High Abundance of Haemoproteus Parasites in Culicoides (Diptera, Ceratopogonidae), with a Confirmation of Culicoides reconditus as a New Vector of These Avian Blood Parasites.

Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors.

First experimental observation on biology of the avian malaria parasite Plasmodium (Novyella) homonucleophilum (lineage pSW2), with remarks on virulence and distribution.

Species of subgenus Novyella remain most fragmentarily studied amongst avian malaria agents. Transmission of the recently described Plasmodium (Novyella) homonucleophilum (lineage pSW2) occurs broadly in the Old World, including Europe, however biology of this pathogen remains insufficiently investigated. This study provided the first data on the development of P. homonucleophilum in the experimentally infected Eurasian siskins Spinus spinus exposed by inoculation of infected blood. The parasite strain was isolated from a naturally infected song thrush Turdus philomelos, multiplied in vivo, and inoculated to six Eurasian siskins. The same number of birds were used as negative controls. All exposed birds were susceptible, and the controls remained uninfected during the entire study (172 days). Prepatent period was 8-12 days post exposure (dpe). Maximum parasitaemia reached 50-90 % of infected erythrocytes between 20 and 44 dpe. Then, parasitaemia decreased but remained relatively high during the entire observation. Three of six exposed birds died, indicating high virulence of this infection. The parasitaemia increase coincided with a decline of haematocrit value, indicating anaemia. Polychromasia was evident in all infected birds but not in controls. Body mass of exposed birds increased, coinciding with increased food intake. The latter probably is an adaptation to compensate energy loss of hosts due to the long-lasting parasitism. Exo-erythrocytic stages were not found, suggesting that long-lasting parasitaemia was entirely due to erythrocytic merogony. The lineage pSW2 has been reported broadly in the Old World and is likely a generalist infection. Neglected avian Novyella malaria parasites are worth more attention of researchers due to their cosmopolitan distribution and high virulence.

Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected roadkill fieldfare Turdus pilaris.

BACKGROUND: Species of Plasmodium (Haemosporida, Plasmodiidae) are remarkably diverse haemoparasites. Information on genetic diversity of avian malaria pathogens has been accumulating rapidly, however exo-erythrocytic development of these organisms remains insufficiently addressed. This is unfortunate because, contrary to Plasmodium species parasitizing mammals, the avian malaria parasites undergo several cycles of exo-erythrocytic development, often resulting in damage of various organs. Insufficient knowledge on the exo-erythrocytic development in most described Plasmodium species precludes the understanding of mechanisms of virulence during avian malaria. This study extends information on the exo-erythrocytic development of bird malaria parasites. METHODS: A roadkill fieldfare (Turdus pilaris) was sampled in Switzerland and examined using pathologic, cytologic, histologic, molecular and microbiologic methods. Avian malaria was diagnosed, and erythrocytic and exo-erythrocytic stages of the parasite were identified using morphologic characteristics and barcode DNA sequences of the cytochrome b gene. The species-specific characteristics were described, illustrated, and pathologic changes were reported. RESULTS: An infection with Plasmodium matutinum lineage pLINN1 was detected. Parasitaemia was relatively low (0.3%), with all erythrocytic stages (trophozoites, meronts and gametocytes) present in blood films. Most growing erythrocytic meronts were markedly vacuolated, which is a species-specific feature of this parasite's development. Phanerozoites at different stages of maturation were seen in leukocytes, macrophages, and capillary endothelial cells in most organs examined; they were particularly numerous in the brain. Like the erythrocytic meronts, growing phanerozoites were markedly vacuolated. Conspicuous exo-erythrocytic development and maturation in leucocytes suggests that this fieldfare was not adapted to the infection and the parasite was capable to escape from cellular immunity. CONCLUSIONS: This is the first report of exo-erythrocytic development of the malaria parasite lineage pLINN1 during single infection and the first report of this lineage in the fieldfare. The findings of multiple phanerozoites in brain, skeletal muscle, and eye tissue in combination with signs of vascular blockage and thrombus formation strongly suggest an impaired vision and neuromuscular responsiveness as cause of the unexpected collision with a slowly moving car. Further studies on exo-erythrocytic stages of haemosporidian parasites are pivotal to understand the true level of populational damage of avian malaria in wild birds.

Immunophenotyping of progenitor cells from articular cartilage of New Zealand Rabbits (Oryctolagus cuniculus).

