Babesia bovis: lipids from virulent S2P and attenuated R1A strains trigger differential signalling and inflammatory responses in bovine macrophages.

The intra-erythrocytic protozoan Babesia bovis is an economically important pathogen that causes an acute and often fatal infection in adult cattle. Babesiosis limitation depends on the early activation of macrophages, essential cells of the host innate immunity, which can generate an inflammatory response mediated by cytokines and nitric oxide (NO). Herein, we demonstrate in bovine macrophages that lipids from B. bovis attenuated R1A strain (LA) produced a stronger NO release, an early TNFα mRNA induction and 2-fold higher IL-12p35 mRNA levels compared to the lipids of virulent S2P strain (LV). Neither LA nor LV induced anti-inflammatory IL-10. Regarding signalling pathways, we here report that LA induced a significant phosphorylation of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) whereas LV only induced a reduced activation of ERK1/2. Besides, NF-κB was activated by LA and LV, but LA produced an early degradation of the inhibitor IκB. Interestingly, LV and the majority of its lipid fractions, exerted a significant inhibition of concanavalin A-induced peripheral blood mononuclear cell proliferation with respect to LA and its corresponding lipid fractions. In addition, we determined that animals infected with R1A developed a higher increase in IgM anti-phosphatidylcholine than those inoculated with S2P. Collectively, S2P lipids generated a decreased inflammatory response contributing to the evasion of innate immunity. Moreover, since R1A lipids induced a pro-inflammatory profile, we propose these molecules as good candidates for immunoprophylactic strategies against babesiosis.

Involvement of TLR6 in the induction of COX-2, PGE2 and IL-10 in macrophages by lipids from virulent S2P and attenuated R1A Babesia bovis strains.

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.

Temperature acclimation of Trypanosoma cruzi epimastigote and metacyclic trypomastigote lipids.

This study examines the changes in cellular lipids that take place when Trypanosoma cruzi epimastigotes and metacyclic trypomastigotes are transferred from 28 to 37 degrees C. We found a rise in the sterol to phospholipid ratio, as well as in the triacylglycerol and steryl ester cellular content in T. cruzi epimastigotes. In addition, saturated to unsaturated fatty acid ratios in phospholipids increase. This latter effect appears to be due to two concurrent processes. Firstly, fatty acyl delta9 and, especially, delta12 desaturations are significantly diminished at 37 degrees C. Secondly, triacylglycerols and steryl esters undergo changes in their fatty acyl composition opposite to those simultaneously observed in phospholipids, i.e. the ratio of saturated to unsaturated fatty acids markedly decreases. Similar alterations in each of the lipid classes and in the fatty acid composition of polar and neutral lipids were found in cultured metacyclic trypomastigotes on exposure to the same shift-up. These observations suggest that a global remodeling of cellular lipids that involves extensive fatty acid exchange between neutral and polar lipid pools represents a novel and important mechanism of adaptation of the parasites to the temperature changes they encounter in their life cycle.

Rapid evolution of kinetoplast DNA mini-circle subpopulations in Trypanosoma cruzi.

Nine Trypanosoma cruzi isolates not examined previously for kDNA structure were characterized by (a) endonuclease restriction analysis of mini-circles, followed by agarose gel-electrophoresis of digests, and (b) hybridization of mini- and maxi-circle fragments with four 32P-labeled cloned mini-circles from T. cruzi (pTck-1, 12, 13 and 14) or with 32P-labeled maxi-circles from T. brucei, respectively. The gel electrophoresis patterns demonstrated significant differences between isolates, which were confirmed and extended by the hybridization assay. When using pTck-1 and pTck-12 as probes, widely distributed heterogeneous mini-circle subpopulations were demonstrated in all the examined isolates, despite the occurrence of extensive homologies. pTck-14, assayed under high stringent conditions, detected an almost homogeneous mini-circle subpopulation in only three isolates, although under relaxed conditions, pTck-14 shared sequence homologies with most of the mini-circle subpopulations from all isolates. Rapidly evolving mini-circle regions were also detected using as probe pTck-13, a small mini-circle fragment. Preliminary maxi-circle characterization revealed polymorphic restriction endonuclease sites in the different T. cruzi isolates. These results were consistent with those obtained with mini-circles subjected to the same treatment.

