Macrophages, IL-10, and nitric oxide increase, induced by hyperglycemic conditions, impact the development of murine melanoma B16F10-Nex2.

Epidemiological studies show a strong correlation between diabetes and the increased risk of developing different cancers, including melanoma. In the present study, we investigated the impact of a streptozotocin (STZ)-induced hyperglycemic environment on B16F10-Nex2 murine melanoma development. Hyperglycemic male C57Bl/6 mice showed increased subcutaneous tumor development, partially inhibited by metformin. Tumors showed increased infiltrating macrophages, and augmented IL-10 and nitric oxide (NO) concentrations. In vivo neutralization of IL-10, NO synthase inhibition, and depletion of macrophages reduced tumor development. STZ-treated TLR4 KO animals showed delayed tumor development; the transfer of hyperglycemic C57Bl/6 macrophages to TLR4 KO reversed this effect. Increased concentrations of IL-10 present in tumor homogenates of hyperglycemic mice induced a higher number of pre-angiogenic structures in vitro, and B16F10-Nex2 cells incubated with different glucose concentrations in vitro produced increased levels of IL-10. In summary, our findings show that a hyperglycemic environment stimulates murine melanoma B16F10-Nex2 primary tumor growth, and this effect is dependent on tumor cell stimulation, increased numbers of macrophages, and augmented IL-10 and NO concentrations. These findings show the involvement of tumor cells and other components of the tumor microenvironment in the development of subcutaneous melanoma under hyperglycemic conditions, defining novel targets for melanoma control in diabetic patients.

High fat diet causes distinct aberrations in the testicular proteome.

Diet has important effects on normal physiology and the potential deleterious effects of high fat diets and obesity on male reproductive health are being increasingly described. We conducted a histological review of the effects of chronic high fat (HF) diet (using a mouse model fed a 45% fat diet for 21 weeks) with a discovery proteomic study to assess for changes in the abundance of proteins in the testis. Mice on a HF diet became obese and developed glucose intolerance. Using mass spectrometry, we identify 102 proteins affected in the testis of obese mice. These included structural proteins important for the blood testis barrier (filamin A, FLNA), proteins involved in oxidative stress responses (spermatogenesis associated 20, SPATA-20) and lipid homoeostasis (sterol regulatory element-binding protein 2, SREBP2 and apolipoprotein A1, APOA1). In addition, an important regulator protein paraspeckle component 1, PSPC-1, which interacts with the androgen receptor was significantly downregulated. Proteomic data was validated using both Western blotting and immunostaining which confirmed and localised protein expression in both mouse and human testis using biopsy specimens. This study focused mainly on the abnormalities that occurred at the protein level and as a result, we have identified several candidate proteins and conducted pathway analysis around the effects of HF diet on the testis providing novel insights not previously described. Some of the identified targets could be targeted therapeutically and future work is directed in this area.

Treatment of semen samples with α-chymotrypsin alters the expression pattern of sperm functional proteins-a pilot study.

Semen hyperviscosity delays the liquefaction of semen sample and is subjected to limited proteolysis by addition of α-chymotrypsin to reduce the viscosity. α-Chymotrypsin is a proteolytic enzyme involved in degradation of the proteins and polypeptides. Even though α-chymotrypsin improves the handling of hyperviscous samples, its effect on the sperm proteins is not clear. This study was aimed to evaluate the alteration in the expression of sperm functional proteins in samples treated with α-chymotrypsin. Among all the proteins examined in both donor and patient samples, HSPA2 (70 KDa), BAG6 (150 KDa), HIST1H2BA (14 KDa), SPA17 (17 KDa formed after cleavage of C-terminal calmodulin-binding domain), and OXPHOS complexes were undetectable in α-chymotrypsin-treated samples, while the expression of the native SPA17 (20 KDa) was significantly decreased in the α-chymotrypsin-treated samples in comparison with controls. The use of α-chymotrypsin for liquefaction of hyperviscous samples degrades functional proteins of spermatozoa. Intracellular proteins, such as OXPHOS complexes and HIST1H2BA, and sperm surface proteins (HSPA2, BAG6, and SPA17) were degraded in all treated samples. Whether treatment of samples with α-chymotrypsin affects the global proteomic outcome is unclear. More in-depth calibration studies are required to determine the appropriate concentration of α-chymotrypsin for processing hyperviscous semen samples without compromising its protein expression and function. Similarly, the effects of altered protein function on assisted reproductive techniques (ART), such as intrauterine insemination (IUI) and in vitro fertilization (IVF) outcome, are not known and require further research.

Towards the identification of reliable sperm biomarkers for male infertility: A sperm proteomic approach.

