Simone Odília Antunes Fernandes

Antiglaucomatous effects of the activation of intrinsic Angiotensin-converting enzyme 2.

PURPOSE To evaluate the effects of the activation of endogenous angiotensin-converting enzyme 2 (ACE2) using the compound diminazene aceturate (DIZE) in an experimental model of glaucoma in Wistar rats. METHODS DIZE (1 mg/kg) was administered daily, either systemically or topically, and the IOP was measured weekly. To examine the role of the Mas receptor in the effects of DIZE, the Ang-(1-7) antagonist A-779 was co-administered. Drainage of the aqueous humor was evaluated by using scintigraphy. The analysis of ACE2 expression by immunohistochemistry and the counting of retinal ganglion cells (RGCs) were performed in histologic sections. Additionally, the nerve fiber structure was evaluated by transmission electron microscopy. RESULTS The systemic administration and topical administration (in the form of eye drops) of DIZE increased the ACE2 expression in the eyes and significantly decreased the IOP of glaucomatous rats without changing the blood pressure. Importantly, this IOP-lowering action of DIZE was similar to the effects of dorzolamide. The antiglaucomatous effects of DIZE were blocked by A-779. Histologic analysis revealed that the reduction in the number of RGCs and the increase in the expression of caspase-3 in the RGC layer in glaucomatous animals were prevented by DIZE. This compound also prevented alterations in the cytoplasm of axons in glaucomatous rats. In addition to these neuroprotective effects, DIZE facilitated the drainage of the aqueous humor. CONCLUSIONS Our results evidence the pathophysiologic relevance of the ocular ACE2/Ang-(1-7)/Mas axis of the renin-angiotensin system and, importantly, indicate that the activation of intrinsic ACE2 is a potential therapeutic strategy to treat glaucoma.

The role of immunomodulators on intestinal barrier homeostasis in experimental models.

The intestinal epithelium is composed of specialized epithelial cells that form a physical and biochemical barrier to commensal and pathogenic microorganisms. However, dysregulation of the epithelial barrier function can lead to increased intestinal permeability and bacterial translocation across the intestinal mucosa, which contributes to local and systemic immune activation. The increase in these parameters is associated with inflammatory bowel disease, physical exercise under heat stress, intestinal obstruction, ischemia, and mucositis, among other conditions. Lately, there has been growing interest in immunomodulatory nutrients and probiotics that can regulate host immune and inflammatory responses and possibly restore the intestinal barrier. Immunomodulators such as amino acids (glutamine, arginine, tryptophan, and citrulline), fatty acids (short-chain and omega-3 fatty acids and conjugated linoleic acids), and probiotics (Bifidobacterium, Saccharomyces, and Lactobacillus) have been reported in the literature. Here, we review the critical roles of immunomodulatory nutrients in supporting gut barrier integrity and function.

l-Arginine Supplementation Prevents Increases in Intestinal Permeability and Bacterial Translocation in Male Swiss Mice Subjected to Physical Exercise under Environmental Heat Stress

Dietary supplementation with l-arginine has been shown to improve the intestinal barrier in many experimental models. This study investigated the effects of arginine supplementation on the intestinal permeability and bacterial translocation (BT) induced by prolonged physical exercise under heat stress. Under anesthesia, male Swiss mice (5-wk-old) were implanted with an abdominal sensor to record their core body temperature (T(core)). After recovering from surgery, the mice were divided into 3 groups: a non-supplemented group that was fed the standard diet formulated by the American Institute of Nutrition (AIN-93G; control), a non-supplemented group that was fed the AIN-93G diet and subjected to exertional hyperthermia (H-NS), and a group supplemented with l-arginine at 2% and subjected to exertional hyperthermia (H-Arg). After 7 d of treatment, the H-NS and H-Arg mice were forced to run on a treadmill (60 min, 8 m/min) in a warm environment (34°C). The control mice remained at 24°C. Thirty min before the exercise or control trials, the mice received a diethylenetriamine pentaacetic acid (DTPA) solution labeled with technetium-99m ((99m)Tc-DTPA) or (99m)Tc-Escherichia coli by gavage to assess intestinal permeability and BT, respectively. The H-NS mice terminated the exercise with T(core) values of ∼40°C, and, 4 h later, presented a 12-fold increase in the blood uptake of (99m)Tc-DTPA and higher bacterial contents in the blood and liver than the control mice. Although supplementation with arginine did not change the exercise-induced increase in T(core), it prevented the increases in intestinal permeability and BT caused by exertional hyperthermia. Our results indicate that dietary l-arginine supplementation preserves the integrity of the intestinal epithelium during exercise under heat stress, acting through mechanisms that are independent of T(core) regulation.

