Muscle-derived stem cells in silk fibroin hydrogels promotes muscle regeneration and angiogenesis in sheep models: an experimental study.

OBJECTIVE: Silk fibroin (SF) hydrogels are of high interest in tissue engineering. However, angiogenesis is one of the major challenges in tissue regeneration and repair. In this study, we present a simple and effective method to develop a 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG)-SF hydrogel. The SF hydrogels had no immunogenicity and approached natural tissues. MATERIALS AND METHODS: The SF scaffolds were first prepared from Bombyx mori silkworms and DMPG. The SF scaffold was seeded with muscle-derived stem cells derived from sheep embryo and implanted in the tibialis anterior muscle of mature sheep. Gelation time, H&E staining, and histochemistry were conducted and observed. The suitability of the hydrogels for 3D cell culture was assessed by living cell stain CM-Dil. RESULTS: The results showed that the SF hydrogels resembled the mechanical properties of natural soft tissues better. The results of H&E staining and histochemistry revealed that the degradation rate showed an S-type change, and muscle regeneration and angiogenesis were clearly visible. Adverse effects were not observed in the sheep models. CONCLUSIONS: DMPG-induced SF hydrogels can be successfully used for in situ cell encapsulation. It provides promising opportunities in biomedical applications, such as in tissue engineering and regeneration.

[Teleost Type 2 Interleukin-1 Receptor (IL-1R2) from the Spotted Halibut (Verasper variegatus): 3D Structure and a Role in Immune Response].

The type 2 interleukin-1 receptor (IL-1R2) is one of natural IL-1ß singling inhibitors in mammals. We cloned and sequenced the IL-1R2 gene in V. variegatus (VvIL-1R2). The phylogenetic analysis showed that the molecular structure VvIL-1R2 is similar to that of its orthologues in other vertebrates. The expression levels of VvIL-1R2 are relatively high in the peripheral blood leukocytes (PBLs), gill, and spleen. In addition, peculiar expression patterns for his molecule were detected at various developmental stages, implying that in flatfishes the IL-1R2 may have be important for embryonic development and metamorphosis. In PBLs, the treatment with pathogen-associated molecular patterns (PAMPs) induced a significant and rapid up-regulation of VvIL-1R2, pointing at its involvement in the immune responses against bacterial and viral pathogens.

[Transplantation of cardiac stem cells overexpressing integrin-linked kinase improves cardiac function in a rat model of acute myocardial infarction].

Objective: To investigate the effect of cardiac stem cells (CSC) overexpressing integrin-linked kinase (ILK-CSC) transplantation on cardiac function after myocardial infarction (MI) and related mechanism. Methods: CSCs were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and ILK-CSC were established by transfecting recombinant adenoviral vector harboring human wild-type ILK cDNA. Forty 8-week-old rats were randomly divided into 4 groups (n=10 each group): sham group, MI plus saline injection group(saline group), MI plus CSC transfected with null vector injection group (Ad-null-CSC group), and MI plus ILK-CSC injection group(ILK-CSC group). MI was induced through coronary artery ligation, and after 15 minutes, 30 µl saline, Ad-null-CSC (1×10(5) cells/30 µl) or ILK-CSC (1×10(5) cells/30 µl) were injected into the hearts of MI rats at 3 different points in infracted zone and infarct border zone. After 4 weeks, left ventricular (LV) function was examined by echocardiography, LV fibrosis was detected by HE and Masson staining, and myocardial protein expression of Ki-67 and p-H3 was evaluated by immuohistochemistry and mRNA expression of cyclinD1 and PCNA was detected by real-time RT-PCR. Results: (1) Thirty-seven rats (sham group=10, saline group=8, Ad-null-CSC group=9 and ILK-CSC group=10) survived at 4 weeks after operation. Left ventricular end-systolic dimension (LVESD, P=0.009) and left ventricular end-diastolic dimension (LVEDD, P=0.002) were significantly increased, and left ventricular ejection fraction(LVEF, P=0.006) was decreased in saline group compared with those of sham group.In Ad-null-CSC group LVESD (P=0.005) and LVEDD (P=0.003) were decreased, but LVEF remained unchanged (P=0.113) compared with those of saline group. LVESD (P=0.004) and LVEDD (P=0.000 1) of ILK-CSC group were significantly decreased, and LVEF (P=0.004) was significantly increased compared with those of Ad-null-CSC group. (2) LV histology and myocardial fibrosis: there were marked myocyte loss and significant increase of myocardial fibrosis in the saline group((70.6±5.1) %, P=0.002) and Ad-null-CSC group((57.7±3.4) %, P=0.001) compared with sham group ((8.2±2.2) %), while the fibrosis was significantly attenuated post injection of ILK-CSC ((30.6±7.0)%, P=0.005 vs. Ad-null-CSC). (3) Protein levels of mitotic genes: the results of immuohistochemistry showed that the brown positive particles were presented in the nuclei of cardiac myocytes in saline, Ad-null-CSC and ILK-CSC groups, but they were negative in sham group. The protein expression of Ki-67 (P=0.007) and phosphohistone-H3 (p-H3) (P=0.003) in ILK-CSC group was significantly higher than in Ad-null-CSC group. (4) mRNA levels of mitotic genes: the results of real-time RT-PCR showed that the mRNA levels of cyclinD1 and proliferating cell nuclear antigen (PCNA) in saline and Ad-null-CSC groups were similar as in sham group (all P>0.05). However, in ILK-CSC group they were significantly increased compared with those in Ad-null-CSC group (cyclinD1, P=0.003; PCNA, P=0.004). Conclusion: Our results suggest that myocardial transplantation of CSC overexpressing ILK improves cardiac function in the post-MI rats, and this beneficial effect may be related to the enhanced proliferation of cardiac myocytes and attenuated myocardial fibrosis.

