Concentrations of and application protocols for hydrogen peroxide bleaching gels: Effects on pulp cell viability and whitening efficacy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of Delivery Method on Neuroprotection by Bone Marrow Mononuclear Cell Therapy following Ventral Root Reimplantation with Fibrin Sealant

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Synaptic plasticity and sensory-motor improvennent following fibrin sealant dorsal root reimplantation and mononuclear cell therapy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mesenchymal stem cells engrafted in a fibrin scaffold stimulate Schwann cell reactivity and axonal regeneration following sciatic nerve tubulization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of biliary anastomosis on recovery from secondary biliary cirrhosis

Objective The influence of choledochoduodenostomy and choledochojejunostomy on the repair of hepatic lesions secondary to biliary obstruction is not well known. The aim of the present study was to compare the effects of choledochoduodenostomy and choledochojejunostomy on the recovery of these lesions in rats with biliary obstruction. Methods Rats subjected to 4 weeks of biliary obstruction underwent choledochoduodenostomy (n=10) or choledochojejunostomy (n=10). The following variables were measured: total bilirubin, alkaline phosphatase, aminotransferases, and albumin. Hepatic mitochondrial energy metabolism was evaluated by calculating the respiratory control ratio and the oxidative phosphorylation index. Hepatic morphometry was used to estimate the mass of the hepatocytes, bile ducts, and fibrosis, as well as the hepatic stellate cell count. Results After choledochoduodenostomy and choledochojejunostomy, there was a regression in cholestasis and a reduction in the oxidative phosphorylation index. However, the total bilirubin, alkaline phosphatase, albumin, and respiratory control ratio values improved only after choledochojejunostomy. The mass of the liver, spleen, and fibrosis was reduced after both choledochoduodenostomy and choledochojejunostomy, but the number of hepatic stellate cells increased. After choledochojejunostomy, the hepatic mass recovered completely, and the spleen mass was significantly reduced compared with that after choledochoduodenostomy. After both choledochoduodenostomy and choledochojejunostomy, enterobiliary reflux, biliary contamination, and an exacerbation in hepatic inflammation developed. Conclusion Choledochojejunostomy was more effective than choledochoduodenostomy, but both techniques induced enterobiliary reflux and biliary contamination, which may explain the maintenance of hepatic alterations, especially after choledochoduodenostomy. Eur J Gastroenterol Hepatol 24: 1039-1050 (C) 2012 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Overexpression of inducible 70-kDa heat shock protein in mouse improves structural and functional recovery of skeletal muscles from atrophy

Heat shock proteins play a key regulatory role in cellular defense. To investigate the role of the inducible 70-kDa heat shock protein (HSP70) in skeletal muscle atrophy and subsequent recovery, soleus (SOL) and extensor digitorum longus (EDL) muscles from overexpressing HSP70 transgenic mice were immobilized for 7 days and subsequently released from immobilization and evaluated after 7 days. Histological analysis showed that there was a decrease in cross-sectional area of type II myofiber from EDL and types I and II myofiber from SOL muscles at 7-day immobilization in both wild-type and HSP70 mice. At 7-day recovery, EDL and SOL myofibers from HSP70 mice, but not from wild-type mice, recovered their size. Muscle tetanic contraction decreased only in SOL muscles from wild-type mice at both 7-day immobilization and 7-day recovery; however, it was unaltered in the respective groups from HSP70 mice. Although no effect in a fatigue protocol was observed among groups, we noticed a better contractile performance of EDL muscles from overexpressing HSP70 groups as compared to their matched wild-type groups. The number of NCAM positive-satellite cells reduced after immobilization and recovery in both EDL and SOL muscles from wild-type mice, but it was unchanged in the muscles from HSP70 mice. These results suggest that HSP70 improves structural and functional recovery of skeletal muscle after disuse atrophy, and this effect might be associated with preservation of satellite cell amount.

