Seed dormancy and germination responses of nine Australian fire ephemerals

Fire ephemerals are short-lived plants with seeds that persist in the soil and germinate after a fire or physical soil disturbance. Ex situ germination of many Australian fire ephemerals has previously been difficult. Dormancy was present in most of the nine fire ephemerals examined. Alyogyne hakeifolia (Giord.) Alef. and Alyogyne huegelii (Endl.) Fryxell (Malvaceae) seeds had physical and possibly also physiological dormancy, Actinotus leucocephalus Benth. (Apiaceae) seeds had morphophysiological dormancy, Austrostipa compressa (R.Br.) S.W.L. Jacobs & J. Everett and Austrostipa macalpinei (Reader) S.W.L. Jacobs & J. Everett (Poaceae) seeds were either non-dormant or possessed physiological dormancy, and seeds of all remaining species possessed physiological dormancy. A proportion of the Alyogyne hakeifolia, Alyogyne huegelii, Austrostipa compressa and Austrostipa macalpinei seed populations were non-dormant because some seeds could germinate at the various incubation temperatures without further treatment. At 20 degrees C, artificial methods of inducing germination such as manual or acid scarification were among the optimal treatments for Austrostipa compressa, Austrostipa macalpinei, Alyogyne huegelii, Actinotus leucocephalus and Grevillea scapigera A.S. George (Proteaceae), and gibberellic acid induced maximum germination of Tersonia cyathiflora (Fenzl) J.W. Green (Gyrostemonaceae) seeds. Heat (70 degrees C for 1 h) and smoke water was one of the most effective treatments for germinating Actinotus leucocephalus and Codonocarpus cotinifolius (Desf.) F. Muell. (Gyrostemonaceae) seeds. Germination of Grevillea scapigera, Codonocarpus cotinifolius, Gyrostemon racemiger H. Walter (Gyrostemonaceae) and Tersonia cyathiflora did not exceed 40% and may require other treatments to overcome dormancy. Although the nine fire ephemerals examined require fire to germinate under natural conditions, a range of germination responses and dormancy types was observed.

Overcoming original antigenic sin to generate effective immune responses in antigen primed host

Original antigenic sin is failure to mount effective immunity to virus variants in a previously virus infected host. We have previously shown that prior immunity to a virus capsid protein inhibits induction from naive CD8 T cells of an IFN-g response to a MHC class I restricted epitope linked to the capsid protein, following immunisation with a capsid expressing the class I restricted epitope. The inhibition is independent of pre-existing antibody to the viral capsid, and the inhibition is observed in animal lacking B cells. CD8 restricted viral capsid specific T cell responses are also not required, but the inhibition is not observed in IL10 knockout mice. We now demonstrate that capsid antigen primed CD4+ T cells secrete IL10 in response to capsid antigen presented by DC, and deviate CD8 cells specific for the linked MHC Class I restricted epitope from IFN-g production to IL-5 production. Neutralizing IL10, either in vitro or in vivo, restores induction following immunisation of an antigen specific IFN-g response to an MHC Class I restricted epitope. This finding demonstrates a strategy for overcoming bias towards a Tc2 response to MHC Class I epitopes upon immunisation of a host already primed to antigen, facilitating immunotherapy for chronic viral infection or cancer

Prolonged recruitment manoeuvre improves lung function with less ultrastructural damage in experimental mild acute lung injury

The effects of prolonged recruitment manoeuvre (PRM) were compared with sustained inflation (SI) in paraquat-induced mild acute lung injury (ALI) in rats. Twenty-four hours after ALI induction, rats were anesthetized and mechanically ventilated with VT = 6 ml/kg and positive end-expiratory pressure (PEEP) = 5 cmH(2)O for 1 h. SI was performed with an instantaneous pressure increase of 40 cmH(2)O that was sustained for 40 s, while PRM was done by a step-wise increase in positive inspiratory pressure (PIP) of 15-20-25 cmH(2)O above a PEEP of 15 cm H(2)O (maximal PIP = 40 cmH(2)O), with interposed periods of PIP = 10 cmH(2)O above a PEEP = 15 cmH(2)O. Lung static elastance and the amount of alveolar collapse were more reduced with PRM than SI, yielding improved oxygenation. Additionally, tumour necrosis factor-alpha, interleukin-6, interferon-gamma, and type III procollagen mRNA expressions in lung tissue and lung epithelial cell apoptosis decreased more in PRM. In conclusion, PRM improved lung function, with less damage to alveolar epithelium, resulting in reduced pulmonary injury. (C) 2009 Elsevier BLV. All rights reserved.