Downregulation of long non-coding RNA LINC01133 is predictive of poor prognosis in colorectal cancer patients.

OBJECTIVE: This study aims to investigate long non-coding RNA LINC01133 (LINC01133) expressions in colorectal cancer (CRC) patients, and discuss its correlation with CRC clinicopathological features and prognosis. PATIENTS AND METHODS: qRT-PCR was performed to measure expression levels of LINC01133 in CRC tissues. The chi-square test was used to assess LINC01133 expression with respect to clinicopathological parameters. Kaplan-Meier analysis and the log-rank test were performed to identify survival differences in CRC patients. Univariate and multivariate analysis were performed using the Cox proportional hazard analysis. RESULTS: LINC01133 was significantly down-regulated in CRC tissues compared to normal tissue samples (p < 0.001), and a low expression of LINC01133 was found to be significantly associated with lymph node metastasis (p = 0.004), distant metastasis (p = 0.043), N classification (p = 0.022) and TNM stage (p = 0.011). Moreover, Kaplan-Meier survival analysis revealed that high LINC01133 expression predicted significantly better overall survival (p = 0.0093). Finally, multivariate analysis results indicated that LINC01133 was an independent prognostic factor in CRC. CONCLUSIONS: Our results indicated that reduced LINC01133 expression contributed to CRC metastasis and poor prognosis. Thus, LINC01133 might serve as a promising biomarker for prognosis of CRC.

[Salubrinal improves cardiac function in rats with heart failure post myocardial infarction through reducing endoplasmic reticulum stress-associated apoptosis].

