Efeito do tratamento com a proteína quimérica IL-13PE sobre a inflamação pulmonar induzida por partículas de sílica em camundongos

A interleucina 13 (IL-13) tem sido apontada como um dos principais mediadores em processos de ativação de fibroblastos e indução de fibrose pulmonar, sendo, portanto, considerada como um alvo terapêutico importante. A silicose é uma doença pulmonar inflamatória crônica, de caráter ocupacional, caracterizada por uma intensa resposta fibrótica e granulomatosa. Com base nestas observações, tivemos por objetivo investigar o potencial efeito da administração da imunotoxina IL-13-PE38QQR (IL-13PE) sobre o modelo de silicose em camundongos. Camundongos Swiss-Webster foram anestesiados e instilados intranasalmente com partículas de sílica (10 mg), sendo a administração da IL-13PE (200ng/dia) realizada por via intranasal, uma vez ao dia em dias alternados no período entre 21 a 27 dias após a provocação. Analisamos o componente inflamatório, a deposição de colágeno e a área de granuloma avaliados através de técnicas clássicas de histologia, incluindo coloração com H&E e Picrus-sirius, ou ainda a quantificação do conteúdo de colágeno por Sircol. Os componentes de matriz extracelular fibronectina e laminina foram avaliados através de imunohistoquímica. Citocinas e quimiocinas foram quantificadas por sistema de ELISA. As medidas de função pulmonar e resposta de hiperreatividade foram realizadas através do sistema de pletismografia de corpo inteiro invasiva. Verificamos que o tratamento curativo com a IL-13PE inibiu de forma acentuada o comprometimento da função pulmonar nos camundongos silicóticos, incluindo tanto aumento da resistência como da elastância, assim como a resposta de hiperratividade das vias aéreas ao agente broncoconstrictor metacolina. De forma coerente, os animais silicóticos quando submetidos ao tratamento com IL-13PE apresentaram marcada redução do componente inflamatório pulmonar e da resposta fibrótica, atestado pela diminuição na produção de colágeno, laminina e fibronectina e redução importante da área de granuloma. De forma semelhante, as citocinas (TNF-α e TGF-) e quimiocinas (MIP-1α, MIP-2, TARC, IP-10, MDC) detectadas em quantidade aumentada no pulmão de animais silicóticos foram reduzidas pelo tratamento com a IL-13PE. Em conclusão, nossos resultados mostram que a administração curativa da IL-13PE foi capaz de inibir os componentes inflamatórios e fibróticos da fase crônica do quadro silicótico em camundongos, o que se refletiu de forma clara na melhora da função pulmonar. Em conjunto, nossos achados indicam que a utilização da IL13PE parece constituir uma abordagem terapêutica extremamente promissora para aplicação em casos de doenças crônicas de natureza fibrótica como a silicose.

Rosuvastatina, doença hepática gordurosa não alcoólica e ativação das células estreladas hepáticas em camundongos

Introdução: A atual epidemia de obesidade tem chamado a atenção para a doença hepática gordurosa não alcoólica (DHGNA). Atualmente não existe um medicamento para o tratamento da esteatose hepática, embora as estatinas sejam muito prescritas para pacientes obesos, este medicamento destina-se ao tratamento da hipercolesterolemia. Este trabalho teve como objetivo investigar os efeitos da rosuvastatina em um modelo de obesidade induzida por dieta, como foco principal a DHGNA e os marcadores hepáticos da lipogênese e beta-oxidação e ativação de células estreladas hepáticas (CEHs) em camundongos. Métodos: Camundongos machos C57BL/6 receberam dieta padrão (SC, 10% de energia como lipídios) ou dieta rica em gorduras (HF, 50% de energia como lipídios) durante 12 semanas. Em seguida, 7 semanas de tratamento, foram feitas, formando os grupos: SC, SCR (SC + rosuvastatina), HF e HFR (HF + rosuvastatina). As análises bioquímicas e técnicas moleculares foram aplicadas para abordar os resultados plasmáticos e moleculares. Resultados: O grupo HF apresentou maiores valores de insulina, colesterol total, triglicerídeos e leptina que o grupo SC, todos os quais foram reduzidos significativamente após o tratamento com Rosuvastatina no grupo HFR. O grupo HF apresentou maior percentual de esteatose, assim como maior ativação das CEHs, enquanto que a rosuvastatina provocou uma redução de 21% na esteatose hepática e atenuou a ativação das CEHs no grupo HFR. Em concordância com os achados histológicos, as expressões de SREBP-1 e PPAR-gama foram aumentados nos animais HF e reduzido após o tratamento no grupo HFR. Por outro lado, a expressão reduzida de PPAR-alfa e CPT-1 foram encontrados nos animais HF, sendo tais parâmetros restaurados após o tratamento no grupo HFR. Conclusão: A rosuvastatina atenua significativamente a ativação das células estreladas na obesidade induzida por dieta, afetando o equilíbrio dos PPARs na lipotoxicidade. Diante desses achados a Rosuvastatina pode ser indicada como alternativa para auxiliar o tratamento da esteatose hepática.

