Expansion of CD4+CD25+FoxP3+ Regulatory T Cells in Chronic Hepatitis C Virus Infection

Background: Regulatory T cells (Tregs) have been involved in impaired immunity and may have a pivotal role in persistence of viral infections. Objective: To develop a simple and reliable in-house three color flow cytometery of peripheral blood to understand the role of HCV infection in the increase of Tregs. Methods: The level of naturally occurring CD4+CD25+FoxP3+ regulatory T cells (nTregs) in 20 chronically infected with hepatitis C virus (HCV) patients was compared to those of 15 healthy individuals by flowcytometry. In a different approach we performed permeabilization and intracellular staining before surface staining which allows the preservation of the surface molecules in the combined detection process and results in the normal frequency of nTregs in blood. Results: Using the optimized method, it was shown that a significantly higher proportion of nTregs in the total CD4+ T cell population was seen in the peripheral blood of chronic HCV patients (0.83 ± 0.21%, p=0.05) as compared to controls (0.26 ± 0.1, p=0.05). Conclusions: In accordance with other studies, we showed that HCV infection induces a dramatic increase in Tregs, which might contribute to the immune response failure during HCV infection.     

RUNX3蛋白在人结直肠癌中的表达及其临床意义

背景:Runx3(runt相关转录因子3)基因是一种新发现的抑癌基因,近年研究发现Runx3异常表达与人类多种消化系肿瘤的发生密切相关。目的:研究人结直肠癌中RUNX3蛋白的表达,分析其表达与结直肠癌临床病理特征的关系。方法:以免疫组化方法检测90例结直肠癌患者的癌组织和癌旁组织中RUNX3蛋白的表达。结果:结直肠癌组织中RUNX3蛋白的阳性表达率(47.8%,43/90)显著低于癌旁结直肠黏膜组织(100%,P<0.05)。RUNX3蛋白的表达与结直肠癌患者的性别、年龄、肿瘤部位、大小和组织学类型无关(P>0.05),与肿瘤浸润深度、分化程度、Dukes分期和有无淋巴结转移有关(P<0.05),浸润越深、分化程度越低、Dukes分期越晚和有淋巴结转移的癌组织,RUNX3蛋白低表达越明显。结论:RUNX3蛋白的表达可能与结直肠癌的浸润、分化和转移相关,提示RUNX3蛋白表达下调可能对结直肠癌的发生、发展具有重要作用。     

Metastatic Testicular Carcinoma From the Colon With Clinical, Immunophenotypical, and Molecular Characterization: Report of a Case

We report a case of testicular metastasis from a colonic adenocarcinoma. The presentation of testicular metastasis, diagnosis, management, and possible modes of spread are reported. In addition to conventional investigations and histopathologic techniques, a molecular study of the testicular metastasis and colon primary, using microsatellite analysis, was performed to confirm the primary origin. Its potential uses are discussed.Presented at UroFair 2003, Singapore, February 14 to 16, 2003.     

Thymic retention of CD4+CD25+FoxP3+ T regulatory cells is associated with their peripheral deficiency and thrombocytopenia in a murine model of immune thrombocytopenia

Immune thrombocytopenia (ITP) is a bleeding disorder in which antibodies and/or T cells lead to enhanced peripheral platelet destruction and reduced bone marrow platelet production. Several reports have observed that ITP is associated with a peripheral deficiency of tolerance-inducing CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs). Using a murine model of ITP, we analyzed Tregs in the spleen and thymus. CD61 knockout mice were immunized against wild-type (CD61(+)) platelets, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice. Compared with SCID mice receiving naive splenocytes, within 2 weeks after transfer, the ITP SCID mice became thrombocytopenic (< 200 × 10(9) platelets/L) and had increased serum anti-CD61 antibodies. The quantity of thymic Tregs by 2 weeks after transfer was significantly elevated, whereas Tregs in the spleens were significantly reduced. Treatment of the ITP mice with 2 g/kg intravenous immunoglobulin raised the platelet counts, reduced antibody production, and normalized the thymic and splenic Treg populations. Compared with thymocytes from ITP mice treated with intravenous immunoglobulin, thymocytes from untreated ITP mice delayed the onset of ITP when administered before engraftment with immune splenocytes. These results suggest that ITP in mice is associated with a peripheral Treg deficiency because of thymic retention and therapy normalizes the Tregs.     

