Impact of autologous whole blood administration upon experimental mouse models of acute Trypanosoma cruzi infection.

BACKGROUND: Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection. METHODS: The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile. RESULTS: No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 µL AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases (< 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and IL-6 were detected at 9 dpi in all infected animals as compared to uninfected mice but only Bz displayed a statistically significant diminution (p = 0.02) in TNF-alpha levels than infected and untreated mice. CONCLUSIONS: This study revealed that the use of autologous whole blood (AWB) in the acute model employed was unable to reduce the parasitic load of infected mice, providing only a minor decrease in parasitemia levels (up to 30%) but without protecting against animal mortality. Further in vivo studies will be necessary to elucidate the effective impact of this procedure.

Impact of autologous whole blood administration upon experimental mouse models of acute Trypanosoma cruzi infection

Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection. Methods: The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile. Results: No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 µL AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases (< 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and IL-6 were detected at 9 dpi in all infected animals as compared to uninfected mice but only Bz displayed a statistically significant diminution (p= 0.02) in TNF-alpha levels than infected and untreated mice. Conclusions: This study revealed that the use of autologous whole blood (AWB) in the acute model employed was unable to reduce the parasitic load of infected mice, providing only a minor decrease in parasitemia levels (up to 30%) but without protecting against animal mortality. Further in vivo studies will be necessary to elucidate the effective impact of this procedure.(AU)

Impact of autologous whole blood administration upon experimental mouse models of acute Trypanosoma cruzi infection

Background: Autologous whole blood (AWB) administration is described as alternative/complementary medical practice widely employed in medical and veterinary therapy against infections, chronic pathologies and neoplasias. Our aim is to investigate in vivo biological effect of AWB using healthy murine models under the course of Trypanosoma cruzi acute infection. Methods: The first set of studies consisted of injecting different volumes of AWB and saline (SAL) into the posterior region of quadriceps muscle of healthy male Swiss mice under distinct therapeutic schemes evaluating: animal behavior, body and organ weight, hemogram, plasmatic biochemical markers for tissue damage and inflammatory cytokine levels and profile. To assess the impact on the experimental T. cruzi infection, different schemes (prior and post infection) and periods of AWB administration (from one up to 10 days) were conducted, also employing heterologous whole blood (HWB) and evaluating plasma cytokine profile. Results: No major adverse events were observed in healthy AWB-treated mice, except gait impairment in animals that received three doses of 20 L AWB in the same hind limb. AWB and SAL triggered an immediate polymorphonuclear response followed by mononuclear infiltrate. Although SAL triggered an inflammatory response, the kinetics and intensity of the histological profile and humoral mediator levels were different from AWB, the latter occurring earlier and more intensely with concomitant elevation of plasma IL-6. Inflammatory peak response of SAL, mainly composed of mononuclear cells with IL-10, was increased at 24 h. According to the mouse model of acute T. cruzi infection, only minor decreases ( 30%) in the parasitemia levels were produced by AWB and HWB given before and after infection, without protecting against mortality. Rises in IFN-gamma, TNF-alpha and...

Kinetic Study of Yellow Fever 17DD Viral Infection in Gallus gallus domesticus Embryos.

Yellow fever continues to be an important epidemiological problem in Africa and South America even though the disease can be controlled by vaccination. The vaccine has been produced since 1937 and is based on YFV 17DD chicken embryo infection. However, little is known about the histopathological background of virus infection and replication in this model. Here we show by morphological and molecular methods (brightfield and confocal microscopies, immunofluorescence, nested-PCR and sequencing) the kinetics of YFV 17DD infection in chicken embryos with 9 days of development, encompassing 24 to 96 hours post infection. Our principal findings indicate that the main cells involved in virus production are myoblasts with a mesenchymal shape, which also are the first cells to express virus proteins in Gallus gallus embryos at 48 hours after infection. At 72 hours post infection, we observed an increase of infected cells in embryos. Many sites are thus affected in the infection sequence, especially the skeletal muscle. We were also able to confirm an increase of nervous system infection at 96 hours post infection. Our data contribute to the comprehension of the pathogenesis of YF 17DD virus infection in Gallus gallus embryos.

