ABSTRACT
Toll like receptors (TLRs) are involved in the modulation of diverse host genes expression through a complex network of signalling events that allow for an appropriate response to a microbial pathogen. In the present work we used TLR6KO mice in order to study the role of TLR6 in the immune discrimination of lipids from two Babesia bovis strains, attenuated R1A (LA) and virulent S2P (LV), and the consequent macrophage activation. We demonstrated that TLR6 is required for lipid body induction in murine peritoneal macrophages by both LA and LV. Interestingly, as regards IL-10 and COX-2/PGE2 pathway induction by LA and LV, we observed differences in the biological effects produced by these lipid extracts. Our results indicate a role of TLR6 in the down-modulation of these immunoregulators only in the case of LA, whereas this receptor was not implicated in pro-inflammatory TNFα, IL-6 and KC release induced by LA. Remarkably, LV did not exert the down-modulatory effect observed for LA, supporting the notion that LA and LV possess different lipid composition that could correlate with the polar pathogenic effect of both B. bovis strains.
Subject(s)
Babesia bovis/metabolism , Cyclooxygenase 2/metabolism , Dinoprostone/metabolism , Interleukin-10/metabolism , Macrophages, Peritoneal/drug effects , Toll-Like Receptor 6/metabolism , Animals , Babesia bovis/pathogenicity , Cyclooxygenase 2/genetics , Dinoprostone/genetics , Interleukin-10/genetics , Lipid Droplets/physiology , Lipid Metabolism/drug effects , Macrophages, Peritoneal/metabolism , Mice , Mice, Knockout , Toll-Like Receptor 6/genetics , VirulenceABSTRACT
Babesia bovis is an intraerythrocytic apicomplexan protozoa of cattle that causes an acute infection with parasite persistence. Babesiosis limitation depends on macrophages, essential effector cells of the host innate defense, which generate inflammatory cytokines and nitric oxide. Herein, we report quantitative differences in the lipid composition of merozoites from two B. bovis strains with polar behaviour: attenuated R1A and virulent S2P. Accordingly, we observed a distinct inflammatory response induced by the total lipids of R1A (L(A)) and S2P (L(V)) in murine peritoneal macrophages. L(A) and particularly its fractions phosphatidic acid and phosphatidylserine+phosphatidylinositol (PS+PI), produced a strong activation of these cells with lipid body formation, cyclooxygenase-2 expression and pro-inflammatory TNFalpha, IL-6 and KC secretion. Although L(V) did not activate these cells, the corresponding PS+PI fraction induced TNFalpha, IL-6 and KC release. Therefore, these facts might be suggesting the presence of an inhibitor in L(V). Furthermore, the employment of wild type and toll like receptor 2 knockout (TLR2KO) mice allowed us to demonstrate that macrophage activation by the stimulating lipid fractions was mediated through TLR2. Interestingly, only L(A) activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Inhibitory studies employing UO126, indicated that the ERK pathway was required for TNFalpha, IL-6 and KC release. In conclusion, the absence of inflammatory response observed with the lipids of S2P virulent strain could constitute an evasion mechanism of the innate immune response enabling parasite establishment in the host.
Subject(s)
Babesia bovis/immunology , Babesia bovis/pathogenicity , Lipids/pharmacology , Macrophage Activation/drug effects , Toll-Like Receptor 2/immunology , Animals , Babesia bovis/drug effects , Cyclooxygenase 2/metabolism , Cytokines/metabolism , Dinoprostone/biosynthesis , Enzyme Induction/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Signaling System/drug effects , Macrophages/drug effects , Macrophages/enzymology , Macrophages/metabolism , Macrophages/parasitology , Merozoites/drug effects , Merozoites/immunology , Mice , Mice, Inbred C57BL , Virulence/drug effectsABSTRACT
In this work, we evaluate the effects of adenosine 5' triphosphate (ATP) on hepatic lesions caused by ischemia/reperfusion (I/R) in liver rabbit. Rabbits were pretreated with ATP (15 mg/kg IV) or saline solution 0.9% (SS), before the hepatic I/R procedure. We evaluated the effects of ATP on hepatic injury before and after I/R. The warm hepatic I/R procedure caused profound acute liver injury, as indicated by elevated serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, as well as a high apoptotic cell count. All these changes were attenuate by ATP treatment before the hepatic I/R procedure. These results suggested that ATP exerted protective effects on hepatic I/R lesions in the rabbit. This ATP effect may be related to improved energy metabolism during reperfusion in ischemic livers protecting against functional damage of cellular and subcellular membranes during lipid peroxidation.