New delivery systems for amphotericin B applied to the improvement of leishmaniasis treatment

Leishmaniasis is one of the six major tropical diseases targeted by the World Health Organization. It is a life-threatening disease of medical, social and economic importance in endemic areas. No vaccine is yet available for human use, and chemotherapy presents several problems. Pentavalent antimonials have been the drugs of choice to treat the disease for more than six decades; however, they exhibit high toxicity and are not indicated for children, for pregnant or breastfeeding women or for chronically ill patients. Amphotericin B (AmpB) is a second-line drug, and although it has been increasingly used to treat visceral leishmaniasis (VL), its clinical use has been hampered due to its high toxicity. This review focuses on the development and in vivo usage of new delivery systems for AmpB that aim to decrease its toxicity without altering its therapeutic efficacy. These new formulations, when adjusted with regard to their production costs, may be considered new drug delivery systems that promise to improve the treatment of leishmaniasis, by reducing the side effects and the number of doses while permitting a satisfactory cost-benefit ratio.

Mitochondrial calcium regulates rat liver regeneration through the modulation of apoptosis.

Subcellular Ca2+ signals control a variety of responses in the liver. For example, mitochondrial Ca2+ (Ca  mit2+ ) regulates apoptosis, whereas Ca2+ in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca  mit2+ also affects liver regeneration. The Ca2+‐buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin–mitochondrial targeting sequence–green fluorescent protein (PV‐MITO‐GFP). This construct properly localized to and effectively buffered Ca2+ signals in the mitochondrial matrix. Additionally, the expression of PV‐MITO‐GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl‐2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2–associated X protein (bax), apoptotic peptidase activating factor 1, and caspase‐6]. PV‐MITO‐GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca  mit2+ buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl‐2 and the decreased expression of bax. Conclusion: Together, these results reveal an essential role for Ca  mit2+ in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis. (HEPATOLOGY 2011;)

Bimatoprost-Loaded Ocular Inserts as Sustained Release Drug Delivery Systems for Glaucoma Treatment: In Vitro and In Vivo Evaluation

The purpose of the present study was to develop and assess a novel sustained-release drug delivery system of Bimatoprost (BIM). Chitosan polymeric inserts were prepared using the solvent casting method and characterized by swelling studies, infrared spectroscopy, differential scanning calorimetry, drug content, scanning electron microscopy and in vitro drug release. Biodistribution of 99mTc-BIM eye drops and 99mTc-BIM-loaded inserts, after ocular administration in Wistar rats, was accessed by ex vivo radiation counting. The inserts were evaluated for their therapeutic efficacy in glaucomatous Wistar rats. Glaucoma was induced by weekly intracameral injection of hyaluronic acid. BIM-loaded inserts (equivalent to 9.0 µg BIM) were administered once into conjunctival sac, after ocular hypertension confirmation. BIM eye drop was topically instilled in a second group of glaucomatous rats for 15 days days, while placebo inserts were administered once in a third group. An untreated glaucomatous group was used as control. Intraocular pressure (IOP) was monitored for four consecutive weeks after treatment began. At the end of the experiment, retinal ganglion cells and optic nerve head cupping were evaluated in the histological eye sections. Characterization results revealed that the drug physically interacted, but did not chemically react with the polymeric matrix. Inserts sustainedly released BIM in vitro during 8 hours. Biodistribution studies showed that the amount of 99mTc-BIM that remained in the eye was significantly lower after eye drop instillation than after chitosan insert implantation. BIM-loaded inserts lowered IOP for 4 weeks, after one application, while IOP values remained significantly high for the placebo and untreated groups. Eye drops were only effective during the daily treatment period. IOP results were reflected in RGC counting and optic nerve head cupping damage. BIM-loaded inserts provided sustained release of BIM and seem to be a promising system for glaucoma management.

An optimized nanoparticle delivery system based on chitosan and chondroitin sulfate molecules reduces the toxicity of amphotericin B and is effective in treating tegumentary leishmaniasis.

