Decreased CTLA4+ and Foxp3+ CD25highCD4+ Cells in Induced Sputum from Patients with Mild Atopic Asthma

BackgroundDetails of the comparisons between airway and peripheral blood regulatory T cells (Tregs) in patients with atopic asthma are still unclear. The objective of this study is to investigate the profiles of both airway and circulating Tregs in atopic asthma.MethodsWe measured the numbers of Tregs and eosinophils in induced sputum and peripheral blood in 28 patients with mild atopic asthma and compared these with numbers in 18 healthy controls. The frequency (%) of Tregs (surface CTLA4⁺, intracellular Foxp3⁺, and CTLA4⁺Foxp3⁺ on CD25^highCD4⁺ T cells) in sputum and blood was determined by intracellular 5-color flow cytometry. We also correlated the numbers with the level of airway hyperresponsiveness (AHR) in asthmatics.ResultsThe mean frequencies of cells expressing CTLA4⁺ (19.4 ± 2.1%, p = 0.075), Foxp3⁺ (16.4 ± 3.3%, p = 0.001), and CTLA4⁺Foxp3⁺ (7.0 ± 1.1%, p = 0.008) in induced sputum from asthmatics were significantly lower than controls (27.2 ± 3.7%, 37.4 ± 4.7%, and 18.2 ± 3.6%, respectively), whereas in peripheral blood, there was no inter-group difference in the frequencies of cells expressing CTLA4⁺ (7.1 ± 1.5% vs 5.7 ± 1.7%, p > 0.05), Foxp3⁺ (35.7 ± 3.2% vs 21.1 ± 3.9%, p > 0.05), and CTLA4⁺Foxp3⁺ (6.6 ± 1.5% vs 4.2 ± 1.0%, p > 0.05). Moreover, the frequency of CD25^highCD4⁺ cells expressing CTLA4⁺, but not Foxp3⁺, in induced sputum was associated with AHR (r = 0.60, p = 0.009) and airway eosinophilic inflammation (r = ? 0.60, p = 0.008) in asthmatics.ConclusionsAirway, but not circulating, Tregs are decreased in mild atopic asthmatics, and are negatively correlated to an increase of airway eosinophilic inflammation and AHR.     

Activated platelets in subjects at increased risk of IDDM

Summary Activated platelets respond to activated leukocytes and endothelial cells via adhesion molecules linking inflammation and thrombosis. Platelets of recent-onset insulin-dependent diabetic (IDDM) patients have been shown to be activated independent of metabolic control. This study evaluates the levels of circulating activated platelets exposing adhesion molecules in healthy subjects at increased risk of IDDM (surface markers were: P-selectin (CD62), thrombospondin, lysosomal GP53 (CD63) ). From the DENIS and the ENDIT screening programmes 19 identified islet cell antibody positive (titre ≥ 20 Juvenile Diabetes Foundation units) first degree relatives of IDDM patients (male/female 9/10; age 22 ± 15 years; body mass index (BMI): 20.0 ± 4.3 kg/m²) with clearly normal metabolism (HbA₁: 6.1 ± 0.8 %; fasting blood glucose: 4.95 ± 0.67 mmol/l) were available for this investigation. Platelet CD62 as well as thrombospondin and CD63 expression were determined by flow cytometry. We matched 50 normal volunteers for age (29 ± 6 years), anthropometric measures (male/female 26/24; BMI: 22.3 ± 2.8 kg/m²) and metabolic parameters (HbA₁: 5.8 % ± 0.3; fasting blood glucose: 4.41 ± 0.53 mmol/l) served as control subjects. The mean number of CD62⁺ platelets was increased 3.2-times in prediabetic patients: 1.94 × 2.91^±1 vs 0.60 × 1.83^±1 %, p < 0.0001. Thrombospondin⁺ and CD63⁺ platelet levels were concomitantly increased (1.45 × 2.38^±1/5.97 × 2.89^±1 % vs 0.52 × 2.01^±1/1.64 × 2.26^±1 %, p < 0.0001 for both comparisons). Thus, intravasal platelet activation is already present in potentially prediabetic subjects representing an antecedent, potentially pathogenic feature of IDDM. [Diabetologia (1997) 40: 573–577] Received: 6 September 1996 and in revised form: 16 January 1997     

Abnormal surface markers expression on bone marrow CD34<Superscript>+</Superscript> cells and correlation with disease activity in patients with systemic lupus erythematosus

