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CD4+ T Lymphocytes (cd4+ t + lymphocyte)
Selected AbstractsMathematical modelling of the impact of haematopoietic stem cell-delivered gene therapy for HIVTHE JOURNAL OF GENE MEDICINE, Issue 12 2009John M. Murray Abstract Background Gene therapy represents a new treatment paradigm for HIV that is potentially delivered by a safe, once-only therapeutic intervention. Methods Using mathematical modelling, we assessed the possible impact of autologous haematopoietic stem cell (HSC) delivered, anti-HIV gene therapy. The therapy comprises a ribozyme construct (OZ1) directed to a conserved region of HIV-1 delivered by transduced HSC (OZ1+HSC). OZ1+HSC contributes to the CD4+ T lymphocyte and monocyte/macrophage cell pools that preferentially expand under the selective pressure of HIV infection. The model was used to predict the efficacy of OZ1 in a highly active antiretroviral therapy (HAART) naïve individual and a HAART-experienced individual undergoing two structured treatment operations. In the standard scenario, OZ1+HSC was taken as 20% of total body HSC. Results For a HAART-naïve individual, modelling predicts a reduction of HIV RNA at 1 and 2 years post-OZ1 therapy of 0.5 log10 and 1 log10, respectively. Eight years after OZ1 therapy, the CD4+ T-lymphocyte count was 271 cells/mm3 compared to 96 cells/mm3 for an untreated individual. In a HAART-experienced individual HIV RNA was reduced by 0.34 log10 and 0.86 log10 at 1 and 2 years. The OZ1 effect was maximal when both CD4+ T lymphocytes and monocytes/macrophages were protected from successful, productive infection by OZ1. Conclusions The modelling indicates a single infusion of HSC cell-delivered gene therapy can impact on HIV viral load and CD4 T-lymphocyte count. Given that gene therapy avoids the complications associated with HAART, there is significant potential for this approach in the treatment of HIV. Copyright © 2009 John Wiley & Sons, Ltd. [source] Allergic contact dermatitis: the cellular effectorsCONTACT DERMATITIS, Issue 1 2002Ian Kimber Contact hypersensitivity reactions are mediated by lymphocytic effector cells. Until recently it was believed that the most important of these were CD4+ T lymphocytes. However, there is growing evidence that in many instances the predominant effector cell may be a CD8+ T lymphocyte, with in some instances CD4+ cells performing a counter-regulatory function. Here we review the roles of CD4+ T helper (Th) cells and CD8+ T cytotoxic (Tc) cells, and their main functional subpopulations (respectively, Th1 and Th2 cells and Tc1 and Tc2 cells) in the elicitation of contact hypersensitivity reactions and consider the implications of effector cell selectivity for the biology of allergic contact dermatitis. [source] Quality control of CD4+ T-lymphocyte enumeration: Results from the last 9 years of the United Kingdom national external quality assessment scheme for immune monitoring (1993,2001)CYTOMETRY, Issue 2 2002Liam Whitby Abstract The human immunodeficiency virus (HIV) global epidemic has necessitated the routine enumeration of T-lymphocyte subsets, which has created a need for external quality assurance (EQA). The United Kingdom National External Quality Assessment Scheme (UK NEQAS) for Immune Monitoring provides EQA for 296 laboratories in 40 countries. In 1993, UK NEQAS developed and incorporated into its program stabilized whole blood that enables the accurate monitoring of laboratory performance. Overall, the mean interlaboratory coefficient of variation (CV) for percentage CD4+ T-lymphocyte subset enumeration has fallen from 15% to less than 5%, as a direct result of the increased use of CD45/ side scatter (SSC) gating. Laboratories using alternative gating strategies (i.e., CD45/CD14 or forward scatter [FSC]/SSC) were about 7.4 times more likely to fail an EQA exercise. Furthermore, the adoption of single-platform technology resulted in a reduction of the overall mean interlaboratory CV for absolute CD4+ T lymphocytes from 56% (prior to the widespread use of single-platform technology) to 9.7%. Individual laboratory deficiencies were also identified using a performance monitoring system and, through re-education by collaboration with the coordinating center, satisfactorily resolved. In conclusion, during the last 9 years, the UK NEQAS for Immune Monitoring program has highlighted the significant technological advances made by laboratories worldwide that undertake lymphocyte subset enumeration. Cytometry (Clin. Cytometry) 50:102,110, 2002. © 2002 Wiley-Liss, Inc. [source] Impaired nutritional status in common variable immunodeficiency patients correlates with reduced levels of serum IgA and of circulating CD4+ T lymphocytesEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 6 2001M. Muscaritoli Background Common variable immunodeficiency (CVI) is a primary defect of the immune system. Infections, persistent diarrhoea and malabsorption may result in malnutrition, which may in turn contribute to increased morbidity. In this paper, the prevalence of malnutrition in CVI was evaluated. Patients and methods Forty CVI patients (20 male, 20 female, aged 17,75 years) underwent anthropometric measurements from which body mass index, arm fat and muscle area were calculated. Body mass index values <,18·5 and arm fat and muscle area values <,10th percentile were considered indicative of malnutrition. Patients were divided into four groups according to circulating CD4+ T cells (lower or greater than 300 µL,1) and serum immunoglobulin A (IgA) levels (detectable and undetectable). Results Body mass index <,18·5, arm fat and muscle area <,10th percentile were observed in 23%, 58% and 44%, respectively, of patients. Lower values of body mass index, arm fat and muscle area were more frequent in patients with low CD4+ cells and undetectable IgA. Low arm fat values were more frequent in patients with diarrhoea (P = 0·03). Infectious episodes were more frequent in undetectable IgA than in detectable IgA patients (P = 0·04). Conclusions Anthropometric measurements revealed an increased rate of malnutrition in CVI patients, particularly in those with low CD4+ and undetectable IgA, suggesting that selected CVI subjects could be considered for standard or specialized nutritional support. [source] Expression and function of NKG2D in CD4+ T cells specific for human cytomegalovirusEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2006Andrea Sáez-Borderías Abstract The human NKG2D killer lectin-like receptor (KLR) is coupled by the DAP10 adapter to phosphoinositide 3-kinase (PI3,K) and specifically interacts with different stress-inducible molecules (i.e. MICA, MICB, ULBP) displayed by some tumour and virus-infected cells. This KLR is commonly expressed by human NK cells as well as TCR,,+ and TCR,,+CD8+ T lymphocytes, but it has been also detected in CD4+ T cells from rheumatoid arthritis and cancer patients. In the present study, we analysed NKG2D expression in human cytomegalovirus (HCMV)-specific CD4+ T lymphocytes. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from healthy seropositive individuals with HCMV promoted variable expansion of CD4+NKG2D+ T lymphocytes that coexpressed perforin. NKG2D was detected in CD28, and CD28dull subsets and was not systematically associated with the expression of other NK cell receptors (i.e. KIR, CD94/NKG2 and ILT2). Engagement of NKG2D with specific mAb synergized with TCR-dependent activation of CD4+ T cells, triggering proliferation and cytokine production (i.e. IFN-, and TNF-,). Altogether, the data support the notion that NKG2D functions as a prototypic costimulatory receptor in a subset of HCMV-specific CD4+ T lymphocytes and thus may have a role in the response against infected HLA class II+ cells displaying NKG2D ligands. [source] Infiltrating cells and related cytokines in lesional skin of patients with chronic idiopathic urticaria and positive autologous serum skin testEXPERIMENTAL DERMATOLOGY, Issue 5 2003M. Caproni Abstract:, In approximately one-third of patients with chronic idiopathic urticaria (CIU), autoantibodies against the high-affinity IgE receptor and/or against IgE can be detected and a wheal-and-flare response can be provoked by the intradermal injection of autologous serum (ASST). In this study we aimed to further characterize the inflammatory response observed in the subgroup of CIU patients with positive ASST and serum-evoked histamine-release in vitro from basophils in comparison with unaffected skin and healthy donors. An immunohistochemical analysis of infiltrating cells (CD4, MPO, EG1, EG2, tryptase), cytokines (IL-4, IL-5, IFN-,), chemokines and chemokine receptors (IL-8, CCR3, CXCR3), and adhesion molecules (ICAM-1, VCAM-1, ELAM-1) was performed on seven selected patients (four males and three females; median age: 45 years; range: 22,57) and five healthy donors. Cytokine evaluation was also performed in five psoriatic patients to obtain an additional control. In spontaneous wheals we observed an increased number of CD4+ T lymphocytes when compared with the controls, and an increased number of neutrophils and eosinophils, whereas mast cells did not show a significant variation. A significant expression for IL-4 and IL-5 could only be observed in lesional skin, while IFN-, showed a slight expression in the same site. Chemokine receptors CCR3 and CXCR3 did not show a defined polarized response in either lesional or unaffected skin. An increased expression of all cellular adhesion molecules (CAMs) studied was detected in spontaneous wheals. The lack of a significant difference in the expression of tryptase + mast cells, T lymphocytes, IL-8, CXCR3 and CCR3, a few CAMs between the lesional and unaffected skin of CIU patients suggests a wide immunological activation that involves not only lesional tissues, but possibly extends to the whole of the skin's immune system. [source] The interleukin-17 pathway is involved in human alcoholic liver disease,,HEPATOLOGY, Issue 2 2009Arnaud Lemmers Immune dysregulations in alcoholic liver diseases are still unclear, especially regarding alcoholic hepatitis inflammatory burst. Interleukin-17 (IL-17) is known to enhance neutrophil recruitment. We studied the IL-17 pathway in alcoholic cirrhosis and alcoholic hepatitis. Patients with alcoholic liver disease were compared with patients with chronic hepatitis C virus (HCV) infection or autoimmune liver disease and with healthy controls. IL-17 plasma levels and peripheral blood mononuclear cell secretion were assessed by enzyme-linked immunosorbent assay (ELISA) and T cell phenotype by flow cytometry. IL-17 staining and co-staining with CD3 and myeloperoxidase were performed on liver biopsy specimens. IL-17 receptor expression was studied on liver biopsies and in human hepatic stellate cells as well as their response to recombinant IL-17 by chemotaxis assays. IL-17 plasma levels were dramatically increased in alcoholic liver disease patients. Peripheral blood mononuclear cells of patients with alcoholic liver disease produced higher amounts of IL-17, and their CD4+ T lymphocytes disclosed an IL-17,secreting phenotype. In the liver, IL-17,secreting cells contributed to inflammatory infiltrates in alcoholic cirrhosis, and alcoholic hepatitis foci disclosed many IL-17+ cells, including T lymphocytes and neutrophils. In alcoholic liver disease, liver IL-17+ cells infiltrates correlated to model for end-stage liver disease score, and in alcoholic hepatitis to modified discriminant function. IL-17 receptor was expressed in alcoholic liver disease by hepatic stellate cells, and these cells recruited neutrophils after IL-17 stimulation in a dose-dependent manner through IL-8 and growth related oncogen , (GRO-,) secretion in vitro. Conclusion: Human alcoholic liver disease is characterized by the activation of the IL-17 pathway. In alcoholic hepatitis, liver infiltration with IL-17,secreting cell infiltrates is a key feature that might contribute to liver neutrophil recruitment. (Clinical trials number NCT00610597). (HEPATOLOGY 2009;49:646,657.) [source] Characteristic appearances of the bone marrow in T-cell large granular lymphocyte leukaemiaHISTOPATHOLOGY, Issue 5 2007N Osuji Aims:, To augment the limited literature on bone marrow (BM) appearances in T-cell large granular lymphocyte (LGL) leukaemia and to identify a histological signature to aid in diagnosis of this condition. Methods and results:, A descriptive analysis of the histology of the BM in T-cell LGL leukaemia was performed (n = 38). Antibodies against CD3, CD4, CD5, CD8, CD16, CD56, CD57 and CD20 or CD79a were employed. Antibodies against CD68 (macrophages) and CD34 (sinusoids) were also included. BM was normocellular or hypercellular in the majority of cases, with interstitial lymphoid infiltration in 97%. Lymphoid nodules were present in 55% and intrasinusoidal