OBJECTIVE: Endoplasmic reticulum (ER) stress plays an important role in ischemia-mediated cell death. The aim of the current study is to investigate the effects of salubrinal (Sal), a selective eIF2a dephosphorylation inhibitor, on heart failure rats and related mechanisms. METHODS: Heart failure was induced by coronary artery ligation (MI) in adult male Sprague-Dawley rats. To ensure comparable MI sizes post coronary artery ligation on various groups, echocardiography examination was performed before and 30 minutes after ligation in MI groups. Then rats were randomly assigned to 4 groups: Sham group (n=12), MI group (n=10), MI plus vehicle injections group (DMSO group, n=12) and MI plus Sal injection group (Sal group, n=12). Sal (1 mg/kg) or DMSO was injected via the tail vein daily for the first 3 days (starting at 30 minutes after ligation of the left coronary artery), followed by subcutaneous injections twice per week for 8 weeks. Cardiac function was assessed by echocardiography and cell apoptosis assessed by flow cytometric analysis after 8 weeks. Protein and mRNA levels of ER stress markers were evaluated by immunohistochemistry and real time RT-PCR respectively. RESULTS: Eight weeks later, LVEF was significantly higher, while LVESD and LVEDD values were significantly lower in Sal group compared to MI and DMSO groups (all P<0.05); LV/BW ratio was significantly higher in MI group than in Sham group ((2.30±0.40) mg/g vs.(1.78±0.31) mg/g, P<0.05), which was significantly reduced in Sal group ((1.88±0.25) mg/g), but not in DMSO group((2.25±0.36) mg/g, P<0.05 vs. MI). In addition, flow cytometric analysis showed that Sal treatment significantly reduced apoptosis but not necrosis in post MI. Immunohistochemistry and real time PCR analysis showed that the myocardial protein and mRNA expression of ER stress markers were significantly lower in Sal group than in MI group, myocardial caspase-12 expression was significantly upregulated in MI group and significantly reduced by Sal treatment. CONCLUSIONS: Our results suggest that reduction of ER stress and myocardial apoptosis through inhibition of eIF2α dephosphorylation may serve as the potential mechanisms for the improved cardiac function and attenuated cardiac remodeling post Sal treatment in this heart failure rat model.

A new strategy for complete identification of sea buckthorn cultivars by using random amplified polymorphic DNA markers.

DNA fingerprinting is both a popular and important technique with several advantages in plant cultivar identification. However, this technique has not been used widely and efficiently in practical plant identification because the analysis and recording of data generated from fingerprinting and genotyping are tedious and difficult. We developed a novel approach known as a cultivar identification diagram (CID) strategy that uses DNA markers to separate plant individuals in a more efficient, practical, and referable manner. A CID was manually constructed and a polymorphic marker was generated from each polymerase chain reaction for sample separation. In this study, 67 important sea buckthorn cultivars cultivated in China were successfully separated with random amplified polymorphic DNA markers using the CID analysis strategy, with only seven 11-nucleotide primers employed. The utilization of the CID of these 67 sea buckthorn cultivars was verified by identifying 2 randomly chosen groups of cultivars among the 67 cultivars. The main advantages of this identification strategy include fewer primers used and separation of all cultivars using the corresponding primers. This sea buckthorn CID was able to separate any sea buckthorn cultivars among the 67 studied, which is useful for sea buckthorn cultivar identification, cultivar-right-protection, and for the sea buckthorn nursery industry in China.

Management and outcomes of axial isolated distal deep vein thrombosis at North Shore Hospital, New Zealand: a retrospective audit.

BACKGROUND: It is standard of care to treat proximal vein deep vein thrombosis (DVT) for a minimum of 3 months. Conversely, management of isolated distal DVT (IDDVT) is controversial, with options including observation and repeat ultrasound scan within 1 week to detect and anticoagulate those with proximal propagation, or anticoagulation for periods of up to 3 months. AIM: The aim was to assess the rates of proximal propagation and venous thromboembolism (VTE) recurrence within 3 months of diagnosis of IDDVT, and to examine how the duration of treatment might influence this. METHODS: Study patients were identified by retrospective audit of data from the North Shore Hospital VTE database. All patients presenting with established axial vein distal DVT from July 2007 to June 2012 were included. A 6-week treatment duration cut-off was used to separate the treatment arms (<6 weeks vs 6 weeks vs >6 weeks), and Fisher's exact or Pearson's Chi-squared tests were used to assess between-group comparisons. RESULTS: Five hundred and seven patients were included in the study, mean age 59.7 years; 53% female. There were three cases of proximal propagation, all occurring in those receiving <6 weeks treatment. There were six VTE recurrences, three in the <6 week and three in the ≥6 week treatment groups respectively. Malignancy was the only significant predictor of VTE recurrence (P = 0.001). CONCLUSION: A 6-week duration of anticoagulation appears to be an effective and safe treatment for isolated axial distal DVT, with low rates of VTE recurrence and proximal propagation.

In vitro and in vivo evaluation of cypermethrin, amitraz, and piperonyl butoxide mixtures for the control of resistant Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in the Mexican tropics.

