The role of GNLY gene polymorphisms in psoriasis pathogenesis

Abstract BACKGROUND: Psoriasis is a systemic inflammatory disorder that involves complex pathogenic interactions between the innate and adaptive immune systems. The most accepted mechanism in the etiopathogenesis of psoriasis is the induction of inflammation with keratinocyte hyperproliferation. Granulysin (GNLY) is a cytolytic antimicrobial peptide (AMP) that is secreted together with granzyme and perforin from the granules of human cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It has been immunohistochemically proven that the expression of granulysin is increased in lesions of psoriasis. OBJECTIVE: This study aimed to investigate the relationship between psoriasis disease and granulysin gene polymorphisms. METHODS: GNLY rs7908 and rs10180391 polymorphisms were studied by PCR-RFLP in 100 psoriasis patients under treatment in the Dermatology Polyclinic of Bulent Ecevit University. In addition, 100 healthy individuals with similar age and sex distribution were used as a control group. RESULTS: In the control group, GNLY rs7908 CC genotype was significantly higher than in psoriasis patients (P= 0.031; OR= 0.305; Cl= 0.305 (0.121 - 0.773). In our study, the genotype distributions in patients and control groups were GNLY rs7908 (SNP) GG (51%, 37%), GC (41%, 44%), CC (8%, 19%); GNLY rs10180391 (SNP) from the CC (41%, 44%), CT (42%, % 41), TT (17%, 15%). STUDY LIMITATIONS: The study only included Turkish patients. CONCLUSION: Our findings showed that GNLY rs7908 CC genotype and C allele had a protective effect against psoriasis and decreased the disease severity (according to PASI score), whereas rs10180391 SNP did not show any effective role in psoriasis pathogenesis.

CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice

Abstract Background: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4+ CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Alemtuzumab in refractory Sézary syndrome

Abstract: Sézary syndrome is a primary cutaneous T-cell lymphoma characterized by the triad of erythroderma, lymphadenopathy and circulating atypical cells. The emergence of new molecular targets has enabled the development of drugs such as alemtuzumab, an anti-CD52 monoclonal antibody, which has shown promising results in the treatment of this entity. We report the case of a 70-year-old male with refractory Sézary syndrome in whom treatment with alemtuzumab achieved an 80% skin lesion clearance with complete haematologic and radiologic response. The treatment was discontinued after 4 months due to adverse effects, with the patient showing a sustained response without disease progression after 13 months of follow-up.

Diagnostic value of STAT6 immunohistochemistry in solitary fibrous tumor/meningeal hemangiopericytoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the diagnostic role of STAT6 immunohistochemistry in solitary fibrous tumors (SFT)/meningeal hemangiopericytomas (HPC).</p><p><b>METHOD</b>Evaluated the expression of STAT6, vimentin, CD34, EMA, PR, S-100, CD56, GFAP and Ki-67 in a cohort of 37 SFT/meningeal HPC, 30 meningiomas and 30 schwannomas by immunohistochemistry staining.</p><p><b>RESULTS</b>All SFT/meningeal HPC demonstrated nuclear positivity for STAT6, and the proportion of positive tumor cells ranged from 60% to 95%, with no significant difference cases.Vimentin was strongly positive in all cases. CD34, EMA and PR positivity was found in 32 cases, 1 case and 4 cases, respectively.S-100 protein, CD56 and GFAP were negative; Ki-67 labeling index was 1%-8%. However, the meningiomas and schwannomas were negative for STAT6.</p><p><b>CONCLUSIONS</b>STAT6 is a relatively specific biomarker for SFT/meningeal HPC, and may be used in the diagnosis and differential diagnosis of SFT/meningeal HPC, especially for the atypical cases, and allows the precise pathologic diagnosis of SFT/meningeal HPC.</p>

Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production.Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the differentA. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation.Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the differentA. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels.Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b.Conclusion A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b ofA. actinomycetemcomitans.

Construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>First, 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Inhibitory effect of arctigenin on lymphocyte activation stimulated with PMA/ionomycin / 药学学报

This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.

