Therapeutic Applications of Physalins: Powerful Natural Weapons.

Physalins, or 16,24-cyclo-13,14-seco steroids, are compounds belonging to the class of withanolides that can be found in plants of Solanaceae family, mainly in species belonging to the genus Physalis spp., which are annual herbaceous plants widely distributed in tropical and subtropical regions of the world. Physalins are versatile molecules that act in several cell signaling pathways and activate different mechanisms of cell death or immunomodulation. A number of studies have shown a variety of actions of these compounds, including anticancer, anti-inflammatory, antiparasitic, antimicrobial, antinociceptive, and antiviral activities. Here we reviewed the main findings related to the anticancer, immunomodulatory, and antiparasitic activities of physalins and its mechanisms of action, highlighting the \challenges and future directions in the pharmacological application of physalins.

A Betulinic Acid Derivative, BA5, Induces G0/G1 Cell Arrest, Apoptosis Like-Death, and Morphological Alterations in Leishmania sp.

Leishmaniasis are endemic diseases caused by different species of intracellular parasites of the genus Leishmania. Due to the high toxicity and drug resistance of current antileishmanial drugs, it is necessary to identify new and more effective drugs. Previously, we investigated the immunomodulatory and anti-Trypanosoma cruzi action of BA5, a derivative of betulinic acid. In the present study, we investigated the in vitro activity of BA5 against different species of Leishmania and their action mechanism. BA5 exhibited low cytotoxicity against macrophages and inhibited the proliferation of promastigote forms of Leishmania amazonensis (IC50 = 4.5 ± 1.1 µM), Leishmania major (IC50 = 3.0 ± 0.8 µM), Leishmania braziliensis (IC50 = 0.9 ± 1.1 µM) and Leishmania infantum (IC50 = 0.15 ± 0.05 µM). Incubation with BA5 reduced the percentage of Leishmania amazonensis-infected macrophages and the number of intracellular parasites (IC50 = 4.1 ± 0.7 µM). To understand the mechanism of action underlying BA5 antileishmanial activity (incubation at IC50/2, IC50 or 2xIC50 values of the drug), we investigated ultrastructural changes by scanning electron microscopy and evaluated cell cycle, membrane mitochondrial potential, and cell death against promastigote forms of Leishmania amazonensis by flow cytometry. Promastigotes incubated with BA5 presented membrane blebbing, flagella damage, increased size, and body deformation. Flow cytometry analysis showed that parasite death is mainly caused by apoptosis-like death, arrested cell cycle in G0/G1 phase and did not alter the membrane mitochondrial potential of Leishmania amazonensis. Surprisingly, the combination of BA5 and amphotericin B, an assay used to determine the degree of drug interaction, revealed synergistic effects (CI = 0.15 ± 0.09) on promastigotes forms of Leishmania amazonensis. In conclusion, BA5 compound is an effective and selective antileishmanial agent.

Structural Design, Synthesis and Antioxidant, Antileishmania, Anti-Inflammatory and Anticancer Activities of a Novel Quercetin Acetylated Derivative.

Quercetin (Q) is a bioflavonoid with biological potential; however, poor solubility in water, extensive enzymatic metabolism and a reduced bioavailability limit its biopharmacological use. The aim of this study was to perform structural modification in Q by acetylation, thus, obtaining the quercetin pentaacetate (Q5) analogue, in order to investigate the biological potentials (antioxidant, antileishmania, anti-inflammatory and cytotoxicity activities) in cell cultures. Q5 was characterized by FTIR, 1H and 13C NMR spectra. The antioxidant potential was evaluated against the radical ABTS•+. The anti-inflammatory potential was evaluated by measuring the pro-inflammatory cytokine tumor necrosis factor (TNF) and the production of nitric oxide (NO) in peritoneal macrophages from BALB/c mice. Cytotoxicity tests were performed using the AlamarBlue method in cancer cells HepG2 (human hepatocarcinoma), HL-60 (promyelocytic leukemia) and MCR-5 (healthy human lung fibroblasts) as well as the MTT method for C6 cell cultures (rat glioma). Q and Q5 showed antioxidant activity of 29% and 18%, respectively, which is justified by the replacement of hydroxyls by acetyl groups. Q and Q5 showed concentration-dependent reductions in NO and TNF production (p < 0.05); Q and Q5 showed higher activity at concentrations > 40µM when compared to dexamethasone (20 µM). For the HL-60 lineage, Q5 demonstrated selectivity, inducing death in cancer cells, when compared to the healthy cell line MRC-5 (IC50 > 80 µM). Finally, the cytotoxic superiority of Q5 was verified (IC50 = 11 µM), which, at 50 µM for 24 h, induced changes in the morphology of C6 glioma cells characterized by a round body shape (not yet reported in the literature). The analogue Q5 had potential biological effects and may be promising for further investigations against other cell cultures, particularly neural ones.

