Investigation on the endemic characteristics of Metorchis orientalisin Huainan area, China

OBJECTIVE: To investigate the endemic characteristics of Metorchis orientalis (M. orientalis)in the Huainan area, Anhui province, China. METHODS: The first-intermediate host, second-intermediate host and reservoir hosts were collected, and the endemic characteristics of M. orientalis were examined through field investigation and artificial infection. RESULTS: Investigation was completed in 89 domestic ducks, 156 domestic chicken, 41 domestic geese, 20 domestic cats and 19 dogs. The infection rate of M. orientaliswas 18.0% (16/89) in ducks, 12.2% (19/156) in chicken, 9. 8% (4/41) in geese, 5.0% (1/20) in cats and 5.3% (1/19) in dogs. Sixty-seven cercariae of M. orientaliswere identified in 1,000 Parafossarulu s striatulus,with a natural infection rate of 6.7%, and 19 cercariae occurred in 300 Pseudorasbora parva, with a natural infection rate of 6.33%. The activity of the cercariae of M. orientaliswas associated with light intensity and temperature. The full life cycle of M. orientalisranged from 120 to 140 days; it occurred approximately in 89 days in snails, 28 days in fish and 20 days in ducks. CONCLUSION: M. orientalisis prevalent in the Huainan area, and it may complete its life cycle in Parafossarulus striatulus, Pseudorasbora parva and natively rais ed ducks.

Composition and diversity of acaroids mites (Acari: Astigmata) community in the stored rhizomatic traditional Chinese medicinal materials.

OBJECTIVE: To investigate the species and breeding density of ac a roid mites in the stored rhizomatic traditional Chinese medicinal materials in Anhui province, China, in order to supply evidences for control and prevention of such species. METHODS: The stored traditional Chinese medicinal materials of root-stock origins were collected in 30 herb stores and warehouses in 17 cities across Anhui province. Mites were collected by using Tullgren funnel and directicopy, and identified under microscopy. RESULTS: Twenty-two species of acaroid mites, belonging to 15 genera under 5 families, were identified from the total 47 stored samples, in which Tyrophagus putrescentiae, Acarus farinae, Carpoglyphus lactis, and Cologlyplus berlesei were predominant. CONCLUSION: Breeding density of acaroid mites was high in the stored rhizomatic traditional Chinese medicinal materials in Anhui province. This indicates that the traditional Chinese medicinal herbs of root-stock origins in storage are seriously contaminated by the acaroid mites, and such infestation should be positively controlled to reduce the potential harm to public health.

Trematode Aspidogastrea found in the freshwater mussels in the Yangtze River basin.

OBJECTIVE: To investigate the prevalence of trematode Aspidogastrea in the freshwater mussels in the Yangtze River basin within Anhui province, China. METHODS: We initially harvested the freshwater mussels living in the Yangtze River running through Anhui area, and labeled them with corresponding number. Then the samples were dissected for isolating the flukes, which were identified by conventional staining. RESULTS: Infection rate of trematode Aspidogastrea in freshwater mussels in the Yangtze River basin within the territory of Anhui province was 30.38% (103/339) in general, and a total of 912 flukes of Aspidogastrea were detected in the 103 mussels, with average infection rate of 8.85 for each mussel. CONCLUSION: Trematode Aspidogastrea is prevalent in the freshwater bivalves living in the Yangtze River basin running through Anhui area, and the treamatode was identified as Aspidogaster sp. belong to Aspidogaste under Aspidogastridae of Aspidogastrea.

Carpoglyphus lactis (Carpoglyphidae) infestation in the stored medicinal Fructus Jujubae.

OBJECTIVE: To investigate the breeding rate and breeding density of Carpoglyphus lactisin stored Fructus Jujubaein Anhui, China in order to provide a scientific basis for prevention of Carpoglyphus lactis (C. lactis)from spoiling the dried fruit of such category. METHODS: By the breeding nature of C. lactis,we collected samples of Fructus Jujubae, which were kept over 6 months in general, from the dried fruit shop and (or) Chinese herbal medicine warehouse, and isolated C. lactisfrom those samples. The mite specimens were prepared, and microscopically and morphologically identified. RESULTS: C. lactiswas identified in 19 of the 300 samples, with breeding density and breeding rate of 6.52 heads/g and 6.33%. Constitute ratio at distinct developmental phase was associated with adult (including nymph, 85.71%), larva (12.27%), hypopus (0.56%) and egg (1.45%), respectively. The richness index, diversity index and evenness index was 1.644, 1.644 and 0.923, respectively. CONCLUSION: Carpoglyphus lactis appears infesting in large quantity in the Fructus Jujubaestored in the above places in Anhui province, and the density is higher. Therefore, it is urgent to take effective measures to prevent C. lactisfrom spreading over other dried products stored in the same room and potential human intestinal acariasis as a result of the biological contamination.

