Neolignan Licarin A presents effect against Leishmania (Leishmania) major associated with immunomodulation in vitro

Leishmaniasis’ treatment is based mostly on pentavalent antimonials or amphotericin B long-term administration, expensive drugs associated with severe side effects. Considering these aforementioned, the search for alternative effective and safe leishmaniasis treatments is a necessity. This work evaluated a neolignan, licarin A anti-leishmanial activity chemically synthesized by our study group. It was observed that licarin A effectively inhibited Leishmania (Leishmania) major promastigotes (IC₅₀ of 9.59 ± 0.94 μg/mL) growth, by inducing in these parasites genomic DNA fragmentation in a typical death pattern by apoptosis. Additionally, the neolignan proved to be even more active against intracellular amastigotes of the parasite (EC₅₀ of 4.71 ± 0.29 μg/mL), and significantly more effective than meglumine antimoniate (EC₅₀ of 216.2 ± 76.7 μg/mL) used as reference drug. The antiamastigote activity is associated with an immunomodulatory activity, since treatment with licarin A of the infected macrophages induced a decrease in the interleukin (IL)-6 and IL-10 production. This study demonstrates for the first time the antileishmanial activity of licarin A and suggests that the compound may be a promising in the development of a new leishmanicidal agent.     

A comparative analysis of structure and spatial distribution of decorin in human leiomyoma and normal myometrium

Leiomyoma is a benign smooth muscle tumor of the uterus that affects many women in active reproductive life. It is composed by bundles of smooth muscle cells surrounded by extracellular matrix. We have recently shown that the glycosylation of extracellular matrix proteoglycans is modified in leiomyoma: increased amounts of galactosaminoglycans with structural modifications are present. The data here presented show that decorin is present in both normal myometrium and leiomyoma but tumoral decorin is glycosylated with longer galactosaminoglycan side chains. Furthermore, these chains contain a higher ratio D-glucuronate/L-iduronate, as compared to normal tissue. To determine if these changes in proteoglycan glycosylation correlates with modifications in the extracellular matrix organization, we compared the general structural architecture of leiomyoma to normal myometrium. By histochemical and immunofluorescence methods, we found a reorganization of muscle fibers and extracellular matrix, with changes in the distribution of glycoproteins, proteoglycans, and collagen. Thin reticular fibers, possibly composed by types I and III collagen, were replaced by thick fibers, possibly richer in type I collagen. Type I collagen colocalized with decorin both in leiomyoma and normal myometrium, in contrast to type IV collagen that did not. The relative amount of decorin was increased and the distribution of decorin and collagen was totally modified in the tumor, as compared to the normal myometrium. These findings reveal that not only decorin structure is modified in leiomyoma but also the tissue architecture changed, especially concerning extracellular matrix.     

Role of FAN in tumor necrosis factor-alpha and lipopolysaccharide-induced interleukin-6 secretion and lethality in D-galactosamine-sensitized mice

Tumor necrosis factor (TNF) alpha-induced neutral sphingomyelinase-mediated generation of ceramide, a bioactive lipid molecule, is transduced by the adaptor protein FAN, which binds to the intracellular region of the CD120a TNFalpha receptor. FAN-deficient mice do not exhibit any gross abnormality. To further explore the functions of FAN in vivo and because CD120a-deficient mice are resistant to endotoxin-induced liver failure and lethality, we investigated the susceptibility of FAN-deficient animals to lipopolysaccharide (LPS). We show that after d-galactosamine sensitization, FAN-deficient mice were partially resistant to LPS- and TNFalpha-induced lethality. Although LPS challenge resulted in a hepatic ceramide content lower in mutant mice than in control animals, it triggered similar histological alterations, caspase activation, and DNA fragmentation in the liver. Interestingly, LPS-induced elevation of IL-6 (but not TNFalpha) serum concentrations was attenuated in FAN-deficient mice. A less pronounced secretion of IL-6 was also observed after LPS or TNFalpha treatment of cultured peritoneal macrophages and embryonic fibroblasts isolated from FAN-deficient mice, as well as in human fibroblasts expressing a mutated FAN. Finally, we show that d-galactosamine-sensitized IL-6-deficient mice were partially resistant to endotoxin-induced liver apoptosis and lethality. These findings highlight the role of FAN and IL-6 in the inflammatory response initiated by endotoxin, implicating TNFalpha.     

