Single-nucleotide polymorphism and haplotypes of TNIP1 associated with systemic lupus erythematosus in a Chinese Han population.

OBJECTIVE: To determine the association of systemic lupus erythematosus (SLE) with single-nucleotide polymorphisms (SNP) in the TNIP1 gene and compare the expression of this gene in cases and controls from a Chinese Han population in this replication study. METHODS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to genotype 19 SNP in TNIP1 in Chinese Han patients with SLE (n = 341) and controls (n = 356). Genotypes were analyzed by codominant, dominant, and recessive models. Analysis of allele frequencies and linkage disequilibrium was also performed. Western blotting and qRT-PCR were used to measure the expression of these genes in peripheral blood mononuclear cells of SLE cases and controls. RESULTS: Seven SNP loci were significantly associated with SLE in our population (p < 0.05 for all comparisons). Two TNIP1 gene haplotypes (ATTGCGC and GTCCTAT) were associated with SLE (p = 0.0246 and p = 0.0024, respectively). Western blotting and qRT-PCR results provide evidence that patients with SLE had significantly reduced expression of TNIP1/ABIN-1 relative to controls. CONCLUSION: Analysis of SNP in the TNIP1 gene and expression of this gene in peripheral blood lymphocytes indicated these SNP were associated with the occurrence of SLE in Han Chinese patients. Future studies should examine the roles of these SNP in the pathogenesis of SLE.

Detection of invasive Candida albicans infection using a specific (99m)Tc-labeled monoclonal antibody for the C. albicans germ tube.

Accurate diagnosis is critical for effective treatment of the invasive infection by Candida albicans. Here, we investigated whether a (99m) technetium (Tc)-labeled Fab' fragment of the monoclonal antibody specific for the C. albicans germ tube could specifically identify an invasive C. albicans infection. The germ tube of C. albicans was used as an immunogen to obtain monoclonal antibodies and the Fab' fragment of MAb03.2 C1-C2 with highest affinity and specificity was labeled with (99m)Tc. In vitro binding assays showed that the labeled Fab' preferentially bound to the germ tubes of C. albicans (4.23 ± 0.17 × 10(2) Bq per 1 × 10(7) cells). These values were significantly higher than those for blastospores of C. albicans, blastospores of heat-killed C. albicans, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli (P < 0.05). By using in vivo biodistribution and planar imaging with single photon emission computed tomography, we demonstrated a significant specific accumulation of radioactivity in C. albicans-infected tissues. In summary, (99m)Tc-MAb03.2 C1-C2 Fab' is able to specifically accumulate in C. albicans-infected tissues, but not in tissue infected with A. fumigatus or bacteria or in a sterile inflammation. This study provides a new and specific radiopharmaceutical for the diagnosis of invasive C. albicans infections.

Study of the biological function and penetration pathways of the mouse epidermal growth factor ethosomal delivery system.

Biological agents are becoming increasingly popular for therapeutic applications in epidermal diseases. Ethosomes facilitate the transdermal/topical delivery of biological macromolecules. The mouse epidermal growth factor (mEGF) was selected as the model biological agent. The aim of this experiment was to determine the penetration pathways and biological functions of the mEGF ethosomal delivery system after its topical application. The mEGF ethosomal delivery system was topically applied on the dorsal skin of C57BL/6 mice at different time points. Freshly excised skin samples were obtained by skin biopsies and shock-frozen, and immunofluorescence was performed. The results showed that penetration of mEGF ethosomes was mainly through the pilosebaceous unit and partly through the intercellular domain. Biological agents encapsulated in the ethosomal delivery system could reach each site of the pilosebaceous unit. We also found that mEGF ethosomes had caused successful transition of the hair follicles from the telogen to the anagen phase of the hair cycle.

Melanocortin receptor type 2 (MC2R, ACTH receptor) expression in patients with alopecia areata.