Whole blood genome-wide gene expression profile in males after prolonged wakefulness and sleep recovery

Pellegrino R, Sunaga DY, Guindalini C, Martins RC, Mazzotti DR, Wei Z, Daye ZJ, Andersen ML, Tufik S. Whole blood genome-wide gene expression profile in males after prolonged wakefulness and sleep recovery. Physiol Genomics 44: 1003-1012, 2012. First published September 4, 2012; doi: 10.1152/physiolgenomics.00058.2012.-Although the specific functions of sleep have not been completely elucidated, the literature has suggested that sleep is essential for proper homeostasis. Sleep loss is associated with changes in behavioral, neurochemical, cellular, and metabolic function as well as impaired immune response. Using high-resolution microarrays we evaluated the gene expression profiles of healthy male volunteers who underwent 60 h of prolonged wakefulness (PW) followed by 12 h of sleep recovery (SR). Peripheral whole blood was collected at 8 am in the morning before the initiation of PW (Baseline), after the second night of PW, and one night after SR. We identified over 500 genes that were differentially expressed. Notably, these genes were related to DNA damage and repair and stress response, as well as diverse immune system responses, such as natural killer pathways including killer cell lectin-like receptors family, as well as granzymes and T-cell receptors, which play important roles in host defense. These results support the idea that sleep loss can lead to alterations in molecular processes that result in perturbation of cellular immunity, induction of inflammatory responses, and homeostatic imbalance. Moreover, expression of multiple genes was downregulated following PW and upregulated after SR compared with PW, suggesting an attempt of the body to re-establish internal homeostasis. In silico validation of alterations in the expression of CETN3, DNAJC, and CEACAM genes confirmed previous findings related to the molecular effects of sleep deprivation. Thus, the present findings confirm that the effects of sleep loss are not restricted to the brain and can occur intensely in peripheral tissues.

In search of mechanisms associated with mesenchymal stem cell-based therapies for acute kidney injury

Acute kidney injury (AKI) is classically described as a rapid loss of kidney function. AKI affects more than 15% of all hospital admissions and is associated with elevated mortality rates. Although many advances have occurred, intermittent or continuous renal replacement therapies are still considered the best options for reversing mild and severe AKI syndrome. For this reason, it is essential that innovative and effective therapies, without side effects and complications, be developed to treat AKI and the end-stages of renal disease. Mesenchymal stem cell (MSC) based therapies have numerous advantages in helping to repair inflamed and damaged tissues and are being considered as a new alternative for treating kidney injuries. Numerous experimental models have shown that MSCs can act via differentiation-independent mechanisms to help renal recovery. Essentially, MSCs can secrete a pool of cytokines, growth factors and chemokines, express enzymes, interact via cell-to-cell contacts and release bioagents such as microvesicles to orchestrate renal protection. In this review, we propose seven distinct properties of MSCs which explain how renoprotection may be conferred: 1) anti-inflammatory; 2) pro-angiogenic; 3) stimulation of endogenous progenitor cells; 4) anti-apoptotic; 5) anti-fibrotic; 6) anti-oxidant; and 7) promotion of cellular reprogramming. In this context, these mechanisms, either individually or synergically, could induce renal protection and functional recovery. This review summarises the most important effects and benefits associated with MSC-based therapies in experimental renal disease models and attempts to clarify the mechanisms behind the MSC-related renoprotection. MSCs may prove to be an effective, innovative and affordable treatment for moderate and severe AKI. However, more studies need to be performed to provide a more comprehensive global understanding of MSC-related therapies and to ensure their safety for future clinical applications.

UVB-induced cell death signaling is associated with G1-S progression and transcription inhibition in primary human fibroblasts

DNA damage induced by ultraviolet (UV) radiation can be removed by nucleotide excision repair through two sub-pathways, one general (GGR) and the other specific for transcribed DNA (TCR), and the processing of unrepaired lesions trigger signals that may lead to cell death. These signals involve the tumor suppressor p53 protein, a central regulator of cell responses to DNA damage, and the E3 ubiquitin ligase Mdm2, that forms a feedback regulatory loop with p53. The involvement of cell cycle and transcription on the signaling to apoptosis was investigated in UVB-irradiated synchronized, DNA repair proficient, CS-B (TCR-deficient) and XP-C (GGR-deficient) primary human fibroblasts. Cells were irradiated in the G1 phase of the cell cycle, with two doses with equivalent levels of apoptosis (low and high), defined for each cell line. In the three cell lines, the low doses of UVB caused only a transient delay in progression to the S phase, whereas the high doses induced permanent cell cycle arrest. However, while accumulation of Mdm2 correlated well with the recovery from transcription inhibition at the low doses for normal and CS-B fibroblasts, for XP-C cells this protein was shown to be accumulated even at UVB doses that induced high levels of apoptosis. Thus, UVB-induced accumulation of Mdm2 is critical for counteracting p53 activation and apoptosis avoidance, but its effect is limited due to transcription inhibition. However, in the case of XP-C cells, an excess of unrepaired DNA damage would be sufficient to block S phase progression, which would signal to apoptosis, independent of Mdm2 accumulation. The data clearly discriminate DNA damage signals that lead to cell death, depending on the presence of UVB-induced DNA damage in replicating or transcribing regions.

