T cell maturation is significantly affected by SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). An adequate T cell response is essential not only for fighting disease but also for the creation of immune memory. Thus, the present study aims to evaluate the T cells of patients with moderate, severe and critical COVID-19 not only at the time of illness but also 2 months after diagnosis to observe whether changes in this compartment persist. In this study, 166 COVID-19 patients were stratified into moderate/severe and critical disease categories. The maturation and activation of T cells were evaluated through flow cytometry. In addition, Treg cells were analysed. Until 15 days after diagnosis, patients presented a reduction in absolute and relative T lymphocyte counts. After 2 months, in moderate/severe patients, the counts returned to a similar level as that of the control group. In convalescent patients who had a critical illness, absolute T lymphocyte values increased considerably. Patients with active disease did not show differentiation of T cells. Nonetheless, after 2 months, patients with critical COVID-19 showed a significant increase in CD4+ EMRA (CD45RA+ effector memory) T lymphocytes. Furthermore, COVID-19 patients showed delayed T cell activation and reduced CD8+ suppressor T cells even 2 months after diagnosis. A reduction in CD4+ Treg cells was also observed, and their numbers returned to a similar level as that of healthy controls in convalescent patients. The results demonstrate that COVID-19 patients have a delayed activation and differentiation of T cells. In addition, these patients have a great reduction of T cells with a suppressor phenotype.

Anti-Protozoan Activities of Polar Fish-Derived Polyalanine Synthetic Peptides.

Chagas disease, sleeping sickness and malaria are infectious diseases caused by protozoan parasites that kill millions of people worldwide. Here, we performed in vitro assays of Pa-MAP, Pa-MAP1.9, and Pa-MAP2 synthetic polyalanine peptides derived from the polar fish Pleuronectes americanus toward Trypanosoma cruzi, T. brucei gambiense and Plasmodium falciparum activities. We demonstrated that the peptides Pa-MAP1.9 and Pa-MAP2 were effective to inhibit T. brucei growth. In addition, structural analyses using molecular dynamics (MD) studies showed that Pa-MAP2 penetrates deeper into the membrane and interacts more with phospholipids than Pa-MAP1.9, corroborating the previous in vitro results showing that Pa-MAP1.9 acts within the cell, while Pa-MAP2 acts via membrane lysis. In conclusion, polyalanine Pa-MAP1.9 and Pa-MAP2 presented activity against bloodstream forms of T. b. gambiense, thus encouraging further studies on the application of these peptides as a treatment for sleeping sickness.

The relationship between peripheral immune response and disease severity in SARS-CoV-2-infected subjects: A cross-sectional study.

Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and marked by an intense inflammatory response and immune dysregulation in the most severe cases. In order to better clarify the relationship between peripheral immune system changes and the severity of COVID-19, this study aimed to evaluate the frequencies and absolute numbers of peripheral subsets of neutrophils, monocytes, and dendritic cells (DCs), in addition to quantifying the levels of inflammatory mediators. One hundred fifty-seven COVID-19 patients were stratified into mild, moderate, severe, and critical disease categories. The cellular components and circulating cytokines were assessed by flow cytometry. Nitric oxide (NOx) and myeloperoxidase (MPO) levels were measured by colourimetric tests. COVID-19 patients presented neutrophilia, with signs of emergency myelopoiesis. Alterations in the monocytic component were observed in patients with moderate to critical illness, with an increase in classical monocytes and a reduction in nonclassical monocytes, in addition to a reduction in the expression of HLA-DR in all subtypes of monocytes, indicating immunosuppression. DCs, especially plasmacytoid DCs, also showed a large reduction in moderate to critical patients. COVID-19 patients showed an increase in MPO, interleukin (IL)-12, IL-6, IL-10, and IL-8, accompanied by a reduction in IL-17A and NOx. IL-10 levels ≥14 pg/ml were strongly related to the worst outcome, with a sensitivity of 78·3% and a specificity of 79·1%. The results of this study indicate the presence of systemic effects induced by COVID-19, which appear to be related to the pathophysiology of the disease, highlighting the potential of IL-10 as a possible prognostic biomarker for COVID-19.

