Recruitment and mortality of Rhizophora mangle L. seedlings in the Tropical Southwestern Atlantic mangrove.

Studies in the long-term recruitment and mortality of mangrove seedlings can help to understand mangrove demography and its relationship with climatic variables, environmental restoration and advances in the ecology of this ecosystem. A seven-year population dynamics study of seedling recruitment and mortality in cohorts of Rhizophora mangle L. was carried out to identify expansion processes and patterns of survival in the understory of mangrove forests on the Atlantic coast of Brazil. The present study aimed to evaluate the relationship between recruitment and mortality R. mangle seedlings at the population level, salinity, and climatic variables (precipitation, temperature and humidity). On an annual scale, seedling recruitment was positively correlated with mean temperature. Seedling density was negatively correlated with the number of recruits and positively with the number of deaths. The number of recruits was associated with dead seedlings, temperature and precipitation considering a population scale, without grouping the data. The seedling density in the stands increased with the number of dead seedlings. Our findings described the relationship between climate variability (durability and magnitude of the dry/rainy season) and the long-term population dynamics of R. mangle seedlings in a poorly studied region and from what moment, on a monthly and annual time scale, did this relationship become significant and changes occur. The findings of this study provide information on the population dynamics of the species that will help in understanding mangrove demography. These results have important implications for projections about the recruitment and survival of the species thinking about to long-term climate change that will modify current weather patterns and mangrove conservation efforts.

Ant pollination of Paepalanthus lundii (Eriocaulaceae) in Brazilian savanna.

BACKGROUND AND AIMS: Ant-plant associations are widely diverse and distributed throughout the world, leading to complex ecological networks. Regarding ant-plant mutualism, ant pollination is a very rare interaction and few studies have shown the role of ants as pollinators. Therefore, we aimed to evaluate the role of ants as effective pollinators of Paepalanthus lundii (Eriocaulaceae) in a Brazilian savanna. METHODS: Fieldwork with experimental manipulation was conducted to evaluate the fitness of P. lundii, considering potential pollinators. For this, we mainly observed the number of seeds produced in different conditions: control, ant exclusion, exclusion of flying insects, and exclusion (entomophily test) of both ants and flying insects. Furthermore, we evaluated all floral visitors throughout the day, stigma receptivity, the numbers of male and female flowers, and patterns of species co-occurrence, which can indicate the presence of different pollinators in the plants at the same time. KEY RESULTS: We observed a relation between seed production and ant visits; Camponotus crassus was the most frequent floral visitor and the most effective pollinator. Also, we observed a statistical difference between the numbers of male and female flowers produced, with a greater number of male flowers. Furthermore, P. lundii presented flowering asynchrony, with 12 different types of maturation sequence, which indicates a cross-pollination system. Lastly, we observed an overlap of the greatest abundance of C. crassus and the time of plant stigmatic receptivity, and a pattern of non co-occurrence of ants, which shows the pollinator role of this ant. CONCLUSIONS: Our data provide evidence that previous generalizations neglecting the importance of ants as pollinators are wrong. Brazilian savanna can reveal a lot about the ant-pollination syndrome, since this environment presents peculiar characteristics related to this association. Thus, this study has great significance for the understanding of the ant-pollination syndrome, and for the understanding of the complex ecological networks present in these dry arid systems.

Dissemination of bla OXA-370 gene among several Enterobacteriaceae species in Brazil.