Synthesis, crystal structure and leishmanicidal activity of new trimethoprim Ru(III), Cu(II) and Pt(II) metal complexes.

Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania, which has very limited treatment options and affects poor and underdeveloped populations. The current treatment is plagued by many complications, such as high toxicity, high cost and resistance to parasites; therefore, novel therapeutic agents are urgently needed. Herein, the synthesis, characterization and in vitro leishmanicidal potential of new complexes with the general formula [RuCl3(TMP)(dppb)] (1), [PtCl(TMP)(PPh3)2]PF6 (2) and [Cu(CH3COO)2(TMP)2]·DMF (3) (dppb = 1,4-bis(diphenylphosphino)butane, PPH3 = triphenylphosphine and TMP = trimethoprim) were evaluated. The complexes were characterized by infrared, UV-vis, cyclic voltammetry, molar conductance measurements, elemental analysis and NMR experiments. Also, the geometry of (2) and (3) were determined by single crystal X-ray diffraction. Despite being less potent against promastigote L. amazonensis proliferation than amphotericin B reference drug (IC50 = 0.09 ± 0.02 µM), complex (2) (IC50 = 3.6 ± 1.5 µM) was several times less cytotoxic (CC50 = 17.8 µM, SI = 4.9) in comparison with amphotericin B (CC50 = 3.3 µM, SI = 36.6) and gentian violet control (CC50 = 0.8 µM). Additionally, complex (2) inhibited J774 macrophage infection and amastigote number by macrophages (IC50 = 6.6 and SI = 2.7). Outstandingly, complex (2) was shown to be a promising candidate for a new leishmanicidal therapeutic agent, considering its biological power combined with low toxicity.

In Vitro, In Vivo and In Silico Effectiveness of LASSBio-1386, an N-Acyl Hydrazone Derivative Phosphodiesterase-4 Inhibitor, Against Leishmania amazonensis.

Leishmaniasis are group of neglected diseases with worldwide distribution that affect about 12 million people. The current treatment is limited and may cause severe adverse effects, and thus, the search for new drugs more effective and less toxic is relevant. We have previously investigated the immunomodulatory effects of LASSBio-1386, an N-acylhydrazone derivative. Here we investigated the in vitro and in vivo activity of LASSBio-1386 against L. amazonensis. LASSBio-1386 inhibited the proliferation of promastigotes of L. amazonensis (EC50 = 2.4 ± 0.48 µM), while presenting low cytotoxicity to macrophages (CC50 = 74.1 ± 2.9 µM). In vitro incubation with LASSBio-1386 reduced the percentage of Leishmania-infected macrophages and the number of intracellular parasites (EC50 = 9.42 ± 0.64 µM). Also, in vivo treatment of BALB/c mice infected with L. amazonensis resulted in a decrease of lesion size, parasitic load and caused histopathological alterations, when compared to vehicle-treated control. Moreover, LASSBio-1386 caused ultrastructural changes, arrested cell cycle in G0/G1 phase and did not alter the membrane mitochondrial potential of L. amazonensis. Aiming to its possible molecular interactions, we performed docking and molecular dynamics studies on Leishmania phosphodiesterase B1 (PDB code: 2R8Q) and LASSBio-1386. The computational analyses suggest that LASSBio-1386 acts against Leishmania through the modulation of leishmanial PDE activity. In conclusion, our results indicate that LASSBio-1386 is a promising candidate for the development of new leishmaniasis treatment.