Yellow Fever 17DD Vaccine Virus Infection Causes Detectable Changes in Chicken Embryos.

The yellow fever (YF) 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system.

Histological analyses demonstrate the temporary contribution of yolk sac, liver, and bone marrow to hematopoiesis during chicken development.

The use of avian animal models has contributed to the understanding of many aspects of the ontogeny of the hematopoietic system in vertebrates. However, specific events that occur in the model itself are still unclear. There is a lack of consensus, among previous studies, about which is the intermediate site responsible for expansion and differentiation of hematopoietic cells, and the liver's contribution to the development of this system. Here we aimed to evaluate the presence of hematopoiesis in the yolk sac and liver in chickens, from the stages of intra-aortic clusters in the aorta-genital ridges-mesonephros (AGM) region until hatching, and how it relates to the establishment of the bone marrow. Gallus gallus domesticus L. embryos and their respective yolk sacs at embryonic day 3 (E3) and up to E21 were collected and processed according to standard histological techniques for paraffin embedding. The slides were stained with hematoxylin-eosin, Lennert's Giemsa, and Sirius Red at pH 10.2, and investigated by light microscopy. This study demonstrated that the yolk sac was a unique hematopoietic site between E4 and E12. Hematopoiesis occurred in the yolk sac and bone marrow between E13 and E20. The liver showed granulocytic differentiation in the connective tissue of portal spaces at E15 and onwards. The yolk sac showed expansion of erythrocytic and granulocytic lineages from E6 to E19, and E7 to E20, respectively. The results suggest that the yolk sac is the major intermediate erythropoietic and granulopoietic site where expansion and differentiation occur during chicken development. The hepatic hematopoiesis is restricted to the portal spaces and represented by the granulocytic lineage.

Eosinophils involved in fulminant hepatic failure are associated with high interleukin-6 expression and absence of interleukin-5 in liver and peripheral blood.

BACKGROUND/AIMS: Although eosinophils are considered to play an important role in the pathogenesis of various parasitic, allergic and autoimmune digestive diseases, their role in fulminant hepatic failure (FHF) is unknown. Our contribution was to identify and quantify eosinophils and cytokine levels [interleukin (IL)-6, IL-5 and macrophage inflammatory protein (MIP)-1alpha] in liver parenchyma and peripheral blood from FHF patients at pre- and post-transplantation steps. METHODS: Histochemical methods were used to identify/quantify eosinophils in liver samples. Liver and plasma cytokine levels were quantified using immunofluorescence methods. RESULTS: Fulminant hepatic failure patients showed a high number of intrahepatic eosinophils concomitant with an increased expression of IL-6, besides the IL-6-positive eosinophils associated with the lack of IL-5. Also, an increased number of eosinophils and soluble IL-6 and MIP-1alpha with a low expression of IL-5 in peripheral blood at the pretransplantation step was observed. CONCLUSIONS: The increased number of intrahepatic eosinophils, besides the high production of IL-6, may be involved in liver dysfunction. In addition, the low presence of IL-5 in liver and peripheral blood may represent a particular pattern of eosinophil behaviour in human liver failure, which may also involve MIP-1alpha. Further ex vivo studies are necessary to evaluate the specific role of eosinophils in FHF.

Angiostrongylus costaricensis (Nematoda: Protostrongylidae): migration route in experimental infection of Omalonyx sp. (Gastropoda: Succineidae).