Effect of vector on infectivity of Trypanosoma cruzi.

A comparative study was carried out on the interaction between Triatoma infestans and bloodstream forms (BSF) of Trypanosoma cruzi isolates RA and UP, both lethal for mouse, and CA-I nonlethal for this host. Parasite duplication was readily detected in triatomes fed with CA-I, metacyclic (Mtc) differentiation reaching a maximum at an optimum ingestion level of 250 BSF/insect. Progress and differentiation proved much lower for RA, but reached intermediate values for the UP isolate. Assays for infectivity for each isolate after bug passage revealed a drop for the RA and UP, whereas for CA-I an increase was observed indicating that virulence of BSF and Mtc differs. Our results suggest that parasite selection by insect passage modulates infectivity of a given parasite population; however, virulence was independent of the absolute number of Mtc in the insect's feces.

Trypanosoma cruzi: comparative studies of infectivity of parasites ingested by Triatoma infestans and those present in their feces.

Artificial feeding of the insects with whole blood containing trypomastigotes resulted in triatome infection; the parasites were present in the host feces after 15--30 days and, when inoculated into mice, elicited parasitemia in 100% of the cases and deaths in 20--70%. This mortality indicates a reduction of the original infective capacity of the bloodstream form, which kills 100% of the mice even when inoculated with a single trypomastigote. When triatomes were fed whole normal mouse blood containing culture forms of low infective capacity (TulL), mice inoculated with feces containing the progeny of the culture forms failed to develop a parasitemia. The absence of any infective capacity of these parasites was proven when the mice challenged with lethal doses of trypomastigotes 30 days after the fecal inoculations died at the same time as the controls. Mice injected with feces from triatomes fed culture forms with high infectivity for mice (TulB) developed patent parasitemia, indicating that triatomes may be infected by parasite forms other than bloodstream trypomastigotes. Experiments on infectivity of parasites present in the feces of triatomes fed TulL and TulB culture forms, also showed a single passage through the digestive tract of the insects did not significantly modify the infective capacity of the parasite population. When stomachs and intestines from insects fed TulB were processed separately, parasites obtained from the latter showed higher infectivity for mice than those obtained from the former.

Influence of organ extracts of Triatoma infestans on differentiation of Trypanosoma cruzi.

The influence that extracts of different T. infestans organs possess to induce Trypanosoma cruzi differentiation from epimastigotes to metacyclic forms in Grace's and modified Grace's media was analyzed. Growth of epimastigotes was adequate in Grace's medium when homogenates of stomach, testes, and ovaries from recently fed insects were added. Growth of epimastigotes was adequate when testes or ovaries obtained from recently fed triatomes were added to modified Grace's medium. Similar results were obtained in Grace's or modified Grace's media supplemented with stomach or intestinal homogenates obtained from "starved" triatomes. None of the above culture conditions induced differentiation of epimastigotes to the metacyclic stage. When extracts of intestine or stomach obtained from triatomes fed 24 to 48 hr previously were used to supplement the modified Grace's medium, growth and differentiation to the metacyclic stage was induced in a high percentage of the population. When extracts of intestine obtained from recently fed insects were used to supplement Grace's medium, differentiation and growth was an in modified Grace's medium. No important differences were observed if cultures were prepared at pH 6.4 or 7.0. The triatome food source did not seem to be important for the differentiation. The parasite population after differentiation to metacyclic type forms showed a higher degree of infectivity than the original population constituted mostly by epimastigotes. These studies suggest the importance of factors from the insect vector in differentiation of T. cruzi.

Severe gastrointestinal bleeding in a uremic patient treated with estrogen-progesterone therapy.

