Differentially enhanced cytokine gene expression in CD4+ and CD8+ T cells in mesenteric lymph nodes in rats infected with Nippostrongylus brasiliensis

Autoinfective strongyloidiasis is potentially fatal, yet the majority of infected individuals harbour asymptomatic and chronic infections. The role of humoral responses in modulating autoinfection was assessed by examining antibody isotype responses to filariform larval antigens amongst chronically infected ex-Far East Prisoners of War (exFEPOWs) with longstanding (> 30 years) infection. Serum immunoglobulin (Ig)G1, IgG4, IgE and IgA responses to whole Strongyloides stercoralis L3 extracts and their constituent antigenic components were characterized by ELISA and quantitative immunoblotting. Comparison of two groups of S. stercoralis infected exFEPOWs with and without detectable larvae in stool demonstrated novel trends. Significantly enhanced recognition of six immunodominant antigenic components by IgA was associated with undetectable larval output, as was enhanced IgE recognition of several components. Additionally, IgE and IgG4 exhibited parallel antigen recognition patterns. These findings are consistent with roles for IgA in modulating larval output, for IgE in regulating autoinfection, and for IgG4 in blocking IgE-mediated responses in human strongyloidiasis.     

HIV感染者外周浅表淋巴结中CD4~+T淋巴细胞计数与胶原沉积的关系研究

目的通过对不同感染阶段HIV感染者外周浅表淋巴结中CD4+T淋巴细胞、胶原蛋白、白细胞介素(interleukin,IL)-7的检测,以及CD4+T淋巴细胞计数与胶原沉积的相关性分析,探讨HIV感染后胶原沉积对CD4+T淋巴细胞的影响。方法选择HIV感染者43例,分为HIV感染无症状组和AIDS组,留取外周浅表淋巴结活体组织检查(活检)组织;另外选择非HIV感染者12名为健康对照组,同样留取其外周浅表淋巴结活检组织。利用免疫组织化学方法检测研究对象淋巴结中CD4+T淋巴细胞、Ⅰ型胶原蛋白和IL-7定量及分布情况。结果 1随着病程进展,HIV感染者外周浅表淋巴结中胶原沉积逐渐增加,AIDS组高于无症状组,无症状组高于健康对照组,差异均有统计学意义(P均0.05);2HIV感染无症状组外周浅表淋巴结中CD4+T淋巴细胞计数与健康对照组相比差异无统计学意义(P0.05),而AIDS组则显著减少(P0.01);3HIV感染者外周浅表淋巴结中CD4+T淋巴细胞计数与胶原沉积量呈负相关(R2=0.724,P=0.000),与外周血中CD4+T淋巴细胞计数呈正相关(R2=0.702,P=0.000);43组IL-7的表达水平差异无统计学意义(P0.05),而AIDS组部分患者淋巴结中IL-7呈局部聚集性分泌。结论 HIV感染后外周浅表淋巴结中胶原沉积逐渐增加导致结构破坏,可能是CD4+T淋巴细胞进行性减少的一个重要原因,虽然IL-7有随病程进展而分泌增加的趋势,但仍不足以弥补淋巴结结构破坏对CD4+T淋巴细胞的影响。     

CXC chemokine receptor 3 expression increases the disease-inducing potential of CD4+ CD25- T cells in adoptive transfer colitis

