Dust mite allergen Der f 4: Expression, characterization, and IgE binding in pediatric asthma.

BACKGROUND: House dust mite hypersensitivity affects millions of people worldwide, and although many allergens produced by house dust mite species have been identified, some of the less potent allergens remain to be studied. METHODS: The full-length cDNA encoding the group 4 allergen of the house dust mite species Dermatophagoides farinae (Der f 4) was generated through degenerate primer-based PCR, 5' RACE, and 3' RACE, and the cDNA fragment was cloned into an expression vector for nucleotide sequencing. Following codon optimization and removal of the signal peptide sequence, the mature gene fragment was subcloned into pET-28b (+) and transfected into E. coli BL21 cells for expression. The recombinant protein was purified by nickel affinity chromatography, identified by SDS-PAGE, Western blotting, and MALDI-TOF, and tested by ELISA for IgE reactivity with sera from individuals with asthma. Bioinformatics analyses were used to identify features of Der f 4. RESULTS: SDS-PAGE and Western blotting of the codon-optimized expression product showed a specific band. The mature recombinant Der f 4 was characterized as a stable and hydrophilic 57.9-kDa protein, and its secondary structure comprised alpha helix (25.3%), extended strand (22.51%), and random coils (52.19%). The structure of the recombinant protein was consistent with that of α-amylase. Among 27 pediatric asthma patients, 40.74% exhibited reactivity to rDer f 4 by ELISA. CONCLUSIONS: This initial cloning and characterization of the Der f 4 allergen serves as a foundation for future studies into the clinical importance and application of this protein for house dust mite allergy.

[Cloning, expression and analysis of Der f Mag 29 allergen of Dermatophagoides farinae].

The full-length Mag 29 gene of Dermatophagoides farinae was amplified by RT-PCR with a pair of specific primers. The PCR product was cloned into pCold TF DNA vector. The constructed plasmid pCold TF-Mag 29 was transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE. The full-length Mag 29 gene was 429 bp. A specific band (Mr 63,000) were detected in the whole cells, the supernatant, and the precipitate. Bioinformatics analysis revealed that Mag 29 protein was composed with 142 amino acid residues with a calculated molecular weight of Mr 15,100, and its secondary structure was composed of alpha helix (55.63%), extended strand (3.52%), and random coil (40.85%). The Mag 29 allergen was a hydrophilic and cytoplasmic protein, and shared a high degree homology with the heat shock protein 70 family.

IL-13 Regulates Orai1 Expression in Human Bronchial Smooth Muscle Cells and Airway Remodeling in Asthma Mice Model via LncRNA H19.

Background: Increased proliferation and hypertrophy of airway smooth muscle cells (ASMCs) contribute substantially to airway remodeling in asthma. Interleukin (IL)-13 regulates ASMC proliferation by increasing Orai1 expression, the pore-forming subunit of store-operated Ca2+ entry (SOCE). The underlying mechanisms of this effect are not fully understood. Methods: Bioinformatic analysis identified an interaction between microRNA 93-5p (miR-93-5p) and long non-coding RNA (lncRNA) H19, and between miR-93-5p and Orai1. RNA interference was used to investigate H19 knockdown on IL-13-induced proliferation and migration of in vitro cultured human bronchial smooth muscle cells (hBSMCs). Functional relevance of H19 in airway inflammation and airway remodeling was investigated in murine models of acute and chronic asthma. Results: IL-13 concentration-dependently increased the expression of H19 and Orai1 and decreased the expression of miR-93-5p in hBSMCs. H19 knockdown partly reversed the effects of IL-13 on the expression of miR-93-5p and Orai1 and attenuated the proliferation and migration of hBSMCs promoted by IL-13. IL-13-promoted expression of Orai1 was attenuated by miR-93-5p mimic and increased by miR-93-5p inhibitor. IL-13-promoted proliferation of hBSMCs was increased by miR-93-5p inhibitor but not affected by miR-93-5p mimic, whereas IL-13-promoted migration of hBSMCs was increased by miR-93-5p inhibitor and attenuated by miR-93-5p mimic. The inhibiting effect of H19 knockdown on IL-13-induced Orai1 expression and the proliferation and migration of hBSMCs was counteracted by miR-93-5p inhibitor but only marginally or not impacted by miR-93-5p mimic. The expression of H19 and Orai1 was higher in the lungs of asthmatic mice than in control mice. In asthmatic mice, H19 siRNA reduced Orai1 expression, inflammatory cell infiltration, goblet cell hyperplasia, collagen deposition and smooth muscle mass in the lungs. Conclusion: H19 may mediate the effects of IL-13 on Orai1 expression by inhibition of miR-93-5p in hBSMCs. H19 may be a therapeutic target for airway inflammation and airway remodeling.

