Kun Yin

Epidemiological Investigation of Asymptomatic Dogs with Leishmania Infection in Southwestern China Where Visceral Leishmaniasis is Intractable

Heishui county, located in northwest Sichuan province, southwestern China, is an endemic area of zoonotic visceral leishmaniasis (VL) and is the most intractable area. VL is never destroyed in it. Asymptomatic dogs (Leishmania parasites have been diagnosed but clinically healthy) are considered to be a potential reservoir host in zoonotic VL area, and most can lead to infection of individuals, that is a new challenge for controlling VL in humans. The present study aimed to assess the Leishmania infection rate of asymptomatic dogs in Heishui county. Total 105 asymptomatic domestic dogs were gathered from 4 districts in Heishui county to investigate the infection rate with serological and molecular methods based on ELISA and kinetoplast minicircle DNA(kDNA) PCR, respectively. Out of 105 dogs, 44 (41.9%) were positive by more than 1 method; 21 (20.0%) were positive by ELISA, and 30 (28.6%) were positive by kDNA-PCR. Our study showed that Leishmania infection of domestic dogs which is clinically healthy is prevalent in the studied district, and the asymptomatic dogs infected by Leishmania may be the primary reason for the prevalence of visceral leishmaniasis in the area.

Characteristics of Imported Malaria and Species of Plasmodium Involved in Shandong Province, China (2012-2014)

Malaria remains a serious public health problem in Shandong Province, China; therefore, it is important to explore the characteristics of the current malaria prevalence situation in the province. In this study, data of malaria cases reported in Shandong during 2012-2014 were analyzed, and Plasmodium species were confirmed by smear microscopy and nested-PCR. A total of 374 malaria cases were reported, 80.8% of which were reported from 6 prefectures. Of all cases, P. falciparum was dominant (81.3%), followed by P. vivax (11.8%); P. ovale and P. malariae together accounted for 6.4% of cases. Notably, for the first time since 2012, no indigenous case had been reported in Shandong Province, a situation that continued through 2014. Total 95.2% of cases were imported from Africa. The ratio of male/female was 92.5:1, and 96.8% of cases occurred in people 20-54 years of age. Farmers or laborers represented 77.5% of cases. No significant trends of monthly pattern were found in the reported cases. All patients were in good condition after treatment, except for 3 who died. These results indicate that imported malaria has increased significantly since 2012 in Shandong Province, especially for P. falciparum, and there is an emergence of species diversity.

Inhibition of RhoA expression by adenovirus-mediated siRNA combined with TNF-α induced apoptosis of hepatocarcinoma cells

Tumor necrosis factor-alpha (TNF-α) has been used as an effective treatment for Hepatocellular Carcinoma, however, inducing tumor cell apoptosis by TNF-α alone is still unsatisfactory. RhoA is highly expressed in hepatocarcinoma cells and can be activated by TNF-α. The activation of RhoA directly leads to a poor prognosis of HCC. Therefore, we propose to investigate the therapeutic effect of TNF-α together with RhoA siRNA. RhoA inhibition was accomplished by constructing a recombinant adenovirus that can efficiently express RhoA siRNA in HepG2 cells. The recombinant adenovirus AdshRNA-RhoA and AdU6-control were generated by adenovirus-mediated siRNA expression system. The inhibition effects were detected by RT-PCR in addition to immunoblot to quantify the decreased levels of RhoA expression, and the therapeutic effect for HCC was demonstrated by the proliferation and apoptosis ratios of HepG2 cells. The inhibition effects of RhoA by AdshRNA-RhoA were significant at both mRNA and protein levels: the transcription of RhoA mRNA decreased by 74.46%, and the expression of protein decreased by 76.48%. The proliferation rate of HepG2 cells detected by MTT showed that a treatment of AdshRNA-RhoA and TNF-α together could strengthen the suppression ability of TNF-α to HepG2 cells, resulting in approximately 14.2% more than those treated with only TNF-α. FCA and TUNEL assays results revealed that the combined treatment can induce apoptosis in approximately 52.14%-65% of the HepG2 cells, whereas this ratio in the TNF-α-alone group was only 21.91%-32%. Our results showed that AdshRNA-RhoA can efficiently enhance the TNF-α-induced apoptosis of hepatocarcinoma cells. This method might be a useful therapeutic route in HCC and other tumors.