Inshore spawning of cobia (Rachycentron canadum) in South Carolina

We documented inshore spawning of the recreationally important cobia (Rachycentron canadum) in Port Royal Sound (PRS) and St. Helena Sound (SHS), South Carolina, during the period from April to June in both 2007 and 2008. Histological analysis of ovaries confirmed the presence of actively spawning females inshore, and gonadosomatic index (GSI) values from females collected inshore (mean=7.8) were higher than the values from females caught offshore (mean=5.6); both of these mean values indicate that spawning occurred locally. Additionally, we conducted an ichthyoplankton survey in 2008 and found cobia eggs and larvae as far as 10 and 15 km inshore from the mouths of SHS and PRS, respectively. A study of egg development that we conducted in 2007 and 2008 using hatchery-reared cobia eggs provided descriptions of embryological development of cobia. Comparison of visual and quantitative characteristics of the field-collected eggs with those of the hatchery-reared eggs allowed positive identification of eggs collected in plankton samples. The ages of field-collected eggs and presence of females with hydrated oocytes in PRS and SHS observed in our ichthyoplankton survey and histological analysis indicated that wild cobia spawn in the afternoon and early evening. The inshore migration of cobia from April to June, the presence of actively spawning females, significantly higher GSI values, and the collection of eggs inside PRS and SHS all confirm that these estuaries provide spawning habitat for cobia. Because of the potential for heavy exploitation by recreational anglers as cobia move inshore to spawn in South Carolina, current management strategies may require review.

Staging ovaries of Haddock (Melanogrammus aeglefinus): implications for maturity indices and field sampling practices

We build on recent efforts to standardize maturation staging methods through the development of a field-proof macroscopic ovarian maturity index for Haddock (Melanogrammus aeglefinus) for studies on diel spawning periodicity. A comparison of field and histological observations helped us to improve the field index and methods, and provided useful insight into the reproductive biology of Haddock and other boreal determinate fecundity species. We found reasonable agreement between field and histological methods, except for the regressing and regenerating stages (however, differentiation of these 2 stages is the least important distinction for determination of maturity or reproductive dynamics). The staging of developing ovaries was problematic for both methods partly because of asynchronous oocyte hydration during the early stage of oocyte maturation. Although staging on the basis of histology in a laboratory is generally more accurate than macroscopic staging methods in the field, we found that field observations can uncover errors in laboratory staging that result from bias in sampling unrepresentative portions of ovaries. For 2 specimens, immature ovaries observed during histological examination were incorrectly assigned as regenerating during macroscopic staging. This type of error can lead to miscalculation of length at maturity and of spawning stock biomass, metrics that are used to characterize the state of a fish population. The revised field index includes 3 new macroscopic stages that represent final oocyte maturation in a batch of oocytes and were found to be reliable for staging spawning readiness in the field. The index was found to be suitable for studies of diel spawning periodicity and conforms to recent standardization guidelines.