Characterization of inflammatory responses to eccentric exercise in humans

Eccentric exercise commonly results in muscle damage. The primary sequence of events leading to exercise-induced muscle damage is believed to involve initial mechanical disruption of sarcomeres, followed by impaired excitation-contraction coupling and calcium signaling, and finally, activation of calcium-sensitive degradation pathways. Muscle damage is characterized by ultrastructural changes to muscle architecture, increased muscle proteins and enzymes in the bloodstream, loss of muscular strength and range of motion and muscle soreness. The inflammatory response to exercise-induced muscle damage is characterized by leukocyte infiltration and production of pro-inflammatory cytokines within damaged muscle tissue, systemic release of leukocytes and cytokines, in addition to alterations in leukocyte receptor expression and functional activity. Current evidence suggests that inflammatory responses to muscle damage are dependent on the type of eccentric exercise, previous eccentric loading (repeated bouts), age and gender. Circulating neutrophil counts and systemic cytokine responses are greater after eccentric exercise using a large muscle mass (e.g. downhill running, eccentric cycling) than after other types of eccentric exercise involving a smaller muscle mass. After an initial bout of eccentric exercise, circulating leukocyte counts and cell surface receptor expression are attenuated. Leukocyte and cytokine responses to eccentric exercise are impaired in elderly individuals, while cellular infiltration into skeletal muscle is greater in human females than males after eccentric exercise. Whether alterations in intracellular calcium homeostasis influence inflammatory responses to muscle damage is uncertain. Furthermore, the effects of antioxidant supplements are variable, and the limited data available indicates that anti-inflammatory drugs largely have no influence on inflammatory responses to eccentric exercise. In this review, we compare local versus systemic inflammatory responses, and discuss some of the possible mechanisms regulating the inflammatory responses to exercise-induced muscle damage in humans.

Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

Phosphorus overload and PTH induce aortic expression of Runx2 in experimental uraemia

Background. Vascular calcification (VC) is commonly seen in patients with chronic kidney disease (CKD). Elevated levels of phosphate and parathormone (PTH) are considered nontraditional risk factors for VC. It has been shown that, in vitro, phosphate transforms vascular smooth muscle cells (VSMCs) into calcifying cells, evidenced by upregulated expression of runt-related transcription factor 2 (Runx2), whereas PTH is protective against VC. In addition, Runx2 has been detected in calcified arteries of CKD patients. However, the in vivo effect of phosphate and PTH on Runx2 expression remains unknown. Methods. Wistar rats were submitted to parathyroidectomy, 5/6 nephrectomy (Nx) and continuous infusion of 1-34 rat PTH (at physiological or supraphysiological rates) or were sham-operated. Diets varied only in phosphate content, which was low (0.2%) or high (1.2%). Biochemical, histological, immunohistochemistry and immunofluorescence analyses were performed. Results. Nephrectomized animals receiving high-PTH infusion presented VC, regardless of the phosphate intake level. However, phosphate overload and normal PTH infusion induced phenotypic changes in VSMCs, as evidenced by upregulated aortic expression of Runx2. High-PTH infusion promoted histological changes in the expression of osteoprotegerin and type I collagen in calcified arteries. Conclusions. Phosphate, by itself is a potential pathogenic factor for VC. It is of note that phosphate overload, even without VC, was associated with overexpression of Runx2 in VSMCs. The mineral imbalance often seen in patients with CKD should be corrected.

Executive "brake failure" following deactivation of human frontal lobe

In the course of daily living, humans frequently encounter situations in which a motor activity, once initiated, becomes unnecessary or inappropriate. Under such circumstances, the ability to inhibit motor responses can be of vital importance. Although the nature of response inhibition has been studied in psychology for several decades, its neural basis remains unclear. Using transcranial magnetic stimulation, we found that temporary deactivation of the pars opercularis in the right inferior frontal gyrus selectively impairs the ability to stop an initiated action. Critically, deactivation of the same region did not affect the ability to execute responses, nor did it influence physiological arousal. These findings confirm and extend recent reports that the inferior frontal gyrus is vital for mediating response inhibition.

In situ immune responses to interstitial pneumonitis in human visceral leishmaniasis

Lung disease during active human visceral leishmaniasis is frequently reported. As such, studies have associated pulmonary symptoms to interstitial pneumonitis with a mononuclear infiltrate. However, the immune response in this condition has never been described before. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of lung involvement (IPL) in human visceral leishmaniasis. Quantitative methods of analysis were performed using immunohistochemistry, and were compared with a control group of normal lung. Interstitial macrophages and cd8 cells were increased in IPL, and IL-4 as well as TNF-alpha displayed increased expression when compared to the control group. This inflammatory process with a Th2 pattern, as suggested by increased IL-4 and low IFN-gamma expression, is consistent with the immune response in other organs of visceral leishmaniasis. The microenvironment of the immune response in this condition is associated with lung disease in patients with interstitial pneumonitis related to visceral leishmaniasis, increasing the chance of bacterial infection.

