FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles from neonatal rat ovaries.

Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles. However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation.

Intercellular adhesion molecule-1 gene K469E polymorphism and ischemic stroke: a case-control study in a Chinese population.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) was involved in the pathogenetic mechanisms responsible for ischemic stroke (IS). Population-based sample have revealed gene-gender interaction in blood pressure which is major risk for IS. We sought to evaluate whether ICAM-1 K469E polymorphism was involved in the causation of IS and whether it was different between female and male. METHODS: A 1:1 case-control study was conducted. The K469E polymorphism of ICAM-1 gene were analyzed by polymerase chain reaction (PCR) and restriction enzyme analysis in Chinese patients with IS (n = 309) and elderly subjects without IS (n = 309). RESULTS: ICAM-1 K469E polymorphism was significantly associated with IS. Interestingly, a further analysis stratified by sex found that there was significance between 469E genotypes and IS in female, but not in male. Multiple regression analysis revealed that ICAM-1 K469E polymorphism was still significantly associated with IS, compared with ICAM-1 KK genotype in all population (OR = 1.60, P = 0.030). Stratified by sex, EE combined EK was contributory factor to IS in female (OR = 3.03, P = 0.004), but not in male. After adjustment for confounding factors, the interaction between female and ICAM-1 EK/EE genotypes was found (OR = 3.54, P = 0.001). CONCLUSIONS: It is suggested that the ICAM-1 469E allele may be important in the pathogenesis of ischemic stroke, especially in female but not in male.

Differentially expressed genes between female and male adult Anopheles anthropophagus.

The aim of the present study was to identify sex-specific genes in adult Anopheles anthropophagus. As the major malaria vector and Brugia malayi vector in the Asian continent, female Anopheles mosquitoes take blood meals and transmit pathogens through this pathway, while males are nectar feeders. This complex behavior is controlled at several levels, but is probably initiated by the genetic background difference between these two groups. In our study, a subtractive cDNA library for female A. anthropophagus was constructed using the suppression subtractive hybridization (SSH) technique and then 3,074 clones from the female SSH library were analyzed using a microarray-based survey. Genes that were expressed differentially according to sex in A. anthropophagus were screened using real-time polymerase chain reaction and reverse transcription polymerase chain reaction. In our results, we report a series of genes which may be involved in female-specific mosquito behavior, including an inorganic phosphate transporter, a serine protease, the salivary protein GP35-2, and the D7 cluster salivary protein. These findings will provide clues to the nature of insect vectors and open up unprecedented opportunities to develop novel strategies for the control of mosquito-borne diseases.

[Study on immuno-effect with GRA4 or SAG2 gene recombinant BCG vaccine of Toxoplasma gondii].

OBJECTIVE: To compare the immuno-protection induced by the recombinant BCG vaccine of Toxoplasma gondii GRA4 gene (rBCG-GRA4) and SAG2 gene (rBCG-SAG2) in BALB/c mice. METHODS: 108 SPF BALB/c mice were divided into 6 groups: PBS, BCG, rBCG, rBCG-GRA4, rBCG-SAG2 and rBCG-GRA4+SAG2, each with 18 mice. Each mouse was injected by 100 microl corresponding materials for 2 times. Blood was taken from tail vein before inoculation. 4,6 and 8 weeks after inoculation, spleen was moved and blood was taken from orbit vein of 3 mice from each group for the detection of cytokines, IgG and IgM antibodies, T lymphocyte subgroups and transformation efficiency. 3 weeks after the last inoculation, 9 mice from each group were challenged intraperitoneally with 50 tachyzoites of T. gondii RH strain and their survival time was observed. RESULTS: rBCG vaccine of T. gondii induced immune response. The value of CD3+ CD4+/CD3+CD8+ of group BCG-GRA4+SAG2 was the highest (14.06+/-1.17) in the 4th week; the IgG titer in the BCG-GRA4+SAG2 group was the highest (0.18+/-0.02) in the 6th week and the IgM titer in the BCG-SAG2 group was the highest (0.82+/-0.05) in the 8th week. The average survival time of the mice in BCG-SAG2 group was about 8.61 days after challenged with tachyzoites, and that of the PBS control group, 7.33 days. The average survival time in the 3 immunized groups was one day longer than that of the control. CONCLUSION: The rBCG vaccine of T. gondii shows certain immuno-protection in mice.