permeation in 58%. Apoptotic figures and haemosiderin deposition were common. All cases showed trilinear haematopoiesis with normal or increased megakaryopoiesis and erythropoiesis, but normal/reduced myelopoiesis. Reticulin was increased (Grade II,III). Immunohistochemistry revealed interstitial infiltration in all cases and helped to identify lymphoid nodules in two-thirds of cases. Preferential localization of CD8+ T lymphocytes to the interstitium and CD4+ T lymphocytes to the periphery of CD20+ B-cell nodules was seen in almost 90% of cases. Conclusions:, Nodules with non-clonal B-cell centres surrounded by CD4+ cells, with interstitial CD8+ cells, are a characteristic finding in T-cell LGL leukaemia and may represent a histological signature for this condition. [source] Expression of CD8, identifies a distinct subset of effector memory CD4+ T lymphocytesIMMUNOLOGY, Issue 2 2006Iole Macchia Summary Circulating CD4+ CD8+ T lymphocytes have been described in the peripheral blood of humans and several animal species. However, the origin and functional properties of these cells remain poorly understood. In the present study, we evaluated the frequency, phenotype and function of peripheral CD4+ CD8+ T cells in rhesus macaques. Two distinct populations of CD4+ CD8+ T cells were identified: the dominant one was CD4hi CD8lo and expressed the CD8,, homodimer, while the minor population was CD4lo CD8hi and expressed the CD8,, heterodimer. The majority of CD4hi CD8,lo T cells exhibited an activated effector/memory phenotype (CCR5lo CD7, CD28, HLA-DR+) and expressed relatively high levels of granzyme B. Intracellular cytokine staining assays demonstrated that the frequency of cytomegalovirus-specific T cells was enriched five-fold in CD4hi CD8,lo T cells compared to single-positive CD4+ T cells, whereas no consistent enrichment was observed for simian immunodeficiency virus (SIV)-specific T cells. Cross-sectional studies of SIV-infected animals demonstrated that the frequency of CD4hi CD8,lo T cells was lower in wild-type SIV-infected animals compared to uninfected controls, although prospective studies of SIV-infected animals demonstrated depletion of CD4hi CD8,lo lymphocytes only in a subset of animals. Taken together, these data suggest that CD4+ T cells expressing CD8, represent an effector/memory subset of CD4+ T cells and that this cell population can be depleted during the course of SIV infection. [source] Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-GuérinIMMUNOLOGY, Issue 1 2004Semih Esin Summary The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3, cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon-, (IFN-,)-producing cells were NK cells, with a peak IFN-, production at 24,30 hr. Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non-stimulated NW cells, the NW cells incubated for 16,20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN-, production and cytotoxic activity, on negatively selected CD56+ CD3, cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections. [source] CD4+ T lymphocytes mediate colitis in HLA-B27 transgenic rats monoassociated with nonpathogenic Bacteroides vulgatusINFLAMMATORY BOWEL DISEASES, Issue 3 2007Frank Hoentjen MD Abstract Background: HLA-B27/,2 microglobulin transgenic (TG) rats develop spontaneous colitis when raised under specific pathogen-free (SPF) conditions or after monoassociation with Bacteroides vulgatus (B. vulgatus), whereas germ-free TG rats fail to develop intestinal inflammation. SPF HLA-B27 TG rnu/rnu rats, which are congenitally athymic, remain disease free. These results indicate that commensal intestinal bacteria and T cells are both pivotal for the development of colitis in TG rats. However, it is not known if T cells are also required in the induction of colitis by a single bacterial strain. The aim of this study was therefore to investigate the role of T cells in the development of colitis in B. vulgatus,monoassociated HLA-B27 TG rats. Methods: HLA-B27 TG rnu/rnu and rnu/+ rats were monoassociated with B. vulgatus for 8,12 weeks. CD4+ T cells from mesenteric lymph nodes (MLNs) of B. vulgatus,monoassociated rnu/+ TG donor rats were transferred into B. vulgatus,monoassociated rnu/rnu TG recipients. Results:B. vulgatus,monoassociated rnu/+ rats showed higher histologic inflammatory scores and elevated colonic interferon-, mRNA, cecal myeloperoxidase, and cecal IL-1, levels compared to those in rnu/rnu TG rats that did not contain T cells. After transfer of CD4+ cells from colitic B. vulgatus,monoassociated rnu/+ TG donor rats, B. vulgatus,monoassociated rnu/rnu TG recipients developed colitis that was accompanied by B. vulgatus- induced IFN-, production by MLN cells in vitro and inflammatory parameters similar to rnu/+ TG rats. Conclusions: These results implicate CD4+ T cells in the development of colitis in HLA-B27 TG rats monoassociated with the nonpathogenic bacterial strain B. vulgatus. (Inflamm Bowel Dis 2007) [source] Chemokine receptor CXCR3 expression in inflammatory bowel diseaseINFLAMMATORY BOWEL DISEASES, Issue 4 2001Yu-Hong Yuan Abstract CD4+ T lymphocytes in the lamina propria (LP) of the gut play a central role in the immune response in inflammatory bowel disease (IBD). CXCR3 is a chemokine receptor expressed on activated T lymphocytes, and a key component for the recruitment of T helper (Th1) effector cells to the site of inflammation. To determine if CXCR3 is involved in localization of T cells to the gut in IBD patients, we investigated the expression of CXCR3 on CD4+ T lymphocytes in the LP and in the submucosa of resection specimens from 51 IBD patients and 15 control patients. Positive cells were microscopically scored using a semiquantitative analysis on a five-point scale. We found that CD4+ T cells, CXCR3+ cells, and CD4+CXCR3+ T cells in the LP were slightly increased in both IBD groups compared with control non-IBD specimens. In addition, CD4+ and CXCR3+ cells in the submucosa were significant increased in the CD group compared with the control group. CD4+ and CXCR3+ expression was not statistically different between CD and UC. Flow cytometry was used to analyze the percentage of CXCR3+ cells within the CD4+ T-cell population isolated from biopsy specimens and peripheral blood from IBD patients and control patients. There was no difference in the percentage of CD4+CXCR3+ cells between the different groups in the gut as well as in the