OBJECTIVETo explore the clinicopathologic features, immunophenotype, differential diagnosis and gene mutation status of the Erdheim-Chester disease (ECD).

METHODSClinical and pathologic findings of 3 ECD cases were examined by gross, microscopic, immunohistochemical methods and BRAF V600E mutation. Related literatures were reviewed.

RESULTSTwo male patients and one female patient presented clinically with multiple skin nodules, bone pain and bony lesions by imaging study. Microscopically, the lesions were composed of spindle-shaped fibroblasts, foamy histiocytes and scattered Touton-type giant cells embedded in reactive fibrous tissue. Lymphocytes, plasma cells, and multinucleated giant cells were also found. Immunohistochemically, all histiocytes were positive for CD68, none of which expressed CD1a, although 2 cases focally expressed weak S-100 stain. In 2 cases,BRAF V600E mutation was detected.

CONCLUSIONSECD is a rare disease of xanthogranulomatous histiocytosis.Its diagnosis relies on pathological and immunohistochemical findings, but correlation with clinical information, especially radiographic findings should be performed.No effective treatment of the disease is currently available.

Antigens, CD ; analysis ; Antigens, CD1 ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Diagnosis, Differential ; Erdheim-Chester Disease ; genetics ; immunology ; pathology ; Female ; Humans ; Male ; Mutation ; S100 Proteins ; analysis ; Treatment Outcome

Acute Disease ; Antigens, CD ; analysis ; Biomarkers, Tumor ; analysis ; Chromosome Aberrations ; Diagnosis, Differential ; Humans ; Immunophenotyping ; Leukemia ; classification ; diagnosis ; genetics ; immunology ; Leukemia, Biphenotypic, Acute ; classification ; diagnosis ; genetics ; immunology ; Phenotype

OBJECTIVETo investigate the effects of Tpo and/or IL-11 gene modified stromal cells on the expansion of CD(34)(+) hematopoietic stem/progenitor cells in cord blood.

METHODSRetroviral vectors containing Tpo or IL-11 gene were constructed and used to transfect the stromal cell line HFCL. Tpo and/or IL-11 mRNA was assayed by Northern blot. Non-modified stromal cells were used, CD(34)(+) hematopoietic stem/progenitor cells from cord blood were expanded on gene-modified stromal cells for 7 days. The phenotype of CD(34)(+)CD(38)(-) primitive progenitors was detected by flow cytometry.

RESULTSHFCL expressed Tpo and/or IL-11 mRNA after transfected by the retroviral vectors. The percentages of CD(34)(+)CD(38)(-) primitive progenitors in the cultures of Tpo, IL-11 and Tpo + IL-11 modified HFCL were (1.8 +/- 0.24)%, (1.62 +/- 0.23)%, and (2.45 +/- 0.28)%, respectively, which were higher than that in the control [(0.8 +/- 0.23)%].

CONCLUSIONThe stromal cells modified by Tpo and/or IL-11 gene were able to enhance ex vivo expansion of CD(34)(+) and CD(34)(+)CD(38)(-) hematopoietic stem/progenitor cells from cord blood.

ADP-ribosyl Cyclase ; analysis ; ADP-ribosyl Cyclase 1 ; Antigens, CD ; analysis ; Antigens, CD34 ; analysis ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; physiology ; Humans ; Infant, Newborn ; Interleukin-11 ; genetics ; Membrane Glycoproteins ; Stromal Cells ; physiology ; Thrombopoietin ; genetics

OBJECTIVEGene therapy of leukemia is a new and effective method. It is known to all that the pathogenesis and development of leukemia are related to a variety of genes. Survivin is a member of inhibitors of apoptotic proteins (IAP). Its cDNA was cloned from target cell protease receptor-1 (EPR-1). It is expressed in common tumors, but there is no expression in normal and mature tissues. High expression of survivin was detected in leukemic cells. The present study was conducted to examine the role of survivin in the differentiation of leukemic cells by using antisense-oligonucleotides.

METHODSHuman leukemic cell K562 was used as the model for the study. K562 cells were divided into 4 groups randomly: antisense oligonucleuotide (ASON) group, nonsense oligonucleotide (NSON) group, lipofectin group and control group. There were 5 samples in each group, and the experiment was repeated for three times. ASON was designed with the reference to targeting survivin mRNA. K562 cells were cultured in RPMI1640 contained fetal cattle serum at a concentration of about 10 percent. Cell transfection was induced by lipofectin. Forty-eight hours after thansfection, the morphology and ultrastructure were observed. Twenty-four hours and 48 hours after thansfection, the function of K562 cells was detected by benzidine staining, POX staining and NBT staining, respectively. The mean fluorescence intensity of CD33 was determined by flow cytometry. The method of immunohistochemistry was used to examine the protein level of survivin.