Gastrointestinal bleeding is a frequent complication in hemodialysis patients; angiodysplasia is a potential cause, with a higher incidence in uremic patients. We describe a case of severe anemia (Hemoglobin up to 3.5 g/dl) secondary to diffuse angiodysplastic lesions in a hemodialysis patient with mixed connective tissue disease. The case is characterised both by the severity of the clinical picture (extension and entity of angiodysplastic lesions, frequency of bleeding episodes) and by the patient's religious faith which made her reject blood transfusions. We underline the efficacy of estrogen-progesterone therapy in view of the modest results obtained with other therapeutic strategies on bleeding.

Effects of dialysis membrane nature on intradialytic phagocytizing activity.

Blood-membrane contact in the extracorporeal circuit affects the activation of many biological systems. Among these, phagocytizing activity has been reported to be influenced by the nature of the hemodialysis membrane used, whether cellulosic or synthetic. This work reports on an ex-vivo, comparative test between cellulosics and synthetics concerning the effects of blood-membrane contact on the polymorphonucleate and monocyte phagocytizing function, both during and after the hemodialysis session. By means of flow cytometry, we evaluated the capacity for phagosoma formation and oxidative burst both in polymorphonucleates and monocytes. Ten hemodialysis patients were included in the study. Six separate dialysis procedures for each patient were considered, one per dialyzer (3 cellulosic and 3 synthetic membranes). Tests were performed at 15', 60', 210' and 4 hours after the session end. Comparative evaluation was made according to Student's t test. Polymorphonucleate phagocytosis and oxidative burst activation were globally more marked for synthetic than cellulosic membranes, tending to level out in the post-dialysis. This result could be affected by their functional exhaustion following pulmonary sequestration. Monocyte intradialytic phagocytosis and oxidative burst proved more activated by cellulosic membrane. All differences tended to vanish in the post-dialysis.

Acute gonococcal urethritis: treatment with a single dose of rifampicin.

A total of 103 patients with acute gonococcal urethritis were treated with a single 1,200 mg dose of rifampicin. A 91 . 3% cure rate was obtained, as proved by the negative bacteriological controls effected on the 7th and 14th days after the initiation of therapy. Three pharyngeal infections and one ano-rectal infection responded successfully to the treatment. No signs of drug intolerance were detected with the stated dose. Reactivity to the VDRL test was not impaired during the duration of the study and three reactive cases were discovered. In previous studies of gonorrhoea we had observed a significant discrepancy between urine cultures and the urethral smears and, in view of this, it was decided to adopt the latter alone as a routine procedure. The proposed dose does not originate resistance to rifampicin in either the Hansen or Koch bacilli.

Trypanosoma cruzi: conditions required to improve metacyclic differentiation in axenic culture.

This work is a study on the behaviour of Epi from various T. cruzi strains and clones grown in media stimulating differentiation such as M 16, TAUP, TAUS, LIT-hemin and G-IH. Results showed that in our experimental conditions the G-IH medium was the only suitable one to induce Epi-Mtc morphogenesis. Optimal temperature for both multiplication and differentiation in this medium was 28 degrees C. The use of G-IH medium after nutrient depletion by dilution failed to induce differentiation. Parasites obtained from biphasic medium at 24-48 h culture proved the best to achieve morphogenesis. External Ca++ supply blockage affected the differentiation capacity of Tul and RA strains to a variable degree.

Trypanosoma cruzi: incorporation of macromolecule precursors during the morphogenic process.

