A marked paucity of granule cells in the developing cerebellum of the Npc1(-/-) mouse is corrected by a single injection of hydroxypropyl-ß-cyclodextrin.

In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1(-/-) mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%-25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8-PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1(-/-) mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1(-/-) mice. A single injection of hydroxypropyl-ß-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size. To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum.

FXR activation improves myocardial fatty acid metabolism in a rodent model of obesity-driven cardiotoxicity.

BACKGROUND AND AIMS: Obesity-driven lipotoxicity is a risk factors for cardiovascular disease. The Farnesoid X Receptor (FXR) is a bile acids sensor and member of the nuclear receptor superfamily. Activation of FXR lowers plasma triacylglycerols and glucose levels through a mechanism that involves both the repression of key regulatory genes in the liver and the modulation of insulin sensitivity in peripheral tissues. In the present study we have investigated whether administering obese (fa/fa) Zucker rats, a genetic model of obesity associated with dyslipidemia and insulin resistance, with an FXR ligand protects against lipid-induced cardiomyopathy. METHODS AND RESULTS: FXR is expressed in neonatal cardiomyocytes and the treatment with FXR agonists, chenodeoxycholic acid (CDCA), and GW4064, increased the mRNA expression of FXR and its canonical target gene, the small heterodimer partner (SHP), as well as proliferator-activated receptor alpha PPARα, acyl-CoA oxidase (AOX) and pyruvate dehydrogenase kinase (PDK-4). Feeding obese fa/fa rats with CDCA, 12 weeks, reduced hyperinsulinemia and hyperlipidaemia. The histological-pathological analysis of hearts demonstrated that treatment with the FXR ligand reduced lipid heart content decreased the rate of apoptosis, fibrosis scores and restored heart insulin signalling. Chronic CDCA administration, in the heart, induced PPARα and PPARα-regulated genes involved in ß-oxidation. CONCLUSION: FXR agonism exerts beneficial effects in a genetic model of lipid-induced cardiomyopathy. The striking benefit of this therapy on cardiac function in this model warrants an effort to determine whether a counterpart of this activity translates in human settings.

Effect of endothelin inhibition on lung fibroblasts on patients with systemic sclerosis.

AIM: The aim of the study was to investigate the effect of selective ETRA Sitaxsentan on viability and differentiation into myofibroblasts of lung fibroblasts derived from SSc-ILD patients and the ability of this drug to modify the lung fibroblast synthesis of VEGF, type I collagen and fibronectin. METHODS: Primary human lung fibroblast cultures were obtained from BAL of SSc-ILD patients. Cell cultures were exposed for 48 h to crescent concentrations of Sitaxsentan (10 -6M to 10 -4M). In these experimental conditions we evaluated cell viability through crystal violet staining, the production and mRNA expression of VEGF, fibronectin and type I collagen respectively through ELISA and real-Time PCR. Further, we detected alpha-Smooth Muscle Actin (α-SMA) through immunocytochemical assay. RESULTS: The lowest concentration of sitaxsentan (10-6M) did not affect fibroblasts viability; conversely at higher concentrations, sitaxsentan induced a significant inhibition of cell viability. Synthesis and mRNA expression of VEGF, type 1 collagen and fibronectin were significantly reduced in treated lung fibroblasts compared to the untreated ones, in a dose-dependent manner. At higher concentrations, Sitaxsentan reduced the expression of α-SMA. CONCLUSION: The results of this study show that sitaxentan is able in vitro to reduce both cell viability than production of VEGF and extra-cellular matrix components in SSc lung fibroblasts, confirming the anti-fibrotic potential of ETRA in SSc. The decreased expression of α-SMA in treated cells indicate that sitaxsentan may inhibit the fibroblast differentiation toward a myo-fibroblast phenotype and further support the hypothesis that the selective ETRAs may be beneficial in patients with SSc-ILD as anti fibrotic agents.

Influence of body lesion severity on oxidative status and gut microbiota of weaned pigs.

