Objective: We have studied the role of lymphoproliferative tumors in the pathogenesis of autoimmune and the origin of the autoantibodies in paraneoplastic pemphigus (PNP) in recent years. A Castleman’s tumor from a patient was identified to produce autoantibody. To identify the relationship between the tumor and pathogenesis of the disease, we analyzed the rearrangement of immunoglobulin variable region gene and its hypermutation in B cells of Castleman’s tumor from a patient who was diagnosed of paraneoplastic pemphigus. Methods: The surface-markers of cultured tumor lymphocytes were assessed with immunochemistry staining. After total RNA of the tumor cells were isolated, the mRNA was reversely transcribed into cDNA. V H and V L genes were cloned and their sequences were analyzed. Results: Immunochemistry staining and flow cytometer analysis showed that the tumor cells were CD20, HLA-DR, smIgM, and smIgG positive. The cloned IgV H and IGHV3-9*01 germ-line gene are homologous and so are the Ig V L and the IGKV4-1*01 germ-line gene. More nucleotide changes in the V H or V L occurred in CDRs than those in FRs. Conclusion: In this reported case, a clone of specific B-lymphocyte in the Castleman’s tumor carrying functional rearranged immunoglobulin heavy and light chain genes was found to have experienced switch recombination and was possible to produce IgG autoantibody.

Objective:To investigate if the combined use of CDAⅡ and sodium butyrate can induce demethylation and re-expression of retinoic acid receptor?2(RAR?2)gene in cultured human breast cancer cells MCF7.To explore if the two drugs can inhibit cell growth and induce cell apoptosis synergetically.Methods:MCF7 cell line was treated with CDAⅡ,sodium butyrate,combination of the two drugs respectively.Methylation was assessed by methylation-specific polymerase chain reaction(MSP)for RAR?2 gene.Gene expression was evaluated by reverse transcription polymerase chain reaction(RT-PCR).Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling(TUNEL)and Hoechst33342/propidiumiodide(PI)staining.Cell growth inhibition was measured by MTT assay.Results:Neither CDAⅡ nor sodium butyrate induced demethylation and re-expression of RAR?2 gene,Combination of the two drugs partially demethylated gene promoter accompanied by re-expression of RAR?2.The apoptotic cells in the double-drug group were obvious following Hoechst33342/PI staining.The percentage of apoptotic cells in the double-drug group was significantly higher than that of the two single-drug group(39.5% vs 5.2%,8.1%)(P

Objective To analyze the genetic variation of low density lipoprotein receptor(LDLR)gene in patients with familial hypercholesterolemia(FH).Methods Polymerase chain reaction(PCR)was used to amplify the promoter region and the 18 exons of LDLR gene family with the patients' genomic DNA as templates.DNA sequencing of the PCR production was used to find mutations in patients with FH.Results A novel missense mutation(G→T)in exon 4 was identified by DNA sequencing in the proband and his mother,and the mutation was characterized at nucleotide site of 385 in exons of coding region,with tyrosine substitute for aspartate at amino acid site of 108.Conclusion Characterization of the novel mutation provides an example of the genetic basis of LDLR causing FH.

Objective:To explore the expression levels of MHCⅡ molecules and its regulator genes CⅡTA on bleomycin-induced pulmonary fibrosis in rats,and to investigate the underlying immunologic mechanisms of pulmonary fibrosis.Methods:The rats were treated with either a single intratracheal bleomycin injection(fibrosis group)or a normal saline injection(control group).The pathologic changes of lung tissues stained with HE and Masson were observed,and the contents of hydroxyproline were detected on the 7th and 28th day respectively after bleomycin administration.The expression of MHCⅡ molecules in the lung tissues was evaluated with immunohistochemistry techniques,and the percentage of MHCⅡ+ cells was measured.The amounts of total CⅡTA and typeⅠ,Ⅲ and Ⅳ CⅡTA mRNA of lung tissues were measured by real-time PCR using Taqman probe.Results:(1)The percentage of MHCⅡ+ cells in lung tissues increased significantly in fibrosis group compared with that of control group on the 7th day and the 28th day[(0.10?0.03)vs(0.06?0.02),P0.05];In fibrosis group,type Ⅳ CⅡTA mRNA was 667.3% [(2.98?0.92)vs(0.39?0.15),P

Objective: To screen and identify gene mutations of 11 Chinese patients with Hailey Hailey disease (HHD). Methods: Cases of HHD were diagnosed by history, clinical menifestations and pathology. Then genomic DNA samples of patients were extracted from perpheral blood leukocytes, and polymerase chain reaction(PCR), DNA sequencing were performed. Results: We found five mutations in ATP2C1 gene including 3 nonsense mutations and 2 splicing mutations. Four of them were novel mutations. Conclusion: Both nonsense mutation and splicing mutation could affect the rusult of transcription,translation, and the functions of protein encoded by ATP2C1 gene, so the mutations reported in this study is the underlying cause of HHD.

