Aim To investigate the effects of multi-gly-cosides of Tripterygium wilfordii(GTW)on mitochon-drial dynamics-related proteins and the mechanism of nephroprotective effects in IgA nephrophathy(IgAN)rats.Methods SPF grade male SD rats were random-ly divided into the Control group,modelling group,prednisone group(6.25 mg·kg·d-1)and GTW group(6.25 mg·kg·d-1).The IgAN rat model was established by the method of"bovine serum albumin(BSA)+carbon tetrachloride(CCl4)+lipopolysac-charide(LPS)".The total amount of urinary protein(24 h-UTP)and erythrocyte count in urine were meas-ured in 24 h urine.Blood biochemistry of serum albu-min(ALB),alanine aminotransferase(ALT),urea ni-trogen(BUN),and creatinine(Scr)were measured in abdominal aorta of the rats;immunofluorescence and HE staining were used to observe the histopathology of the kidneys;RT-PCR and Western blotting were used to detect the mRNA and protein expression levels of key proteins regulating mitochondrial division and fu-sion:dynamin-related protein 1(Drp1),mitochondrial fusion protein 1(Mfn1),and mitochondrial fusion pro-tein 2(Mfn2),and PTEN-induced putative kinase 1(Pink1),in the kidney tissue of rats.Results GTW significantly reduced urinary erythrocyte count and 24 h-UTP,decreased serum ALT,BUN and Scr levels,in-creased serum ALB levels,improved renal histopatho-logical status in IgAN rats,increased the protein and mRNA expression levels of Mfn1,Mfn2,and Pink1,and decreased the protein and mRNA expression levels of Drp1 in renal tissues.Conclusions GTW may regu-late mitochondrial structure and maintain the dynamic balance of mitochondrial dynamics by promoting the ex-pression of Mfn1,Mfn2,Pink1 and decreasing Drp1.This may result in a reduction in urinary erythrocyte counts and proteinuria,and an improvement in renal function.

Aim To explore the mechanism of action of Tripterygium glycosides tablets on kidney of rats with IgA nephropathy based on inflammation-related path-ways.Methods Forty-five male SD rats of SPF grade were randomly divided into control group and modeling group.In addition to the blank group,the modeling group used the combination of bovine serum albumin(BSA)+carbon tetrachloride(CC14)+lipopolysac-charide(LPS)to establish the IgA nephropathy rat model.Successfully modeled rats were randomly divid-ed into the model group,the prednisone group and Tripterygium glycosides tablets group,and the treat-ment group was given the drug by gavage from the 13 th week,and the 24 hours urine,blood and kidney tis-sues of the rats were collected and examined after 4 weeks of the administration of the drug.Urine erythro-cyte count,quantitative 24-h urine protein(24 h-UTP),urea nitrogen(BUN),and blood creatinine(Scr)were detected in each group;serum interleukin 1β(IL-1β)and tumor necrosis factor α(TNF-α)were detected by enzyme-linked immunosorbent assay(Elisa);the pathological changes in the renal tissues of the rats in each group were observed by horizontal hematoxylin-eosin(HE)staining;and the renal tis-sues in each group were observed by Western blotting.The expressions of LIGHT,HVEM,LTβR proteins and their mRNAs in rat kidney tissue were detected by Western blot and real-time fluorescence quantitative polymerase chain reaction(RT-PCR).Results Tripterygium glycosides tablets significantly reduced the levels of urinary erythrocyte count,24 h-UTP,BUN,and Scr in IgA nephropathy rats(P＜0.01),improved renal histopathology,lowered the levels of se-rum inflammatory factors IL-1β and TNF-α(P＜0.01),and lowered the levels of LIGHT,HVEM,LTβR proteins and their mRNA expression in renal tis-sues(P＜0.01).Conclusions Tripterygium glyco-sides tablets may inhibit the immune response and re-duce the release of inflammatory factors by down-regu-lating the LIGHT-HVEM/LT(3R pathway,thus reduc-ing the inflammatory response,lowering the urinary e-rythrocytes and urinary proteins,improving the renal nephron pathologic injury,and protecting the renal function.

To investigate the relationship between the LNK(SH2B3) gene single nucleotide polymorphism and risk of acute leukemia (AL) in Chinese population.