Defects of hematopoietic stem cells (HSCs) have been suggested to contribute to the development of systemic lupus erythematosus (SLE). The aim of this study was to investigate the phenotypic characteristics of bone marrow (BM) CD34⁺ cells in patients with SLE and its relationship with SLE disease activity. Ten SLE patients and 10 healthy subjects were recruited and their BM CD34⁺ cells were analyzed by flow cytometric analysis with CD45/SSC gating for the expression of CD90, CD95, CD117, CD123, CD164, CD166, FAS-L, and HLA-DR. The percentage of BM CD34⁺ cells was significantly decreased in active SLE patients (1.48 ± 0.41%, n = 7) compared to the healthy controls (2.31 ± 0.75%, n = 10, p < 0.01), but no significant difference was found between the inactive patients (2.04 ± 0.44%, n = 3) and the controls. The expression of CD95, CD123, and CD166 on BM CD34⁺ cells were significantly increased in SLE patients (48.31 ± 10.59%, 44.9 ± 21.5%, 30.9 ± 19.54%, respectively, n = 10) when compared with the control subjects (24.33 ± 11.1%, 19.5 ± 4.4%, 10.7 ± 5.5%, respectively, n = 10, p < 0.05). The increased CD123 expression was negatively correlated with the number of peripheral white blood cells (r = −0.700, p < 0.05, n = 10). The percentage of CD166 expression was found significantly correlated with the index of SLE disease activity (r = 0.472, p < 0.05, n = 10) and 24 h proteinuria (r = 0.558, p < 0.05, n = 10), but negatively correlated with serum C3 level (r = −0.712, p < 0.01, n = 10). Our study found that the surface marker expression of CD95, CD123, and CD166 on BM CD34⁺ cells were significantly increased in patients. This supports the hypothesis that there are abnormalities of the HSC in SLE. Since CD166 and CD123 correlated with the overall lupus activity, their role as a biomarker of inflammatory disease activity also requires further study.     

The expression kinetics of CD137 in chronic hepatitis C patients treated with pegylated-interferon and ribavirin

Abstract

Objective. CD137, a member of the tumor necrosis factor receptor family, generates co-stimulatory signals leading to T-cell activation and proliferation for viral eradication. We examined the expression kinetics of CD137 to validate whether it can affect treatment outcomes of chronic hepatitis C (CHC) patients. Methods. The expression of CD137 on peripheral blood mononuclear cells (PBMC) from 50 CHC patients and 20 healthy controls was analyzed by flow cytometry. CD137 expression levels were examined before treatment, and every 4 weeks during treatment until week 24 or 48, and at the 24-week follow-up. Results. CD137 expression on PBMC was significantly lower in CHC patients than controls (15.5 ± 7.8% vs 23.4 ± 5.2%; p < 0.05). Patients with sustained virological response (SVR) showed higher level of CD137 expression on PBMC than treatment failures at week 4 (20.11% vs 10.97%; p < 0.05) and week 12 (15.48% vs 5.74%; p < 0.01). CD137 expression on CD4 T cells was also higher in patients with SVR at week 8 (7.75% vs 3.29%; p < 0.05). CD137 expression on PBMC from patients with SVR recovered to the control level at the 24-week follow-up. In multivariate analysis, the increased expression of CD137 at week 4 and genotype non-1 were significantly associated with SVR. Conclusions. The increased expression of CD137 within 12 weeks after the initiation of interferon therapy might be associated with a successful treatment outcome. Modulation to improve expression of CD137 might improve efficacy of CHC treatment.     

Magnesium Affects the Cytokine Secretion of CD4+ T Lymphocytes in Acute Asthma

Introduction. Magnesium (Mg) administration has been shown to promote bronchodilation and to improve lung function in asthma. It also plays an additional role in modulating the immune responses. This study was initiated to explore if Mg supplementation could affect the secretion of cytokines in acute asthmatic CD4⁺ T cells. Methods. Total serum Mg concentrations of the acute asthmatic patients and healthy controls were determined. CD4⁺ T cells were isolated from the blood of the acute asthmatic patients. They were cultured in various concentrations of Mg-supplemented (0.8, 5, 10, 15, and 20 mmol/l) medium. Cytokine (IL-5, IL-13, and IFN-γ) levels were determined by Enzyme-Linked Immunosorbnent Assay (ELISA). Results. Serum Mg concentration was lower in the acute asthmatic patients than that in the healthy controls (p < .05). The secretion of IL-5 and IL-13 was decreased, while the acute asthmatic CD4⁺ T cells were cultured in 10 and 15 mmol/l Mg-supplemented medium, respectively, as compared to the 0.8 mmol/l Mg group (p < .05). The secretion of IFN-γ increased in the 10 mmol/l Mg group (p < .05). Conclusion. Mg supplementation was able to modulate the immune responses of acute asthmatic CD4⁺ T cells and decrease the secretion of type 2 CD4⁺ T lymphocytes cytokines.     