Monoclonal antibody 12D5 inhibits eosinophil infiltration in the brain of Angiostrongylus cantonensis-infected BALB/c mice.

Each of BALB/c mice was infected with 50 Angiostrongylus cantonensis larvae. One group of mice received an intraperitoneal injection of 50 µg 12D5 monoclonal antibody (mAb) against a 98 kDa antigen of adult worms at 10 days post-infection (dpi), with a booster injection of 25 µg at 12 dpi. Five mice from each group were sacrificed at 14 dpi for pathological examination and RNA extraction. The infiltration of eosinophils and severity of eosinophilic meningitis were reduced in 12D5 mAb-treated mice compared with the infected mice without 12D5 treatment. The levels of eotaxin mRNA expression in spleen significantly increased and the expression of the Th2-type cytokine IL-5 significantly decreased. However, the expression of IL-4 was not changed. 12D5 mAb can observably enhance the survival rate of infected mice and reduce symptoms of angiostrongyliasis. A. cantonensis infection is a major cause of eosinophilic meningoencephalitis. The results of this study could be helpful for the development of treatment of human angiostrongylosis.

Seroprevalence of Angiostrongylus cantonensis infection in humans in China.

A seroepidemiological survey was carried out in China during 2009-2010 to determine the extent of circulating antigens (CAg) for Angiostrongylus cantonensis in the Chinese population using the gold immunochromatographic assay, with the objective of elucidating the nationwide prevalence of angiostrongyliasis in China. A total of 1,730 blood samples was collected and assayed from the general adult population (the "general group"), and those involved in aquaculture or processing of snails Achatina fulica and Pomacea canaliculat (the "occupational group") from 5 provinces (Fujian, Hunan, Guangdong, Guangxi, and Zhejiang) and 1 municipal city (Beijing). The overall seroprevalence for the "occupational group" was 7.4% (40/540), which was significantly higher (P < 0.001) than that of the "general group" (0.8%, 9/1,190). The seroprevalence in males (9.5%) was significantly higher than in females (4.2%) (P < 0.05). These results demonstrate that angiostrongyliasis represents a significant zoonotic disease in China, requiring the strengthening of food safety for control of this food-borne disease.

Level of CYP4G19 Expression Is Associated with Pyrethroid Resistance in Blattella germanica.

German cockroaches have become a large problem in the Shenzhen area because of their pesticide resistance, especially to pyrethroid. A pyrethroid called "Jia Chong Qing" to prevent pests for a long time were found to be resistant to "Jia Chong Qing" with resistance index of 3.88 measured using RT-PCR and immunohistochemistry analysis showed that both CYP4G19 mRNA and CYP4G19 protein expression levels in the wild strain were substantially higher than that of a sensitive strain. dsRNA segments derived from the target gene CYP4G19 were prepared using in vitro transcription and were microinjected into abdomens of the wild strain. Two to eight days after injection, the result showed that CYP4G19 mRNA expressions were significantly reduced in the groups injected with dsRNAs.

[Detection of circulating antigen of Angiostrongylus cantonensis by 12D5 and 21B7 monoclonal antibodies].

