Up-regulation Of Dnam-1 And Nkp30, Associated With Improvement Of Nk Cells Activation After Long-term Culture Of Mononuclear Cells From Patients With Ovarian Neoplasia.

This study aimed at evaluating the functional activation and activating receptors expression on resting, short- and long-term NK and NK-like T cells from blood of ovarian neoplasia patients. Blood from patients with adnexal benign alterations (n = 10) and ovarian cancer (grade I-IV n = 14) were collected after signed consent. Effector cells activation was evaluated by the expression of the CD107a molecule. Short-term culture was conducted overnight with IL-2 and long-term culture for 21 days, by a method designed to expand CD56(+) lymphocytes. Short-term culture significantly increased NK cells activation compared to resting NK cells (p<0.05), however, the long-term procedure supported an even higher increase (p<0.001). Resting NK-like T cells showed poor activation, which was not altered by the culture procedures. The long-term culture effectively increased the expression of the activating receptors on NK and NK-like T cells, either by increasing the number of cells expressing a given receptor and/or by up-regulating their expression intensity. As a conclusion, the long-term culture system employed, resulted in a high number of functional NK cells. The culture system was particularly efficient on the up-regulation of NKp30 and DNAM-1 receptors on NK cells.

ABCA1 expression and statins: inhibitory effect in peripheral blood mononuclear cells

The ATP-binding cassette transporter A1 (ABCA1) has an essential role in the formation of nascent high-density lipoprotein particles and also participates in the cholesterol efflux from macrophages in the artery wall. Several substances, such as statins, or even gene variants are able to modulate ABCA1 expression. There is strong evidence that statin treatment downregulates the ABCA1 expression in nonloaded macrophages. Interestingly, in cholesterol-loaded macrophages, which are more relevant to atherogenesis, this effect is lost. We observed an inhibitory effect of atorvastatin in peripheral blood mononuclear cells of hypercholesterolemic individuals. Moreover, in these individuals, the ABCA1 -14C > T polymorphism was associated with high baseline gene-expression levels. Other studies are needed to evaluate how relevant these findings are to the formation of arterial foam cells in vivo.

Serum amyloid A induces CCL20 secretion in mononuclear cells through MAPK (p38 and ERK1/2) signaling pathways

Although the serum levels of SAA had been reported to be upregulated during inflammatory/infectious process, the role of this acute-phase protein has not been completely elucidated. In previous studies, we demonstrated that SAA stimulated the production of TNF-alpha, IL-1 beta, IL-8, NO, and ROS by neutrophils and/or mononuclear cells. Herein we demonstrate that SAA induces the expression and release of CCL20 from Cultured human blood mononuclear cells. We also focus on the signaling pathways triggered by SAA. in THP-1 cells SAA promotes phosphorylation of p38 and ERK1/2. Furthermore, the addition of SB203580 (p38 inhibitor) and PD98059 (ERK 1/2 inhibitor) inhibits the expression and release of CCL20 in mononuclear cells treated with SAA. Our results point to SAA as an important link of innate to adaptive immunity, once it might act on the recruitment of mononuclear cells. (C) 2008 Elsevier B.V. All rights reserved.

Atorvastatin effects on SREBF1a and SCAP gene expression in mononuclear cells and its relation with lowering-lipids response

Background: The transcription factors SREBP1 and SCAP are involved in intracellular cholesterol homeostasis. Polymorphisms of these genes have been associated with variations on serum lipid levels and response to statins that are potent cholesterol-lowering drugs. We evaluated the effects of atorvastatin on SREBF1a and SCAP mRNA expression in peripheral blood mononuclear cells (PBMC) and a possible association with gene polymorphisms and lowering-cholesterol response. Methods: Fifty-nine hypercholesterolemic patients were treated with atorvastatin (10 mg/day for 4 weeks). Serum lipid profile and mRNA expression in PBMC were assessed before and after the treatment. Gene expression was quantified by real-time PCR using GAPD as endogenous reference and mRNA expression in HepG2 cells as calibrator. SREBF1 -36delG and SCAP A2386G polymorphisms were detected by PCR-RFLP. Results: Our results showed that transcription of SREBF1a and SCAP was coordinately regulated by atorvastatin (r=0.595, p<0.001), and that reduction in SCAP transcription was associated with the 2386AA genotype (p=0.019). Individuals who responded to atorvastatin with a downregulation of SCAP had also a lower triglyceride compared to those who responded to atorvastatin with an upregulation of SCAP. Conclusion: Atorvastatin has differential effects on SREBF1a and SCAP mRNA expression in PBMC that are associated with baseline transcription levels, triglycerides response to atorvastatin and SCAP A2386G polymorphism. (c) 2008 Elsevier B.V. All rights reserved.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. one hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p < 0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p < 0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p = 0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4 +/- 12.4%) and apolipoprotein B (apoB) (17.0 +/- 31.3%) when compared with the higher quartile (>0.085: LDL-c = 40.3 +/- 14.3%; apoB = 32.5 +/- 10.7%; p < 0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism. and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin. (C) 2008 Elsevier Inc. All rights reserved.