Protective immune response against Toxoplasma gondii elicited by a novel yeast-based vaccine with microneme protein 16

Toxoplasma gondii is an obligate intracellular protozoan that can invade all eukaryotic cells and infect all warm-blood animals, causing the important zoonosis toxoplasmosis. Invasion of host cells is the key step necessary for T. gondii to complete its life cycle and microneme proteins play an important role in attachment and invasion of host cells. Microneme protein 16 (TgMIC16) is a new protective protein in T. gondii and belongs to transmembrane microneme proteins (TM-MIC). The TM-MICs are released onto the parasites surface as complexes capable of interacting with host cell receptors. In the present study, we expressed the TgMIC16 protein on the surface of Saccharomyce cerevisiae (pCTCON2-TgMIC16/EBY100) and evaluated it as a potential vaccine for BALB/c mice against challenge infection with the RH strain of T. gondii. We immunized BALB/c mice both orally and intraperitoneally. After three immunizations, the immune response was evaluated by measuring antibody levels, lymphocyte proliferative responses, percentages of CD4+ and CD8+ T lymphocytes, cytokine production, and the survival times of challenged mice. The results showed that the pCTCON2-TgMIC16/EBY100 vaccine stimulated humoral and cellular immune responses. In addition, mice immunized with the pCTCON2-TgMIC16/EBY100 vaccine showed increased survival times compared with non-immunized controls. In summary, TgMIC16 displayed on the cell surface of S. cerevisiae could be used as potential vaccine against toxoplasmosis.

Surveillance of Antimalarial Resistance Pfcrt , Pfmdr1 , and Pfkelch13 Polymorphisms in African Plasmodium falciparum imported to Shandong Province, China

Antimalarial drug resistance is a major public health problem in China. From 2012 to 2015, more than 75% of malaria cases in Shandong Province were P. falciparum returned from Africa. However, molecular marker polymorphisms of drug resistance in imported P. falciparum cases have not been evaluated. In this study, we analyzed polymorphisms of the Pfcrt, Pfmdr1, and Pfkelch13 genes in 282 P. falciparum cases returned from Africa to Shandong between 2012 and 2015. Among the isolates, polymorphisms were detected in codons 74–76 of Pfcrt and 86, 184, 1246 of Pfmdr1, among which K76T (36.6%) and Y184F (60.7%) were the most prevalent, respectively. Six Pfcrt haplotypes and 11 Pfmdr1 haplotypes were identified and a comparison was made on the prevalence of haplotypes among East Africa, West Africa, Central Africa and South Africa. One synonymous and 9 nonsynonymous mutations in Pfkelch13 were detected in the isolates (4.6%), among which a candidate artemisinin (ART) resistance mutation P553L was observed. The study establishes fundamental data for detection of chloroquine resistance (CQR) and ART resistance with molecular markers of the imported P. falciparum in China, and it also enriches the genetic data of antimalarial resistance for the malaria endemic countries in Africa.

Cysticercosis in Shandong Province, Eastern China

We analyzed demographic and clinical data and estimated the incidence of cysticercosis in Shandong Province, China, during 1975–2014. Our analyses showed that a cysticercosis-endemic area is present in Shandong Province, especially in its western regions. Improved surveillance and control are needed to address the elevated risk for cysticercosis in this region.

Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction

Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat’s small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5–2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

Overexpression of C35 in breast carcinomas is associated with tumor progression and lymphnode metastasis.

To investigate C35 protein expression in breast carcinoma and to investigate its clinicopathological significance, a total of 68 cases of breast carcinoma and 20 cases of normal breast tissue samples were obtained from the clinic. Protein expression of C35, ER, PR and HER-2 were determined using immunohistochemistry. The correlations between C35 expression and clinicopathological parameters were analyzed on the basis of individual clinicopathologic records. Overexpression of C35 was detected in 56 of 68 (82.35%) breast carcinoma samples and only 3 of 20 (15%) normal breast tissue samples, and frequency of C35 expression was significantly associated with clinical Tumor Node Metastasis staging and Scarff-Bloom-Richardson grade (p < 0.05), but was not related to patients age, menstrual status and tumor diameter. C35 expression was positively related with the expression of HER-2 (r = 0.207), whereas negatively related with the expression of ER and PR. Further, C35 was prevalent in all four molecular subtypes of breast carcinoma with no significant difference of expression frequency. However, they have significant differences in lymphatic metastasis cases compared to the non-metastasis cases (p < 0.05). Since C35 protein was extensively expressed in all stages of breast carcinoma, and was closely associated with tumor progression and lymph node metastasis, it might be used as a reliable biomarker or therapeutic target for diagnosis and treatment.