Early short-term antiretroviral therapy is associated with a reduced prevalence of CD8(+)FoxP3(+) T cells in simian immunodeficiency virus-infected controller rhesus macaques

Regulatory T cells contain a mix of CD4 and CD8 T cell subsets that can suppress immune activation and at the same time suppress immune responses, thereby contributing to disease progression. Recent studies have shown that an increased prevalence of CD8(+)FoxP3(+) T regulatory cells was associated with immune suppression and diminished viral control in simian immunodeficiency virus (SIV)-infected rhesus macaques. Preventing an increase in the prevalence of CD8 T regulatory subsets is likely to lead to a better long-term outcome. Here we show that short-term antiretroviral therapy initiated within 1 week after SIV infection was associated with lower viral set point and immune activation after withdrawal of therapy as compared to untreated animals. Early short-term treated controller animals were found to have better SIV-specific immune responses and a significantly lower prevalence of immunosuppressive CD8(+)FoxP3(+) T cells. Lower levels of CD8(+)FoxP3(+) T cells coincided with preservation of CD4(+)FoxP3(+) T cells at homeostatic levels, and significantly correlated with lower immune activation, suggesting a role for viral infection-driven immune activation in the expansion of CD8(+)FoxP3(+) T cells. Interestingly, initiation of continuous therapy later in infection did not reduce the increased prevalence of CD8(+)FoxP3(+) T cells to homeostatic levels. Taken together, our results suggest that early antiretroviral therapy preserves the integrity of the immune system leading to a lower viral set point in controller animals, and prevents alterations in the homeostatic balance between CD4(+) and CD8(+) T regulatory cells that could aid in better long-term outcome.     

Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP

Gene expression studies have identified the microenvironment as a prognostic player in diffuse large B-cell lymphoma. However, there is a lack of simple immune biomarkers that can be applied in the clinical setting and could be helpful in stratifying patients. Immunohistochemistry has been used for this purpose but the results are inconsistent. We decided to reinvestigate the immune microenvironment and its impact using immunohistochemistry, with two systems of image analysis, in a large set of patients with diffuse large B-cell lymphoma. Diagnostic tissue from 309 patients was arrayed onto tissue microarrays. Results from 161 chemoimmunotherapy-treated patients were used for outcome prediction. Positive cells, percentage stained area and numbers of pixels/area were quantified and results were compared with the purpose of inferring consistency between the two semi-automated systems. Measurement cutpoints were assessed using a recursive partitioning algorithm classifying results according to survival. Kaplan-Meier estimators and Fisher exact tests were evaluated to check for significant differences between measurement classes, and for dependence between pairs of measurements, respectively. Results were validated by multivariate analysis incorporating the International Prognostic Index. The concordance between the two systems of image analysis was surprisingly high, supporting their applicability for immunohistochemistry studies. Patients with a high density of CD3 and FoxP3 by both methods had a better outcome. Automated analysis should be the preferred method for immunohistochemistry studies. Following the use of two methods of semi-automated analysis we suggest that CD3 and FoxP3 play a role in predicting response to chemoimmunotherapy in diffuse large B-cell lymphoma.     

Human mast cells derived from fetal liver cells cultured with stem cell factor express a functional CD51/CD61 (alpha v beta 3) integrin

Human fetal livers contain progenitor cells that become mast cells after 4 weeks of culture with recombinant human stem cell factor. Expression of cell surface CD29 (beta 1), CD18 (beta 2), CD61 (beta 3), and beta 5 integrins was investigated on such cells by flow cytometry and adhesion measurements. High surface expression of CD49e, CD51, and CD61 along with kit was apparent by 4 weeks of culture, whereas expression of each at day 0 was low to undetectable. CD29 and CD49d were detected on cells from day 0 to 4 weeks of culture; CD49b, CD49c, CD49f, CD18, and CD54 expression was negligible. The fetal liver- derived mast cells spontaneously adhered to vitronectin. No evidence for degranulation was found during vitronectin-dependent adhesion. Adhesion occurred in part through the CD61/CD51 receptor. No evidence for adhesion to vitronectin through CD29 and beta 5 integrins was obtained. Almost all of the vitronectin-adherent cells expressed CD51, CD61, kit, and tryptase, and exhibited metachromasia with toluidine blue. Thus, among the fetal liver-derived cells, developing mast cells were selectively adherent to vitronectin. These mast cells and the other cell types present also adhere spontaneously to fibronectin and to laminin, this adhesion being partially inhibited by antibodies against CD61 and CD29 integrins. In conclusion, human mast cells acquire functional vitronectin receptors as they develop from fetal liver progenitors under the influence of rhSCF. This may be important for the recruitment, localization, and retention of developing mast cells.     

