Increase of reactive oxygen species by desferrioxamine during experimental Chagas' disease.

Oxidative stress is common in inflammatory processes associated with many diseases including Chagas' disease. The aim of the present study was to evaluate, in a murine model, biomarkers of oxidative stress together with components of the antioxidant system in order to provide an overview of the mechanism of action of the iron chelator desferrioxamine (DFO). The study population comprised 48 male Swiss mice, half of which were treated daily by intraperitoneal injection of DFO over a 35-day period, while half were administered sterile water in a similar manner. On the 14th day of the experiment, 12 DFO-treated mice and an equal number of untreated mice were experimentally infected with Trypanosoma cruzi. Serum concentrations of nitric oxide and superoxide dismutase and hepatic levels of total glutathione, thiobarbituric acid reactive species and protein carbonyl, were determined on days 0, 7, 14 and 21 post-infection. The results obtained revealed that DFO enhances antioxidant activity in the host but also increases oxidative stress, indicating that the mode of action of the drug involves a positive contribution to the host together with an effect that is not beneficial to the parasite

Trypanosoma cruzi: desferrioxamine decreases mortality and parasitemia in infected mice through a trypanostatic effect.

Desferrioxamine (DFO) is a potent iron chelator that is also known to modulate inflammation and act as an efficient antioxidant under normal conditions and under oxidative stress. Many in vitro and in vivo studies have shown the efficacy of DFO in the treatment of viral, bacterial and protozoan infections. DFO is known to reduce the intensity of Trypanosoma cruzi infections in mice even during a course of therapy that is not effective in maintaining anaemia or low iron levels. To further clarify these findings, we investigated the action of DFO on mouse T. cruzi infection outcomes and the direct impact of DFO on parasites. Infected animals treated with DFO (5 mg/animal/day) for 35 days, beginning 14 days prior to infection, presented lower parasitemia and lower cumulative mortality rate. No significant effect was observed on iron metabolism markers, erythrograms, leukograms or lymphocyte subsets. In the rapid method for testing in vivo T. cruzi susceptibility, DFO also induced lower parasitemia. In regard to its direct impact on parasites, DFO slightly inhibited the growth of amastigotes and trypomastigotes in fibroblast culture. Trypan blue staining showed no effects of DFO on parasite viability, and only minor apoptosis in trypomastigotes was observed. Nevertheless, a clear decrease in parasite mobility was detected. In conclusion, the beneficial actions of DFO on mice T. cruzi infection seem to be independent of host iron metabolism and free of significant haematological side effects. Through direct action on the parasite, DFO has more effective trypanostatic than trypanocidal properties

Low Density Lipoproteins at 2% Concentration Can Replace Whole Egg Yolk in TES-Tris-Milk Extender for Freezing Buffalo Sperm Cells

Background: Over the years, the most commonly used extenders for semen cryopreservation contain egg yolk as cryoprotectant. However, more recent studies have used the low density lipoproteins, extract of hen egg yolk which is responsible for the cryoprotective effect. Nevertheless, little was known about its required minimum concentration as well as its interaction with other extra cellular cryoprotectants, like skimmed milk. The present study aimed at investigating the effect of replacing whole egg yolk by adding low density lipoproteins at low concentrations, in TES-Tris-skim milk based extender, on the post-thaw quality of buffalo bull sperm.Materials, Methods & Results: Eighteen ejaculates were collected from six buffalo bulls and diluted with TES-Tris-skim milk based extender containing LDL, extracted from hen egg yolks, at the concentrations of 2%, 4%, 8% and 14%, against a control extender containing 20% fresh egg yolk. After semen collection, analyses of subjective motility, vigor, force tourbillon, sperm concentration (Neubauer chamber) and sperm morphology (phase contrast microscopy) were performed. The diluted semen was packaged in 0.25 mL straws, and cooling was performed on computerized machine (TK 4000®), using a cooling rate of -0.25°C/min to 5°C. Semen was kept in balance at 5°C for 4 h. The straws were frozen in an ice chest, kept at 5 cm from the surface of liquid nitrogen for 20 min and then immersed in liquid nitrogen. The samples were kept in cryogenic container until thawing. Post-thaw kinetic parameters during incubation at 37°C (CASA), sperm membrane integrity (SYBR-14/PI), membrane functionality (hypo-osmotic swelling test) and DNA fragmentation (%DFI - SCSA) were evaluated after thawing. Immediately post-thaw, total motility was higher in the control (56.53 ± 9.73) than in the tested extenders; however, after 30 min the difference was no longer detected. All other kinetic parameters presented significantly better results in extenders containing 2%, 4% and 8% LDL, compared with the control. There was no difference between treatments regarding the integrity of membranes or fragmentation of the DNA after freezing/thawing procedures.Discussion: The molecules of low density lipoproteins of egg yolk have the known action of sequestering BSP ( binder of sperm proteins) protein, due to the chemical affinity of their main components, phosphatidylcholine and phosphatidylethanolamine. The BSP are responsible for removing cholesterol from the plasma membrane of the sperm, preparing it for the sperm capacitation phase. Low density lipoproteins by inhibiting the action of the BSP increase the stability of the plasma membrane during the freeze-thaw process. The milk caseins micelles have a similar ability to bind the BSP, but with lower affinity. The present study has shown that the use of purified low density lipoproteins has an advantage over the use of whole egg yolk, especially with regard to the kinetic parameters of the spermatozoa after thawing. Furthermore, it was observed that low concentrations of low density lipoproteins, such as 2%, in extenders containing skim milk, could preserve the sperm cell during the freeze-thawing process like that higher LDL concentrations and whole egg yolk. Further studies are needed to determine if the cryoprotectant action found in this study was a result of the synergistic action of skim milk with lipoproteins or even at low concentrations like 2% the lipoproteins can provide protection to the buffalo spermatozoa during freezing, as it has been reproduced in other domestic species. In conclusion, our results indicated that the extender TES-Tris-skim milk containing 2% LDL extracted from egg yolk could be used successfully in the cryopreservation of buffalo sperm cells

