Diagnosis of Fasciola gigantica infection using a monoclonal antibody-based sandwich ELISA for detection of circulating cathepsin B3 protease

A reliable monoclonal antibody (MoAb)-based sandwich enzyme-linked immunosorbent assay (sandwich ELISA) was developed for the detection of circulating cathepsin B3 protease (CatB3) in the sera from mice experimentally infected with Fasciola gigantica and cattle naturally infected with the same parasite. The MoAb 2F9 and biotinylated rabbit polyclonal anti-recombinant CatB3 antibody were selected due to their high reactivities and specificities to F. gigantica CatB3 antigen based on indirect ELISA and immunoblotting. The lower detection limit of the sandwich ELISA assay was 10, 100 and 400 pg/ml, when applied for the detection of rCatB3 antigen and CatB3 in whole body (WB) of newly excysted juveniles (NEJ) and metacercariae (Met) of F. gigantica, respectively. This sandwich ELISA assay could detect F. gigantica infection from day 1 to 35 post infection and revealed that circulating level of CatB3 peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using serum samples from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as normal mice and hamsters. In addition, sera from cattle infected with Paramphistomum cervi, Strongylid, Trichuris sp. and Strongyloides sp., as well as sera from normal cattle were also assessed. In experimental mice, the diagnostic sensitivity, specificity, positive predictive value, negative predictive value, false positive rate, false negative rate and accuracy of ELISA were 95%, 100%, 100%, 97.9%, 0%, 5.3% and 98.5%, while in natural cattle they were 96.7%, 100%, 100%, 98.5%, 0%, 3.4% and 98.9%, respectively. Hence, this assay method showed high efficient and precision for early diagnosis of fasciolosis by F. gigantica.     

Clinical significance of autoantibodies to the pericentromeric heterochromatin protein 1a protein

OjectiveThe objective of the study is to determine the frequency and the clinical significance of autoantibodies to the pericentromeric heterochromatin protein 1 (HP1). So far this antinuclear antibody specificity has been mainly reported in patients with the CREST syndrome.MethodsWe screened the sera of 199 individuals, including patients suffering from various autoimmune disorders (Group I, n = 145) and non autoimmune diseases (Group II, n = 44 patients) as well as healthy individuals (Group III, n = 30). The sera were systematically tested by Western blot and ELISA using a GST–HP1α fusion protein as an antigen.ResultsAnti-HP1 antibodies were detected in 32% of patients in Group I, 11.3% in Group II and 3.3% of individuals in Group III. They could be detected in sera containing or not antinuclear antibodies detectable by indirect immunofluorescence. Anti-HP1 antibodies were mostly associated with the CREST and Sjogren's syndromes (70% and 44.4%, respectively). They could also be detected in 22.2% of patients suffering from various other autoimmune diseases. However, their negative predictive value was 94% in the CREST syndrome.ConclusionAnti-HP1 autoantibodies are associated with a large spectrum of disorders. However, they have a diagnostic value in the CREST syndrome.     

Molecular cloning and characterization of heat shock protein 70 from Trichinella spiralis

A cDNA encoding heat shock protein 70 of Trichinella spiralis (Ts-Hsp70) was identified by immunoscreening the adult T. spiralis cDNA library with rabbit antisera against T. spiralis adult extracts. The open reading frame of Ts-Hsp70 cDNA encoded a 623-amino acid peptide with a predicted molecular weight of 68.7 kDa, which shares a high degree of sequence conservation with HSP70s from other parasites. Recombinant Ts-Hsp70 was expressed in Escherichia coli and purified with nickel column chromatography. Western blot analysis showed that recombinant Ts-Hsp70 could be recognized not only by trichinellosis patient sera, but also by T. spiralis-infected sera from rabbits, swine, and mice. Mice vaccinated with recombinant Ts-Hsp70 formulated with Freund's adjuvant exhibited strong humoral immune responses indicated by high titer of IgG antibody and significant muscle larval reduction (37%) after being challenged with T. spiralis larvae. The present results indicate that Ts-Hsp70 is a possible candidate vaccine against T. spiralis infection.     