The Ruthenium Complex cis-(Dichloro) Tetraammineruthenium(III) Chloride Presents Immune Stimulatory Activity on Human Peripheral Blood Mononuclear Cells

Ruthenium compounds in general are well suited for medicinal applications. They have been investigated as immunosuppressants, nitric oxide scavengers, antimicrobial agents, and antimalarials. The aim of this study is to evaluate the immunomodulatory activity of cis-(dichloro) tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) on human peripheral blood mononuclear cells (PBMC). The cytotoxic studies performed here revealed that the ruthenium( III) complex presents a cytotoxic activity towards normal human PBMC, only at very high concentration. Results also showed that cis-[ RuCl(2)(NH(3))(4)] Cl presents a dual role on PBMC stimulating proliferation and interleukin-2 (IL-2) production at low concentration and inducing cytotoxicity, inability to proliferate, and inhibiting IL-2 production at high concentration. The noncytotoxic activity of cis-[RuCl(2)(NH(3))(4)] Cl at low concentration towards PBMC, which correlates with the small number of annexin V positive cells and also the absence of DNA fragmentation, suggest that this compound does not induce apoptosis on PBMC. For the first time, we show that, at low concentration (10-100 mu g L(-1)), the cis-[ RuCl(2)(NH(3))(4)] Cl compound induces peripheral blood lymphocytes proliferation and also stimulates them to IL-2 production. These results open a new potential applicability of ruthenium(III) complexes as a possible immune regulatory compound acting as immune suppressor at high concentration and as immune stimulator at low concentration.

Lactic acid stimulates interleukin-23 production by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide

Lactic acid is the predominant acid present in the vagina. We evaluated the consequences of lactic acid, at physiological levels present in the vagina, on cytokine responses of peripheral blood mononuclear cells (PBMCs) obtained from 10 individuals in the presence or absence of bacterial lipopolysaccharide. Preincubation of PBMCs in 15 mM lactic acid before the addition of lipopolysaccharide resulted in a 246% mean increase in interleukin-23 (IL-23) secretion over that released in the presence of lipopolysaccharide alone (P=0.0068). The lipopolysaccharide-induced production of tumor necrosis factor-alpha, IL-6, IL-10 and IL-12 was unaffected by lactic acid. IL-23 stimulation was not observed if the lactic acid was neutralized before its addition to the culture medium or if hydrochloric acid was substituted for lactic acid. In the absence of lipopolysaccharide, lactic acid did not stimulate the production of IL-23 or any of the other cytokines. The increase in IL-23 production was proportional to the lactic acid concentration over a 15-60 mM range. We conclude that at body sites characterized by lactic acid accumulation, such as in the human vagina, exposure to gram-negative bacteria results in selective IL-23 production, leading to a subsequent preferential stimulation of the Th17 T lymphocyte pathway.

Altered adhesion molecules expression on peripheral blood mononuclear cells from patients with systemic sclerosis and clinical correlations

The aim of the study was to evaluate the expressions of adhesion molecules (AM) on peripheral blood mononuclear cells (PBMNC) from systemic sclerosis (SSc) patients. Thirty-one SSc patients (ACR) and 20 normal subjects were selected for the study. PBMNC were analyzed for LFA-1 alpha, LFA-1 beta, ICAM-3, ICAM-1, and l-selectin expressions. ICAM-3 expression was decreased while ICAM-1 was increased on SSc PBMNC, compared to controls (p = 0.04 and 0.003, respectively). A positive association was found between LFA-1 alpha (r = 0.37, p = 0.03), LFA-1 beta (r = 0.38, p = 0.002), ICAM-3 (r = 0.42, p = 0.01), and l-selectin (r = 0.38, p = 0.03) expressions and greater number of immunosuppressive drugs taken by SSc patients. Also, anti-centromeric positive SSc patients had lower expressions of LFA-1 alpha, LFA-1 beta, ICAM-3, and l-selectin. Lower expression of ICAM-3 and higher expression of ICAM-1 suggest that AMs may be involved in the pathogenesis of scleroderma.