Investigation on the zoonotic trematode species and their natural infection status in Huainan areas of China.

BACKGROUND: To investigate the species of zoonotic trematodes and the endemic infection status in the domestic animals in Huainan areas, north Anhui province of China, we intent to provide evidences for prevention of the parasitic zoonoses. METHODS: The livestock and poultry (definitive hosts) were purchased from the farmers living in the water areas, including South Luohe, Yaohe, Jiaogang and Gaotang Lakes, and dissected the viscera of these collected hosts to obtain the parasitic samples. Then the specimens were microscopically identified, with reference to the descriptions in previous literatures for counting the zoonotic species found in these areas. RESULTS: A total of 41 species were detected in the domestic samples, in which 23 were zoonotic trematodes, and 18 were internal trematodes of animals. Of the 41 species, 38 were novel records in Huainan areas, and 12 were newly detected in Anhui province, including Metorchis anatinus, Echinostoma hortense, E. cinetorchis, E. angustitestis, E. lindoensis, E. nordiana, E. ilocanum, Metagonimus yokogawai, Prosthogonimus gracilis, P. skrjabini, P. anatinusand Trichobilharzia sp. which generally occurred in definitive hosts of chicken, ducks, geese, dogs, cattle, buffaloes, sheep, goats and pigs, respectively. CONCLUSION: A large quantity of livestock and poultry are fed by the local farmers living along the river banks in Huainan area, suggesting that the population in that area are at higher risks of natural focus of zoonotic infections, since these animals are favorable definitive hosts to the zoonotic trematodes.

[The effect of Dermatophagoides pteronyssinus group 2 allergen T cell fusion epitope on STAT6 signaling in mice with asthma].

Objective: To investigate the alterations of signal transducer and activator of transcription factor 6(STAT6) signaling in a mouse model of asthma receiving treatment with Dermatophagoides pteronyssinus group 2 allergen(Der p 2) T cell fusion epitope and the mechanisms of the specific immunotherapy. Methods: Thirty mice were randomly divided into three groups by the random number table method: the asthma group, the treatment group receiving immunotherapy with Der p 2 T cell fusion epitope, and the negative control group (PBS group)(n = 10 in each group). Mice in the asthma and the treatment groups received intraperitoneal (i. p.) injection of 100 µl Der p 2 solution [PBS containing 100 µg/ml Der p 2 and 2% Al(OH)3] on days 0,7 and 14, respetively, while mice in the PBS group received same volume of PBS containing 2% Al(OH)3. From day 21, 30-min steam inhalation of 0.5 µg/ml Der p 2 was applied to the asthma and treatment groups (once daily for 7 successive days), and the PBS group inhaled same volume of PBS. From day 25 to day 27, the mice in the treatment group received i. p. injection of 200 µl of Der p 2 T cell fusion epitope (100 µg/ml) while the PBS and the asthma groups received the same volume of PBS. Mice were sacrificed at 24 h after the last inhalation, the bronchoalveolar lavage fluid (BALF) collected, and the total protein was extracted from the lung tissue. The levels of IFN-γ, IL-4 and IL-13 in BALF were determined by ELISA. The expression of STAT6 and phosphorylated STAT6 (p-STAT6) in the lung tissue was detected by Western blotting. Data were analyzed with the one-way variance analysis (ANOVA) method. Results: The level of IFN-γ in the treatment group[(234.40 ± 24.46) pg/ml] was significantly higher than that in the asthma group[(155.80 ± 20.53) pg/ml](P < 0.01). The levels of IL-4 and IL-13 in the treatment group [(30.00 ± 5.50) pg/ml and (174.50 ± 25.99) pg/ml, respectively] were both significantly lower than those in the asthma group[(53.28 ± 8.26) pg/ml and (308.10 ± 28.32) pg/ml, respectively](P < 0.01). Similarly, the levels of STAT6 and p-STAT6 in the treatment group(0.803 ± 0.221 and 0.966 ± 0.323, respectively) were both significantly lower than those in the asthma group (1.669 ± 0.296 and 1.735 ± 0.298, respectively)(P < 0.01). Conclusion: The Der p 2 T cell fusion epitope may function through inhibiting STAT6 to treat asthma in mice.