Expansion of CD4+CD25+FoxP3+ regulatory T cells in hepatitis C virus‐related chronic hepatitis,cirrhosis and hepatocellular carcinoma

Aim: Regulatory T (Treg) cells may play a pivotal role in the persistence of hepatitis C virus (HCV) infection and the development of hepatocellular carcinoma (HCC). Therefore, we examined their frequency in peripheral blood from patients with HCV‐positive chronic hepatitis (CH), cirrhosis (LC) and HCC. Methods: Treg cells were identified as CD4⁺, CD25⁺ and FoxP3⁺ T lymphocytes using three‐color FACS. The frequency of Treg cells was expressed as a percentage of the total CD4⁺ T lymphocytes, and the phenotype of Treg cells was examined using CD45RA. Results: Treg cells were significantly increased in CH (5.88 ± 0.19%, n = 76; P < 0.01), LC (6.10 ± 0.28%, n = 40; P < 0.001) and HCC (6.80 ± 0.30%, n = 57; P < 0.0001) compared to healthy control (5.13 ± 0.25%, n = 31). However, Treg cells were not increased with the progression of fibrosis or the grade of inflammations. Treg cells were slightly increased in early‐stage HCC (6.91 ± 0.40%) compared with advanced‐stage HCC (6.58 ± 0.39%), but these results were not statistically significant. In a serial examination, a distinct increase in Treg cells after local therapy for early‐stage HCC was a hallmark of early recurrence. Most expanded Treg cells in HCC were CD45RA^‐, suggesting that a memory‐type Treg population had differentiated in the periphery and not in the thymus. Conclusion: We observed an increase in Treg cells in HCV‐related chronic liver disease, particularly in HCC, and these cells were shown to be memory‐type Treg cells.     

CD4+Foxp3+ T cells,interleukin-35 (IL-35) and IL-10 in systemic lupus erythematosus patients: Relation to disease activity

Aim of the workTo evaluate three subtypes of CD4⁺Foxp3⁺ T cells, interleukin-35 (IL-35) and IL-10 in systemic lupus eryhtematosus (SLE) patients and study their relation to disease activity.Patients and methodsFifty SLE patients were included and divided according to the SLE disease activity index (SLEDAI) into 2 equal groups with activity or in remission. Twenty healthy subjects were included as controls. All subjects underwent flow cytometric analysis of CD4, CD25, CXCR5 and Foxp3 expression on T cells. Serum IL-35 and IL-10 levels were measured by ELISA.ResultsPatients were 46 females and 4 males with a mean age of 38.0 ± 10.0 years, disease duration of 9.2 ± 6.0 years. The mean SLEDAI was 6.8 ± 3.7 in active ones. SLE patients especially those with activity had significantly reduced percents of CD4⁺CD25⁺Foxp3⁺ and CD4⁺CXCR5⁺Foxp3⁺ T cells, but increased percents of CD4⁺CD25⁻Foxp3⁺ T cells. This was accompanied by significant higher levels of serum IL-35 and IL-10 (p < 0.0001). The SLEDAI in active patients significantly correlated with CD4⁺CD25⁻Foxp3⁺ T cell percent, serum IL-35 and IL-10 levels (p < 0.05) and inversely with the CD4⁺CD25⁺Foxp3⁺ and CD4⁺CXCR5⁺Foxp3⁺ T cell percents (p < 0.05). At cut-off values of 3.29% for CD4⁺CD25⁺Foxp3⁺ T cell, 7.62% for CD4⁺CD25⁻Foxp3⁺ T cell, 1.77% for CD4⁺CXCR5⁺Foxp3⁺ T cell, 22.04 pg/ml for IL-35 level and 30.51 pg/ml for serum IL-10 level were found to be highly sensitive and specific for detecting lupus activity.ConclusionCD4⁺Foxp3⁺ T cells, IL-35 and IL-10 showed high sensitivity and specificity for detecting SLE activity and may be considered as potentially promising therapeutic targets.     

Expression of interferon-g and interleukin-4 production in CD4+ T cells in patients with chronic heart failure

The prevalence of inflammation is high among patients with chronic heart failure (CHF). Reduced ejection fraction was associated with frequency of CD4⁺ T cells of leukocytes. Therefore, we investigated inflammatory cytokines of expression markers in CD4⁺ T cells in patients with CHF. Blood samples were obtained from 103 patients with CHF, from 83 patients with stable angina (SA), and from 57 controls. Interferon-γ (IFN-γ)-positive CD4⁺ T cells and interleukin-4 (IL-4)-positive CD4⁺ T cells were analyzed using 3-color flow cytometry. The frequency (%) of IFN-γ-positive CD4⁺ T cells increased in patients with CHF compared with those with SA and controls (CHF: 28.3 ± 13.8, SA: 23.50 ± 10.38, controls: 19.00 ± 7.45, P < 0.001). There was no significant difference in the frequency of IL-4-positive CD4⁺ T cells among the three groups. The frequencies of CD4⁺ T cells that stained for IFN-γ decreased from 32.37% ± 16.40% on admission to 26.91% ± 12.53% after 2 weeks in 26 patients with CHF. B-type natriuretic peptide (pg/ml) and high-sensitivity C-reactive protein (mg/dl) levels decreased from 251.7 ± 150.4 and 0.64 ± 0.78 on admission to 208.2 ± 166.4 and 0.36 ± 0.34 after 2 weeks in the 26 patients with CHF. We have demonstrated expression of IFN-γ production of CD4⁺ T cells during CHF. Prevention of unwanted T cell activation could represent a new target in the treatment of CHF.     