Cloning, expression, and characterization of Der f 7, an allergen of Dermatophagoides farinae from China.

A full-length cDNA encoding house dust mite allergen Der f 7 from Dermatophagoides farina (Acari: Pyroglyphidae) from China was cloned, sequenced, and successfully expressed. A reference sequence (GenBank accession AY283292) was used to design polymerase chain reaction primers. Analysis revealed eight mismatched nucleotides in five Der f 7 cDNA clones, and the projected amino acid sequence contained six incompatible residues. These results suggest that the sequence of Der f 7 may be polymorphic. Further bioinformatic analysis revealed that the mature Der f 7 allergen had a molecular mass of approximately 21.88 kDa and a theoretical isoelectric point of 4.90. Der f 7 protein secondary structure was composed of a helix (56.63%), extended strand (5.10%), and random coil (38.27%). Group 7 allergens are present in Pyroglyphidae, Acaridae, and Glycyphagidae families, and homology analysis revealed a 86% similarity between Der f 7 and Der p 7. Furthermore, a phylogenetic tree constructed of group 7 allergens from different mite species revealed that Der f 7 and Der p 7 clustered with 100% bootstrap support. Bioinformatics-driven characterization of Der f 7 allergen as conducted in this study may contribute to diagnostic and therapeutic applications for dust mite allergies.

Clinical significance of neutrophil-lymphocyte ratio and monocyte-lymphocyte ratio in women with hyperglycemia.

Objective: Abnormal pro-inflammatory regulation of the immune system might contribute to the pathogenesis of hyperglycemia during pregnancy. We examined the correlations of neutrophil-lymphocyte ratio (NLR) and monocyte-lymphocyte ratio (MLR) with disease severity and assessed their predictive values. Methods: This retrospective case-control study included 311 cases of hyperglycemia first detected during pregnancy (HFDP) [153 with gestational diabetes mellitus (GDM) and 158 with diabetes in pregnancy (DIP)] and, as a control group, 172 pregnant women with normal glucose tolerance. The NLRs and MLRs were calculated from the blood test data. Results: The absolute leukocyte, neutrophil, monocyte, and lymphocyte counts as well as the NLR and MLR values of HFDP patients significantly differed from control values, but no significant differences were detected in the leukocyte, neutrophil, and monocyte counts of the GDM and DIP groups. Significantly different metrics were selected, binary analysis performed, and odds ratios calculated to identify risk factors. Age, BMI, NLR, and MLR were found to be risk factors for HFDP, and high systolic blood pressure (SBP) at triage and MLR related to the occurrence of DIP. Receiver operating characteristics curve analysis showed that NLR and MLR had better diagnostic accuracy in distinguishing HFDP from controls [NLR area under the curve (AUC) = 0.78; MLR AUC = 0.72] than age and BMI. Values for NLR > 4.394 or MLR > 0.309 correlated with the severity of maternal clinical symptoms and perinatal infant outcomes. MLR was the best predictor of DIP (AUC = 0.72) and MLR values > 0.299 could identify patients at risk for developing DIP and having poor fetal outcomes. Conclusion: Metrics derived from peripheral blood neutrophil, monocyte, and lymphocyte counts are thought to reflect systemic immune-inflammation. Elevated MLR and NLR may be unfavorable prognostic factors for clinical outcomes in patients with hyperglycemia during pregnancy.

Molecular Dynamics Simulations of Mite Aquaporin DerfAQP1 from the Dust Mite Dermatophagoides farinae (Acariformes: Pyroglyphidae).

