Effects of antifreeze protein from Lolium perenne L. (LpAFP) in the vitrification of in vitro-produced bovine embryos.

In the present study, the cryoprotective effects of Lolium perenne antifreeze protein (LpAFP) on the vitrification of bovine embryos were evaluated. In vitro-produced blastocysts were divided into two groups: the control group (CG) without the addition of LpAFP and the treatment group (TG) with the addition of 500 ng/ml of LpAFP in the equilibrium and vitrification solution. Vitrification was carried out by transferring the blastocysts to the equilibrium solution [7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO)] for 2 min and then to the vitrification solution (15% EG, 15% DMSO and 0.5M sucrose). The blastocysts were deposited on a cryotop device and submerged in liquid nitrogen. Warming was carried out in three steps in solutions with different sucrose concentrations (1.0, 0.5, and 0.0 M, respectively). Embryos were evaluated for re-expansion/hatching, the total cell count, and ultrastructural analysis. There was no significant difference in the re-expansion rate 24 h after warming; however, there was variation (P < 0.05) in the hatching rate in the TG and the total number of cells 24 h after warming was higher in the TG (114.87 ± 7.24) when compared with the CG (91.81 ± 4.94). The ultrastructural analysis showed changes in organelles related to the vitrification process but, in the TG, there was less damage to mitochondria and rough endoplasmic reticulum compared with the CG. In conclusion, the addition of 500 ng/ml of LpAFP during the vitrification of in vitro-produced bovine embryos improved the hatching rate and total cell number of blastocysts after warming and mitigated intracellular damage.

Effects of dexamethasone on growth, viability and ultrastructure of bovine secondary follicles cultured in vitro.

This study aimed to evaluate the effects of dexamethasone on development, viability, antrum formation and ultrastructural integrity of bovine secondary follicles cultured in vitro for 18 days. Bovine ovaries were obtained from slaughterhouses and secondary follicles of ~150-200 µm diameter were isolated and cultured in the laboratory in TCM-199+ alone or supplemented with different concentrations of dexamethasone (1, 10, 100 and 1000 ng/ml). Follicle viability was evaluated after the culture period, using calcein-AM (viable) and ethidium homodimer (nonviable). Follicle diameters and antrum formation were evaluated at days 0, 6, 12 and 18. Before or after in vitro culture, follicles were fixed for histological and ultrastructural analysis. Follicle diameters were evaluated using analysis of variance and Kruskal-Wallis test, while chi-squared test was used to evaluate the percentage of viable follicles and antrum formation (P < 0.05). Follicles cultured for 6 days with all treatments increased their diameters significantly, but there was no significant difference between treatments at the end of the culture period. In vitro cultured follicles showed antral cavity formation at the end of the culture period, but no influence of dexamethasone was seen. Ultrastructural analysis showed that follicles cultured with dexamethasone (1, 10, 100 and 1000 ng/ml) had well preserved granulosa cells. However, oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone showed signs of degeneration. It can be concluded that follicles cultured in vitro in the presence of dexamethasone demonstrated continuous in vitro growth, but oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone had poor ultrastructure.

Expression of angiotensin II receptors in the caprine ovary and improvement of follicular viability in vitro.

This study aimed to evaluate mRNA levels of angiotensin II (ANG II) receptors (AGTR1 and AGTR2) in caprine follicles and to investigate the influence of ANG II on the viability and in vitro growth of preantral follicles. Real-time polymerase chain reaction (PCR) was used to quantify AGTR1 and AGTR2 mRNA levels in the different follicular stages. For culture, caprine ovaries were collected, cut into 13 fragments and then either directly fixed for histological and ultrastructural analysis (fresh control) or placed in culture for 1 or 7 days in α-minumum essential medium plus (α-MEM+) with 0, 1, 5, 10, 50 or 100 ng/ml ANG II. Then, the fragments were destined to morphological, viability and ultrastructural analysis. The results showed that primordial follicles had higher levels of AGTR1 and AGTR2 mRNA than secondary follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA than their respective cumulus-oocyte complex (COCs). After 7 days of culture, ANG II (10 or 50 ng/ml) maintained the percentages of normal follicles compared with α-MEM+. Fluorescence and ultrastructural microscopy confirmed follicular integrity in ANG II (10 ng/ml). In conclusion, a high expression of AGTR1 and AGTR2 is observed in primordial follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA. Finally, 10 ng/ml ANG II maintained the viability of caprine preantral follicles after in vitro culture.