Mesenchymal stem cell (MSC) have immunomodulatory and anti-inflammatory effects, allowing its application in the therapy of different diseases, including articular cartilage injuries, which induce the establishment of a pro-regenerative microenvironment in the injured tissue. Therefore, our objective was to isolate, characterize and differentiate cartilage cells from different joints of New Zealand rabbit (Oryctolagus cuniculus), in order to verify their potential as MSC for future clinical use. For this, cartilage fragments were isolated from the humerus-radio-ulnar joints, humeral scapula, femoro-tibio-patellar, and lame femoris from rabbits. The results showed that the cells were rounded in the center of the plate and fibroblastoids in the periphery. After thawing, the cells did not change their growth time in culture, nor their morphology. The cells showed labeling for mesenchymal stem cell, cytoskeleton, pluripotency and cell proliferation, but not for hematopoiesis markers (CD105+ and CD34-). We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. After application of these cells in nude mice, no tumor growth was observed in spleen, kidney, liver, lung and heart. Therefore, we conclude that cells isolated from the articular cartilage of rabbits present characteristics of MSC with potential for future clinical applications.

Antioxidant characteristics and antibacterial activity of native woody species from Catamarca, Argentina.

Numerous reports describe the antioxidant and antimicrobial activity of polyphenols-rich plant extracts. The aim of this study was to determine the total polyphenols content (TPC), and the in vitro (DPPH, FRAP and TEAC) antioxidant and antibacterial activity of leaves and wood of six native woody species (Aspidosperma quebracho-blanct, Sarcomphalus mistol, Geoffroea decorticans, Prosopis chilensis, Larrea divaricata and Larrea cuneifolia) from Catamarca. Also, the phenolic profile was determined in the species with higher activity. L. cuneifolia leaf extracts showed the highest antioxidant activity followed by L. divaricata and S. mistol, while S. mistol wood extracts showed the highest. Furthermore, Larrea species showed antibacterial activity against S. aureus and E. faecalis strains showing cidal effects mainly against S. aureus. Fifty-nine polyphenols were identified in leaves and wood of Larrea and S. mistol species, which are likely to be responsible for the different activities observed.

NF-кB c-Rel modulates pre-fibrotic changes in human fibroblasts.

Skin fibrosis is one central hallmark of the heterogeneous autoimmune disease systemic sclerosis. So far, there are hardly any standardized and effective treatment options. Pathogenic mechanisms underlying fibrosis comprise excessive and uncontrolled myofibroblast differentiation, increased extracellular matrix protein (ECM) synthesis and an intensification of the forces exerted by the cytoskeleton. A deeper understanding of fibroblast transformation could help to prevent or reverse fibrosis by specifically interfering with abnormally regulated signaling pathways. The transcription factor NF-κB has been implicated in the progression of fibrotic processes. However, the cellular processes regulated by NF-κB in fibrosis as well as the NF-κB isoforms preferentially involved are still completely unknown. In an in vitro model of fibrosis, we consistently observed the induction of the c-Rel subunit of NF-κB. Functional abrogation of c-Rel by siRNA resulted in diminished cell contractility of dermal fibroblasts in relaxed, but not in stressed 3D collagen matrices. Furthermore, directed migration was reduced after c-Rel silencing and total N-cadherin expression level was diminished, possibly mediating the observed cellular defects. Therefore, NF-кB c-Rel impacts central cellular adhesion markers and processes which negatively regulate fibrotic progression in SSc pathophysiology.

Massive Infection of Lungs with Exo-Erythrocytic Meronts in European Robin Erithacus rubecula during Natural Haemoproteus attenuatus Haemoproteosis.

Haemoproteus species are widespread avian blood parasites belonging to Haemoproteidae (Haemosporida). Blood stages of these pathogens have been relatively well-investigated, though exo-erythrocytic (tissue) stages remain unidentified for the majority of species. However, recent histopathological studies show that haemoproteins markedly affect bird organs during tissue merogony. This study investigated the exo-erythrocytic development of Haemoproteus (Parahaemoproteus) attenuatus (lineage hROBIN1), the common parasite of flycatchers (Muscicapidae). Naturally infected European robins Erithacus rubecula were examined. Parasite species and lineage were identified using microscopic examination of blood stages and DNA sequence analysis. Parasitaemia intensity varied between 0.8 and 26.5% in seven host individuals. Organs of infected birds were collected and processed for histological examination. Tissues stages (meronts) were seen in six birds and were present only in the lungs. The parasites were usually located in groups and were at different stages of maturation, indicating asynchronous exo-erythrocytic development. In most parasitized individuals, 100 meronts were observed in 1 cm2 section of lungs. The largest meronts reached 108 µm in length. Mature meronts contained numerous roundish merozoites of approximately 0.8 µm in diameter. Megalomeronts were not observed. Massive merogony and resulting damage of lungs is a characteristic feature during H. attenuatus infections and might occur in related parasite lineages, causing haemoproteosis.