Body lesions in pigs are a common welfare concern, particularly during the weaning period. These lesions can lead to pain, infection, and impaired mobility, resulting in reduced growth performance and increased mortality. Moreover, weaning stress can affect gut microbiota, immune response and increase the oxidative stress of piglets during this transition period. It has been hypothesised that social stress and body lesions could contribute to affect the gut microbiota, physiological and immune response of piglets. The study aims to evaluate the impact of the body lesions due to social stress on microbial profile, immune response, and oxidative status of weaned piglets. Lesion score (LS) on skin, tail, ear, neck, middle trunk, and hind quarters was measured 1 week (28 days of age, T1) and 7 weeks postweaning (T2) on 45 tail-docked pigs according to the method suggested from the Walfer Quality® (2009) on a scale from 0 to 2. Based on the LS, at T1, piglets were classified as High LS (n = 16), when LS was >1 in at least two of the areas considered, or Low LS (n = 29). At T2, based on the same scoring system and to the LS observed at T1, piglets were divided into four groups: High to Low LS (H-L, n = 11), High to High LS (H-H, n = 5), Low to Low LS (L-L, n = 21) and Low to High LS (L-H, n = 8). Blood and faecal samples were collected at T1 and T2. At T1, pigs with a high LS had a lower biological antioxidant potential compared with the L group (P < 0.02). At T2, the L-H group had a lower Reactive Oxygen Metabolites concentration compared with the H-H group (P = 0.03) while the L-L group had a lower concentration of Immunoglobulin A compared with H-H and L-H groups (P = 0.02 and P = 0.04, respectively). At T1, piglets with high LS had a different microbiota compared to piglets with low LS (R2 = 0.04, P < 0.01). Low LS pigs were characterised by a higher abundance of Firmicutes, Blautia, Eubacterium coprostanoligenes, Faecalibacterium, Megasphaera, Subdoligranulum (P.adj < 0.05), while pigs with high LS were characterised by higher abundance of Bacteroidota, Rikenellaceae RC9, Prevotellaceae UCG-003, uncultured-Lachnospiraceae and uncultured-Oscillospiraceae (P.adj < 0.05). At T2, the H-H group were characterised by Oscillospirales-UCG-010, H-L by Agatobachter and L-L by Alloprevotella (P.adj < 0.05). Overall, this study provides valuable insights into the relationship between body lesions, oxidative stress, and gut microbiota in weaned pigs.

Menopausal asthma: a new biological phenotype?

BACKGROUND: Female hormones play an important role in women's lung health, especially in asthma pathophysiology. Although a growing interest has recently been aroused in asthma related to short-term reproductive states, menopausal asthma has been little studied in the past. The aim of the present study was to explore airway inflammation in menopausal asthmatic women in a noninvasive manner. METHODS: Forty consecutive women with menopausal asthma, 35 consecutive women with premenopausal asthma and 30 age-matched healthy controls were enrolled in the study. Urinary LTE-4, induced sputum inflammatory cells, and exhaled LTE-4, IL-6, pH, and NO levels were measured in all the subjects enrolled. RESULTS: Women with menopausal asthma showed decreased estradiol concentrations, high sputum neutrophils, and exhaled IL-6. Women with premenopausal asthma presented instead an essentially eosinophilic inflammatory pattern. Higher urine and breath condensate LTE-4 concentrations were found in premenopausal and menopausal asthma compared to controls. CONCLUSION: Our results substantiate the existence of a new biological phenotype of menopausal asthma that is mainly characterized by neutrophilic airways inflammation and shares several characteristics of the severe asthma phenotype.

Early Prediction of Cardiovascular Disease in Kidney Transplant Recipients.