Canine visceral leishmaniasis: Asymptomatic infected dogs as a source of L. infantum infection

Clinically infected dogs have been identified as the main reservoir hosts of visceral leishmaniasis (VL) caused by Leishmania infantum in the Mediterranean region. The objective of this study was to determine the potential of asymptomatic infected dogs compared with symptomatic ones as a source of L. infantum infection to golden hamster.For this purpose, anti-Leishmania antibodies were detected with direct agglutination test (DAT) in 13 symptomatic (7 seropositive = ≥1:320) and 53 asymptomatic (9 seropositive = ≥1:320 and 44 seronegative =  <1:320) ownership dogs. DNA of Leishmania sp. was extracted from skin and peripheral blood tissues of each dog and tested by PCR. Sixty-six Syrian golden hamsters (Mesocricetus auratus) were used for the determination of infectivity and pathogenicity of L. infantum, isolated from the dogs. We used the internal transcribed spacer 2 (ITS 2) rDNA sequence analysis. The results showed that 22 and 11 out of 66 inoculated golden hamsters were positive by PCR and parasitological examinations, respectively. From 22 PCR positive hamsters, 17 were related to asymptomatic dogs and 5 were from symptomatic ones. There was no significant difference between symptomatic and asymptomatic dogs in producing Leishmania infection in the susceptible animal model (P = 0.66). Smears and cultures of 5 dogs from 13 symptomatic dogs (38.5%) and 6 dogs from 53 asymptomatic ones (11.3%) were found to be positive at parasitological examination.All the L. infantum isolates from symptomatic and asymptomatic dogs were similar in sequencing.In conclusion, asymptomatic infected dogs as well as symptomatic ones can harbor L. infantum in their blood and skins which are virulent and infectious for inoculated golden hamster.     

Can Helicobacter pylori infection influence human reproduction?

Helicobacter pylori(H.pylori)infection could be associated with extra-digestive diseases.Here,we report the evidences concerning the decrease in reproductive potential occurring in individuals infected by H.pylori,especially by strains expressing CagA.This infection is more prevalent in individuals with fertility disorders.Infected women have anti-H.pylori antibodies in cervical mucus and follicular fluid that may decrease sperm motility and cross react immunologically with spermatozoa,conceivably hampering the oocyte/sperm fusion.Infection by CagA positive organisms enhances the risk of preeclampsia,which is a main cause of foetus death.These findings are supported by the results of experimental infections of pregnant mice,which may cause reabsorption of a high number of foetuses and alter the balance between Th1 and Th2 cell response.Infected men have decreased sperm motility,viability and numbers of normally shaped sperm and augmented systemic levels of inflammatory cytokines,such as TNF-α,which may damage spermatozoa.In countries where parasitic infestation is endemic,detrimental effects of infection upon spermatozoa may not occur,because the immune response to parasites could determine a switch from a predominant Th1 type to Th2 type lymphocytes,with production of anti-inflammatory cytokines.In conclusion,the evidences gathered until now should be taken into consideration for future studies aiming to explore the possible role of H.pylori infection on human reproduction.     

Opisthorchis viverrini: Evaluation of 28 kDa glutathione S-transferase as diagnostic tool in human opisthorchiasis

The liver fluke Opisthorchis viverrini is the agent of human opisthorchiasis in Thailand with a high prevalence observed in the rural population of north and northeastern regions of the country. A focus of research has therefore been the development of diagnostic tools to indicate infection by this parasite. In the present study, a 28 kDa glutathione S-transferase of O. viverrini (OV28GST), which is found in the excretion/secretion product of the parasite, was evaluated for its application in diagnosis of human opisthorchiasis. Bacterially expressed and functionally active rOV28GST was used in immunoblots and indirect ELISA to detect anti-OV28GST antibody in sera of infected individuals. Crude whole worm extract, sera of uninfected individuals and a rabbit anti-rOV28GST antiserum were used as controls in the assays while positivity for parasite DNA by PCR and egg count in faeces were used as primary indicators of infection. The results showed weak or absent reactivity of the infected sera to immunoblotted rOV28GST and no significant difference in absorbance values when compared to uninfected sera in ELISA. In addition, a glutathione capture ELISA which was performed to test for circulating OV28GST in human and hamster sera showed negative results. In conclusion, OV28GST is not applicable as a diagnostic tool in established infections due to low specific antibody titre and abundance as circulating antigen.     