Trypanosoma cruzi infection increases atherosclerotic lesion in ApoE-deficient mice.

Apolipoprotein E (ApoE) is the major ligand for the transporting and removal of chylomicrons and lipoproteins by the liver. Since the creation of the ApoE-knockout mice, it is well established that ApoE deficiency results in spontaneous atherosclerosis in aged animals. Atherosclerosis is also observed in animals infected with Trypanosoma cruzi, a protozoan that elicits a systemic inflammatory response in mammalian hosts, culminating in damage to cardiac, neuronal, and endothelial cells. Pro-atherogenic effects related to the experimental infection with T. cruzi may be induced by inflammatory components affecting the vascular wall. Herein, we evaluated whether infection with different strains of T. cruzi worsened the atherogenic lesions observed in aged ApoE-/- mice. After four weeks of infection with Berenice-78 (Be-78) or Colombian (Col) strains of the parasite, mice presented increased CCL2 and CCL5 production and high migration of inflammatory cells to cardiac tissue. Although the infection with either strain did not affect the survival rate, only the infection with Col strain caused abundant parasite growth in blood and heart and increased aortic root lesions in ApoE-/- mice. Our findings show, for the first time that ApoE exerts a protective anti-atherosclerotic role in the aortic root of mice in the acute phase of experimental infection with the Col strain of T. cruzi. Further studies should target ApoE and nutritional interventions to modulate susceptibility to cardiovascular disabilities after T. cruzi infection, minimizing the risk of death in both experimental animals and humans.

Perinatal exposure to a glyphosate pesticide formulation induces offspring liver damage.

The present study investigated the effects of perinatal exposure to glyphosate-based herbicide (GBH) in offspring's liver. Pregnant Wistar rats were exposed to GBH (70 mg glyphosate/Kg body weight/day) in drinking water from gestation day 5 to postnatal day 15. The perinatal exposure to GBH increased 45Ca2+ influx in offspring's liver. Pharmacological tools indicated a role played by oxidative stress, phospholipase C (PLC) and Akt pathways, as well as voltage-dependent Ca2+ channel modulation on GBH-induced Ca2+ influx in offspring's liver. In addition, changes in the enzymatic antioxidant defense system, decreased GSH content, lipid peroxidation and protein carbonylation suggest a connection between GBH-induced hepatotoxic mechanism and redox imbalance. The perinatal exposure to GBH also increased the enzymatic activities of transaminases and gamma-glutamyl transferase in offspring's liver and blood, suggesting a pesticide-induced liver injury. Moreover, we detected increased iron levels in liver, blood and bone marrow of GBH-exposed rats, which were accompanied by increased transferrin saturation and decreased transferrin levels in blood. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were increased in the liver of rats perinatally exposed to GBH, which were associated with. Increased phospho-p65NFκB immunocontent. Therefore, we propose that excessive amounts of iron in offspring's liver, blood and bone marrow induced by perinatal exposure to GBH may account for iron-driven hepatotoxicity, which was associated with Ca2+ influx, oxidative damage and inflammation. Further studies will clarify whether these events can ultimately impact on liver function.

Involvement of the NF-κB and PI3K/Akt/mTOR pathways in cell death triggered by stypoldione, an o-quinone isolated from the brown algae Stypopodium zonale.