Male infertility evaluation is mainly based on semen analysis. Thus, identification of additional diagnostic methods is valuable. The aim of this study was to analyse the sperm proteome of infertile men to identify the underlying mechanisms and reliable diagnostic biomarkers. This cross-sectional study consisted of 16 infertile men and seven proven fertile men. An LC-MS/MS approach was performed in five pooled samples of each group (proven fertile men, primary infertility and secondary infertility). Differentially expressed proteins were used for functional enrichment analyses, and the most central proteins involved in altered functions in both infertile groups and the testis-specific proteins were validated using Western blotting and immunocytochemistry. In total, 1,305 sperm proteins were identified, of which 102 were underexpressed and 15 were overexpressed proteins in both infertile groups. Underexpressed proteins were mostly related to protein post-translational modification and folding, especially BAG6, HSPA2 and SPA17. Validation analysis revealed an underexpression of BAG6 in infertile men, whereas HSPA2 and SPA17 expressions did not differ between the groups. No differences were observed in the sperm localisation of these proteins. An overexpression of HIST1H2BA-a testis-specific protein-was observed in both proteomic approaches. Therefore, BAG6 and HIST1H2BA are potential candidates for male infertility biomarkers.

Outbreak of Zika Virus Infection, Chiapas State, Mexico, 2015, and First Confirmed Transmission by Aedes aegypti Mosquitoes in the Americas.

BACKGROUND: After decades of obscurity, Zika virus (ZIKV) has spread through the Americas since 2015 accompanied by congenital microcephaly and Guillain-Barré syndrome. Although these epidemics presumably involve transmission by Aedes aegypti, no direct evidence of vector involvement has been reported, prompting speculation that other mosquitoes such as Culex quinquefasciatus could be involved. METHODS: We detected an outbreak of ZIKV infection in southern Mexico in late 2015. Sera from suspected ZIKV-infected patients were analyzed for viral RNA and antibodies. Mosquitoes were collected in and around patient homes and tested for ZIKV. RESULTS: Of 119 suspected ZIKV-infected patients, 25 (21%) were confirmed by RT-PCR of serum collected 1-8 days after the onset of signs and symptoms including rash, arthralgia, headache, pruritus, myalgia, and fever. Of 796 mosquitoes collected, A. aegypti yielded ZIKV detection by RT-PCR in 15 of 55 pools (27.3%). No ZIKV was detected in C. quinquefasciatus ZIKV sequences derived from sera and mosquitoes showed a monophyletic relationship suggestive of a point source introduction from Guatemala. CONCLUSIONS: These results demonstrate the continued, rapid northward progression of ZIKV into North America with typically mild disease manifestations, and implicate A. aegypti for the first time as a principal vector in North America.

Susceptibility to Temephos and Spinosad in Aedes aegypti (Diptera: Culicidae) From Puerto Rico.

We examined the susceptibility to temephos and spinosad (Natular EC) of eight Aedes aegypti (L.) populations from Puerto Rico, following WHO method (WHO 2005). Enzyme activity was measured for alpha- and beta-esterases, multiple function oxidases, glutathione-s-transferases, and insensitive acetylcholinesterase and was tested for correlation with the susceptibility level. The results showed that larval populations from Puerto Rico obtained during 2014 were found to be susceptible to both larvicides, with low (resistance factor) RRLC50 values (<5 fold) and altered and incipiently altered enzyme expression for all populations, except the insensitive acetylcholinesterase enzyme, where only the population of Ponce showed overexpression (53.3%) above the threshold established with the New Orleans susceptible strain. We recommend the use of both larvicides for mosquito control in the study area and encourage further susceptibility monitoring.

Anti-metastatic immunotherapy based on mucosal administration of flagellin and immunomodulatory P10.

Current therapies against malignant melanoma generally fail to increase survival in most patients, and immunotherapy is a promising approach as it could reduce the dosage of toxic therapeutic drugs. In the present study, we show that an immunotherapeutic approach based on the use of the Toll-like receptor (TLR)-5 ligand flagellin (Salmonella Typhimurium FliCi) combined with the major histocompatibility complex class II-restricted P10 peptide, derived from the Paracoccidioides brasiliensis gp43 major surface protein, reduced the number of lung metastasis in a murine melanoma model. Compounds were administered intranasally into C57Bl/6 mice intravenously challenged with syngeneic B16F10-Nex2 melanoma cells, aiming at the local (pulmonary) immune response modulation. Along with a marked reduction in the number of lung nodules, a significant increase in survival was observed. The immunization regimen induced both local and systemic proinflammatory responses. Lung macrophages were polarized towards a M1 phenotype, lymph node cells, and splenocytes secreted higher interleukin-12p40 and interferon (IFN)-γ levels when re-stimulated with tumor antigens. The protective effect of the FliCi+P10 formulation required TLR-5, myeloid differentiation primary response gene 88 and IFN-γ expression, but caspase-1 knockout mice were only partially protected, suggesting that intracellular flagellin receptors are not involved with the anti-tumor effect. The immune therapy resulted in the activation of tumor-specific CD4(+) T lymphocytes, which conferred protection to metastatic melanoma growth after adoptive transfer. Taken together, our results report a new immunotherapeutic approach based on TLR-5 activation and IFN-γ production capable to control the metastatic growth of B16F10-Nex2 melanoma, being a promising alternative to be associated with chemotherapeutic drugs for an effective anti-tumor responses.