RESULTSAfter thansfection with ASON, the size of K562 cells was reduced, but the cytoplasm was increased. The metarubricyte, segment granulocyte, apoptotic cells could be found. Morphologically and ultrastructurally, erythroid and myelocytic differentiation was observed. The positive level of benzidine staining in ASON group (11.90 +/- 2.30 at 24 h and 18.20 +/- 2.93 at 48 h) was higher than that of NSON group, lipofectin group and control group, respectively. The positive level of POX staining in ASON group (17.40 +/- 3.54 at 24 h and 29.40 +/- 3.70 at 48 h) was also higher than that of any other groups. The positive level of NBT staining in ASON group (7.50 +/- 2.26 at 24 h and 12.10 +/- 2.63 at 48 h) was significantly higher than that of NSON group, lipofectin group and control group, respectively (P < 0.01). In ASON group, the mean fluorescence intensity of CD33 (21.43 +/- 1.61 at 24 h and 14.86 +/- 1.20 at 48 h) was significantly lower than that of any other groups (P < 0.01). After thansfection for 24 h, the protein level of survivin in ASON group was decreased significantly compared to that of control group. There was no difference in survivin protein level amongst ASON group, NSON group and lipofectin group at 24 h (P > 0.05). Forty-eight hours after thansfection, the protein level of survivin was decreased significantly.

CONCLUSIONSASON targeting survivin can induce K562 to erythroid and myelocytic differentiation. Survivin is related to differentiation of K562 cells, and it can be a target of gene therapy for leukemia.

Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Cell Differentiation ; Humans ; Inhibitor of Apoptosis Proteins ; K562 Cells ; Microtubule-Associated Proteins ; analysis ; antagonists & inhibitors ; genetics ; physiology ; Oligonucleotides, Antisense ; genetics ; Sialic Acid Binding Ig-like Lectin 3 ; Transfection

As one important member of B7/CD28/CTLA-4 costimulatory signal pathway, B7-2 molecule plays a critical role in regulating T-cell response. In order to further explore its effects on regulation of T cell activation, proliferation and associated signal pathways, the cDNA encoding extracellular region of human B7-2 was amplified via PCR and subcloned into some prokaryotic expression vectors to express target protein in host strains. The expressed protein was identified with Western blot and MTT. Results showed that after screening, the expression level of the protein of interest attained the yield of over 20% total bacterial protein by using pGEX-4T-2 vector and E. coli BL21 (DE3)-CodonPlus-RIL host cells. The recombinant protein could specially react with B7-2 McAb and could stimulate T-cell proliferation combined with anti-CD3 antibody. In conclusion, the recombinant protein was bioactive, therefore the study will make it possible for the research of relationship between B7-2 structure and its function.
Antigens, CD ; biosynthesis ; genetics ; pharmacology ; B7-2 Antigen ; Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; analysis ; Humans ; Membrane Glycoproteins ; biosynthesis ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

Acanthosis Nigricans ; diagnosis ; genetics ; pathology ; Adolescent ; Antigens, CD ; genetics ; Biomarkers ; blood ; DNA Mutational Analysis ; Donohue Syndrome ; diagnosis ; genetics ; pathology ; Humans ; Insulin Resistance ; genetics ; Male ; Mutation ; genetics ; Polymerase Chain Reaction ; Receptor, Insulin ; genetics ; Sequence Analysis, DNA

CD99 is characteristically expressed in Ewing's sarcoma/primitive neuroectodermal tumor. Recently its immunoreactivity has also been reported in other tumors. However, the significance of CD99 isoforms expressed in these tumors has not been elucidated. In this study, we evaluated the expression of CD99 isoforms and its relationship with histopathologic parameters in gastric adenocarcinomas. Paraffin sections of 46 gastric adenocarcinomas were stained with an anti-CD99 monoclonal antibody, YG32. Twelve (26.1%) cases of 46 gastric adenocarcinomas showed immunoreactivity to YG32. The CD99 expression was also seen both in non-neoplastic foveolar epithelial cells and infiltrating lymphocytes. In addition, Western blot and RT-PCR analyses revealed that the type I is the predominant isoform of CD99 in non-neoplastic and neoplastic gastric tissues. The CD99 expression was usually seen in the intestinal type adenocarcinoma, while rarely in the diffuse type. The CD99 immunoreactivity decreased in MMP-2-overexpressing adenocarcinomas (p=0.028). Our results suggest that the type I is the major isoform of CD99 expressed in non-neoplastic gastric mucosa and gastric adenocarcinomas and its downregulation in gastric adenocarcinoma may be associated with cellular dedifferentiation and/or MMP-2 overexpression.
Adenocarcinoma/*immunology/pathology ; Adult ; Aged ; Antigens, CD/*analysis/genetics ; Cell Adhesion Molecules/*analysis/genetics ; Female ; Gastric Mucosa/cytology/immunology ; Humans ; Male ; Matrix Metalloproteinases/metabolism ; Middle Aged ; Protein Isoforms/analysis/genetics ; RNA, Messenger/genetics/metabolism ; Stomach Neoplasms/*immunology/pathology

OBJECTIVETo construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector.

METHODSFirst, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells.

RESULTSThe construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69.

CONCLUSIONThe construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.