Cardiovascular disease (CVD) is frequent after kidney transplantation (KT). This study investigated CVD prediction in KT by information available before KT or within 6 months after KT. The study cohort consisted of 629 patients with KT in 2005-10 and with adult age at KT. The end point was incidence up to 2015 of CVD (coronary heart disease, cerebrovascular disease, peripheral artery disease). Graft failure, non-CVD death with functioning graft, and loss to follow-up were considered competing events. CVD prediction was investigated for 34 variables by means of competing-risks regression. Follow-up range was 0.28-10.00 years (mean ± SD, 7.30 ± 3.10). First incident event was CVD in 103 patients and competing events in 146 patients. In the multivariable model for pre-KT variables only, CVD predictors were male sex (hazard ratio [HR], 1.68; 95% confidence interval [CI], 1.06-2.66), diabetic nephropathy (HR, 6.63; 95% CI, 1.81-24.35), pre-KT dialysis for ≥5 years (HR, 1.52; 95% CI, 1.02-2.27), pre-KT CVD (HR, 4.87; 95% CI, 2.84-8.35), and age at KT ≥45 years (HR, 2.98; 95% CI, 1.83-4.87). In the model for pre-KT and post-KT variables together, the sole post-KT CVD predictor was estimated glomerular filtration rate <60 mL/min at the 6-month visit (HR, 1.75; 95% CI, 1.11-2.77). Diabetic nephropathy, pre-KT dialysis, pre-KT CVD, and age at KT predicted 91.2% of incident CVD. Early available information effectively predicted CVD in KT independently from competing events.

Mass spectrometric analysis of tobacco-specific nitrosamine hemoglobin adducts in snuff dippers, smokers, and nonsmokers.

Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and N'-nitrosonornicotine were quantified in blood samples collected from snuff dippers, smokers, and nonsmokers. Mild base treatment of hemoglobin adducted by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N'-nitrosonornicotine releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). HPB was enriched by solvent partitioning and derivatized to its pentafluorobenzoate. After purification by high performance liquid chromatography, HPB-pentafluorobenzoate was analyzed by capillary column gas chromatography with detection by negative ion chemical ionization mass spectrometry and selected ion monitoring. [4,4-D2]HPB was used as internal standard. The detection limit for HPB-pentafluorobenzoate was approximately 100 amol/injection or 5 fmol/g hemoglobin. Mean adduct levels (fmol HPB/g hemoglobin) were 517 +/- 538 (SD) in snuff dippers, 79.6 +/- 189 in smokers, and 29.3 +/- 25.9 in nonsmokers. Adduct levels in snuff dippers and in a subgroup of smokers were higher than would have been predicted solely based on estimates of exposure to tobacco-specific nitrosamines. The results of this study provide the first measurements of tobacco-specific nitrosamine hemoglobin adducts in humans and suggest new approaches to understanding the metabolic activation of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine in humans.

Brain metabolism during increased intracranial pressure as assessed by niroscopy.

Intracranial pressure was increased in cats by infusion of "mock" cerebrospinal fluid into the cisterna magna. This condition was then treated by hyperventilation. In addition to direct measurement of intracranial pressure, cerebral metabolism was assessed by near infrared spectrophotometric (niroscopic) measurement of cytochrome a,a3 redox state and the quantity of reduced and oxygenated hemoglobin in the illuminated brain. Cerebral perfusion was assessed by injection of Cardio-Green. Increased intracranial pressure resulted in the expected reduction in cytochrome a,a3,a decrease in HbO2, an increase in Hb, and a reduction of blood flow. The vasoconstriction produced by hyperventilation, while reducing intracranial pressure, produced a further reduction in cytochrome a,a3 and HbO2, with no improvement in blood flow. The data illustrate the fallacy of governing therapy solely by intracranial pressure and demonstrate the need for a direct assessment of brain metabolism. The data also demonstrate the strengths and weaknesses of niroscopy as a noninvasive monitor of the brain metabolism if applied to patients at risk for increased intracranial pressure.

Generation of effector cytotoxic T cells from cytotoxic T cell precursors: role of soluble factors.