An updated meta-analysis of transforming growth factor-ß1 gene: Three well-characterized polymorphisms with asthma.

The association between TGF-ß1 polymorphisms and asthma risk has been widely reported, but results were controversial. We performed this meta-analysis based on the Preferred Reporting Items for Systematic Reviews and meta-analyses statement (PRISMA). Electronic database of Pub Med, Web of Science, CBM, and CNKI were searched for eligible articles published up to September, 2013. The effect summary odds ratio (OR) and 95% confidence intervals were obtained. Finally, a total of 20 articles were identified, 17 studies with 3694 cases and 5613 controls for C-509T polymorphism, 7 studies with 1109 cases and 1098 controls for T869C polymorphism and 5 studies with 849 cases and 829 controls for G915C polymorphism. For C-509T, significant associations with asthma were found in Asians (TT+TC vs. CC: P=0.004, OR=1.43, 95%CI=1.12-1.81, Pheterogeneity=0.001) and in Caucasians (P=0.05, OR=1.16, 95%CI=1.00-1.34, Pheterogeneity=0.36). With respect to T869C, a small significant association was observed in overall analysis of allele contrasts(C vs. T: OR=1.14, 95%CI: 1.01-1.29, P=0.03) and homozygote comparison (CC vs. TT: OR=1.29, 95%CI: 1.00-1.65, P=0.05), but no significant risks were found among Caucasian population and Asian population. For G915C polymorphism, no significant association with asthma risk was demonstrated in overall analysis and subgroup analyses according to ethnicity for all genetic models. This meta-analysis suggested that TGF-ß1 C-509T and T869C polymorphisms may be risk factors for asthma.

[Morphological observation on hypopus of Caloglyphus berlesei by optical microscope].

OBJECTIVE: To understand the structure characteristics of hypopus of Caloglyphus berlesei. METHODS: The hypopus of C. berlesei was collected from the feed of Chinese Polyphaga, and was made into the conventional glass specimens. The structure characteristics of hypopus of C. berlesei were observed by an optical microscope. RESULTS: The hypopus of C. berlesei had 4 pairs of legs, and the foot claws and tarsus were well-developed. The structural features were shown, such as the setae of tibia and setae of genu. Genital plates were obviously ossified. CONCLUSION: The research on hypopus of Caloglyphus berlesei provides the reference for its further scientific classification and research on the life cycle.

[Immunotherapeutic Effect of Dermatophagoides pteronyssinus Group 1 Allergen T Cell Epitope Peptide Against Allergic Asthma in Mice].