Ultrastructure evaluation of goat spermatozoa after freezing in a skim milk-based extender with Trolox supplementation.

The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120 nmol ml(-1) ] and frozen in an automated system. Thawed samples (37 °C/30 s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post-thawed semen. No significant difference (P > 0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120 nmol ml(-1) Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P > 0.05) before being frozen. A higher percentage (P < 0.05) of spermatozoa with intact mitochondria was observed in fresh semen, control and Trolox 60 nmol ml(-1) groups than in the other groups. Addition of Trolox to skim milk extender at 60 nmol ml(-1) ultrastructurally preserves the plasma membrane and mitochondrial sheath integrity in goat spermatozoa after cryopreservation.

Extract of Cimicifuga racemosa (L.) Nutt protects ovarian follicle reserve of mice against in vitro deleterious effects of dexamethasone.

The present study aims to investigate if Cimicifuga racemosa (L.) Nutt extract (CIMI) reduces deleterious effects of dexamethasone (DEXA) in ovaries cultured in vitro. Mouse ovaries were collected and cultured in DMEM+ only or supplemented with 5 ng/mL of CIMI, or 4 ng/mL DEXA, or both CIMI and DEXA. The ovaries were cultured at 37.5°C in 5% CO2 for 6 days. Ovarian morphology, follicular ultrastructure, and the levels of mRNA for Bax, Bcl-2, and Caspase-3 were evaluated. The results showed that DEXA reduced the percentage of morphologically normal follicles, while CIMI prevented the deleterious effects caused by DEXA. In addition, DEXA negatively affected the stromal cellular density, while CIMI prevented these adverse effects. Ovaries cultured with DEXA and CIMI showed similar levels of mRNA for Bax, Bcl-2, and Caspase-3 compared to those cultured in control medium, while ovaries cultured with DEXA had increased expression of the above genes. Additionally, the ultrastructure of the ovaries cultured with CIMI was well preserved. Thus, the extract of CIMI was able to prevent the deleterious effects caused by DEXA on cultured mouse ovaries.

The effects of insulin and follicle-simulating hormone (FSH) during in vitro development of ovarian goat preantral follicles and the relative mRNA expression for insulin and FSH receptors and cytochrome P450 aromatase in cultured follicles.

The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 µg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

Dynamic medium containing kit ligand and follicle-stimulating hormone promotes follicular survival, activation, and growth during long-term in vitro culture of caprine preantral follicles.

The aim of this study was to evaluate the effects of a dynamic medium containing kit ligand (KL) and follicle-stimulating hormone (FSH) on the in vitro culture of caprine preantral follicles for 16 days. Ovarian fragments were cultured in α-MEM(+) containing or not containing KL (50 ng/ml) and/or FSH (50 ng/ml) added during the first (days 0-8) and/or second half (days 8-16) of the culture period. Noncultured (control) and cultured fragments were processed for histological and ultrastructural evaluation. After 1 day of culture, only the treatments performed with KL or FSH maintained a percentage of normal follicles similar to that of the control. After 16 days, all treatments using KL until day 8 (KL/KL, KL/FSH, and KL/FSH+KL) and only FSH during the entire culture period (FSH/FSH) showed higher rates of follicular survival compared to α-MEM(+) alone. After 1 and 8 days, the treatments initially cultured with KL increased the percentage of follicular activation in comparison to α-MEM(+) alone and other treatments. The highest follicular diameter after 16 days was observed in follicles cultured with KL until day 8 followed by FSH (KL/FSH). Furthermore, this treatment promoted, as early as after 1 day of culture, an increase in oocyte growth compared to α-MEM(+) alone. Ultrastructural analysis confirmed the integrity of follicles cultured in KL/FSH after 16 days. In conclusion, a dynamic medium containing KL and FSH maintained follicular integrity and promoted follicular activation and growth during the long-term in vitro culture of caprine preantral follicles.