Algal wastewater treatment and biomass producing potential: nutrient removal efficiency and cell physiological responses

Microalgae are sun - light cell factories that convert carbon dioxide to biofuels, foods, feeds, and other bioproducts. The concept of microalgae cultivation as an integrated system in wastewater treatment has optimized the potential of the microalgae - based biofuel production. These microorganisms contains lipids, polysaccharides, proteins, pigments and other cell compounds, and their biomass can provide different kinds of biofuels such as biodiesel, biomethane and ethanol. The algal biomass application strongly depends on the cell composition and the production of biofuels appears to be economically convenient only in conjunction with wastewater treatment. The aim of this research thesis was to investigate a biological wastewater system on a laboratory scale growing a newly isolated freshwater microalgae, Desmodesmus communis, in effluents generated by a local wastewater reclamation facility in Cesena (Emilia Romagna, Italy) in batch and semi - continuous cultures. This work showed the potential utilization of this microorganism in an algae - based wastewater treatment; Desmodesmus communis had a great capacity to grow in the wastewater, competing with other microorganisms naturally present and adapting to various environmental conditions such as different irradiance levels and nutrient concentrations. The nutrient removal efficiency was characterized at different hydraulic retention times as well as the algal growth rate and biomass composition in terms of proteins, polysaccharides, total lipids and total fatty acids (TFAs) which are considered the substrate for biodiesel production. The biochemical analyses were coupled with the biomass elemental analysis which specified the amount of carbon and nitrogen in the algal biomass. Furthermore photosynthetic investigations were carried out to better correlate the environmental conditions with the physiology responses of the cells and consequently get more information to optimize the growth rate and the increase of TFAs and C/N ratio, cellular compounds and biomass parameter which are fundamental in the biomass energy recovery.

Continuos Flow Single Cell Separation into Open Microwell Arrays

A novel design based on electric field-free open microwell arrays for the automated continuous-flow sorting of single or small clusters of cells is presented. The main feature of the proposed device is the parallel analysis of cell-cell and cell-particle interactions in each microwell of the array. High throughput sample recovery with a fast and separate transfer from the microsites to standard microtiter plates is also possible thanks to the flexible printed circuit board technology which permits to produce cost effective large area arrays featuring geometries compatible with laboratory equipment. The particle isolation is performed via negative dielectrophoretic forces which convey the particles’ into the microwells. Particles such as cells and beads flow in electrically active microchannels on whose substrate the electrodes are patterned. The introduction of particles within the microwells is automatically performed by generating the required feedback signal by a microscope-based optical counting and detection routine. In order to isolate a controlled number of particles we created two particular configurations of the electric field within the structure. The first one permits their isolation whereas the second one creates a net force which repels the particles from the microwell entrance. To increase the parallelism at which the cell-isolation function is implemented, a new technique based on coplanar electrodes to detect particle presence was implemented. A lock-in amplifying scheme was used to monitor the impedance of the channel perturbed by flowing particles in high-conductivity suspension mediums. The impedance measurement module was also combined with the dielectrophoretic focusing stage situated upstream of the measurement stage, to limit the measured signal amplitude dispersion due to the particles position variation within the microchannel. In conclusion, the designed system complies with the initial specifications making it suitable for cellomics and biotechnology applications.