Aquaporins are a large family of transmembrane channel proteins that facilitate the passive but highly selective transport of water and other small solutes across biological membranes. House dust mite (Dermatophagoides farinae) is the major source of household immunogens, and we have recently reported six cDNA sequence encoding aquaporins from this mite species. To better understand the structure and role of mite aquaporin, we constructed a tertiary structure for DerfAQP1 by homology modeling from the X-ray structure of malaria aquaporin PfAQP (Protein Data Bank code No. 3C02) and conducted molecular dynamics simulation. The simulation arranged seven water molecules in a single file through the pores of the DerfAQP1. Further, two conserved Asn-Pro-Ala motifs were located on Asn203 and Asn77; residues Arg206, Trp57, Met190, Gly200, and Asp207 constituted an extracellular vestibule of the pore; and residues His75, Val80, Ile65, and Ile182 constituted the cytoplasmic portions. The overall free energy profile for water transport through DerfAQP1 revealed an energy barrier of ~2.5 kcal/mol. These results contribute to the understanding of mite physiology and pathology.

Assessment efficacy of neutrophil-lymphocyte ratio and monocyte-lymphocyte ratio in preeclampsia.

OBJECTIVE: Abnormal changes in immune-mediated inflammation contribute to the pathogenesis of preeclampsia (PE). We aim to investigate the value of systemic immune inflammation indices-neutrophil-lymphocyte ratio (NLR) and monocyte-lymphocyte ratio (MLR)-to identify and evaluate the prognosis of patients with PE. METHODS: This study reviewed clinical records of 367 PE patients (162 with mild PE and 205 with severe PE), in addition to a control group of 172 normal pregnancies. Blood cell counts were performed at the first diagnosis of PE, and NLR and MLR were calculated by absolute cell count. RESULTS: Absolute neutrophil, lymphocyte, and monocyte counts and NLR and MLR values in PE were significantly different from controls, although monocyte counts did not significantly differ between mild and severe PE. Receiver operating characteristics curve (ROC) analysis showed NLR and MLR had better diagnostic accuracy in distinguishing PE from controls [NLR area under the curve (AUC) = 0.70; MLR AUC = 0.78]. Further, NLR was the best predictor of disease severity (AUC = 0.71). Cutoff values of NLR > 4.198 or MLR > 0.325 for control and PE groups or a cutoff value of NLR > 4.182 for PE groups indicated that patients were more likely to encounter preterm delivery, have shorter admission-to-delivery interval, and develop maternal and neonatal complications. CONCLUSION: Secondary analyses of white blood cell differential count parameters effectively evaluate the systemic inflammatory/immune state. Compared with absolute cell counts, NLR and MLR offer more effective indicators of clinical assessment, disease severity evaluation, and prognosis evaluation of PE.

[Preliminary investigation on phylogenetic relationship among three common species of house dust mites].

Dermatophagoides farinae, D. pteronyssinus and Euroglyphus maynei were used for the investigation. The cDNA fragment coding for Der f1 and Der f2 were amplified by PCR, cloned and sequenced. By bioinformatics softwares, the amino acid sequences for Der f1 and Der f2 were deduced and compared with those for the groups 1 and 2 allergens of D. pteronyssinus and E. maynei available in GenBank. Amino acid sequence similarity analysis showed that Der p1 shared 84% identical residues with Eur m1 and 83% with Der f1. Similarly, Der p2 shared 87% identical residues with Eur m2 and 68% with Der f2. In the two phylogenetic trees constructed with group 1 and 2 allergens, D. pteronyssinus was clustered with E. maynei but not with D. farinae, although D. pteronyssinus and D. farinae belong to the same genus. D. pteronyssinus should be more similar to E. maynei than to D. farinae at evolutional level, which was not consistent with the conventional taxonomical relationship based on their morphological characteristics.

[Investigation on species composition of cockroaches and bacteria-carrying on their bodies in five cities of Hainan].