丁酸钠和1，25-（OH）2D3对人结肠癌细胞增殖和hTERT表达的影响

背景：端粒酶在肿瘤细胞永生化过程中起重要作用．人端粒酶逆转录酶（hTERT）是调节端粒酶活性的关键因素。有研究发现生物活性制剂丁酸钠和1n,25-二羟维生素D3[1．25-（OH）,D3]具有潜在抗肿瘤效应。目的：观察丁酸钠和1,25-（OH）：D3对人结肠癌细胞增殖的影响及其可能机制。方法：以不同浓度丁酸钠（0．5～2．0mmol／L）、1,25-（0H）2D3（10^-6～10^-6mol/L）或两者联合[1．0mmol／L丁酸钠＋10^-7mol／L1,25-（OH）2D3]诱导人结肠癌细胞株HT29,MTT法检测细胞生长抑制率,流式细胞术检测细胞周期和细胞凋亡,RT—PCR检测hTERTmRNA表达。结果：丁酸钠和1,25-（OH）2D3能剂量和时间依赖性地抑制HT29细胞生长：1．0mmol／L丁酸钠和10^-7mol／L 1．25-（OH）2D3能诱导HT29细胞G0／G1期阻滞和细胞凋亡,抑制hTERTmRNA表达。联合用药组的上述作用较两者单用更为明显（P〈0．05）。结论：丁酸钠和1,25-（OH）2D3抑制人结肠癌细胞增殖的作用与其通过下调hTERT基因表达而抑制端粒酶活性、阻滞细胞周期、诱导细胞凋亡有关：两者联合可产生协同作用．     

Anaplastic large cell lymphoma (CD30+/Ki-1+): results of a prospective clinico-pathological study of 69 cases

Summary. Sixty-nine anaplastic large cell lymphomas (ALCLs) were selected from an Italian comparative trial on MACOP-B and F-MACHOP. As no significant difference in effectiveness of the protocols emerged, they were considered homogenously treated. The ALCLs were divided into two groups according to previously defined criteria: 41 were common type (ALCLs-CT) and 28 Hodgkin-related (ALCLs-HR). T-cell phenotype was most common (58%), while B-cell, null and hybrid forms accounted for 27%, 13% and 2%. Clinically, ALCLs CT and HR differed as to mean age (27 v 34·3 years) and presentation; all ALCLs-HR showed mediastinal involvement, with bulky disease in 57%, and more frequent occurrence in stage II. In contrast, ALCLs-CT showed mediastinal masses in 58·5%, infrequently revealed bulky disease (24%), and were not specifically associated to stage. Among the ALCLs-CT, 68·4% achieved complete remission (CR), 24·4% partial remission (PR), one (2·4%) was resistant to therapy, and two (4·8%) had fatal drug toxicity. Of the ALCLs-HR, 67·8% reached CR, 14·3% PR, and 17·9% did not respond. In CR, ALCLs-CT showed a greater tendency to relapse (32·1% v 14·2%). At present, 65·8% of ALCLs-CT and 67·8% of ALCLs-HR are alive with overall survival/disease-free survival averages of 31/27 and 29/24 months respectively. Our data emphasize that, independently of subtype, ALCLs benefit from the application of third-generation protocols for high-grade non-Hodgkin's lymphomas.     

The Atypical Zeta (ζ) Isoform of Protein Kinase C Regulates CD11b/CD18 Activation in Human Neutrophils

Objective: The objective of this study was to examine the role of protein kinase C zeta (PKCζ) in interleukin (IL)‐8‐mediated activation of Mac‐1 (CD11b/CD18) in human neutrophils. Materials and Methods: Neutrophils were stimulated with IL‐8 in the presence or absence of pharmacologic inhibitors of PKC or a myristoylated PKCζ pseudosubstrate. The resulting changes in Mac‐1 surface expression, affinity, and avidity, as measured by clustering, were determined by using a combination of flow cytometry and immunofluorescence (IF). Colocalization of Mac‐1 with PKCζ was also probed using IF. Finally, neutrophil adhesion to matrix proteins was examined under static conditions and adhesion to tumor necrosis factor‐alpha‐stimulated human umbilical vein endothelial cells was examined under flow conditions, using a parallel‐plate flow chamber. Results: PKCζ and Mac‐1 colocalized following stimulation with IL‐8. Blocking PKCζ prevented IL‐8‐induced Mac‐1 clustering while simultaneously increasing Mac‐1 affinity. To determine the relative contribution of affinity versus avidity in neutrophil adhesion, we examined adhesion under both static and flow conditions, and found that blocking PKCζ prevented neutrophil adhesion, despite increased affinity of Mac‐1. Conclusions: These data suggest that PKCζ is a negative regulator of Mac‐1 affinity and a positive regulator of Mac‐1 avidity. Further, Mac‐1 avidity is more important than increased affinity alone in regulating neutrophil firm adhesion.     

Evidence for a human immunodeficiency virus type 1-mediated suppression of uninfected hematopoietic (CD34+) cells in AIDS patients.