Dual role of Rac in the assembly of NADPH oxidase, tethering to the membrane and activation of p67phox: a study based on mutagenesis of p67phox-Rac1 chimeras

NADPH oxidase activation involves the assembly of membrane-localized cytochrome b559 with the cytosolic components p47phox, p67phox, and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67phox and full-length Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47phox. We subjected chimera p67phox-(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67phox and Rac1, in the p67phox-(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67phox. This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67phox to the membrane; the other is to induce an "activating" conformational change in p67phox.     

Sensibility of the abdomen after abdominoplasty

Abdominal skin hypesthesia may occur after abdominoplasty. The purpose of this study was to find out (1) which sensibility modalities are decreased and (2) which areas of the abdominal wall are affected, so that patients can be warned preoperatively about this condition. Forty patients were divided in two groups of 20 patients each. In the control group, patients had no previous abdominal incisions. The sensibility evaluation of patients from the experimental group was made from 12 to 60 months after abdominoplasty, with an average of 31.5 months. These patients were divided into two groups of 10 patients each, a short-term follow-up group (12 to 30 months postoperatively) and a long-term follow-up group (31 to 60 months postoperatively). The abdominal skin was divided into 12 areas; nine were above the abdominoplasty incision and three were below it. Sensibility to superficial touch, superficial pain, and hot and cold modalities was recorded as positive in all areas by a variable number of patients of the experimental group. However, in area 8 (hypogastric area), a statistically significant number of patients had decreased sensibility in all sensibility modalities (Fisher's test and t test). Patients in the experimental group also showed decreased sensibility to hot and cold temperature in area 11 (pubic area). Sensibility to pressure decreased significantly in all areas of the abdomen when compared with the control group (t test). When patients of the short-term follow-up group were compared with those of the long-term follow-up group, there was no statistically significant difference for all modalities of sensibility in the areas studied, except for area 5. In this area it was found that long-term follow-up patients recovered sensibility to cold and hot temperatures. These findings help plastic surgeons to orient their patients about possible risk of exposure to injuries in the areas with decreased sensibility after abdominoplasty. Most importantly, as these patients have decreased sensibility to pressure and hot temperature in a more extensive area of the abdomen, they are exposed to a higher risk of burn injury.     

NK cells eliminate Cryptococcus neoformans by potentiating the fungicidal activity of macrophages rather than by directly killing them upon stimulation with IL-12 and IL-18

In the present study, we examined whether natural killer (NK) cells have direct fungicidal activity against Cryptococcus neoformans. Splenic NK cells were obtained from SCID mice and stimulated with a combination of interleukin (IL)-12 and IL-18 in flat culture plates or round tubes. They were then or at the same time cultured with the yeast cells and the number of viable yeast cells was examined. We could not detect direct fungicidal activity by NK cells under any culture condition, although they produced a large amount of IFN-gamma and exerted marked cytotoxic activity against YAC-1 cells. On the other hand, NK cells significantly potentiated the nitric oxide-mediated cryptococcocidal activity of thioglycolate-elicited peritoneal macrophages obtained from SCID mice upon stimulation with IL-12 and IL-18. The culture supernatants of NK cells stimulated with IL-12 and IL-18 provided similar results when used in place of NK cells. The induction of macrophage anticryptococcal activity by NK cells and NK cell culture supernatants were both mediated by IFN-gamma because the specific mAb almost completely abrogated such effect. Considered collectively, our results suggested that NK cells may play a regulatory role in potentiating macrophage-mediated fungicidal mechanisms in host resistance to infection with C. neoformans rather than exerting a direct killing activity against the fungal pathogen.     

Effects of methotrexate upon inflammatory parameters induced by carrageenan in the mouse model of pleurisy

BACKGROUND: The model of pleurisy induced by carrageenan exhibits a biphasic response (4 and 48 h) and permits the quantification of exudate, cell migration and certain enzymes such as myeloperoxidase (MPO) and adenosine-deaminase (ADA) that are markers of activated leukocytes. AIMS: The present study evaluates whether there exists, in the pleurisy model, a significant inhibition of ADA and MPO enzymes, leukocyte kinetics and other markers of inflammation [nitric oxide (NO) levels, exudation] caused by methotrexate treatment by the intraperitoneal (i.p.) route. METHODS: The pleurisy was induced by carrageenan (1%) in mice, and the parameters were analyzed 4 and 48 h after. RESULTS: After the induction of inflammation (4 h), methotrexate (20 mg/kg, i.p., 24 h before pleurisy induction) inhibited the leukocyte infiltration (p < 0.05), NO levels and MPO activity (p < 0.01), but not ADA activity and fluid leakage (p > 0.05). Regarding the second phase of pleurisy (48 h), methotrexate (40 mg/kg, i.p., 0.5 h before pleurisy induction) inhibited the leukocyte infiltration (p < 0.05), fluid leakage, NO levels (p < 0.01), and ADA and MPO activity (p < 0.05). CONCLUSIONS: These findings support the evidence that the acute administration of methotrexate has an important systemic anti-inflammatory activity in the studied inflammatory model. This effect was due to a significant inhibition on both neutrophil and mononuclear cells, being less marked in relation to exudation 48 h after. In relation to the enzymes studied and to NO levels, the findings support the evidence that methotrexate inhibits both enzymes (MPO and ADA) from leukocytes at the site of injury, thus reflecting the activation of both neutrophils and lymphocytes, respectively. Furthermore, the inhibiting effect on NO in both phases of pleurisy induced by carrageenan (4 and 48 h) indicates that methotrexate acts on constitutive and/or inducible NO synthases by means of different cells of the pleural cavity.     