"ZA-type" cages were used to capture cockroaches in 267 sites of 5 cities in Hainan. Species were identified and bacteria were isolated by routine method. 441 cockroaches were collected and identified as five species belonging to two genera, 75.3% being Periplaneta americana. More cockroaches were found in sewerage. Bacteria were detected from 82.4% of cockroaches, including Escherichia coli, Pseudomonas aeruginosa, Salmonella sp, Staphylococcus aureus, Shigella sp, Bacillus proteus and sort of mycetes. Therefore, the dominant species is Periplaneta americana in Hainan, and the high bacteria-carrying behavior of cockroaches indicates its importance in disease transmission.

The Dermatophagoides farinae group 22 allergen: cloning and expression in Escherichia coli.

BACKGROUND: Dermatophagoides farinae (Hughes) (Acari: Pyroglyphidae) and other domestic mites produce allergens that affect people worldwide. Here, the complementary DNA (cDNA) coding for group 22 allergen of D. farinae (Der f 22) from China was cloned, sequenced, and expressed successfully. METHODS: The cDNA encoding Der f 22 was synthesized by reverse transcription polymerase chain reaction (RT-PCR), then ligated to the pCold-TF for expression in Escherichia coli BL21 cells. The purified recombinant fusion protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western-blotting, and tandem matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF). RESULTS: The full-length cDNA comprised 468 nucleotides and was 99.57% (466/468) identical with the reference sequence (GenBank: DQ643992). After the plasmid pCold-TF-Der f 22 was transformed into E. coli BL21 and expressed with the induction of IPTG, SDS-PAGE showed a specific band for the recombinant fusion protein. The recombinant fusion protein, which was purified by chromatography, bound with a His-tagged antibody by Western blotting. MALDI-TOF/TOF mass spectrometry revealed that the structure of the recombinant protein was identical to the predicted Der f 22 structure. The hydrophilic protein contains a signal peptide of 20 amino acids, and the mature Der f 22 consists of 135 amino acid residues with a molecular weight of 14.7 kDa and theoretical isoelectric points (pI) of 6.38. Its secondary structure comprises an alpha helix (38.5%), beta-sheet (45.9%), random coils (11.85%), and beta-turns (11.1%). CONCLUSION: This work represents the first reported full-length sequence and successful cloning of Der f 22 from D. farinae in China; bioinformatics analysis can be used to further study the allergenicity and clinical utility of the recombinant Der f 22.

Diagnosis of intestinal acariasis with avidin-biotin system enzyme-linked immunosorbent assay.

AIM: To explore the value of avidin-biotin system enzyme-linked immunosorbent assay (ABC-ELISA) in diagnosis of intestinal acariasis. METHODS: Mite-specific IgG levels in serum of 48 patients with intestinal acariasis were measured with ABC-ELISA. The sensitivity of this method was compared with that of staphylococcal protein A enzyme-linked immunosorbent assay (SPA-ELISA). RESULTS: The positive rate of mite-specific IgG detected with ABC-ELISA and SPA-ELISA was 89.58% (43/48) and 56.25% (27/48), respectively. The positive rate with ABC-ELISA was statistically higher than that with SPA-ELISA (chi2=13.50, P<0.01). CONCLUSION: ABC-ELISA is an effective method for the diagnosis of intestinal acariasis.

Epidemiological survey of Blastocystis hominis in Huainan City, Anhui Province, China.