CD34+ and CFU-GM progenitors are significantly decreased in sivsmm9 infected rhesus macaques with minimal evidence of direct viral infection by polymerase chain reaction

Hematologic abnormalities in the peripheral blood and bone marrow are associated with human immunodeficiency and simian immunodeficiency virus (HIV, SIV) infection. The reasons for these abnormalities remain unclear. Bone marrow specimens collected from uninfected animals (Group A, Controls) and from rhesus macaques experimentally infected with SIVsmm9 during the asymptomatic stage (Group B, SIV⁺ “well”) and during the clinically ill stage (Group C, SIV⁺ “sick”), underwent a variety of in vitro assays of hematopoiesis. Colony forming unit-granulocyte/macrophage (CFU-GM) per plate growth was 46.7 ± 7.7, 31.9 ± 8.4 and 6.5 ± 5.0 (means ± sd, P <.02 each mean compared to the others) in the 3 groups respectively. Burst forming unit-erythroid (BFU-E) growth was similarly decreased in bone marrow samples from the SIV⁺ animals. There was no change in the number of CFU-GM per plate with the removal of plastic adherent or T-cell mononuclear cell fractions. There was no increase in CFU-GM per plate growth with the addition of low dose GM-CSF (1 or 5 ng/mL) though there was a near 67% increase (48 to 80 CFU-GM per plate) with the addition of 100 ng/mL recombinant rhesus IL-3 and 100 ng/mL GM-CSF in SIV⁺ 'sick' animals. Variation in frequency of CD34⁺ progenitor cells in SIV⁺ animals was seen, with 3.0% CD34 cells in SIV^? controls, 4.9% CD34⁺ cells in SIV⁺ 'well' animals and 0.5% CD34⁺ progenitor cells in SIV⁺ 'sick' monkeys (P <.01, each mean compared to the others). Finally, there was minimal evidence of SIV sequences by polymerase chain reaction in pooled cultured CFU-GM, and no evidence in flowcytometrically sorted CD34⁺ progenitor cells from selected animals. Thus, the SIV seropositive rhesus monkey appears to have similar hematopoietic aberrations as are found in HIV infected human subjects and may be an excellent model for studying the interaction of lentiviruses on the kinetics of blood formation.     

A transforming growth factor-β1-mediated bystander immune suppression could be associated with remission of chronic idiopathic thrombocytopenic purpura

 Bystander immune suppression has been demonstrated in experimental models of oral immune tolerance induction. This phenomenon is associated with expression of transforming growth factor (TGF)-β1 and T-helper cell (Th) 2 cytokines. We have studied serum levels of Th cytokines and B- and T-lymphocyte subsets in chronic idiopathic thrombocytopenic purpura (ITP), a disorder in which the production of platelet autoantibodies might be caused by a cytokine network dysregulation. Forty-six patients with ITP were separated into three groups depending on the platelet count (pltc): (1) <50×10⁹/l, (2) 50–150×10⁹/l and (3) >150×10⁹/l. We found significantly elevated plasma levels of the Th3 cytokine TGF-β1 in patients with pltc >150×10⁹/l (23.5±2.8 ng/ml), compared with patients with pltc <50×10⁹/l (2.3±0.6 ng/ml;P<0.0001), patients with pltc 50–150×10⁹/l (7.2±1.7 ng/ml;P<0.0001) and healthy volunteers (9.8±1.3 ng/ml;P<0.01). The serum levels of the Th1 cytokines interleukin (IL)-2 and interferon (IFN)-γ were below the detection limits of the assays. Likewise, the Th2 cytokine IL-4 was not detectable or was very low both in patients and controls. The serum levels of IL-10, a Th2 cytokine, were within the assay range and patients with pltc <50×10⁹/l had significantly lower levels (0.6±0.1 pg/ml) than both patients with pltc 50–150×10⁹/l (1.8±0.1 pg/ml;P<0.005) and healthy volunteers (1.4±0.1 pg/ml;P<0.005). Furthermore, patients with pltc <50×10⁹/l and splenectomised patients had significantly higher levels of CD4+CD25+ activated T cells [26.2±14.8% (P<0.05) and 26.7±11.9% (P<0.005), respectively] than healthy controls (16.5±4.0%). Also, the number of natural killer (NK) cells among patients with pltc >150×10⁹/l were significantly elevated (26.6±16.0%;P<0.05) compared with controls (17.4±7.6%). In conclusion, our data corroborate previous findings of elevated numbers of activated T cells in chronic ITP patients with active disease, but neither a clear-cut Th1 nor a Th2 serum cytokine profile could be established. However, ITP in remission was associated with elevated TGF-β1, which might be a part of a bystander immune suppression. We propose that the effect of possible expression of TGF-β1 by oral immune tolerance induction deserves to be explored in ITP patients with an active disease. Received: 29 September 1999 / Accepted: 25 January 2000     