Subject(s)
Liver Diseases/physiopathology , Purines/metabolism , Reperfusion Injury/physiopathology , Adenosine Triphosphate/therapeutic use , Alanine Transaminase/drug effects , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/drug effects , Aspartate Aminotransferases/metabolism , Ischemia/physiopathology , L-Lactate Dehydrogenase/drug effects , L-Lactate Dehydrogenase/metabolism , Liver/drug effects , Liver/physiopathology , Liver Diseases/prevention & control , Male , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Rabbits , Reperfusion Injury/prevention & controlABSTRACT
We evaluated the effects of a substrate in the biosynthesis of nitric oxide (NO)-l-arginine (LARG)-on hepatic lesions caused by ischemia/reperfusion (I/R) injury in rabbit livers. Rabbits were pretreated with LARG (150 mg/kg IV) or saline solution 0.9% (SS) before the hepatic I/R procedure. The effects of LARG on hepatic injury were evaluated before and after I/R. The warm hepatic I/R procedure produced profound acute liver injury, as indicated by elevated values of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH), as well as a high apoptotic cell count. All changes were attenuated by treatment with LARG before the hepatic I/R procedure. These results suggested that LARG produced protective effects on hepatic I/R lesions. This protective effect of LARG was probably associated with blocking generation of superoxide anions during the hepatic I/R procedure.
Subject(s)
Arginine/therapeutic use , Liver Diseases/prevention & control , Reperfusion Injury/prevention & control , Alanine Transaminase/blood , Alanine Transaminase/drug effects , Animals , Aspartate Aminotransferases/blood , Aspartate Aminotransferases/drug effects , L-Lactate Dehydrogenase/blood , L-Lactate Dehydrogenase/drug effects , Liver Circulation/drug effects , Male , Nitric Oxide/metabolism , Rabbits , Reperfusion Injury/enzymology , Reperfusion Injury/pathology , Vasoconstriction/drug effectsABSTRACT
Because the role of heparin (HEP) in hepatic ischemia/reperfusion (I/R) injury is still not fully understood, we investigated the effects of treatment with HEP on hepatic I/R injury in rabbits. For I/R procedures, the portal vein and hepatic artery were occluded by a metallic clamp to promote ischemia. The clamp was removed after 30 minutes to allow reperfusion. Rabbits undergoing the I/R procedure were treated with HEP (100 U/kg) or saline solution 0.9% (SS). When compared with levels before I/R, the serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, levels were increased by the hepatic I/R procedure, among rabbits treated with SS or HEP. However, the increase in these enzymes was lower among rabbits treated with HEP. Histologic analysis of hepatic tissue of rabbits undergoing I/R and treated with SS showed marked lesions in the central lobule with significant inflammatory infiltration. In contrast, a significant reduction in lesions caused by I/R was observed in the livers of rabbits treated with HEP. After starting reperfusion, we visualized apoptotic cells with nuclear staining among rabbits submitted to I/R and treated with SS, but not those treated with HEP. These results suggested that HEP was able to attenuate hepatic lesions caused by I/R in the livers of rabbits.
Subject(s)
Heparin/therapeutic use , Ischemia/drug therapy , Liver Diseases/drug therapy , Reperfusion Injury/prevention & control , Alanine Transaminase/drug effects , Alanine Transaminase/metabolism , Animals , Aspartate Aminotransferases/drug effects , Aspartate Aminotransferases/metabolism , Disease Models, Animal , Fibrinolytic Agents/therapeutic use , Ischemia/enzymology , L-Lactate Dehydrogenase/drug effects , L-Lactate Dehydrogenase/metabolism , Liver/enzymology , Liver Diseases/enzymology , Male , Rabbits , Reperfusion Injury/enzymologyABSTRACT
In this work, we evaluated the effects of allopurinol (ALO), an inhibitor of xanthine oxidase (XO), on hepatic lesions caused by ischemia/reperfusion (I/R) in the rabbit liver. Rabbits were pretreated with ALO (10 mg/kg IV) or saline solution 0.9% before the hepatic I/R procedure. The effects of ALO on hepatic injury were evaluated before and after I/R. A standard, warm hepatic I/R procedure caused profound acute liver injury, as indicated by elevated serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, as well as a high apoptotic cell count. All of these changes were reversed by the administration of ALO before the hepatic I/R procedure. In conclusion, ALO exerted protective effects on hepatic I/R lesions. This protective effect of ALO was probably associated with blocking the generation of superoxide anions during the hepatic I/R procedure by inhibiting XO activity.