Characterization of a Trichinella spiralis 31 kDa protein and its potential application for the serodiagnosis of trichinellosis

The Trichinella spiralis 31 kDa protein (Ts31) was screened from the excretory-secretory (ES) proteins of muscle larvae (ML) by immunoproteomics using serum from mice infected with T. spiralis at 18 days post infection (dpi). The aim of this study was to characterize the Ts31 protein and to evaluate the potential of the recombinant Ts31 protein (rTs31) for serodiagnosis of human trichinellosis. Ts31 gene was cloned and rTs31 was produced in an E. coli expression system. An anti-rTs31serum recognized the native protein migrating in a 25–55 kDa range by Western blotting of ML crude or ES antigens. Expression of Ts31 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and ML). An immunolocalization analysis identified Ts31 in the cuticle and stichocytes of the parasite. The sensitivity of rTs31-ELISA and ES antigen ELISA for detecting anti-Trichinella IgG antibodies in sera of patients with trichinellosis was 97.83% (45/46) and 86.78% (39/46), respectively (P > 0.05); The specificity of rTs31-ELISA was 99.13% (114/115), which was significantly higher than 85.22% (98/115) of ES antigen ELISA (P < 0.01). The rTs31 protein of T. spiralis could be considered as a potential diagnostic antigen for trichinellosis.     

Tropomyosin implicated in host protective responses to microfilariae in onchocerciasis

A cDNA from adult female Onchocerca volvulus encoding the C-terminal portion of a tropomyosin isoform (termed MOv-14) has been shown previously to confer protective immunity in rodent models of onchocerciasis. The full-length sequence (designated Ov-tmy-1) obtained by PCR amplification, codes for a protein of 33 kDa and shares 91% identity with tropomyosins from other nematodes, falling to 57% identity with human α-tropomyosin. Ov-TMY-1 migrates with an apparent molecular mass of 42 kDa on SDS/PAGE and is present in all life-cycle stages, as determined by immunoblotting. Immunogold electron microscopy identified antigenic sites within muscle blocks and the cuticle of microfilariae and infective larvae. Anti-MOv14 antibodies were abundant in mice exhibiting serum-transferable protection against microfilariae conferred by vaccination with a PBS-soluble parasite extract. In contrast, little or no MOv14-specific antibody was present in mice inoculated with live microfilariae, in which resistance is mediated by antibody-independent mechanisms. In human infections, there was an inverse correlation between anti-tropomyosin IgG levels and densities of microfilariae in the skin. Seropositivity varied with the relative endemicity of infection. An immunodominant B cell epitope within Ov-TMY-1 (AQLLAEEADRKYD) was mapped to the N terminus of the MOv14 protein by using sera from protectively vaccinated mice. Intriguingly, the sequence coincides with an IgE-binding epitope within shrimp tropomyosin, believed to be responsible for hypersensitivity in individuals exhibiting allergy to shellfish. IgG and IgE antibodies reacting with the O. volvulus epitope were detected in human infections. It is concluded that antibody responses to tropomyosin may be important in limiting microfilarial densities in a proportion of individuals with onchocerciasis and have the potential to mediate hypersensitivity reactions to dead microfilariae, raising the possibility of a link with the immunopathology of infection.     

IgG subclasses pattern and high-avidity antibody to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in an unstable hypoendemic region in Iran

The C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP-1₁₉) is a leading vaccine candidate for inclusion in a polyvalent malaria vaccine. In the present study, the IgG subclasses profile and the avidity of IgG to PvMSP-1₁₉ were evaluated in individuals (n = 94) naturally exposed to P. vivax parasite in malaria endemic areas in Chabahar districts, Iran. In individuals with patent P. vivax malaria, 86.1% was sero-positive to PvMSP-1₁₉ and IgG1 (81.9%) was the predominant subclass. In addition, to determine the persistence of specific IgG, IgG1 and IgG3 antibodies to PvMSP-1₁₉, the frequency of antibodies was determined in the infected subjects (n = 74) after treatment with standard chloroquine and it was detected that the frequency of responders was significantly reduced to 51.3%, 51% and 16.2%, respectively. The antigen-binding avidity of IgG antibodies to PvMSP-1₁₉ was measured in sero-positive sera and the high-avidity of IgG, IgG1 and IgG3 was found in 66.6%, 61% and 47% of the infected subjects with P. vivax, respectively. The present result shows that individuals who exposed to vivax malaria in the endemic region in Iran develop antibodies with high-avidity to PvMSP-1₁₉. These results could help to understand the interactions between the host and P. vivax parasite in development of MSP-1₁₉-based vaccine.     