Objective To detect the proteins structure encoded by COL4A4 gene with different missense mutations of thin basement membrane nephropathy (TBMN) and to analyze the effect of gene mutation on the secondary structure of α4 (Ⅳ) chain and its association with phenotype. Methods A COL4A4-linked TBMN patient with FSGS by a missense mutation (g. 1214G>A resulting in p. G405E) diagnosed by clinical manifestations, family history and renal biopsy examination, as well as two controls (one healthy, one pure TBMN carrying a g. 1550G>A mutation resulting in p. G448S) were enrolled in this study. The fragments of cDNA with the two mutations and that of corresponding cDNA from the healthy control were expressed in E. coll. The secondary structures of recombinant polypeptides were analyzed by circular dichroism (CD) spectroscopy. Results CD spectra of healthy control exhibited a negative peak near 208 nm whereas that of TBMN patient with FSGS exhibited a negative peak near 220 nm. Furthermore, the magnitude of the negative peak of this patient decreased as compared with that of healthy control. CD spectra of pure TBMN control was slightly changed with the negative peak remaining near 208 run and the magnitude slightly decreased as compared with that of healthy control. In addition, the secondary structure of pelypeptide from healthy control was composed of about 1/4 α-helix and 1/4 β-sheet, whereas that from the patient presented about 1/3 α-helix without any β-sheet. The secondary structure of polypeptide from pure TBMN control was almost the same as the healthy control, except a shght reduction of α-helix and a slight increase of β-sheet. Conclusions Although the glycine substitutions exists in the nearby domain of α4 (Ⅳ)chain, the TBMN patient complicating FSGS with severe phenotype and g. 1214G>A mutation and the pure TBMN control with the mild phenotype and g. 1550G>A mutation are revealed with different secondary structures of α4 (Ⅳ)chain. Moreover, the secondary structure change of α4 (Ⅳ) chain is consistent with their corresponding phenotype severity.

Objective To explore possible impact of endogenous hydrogen sulfide(H2S) on connective tissue growth factor(CTGF) expression in rat pulmonary artery with high pulmonary blood flow.Methods Thirty-two male SD rats,weighing 120～140 g,were randomly divided into 4 groups:shunt group,shunt+PPG group,sham group and sham+PPG group.After 4 weeks of experiment,Rat lung tissue H2S content was determined by a modified sulfide electrode method.Plasma ET-1 concentration was detected by radioimmunoactivity,and lung tissue ET-1 mRNA expression of rat was determined by quantitative competitive reverse transcriptional polymerase chain reaction(RT-PCR).Pulmonary artery connective tissue growth factor(CTGF) protein expression of rats was investigated by immunohistochemistry.Results After 4 weeks of experiment,lung tissue H2S content plasma ET-1,lung tissue ET-1 mRNA and CTGF expression increased significantly in rats of shunt group as compared with that of sham group(P

Objective To determine XPAC gene mutation in a Chinese pedigree with xeroderma pigmentosum group A.Methods All exons of XPAC gene were analyzed by PCR-DNA sequencing in the pedigree.The mutations were confirmed by restriction fragment length polymorphism(RFLP).Results By PCR-DNA sequencing,a nonsense mutation of C631T was identified which caused R211X substitution in exon5of XPAC gene.The mutated gene encoded a defective XPA protein truncated by63amino acids in C-terminus.The parents were all heterozygotes.The results were confirmed by RFLP.Conclusions A nonsense mutation is found in XPAC gene of a pedigree of xeroderma pigmentosum group A.This mutation may impair XPA protein function of DNA repair,and as a consequence,cause skin aging and carcinogenesis.

Objective To investigate the role of Castleman′s disease in the pathogenesis of paraneoplastic pemphigus (PNP). Methods In six PNP patients associated with Castleman′s disease, routine immunohistochemistry was performed on tumor tissue. Reverse transcription - PCR, DNA sequencing of cloned PCR product and in situ hybridization (ISH) were used to estimate the clonality of the B-cells in the tumors. The expression of the specific tumor B-cell clones was evaluated by Northern blot. Six patients with Castleman′s disease without mucocutaneous lesion and 3 patients with reactive lymphadenopathy were used as the controls. Results Immunohistochemistry showed that CD20-positive B-cells in high density located in lymphoid follicles. The PCR produced one discrete band of about 128 bp in every paraneoplastic pemphigus patients. After sequencing the cloned PCR product, only two kinds of highly homologous sequences were found in all of the PNP patients. The 128 bp sequences were the major clones seen in all patients, and the 122 bp sequences were the relatively minor one seen in 4 patients. Anti-sense RNA probe transcribed from a clone of 128 bp was used in ISH. Signals of ISH located in cytoplasm of the cells in follicles of the tumors. Furthermore, this probe was also used for Northern blot and showed a strong signal in PNP patients. Conclusions Castleman′s disease associated with PNP share a major B-cell clone. The B-cell clone is expressed and maybe produces functional antibody initiating the mucocutaneous immune injury.

Objective To identify features of antibodies in the supernatants of cultured Castleman's disease cells.Methods Lymphocytes of Castleman's disease were isolated and cultured.Immunofluorescence and immunoblot assays were performed with IgG extracted from culture supernatants.The immunoglobulin heavy chaingene of cultured tumor B cells was analyzed by RT-PCR,cloning and sequencing.ResultsIg Gextracted from culture supernatant scouldattachtotheepithelialcellsurfacesofmousebladdertissues.Theantibodycouldalsoidentifytwoantigencomponents,210000and190000,ofnormalhumanepidermaltis-sues.ThesequencesimilaritywasfoundinimmunoglobulinheavychaingeneofculturedtumorBcellscom-paredwiththatof6patientswithCastleman'sdiseasepreviouslyreported.Conclusions Castleman's tumor associated with paraneoplastic pemphigus can secret autoantibody with similar features to that found in patients'sera.