Demonstration of inhibition of mediator release from human mast cells by azatadine base. In vivo and in vitro evaluation

In vitro experimentation using dispersed human lung mast cells demonstrated that azatadine base, a compound with known H1-antihistamine properties, inhibited anti-IgE-induced release of histamine and leukotriene C4 by 45% and 85%, respectively. To assess the clinical relevance of these findings and to compare in vitro mast cell data with results obtained in vivo, nasally instilled azatadine was tested in a double-blind, placebo-controlled clinical trial in which nasal challenges with antigen were performed on eight allergic individuals. Pretreatment with azatadine significantly suppressed the number of sneezes following antigen challenge and inhibited the associated elevations in histamine, kinins, and enzyme(s) hydrolyzing the artificial substrate N-alpha-tosyl-L-arginine-methyl-ester in nasal secretions, whereas placebo was inactive. Hence, we showed agreement between our in vitro and in vivo experimental models of the allergic reaction. Topical application of azatadine base has the potential to become an effective antiallergic treatment.     

Renal aquaporin-4 associated pathology in TG-26 mice

Human immunodeficiency virus-associated nephropathy (HIVAN) is a leading cause of end-stage renal disease in HIV patients, which is characterized by glomerulosclerosis and renal tubular dysfunction. Aquaporin-4 (AQP-4) is a membrane bound water channel protein that plays a distinct role in water reabsorption from renal tubular fluid. It has been proven that failure of AQP-4 insertion into the renal tubular membrane leads to renal dysfunction. However, the role of AQP-4 in HIVAN is unclear. We hypothesize that impaired water reabsorption leads to renal injury in HIVAN, where AQP-4 plays a crucial role. Renal function is assessed by urinary protein and serum blood urea nitrogen (BUN). Kidneys from HIV Transgenic (TG26) mice (HIVAN animal model) were compared to wild type mice by immunostaining, immunoblotting and quantitative RT-PCR. TG26 mice had increased proteinuria and BUN. We found decreased AQP-4 levels in the renal medulla, increased endothelin-1, endothelin receptor A and reduced Sirtuin1 (SIRT-1) levels in TG26 mice. Also, oxidative and endoplasmic reticulum stress was enhanced in kidneys of TG26 mice. We provide the first evidence that AQP-4 is inhibited due to induction of HIV associated stress in the kidneys of TG26 mice which limits water reabsorption in the kidney which may be one of the cause associated with HIVAN, impairing kidney physiology. AQP-4 dysregulation in TG26 mice suggests that similar changes may occur in HIVAN patients. This work may identify new therapeutic targets to be evaluated in HIVAN.     

Membrane phospholipid abnormalities of basal ganglia in never-treated schizophrenia: a 31P magnetic resonance spectroscopy study.

BACKGROUND: Studies in schizophrenia using in vivo (31)P magnetic resonance spectroscopy (MRS) have mostly focused on the association cortices, including the frontal and temporal lobes. Striatum has also been implicated in schizophrenia, although neuroleptic exposure in the patients is a potential confound limiting interpretation of earlier studies. We examined membrane phospholipid abnormality in the basal ganglia using (31)P MRS in neuroleptic-naive schizophrenia patients. METHODS: Never-treated, DSM-IV schizophrenia patients (n=20) and age- and gender-matched healthy control subjects (n=30) underwent in vivo 1-D 31P MRS of both basal ganglia using an image-selected technique on a 1.5-T magnetic resonance imaging scanner. A neuroradiologist blind to clinical data measured the phosphomonoester (PME) and phosphodiester (PDE) from the spectra. RESULTS: The schizophrenia patients showed significantly and bilaterally elevated levels of PME/PDE ratios in basal ganglia as compared with control subjects. There were no significant differences in the ratios between the two sides in either patient or control groups. Phosphomonoester/phosphodiester ratio did not correlate with illness duration. Lower Positive and Negative Syndrome Scale scores were associated with lower PME/PDE ratio. CONCLUSIONS: The basal ganglia of never-treated schizophrenia patients show features suggestive of reduced breakdown and/or increased synthesis of membrane phospholipids. Lack of correlation between illness duration and the membrane phosphorus moiety ratio may be consistent with a nonprogressive, possibly neurodevelopmental etiopathogenesis.     

A novel in vitro ELISA for estimation of glycoprotein content in human rabies vaccines

In vitro methods for quantification of immunodominant glycoprotein in the rabies vaccine formulations serve as good alternative to the cumbersome and variable mice potency assay as a batch release test for the vaccine. The present study presents the development of a sandwich ELISA with optimal concentrations of a high affinity recombinant diabody (D06) and a specific monoclonal antibody (M5B4) against rabies glycoprotein for its quantification in the vaccine formulations. The glycoprotein estimate correlated linearly (r² = 0.8) to the in vivo potency estimate for the vaccine formulations. This ELISA promises a good forecast of the mice potency values and thereby can serve as a simple, yet effective batch release test for the rabies vaccines replacing the in vivo assay.     