Angiostrongylus costaricensis can infect several mollusks, and its migration route in intermediate hosts has been studied only in Sarasinula marginata. To verify the susceptibility of Omalonyx sp. as an intermediate host of A. costaricensis and to analyze the nematode migration route, individuals were infected with stage 1 larvae. Obtained stage 3 larvae were orally inoculated in mice, and after 30 days, adult worms and stage 1 larvae were recovered, demonstrating Omalonyx susceptibility and suitability to infection. To define the parasite migration routes, specimens of Omalonyx with 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 2 days, 5 days, 10 days, 12 days, 15 days, 20 days, 21 days, 25 days, 28 days, and 30 days of infection were fixed and serially sectioned. Histological sections were stained with hematoxylin-eosin. The results were compared to those described in S. marginata. Oral and cutaneous infections were noted. After the penetration, larvae were retained, mainly in the fibromuscular tissue, by hemocytes, or they spread to the whole organism through the circulation, following the anatomical structure of the vasculature. The perilarval hemocyte reaction in Omalonyx was more intense until stage 2 larva instar, decreasing in the presence of stage 3 larvae. Differences in some aspects of hemocyte reaction between S. marginata and Omalonyx exemplify interspecific peculiarities in snail response to the same parasite.

Expression and arrangement of extracellular matrix proteins in the lungs of mice infected with Paracoccidioides brasiliensis conidia.

Extracellular matrix (ECM) proteins are important modulators of migration, differentiation and proliferation for the various cell types present in the lungs; they influence the immune response as well as participate in the adherence of several fungi including Paracoccidioides brasiliensis. The expression, deposition and arrangement of ECM proteins such as laminin, fibronectin, fibrinogen, collagen and proteoglycans in the lungs of mice infected with P. brasiliensis conidia has been evaluated in this study, together with the elastic fibre system. Lungs of BALB/c mice infected with P. brasiliensis conidia were analysed for the different ECM proteins by histological and immunohistochemical procedures at different times of infection. In addition, laser scanning confocal microscopy and scanning electron microscopy were used. During the early periods, the lungs of infected animals showed an inflammatory infiltrate composed mainly of polymorphonuclear neutrophils (PMNs) and macrophages, while during the later periods, mice presented a chronic inflammatory response with granuloma formation. Re-arrangement and increased expression of all ECM proteins tested were observed throughout all studied periods, especially during the occurrence of inflammatory infiltration and formation of the granuloma. The elastic fibre system showed an elastolysis process in all experiments. In conclusion, this study provides new details of pulmonary ECM distribution during the course of paracoccidioidomycosis.

Expression of adhesion molecules in lungs of mice infected with Paracoccidioides brasiliensis conidia.

In infected tissues, leukocyte recruitment is mediated by interactions between adhesion molecules, expressed on activated vascular endothelial cells, and ligands present on circulating cells. We evaluated the inflammatory response and the expression of cellular adhesion molecules (ICAM-1, VCAM-1, CD18, LFA-1 and Mac-1) in lungs of BALB/c mice infected with Paracoccidioides brasiliensis conidia. When compared with uninfected animals, infected mice had a significant increase in the inflammatory response during the first 4 days, peaking 2-3 days post-challenge, 40.3% vs. 0.0% and 41.8% vs. 0.7%, respectively. This inflammatory infiltrate was composed mainly of neutrophils and macrophages with a few eosinophils and lymphocytes. An increase in the intensity of immunofluorescence (IF) for ICAM-1 was also observed during days 1-4. ICAM-1 was present in bronchiolar epithelium, type II pneumocytes, and macrophages, as well as on vascular endothelium. The control animals presented ICAM-1 constitutively. In infected mice, VCAM-1 was only observed on vascular endothelium during the first 2 days, with some macrophages expressing this molecule throughout the study periods. CD18 and Mac-1 but not LFA-1 were expressed with a high intensity on neutrophils and macrophages present in the inflammatory infiltrate. In addition, we observed a significant decrease in Colony forming units (CFUs) after the first 2 days post-challenge. These findings suggest that during these early stages, up-regulation of ICAM-1, VCAM-1, CD18 and Mac-1 expression occurs, participating in the inflammatory process and as such, in the pathogenesis of paracoccidioidomycosis (PCM).

The equine thymus microenvironment: a morphological and immunohistochemical analysis.