Human skin expresses elements of the hypothalamo-pituitary-adrenal (HPA) axis that function as a local stress response system. Because adrenocorticotropic hormone (ACTH) is an intermediate in the HPA axis from corticotropin-releasing hormone (CRH) signal to cortisol secretion, MC2R that binds only ACTH may be important in the stress response of skin. We investigated the local expression of MC2R by immunohistochemistry to identify the role of ACTH/MC2R in stress-associated alopecia areata (AA). MC2R appeared to be highly compartmentalized in scalp skin including the epidermal cells of hair follicles and epidermis, sebaceous and eccrine glands, as well as dermal fibroblasts. The expression of MC2R was lower in AA lesions than in normal scalp tissue in almost all scalp skin cells, especially in epithelial cells. These findings demonstrate that MC2R expression is aberrant in AA and suggest a deficit in ACTH/MC2R activity may play an important role in the pathophysiology of AA.

Phenotypic and molecular characterization of Madurella pseudomycetomatis sp. nov., a novel opportunistic fungus possibly causing black-grain mycetoma.

A case of black-grain mycetoma occurring on the lower jaw with an odontogenic origin, which to our knowledge is the first case reported in China, is presented here. The clinical manifestation, histopathological morphology, and microbiological features are described. The new species, Madurella pseudomycetomatis, isolated from the black grains discharged by this patient, was analyzed using sequence data of the multiloci of ribosomal DNA (rDNA) and its ability to ferment carbohydrate as well as morphology. The analyses of the internal transcribed spacer (ITS) region and the D1/D2 hypervariable region of the 28S ribosomal gene sequences support a new species designation. Antifungal susceptibility testing was conducted, indicating that Madurella pseudomycetomatis was highly susceptible to itraconazole, voriconazole, and amphotericin B; moderately susceptible to terbinafine; and resistant to fluconazole and flucytosine.

[Induction of oral immune tolerance in systemic lupus erythematosus-like murine model by recombinant nucleosomal universal Th cell epitope].

OBJECTIVE: To explore the feasibility of oral immune tolerance of systemic lupus erythematosus (SLE)-like model induced by nucleosomal Th cell epitope via the attenuated Salmonella typhimurium. METHODS: SLE-like murine model was established by immunization with apoptotic syngeneic lymphocytes. The recombinant strains were orally administrated to induce immune tolerance. The levels of serum autoantibodies, such as anti-ANA, ds-DNA, and antinucleosome antibody, leukopenia, proteinuria and kidney injuries were evaluated. RESULTS: SLE-like murine model was successfully established. Compared with controls, it was shown that CTLA4-Ig-H2B group could dramatically reduce the levels of serum autoantibodies, such as anti-ANA, ds-DNA and antinucleosome antibody and ameliorate leukopenia and proteinuria (all P < 0.05). Immune complex deposits of IgG in glomeruli were lower in CTLA4-Ig-H2B (1.35 +/- 0.16) than in CTLA4-Ig (1.66 +/- 0.23) and H2B (1.69 +/- 0.24) (both P < 0.05). The score of glomeruli lesion of CTLA4-Ig-H2B (1.26 +/- 0.14) was significantly lower than those of CTLA4-Ig (1.73 +/- 0.25) and H2B (1.71 +/- 0.20) (both P < 0.05). CONCLUSION: Combined with CTLA4-Ig, it is feasible to induce oral immune tolerance of SLE models with nucleosomal Th cell epitope via the attenuated Salmonella typhimurium. This may provide a novel way to prevent and treat SLE by oral immune tolerance.

HPV16E7 tumor antigen modified by KDEL sequence induce specific cytotoxic T lymphocytes-dependent antitumor immunity.