OBJECTIVE: To detect the rate of Angiostrongylus cantonensis infection and to study the effects of treatment so as to prepare monoclonal antibodies (McAbs), and gold immunochromatography assay (GICA) with 12D5 and 21B7 McAbs could be prepared in advance. METHODS: Two McAbs (12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA. Either 12D5 or 21B7 McAbs was used as antibody and protein A was conjugated with colloid gold as the detection marker. A special pad for GICA was designed according to the reaction procedure, and CAg were detected by GICA in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively. RESULTS: 12D5 McAb was identified as IgG1 and 21B7 McAb was IgM. Results from Western blotting showed that two McAbs could be used to identified 55 KD protein of adult worms of A. cantonensis. The detection rates of CAg in the sera of infected rats was 100% (48/48) and the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonorchiasis, fasciolopsiasis, ancylostomiasis, ancylostomiasis, anisakiasis as well as schistosomiasis wee seen and normal sera did not react with 12D5 and 21B7 McAbs. CONCLUSION: Results from sandwich ELISA and GICA with 12D5 and 21B7 McAbs showed high specificity and acting as detecting CAg of A. cantonensis in sera of infected animals and patients. We noticed that GICA with 12D5 and 21B7 was not only rapid and simple that without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.

[Study on the relationship between polymorphism of adiponectin gene and risk of ischemic stroke among Han population in the Northern parts of China].

OBJECTIVE: To explore the association between polymorphisms of adiponectin gene and the risk of ischemic stroke in Han population from the Northern parts of China. METHODS: TaqMan probe of RT-PCR was applied to detect the genotype frequency of single nucleotide polymorphism (SNPs) (rs266729 and rs2241766) of adiponectin gene in 357 ischemic stroke cases who developed the episode at first time and with 345 healthy controls. Logistic regression analysis was used to evaluate the relationship of each genotype of SNPs and ischemic stroke. RESULTS: Mutation of rs2241766 (T > G) increased the risk of ischemic stroke among all the samples (OR = 1.55, P = 0.01) and it was still the risk factor of ischemic stroke when analyzed by multi-factors logistic regression after each factor was adjusted (OR = 1.55, P = 0.00). The polymorphism of rs266729 was not related to the risk of ischemic stroke among all the samples (OR = 1.13, P = 0.57). However, the genotype GG of rs266729 increased the risk of ischemic stroke among female population (OR = 3.25, P = 0.04). CONCLUSION: The variance of rs2241766 in adiponectin gene was related to the risk of ischemic stroke in Han population from the Northern parts of China and the genotype GG of rs266729 could possibly increase the risk of ischemic stroke in women of Han population from the Northern parts of the country.

Monoclonal antibodies against excretory/secretory antigens of Angiostrongylus cantonensis.

In the present study, four murine monoclonal antibodies (MAbs) were generated against the excretory/secretory (ES) products of Angiostrongylus cantonensis adult worms; two represented IgG1 and two represented IgM MAbs, and they were designated 12D5, 15F8, 21B7 and 14G10, respectively. Immunoblotting revealed that all of the MAbs predominantly recognized a 98 kDa antigen in the ES products of A. cantonensis adult worms, and no cross reactions were found with the whole worm antigens of some other common parasites, namely, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani, Ascaris lumbricoides, Trichinella spiralis, Anisakis sp., Echinococcus granulosus, Taenia solium, and Spirometra erinacei. Immunolocalization showed that all of the four MAbs reacted with the cuticle of the adult parasite, the external surface of its intestinal canal and reproductive organs, and its egg and first-stage larvae in the lungs of rats experimentally infected with A. cantonensis. The generation and characterization of four specific MAbs against A. cantonensis ES antigens provide foundation for the development of specific immunological diagnostic techniques for human infections with A. cantonensis.

[Study on the epidemiological characteristics and natural infectious focus of Angiostrongylus cantonensis in Shenzhen area of Zhujiang Delta in China].