Potent immunosuppressive activity of a phosphodiesterase-4 inhibitor N-acylhydrazone in models of lipopolysaccharide-induced shock and delayed-type hypersensitivity reaction.

Immunosuppressive drugs are widely used for the treatment of immune-mediated diseases and inflammation, but the toxicity and side effects of the available immunosuppressors make the search of new agents of great relevance. Here, we evaluated the immunomodulatory activity of an N-acylhydrazone derivative, (E)-N'-(3,4-dimethoxybenzylidene)-4-methoxybenzohydrazide (LASSBio-1386), a phosphodiesterase-4 (PDE-4) inhibitor. LASSBio-1386 inhibited lymphocyte activation in a concentration-dependent fashion, decreasing lymphoproliferation and IFN-γ and IL-2 production stimulated by anti-CD3/CD28 mAbs or concanavalin A (Con A) and inducing cell-cycle arrest in the G0/G1 phase. These effects were not blocked by RU486, a glucocorticoid receptor (GR) antagonist, indicating an effect independent of glucocorticoid receptor activation. Combination index-isobologram analysis indicates a synergistic effect between LASSBio-1386 and dexamethasone in lymphoproliferation inhibition. LASSBio-1386 presented immunomodulatory action in macrophage cultures, as observed by a significant and concentration-dependent decrease in NO and TNF-α production, an effect achieved by reducing IĸB expression and NF-κB activation. In the mouse model of endotoxic shock, LASSBio-1386 at 50 and 100 mg/kg protected 50 and 85% of mice against LPS-induced lethality, respectively. In agreement to its in vitro action, treatment with 100 mg/kg of LASSBio-1386 reduced TNF-α and IL-1ß serum levels, while increased IL-6 and IL-10. Finally, LASSBio-1386 reduced the paw edema in a BSA-induced delayed-type hypersensitivity model. These findings demonstrate the immunomodulatory and immunosuppressant effects of LASSBio-1386 and indicate this molecule is a promising pharmacologic agent for immune-mediated diseases.

Betulinic acid derivative BA5, a dual NF-kB/calcineurin inhibitor, alleviates experimental shock and delayed hypersensitivity.

Betulinic acid (BA) is a naturally occurring triterpenoid with several biological properties already described, including immunomodulatory activity. Here we investigated the immunomodulatory activity of eight semi-synthetic amide derivatives of betulinic acid. Screening of derivatives BA1-BA8 led to the identification of compounds with superior immunomodulatory activity than BA on activated macrophages and lymphocytes. BA5, the most potent derivative, inhibited nitric oxide and TNFα production in a concentration-dependent manner, and decreased NF-κB activation in Raw 264.7 cells. Additionally, BA5 inhibited the proliferation of activated lymphocytes and the secretion of IL-2, IL-4 IL-6, IL-10, IL-17A and IFNÉ£, in a concentration-dependent manner. Flow cytometry analysis in lymphocyte cultures showed that treatment with BA5 induces cell cycle arrest in pre-G1 phase followed by cell death by apoptosis. Moreover, BA5 also inhibited the activity of calcineurin, an enzyme that plays a critical role in the progression of cell cycle and T lymphocyte activation. BA5 has a synergistic inhibitory effect with dexamethasone on lymphoproliferation, showing a promising profile for drug combination. Finally, we observed immunosuppressive effects of BA5 in vivo in mouse models of lethal endotoxemia and delayed type hypersensitivity. Our results reinforce the potential use of betulinic acid and its derivatives in the search for potent immunomodulatory drugs.

Antiparasitic evaluation of betulinic acid derivatives reveals effective and selective anti-Trypanosoma cruzi inhibitors.