Gonadal differentiation during embryonic and fetal development of male New Zealand rabbits (Oryctolagus cuniculus) / Diferenciación gonadal durante el desarrollo embrionario y fetal de conejos machos neozelandeses (Oryctolagus cuniculus)

SUMMARY: The rabbit is considered an ideal animal model for studies that describe abnormalities in the testicles due to the similar morphogenetic mechanisms of sexual development and diseases commonly found in humans. The aim of this study was to determine the male sexual differentiation of the New Zealand rabbit (Oryctolagus cuniculus) through development. The gestational age was estimated and classified as 9, 12, 14, 16, 18, 20, 23 and 28 gestational days. The morphological and sexual determination were performed by histological analysis of the reproductive tract in the embryos and fetuses (9-28 days) as well as by immunohistochemistry- Desert hedgehog-Dhh- (testis-specific protein on Y chromosome- 16, 20, 23 days and adult rabbits). Gonads were observed from the 14th day in an undifferentiated stage and with homogeneous aspect. Sexual differentiation was observed from the 16th day with presence of cells forming gonadal cords and Dhh+ cells in the gonadal parenchyma. From the 18th gestational day testicular cords were observed, which evolved into organized seminiferous tubules. The formation of the efferent ducts and ductus deferens and epididymis was observed on the 20th and 23rd days, respectively. The differentiation of the external genitalia occurred from the 23rd days from the anogenital distance and was identified to identify the penile structures. In summary, the features of the sexual differentiation were determined by observation of the Dhh+ protein in embryos from the 16th day to adulthood, and the morphological particularities observed from the 18th gestational day, determined by differentiation of the external genitalia from the 23rd day.

RESUMEN: El conejo se considera un modelo animal ideal para estudios que describen anomalías a nivel testícular debido a que presenta mecanismos morfogenéticos similares al desa- rrollo sexual y enfermedades que se encuentran comúnmente en los seres humanos. El objetivo de este estudio fue determinar la diferenciación sexual masculina del conejo de Nueva Zelanda (Oryctolagus cuniculus) a través del desarrollo. La edad gestacional se estimó y clasificó en 9, 12, 14, 16, 18, 20, 23 y 28 días gestacionales. La determinación morfológica y sexual se realizó mediante análisis histológico del tracto reproductivo en los embriones y fetos (9 - 28 días) así como mediante inmunohistoquímica -Desert hedgehog-Dhh- (proteína testicular específica en el cromosoma Y- 16, 20, 23 días y conejos adultos). Las gónadas se observaron a partir del día 14 en un estadio indiferenciado y con aspecto homogéneo. Se observó diferenciación sexual a partir del día 16 con presencia de células formadoras de cordones gonadales y células Dhh+ en el parénquima gonadal. A partir del día 18 de gestación se observaron cordones testiculares, que evolucionaron a túbulos seminíferos organizados. La formación de los conductos eferentes, deferentes y del epidídimo se observó a los 20 y 23 días, respectivamente. La diferenciación de los genitales externos ocurrió a partir del día 23 desde la distancia anogenital y se utilizó para identificar las estructuras del pene. En conclusión, las características de la diferenciación sexual se determinaron mediante la observación de la proteína Dhh en embriones desde el día 16 hasta la edad adulta, y las particularidades morfológicas observadas a partir del día 18 de gestación, determinadas por diferenciación de los genitales externos a partir del día 23.

Stereological analysis of metanephros from domestic cat (Felis catus, Linnaeus 1798) embryos and fetus.