Metabolic signatures imaged in cancer-induced cachexia

Cancer-induced cachexia is a complex and poorly understood life-threatening syndrome that is characterized by progressive weight loss due to metabolic alterations, depletion of lipid stores, and severe loss of skeletal muscle protein. Gaining the ability to noninvasively image the presence or onset of cachexia is important to better treat this condition, to improve the design and optimization of therapeutic strategies, and to detect the responses to such treatments. In this study, we employed noninvasive magnetic resonance spectroscopic imaging (MRSI) and [(18)F]fluoro-2-deoxy-D-glucose ((18)FDG) positron emission tomography (PET) to identify metabolic signatures typical of cachectic tumors, using this information to analyze the types and extents of metabolic changes induced by the onset of cachexia in normal tissues. Cachexia was confirmed by weight loss as well as analyses of muscle tissue and serum. In vivo, cachexia-inducing murine adenocarcinoma (MAC)16 tumors were characterized by higher total choline (tCho) and higher (18)FDG uptake than histologically similar noncachectic MAC13 tumors. A profound depletion of the lipid signal was observed in normal tissue of MAC16 tumor-bearing mice but not within the tumor tissue itself. High-resolution (1)H magnetic resonance spectroscopy (MRS) confirmed the high tCho level observed in cachectic tumors that occurred because of an increase of free choline and phosphocholine. Higher succinate and lower creatine levels were also detected in cachectic tumors. Taken together, these findings enhance our understanding of the effect of cancer on host organs and tissues as well as promote the development of noninvasive biomarkers for the presence of cachexia and identification of new therapeutic targets.     

Efficacy of preoxygenation using tidal volume and deep breathing techniques with and without prior maximal exhalation

PURPOSE: We evaluated the influence of prior maximal exhalation on preoxygenation in 15 adult volunteers using tidal volume breathing (TVB) for five minutes and deep breathing (DB) for two minutes with and without prior maximal exhalation. METHODS: Inspired and end-tidal oxygen, nitrogen and carbon dioxide were monitored continuously and recorded during room air breathing and at 30-sec intervals during 100% oxygen TVB or DB (rate of 8 breaths.min(-1)). RESULTS: Tidal volume breathing with prior maximal exhalation resulted in an end-tidal oxygen concentration (ETO(2)) slightly higher (P = 0.028) at 0.5 and 1.0 min as compared with TVB without prior maximal exhalation at the same time periods. Regardless of whether TVB was preceded by maximal exhalation or not, 2.5 min was required to reach a mean ETO(2) value of 90% or higher. With DB, there were no differences in ETO(2) values at any time period and 1.5 min was required to reach an ETO(2) of 90% or greater, with or without prior maximal exhalation. CONCLUSIONS: Maximal exhalation prior to TVB slightly steepens the initial rise in ETO(2) during the first minute, but confers no real benefit if maximal preoxygenation is the goal. Maximal exhalation prior to DB has no added value in enhancing preoxygenation.     

Chemical modification of SWNT alters in vitro cell-SWNT interactions

Single-walled carbon nanotubes (SWNT) have been the focus of considerable attention as a material with extraordinary mechanical and electrical properties. SWNT have been proposed in a number of biomedical applications, including neural, bone, and dental tissue engineering. In these applications, it is clear that surrounding tissues will come into surface contact with SWNT composites, and compatibility between SWNT and host cells must be addressed. This investigation describes the gross physical and chemical effects of different SWNT preparations on in vitro cell viability and metabolic activity. Three different SWNT preparations were analyzed: as purchased (AP-NT), purified (PUR-NT), and functionalized with glucosamine (GA-NT), over concentrations of 0.001-1.0% (wt/vol). With the exception of the lowest SWNT concentrations, increasing concentrations of SWNT resulted in a decrease of cell viability, which was dependent on SWNT preparation. The metabolic activity of 3T3 cells was also dependent on SWNT preparation and concentration. These investigations have shown that these SWNT preparations have significant effects on in vitro cellular function that cannot be attributed to one factor alone, but are more likely the result of several unfavorable interactions. Effects, such as destabilizing the cell membrane, soluble toxic contaminants, and limitations in mass transfer as the SWNT coalesce into sheets, may all play a role in these interactions. Using comprehensive purification processes and modifying the NT-surface chemistry to introduce functional groups or reduce hydrophobicity or both, these interactions can be significantly improved.