Betulinic acid is a pentacyclic triterpenoid with several biological properties already described, including antiparasitic activity. Here, the anti-Trypanosoma cruzi activity of betulinic acid and its semi-synthetic amide derivatives (BA1-BA8) was investigated. The anti-Trypanosoma cruzi activity and selectivity were enhanced in semi-synthetic derivatives, specially on derivatives BA5, BA6 and BA8. To understand the mechanism of action underlying betulinic acid anti-T. cruzi activity, we investigated ultrastructural changes by electron microscopy. Ultrastructural studies showed that trypomastigotes incubated with BA5 had membrane blebling, flagella retraction, atypical cytoplasmic vacuoles and Golgi cisternae dilatation. Flow cytometry analysis showed that parasite death is mainly caused by necrosis. Treatment with derivatives BA5, BA6 or BA8 reduced the invasion process, as well as intracellular parasite development in host cells, with a potency and selectivity similar to that observed in benznidazole-treated cells. More importantly, the combination of BA5 and benznidazole revealed synergistic effects on trypomastigote and amastigote forms of T. cruzi. In conclusion, we demonstrated that BA5 compound is an effective and selective anti-T. cruzi agent.

In vitro and in vivo antiparasitic activity of Physalis angulata L. concentrated ethanolic extract against Trypanosoma cruzi.

BACKGROUND: The current treatment of Chagas disease, endemic in Latin America and emerging in several countries, is limited by the frequent side effects and variable efficacy of benznidazole. Natural products are an important source for the search for new drugs. AIM/HYPOTHESIS: Considering the great potential of natural products as antiparasitic agents, we investigated the anti-Trypanosoma cruzi activity of a concentrated ethanolic extract of Physalis angulata (EEPA). METHODS: Cytotoxicity to mammalian cells was determined using mouse peritoneal macrophages. The antiparasitic activity was evaluated against axenic epimastigote and bloodstream trypomastigote forms of T. cruzi, and against amastigote forms using T. cruzi-infected macrophages. Cell death mechanism was determined in trypomastigotes by flow cytometry analysis after annexin V and propidium iodide staining. The efficacy of EEPA was examined in vivo in an acute model of infection by monitoring blood parasitaemia and survival rate 30 days after treatment. The effect against trypomastigotes of EEPA and benznidazole acting in combination was evaluated. RESULTS: EEPA effectively inhibits the epimastigote growth (IC50 2.9 ± 0.1 µM) and reduces bloodstream trypomastigote viability (EC50 1.7 ± 0.5 µM). It causes parasite cell death by necrosis. EEPA impairs parasite infectivity as well as amastigote development in concentrations noncytotoxic to mammalian cells. In mice acutely-infected with T. cruzi, EEPA reduced the blood parasitaemia in 72.7%. When combined with benznidazole, EEPA showed a synergistic anti-T. cruzi activity, displaying CI values of 0.8 ± 0.07 at EC50 and 0.83 ± 0.1 at EC90. CONCLUSION: EEPA has antiparasitic activity against T. cruzi, causing cell death by necrosis and showing synergistic activity with benznidazole. These findings were reinforced by the observed efficacy of EEPA in reducing parasite load in T. cruzi-mice. Therefore, this represents an important source of antiparasitic natural products.

Design, synthesis and structure-activity relationship of phthalimides endowed with dual antiproliferative and immunomodulatory activities.

The present work reports the synthesis and evaluation of the antitumour and immunomodulatory properties of new phthalimides derivatives designed to explore molecular hybridization and bioisosterism approaches between thalidomide, thiosemicarbazone, thiazolidinone and thiazole series. Twenty-seven new molecules were assessed for their immunosuppressive effect toward TNFα, IFNγ, IL-2 and IL-6 production and antiproliferative activity. The best activity profile was observed for the (6a-f) series, which presents phthalyl and thiazolidinone groups.

Potent anti-inflammatory activity of betulinic acid treatment in a model of lethal endotoxemia.