A study was conducted to evaluate the efficacy of cypermethrin, amitraz, and piperonyl butoxide (PBO) mixtures, through in vitro laboratory bioassays and in vivo on-animal efficacy trials, for the control of resistant Rhipicephalus (Boophilus) microplus on cattle in the Mexican tropics. Also, to examine mechanisms of resistance to cypermethrin in this tick population, the frequency of a mutated sodium channel gene (F1550I) was determined using a PCR assay. Results of laboratory bioassays using modified larval packet tests revealed that cypermethrin toxicity was synergized by PBO (from 46.6-57.0% to 83.7-85.0% larval mortality; P<0.05). The cypermethrin and amitraz mixture showed an additive effect (from 46.6-57.0% to 56.0-74.3% larval mortality). Strong synergism was observed with the mixture of cypermethrin+amitraz+PBO and this mixture was the most effective killing resistant tick larvae in vitro (96.7-100% of larval mortality). Tick larvae surviving exposure to cypermethrin or mixtures either with amitraz and PBO in vitro showed 2.9-49.6 higher probability to present the mutated allele than those killed by acaricide treatment (P<0.05). In the in vivo trial, the mixtures containing cypermethrin+PBO (80.6-97.3%), and cypermethrin+amitraz (87.0-89.7%) were more efficacious than cypermethrin alone (76.3-80.5%). The highest level of efficacy was obtained with the mixture of cypermethrin+amitraz+PBO, which yielded >95% control that persisted for 28 days post-treatment against R. microplus infesting cattle when tested under field conditions in the Mexican tropics. Although this mixture is a potentially useful tool to combat pyrethroid resistance, a product based on an acaricide mixture like the one tested in this study has to be used rationally.

A sero-epidemiological survey of Chinese Babesia motasi for small ruminants in China.

Babesia motasi BQ1 (Lintan) was first isolated from Haemaphysalis qinghaiensis collected in Gannan Tibet Autonomous Region, Gansu province in April 2000. In this study, a total of 3,204 serum samples from small ruminants in 22 provinces located in different districts of China were tested for antibodies against merozoite antigens from cultured B. motasi BQ1 (Lintan) by enzyme-linked immunosorbent assay. This method can survey the prevalence of low-pathogenic Chinese B. motasi. The results of this survey indicated that the average positive rate was 43.5 %, and the positive rates of investigated provinces were significantly different from 6.1 to 91.0 %, and the infections had been found in all provinces investigated. Our data provide large important information regarding the current sero-prevalence of B. motasi in China.

Traditional Chinese medicine and related active compounds: a review of their role on hepatitis B virus infection.

Since the significant public health hazard of Hepatitis B virus (HBV) infection and obvious drug resistance and dose-dependent side effects for common antiviral agents (e.g., interferon α, lamivudine, and adefovir), continuous development of agents to treat HBV infection is urgently needed. Traditional Chinese medicine (TCM) is an established segment of the health care system in China. Currently, it is widely used for chronic hepatitis B (CHB) in China and many parts of the world. Over a long period of time in clinical practice and in basic research progress, the effectiveness and beneficial contribution of TCM on CHB have been gradually known and confirmed. Based upon our review of related papers and because of our prior knowledge and experience, we have selected some Chinese medicines, including Chinese herbal formulas (e.g., Xiao-Chai-Hu-Tang, Xiao-Yao-San, and Long-Dan-Xie-Gan-Tang), single herbs (e.g., Phyllanthus niruri, Radix astragali, Polygonum cuspidatum, Rheum palmatum, and Salvia miltiorrhiza) and related active compounds (e.g., wogonin, artesunate, saikosaponin, astragaloside IV, and chrysophanol 8-O-beta-Dglucoside) and Chinese medicine preparations (e.g., silymarin, silibinin, kushenin, and cinobufacini), which seem effective and worthy of additional and indepth study in treating CHB, and we have given them a brief review. We conclude that these Chinese herbal medicines exhibit significant anti-HBV activities with improved liver function, and enhanced HBeAg and HBsAg sero-conversion rates as well as HBV DNA clearance rates in HepG2 2.2.15 cells, DHBV models, or patients with CHB. We hope this review will contribute to an understanding of TCM and related active compounds as an effective treatment for CHB and provide useful information for the development of more effective antiviral drugs.

Effects of pyriproxyfen and buprofezin on immature development and reproduction in the stable fly.