OXA-370 is a recently described OXA-48 variant that has only been described in a few Enterobacter spp. and Klebsiella pneumoniae isolates. The purpose of this study is to assess the prevalence of OXA-370-producing isolates in carbapenem-nonsusceptible Enterobacteriaceae recovered from 28 hospitals from Brazil. Real-time PCR was used to determine the presence of bla NDM-1, bla KPC-2, bla VIM-type, bla GES-type, bla OXA-48-like, and bla IMP-type genes. A total of 4,451 Enterobacteriaceae were screened. The gene bla OXA-48-like was detected in 74 (2.5%) isolates, mostly of Enterobacter spp. (44.6% E. cloacae and 2.7% E. aerogenes) and Klebsiella spp. (31.1% K. pneumoniae and 6.7% K. oxytoca), followed by Escherichia coli, (6.7%), Morganella morganii, (2.7%), Citrobacter freundii (1.3%), Proteus mirabilis (1.3%), Providencia stuartii (1.3%), and Serratia spp. (1.3%). These isolates were from five hospitals, 67 (90.5%) from the hospital where the bla OXA-370 was first described. Sequencing of bla OXA-48-like was performed in 52 isolates, including E. cloacae, E. aerogenes, K. pneumoniae, K. oxytoca, E. coli, and C. freundii; all presenting 100% identity with bla OXA-370. PFGE revealed the presence of distinct clones among K. pneumoniae, E. cloacae, K. oxytoca, and E. coli. Susceptibility rates to meropenem, imipenem, and ertapenem among OXA-370-producing isolates were 92.3%, 78.8%, 7.7% respectively; the MIC50 /MIC90 were 0.38/2 mg/L and 1/3 mg/L for meropenem and imipenem respectively. Overall, antimicrobial susceptibility analysis suggests that OXA-370 lacks carbapenemase activity. Our study demonstrated that the bla OXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination.

Mesophotic fishes of the Abrolhos Shelf, the largest reef ecosystem in the South Atlantic.

Fishes inhabiting rhodolith beds and reefs at mesophotic depths on the Abrolhos Shelf, which encompasses the largest and richest coral reef formation in the South Atlantic Ocean, were assessed through technical diving and remotely operated vehicles (ROVs). A total of 74 fish species were recorded, including at least one new species, one new record for the south-western Atlantic and six new records for the Abrolhos region. Overfishing, mining and port activities are already threatening many endangered and commercially important species recorded on the mesophotic reefs of Abrolhos Shelf, and the establishment of marine protected areas and off-reserve fisheries regulations are urgently needed.

Note: Cold spectra of the electronic transition A(2)Σ(+)-X(2)Π of N2O(+) radical: High resolution analysis of the bands 000-100, 100-100, and 001-101.

In this note, three vibrational bands of the electronic transition A(2)Σ(+)-X(2)Π of the N2O(+) radical (000-100, 100-100, and 001-101) were theoretically analysed. Starting from Hamiltonian models proposed for this kind of molecule, their parameters were calculated using a Levenberg-Marquardt fit procedure in order to reduce the root mean square deviation from the experimental transitions below to 0.01 cm(-1). The main objective of this work is to obtain new and reliable values for rotational constant B″ and the spin-orbit interaction parameter A of the analysed vibrational levels of the X(2)Π electronic state of this molecule.

Mechanisms underpinning inattention and hyperactivity: neurocognitive support for ADHD dimensionality.

BACKGROUND: Taxometric and behavioral genetic studies suggest that attention deficit hyperactivity disorder (ADHD) is best modeled as a dimension rather than a category. We extended these analyses by testing for the existence of putative ADHD-related deficits in basic information processing (BIP) and inhibitory-based executive function (IB-EF) in individuals in the subclinical and full clinical ranges. Consistent with the dimensional model, we predicted that ADHD-related deficits would be expressed across the full spectrum, with the degree of deficit linearly related to the severity of the clinical presentation. METHOD: A total of 1547 children (aged 6-12 years) participated in the study. The Development and Well-Being Assessment (DAWBA) was used to classify children into groups according to levels of inattention and hyperactivity independently: (1) asymptomatic, (2) subthreshold minimal, (3) subthreshold moderate and (4) clinical ADHD. Neurocognitive performance was evaluated using a two-choice reaction time task (2C-RT) and a conflict control task (CCT). BIP and IB-EF measures were derived using a diffusion model (DM) for decomposition of reaction time (RT) and error data. RESULTS: Deficient BIP was found in subjects with minimal, moderate and full ADHD defined in terms of inattention (in both tasks) and hyperactivity/impulsivity dimensions (in the 2C-RT). The size of the deficit increased in a linear manner across increasingly severe presentations of ADHD. IB-EF was unrelated to ADHD. CONCLUSIONS: Deficits in BIP operate at subclinical and clinical levels of ADHD. The linear nature of this relationship provides support for a dimensional model of ADHD in which diagnostic thresholds are defined in terms of clinical and societal burden rather than representing discrete pathophysiological states.

Specificity of basic information processing and inhibitory control in attention deficit hyperactivity disorder.