Effect of MHSP65-TCL anti-melanoma vaccine on the activity of immunocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the therapeutic effect of MHSP65-TCL on melanoma and its effect on the activity of the immunocytes.</p><p><b>METHODS</b>MHSP65-TCL was prepared by mixing MHSP65 with TCL derived from B16 melanoma cell lysate by repeated freezing and thawing. The MHSP65-TCL vaccine was administered in mice bearing B16 melanoma, and the changes in melanoma growth was observed. To investigate the influence of TCL in MHSP65-TCL on the activity of the immunocytes, we co-cultured TCL and mouse spleen cells in vitro, and analyzed CD69 expression on the cells, cell apoptosis, and levels of IL-10 and IFN-γ in the cell culture supernatant.</p><p><b>RESULTS</b>The MHSP65-TCL vaccine showed an anti-melanoma effect in the tumor-bearing mice. In the in vitro experiment, TCL in MHSP65-TCL strongly stimulated the activation of mouse spleen cells while causing apoptosis in some of the immunocytes and promoting cellular IL-10 secretion, but not IFN-γ.</p><p><b>CONCLUSIONS</b>MHSP65-TCL derived from B16 melanoma cells has an anti-melanoma effect mediated by the activation of immunocytes. TCL in MHSP65-TCL also has immunosuppressive effect on immunocytes possibly due to the presence of suppressive components in TCL, and identifying and eliminating these components may potentially improve the anti-tumor actovoty of MSHP65-TCL vaccine.</p>

CD69+NK cells contribute to the murine hepatitis virus strain 3-induced murine hepatitis / 华中科技大学学报（医学）（英德文版）

The role of hepatic CD69+ natural killer (NK) cells in virus-induced severe liver injury and subsequent hepatic failure is not well defined. In this study, a mouse model of fulminant liver failure (FHF) induced by murine hepatitis virus strain 3 (MHV-3) was used to study the role of hepatic CD69+NK cells in the development of FHF. The CD69 expression in NK cells in the liver, spleen, bone marrow and peripheral blood was detected by using flow cytometry. The correlation between the CD69 level in hepatic NK cells and liver injury was studied. The functional marker (CD107a), and activating and inhibitory receptor (NKG2D and NKG2A) expressed on CD69+NK cells and CD69-NK cells were detected by using flow cytometry. Pro-inflammatory cytokines (IL-9, IFN-γ and TNF-α) were also examined by using intracellular staining. After MHV-3 infection, the number of CD69+NK cells in the liver of BALB/cJ mice was increased markedly and peaked at 72 h post-infection. Similar changes were also observed in the spleen, bone marrow and peripheral blood. Meanwhile, the CD69 expression in hepatic NK cells was highly correlated with the serum level of ALT and AST. The expression of CD107a and NKG2D, as well as the production of TNF-α, IFN-γ and IL-9 in hepatic CD69+NK cells was all significantly up-regulated during 48-72 h post-infection. In contrast, the NKG2A expression was increased in hepatic CD69-NK cells but not in CD69+NK cells. These results suggested that hepatic CD69+NK cells play a pivotal role in the pathogenesis of FHF by enhancing degranulation and cytotoxic ability of NK cells and increasing the production of pro-inflammatory cytokines.

Mechanism of Allergic Asthma Pathogenesis by Protease Allergen / 한양의대학술지

Asthma is a complex immune mediated chronic inflammatory lung disease characterized by chronic inflammation of the airways, airway hyper-responsiveness and airway obstruction, and the prevalence of this disease has increased in recent years. It is well known that many features of allergic asthma are consequences of Th2 cell dominated immune responses against allergens, thus allergen specific Th2 cells play a critical role in the pathogenesis. In this review, we will discuss the properties of common indoor and outdoor allergens including house dust mite, fungus, pollen and cockroach, the activation and differentiation of naive CD4 T cells by protease allergens, how specific allergens modify host's immune system to mediate immune evasion, and regulation of homing receptor expression and trafficking of allergen specific Th2 cells. Lastly, we will also overview the general course of pathogenesis of allergic asthma and discuss prospects of development of novel immuno-therapies to asthma.

