A model for preservation of thymocyte-depleted thymus.

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

A model for preservation of thymocyte-depleted thymus

DiGeorge syndrome is a disorder caused by a microdeletion on the long arm of chromosome 22. Approximately 1% of patients diagnosed with DiGeorge syndrome may have an absence of a functional thymus, which characterizes the complete form of the syndrome. These patients require urgent treatment to reconstitute T cell immunity. Thymus transplantation is a promising investigational procedure for reconstitution of thymic function in infants with congenital athymia. Here, we demonstrate a possible optimization of the preparation of thymus slices for transplantation through prior depletion of thymocytes and leukocyte cell lineages followed by cryopreservation with cryoprotective media (5% dextran FP 40, 5% Me2SO, and 5% FBS) while preserving tissue architecture. Thymus fragments were stored in liquid nitrogen at -196°C for 30 days or one year. The tissue architecture of the fragments was preserved, including the distinction between medullary thymic epithelial cells (TECs), cortical TECs, and Hassall bodies. Moreover, depleted thymus fragments cryopreserved for one year were recolonized by intrathymic injections of 3×106 thymocytes per mL, demonstrating the capability of these fragments to support T cell development. Thus, this technique opens up the possibility of freezing and storing large volumes of thymus tissue for immediate transplantation into patients with DiGeorge syndrome or atypical (Omenn-like) phenotype.

Effects of aerobic exercise on molecular aspects of asthma: involvement of SOCS-JAK-STAT.

BACKGROUND: Aerobic training (AT) decreases airway inflammation in asthma, but the underlying cellular and molecular mechanisms are not completely understood. Thus, this study evaluated the participation of SOCS-JAK-STAT signaling in the effects of AT on airway inflammation, remodeling and hyperresponsiveness in a model of allergic airway inflammation. METHODS: C57Bl/6 mice were divided into Control (Co), Exercise (Ex), HDM (HDM), and HDM+Exercise (HDM+ Ex). Dermatophagoides pteronyssinus (100ug/mouse) were administered oro-tracheally on days 0, 7, 14, 21, 28, 35, 42 and 49. AT was performed in a treadmill during 4 weeks in moderate intensity, from day 24 until day 52. RESULTS: AT inhibited HDM-induced total cells (p<0.001), eosinophils (p<0.01), neutrophils (p<0.01) and lymphocytes (p<0.01) in BAL, and eosinophils (p<0.01), neutrophils (p<0.01) and lymphocytes (p<0.01) in peribronchial space. AT also reduced BAL levels of IL-4 (p<0.001), IL-5 (p<0.001), IL-13 (p<0.001), CXCL1 (p<0.01), IL-17 (p<0.01), IL-23 (p<0.05), IL-33 (p<0.05), while increased IL- 10 (p<0.05). Airway collagen fibers (p<0.01), elastic fibers p<0.01) and mucin (p<0.01) were also reduced by AT. AT also inhibited HDM-induced airway hyperresponsiveness (AHR) to methacholine 6,25mg/ml (p<0.01), 12,5mg/mL (p<0.01), 25mg/mL (p<0.01) and 50mg/mL (p<0.01). Mechanistically, AT reduced the expression of STAT6 (p<0.05), STAT3 (p<0.001), STAT5 (p<0.01) and JAK2 (p<0.001), similarly by peribronchial leukocytes and by airway epithelial cells. SOCS1 expression (p<0.001) was upregulated in leukocytes and in epithelial cells, SOCS2 (p<0.01) was upregulated in leukocytes and SOCS3 down-regulated in leukocytes (p<0.05) and in epithelial cells (p<0.001). CONCLUSIONS: AT reduces asthma phenotype involving SOCSJAK- STAT signaling.

Structural alterations in adult rat carotid bodies exposed to hyperbaric oxygenation.