Microscopic colitis patients have increased proportions of Ki67+ proliferating and CD45RO+ active/memory CD8+ and CD4+8+ mucosal T cells

BackgroundCollagenous colitis (CC) and lymphocytic colitis (LC) are chronic inflammatory bowel disorders of unknown etiology. This study investigated phenotypic characteristics of the mucosal lymphocytes in CC and LC.MethodsLamina propria and intraepithelial lymphocytes (LPLs, IELs) isolated from mucosal biopsies from CC (n = 7), LC (n = 6), as well as LC or CC patients in histopathological remission, (LC-HR) (n = 6) and CC-HR (n = 4) and non-inflamed controls (n = 10) were phenotypically characterized by four-color flow cytometry.ResultsThe proportions of CD8⁺ IELs were increased in CC and LC (p < 0.01) compared to controls. Increased proportions of CD45RO⁺CD8⁺ IELs and LPLs were observed in LC and even more in CC patients (p < 0.01). Both CC (p < 0.05) and LC patients had elevated proportions of CD4⁺8⁺ IELs and LPLs compared to controls. The proportions of CD45RO⁺ cells were increased in CD4⁺8⁺ IELs and LPLs (p < 0.05) in CC and LC patients compared to controls. Both CC (p < 0.05) and LC patients had higher proportions of Ki67⁺CD8⁺ IELs and LPLs compared to controls.In contrast, decreased proportions of CD4⁺ LPLs were observed in CC and LC as well as CD4⁺ IELs in LC compared to controls. Increased proportions of Ki67⁺CD4⁺ IELs and LPLs (p < 0.05) were observed in CC and LC patients.CC-HR but not LC-HR patients demonstrated normalized proportions of both IELs and LPLs compared to CC and LC patients respectively.ConclusionLC and CC patients have differences in mucosal lymphocyte subsets, with increased proportions of Ki67⁺ and CD45RO⁺ CD8⁺ and CD4⁺8⁺ mucosal T cells.     

Clinical predictors of delayed engraftment in autologous hematopoietic cell transplant recipients

Objective/backgroundClinical predictors of delayed engraftment following autologous hematopoietic cell transplantation (AHCT) are poorly described in the literature. The purpose of this study was to identify pretransplant characteristics contributing to delayed engraftment (DE) following AHCT.MethodsA retrospective, single institution study of 1162 consecutive patients undergoing AHCT from January 1996 to August 2016 was studied for DE. DE was defined as platelet count ≤ 50,000/µl, hemoglobin ≤ 8 g/dL, or absolute neutrophil count ≤ 1000/mm³.ResultsOf the 1162 AHCT recipients, 263 (22.6%) were identified as having DE at 30-days post-AHCT with 80.0% being solely due to delayed platelet engraftment. Patients with Non-Hodgkin lymphoma (NHL) represented 18% of the original cohort, but accounted for 45% of those with DE, whereas multiple myeloma patients represented 59% of the initial cohort, but only 29% of those that had DE. At 3 months post-AHCT, transfusion dependence (p = .0083) prior to AHCT, low-infused CD34⁺ cell dose < 3 × 10⁶/kg (p = .0012), and low preAHCT platelet count < 150 × 10³/µL (p = .0027) were significantly associated with delayed engraftment.ConclusionTransfusion dependence prior to AHCT, pre-AHCT platelet count, and CD34⁺ cell dose were the strongest predictors of delayed engraftment in patients undergoing AHCT.     