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is one of the most significant biting flies that affect cattle. The use of traditional insecticides for stable fly control has only a limited success owing to the insect's unique feeding behaviours and immature development sites. A laboratory study was conducted to evaluate the effects of two insect growth regulator (IGR) products, pyriproxyfen and buprofezin, on the development of the immature stages of the stable fly and the effects of pyriproxyfen on oviposition and egg hatch. Both pyriproxyfen and buprofezin had significant inhibitory effects on immature development. The LC(50) s of pyriproxyfen and buprofezin were 0.002 and 18.92 p.p.m., respectively. Topical treatment of adult females with different doses of pyriproxyfen had significant negative effects on both female oviposition and egg hatching when 1- and 3-day-old females were treated, and the effects were dose dependent. A significant reduction in the mean number of eggs laid was observed only at the highest pyriproxyfen dose (8 µg/fly) and egg hatch was unaffected by pyriproxyfen treatment when 5-day-old females were treated. Results from the present study indicate that pyriproxyfen has the potential to be used as part of an integrated stable fly management programme.

High levels of insecticide resistance in introduced horn fly (Diptera: Muscidae) populations and implications for management.

The horn fly, Haematobia irritans (L.) (Diptera: Muscidae), was introduced to Chile in the beginning of the 1990s. Since its introduction, farmers have controlled this pest almost exclusively with insecticides. To understand the consequences of different control strategies on the development of insecticide resistance and their persistence, a field survey was conducted at eight farms in the south of Chile to characterize insecticide resistance in field populations and resistance mechanisms. Horn fly samples were assayed to determine levels of resistance to pyrethroids and diazinon, genotyped for kdr and HialphaE7 mutations, and tested for general esterase activity. All field populations, including ones that were not treated with insecticides for the past 5 yr, showed high levels of cypermethrin resistance and high frequencies of the kdr mutation. None of the fly populations demonstrated resistance to diazinon and the HialphaE7 mutation was not detected in any of the fly samples. Esterase activities in all populations were comparable to those found in the susceptible reference strain. The findings of high frequencies of homozygous resistant and heterozygous individuals both in insecticide treated horn fly populations and in the untreated fly populations suggests complex interactions among field populations of the horn fly in Chile.

Glial cell line-derived neurotrophic factor is a key neurotrophin in the postnatal enteric nervous system.

BACKGROUND: The enteric nervous system (ENS) continues its structural and functional growth after birth, with formation of ganglia and the innervation of growing smooth muscle. However, little is known about factors in the postnatal intestine that influence these processes. METHODS: We examined the presence and potential role of glial cell line-derived nerve growth factor (GDNF) in the rat postnatal ENS using neonatal tissue, primary co-cultures of the myenteric plexus, smooth muscle, and glial cells as well as cell lines of smooth muscle or glial cells. KEY RESULTS: Western blot analysis showed that GDNF and its co-receptors rearranged during transfection (RET) and GDNF family receptor alpha-1 were expressed in the muscle layer of the neonatal and adult rat intestine. Immunohistochemistry localized the receptors for GDNF to myenteric neurons, while GDNF was localized to smooth muscle cells. In a co-culture model, GDNF but not nerve growth factor, brain derived neurotrophic factor or neurotrophin-3 significantly increased neuronal survival and more than doubled the numbers of neurites in vitro. RT-PCR, qPCR, Western blotting, ELISA, and immunocytochemistry as well as bioassays of neuronal survival and of RET phosphorylation all identified intestinal smooth muscle as the source of GDNF in vitro. GDNF also induced morphological changes in the structure and organization of neurons and axons, causing marked aggregation of neuronal cell bodies and collinear development of axons. As well, GDNF (50-150 ng mL(-1)) significantly increased [(3)H]-choline uptake and stimulated [(3)H]-acetylcholine release. CONCLUSIONS & INFERENCES: We conclude that GDNF derived from intestinal smooth muscle cells is a key factor influencing the structural and functional development of postnatal myenteric neurons.

Traditional Chinese medicine Ningdong granule: the beneficial effects in Tourette's disorder.