METHODSThe bone marrow and peripheral blood samples from 31 cases of acute lymphoblastic leukemia, 70 cases of acute myeloid leukemia and 130 healthy persons as the controls were collected. Genotype of LNK SNP Rs3184504(c.784T>C) and Rs78894077(c.724C>T) were determined by PCR-RFLP, and were confirmed by gel electrophoresis and sequencing. The NB4, THP-1 and Raji leukemia cell line models were cultured, the leukemia cell line LNK Rs3184504 and Rs78894077 polymorphism were detected by using direct sequencing.

RESULTSThe CC genotype frequencies of Rs3184504 SNP were higher in ALL and AML patients than those in control (P<0.01), but there was no different between the groups in AML and ALL. The frequency of LNK gene Rs3184504 C allele was higher in AL as compared with control (P<0.01). The LNK gene Rs78894077 locus genotype distribution was not significantly different between the AL and the normal control group (P>0.05). Both Rs3184504 and Rs78894077 sites were detected as CC genotype in NB4, THP-1 and Raji cells.

CONCLUSIONThe persons carrying C allele of LNK gene Rs3184504 are more prone to develop acute leukemia.

OBJECTIVETo study the relationship between surface markers of CD56 and CD19 and karyotypes and prognosis in multiple myeloma.

METHODSA total of 126 cases of newly diagnosed multiple myeloma in the first hospital of Peking university from 2011 to 2015 were enrolled in this study. Cytogenetic abnormalities and immunophenotypes were detected by using fluorescence in situ hybridization and flow cytometry respectively before chemotherapy. Bone marrow smear was used for detection of abnormal plasma cell infiltration. By combining with their basic data, the relationship between immunophenotypes, cytogenetics and prognosis of MM was analyzed.

RESULTS(1) The median of myeloma cells in the 126 patients was 0.24（0.01-0.97）; the median of myeloma cells in 116 patients who have immunophenotype datas was 0.25（0.01-0.97）； the median of myeloma cells in CD19 positive patients was 0.11（0.01-0.53）; the median of myeloma cells in CD19 negative patients was 0.26（0.01-0.97）. The median of myeloma cells in CD19 positive patients was much lower than that in CD19 negative patients（P=0.036）. (2)In 116 patients detected by the immunophenotype, the myeloma cells expressed CD19,CD20,CD56 and CD117. Compared with CD56 negative patients(45/116,38.79%),CD56 positive patients(71/116,61.21%) had a clearly favorable disease outcome（OS was 53.0 month vs 31.0 month,P=0.016; PFS was 37.5 months vs 18.4 months, P=0.036）. (3)CD19 positive patients was 16.38%（19/116）,CD19 negative patients was 83.62%（97/116）； CD19 positive MM and CD19 negative MM had no difference in OS and PFS. (4)CD117 positive rate in CD19 positive patients was 42.11%(8/19), the CD117 positive rate in CD19 negative patients was 18.57%(18/97), the CD19 expression positively correlated with CD117 expression. (5)FISH detection was done for 67 newly diagnosed MM patients, 8 patients showed normal karyotypes(11.94%), 59 patients had abnormal karyotypes(88.06%). The most common abnormal karyotypes were IgH rearragement which occurred in 47 patients(70.15%). Other abnormal karyotypes included 1q21+, del(13q14),del(13q14.3),del(17p13) . These abnormal karyotypes occurred in 37 patients（55.22%）,31 patients（46.27%）,33 patients（49.25%） and 13 patients（19.40%） respectively. In comparison with CD19 negative MM patients, the incidence rate of 1q21+ and del(13q14.3) was significantly lower in CD19 positive patients(1q21+:33.33% vs 61.54%,P=0.016; del(13q14.3): 33.33% vs 53.85%,P=0.043）.

CONCLUSIONThe prognosis of CD56 positive MM patients is better than that of CD56 negative MM patients, CD19 negative MM has more abnormal karyotypes and bone marrow infiltration,but they have no statistical prognostic differences.

Chromosome Aberrations ; Chromosome Deletion ; Flow Cytometry ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Karyotyping ; Multiple Myeloma ; Prognosis

OBJECTIVETo analyse the clinical features and prognostic significance of cross-lineage antigen expression in patients with acute myeloid leukemia(AML) in order to establish individualized treatment for a better outcome and prognosis.

METHODSA total of 227 cases (exduding M3) were detected by flow cytometry for immune phenotype. The CD7(-)CD56(-)CD19(-) AML served as control. The clinical features, treatment response and prognosis of CD7(+) group, CD56(+) group and CD19(+) group were compared.