Objective: To study the specific immunotherapeutic effect of Dermatophagoides pteronyssinus group 1 major allergen T-cell fusion epitope peptide vaccine TAT-IhC-DPTCE against allergic asthma. Methods: One hundred and twenty SPF-grade BALB/c mice were randomized into PBS group (group A), asthma group （group B）, and immune treatment groups respectively receiving intraperitoneal (i.p.) injections of ProDer p 1 allergen (group C), DPTCE （group D）, TAT-DPTCE (group E) or TAT-IhC-DPTCE (group F) （n=20 in each group）. In detail, PBS （group A） or allergen extract derived from Dermatophagoides pteronyssinus （groups B-F, 10 µg） was intraperitoneally injected on days 0, 7 and 14, and was continued by aerosol inhalation from day 21 for 7 consecutive days （0.5 µg/ml, once/day, 30 min each time）. The mice in groups C-F received i.p. injections of 100 µg/ml ProDer p 1, DPTCE, TAT-DPTCE and TAT-IhC-DPTCE respectively 30 min prior to inhalation challenge on days 25-27 as a specific immunotherapy, while those in groups A and B received 200 µl PBS. Twenty-four hours after the last inhalation challenge, all the mice were sacrificed. The lung histopathological changes were examined by HE staining. The levels of IFN-γ, IL-13, IL-10 and TGF-ß in the bronchoalveolar lavage fluid (BALF) was determined with ELISA, and eosinophils in the BALF were counted (n=20 mice in each group). The serum level of IgE, IgG1 and IgG2a in orbital blood was determined by ELISA（n=5 mice in each group）. Results: HE staining revealed increased BALF eosinophils and decreased pulmonary inflammation in group F compared with group B. The IFN-γ level in group F ［（298.75±26.09） pg/mlï¼½ was significantly higher than those in groups B［（158.71±20.89） pg/mlï¼½, C［（210.38±18.92） pg/mlï¼½, D ［（229.44±13.00） pg/mlï¼½ and E［（233.24±20.39） pg/mlï¼½ （all P<0.01）. Similar results were also found for IL-10 and TGF-ß, while the IL-13 levels in groups C ［（47.35±4.71） pg/mlï¼½, D ［（41.90±4.28） pg/mlï¼½, E［（41.05±6.50） pg/mlï¼½ and F［（18.53±5.67） pg/mlï¼½ were all significantly lower than that in group B ［（66.68±6.63） pg/ml］（all P<0.01）. The number of BALF eosinophils in group B ï¼»5.65±0.91ï¼½×105/mlï¼½ was significantly higher than that in group A ［（0.45±0.39）×105/mlï¼½ （P<0.01）, while the BALF eosinophils in groups C ［（4.00±0.59）×105/mlï¼½, D ［（3.39±0.63）×105/mlï¼½, E ［（3.24±0.69）×105/mlï¼½ and F ［（1.42±0.49）×105/mlï¼½ decreased after immune treatment (all P<0.01). ELISA results showed that the serum IgE level in group F ［（5.26±1.72） ng/mlï¼½ was significantly lower than those in group B ［（32.81±2.98） ng/mlï¼½ and the other 3 treatment groups [group C, （20.06±3.17） ng/ml; D, （17.06±3.18） ng/ml; E, （16.23±3.61） ng/mlï¼½. Similar results were also obtained for IgG1. In contrast, the serum IgG2a level in group F［（43.10±1.34） ng/mlï¼½ was significantly higher than those in group B［（12.61±1.87） ng/mlï¼½ and the other 3 treatment groups ï¼»group C, （23.37±2.67） ng/ml; D, （25.60±2.10） ng/ml; E, （25.91±1.33） ng/mlï¼½ （all P<0.01）. Conclusion: Immunotherapy with chimeric TAT-IhC-DPTCE can effectively ameliorate the allergic airway response and pulmonary inflammation in mice.

Characterization of the complete mitochondrial genome of the storage mite pest Tyrophagus longior (Gervais) (Acari: Acaridae) and comparative mitogenomic analysis of four acarid mites.

Mites of the genus Tyrophagus are economically important polyphagous pest commonly living on stored products and also responsible for allergic reactions to humans. Complete mitochondrial genomes (mitogenomes) and the gene features therein are widely used as molecular markers in the study of population genetics, phylogenetics as well as molecular evolution. However, scarcity on the sequence data has greatly impeded the studies in these areas pertaining to the Acari (mites and ticks). Information on the Tyrophagus mitogenomes is quite critical for phylogenetic evaluation and molecular evolution of the mitogenomes within Acariformes. Herein, we reported the complete mitogenome of the allergenic acarid storage mite Tyrophagus longior (Astigmata: Acaridae), an important member of stored food pests, and compared with those of other three acarid mites. The complete mitogenome of T. longior was a circular molecule of 13,271 bp. Unexpectedly, only 19 transfer RNA genes (tRNAs) were present, lacking trnF, trnS1 and trnQ. Furthermore, it also contained 13 protein-coding genes (PCGs) and 2 genes for rRNA (rrnS and rrnL) commonly detected in metazoans. The four mitogenomes displayed similar characteristics with respect to the gene content, nucleotide comparison, and codon usages. Yet, the gene order of T. longior was different from that in other Acari. The J-strands of the four mitogenomes possessed high A+T content (67.4-70.0%), and exhibited positive GC-skews and negative AT-skews. Most inferred tRNAs of T. longior were extremely truncated, lacking either a D- or T-arm, as found in other acarid mites. In T. longior mitogenome the A+T-rich region was just 50 bp in length and can be folded as a stable stem-loop structure, whereas in the region some structures of microsatellite-like (AT)n and palindromic sequences was not present. Besides, reconstructing of the phylogenetic relationship based on concatenated amino acid sequences of 13 PCGs supported that monophyly of the family Acaridae and the order Astigmata, to which the former belongs. Our results were consistent with the traditional classifications.

[Exploration on the Technology of Counter Staining of Fluke].

