Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients

ObjectiveTo investigate the expression of TNFα membrane-bound receptors: the percentage of cells expressing these receptors and the number of molecules expressed on different immune cell subsets, and to evaluate serum concentrations of soluble TNFα and its receptors (sTNFRI and sTNFRII) in patients with rheumatoid arthritis in acute stage and after response to treatment compared to healthy donors.MethodsThe objects of the study are peripheral blood mononuclear cells (PBMC) of healthy donors (n = 150) and RA patients (n = 40) subjected to hospital treatment with either biological agents (Rituximab) or glucocorticosteroids (methylprednisolone). To determine PBMC phenotype antibodies anti-hCD3-APC, anti-hCD19 PECy7, anti-hCD14 FITC (eBioscience), as well as anti-hTNFRI-PE and anti-hTNFRII-PE (R&D Systems) were used. To determine receptor number on the cells Quantibrite PE Beads (BD) were used.ResultsCells obtained from patients who responded to therapy and achieved disease remission exhibited either an increase in the percentage of TNFRI+ cells or elevated expression density of this receptor type.ConclusionSubsets of immunocompetent cells from RA patients show variation in the percentage of membrane-bound receptor positive cells and receptor expression density, which influences the development and progression of the pathological processes in RA. Response to therapy and achievement of disease remission are associated with an increase of TNFRI expression.     

Tumor necrosis factor α release in peripheral blood mononuclear cells of cutaneous lupus and dermatomyositis patients

Introduction  

Several studies have reported that TNFα is substantially increased within skin lesions of patients with discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE) and dermatomyositis (DM) compared to controls. Elevated TNFα has been reported in the sera of some patients with systemic lupus erythematosus, DLE and SCLE, but not in the sera of patients with DM. Because of the key pathogenic role of autoimmunity in these diseases, in this study we sought to evaluate TNFα production by a readily available source of immune cells (namely, peripheral blood mononuclear cells (PBMCs)) taken from controls and from patients with cutaneous lupus or DM.     

Different pattern of T cell receptor δ gene rearrangement in tumour-infiltrating lymphocytes and peripheral blood in patients with solid tumours

Tumor-infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) from four patients with renal-cell carcinoma (three paired with blood), two colon carcinomas (both paired with blood) and two melanomas (blood was not available) were analysed for the T cell receptor (TCR) gene repertoire. Polymerase chain reaction analysis, employing a panel of specific primers for TCR gene segments, showed different gene rearrangement patterns in TIL and PBL in all patients. Simultaneous analysis of TIL and PBL revealed the presence of lymphoid cells in the tumour tissue that were not present in the periphery. These results demonstrate that, although tumor-infiltrating lymphocytes contain / T cells within the range observed in peripheral blood, these cells differ from those in peripheral blood in their gene repertoire and this may account for selective accumulation or/and in situ amplification of / lymphocytes at the tumour site, indicating a unique type of host reaction against tumors.     

Breakage of the T cell receptor α chain locus in non malignant clones from patients with ataxia telangiectasia

Summary Patients with ataxia telangiectasia (A-T) develop specific chromosome translocations, which may confer a proliferative advantage, resulting in the appearance of large clones in the peripheral blood lymphocytes. These lymphocytes are not malignant. Using in situ hybridisation techniques we have investigated a consistent 14q11 translocation break-point observed in a t(X;14)(q28;q11) translocation clone from each of two different patients and a t(14;14)(q11;q32) clone from a third patient. In all cases the chromosome translocation involved breakage within the chain locus of the T cell receptor (TCR), between the variable and constant regions, at 14q11. Chromosome rearrangement involving breakage within TCR can therefore precede the development of malignancy. Further chromosomal rearrangement may be required in these patients, for progression to the leukaemic state.     