BACKGROUND: Both inhibitory-based executive functioning (IB-EF) and basic information processing (BIP) deficits are found in clinic-referred attention deficit hyperactivity disorder (ADHD) samples. However, it remains to be determined whether: (1) such deficits occur in non-referred samples of ADHD; (2) they are specific to ADHD; (3) the co-morbidity between ADHD and oppositional defiant disorder/conduct disorder (ODD/CD) has additive or interactive effects; and (4) IB-EF deficits are primary in ADHD or are due to BIP deficits. METHOD: We assessed 704 subjects (age 6-12 years) from a non-referred sample using the Development and Well-Being Assessment (DAWBA) and classified them into five groups: typical developing controls (TDC; n = 378), Fear disorders (n = 90), Distress disorders (n = 57), ADHD (n = 100), ODD/CD (n = 40) and ADHD+ODD/CD (n = 39). We evaluated neurocognitive performance with a Two-Choice Reaction Time Task (2C-RT), a Conflict Control Task (CCT) and a Go/No-Go (GNG) task. We used a diffusion model (DM) to decompose BIP into processing efficiency, speed-accuracy trade-off and encoding/motor function along with variability parameters. RESULTS: Poorer processing efficiency was found to be specific to ADHD. Faster encoding/motor function differentiated ADHD from TDC and from fear/distress whereas a more cautious (not impulsive) response style differentiated ADHD from both TDC and ODD/CD. The co-morbidity between ADHD and ODD/CD reflected only additive effects. All ADHD-related IB-EF classical effects were fully moderated by deficits in BIP. CONCLUSIONS: Our findings challenge the IB-EF hypothesis for ADHD and underscore the importance of processing efficiency as the key specific mechanism for ADHD pathophysiology.

Threat bias in attention orienting: evidence of specificity in a large community-based study.

BACKGROUND: Preliminary research implicates threat-related attention biases in paediatric anxiety disorders. However, major questions exist concerning diagnostic specificity, effects of symptom-severity levels, and threat-stimulus exposure durations in attention paradigms. This study examines these issues in a large, community school-based sample. Method A total of 2046 children (ages 6-12 years) were assessed using the Development and Well Being Assessment (DAWBA), Childhood Behavior Checklist (CBCL) and dot-probe tasks. Children were classified based on presence or absence of 'fear-related' disorders, 'distress-related' disorders, and behavioural disorders. Two dot-probe tasks, which differed in stimulus exposure, assessed attention biases for happy-face and threat-face cues. The main analysis included 1774 children. RESULTS: For attention bias scores, a three-way interaction emerged among face-cue emotional valence, diagnostic group, and internalizing symptom severity (F = 2.87, p < 0.05). This interaction reflected different associations between internalizing symptom severity and threat-related attention bias across diagnostic groups. In children with no diagnosis (n = 1411, mean difference = 11.03, s.e. = 3.47, df = 1, p < 0.001) and those with distress-related disorders (n = 66, mean difference = 10.63, s.e. = 5.24, df = 1, p < 0.05), high internalizing symptoms predicted vigilance towards threat. However, in children with fear-related disorders (n = 86, mean difference = -11.90, s.e. = 5.94, df = 1, p < 0.05), high internalizing symptoms predicted an opposite tendency, manifesting as greater bias away from threat. These associations did not emerge in the behaviour-disorder group (n = 211). CONCLUSIONS: The association between internalizing symptoms and biased orienting varies with the nature of developmental psychopathology. Both the form and severity of psychopathology moderates threat-related attention biases in children.

A new record of whale shark Rhincodon typus in Brazilian waters: a report of association with Caranx crysos.

In May 2011, a Rhincodon typus was sighted on the continental shelf of the central Brazilian coast, in the vicinity of a gas platform. During the video record, an interspecific following association was observed between a Caranx crysos school and the R. typus.

Vero cells infected with the Lederle strain of canine distemper virus have increased Fas receptor signaling expression at 15 h post-infection.