Kidney diseases are the most common illness for cats with a prevalence seven times higher than in dogs. Metanephros is the last of three renal systems to be formed during the embryonic period, which then becomes the permanent kidney. The current work aimed to analyse the morphology and to quantify the structures present in the development of metanephros from domestic cat (Felis catus) embryos and foetuses. For this purpose, the evaluation of the biometric parameters of metanephros from cat embryos and foetuses was performed in addition to the quantification of renal corpuscles and volume of cortical and medullary layers by stereological analysis. The evaluated biometric parameters were weight, width, height, thickness and volume. The values of the measured biometric parameters increased throughout the gestational stages. The quantity of renal corpuscles gradually increased following the embryo-foetal development, mainly during the middle of the gestational stage. It was during this phase that morphologically, a complete corticomedullary division was observed. Although the difference in the quantity of renal corpuscles between the middle and the end of the gestational stages was not statistically significant, there was an increase in the volume of the medullary layer and a decrease in the volume of the cortical layer between these two stages. These findings suggest that the metanephros presents a progressive growth with the renal corpuscles following this development until the middle of the gestational stage. Starting from this phase, the differentiation of the corticomedullary layers can be seen with a significant increase in the medullary layer.

Evaluation of immunohistopathological profile of tubular and solid canine mammary carcinomas.

Breast cancer is the most common cancer in women, but the incidence of mammary carcinoma in female dogs is even higher than in humans. These two tumors have similarities that can be seen by its biological behavior, molecular genetic alterations, and histology. This suggest that female dogs can be an excellent model for preclinical oncological studies. And the mammary carcinoma most frequently found in this species is the tubular and solid carcinomas. The extracellular matrix (ECM) has an important role in the progression of these tumors. Because of that we proposed to evaluate the ECM components of these carcinomas through histology with specific stains such as Masson's Trichrome, Picrosirius Red and the technique of scanning electron microscopy. With that, we found the presence of collagen fibers in the tubular carcinoma and around its parenchyma. On the other hand, the solid carcinoma presented collagen fibers throughout the parenchyma and around each tumor cell. With the transmission electron microscopy, we observed the presence of mitochondrias and rough endoplasmic reticulum in both tumors. And finally, we evaluated the expression of proteins through the immunohistochemistry, in which we found a high expression of VEGF, PCNA, CK-18 and vimentin in solid carcinoma, and a positive mark in the tubular and solid carcinoma for collagen I, III and fibronectin. Thus, we demonstrated some differences in the ECM of these mammary carcinomas, allowing a better understanding of its histological characteristics, and these data may contribute to future studies about therapies focused on tumors ECM.

Ligação de um instrumento de avaliação clínica de exposição ao vírus Zika com a Classificação Internacional de Funcionalidade, Incapacidade e Saúde / Linking a clinical assessment instrument for Zika virus exposure with the International Classification of Functioning, Disability and Health

Instrumentos de avaliação clínica foram criados a partir de experiências prévias de cuidados para outras condições crônicas complexas da infância, de modo a planejar e sistematizar ações. Contudo, é importante que tais instrumentos tenham validação externa e contemplem o atual conceito de saúde biopsicossocial. Uma interlocução com a Classificação Internacional de Funcionalidade, Incapacidade e Saúde (CIF) poderia dar subsídio teórico e validação aos instrumentos criados. Objetivo: Identificar o conteúdo comum da CIF com o instrumento de avaliação clínica aplicado em população exposta ao vírus Zika em um ambulatório de doenças infecciosas em pediatria de um hospital de referência no Estado do Rio de Janeiro. Métodos: A Ligação com a CIF foi realizada por dois revisores independentes, segundo a proposta de Cieza e colaboradores. O coeficiente Kappa foi utilizado para análise da concordância interobservadores. Resultados: O instrumento de avaliação clínica utilizado no ambulatório de referência é composto principalmente por categorias da CIF de estruturas do corpo (46,4%). Observou-se poucas categorias relacionadas aos fatores contextuais (13,1%). E, não foram encontrados itens relacionados às categorias de atividade e participação. Conclusão: A ferramenta de avaliação apresenta principalmente informações sobre as funções e estruturas do corpo, voltando-se a um olhar puramente restrito as funções fisiológicas e as estruturas anatômicas. A inexistência de categorias de atividade e participação pode comprometer a percepção das experiências vividas pelas crianças que foram expostas ao vírus Zika