Fixed samples of Clonorchis sinensis and Fasciolopsis buski were stained with acetocarmine and malachite green, or stained with acetocarmine only. The samples displayed three different colors after staining with acetocarmine and malachite green. The digestive system, excretory system and the surrounding muscle tissue were stained reddish, the uterus was bright green, and the vitellarium at each side of the worm was tan. Staining with the two dyes resulted in clear structure and moderate degree of staining, and allowed three-dimensional observation, while staining with acetocarmine highlighted the testis tissue. Therefore, combination of the two staining methods is recommended in teaching and research to more effectively facilitate observation.

EPITOPE-BASED VACCINE FOR THE TREATMENT OF DER F 3 ALLERGY.

INTRODUCTION: mites allergic asthma is caused by exposure to home dust mite (HDM). Der f 3 is believed to be one of the major allergens in mites allergic asthma. The work was to identify the immune characteristics of Der f 3 epitope-based vaccine containing T cell and B cell epitopes. METHODS: T cell lines were generated from peripheral blood mononuclear cells of Der f 3 allergic patients. Three T cell epitopes and five B cell epitopes of Der f 3, which we identified previously, were selected to design a polypeptide (named Der f3-peptides). DNA constructions encoding these Der f 3-peptides were expressed in Escherichia coli. The T cell lines were stimulated with the peptides and tested for proliferative capacity and cytokine production. RESULTS: plasmid pET28a (+)-Der f 3-peptides was constructed and expressed in E. coli BL21, and the Der f3-peptides protein was purified and confirmed by Western blotting. The Der f 3-peptides were recognized by the T cell clones from allergic patients. SI value of Der f 3 group and Der f 3-peptides group were both higher than that of PBS group (P<0.05). The Der f 3 and Der f 3 peptides induced secretions of IL-4 and IL-5 were decreased compared with that of PBS group (P<0.05). The capacity of IgE-binding to Der f 3-peptides (41.25±5.67) µg/ml was decreased dramatically compared with that of Der f 3 (83.60 ± 10.92) µg/ml (P < 0.05). CONCLUSIONS: our results demonstrate that several major T cell epitopes and B cell epitopes of Der f 3 can be valuable for designing the peptide-based immunotherapeutics for the mites allergic asthma.

Introducción: el asma alérgica está causada por la exposición a los ácaros del polvo casero (HDM). Der f 3 se cree que es uno de los principales alérgenos en los ácaros del asma alérgica. El trabajo consistió en identificar las características inmunológicas de la vacuna basada en epítopo-Der f 3 que contienen las células T y las células B. Métodos: se generaron líneas de células T a partir de células mononucleares de sangre periférica de pacientes alérgicos a Der f 3. Tres epítopos de células T y cinco epítopos de células B de Der f 3, que hemos identificado previamente, fueron seleccionados para diseñar un polipéptido (denominados péptidos Der f 3). Construcciones de DNA que codifican estos péptidos Der f 3 se expresaron en Escherichia coli. Las líneas de células T se estimularon con los péptidos y se utilizaron en el ensayo por su capacidad proliferativa y la producción de citoquinas. Resultados: el plásmido pET28a (+) - Der f 3-péptidos se construyó y se expresaron en E. coli BL21, y la proteína de Der f 3-péptidos se purificó y se confirmaron mediante transferencia de Western. Los Der f 3-péptidos fueron reconocidos por los clones de células T procedentes de pacientes alérgicos. Valor SI de Der f 3 grupo y f grupo 3-péptidos Der eran tanto mayor que la del grupo de PBS (P.

PROKARYOTIC EXPRESSION AND BIOACTIVITY EVALUATION OF THE CHIMERIC GENE DERIVED FROM THE GROUP 1 ALLERGENS OF DUST MITES.