We evaluated the expression of the Fas receptor gene in Vero cells infected with the Lederle vaccine strain of canine distemper virus using RT-PCR. Vero cells were plated, and after being grown for 24 h in MEM with 5% FBS, 80-90% confluent monolayer cultures were infected with the virus. The cells were harvested at 3, 6, 9, and 15 h post-infection. Uninfected Vero cells were used as a control. Total RNA was isolated from Vero cells using 1 mL Trizol(®) LS, and RT was performed using 2 µg total RNA. Primer pairs for RT-PCR amplification for the canine distemper virus nucleocapsid gene, the S26 reference gene, and the Vero rFas gene were used to analyze expression in Vero cells. RT-PCR results revealed virus activity at 3, 6, 9, and 15 h in the virus-infected Vero cells. The S26 housekeeping gene was amplified in virus infected and control samples. However, expression of the cell death receptor Fas was detected in Vero cells only at 15 h post-infection. We suggest that the Lederle vaccine induces apoptosis by Fas receptor signaling, possibly through caspase-8 signaling rather than through mitochondrial signaling in the infected cells.

Lymphatic vessel density and expressions of lymphangiogenic growth factors in salivary carcinomas.

Nodal metastasis is an important prognostic indicator in head and neck cancers, including salivary carcinomas. In these, the risk for lymph node metastasis is variable and strongly associated with the tumor histologic type. The aim of the current study was to evaluate the lymphatic vessel density (LVD) and expressions of lymphangiogenic growth factors by tumor cells in different histologic types of salivary carcinomas subdivided according to the risk for nodal metastasis. In 15 high-risk (undifferentiated, high-grade mucoepidermoid and salivary duct carcinomas) and 60 low/moderate-risk tumors (adenoid cystic, low/intermediate-grade mucoepidermoid, acinic cell, myoepithelial, epithelial-myoepithelial and polymorphic low-grade carcinomas) the expressions of vascular endothelial growth factor-C (VEGF-C), hepatocyte growth factor (HGF) and D2-40 (for assessing LVD) were examined. No significant differences were encountered between high- and low/moderate/-risk carcinomas regarding LVD and VEGF-C or HGF expressions. Furthermore, the expression of these proteins did not correlate with LVD. Lymphatic vascular invasion was found mainly in high-risk carcinomas. Intratumoral LVD was significantly lower than peritumoral, regardless of the risk for metastasis and primary site of the lesion. The histologic types of salivary carcinomas which are associated with high-risk for nodal metastasis do not present increased LVD or VEGF-C and HGF expressions. The greater tendency for metastasis in these carcinomas seems to be related to their capacity to invade lymph vessels. Further studies on tumor cell interactions with lymphatic endothelial cells are needed to improve our understanding of the metastatic potential of salivary carcinomas.

Apoptose e expressão de Bcl-2 e das caspases 3 e 8 em placenta bovina, em diferentes estádios de gestação / Apoptosis and expression of Bcl-2 and caspases 3 and 8 in placenta of cows at different pregnancy stages

Apoptose e seus mecanismos reguladores são eventos fisiológicos cruciais para a manutenção da homeostase placentária, e o desequilíbrio desses processos pode comprometer a função placentária e, consequentemente, o sucesso da gravidez. Neste estudo, investigou-se a apoptose utilizando-se histomorfometria em lâminas coradas em HE e submetidas à reação de TUNEL. Além disso, avaliou-se a expressão de Bcl-2 e das caspases 8 e 3, pela reação de polimerase em cadeia em tempo real, em placentas saudáveis em diferentes estádios de gestação. Amostras de placentônios de vacas com quatro, seis e nove meses de gestação foram colhidas e processadas. O índice apoptótico aumentou progressivamente com o avanço da gestação. Tanto o Bcl-2 quanto as caspases 3 e 8 foram expressas nos três períodos estudados, sendo a expressão de Bcl-2 menor que a de caspase 8, que é menor que a de caspase 3. Estes resultados indicam que essas moléculas estão envolvidas na via apoptótica ativada na maturação placentária, exibindo um padrão de expressão ao longo da gestação e contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.

Apoptosis and its regulating mechanisms are crucial physiological events for the maintenance of the placental homostasis; and disequilibrium of these processes may compromise placental function and the success of the pregnancy. In this study, apoptosis was investigated by histomorphometry using slides stained with HE and TUNEL reaction. Besides that, Bcl-2 and caspases 8 and 3 expression were evaluated by real time polymerase chain reaction in healthy placentas under different gestacional ages. Samples of placentones of cows at 4th, 6th, and 9th months of gestation were harvested and processed. The apoptotic index gradually increased with the advance of the gestation. Bcl-2 and caspases 3 and 8 were expressed in all the studied periods, being the expression of Bcl-2 lower than that of caspase 8, which was lower than caspase 3. These results indicate that these molecules are involved in the activated apoptotic way in the placental maturation, showing a standard expression throughout the gestation and contributing for the physiological balance of the cellularity and cellular turn over in bovine placenta.