菘蓝组织培养及植株再生研究

本实验通过对菘蓝(Tsatis Japanica）的六种外植体进行培养并诱导分化，研究了不同的外植体在相同培养条件下器官分化的不同状况并以分化周期最短、分化频率最高的根外植物为实验材料，通过改变外源激素的配比和浓度，以及其池培养条件的变化来控制外植体定向分化．对培养根段中不定芽的发生途径，做了完整的组织学和扫描电镜观察，并用不定芽做了人工种子的包埋，初步进行j，菘蓝人工种子的研究。

Oogenesis and spawn formation in the invasive lionfish, Pterois miles and Pterois volitans

The Indo-Pacific lionfish, Pterois miles and P. volitans, have recently invaded the U.S. east coast and the Caribbean and pose a significant threat to native reef fish communities. Few studies have documented reproduction in pteroines from the Indo-Pacific. This study provides a description of oogenesis and spawn formation in P. miles and P. volitans collected from offshore waters of North Carolina, U.S.A and the Bahamas. Using histological and laboratory observations, we found no differences in reproductive biology between P. miles and P. volitans. These lionfish spawn buoyant eggs that are encased in a hollow mass of mucus produced by specialized secretory cells of the ovarian wall complex. Oocytes develop on highly vascularized peduncles with all oocyte stages present in the ovary of spawning females and the most mature oocytes placed terminally, near the ovarian lumen. Given these ovarian characteristics, these lionfish are asynchronous, indeterminate batch spawners and are thus capable of sustained reproduction throughout the year when conditions are suitable. This mode of reproduction could have contributed to the recent and rapid establishment of these lionfish in the northwestern Atlantic and Caribbean.

Translocation as a strategy to rehabilitate the queen conch (Strombus gigas) population in the Florida Keys

Queen conch (Strombus gigas) stocks in the Florida Keys once supported commercial and recreational fisheries, but overharvesting has decimated this once abundant snail. Despite a ban on harvesting this species since 1985, the local conch population has not recovered. In addition, previous work has reported that conch located in nearshore Keys waters are incapable of spawning because of poor gonadal condition, although reproduction does occur offshore. Queen conch in other areas undergo ontogenetic migrations from shallow, nearshore sites to offshore habitats, but conch in the Florida Keys are prevented from doing so by Hawk Channel. The present study was initiated to determine the potential of translocating nonspawning nearshore conch to offshore sites in order to augment the spawning stock. We translocated adult conch from two nearshore sites to two offshore sites. Histological examinations at the initiation of this study confirmed that nearshore conch were incapable of reproduction, whereas offshore conch had normal gonads and thus were able to reproduce. The gonads of nearshore females were in worse condition than those of nearshore males. However, the gonadal condition of the translocated nearshore conch improved, and these animals began spawning after three months offshore. This finding suggests that some component of the nearshore environment (e.g., pollutants, temperature extremes, poor food or habitat quality) disrupts reproduction in conch, but that removal of nearshore animals to suitable offshore habitat can restore reproductive viability. These results indicate that translocations are preferable to releasing hatchery-reared juveniles because they are more cost-effective, result in a more rapid increase in reproductive output, and maintain the genetic integrity of the wild stock. Therefore, translocating nearshore conch to offshore spawning aggregations may be the key to expediting the recovery of queen conch stocks in the Florida Keys.

Testicular development in migrant and spawning bluefin tuna (Thunnus thynnus (L.)) from the eastern Atlantic and Mediterranean