Multiple myeloma (MM) is a clonal plasma cell malignancy that remains incurable to date. Thus, the aims of this study were to evaluate the involvement of the NF-κB and PI3K/Akt/mTOR pathways in the cytotoxicity of stypoldione, an o-quinone isolated from the brown algae Stypopodium zonale, in MM cells (MM1.S). The cytotoxic effect was evaluated in MM1.S cells and peripheral blood mononuclear cells (PBMCs) by MTT assay. The stypoldione reduced the cell viability of MM1.S cells in a concentration and time-dependent manner (IC50 in MM.1S from 2.55 to 5.38 µM). However, it was also cytotoxic to PBMCs, but at a lower range. Additionally, no significant hemolysis was observed even at concentration up to 10 times the IC50 . Apoptotic cell death was confirmed by cell morphology and Annexin V-FITC assay. Stypoldione induced intrinsic and extrinsic apoptosis by increasing FasR expression and reactive oxygen species (ROS) production, inverting the Bax/Bcl-2 ratio, and inducing ΔΨm loss, which resulted in AIF release and caspase-3 activation. It also increased Ki-67 and survivin expression and inhibited the NF-κB and PI3K/Akt/mTOR pathways. These results suggest that stypoldione is a good candidate for the development of new drugs for MM treatment.

Caspase-11-dependent IL-1α release boosts Th17 immunity against Paracoccidioides brasiliensis.

The granulomatous lesion resulting from infection with the fungus Paracoccidioides brasiliensis is characterized by a compact aggregate of mature cells, surrounded by a fibroblast- and collagen-rich content. Granuloma formation requires signaling elicited by inflammatory molecules such as members of the interleukin-1 family. Two members of this family have been thoroughly studied, namely IL-1α and IL-1ß. In this study, we addressed the mechanisms underlying IL-1α secretion and its functional role on the host resistance to fungal infection. We found that, the expression of caspase-11 triggered by P. brasiliensis infection of macrophages depends on IFN-ß production, because its inhibition reduced procaspase-11 levels. Curiously, caspase-11 deficiency did not impair IL-1ß production, however caspase-11 was required for a rapid pore-mediated cell lysis. The plasma membrane rupture facilitated the release of IL-1α, which was necessary to induce NO production and restrict fungal replication. Furthermore, P. brasiliensis-infected macrophages required IL-1α to produce optimal levels of IL-6, a major component of Th17 lymphocyte differentiation. Indeed, IL-1α deficiency accounted for a significant reduction of Th17 lymphocytes in lungs of infected mice, correlating with diminished neutrophil infiltration in the lungs. Strikingly, we identified that IL-1α directly reprograms the transcriptional profile of Th17-committed lymphocytes, increasing cellular proliferation, as for boosting IL-17 production by these cells. Beyond neutrophil chemotaxis in vivo, IL-17 also amplified IL-1α production by infected macrophages in vitro, endorsing a critical amplification loop of the inflammatory response. Therefore, our data suggest that the IFN-ß/caspase-11/IL-1α pathway shapes a protective antifungal Th17 immunity, revealing a molecular mechanism underlying the cross-talk between innate and adaptive immunity.

Synthesis of chalcones derived from 1-naphthylacetophenone and evaluation of their cytotoxic and apoptotic effects in acute leukemia cell lines.

Chalcones and their derivatives have been described as promising compounds with antiproliferative activity against leukemic cells. This study aimed to investigate the cytotoxic effect of three synthetic chalcones derived from 1-naphthylacetophenone (F07, F09, and F10) in acute leukemia cell lines (K562 and Jurkat) and examine the mechanisms of cell death induced by these compounds. The three compounds were cytotoxic to K562 and Jurkat cells, with IC50 values ranging from 1.03 to 31.66 µM. Chalcones induced intrinsic and extrinsic apoptosis, resulting in activation of caspase-3 and DNA fragmentation. F07, F09, and F10 were not cytotoxic to human peripheral blood mononuclear cells, did not produce any significant hemolytic activity, and did not affect platelet aggregation after ADP stimulation. These results, combined with calculations of molecular properties, suggest that chalcones F07, F09, and F10 are promising molecules for the development of novel antileukemic drugs.

Cell Cycle Arrest and Apoptosis Induction by a New 2,4-Dinitrobenzenesulfonamide Derivative In Acute Leukemia Cells.