Toxicity of artemisinin [Artemisia annua L.] in two different periods of pregnancy in Wistar rats.

Artemisinin compounds are important for treating multidrug-resistant malaria; however, the possible resorption and abnormalities observed in animal reproduction studies may contraindicate artemisinin use during the first trimester. To evaluate whether artemisinin interferes with developmental outcomes at different periods of pregnancy, Wistar rats were treated by gavage with increasing doses of 7, 35 and 70 mg/kg/day from gestational day [GD] 7 to 13 or 14 to 20. Viable embryos and post-implantation losses, and progestagens and testosterone levels, were monitored in the former treatment group and pregnancy and outcomes data, post-implantation losses and male and female developmental endpoints of the offspring were evaluated in the latter treatment group. Results indicate toxicity for both periods of treatment, with lower sensitivity at later stages of pregnancy. The results showed that dosing with 35 or 75 mg/kg of artemisinin caused high percentages of post-implantation losses that correlated with a trend to lower maternal progestagens and a significant maternal testosterone decrease. These findings demonstrate that oral administration of artemisinin can adversely effect post-implantation development and pregnancy in the rat.

Reproductive evaluation of aqueous crude extract of Achillea millefolium L. (Asteraceae) in Wistar rats.

The present study was conducted to evaluate the toxicity of the exposure to the aqueous extract from leaves (AE) of Achillea millefolium L. on reproductive endpoints in Wistar rats. Adult male rats were treated daily with yarrow extract (0.3, 0.6 and 1.2 g/kg/day) during 90 days by oral gavage. Endpoints including reproductive organ weights, sperm and spermatid numbers as well as sperm morphology were evaluated. No clinical signs of toxicity were detected over the treatment period, and body weight gain was similar in all groups. A significant increase in the percentage of abnormal sperm in the group treated with the highest dose of yarrow extract was detected with no other important changes in the other reproductive endpoints studied in the male rats. Furthermore, a possible estrogenic/antiestrogenic activity of the yarrow extract screened after a 3-day treatment of immature female rats which did not show any uterotrophic effects.

Pre and postnatal exposure to endosulfan in Wistar rats.

The possible reproductive adverse effects of the pesticide endosulfan on male offspring rats exposed in utero and during lactation were investigated. Dams were treated orally with 0, 0.5 or 1.5 mg of endosulfan/kg 21 days prior to mating, during the mating, pregnancy and lactation. Maternal and reproductive outcome data and male sexual development landmarks (testis descent and preputial separation) were assessed. Reproductive endpoints of the male offspring were examined at adulthood: sex organ weights, daily sperm production, spermatid number, sperm transit, sperm morphology and testosterone level. No signs of maternal toxicity were detected at the dose levels tested. Sexual development landmarks were also unaffected. Moreover, with the exception of a significant increase in the relative epididymis weight seen in the group treated with the lowest dose, we have not found any statistically significant adverse effect in the reproductive endpoints investigated at adulthood. The results of the present study indicate that pre and postnatal exposure to low doses of endosulfan (0.5 and 1.5 mg/kg) do not induce significant adverse effects in the reproductive system of male offspring Wistar rats at adulthood.

Mediation of oxidative stress in HCH-induced neurotoxicity in rat.

Effect of repeated oral administration of hexachlorocyclohexane (HCH) (10 and 20 mg/kg body weight/day for 7 and 30 days) on the antioxidant defense system and lipid peroxidation (LPX) of rat cerebral hemisphere (CH) was evaluated. The level of LPX was elevated after 7 days of treatment in crude homogenate (endogenous and FeSO(4)- and ascorbic acid-stimulated) and subcellular fractions except the nuclear fraction in which induction was seen after 30 days. The pesticide elicited a significant decrease in the activities of cytosolic total and CN(-)-sensitive superoxide dismutase (SOD) after 7 and 30 days of HCH treatment, but failed to evoke any change in CN(-)-resistant SOD. Catalase activity decreased throughout the treatment period. Cerebral glutathione peroxidase activity (both selenium-dependent and -independent isoenzymes) and the level of glutathione content were decreased after 7 and 30 days of treatment, respectively. Activity of glutathione reductase and content of ascorbic acid, however, were enhanced following the pesticide exposure. The results suggest that repeated HCH administration induced oxidative stress in rat CH.