Endurance exercise training increases adipose tissue glucocorticoid exposure: adaptations that facilitate lipolysis

Glucocorticoids (GCs) have long been thought to be lipolytic in nature. Recently, however, increased exposure to GCs in insulin-sensitive tissues has been associated with lipid accumulation and metabolic complications, regardless of plasma concentrations. Intracellular GC action is determined by both 11-β hydroxysteroid dehydrogenase type 1 (11βHSD1) and the GC receptor (GR). We hypothesized that exercise training would increase 11βHSD1 and GR protein in adipose tissue, resulting in increased lipolysis. To test the effects of exercise on adipose tissue GR and 11βHSD1 protein, 2 sets of hamsters were trained for 6 weeks: young, diet-induced obese animals and older, overweight animals. Young (6 week old) hamsters, fructose-fed to induce an obese phenotype, and older (6 month old) hamsters were randomly divided into exercising and sedentary groups. Exercise training decreased adipose tissue mass in both fructose-fed and older hamsters. In addition, exercise training increased 11βHSD1 (31.5% ± 15% and 20.0% ± 7%, fructose-fed and older, respectively) and GR (45.6% ± 14% and 61.1% ± 27%, fructose-fed and older, respectively) protein expression in the perirenal adipose depot and increased 11βHSD1 (16.7% ± 7%, P = .09) and GR (47.4% ± 19%, P < .05) in the subcutaneous adipose depot of the older hamsters. To determine the metabolic effect of increased GC exposure in adipocytes, 3T3-L1 adipocytes were treated with corticosterone for 24 hours; and measures of lipolytic rates were conducted. Low concentrations of GCs (0.01-0.1 μmol/L) increased GR (44.1% ± 18%, P < .05) and 11βHSD1 (95.3% ± 24%) protein expression, as well as lipolytic rates (34.6% ± 6%) as measured by glycerol release. The increased lipolysis was blocked by RU486, a GR antagonist, suggesting that the elevated lipolysis was a direct result of GC action. These results suggest that exercise training amplifies the activity of GCs in adipose tissue of overweight animals through alterations in 11βHSD1 and GR despite differences in age and amounts of adiposity. In vitro, GCs are capable of increasing lipolysis, but depend upon the presence of GR. We propose that GCs play a significant role in changing the phenotype of adipose tissue during exercise training, resulting in decreased fat mass.     

旋毛虫Ts21重组蛋白的免疫诊断价值及免疫保护作用的研究

目的 探讨旋毛虫Ts21重组蛋白的免疫诊断价值及免疫保护作用。 方法 应用旋毛虫Ts21重组蛋白ELISA（Ts21-LISA）与肌幼虫ES抗原ELISA（ES-LISA）对旋毛虫病与其他寄生虫病患者血清及5种旋毛虫（T1、T2、T3、T4和T7）感染小鼠血清进行检测,并观察不同剂量旋毛虫感染小鼠后不同时间的血清抗体水平。将Ts21重组蛋白皮下注射免疫小鼠（20 μg/只,免疫3次,每次间隔10 d）,末次免疫后10 d,每只小鼠用300条旋毛虫肌幼虫经口攻击感染,3.5 d和42 d 后剖杀,观察肠道成虫与肌幼虫数并计算减虫率。 结果 Ts21-LISA检测旋毛虫病、并殖吸虫病、囊尾蚴病及棘球蚴病患者血清的抗体阳性率分别为94.7%（18/19）、15.8%（3/19）、9.1%（1/11）和7.7%（1/13）,与血吸虫病、华支睾吸虫病患者血清及健康人血清无交叉反应;Ts21重组蛋白与ES抗原ELISA检测旋毛虫病患者血清抗体的敏感性与特异性差异均无统计学意义（χ²=0,P＞0.05;χ²=0.358,P＞0.05）。Ts21重组蛋白与ES抗原检测T1感染小鼠血清的敏感性差异无统计学意义（χ²=0.104,P＞0.05）,与T2、T3、T4、T7感染小鼠血清的交叉反应率明显低于ES抗原（χ²=17.069,P＜0.05）。小鼠感染300条旋毛虫后4 周,应用Ts21-LISA检测的血清抗体阳性率为100%（10/10）;小鼠感染5条旋毛虫后6周,血清抗体阳性率为100%（10/10）。Ts21重组蛋白免疫小鼠用旋毛虫攻击感染后3.5 d和42 d,肠道成虫与肌幼虫减虫率分别为42.71%和49.8%。 结论 Ts21重组蛋白可用于旋毛虫病的血清学检测,但不能忽视与并殖吸虫病、囊尾蚴病及棘球蚴病患者血清的交叉反应。     