BACKGROUND: CD4CD25 T cells induce severe colitis when injected into immunodeficient recipients. The migration of disease-inducing cells to the bowel is controlled by adhesion molecules and chemotactic proteins. Chemokine receptors expressed on the T cells are therefore potential targets for anti-inflammatory therapy in inflammatory bowel disease. In this study, we have investigated the role of the chemokine receptor CXCR3 in the development of chronic colitis in a murine model. METHOD: Expression of CXCR3 on CD4 T cell from normal and colitic mice was assessed by flow cytometry. Development of colitis was followed after transfer of either normal or CXCR3CD4CD25T cell into immunodeficient host. In addition, the ability of regulatory T cell to function in vivo in the absence of CXCR3 was tested. RESULTS: We find CXCR3 to be expressed on 80% to 90% of CD4 T cells isolated from colitic mice compared with only 4% to 10% of CD4 T cells in normal na?ve mice. Injecting CD4CXCR3CD25 T cells into immunodeficient hosts results in an ameliorated form of colitis with a lack of clinical symptoms, suggesting that CXCR3 expression is important for enteroantigen priming of CD4 T cells and/or subsequent migration into the gut wall. In contrast, CXCR3 expression does not affect the function of regulatory T cells because CXCR3 regulatory T cells are just as capable as their wild-type counterpart of controlling disease development. The diminished disease-inducing capability of CXCR3 T cells is not caused by the absence of enteroantigen specificity; we also tested the enteroantigen-specific proliferative ability of CD4CD25 T cells from CXCR3 mice in vitro and found that they respond even more strongly than wild-type cells. CONCLUSIONS: The present data indicate that CXCR3 plays an important role in controlling the migration of disease-inducing CD4CD25 T cells into the gut wall. In contrast, lack of CXCR3 expression by regulatory T cells does not compromise their function in this model of colitis.     

Increased IL-4- and IL-17-producing CD8+ cells are related to decreased CD39+CD4+Foxp3+ cells in allergic asthma

Objective: In allergic asthma, regulatory T cell (Treg) number and function are decreased. Antigen-primed CD8⁺ T cells play an indispensable role in the full development of airway inflammation and airway hyper-responsiveness (AHR) occurring in asthma. In this study, we investigated the relationship between subpopulations of CD8⁺ T cells and CD39⁺ Tregs. Methods: Female C57BL/6 mice were used to develop the model of allergic asthma. Experimental mice were immunized with ovalbumin (OVA) by intra-peritoneal (i.p) injection and then challenged with OVA by intra-tracheal administration. Control mice were immunized with vehicle by i.p injection and challenged with OVA. Airway inflammation was determined by histology and AHR was measured by an invasive method. Levels of interferon (IFN)-γ, IL-4, and IL-17 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The frequencies of CD8⁺IFN-γ⁺ cells (Tc1), CD8⁺IL-4⁺ cells (Tc2), CD8⁺IL-17⁺cells (Tc17), and CD39⁺Tregs were measured by flow cytometry. The correlation between CD39⁺Tregs and Tc subsets was analyzed by Pearson’s test. Results: Experimental mice displayed phenotypes of allergic asthma, including inflammatory cell infiltration into the lungs, goblet cell hyperplasia, increased airway resistance, and increased IL-4 and IL-17 in BALF. Compared to control mice, experimental mice displayed lower CD39⁺Tregs and Tc1 but higher Tc2 and Tc17. There was a negative correlation between CD39⁺Tregs and Tc2 or Tc17. Conclusion: In allergic asthma, increased Tc2 and Tc17 are possibly related to insufficient CD39⁺Tregs.     

Rapamycin, not cyclosporine, permits thymic generation and peripheral preservation of CD4+ CD25+ FoxP3+ T cells

Graft-versus-host-disease (GVHD) is the most common cause of poor outcome after allogeneic stem cell transplantation (SCT). Of late, exploitation of FOXP3(+) regulatory T-cell (T(REG)) function is emerging as a promising strategy in suppression of GVHD, while preserving graft-versus-leukemia (GVL). Cyclosporine and rapamycin reduce the expansion of effector T cells by blocking interleukin (IL)-2, but signaling by IL-2 is pivotal for T(REG) homeostasis. The resolution of GVHD is critically dependent on thymus-dependent reconstitution of the immunoregulatory system. Thus, there has been concern about the impact of blocking IL-2 signaling by immunosuppressive agents on T(REG) homeostasis. Here we demonstrate in a mouse model that in contrast to rapamycin, cyclosporine compromises not only the thymic generation of CD4(+)CD25(+)FoxP3(+) T cells but also their homeostatic behavior in peripheral immune compartments. Treatment with cyclosporine resulted in a sharp reduction of peripheral CD25(+)FoxP3(+) T cells in all immune compartments studied. Prolonged rapamycin treatment allowed for thymic generation of CD4(+)FoxP3(+) T cells, whereas treatment with cyclosporine led to a reduced generation of these cells. In conclusion, cyclosporine and rapamycin differentially affect homeostasis of CD4(+)FoxP3(+) T(REG) in vivo. As peripheral tolerance induction is a prerequisite for successful treatment outcome after allogeneic SCT, these findings are of potential clinical relevance.     