AIM: To provide scientific evidence for prevention and controlling of blastocystosis, the infection of Blastocystis homonis and to study its clinical significance in Huainan City, Anhui Province, China. METHODS: Blastocystis homonis in fresh stools taken from 100 infants, 100 pupils, 100 middle school students and 403 patients with diarrhea was smeared and detected with method of iodine staining and hematoxylin staining. After preliminary direct microscopy, the shape and size of Blastocystis homonis were observed with high power lens. The cellular immune function of the patients with blastocystosis was detected with biotin-streptavidin (BSA). RESULTS: The positive rates of Blastocystis homonis in fresh stools taken from the infants, pupils, middle school students and the patients with diarrhea, were 1.0 % (1/100), 1.0 % (1/100), 0 % (0/100) and 5.96 % (24/403) respectively. Furthermore, the positive rates of Blastocystis homonis in the stool samples taken from the patients with mild diarrhea, intermediate diarrhea, severe diarrhea and obstinate diarrhea were 6.03 % (14/232), 2.25 % (2/89), 0 % (0/17) and 12.31 % (8/65) respectively. The positive rates of Blastocystis homonis in fresh stools of male and female patients with diarrhea were 7.52 % (17/226) and 3.95 % (7/177) respectively, and those of patients in urban and rural areas were 4.56 % (11/241) and 8.02 % (13/162) respectively. There was no significant difference between them (P>0.05). The positive rates of CD(3)(+), CD(4)(+), CD(8)(+) in serum of Blastocystis homonis-positive and-negative individuals were 0.64+/-0.06, 0.44+/-0.06, 0.28+/-0.04 and 0.60+/-0.05, 0.40+/-0.05 and 0.30+/-0.05 respectively, and the ratio of CD(4)(+)/CD(8)(+) of the two groups were 1.53+/-0.34 and 1.27+/-0.22. There was significant difference between the two groups (P<0.05, P<0.01). CONCLUSION: The prevalence of Blastocystis hominis as an enteric pathogen in human seems not to be associated with gender and living environment, and that Blastocystis hominis is more common in stool samples of the patients with diarrhea, especially with chronic diarrhea or obstinate diarrhea. When patients with diarrhea infected by Blastocystis hominis, their cellular immune function decreases, which make it more difficult to be cured.

Diarrhea and acaroid mites: a clinical study.

AIM: To explore the characteristics of diarrhea caused by acaroid mites. METHODS: Acaroid mites in fresh stools of 241 patients with diarrhea were separated by flotation in saturated saline. Meanwhile, skin prick test, total IgE and mite-specific IgE were detected in all patients. RESULTS: The total positive rate of mites in stool samples of the patients was 17.01 % (41/241), the positive rates of mites in male and female patients were 15.86 % (23/145) and 18.75 % (18/96), respectively, without significant difference (P>0.05). The percentage of skin prick test as "+++", "++", "+", "+/-" and "-" was 9.13 % (22/241), 7.47 % (18/241), 5.81 % (14/241), 4.98 % (12/241) and 72.61 % (175/241), respectively. The serum levels of total IgE, mite-specific IgE in patients with and without mites in stool samples were (165.72+/-78.55) IU/ml, (132.44+/-26.80) IU/ml and (145.22+/-82.47) IU/ml, (67.35+/-45.28) IU/ml, respectively, with significant difference (P<0.01). The positive rate of mites in stool samples in staffs working in traditional Chinese medicine storehouses or rice storehouses (experimental group) was 26.74 % (23/86), which was significantly higher than that (11.61 %, 18/155) in people engaged in other professions (chi(2)=8.97, P<0.01). CONCLUSION: Acaroid mites cause diarrhea and increase serum levels of total IgE and mite-specific IgE of patients, and the prevalence of diarrhea caused by acaroid mites is associated with occupations rather than the gender of patients.

Clinical and epidemiological features of patients with clonorchiasis.