Bovine dominant follicular fluid promotes the in vitro development of goat preantral follicles.

The aim of this study was to evaluate the effect of follicular fluid collected from bovine dominant follicles (bFF) on the in vitro development of goat preantral follicles and determine the best time to add this supplement to the culture medium. The preantral follicles were isolated and randomly distributed into four treatments in absence (control) or presence of 10% of bFF added on Days 0 (FF0-18), 6 (FF6-18) or 12 (FF12-18) of culture onwards. After 18 days, follicular development was assessed based on follicular survival, antral cavity formation, increased follicular diameter as well as fully grown oocyte (>110 µm) viability and meiosis resumption. The oocytes from the cultured follicles were in vitro-matured and processed for fluorescence or ultrastructural analysis. The results showed that on Day 18 the treatment FF0-18 had a significantly higher (P<0.05) survival than control and FF12-18, but not FF6-18. The addition of bFF at the beginning of culture (FF0-18 and FF6-18) promoted a high percentage of follicular growth, meiosis resumption and early antrum formation. Moreover, this study described for the first time the ultrastructural analysis of caprine oocytes grown in vitro. This evaluation revealed that in the presence of bFF on (FF0-18) the in vitro-grown oocytes presented normal organelle distribution and well-defined, intact plasma and nuclear membranes. In conclusion, bFF originating from dominant follicles maintain the survival and promote the in vitro growth of goat preantral follicles when added at the beginning of culture.

In vitro and in vivo evaluation of ram sperm frozen in tris egg-yolk and supplemented with superoxide dismutase and reduced glutathione.

The aim of the present study was to evaluate the in vitro and in vivo effect of the addition of superoxide dismutase (SOD) and reduced glutathione (GSH) to ram semen freezing extender. Significant differences (p < 0.05) were detected between groups regarding total motility (TM), straightness (STR) and wobble (WOB), for which the GSH 7 mM group had lesser TM and better STR than the other groups and the GSH 5 and 7 mM groups had higher wobble values than the control, SOD 25 and 100 U/ml groups. The ultrastructural analysis revealed that the acrosome was better preserved after freezing in the SOD 100 U/ml and GSH 2 and 5 mM (p < 0.05) groups than the other groups, whereas mitochondria in both the control group and the 7 mM GSH group suffered the greatest damage. The plasma membrane remained preserved after freezing, regardless of the group. For in vivo fertilization, the SOD group achieved better results than the GSH group (p > 0.05). It can therefore be concluded that the addition of SOD 100 U/ml and GSH 2 and 5 mM preserves the acrosome integrity of frozen ram spermatozoa, while the addition of SOD 100 U/ml to Tris egg-yolk extender offers protection to the membranes of sperm cells after thawing.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae).

Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Effects of epidermal growth factor and progesterone on development, ultrastructure and gene expression of bovine secondary follicles cultured in vitro.

The aims of this study were to investigate the effects of epidermal growth factor (EGF) and progesterone on the development, viability and the gene expression of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (∼0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5 °C, with 5% CO2 in air, for 18 days, in TCM-199+ (n = 63) alone (control medium) or supplemented with 10 ng/mL progesterone (n = 64), 10 ng/mL EGF (n = 61) or both EGF and progesterone (n = 66). The effects of these treatments on growth, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and cyclin B1 were evaluated, significantly different (p < 0.05). The results showed that there was a progressive increase in follicular diameter in all treatments, but only follicles cultured in medium supplemented with EGF had increased significantly in diameter when compared to follicles cultured in the control medium at the end of the culture period, significantly different (p < 0.05). A positive interaction between EGF and progesterone was not observed. In addition, the presence of EGF, progesterone or both in culture medium did not influence the rate of follicle survival and antrum formation. However, the presence of only progesterone in cultured medium increased the expression of mRNAs for GDF9 and cyclin B1 in oocytes. EGF also significantly increased the levels of mRNAs for cMOS and GDF9 when compared to follicles cultured in control medium. Ultrastructural analyzes showed that cultured follicles in all treatments maintained the integrity of granulosa cells. In conclusion, the EGF promotes the development of secondary follicles cultured in vitro for 18 days and increases the expression of cMOS and GDF9, while progesterone alone or in association with EGF have not a positive effect on follicular growth. However, progesterone increases the expression of GDF9 and cyclin B1 in oocytes.