During the morphogenic process from epimastigote to metacyclic trypomastigote stage changes at molecular levels are produced. In the present work the incorporation of macromolecule precursors L [3H]-leucine, [3H]-thymidine and [3H]-uridine was used to monitor the differences between parasites growing with and without specific stimulation for morphogenesis (SEpi and NEpi, respectively). The peak of maximum [3H]-uridine incorporation was earlier in the SEpi than in NEpi (3 vs 4 days, respectively) reaching 129% higher values. Even at day 2, SEpi already showed values of [3H]-uridine incorporation slightly higher (29%) than the maximum reached by NEpi. One the other hand the peak of L-[3H]-leucine incorporation was 48 h delayed comparing SEpi vs NEpi with quantitatively similar patterns, while no differences were registered for [3H]-thymidine incorporation. Therefore the [3H]-uridine incorporation may constitute an earlier marker of morphogenesis since the process was optically detected starting day at 6.

[Effect of the hemolymph of Triatoma infestans on the morphogenesis of Trypanosoma cruzi]. / Influencia de la hemolinfa de Triatoma infestans en la morfogénesis del Trypanosoma cruzi.

Growth and differentiation patterns induced by T. infestans whole hemolymph as well as by hemocyte free or hemocyte and quinone free samples were similar; these results indicate that neither hemocytes nor quinones interfered in the duplication of epimastigotes or in their differentiation to metacyclic forms. The minimum amount required to induce differentiation was 2%, lacking inhibitory effect even when assayed at a concentration of 30%. Differentiation was reached simultaneously with the exponential growth period of the culture up to 80%.

Trypanosoma cruzi: immunobiological studies on epimastigotes of several strains.

Epimastigotes from six Trypanosoma cruzi strains showed similar growth pattern and duplication time in a biphasic culture medium as well as similar capability to differentiate into metacyclic forms when grown in the special GM-TIIH culture; interactions of epimastigotes and Concanavalin A, Soy bean agglutinin or Wheat germ agglutinin were also similar for all the strains. On the other hand, differences in the antigenic patterns were detected by double diffusion tests. At least one precipitogen, undetectable in the other strains, was shared by the Y and RA strains. In the RA a second precipitogen was shared with Tul strain. The three strains mentioned, as identified by their IEPh pattern belonged to the C group described by Nussenzweig and Goble in 1966. However, the differences between Y and Tul precipitogens proved that the C group originally described might be made up by several subgroups. As Tul strain had been previously classified twice in the A group and once in the C group, the classic A, B and C antigenic groups seem to be relatively unstable. Due to this, and because of the differences in shape, infective capability surface saccharides, etc. already established for the trypomastigote stage, which correlate making strain grouping feasible, we consider the latter might prove a better stage to search for a strain marker.

[Cytochromes in different stages, strains, and populations of Trypanosoma cruzi]. / Citocromos en diferentes estadios, cepas y poblaciones de Trypanosoma cruzi.

Differential reduced minus oxidized (Red-Ox) or reduced. CO minus reduced (Red. CO-Red) spectra of Trypanosoma cruzi metacyclic trypomastigotes (Tulahuen strain), revealed the presence of cytochromes aa3, b, c5 5 8, o and possibly d (Fig. 1). Mitochondrial membranes from the epimastigote form of the parasite showed similar cytochrome spectra (Fig. 2A). Extraction of the mitochondrial membranes with guanidine and cholate, and spectroscopy of the cytochrome o -cyanide derivative proved that this cytochrome is an integral constituent of the mitochondrial membranes (Figs. 2B and 4, and Table 1). Contamination of the investigated samples with hemoglobin, peroxidase, catalase, cytochrome P-420, cytochrome P-450 or culture medium hemoproteins was ruled out by filtering the cells and mitochondrial membranes on Sephadex G-50, or by differential spectroscopy of pigments, or by differential centrifugations of samples. Investigation of pyridine-hemochromes revealed hemes A, B, C and D (Fig. 3), thus confirming the presence of the postulated cytochromes. Comparative spectroscopy of a series of T. cruzi stocks (including Tulahuen and Y strains), many of them obtained from acute or chronic forms of Chagas disease, revealed significant variability in the cytochrome content (Table 2). Taking cytochromes o and b as standard for comparison, the epimastigotes samples could be grouped as follows (in parenthesis number of passages through the culture medium): 1) stocks with a relatively high content of cytochromes b and o, prevailing the former (stocks Y (116), RA (114), AF, FN, TN and MG (14 y 16); 2) stocks with a relatively low content of both cytochromes: Y (119), AWP and UP; 3) stocks with a low content of cytochrome b, without cytochrome o: CA-I and CA-I (V); 4) stocks without cytochromes: Y(117 and 118) and RA(113). In some strains (e.g. Y and RA), significant variation of cytochrome content in different stocks of the same isolate was observed (Table 2), but the Tulahuen strain proved to be less variable. Comparison of cytochrome distribution and other properties of parasites, namely, lethality for mice and morphology, did not allow to establish positive correlations.