Analysis of T cell receptor β and γ genes from peripheral blood,regional lymph node and tumor-infiltrating lymphocyte clones from melanoma patients

Summary A total of 199 T cell clones from two melanoma patients were derived from progenitor T cells from recurrent melanoma, regional lymph nodes (either involved or uninvolved with malignancy) and peripheral blood by inoculating single cells directly into the wells of microtiter plates before in vitro expansion. The surface marker phenotype of most clones was CD4⁺CD8^–, although some were CD4^–CD8⁺. Genomic DNA prepared from all clones was analyzed by Southern blot hybridization using T cell receptor (TCR) and gene probes, seeking clones with identical TCR gene rearrangement patterns as direct evidence for in vivo progenitor T cell clonal amplification. ProbingHindIII-digested DNA with TCR and TCR probes revealed several clones with identical TCR gene rearrangement patterns. These clones had subsequent probing ofBamHI-digested DNA with TCR and TCR probes, which showed all but 2 clones to have distinct rearrangement patterns. These analyses provide clear molecular evidence for in vivo polyclonal CD4⁺ T cell populations in each of several separate immune compartments in these patients.This investigation was supported by National Institutes of Health, National Research Service Award CA-08 397 from the National Cancer Institute as well as NIH CA-32 685, CA-30 688, DOE FG028 760 502 and American Cancer Society Grant ACS CH-237     

Induction of bacillus-Calmette-Guérin-activated killer cells from human peripheral blood mononuclear cells against human bladder carcinoma cell lines in vitro

Cytotoxicity against two human bladder carcinoma cell lines (BT-A and BT-B) was investigated using human peripheral blood mononuclear cells (PBMC) stimulated with viable bacillus Calmette-Guérin (BCG) or sonicated BCG (s-BCG). We applied a cytotoxicity assay based on radioactive labelling of tumour cells by incorporation ofl[³H]methionine. The results were compared with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interleukin-2 (IL-2) and interferon (IFN). BCG-stimulated PBMC showed a cytotoxic potential against BT-A and BT-B comparable to that of IFN-generated LAK cells, but this did not reach the level of IL-2-generated LAK cells. We termed these cytotoxic effectors BCG-activated killer (BAK) cells. In contrast to their cytotoxicity against bladder tumour cells. BAK cells did not differ from unstimulated PBMC in the killing of K562 cells. Only viable but not sonicated BCG was able to induce cytotoxicity against BT-A and BT-B. We could demonstrate the presence of the cytokines IFN, IL-2, tumour necrosis factor (TNF) and TNFß in the supernatants harvested during the generation of BAK cells. Monoclonal antibodies neutralizing IFN were able to inhibit BCG-mediated cytotoxicity, giving evidence of the involvement of IFN in the induction of BAK cells. Furthermore, we performed experiments to investigate the cytotoxic potential of distinct cell populations. The cells effective in BCG-activated killing of bladder tumour cells could be localized within the CD8+/CD56+ lymphocyte subset. CD4+ cells and macrophages did not exhibit cytolytic activity. Our findings imply that the activation by BCG of CD8+/CD56+ killer cells might be an important antitumoral mechanism during BCG therapy against superficial urothelial bladder cancer.     

Impaired activation of IFN-γCD4 T cells in peripheral blood of patients with dilated cardiomyopathy

Viral persistence and autoantibodies are pathogenic components in patients with idiopathic dilated cardiomyopathy (DCM). The aim was to evaluate T-cell function in DCM using different flow cytometry based detection techniques. Following stimulation, the frequency of IFN-γ-producing CD4⁺ T cells was significantly lower in patients compared with controls. In contrast, the frequency of IL-4 producing CD4⁺ T cells was no different. In supernatants of cultured PBMC, IFN-γ and IL-10 were significantly lower in patients. In addition, lymphocyte proliferation was significantly lower in patients compared with controls, whereas major lymphocyte subsets were not different. IFN-γ and IL-10 are key cytokines in the ability to mount protective immune responses and to maintain self-tolerance. A reduced activation of T-helper 1 (IFN-γ producing) cells and a decreased capacity to produce IL-10, found in the present study, could explain parts of the autoimmune features seen in patients with DCM.     