Betulinic acid (BA) is a lupane-type triterpene with a number of biological activities already reported. While potent anti-HIV and antitumoral activities were attributed to BA, it is considered to have a moderate anti-inflammatory activity. Here we evaluated the effects of BA in a mouse model of endotoxic shock. Endotoxemia was induced through intraperitoneally LPS administration, nitric oxide (NO) and cytokines were assessed by Griess method and ELISA, respectively. Treatment of BALB/c mice with BA at 67 mg/kg caused a 100% survival against a lethal dose of lipopolysaccharide (LPS). BA treatment caused a reduction in TNF-α production induced by LPS but did not alter IL-6 production. Moreover, BA treatment increased significantly the serum levels of IL-10 compared to vehicle-treated, LPS-challenged mice. To investigate the role of IL-10 in BA-induced protection, wild-type and IL-10(-/-) mice were studied. In contrast to the observations in IL-10(+/+) mice, BA did not protect IL-10(-/-) mice against a lethal LPS challenge. Addition of BA inhibited the production of pro-inflammatory mediators by macrophages stimulated with LPS, while promoting a significant increase in IL-10 production. BA-treated peritoneal exudate macrophages produced lower concentrations of TNF-α and NO and higher concentrations of IL-10 upon LPS stimulation. Similarly, macrophages obtained from BA-treated mice produced less pro-inflammatory mediators and increased IL-10 when compared to non-stimulated macrophages obtained from vehicle-treated mice. In conclusion, we have shown that BA has a potent anti-inflammatory activity in vivo, protecting mice against LPS by modulating TNF-α production by macrophages in vivo through a mechanism dependent on IL-10.

Use of autologous mesenchymal stem cells derived from bone marrow for the treatment of naturally injured spinal cord in dogs.

The use of stem cells in injury repair has been extensively investigated. Here, we examined the therapeutic effects of autologous bone marrow mesenchymal stem cells (MSC) transplantation in four dogs with natural traumatic spinal cord injuries. MSC were cultured in vitro, and proliferation rate and cell viability were evaluated. Cell suspensions were prepared and surgically administered into the spinal cord. The animals were clinically evaluated and examined by nuclear magnetic resonance. Ten days after the surgical procedure and MSC transplantation, we observed a progressive recovery of the panniculus reflex and diminished superficial and deep pain response, although there were still low proprioceptive reflexes in addition to a hyperreflex in the ataxic hind limb movement responses. Each dog demonstrated an improvement in these gains over time. Conscious reflex recovery occurred simultaneously with moderate improvement in intestine and urinary bladder functions in two of the four dogs. By the 18th month of clinical monitoring, we observed a remarkable clinical amelioration accompanied by improved movement, in three of the four dogs. However, no clinical gain was associated with alterations in magnetic resonance imaging. Our results indicate that MSC are potential candidates for the stem cell therapy following spinal cord injury.

Structural design, synthesis and structure-activity relationships of thiazolidinones with enhanced anti-Trypanosoma cruzi activity.

Pharmacological treatment of Chagas disease is based on benznidazole, which displays poor efficacy when administered during the chronic phase of infection. Therefore, the development of new therapeutic options is needed. This study reports on the structural design and synthesis of a new class of anti-Trypanosoma cruzi thiazolidinones (4 a-p). (2-[2-Phenoxy-1-(4-bromophenyl)ethylidene)hydrazono]-5-ethylthiazolidin-4-one (4 h) and (2-[2-phenoxy-1-(4-phenylphenyl)ethylidene)hydrazono]-5-ethylthiazolidin-4-one (4 l) were the most potent compounds, resulting in reduced epimastigote proliferation and were toxic for trypomastigotes at concentrations below 10 µM, while they did not display host cell toxicity up to 200 µM. Thiazolidinone 4 h was able to reduce the in vitro parasite burden and the blood parasitemia in mice with similar potency to benznidazole. More importantly, T. cruzi infection reduction was achieved without exhibiting mouse toxicity. Regarding the molecular mechanism of action, these thiazolidinones did not inhibit cruzain activity, which is the major trypanosomal protease. However, investigating the cellular mechanism of action, thiazolidinones altered Golgi complex and endoplasmic reticulum (ER) morphology, produced atypical cytosolic vacuoles, as well as induced necrotic parasite death. This structural design employed for the new anti-T. cruzi thiazolidinones (4 a-p) led to the identification of compounds with enhanced potency and selectivity compared to first-generation thiazolidinones. These compounds did not inhibit cruzain activity, but exhibited strong antiparasitic activity by acting as parasiticidal agents and inducing a necrotic parasite cell death.