Detection of circulating antigen in serum of mice infected with Schistosoma japonicum by immunomagnetic bead ELISA based on IgY

We developed a novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) for detection of circulating antigen (CA) in sera of mice infected with Schistosoma japonicum. The assay involved the use of chicken polyclonal antibodies IgY against soluble egg antigens (SEA) of S. japonicum as a capture antibody and anti-SEA mouse monoclonal antibody NP28-5B labeled horseradish peroxidase (HRP) as a detecting antibody. Two groups of BALB/c mice infected with S. japonicum cercariae were used: lightly infected mice (infected with 10 S. japonicum cercariae) and heavily infected mice (infected with 30 S. japonicum cercariae). The CA was detectable as early as 4 and 5 weeks after infection in the sera of heavily and lightly infected mice, respectively. The CA levels rose rapidly and reached a peak in 8 weeks after infection and then remained a plateau for at least another 6 weeks in both groups. Moreover, the effect of praziquantel on the CA levels was also investigated. The heavily infected mice were treated with praziquantel and the CA levels in sera increased dramatically in the first week post-treatment and then decreased to the control level by 6 weeks after treatment. The novel assay appears to be sensitive for detection of schistosomal antigenemia and valuable to judge the efficacy of chemotherapy in murine schistosomiasis.     

Three-dimensional structure of tropism-switching Bordetella bacteriophage

Bacteriophage BPP-1, which infects Bordetella species, can switch its specificity by mutations to the ligand-binding surface of its major tropism-determinant protein, Mtd. This targeted mutagenesis results from the activity of a phage-encoded diversity-generating retroelement. Purified Mtd binds its receptor with low affinity, yet BPP-1 binding and infection of Bordettella cells are efficient because of high-avidity binding between phage-associated Mtd and its receptor. Here, using an integrative approach of three-dimensional (3D) structural analyses of the entire phage by cryo-electron tomography and single-prticle cryo-electron microscopy, we provide direct localization of Mtd in the phage and the structural basis of the high-avidity binding of the BPP-1 phage. Our structure shows that each BPP-1 particle has a T = 7 icosahedral head and an unusual tail apparatus consisting of a short central tail “hub,” six short tail spikes, and six extended tail fibers. Subtomographic averaging of the tail fiber maps revealed a two-lobed globular structure at the distal end of each long tail fiber. Tomographic reconstructions of immuno-gold-labeled BPP-1 directly localized Mtd to these globular structures. Finally, our icosahedral reconstruction of the BPP-1 head at 7Å resolution reveals an HK97-like major capsid protein stabilized by a smaller cementing protein. Our structure represents a unique bacteriophage reconstruction with its tail fibers and ligand-binding domains shown in relation to its tail apparatus. The localization of Mtd at the distal ends of the six tail fibers explains the high avidity binding of Mtd molecules to cell surfaces for initiation of infection.     

Campylobacter Jejuni and Cytopenias

Background

Leukopenia and thrombocytopenia in a febrile patient are not uncommon and may be a diagnostic clue in patients without an alternative explanation for cytopenias. This has not been reported in Campylobacter jejuni infections.

Methods

A healthy patient with fever, rigors, and an acute diarrheal illness was noted to have a white blood cell count of 2.65 × 10⁹/L and platelet level of 125 × 10⁹/L. Retrospective chart review of all adult C. jejuni stool-positive cases admitted over 1 year revealed leukopenia in 6 of 20 (30%), thrombocytopenia in 5 of 20 (25%), and both in 1 of 20 (5%).

Results

Cytopenias were mild, transient, and not associated with prolonged hospital stay or complications.

Conclusions

Acute C. jejuni infections should be added to the differential diagnosis of acute febrile illnesses that may be associated with leukopenia or thrombocytopenia. Cytopenias can be an important diagnostic clue in febrile illnesses, and their differential is presented.     