Age-related changes in rat testicular oxidative stress parameters by hexachlorocyclohexane.

Effect of repeated oral administration of hexachlorocyclohexane (HCH; 10 and 20 mg/kg body weight per day for 7, 15 and 30 days) on antioxidant defence system and lipid peroxidation (LPX) in the testis was compared between immature (15-day-old) and mature (90-day-old) rats. In both age-groups of rats, the pesticide elicited a significant decrease in the activities of cytosolic superoxide dismutase (SOD; total and CN(-)-resistant) and catalase, and ascorbic acid content together with an increase in the levels of LPX (both in crude homogenate and subcellular fractions) and H2O2. Testicular glutathione peroxidase (GPx; total and non-selenium-dependent) activity was enhanced in both the age-groups of rats while the testicular glutathione content as well as glutathione reductase activity remained unaltered. HCH treatment resulted in a decrease of total epididymal sperm number with a higher incidence of dead and damaged spermatozoa, and sperms having anomalous head. Statistical analyses suggest that the alterations in the testicular antioxidant defence profile in the rat are not only dependent on the duration of pesticide treatment, but also influenced by age.

Changes in rat testicular antioxidant defence profile as a function of age and its impairment by hexachlorocyclohexane during critical stages of maturation.

Age-related changes in rat testicular oxidative stress parameters were investigated. A biphasic pattern was evident for lipid peroxidation and for the activity ratio of superoxide dismutase to catalase and glutathione peroxidase with increasing age. In the first phase of life (birth-7 days), a linear fall in lipid peroxidation was accompanied by a gradual increase in the enzyme ratio which was reversed in the second phase (15-600 days). Glutathione and ascorbic acid levels increased from birth to the 45th day and remained unchanged up till 365 days and then reduced at 600 days of age. The maximum level of H2O2 observed at birth gradually decreased till 90 days and remained unchanged up till 365 days of age; thereafter its level was elevated on day 600. The results suggest that an antioxidant defence system plays a crucial role in development and maturation of the rat testis. When the rats were treated with hexachlorocyclohexane during critical stages of testicular development (6th-30th day) and responses were evaluated on the 46th day of age, elevations in the levels of testicular lipid peroxidation and H2O2 along with reduction in levels of superoxide dismutase, catalase and ascorbic acid were observed. However, no change in glutathione and its metabolizing enzymes was recorded. On the other hand, hexachlorocyclohexane elevated total testicular Ca(2+)-Mg(2+)-ATPase activity. The results advocate for impairment of testicular functions in adult age as a consequence of some permanent lesions induced by hexachlorocyclohexane during critical stages of sexual maturation.

Hexachlorocyclohexane-induced behavioural and neurochemical changes in rat.

Effect of chronic oral exposure (10 and 20 mg kg(-1) body wt. for 7, 15 and 30 days) to hexachlorocyclohexane (HCH) on open-field behaviour and activities of cerebral Na+,K+-ATPase, Mg2+-ATPase and acetylcholinesterase (AChE) of rat was evaluated. Motor and grooming activities were altered, whereas vertical exploratory activity was unaffected by HCH. Activities of Na+,K+-ATPase, Mg2+-ATPase and AChE were inhibited significantly by the pesticide. The results suggest that HCH induces impairment of the enzymes involved in synaptic activity, resulting in behavioural alterations.

Age-related differences of hexachlorocyclohexane effect on hepatic oxidative stress parameters of chicks.

Acute hexachlorocyclohexane (HCH; 50 mg/kg body wt, i.p.) treatment in young (30-day-old) chicks resulted in elevation of lipid peroxidation (LPX) only in nuclear fractions of livers while the same treatment in immature (7-day-old) chicks resulted in elevation of LPX in various liver subcellular fractions. Treatment of various subcellular fractions of livers with HCH in vitro stimulated increased LPX in young chicks than in immature ones. Hepatic cytoplasmic CN-sensitive and -resistant superoxide dismutase activities in immature chicks were inhibited significantly by HCH but no effect was observed in case of young chicks. The pesticide treatment in both immature and young chicks did not influence hepatic catalase activity. The level of glutathione in the liver of young chicks increased in response to the pesticide treatment but remained unaffected in the case of immature chicks. The results of the present study show that HCH-induced changes in hepatic oxidative stress parameters in chicks are age dependent.