Cardiometabolic and immune factors associated with increased common carotid artery intima-media thickness and cardiovascular disease in patients with systemic lupus erythematosus

Background and aimPatients with systemic lupus erythematosus (SLE) have a higher prevalence of subclinical atherosclerosis and higher risk of cardiovascular (CV) events compared to the general population. The relative contribution of CV-, immune- and disease-related risk factors to accelerated atherogenesis in SLE is unclear.Methods and resultsFifty SLE patients with long-lasting disease (mean age 44 ± 10 years, 86% female) and 50 sex- and age-matched control subjects were studied. Common carotid artery intima-media thickness (CCA-IMT) was used as a surrogate marker of atherosclerosis. We evaluated traditional and immune- and disease-related factors, assessed multiple T-cell subsets by 10-parameter-eight-colour polychromatic flow cytometry and addressed the effect of pharmacological therapies on CCA-IMT. In SLE patients, among several cardiometabolic risk factors, only high-density lipoprotein levels (HDL) and their adenosine triphosphate-binding cassette transporter 1 (ABCA-1)-dependent cholesterol efflux capacity were markedly reduced (p < 0.01), whereas the CCA-IMT was significantly increased (p = 0.03) compared to controls. CCA-IMT correlated with systolic blood pressure, low-density lipoprotein (LDL) cholesterol and body mass index (BMI), but not with disease activity and duration. The activated CD4⁺HLA-DR⁺ and CCR5⁺ T-cell subsets were expanded in SLE patients. Patients under hydroxychloroquine (HCQ) therapy showed lower CCA-IMT (0.62 ± 0.08 vs. 0.68 ± 0.10 mm; p = 0.03) and better risk-factor profile and presented reduced circulating pro-atherogenic effector memory T-cell subsets and a parallel increased percentage of naïve T-cell subsets.ConclusionHDL represents the main metabolic parameter altered in SLE patients. The increased CCA-IMT in SLE patients may represent the net result of a process in which ‘classic’ CV risk factors give a continuous contribution, together with immunological factors (CD4⁺HLA-DR⁺ T cells) which, on the contrary, could contribute through flares of activity of various degrees over time. Patients under HCQ therapy present a modified metabolic profile, a reduced T-cell activation associated with decreased subclinical atherosclerosis.     

Activated CD4+ and CD8+ T-cell subsets in Wegener's granulomatosis

Several lines of evidence argue in favour of an involvement of T cells in the pathogenesis of Wegener's granulomatosis (WG). These include the presence of highly specific IgG autoantibodies to proteinase 3, perivascular T-cell infiltrates and elevated amounts of soluble interleukin-2 (IL-2) receptors in patient's serum. In order to further address this question we evaluated by double immunoflourescence and flow cytometry the expression of several cell surface molecules associated with T-cell activation. As compared to healthy controls (n=15), the CD4⁺ subset was significantly diminished, while the percentage of CD8⁺ T cells was elevated in WG patients (n=24). Within the CD4⁺ T-cell subset we found a highly significant increase in activation/memory markers (CD25, CD29, HLA-DR). Within the CD8⁺ T-cell subset the expression of CD11b, CD29 and CD57 was significantly elevated, while the expression of VD28 was reduced. The use of 10 V-, 1 V-and 1 V-specific monoclonal reagents failed to reveal any significant bias in the peripheral T-cell receptor V-gene repertoire of WG patients. There was also no correlation between T-cell activation markers and laboratory parameters [C-reactive protein (CRP), ESR], disease duration or therapy. A significant correlation was found only for the degree of organ involvement and the increase in CD4⁺ T cells coexpressing HLA-DR, as well as the increase in CD57 expression on CD8⁺ T cells. In conclusion, both CD4⁺ and CD8⁺ T-cell subsets were activated in WG. Cytotoxic CD8⁺ CD57⁺ CD11b⁺ CD28^– T cells may directly contribute to damage of vascular endothelium.     

CD14++CD16+ monocyte subset expansion in rheumatoid arthritis patients: Relation to disease activity and interleukin-17

Aim of the workMonocytes are divided into three major subsets based on the expression of the cluster of differentiation CD14 and CD16. The aim of this work was to determine which of the CD16⁺ monocyte subpopulations is expanded in rheumatoid arthritis (RA) and its association with disease activity and interleukin-17 (IL17) levels.Patients and methodsFifty-three RA patients and 20 controls were enrolled in this study. Flow cytometry was performed to detect monocyte subsets and IL17 was measured by ELISA. Disease activity score (DAS28) was assessed.ResultsCD14⁺⁺CD16⁺ monocyte percentage was significantly higher in long standing RA patients compared with early patients and controls (p < 0.01, p < 0.001 respectively). It was significantly higher in patients with RA disease activity and remission compared with the controls (p < 0.001, p < 0.01 respectively). It was not significantly associated with resistance to disease modifying antirheumatic drugs (DMARDs), C-reactive protein, rheumatoid factor and anti-CCP positivity (p > 0.05). It significantly correlated with IL17 (p < 0.002). CD14⁺CD16⁺ monocyte percentage was not significantly correlated with any of the above parameters. IL17 level was significantly higher in patients with early and long standing RA compared to controls (p < 0.01, p < 0.001 respectively). IL17 was higher in RA patients with active disease compared to those in remission and controls (p < 0.01, p < 0.001 respectively). It was higher in RA patients resistant to DMARDs than in responding patients (p < 0.017).ConclusionCD14⁺⁺CD16⁺ monocyte subpopulation was expanded in long standing RA and was correlated with IL17 levels indicating its potential pathogenic importance in RA and may represent an attractive target for future therapeutic interventions.     