circulation. These results suggest that CD4+CXCR3+ T cells migrate to the normal and inflamed intestinal mucosa, indicating a role in maintaining normal gut homeostasis. The selective expression of CXCR3+ cells in the submucosa of CD patients might also indicate that these cells play a role in inflammation. [source] Reciprocal activating interaction between 6-sulfo LacNAc+ dendritic cells and NK cellsINTERNATIONAL JOURNAL OF CANCER, Issue 2 2009Rebekka Wehner Abstract Dendritic cells (DCs) display an extraordinary capacity to induce T-cell responses providing the opportunity of DC-based cancer vaccination strategies. Additional findings indicate that DCs may also play a crucial role for the activation of natural killer (NK) cells, which are important effectors in innate antitumor immunity. However, studies investigating the interaction between native human DCs and NK cells are limited. Recently, we defined 6-sulfo LacNAc (slan) DCs as a major subpopulation of myeloid human blood DCs, which represent principal producers of the proinflammatory cytokines tumor necrosis factor-, and interleukin (IL)-12. Functional data revealed that slanDCs efficiently induce neoantigen-specific CD4+ T cells and activate tumor-reactive cytotoxic T cells. When evaluating the crosstalk between slanDCs and NK cells in this study, we found that lipopolysaccharide (LPS)-activated slanDCs efficiently enhance NK cell CD69 expression and interferon (IFN)-, secretion. NK cell-mediated tumor-directed cytotoxicity was significantly improved by slanDCs. NK cell activation induced by slanDCs was critically dependent on IL-12. When investigating the impact of NK cells on the immunostimulatory capacity of slanDCs, we observed that they promote DC maturation. In addition, NK cells strongly enhanced the secretion of immunomodulatory IL-12 and reduced the release of immunosuppressive IL-10 by slanDCs. IFN-, and cell-to-cell contact contributed to these effects. Furthermore, data revealed that DC-NK cell crosstalk improves slanDC-mediated differentiation of naïve CD4+ T lymphocytes into IFN-,-producing Th1 cells. In conclusion, we demonstrate a reciprocal activating interaction between slanDCs and NK cells, which may play a pivotal role in the regulation of antitumor immunity. © 2008 Wiley-Liss, Inc. [source] Spontaneous regression of bowenoid papulosis in a patient with acquired immunodeficiency syndrome after an increase in peripheral CD4+ T lymphocytesINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 2 2009Akinori Kawakami MD No abstract is available for this article. [source] Quantitative evaluation of Enterocytozoon bieneusi infection in simian immunodeficiency virus-infected rhesus monkeysJOURNAL OF MEDICAL PRIMATOLOGY, Issue 2 2003K. Sestak Abstract: The association of the microsporidia Enterocytozoon bieneusi with chronic diarrhea and wasting in individuals with acquired immunodeficiency syndrome (AIDS) has been demonstrated. The disease caused by E. bieneusi has been linked to decreased levels of circulating CD4+ T lymphocytes. In this study, we investigated the relationship between the extent of excretion of E. bieneusi in feces of simian immunodeficiency virus (SIV)-infected juvenile macaques and the CD4+ T lymphocyte counts in the peripheral blood. Twelve juvenile rhesus monkeys (Macaca mulatta) were intravenously inoculated with the pathogenic molecular clone SIVmac239. Numbers of CD4+ T lymphocytes were assessed by three-color flow cytometry. The presence of E. bieneusi DNA in feces was assessed by nested PCR. In addition, selected samples of feces were examined by competitive quantitative PCR to assess the level of E. bieneusi infection. Low (n = 5) to undetectable (n = 7) quantities of E. bieneusi were present in feces of the twelve animals in prior to inoculation with SIV. After SIV inoculation the number of animals shedding E. bieneusi increased (n = 10) as did the quantity of E. bieneusi shedding in the feces. Of the twelve juvenile animals, five animals died within 8 months post-SIV inoculation with symptoms of AIDS. Four of the five deceased animals showed shedding of E. bieneusi DNA in feces (,100 spores/g) for at least three consecutive months. Increased number of E. bieneusi in feces was accompanied by decreased counts of circulating CD4+ T lymphocytes and increased SIV plasma viral load. [source] Longitudinal follow up of SIVmac pathogenesis in rhesus macaques of Chinese origin: emergence of B cell lymphomaJOURNAL OF MEDICAL PRIMATOLOGY, Issue 4-5 2002B. Ling Abstract: Two subspecies of rhesus (Rh) macaques, the Chinese (Ch) and Indian (Ind) subspecies were infected intravenously with 100TCID50 SIVmac239. CD4+, CD8+ T cells, plasma viral loads, depletion of intestinal lymphocytes with memory phenotype, humoral immune responses and clinical courses were monitored for 600 days. The pathogenesis of SIVmac was also compared with primary human immunodeficiency virus (HIV) infection of humans. Plasma viral loads in Ch Rh were lower in the acute and chronic phases compared with Ind Rh. SIVmac pathogenesis in Ch Rh was closer to virus loads in untreated HIV infected humans. Ch Rh had higher CD4/CD8 ratios, stronger antibody responses and interestingly, less depletion of intestinal memory CCR5+ CD4+ T lymphocytes compared with Ind Rh. One Ch Rh developed B cell origin lymphoma at 570 days post-infection, the first such report in this subspecies. Three of four Ind Rh developed AIDS within 6 months. The findings indicate that Ch Rh are more resistant to SIVmac pathogenesis compared with Ind Rh and that Ch Rh paralleled HIV-1 infections in untreated adult humans. The SIVmac infected Ch Rh subspecies are an acceptable model for HIV/AIDS. [source] Vaccination against hepatitis B in liver transplant recipients: Pilot analysis of cellular immune response shows evidence of HBsAg-specific regulatory T cellsLIVER TRANSPLANTATION, Issue 3 2007Tanja Bauer After liver transplantation for hepatitis-B-related diseases, patients currently receive lifelong treatment with hepatitis B immunoglobulin to prevent endogenous reinfection with hepatitis B virus (HBV). Active immunization with hepatitis B vaccine would be a preferable alternative; however, most attempts to immunize these patients with standard vaccine have failed. A recent study with a new adjuvanted hepatitis B vaccine was exceptionally successful, leading to a high-titered long-lasting antibody response in 80% of all vaccinees. To