Caspase-3/-8/-9, Bax and Bcl-2 expression in the cerebellum, lymph nodes and leukocytes of dogs naturally infected with canine distemper virus.

Canine distemper is an immunosuppressive disease caused by the canine distemper virus (CDV). Pathogenesis mainly involves the central nervous system and immunosuppression. Dogs naturally infected with CDV develop apoptotic cells in lymphoid tissues and the cerebellum, but this apoptotic mechanism is not well characterized. To better understand this process, we evaluated the expression of Bax, Bcl-2, and caspase-3, -8 and -9, by evaluating mRNA levels in the peripheral blood, lymph nodes and cerebellum of CDV-infected (CDV+) and uninfected (CDV-) dogs by real-time polymerase chain reaction (PCR). Blood samples from 12 CDV+ and 8 CDV- dogs, diagnosed by reverse transcription-PCR, were subjected to hematological analysis and apoptotic gene expression was evaluated using real-time-PCR. Tissues from the cerebellum and lymph nodes of four CDV+ and three CDV-dogs were also subjected to real time-PCR. No significant differences were found between CDV+ and CDV- dogs in the hemotological results or in the expression of caspase-3, -8, -9, Bax, and Bcl-2 in the peripheral blood. However, expression of Bax, caspase-3, -8 and -9 was significantly higher in the cerebellum of CDV+ compared to CDV- dogs. Expression of caspase-3 and -8 was significantly higher in the lymph nodes of CDV+ compared to CDV- dogs. We concluded that infection with CDV induces apoptosis in the cerebellum and lymph nodes in different ways. Lymph node apoptosis apparently occurs via caspase-3 activation, through the caspase-8 pathway, and cerebellum apoptosis apparently occurs via caspase-3 activation, through the caspase-8 and mitochondrial pathways.

Expression of the proto-oncogene c-fos and the immunolocalization of c-fos, phosphorylated c-fos and estrogen receptor beta in the human testis.

Spermatogenesis is under the control of a complex endocrine and paracrine system, including estrogen receptor (ER) signaling. In many target cells, ER promotes the transcription of c-fos and other proto-oncogenes to regulate cell growth and differentiation. Thus, in this study we evaluated the expression of the proto-oncogene c-fos and the immunolocalization of c-fos, phosphorylated c-fos and ERbeta proteins in the human testis. Testis tissue samples were obtained from 12 men undergoing orchiectomy as adjuvant treatment for prostate cancer, and were stained by immunohistochemistry for c-fos, phosphorylated c-fos and ERbeta localization. Both forms of c-fos proteins were immunoreactive, mainly in germ cells (spermatogonia, spermatocytes and spermatids) and Sertoli cells, while ERbeta was primarily present in somatic cells (Leydig, Sertoli and myofibrillar cells). In addition, testicular biopsies obtained from infertile men with obstructive azoospermia/normal spermatogenesis (n=8) or non-obstructive azoospermia/severely impaired spermatogenesis (n=12) were evaluated for c-fos and ERbeta mRNA levels using real time polymerase chain reaction. The expression of c-fos mRNA was significantly lower (fold change = 0.08, p<0.05) whereas that of ERbeta mRNA was higher (fold change = 9.43, p<0.05) in the testis of men with non-obstructive azoospermia compared to those with obstructive azoospermia. These findings suggest a complex interrelation between estrogen signaling and c-fos transcriptional activity within the human testis, with the increase of ERbeta mRNA being putatively a compensatory mechanism for lower c-fos expression in infertile men with damaged spermatogenesis.

Stable interference of EWS-FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target.

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS-FLI1 target genes still remain unknown and many have been identified in heterologous model systems. METHODS: We have developed a stable RNA interference model knocking down EWS-FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS-FLI1 and to identify genes that could contribute to tumourigenesis. RESULTS: EWS-FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS-FLI1 inhibition. We showed that TOPK is a new target gene of EWS-FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence. CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.