The Effect of Sodium Depletion and Potassium Supplementation on Vasopressin,Renin and Catecholamines in Hypertensive Men

ABSTRACT Seventeen 50-year-old hypertensive men (157±4/110±2 mmHg, mean ± SE) were given low sodium diet for one week, which was supplemented with potassium the following week. The urinary Na⁺/K⁺ excretion ratio changed from 2:1 to 1:5 and 1:12, respectively, during dietary intervention. Arterial plasma vasopressin decreased by 3.4±1.7 ng/l (0.05>p<0.10) and urinary excretion of vasopressin was reduced by nearly 50% (p<0.001) during sodium depletion, while plasma noradrenaline increased by 38% (p<0.001) and plasma dopamine showed an increase by 58% (p<0.001). Plasma renin concentration increased four-fold during sodium depletion (p<0.001). With combined salt depletion and potassium supplementation, arterial plasma vasopressin decreased by 9.5±4.0 ng/l (p<0.05) compared to control. Urinary excretion of vasopressin together with plasma noradrenaline and dopamine were unchanged during the second week. The reduction of blood pressure was most marked during the first week (143±3/103±2 mmHg, p<0.05), but continued to fall also during the second week. Thus, during sodium restriction in middle-aged hypertensive men, blood pressure reduction occurs concomitantly with inhibited vasopressin release, despite enhanced renin and catecholamine release. Potassium supplementation during sodium restriction induces only minor changes in these variables.     

Differential in vitro CD4+/CD8+ T‐cell response to live vs. killed Leishmania major

Clinical trials of killed Leishmania vaccines showed a limited efficacy compared with leishmanization (LZ). The reason for this difference in protection against cutaneous leishmaniasis (CL) is not known and in vivo studies on T‐cell function may provide valuable information. Nevertheless, there are limited studies on the nature of the stimulatory effects of live vs. killed parasites on human T cells in vitro. A total of nine Leishmanin Skin Test+ volunteers with a history of self‐healing CL (HCL) and seven healthy volunteers were included in this study. 5,6‐carboxyfluroescein diacetate succinimidyl ester‐labelled CD4⁺/CD8⁺ lymphocytes were cultured with killed Leishmania Lysate (Killed LL) or live Leishmania major (Live LM) and analysed for proliferation using flow cytometry. Culture supernatants were used for cytokine titration. In HCL volunteers, upon stimulation with killed LL, the number of proliferated CD4⁺/CD8⁺ cells was significantly more than that of unstimulated (P < 0·001) or live LM stimulated (P < 0·05) cells, or cells from controls (CD4⁺/CD8⁺: P < 0·05/P < 0·001). Stimulation of CD4⁺ cells with Live LM (P < 0·001) or Killed LL (P < 0·05) induced a significantly higher IFN‐γ production compared with that of controls, but Live LM induced significantly (P < 0·05) more IFN‐γ than Killed LL. A significantly (P < 0·05) higher IFN‐γ production was observed when CD8⁺ cells were stimulated with Live LM. Cells from HCL volunteers showed significantly more IL‐10 production to Live LM stimulation compared with that of controls (CD4⁺: P < 0·05 /CD8⁺: P < 0·001) or cells stimulated with Killed LL (CD4⁺/CD8⁺: P < 0·001/P < 0·0005). Whereas Killed LL induced more proliferation response in purified T cells, Live LM induced cytokine production without significant induction of proliferation. The results from healed CL volunteers in this study could be implicated in further studies on T‐cell response in vaccinated individuals.     

Overexpression of CD73 in pancreatic ductal adenocarcinoma is associated with immunosuppressive tumor microenvironment and poor survival