Monocyte derived dendritic cells have reduced expression of co-stimulatory molecules but are able to stimulate autologous T-cells in patients with MDS

Research has implied that the immune system plays a role in the pathogenesis of MDS and that T-cells are reacting to tumour antigen present on the surface of the malignant cells. This could imply that the immune system could be utilized to generate immune based therapy. The aim of this pilot study was to examine the feasibility of studying this further by analysing the interaction of dendritic cells with T-cells in a small cohort of MDS patients. Dendritic cells were generated in 6 MDS patients and 9 controls by culturing monocytes with GMCSF and IL-4. After activation with LPS and TNFalpha, the dendritic cells were analyzed for expression of co-stimulatory and activation antigens. Thereafter, they were co-cultured with T-cells and the T-cell response was examined by measuring the % change in expression of the activation antigen CD69. MDS MoDC had reduced expression of HLA-DR [p=0.006], CD11c [p=0.0004], CD80 [p=0.03] and CD86 [p=0.003], while resting T-cells from MDS patients had higher expression of the activation antigen CD69 on all subsets. The % change in CD69 expression increased significantly for both the control and MDS T-cells after co-culture with allogeneic dendritic cells, however this change was lower in the MDS group. Despite the increased CD69 expression prior to culture, MDS MoDC significantly up-regulated CD69 expression on autologous T-cells to values that were statistically higher than control cells. This initial study suggests that the T-cells in MDS are able to respond to dendritic cells and are therefore probably not part of the malignant clone. It further implies that the dendritic cell population could be capable of presenting antigen and initiating an immune response and therefore further study is both feasible and warranted

CD226 rs763361 [Gly307Ser] polymorphism is associated with susceptibility to rheumatoid arthritis in Zahedan, southeast Iran

Rheumatoid arthritis [RA] is a chronic inflammatory disease with many genetic factors predisposing to disease susceptibility. The aim of the present study was to investigate the impact of CD226 rs727088 and rs763361 polymorphisms and susceptibility to RA in a sample of the Iranian population. This case-control study was carried out on 100 patients with RA and 104 healthy subjects. The polymorphisms were determined using tetra amplification refractory mutation system-polymerase chain reaction assay. The rs763361 [Gly307Ser] polymorphism increased the risk of RA in codominant, dominant and recessive-tested inheritance models [odds ratio [OR] = 3.18, 95% confidence intervals [95% CI] = 1.44-7.02, P = 0.004, CC vs. TT, and OR = 1.98, 95% CI = 1.10-3.57, P = 0.023, CC vs. CT-TT, and OR = 2.61, 95% CI = 1.26-5.37, P = 0.010, CC + CT vs. TT, respectively]. In addition, the rs763361 T allele increased the risk of RA [OR = 2.06, 95% CI = 1.38-3.08, P<0.001]. However, no significant difference was observed among the groups regarding CD226 rs727088 polymorphism [Chi[2] = 3.20, P = 0.202]. Our finding showed that CD226 rs763361, but not rs727088, gene polymorphism increased the risk of RA in a sample of the Iranian population

A Case of Colonic Collision Tumor (Adenocarcinoma and Neuroendocrine Carcinoma) / 대한소화기학회지

Collision tumors of the colon are rare. A 54-year-old man was referred to our hospital for the evaluation of hematochezia. Colonoscopy demonstrated the presence of about 3 cm sized mass in the rectosigmoid junction. After surgical resection, the colonic lesion was histologically composed of two discrete lesions: adenocarcinoma in the superficial layer and poorly differentiated neuroendocrine carcinoma in the deeper layer. We report this case of colonic collision tumor (adenocarcinoma and neuroendocrine carcinoma) with a review of the literature.