Apoptosis in the transplanted canine transmissible venereal tumor during growth and regression phases / Apoptose no tumor venéreo transmissível canino durante as fases de crescimento e regressão

Twelve male, mongrel, adult dogs were subcutaneously transplanted with cells originated from two canine transmissible venereal tumors (TVT). The aim was to demonstrate and to quantify the occurrence of apoptosis in the TVT regression. After six months of transplantation, a tumor sample was obtained from each dog, being six dogs with TVT in the growing phase and six in the regression phase as verified by daily measurements. Samples were processed for histological and ultrastructural purposes as well as for DNA extraction. Sections of 4µm were stained by HE, Shorr, methyl green pyronine, Van Gieson, TUNEL reaction and immunostained for P53. The Shorr stained sections went through morphometry that demonstrated an increase of the apoptotic cells per field in the regressive tumors. It was also confirmed by transmission electron microscopy, which showed cells with typical morphology of apoptosis and by the TUNEL reaction that detected in situ the 3'OH nick end labeling mainly in the regressive tumors. The regressive TVTs also showed an intensified immunostaining for P53 besides a more intense genomic DNA fragmentation detected by the agarose gel electrophoresis. In conclusion, apoptosis has an important role in the regression of the experimental TVT in a way that is P53-dependent.

Doze cães, adultos, machos e sem raça definida foram transplantados subcutaneamente, na região hipogástrica, com células originadas de dois tumores venéreos transmissíveis caninos (TVT). O objetivo do estudo foi demonstrar e quantificar a ocorrência de apoptose na regressão do TVT. Após seis meses, foi obtido um tumor de cada animal, totalizando seis em crescimento e seis em regressão. Fragmentos dos tumores foram processados para avaliação histológica, ultra-estrutural e também para extração de DNA. Cortes de 4µm foram corados em HE, Shorr, verde de metila pironina e Van Gieson e alguns foram submetidos à reação do TUNEL e à imunoistoquímica para P53. Secções coradas pelo Shorr, submetidas à morfometria, demonstram maior índice apoptótico nos tumores em regressão. Esse achado foi confirmado pela microscopia eletrônica de transmissão que evidenciou células com morfologia típica de apoptose e pela reação de TUNEL que marcou mais células nos tumores em regressão que naqueles em crescimento. A imunomarcação para P53 foi mais intensa nos tumores em regressão, assim como a fragmentação internucleossômica do genoma mostrada pela eletroforese em gel de agarose. Concluiu-se que a apoptose tem importante papel na regressão do TVT transplantado experimentalmente, sendo, nesse caso, dependente de P53 para a sua execução.

Apoptose e expressão de VP2 e GAPDH na infecção precoce pelo vírus da doença infecciosa da bursa de Fabricius em pintos SPF / Apoptosis and expression of VP2 and GADPH in an experimental infectious bursal disease in SPF chicks

Vinte e nove pintos SPF de um dia foram inoculados com o vírus da doença infecciosa da bursa de Fabricius (VDIB) para avaliar a ocorrência precoce de apoptose e a expressão da proteína viral 2 (VP2) e da enzima gliceraldeído fosfato dehidrogenase (GAPDH). Os animais foram distribuídos em cinco grupos: 1-controle; e 2 a 5- com 24, 48, 72 e 96 horas pós-inoculação, respectivamente. Fragmentos da bursa de Fabricius foram colhidos para processamento histológico e extração de RNA. Lâminas coradas em HE e TUNEL (marcação in situ da fragmentação do genoma com transferase terminal de deoxinucleotídeo) foram utilizadas na morfometria do índice apoptótico. Amostras de mRNA foram testadas para a expressão dos genes VP2 e GAPDH utilizando-se transcrição reversa e RT-PCR. Utilizou-se um kit SYBR GREEN PCR, e a reação foi desenvolvida em ABI Prism 7000 SDS. Os índices apoptóticos cresceram progressivamente indicando uma relação na atrofia bursal causada pelo VDIB. Paralelamente, os resultados da PCR em tempo real demonstraram queda da carga viral nas células linfóides da bursa nos diferentes intervalos de tempo do experimento. Esses resultados sugerem um papel protetor da apoptose na diminuição da replicação viral.