BACKGROUND: Infection by high-risk HPV (human papillomavirus) is the primary cause of cervical cancer. Dendritic cell-based (DC-based) therapeutic vaccine represents a promising approach to the prevention and treatment of many cancers, including HPV-related cancers, but current strategies have met with only limited success in preclinical and clinical research. It is necessary to find a properly and effective antigen presenting system of DC-based vaccine. OBJECTIVE: To design a new HPV16 therapeutic vaccine using an endoplasmic reticulum (ER) retrieval signal and study its ability to induce the specific CTL activity in vitro and in vivo. METHODS: E7(p)-KDEL and its control peptide were synthesized on solid phase. A series of methods were used, including standard (51)Cr-labeled release assay, enzyme-linked immunospot (ELISPOT) assay and ELISA, to detect the CTL activity induced by different peptides. Prophylactic models and therapeutic models were examined to detect the in vivo effectiveness of E7(p)-KDEL-loaded DCs. RESULTS: The specific CTL activity induced by E7(p)-KDEL-loaded DCs was much stronger than that induced by the other peptide-loaded DCs. Comparing with the control peptides, after incubation with the spleen cells of mice, the E7(p)-KDEL-loaded DCs could induce higher concentration of secreted IFN-gamma and had higher ELISPOT numbers. In animal models, E7(p)-KDEL-loaded DCs vaccines effectively protected mice against fatal TC-1 tumor challenge and cured tumor-bearing mice. CONCLUSIONS: The ER retrieval signal-mediated antigen delivery system may have important clinical application for cancer therapy, even virus infectious disease and autoimmune disease.

[Effects of HS1-associated protein X-1 on the lupus activities: experiment with MRL/lpr lupus-like mice].

OBJECTIVE: To investigate the effects of HS1-associated protein X-1 (HAX-1) on the lupus activity of MRL/lpr lupus-like mice. METHODS: Fifteen MRL/lpr mice were divided into 3 equal groups: Group A, injected with phosphate-buffered saline, Group B, injected intraperitoneally with control virus AdEGFP and Group C, injected intraperitoneally with recombinant AdHAX-1 twice a week for 4 weeks. Peripheral blood samples were collected before the injection, and 2 and 4 weeks after the injection to be detected the white blood cell count, antinuclear antibody (ANA), anti-double strand-DNA antibodies, circulating immune complex (CIC), anti-histone antibodies, and interferon (IFN)-gamma. The level of urine protein was measured, too. Then the mice were killed, a kidney underwent direct immunofluorescence (DIF) to observe the deposition of Immune complexes, and the other kidney underwent periodic acid-Schiff (PAS) staining and pathological examination. MTT method was used to detect the proliferation of the lymphocytes in the spleen. Splenocytes were isolated from the other 15 MRL/lpr mice and then divided into 4 groups: Group, transfected with DMRIE-C without plasmid; Group E, as negative control group; Group F, transfected with blank plasmid pGenesil-1; and Group G, transfected with pGenesil-HAX-1. Forty-eight hours later MTT method was used to detect the proliferation rateof the spleen lymphocytes. RESULTS: The urine protein level of Group C was significantly higher than those of Groups A and B (both P < 0.01). Four weeks later the levels of ANA, anti-double strand DNA antibodies, and IFN-gamma were all significantly higher than those of Groups A and B (all P < 0.01). Hypercellularity and increased deposition of IgG in glomeruli were also observed in Group C. The score of glomeruli lesion of Group C (1.50 +/- 0.34) was significantly higher than those of Groups A (0.67 +/- 0.14) and Groups B (0.81 +/- 0.26) (both P < 0.01). MTT method showed that the growth curve of the spleen lymphocytes of Group C was higher than those of Groups A and B. The spleen lymphocyte proliferation rate and the levels of IFN-gamma of Group G was significantly lower than that of Group F (both P < 0.05). CONCLUSION: One of the important factors in apoptosis regulation of SLE, HAX-1 may be involved in the pathogenesis of SLE, and the silence of HAX-1 may be beneficial for the improvement of SLE.

[Inhibitory effect of short hairpin RNA on expression of Hax-1 gene in HeLa cells].