This 8-week, randomized, placebo-controlled, double-blind clinical study investigated the short-term effectiveness and safety of the traditional Chinese medicine, Ningdong (ND) granule in paediatric subjects (aged 7 - 18 years) with Tourette's disorder (TD). Subjects received either 1 g/kg per day ND granule (n = 33) or placebo (n = 31) and tics were measured at baseline and after 4 and 8 weeks of treatment. Body mass index, electrocardiogram and laboratory measures were recorded at baseline and at week 8. After 8 weeks of treatment with ND granule, subjects exhibited a 41.39% reduction in the total tic score while the placebo group showed a 10.79% decrease. Regarding side-effects, two subjects reported loss of appetite and one experienced constipation in the ND granule group. There were no significant changes in laboratory and physical examinations. These results suggest that ND granule is both effective and safe, and may be a good candidate for the treatment of patients with TD.

Apoptosis-inducing effect of cinobufacini, Bufo bufo gargarizans Cantor skin extract, on human hepatoma cell line BEL-7402.

Cinobufacini, a water-soluble preparation of Chinese medicine, is extracted from the skin of Bufo bufo gargarizans Cantor. The present study sought to investigate the effects of Cinobufacini on apoptosis of the hepatocellular carcinoma cell line BEL-7402. Cell viability was measured by methyl thiazolyl tetrazolium assay. Cell morphology was observed by Hoechst 33258 staining. Western blotting analysis was used to detect Bax and Bcl-2 expression. Results indicated that Cinobufacini inhibited the proliferation of BEL-7402 cells in a dose and time-dependent manner. Marked morphological changes indicative of apoptosis were observed after treatment with different concentrations of Cinobufacini. Western blot analysis showed that Bcl-2 expression was down-regulated while Bax expression was upregulated. Thus, Cinobufacini may have a significant　apoptosis-inducing effect on BEL-7402 cells, and this could prove useful for further anti-cancer research.

Recall of discharge advice given to patients with minor head injury presenting to a Singapore emergency department.

INTRODUCTION: Most patients presenting to the emergency department (ED) with minor head injury (HI) can be discharged, provided a caregiver is present and careful discharge instructions are given. The study ED uses an advice leaflet with verbal reinforcement to patients and caregivers detailing post-discharge instructions and warning symptoms of worsening HI. We aim to evaluate local patients' and caregivers' compliance to discharge instructions and their ability to recall HI advice. METHODS: A prospective study was conducted in an adult ED between April 10, 2006 and May 1, 2006. All patients with minor HI discharged from the ED or its 24-hour observation ward were included in the study. A telephone survey was conducted within 48 hours of discharge using a standardised questionnaire. RESULTS: During the study period, 292 patients had HI, of which 182 were eligible for the study. 71 were uncontactable and one refused to participate, leaving 110 patients in the study. Patients' age ranged between 7 and 109 years (median 41 years). 100 confirmed receiving HI advice (57 percent received by patients, 26 percent caregivers, 16 percent both patients and caregivers). 29 percent of respondents reported non-compliance to discharge advice. Mean HI-symptom recall score was 1.9 (SD 1.6) (total 9 symptoms). 30 percent cited other symptoms not part of the HI advice, which they believed necessitated a return to the ED. Recall scores were not statistically different, regardless of mode of instruction (verbal or printed) or the recipient (patient, caregiver or both). CONCLUSION: Our study raises concerns about the reliability of discharge advice for minor HI patients.

Serial analysis of gene expression in the southern cattle tick following acaricide treatment of larvae from organophosphate resistant and susceptible strains.

Organophosphate resistant and susceptible tick larvae from laboratory strains of the southern cattle tick, Rhipicephalus (Boophilus) microplus were exposed to low doses of the organophosphate (OP) acaricide, coumaphos. Serial analysis of gene expression (SAGE) was used to analyse differential gene expression in response to OP treatment and to compare the responses of OP-treated and -untreated resistant and susceptible tick larvae. An R. microplus Gene Index was used as an EST database to identify genes which corresponded to SAGE tags whose abundance changed in response to acaricide exposure. Relative quantitative RT-PCR was used to confirm the differential expression results from the SAGE experiments. Of particular interest is a SAGE tag which corresponds to a cytochrome P450-like EST in the Gene Index which was more abundant in untreated OP resistant larvae compared to untreated OP susceptible larvae. This SAGE tag was also more abundant in OP resistant larvae treated with OP compared to OP susceptible larvae treated with OP.

