Performance of LBSap Vaccine after Intradermal Challenge with L. infantum and Saliva of Lu. longipalpis: Immunogenicity and Parasitological Evaluation

In the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21⁺) and both CD4⁺ and CD8⁺ T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4⁺ T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-γ and low IL-10 and TGF-β1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions.     

Trypanosoma cruzi: Induction of benznidazole resistance in vivo and its modulation by in vitro culturing and mice infection

Through a continuous in vivo drug pressure protocol, using mice as experimental model, we induced benznidazole resistance in Trypanosoma cruzi stocks. Full resistance was obtained for four out of five T. cruzi stocks analyzed. However, the number of benznidazole doses (40–180), as well as the time (4–18 months) necessary to induce resistance varied among the different T. cruzi stocks. The resistance phenotype remained stable after T. cruzi stocks has been maintained by 12 passages in mice (six months) and in acellular culture for the same time. However, the maintenance of resistant parasite for 12 months in acellular culture induces a reduction in its level of benznidazole resistance, while no alteration was detected in parasite maintained for the same time in mice. The data showed the stability of the resistance acquired by drug pressure, but suggest the possibility of reversible changes in the resistance levels after maintenance for long time in acellular culture.     

Cytokine release and expression induced by OmpA proteins from the Gram-negative anaerobes, Porphyromonas asaccharolytica and Bacteroides fragilis

OmpA proteins from Gram-negative anaerobes Porphyromonas asaccharolytica and Bacteroides fragilis induced release and expression of IL-1alpha, tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-6, and IL-10 from murine splenocytes in vitro in a dose-dependent fashion. The release of the cytokines induced by B. fragilis Bf-OmpA was at much lower levels compared with P. asaccharolytica Omp-PA; Bf-OmpA did not induce release of IL-10. Omp-PA and Bf-OmpA were able to upregulate mRNA expression of the tested cytokines. The results obtained with refolded Bf-OmpA were similar to those with native Bf-OmpA. The data presented in this research demonstrate for the first time that Omps from anaerobic bacteria can induce the release of cytokines, suggesting that Omp-PA and Bf-OmpA may play important roles in the pathogenic processes of these bacteria.     

Branched activation- and catalysis-specific pathways for electron relay to the manganese/iron cofactor in ribonucleotide reductase from Chlamydia trachomatis

A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y (*)) in its R2 subunit for reversible generation of a 3'-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor (specifically, the Mn (IV) ion) in place of the Y (*) for radical initiation. Ct R2 is activated when its Mn (II)/Fe (II) form reacts with O 2 to generate a Mn (IV)/Fe (IV) intermediate, which decays by reduction of the Fe (IV) site to the active Mn (IV)/Fe (III) state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the Mn (IV)/Fe (IV) intermediate by approximately 10-fold and diminishes the ability of ascorbate to accelerate the decay by approximately 65-fold but has no detectable effect on the catalytic activity of the Mn (IV)/Fe (III)-R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 <--> R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli ( Ec) R2], has almost no effect on decay of the Mn (IV)/Fe (IV) intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 <--> R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the Mn (IV)/Fe (IV) intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y (*)-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.     

Host-Parasite Interactions in Chagas Disease: Genetically Unidentical Isolates of a Single Trypanosoma cruzi Strain Identified In Vitro via LSSP-PCR

The present study aims at establishing whether the diversity in pathogenesis within a genetically diverse host population infected with a single polyclonal strain of Trypanosoma cruzi is due to selection of specific subpopulations within the strain. For this purpose we infected Swiss mice, a genetically diverse population, with the polyclonal strain of Trypanosoma cruzi Berenice-78 and characterized via LSSP-PCR the kinetoplast DNA of subpopulations isolated from blood samples collected from the animals at various times after inoculation (3, 6 and 12 months after inoculation). We examined the biological behavior of the isolates in acellular medium and in vitro profiles of infectivity in Vero cell medium. We compared the characteristics of the isolates with the inoculating strain and with another strain, Berenice 62, isolated from the same patient 16 years earlier. We found that one of the isolates had intermediate behavior in comparison with Berenice-78 and Berenice-62 and a significantly different genetic profile by LSSP-PCR in comparison with the inoculating strain. We hereby demonstrate that genetically distinct Trypanosoma cruzi isolates may be obtained upon experimental murine infection with a single polyclonal Trypanosoma cruzi strain.     

Probability graphs resolve components with buoyant-density differences of 0.001 g/cm3 in equilibrium density gradients

A method derived from DNA melting-curve analysis has been modified for determining homogeneity of DNA species centrifuged to equilibrium in a CsCl gradient. DNA species of known density were purified, centrifuged to equilibrium in a Spinco Model E analytical ultracentrifuge, and analyzed by probability distribution. In order to determine the limits of resolution by this procedure, samples with varying buoyant-density separations were obtained by mixing different DNA species. Results indicate this method can detect overlapping mixtures of DNA in relative amounts of 9:1 and resolve two species of DNA of equal concentrations with a buoyant-density separation of 0.001 g/cm³.     

Increased gene expression of plasminogen activators and inhibitors in left ventricular hypertrophy

In the early stages of left ventricular hypertrophy (LVH) acute adaptive changes occur in the coronary vasculature as it remodels. Plasminogen activators (PAs) and inhibitors (PAIs) have the potential effects of proteolytic degradation that is relevant to tissue remodeling and angiogenesis. Our study focused on the possible roles of PAI-1, PAI-2, uPA and tPA in myocyte hypertrophy and angiogenesis in the early and late stages of pressure overload induced left ventricular hypertrophy (LVH). We divided seventeen adult swine, weighing 24.2 ± 6.5 kg, into four groups: control, sham-operated, early LVH and late heart failure LVH group. At surgery we placed a fixed constrictor on the ascending aorta immediately above the aortic valve. This increased LV systolic pressure from 133 ± 15 to 193 ± 24 mm Hg after the surgery. We subdivided the early group into groups of 3 animals each that we euthanized at 8, 24 and 72 h after operation and obtained heart samples for analysis. In the late heart failure group individual animals were euthanized at 55, 59, 62 and 72 days after the detection of congestive heart failure. We also obtained tissue samples from the control and sham-operated swine. Sections for histologic analysis were fixed in 10% buffered formalin. We isolated RNA, size fractionated it using 1% formaldehyde-agarose gel electrophoresis and then did Northern blots. The mRNAs from both PAI-1 and PAI-2 showed a remarkable increase at 8 and 24 h after acute aortic constriction and returned to control by 72 h. Regional differences showed that most of the increases were in the endocardium. Three animals in the late heart failure LVH group were determined to be in congestive heart failure at about 2 months after the onset of aortic constriction. In these animals PAI-1 and PAI-2 were increased in both the left and right ventricles but remained low in an animal of the same elevation in aortic pressure seen by the LV who did not have congestive failure. These data suggest that PA and PAI gene expressions change before morphologic changes occur in the early stages of developing LVH. Also at the time of onset of congestive heart failure this increased expression reappears. PAs and PA inhibitors mRNA levels vary in the different regions of the heart reflecting changing wall stresses. Thus, the PAs and PA inhibitors may play an important role in angiogenesis that occurs during the early stages of LVH. The increased expression in the late stage of LVH may reflect further changes in wall stresses since these animals also showed overt clinical signs of heart failure.