The Zika epidemic and the emergence of a new health condition in Brazil, imposed a rapid organization on services to meet the current demand for care. Clinical assessment instruments were created, based on previous care experiences for other complex chronic conditions in order to plan and systematize actions. However, it is important that these instruments have external validation and include the current concept of health, biopsychosocial. An interlocution with the International Classification of Functioning, Disability and Health (CIF) could provide theoretical support and validation to the instruments created. Objective: To identify the common content among the clinical evaluation instrument applied to the population exposed to the Zika virus in a pediatric infectious disease outpatient clinic in a reference hospital in the State of Rio de Janeiro with the ICF. Methods: The link with the ICF was carried out by two independent reviewers, according to Cieza's proposal and the Kappa coefficient was used for interobserver analysis. Results: It was identified that the instrument is mainly composed of items of body structure (39; 46.4%). There were few categories related to contextual factors (11; 13.1%). And there were no items related to the categories of activity and participation. Conclusion: The assessment tool presents mainly domains of function and structure of the body, turning to a biomedical look. The lack of categories of activity and participation can compromise the perception of health status. A line of care in the light of the biopsychosocial model provides advantages for health services planning and actions

Effects of doxorubicin associated with amniotic membrane stem cells in the treatment of canine inflammatory breast carcinoma (IPC-366) cells.

BACKGROUND: Tumours in mammary glands represent the most common neoplasia in bitches, as in humans. This high incidence results in part from the stimulation of sex hormones on these glands. Among mammary tumours, inflammatory carcinoma is the most aggressive, presenting a poor prognosis to surgical treatment and chemotherapy. One of the most widely used chemotherapy drugs for breast cancer treatment is doxorubicin (DOXO). Alternative therapies have been introduced in order to assist in these treatments; studies on treatments using stem cells have emerged, since they have anti-inflammatory and immunomodulatory properties. The aim of this study was to evaluate the effects of DOXO and canine amniotic membrane stem cells (AMCs) on the triple-negative canine inflammatory mammary carcinoma cell line IPC-366. METHODS: Four experimental groups were analysed: a control group without treatment; Group I with DOXO, Group II with AMC and Group III with an association of DOXO and AMCs. We performed the MTT assay with DOXO in order to select the best concentration for the experiments. The growth curve was performed with all groups (I-III) in order to verify the potential of treatments to reduce the growth of IPC-366. For the cell cycle, all groups (I-III) were tested using propidium iodide. While in the flow cytometry, antibodies to progesterone receptor (PR), estrogen receptor (ER), PCNA, VEGF, IL-10 and TGF-ß1 were used. For steroidogenic pathway hormones, an ELISA assay was performed. RESULTS: The results showed that cells treated with 10 µg/mL DOXO showed a 71.64% reduction in cellular growth after 72 h of treatment. Reductions in the expression of VEGF and PCNA-3 were observed by flow cytometry in all treatments when compared to the control. The intracellular levels of ERs were also significantly increased in Group III (4.67% vs. 27.1%). Regarding to the levels of steroid hormones, significant increases in the levels of estradiol (E2) and estrone sulphate (S04E1) were observed in Groups I and III. On the other hand, Group II did not show differences in steroid hormone levels in relation to the control. We conclude that the association of DOXO with AMCs (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC-366 triple-negative cells. CONCLUSIONS: This treatment promoted ER positive expression, suggesting that the accumulated oestrogen conducted these cells to a synergistic state, rendering these tumour cells responsive to ERs and susceptible to new hormonal cancer therapies.

Effects of land use change (rural-urban) on the diversity and epizootiological parameters of avian Haemosporida in a widespread neotropical bird.

Transformation of natural environments for livestock, agriculture and human settlements modifies the diversity of organisms, usually decreasing in highly disturbed land uses. Like their hosts, parasites have to adapt to novel human impacted landscapes, in which the abiotic and biotic conditions are radically different from those of conserved natural environments. We evaluated the diversity (alpha and beta taxonomic and phylogenetic diversity) of haemosporidians (mtDNA cyt b lineages) in the common chlorospingus (Chlorospingus flavopectus) at five land use types. We further analyzed the response of prevalence, parasitaemia and parasite aggregation to land use types and seasonality. Parasite lineage richness (i.e., haplotypes) and abundance (no. infected hosts) decreased with disturbance. Parasite assemblages were commonly dominated by either one of two lineages, one dominant in the urban greenspace (pBAEBIC02) and the other dominant in well-preserved mountain cloud forest (hCHLFLA01). Beta diversity was mainly explained by lineage turnover. Phylo beta diversity was low (i.e., lineages are closely related). Overall prevalence increased in wet season that coincides with host's breeding season. Haemoproteus and Plasmodium prevalence presented the opposite response to urbanization (negative and positive, respectively). Parasitaemia presented similar values across land uses for both genera and seasons, while Plasmodium aggregation decreased with urbanization. Thus, some parasite lineages (pBAEBIC02) will benefit from the urbanization process, while others will entirely disappear from cities (hCHLFLA01).