Testis histological structure was studied in bluefin tuna (Thunnus thynnus) from the eastern Atlantic and Mediterranean during the reproductive season (from late April to early June). Testicular maturation was investigated by comparing samples from bluefin tuna caught on their eastward reproductive migration off Barbate (Strait of Gibraltar area) with samples of bluefin tuna fished in spawning grounds around the Balearic Islands. Histological evaluations of cross sections showed that the testis consists of two structurally different regions, an outer proliferative region where germ cells develop synchronously in cysts, and a central region made up of a well-developed system of ducts that convey the spermatozoa produced in the proliferative region to the main sperm duct. Ultrastructural features of the different stages of the male germ cell line are very similar to those described in other teleost species. The bluefin tuna testis is of the unrestricted spermatogonial testicular type, where primary spermatogonia are present all along the germinative portion of the lobules. All stages of spermatogenesis were present in the gonad tissue of migrant and spawning bluefin tuna, although spermatids were more abundant in spawning fish. The testis size was found to increase by a factor of four (on average) during migration to the Mediterranean spawning grounds, whereas the fat bodies (mesenteric lipid stores associated with the gonads) became reduced to half their weight, and the liver mass did not change significantly with sexual maturation. Linear regression analysis of the pooled data of migrant and spawning bluefin tuna revealed a significant negative correlation between the gonad index (IG) and the fat tissue index (IF), and a weaker positive correlation between the gonad index (IG) and the liver index (IL). Our analyses indicate that the liver does not play a significant role in the storage of lipids and that mesenteric lipid reserves constitute an important energy source for gametogenesis in bluefin tuna.

Reproductive dynamics of female spotted seatrout (Cynoscion nebulosus) in South Carolina

We describe reproductive dynamics of female spotted seatrout (Cynoscion nebulosus) in South Carolina (SC). Batch fecundity (BF), spawning frequency (SF), relative fecundity (RF), and annual fecundity (AF) for age classes 1−3 were estimated during the spawning seasons of 1998, 1999, and 2000. Based on histological evidence, spawning of spotted seatrout in SC was determined to take place from late April through early September. Size at first maturity was 248 mm total length (TL); 50% and 100% maturity occurred at 268 mm and 301 mm TL, respectively. Batch fecundity estimates from counts of oocytes in final maturation varied significantly among year classes. One-year-old spotted seatrout spawned an average of 145,452 oocytes per batch, whereas fish aged 2 and 3 had a mean BF of 291,123 and 529,976 oocytes, respectively. We determined monthly SF from the inverse of the proportion of ovaries with postovulatory follicles (POF) less than 24 hours old among mature and developing females. Overall, spotted seatrout spawned every 4.4 days, an average of 28 times during the season. A chronology of POF atresia for water temperature >25°C is presented. Length, weight (ovary-free), and age explained 67%, 65%, and 58% of the variability in BF, respectively. Neither RF (number of oocytes/g ovary-free weight) nor oocyte diameter varied significantly with age. However, RF was significantly greater and oocyte diameter was smaller at the end of the spawning season. Annual fecundity estimates were approximately 3.2, 9.5, and 17.6 million oocytes for each age class, respectively. Spotted seatrout ages 1−3 contributed an average of 29%, 39%, and 21% to the overall reproductive effort according to the relative abundance of each age class. Ages 4 and 5 contributed 7% and 4%, respectively, according to predicted AF values.

Reproductive biology of male franciscanas (Pontoporia blainvillei) (Mammalia: Cetacea) from Rio Grande do Sul, southern Brazil

The reproductive biology of male franciscanas (Pontoporia blainvillei), based on 121 individuals collected in Rio Grande do Sul State, southern Brazil, was studied. Estimates on age, length, and weight at attainment of sexual maturity are presented. Data on the reproductive seasonality and on the relationship between some testicular characteristics and age, size, and maturity status are provided. Sexual maturity was assessed by histological examination of the testes. Seasonality was determined by changes in relative and total testis weight, and in seminiferous tubule diameters. Testis weight, testicular index of maturity, and seminiferous tubule diameters were reliable indicators of sexual maturity, whereas testis length, age, length, and weight of the dolphin were not. Sexual maturity was estimated to be attained at 3.6 years (CI 95% =2.7–4.5) with the DeMaster method and 3.0 years with the logistic equation. Length and weight at attainment of sexual maturity were 128.2 cm (CI 95%=125.3–131.1 cm) and 26.4 kg (CI 95% =24.7–28.1 kg), respectively. It could not be verified that there was any seasonal change in the testis weight and in the seminiferous tubule diameters in mature males. It is suggested that at least some mature males may remain reproductively active throughout the year. The extremely low relative testis weight indicates that sperm competition does not occur in the species. On the other hand, the absence of secondary sexual characteristics, the reversed sexual size dimorphism, and the small number of scars from intrassexual combats in males reinforce the hypothesis that male combats for female reproductive access may be rare for franciscana. It is hypothesized that P. blainvillei form temporary pairs (one male copulating with only one female) during the reproductive period.