BACKGROUND: we successfully reconstituted the gene from group 1 allergens of dust mites, and obtained a body of shuffled genes. In order to verify the prediction on the chimeric gene, we tentatively cloned R8 into the vector that was prokaryoticly expressed, purified and assessed for its bio-activities. METHODS: the expressed product was detected by SDSPAGE and the target protein was purified. The purified protein R8 was detected by ELISA. 75 BALB/c mice were divided into 5 groups, namely PBS, rDer f1, rDer p1, R8 and asthma group. The mice were treated with dust mite allergens at 0, 7, 14 day by intraperitoneal injection and inhaled challenge as aerosol on day 21 for 7 days. Specific allergen immunotherapy was performed using rDer f1, rDer p1 and R8 allergens respectively. The level of IFN and IL- 4 in BALF was detected by ELISA. RESULTS: the chimeric gene R8 was expressed with a band of approximately Mr 35000. Compared with groups of rDer f 1 and rDer p 1 [(80.44±15.50) and (90.79±10.38) µg/ml respectively], IgE binding capacity of the protein R8 (37.03±12.46) µg/ml was statistically lower (P<0.001). The level of IFN in sera of R8 group [(343.43±38.79) pg/ml] was higher than that of the PBS and asthma groups [(393.93±50.68) and (208.44±46.11) pg/ml respectively] (P<0.01), but no statistical difference to that of the rDer f 1 and rDer p 1 groups (P>0.05). IL-4 level in R8 group was lower markedly than the others (P<0.05 or P<0.01). CONCLUSIONS: chimeric protein R8 derived from the group 1 allergens of dust mites has been expressed with low allergenicity and high immunogenicity.

Antecedentes: se reconstituyó con éxito el gen del grupo 1 alérgenos de los ácaros del polvo, y obtuvo un conjunto de genes barajadas. Con el fin de verificar la predicción en el gen quimérico, hemos clonado tentativamente R8 en el vector que se expresó prokaryoticly, purificó y se evaluó por sus actividades-bio. Métodos: el producto expresado se detectó por SDS-PAGE y la proteína diana se purificó. La proteína purificada R8 se detectó por ELISA. Setenta y cinco ratones BALB/ c se dividieron en 5 grupos, a saber: PBS, rDer f1, rDer p1, R8 y el grupo de asma. Los ratones fueron tratados con alérgenos de ácaros del polvo a los 0, 7, 14 días mediante inyección intraperitoneal y inhaladas desafío como aerosol en día 21 durante 7 días. La inmunoterapia específica para el alérgeno se realizó utilizando rDer f1, rDer p1 y alérgenos R8, respectivamente. El nivel de IFN e IL-4 en BALF se detectó por ELISA. Resultados: el gen quimérico R8 se expresó con una banda de aproximadamente Mr 35000. En comparación con los grupos de rDer f 1 y rDer p 1 [(80,44 ± 15,50) y (90,79 ± 10,38) µg/ml, respectivamente], la capacidad de unión a IgE de la proteína R8 (37,03 ± 12,46) µg/ml fue estadísticamente inferior (P.

[Cloning and Prokaryotic Expression of Fusion Gene of Group II Allergen Der p2 T Cell Epitope from Dermatophagoides pteronyssinus].

OBJECTIVE: To express and purify the T cell epitope fusion peptide of the major allergen Der p2 from Dermatophagoides pteronyssinus. METHODS: Nucleotide sequences reported to encode four T-cell epitopes (T1-T4) of Der p2 of D. pteronyssinus were linked in the rank of T1-T2-T3-T4. In this way, the chimeric gene was synthesized, named as Der p2 T. The gene of Der p2 T was amplified by PCR, purified, and cloned into the pET-28a (+) vector, forming the prokaryotic recombinant expression vector pET-28a (+)-Der p2 T. This formation was verified by double digestion. The pET-28a (+)-Der p2 T vector was transfected into E. coli strain BL-21, and its expression was induced by addition of IPTG. The recombinant protein was purified and collected by Ni-NTA affinity chromatography, and prepared for SDS-PAGE and Western blotting analysis. ELISA was used to evaluate the binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy. RESULTS: Double digestion results confirmed the construction of the pET-28a (+)-Der p2 T vector. SDS-PAGE revealed the expression of recombinant Der p2 T cell epitope fusion peptide with M, of 10,000. Western blotting confirmed the purification of Der p2 T cell epitope fusion peptide. The binding ability of Der p2 T cell epitope fusion peptide to serum IgE from patients with house dust mite allergy [(37.70±9.89) µg/ml] decreased significantly in comparison to that of Der p2 [(85.89±9.63) µg/ml] (P<0.01). CONCLUSION: The Der p2 T cell epitope fusion peptide is prepared, and its binding ability to serum IgE from patients with house dust mite allergy significantly decreases than that of Der p2.

CONSTRUCTION AND EXPRESSION OF DERMATOPHAGOIDES PTERONYSSINUS GROUP 1 MAJOR ALLERGEN T CELL FUSION EPITOPE PEPTIDE VACCINE VECTOR BASED ON THE MHC II PATHWAY.

UNLABELLED: Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was to construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. METHODS: the nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET- 28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. RESULTS: the recombinant plasmid pET-28a-TAT-IhCDer p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1. CONCLUSION: we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.