Extract of Cimicifuga racemosa (L.) Nutt protects ovarian follicle reserve of mice against in vitro deleterious effects of dexamethasone

The present study aims to investigate if Cimicifuga racemosa (L.) Nutt extract (CIMI) reduces deleterious effects of dexamethasone (DEXA) in ovaries cultured in vitro. Mouse ovaries were collected and cultured in DMEM+ only or supplemented with 5 ng/mL of CIMI, or 4 ng/mL DEXA, or both CIMI and DEXA. The ovaries were cultured at 37.5°C in 5% CO2 for 6 days. Ovarian morphology, follicular ultrastructure, and the levels of mRNA for Bax, Bcl-2, and Caspase-3 were evaluated. The results showed that DEXA reduced the percentage of morphologically normal follicles, while CIMI prevented the deleterious effects caused by DEXA. In addition, DEXA negatively affected the stromal cellular density, while CIMI prevented these adverse effects. Ovaries cultured with DEXA and CIMI showed similar levels of mRNA for Bax, Bcl-2, and Caspase-3 compared to those cultured in control medium, while ovaries cultured with DEXA had increased expression of the above genes. Additionally, the ultrastructure of the ovaries cultured with CIMI was well preserved. Thus, the extract of CIMI was able to prevent the deleterious effects caused by DEXA on cultured mouse ovaries.

mRNA expression profile of the TNF-α system in LH-induced bovine preovulatory follicles and effects of TNF-α on gene expression, ultrastructure and expansion of cumulus-oocyte complexes cultured in vitro.

This study evaluated (1) the effects of in vivo GnRH treatment on mRNA expression of TNF-α system (TNF-α, TNFR1 and TNFR2) in granulosa cells of bovine preovulatory follicles, (2) the in vitro influence of gonadotropins on mRNA expression of TNF-α system in cultured cumulus cells, (3) the protein expression of the TNF-α system in late antral follicles and, (4) the influence of TNF-α on cumulus cells expansion, ultrastructure and on expression of HAS2, CASP3 and CASP6 in follicular cells cultured for 24 h. An increased expression of TNF-α and TNFR1 was observed after 3, 6 and 12 h of GnRH treatment when compared to 0 and 24h. Higher TNFR2 mRNA levels were observed 3, 6 and 12 h after GnRH, when compared to 0 and 24 h. Proteins of TNF-α system were also expressed in late antral follicles. In vitro, TNF-α did not affect cumulus cells expansion, but reduced the HAS2, CASP3 and CASP6 mRNA levels in cumulus cells after 12 h. After 24 h of culture, TNF-α increased the mRNA levels for CASP6 in mural granulosa cells, while the TNF-α, TNFR1 and TNFR2 mRNA levels were increased in cumulus-oocyte complexes (COCs) cultured for 12 h with gonadotropins, but not after 24 h. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α. In conclusion, TNF-α system members are present in bovine antral follicles and expression of TNF-α is influenced by gonadotropins in vivo and in vitro. In vitro, TNF-α maintained cumulus cells ultrastructure during COC culture.

Expression of TNF-α system members in bovine ovarian follicles and the effects of TNF-α or dexamethasone on preantral follicle survival, development and ultrastructure in vitro.