Chemical composition and anti-Trypanosoma cruzi activity of essential oils obtained from leaves of Xylopia frutescens and X. laevigata (Annonaceae).

Essential oils from leaves of Xylopia frutescens (XFMJ) and two specimens of Xylopia laevigata (XLMC and XLSI) were obtained by hydrodistillation using a Clevenger-type apparatus, and analyzed by GC-MS and GC-FID. Sesquiterpenes dominated the essential oils. The main constituents of XFMJ were (E)-caryophyllene (24.8%), bicyclogermacrene (20.8%), germacrene D (17.0%), beta-elemene (7.9%), and (E)-beta-ocimene (6.8%). XLMC contained significant quantities of germacrene D (18.9%), bicyclogermacrene (18.4%), beta-elemene (9.5%), delta-selinene (9.2%), (E)-caryophyllene (8.5%), germacrene B (5.7%) and gamma-muurolene (5.7%), while germacrene D (27.0%), bicyclogermacrene (12.8%), (E)-caryophyllene (8.6%), gamma-muurolene (8.6%), delta-cadinene (6.8%), and germacrene B (6.0%) were the main components of XLSI. The essential oils had trypanocidal activity against the Y strain of Trypanosoma cruzi, with IC50 values lower than 30 microg x mL(-1) and 15 microg x mL(-1) against epimastigote and trypomastigote forms of T. cruzi, respectively, and were also able to reduce the percentage in vitro of T. cruzi-infected macrophages and the intracellular number of amastigotes at concentrations that were non-cytotoxic to macrophages.

Activity of antimalarial drugs in vitro and in a murine model of cutaneous leishmaniasis.

The currently used treatments for leishmaniasis, a neglected parasitic disease, are associated with several side effects, high cost and resistance of the Leishmania parasites. Here we evaluated in vitro and in vivo the antileishmanial activity of five antimalarial drugs against Leishmania amazonensis. Mefloquine was effective against promastigotes in axenic cultures and showed an IC50 (concentration giving half-maximal inhibition) value of 8.4±0.7 µM. In addition, mefloquine, chloroquine and hydroxychloroquine were active against intracellular amastigotes in macrophage-infected cultures, presenting IC50 values of 1.56±0.19 µM, 0.78±0.08 µM and 0.67±0.12 µM, respectively. The ultrastructural analysis of chloroquine- or mefloquine-treated amastigotes showed an accumulation of multivesicular bodies in the cytoplasm of the parasite, suggesting endocytic pathway impairment, in addition to the formation of myelin-like figures and enlargement of the Golgi cisternae. CBA mice were infected with L. amazonensis in the ear dermis, and treated by oral and/or topical routes with chloroquine and mefloquine. Treatment of L. amazonensis-infected mice with chloroquine by the oral route reduced lesion size, which was associated with a decrease in the number of parasites in the ear, as well as the parasite burden in the draining lymph nodes. In contrast, mefloquine administration by both routes decreased the lesion size in infected mice without causing a reduction in parasite burden. Our results revealed a promising antileishmanial effect of chloroquine and suggest its use in cutaneous leishmaniasis treatment.

Transplantation of stem cells obtained from murine dental pulp improves pancreatic damage, renal function, and painful diabetic neuropathy in diabetic type 1 mouse model.

Diabetes mellitus (DM) is one of the most common and serious chronic diseases in the world. Here, we investigated the effects of mouse dental pulp stem cell (mDPSC) transplantation in a streptozotocin (STZ)-induced diabetes type 1 model. C57BL/6 mice were treated intraperitoneally with 80 mg/kg of STZ and transplanted with 1 × 10(6) mDPSCs or injected with saline, by an endovenous route, after diabetes onset. Blood and urine glucose levels were reduced in hyperglycemic mice treated with mDPSCs when compared to saline-treated controls. This correlated with an increase in pancreatic islets and insulin production 30 days after mDPSC therapy. Moreover, urea and proteinuria levels normalized after mDPSC transplantation in diabetic mice, indicating an improvement of renal function. This was confirmed by a histopathological analysis of kidney sections. We observed the loss of the epithelial brush border and proximal tubule dilatation only in saline-treated diabetic mice, which is indicative of acute renal lesion. STZ-induced thermal hyperalgesia was also reduced after cell therapy. Three days after transplantation, mDPSC-treated diabetic mice exhibited nociceptive thresholds similar to that of nondiabetic mice, an effect maintained throughout the 90-day evaluation period. Immunofluorescence analyses of the pancreas revealed the presence of GFP(+) cells in, or surrounding, pancreatic islets. Our results demonstrate that mDPSCs may contribute to pancreatic ß-cell renewal, prevent renal damage in diabetic animals, and produce a powerful and long-lasting antinociceptive effect on behavioral neuropathic pain. Our results suggest stem cell therapy as an option for the control of diabetes complications such as intractable diabetic neuropathic pain.