Antecedentes y objetivo: el Dermatophagoides peteronyssinus es uno de los principales ácaros del polvo doméstico responsables del asma alérgica que se pueden administrar provisionalmente para una inmunoterapia específica. El presente estudio busca construir un vector que codifique epítopos de células T del grupo de alérgenos principal, el Grupo 1 de Dermatophagoides pteronyssinus como una vacuna suministrada mediante la vía MHC de clase II. Métodos: se sintetizaron las secuencias de nucleótidos de los 3 genes objetivo, incluyendo TAT, IhC y el fragmento recombinante de Der p 1 encargado de codificar 3 epítopos de célula T. Después de la amplificación de los 3 fragmentos objetivo por PCR y digestión con endonucleasas de restricción correspondientes, el gen recombinante TAT-IhC-Der p 1-3T se ligó usando T4 DNA ligasa y se insertó en el vector de expresión procariota pET28a (+) para construir el plásmido recombinante pET 28a (+)-TAT-IHC-Der p 1-3T, que se confirmó por digestión con endonucleasas de restricción y secuenciación. El vector recombinante se transformó en E. coli cepa BL21 (DE3) y se indujo con IPTG, y la proteína inducida TATIHC- Der p1-3T se detectó mediante SDS-PAGE. Después de la purificación, la proteina recombinante se confirmó por análisis de inmunotransferencia (Western blot) y se probó su alergenicidad usando el ensayo de unión a IgE. Resultados: el plásmido recombinante pET-28a-TATIHCDer p1-3T se construyó con éxito, se confirmó por digestión con endonucleasas de restricción y la secuenciación y la expresión de la proteína recombinante TAT-IHCDer p1-3T fue inducida en E. coli. Purificación con éxito verificada mediante Western blot de la proteína objetivo, que mostró una capacidad de unión a IgE más fuerte que Der p1. Conclusión: hemos construido con éxito el vector de expresión recombinante pET-28a-TAT-IHC-Der p1-3T que expresa una vacuna de epítopo de células T administrada por vía MHC II con fuerte capacidad de union a IgE. Este trabajo proporciona una base para seguir estudiando la inmunoterapia específica mediante la vía MHC de clase II.

[Biological toxicity of heavy metals to Caenorhabditis elegans].

OBJECTIVE: To evaluate the biological toxicity of heavy metals by using Caenorhabditis elegans. METHODS: The C. elegans at L4 stage were exposed to CdCl, CrCl3, As2O3, PbCh2, HgCl2 with low concentrations and M9 buffer (the control group) for 72 h, respectively, and the effects of heavy metals with different concentrations on the survival time and reproduction of C. elegans were evaluated. RESULTS: After exposure to 2.5, 10 µmol/L HgCl2 and PbCl2, 10 µmol/L CdCl2, and 50 µmol/L CrCl3 for 72 h, respectively, the life spans and survival curves of the C. elegans were different from those in the control group, the differences were statistically significant (all P < 0.05). After exposure to CdCl2, CrCl3, As2O3, PbCl2 and HgCl2 with the con- centrations of 2.5, 50, 100 µmol/L for 72 h, respectively, the generational time and brood size of C. elegans were all different from those in the control group (all P < 0.01). Among the 5 heavy metals at low concentrations, the reproduction toxicity of Hg was bigger than Pb, Cd, Cr, and the toxicity of As was the weakest. CONCLUSION: Heavy metal exposure can affect the life span and reproductive toxicity of C. elegans.

[Metorchis orientalis founded in basin of Wuhu City, Anhui Province].

OBJECTIVE: To investigate the epidemic situation of Metorchis orientalis in Wuhu City, Anhui Province. METHODS: Three lakes (Fengming, Longwo and Kui lakes) were selected in Wuhu City, and the poultries around the lakes and freshwater fishes in the lakes were captured to test the infection of M. orientalis, and the infection rates were calculated. RESULTS: The adult worms of M. orientalis were found in gall bladder and hepatic duct of domestic ducks with infection rate of 16.7%. The metacercariae of M. orientalis were discovered in the muscle of Pseudorasbora parva and Pseudogobio rivularis with infection rates of 7.13% and 3.38% respectively. CONCLUSION: M. orientalis is endemic in Wuhu City, which should be paid enough attention to the animal husbandry, aquaculture and medicine.