Identification of a novel 19 kDa Echinococcus granulosus antigen

By screening an Echinococcus granulosus cDNA library with IgG4 from patients with active cystic echinococcosis (CE), we identified a cDNA encoding a protein of 19.0 kDa (Eg19). Eg19, in 12% SDS-PAGE in reducing and non-reducing conditions, showed several bands between 19 and 100 kDa. Immunoblotting (IB) analysis detected total IgG, IgG1 and IgG4 specific to the 38/40 kDa band of Eg19 in the 10% of patients’ sera. The percentage of total IgG, IgG1 and IgG4-positive sera were significantly higher in sera from patients with active disease and cyst in multiple sites than from patients with inactive disease and cyst in the liver (P < 10⁻⁴). ELISA analysis disclosed that during the follow-up anti-Eg19 antibody concentration decreased over the course of treatment in sera from patients with cured disease. Even if Eg19 appear to have no benefit in the diagnosis of the disease, our data, confirming the presence of antigens inducing both IgG1 and IgG4 during active development of CE, suggest that Eg19 might be a marker of disease status.     

Hydrogen-rich water protects against acetaminopheninduced hepatotoxicity in mice

AIM: To investigate the hepatoprotective effects and mechanisms of hydrogen-rich water(HRW) in acetaminophen(APAP)-induced liver injury in mice.METHODS: Male mice were randomly divided into the following four groups: normal saline(NS) control group, mice received equivalent volumes of NS intraperitoneally(ip); HRW control group, mice were given HRW(same volume as the NS group); APAP + NS group, mice received NS ip for 3 d(5 mL /kg body weight, twice a day at 8 am and 5 pm) after APAP injection; APAP + HRW group, mice received HRW for 3 d(same as NS treatment) after APAP challenge.In the first experiment, mice were injected ip with a lethal dose of 750 mg/kg APAP to determine the 5-d survival rates.In the second experiment, mice were injected ip with a sub-lethal dose of 500 mg/kg.Blood and liver samples were collected at 24, 48, and 72 h after APAP injection to determine the degree of liver injury.RESULTS :Treatment with HRW resulted ina significant increase in the 5-d survival rate compared with the APAP + NS treatment group(60% vs 26.67%, P 0.05).HRW could significantly decrease the serum alanine aminotransferase level(24 h: 4442 ± 714.3 U/L vs 6909 ± 304.8 U/L, P 0.01; 48 h: 3782 ± 557.5 U/L vs 5111 ± 404 U/L, P 0.01; and3255 ± 337.4 U/L vs 3814 ± 250.2 U/L, P 0.05, respectively) and aspartate aminotransferase level(24 h: 4683 ± 443.4 U/L vs 5307 ± 408.4 U/L, P 0.05; 48 h: 3392 ± 377.6 U/L vs 4458 ± 423.6 U/L, P 0.01; and 3354 ± 399.4 U/L vs 3778 ± 358 U/L, respectively) compared with the APAP treatment group.The alkaline phosphatase, total bilirubin and lactate dehydrogenase levels had the same result.Seventy-two hours after APAP administration, liver samples were collected for pathological examination and serum was collected to detect the cytokine levels.The liver index(5.16% ± 0.26% vs 5.88% ± 0.073%, P 0.05) and percentage of liver necrosis area(27.73% ± 0.58% vs 36.87% ± 0.49%, P 0.01) were significantly lower in the HRW-treated animals.The malonyldialdehyde(MDA) contents were significantly reduced in the HRW pretreatment group, but they were increased in the APAP-treated group(10.44 ± 1.339 nmol/mg protein vs 16.70 ± 1.646 nmol/mg protein, P 0.05).A decrease in superoxide dismutase(SOD) activity in the APAP treatment group and an increase of SOD in the HRW treatment group were also detected(9.74 ± 0.46 U/mg protein vs 12.1 ± 0.67 U/mg protein, P 0.05).Furthermore, HRW could significantly increase the glutathione(GSH) contents(878.7 ± 76.73 mg/g protein vs 499.2 ± 48.87 mg/g protein) compared with the APAP treatment group.Meanwhile, HRW could reduce the inflammation level(serum TNF-α: 399.3 ± 45.50 pg/L vs 542.8 ± 22.38 pg/L, P 0.05; and serum IL-6: 1056 ± 77.01 pg/L vs 1565 ± 42.11 pg/L, P 0.01, respectively).In addition, HRW could inhibit 4-HNE, nitrotyrosine formation, JNK phosphorylation, connexin 32 and cytochrome P4502 E expression.Simultaneously, HRW could facilitate hepatocyte mitosis to promote liver regeneration.CONCLUSION: HRW has significant therapeutic potential in APAP-induced hepatotoxicity by inhibiting oxidative stress and inflammation and promoting liver regeneration.     