We characterized herein the microarchitecture of the equine thymus along with post-natal development (6 months-->18 years). Thymuses showed an involutional process, beginning before the puberty and defined by five histological grades, which consider the progressive cortical thymocyte depletion, shrinkage and rearrangement of the epithelial network and increase in extracellular matrix (ECM). A second feature of the equine thymus was the presence of eosinopoiesis, erythropoiesis, mastocytopoiesis and plasmacytogenesis. Additionally, lymphatic vessels, full of lymphocytes, were particularly prominent. Distribution of ECM proteins was heterogeneous, being denser in the medulla, as well as basement membranes of capsule, septa and perivascular spaces, thus similar to the patterns seen in other mammals. In vitro, horse thymic nurse cells produce ECM proteins, which are relevant in thymocyte/epithelial cell interactions. In conclusion, the equine thymus presents morphological and involutional characteristics similar to other mammals, exhibiting particular features, as prominent non-lymphoid hematopoiesis and lymphatic vessels.

Angiostrongylus costaricensis and experimental infection of Sarasinula marginata II: elimination routes

Angiostrongylus costaricensis intermediate hosts are terrestrial mollusks mostly belonging to the Veronicellidae family. In the present investigation we focused on the mechanisms of larval expulsion from Sarasinula marginata infected with A. costaricensis. Twenty-five mollusks were individually infected with 5000 L1 and sacrificed at 30 min and 1, 2, 4, 6, and 8 h post-infection and at days 1, 2, 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 20, 21, 22, 25, 26, 28, and 30 post-infection; the mollusks were then fixed and stained. Diverse organs involved throughout the course of the migratory routes of larvae from oral penetration on were specified and the mechanisms of larval access to the fibromuscular layer through the kidney, rectum, and vascular system were defined. The elimination of L3, derived from oral and/or cutaneous infections, appears to depend on granulomas located close to the excretory ducts of mucous cells.

Angiostrongylus costaricensis and experimental infection of Sarasinula marginata II: elimination routes.

Angiostrongylus costaricensis intermediate hosts are terrestrial mollusks mostly belonging to the Veronicellidae family. In the present investigation we focused on the mechanisms of larval expulsion from Sarasinula marginata infected with A. costaricensis. Twenty-five mollusks were individually infected with 5000 L1 and sacrificed at 30 min and 1, 2, 4, 6, and 8 h post-infection and at days 1, 2, 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 20, 21, 22, 25, 26, 28, and 30 post-infection; the mollusks were then fixed and stained. Diverse organs involved throughout the course of the migratory routes of larvae from oral penetration on were specified and the mechanisms of larval access to the fibromuscular layer through the kidney, rectum, and vascular system were defined. The elimination of L3, derived from oral and/or cutaneous infections, appears to depend on granulomas located close to the excretory ducts of mucous cells.

Penetration Sites and Migratory Routes of Angiostrongylus costaricensis in the Experimental Intermediate Host (Sarasinula marginata)

The intermediate hosts of Angiostrongylus costaricensis are terrestrian molluscs, mostly of the family Veronicellidae. The present work aimed at clarifying more accurately the sites of penetration and the migratory routes of A. costaricensis in the tissue slugs and at verifying the pattern of the perilarval reaction at different times of infection. Slugs were individually infected with 5,000 L1, and killed from 30 min to 30 days after infection. From 30 min up to 2 hr after infection, L1 were found within the lumen of different segments of the digestive tube having their number diminished in more advanced times after exposition until complete disappearance. After 30 min of exposition, percutaneous infection occurred, simultaneously to oral infection. Perilarval reaction was observed from 2 hr of infection around larvae in fibromuscular layer, appearing later (after 6 hr) around larvae located in the viscera. A pre-granulomatous reaction was characterized by gradative concentration of amebocytes around larvae, evolving two well-organized granulomas. In this work we confirmed the simultaneous occurrence of oral and percutaneous infections. Perilarval reaction, when very well developed, defined typical granulomatous structure, including epithelioid cell transformation. The infection also caused a systemic mobilization of amebocytes and provoked amebocyte-endothelium interactions