STUDY OF ACAROID MITES POLLUTION IN STORED FRUIT-DERIVED CHINESE MEDICINAL MATERIALS.

OBJECTIVE: to investigate the species and breeding density of acaroid mites in stored fruit-derived Chinese medicinal materials in Anhui province. METHODS: samples of stored fruit-derived Chinese medicinal materials were collected from 30 herb stores and storehouses in 17 Anhui cities, where the breeding acaroids mites were detected. RESULTS: 20 species of acaroids mites were found in 33 samples, belonging to 15 genus, 5 families of the acaridae respectively, among which T. putrescentiae, A. farinae, C. lactis, and C. berlesei are predominant species. CONCLUSION: stored fruit-derived Chinese medicinal materials in Anhui areas suffer from serious acaroid mites pollution. Therefore, proactive measures should be taken to control acaroid mites from breeding in an effort to reduce the harm on medicinal materials.

Objetivo: investigar las especies y la densidad de reproducción de ácaros en productos medicinales chinos almacenados derivados de la fruta en la provincia de Anhui. Métodos: muestras de productos medicinales chinos almacenados derivados de la fruta fueron recogidos a partir de 30 herbolarios y almacenes en 17 ciudades de Anhui, donde se detectó la reproducción de ácaros. Resultados: se detectaron 20 especies de ácaros en 33 muestras, pertenecientes a 15 géneros, 5 familias de ácaros respectivamente, entre los cuales T. putrescentiae, A. farinae, C. lactis y C. berlesei son las especies predominantes. Conclusión: los productos medicinales chinos almacenados derivados de la fruta en la zona de Anhui sufren una grave contaminación por ácaros. Por lo tanto, se deben tomar medidas dinámicas para controlar la reproducción de ácaros en un esfuerzo por reducir los daños en los productos medicinales.

ORIBATID MITE INFESTATION IN THE STORED CHINESE HERBAL MEDICINES.

OBJECTIVE: to investigate the breeding status of oribatid mites and its related biological traits in the stored traditional Chinese herbal medicines. METHODS: sixty-three sorts of stored traditional Chinese herbal medicines of root and stem origins were collected from Huainan City in Anhui Province. The mites were isolated by Shakesieve and Tullgren, and identified and counted under the light microscope. RESULTS: thirteen samples in 63 were infested with the oribatid mites, which accounted for 21.67 %(13/60). A total of 11 species of oribatid mites, belonging to 6 families, were found in the 13 samples, and the most abundant species were Scheloribates laevigatus (36.19%), Scheloribates latipes (28.35%) and Trhypochthpnius japonicus (19.72%). The average breeding density of oribatid mites was about 4.51 individuals/g; the index of species richness, the diversity index, and the evenness index of species were 1.14, 1.581, and 0.212, respectively. CONCLUSION: oribatid mites were found in stored Chinese herbal medicines from Huainan city in Anhui province of China, suggesting that the conventional storage and processing technique should be improved in order to ensure the quality and safety of the herbal medicines.

Objetivo: investigar el estado reproductivo de los ácaros oribátidos y sus rasgos biológicos en el almacenamiento de medicinas a base de hierbas chinas tradicionales. Métodos: sesenta y tres tipos de medicinas a base de hierbas chinas tradicionales extraídas de raíces y tallos almacenadas fueron recopilados en Huainan City, en la provincia de Anhui. Los ácaros fueron aislados por Shakesieve y Tullgren, y su identificación y recuento se realizó mediante microscopio de luz. Resultados: trece de cada 63 muestras estaban infestadas por los ácaros oribátidos, lo que supone un 21,67% (13/60). Un total de 11 especies de ácaros oribátidos, pertenecientes a 6 familias, fueron encontrados en las 13 muestras, y las especies más abundantes fueron: Scheloribates laevigatus (36,19%), Scheloribates latipes (28,35%) y Trhypochthpnius japonicus (19,72%). El promedio de densidad reproductiva de los ácaros oribátidos fue de alrededor de 4,51 individuos/g; el índice de riqueza de especies, el índice de diversidad y la uniformidad del índice de especies fue 1,14, 1,581 y 0,212, respectivamente. Conclusión: se detectó la presencia de ácaros oribátidos en las hierbas medicinales chinas almacenadas de Huainan City, en la provincia de Anhui, China, lo que sugiere que la técnica convencional de almacenamiento y procesamiento debe mejorarse a fin de garantizar la calidad y la seguridad de los medicamentos a base de hierbas.