OBJECTIVE: To delimit the natural infectious focus, including the distribution of wildlife, species, ecology of intermediate hosts and final host of Angiostrongylus cantonensis, as well as the routes of transmission and epidemiological characteristics and wildlife of human Angiostrongylus cantonensis, based on human diverging cases identified in Shenzhen, southern area of China. METHODS: Data including rate of infection and density of Angiostrongylus cantonensis among different hosts in 12 different areas in Shenzhen was collected, using microscope to inspect homogenate liquids of snails. Wild mice were captured with mouse cage to examine the adult Angiostrongylus cantonensis. Using larva isolated from wild-snails-infected rats to observe the life cycle of Angiostrongylus cantonensis. RESULTS: Wild life of Angiostrongylus cantonensis existed in the southwest part of Shenzhen with its majority intermediate hosts as Achatina fulica. The overall rate of infection was 31% in wildlife and final host was found to be Rattus andersoni, Achatina fulica which were extensively distributed in the shrub region of Shenzhen because of suitable climate, humidity and vegetation for generating the life cycle of Achatina fulica. Human infected Angiostrongylus cantonensis was mainly due to eating raw snails or vegetables contaminated by larva of Angiostrongylus cantonensis. The peak of infection was seen from April to November in Shenzhen area. CONCLUSION: Wildlife of Angiostrongylus cantonensis existed in the southwest part of Shenzhen with major wildlife reservoir including fresh water snail and wild mouse. The existence of natural focus Angiostrongylus cantonensis was now recognized as an important source of human angiostrongyliasis in Shenzhen area.

[Epidemiological studies on Clonorchis sinensis infection along the Zhujiang River in Lou village of Shenzhen].

OBJECTIVE: To study the transmission route and epidemiological features of Clonorchis sinensis infection in Shenzhen area--the biggest immigration city of Southern China. METHODS: In this study, we examined 1473 individuals (710 males and 763 females) to determine the current status of C. sinensis infection among the people in one village in Zhujiang river region, Guangdong province, China. Blood samples were detected on antibody of C. sinensis with enzyme linked immunosorbent assay,and stool specimens from sera positive cases were examined by modified Kato-Katz thick smear to confirm the density of infection. People were interviewed on their life styles under the structured questionnaire which was administered by trained staff members. Major content of the questionnaire included eating raw fish, using the same utensils for both raw fish and cooked food, using feces of domestic animals and human feces to feed fish and so on. RESULTS: Among 1473 people examined, 70 (4.75%) were found infected with C. sinensis. By counting eggs per gram feces (EPG), it was found that heavy intensities of infection in males was stronger than that of females,and the overall average EPG was 41.87. Of 1473 interviewees, 54% of them did not know about fluke disease or its transmission route, 12% of those who knew about the fluke but believed that the infection caused no harm or only slight harm to their health. 27% of the interviewees ate raw fish at least 1-2 times per months with 5% of the families using the same utensils for both raw fish and cooked food. 40% of the fish ponds owners fed their fish with the feces of domestic animals and human feces. CONCLUSION: Together with these results, unhealthy behaviors, poor knowledge, inappropriate farming/fishery practices, eating raw fish were important factors influencing the C. sinensis prevalence in humans.

Identification of differentially expressed genes in female Culex pipiens pallens.

Culex pipiens pallens is the mosquito vector of a number of human pathogens such as Wuchereria bancrofti, Brugia malayi, and epidemic encephalitis B virus. Female C. pipiens pallens play an important role in transmitting pathogens by sucking blood, which is essential for reproduction. In the present study, a subtractive cDNA library for female C. pipiens pallens was constructed by the suppression subtractive hybridization (SSH) technique and then 100 clones from the female SSH library were sequenced and analyzed. Female-differentially expressed genes in C. pipiens pallens were screened using semiquantitative RT-PCR. The full-length cDNA of an EST sequence (fs68) that was specifically expressed in female C. pipiens pallens was characterized by 3' and 5' rapid amplification of cDNA ends (RACE). The characteristics of the female-specific gene were further analyzed using bioinformatics and Northern blot. It was shown that the female-specific gene was a previously uncharacterized gene and may encode a salivary peptide. This putative salivary peptide could be a very important molecule in the blood feeding of female C. pipiens pallens.