identify the immunological mechanisms behind these unexpected results, the successfully vaccinated participants were tested for hepatitis B surface antigen (HBsAg)-specific T and B cells, and their cellular responses to revaccination with conventional vaccine were studied. HBsAg-specific CD4+ T lymphocytes could be detected in 13 of 16 patients after immunization with the new vaccine. Unexpectedly, these T cells produced almost exclusively interleukin (IL)-10 and had a CD4+/CD25+ phenotype. They were functionally active, suppressing cytokine secretion in HBsAg-specific (Th1) cells, thus representing antigen-specific regulatory T cells (TReg). Following a booster dose with conventional vaccine 22-31 months after completion of the initial vaccination series, the T-cell pattern in the revaccinated individuals changed substantially: 7 days after revaccination 9 of 11 individuals showed a switch to a Th1-type immune response with HBsAg-specific T cells secreting IL-2, interferon gamma and tumor necrosis factor alpha as observed in healthy controls. Four weeks after the booster, 4 patients still showed a Th1-type cytokine pattern, whereas in 5 patients only IL-10-secreting cells were detectable. After 1 year, in 3 of 4 revaccinated individuals only IL-10-secreting cells could be found, whereas the specific T cells of the fourth patient still showed a Th1-type of response. HBsAg-specific TReg cells could be demonstrated in HBV-positive liver transplant recipients successfully immunized with a new adjuvanted vaccine. Revaccination led to immediate disappearance of the these cells and the appearance of HBsAg-specific T cells with a Th1-type cytokine profile, which in most cases were replaced by the IL-10-secreting regulatory cells during the following months. The specific induction of TReg cells could contribute to the poor response of liver transplant recipients to conventional vaccine. In conclusion,, for successful vaccination of these patients, a vaccine with a strong inhibitory effect on TReg cells would be desirable. Liver Transpl 13:434,442, 2007. © 2007 AASLD. [source] Correlation between radiculalgia and counts of T lymphocyte subsets in the peripheral blood of patients with lumbar disc herniationORTHOPAEDIC SURGERY, Issue 4 2009Peng Tian MD Objective:, To determine the correlation between the degree of radiculalgia and counts of T lymphocyte subsets in the peripheral blood of patients with lumbar disc herniation. Methods:, Forty-nine patients with lumbar disc herniation (group A) were divided into three subgroups according to Visual Analogue Scale (VAS) pain scores (group A1: n= 12, VAS 0,4.0; A2: n= 24, VAS 4.1,7.0; A3: n= 13, VAS 7.1,10.0. Twenty health blood donors who volunteered to be involved in the study comprised the control group (group B). Peripheral blood counts of various T lymphocyte subsets were measured in each group. Results:, (i) The counts of CD4+ T and CD4+/CD8+ lymphocytes were higher in group A than in group B, and the difference between the two groups was statistically significant (P < 0.05). There were also statistically significant differences between group A and group B in the counts of CD3+ and CD8+ T lymphocytes (P < 0.05); (ii) There was no correlation between the VAS scores and the counts of CD3+ T lymphocytes (r= 0.194, P > 0.05). A strong significant correlation was observed between the VAS scores and counts of CD4+ T lymphocytes (r= 0.542, P < 0.05), CD4+/CD8+ (r= 0.468, P < 0.05), which increased with increasing VAS scores in the three subgroups of group A (P < 0.05). However there was a significant negative linear correlation between CD8+ T lymphocyte counts and pain scores (r=,0.462, P < 0.05). Conclusion:, Our results suggest that changes in T lymphocyte subsets in peripheral blood take place after prolapse of lumbar intervertebral discs. The current results may provide support for involvement of immunologic mechanisms in low back pain secondary to herniation of the lumbar disc. T lymphocytes may play an important role in the development of symptoms in patients with lumbar intervertebral disc herniation. [source] Abomasal lymph node responses to Haemonchus contortus intestinal antigens established in kid goats by infection or immunization with intestinal antigensPARASITE IMMUNOLOGY, Issue 2 2003Douglas P. Jasmer SUMMARY Immune responses to Haemonchus contortus intestinal antigens were evaluated using abomasal lymph node (ALN) lymphocytes from kid goats protected against challenge infection by immunization with parasite intestinal antigen, and from kids that were challenged after immunization with ovalbumin. ALN lymphocytes from the intestinal antigen-immunized group produced significantly higher antibody levels against intestinal antigens than the ovalbumin group, supporting the theory that immunization contributed to that ALN response. In contrast, intestinal lysates and membrane enriched preparations from intestinal cells stimulated significant proliferation of ALN lymphocytes in both groups. The proliferation was antigen-dependent, since intestinal antigens failed to stimulate proliferation in ALN lymphocytes from unimmunized and uninfected kids. For both the intestinal antigen and ovalbumin immunized groups, CD4+ T lymphocytes predominated in ALN lymphocytes that were stimulated to proliferate by intestinal antigens. The results indicate that H. contortus infection alone can induce ALN lymphocyte responses to intestinal antigens. In contrast to ALN lymphocyte responses, serum antibody against intestinal antigens was generally low to undetectable in ovalbumin-immunized kids following infection. Abomasal mucus from an H. contortus infected lamb was probed with a monoclonal antibody that binds to a periodate sensitive determinant on numerous H. contortus intestinal membrane and secreted proteins. Numerous bands of reactivity were detected, indicating that multiple parasite intestinal antigens were released into abomasal mucus during infection. The results, challenge the general concept that H. contortus intestinal antigens are ,hidden' from the host immune system during an infection. On the contrary, parasite intestinal proteins may be relatively abundant antigens presented to the host during infection. In addition, ALN T lymphocytes appear to provide a more sensitive measure than serum antibody to detect presentation of these antigens to the host immune system. [source] Mathematical modelling of the