UNLABELLED: Inhibition of carotid body (CB) function is the main mechanism involved in the attenuation of respiratory drive observed during hyperoxia. However, only a few studies at 5.0 atmospheres absolutes (ATA) have analyzed carotid body structure or function in hyperbaric oxygenation (HBO2) situations. We hypothesized that rats will present CB structural alterations when exposed to different lower hyperbaric oxygen doses enough to alter their chemosensory response to hypoxia. METHODS: Twenty-one adult male Wistar rats, divided into three groups, were maintained in room air or exposed to O2 at 2.4 or 3.0 ATA for six hours. Histological, ultrastructural and immunohistochemical analyses for neuronal nitric oxide synthase (nNOS) and F2-isoprostane were performed in the excised CBs. RESULTS: Histological analyses revealed signs of intracellular edema in animals exposed to both conditions, but this was more marked in the 3.0 ATA group, which showed ultrastructural alterations at the mitochondrial level. There was a significant increase in the volume density of intraglomic-congested capillaries in the 3.0 ATA group associated with an arteriolar vasoconstriction. In the 2.4 ATA group, there was a relative increase of glomic light cells and a decrease of glomic progenitor cells. Additionally, there was a stronger immunoreactivity for F2-isoprostane in the 3.0 ATA O2-exposed carotid bodies. The glomic cells stained positive for nNOS, but no difference was observed between the groups. Our results show that high O2 exposures may induce structural alterations in glomic cells with signs of lipid peroxidation. We further suggest that deviation of blood flow toward intraglomic capillaries occurs in hyperbaric hyperoxia.

Baroreflex deficit blunts exercise training-induced cardiovascular and autonomic adaptations in hypertensive rats.

1. Baroreceptors regulate moment-to-moment blood pressure (BP) variations, but their long-term effect on the cardiovascular system remains unclear. Baroreceptor deficit accompanying hypertension contributes to increased BP variability (BPV) and sympathetic activity, whereas exercise training has been associated with an improvement in these baroreflex-mediated changes. The aim of the present study was to evaluate the autonomic, haemodynamic and cardiac morphofunctional effects of long-term sinoaortic baroreceptor denervation (SAD) in trained and sedentary spontaneously hypertensive rats (SHR). 2. Rats were subjected to SAD or sham surgery and were then further divided into sedentary and trained groups. Exercise training was performed on a treadmill (five times per week, 50-70% maximal running speed). All groups were studied after 10 weeks. 3. Sinoaortic baroreceptor denervation in SHR had no effect on basal heart rate (HR) or BP, but did augment BPV, impairing the cardiac function associated with increased cardiac hypertrophy and collagen deposition. Exercise training reduced BP and HR, re-established baroreflex sensitivity and improved both HR variability and BPV. However, SAD in trained SHR blunted all these improvements. Moreover, the systolic and diastolic hypertensive dysfunction, reduced left ventricular chamber diameter and increased cardiac collagen deposition seen in SHR were improved after the training protocol. These benefits were attenuated in trained SAD SHR. 4. In conclusion, the present study has demonstrated that the arterial baroreflex mediates cardiac disturbances associated with hypertension and is crucial for the beneficial cardiovascular morphofunctional and autonomic adaptations induced by chronic exercise in hypertension.

Early short-term versus prolonged low-dose methylprednisolone therapy in acute lung injury.

The present study compared the effects of early short-term with prolonged low-dose corticosteroid therapy in acute lung injury (ALI). In total, 120 BALB/c mice were randomly divided into five groups. In the control group, saline was intratracheally (i.t.) instilled. In the ALI group, mice received Escherichia coli lipopolysaccharide (10 microg i.t.). ALI animals were further randomised into four subgroups to receive saline (0.1 mL i.v.) or methylprednisolone (2 mg x kg(-1) i.v.) at 6 h, 24 h or daily (for 7 days, beginning at day 1). At 1, 3 and 8 weeks, in vivo and in vitro lung mechanics and histology (light and electron microscopy), collagen and elastic fibre content, cytokines in bronchoalveolar lavage fluid and the expression of matrix metalloproteinase (MMP)-9 and -2 were measured. In vivo (static elastance and viscoelastic pressure) and in vitro (tissue elastance and resistance) lung mechanics, alveolar collapse, cell infiltration, collagen and elastic fibre content and the expression of MMP-9 and MMP-2 were increased in ALI at 1 week. Methylprednisolone led to a complete resolution of lung mechanics, avoided fibroelastogenesis and the increase in the expression of MMP-9 and MMP-2 independent of steroid treatment design. Thus, early short-term, low-dose methylprednisolone is as effective as prolonged therapy in acute lung injury.