Factors that influence the prevalence of acaricide resistance and tick-borne diseases.

This manuscript provides a summary of the results presented at a symposium organized to accumulate information on factors that influence the prevalence of acaricide resistance and tick-borne diseases. This symposium was part of the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP), held in New Orleans, LA, USA, during August 10-14, 2003. Populations of southern cattle ticks, Boophilus microplus, from Mexico have developed resistance to many classes of acaricide including chlorinated hydrocarbons (DDT), pyrethroids, organophosphates, and formamidines (amitraz). Target site mutations are the most common resistance mechanism observed, but there are examples of metabolic mechanisms. In many pyrethroid resistant strains, a single target site mutation on the Na(+) channel confers very high resistance (resistance ratios: >1000x) to both DDT and all pyrethroid acaricides. Acetylcholine esterase affinity for OPs is changed in resistant tick populations. A second mechanism of OP resistance is linked to cytochrome P450 monooxygenase activity. A PCR-based assay to detect a specific sodium channel gene mutation that is associated with resistance to permethrin has been developed. This assay can be performed on individual ticks at any life stage with results available in a few hours. A number of Mexican strains of B. microplus with varying profiles of pesticide resistance have been genotyped using this test. Additionally, a specific metabolic esterase with permethrin-hydrolyzing activity, CzEst9, has been purified and its gene coding region cloned. This esterase has been associated with high resistance to permethrin in one Mexican tick population. Work is continuing to clone specific acetylcholinesterase (AChE) and carboxylesterase genes that appear to be involved in resistance to organophosphates. Our ultimate goal is the design of a battery of DNA- or ELISA-based assays capable of rapidly genotyping individual ticks to obtain a comprehensive profile of their susceptibility to various pesticides. More outbreaks of clinical bovine babesisois and anaplasmosis have been associated with the presence of synthetic pyrethroid (SP) resistance when compared to OP and amidine resistance. This may be the result of differences in the temporal and geographic patterns of resistance development to the different acaricides. If acaricide resistance develops slowly, herd immunity may not be affected. The use of pesticides for the control of pests of cattle other than ticks can affect the incidence of tick resistance and tick-borne diseases. Simple analytical models of tick- and tsetse-borne diseases suggest that reducing the abundance of ticks, by treating cattle with pyrethroids for example, can have a variety of effects on tick-borne diseases. In the worst-case scenario, the models suggest that treating cattle might not only have no impact on trypanosomosis but could increase the incidence of tick-borne disease. In the best-case, treatment could reduce the incidence of both trypanosomosis and tick-borne diseases Surveys of beef and dairy properties in Queensland for which tick resistance to amitraz was known were intended to provide a clear understanding of the economic and management consequences resistance had on their properties. Farmers continued to use amitraz as the major acaricide for tick control after the diagnosis of resistance, although it was supplemented with moxidectin (dairy farms) or fluazuron, macrocyclic lactones or cypermethrin/chlorfenvinphos.

[Experimental studies of antitumor effect of artesunate on liver cancer].

OBJECTIVE: To observe the inhibiting effect of Artesunate on liver cancer in vitro and in vivo. METHOD: The mice bearing H22 solid and ascitic liver tumor were applied in vivo experiments. Microculture tetrazolium assay and colony-forming unit assay were applied to test the cytotoxicity to human hepatocarcinoma SMMC-7721 cell line in vitro. RESULT: The growth of solid tumor were obviously inhibited by Artesunate at the dose of 300 mg.kg-1.d-1 ig for 7 days. The tumor inhibiting rates of Artesunate were 49.1%, 48.7%, 46.6% in 3 experiments respectively. After administration of Artesunate, the survival rate of the mice bearing H22 ascitic liver tumor were increased to 45%. Compared with the control groups, the difference was statistically significant (P < 0.01). In additional, Artesunate can synergize the antitumor activity of 5-fluorouracil. Artesunate showed evident cytotoxicity to human hepatocarcinoma SMMC-7721 cells, the IC50 of Artesunate being 2.07 micrograms.ml-1 in MTT experiment and 2.48 micrograms.ml-1 in colony-forming unit experiment. CONCLUSION: Artesunate has marked antitumor activity in vitro and in vivo.