Clinical value of anti-Leishmania (Leishmania) chagasi IgG titers detected by flow cytometry to distinguish infected from vaccinated dogs.

Leishmune® vaccination covers a broader number of endemic areas of canine visceral leishmaniasis (CVL) and therefore the development of new serological devices able to discriminate CVL from Leishmune® vaccinees becomes an urgent need considering the post-vaccine seroconversion detected throughout conventional methodologies. Herein, we have described the establishment of a flow cytometry based methodology to detect anti-fixedL. ( L. ) chagasipromastigotes antibodies (FC-AFPA-IgG, FC-AFPA-IgG1 and FC-AFPA-IgG2) in sera samples from Leishmania ( Leishmania ) chagasiinfected dogs and Leishmune® vaccinees. The results of FC-AFPA were reported along the sera titration curve (1:128–1:524,288), as percentage-of-positive-fluorescent-parasite (PPFP). The use of PPFP = 20% as a cut-off edge to segregate negative and positive results at sera dilution 1:2048 revealed outstanding performance indexes that elect FC-AFPA-IgG and IgG2 (both detected by polyclonal FITC-labeled second step reagent) applicable to the serological diagnosis of CVL, with 100% of specificity for both IgG and IgG2 and 97 and 93% of sensitivity, respectively. Moreover, FC-AFPA-IgG, applied at sera dilution 1:2048, also appeared as a useful tool to discriminate L. chagasiinfected dogs from Leishmune® vaccinees, with 76% of specificity. Outstanding likelihood indexes further support the performance of FC-AFPA-IgG for exclusion diagnosis of CVL in Leishmune® vaccinees. Analysis of FC-AFPA-IgG at sera dilution 1:8192 revealed the most outstanding indexes, demonstrating that besides the ability of PPFP 20% to exclude the diagnosis of CVL, a PPFP values higher 80%, mostly observed for infected dogs (INF) have a minimal change to come from a non-infected animal (NI) or Leishmune®

Clinical value of anti-Leishmania (Leishmania) chagasi IgG titers detected by flow cytometry to distinguish infected from vaccinated dogs.

Leishmune® vaccination covers a broader number of endemic areas of canine visceral leishmaniasis (CVL) and therefore the development of new serological devices able to discriminate CVL from Leishmune® vaccinees becomes an urgent need considering the post-vaccine seroconversion detected throughout conventional methodologies. Herein, we have described the establishment of a flow cytometry based methodology to detect anti-fixedL. ( L. ) chagasipromastigotes antibodies (FC-AFPA-IgG, FC-AFPA-IgG1 and FC-AFPA-IgG2) in sera samples from Leishmania ( Leishmania ) chagasiinfected dogs and Leishmune® vaccinees. The results of FC-AFPA were reported along the sera titration curve (1:128–1:524,288), as percentage-of-positive-fluorescent-parasite (PPFP). The use of PPFP = 20% as a cut-off edge to segregate negative and positive results at sera dilution 1:2048 revealed outstanding performance indexes that elect FC-AFPA-IgG and IgG2 (both detected by polyclonal FITC-labeled second step reagent) applicable to the serological diagnosis of CVL, with 100% of specificity for both IgG and IgG2 and 97 and 93% of sensitivity, respectively. Moreover, FC-AFPA-IgG, applied at sera dilution 1:2048, also appeared as a useful tool to discriminate L. chagasiinfected dogs from Leishmune® vaccinees, with 76% of specificity. Outstanding likelihood indexes further support the performance of FC-AFPA-IgG for exclusion diagnosis of CVL in Leishmune® vaccinees. Analysis of FC-AFPA-IgG at sera dilution 1:8192 revealed the most outstanding indexes, demonstrating that besides the ability of PPFP 20% to exclude the diagnosis of CVL, a PPFP values higher 80%, mostly observed for infected dogs (INF) have a minimal change to come from a non-infected animal (NI) or Leishmune®