Animals ; Antigens, CD ; genetics ; Antigens, Differentiation, T-Lymphocyte ; genetics ; DNA, Complementary ; Genetic Vectors ; Genotype ; Green Fluorescent Proteins ; genetics ; Lectins, C-Type ; genetics ; Mice ; Mice, Transgenic ; Plasmids ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transfection

OBJECTIVEIt is well known that vitamin A can improve mucosal immunity and anti-infection immunity. But the mechanisms thereof remain to be clarified. Previous studies on the role of vitamin A in immune regulation focused on lymphocytes, whereas little had been done about dendritic cells, which play very important roles in immune response. The objective of this study was to understand the effects of retinoic acid (RA), the metabolic product of vitamin A in vivo,on the differentiation, maturation and functions of dendritic cells from cord blood.

METHODSCord blood samples were collected from nine well-nourished full-term neonates. Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and cultured in the presence of 1000 u/ml GM-CSF, 500 u/ml IL-4 for 6 days, then TNF-alpha 20 ng/ml was added into the medium and cultured for another 3 days. The cells were incubated with or without 1 x 10(-6) MRA. Expression of surface molecules, CD1a, CD83, HLA-DR on DC was measured by flow cytometry. The ability of DC derived from the culture to induce proliferation of T cells in the mixed lymphocyte reaction (allo-MLR) was used for the evaluation of their function. IL-12, IFN-gamma, IL-4 and IL-10 were detected at mRNA levels by RT-PCR to understand the roles of DC treated with RA in regulation of Th1/Th2 balance.

RESULTSOn the sixth day of cell culture, the percentage of DC incubated with RA (57.28 +/- 9.22) was much lower than that without RA (79.57 +/- 11.85) (P < 0.001), but on the ninth day, there were no differences between the presence or absence of RA (76.18 +/- 10.27 vs. 73.72 +/- 15.58). When RA was added to the medium and the culture was continued for nine days, the percent of immature DC (CD1a + HLA-DR+) was much higher than that of the control (absence of RA) (58.93 +/- 4.70 vs. 45.80 +/- 7.88, t = 6.575, P < 0.001); whereas, mature DC (CD83 + HLA-DR+) percentage was markedly lower than that of the control (17.25 +/- 8.49 vs. 27.92 +/- 13.94, t = 4.435, P = 0.002). The T lymphocytes proliferation induced by the DC treated with RA was reduced from 16 857 +/- 3 643 to 11 924 +/- 2 576 cpm (t = 5.598, P < 0.001) in allo-MLR. Expression of mRNA for IL-12p35, IL-12p40, IFN-gamma in the cells that had been incubated with RA declined, but IL-10, IL-4 increased significantly.

CONCLUSIONVitamin A inhibited the differentiation and maturation of cord blood DC, reduced it's ability to stimulate allo-T lymphocytes proliferation, and down-regulated Th1 cytokines, up-regulated Th2 cytokines, consequently made immune response inclined to Th2.

Antigens, CD ; Antigens, CD1 ; analysis ; Cell Differentiation ; drug effects ; genetics ; immunology ; Cytokines ; genetics ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Fetal Blood ; cytology ; immunology ; Flow Cytometry ; Gene Expression ; drug effects ; HLA-DR Antigens ; analysis ; Humans ; Immunoglobulins ; analysis ; Interferon-gamma ; genetics ; Interleukin-10 ; genetics ; Interleukin-12 ; genetics ; Interleukin-4 ; genetics ; Membrane Glycoproteins ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Vitamin A ; pharmacology

OBJECTIVE: Our previous study revealed many genes differentially expressed in human laryngeal squamous cell carcinoma tissues (LSCC) and related adjacent normal tissues. We verificated the differentiated expressions of target genes, possibly related to LSCC. And these results can be foundations of further study of these target genes function. METHOD: SENP1, CD109, Laminin alpha 2, Laminin alpha 3 were selected according to the cDNA microarray results. The expression of these genes mRNA and protein were detected by RT-PCR and Western blot in 12 cases of LSCC and related adjacent normal tissues. RESULT: The mRNA expression of SENP1, CD109 and Laminin alpha 3 were significantly higher while Laminin alpha 2 were significantly lower in LSCC tissues than in corresponding adjacent normal tissues by Semiquantitative RT-PCR. Western blot analysis revealed SENP1, CD109 protein expression were significantly higher in LSCC tissues than in corresponding adjacent normal tissues. CONCLUSION SENP1, CD109, Laminin alpha 2 and Laminin alpha 3 may correlated with tumorigenesis and development of LSCC and can provide beneficial clue for study pathogenesis of LSCC in molecular level.
Aged ; Aged, 80 and over ; Antigens, CD ; genetics ; Carcinoma, Squamous Cell ; genetics ; Cysteine Endopeptidases ; Endopeptidases ; genetics ; GPI-Linked Proteins ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Laminin ; genetics ; Laryngeal Neoplasms ; genetics ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; Oligonucleotide Array Sequence Analysis