This study was conducted to detect the protein expression of TNF-α system members (TNF-α/TNFR1/TNFR2) in bovine ovarian follicles and to evaluate the effects of TNF-α or dexamethasone on the survival and growth of primordial follicles in vitro, as well as on gene expression in cultured ovarian tissue. It was hypothesized that TNF-α induces follicular atresia in ovarian tissues cultured in vitro, and that dexamethasone suppresses the production of endogenous TNF-α, which can improve follicle viability in vitro. Ovarian fragments were cultured for 6days in α-MEM+ supplemented with TNF-α (0, 1, 10, 100 or 200ng/ml) or dexamethasone (0, 1, 10, 100 or 200ng/ml). After culture, the expression of mRNAs for BCL-2, BAX, P53, TNF-α, and CASP3 and CASP6 were evaluated. Immunohistochemical results showed that the TNF-α system members, were detected in bovine preantral and antral follicles. After 6days, the TNF-α (10ng/ml) treatment reduced the percentage of normal preantral follicles and increased the number of TUNEL-positive cells in cultured tissue. Dexamethasone (10ng/ml) during 6days of culture did maintain the percentage of normal follicles and the ultrastructure of follicles, while the presence of TNF-α or dexamethasone did not influence primordial follicle activation. However, TNF-α or dexamethasone had no effect on the levels of mRNA for P53, BCL-2, BAX and CASP6, in cultured tissues, but the presence of dexamethasone reduced the levels of CASP3 compared to ovarian slices cultured in control medium (α-MEM+). In conclusion, proteins of the TNF-α system are expressed at different bovine follicle stages. The addition of TNF-α in culture reduces follicle survival and increases the number of apoptotic cells in ovarian tissue, while the presence of dexamethasone maintains follicle ultrastructure in cultured tissue.

Identification and location of albumin-like antigens in third-stage larva of W. bancrofti, in adult forms of Litomosoides chagasfilhoi and in the free-living nematode Caenorhabditis elegans.

Antigens resembling those of host proteins have been identified on the surface of several filarial parasites, such as immunoglobulins and serum albumins. The origin of albumin-like antigens on filarial parasites remains unclear. Several authors suggested that they have been adsorbed, or that they were metabolic waste products from nutritional utilization of human albumin, or perhaps a contamination with human products. This study searched for human albumin-like antigens by Western blot and ultrastructural analyses on filarial parasites, third stage of W. bancrofti and adult females of Litomosoides chagasfilhoi, and on the free-living Caenorhabditis elegans nematode. Our results showed approximately 67kDa proteins recognized by anti-human albumin antibodies on extracts and excretory-secretory (ES) products of the third-stage W. bancrofti. Similar albumin-like proteins were also detected on the filarial parasite L. chagasfilhoi and on C. elegans extracts. The immunocytochemistry analysis showed human albumin-like antigens on similar tissues of these nematodes. These results provide evidence that these proteins have antigenic similarity and similar distribution in nematodes tissues. Our observations suggest that albumin-like antigens presented on filarial parasites are not acquired from the host, but rather are shared antigenic determinants found even in the third-stage larvae recovered from the invertebrate host.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae) / Isolados de Bacillus thuringiensis de biomas maranhenses com potencial ação inseticida contra larvas de Aedes aegypti (Diptera, Culicidae)

Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

The ultrastructure of infective larvae (L3) of Wuchereria bancrofti after treatment with diethylcarbamazine.

Although the large use of diethylcarbamazine (DEC), as the major anti-filaricide drug, its mechanism of action remains a matter of controversy. Several authors defend the hypothesis that DEC has no direct effect on nematodes. This study demonstrated that infective larvae (L3) of Wuchereria bancrofti treated in vitro with DEC presented several behaviour and morphological changes. The first alteration produced by treatment for 2 h with 3, 5, 10 microg/ml of DEC was the reduction of motility. Larvae treated with 5, 10 microg/ml DEC showed severely affected organelles, formation of several vacuoles, mainly in neurocytes and in the muscle cells, and dissolution of cytoplasm. Some larvae showed extreme cellular disorganization with abundance of large and dense mitochondria and numerous large vacuoles containing residual organelles. Lamellar bodies, probably related to an assembly of hipodermal membranes, were also observed in some damaged larvae. Thus, undoubtedly in vitro treatment with concentrations of DEC similar to therapeutic conditions, which are 1-5 microg/ml (Hawking, 1979), had a direct effect on infective larvae of W. bancrofti by causing, primarily neuromuscular alterations with subsequent damage to organelles.

Ultrastructural characterization of the hemocytes of Culex quinquefasciatus (DIPTERA: Culicidae).