Seasonal egg production of whitemouth croaker (Micropogonias furnieri) in the Río de la Plata estuary, Argentina-Uruguay

The reproductive biology of the whitemouth croaker (Micropogonias furnieri) inhabiting the estuarine waters of the Río de la Plata (Argentina-Uruguay) was studied by using histological analysis of the ovaries. Samples were collected during the spawning peak and the end of two breeding seasons (November 1995–Feb-ruary 1996 and November 1997–March 1998). Micropogonias furnieri is a multiple spawner with indeterminate annual fecundity. Spawning frequency, determined by using the percentage of females with postovulatory follicles, was about 31% in November 1995 and 25% in February 1996. At these frequencies, a female on average spawned a new batch of eggs every 3–4 days during the spawning season. Batch fecundity was fitted to a power function of length and a linear function of ovary-free female weight. The number of hydrated oocytes decreased at the end of the breeding season, coinciding with an increase of atresia. Annual egg production for a 40-cm-TL female was estimated to be between 3,300,000 and 7,300,000 eggs. In addition to the seasonal decrease in fecundity and spawning activity, a decline in egg size and weight toward the end of the breeding season was also observed.

Reproduction in the protogynous black grouper Mexico (Mycteroperca bonaci (Poey)) from the southern Gulf of Mexico

An analysis was made of sexual pattern, spawning season, sizes at sexual maturation, and sex change in black grouper (Mycteroperca bonaci) from the southern Gulf of Mexico. Samples were taken between 1996 and 2000, from industrial and small-craft commercial fi sheries, in offshore and inshore waters of the continental shelf of the Yucatan Peninsula (Campeche Bank), including the shallow waters of National Marine Park Alacranes Reef. For all collected specimens (n=1229), sex and maturation condition were determined by histological analysis of the gonads. The offshore sample consisted of 75.1% females, 24.3% males, and 0.6% transitional-stage fish. All individuals collected from inshore waters were females. Gonadal structure and population structure characteristics for Campeche Bank black grouper were consistent with the characteristics of monandric protogynous hermaphrodism for a serranid fish. Sexually active males and females were observed year-round, although ripening females, with stage-III, -IV, and -V vitellogenic oocytes in the ovaries, dominated in samples taken between December and March. In addition, peak occurrence of ripe-running females with hyaline oocytes or postovulatory follicles (or both) in the ovaries was recorded in January and February. A few precocious females began spawning in October and November, and others were still in spawning condition in May and June. Fifty percent maturity of females was attained at 72.1 cm fork length (FL). Median size at sexual inversion was 103.3 cm FL, and 50% of the females measuring 111.4 cm FL had transformed into males. The southern Gulf of Mexico grouper fishery was considered deteriorated and lacked a well-defined management strategy. Results of the present study provide helpful information on black grouper reproduction in this area and could help Mexican authorities choose appropriate management strategies for this fishery, such as minimum size limit, closed fishing season, and protection of spawning aggregations.

Spawning cycles and habitats for ballyhoo (Hemiramphus brasiliensis) and balao (H. balao) in south Florida