RESULTSThe detection rate of CD56(+),CD7(+) and CD19(+) in AML was 15.9%, 25.1% and 11.0%, respectively. There were no differences between CD56(+) AML, CD7(+) AML, CD19(+) AML, and CD56(-)CD7(-)CD19(-) AML in the proportion of blast cells, white blood cell count, hemoglobin level, platelet count and MDS transformed AML rate. The CR after the first course chemotherapy and cumulative CR in CD56(+) AML patients were lower than those in the control group (20.0% vs 58.1%, P=0.0099; 73.3% vs 87.5%, P=0.04). The median time of CR in CD56(+) AML was longer than that in the control group (118 days vs 46 days, P=0.04). The PFS time and OS time of CD56(+) AML were shorter than those in the control group (245 days vs 580 days, P=0.037; 494 days vs 809 days, P=0.04). The CR after the first course chemotherapy and cumulative CR in CD19(+) AML patients were higher than those in the control group(75.0% vs 58.1%, P=0.46; 100% vs 87.5%, P=0.02). The median time of CR in CD19(+) AML was shorter than that in the control group (28 days vs 46 days, P=0.02). The PFS time and OS time of CD19(+) AML tended to be longer than those in the control group (P=0.13, P=0.07, respectively). The median PFS and OS were not reached at the time of last follow-up. The CR after the first course chemotherapy, cumulative CR and median time to CR in CD7(+) AML patients were not different from those in the control group (53.1% vs 58.1%, P=0.67; 87.1% vs 87.5%, P=0.44; 50 days vs 46 days, P=0.44). No differences of PFS and OS were observed between CD7(+) AML and the control.

CONCLUSIONCD56(+) AML patients respond poorly to treatment, frequently relapse after complete remission and have a low survival rate. These patients need more intensive chemotherapy or in combination with other treatments. The interval of MRD detection should be shortened to find out relapse earlier. CD19(+) AML patients have a good treatment outcome and often accompanies with AML1/ETO fusion gene, which is known to be a good prognostic marker. Aberrant expression of CD7 on AML cells is not a poor prognostic factor in this study.

Antigens, CD ; Flow Cytometry ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; Prognosis ; Remission Induction ; Survival Rate

This study was purposed to investigate the intercellular cell adhesion molecule-1 (ICAM-1) gene K469E (A/G) (rs5498) and K56M (A/T) (rs5491) single nucleotide polymorphisms (SNP) and soluble ICAM-1 (sICAM-1) levels in plasma in three Chinese populations of Yugur, Tibetan and Han nationalities, to analyze comparatively the genotypes and allele frequencies distribution in different ethnic groups, and to explore the effects of ICAM-1 K469E and K56M polymorphism and sICAM-1 levels in plasma. EDTA-anticoagulant venous blood from Yugur(327 cases), Tibetan (400 cases) and Han (126 cases) people was collected, the DNA was extracted by using whole blood genomic DNA extraction kit, DNA SNP were analyzed by PCR-RFLP, genotype was judged by gel scan imaging system after agarose gel electrophoresis, the gene sequence was determined and the distribution of ICAM-1 genotypes and allele frequencies were compared among different ethnic groups, besides, the group representativeness was tested via the Hardy-Weinberg genetic equilibrium. Finally, the human sICAM-1 plasma levels were detected by using human ICAM-1 ELISA kit. The results showed that DNA sequencing result was consistent with PCR-RFLP analysis. In Yugur, Tibetan and Han nationalities, the KK, KE and EE three genotypes at ICAM-1 K469E gene locus were detected, the genotype distribution was not statistically significantly different, while the K, E allele frequency distribution was statistically significantly different (P < 0.05). Both of genotype and allele frequency distribution between Yugur, Tibetan and Han nationalities were statistically significantly different (P < 0.05). In K56M site only KK, KM two genotypes were detected, but the MM genotype was not detected in the three ethnic groups; the difference of two genotypes and K, M allele frequencies between Yugur and Han population was statistically significantly different (P < 0.05). Among three ethnic groups, the sex ratio and age distribution of K469E, K56M genotypes and allele frequencies of ICAM-1 gene were not significantly different, and distribution was in accordance with Hardy-Weinberg genetic equilibrium (P > 0.05). The plasma sICAM-1 level at ICAM-1 K469E allele locus in K individuals [(253 ± 122), (185 ± 97) µg/L] was higher than that at non-K allele [(145 ± 110) µg/L, P < 0.01]; the plasma sICAM-1 level of ICAM-1 K56M sites with KK genotype [(253 ± 122) µg/L] was higher than that of the KM genotypes [(168 ± 103) µg/L, P < 0.01]. In Yugur and Tibetan groups, the plasma sICAM-1 levels [(224 ± 80), (214 ± 111) µg/L] were higher than that in the Han group [(175 ± 125)µg/L, P < 0.05]. Pairwise comparison indicated that the plasma sICAM-1 levels between Yugur and Han group were statistically significantly different (P < 0.01), that was significantly different between Tibetan and Han group (P < 0.05). It is concluded that in Yugur, Tibetan and Han population, the genotypes and gene frequencies of two amino acid sites K469E and K56M in ICAM-1 were KK/KE-type, KK-type and K allele, moreover, the ratio of them in Yugur and Tibetan group was higher than that in Han, while there is not significant difference in sex ratio and age distribution, therefore, ICAM-1 genotype and allele frequency distribution in this study had ethnic representativeness. ICAM-1 gene K469E and K56M polymorphisms were likely to affect the plasma sICAM-1 expression level. K469E gene K allele may be a genetic risk factor, while K56M gene M allele a may be genetic protective factor for some diseases.
Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Ethnic Groups ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Intercellular Adhesion Molecule-1 ; blood ; genetics ; Male ; Middle Aged ; Plasma ; metabolism ; Polymorphism, Genetic ; Young Adult