AIM: To construct the short hairpin RNA (shRNA) eukaryotic expression vector specific for HS1-associated protein X-1 (Hax-1) and investigate the inhibitory effect of it on Hax-1. METHODS: According to the design rules of shRNA, the specific sequence of 19 nucleotides were selected from Hax-1 cDNA sequence and designed as the cDNA template of siRNA. ShRNA vector pGenesil-Hax-1 was constructed by recombining the synthesized specific sequence with siRNA expression vector pGensil-1. After the recombinant plasmids were transfected into HeLa cells, RT-PCR technique and Western blot were applied to analyze mRNA and protein expression of Hax-1. RESULTS: The results of RT-PCR showed that the down-regulation of Hax-1 mRNA expression was found in the pGenesil-Hax-1 transfected group, but not in the pGenesil-1 transfected group or the negative control group (P<0.01). The expression of Hax-1 protein decreased by 70% in the pGenesil-Hax-1 transfected group compared with the negative control group. CONCLUSION: Hax-1 gene expression can be inhibited markedly by specific shRNA in HeLa cells, which establishes the experimental foundation for further study on the biological functions of Hax-1 in HeLa cells.

Hair follicle reformation induced by dermal papilla cells from human scalp skin.

To investigate the possibility of hair follicle reformation induced by dermal papilla cells in vivo and in vitro. Dermal papilla cells, dermal sheath cells obtained from human scalp skin by enzyme digestion were mixed with collagen to form mesenchymal cell-populated collagen gels. Superior and inferior epithelial cells and bulb matrical cells were then cultured on these gels by organotypic culture to recombine bilayer artificial skins. Dermal papilla cells and outer root sheath keratinocytes were mingled together and transplanted under subcutaneous tissue of the dorsal skin of nude mice. The results of histologic examination was observed with HE stain. These recombinants by organotypic culture all reformed bilayer structure like nature skin. Hair follicle-like structure reformation was found in dermal sheath cell-populated collagen gel when combined with superior or inferior epithelial cells. Dermal papilla cells also induced superior and inferior epithelial cells to form hair follicle on nude mice. Low passage dermal papilla cells mixed with hair follicle epithelial cells reformed many typical hair follicle structures and produced hair fibres after transplantation on nude mice. The dermal part of hair follicle, such as dermal papilla cells and dermal sheath cells, has the ability to induce hair follicle formation by interaction with the epithelial cells of hair follicle.

[Clinical analysis of the infection with human papillomavirus in women].

BACKGROUND: To determine the relationship between human papillomavirus infection, cervical carcinoma, pre-cancerous lesion and condyloma acuminatum. METHODS: From January 2004 to August 2005, 1086 inpatients in department of dermatology and department of gynaecology and obstetrics in Southwest Hospital and No. 302 Hospital with cervical lesions and condyloma were reviewed. All specimens were detected for HPV-DNA using techniques of Gene Array and fluorogenic quantitative polymerase chain reaction (FQ-PCR). All detections of HPV-DNA were performed in the first admission before the patients underwent any examination or treatment. RESULTS: The positive rates of HPV-DNA detection were 100% in cervical intraepithelial neoplasia (CIN) I, and II and cervical carcinoma. Among these, the main subtype was HPV 16. But some of the patients were found to be positive for more than 2 subtypes of HPV. While the commonest HPV subtype was HPV 18 in endometrial cancer. Some of the patients were detected to be positive for more than 2 subtypes of HPV. In 636 female patient with condyloma acuminatum, the infection rates of HPV6, HPV11 accounted for 44.97% and 29.40%, respectively, HPV 16 and/or HPV 18 infection constituted a small percentage. In a few cases, infection with more than 2 subtypes was detected. CONCLUSION: Cervical carcinoma including pre-cancerous lesion differs from condyloma acuminatum in dominate infectious subtype of HPV. The former is mainly associated with HPV 16 and HPV 18 infections, and the latter mainly associated with HPV 6 and HPV 11 infections. But in both of the above lesions, a mixed infection with more than 2 types may occur and make the pathological changes and clinical treatment more complicated. The early diagnosis and supervision of HPV infection may be of great value for improvement of prognosis and quality of life.

[Retrospective analysis of 1905 patients with skin cancer from two general hospitals in western China from 1981 to 2000].

