exhibited no effect on the activities of the aforementioned parameters of the hepatic drug-metabolizing enzyme system. However, when the intragastric and intraperitoneal doses were increased to 800 mg/kg and 100 mg/kg, respectively, the black pepper alkaloid produced a significant decrease in the levels of cytochrome P-450, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h after treatment. None of the treatments significantly elevated the activities of serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and isocitrate dehydrogenase (ICD), suggesting that piperine is not a hepatotoxic agent.

Differences in the effects of piperine and piperonyl butoxide on hepatic drug-metabolizing enzyme system in rats.

An i.p. administration of rats with piperine (100 mg/kg) and piperonyl butoxide (400 mg/kg) produced a significant decrease in hepatic cytochrome P-450, and activities of benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 1 hr after the treatment. Twenty-four hr later, these parameters along with cytochrome b5 and NADPH-cytochrome c reductase remained depressed only in piperine-treated rats. In contrast, piperonyl butoxide caused a significant induction of these parameters with the exception of cytochrome b5 and aminopyrine N-demethylase, which were up by 36 and 33% over their respective controls but not significantly. These results point up that effect of piperine on hepatic mixed-function oxidases is monophasic while that of piperonyl butoxide is biphasic.