Pedro Paulo de Abreu Manso

Four whole-istic aspects of schistosome granuloma biology: fractal arrangement, internal regulation, autopoietic component and closure.

This paper centers on some whole-istic organizational and functional aspects of hepatic Schistosoma mansoni granuloma, which is an extremely complex system. First, it structurally develops a collagenic topology, originated bidirectionally from an inward and outward assembly of growth units. Inward growth appears to be originated from myofibroblasts derived from small portal vessel around intravascular entrapped eggs, while outward growth arises from hepatic stellate cells. The auto-assembly of the growth units defines the three-dimensional scaffold of the schistosome granulomas. The granuloma surface irregularity and its border presented fractal dimension equal to 1.58. Second, it is internally regulated by intricate networks of immuneneuroendocrine stimuli orchestrated by leptin and leptin receptors, substance P and Vasoactive intestinal peptide. Third, it can reach the population of +/- 40,000 cells and presents an autopoietic component evidenced by internal proliferation (Ki-67+ Cells), and by expression of c-Kit+ Cells, leptin and leptin receptor (Ob-R), granulocyte-colony stimulating factor (G-CSF-R), and erythropoietin (Epo-R) receptors. Fourth, the granulomas cells are intimately connected by pan-cadherins, occludin and connexin-43, building a state of closing (granuloma closure). In conclusion, the granuloma is characterized by transitory stages in such a way that its organized structure emerges as a global property which is greater than the sum of actions of its individual cells and extracellular matrix components.

Localization of hematopoietic cells in the bullfrog (Lithobates catesbeianus).

Amphibians represent the first phylogenetic group to possess hematopoietic bone marrow. However, adult amphibian hematopoiesis has only been described in a few species and with conflicting data. Bone marrow, kidney, spleen, liver, gut, stomach, lung, tegument, and heart were therefore collected from adult Lithobates catesbeianus and investigated by light microscopy and immunohistochemical methods under confocal laser microscopy. Our study demonstrated active hematopoiesis in the bone marrow of vertebrae, femur, and fingers and in the kidney, but no hematopoietic activity inside other organs including the spleen and liver. Blood cells were identified as a heterogeneous cell population constituted by heterophils, basophils, eosinophils, monocytes, erythrocytic cells, lymphocytes, and their precursors. Cellular islets of the thrombocytic lineage occurred near sinusoids of the bone marrow. Antibodies against CD34, CD117, stem cell antigen, erythropoietin receptor, and the receptor for granulocyte colony-stimulating factor identified some cell populations, and some circulating immature cells were seen in the bloodstream. Thus, on the basis of these phylogenetic features, we propose that L. catesbeianus can be used as an important model for hematopoietic studies, since this anuran exhibits hematopoiesis characteristics both of lower vertebrates (renal hematopoiesis) and of higher vertebrates (bone marrow hematopoiesis).

Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus

Zika virus (ZIKV) is a member of the flavivirus genus, and its genome is approximately 10.8 kilobases of positive-strand RNA enclosed in a capsid and surrounded by a membrane. Studies on the replication dynamics of ZIKV are scarce, which limits the development of antiviral agents and vaccines directed against ZIKV. In this study, Aedes albopictus mosquito lineage cells (C6/36 cells) and African green monkey kidney epithelial cells (Vero cells) were inoculated with a ZIKV sample isolated from a Brazilian patient, and the infection was characterized by immunofluorescence staining, phase contrast light microscopy, transmission electron microscopy and real-time RT-PCR. The infection was observed in both cell lineages, and ZIKV particles were observed inside lysosomes, the rough endoplasmic reticulum and viroplasm-like structures. The susceptibility of C6/36 and Vero cells to ZIKV infection was demonstrated. Moreover, this study showed that part of the replicative cycle may occur within viroplasm-like structures, which has not been previously demonstrated in other flaviviruses.

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 livers 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, Lennerts 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.

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.

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.

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.