AIM: To study the clinical and epidemiological features of patients with clonorchiasis so as to provide scientific evidences for the diagnosis and prevention of clonorchiasis. METHODS: Stools from 282 subjects suspected of having clonorchiasis were examined for helminth eggs with modified Kato's thick smear and sedimentation methods, and their sera were tested for HAV-DNA, HBV-DNA, HCV-RNA, HDV-RNA and HEV-RNA with polymerase chain reaction (PCR). Clinical symptoms of patients with clonorchiasis only were analyzed, and their blood samples were tested for circulating antigen (CAg) with Dot-ELISA, eosinophilic granulocyte count, and alanine aminotransferase (ALT). Meanwhile, they were asked to provide data of occupation, eating habit, hygienic habit and knowledge of clonorchiasis. In addition, the ecosystem of the environment in epidemic areas was surveyed. RESULTS: Among the 282 patients, 61 (21.43%) were infected with clonorchis sinensis only, 97 (34.64%) were co-infected with clonorchis sinensis and other pathogens, 92 (32.86%) were infected with hepatitis virus only and 31 (11.07%) neither with clonorchis sinensis nor hepatitis virus. Among the 61 patients with clonorchiasis only, there were 14 (22.95%) subjects with discomfort over hepatic region or epigasfrium, 12 (19.67%) with general malaise or discomfort and inertia in total body, 6 (9.84%) with anorexia, indigestion and nausea, 4 (6.56%) with fever, dizziness and headache (6.56%), and 25 (40.98%) without any symptoms; sixty one (100%) with CAg (+), 98.33% (59/60) with eosinophilic granulocytes increased and 65.00% (39/60) with ALT increased. B-mode ultrasonography revealed 61 cases with dilated and thickened walls of intrahepatic bile duct, and blurred patchy echo acoustic image in liver. Twenty-six cases had stones in the bile duct, 39 cases had slightly enlarged liver with diffuse coarse spots in liver parenchyma. Twenty cases had enlarged gallbladder with thickened coarse wall and image of floating plagues, 9 cases had slightly enlarged spleen. By analysis of epidemiological data, we found that the ecologic environment was favorable for the epidemiology of clonorchiasis. Most patients with clonorchiasis were lack of knowledge about the disease. Their living environment, hygienic habits, eating habits and their occupations were the related factors that caused the prevalence of the disease. CONCLUSION: The clinical symptoms of clonorchiasis are non-specific, and the main evidences for diagnosis of clonorchiasis should be provided by etiologic examination, B-mode ultrasonography and clinical history. The infection of clonorchis sinensis is related to occupations, bad eating habits and lack of knowledge about prevention of the disease.

Acaroid mite, intestinal and urinary acariasis.

AIM: To investigate epidemiology and pathogenic mite species of intestinal and urinary acariasis in individuals with different occupations. METHODS: A total of 1994 individuals were tested in this study. History collection, skin prick test and pathogen identification were conducted. The mites were isolated from stool and urine samples by saturated saline flotation methods and sieving following centrifugation, respectively. RESULTS: Among the 1994 individuals examined, responses to the skin prick test of "+++", "++", "+","+/-" and "-" were observed at frequencies of 3.96 % (79), 3.21 % (64), 2.31 % (46), 1.25 % (25) and 89.27 % (1780), respectively. A total number of 161 (8.07 %) individuals were shown to carry mites, with 92 (4.61 %) positive only for stool samples, 37 (1.86 %) positive only for urine samples and 32 (1.60 %) for both. The positive rate of mites in stool samples was 6.22 % (124/1994), being 6.84 % (78/1140) for males and 5.39 % (46/854) for females. No gender difference was observed in this study (chi(2)=1.77, P>0.05). The mites from stool samples included Acarus siro, TyroPhagus putrescentiae, Dermatophagoides farinae, D. pteronyssinus, Glycyphagus domesticus, G. ornatus, Carpoglyphus lactis and Tarsonemus granaries. The positive rate of mites in urine samples was 3.46 % (69/1994). The positive rates for male and female subjects were found to be 3.95 % (45/1140) and 2.81 % (24/854) respectively, with no gender difference observed (chi(2)=1.89, P>0.05). Mites species in urine samples included Acarus siro, Tyrophagus putrescentiae, T. longior, Aleuroglyphus ovatus, Caloglyphus berlesei, C. mycophagus, Suidasia nesbitti, Lardoglyphus konoi, Glycyphagus domesticus, Carpoglyphus lactis, Lepidoglyphus destructor, Dermatophagoides farinae, D. pteronyssinus, Euroglyphus magnei, Caloglyphus hughesi, Tarsonemus granarus and T. hominis. The species of mites in stool and urine samples were consistent with those separated from working environment. A significant difference was found among the frequencies of mite infection in individuals with different occupations (chi(2)=82.55, P<0.001), with its frequencies in those working in medicinal herb storehouses, those in rice storehouse or mills, miners, railway workers, pupils and teachers being 15.89 % (68/428), 12.96 % (53/409), 3.28 % (18/549), 2.54 % (6/236), 5.10 % (13/255) and 2.56 % (3/117), respectively. CONCLUSION: The prevalence of human intestinal and urinary acariasis was not associated with gender, and these diseases are more frequently found in individuals working in medicinal herb, rice storehouses or mills and other sites with high density of mites. More attention should be paid to the mite prevention and labor protection for these high-risk groups.

L-forms of H. pylori.