BACKGROUND: Current therapies for acute leukemias (ALs) are associated with severe adverse reactions and high relapse rates, which makes the search for new antileukemic agents a necessity. Therefore, the aim of this study was to evaluate the effects of a new sulfonamide, S1, in AL cells K562 and Jurkat. METHODS: The cytotoxic activity of S1 was assessed using MTT method. The involvement of apoptosis in the mechanism of cell death was assessed by flow cytometry and fluorescence microscopy. RESULTS: Our results demonstrated that S1 induced morphological changes suggestive of apoptosis in both K562 and Jurkat cells. Additionally, S1 was not cytotoxic to normal erythrocytes and mononuclear cells and had a highly selective cytotoxicity for AL lineages. The mechanisms of cell death induced by S1 in K562 cells involves cell cycle arrest at G2/M phase and the activation of both extrinsic and intrinsic apoptosis, with an increased FasR and AIF expression and the loss of mitochondrial potential. As for Jurkat, we observed cell cycle blockade at G0/G1 phase, phosphatidylserine exposure and the involvement of intrinsic apoptosis only, with mitochondrial potential loss and a reduced expression of Survivin.  Although sulfonamide S1 did not altered Bcl-2 and Bax expression in AL cell lines, it was able to activate caspase-3 in K562 cells. CONCLUSION: Our results suggest that sulfonamide S1 may be a promising candidate for the development of new drugs for the treatment of ALs.

Immune response of Brazilian farmers exposed to multiple pesticides.

Occupational exposure to pesticides has been identified as a factor that predisposes to disorders of the immune system. Immunosuppression, autoimmunity, cancer of various organs and other diseases in people who apply these products have been reported by the studies. This study aimed to investigate the relationship between occupational exposure to pesticides and the immunological profile in 43 farmers exposed to mixtures of pesticides for at least 15 years. A control group composed of 30 individuals without a history of occupational exposure to pesticides was also evaluated. Peripheral blood samples were processed by flow cytometry and cells were labelled with an 8-color monoclonal antibody panel. Plasma cytokines were also measured. Significant increase in classical monocytes (p < 0.001) and dendritic cells (p < 0.001) in the exposed group was observed as well in total T cells (p = 0.04), central memory CD8 T cells (p = 0.02) and effector memory CD8 T cells (p = 0.01). On the other hand, the activation markers of T cells as the expression of CD57, HLA-DR, CD25 and CD28 were evaluated and no difference was found between groups. When the B cells were analyzed, a significant decrease in total B cells (p = 0.01), regulatory B cells (p < 0.001) and plasmablasts (p < 0.001) in the exposed group, compared to healthy controls, was observed. Pro-inflammatory IL-6 was significantly elevated (p = 0.04) in the plasma of farmers compared to that of controls. The constant antigenic stimulus that occurs during exposure to pesticides can favor the recruitment of dendritic cells and macrophages (APCs) presents in the skin and respiratory tract. In the secondary lymphoid organs, the CD4 T and B cells that process such antigens are possibly undergoing proliferative exhaustion, with the consequent depletion of all mature B subpopulations. The resulting drop in humoral immunity may be offset by an increase in the number of circulating CD8 T lymphocytes due to their cytotoxic action.

Modulation of ERG Genes Expression in Clinical Isolates of Candida tropicalis Susceptible and Resistant to Fluconazole and Itraconazole.

Candida tropicalis is a non-albicans Candida specie that causes candidosis in several countries, including Brazil. However, little is known about the mechanisms of drug resistance in C. tropicalis infections. In this study, we used clinical isolates of C. tropicalis susceptible as well as resistant to either Fluconazole or Itraconazole to assess the relationship between drug resistance and the expression of ERG and efflux pump genes. Our results showed that the main mechanism of resistance against both Fluconazole and Itraconazole in this specie is through the up-regulation of ERG rather than that of the efflux pump genes. We demonstrated that, although pre-treatment with azole drugs increases the expression of both ERG6 and ERG11 genes, the resistant or susceptible dose-dependent (SDD) samples are able to maintain high expression levels of these genes for longer periods of time than the susceptible samples.

MICONIDINE acetate, a new selective and cytotoxic compound with synergic potential, induces cell cycle arrest and apoptosis in leukemia cells.