The aim of this study was to investigate the similarities and differences of A1381T (rs216311) and -1793G/C (rs7966230) single nucleotide polymorphisms (SNP) in Chinese Yugur, Tibetan, and Han nationalities and their influence on plasma vWF concentration in order to explore the sensitivity of these 3 nationalities to vWF-related diseases. Peripheral venous blood was obtained from 322 Yugur, 399 Tibetan, and 120 Han healthy people. The DNA were then extracted. vWF gene A1381T and -1793G/C polymorphisms were analyzed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequenced when it was necessary. The vWF:Ag level in plasma was determined by ELISA. The results showed that the genotype distribution of vWF gene at both A1381T and -1793G/C loci in Yugur, Tibetan and Han nationalities was different with statistically significance (P < 0.05). GG genotype of A1381T locus accounted for 69.9% in Yugur nationality, which was much higher than 56.6% and 53.3% in Tibetan and Han nationalities respectively(P < 0.01); AA genotype of A1381T locus expressed a low level of vWF in plasma. For the -1793G/C locus, the proportion of CG genotype in Yugur was much higher than that in Han, CC genotype expressed a high level of vWF in plasma. The plasma vWF levels with different nationalities and the polymorphism of vWF gene were significantly different. It is concluded that the polymorphisms of vWF gene at both A1381T and -1793G/C loci in Yugur, Tibetan and Han are significantly different; the polymorphism of vWF gene influences the plasma vWF level; the plasma vWF levels in Yugur and Tibetan are significantly higher than that in Han, which may be associated with the living environment and habits.
Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; China ; Ethnic Groups ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Plasma ; chemistry ; Polymorphism, Genetic ; Young Adult ; von Willebrand Factor ; genetics ; metabolism

Different cytokines are needed in the course of culturing cells to do adoptive immunotherapy. This study was aimed to investigate the differentiation directions of lymphocytes and related gene expression characteristics after combined stimulation of lymphocytes by different cytokines or EBV antigen peptide combined with cytokines. The experiment was divided into 4 groups. The levels of total T lymphocytes (CD3(+)), T helper lymphocytes (CD3(+)CD4(+)), cytotoxic T-lymphocyte (CD3(+)CD8(+)), memory T cells (CD3(+)CD8(+)CD45RO(+)), naive T cells (CD3(+)CD8(+)CD45RA(+)), Th2 cells (CD3(+)CD30(+)), B cells (CD19(+)), NK cells (CD56(+)), naive T regulatory cells (CD4(+)CD25(+)), precise T regulatory cells (CD4(+)CD25(+)FOXP3(+)) were detected by flow cytometry. The expression levels of house-keeping gene (mad1, pten), T helper cells transcriptional regulatory gene t-bet (Th1), gata3 (Th2), cytokine IFN-γ(Th1), IL-4(Th2) were detected by using RT-PCR. The results showed that CTL in EBV polypeptide group were dominant cells with certain clinical effects. Comparison of result of EBV polypeptide group with other 3 different cytokine stimulating groups demonstrated that EBV antigen peptide had much more effects on stimulating CTL generation. The expression of IFN-γ gene was significantly increased; the T helper differentiation-related gene t-bet, gata3 also increased evidently, while expression change of house-keeping gene mad1 and pten were not evident. Addition of different cytokines and antigen peptides in culture may be much more effective on stimulating CTL generation. It is concluded that specific CTL can be obtained by using the lymphocytes co-cultured with EBV and cytokines, and the different cytokines play different roles in cell differentiation.
Cells, Cultured ; Cytokines ; immunology ; metabolism ; Epstein-Barr Virus Nuclear Antigens ; genetics ; Flow Cytometry ; Humans ; Immunotherapy, Adoptive ; Lymphocyte Count ; Lymphoma, Extranodal NK-T-Cell ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