Subject(s)
Allopurinol/therapeutic use , Liver Diseases/prevention & control , Reperfusion Injury/prevention & control , Alanine Transaminase/blood , Alanine Transaminase/drug effects , Animals , Aspartate Aminotransferases/blood , Aspartate Aminotransferases/drug effects , Enzyme Inhibitors/therapeutic use , L-Lactate Dehydrogenase/blood , L-Lactate Dehydrogenase/drug effects , Male , Rabbits , Xanthine Oxidase/antagonists & inhibitorsABSTRACT
Due to difficulties concerning morphological identification of planorbid snails of the genus Biomphalaria, and given a high variation of characters and in the organs with muscular tissue, we designed specific polymerase chain reaction (PCR) primers for Brazilian snail hosts of Schistosoma mansoni from available sequences of internal transcribed spacer 2 (ITS2) of the ribosomal RNA gene. From the previous sequencing of the ITS2 region, one primer was designed to anchor in the 5.8S conserved region and three other species-specific primers in the 28S region, flanking the ITS2 region. These four primers were simultaneously used in the same reaction (Multiplex-PCR), under high stringency conditions. Amplification of the ITS2 region of Biomphalaria snails produced distinct profiles (between 280 and 350 bp) for B. glabrata, B. tenagophila and B. straminea. The present study demonstrates that Multiplex-PCR of ITS2-DNAr showed to be a promising auxiliary tool for the morphological identification of Biomphalaria snails, the intermediate hosts of S. mansoni.
Subject(s)
Biomphalaria/genetics , DNA, Ribosomal Spacer/genetics , Disease Vectors , Polymerase Chain Reaction/methods , Schistosoma mansoni/isolation & purification , Animals , Biomphalaria/classification , Brazil , DNA Primers , Schistosomiasis/prevention & control , Silver Staining , Species SpecificityABSTRACT
Due to difficulties concerning morphological identification of planorbid snails of the genus Biomphalaria, and given a high variation of characters and in the organs with muscular tissue, we designed specific polymerase chain reaction (PCR) primers for Brazilian snail hosts of Schistosoma mansoni from available sequences of internal transcribed spacer 2 (ITS2) of the ribosomal RNA gene. From the previous sequencing of the ITS2 region, one primer was designed to anchor in the 5.8S conserved region and three other species-specific primers in the 28S region, flanking the ITS2 region. These four primers were simultaneously used in the same reaction (Multiplex-PCR), under high stringency conditions. Amplification of the ITS2 region of Biomphalaria snails produced distinct profiles (between 280 and 350 bp) for B. glabrata, B. tenagophila and B. straminea. The present study demonstrates that Multiplex-PCR of ITS2-DNAr showed to be a promising auxiliary tool for the morphological identification of Biomphalaria snails, the intermediate hosts of S. mansoni
Subject(s)
Animals , Biomphalaria , Polymerase Chain Reaction , Schistosoma mansoni , Biomphalaria , Brazil , Disease Vectors , DNA Primers , Schistosomiasis , Silver StainingABSTRACT
The intermediate host of Fasciola hepatica, Lymnaea columella, collected in Belo Horizonte, Minas Gerais, Brazil, was reared in our laboratory. The aim of the current study was to standardize a rearing and maintenance technique. Two kinds of diet were tested: fresh lettuce (A) and rodent ration + 10% CaCO3 plus fresh lettuce (B). The age for the beginning of oviposition ranged from 27 to 57 days. Ten days after oviposition at 24.7 degrees C, 100% eclosion occurred. The complete life cycle varied from 37 to 67 days. The average numbers of eggs per egg mass were 26.3 and 31.1 with diets (A) and (B), respectively. The lettuce and ration fed snails presented a increased growth although the difference was not statistically significant (p > 0.05). The mortality rate varied from 40 to 64% after 90 days. The maximum longevity was 183 days, 21.5 mm length and 11 mm wide. The methodology to mass breed and maintain these snails was found to be suitable in the laboratory