Bile duct ligation in neonatal rats: Is it a valid experimental model for biliary atresia studies?

BA is the most important disease requiring liver transplantation in children. Common BDL in rats is a classic experimental model to study biliary obstruction. The response of the neonatal animal to BDL has yet to be completely understood and few reports have focused on the behavioral differences of the liver between neonatal and adult animals. Ninety newborn Wistar rats aged six days, weighing 8.0-13.9 g, and 90 adult Wistar rats weighing 199.7-357.0 g, were submitted to BDL. After surgery, they were randomly divided and killed on the 3rd, 5th, 7th, 14th, 21st and 28th day post-BDL. Hepatic biopsies were obtained and the following were measured: (i) semiquantification of the bile ductule proliferation and inflammatory infiltrate by HE stain, (ii) quanti. cation of portal and periportal fibrosis with the Sirius-red stain. Although the initial response of ductule proliferation and inflammatory infiltrate were less intense in the newborn animal, the portal and periportal fibrosis were higher when compared with adult animals (p < 0.0491). These findings may contribute to the understanding of the pathophysiology of BA.

Less inhibited with age? Larval age modifies responses to natural settlement inhibitors

As larvae of marine invertebrates age, their response to settlement cues can change. This change can have significant consequences to both the ecology of these organisms, and to their response to antifouling coatings. This study examines how larval age affects the settlement response of larvae to two naturally derived settlement inhibitors, non-polar extracts from the algae Delisea pulchra and Dilophus marginatus, the former of which contains compounds that are in commercial development as antifoulants. Two species of marine invertebrates with non-feeding larvae were investigated: the bryozoans Watersipora subtorquata and Bugula neritina. Larval age strongly affected larval settlement, with older larvae settling at much higher rates than younger larvae. Despite having strong, inhibitory effects on young larvae, the non-polar extracts did not inhibit the settlement of older larvae to the same degree for both species studied. The results show that the effects of ecologically realistic settlement inhibitors are highly dependent on larval age. Given that the age of settling larvae is likely to be variable in the field, such age specific variation in settlement response of larvae may have important consequences for host-epibiont interactions in natural communities.

In vivo biological performance of a novel highly bioactive glass-ceramic (Biosilicate (R)): A biomechanical and histomorphometric study in rat tibial defects

This study aimed to investigate bone responses to a novel bioactive fully crystallized glass-ceramic of the quaternary system P(2)O(5)-Na(2)O-CaO-SiO(2) (Biosilicates (R)). Although a previous study demonstrated positive effects of Biosilicate (R) on in vitro bone-like matrix formation, its in vivo effect was not studied yet. Male Wistar rats (n = 40) with tibial defects were used. Four experimental groups were designed to compare this novel biomaterial with a gold standard bioactive material (Bioglass (R) 45S5), unfilled defects and intact controls. A three-point bending test was performed 20 days after the surgical procedure, as well as the histomorphometric analysis in two regions of interest: cortical bone and medullary canal where the particulate biomaterial was implanted. The biomechanical test revealed a significant increase in the maximum load at failure and stiffness in the Biosilicate group (R) (vs. control defects), whose values were similar to uninjured bones. There were no differences in the cortical bone parameters in groups with bone defects, but a great deal of woven bone was present surrounding Biosilicate (R) and Bioglass (R) 45S5 particulate. Although both bioactive materials supported significant higher bone formation; Biosilicate (R) was superior to Bioglass (R) 45S5 in some histomorphometric parameters (bone volume and number of osteoblasts). Regarding bone resorption, Biosilicate (R) group showed significant higher number of osteoclasts per unit of tissue area than defect and intact controls, despite of the non-significant difference in the osteoclastic surface as percentage of bone surface. This study reveals that the fully crystallized Biosilicate (R) has good bone-forming and bone-bonding properties. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 978: 139-147, 2011.

Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells

The main aim of this study is to evaluate the capacity of human dental pulp stem cells (hDPSC), isolated from deciduous teeth, to reconstruct large-sized cranial bone defects in nonimmunosuppressed (NIS) rats. To our knowledge, these cells were not used before in similar experiments. We performed two symmetric full-thickness cranial defects (5 x 8 mm) on each parietal region of eight NIS rats. In six of them, the left side was supplied with collagen membrane only and the right side (RS) with collagen membrane and hDPSC. In two rats, the RS had collagen membrane only and nothing was added at the left side (controls). Cells were used after in vitro characterization as mesenchymal cells. Animals were euthanized at 7, 20, 30, 60, and 120 days postoperatively and cranial tissue samples were taken from the defects for histologic analysis. Analysis of the presence of human cells in the new bone was confirmed by molecular analysis. The hDPSC lineage was positive for the four mesenchymal cell markers tested and showed osteogenic, adipogenic, and myogenic in vitro differentiation. We observed bone formation 1 month after surgery in both sides, but a more mature bone was present in the RS. Human DNA was polymerase chain reaction-amplified only at the RS, indicating that this new bone had human cells. The us e of hDPSC in NIS rats did not cause any graft. rejection. Our findings suggest that hDPSC is an additional cell resource for correcting large cranial defects in rats and constitutes a promising model for reconstruction of human large cranial defects in craniofacial surgery.

Effectiveness of Thalidomide and Tamoxifen in Preventing Neointimal Hyperplasia in Experimental Vascular Injury in Rats

Introduction. Chronic allograft vasculopathy is an important cause of graft loss. Considering the inflammatory response in the development of chronic vascular lesions, therapeutic approaches to target the inflammatory process may be useful. We sought to investigate the possible protective effects on balloon catheter-induced vascular injury of thalidomide and tamoxifen, 2 drugs with powerful anti-inflammatory, immunomodulatory, and antifibrotic effects, using an animal model that mimics the morphologic features of chronic allograft vasculopathy. Methods. Male Wistar rats subjected to balloon catheter carotid injury (INJ) were treated with thalidomide (100 mg/kg), or tamoxifen (10 mg/kg), or vehicle. Contralateral right carotid arteries were used as uninjured controls. Morphometric and immunohistochemical analyses were performed at 14 days postinjury. Results. Injured carotid arteries showed marked neointimal hyperplasia, which was significantly inhibited among animals treated with thalidomide or tamoxifen: neointimal/media ratios of 1.4 +/- 0.4 versus 0.2 +/- 0.1 versus 0.4 +/- 0.2, for INJ, INJ + Thalid, and INJ + Tamox; respectively (P < .001). The endothelial cell loss was significantly less pronounced among animals subjected to carotid balloon injury that were treated with thalidomide (24 +/- 14 vs 1 +/- 1 cells per section in INJ, respectively (P < .05). Therapy with either thalidomide or tamoxifen effectively maintained alpha-smooth muscle actin expression in the media, similar to uninjured arteries. In this setting, tamoxifen was additionally effective to prevent the migration of myofibroblasts in to the intima. Conclusion. Thalidomide and tamoxifen were effective to reduce neointimal hyperplasia secondary to vascular damage. The vasculoprotective effects of thalidomide were more pronounced to preserve endothelial cells, whereas tamoxifen inhibited smooth muscle cell migration and proliferation. A possible beneficial effect of combined therapy with thalidomide plus tamoxifen should be addressed in future studies.

Impact of Stent-Graft Oversizing on the Thoracic Aorta: Experimental Study in a Porcine Model

Purpose: To analyze in an experimental animal model the effect of 4 different levels of stents-graft oversizing on non-atherosclerotic aortas such as those found in young individuals who undergo stent-graft repair for traumatic aortic injuries. Methods: The diameter of the porcine thoracic aorta is similar to the aorta of young adults (18-20 mm), so 25 pigs were randomized into 5 groups: 1 control (without stent-graft) and 4 oversizing groups (A: 10%-19%, B: 20%-29%, C: 30%-39%, and D: >40%). Two types of biomechanical tests were performed on all aortas 4 weeks after endoprosthesis deployment. Results: The results of the detachment test, which analyzed the strength necessary to remove the stent-graft from the aorta, were similar in the 4 groups (A: 42 N, B: 41 N, C: 46 N, and D: 46 N). However, 2 aortas ruptured during the tests (groups C and D). The second test was performed in 3 aortic segments. Maximum shear strength, maximum stress, and maximum tension supported by the aortic wall had a negative and linear correlation with oversizing. There were significant differences in all 4 groups when compared with the control group. Strain, which reflects the elastic properties of the aortic wall, was very similar in all 4 groups, but a great difference was found when compared with the control group (p<0.0001). Conclusion: The study showed an important subacute change in the biomechanical properties of the aortic wall after implantation of an oversized endoprosthesis. This weakness of the aortic wall was confirmed by 2 ruptures during the detachment test. These results partially explain the interaction of stent-grafts with non-atherosclerotic thoracic aortas and may serve as a basis for further studies and the development of specific material to be used in vascular trauma and young patients. J Endovasc Ther. 2011; 18: 576-584