Hematopoietic progenitor (CD34+) cells were purified from the bone marrow of 6 human immunodeficiency virus (HIV) type 1-seropositive cytopenic patients and 10 healthy donors. HIV-1-seropositive patients showed a reduced number of granulocyte/macrophage, erythroid, and megakaryocyte progenitors and also a progressive and significant decline of numbers of CD34+ cells in liquid culture, which did not result from a productive or latent HIV-1 infection of CD34+ cells. However, all HIV-1-seropositive patients showed signs of active viral replication at the bone marrow level. Moreover, virus isolates from 3 HIV-1-seropositive patients showed a dose-dependent inhibition on growth of normal CD34+ cells. This suppressive activity was almost completely reversed by incubating the virus isolates with an anti-gp120 polyclonal antibody before adding to normal CD34+ cells.     

Enhancement of proliferation and differentiation of erythroid progenitors by co-transduction of erythropoietin receptor and H-ras cDNAS into single CD34(3+) cord blood cells

Our previous studies have demonstrated that retrovirus-mediated gene transduction of either the human erythropoietin receptor (EpoR) or H-ras cDNA into single purified hematopoietic progenitor (HPC), CD34(3+), cells from cord blood (CB) resulted in increased numbers and sizes of erythroid cell containing colonies. We therefore evaluated if there were further effects when H-ras and EpoR genes were co-transduced into the same progenitor cells. Highly purified single sorted CD34(3+) CB cells were transduced with retroviral vectors encoding EpoR or H-ras cDNA. At the single cell level, and in response to stimulation by a combination of growth factors, including Epo, the number of colonies formed by BFU-E and CFU-GEMM was significantly increased in cells transduced with either single H-ras or EpoR cDNA compared to mock virus-transduced cells as previously described. Increased numbers of BFU-E, but not CFU-GEMM, colonies were produced from cells simultaneously co-transduced with both EpoR and Hras genes. Little or no growth was seen in transduced cells without exogenously added cytokines. The size of all types of colonies including CFU-GM was increased in cells transduced with H-ras and/or EpoR cDNAs, and the greatest increase was noticed in cells co-transduced with both genes. Integration and expression of either gene in individual colonies as assessed by PCR and RT-PCR analysis were 45-62% and 48-58%, respectively, with approximately 31% of the cells containing and expressing both genes. These results add to information suggesting an enhancing interacting role of H-ras and EpoR in erythroid proliferation/differentiation.     

Total cell content of CR3 (CD11b/CD18) and LFA-1 (CD11a/CD18) in neonatal neutrophils: relationship to gestational age

Neonatal neutrophils (PMN) exhibit a well-documented defect in chemotaxis that is associated with several abnormalities of PMN structure and function, including deficient surface expression of CR3 (CD11b/CD18), a critical adhesion molecule, on chemoattractant- activated PMN. We recently documented that deficient surface expression of CR3 on stimulated neonatal PMN is due principally to a deficiency in total cell content of CR3. In the current studies, we tested the hypothesis that total cell CR3 content of PMN is even more profoundly deficient in premature infants and that PMN CR3 content is directly related to gestational age. A sandwich enzyme-linked immunosorbent assay for CR3 showed that PMN lysates from term neonates ( > or = 37 weeks gestation) contain about 60% of adult PMN levels of CR3, whereas PMN from premature infants (range of 27 to 36 weeks gestation) contained a mean of about 30%, ranging from 10% to 48% (P < .001 for term [n = 6] v premature [n = 11] by unpaired t-test). When the relationship between total cell CR3 and gestational age (n = 15) was analyzed, the correlation coefficient was .94 by linear regression, and the Spearman rank correlation was significant with P < .001. PMN content of LFA-1 (CD11a/CD18) was similarly measured for 14 neonates. Term neonates were equivalent to adults in LFA-1 content of their PMN (99.4% +/- 3.2% of paired adult values, n = 6), whereas prematures (28 to 36 weeks gestation) were deficient, overall (69.1% +/- 10.4%, n = 8, P = .035). Below 35 weeks gestation, LFA-1 values ranged from 26% to 65% of paired adult control values, but no infant of > or = 35 weeks gestation had PMN LFA-1 content that was less than 85% of its adult control. We concluded that CR3 total cell content is more profoundly deficient in premature than in term neonates, that at birth there is a direct relationship between PMN CR3 content and gestational age, and that LFA-1 is deficient only in prematures less than 35 weeks of gestational age. Below 30 weeks gestation, CR3 content of PMN approached that seen in genetic deficiency of the CD18 family of leukocyte integrins, or type 1 leukocyte adhesion deficiency, underscoring the severity of this host impairment in very early preterm neonates.