Metallothionein Expression and its Influence on the In Vitro Biological Behavior of Mucoepidermoid Carcinoma.

Mucoepidermoid carcinoma (MEC) is the most common tumor in the salivary glands, often presenting with recurrence and metastasis due to its high invasive capacity. Metallothionein (MT), a zinc storage protein that supplies this element for protease activity, is probably related to mucoepidermoid carcinoma behavior. This prompted us to characterize a cell line derived from mucoepidermoid carcinoma and to correlate metallothionein expression with transforming growth factor-α (TGF-α), tumor necrosis factor-α (TNF-α) and matrix metalloproteinases (MMPs). Transcriptomic analysis and cytogenetic assays were performed to detect the expression of genes of interest and cellular chromosomal alterations, respectively. MEC cells with a depleted metallothionein 2A (MT2A) gene were subjected to Western blot to correlate metallothionein expression with growth factors and MMPs. Additionally, cells with depleted MT were subjected to migration and invasion assays. The transcriptomic study revealed reads mapped to cytokeratins 19 and AE1/AE3, α-smooth muscle actin, vimentin, and fibronectin. Cytogenetic evaluation demonstrated structural and numerical alterations, including the translocation t(11;19)(q21;p13), characteristic of MEC. Metallothionein depletion was correlated with the decreased expression of TGF-α and MMP-9, while TNF-α protein levels were augmented. Migration and invasion activity were diminished after metallothionein silencing. Our findings suggest an important role of MT in MEC invasion, through the regulation of proteins involved in this process.

Characterization of the extracellular matrix in the chorioallantoic membrane of water buffalo (Bubalus bubalis) in early gestation.

Placenta is formed by a parenchyma rich in extracellular matrix (ECM), and this structure guarantees the proper development of the embryo and placental functioning. Recently, studies have focused on the characterization of ECM in the placenta and foetal membranes of different species. This work aimed to analyse the composition of the ECM and to quantify the types of collagens in its composition. For this, 33 chorioallantoic membranes were used at different gestational ages, which were grouped into five groups. Subsequently, haematoxylin-eosin staining, Masson trichrome and picrosirius were performed for histological analysis. Through the technique of polarized light, it was possible to quantify the total collagen present in the membranes and finally the immunohistochemical technique was performed to verify the presence of collagens and glycoproteins. It was possible to verify that the chorioallantoic membranes have, in all the gestational periods of the initial third of gestation, the same histological structures, being the most significant difference the membrane thickening that occurs gradually during the gestation. However, we notice the appearance of binucleate cells only from group II. In addition, it was verified that a gradual increase of collagen occurs until the group IV, yet from the group V begins to occur a decrease of this protein. In addition, collagen I, collagen III, fibronectin and laminin were present in all membranes. With this, we concluded that the buffalo chorioallantoic membrane presents ECM in constant remodelling at the beginning of gestation and can be used as biomaterial in works on regenerative biology.

Canine amniotic membrane mesenchymal stromal/stem cells: Isolation, characterization and differentiation.

The amniotic membrane can be considered as one of the sources of isolation of these cells, since it is found in the fetal maternal interface and has low immunogenicity. Mesenchymal stromal/stem cells (MSCs) have not been identified in canine amniotic membrane (AMC). Therefore, our objective was to isolate, culture, characterize and differentiate cells derived from canine amniotic membrane (AMC) and to verify its immunological and tumorigenic potential. For this, 12 dogs fetuses of each gestational age 32, 43 and 55 days were used, and the isolation and culture of the AMC were performed. We observed that the cells presented fibroblastoid morphology and high confluence even after freezing. We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells, as well as being CD34- and CD105+. Regarding the immunological markers, we found that IL-1, IL-2, IL-6, IL-10 and MHC II were not expressed, whereas MHC I was expressed. After application of AMC cells in nude mice we can verify that there was no tumor formation. Based on this, we conclude that canine amniotic membrane is a good and accessible source for obtaining MSCs of low immunogenic and tumorigenic potential for veterinary therapeutic applications.