HIV感染后不同病程阶段淋巴结中CD4+T淋巴细胞各亚群频率的改变

目的 评价淋巴结(lymph nodes,LNs)中初始、中枢记忆性、效应记忆性和效应CD4+T淋巴细胞频率的改变,探讨伴随疾病进展,LNs中CD4+T淋巴细胞各亚群频率改变的可能原因.方法 选取HIV感染者71例,根据外周血CD4+T淋巴细胞计数和临床症状,将其分为HIV感染无症状组和AIDS患者组,并选取28例非HIV感染者作为健康对照组,留取以上3组LNs组织行活体组织检查,分离其中淋巴细胞,利用流式细胞术检测各细胞群的频率.结果 ①AIDS患者组LNs中初始CD4+T淋巴细胞频率与无症状组及健康对照组相比显著升高,而无症状组LNs中初始CD4+T淋巴细胞频率与健康对照组相比下降,但差异     

Function of CD4+CD3- cells in relation to B- and T-zone stroma in spleen

Lymphocytes from lymphotoxin (LT) alpha-deficient mice, which lack segregation of their B- and T-cell areas, acquire normal organization following adoptive transfer into RAG-deficient recipients, identifying a non-B non-T cell in the segregation process. Here we show that a CD4+CD3- accessory cell is tightly associated with discrete VCAM-1-expressing stromal cells in B- and T-cell areas of the mouse spleen. CD4+CD3- cells express high levels of LTalpha, LTbeta, and tumor necrosis factor (TNF) alpha, which are the ligands for the LTbeta receptor and TNFR1 expressed by stromal cells. The expression of these ligands is functional, as transferring CD4+CD3- cells derived from either embryonic or adult tissues into LTalpha-deficient mice organizes B/T segregation and up-regulates CCL21 protein expression in areas where T cells are segregated from B cells. We propose that the function of CD4+CD3- cells is to form a link between primed CD4 T cells and the underlying stromal elements, creating distinct microenvironments in which they enable effector responses.     

CD4+CD25+ Foxp3+调节性T细胞对ox-LDL诱导内皮细胞炎性因子表达的影响

目的:探讨CD4+CD25+调节性T细胞(Tregs)对氧化型低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HUVECs)炎性因子表达的影响.方法:磁性细胞分离器(MACS)分离CD4+CD25+T细胞及CD4+CD25-T细胞.在ox-LDL作用下,将HUVECs分别与anti-CD3 mAb 激活的CD4+CD25+T细胞、CD4+CD25-T细胞共培养24 h.分别应用流式细胞术、ELISA、real-time PCR测定Tregs对ox-LDL诱导损伤HUVECs炎性因子VCAM-1、MCP-1、IL-6表达的影响.应用凝胶电泳迁移率实验(EMSA)方法观察Tregs对ox-LDL诱导损伤HUVECs NF-κB激活的影响.结果:与对照组比较,Tregs细胞可显著抑制ox-LDL诱导损伤HUVECs炎性因子(VCAM-1、MCP-1、IL-6)及NF-κB的结合活性.结论:Tregs细胞可显著抑制ox-LDL诱导损伤HUVECs炎性因子的表达,其作用机制可能为下调了NF-κB的结合活性.     