Cytotoxic T lymphocytes (CTL) have long been recognized as playing a major role in the immune response to alloantigens and viral antigens as well as tumor antigens. The progress of the last decade in the identification and characterization of soluble factors involved in the regulation of the immune response has greatly improved our knowledge of the mechanisms of CTL activation and regulation. This review will summarize the data available in the literature regarding different lymphokines and their specific activity on CTL. In addition it will point out a few of the elements of the systems that hamper its full understanding and it will suggest possible directions for future research.

Influenza subunit vaccine coated microneedle patches elicit comparable immune responses to intramuscular injection in guinea pigs.

Delivery of influenza vaccine using innovative approaches such as microneedles has been researched extensively in the past decade. In this study we present concentration followed by formulation and coating of monobulks from 2008/2009 seasonal vaccine on to 3M's solid microstructured transdermal system (sMTS) by a GMP-scalable process. The hemagglutinin (HA) in monobulks was concentrated by tangential flow filtration (TFF) to achieve HA concentrations as high as 20mg/ml. The stability of the coated antigens was evaluated by the functional assay, single radial immunodiffusion (SRID). The data generated show stability of the coated antigen upon storage at 4°C and room temperature in the presence of desiccant for at least 8 weeks. Freeze-thaw stability data indicate the stability of the coated antigen in stressed conditions. The vaccine coated microstructures were evaluated in vivo in a guinea pig model, and resulted in immune titers comparable to the traditional trivalent vaccine administered intramuscularly. The data presented indicate the potential use of the technology in delivery of influenza vaccine. This paper also addresses the key issues of stability of coated antigen, reproducibility and scalability of the processes used in preparation of influenza vaccine coated microneedle patches that are important in developing a successful product.

Serological evidence of human infections with avian influenza viruses. Brief report.

Two hundred ninety-four subjects from Milan were tested for serum hemagglutination-inhibiting (HI) and neuraminidase-inhibiting (NI) antibodies to five avian influenza viruses. No HI antibodies were found in all the serum samples. On the contrary, NI antibodies to each strain were detected depending on the year of birth of the subjects.

Activity of CD4+ T-cell clones of type 1 and type 2 in generation of influenza virus-specific cytotoxic responses in vitro.

The activity of distinct CD4+ T-helper cell (Th) clones in promoting secondary A/PR/8/34/Mt.S.(H1N1) (A/PR8) influenza virus-specific, class I-restricted cytotoxic T-lymphocyte (CTL) responses in vitro was examined. CD8+ T cells which had been purified by fluorescence-activated cell sorter from spleen cells of A/PR8-primed mice were used as responders. On their own, purified CD8+ T cells were unable to generate cytotoxic activity upon in vitro culture with A/PR8-infected stimulator cells. Significant cytotoxic activity was generated in cultures that were additionally supplemented with A/PR8-specific Th clones or cell-free supernatant from these clones. Although there were large differences among individual Th clones in this function, Th clones of type 1 (Th1) promoted, on average, significantly stronger cytotoxic responses than Th clones of type 2 (Th2). The differences in promotion of a cytotoxic response correlated with the amount of interleukin-2 (IL-2) or IL-4 secreted by individual Th clones. These two lymphokines accounted for the CTL-promoting activity of the respective Th clones, since addition of recombinant IL-2 (IL-2) or rIL-4 to Th-free cultures substituted fully for the respective Th clones. As observed with Th clones, rIL-2 was significantly more effective than rIL-4 in promoting a cytotoxic response. When used in combination, Th2 clones had an antagonistic effect on the generation of a CTL response by Th1 clones. This effect could be partially transferred with cell-free supernatant from activated Th2 clones and could be reversed by addition of excess rIL-2. Both consumption of IL-2 by Th2 and secretion of an inhibitory factor(s) appear to be involved in this phenomenon.

Virus-neutralizing antibodies of immunoglobulin G (IgG) but not of IgM or IgA isotypes can cure influenza virus pneumonia in SCID mice.