Immunomodulatory activity of a lupane triterpenoid ester isolated from the eastern Nigeria mistletoe, Loranthus micranthus (Linn)

OBJECTIVE@#To provide further evidence for the ethnomedicinal use of the Eastern Nigeria mistletoe, Loranthus micranthus (L. micranthus), as an immunostimulant.@*METHODS@#Solvent fractions from the crude extract of the mistletoe plant was obtained and screened by the cell mediated delayed type hypersensitivity reaction (DTHR) model in mice. Then the immunomodulatory potentials of a major lupane triterpenoid ester isolated from an active hexane fraction of the Eastern Nigeria mistletoe was investigated. Three lupeol-based triterpenoid esters: 7β 15α-dihydroxyl-lup-20(29)-ene-3β-palmitate (I), 7β, 15α-dihydroxyl-lup-20(29)-ene-3β-stearate (II) and 7β, 15α-dihydroxyl-lup-20(29)-ene-3β-decadecanoate (III) were isolated from the plant leaves epiphyting on a local kola nut tree and were characterized. Compound 1 was subjected to cell proliferation studies using C57Bl/6 splenocytes at three dose levels (5, 25 and 100 μg/mL) in presence of controls. Furthermore, the effect of this compound on IL-8 receptor expression was evaluated at three doses (1, 5 and 10 μg/mL) using the real time polymerase chain reaction assay.@*RESULTS@#This triterpenoid ester produced some enhancement of the splenocytes at the tested doses but at doses higher than 5 μg/mL caused inhibition of the IL-8 receptor expression.@*CONCLUSIONS@#The present findings support the ethnomedicinal use of the Eastern Nigeria Mistletoe in the management of diseases affecting the immune system. The triterpenoid(s) have some immunomodulatory abilities on splenocytes and IL-8 receptors and may partly account for the overall immunomodulatory activity of this plant.

Expression of co-stimulators in ulcerative colitis and its pathologic significance / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the expression and localization of co-stimulators in the mucosa of patients with ulcerative colitis (UC), and to explore its role in the pathogenesis of UC.</p><p><b>METHODS</b>Expression of co-stimulators CD86 and inducible co-stimulator (ICOS) was studied by immunohistochemistry on paraffin-embedded mucosal tissue from patients with active UC (64 cases), inactive UC (51 cases) and normal controls (20 cases). Immunostaining for CD28 was also carried out on frozen fresh mucosal tissue sampled from patients with active UC (7 cases), inactive UC (2 cases) and normal controls (5 cases). In addition, expression of CD4, CD8 and CD20 were also examined.</p><p><b>RESULTS</b>In active UC, increased expression of CD86 was not only observed in lamina propria mononuclear cells but also in the intestinal epithelial cells, as compared with inactive UC and the normal controls (P < 0.01). Increased ICOS expression in lamina propria mononuclear cells was detected in active UC, as compared with inactive UC and the normal controls (P < 0.01). Increased ICOS expression in intestinal epithelial cells was also seen in active UC, as compared with that of inactive UC (P < 0.01). The expression of CD86 was higher in inactive UC than in the normal controls (P < 0.05 or P < 0.01). However, the expression of ICOS showed no statistically significant difference between inactive UC and normal controls. Increased expression of CD28 in active UC, compared with that in inactive UC and normal controls, was also noticed (P < 0.05 or P < 0.01). The number of CD4 or CD8-positive intraepithelial lymphocytes and lymphocytes infiltrating in the lamina propria and small vessel walls was much higher in active UC than in inactive UC and normal controls (P < 0.01). Moreover, the ratio of CD4/CD8 was highest in active UC (P < 0.01). The number of CD20-positive B lymphocytes in lamina propria was also higher in active UC than in inactive UC and normal controls (P < 0.01).</p><p><b>CONCLUSIONS</b>In active UC, CD86 and ICOS were over-expressed in the intestinal epithelial cells and lamina propria mononuclear cells. The phenomenon suggests that abnormal expression of co-stimulators may contribute to the deregulation of acquired immune responses in UC.</p>

