HOUSE DUST MITE ALLERGEN (Derp1 AND Blot5) LEVELS IN ASTHMATICS' HOME IN HONGKONG

To measure Derp1 and Blot5 allergen levels in asthmatics’ homes in Hongkong. Methods Seventy houses were enrolled for a mite indoor environment study. Dust samples were obtained from two sites of each patients’ house: bed and floor. Derp1 and Blot5 levels were quantified by a two-site monoclonal antibody-based ELISA technique. Results The levels of Derp1 allergens found in bed (geometric mean (GM) 3.43 μg/g of dust; 95%CI, 1.89-4.96 μg/g) and on the floor (GM 1.12 μg/g of dust; 95%CI, 0.71-1.53 μg/g) indicated significant differences (P = 0.005). However, the levels of Blot5 allergens found in bed (GM 19.00 μg/g of dust; 95%CI, 0.89-38.90 μg/g) and on the floor (GM 6.14 μg/g of dust; 95%CI, 0.40-11.90 μg/g) showed no statistically significant difference. In addition, in regards to the exposure index for Derp1 and Blot5 allergens found in bed and on the floor, 17.6% in bed and 8.6% on the floor had levels of Blot5 ≥ 10 μg/g of dust, higher than those obtained for Derp1 (7.2% and 0% in bed and on the floor respectively, P < 0.05); higher percentages in bed and on the floor (25.0% and 35.7%) were observed for levels of Blot5 = 0 μg/g of dust as compared with Derp1 in bed and on the floor (4.3% and 14.5% respectively, P < 0.05). Conclusions Derp1 and Blot5 are the major allergens found in this regional study, Blot5 is a more potent allergen in Hongkong, probably reflecting the high level of exposure to Blomia tropicalis (Bt). Bt and Dermatophagoides pteronyssinus(Dp) allergens should be included for precise diagnosis and effective immuno-therapeutic treatment of mite allergy in Hong-kong.

DNA vaccine encoding Der p 2 allergen generates immunologic protection in recombinant Der p 2 allergen-induced allergic airway inflammation mice model

Background DNA immunization is a promising novel type of immunotherapy against allergy. An estimated 79.2% patients with asthma, wheezing and/or rhinitis suffer from Dermatophagoides pteronyssinus group 2 (Der p 2) allegen. The aim of the present study was to determine whether DNA vaccine encoding Der p 2 could generate immunologic protection in recombinant Der p 2 (rDer p 2) allergen-induced allergic airway inflammation mice model and to understand the role of DNA vaccination in specific-allergen immunotherapy for asthma. Methods After DNA vaccination, BALB/c mice were sensitized by intraperitoneal injection (i.p) and challenged by intranasal instillation of rDer p 2. The lung tissues were assessed using hematoxylin and eosin. Mucus-producing goblet cells were identifed using periodic acid-Schiff(PAS)/alcian blue. The total cell number and composition of bronchoalveolar lavage samples were determined. The levels of the cytokines IL-4 and IFN-γ, as well as IgE and IgG_(2a)in the serum were determined by enzyme-linked immunosorbent assay. Allergen-specific IL-4 and IFN-γ production by spleen cells were also measured by enzyme-linked immunosorbent assay. Expression of signal transducer and activator of transcription 6 (STAT6) in splenocytes were determined by Western blot. Results DNA vaccine encoding Der p 2 allergen inhibited extensive infiltration of inflammatory cells and production of mucin induced by allergen. The influx of eosinophils into the lung interstitium was significantly reduced after administration of DNA vaccine. Significant reductions of IL-4 and increase in levels of IFN-γ in bronchoalveolar lavage fluid were observed. The allergen-specific IgE was markedly decreased in mice receiving DNA vaccination. Allergen could induce higher IFN-γ, weaker IL-4 in cultured spleen cells from mice receiving DNA vaccine. DNA vaccination inhibited STAT6 expression of spleen cells induced by allergen. Conclusion These results indicated that DNA vaccine encoding Der p 2 allergen generates immunologic protection in recombinant Der p 2 allergen-induced allergic airway inflammation mice model with regulating the immune response towards a Th1-type reaction.

The protein X4 of severe acute respiratory syndrome-associated coronavirus is expressed on both virus-infected cells and lung tissue of severe acute respiratory syndrome patients and inhibits growth of Balb/c 3T3 cell line

Background The genome of the severe acute respiratory syndrome-associated coronavirus ( SARS-CoV) includes sequences encoding the putative protein X4 ( ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection. Methods The prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles. Results We expressed and purified soluble recombinant protein X4 from E. coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the tung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 313 cells as determined by cell counting and MTT assays. Conclusion The results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.

Activation of Signal Transducer and Activator of Transcription 5 (STAT5) in Splenocyte Proliferation of Asthma Mice Induced by Ovalbumin

To investigate the role of signal transducer and transcriptional activator 5(STAT5)activated in ovalbumin (OVA)-induced splenocyte proliferation of asthma mice,an asthma mouse model was set up by intraperitoneal injection and aspiration of OVA with nebulizer.The proliferation of splenocytes isolated from the asthma mice was detected by [~3H]thymidine incorporation.The phosphorytation of STAT5 was examined by Western blotting and STAT5-DNA binding was measured by electrophoretic mobility shift assay(EMSA).OVA could pronouncedly induce the splenocyte proliferation of asthma mice in a dose-dependent manner compared with control groups.Phosphorylation of STAT5 and STAT5-DNA binding were observed in splenocytes from asthma mice induced by OVA at 1 h and 3 h.These results indicated that STAT5 signal pathway played an important role in lymphocyte proliferation of asthma mice induced by OVA.Cellular & Molecular Immunology. 2004;1(6):471-474.

Evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus

Background Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients’ sera.Methods Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. Results The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. Conclusion The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

Primary endotracheal non-Hodgkin’s lymphoma in a Chinese woman:a case report

Most patients with non-Hodgkin’s lymphoma (NHL) present with peripheral lymph node enlargement, with or without systemic symptoms. NHL may also involve mediastinal, intra-abdominal and pelvic lymph nodes with resulting symptoms. They may involve only an extranodal site, such as part of the gastrointestinal tract, lung, brain or testis. Extranodal presentation is more common in NHL than in Hodgkin’s disease. Primary endotracheobronchial involvement in non-Hodgkin’s lymphoma is a rare presentation. From 1989 to the present, only 3 cases of primary tracheal NHL were reported in Medline.

Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model

Background The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL pro , following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain. Methods The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein.Results The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed. Conclusions The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL pro of SARS-CoV urbani strain, 3CL pro of transmissible gastroenteritis virus and 3CL pro of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL pro .