Six hemocytes cell types from Culex quinquefasciatus were identified by light and transmission electron microscopy: They are prohemocytes (9.3%), spherulocytes (1.6%), adipohemocytes (0.8%), oenocytoids (4.6%), plasmatocytes (43.4%) and granulocytes (40.3%). The prohemocytes were the smallest hemocytes encountered in the hemolymph, displaying a large and centrally located nucleus, almost filling the whole cell. The spherulocytes, which were small hemocytes, presented small and numerous spherules with a lamellar pattern and an electron-dense core. Rare adipohemocytes were observed in the C. quinquefasciatus hemolymph, presenting large nucleus with an evident nucleolus, cytoplasm containing rough endoplasmic reticulum (RER), mitochondriae and lipid inclusions. C. quinquefasciatus oenocytoids showed homogeneous cytoplasm with several granules, completely or partially filled with amorphous material. These cells showed abundant smooth endoplasmic reticulum (SER) and dense mitochondriae. By light microscopy analysis we identified two morphological types of plasmatocytes, granular and agranular. However, ultrastructural investigation revealed that the granular cells contained lipid inclusion between RER membranes, instead of membrane-bounded granules. The granulocytes presented a fusiform or circular profile and displayed a unique and very complex process of granules formation, including organization of polysomes inside vesicles that protrude from the Golgi system, synthesis of a proteinaceous material, condensation of the granule matrix and recycling of endoplasmic membranes. Intense endocytic pathways were also observed in the granulocytes.

The effects of diethylcarbamazine on the ultrastructure of lung cells in vivo.

The pulmonary surfactant synthesis is disturbed in experimentally induced asthma, as are the intracellular storage capacity and its physical activity. These alterations may also be present in chronic asthmatic patients, and therefore the dysfunction of the pulmonary surfactant system may play an important role in the pathophysiology of asthma. Some clinical reports have described favorable results with the use of diethylcarbamazine (DEC) in patients with bronchial asthma showing that DEC is effective in terminating acute attacks of bronchial asthma. The present study aimed to analyze the ultrastructural alterations of lung cells after treatment in vivo with diethylcarbamazine. After 12 days of treatment with DEC, when compared with control samples, type II pneumocytes showed active nuclei with abundant euchromatin and evident nucleoli, and a substantially greater number of mature secretion vesicle. On the other hand, type I pneumocytes showed no morphological alterations. After DEC treatment, lung macrophages also presented several characteristics of cellular activation such as nuclei with a prominence of euchromatin and central nucleoli as well as an abundance of early and late endossomes distributed throughout the cytoplasm. These results confirm that DEC exerts a role in the activation of important pulmonary cellular pathways, which are probably related to the clinical improvement of asthma symptoms after DEC treatment.

Sildenafil (Viagra®) blocks inflammatory injury in LPS-induced mouse abortion: A potential prophylactic treatment against acute pregnancy loss?

INTRODUCTION: Recurrent pregnancy losses (RPL) are common women's health issues. Inflammatory and thrombotic events have been associated with RPL including excessive production of cytokines, in particular TNF-α. However, mechanisms behind gestational losses are not yet fully understood. Sildenafil inhibits phosphodiesterase Type-5 (PDE5). This drug increases intracellular cyclic guanosine monophosphate, having vasodilatory and, more recently described, anti-inflammatory properties. PDE5 is present in murine and human uterus and placenta. Sildenafil is already used clinically for treatment of human fetal growth restriction (FGR). Our objective was to determine if Sildenafil alone or in combination with Heparin had protective effects in pregnant Swiss albino challenged to abort by lipopolysaccharide (LPS). METHODS: Treatments (Sildenafil (50 mg/kg/day), Heparin (500 IU/Kg/day) or Sildenafil + Heparin at the same doses) were initiated the morning of copulation plug detection (gestational day (gd0)). On the 15th day of pregnancy, an intra-peritoneal injection of LPS (100 µg/kg) was administered. Untreated, pregnant mice challenged by LPS served as controls. RESULTS: Assessments at 48 h after LPS revealed that Sildenafil + Heparin prevented fetal loss. Early assessments at 2 h after LPS indicated that the pretreatments prevented induction of inflammatory cytokine production (TNF-α, IL-1ß/NF-κß) and preserved placental histopathology. DISCUSSION: Combined Sildenafil + Heparin therapy was superior to either treatment alone in most analyses. The known safety of Sildenafil and Heparin in human pregnancy suggests that usage of these combined agents may be of value for treatment of patients with impending pregnancy loss or prophylactically in women with a history of recurrent miscarriages.