The ability of monoclonal antibodies (MAbs) to passively cure an influenza virus pneumonia in the absence of endogenous T- and B-cell responses was investigated by treating C.B-17 mice, homozygous for the severe combined immunodeficiency (SCID) mutation, with individual monoclonal antiviral antibodies 1 day after pulmonary infection with influenza virus PR8 [A/PR/8/34 (H1N1)]. Less than 10% of untreated SCID mice survived the infection. By contrast, 100% of infected SCID mice that had been treated with a single intraperitoneal inoculation of at least 175 micrograms of a pool of virus-neutralizing (VN+) antihemagglutinin (anti-HA) MAbs survived, even if antibody treatment was delayed up to 7 days after infection. The use of individual MAbs showed that recovery could be achieved by VN+ anti-HA MAbs of the immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 isotypes but not by VN+ anti-HA MAbs of the IgA and IgM isotypes, even if the latter were used in a chronic treatment protocol to compensate for their shorter half-lives in vivo. Both IgA and IgM, although ineffective therapeutically, protected against infection when given prophylactically, i.e., before exposure to virus. An Fc gamma-specific effector mechanism was not an absolute requirement for antibody-mediated recovery, as F(ab')2 preparations of IgGs could cure the disease, although with lesser efficacy, than intact IgG. An anti-M2 MAb of the IgG1 isotype, which was VN- but bound well to infected cells and inhibited virus growth in vitro, failed to cure. These observations are consistent with the idea that MAbs of the IgG isotype cure the disease by neutralizing all progeny virus until all productively infected host cells have died. VN+ MAbs of the IgA and IgM isotypes may be ineffective therapeutically because they do not have sufficient access to all tissue sites in which virus is produced during influenza virus pneumonia.

Heterosubtypic immunity to influenza type A virus in mice. Effector mechanisms and their longevity.

Immunity that cross-reacts between influenza type A viruses of distinct subtypes is called hetero(sub)typic (Het-I). We have studied Het-I by challenging PR8-immune mice with the heterosubtypic virus X31. Het-I did not prevent infection by X31 but, at its height, strongly aided in recovery. The nature of the effector mechanisms involved was investigated by simultaneous challenge with X31 and an immunologically unrelated influenza type B virus and by depleting individual lymphocyte subsets in PR8-immune mice before challenge. The study showed the following: 1) The effector mechanisms were intimately associated with immune recognition events. 2) In the nose, depletion of CD8+ or CD4+ T cells led to partial reduction of Het-I, and simultaneous depletion of both T cell subsets abrogated Het-I almost completely. This T cell-mediated immunity was short lived and had disappeared 4 to 5 mo after induction. 3) In trachea and lung, depletion of CD8+ T cells led to a partial reduction of Het-I, whereas depletion of CD4+ T cells was without significant effect. The CD8-mediated component appeared short lived, whereas the residual immunity (in CD4/8-depleted mice) was long lived and persisted past 7 mos after induction. 4) Depletion of NK cells did not significantly reduce the strength of Het-I in either nose or lung. In conclusion, the study shows that Het-I in this system is mediated by a complex combination of immune mechanisms that differ, in part, between upper and lower respiratory tract.

Effect of intravenous epinephrine on cerebral cortical blood flow, oxygen tension, and high-energy phosphate metabolism after hypoxic hypotension.

An experimental model was examined in which rats were subjected to 30 minutes of hemorrhagic hypotension and hypoxia. In addition to resuscitation by infusion of blood and saline and increasing the fraction of inspired oxygen (FIO2), the possible beneficial effects of including intravenous epinephrine as part of the resuscitative regimen were studied. Mean arterial pressure, cortical microflow, and brain tissue oxygen tension were increased after epinephrine (P less than 0.05). Cortical ATP concentration was decreased after hypoxic hypotension and failed to improve after epinephrine.

ATP-MgCl2 treatment prior to hypoxic-hypotension.