Association of reduced heme oxygenase-1 with excessive Toll-like receptor 4 expression in peripheral blood mononuclear cells in Behçet's disease

Introduction

Toll-like receptors (TLRs) mediate signaling that triggers activation of the innate immune system, whereas heme oxygenase (HO)-1 (an inducible heme-degrading enzyme that is induced by various stresses) suppresses inflammatory responses. We investigated the interaction between TLR and HO-1 in an inflammatory disorder, namely Behçet's disease.

Methods

Thirty-three patients with Behçet's disease and 30 healthy control individuals were included in the study. Expression levels of HO-1, TLR2 and TLR4 mRNA were semiquantitatively analyzed using a real-time PCR technique, and HO-1 protein level was determined by immunoblotting in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes. In some experiments, cells were stimulated with lipopolysaccharide or heat shock protein-60; these proteins are known to be ligands for TLR2 and 4.

Results

Levels of expression of HO-1 mRNA were significantly reduced in PBMCs from patients with active Behçet's disease, whereas those of TLR4, but not TLR2, were increased in PBMCs, regardless of disease activity. Moreover, HO-1 expression in PBMCs from patients with Behçet's disease was repressed in the presence of either lipopolysaccharide or heat shock protein-60.

Conclusion

The results suggest that upregulated TLR4 is associated with HO-1 reduction in PBMCs from patients with Behçet's disease, leading to augmented inflammatory responses.     

Calreticulin transacetylase catalyzed modification of the TNF-α mediated pathway in the human peripheral blood mononuclear cells by polyphenolic acetates

Polyphenols, coumarin (1,2-benzopyrone) and chromone (1,4-benzopyrone), are naturally occurring constituent of variety of plant species. They have attracted immense interest because of their diverse pharmacological activities. Not much was known about biological activities of acetyl derivative (polyphenolic acetates) of parent polyphenols. In previous investigations, we have conclusively established calreticulin transacetylase catalyzed activation of endothelial nitric oxide synthase (eNOS) by polyphenolic acetates. In the present work, calreticulin transacetylase of human peripheral blood mononuclear cells was characterized with respect to specificity for various polyphenolic acetates and its role in the activation of TNF-α induced nitric oxide synthase (iNOS). Peripheral blood mononuclear cells incubated with a model polyphenolic acetate, 7,8-diacetoxy-4-methylcoumarin (DAMC), along with l-arginine caused activation of NOS. The incubation of peripheral blood mononuclear cells with TNF-α and DAMC resulted in increased production of NO as compared to TNF-α alone. This increased NO production was attenuated by l-Nω-nitro-l-arginine methyl ester (l-NAME), a well known non-specific inhibitor of NOS, and 1400W (N-[3-(aminomethyl) benzyl] acetamidine), a specific inhibitor of human iNOS. These results substantiate the CRTAase catalyzed activation of iNOS. Further, expression of NOS isoforms by semi-quantitative PCR and real-time RT-PCR confirms the preponderance of iNOS in TNF-α treated peripheral blood mononuclear cells over the untreated one. It was also observed that polyphenolic acetates inhibit TNF-α mediated release of IL-6 from peripheral blood mononuclear cells.     

The influence of μ-opioid receptor agonist and antagonist peptides on peripheral blood mononuclear cells (PBMCs)

Milk is one of the main source of biologically-active peptides that may function as regulatory substances called food hormones. After passing the gut-blood barrier, the μ-opioid receptor agonist and antagonist peptides may become the new factors influencing various functions of the human organism. The aim of the conducted research was to determine the influence of μ-opioid receptor agonist peptides: human and bovine β-casomorphin-7 (h/bBCM-7) and antagonistic peptides: casoxin-6 and- D (CXN-6/D) on proliferation and cytokine secretion of human peripheral blood mononuclear cells (PBMCs). The PBMCs proliferation was measured by the use of the BrdU test, which assesses the DNA synthesis activity and the WST-1 test which assesses the activity of mitochondrial dehydrogenase enzymes. The influence of all the investigated peptides on secretion of IL-4, IL-8, IL-13 and IFN-γ was determined by the use of the ELISA tests. Incubating the cells with the peptides has not caused any changes to their enzymatic activity, which has been proved by a WST-1 test. When using a BrdU test, however, it has been observed that there appear changes to proliferation of PBMCs correlated to amounts of bromodeoxyuridine incorporated into the cellular DNA. Moreover, changes to secretion of IL-4 and IL-13 by the cells under the influence of agonists were detected, as well as changes to secretion of IFN-gamma under the influence of all the examined substances. The obtained results provide information on immunomodulatory effects of food-derived opioid peptides, which may be of clinical significance especially in the case of allergic diseases in newborns.     