Comparison of hexachlorocyclohexane-induced oxidative stress in the testis of immature and adult rats.

1. The acute effect of hexachlorocyclohexane (HCH) administration (i.p.) on testicular antioxidant system and lipid peroxidation (LPX) in immature and mature rats (15- and 90-day-old, respectively) were compared. 2. In both the age groups, the level of LPX in crude homogenate of testis (endogenous, as well as FeSO4, and ascorbic acid-stimulated) was increased after 6 hr of HCH treatment and remained high till 24 hr. However, FeSO4 and ascorbic acid-stimulated LPX was higher in 90-day-old rats in comparison to 15-day-old rats. HCH treatment also resulted in elevation of LPX level in testicular subcellular (nuclear, mitochondrial and microsomal) fractions by 6 hr of treatment. However, the magnitude of increase was greater in case of 90-day-old rats. 3. Activities of testicular cytosolic superoxide dismutases (total and CN(-)-resistant) of rats of 15- and 90-day-old age groups decreased significantly after 6 hr of HCH treatment, and remained decreased till 24 hr of the pesticide treatment. The percentage of decrease was higher in 15-day-old rats than 90-day-old rats. CN(-)-sensitive SOD activity of testis was found to decrease by 12 and 24 hr after the pesticide treatment in 15- and 90-day-old rats, respectively. The activity of catalase decreased 6 hr after the pesticide treatment in both the age groups. However, the magnitude of decrease was similar for both age groups of rats. 4. Testicular glutathione content, as well as levels of glutathione metabolizing enzymes (glutathione peroxidase and glutathione reductase), did not change in response to HCH treatment, whereas ascorbic acid content decreased by 12 and 6 hr after HCH treatment in 15- and 90-day-old rats, respectively. The level of H2O2 was found to be elevated after 6 hr of the pesticide treatment in both age groups. 5. Total epididymal sperm number was comparable in all experimental groups. However, the percentage of dead and damaged spermatozoa was significantly enhanced in HCH treated rats. 6. Acute HCH administration to rats results in induction of oxidative stress in the testis which depends upon the age of the animal.

Testosterone-induced changes in testicular antioxidant system.

In order to investigate the role of testosterone propionate (TP) on the antioxidant system of the rat testis, lipid peroxidation (LPX) and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) of the testis of testosterone-treated and control rats were compared. The results indicate that TP administration to intact adult rats resulted in a significant decline in protein content of various subcellular fractions. This is accompanied with significant elevation in LPX levels of various subcellular fractions suggesting induction of oxidative stress. Activities of three enzymes related to the metabolism of superoxide radical (SOD) and hydrogen peroxide (CAT and GPx) of testis, were found to be significantly decreased in response to TP treatment. The role of testosterone in regulating testicular spermatogenesis through oxidative stress is discussed.

Effect of aluminum on superoxide dismutase, catalase and lipid peroxidation of rat liver.

Results of the present study suggest that aluminum treatment to rats resulted in elevation of microsomal lipid peroxidation along with inhibition of catalase activity in the liver. Aluminum treatment to rats has no significant effect on rat liver superoxide dismutase activity. A 25% inhibition in catalase activity was observed when liver supernatant was incubated with A1C13 at 5 x 10(-3) M concentration and above.

Hexachlorocyclohexane-induced changes in lipid peroxidation, superoxide dismutase and catalase activities and glutathione content in chick liver.

An elevation in the level of lipid peroxides in nuclear, mitochondrial and microsomal fractions of chick liver was recorded 6 hr after hexachlorocyclohexane (HCH; 50 mg/kg body wt., ip) treatment. The magnitude of increase remained more or less same even 24 hr after the pesticide treatment. Total glutathione content also increased by 6 hr of HCH treatment and did not change even 24 hr after the pesticide treatment. Protein content of crude homogenate and 10000 g supernatant decreased significantly 6 hr after the pesticide treatment. The magnitude of decrease was more or less same even 24 hr after the pesticide treatment. Although cytoplasmic superoxide dismutase and catalase activities (expressed as units/mg protein) did not change 24 hr after HCH treatment, a small but significant increase in superoxide dismutase activity was recorded 6 hr after HCH treatment. On the other hand when activities were expressed as units/mg tissue wt., a significant decrease in the activities of both the enzymes was recorded 6 and 24 hr after HCH treatment. Therefore, the decrease in the activities of both the enzymes in response to HCH in chick liver may be due to decrease in tissue protein content in general rather than specific decrease in the activities of the enzymes.