VEGFR-3 and CD133 identify a population of CD34+ lymphatic/vascular endothelial precursor cells

Human CD133 (AC133)(+)CD34(+) stem and progenitor cells derived from fetal liver and from bone marrow and blood incorporate a functional population of circulating endothelial precursor cells. Vascular endothelial growth factor receptor 3 (VEGFR-3) regulates cardiovascular development and physiological and pathological lymphangiogenesis and angiogenesis. However, the origin of VEGFR-3(+) endothelial cells (ECs) and the mechanisms by which these cells contribute to postnatal physiological processes are not known, and the possible existence of VEGFR-3(+) lymphatic or vascular EC progenitors has not been studied. Using monoclonal antibodies to the extracellular domain of VEGFR-3, we show that 11% +/- 1% of CD34(+) cells isolated from human fetal liver, 1.9% +/- 0.8% CD34(+) cells from human cord blood, and 0.2% +/- 0.1% of CD34(+) cells from healthy adult blood donors are positive for VEGFR-3. CD34(+)VEGFR-3(+) cells from fetal liver coexpress the stem/precursor cell marker CD133 (AC133). Because mature ECs do not express CD133, coexpression of VEGFR-3 and CD133 on CD34(+) cells identifies a unique population of stem and progenitor cells. Incubation of isolated CD34(+)VEGFR-3(+) cells in EC growth medium resulted in a strong proliferation (40-fold in 2 weeks) of nonadherent VEGFR-3(+) cells. Plating of these cells resulted in the formation of adherent VEGFR-3(+)Ac-LDL(+) (Ac-LDL = acetylated low-density lipoprotein) EC monolayers expressing various vascular and lymphatic endothelial-specific surface markers, including CD34, VE-cadherin, CD51/61, CD105, LYVE-1, and podoplanin. These data demonstrate that human CD34(+)CD133(+) cells expressing VEGFR-3 constitute a phenotypically and functionally distinct population of endothelial stem and precursor cells that may play a role in postnatal lymphangiogenesis and/or angiogenesis.     

CD3+ and CD4+ cells adoptively transfer experimental hypersensitivity pneumonitis.

To characterize the cells responsible for transfer of adoptive murine experimental hypersensitivity pneumonitis (EHP), we depleted Micropolyspora faeni (M. faeni)-sensitized C3H/HeJ spleen cell (SC) cultures of CD3+, CD4+, or CD8+ cells before administration to recipients. We determined the length of time sensitization persists, the ability of cultured lung-associated lymph node (LALN) cells to transfer EHP, and the ability of cultured SC from animals subjected to two, four, or eight weekly intratracheal challenges of M. faeni to transfer EHP. The extent of pulmonary inflammatory response after challenge with intratracheal M. faeni was used to determine adoptive transfer. Depletion reduced the proportion of CD3+ cells from 21 to 1%, CD4+ cells from 15 to 3%, and CD8+ cells from 7 to 1% in the cultured SC population. The proportion of B cells exhibited reciprocal changes. Cultured SC could transfer EHP. Depletion of CD3+ and CD4+, but not CD8+ cells, ablated or diminished the capacity to transfer EHP. Sensitized cells persisted in recipients for at least 8 wk. Cultured LALN cells could transfer EHP. Recipients of cultured SC from 4- and 8-wk donors exhibited less extensive pulmonary abnormalities than recipients of cultured SC from 2-wk donors. The proportion of CD3+, CD4+, CD8+, B cells, and macrophages was the same in cultured cells from 2-, 4-, and 8-wk donors. We conclude that the active cells in SC cultures are CD3+, CD4+, and CD8- T cells, and there are differences in the ability of cultured cells to adoptively transfer EHP that are dependent on the nature of the donor but not on the phenotype of the cell population.     

CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells

Foxp3 expression was initially thought to be restricted to the CD4(+)CD25(+) regulatory T-cell population. However, recent studies suggest that forkhead box P3 (Foxp3) is expressed in CD4(+)CD25(-) T cells in aged mice. In the present study in B-cell non-Hodgkin lymphoma (NHL), we found that a subset of intratumoral but not peripheral blood CD4(+)CD25(-) T cells, comprising about 15% of intratumoral CD4(+) T cells, express Foxp3 and are capable of suppressing the proliferation of autologous infiltrating CD8(+) T cells. In vitro activation with OKT3/anti-CD28 antibody (Ab) or dendritic cells (DCs) induced Foxp3 expression in a subset of these CD4(+)CD25(-)Foxp3(-) T cells. We found that the presence of lymphoma B cells during activation augmented activation-induced Foxp3 expression in CD4(+)CD25(-) T cells. We also found that CD70(+) lymphoma B cells significantly contributed to the activation-induced Foxp3 expression in intratumoral CD4(+)CD25(-) T cells. Furthermore, the blockade of CD27-CD70 interaction by anti-CD70 Ab abrogated lymphoma B-cell-mediated induction of Foxp3 expression in intratumoral CD4(+)CD25(-) T cells. Taken together, these studies reveal a novel role for NHL B cells in the development of intratumoral regulatory T cells.     

Defective proliferation and regulatory function of CD4+ T cells bearing Leu-8 homing receptor in primary biliary cirrhosis

The majority of circulating CD4⁺ T cells express the Leu-8 peripheral lymph node homing receptor, and these cells have previously been shown to have suppressor-inducer and suppressor function. In the present study, it was found that CD4⁺, Leu-8⁺ T cells from patients with primary biliary cirrhosis (PBC) have a significantly (P<0.01) lower proliferative response when stimulated with phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM) compared to normal controls. The proliferative response of CD4⁺, Leu-8^– T cells was similar in patients and controls. However, the proliferative responses of CD4⁺, Leu-8⁺ from patients with PBC was normal when cells were stimulated with PHA, Con A, anti-CD3 monoclonal antibody, or ionomycin in combination with phorbol myristate acetate (PMA). CD4⁺ T cells from patients with PBC mediated normal helper function for PWM-stimulated immunoglobulin synthesis at high T/B ratios and their regulatory function was similar to that of normal CD4⁺ T cells that had been irradiated to inactivate their suppressor activity. When CD4⁺ T cells from patients with PBC were precultured with the combination of Con A and PMA, they mediated potent inhibitory activity similar to that of normal CD4⁺ T cells. Thus, CD4⁺, Leu-8⁺ T cells from patients with PBC have a defect of proliferation and suppressor function that is reversed by coculture with PMA. This finding suggests that impairment of a PMA-inducible lymphocyte activation pathway contributes to abnormal lymphocyte function in PBC.     

IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3(+) regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2'-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.     

HIV-1 infection and expression in human colonic cells: infection and expression in CD4+ and CD4- cell lines

Three human colonic epithelial cell lines, SW620, HT29, and T84, were characterized with respect to HIV-1 infection and gene expression. SW620 and HT29, but not T84, could be infected with HIV-1. CD4 messenger RNA and its protein product were identified in SW620 cells but not in HT29 or T84 cells. Anti-CD4 antibody blocked infection of SW620 cells but had no effect on infection of HT29 cells. In SW620 and HT29 cells transfected with the HIV-1 long terminal repeat (LTR) linked to the chloramphenicol acetyl transferase (CAT) reporter gene, an intact HIV-1 enhancer element was required for stimulation of CAT activity by tumor necrosis factor alpha (TNF alpha) and phorbol ester. T84 was not able to mediate a TNF alpha or phorbol ester response. These studies provide further evidence that HIV-1 can infect cells by mechanisms other than those mediated by the CD4 receptor and describe complementary models for analyzing HIV-1 infection and expression in colonic epithelial cells.     