Interactions between the gut microbiota, short-chain fatty acids and the immune system in pediatric patients undergoing hematopoietic stem cell transplantation

The gut microbiota (GM) is essential for human health and contributes to several diseases; indeed it can be considered an extension of the self and, together with the genetic makeup, determines the physiology of an organism. In this thesis has been studied the peripheral immune system reconstitution in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (aHSCT) in the early phase; in parallel, have been also explored the gut microbiota variations as one of the of primary factors in governing the fate of the immunological recovery, predisposing or protecting from complications such as the onset of acute graft-versus-host disease (GvHD). Has been demonstrated, to our knowledge for the first time, that aHSCT in pediatric patients is associated to a profound modification of the GM ecosystem with a disruption of its mutualistic asset. aGvHD and non-aGvHD subjects showed differences in the process of GM recovery, in members abundance of the phylum Bacteroidetes, and in propionate fecal concentration; the latter are higher in the pre-HSCT composition of non-GvHD subjects than GvHD ones. Short-chain fatty acids (SCFAs), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients and distribute systemically from the gut to blood. For this reason, has been studied their effect in vitro on human DCs, the key regulators of our immune system and the main player of aGvHD onset. Has been observed that propionate and, particularly, butyrate show a strong and direct immunomodulatory activity on DCs reducing inflammatory markers such as chemokines and interleukins. This study, with the needed caution, suggests that the pre-existing GM structure can be protective against aGvHD onset, exerting its protective role through SCFAs. They, indeed, may regulate cell traffic within secondary lymphoid tissues, influence T cell development during antigen recognition, and, thus, directly shape the immune system.

Chlamydiae host cell interaction and immune evasion strategies

Chlamydiae are obligate intracellular bacteria with a strong global prevalence. They cause infections of the eye, lung and the genital tract and can either replicate in inclusion compartments or persist inside their host cell. In this thesis we focused on two aspects of chlamydiae infection. We hypothesize that transcription factor AP-1 is crucial for a replicative chlamydiae infection in epithelial cells. In addition we suggest that chlamydiae hide inside apoptotic blebs for a silent uptake by macrophages as immune evasion strategy.rnFocusing on AP-1, we could demonstrate that during Chlamydia pneumoniae infection, protein expression and phosphorylation of the AP-1 family member c-Jun significantly increased in a time and dose dependent manner. A siRNA knockdown of c-Jun in HEp-2 cells reduced chlamydial load, resulting in smaller inclusions and a significant lower chlamydial recovery. Furthermore, inhibition of the c-Jun containing AP-1 complexes, using Tanshinone IIA, changed the replicative infection into a persistent phenotype, characterized by (i) smaller, aberrant inclusions, (ii) a strong decrease in chlamydial load, as well as by (iii) its reversibility after removal of Tanshinone IIA. As chlamydiae are energy parasites, we investigated whether Tanshinone IIA interferes with energy/metabolism related processes. rnA role for autophagy or gene expression of glut-1 and c-jun in persistence could not be determined. However we could demonstrate Tanshinone IIA treatment to be accompanied by a significant decrease of ATP levels, probably causing a chlamydiae persistent phenotype.rnRegarding the chlamydial interaction with human primary cells we characterized infection of different chlamydiae species in either pro-inflammatory (type I) or anti-inflammatory (type II) human monocyte derived macrophages (hMDM). We found both phenotypes to be susceptible to chlamydiae infection. Furthermore, we observed that upon Chlamydia trachomatis and GFP-expressing Chlamydia trachomatis infection more hMDM type II were infected. However the chlamydial load was higher in hMDM type I and correspondingly, more replicative-like inclusions were found in this phenotype. Next, we focused on the chlamydial transfer using a combination of high speed live cell imaging and GFP-expressing Chlamydia trachomatis for optimal visualization. Thereby, we could successfully visualize the formation of apoptotic, chlamydiae-containing blebs and the interaction of hMDM with these blebs. Moreover, we observed the development of a replicative infection in hMDM. rnIn conclusion, we demonstrated a crucial role of AP-1 for C. pneumoniae development and preliminary time lapse data suggest that chlamydiae can be transferred to hMDMs via apoptotic blebs. In all, these data may contribute to a better understanding of chlamydial infection processes in humans.rn

Optimizing the success of cell transplantation therapy for stroke

Stem cell transplantation has evolved as a promising experimental treatment approach for stroke. In this review, we address the major hurdles for successful translation from basic research into clinical applications and discuss possible strategies to overcome these issues. We summarize the results from present pre-clinical and clinical studies and focus on specific areas of current controversy and research: (i) the therapeutic time window for cell transplantation; (ii) the selection of patients likely to benefit from such a therapy; (iii) the optimal route of cell delivery to the ischemic brain; (iv) the most suitable cell types and sources; (v) the potential mechanisms of functional recovery after cell transplantation; and (vi) the development of imaging techniques to monitor cell therapy.