Amphotericin B (AmpB) is active against leishmaniasis, but its use is hampered due to its high toxicity observed in patients. In this study, a nanoparticles-delivery system for AmpB (NQC-AmpB), containing chitosan and chondroitin sulfate molecules, was evaluated in BALB/c mice against Leishmania amazonensis. An in vivo biodistribution study, including biochemical and toxicological evaluations, was performed to evaluate the toxicity of AmpB. Nanoparticles were radiolabeled with technetium-99m and injected in mice. The products presented a similar biodistribution in the liver, spleen, and kidneys of the animals. Free AmpB induced alterations in the body weight of the mice, which, in the biochemical analysis, indicated hepatic and renal injury, as well as morphological damage to the kidneys of the animals. In general, no significant organic alteration was observed in the animals treated with NQC-AmpB. Mice were infected with L. amazonensis and treated with the nanoparticles or free AmpB; then, parasitological and immunological analyses were performed. The NQC-AmpB group, as compared to the control groups, presented significant reductions in the lesion size and in the parasite burden in all evaluated organs. These animals presented significantly higher levels of IFN-γ and IL-12, and low levels of IL-4 and IL-10, when compared to the control groups. The NQC-AmpB system was effective in reducing the infection in the animals, and proved to be effective in diminishing the toxicity evoked by AmpB, which was observed when it was administered alone. In conclusion, NQC-AmpB could be considered a viable possibility for future studies in the treatment of leishmaniasis.

Pretreatment with Saccharomyces boulardii does not prevent the experimental mucositis in Swiss mice.

BackgroundThe antimetabolite chemotherapy 5-Fluorouracil is one of the most commonly prescribed drugs in clinical cancer treatment. Although this drug is not specific for cancer cells and also acts on healthy cells, it can cause mucositis, a common collateral effect. Dysbiosis has also been described in 5-fluorouracil-induced mucositis and is likely to contribute to the overall development of mucositis. In light of this theory, the use of probiotics could be a helpful strategy to alleviate mucositis. So the aim of this study was evaluate the impact of the probiotic Saccharomyces boulardii in a model of mucositis.ResultsAfter induced of mucositis, mice from the Mucositis groups showed a decrease in food consumption (p < 0.05) and therefore had a greater weight loss (p < 0.05). The treatment with Saccharomyces boulardii did not reverse this effect (p > 0.05). Mucositis induced an increase in intestinal permeability and intestinal inflammation (p < 0.05). There were no differences in mucosal lesions, intestinal permeability and sIgA secretion (p > 0.05) in mice pretreated with S. boulardii.ConclusionsS. boulardii was not able to prevent the effects of experimental mucositis induced by 5- Fluorouracil.

Technetium-99m-labeled ceftizoxime loaded long-circulating and pH-sensitive liposomes used to identify osteomyelitis

Osteomyelitis is an infectious disease located in the bone or bone marrow. Long-circulating and pH-sensitive liposomes containing a technetium-99m-labeled antibiotic, ceftizoxime, (SpHL-(99m)Tc-CF) were developed to identify osteomyelitis foci. Biodistribution studies and scintigraphic images of bone infection or non infection-bearing rats that had been treated with these liposomes were performed. A high accumulation in infectious foci and high values in the target-non target ratio could be observed. These results indicate the potential of SpHL-(99m)Tc-CF as a potential agent for the diagnosis of bone infections.

Identification of Staphylococcus aureus infection by aptamers directly radiolabeled with technetium-99m

INTRODUCTION Aptamers are oligonucleotides that have high affinity and specificity for their molecular targets which are emerging as a new class of molecules for radiopharmaceuticals development. In this study, aptamers selected to Staphylococcus aureus were evaluated for bacterial infection identification. METHODS Anti S. aureus aptamers were labeled with (99m)Tc by the direct method. The radiolabel yield and complex stability were assessed by thin-layer chromatography (TLC). Three groups of Swiss mice containing 6 animals each were used. The first group was infected intramuscularly in the right thigh with S. aureus. The second group was infected in the same way with C. albicans and the third group was injected with zymosan to induce aseptic inflammation. After 24 h, radiolabeled aptamers (22.2 MBq) were injected by the tail vein. The mice were euthanized 4 h post injection and tissue sample activities measured in a gamma counter. RESULTS The (99m)Tc labeled aptamers were stable in saline, plasma and cystein excess. Radiolabeled aptamers showed increased uptake in the kidneys for all groups indicating a main renal excretion, which is consistent with the hydrophilic nature and small size of aptamers. The radiopharmaceutical showed rapid blood clearance indicated by a reduced dose (% ID/g) in the blood. The biodistribution showed that aptamers were able to identify the infection foci caused by S. aureus displaying a target/non-target ratio of 4.0±0.5. This ratio for mice infected with C. albicans was 2.0±0.4 while for mice with aseptic inflammation was 1.2±0.2. Histology confirmed the presence of infection in groups 1 and 2, and inflammation in group 3. CONCLUSIONS The biodistibution study demonstrated a statistically higher uptake in the S. aureus foci relative to inflammation and C. albicans infected areas. These results highlight the potential of aptamers labeled directly with (99m)Tc for bacterial infection diagnosis by scintigraphy.