Twenty-nine SPF 1-day-old chicks were inoculated with infectious bursal disease virus (IBDV) to evaluate early apoptosis and the expression of viral protein 2 (VP2) and glyceraldehyde-3-phosphate dehydrogenease (GAPDH). Five groups were formed: G1-control -and G2 to G5, - 24, 48, 72 and 96 hours post inoculation, respectively. Half of each BF was fixed and processed by routine techniques. To quantify apoptosis, 5µm-thick sections were stained with HE and submitted to TUNEL (terminal transferase UDP nick end labeling) technique. mRNA was extracted from pooled samples of 3 animals/group and used for the expression of VP2 and GADPH genes using the reverse transcription and real-time polymerase chain reaction (RT-PCR). A SYBR GREEN PCR kit was used and the reaction was carried out in an ABI Prism 7000 SDS. Apoptotic indexes progressively increased indicating a role of IBDV in inducing hypotrophy of the BF. Also, it was showed that as long as apoptosis increased, viral protein expression decreased, which suggests that apoptosis plays a role as a defense mechanism against viral replication.

Morphometric analysis of the thymus of puppies infected with the Snyder Hill Strain of canine distemper virus / Análise morfométrica do timo de cães filhotes infectados com a amostra Snyder Hill do vírus de cinomose canina

The thymic morphometry analysis was used for determining apoptosis and atrophy of the thymus of eight puppies inoculated with canine distemper virus (CDV). Three healthy dogs were used as negative controls. Sections, 5æm thick, were stained by HE and Shorr, and the latter were evaluated by morphometry. CDV nucleoprotein was detected by immunohistochemistry. Morphometric results confirmed lymphoid hypotrophy in CDV inoculated dog thymuses, more stroma, less parenchyma and higher apoptotic index/field than negative control (not inoculated) puppies. Apoptosis plays a role in the mechanism of thymus atrophy that develops in canine distemper.

Determinaram-se a apoptose e a atrofia no timo de oito cães novos, inoculados experimentalmente com o vírus de cinomose. Três cães saudáveis foram usados como controle negativo. Secções coradas pelo Shorr foram avaliadas por morfometria. A nucleoproteína viral foi detectada por imunoistoquímica. Os resultados morfométricos confirmaram a hipotrofia e mostraram que o timo dos cães inoculados tinha mais estroma, menos parênquima e maior índice apoptótico/campo que o dos animais-controle. Pode-se concluir que a apoptose desempenha importante papel no mecanismo de hipotrofia tímica que se desenvolve na cinomose.

Expressão gênica de caspases 3 e 8 em timo e baço de ratas recém-desmamadas e imunossuprimidas por glicocorticóide / Genic expression of caspases 3 and 8 in thymus and spleen of glicocorticoid-immunossupressed weaned rats

Determinou-se a expressão gênica das caspases 3 e 8 mediante transcrição reversa de mRNA total e reação em cadeia da polimerase (RT-PCR) para avaliar a apoptose em timo e baço de ratas imunossuprimidas por glicocorticóides. Utilizou-se dexametasona para indução da apoptose e atrofia linfóide. Quarenta e cinco fêmeas Wistar recém-desmamadas foram separadas em três grupos: as ratas de A (n=18) e B (n=18) foram tratadas com 250 e 500mg de glicocorticóide, via intramuscular, respectivamente, e as do C (n=9) não foram tratadas. Após 24, 48 e 72 horas, seis animais de cada grupo tratado e três do controle foram anestesiados, pesados e sacrificados. O baço e o timo foram coletados e pesados. Fragmentos dos órgãos foram fixados em formol tamponado a 10 por cento e processados segundo técnica para inclusão em parafina. Os blocos foram seccionados em 5æm, e os cortes corados em hematoxilina e eosina. A análise histopatológica aliada ao peso dos órgãos nas diferentes doses e tempos demonstrou que a dexametasona induziu hipotrofia linfóide, que ocorreu com maior intensidade no tempo de 72 horas em animais do grupo B. Fragmentos de timo e de baço foram imediatamente congelados em nitrogênio líquido para extração de mRNA e DNA. Para a padronização da técnica de RT-PCR, utilizaram-se pool de amostras de mRNA dos animais-controle e pool de mRNA de animais tratados em cada tempo de experimento. A técnica de RT-PCR foi sensível o suficiente para a detecção dos mRNAs que codificam as caspases 3 e 8, e ambas participaram do processo de apoptose induzido por dexametasona.