impact of haematopoietic stem cell-delivered gene therapy for HIVTHE JOURNAL OF GENE MEDICINE, Issue 12 2009John M. Murray Abstract Background Gene therapy represents a new treatment paradigm for HIV that is potentially delivered by a safe, once-only therapeutic intervention. Methods Using mathematical modelling, we assessed the possible impact of autologous haematopoietic stem cell (HSC) delivered, anti-HIV gene therapy. The therapy comprises a ribozyme construct (OZ1) directed to a conserved region of HIV-1 delivered by transduced HSC (OZ1+HSC). OZ1+HSC contributes to the CD4+ T lymphocyte and monocyte/macrophage cell pools that preferentially expand under the selective pressure of HIV infection. The model was used to predict the efficacy of OZ1 in a highly active antiretroviral therapy (HAART) naïve individual and a HAART-experienced individual undergoing two structured treatment operations. In the standard scenario, OZ1+HSC was taken as 20% of total body HSC. Results For a HAART-naïve individual, modelling predicts a reduction of HIV RNA at 1 and 2 years post-OZ1 therapy of 0.5 log10 and 1 log10, respectively. Eight years after OZ1 therapy, the CD4+ T-lymphocyte count was 271 cells/mm3 compared to 96 cells/mm3 for an untreated individual. In a HAART-experienced individual HIV RNA was reduced by 0.34 log10 and 0.86 log10 at 1 and 2 years. The OZ1 effect was maximal when both CD4+ T lymphocytes and monocytes/macrophages were protected from successful, productive infection by OZ1. Conclusions The modelling indicates a single infusion of HSC cell-delivered gene therapy can impact on HIV viral load and CD4 T-lymphocyte count. Given that gene therapy avoids the complications associated with HAART, there is significant potential for this approach in the treatment of HIV. Copyright © 2009 John Wiley & Sons, Ltd. [source] ORIGINAL ARTICLE: The Effect of High Gravidity on the Carcinogenesis of Mammary Gland in TA2 MiceAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 5 2010Xuan Wang Citation Wang X, Huang C, Sun B, Gu Y, Cui Y, Zhao X, Li Y, Zhang S. The effect of high gravidity on the carcinogenesis of mammary gland in TA2 mice. Am J Reprod Immunol 2010 Problem Spontaneous breast cancer in Tientsin Albinao 2 (TA2) mice, like human pregnancy-associated breast cancer (PABC), often occurs in pregnancy and puerperium, especially in mice with high gravidity. We hypothesized that the dysfunction of cellular immunity caused by the increase of 17,-estradiol (E2) and progesterone (P) might be one of the reasons for carcinogenesis of mammary gland. Method of study We investigated the T lymphocyte subsets and the concentration of serum hormone and cytokines in cancer-bearing, pregnant or postpartum TA2 mice using flow cytometry, chemiluminescent immunoassay, and enzyme-linked immunosorbent assay (ELISA), respectively. Results The number of T lymphocytes and the concentration of E2, P, interleukin-2 (IL-2), IL-4, and interferon-gamma (IFN-,) changed with the increase of pregnancy and puerperium. During four pregnancies, elevated E2 and P resulted in a decrease in the number of CD3+, CD4+ T lymphocytes, CD4+/CD8+ ratio, and the concentration of IL-2, IL-4, and IFN-,. Data in the fourth pregnancy were the closest to those of cancer-bearing mice. Conclusion T lymphocyte subsets and concentration of IL-2, IL-4, and IFN-, are affected by E2 and P during multiple pregnancy and delivery to some degree, which may contribute to the genesis of spontaneous breast cancer in TA2 mice. [source] ORIGINAL ARTICLE: The Characterization of the Subpopulation of Suppressive B7H4+ Macrophages and the Subpopulation of CD25+ CD4+ and FOXP3+ Regulatory T-cells in Decidua during the Secretory Cycle Phase, Arias Stella Reaction, and Spontaneous Abortion , A Preliminary ReportAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2009Lukasz Wicherek Problem, The presence of immunosuppressive cells within the endometrium and decidua is crucial for establishing maternal immune tolerance against fetal antigens. We decided to evaluate the subpopulations of Treg cells and B7H4 macrophages in eutopic endometrium typified by Arias Stella reaction during the development of Fallopian tube pregnancy as well as in decidua at the time of spontaneous abortion (SA), and to compare these findings to those observed in the endometrium during the secretory cycle phase of healthy women. Method of study, The decidual tissue samples evaluated in our study were obtained from 26 women who underwent curettage as a result of the following circumstances: five of the women because of a laparoscopic procedure necessitated by Fallopian tube pregnancy, and 11 of them because of SA. The control group consisted of 10 patients on whom curettage was preformed as an additional procedure during laparoscopic myomectomy. The presence of regulatory T-cells and B7H4-positive macrophages in the samples was analysed by fluorescence-activated cell sorter (FACScan). Results, Both the percentages of FOXP3+ cells in the subpopulation of CD25+ CD4+ T lymphocytes and the percentage of B7H4-positive cells in the macrophage subpopulation found in the deciduae of patients suffering SA were higher than those found in eutopic endometrium with Arias Stella reaction. No such differences in the percentages of these cells were observed when the tissue samples from patients with SA were compared with those from the control group. The percentage of B7H4-positive macrophages, however, was found to be significantly lower in endometrium with Arias Stella reaction in comparison to that observed in secretory endometrium. Conclusion, The alterations in both the Treg cell and suppressive B7H4+ macrophage subpopulations would seem to be related to the suppression of maternal immune cells in the endometrium at the beginning of decidualization. [source] ORIGINAL ARTICLE: TH1 , TH2 Response and the Atopy Risk in Patients with Reproduction FailureAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2009Jana Hanzlikova Problem, Enhanced TH2 activity is characteristic for atopic diseases and is observed also in physiological pregnancy. The immune causes of repeated pregnancy losses and/or repeated in vitro fertilization failure may be associated with TH2 hypoactivity. The association with frequency of atopic diseases is unclear. Method of Study, Intracellular production of IL-4 and IFN-, by peripheral CD4+ T lymphocytes was studied, as well as serum levels of