AIM: To study the occurrence of L-forms of H. pylori infection in patients with peptic ulcers and its association with possible changes of cellular immune function in the patients. METHODS: Endoscopic biopsy specimens of gastric antrum and gastric corpus were taken from 228 patients with peptic ulcers and inoculated into Skirrow selective medium for H. pylori vegetative forms and special medium for H. pylori L-forms, followed by bacterial isolation and identification. And peripheral venous blood of the patients was taken to detect the percentage of CD3(+), CD4(+) and CD8(+) with biotin-streptavidin (BSA) and the level of IL-2, IL-6 and IL-8 with ELISA. RESULTS: (1) The detection rates of H. pylori L-forms and vegetative forms in the patients were 50.88 % (116/228) and 64.91 % (148/228) respectively, and the co-infection rate of H. pylori L-forms and vegetative forms was 78.38 % (116/148). To be more exact, the detection rates of H. pylori L-forms in male and female patients were 57.04 % (77/135) and 41.94 % (39/93) respectively, and statistics found significant difference between them (P<0.05). Furthermore, the detection rates of H. pylori L-forms in patients aged 14 years-, 30 years-, 40 years- and 50 years- were 31.91 % (15/47), 42.86 % (24/56), 56.94 % (41/72) and 67.92 % (36/53) respectively, and there was significant difference between them (P<0.01). (2) The percentages of CD3(+), CD4(+), CD8(+), the ratio of CD4(+)/CD8(+), and the level of IL-2, IL-6, IL-8 in H. pylori-positive patients were (52.59+/-5.44) %, (35.51+/-5.74) %, (27.77+/-8.64) %, (1.56+/-0.51), (2.66+/-0.47) mg/L, (108.62+/-5.85) ng/L and (115.79+/-7.18) ng/L respectively, compared with those in H. pylori-negative patients, the percentages of CD3(+), CD4(+) and the ratio of CD4(+)/CD8(+) decreased, but the level of IL-2, IL-6 increased, and the difference was significant (P<0.001-P<0.01). Moreover, the percentages of CD3(+), CD4(+), CD8(+), the ratio of CD4(+)/CD8(+), and the level of IL-2, IL-6, IL-8 in the patients with mixed infection of both H. pylori L-forms and vegetative forms were (51.69+/-5.28) %, (34.75+/-5.89) %, (27.15+/-7.45) %, (1.48+/-0.47), (2.16+/-0.38) mg/L, (119.45+/-5.44) ng/L and (123.64+/-6.24) ng/L respectively, compared with those in patients with simple infection of H. pylori vegetative forms, the percentage of CD4(+), the ratio of CD4(+)/CD8(+) and the level of IL-2 increased, but the level of IL-6 and IL-8 decreased, statistical difference was found between them (P<0.001-P<0.05). CONCLUSION: L-forms variation often occurs in patients with peptic ulcers who are infected by H. pylori, which is commonly found in male patients and related to ages. The L-forms variation of H. pylori can be an important factor causing disorder of cellular immune function in the patients with peptic ulcers who are infected by H. pylori.

Detection of serum anti-Helicobacter pylori immunoglobulin G in patients with different digestive malignant tumors.

AIM: To investigate the seroprevalence of Helicobacter pylori infection in patients with different digestive malignant tumors. METHODS: Enzyme linked immunosorbent assay (ELISA) was used to detect serum anti-Helicobacter pylori IgG antibody in 374 patients with different digestive malignant tumors and 310 healthy subjects (normal control group). RESULTS: The seroprevalence of Helicobacter pylori infection was 61.50% (230/374) and 46.77% (145/310), respectively, in patients with digestive tumors and normal controls (P<0.05). The seroprevalence was 52.38% (33/63), 86.60% (84/97), 83.14% (84/101), 45.24 (19/42), 51.13% (18/35) and 44.44% (16/36), respectively in patients with carcinomas of esophagus, stomach, duodenum, rectum, colon and liver (P<0.01). In patients with intestinal and diffuse type gastric cancers, the seroprevalence was 93.75% (60/64) and 72.73% (24/33), respectively (P<0.05). In patients with gastric antral and cardiac cancers, the seroprevalence was 96.43% (54/56) and 73.17% (30/41), respectively (P<0.05). In patients with ulcerous and proliferous type duodenal cancers, the seroprevalence of H pylori infection was 91.04% (61/67) and 52.27% (23/44), respectively (P<0.05). In patients with duodenal bulb and descending cancers, the seroprevalence was 94.20% (65/69) and 45.20% (19/42), respectively (P<0.05). CONCLUSION: H pylori infection is associated with occurrence and development of gastric and duodenal carcinomas. Furthermore, it is also associated with histological type and locations of gastric and duodenal carcinomas.