Plants are important sources of biologically active compounds and they provide unlimited opportunities for the discovery and development of new drug leads, including new chemotherapeutics. Miconidin acetate (MA) is a hydroquinone derivative isolated from E. hiemalis. In this study we demonstrated that MA was cytotoxic against acute leukemia (AL), solid tumor cells and cancer stem cells, with the strongest effect exhibited against AL. Furthermore, it was non-cytotoxic against non-tumor cells and did not cause significant hemolysis. MA blocks the G2/M phase and causes cytostatic effects, acting in a similar way to dexamethasone by increasing PML expression. The compound also triggered intrinsic and extrinsic apoptosis by modulating Bax, FasR and survivin expression. This led to an extensive mitochondrial damage that resulted in AIF, cytochrome c and endonuclease G release, caspase-3 and PARP cleavage and DNA fragmentation. We have further demonstrated that MA was strongly cytotoxic against neoplastic cells collected from patients with different AL subtypes. Interestingly, MA increased the cytotoxic effect of chemotherapeutics cytarabine and vincristine. This study indicates that MA may be a new agent for AL and highlights its potential as a new source of anticancer drugs. Graphical abstract MA blocks G2/M phase with PML expression and KI67 inhibition, ROS generation and intrinsic and extrinsic apoptosis, leading to mitochondrial damage, caspase 3 and PARP cleavage and DNA fragmentation.

Synthesis of novel pyrazoline derivatives and the evaluation of death mechanisms involved in their antileukemic activity.

Malignant neoplasms are one of the leading causes of death worldwide and hematologic malignancies, including acute leukemia (AL) is one of the most relevant cancer types. Current available chemotherapeutics are associated with high morbidity and mortality rates, therefore, the search for new molecules with antitumor activity, specific and selective for neoplastic cells, became a great challenge for researchers in the oncology field. As pyrazolines stand out in the literature for their great variety of biological activities, the aim of this study was to synthesize and evaluate the antileukemic activity of five new pyrazoline derivatives. All pyrazolines showed adequate physicochemical properties for a good oral bioavailability. The two unpublished and most effective pyrazoline derivatives have been selected for further experiments. These compounds are highly selective for leukemic cells when compared to non-neoplastic cells and did not cause lysis on human red blood cells. Additionally, selected pyrazolines induced cell cycle arrest at G0/G1 phase and decreased cell proliferation marker KI67. Apoptotic cell death induced by selected pyrazolines was confirmed by morphological analysis, assessment of phosphatidylserine residue exposure and DNA fragmentation. Several factors indicate that both intrinsic and extrinsic apoptosis occurred. These were: increased FasR expression; the predominance of Bax in relation to Bcl-2; the loss of mitochondrial membrane potential; AIF release; decreased expression of survivin (an antiapoptotic protein); and the activation of caspase-3. The selected pyrazolines were also found to be cytotoxic against neoplastic cells collected from the peripheral blood and bone marrow of patients with different subtypes of acute leukemia.

Increased in vitro leishmanicidal activity of octyl gallate loaded poly(methyl methacrylate) nanoparticles.

The current paucity of effective and affordable drugs for the treatment of leishmaniasis renders the search for new therapeutic alternatives a priority. Gallic acid-related compounds display anti-parasitic activities and their incorporation into drug carrier systems, such as polymeric nanoparticles may be a viable alternative for leishmaniasis treatment. Therefore, this study focused on the synthesis and characterization of octyl gallate (G8) loaded poly(methyl methacrylate) (PMMA) nanoparticles via miniemulsion polymerization in order to increase the leishmanicidal activity of this compound. G8 loaded PMMA nanoparticles presented a spherical morphology with a mean size of 108 nm, a negatively charged surface (-33 ± 5 mV) and high encapsulation efficiency (83% ± 5). Fourier-transform infrared spectroscopy and X-ray diffraction analysis confirmed that G8 was encapsulated in PMMA nanoparticles and presented a biphasic release profile. The G8 loaded PMMA nanoparticles did not present cytotoxic effect on human red blood cells. G8 loaded PMMA nanoparticles displayed a leishmanicidal activity almost three times higher than free G8 while the cytotoxic activity against human THP-1 cells remained unchanged.