APC gene is modulated by hsa-miR-135b-5p in both diffuse and intestinal gastric cancer subtypes.

BACKGROUND: Several genetic and epigenetic alterations are related to the development and progression of Gastric Cancer (GC), one of those being the deregulated microRNA (miRNA) expression profile. miRNAs are small noncoding RNAs that negatively regulate the expression of thousands of genes, including oncogenes and tumor suppressor genes. Our group identified, in previous studies, some miRNAs that are differentially expressed in GC when compared to the gastric mucosa without cancer, including hsa-miR-29c and hsa-miR-135b. The aim of the study was to modulate the expression of the miRNAs hsa-miR-29c-5p and hsa-miR-135b-5p and evaluate the expression of their target genes in 2D and 3D cell cultures. METHODS: hsa-miR-29c-5p and hsa-miR-135b-5p expression profiles were modulated by transfecting mimics and antimiRs, respectively, in 2D and 3D cell cultures. The expression of the proteins coded by the genes CDC42, DNMT3A (target genes of hsa-miR-29c-5p) and APC (target gene of hsa-miR-135b-5p) were measured by Western Blot. RESULTS: Results showed that mimics and antimiRs transfection significantly altered the expression of both miRNAs, increasing the expression of hsa-miR-29c-5p and reducing the expression of hsa-miR-135b-5p, especially in the 3D culture of the cell lines. When analyzing the proteins expression, we observed that AGP01 and AGP03 cell lines transfected with mimics had a reduction in the levels of CDC42 and DNMT3A and all three cell lines transfected with antimiRs had an increase in the expression of the protein APC. CONCLUSION: We concluded that three-dimensional culture can be a more representative in vitro model that resembles better the in vivo reality. Our results also showed that hsa-miR-29c-5p is an important regulator of CDC42 and DNMT3A genes in the intestinal subtype gastric cancer and hsa-miR-135b-5p regulates the APC gene in both intestinal and diffuse subtypes of GC. Dysregulation in their expression, and consequently in their respectively signaling pathways, shows how these miRNAs can influence the carcinogenesis of different histological subtypes of gastric cancer.

Combining morphological and molecular data to reconstruct the phylogeny of avian Haemosporida.

The traditional classification of avian Haemosporida is based mainly on morphology and life history traits. Recently, molecular hypotheses have challenged the traditional classification, leading to contradictory opinions on whether morphology is phylogenetically informative. However, the morphology has never been used to reconstruct the relationships within the group. We inferred the phylogeny of avian Haemosporida from 133 morphological characters present in blood stages. We included all species with at least one mitochondrial gene characterized (n = 93). The morphological hypothesis was compared with the one retrieved from mitochondrial DNA (mtDNA) nucleotide sequences and a hypothesis that used a combination of morphological and molecular data (i.e., total evidence). In order to recover the evolutionary history and identify phylogenetically and taxonomically informative characters, they were mapped on the total evidence phylogeny. The morphological hypothesis presented more polytomies than the other two, especially within Haemoproteus. In the molecular hypothesis, the two Haemoproteus subgenera are paraphyletic, and some relationships within Parahaemoproteus were resolved. By combining the morphological and molecular data, we were able to resolve the majority of polytomies and posterior probabilities increased. We identified a unique combination of morphological traits, clearly differentiating avian Haemosporida genera, sub-genera of Leucocytozoon and Haemoproteus, and some Plasmodium sub-genera. Plasmodium had the highest number of synapomorphies. Furthermore, 86% of the species presented a unique combination of taxonomically informative characters. A limiting factor was the mismatch of traits characterized in species descriptions, leading to a morphological matrix with a considerable amount of missing data, particularly for the stages of early young and young gametocytes (67% of all missing data). Characters lacking information for the majority of species included the colour of pigment granules, the cytoplasm appearance, and the presence and dimensions of vacuoles. According to our results, the combination of morphology and mtDNA proved to be a robust alternative to reconstruct the relationships among avian Haemosporida, obtaining a resolution and support similar to that obtained using full mitochondrial genome sequences for over 100 lineages.

Morphology of the Oligoryzomys nigripes respiratory system.