Expression profile of immune effector molecules in natural killer cells in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expression profile of immune effector molecules in peripheral natural killer cells (NK) in patients with chronic hepatitis virus B.</p><p><b>METHODS</b>According to the infection status, patients were divided into four experiment groups: normal hepatic function and high HBV DNA level group, normal hepatic function and low HBV DNA level group, abnormal hepatic function and high HBV DNA level group and abnormal hepatic function and low HBV DNA level group. The expression of perforin (PF), granzyme B (Gr B), granulysin (GNLY), tumor necrosis factor alpha (TNFa) and interferon gamma (IFNr) in NK cells were detected by flow cytometer.</p><p><b>RESULTS</b>Compared with control group (31.50%+/-27.64%), the expression of GNLY was significantly increased in normal hepatic function and high HBV DNA level group (59.74%+/-30.82%) and normal hepatic function and low HBV DNA level group (61.89%+/-33.30%); the expression of IFNr in normal hepatic function and high HBV DNA level group (39.89%+/-21.30%) and abnormal hepatic function and high HBV DNA level group (37.54%+/-18.79%) was lower than that in normal control group (57.38%+/-23.69%); the expression of PF, GrB, GNLY in abnormal hepatic function and high HBV DNA level group (35.47%+/-29.64%, 66.55%+/-22.92%, 42.03%+/-33.17%) was lower than that in normal hepatic function and high HBV DNA level group (56.98%+/-38.34%, 81.53%+/-19.58%, 59.74%+/-30.82%) and normal hepatic function and low HBV DNA level groups (62.95%+/-31.98%, 84.51%+/-14.57%, 61.89%+/-33.3%); there were positive correlations between ef PF, Gr B, GNLY, TNFa, and IFNr.</p><p><b>CONCLUSION</b>The expression of IFNr in NK cells from patients with high HBV DNA replication level is lower than that in normal control group; the expression of PF, Gr B and GNLY in NK cells from patients with normal hepatic function is higher than that in NK cells from patients with abnormal hepatic function.</p>

Anti-inflammatory and immunosuppressive effect of phloretin / 药学学报

This study investigated the effect of phloretin (Ph) on the proliferation, activation, and cell-cycle distribution of mouse T lymphocytes and NO production and phagocytosis of macrophages. Carboxyfluorescein diacetatesuccinimidyl ester (CFDA-SE) staining plus flow cytometry assay was employed to obtain the proliferation-related index (PI) of lymphocytes. The expression levels of CD69 and CD25 on T lymphocytes stimulated with Con A were evaluated with flow cytometry after staining with fluorescent monoclonal antibody. Cell-cycle distribution of T lymphocytes was analyzed by propidium iodide staining. Griess kit was used to evaluate the NO production and fluorescent microbeads were used to analyze the phagocytosis ability of macrophages. Our results showed that phloretin (40, 60, and 80 micromol x L(-7)) significantly inhibited the proliferation of T lymphocytes and the PI reduced from 1.41 +/- 0.13 to 1.34 +/- 0.16, 1.19 +/- 0.12 and 1.07 +/- 0.06, respectively. Phloretin significantly inhibited the expression of CD69 and CD25 (P < 0.01). The cell cycle distribution analysis showed that phloretin could induce a cell cycle arrest at G0/G1 phase. NO production of LPS +IFN-gamma group of macrophages was (26.72 +/- 3.57) micromol x L(-1), and was significantly reduced by phloretin (P < 0.01). And phagocytosis rate of macrophages was significantly reduced by phloretin (P < 0.01). The results demonstrate that phloretin might be developed into a new immuosuppressive drug.