Atopic dermatitis in adults: evaluation of peripheral blood mononuclear cells proliferation response to Staphylococcus aureus enterotoxins A and B and analysis of interleukin-18 secretion

Atopic dermatitis (AD) is a chronic, inflammatory skin disease with a high prevalence and complex pathogenesis. The skin of AD patients is usually colonized by Staphylococcus aureus (S. aureus); its exotoxins may trigger or enhance the cutaneous inflammation. Several mediators are related to the AD immune imbalance and interleukin-18 (IL-18), an inflammatory cytokine, may play a role in the atopic skin inflammation. To evaluate peripheral blood mononuclear cells (PBMC) proliferation response to staphylococcal enterotoxins A (SEA) and B (SEB) and the levels of IL-18 in adults with AD. Thirty-eight adult patients with AD and 33 healthy controls were analysed. PBMC were stimulated with SEA and SEB, phytohemaglutinin (PHA), pokeweed (PWM), tetanus toxoid (TT) and Candida albicans (CMA). IL-18 secretion from PBMC culture supernatants and sera were measured by ELISA. A significant inhibition of the PBMC proliferation response to SEA, PHA, TT and CMA of AD patients was detected (P <= 0.05). Furthermore, increased levels of IL-18 were detected both in sera and non-stimulated PBMC culture supernatants from AD patients (P <= 0.05). A decreased PBMC proliferation response to distinct antigens and mitogens (TT, CMA, SEA and PHA) in adults with AD suggest a compromised immune profile. IL-18 secretion from AD upon stimulation was similar from controls, which may indicate a diverse mechanism of skin inflammation maintained by Staphylococcus aureus. On the other hand, augmented IL-18 secretion from AD sera and non-stimulated cell culture may enhance the immune dysfunction observed in AD, leading to constant skin inflammation.

Effects of bone marrow-derived mononuclear cells on airway and lung parenchyma remodeling in a murine model of chronic allergic inflammation

We hypothesized that bone marrow-derived mononuclear cells (BMDMC) would attenuate the remodeling process in a chronic allergic inflammation model. C57BL/6 mice were assigned to two groups. In OVA, mice were sensitized and repeatedly challenged with ovalbumin. Control mice (C) received saline under the same protocol. C and OVA were further randomized to receive BMDMC (2 x 10(6)) or saline intravenously 24 h before the first challenge. BMDMC therapy reduced eosinophil infiltration, smooth muscle-specific actin expression, subepithelial fibrosis, and myocyte hypertrophy and hyperplasia, thus causing a decrease in airway hyperresponsiveness and lung mechanical parameters. BMDMC from green fluorescent protein (GFP)-transgenic mice transplanted into GFP-negative mice yielded lower engraftment in OVA. BMDMC increased insulin-like growth factor expression, but reduced interleukin-5, transforming growth factor-beta, platelet-derived growth factor, and vascular endothelial growth factor mRNA expression. In conclusion, in the present chronic allergic inflammation model, BMDMC therapy was an effective pre-treatment protocol that potentiated airway epithelial cell repair and prevented inflammatory and remodeling processes. (C) 2010 Elsevier B.V. All rights reserved.

Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than CKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with CKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity. (C) 2009 Elsevier B.V. All rights reserved.

Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic, pathogenic and treatment features

This study aimed to evaluate the association between the differential gene expression profiling of peripheral blood mononuclear cells of rheumatoid arthritis patients with their immunogenetic (human leucocyte antigen shared-epitope, HLA-SE), autoimmune response [anti-cyclic citrullinated peptide (CCP) antibodies], disease activity score (DAS-28) and treatment (disease-modifying antirheumatic drugs and tumour necrosis factor blocker) features. Total RNA samples were copied into Cy3-labelled complementary DNA probes, hybridized onto a glass slide microarray containing 4500 human IMAGE complementary DNA target sequences. The Cy3-monocolour microarray images from patients were quantified and normalized. Analysis of the data using the significance analysis of microarrays algorithm together with a Venn diagram allowed the identification of shared and of exclusively modulated genes, according to patient features. Thirteen genes were exclusively associated with the presence of HLA-SE alleles, whose major biological function was related to signal transduction, phosphorylation and apoptosis. Ninety-one genes were associated with disease activity, being involved in signal transduction, apoptosis, response to stress and DNA damage. One hundred and one genes were associated with the presence of anti-CCP antibodies, being involved in signal transduction, cell proliferation and apoptosis. Twenty-eight genes were associated with tumour necrosis factor blocker treatment, being involved in intracellular signalling cascade, phosphorylation and protein transport. Some of these genes had been previously associated with rheumatoid arthritis pathogenesis, whereas others were unveiled for future research.