Rodents are important in the transmission of infectious diseases that affect the respiratory tract, including simple infections and those caused by specific pathogens. These animals are natural reservoirs of zoonoses that cause many public health diseases. Basic knowledge on the morphology of these animals is important as basic research is useful for applied studies, such as the development of clinical, therapeutic, surgical and clinical models. Morphological data of respiratory tract in Oligoryzomys nigripes are absent in the literature. Therefore, the aim of this study was to perform a morphological analysis of the respiratory tract of O. nigripes. Five adult females from the environmental reserve in São Joaquim da Barra, São Paulo were used, donated to the Museum of Veterinary Anatomy (FMVZ/USP). Several morphological features follow the same pattern seen in rodents; however, this species showed some differences such as the presence of three lobar bronchi, nonlobed left lung and the right lung constituted by two lobes. Respiratory epithelium lined the whole respiratory tract and was seen using scanning electron microscopy the oval shape of the parenchyma and alveoli.

Sexual determination and differentiation during embryonic and fetal development of New Zealand rabbit females / Determinación y diferenciación sexual durante el desarrollo embrionario y fetal de conejos hembras de Nueva Zelanda

The aim of this study was to know the embryonic and fetal development of the female rabbit genital system (Oryctolagus cuniculus), describing its main phases and the moment of sexual differentiation. Eleven pregnant New Zealand female rabbits were used in different gestational phases. The day of coitus was determined as day 0. For each stage a minimum of two animals was considered. The samples were obtained every two days from the ninth day post-coitus (dpc) until the 28th dpc. The gestational period was divided in two: animals with undifferentiated sex (group 1) and animals with differentiated sex (group 2). The ages of embryos and fetuses were estimated through the crown-rump method. Subsequently, embryos and fetuses were dissected, fixed and processed to be embedded in paraffin (Histosec). The histological analysis was performed on sections stained with hematoxylin and eosin. Immunohistochemical analysis to determine sexual differentiation was performed on samples from the 16th, 18th and 28th dpc. Desert Hedgehog (Dhh) and Indian Hedgehog (Ihh) primary antibodies, respectively, were used to identify cells of the male and female germinal epithelium. The immunohistochemical results showed that at the 16th dpc, female sexual differentiation was evident, since positive expression of the Ihh protein was observed. Sexual differentiation was obtained through histological analysis on the 18th dpc and through anatomical observation of the external genitalia on the 24th dpc. Knowing the characteristics of the embryonic and fetal development of the female rabbit genital system as well as the moment of sexual differentiation make it possible to establish bases for future research that address the physiology and pathology of these organs. Thus, any alteration in the chain of events of sexual determination and differentiation must search for an explanation from the knowledge of the possible normal mechanisms affected.

El objetivo de esta investigación fue conocer el desarrollo embrionario y fetal del sistema genital femenino de conejo (Oryctolagus cuniculus), describiendo sus principales fases y el momento de la diferenciación sexual. Se utilizaron 11 conejos hembras gestantes neozelandesas, en diferentes fases gestacionales. El día del coito se determinó como día 0. Para cada etapa fue considerado un mínimos de dos animales. Las muestras fueron obtenidas cada dos días, a partir del noveno día post-coito (dpc) hasta el 28 dpc. El periodo gestacional fue dividido en dos: animales con sexo indiferenciado (grupo 1) y, animales con sexo diferenciado (grupo 2). Las edades de los embriones y los fetos fueron estimadas a través del método de crown-rump. Posteriormente, embriones y fetos fueron disecados, fijados y procesados para su inclusión en parafina (Histosec). El análisis histológico se realizó en secciones teñidas con Hematoxilina y Eosina. El análisis inmunohistoquímico para determinar la diferenciación sexual fue realizado en muestras de 16, 18 y 28 dpc. Para identificar células del epitelio germinativo masculino y feminino se utilizaron los anticuerpos primarios Desert Hedgehog (Dhh) e Indian Hedgehog (Ihh), respectivamente. Los resultados inmunohistoquímicos mostraron que a los 16 dpc se evidenció diferenciación sexual femenina, ya que se observó expresión positiva de la proteína Ihh. La diferenciación sexual, a través del análisis histológico fue obtenida a los 18 dpc y a través de la observación anatómica de los genitales externos a los 24 dpc. Conocer las características del desarrollo embrionario y fetal del sistema genital femenino de conejo, así como, el momento de la diferenciación sexual, permiten sentar bases para futuras investigaciones que aborden la fisiología y patología de estos órganos. Así, cualquier alteración en la cadena de eventos de la determinación y diferenciación sexual deberá buscar una explicación a partir del conocimiento de los posibles mecanismos normales afectados.