Effect of mesenchymal stem cells on expression of CD69 in cord blood CIK/NK cells and quantity ratio of T regulatory cells in CIK/NK cell culture / 中国实验血液学杂志

This study was purposed to explore the effect of bone marrow derived mesenchymal stem cells (MSCs) on the expression of CD69 on cytokine-induced killer (CIK)/natural killer (NK) cells derived from cord blood and on the quantity ratio of CD4+CD25+ T regulatory cells in CIK/NK cell culture system using Transwell non-contact cell culture system. The experiments were divided into two groups: Transwell non-contact culture and mixture culture. The ratio of MSC to CIK/ NK cells was 1:20, 1:50 and 1:100. In mixture culture groups, MSC and CIK/NK cells were co-cultured by together contact as the same ratio of Transwell non-contact culture groups. The expression of CD69 on CIK/NK cells, as well as the quantity ratio of CD4+CD25+ T regulatory cells in CIK/NK cell culture were evaluated by flow cytometry. The results showed that the expression of CD69 on CIK/NK cells in experimental groups were significantly lower than that in control group (p<0.001). As to Transwell groups, CD69 expression on the CIK/NK cells at 1:20 ratio of MSC and CIK/NK was significantly lower than that at 1:50 and 1:100 ratio. There were no differences in the expression of CD69 on CIK cells in mixture groups with various MSC ratios, whereas the expression of CD69 on NK cells at 1:20 ratio was significantly lower than that at 1:50 and 1:100. The quantity ratio of CD4+CD25+ cells in CIK/NK cell culture system of experimental groups with MSC co-culture was significantly higher than that in control. As to Transwell groups, the ratio of CD4+CD25+ cells in CIK/NK cell culture system at 1:20 and 1:50 was significantly higher than that at 1:100. The quantity ratio of CD4+CD25+ cells in CIK/NK cell culture system showed significant differences in various mixture groups. As to 1:20 ratio the amount of CD4+CD25+ cells in CIK/NK cell culture system of mixture groups was significantly higher than that in Transwell groups, while there were no differences of the quantity ratio of CD4+CD25+ cells in CIK/NK cell culture at 1:50 and 1:100. It is concluded that either by non-contact Transwell or mixed co-culture, the MSC can suppress the activation of allogeneic CB-CIK/NK cells, which maybe relate to up-regulating the ratio of CD4+CD25+ T regulatory cells in CIK/NK cell culture system in dose-dependent manner.

Efficient fusion expression of G13 domain derived from granulysin in Escherichia coli / 生物工程学报

The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.

Blocking ICOS-B7h signal pathway by ICOS-Ig fusion protein inhibits function of allogeneic T lymphocytes / 中国实验血液学杂志

Inducible costimulatory molecule (ICOS), a CD28 family member expressed on activated T cells, plays an important roles in T cell activation and effector function. This study was purposed to investigate the effect of blocking ICOS-B7h signal pathway by ICOS-Ig fusion protein on allogeneic reactive T cells and its mechanism. CHO cells stably and highly expressing ICOS-Ig were established, while the human ICOS-Ig fusion protein was harvested and purified from supernatant of CHO cells transfected with pSecTag2/Hygro A-ICOS-Ig. The CD4(+) cells from spleen of C57BL/6 mice were used as reactive cells, the bone marrow derived dendritic cells (DCs) from BALB/C mice were used as stimulatory cells, these cells were treated with different concentrations of ICOS-Ig or human Ig (h-Ig) as control. The results showed that the target protein with molecular weigh 54 kD and endotoxin level < 10 EU/ml was gained. The ICOS-Ig (> or = 10 microg/ml) could significantly inhibited the proliferative effect of allogeneic reactive T cells resulting from stimulation of DCs (p < 0.01). ICOS-Ig did not influence the activation of CD4(+) T cells. ICOS-Ig concentration positively related to the apoptosis of CD4(+) T cells. The percentages of CD4(+) Annexin V(+)PI(-) cells in simple stimulated group, ICOS-Ig 10 microg/ml group and ICOS-Ig 20 microg/ml group were 15.1%, 26.4% and 33.6% respectively. ICOS-Ig decreased secretion of TNFalpha and increased secretion of IL-4. It is concluded that the ICOS-Ig fusion protein has bioactivity of inhibiting T cell proliferation and altering the polarization of T helper cells to Th2 cells which promotes the apoptosis of allogeneic reactive T cells but had no effect on the activation of allo-reactive CD4(+) T cells.