BACKGROUNDThe presence of autoantibodies against multiple epidermal proteins is an important feature in paraneoplastic pemphigus (PNP). Circulating anti-desmoglein 3 autoantibody, the major pathogenic autoantibody in pemphigus vulgaris (PV), has been proved pathogenic in PNP. Because of many clinical differences between PNP and PV, we speculate about the involvement of other autoantibodies in the pathogenesis of PNP. Envoplakin (EPL) and periplakin (PPL) are recognized by most PNP sera. Their linker subdomains are highly homologous and necessary for the association of intermediate filaments.

METHODSWe characterized the autoantibodies against the linker subdomains of EPL and PPL in PNP patients' sera and their associated tumors by enzyme-linked immunosorbent assay (ELISA) and immunofluorence. We also applied the purified autoantibodies against EPL and PPL from PNP sera to cultured human epidermal keratinocytes (HEK), to evaluate the changes of cell-cell adhesion.

RESULTSAutoantibodies against EPL and PPL were detected in most PNP patients by ELISA, and the decrease of these autoantibodies after removal of the tumors was roughly comparable to the improvement of clinical symptoms. Cultured tumor cells from PNP patients secreted these autoantibodies. Specific immunoglobulin receptors for EPL and PPL were found on B lymphocytes in tumors from PNP. Furthermore, purified anti-EPL and anti-PPL autoantibodies from PNP sera were capable of dissociating cultured human epidermal keratinocytes.

CONCLUSIONAutoantibodies against EPL and PPL may also be pathogenic in PNP.

Adolescent ; Adult ; Autoantibodies ; immunology ; pharmacology ; Cell Adhesion ; drug effects ; Cells, Cultured ; Desmoglein 3 ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epidermis ; cytology ; Female ; Humans ; Keratinocytes ; cytology ; drug effects ; Male ; Membrane Proteins ; immunology ; Middle Aged ; Paraneoplastic Syndromes ; immunology ; metabolism ; Pemphigus ; immunology ; metabolism ; Plakins ; immunology ; Protein Precursors ; immunology ; Young Adult

Objective We investigated the in vivo effects of recombinant adenovirus-associated virus type-2(AAV-2)mediated interleukin-10(IL-10)gene transfer on the expression of matrix metalloproteinase(MMP)-2,9,tissue inhibitor of metalloproteinase(TIMP)-1,collagen type Ⅰ and type Ⅲ in a rat acute myocardial infarction model.Method Male Sprague-Dawley(SD)rats were randomly divided into three groups(each n=6):sham operation group,MI/AAV2 group,and MI/AAV2-IL-10 group(1010 vg/ml×0.1 ml injection at peri-infarct regions immediately post MI).Five days later,the expressions of MMP-2 and MMP-9 were measured by RT-PCR, Western blot and zymography.The expression of TIMP-1 was measured by RT-PCR and Western blot.Collagen type Ⅰ and type Ⅲ were assessed bv RT-PCR and immunohistochemical stain.Results The myocardial expressions of MMP-2,MMP-9 and collagen contents in MI/AAV2 group were significantly increased than those in sham operation group.Myocardial expressions of MMP-2,MMP-9 were significantly decreased and the expression of TIMP-1 significantly increased in the MI/AAV2-IL-10 group than those in MI/AAV2 group. Moreover,the expressions of collagen type Ⅰ,collagen type Ⅲ and the ratio of Ⅰ/Ⅲ collagen in border zones of infarcted myocardium were decreased by 47.6%(P＜0.01),23.6%(P＜0.05),and 17.9%(P＜0.05)respectively,while the expression of TIMP-1 increased by 73.1%(P＜0.05)in MI/AAV2-IL-10 group compared to MI/AAV2 group.Conclusion In vivo myocardial IL-10 transfer reduced myocardial MMP and collagen expression and increasing the TIMP expression.