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.     

Role of adhesion molecules in recruitment of Vδ1 T cells from the peripheral blood to the tumor tissue of esophageal cancer patients

 The mechanism responsible for tissue specific localization of γδ T cell subsets is not well understood. In order to explain the sequestration of specific γδ T cell subsets in the peripheral blood and tumor tissue of patients with esophageal cancer, we examined the function and expression of adhesion molecules on these cells. A hierarchy in the expression of adhesion molecules was observed. In vitro activated γδ T cells showed dominant expression of LFA-1 (CD11a), VLA-α4 (CD49d), intermediate expression of VLA-α5 (CD49e) and L-selectin (CD62L), but low expression of CD44v6 and α_Eβ₇ (CD103). It was observed that the γδ T cells use LFA-1, L-selectin and CD44v6 to bind to squamous cell carcinoma (SCC) cells, whereas they adhere to fibroblast cells using LFA-1, VLA-α4 and VLA-α5. Vδ1 T cell subsets from the peripheral blood γδ T cells utilize a larger array of adhesion molecules, namely LFA-1, VLA-α4, VLA-α5, L-selectin and α_Eβ_7, to bind to SCC cells compared to the restricted usage of LFA-1, L-selectin and CD44v6 by the Vδ2 T cells. Flow cytometric analysis of tumor infiltrating lymphocytes from the esophageal tumors confirmed the selective accumulation of Vδ1⁺γδ T cells in the tumor compartment. It thus appears that adhesion molecules expressed on these lymphocytes play an important role in the recruitment and retention of Vδ1 T cells in the tumor milieu. Received: 27 November 2000 / Accepted: 1 March 2001     

Bm1-Bm5 classification of peripheral blood B cells reveals circulating germinal center founder cells in healthy individuals and disturbance in the B cell subpopulations in patients with primary Sjögren's syndrome

Analyses of B cells in the bone marrow and secondary lymphoid tissues have revealed a broad range of cell surface markers defining B cell subpopulations, but only a few of these have been used to analyze B cell subpopulations in peripheral blood (PB). We report here the delineation of circulating PB B cell subpopulations by staining for CD19, CD38, and IgD in combination with CD10, CD44, CD77, CD95, CD23, IgM, and the B cell memory marker CD27. The utility of this approach is shown by the demonstration of disturbances of circulating B cell subpopulations in patients with autoimmune disease. Five mature B cell (Bm) subpopulations were identified in normal PB that were comparable with the tonsillar Bm1, Bm2, early Bm5, Bm5 subpopulations and, surprisingly, to the germinal center (GC) founder cell subpopulation (Bm2' and Bm3delta-4delta), suggesting that some GC founder cells are circulating. No PB B cells resembled the Bm3 and Bm4 GC cells. Remarkably, some cells with the CD38-IgD+ phenotype, previously known as naive Bm1 cells, expressed CD27. The CD38-IgD+ subpopulation therefore includes both naive Bm1 cells and IgD+ memory B cells. This new classification of B cell developmental stages reveals disturbances in the proportions of B cell subpopulations in primary Sj?gren's syndrome (pSS) patients compared with healthy donors and rheumatoid arthritis patients. Patients with pSS contained a significantly higher percentage of B cells in two activated stages, which might reflect a disturbance in B cell trafficking and/or alteration in B cell differentiation. These findings could be of diagnostic significance for pSS.     