[On-line haemodiafiltration with sorbent-regenerated ultrafiltrate as replacement fluid: Beta2-microglobulin removal versus filtration fraction]. / Emodiafiltrazione on-line con reinfusione endogena (HFR): rimozione di Beta2-microglobulina (Beta2-m) vs frazione di filtrazione (FF%).

PURPOSE: In order to encourage the removal of middle molecules in hemodiafiltration (HDF) techniques an attempt is made to maximize convective clearance by increasing the ultrafiltration rate. However, convective clearance is limited by the maximum filtration fraction (FF%) obtainable, by the pre- or post reinfusion method and by the convective surface and the capacity of the filter used. This study aimed to evaluate the effect of the FF% in the removal of Beta2-microglobulin (Beta2-m) during hemodiafiltration reinfusion (HFR) to identify the best ultrafiltration strategies; and therefore, a better removal of medium molecular weight solutes in this hemodiafiltrative technique recently introduced in clinical practice. METHODS: Ten chronic uremic patients (eight males, two females; age 66 +/- 18 yrs) already on renal dialysis therapy (RDT) for 80 +/- 36 months, were subjected to four HFR sessions, with Td=240 +/- 10 min, Qb=312 +/- 18 and Qd=500 mL/min; the reinfusion rates (Qr) used were 43.6 +/- 7.2 mL/min (25-58) with FF% rates varying from 20-34 (24.2 +/- 3.8) for hematocritic levels of 34.6 +/- 4.2% at the start of the dialysis session. For each session the intradialytic reduction rates (RR%) of urea, creatinine (Cr), phosphate, uric acid and Beta2-m and its average clearance (KBeta2-m mL/min) were evaluated. RESULTS: The results obtained gave a RR% for urea of 69.4 +/- 5 (Kt/Veq=1.23 +/- 0.2) and for Cr, phosphate and uric acid values of, respectively, 61.2 +/- 5.4, 47.5 +/- 10 and 75.8 +/- 6.7. The intradialytic reductions in Beta2-m were 49.3 +/- 10.3% with a variability range from 29-69% and with average KBeta2-m values of 63.8 +/- 13.5 mL/min. The RR% of ss2-m and KBeta2-m were inversely correlated (p<0.01) to the FF% rate applied during the treatment; 75% of the HRF sessions in which we observed a reduction in Beta2-m levels >40% were those where a FF% between 20 and 26% was applied. CONCLUSIONS. From our study, it appears that in HFR the best ultrafiltration strategy from the convective sector in removing Beta2-m has FF% values in the range 20-26%. The occurrence of lower intradialytic reductions of Beta2-m with increasing FF% can be interpreted as a consequence of phenomena related to high intradialytic hemoconcentrations, to the excessive increase in the TMP and/or the increase in the protein cake with a consequent reduction in permeability and mass transfer. Although using a limited convective surface with a limited possibility of increasing the FF%, nevertheless, HFR seems capable of ensuring a satisfactory uremic toxin removal of low and medium molecular weight, which combined with the high biocompatibility deriving from the use of reinfused endogen, can be considered an effective dialytic strategy for preventing or retarding the complications in dialytic patients.

Lipids from attenuated and virulent Babesia bovis strains induce differential TLR2-mediated macrophage activation.

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.