Chronic liver fluke disease with dyspepsia: a case report.

BACKGROUND: Chronic liver fluke disease with dyspepsia is rarely seen clinically. In this study, we assessed the etiological factors, symptoms, physical signs and diadynamic methods in a case of chronic liver fluke disease with dyspepsia. METHODS: Physical examination, laboratory studies, ultrasonography and CT scan were performed before pathogen examination. The eggs of fluke found with the inverted sedimentation method were also observed under a microscopy. They were diagnosed as the eggs of Clonorchis sinensis. RESULTS: The patient was diagnosed as having chronic liver fluke disease, and his appetite recovered after three courses of treatment with praziquantel. CONCLUSION: Eating fresh fish and shrimp might cause liver fluke disease. The symptoms of this disease with dyspepsia can be anorexia, abdominal distention, bellyache, and loose stools.

Expression, cloning, and IgE-binding of the full-length dust mite allergen Der f 8.

Dermatophagoides farinae, a domestic mite species, produces some of the most potent allergens that contribute to allergy in China and worldwide. We sought to clone and express the group 8 allergen of D. farinae (Der f 8) to investigate its IgE-binding reactivity. The full-length cDNA encoding Der f 8 was generated by using RT-PCR and 5' RACE, cloned into pCold-TF expression vector, confirmed by nucleotide sequencing, sub-cloned into pET-28b (+), and transfected into E. coli BL21 cells for expression. After purification by nickel affinity chromatography and identified by SDS-PAGE, the recombinant Der f 8 bound with sera from 40.9 % (9/22) of mite-allergic patients according to ELISA testing. Analysis of the recombinant DNA sequence revealed a 231 amino acid open reading frame encoding a protein with a derived molecular mass of 26.4 kDa and an isoelectric point of 6.84. The deduced amino acid sequence has nine phosphorylation sites, displaying strong homology with glutathione S-transferase, and its secondary structure comprises alpha helix (45.5 %), extended strand (11.3 %), and random coils (43.3 %). BLAST through the National Center for Biotechnology Information database and alignment identified similarity with group 8 allergens or glutathione S-transferases of Dermatophagoides pteronyssinus, Suidasia medanensis, Lepidoglyphus destructor, Glycyphagus domesticus, and Aleuroglyphus ovatus (64, 65, 53, 53, and 50 %, respectively). The first recombinant Der f 8 protein produced in full length successfully bound with patient IgE, demonstrating the importance of Der f 8 in mite allergy.

Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification.

A cDNA fragment encoding the S-layer protein SllB cloned from Bacillus sphaericus ATCC 14577 was expressed on the surface of E. coli BL21 (DE3) cells and confirmed by the square lattice structure at the nanoscale level. The amplified gene fragment designed with PCR primers from a specified reference sequence (GenBank accession no. AJ849550) showed a high degree of sequence identity with the known sequences for S-layer protein. The best alignment scores were seen in B. sphaericus strains JG-A12 and NCTC9602, which code for a pre-form protein with a predicted cleavage site located between the two alanine residues 31 and 32. After this signal peptide sequence was removed, the mature protein had a molecular mass of 116.2613 kDa and a theoretical pI of 5.40. Further bioinformatic analysis revealed three S-layer homology (SLH) domains in the N-terminus of the mature protein, positioned at the 1-61, 63-128 and 137-197 residues. The mature S-layer protein was composed of alpha helices (24.86%), extended strands (27.01%), and rich random coils (48.13%). Bioinformatics-driven characterization of SllB may provide scientific evidence for further application of this gene in the fields of nanobiotechnology and biomimetics in the future.