BackgroundCD73, a newly recognized immune checkpoint mediator, is expressed in several types of malignancies. However, CD73 expression and its impact on tumor microenvironment and clinical outcomes in pancreatic ductal adenocarcinoma (PDAC) remain unclear.MethodsThis study included two cohorts: 138 patients from our institution (MDA) and 176 patients from TCGA dataset. CD73 expression, CD4⁺, CD8⁺, CD21⁺ and CD45RO + tumor infiltrating lymphocytes (TILs) were evaluated by immunohistochemistry using tissue microarrays. The results of CD73 expression were correlated with clinicopathologic parameters, survival and TILs.ResultsCD73 overexpression correlated with poor differentiation (P = 0.002) and tumor size (P = 0.049). For CD73-low group, median overall survival (OS) and recurrence-free survival (RFS) were 26.9 ± 3.8 months and 12.6 ± 2.6 months, respectively, compared to 16.9 ± 4.4 months (P = 0.01) and 7.9 ± 1.2 months (P = 0.01), respectively, in CD73-high group. CD73 was an independent predictor for both RFS (P = 0.02) and OS (P = 0.01) by multivariate variate analysis. Similarly, CD73-high tumors had significantly shorter OS than CD73-low tumors in TCGA dataset (P < 0.0001). CD73-high correlated with decreased CD4⁺ TILs in MDA cohort and decreased CD8A and CR2 (CD21) expression in TCGA cohort.ConclusionsCD73 overexpression is associated with poor differentiation, tumor size, and shorter survival, and is an independent prognostic factor in PDAC patients. CD73 overexpression is associated with decreased CD4⁺, CD8⁺ and CD21⁺ TILs. Our data support that CD73 plays an important role in immunosuppressive tumor microenvironment and promote tumor progression in PDAC.     

Potentiation of Pulmonary Arteriolar Vasoconstriction to Endothelin-1 by Inhibition of Nitric Oxide Synthesis in the Intact Lung

Objective : To observe pulmonary arteriolar effects of endothelin-1 (ET-1) in the intact lung and determine if constriction to ET-1 is potentiated by inhibition of nitric oxide (NO) synthesis. Methods : In anesthetized male Sprague-Dawley rats with open chest, the lungs were ventilated with air through the lower trachea and in vivo responses of pulmonary arterioles were examined by video microscopy. Observations were made when the lungs were statically inflated with oxygen to a pressure of approximately 10 cm H₂O for brief periods. A lens with a dipping cone was held at the pleural surface. ET-1 (10^?7–10^?5 M; approximately 0.1 ml) was applied topically to the fluid layer under the dipping cone. Results : ET-1 (10^?6 M) constricted parent arterioles 60 ± 5 µm in diameter by 52 ± 12% (range: 20–100%) and branches 45 ± 3 µm in diameter by 36 ± 4% (19–48%). Constriction persisted and there was a dramatic long-lasting decrease in flow. Alveolar walls quickly became pale, indicating reduced capillary perfusion. A lower concentration of ET-1 (10^?7 M) constricted (p < 0.05) parent arterioles 61 ± 4 µm in diameter by 7 ± 3% initially, and by 13 ± 8% after 14 ± 2 minutes, while smaller branches did not respond. In separate experiments, infusion of the NO synthase inhibitor L-NAME (1 mg/kg per minute), modestly (10 ± 3%) decreased (p < 0.05) baseline parent arteriolar diameter from 72 ± 7 µm to 64 ± 5 µm. Branch diameter changed insignificantly from 42 ± 7 µm to 38 ± 7 µm. After l -NAME, ET-1 (10^?7 M) constricted (p < 0.05) parent arterioles by 17 ± 4% initially and 40 ± 14% after 14 ± 2 minutes. Concurrently, branches constricted (p < 0.05) by 14 ± 4% and 26 ± 15%. Conclusions : Arterioles less than 80 µm in diameter were very responsive to ET-1, which could be a factor in altering pulmonary microvascular resistance. Inhibition of NO synthesis appears to potentiate constriction to ET-1.     

Increased Risk of Developing Atrophic Gastritis in Patients Infected with CagA+Helicobacter pylori

Background: To clarify the possible role of CagA positive (CagA⁺) Helicobacter pylori strains in the development of atrophic gastritis, the prevalence of antibodies to H. pylori and CagA (120 kD protein) was studied among subjects with atrophic and non-atrophic gastritis. Methods: The study population was randomly selected among 12,252 Finnish men who were screened for atrophic corpus gastritis with serum pepsinogen I-assay (S-PGI). S-PGI level was used as a selection criterion. Group A consisted of 295 subjects with S-PGI &lt;25 µg/l (low), group B of 320 subjects with S-PGI 25-100 µg/l (normal) and group C of 338 subjects with S-PGI &gt;100 µg/l (high). Antibodies to H. pylori were measured with EIA and immunoblot analysis and antibodies to CagA with immunoblot analysis. Endoscopical and histological examinations were performed for 203 patients from group A. Results: The prevalence of antibodies to H. pylori was significantly lower in group B than in groups A or C (P &lt; 0.0001, chi-squared test). There was a significant association between the prevalence of antibodies to CagA and the lowered level of S-PGI (P &lt; 0.0001, Jonckheere-Terpstra trend test). There was also a linear decrease in the prevalence of antibodies to CagA as the atrophic corpus gastritis became more severe (P &lt; 0.0001, linear-by-linear trend test). Conclusion: The presence of antibodies to CagA seems to be associated with development of atrophic corpus gastritis.