Effects of 1,3,5-triphenyl-4,5-dihydro-1H-pyrazole derivatives on cell-cycle and apoptosis in human acute leukemia cell lines.

Pyrazoline is an important 5-membered nitrogen heterocycle that has been extensively researched. Ten derivatives were synthesized and tested for antileukemic effects on 2 human acute leukemia cell lines, K562 and Jurkat. The most cytotoxic of these derivatives, compound 21, was chosen for investigation of cytotoxicity mechanisms. The results obtained with selectivity calculations revealed that compound 21 is more selective for acute leukemia (K562 and Jurkat cell lines) than for other tumor cell lines. Moreover, compound 21 was not cytotoxic to normal cell lines, indicating a potential use in clinical tests. Compound 21 caused a significant cell cycle arrest in the S-phase in Jurkat cells and increased the proportion of cells in the sub G0/G1 phase in both cell lines. Cells treated with compound 21 demonstrated morphological changes characteristic of apoptosis in the EB/AO assay, confirmed by externalization of phosphatidylserine by the annexin V - fluorescein isothiocyanate method and by DNA fragmentation. An investigation of cytotoxicity mechanisms suggests the involvement of an intrinsic apoptosis pathway due to mitochondrial damage and an increase in the ratio of mitochondrial Bax/Bcl2. Pyrazoline 21 obeyed Lipinski's "rule of five" for drug-likeness. Based on these preliminary results, the antileukemic activity of compound 21 makes it a potential anticancer agent.

Investigation of cellular mechanisms involved in apoptosis induced by a synthetic naphthylchalcone in acute leukemia cell lines.

We have previously reported the cytotoxic effects of chalcone A1, derived from 1-naphthaldehyde, in leukemia cell lines. On the basis of these findings, the main aim of this study was to elucidate some of the molecular mechanisms involved in apoptosis induced by chalcone A1 toward K562 and Jurkat cells. In both cell lines, chalcone A1 decreased the mitochondrial membrane potential, increased the expression of Bax proapoptotic protein, and decreased the expression of Bcl-2 antiapoptotic protein (resulting in the inversion of the Bcl-2/Bax ratio), which indicates the involvement of the intrinsic pathway. In addition, chalcone A1 increased the expression of FasR in Jurkat cells, which also indicates the involvement of the extrinsic pathway in this cell line. The results also showed an increased expression of effector caspase-3 and cleaved PARP-1 and a decreased expression of IAP protein survivin, which are consistent with apoptotic cell death. The decreased expression of Ki67 suggests that the mechanism involved in cell death induced by chalcone A1 also involves a decrease in cell proliferation. In ex-vivo experiments, chalcone A1 reduced the cell viability of blast cells collected from eight patients with different types of acute leukemia, confirming the cytotoxicity results found in vitro. The results obtained so far are very promising and further studies need to be carried out so that chalcone A1 can be used as a prototype for the development of new antileukemia agents.

Oral Delivery of a High Quercetin Payload Nanosized Emulsion: In Vitro and In Vivo Activity Against B16-F10 Melanoma.