Effect of the glycemic control on intracellular cytokine production from peripheral blood mononuclear cells of type 1 and type 2 diabetic patients

Aims: To evaluate the intracellular production of tumor necrosis factor (TNF-alpha), interleukine-6 (IL-6), INF-gamma, IL-8 and IL-10 in peripheral blood lympbomononuclear cells from type 1 and type 2 diabetic patients, stratified according to the glycemic control. Methods: Thirty-five diabetic patients (17 type 1 and 18 type 2) and nine healthy individuals paired to patients in terms of sex and age were studied. Nine patients of each group were on inadequate glycemic controls. Intracellular cytokines were evaluated using flow cytometry. Cell cultures were stimulated with LPS to evaluate TNF-alpha and IL-6 or with PMA and lonomycin to evaluate IFN-gamma, IL-8 and IL-10 intracellular staining. Results: The percentages of CD33(+) cells bearing TNF-alpha and CD3(+) cells bearing IL-10 were increased in type 1 diabetic patients with inadequate glycemic control in relation to those with adequate control. In contrast, the percentage of CD3(+) cells bearing IL-8 was decreased in type 2 patients under inadequate glycemic control. Conclusions: The glycemic control is important for the detection of intracellular cytokines, and may contribute towards the susceptibility to infections in diabetic patients. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Evaluation of Cytokine Production from Peripheral Blood Mononuclear Cells of Type 1 Diabetic Patients

This study aims to evaluate the production of cytokines, tumor necrosis factor (TNF), and interleukin 10 (IL-10) in peripheral blood mononuclear cells (PBMCs) from type 1 diabetic (T1D) patients by means of intracellular staining, flow cytometry, and ELISA and to correlate it with inadequate (IN) and adequate (A) metabolic controls. We studied 28 patients with T1D and 20 healthy individuals (C) paired by sex and age. T1D patients were divided in patients with IN and A metabolic control. PBMC cultures were stimulated with LPS to evaluate TNF or were stimulated with PMA/ionomycin or concanavalin A to evaluate IL-10. The TNF levels in supernatant of stimulated cultures, evaluated by ELISA, of diabetic patients were similar to those of healthy individuals, although the percentage of CD 33(+) cells that were positive for TNF was higher in the T1D IN group compared to the T1D A group (P = 0.01). Similarly, the IL-10 levels evaluated by ELISA in stimulated cultures of T1D patients were not different from those in the control group; moreover, the percentage of CD3(+) cells positive for intracellular IL-10 were higher in the T1D IN group compared to C groups (P = 0.007). The increased levels of cytokines in T1D IN diabetic patients, with reduction in the A group, suggests that hyperglycemia stimulates an inflammatory state that can result in a deficient immune cellular response. The data suggest that assessment by intracellular staining seems to be more accurate than the ELISA technique in evaluating diabetic patients.

INTRAVITREAL INJECTION OF AUTOLOGOUS BONE MARROW-DERIVED MONONUCLEAR CELLS FOR HEREDITARY RETINAL DYSTROPHY A Phase I Trial

Purpose: To evaluate the short-term (10 months) safety of a single intravitreal injection of autologous bone marrow-derived mononuclear cells in patients with retinitis pigmentosa or cone-rod dystrophy. Methods: A prospective, Phase I, nonrandomized, open-label study including 3 patients with retinitis pigmentosa and 2 patients with cone-rod dystrophy and an Early Treatment Diabetic Retinopathy Study best-corrected visual acuity of 20/200 or worse. Evaluations including best-corrected visual acuity, full-field electroretinography, kinetic visual field (Goldman), fluorescein and indocyanine green angiography, and optical coherence tomography were performed at baseline and 1, 7, 13, 18, 22, and 40 weeks after intravitreal injection of 10 X 10(6) autologous bone marrow-derived mononuclear cells (0.1 mL) into 1 study eye of each patient. Results: No adverse event associated with the injection was observed. A 1-line improvement in best-corrected visual acuity was measured in 4 patients 1 week after injection and was maintained throughout follow-up. Three patients showed undetectable electroretinography responses at all study visits, while 1 patient demonstrated residual responses for dark-adapted standard flash stimulus (a wave amplitude approximately 35 mu V), which remained recordable throughout follow-up, and 1 patient showed a small response (a wave amplitude approximately 20 mu V) recordable only at Weeks 7, 13, 22, and 40. Visual fields showed no reduction (with a Goldman Standard V5e stimulus) for any patient at any visit. No other changes were observed on optical coherence tomography or fluorescein and indocyanine green angiograms. Conclusion: Intravitreal injection of autologous bone marrow-derived mononuclear cells in eyes with advanced retinitis pigmentosa or cone-rod dystrophy was associated with no detectable structural or functional toxicity over a period of 10 months. Further studies are required to investigate the role, if any, of autologous bone marrow-derived mononuclear cell therapy in the management of retinal dystrophies. RETINA 31: 1207-1214, 2011