Inhibition of the AnxA1/FPR1 autocrine axis reduces MDA-MB-231 breast cancer cell growth and aggressiveness in vitro and in vivo

Breast Cancer (BC) is a highly heterogeneous disease whose most aggressive behavior is displayed by triple-negative breast cancer (TNBC), which lacks an efficient targeted therapy. Despite its controversial role, one of the proteins that having been linked with BC is Annexin A1 (AnxA1), which is a Ca⁺² binding protein that acts modulating the immune system, cell membrane organization and vesicular trafficking. In this work we analyzed tissue microarrays of BC samples and observed a higher expression of AnxA1 in TNBCs and in lymph node metastasis. We also observed a positive correlation in primary tumors between expression levels of AnxA1 and its receptor, FPR1. Despite displaying a lesser strength, this correlation also exists in BC lymph node metastasis. In agreement, we have found that AnxA1 was highly expressed and secreted in the TNBC cell line MDA-MB-231 that also expressed high levels of FPR1. Furthermore, we demonstrated, by using the specific FPR1 inhibitor Cyclosporin H (CsH) and the immunosuppressive drug Cyclosporin A (CsA), the existence of an autocrine signaling of AnxA1 through the FPR1. Such signaling, elicited by AnxA1 upon its secretion, increased the aggressiveness and survival of MDA-MB-231 cells. In this manner, we demonstrated that CsA works very efficiently as an FPR1 inhibitor. Finally, by using CsA, we demonstrated that FPR1 inhibition decreased MDA-MB-231 tumor growth and metastasis formation in nude mice. These results indicate that FPR1 inhibition could be a potential intervention strategy to manage TNBCs displaying the characteristics of MDA-MB-231 cells. FPR1 inhibition can be efficiently achieved by CsA.     

Signature of Aberrantly Expressed microRNAs in the Striatum of Rotenone-Induced Parkinsonian Rats

Parkinson’s disease (PD) is a highly complex brain disorder regarding clinical presentation, pathogenesis, and therapeutics. The cardinal motor signs, i.e., rigidity, bradykinesia, and unilateral tremors, arise in consequence of a progressive neuron death during the prodromal phase. Although multiple transmission systems are involved in disease neurobiology, patients will cross the line between the prodromal and early stage of diagnosed PD when they had lost half of the dopaminergic nigrostriatal cells. As the neurons continue to die ascending the neuroaxis, patients will face a more disabling disease with motor and nonmotor signs. Shedding light on molecular mechanisms of neuron death is an urgent need for understanding PD pathogenesis and projecting therapeutics. This work examined the expression of microRNAs in the striatum of parkinsonian rats chronically exposed to rotenone (2.5 mg/Kg, i.p., daily for 10 days). Rotenone caused motor deficits, the loss of TH(+) cells in the nigrostriatal pathway, and a marked microgliosis. This parkinsonian rat striatum was examined at 26 days after the last rotenone injection, for quantification of microRNAs, miR-7, miR-34a, miR-26a, miR-132, miR-382, and Let7a, by qPCR. Parkinsonian rats presented a significant increase in miR-26a and miR-34a (1.5 and 2.2 fold, respectively, P?<?0.05), while miR-7 (0.5 fold, P?<?0.05) and Let7a were downregulated. This work reports for first time microRNAs aberrantly expressed in the striatum of rotenone-induced parkinsonian rats, suggesting that this dysregulation may contribute to PD pathogenesis. Beyond revealing new clues of neurodegeneration, our findings might prime further studies targeting miRNAs for neuroprotection or even for diagnosis proposal.