Structural investigation of anti-Trypanosoma cruzi 2-iminothiazolidin-4-ones allows the identification of agents with efficacy in infected mice.

We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6-24. Compounds with a phenyl at position N3, 15-19, 22-24, exhibited better inhibitory properties for cruzain and against the parasite than 2-iminothiazolidin-4-one 5. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which inhibited the activity of cruzain and the proliferation of epimastigotes and was cidal for trypomastigotes but was not toxic for splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26-45 and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.

Optimization of anti-Trypanosoma cruzi oxadiazoles leads to identification of compounds with efficacy in infected mice.

We recently showed that oxadiazoles have anti-Trypanosoma cruzi activity at micromolar concentrations. These compounds are easy to synthesize and show a number of clear and interpretable structure-activity relationships (SAR), features that make them attractive to pursue potency enhancement. We present here the structural design, synthesis, and anti-T. cruzi evaluation of new oxadiazoles denoted 5a-h and 6a-h. The design of these compounds was based on a previous model of computational docking of oxadiazoles on the T. cruzi protease cruzain. We tested the ability of these compounds to inhibit catalytic activity of cruzain, but we found no correlation between the enzyme inhibition and the antiparasitic activity of the compounds. However, we found reliable SAR data when we tested these compounds against the whole parasite. While none of these oxadiazoles showed toxicity for mammalian cells, oxadiazoles 6c (fluorine), 6d (chlorine), and 6e (bromine) reduced epimastigote proliferation and were cidal for trypomastigotes of T. cruzi Y strain. Oxadiazoles 6c and 6d have IC(50) of 9.5 ± 2.8 and 3.5 ± 1.8 µM for trypomastigotes, while Benznidazole, which is the currently used drug for Chagas disease treatment, showed an IC(50) of 11.3 ± 2.8 µM. Compounds 6c and 6d impair trypomastigote development and invasion in macrophages, and also induce ultrastructural alterations in trypomastigotes. Finally, compound 6d given orally at 50mg/kg substantially reduces the parasitemia in T. cruzi-infected BALB/c mice. Our drug design resulted in potency enhancement of oxadiazoles as anti-Chagas disease agents, and culminated with the identification of oxadiazole 6d, a trypanosomicidal compound in an animal model of infection.

Reduction of galectin-3 expression and liver fibrosis after cell therapy in a mouse model of cirrhosis.

BACKGROUND AIMS: Cirrhosis, end-stage liver disease, is caused by different mechanisms of injury, associated with persistent inflammation. Galectin-3 is an important regulator of fibrosis that links chronic inflammation to fibrogenesis. We investigated the role of bone marrow cell (BMC) transplantation in chronic inflammation and hepatic fibrosis. METHODS: Liver cirrhosis was induced by administration of carbon tetrachloride and ethanol to wild-type C57BL/6 or bone marrow chimeric mice. Bone marrow chimeras were generated by lethal irradiation and transplantation with BMC obtained from green fluorescent protein (GFP(+) )donors. Wild-type cirrhotic mice were transplanted with BMC without irradiation. Livers from chimeras and cirrhotic transplanted mice were obtained for evaluation of inflammation, fibrosis and regulatory factors [galectin-3, matrix metallopeptidase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1 and transforming growth factor (TGF)-ß]. RESULTS: The development of cirrhosis was associated with increased expression of galectin-3 by F4/80(+) cells and intense migration of BMC to the liver. Furthermore, when transplanted after the establishment of cirrhosis, BMC also migrated to the liver and localized within the fibrous septa. Two months after BMC therapy, cirrhotic mice had a significant reduction in liver fibrosis and expression of type I collagen. We did not find any difference in levels of TGF-ß, TIMP-1 and MMP-9 between saline and BMC groups. However, the numbers of inflammatory cells, phagocytes and galectin-3(+) cells were markedly lower in the livers of cirrhotic mice treated with BMC. CONCLUSIONS: Our results demonstrate an important role for BMC in the regulation of liver fibrosis and that transplantation of BMC can accelerate fibrosis regression through modulatory mechanisms.