total and allergen specific IgE. Simultaneously skin prick tests with inhalant allergens were performed, and clinical features of atopy were registered by means of a questionnaire. Results, Lower intracellular production of IL-4 by peripheral CD4+ T cells and lower frequency of elevated total and allergen specific IgE were found in women with reproduction failure compared to controls, as well as lower frequency of some symptoms possibly associated with atopy. Conclusion, Our study showed the presence of TH2 hypoactivity in women with reproduction failure, which may be associated with lower occurrence of atopic diseases. [source] ORIGINAL ARTICLE: Changes in the Subpopulation of CD25+ CD4+ and FOXP3+ Regulatory T Cells in Decidua with Respect to the Progression of Labor at Term and the Lack of Analogical Changes in the Subpopulation of Suppressive B7-H4+ Macrophages , A Preliminary ReportAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 2 2009Krystyna Galazka Problem, The initiation of labor is accompanied by alterations in the level of maternal immune tolerance toward fetal antigens. It is a complex molecular response leading to a brief activation of the maternal immune system with an accompanying capacity to restrict this same activation. The aim of our study was to evaluate the subpopulation of regulatory T cells (Tregs) and B7-H4 macrophages in the decidua basalis during cesarean sections performed on patients in various stages of labor. Method of study, The decidual tissue samples evaluated in our study were obtained from 23 pregnant women who underwent cesarean sections at term. Moreover, the patients were divided into three subgroups according to the progression of labor at the time of the cesarean. The presence of Treg cells and B7-H4 positive macrophages were analysed by fluorescence-activated cell sorter. Results, The percentages of FOXP3+ cells in the subpopulation of CD25+ CD4+ T lymphocytes found in the deciduas of patients decreased with the successive stages of labor, while the percentages of B7-H4 positive cells in the macrophage subpopulation remained almost constant. Conclusion, These changes in the Treg cell subpopulation in the decidua would seem to be related to a brief activation of the maternal immune system as labor begins and lack of analogical changes in the subpopulation of decidual suppressive B7-H4+ macrophages that enable the restriction of this same activation as labor progresses. [source] Pathogenic CD8+ T cells in multiple sclerosis,ANNALS OF NEUROLOGY, Issue 2 2009Manuel A. Friese MD Traditionally, autoimmune pathogeneses have been attributed to CD4+ T lymphocytes, as in multiple sclerosis (MS), rheumatoid arthritis, type 1 diabetes mellitus, and/or to B lymphocytes, as in myasthenia gravis and systemic lupus erythematosus. That is because their primary genetic associations are mostly with certain human leukocyte antigen class II alleles, whose gene products present antigens to CD4+ T cells. Because few autoimmune diseases show stronger associations with major histocompatibility complex class I alleles (ankylosing spondylitis, Behçet's disease, and psoriasis), CD8+ T cells, which interact with major histocompatibility complex class I molecules, have been largely ignored in autoimmunity research. However, a variety of findings has recently revived interest in this population, particularly in MS. First, it shows associations with major histocompatibility complex class I alleles. Second, its lesions show a predominance of CD8+ T cells. Third, these represent effectors that can directly damage central nervous system target cells. Furthermore, several clinical trials of monoclonal antibodies specifically against CD4+ T cells, or the polarizing cytokines on which they depend, have failed to show any therapeutic benefit in MS, unlike broader-spectrum antibodies that deplete all T cells. Here, we review the evidence that CD8+ T cells play a role in MS pathogenesis. Ann Neurol 2009;66:132,141 [source] Recombinant Human Erythropoietin Treatment of Chronic Renal Failure Patients Normalizes Altered Phenotype and Proliferation of CD4-positive T LymphocytesARTIFICIAL ORGANS, Issue 3 2010Katarzyna A. Lisowska Abstract Patients with chronic renal failure (CRF) receive recombinant human erythropoietin (rhEPO) for the correction of anemia. However, rhEPO also has an immunomodulatory effect. Detailed changes of phenotype and function of CD4+ T lymphocytes in CRF patients receiving rhEPO have not been reported yet; their study may bring insight into understanding of this immunomodulatory action of rhEPO. Two groups of CRF patients were included into the study: those treated; and those not receiving rhEPO. The expression of activation markers on CD4+ lymphocytes was measured with flow cytometry, both ex vivo and in vitro. The kinetics of CD4+ T lymphocytes proliferation was calculated using a dividing cells tracing method and numerical approach. Significantly higher percentages of CD4+CD95+, CD4+HLA-DR+ cells, and lower percentages of CD4+CD69+ and CD4+CD28+ cells were observed in both rhEPO-treated and untreated patients when compared with healthy controls. Changes in the proportions of CD4+CD28+ and CD4+HLA-DR+ subpopulations were dependent on the type of rhEPO, being more pronounced for rhEPO,. CD4+ lymphocytes from untreated patients exhibited decreased expression of CD28 and CD69 after stimulation in vitro, whereas the expression of these antigens on lymphocytes of rhEPO-treated patients was similar to that observed in healthy controls. Fewer CD4+CD28+ T lymphocytes of untreated patients proliferated in vitro; these cells had longer G0,G1 time, which negatively correlated with surface expression of CD28. Our study confirms that rhEPO treatment normalizes activation parameters of CD4+ T lymphocytes and their proliferative capacity, which could explain earlier described immunomodulatory effects of rhEPO in patients suffering from CRF. [source] Neuronal FasL Induces Cell Death of Encephalitogenic T LymphocytesBRAIN PATHOLOGY, Issue 3 2000A. Flügel Apoptosis of inflammatory cells plays a crucial role in the recovery from autoimmune CNS disease. However, the underlying mechanisms of apoptosis induction are as yet ill-defined. Here we report on the neuronal expression of FasL and its potential function in inducing T-cell apoptosis. Using a combination of facial nerve axotomy and passive transfer encephalomyelitis, the fate of CD4+ encephalitogenic T cells engineered to express the gene for green fluorescent protein was followed. FasL gene transcripts and FasL protein were detected in neurons by in situ -hybridization and immunohistochemistry. T cells infiltrating preferentially the injured brain parenchyma were found in the immediate vicinity of FasL expressing neurons and even inside their perikarya. In contrast to neurons, T cells rapidly underwent apoptosis. In co-cultures of hippocampal nerve cells and CD4+ T lymphocytes, we confirmed expression of FasL in neurons and concomitant induction of T-cell death. Antibodies blocking neuronal FasL were shown to have a protective effect on T-cell survival. Thus, FasL expression by neurons in neuroinflammatory diseases may constitute a pivotal mechanism underlying apoptosis of encephalitogenic T cells. [source] Infiltrating cells, related cytokines and chemokine receptors in lesional skin of patients with dermatomyositisBRITISH JOURNAL OF DERMATOLOGY, Issue 4 2004M. Caproni Summary Background, There have been only two reports on immunophenotypic characterization in the cutaneous lesions of dermatomyositis (DM) that emphasize the importance of the infiltrating CD4+ T lymphocytes. Objectives, To characterize the immunophenotype of the cells that infiltrate the lesional skin of DM and to evaluate the possible T-helper (Th) polarization Th1/Th2 through detection of specific cytokines, chemokine receptors and markers of cellular activation. Methods, Skin biopsy specimens derived from pathognomonic lesions (Gottron's papules and Gottron's sign) of eight patients with DM were immunostained with a large panel of monoclonal antibodies to CD3, CD4, CD8, myeloperoxidase (MPO), eosinophil cationic protein, tryptase, CD40, CD40 ligand (CD40L), HLA-DR, interleukin (IL)-2, IL-4, IL-5, IL-13, interferon-,, tumour necrosis factor-,, receptor 3 for CXC chemokines (CXCR3) and receptor 3 for CC chemokines, using the alkaline phosphatase,antialkaline phosphatase method. Control specimens were obtained from five healthy subjects and from six patients with discoid lupus erythematosus. Results, Activated CD4+ Th lymphocytes (HLA-DR+ CD40L+) were the principal infiltrating cells in the lesional skin of DM; the CD4/CD8 ratio was approximately 2·5. A mixed Th1/Th2 profile and higher Th1 cytokine production together with significant staining for CXCR3 were detected. Neutrophil granulocytes were the second most abundant population; eosinophil granulocytes were very poorly represented. Conclusions, Activated CD4+ T cells presumably mediate the main pathogenetic mechanisms in pathognomonic skin lesions. The interaction between CD40 and CD40L could be an important mechanism of cellular activation in cutaneous immune-mediated inflammation by induction of secretion of proinflammatory cytokines and chemokines. Neither Th1 nor Th2 clear polarization was found, although there was a slight Th1 prevalence. There was a significant quantity of MPO+ cells (neutrophil granulocytes) in the inflamed tissue, and they might have a role in sustaining the chronic inflammation. [source] Regional Immunity of the EyeACTA OPHTHALMOLOGICA, Issue 3 2010Manabu Mochizuki Abstract. This article reviews molecular mechanism of intraocular inflammation in animal models and in humans, and the immunological defence system of the eye with particular attention to ocular pigment epithelium. In experimental autoimmune uveitis (EAU), T lymphocytes, particularly CD4+ T lymphocytes, play a central role in its immunopathogenic mechanisms. In humans, activated CD4+ T cells also play a central role in the immunopathogenic mechanisms. This notion is demonstrated in two human diseases: one is Vogt,Koyanagi,Harada disease, and the other is human T-cell leukemia virus type 1 (HTLV-1) uveitis. Activated CD4+ T cells infiltrating the eye are harmful to vision-related cells and tissues in the eye and cause sight-threatening conditions. However, the eye has regional defence systems to protect itself from these harmful activated T cells. We focus on ocular pigment epithelium (PE) and demonstrate immunoregulatory activity of iris PE and retinal PE. Iris PE suppresses activated CD4+ T cells by cell-to-cell contact with a crucial role played by B7-2 molecule on iris PE and CTLA4 on T cells. The actual immunosuppressive factor being membrane bound TGF-,. In contrast, retinal PE suppresses activated CD4+ T cells by soluble factors, such as soluble TGF-, and thrombospondin 1. In addition to the direct T-cell suppression by ocular PE, ocular PE has the capacity to promote activated T cells to regulatory T cells and use them as a tool to amplify the immune down regulation in the eye. The molecular mechanisms of generation of T regulatory cells by iris PE and retinal PE is also discussed. [source] Toll-like receptor 2 agonist Pam3CSK4 enhances the induction of antigen-specific tolerance via the sublingual routeCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2008V. Lombardi Summary Background Sublingual immunotherapy (SLIT) has been established in humans as a safe and efficacious treatment for type I respiratory allergies. Objective In this study, we compared three Toll-like receptor (TLR) 2 ligands (Pam3CSK4, Porphyromonas gingivalis lipopolysaccharide and lipoteichoic acid) as potential adjuvants for sublingual allergy vaccines. Methods These molecules were tested in co-cultures of adjuvant-pre-treated dendritic cells (DCs) with murine naïve CD4+ T lymphocytes. Patterns of cytokine production, phenotype, proliferation and gene expression were analysed by ELISA, cytofluorometry and quantitative PCR, respectively. TLR2 ligands were subsequently tested in a model of SLIT in BALB/c mice sensitized with ovalbumin (OVA). Results Among the three TLR2 ligands tested, the synthetic lipopeptide Pam3CSK4 is the most potent inducer of IL-12p35 and IL-10 gene expression in murine bone marrow-derived DCs, as well as in purified oral myeloid DCs. Only Pam3CSK4-treated DCs induce IFN-, and IL-10 secretion by naïve CD4+ T cells. Sublingual administration of Pam3CSK4 together with the antigen in BALB/c mice sensitized to OVA decreases airway hyperresponsiveness as well as OVA-specific T-helper type 2 (Th2) responses in cervical lymph nodes dramatically. Conclusion Pam3CSK4 induces Th1/regulatory T cell responses, and as such, is a valid candidate adjuvant for sublingual allergy vaccines. [source] | |||||||||||||||||||||||||||||||