Abnormal activation of the Akt-GSK3β signaling pathway in peripheral blood T cells from patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease accompanied by the activation and proliferation of T cells and B cells. In this study, we found that the distributions of lymphocytes obtained from patients with SLE or SLE with renal disease (RSLE) were reduced in the G₀/G₁ phase and were elevated in the S phase after phytohemagglutinin treatment. Increased expression of CDK2 and decreased expression of cyclin-dependent kinase inhibitors p27^Kip1 and p21^WAF1/CIP1 were observed in RSLE and SLE lymphocytes. The phosphorylation levels of Akt473 and GSK3β (ser9) were increased in lymphocytes from the patients. Moreover, inhibition of GSK3β with lithium chloride or SB216763 induced T cell proliferation, and the most significant effects were observed in RSLE lymphocytes. These results indicate that upregulation of CDKs and downregulation of p27^Kip1 and p21^WAF1/CIP1 increased the proliferation of T lymphocytes in SLE patients. Abnormal activation of the Akt–GSK3β signaling pathway increased the proliferation of lupus lymphocytes.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

Are T cells at the origin of B cell lymphomas?

Lymphoma pathogenesis is at least in some cases related to transformed B cells (BCs) arising from germinal centre reactions (GCRs). In this article possible deregulations of GCRs are investigated using in silico simulations. It is found that the final differentiation of BCs as regulated by helper T cells (TCs) is the best candidate mechanism for such a deregulation. This shifts the paradigm of BC lymphoma pathogenesis from BC transformations to an emphasized role of TC-BC interactions.     

Anti-inflammatory effect of rosiglitazone is not reflected in expression of NFκB-related genes in peripheral blood mononuclear cells of patients with type 2 diabetes mellitus

Background

The aim of this study is to perform a cost-effectiveness comparison between palpation-guided thyroid fine-needle aspiration biopsies (P-FNA) and ultrasound-guided thyroid FNA biopsies (USG-FNA).

Methods

Each nodule was considered as a case. Diagnostic steps were history and physical examination, TSH measurement, Tc^99m thyroid scintigraphy for nodules with a low TSH level, initial P-FNA versus initial USG-FNA, repeat USG-FNA for nodules with initial inadequate P-FNA or USG-FNA, hemithyroidectomy for inadequate repeat USG-FNA. American Thyroid Association thyroid nodule management guidelines were simulated in estimating the cost of P-FNA strategy. American Association of Clinical Endocrinologists guidelines were simulated for USG-FNA strategy. Total costs were estimated by adding the cost of each diagnostic step to reach a diagnosis for 100 nodules. Strategy cost was found by dividing the total cost to 100. Incremental cost-effectiveness ratio (ICER) was calculated by dividing the difference between strategy cost of USG-FNA and P-FNA to the difference between accuracy of USG-FNA and P-FNA. A positive ICER indicates more and a negative ICER indicates less expense to achieve one more additional accurate diagnosis of thyroid cancer for USG-FNA.

Results

Seventy-eight P-FNAs and 190 USG-FNAs were performed between April 2003 and May 2008. There were no differences in age, gender, thyroid function, frequency of multinodular goiter, nodule location and diameter (median nodule diameter: 18.4 mm in P-FNA and 17.0 mm in USG-FNA) between groups. Cytology results in P-FNA versus USG-FNA groups were as follows: benign 49% versus 62% (p = 0.04), inadequate 42% versus 29% (p = 0.03), malignant 3% (p = 1.00) and indeterminate 6% (p = 0.78) for both. Eleven nodules from P-FNA and 18 from USG-FNA group underwent surgery. The accuracy of P-FNA was 0.64 and USG-FNA 0.72. Unit cost of P-FNA was 148 Euros and USG-FNA 226 Euros. The cost of P-FNA strategy was 534 Euros and USG-FNA strategy 523 Euros. Strategy cost includes the expense of repeat USG-FNA for initial inadequate FNAs and surgery for repeat inadequate USG-FNAs. ICER was -138 Euros.

Conclusion

Universal application of USG-FNA for all thyroid nodules is cost-effective and saves 138 Euros per additional accurate diagnosis of benign versus malignant thyroid nodular disease.

Trial registration

ClinicalTrials.gov, NCT00571090