OBJECTIVE: To investigate the clinical features and changes in the incidence of skin cancer in two hospitals located in western China. METHODS: The patients diagnosed pathologically as skin cancer from 1981 to 2000 were retrospectively collected from the two hospitals. Clinical data of patients with skin cancer were collected and analyzed. RESULTS: (1) Of the 1 905 patients with skin cancer, squamous cell carcinoma accounted for 29.4%(560 patients), basal cell carcinoma 28.0% (534), and cutaneous malignant melanoma(CMM) 16.0% (305). (2) There were 591 patients with skin cancer diagnosed between 1980 and 1990, and 1 314 between 1991 and 2000, and accounted for 0.34% and 0.58% of all biopsy cases, respectively. The number of total biopsy patients increased 1.6% every year during the 20 years. The number of biopsy patients with skin cancer and with CMM increased 3.5% and 3.9% every year,respectively. (3) Of the 305 CMM patients, 63.3% located on the acra. These patients were elder, and have a higher rate of trauma and a higher incidence in the male than that in patients with CMM located on the other sites. (4) Of the 305 CMM patients, 64 (21%) had history of trauma at the primary onset sites, and 47 (15.4%) had history of small congenital nevi at the primary sites. CONCLUSION: There are some differences in the clinical features such as location and age between the skin cancer patients in our study and those in white population. The incidence of skin cancer in the two hospitals had been increasing in the 20 years (between 1981 and 2000). Both trauma and small congenital nevi are important risk factors of CMM.

[Expressions of bFGF, ET-1 and SCF in dermal papilla cells and the relation to their biological properties].

OBJECTIVE: To investigate the expression of bFGF, ET-1 and SCF in different passages of cultured dermal papilla cells (DPC), and their possible effect on biological behaviour of DPC. METHODS: The expression of bFGF, ET-1 and SCF in different passages of cultured DPC was detected by immunocytochemistry and in situ hybridization. RESULT: The expression of ET-1 and SCF in early passages of cultured DPC was stronger, but became negative in late passages (>6 passages). The stronger the expression of ET-1 and SCF in DPC, the higher ability of DPC to induce hair follicle regeneration. CONCLUSION: The expression strength of ET-1 and SCF is related to the ability of DPC inducing hair follicle regeneration.

Identification of differentially expressed genes in anagen dermal papilla by suppression subtractive hybridization.

BACKGROUND: We constructed a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) technique and clone differentially expressed genes related to DPCs in anagen. METHODS: Total mRNA was isolated from DPCs of anagen and telogen follicles. Moreover, single-strand (ss) and double-strand (ds) cDNAs were synthesized in turn using SMART PCR cDNA synthesis technology. ds cDNAs then were digested with Rsa I and divided into two groups, and ligated to the specific adaptor 1 and adaptor 2R, respectively. After cDNAs were hybridized with each other twice and underwent two rounds of nested PCR. PCR products were ligated with arms of T/A plasmid vectors to set up the subtractive library. Selected clones were demonstrated by reverse Northern blot and sequenced. The acquired sequence data were aligned against the Genbank nucleotide database. RESULTS: cDNA subtractive library of DPCs in anagen follicles was set up successfully with high subtractive efficiency. Thirty-five genes were identified in this study with 22 known functional genes and 13 unknown functional genes. CONCLUSIONS: All results confirm the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in hair follicle growth regulation.

[Factors related to collagen gel contraction in hair follicle organotypic culture].

OBJECTIVE: To investigate the effects of rat tail collagen, hair follicle dermal papilla cells and hair follicle epithelium cells on collagen gel contraction in organotypic culture. METHODS: The hair follicle organotypic culture was prepared with different concentrations of rat tail collagen, different number of dermal papilla cells and hair follicle epithelium cells in DMEM medium, after cultured for 10 days the diameter of collagen gel was measured. RESULT: The concentration of rat tail collagen, hair follicle dermal papilla cells and hair follicle epithelium cells significantly influenced on collagen gel contraction in organotypic culture (P<0.01). The contraction of collagen gel was negatively related to the concentration of rat tail collagen, while the concentration of dermal papilla cells and hair follicle epithelium cells was positively related to the contraction of collagen gel. CONCLUSION: The key factor influencing collagen gel contraction in organotypic culture is the concentration of rat tail collagen, hair follicle dermal papilla cells and hair follicle epithelium cells.