MT1M and MT1G promoter methylation as biomarkers for hepatocellular carcinoma

AIM:To investigate the potential of promoter methylation of two tumor suppressor genes(TSGs)as biomarkers for hepatocellular carcinoma(HCC).METHODS:A total of 189 subjects were included in this retrospective cohort,which contained 121 HCC patients without any history of curative treatment,37 patients with chronic hepatitis B(CHB),and 31 normal controls(NCs).DNA samples were extracted from 400μL of serum of each subject and then modified using bisulfite treatment.Methylation of the promoters of the TSGs(metallothionein1M,MT1M;and metallothionein 1G,MT1G)was determined using methylation-specific polymerase chain reaction.The diagnostic value of combined MT1M and MT1G promoter methylation was evaluated using the area under the receiver operating characteristic curves.RESULTS:Our results indicated that the methylation status of serum MT1M(48.8%,59/121)and MT1G(70.2%,85/121)promoters in the HCC group was significantly higher than that in the CHB group(MT1M 5.4%,2/37,P<0.001;MT1G 16.2%,6/37,P<0.001)and NC group(MT1M 6.5%,2/31,P<0.001;MT1G 12.9%,4/27,P<0.001).Aberrant serum MT1M promoter methylation gave higher specificity to discriminate HCC from CHB(94.6%)and NCs(93.5%),whereas combined methylation of serum MT1M and MT1G promoters showed higher diagnostic sensitivity(90.9%),suggesting that they are potential markers for noninvasive detection of HCC.Furthermore,MT1M promoter methylation was positively correlated with tumor size(rs=0.321,P<0.001),and HCC patients with both MT1M and MT1G promoter methylation tended to show a higher incidence of vascular invasion or metastasis(P=0.018).CONCLUSION:MT1M and MT1G promoter methylation may be used as serum biomarkers for noninvasive detection of HCC.     

The Anopheles stephensi odorant binding protein 1 (AsteObp1) gene: A new molecular marker for biological forms diagnosis

Anopheles (Cellia) stephensi Liston 1901 is known as an Asian malaria vector. Three biological forms, namely “mysorensis”, “intermediate”, and “type” have been earlier reported in this species. Nevertheless, the present morphological and molecular information is insufficient to diagnose these forms. During this investigation, An. stephensi biological forms were morphologically identified and sequenced for odorant-binding protein 1 (Obp1) gene. Also, intron I sequences were used to construct phylogenetic trees. Despite nucleotide sequence variation in exon of AsteObp1, nearly 100% identity was observed at the amino acid level among the three biological forms. In order to overcome difficulties in using egg morphology characters, intron I sequences of An. stephensi Obp1 opens new molecular way to the identification of the main Asian malaria vector biological forms. However, multidisciplinary studies are needed to establish the taxonomic status of An. stephensi.     

Alterations in the expression level of a putative aspartic protease in the development of Angiostrongylus cantonensis

Aspartic proteases are a family of proteinases with catalytic aspartate residues in the active site. These enzymes have been reported to initialize the degradation of host hemoglobin in blood-feeding helminths. After identifying an expressed sequence tag representing an aspartic protease from an Angiostrongylus cantonensis young adult dataset, this sequence was found to encode a protein with a predicted molecular mass of 46 kDa. It also showed good homologies to aspartic proteases from Caenorhabditis elegans (50.7% identity), Haemonchus contortus (43.0% identity), Necator americanus (41.5% identity), Strongyloides stercoralis (35.9% identity), and Burgia malayi (29.6% identity). This putative aspartic protease was determined to be expressed in the infective larvae, young adults, and adult worms of A. cantonensis by quantitative real-time PCR. Among male worms, the expression level was determined to increase by 223.0 ± 24.2 fold in young adults relative to the infective larvae and then decreased to 7.1 ± 0.2 fold in adult worms. In female worms, the expression level was observed to increase by 118.5 ± 10.1 fold in young adults and by 277.5 ± 29.2 fold in the adults, when compared with infective larvae. These findings not only indicate that the expression level of aspartic protease gene in A. cantonensis changes with development but also has a sexual difference in individual developmental stages in the final host.