Two halfbeak species, ballyhoo (Hemiramphus brasiliensis) and balao (H. balao), are harvested as bait in south Florida waters, and recent changes in fishing effort and regulations prompted this investigation of the overlap of halfbeak fishing grounds and spawning grounds. Halfbeaks were sampled aboard commercial fishing vessels, and during fishery-independent trips, to determine spatial and temporal spawning patterns of both species. Cyclic patterns of gonadosomatic indices (GSIs) indicated that both species spawned during spring and summer months. Histological analysis demonstrated that specific stages of oocyte development can be predicted from GSI values; for example, female ballyhoo with GSIs >6.0 had hydrated oocytes that were 2.0−3.5 mm diameter. Diel changes in oocyte diameters and histological criteria demonstrated that final oocyte maturation occurred over a 30- to 36-hour period and that ballyhoo spawned at dusk. Hydration of oocytes began in the morning, and ovulation occurred at sunset of that same day; therefore females with hydrated oocytes were ready to spawn within hours. We compared maps of all locations where fish were collected to maps of locations where spawning females (i.e. females with GSIs >6.0) were collected to determine the degree of overlap of halfbeak fishing and spawning grounds. We also used geographic information system (GIS) data to describe the depth and bottom type of halfbeak spawning grounds. Ballyhoo spawned all along the coral reef tract of the Atlantic Ocean, inshore of the reef tract, and in association with bank habitats within Florida Bay. In the Atlantic Ocean, balao spawned along the reef tract and in deeper, more offshore waters than did ballyhoo; balao were not found inshore of the coral reef tract or in Florida Bay. Both halfbeak species, considered together, spawned throughout the fishing grounds of south Florida.

Fecundity and spawning season of striped mullet (Mugil cephalus L.) in South Carolina estuaries

Fecundity in striped mullet (Mugil cephalus) from South Carolina correlated highly with length and weight, but not with age. Oocyte counts ranged from 4.47 × 105 to 2.52 × 106 in 1998 for fish ranging in size from 331 mm to 600 mm total length, 2.13 × 105to 3.89 × 106in 1999 for fish ranging in size from 332 mm to 588 mm total length, and 3.89 × 105 to 3.01 × 106 in 2000 for fish ranging in size from 325 mm to 592 mm total length. The striped mullet in this study had a high degree of variability in the size-at-age relation-ship; this variability was indicative of varied growth rates and compounded the errors in estimating fecundity at age. The stronger relationship of fecundity to fish size allowed a much better predictive model for potential fecundity in striped mullet. By comparing fecundity with other measures of reproductive activity, such as the gonadosomatic index, histological examination, and the measurement of mean oocyte diameters, we determined that none of these methods by themselves were adequate to determine the extent of reproductive development. Histological examinations and oocyte diameter measurements revealed that fecundity counts could be made once developing oocytes reached 0.400 μm or larger. Striped mullet are isochronal spawners; therefore fecundity estimates for this species are easier to determine because oocytes develop at approximately the same rate upon reaching 400 μm. This uniform development made oocytes that were to be spawned easier to count. When fecundity counts were used in conjunction with histological examination, oocyte diameter measurements, and gonadosomatic index, a more complete measure of reproductive potential and the timing of the spawning season was possible. In addition, it was determined that striped mullet that recruit into South Carolina estuaries spawn from October through April.

Temporal and spatial dynamics of spawning, settlement, and growth of gray snapper (Lutjanus griseus) from the West Florida shelf as determined from otolith microstructures

The goal of our study was to understand the spatial and temporal variation in spawning and settlement of gray snapper (Lutjanus griseus) along the West Florida shelf (WFS). Juvenile gray snapper were collected over two consecutive years from seagrass meadows with a benthic scrape and otter trawl. Spawning, settlement, and growth patterns were compared across three sampling regions (Panhandle, Big bend, and Southwest) by using otolith microstructure. Histology of adult gonads was also used for an independent estimate of spawning time. Daily growth increments were visible in the lapilli of snapper 11–150 mm standard length; ages ranged from 38 to 229 days and estimated average planktonic larval duration was 25 days. Estimated growth rates ranged from 0.60 to 1.02 mm/d and did not differ among the three sampling regions, but did differ across sampling years. Back-calculated fertilization dates from otoliths indicated that juveniles in the Panhandle and Big Bend were mainly summer spawned fish, whereas Southwest juveniles had winter and summer fertilization dates. Settlement occurred during summer both years and in the winter of 1997 for the southern portion of the WFS. Moon phase did not appear to be strongly correlated with fertilization or settlement. Histological samples of gonads from adults collected near the juvenile sampling areas indicated a summer spawning period.