We have previously implicated uncoupling of oxidative phosphorylation and the associated decreased concentrations of tissue adenosine triphosphate (ATP) in the liver and brain as major factors contributing to death after hypoxia and hypotension. To determine if intravenously administered ATP-MgCl2 complex would result in increased liver and/or brain concentration of ATP, rats were pretreated with intravenous ATP-MgCl2 prior to exposing them to hypoxic-hypotensive stress. In this preliminary work, pretreatment was selected based on the premise that such an experimental design was most apt to demonstrate an effect if such existed. Baseline cerebral cortical and liver ATP and lactate concentrations were obtained immediately after the intravenous infusion of either saline (control group) or ATP-MgCl2 (treated group). ATP and lactate concentrations were again determined after 30 min hypoxic-hypotension and 20 min after resuscitation. ATP-MgCl2 pretreatment resulted in a modest increase in hepatic ATP concentration when measured after 30 min of hypoxic-hypotension. This increase was not evident 20 min later. No increases in cerebral ATP concentrations were noted at any sample time after ATP-MgCl2 pretreatment.

Failure of autoregulation after closed head injury: an experimental model.

Cats were subjected to a 3.5-atm fluid percussion impact administered to the cerebral cortex. Near-infrared spectrophotometry (NIRS) was used to measure the quantity of oxyhemoglobin and total hemoglobin in the illuminated tissue as well as the cytochrome a, a3 redox state. Corroborative data were obtained by freezing brains with liquid nitrogen and measuring cortical concentrations of ATP, creatine phosphate (CP), and lactate. Immediately postimpact there was a rise in mean arterial pressure with a 38% increase of highly oxygenated blood and a shift toward oxidation in the cytochrome a, a3 redox state. By 4 hours postimpact, cytochrome a, a3 was becoming progressively reduced despite the persistence of hyperemia. This was associated with a significant (p less than 0.01) decrease in ATP and CP concentration. Additional studies in which a 0.5-sec, 100-v electrical seizure was induced before and after fluid percussion demonstrated significant differences in seizure response, indicating a failure of autoregulation.

Formation of cyclic nucleic acid adducts from some simple alpha, beta-unsaturated carbonyl compounds and cyclic nitrosamines.

To determine the structures of the DNA adducts of two cyclic nitrosamines, N-nitrosopyrrolidine and N-nitrosomorpholine, the model compounds, alpha-acetoxy-N-nitrosopyrrolidine, 4-(carbethoxynitrosamino)butanal and 2-(carbethoxynitrosamino)ethoxyacetaldehyde, were allowed to react with deoxyguanosine in the presence of porcine liver esterase or base. These model compounds are stable precursors of intermediates formed upon metabolic alpha-hydroxylation of N-nitrosopyrrolidine and N-nitrosomorpholine. The major adducts formed in these reactions were isolated and characterized, on the basis of ultraviolet absorption, mass spectrometry, proton nuclear magnetic resonance and chromatographic properties, as structurally unique 1,N2-cyclic deoxyguanosine adducts. Reaction of crotonaldehyde and glyoxal with deoxyguanosine at 37 degrees C, pH 7, also led to the formation of 1,N2-cyclic deoxyguanosine adducts identical to those formed from the model compounds for alpha-hydroxylation of N-nitrosopyrrolidine and N-nitrosomorpholine, respectively. The 1,N2-cyclic deoxyguanosine adducts were also formed in DNA, upon incubation with N-nitrosopyrrolidine and rat liver microsomes. Acrolein, the simplest alpha, beta-unsaturated carbonyl compound, reacted readily with deoxyguanosine to form three major adducts. These adducts were characterized as cyclic 1,N2-propanodeoxyguanosine adducts resulting from Michael addition of acrolein to the 1- and N2-positions of deoxyguanosine followed by ring closure. One of these adducts, as well as the corresponding crotonaldehyde adduct, was formed in calf thymus DNA upon reaction with acrolein or crotonaldehyde under physiological conditions. The level of modification of DNA by acrolein was considerably higher than that by either crotonaldehyde or N-nitrosopyrrolidine.