Coleta e cultura de células-tronco obtidas da polpa de dentes decíduos: técnica e relato de caso clínico / Collection and culture of stem cells derived from dental pulp of deciduous teeth: technique and clinical case report

INTRODUÇÃO: as células-tronco (CT) possuem capacidade de induzir a regeneração tecidual e, portanto, apresentam um potencial terapêutico. Assim como a medula óssea e o cordão umbilical, a polpa dentária é uma das fontes disponíveis de CT. O seu fácil acesso e o fato de os dentes decíduos não serem órgãos vitais, que normalmente são descartados após a esfoliação, provêm um atrativo para testes de segurança e viabilidade terapêutica dessas células. OBJETIVOS: descrever a coleta, o isolamento e o cultivo de CT obtidas da polpa de dentes decíduos, assim como a sua caracterização por meio de citometria de fluxo e da indução da diferenciação em linhagens osteogênica e adipogênica. MÉTODOS: as CT foram obtidas de forma relativamente simples e apresentaram boa capacidade proliferativa, mesmo a partir de pouca quantidade de tecido pulpar. RESULTADOS: a análise por citometria de fluxo confirmou as características de CT mesenquimais, com baixos níveis de expressão dos antígenos CD34 e CD45, que são marcadores de células hematopoiéticas, e altos níveis de expressão dos antígenos CD105, CD166, CD90 e CD73, que são marcadores de CT mesenquimais. A plasticidade das células foi confirmada pela identificação de depósitos de cálcio nas culturas que receberam meio osteogênico, e de acúmulo lipídico intracelular nas culturas que receberam meio adipogênico. CONCLUSÕES: as CT de dentes decíduos têm um potencial promissor de aplicação em regeneração tecidual. Sendo assim, é importante difundir entre os cirurgiões-dentistas o conhecimento sobre a existência e as características dessa fonte de CT, discutindo a técnica utilizada, suas limitações e possíveis indicações.

INTRODUCTION: Stem cells (SCs) are capable of inducing tissue regeneration and are, therefore, potentially therapeutic. Similarly to bone marrow and umbilical cords, dental pulp is one of the available sources of SCs. The fact that these cells are easily accessible and that deciduous teeth are not vital organs, and are normally discarded after exfoliation, make them particularly attractive for use in safety and viability tests. OBJECTIVE: To describe the collection, isolation and culture of SCs obtained from the pulp of deciduous teeth as well as their characterization by flow cytometry, and the induction of differentiation into osteogenic and adipogenic lineages. METHODS: SCs were obtained in a relatively straightforward manner and showed good proliferative capacity, even from a small amount of pulp tissue. RESULTS: Analysis by flow cytometry confirmed the characteristics of mesenchymal SCs with low expression of CD34 and CD45 antigens, which are markers for hematopoietic cells, and high levels of expression of CD105, CD166, CD90 and CD73 antigens, which are markers for mesenchymal SCs. Cell plasticity was confirmed by identifying calcium deposits in cultures that received osteogenic medium, and intracellular lipid accumulation in adipogenic cultures that received adipogenic medium. CONCLUSIONS: SCs collected from deciduous teeth show promising potential for application in tissue regeneration. Therefore, it is important that knowledge about the existence and characteristics of this source of stem cells be disseminated among dentists and that the technique, its limitations and possible indications are highlighted and discussed.