Quercetin is a natural compound that has several biological activities including anticancer activity. However, the use of this drug has been limited mostly because of its poor water solubility and low bioavailability. Therefore, the development of quercetin-loaded nanocarrier systems may be considered a promising advance to exploit its therapeutic properties in clinical setting including cancer treatment. This study evaluates the effect of orally administered nanosized emulsion containing quercetin (QU-NE) on the cytotoxicity activity against B16-F10 cells in vitro, and on subcutaneous melanoma in mice inoculated with B16-F1O cells. In vivo experiments, also evaluate the co-administration of quercetin with cisplatin in order to predict synergic effects and the renal and hepatic toxicity. The nanocarriers were prepared through the hot solvent diffusion associated with the phase inversion temperature methods. In vitro study showed reduction of cell viability in a concentration-depend manner for free quercetin and QU-NE. In vivo study, quercetin either as a free drug or colloidal dispersion was administrated at a dose of 5 mg kg(-1) twice a week for 17 days via oral route. Cisplatin was administrated at dose of 1 mg kg(-1) once a week intraperitoneally. Free quercetin and QU-NE reduced tumor growth, however, the reduction observed for QU-NE (P < 0.001 vs. control) was significantly higher than free quercetin (P < 0.05 vs. control). The association of both drugs did not show synergic effect. Besides, no renal or hepatic toxicities were observed after administration of free quercetin and QU-NE. These results suggest that an improvement in the oral bioavailability of quercetin occurred when this compound was dissolved in the oily phase of a nanosized emulsion, indicating that it might have a potential application in the treatment of melanoma.

Doxycycline and Benznidazole Reduce the Profile of Th1, Th2, and Th17 Chemokines and Chemokine Receptors in Cardiac Tissue from Chronic Trypanosoma cruzi-Infected Dogs.

Chemokines (CKs) and chemokine receptors (CKR) promote leukocyte recruitment into cardiac tissue infected by the Trypanosoma cruzi. This study investigated the long-term treatment with subantimicrobial doses of doxycycline (Dox) in association, or not, with benznidazole (Bz) on the expression of CK and CKR in cardiac tissue. Thirty mongrel dogs were infected, or not, with the Berenice-78 strain of T. cruzi and grouped according their treatments: (i) two months after infection, Dox (50 mg/kg) 2x/day for 12 months; (ii) nine months after infection, Bz (3,5 mg/kg) 2x/day for 60 days; (iii) Dox + Bz; and (iv) vehicle. After 14 months of infection, hearts were excised and processed for qPCR analysis of Th1 (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL11), Th2 (CCL1, CCL17, CCL24, and CCL26), Th17 (CCL20) CKs, Th1 (CCR5, CCR6, and CXCR3), and Th2/Th17 (CCR3, CCR4, and CCR8) CKR, as well as IL-17. T. cruzi infection increases CCL1, CCL2, CCL4, CCL5, CCL17, CXCL10, and CCR5 expression in the heart. Dox, Bz, or Dox + Bz treatments cause a reversal of CK and CKR and reduce the expression of CCL20, IL-17, CCR6, and CXCR3. Our data reveal an immune modulatory effect of Dox with Bz, during the chronic phase of infection suggesting a promising therapy for cardiac protection.

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid.

Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Results showed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of the polymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0-1 h) followed by a slow and sustained release, indicating a biphasic release system. Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggests that this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.

Curcumin-Loaded Chitosan-Coated Nanoparticles as a New Approach for the Local Treatment of Oral Cavity Cancer.

Mucoadhesive nanoparticles loaded with curcumin were developed as a new approach to deliver curcumin for the local treatment of oral cancer. PCL nanoparticles coated with chitosan displaying different molar masses were prepared by using the nanoprecipitation technique. The mucoadhesive properties of nanoparticle suspensions were demonstrated by their strong ability to interact with the glycoprotein mucin through electrostatic interactions. Similar permeation profiles of curcumin loaded in uncoated and chitosan-coated nanoparticles across porcine esophageal mucosa were verified. Curcumin concentrations retained in the mucosa suggest the possibility of a local effect of the drug. In vitro studies demonstrated that free curcumin.and curcumin loaded into nanoparticles coated with chitosan caused significant reduction of SCC-9 human oral cancer cell viability in a concentration and time-dependent manner. However, no significant cell death was observed after 24 h of treatment with unloaded nanoparticles coated with chitosan. In addition, curcumin-loaded nanoparticles showed reduced cytotoxicity, when compared with the free drug. Therefore, chitosan-coated PCL nanoparticles may be considered a promising strategy to deliver curcumin directly into the oral cavity for the treatment of oral cancer.