Peripheral decrease and pulmonary homing of CD4+CD45RO+ helper memory T cells in cystic fibrosis

Interstitial lung disease, although of prognostic impact for patients with cystic fibrosis (CF), remains difficult to assess without histopathologic investigations. As changes of peripheral blood lymphocyte subsets (LS) may accompany severe systemic lymphocyte immune responses, we compared peripheral LS of 44 patients with CF, 23 non-CF patients with recurrent pulmonary infections and 83 healthy controls (flow cytometry; CD3, CD19, CD16, CD56, CD4, CD8, CD11b, CD45RA, CD45RO, HLA-DR and CD25 antigens). Additional immunohistochemistry was performed on lung tissue of four CF patients aged 0.5, 12, 17 and 20 years, respectively. Patients with CF showed low absolute counts of CD4+CD45RO+ memory helperT cells, CD16+CD56+ NK cells, CD8+ and interleukin-2 receptor-positive T cells in peripheral blood (P < 0.001). Similar changes were registered in the non-CF patients with pulmonary infections, indicating that those were not specific for CF. Immunohistochemistry showed activation of bronchus-associated lymphoid tissue with interstitial accumulation of CD4+CD45 RO+ T cells in the three older patients. Patients with CF show marked changes of peripheral blood LS which are presumably not CF-specific and may mirror homing to lung tissue in the course of interstitial lung disease. Further research should evaluate its usefulness in monitoring progression of lung disease in CF.     

免疫性肝损伤动物模型建立及4-1BB、CD4+CD25+T淋巴细胞表达

4-1BB是一种主要表达在活化T淋巴细胞上共刺激信号,是神经生长因了/肿瘤坏死因子受体家族成员之一。CD4^＋CD25^＋T淋巴细胞是一种免疫耐受细胞,具有免疫无能性及免疫抑制性两方面功能。本实验对免疫性肝损伤中4 1BB及CD4^＋CD25^＋T淋巴细胞的变化进行研究。[第一段]     

Oxidized LDL differentially regulates MMP-1 and TIMP-1 expression in vascular endothelial cells

We have reported recently that oxidized low-density lipoprotein (oxLDL) stimulates matrix metalloproteinase-1 (MMP-1) expression in human vascular endothelial cells. The present study was conducted to examine the effect of oxLDL on expression of Tissue inhibitor of metalloproteinase-1 (TIMP-1), an endogenous inhibitor of MMPs, in human vascular endothelial cells. Our enzyme-linked immunosorbent assay and Northern blot analysis showed that oxLDL inhibited TIMP-1 secretion and expression by human umbilical vein endothelial cells. In contrast, PMA stimulated TIMP-1 expression and secretion. Both oxLDL and PMA increased MMP-1 expression and secretion significantly as previously reported. Inhibition by oxLDL of TIMP-1 expression was also observed in human aortic endothelial cells. Collagenase activity as detected by an enzymatic activity assay demonstrated, as expected, an increase in collagenase activity in the culture medium from oxLDL-treated cells as compared with that from untreated cells. The presented data indicates that oxLDL differentially regulates TIMP-1 and MMP-1 expression, whereas PMA coordinately regulates TIMP-1 and MMP-1 in vascular endothelial cells. The lack of coordination in the secretion of MMP-1 and TIMP-1 induced by oxLDL leads to an increased collagen-degrading activity that may contribute to destabilization of atherosclerotic plaques.     

Stable changes in CD4+ T lymphocyte miRNA expression after exposure to HIV-1

MicroRNAs (miRNAs) inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4(+) T cells from HIV-1 élite long-term nonprogressors (éLTNPs), naive patients, and multiply exposed uninfected (MEU) patients, and we observed that the éLTNP patients clustered with naive patients, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated éLTNP from MEU patients and 23 miRNAs distinguished naive from MEU patients, whereas only 1 miRNA (miR-155) discriminated éLTNP from naive patients. We proposed that miRNA expression may discriminate between HIV-1-infected and -exposed but negative patients. Analysis of miRNAs expression after exposure of healthy CD4(+) T cells to gp120 in vitro confirmed our hypothesis that a miRNA profile could be the result not only of a productive infection but also of the exposure to HIV-1 products that leave a signature in immune cells. The comparison of normalized Dicer and Drosha expression in ex vivo and in vitro condition revealed that these enzymes did not affect the change of miRNA profiles, supporting the existence of a Dicer-independent biogenesis pathway.