Ex Vivo Treatment with Allogenic Mesenchymal Stem Cells of a Healthy Donor on Peripheral Blood Mononuclear Cells of Patients with Severe Alopecia Areata: Targeting Dysregulated T Cells and the Acquisition of Immunotolerance.

Alopecia areata (AA) is an autoimmune condition related to the collapse of the immune privilege of hair follicles. Certain AA populations present severe clinical manifestations, such as total scalp hair or body hair loss and a treatment refractory property. The aim of this study was to assess the effects of allogenic human mesenchymal stem cells (hMSCs) from healthy donors on the peripheral blood mononuclear cells (PBMCs) of severe AA patients, with a focus on the change in the cell fraction of Th1, Th17, and Treg cells and immunomodulatory functions. PBMCs of 10 AA patients and eight healthy controls were collected. Levels of Th17, Th1, and Treg subsets were determined via flow cytometry at baseline, activation status, and after co-culturing with hMSCs. All participants were severe AA patients with SALT > 50 and with a long disease duration. While the baseline Th1 and Treg levels of AA patients were comparable to those of healthy controls, their Th17 levels were significantly lower than those of the controls. When stimulated, the levels of CD4+IFN-γ+ T cells of the AA patients rose sharply compared to the baseline, which was not the case in those of healthy controls. The cell fraction of CD4+Foxp3+ regulatory T cells also abruptly increased in AA patients only. Co-culturing with allogenic hMSCs in activated AA PBMCs slightly suppressed the activation levels of CD4+INF-γ+ T cells, whereas it significantly induced the differentiation of CD4+Foxp3+ regulatory T cells. However, these changes were not prominent in the PBMCs of health controls. To examine the pathomechanisms, PBMCs of healthy donors were treated with IFN-γ to induce AA-like environment and then treated with allogenic grants and compared with ruxolitinib as a positive treatment control. hMSC treatment was shown to significantly inhibit the mRNA levels of proinflammatory cytokines, such as IFN-γ, TNF-α, IL-1α, IL-2R, IL-15, and IL-18, and chemokines, such as CCR7 and CCR10, in IFN-treated PBMCs. Interestingly, hMSCs suppressed the activation of JAK/STAT signaling by IFN in PBMCs with an effect that was comparable to that of ruxolitinib. Furthermore, the hMSC treatment showed stronger efficacy in inducing Foxp3, IL-10, and TGF-ß mRNA transcription than ruxolitinib in IFN-treated PBMCs. This study suggests that allogenic hMSC treatments have therapeutic potential to induce immune tolerance and anti-inflammatory effects in severe AA patients.

Preliminary results on the interactive effects of deoxynivalenol, zearalenone and fumonisin B1 on porcine lymphocytes.

Fusarium mycotoxins, such as fumonisin B1 (FB1), deoxynivalenol (DON) and zearalenone (ZEN), frequently co-occur in feed raw materials and their presence is ubiquitous. The aims of this study were to determine the concentration that inhibits cell viability by 50% (IC50 values) for each mycotoxin (after 24, 48 and 72 h) and to investigate their combined effects in binary (DON + ZEN: DZ, DON + FB1: DF, FB1 + ZEN: FZ) and ternary (DFZ) mixtures using cyto- and genotoxicity on porcine lymphocytes as endpoints. The potency of cytotoxicity of the three toxins in an increasing order was FB1 < ZEN < DON. The range of IC values depending on the period of exposure was 0.31-0.42 µg/ml and 16.6- 22.9 µg/ml for DON and ZEN, respectively, and 101.15 µg/ml for FB1 (50% viability was reached only after 72 h). The main interaction observed was antagonism regarding cytotoxicity. Lower and higher sets of concentrations were used for the genotoxicity (comet assay) experiments. When lower concentrations were used, antagonism was again the main interaction observed. However, at higher concentrations an antagonism was confirmed only for DFZ, whereas for DZ and FZ a synergism was observed. Interactions of DF were inconsistent in different exposure periods in both series of experiments. Further studies with additional endpoints should be performed (e.g. DNA fragmentation, protein synthesis) in order to elucidate the mechanisms underlying the interactions observed.

Synthesis, Spectral Analysis and Preliminary in Vitro Evaluation of Some Tetrapyrrolic Complexes with 3d Metal Ions.

In this paper, two tetrapyrrolic complexes, Zn(II)-5-(3-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin and Cu(II)-5-(3-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin were synthesized, and characterized from a spectral and biological point of view. The study provided data concerning the behavior of identical external substituents vs. two different core insertions. Some of the properties of the proposed tetrapyrrolic structures were highlighted, having photodynamic therapy of cancer as a targeted biomedical application. Elemental analysis, NMR, FTIR and UV-Vis data in various solvents were provided. A preliminary in vitro study on normal and cancer cultured cells was carried out for biocompatibility assessment in dark conditions. The preliminary in vitro study performed on human peripheral mononuclear cells exposed to tetrapyrrolic compounds (2 µM) showed that the proposed compounds had a convenient cytotoxic profile on human normal peripheral blood mononuclear cells under dark conditions. Meanwhile, the investigated compounds reduced the number of metabolically active breast tumor MCF-7 cells, with the exception of Zn(II) complex-containing a symmetrical ligand. Accordingly, preliminary in vitro data suggest that the proposed tetrapyrrolic compounds are good candidates for PDT, as they limit tumor expansion even under dark conditions, whilst sparing normal cells.

Characterization of the CD40L/Oncostatin M/Oncostatin M receptor axis as an antiviral and immunostimulatory system disrupted in chronic HCV infection.

BACKGROUND & AIMS: Oncostatin M (OSM) is an inflammatory cytokine which interacts with a heterodimeric receptor formed by gp130 and either OSMRß or LIFR. Here we have analysed OSM and its receptors in livers with chronic hepatitis C (CHC) and studied the factors that regulate this system. METHODS: OSM, OSM receptors and OSM-target molecules were studied by immunohistochemistry and/or qPCR analysis in livers from CHC patients and controls. We determined the production of OSM by CD40L-stimulated antigen presenting cells (APC) and its biological effects on HuH7 cells containing HCV replicon (HuH7 Core-3'). RESULTS: OSM was upregulated in livers with CHC and its production was mapped to CD11c+ cells. OSM levels correlated directly with inflammatory activity and CD40L expression. In vitro studies showed that OSM is released by APC upon interaction with activated CD4+ T cells in a CD40L-dependent manner. Culture of HuH7 Core-3' cells with supernatant from CD40L-stimulated APC repressed HCV replication and induced IL-7 and IL-15Rα. These effects were dampened by antibodies blocking OSM or gp130 and by silencing OSMRß. In CHC livers OSMRß and LIFR were significantly downregulated and their values correlated with those of OSM-induced molecules. Experiments in HuH7 cells showed that impaired STAT3 signaling and exposure to TGFß1, two findings in CHC, are factors involved in repressing OSMRß and LIFR, respectively. CONCLUSIONS: OSM is a cytokine possessing vigorous antiviral and immunostimulatory properties which is released by APC upon interaction with CD40L present on activated CD4+ T cells. In livers with CHC, OSM is overexpressed but its biological activity appears to be hampered because of downregulation of its receptor subunits.

Modification of Polyethylene Glycol-Hydroxypropyl Methacrylate Polymeric Micelles Loaded with Curcumin for Cellular Internalization and Cytotoxicity to Wilms Tumor 1-Expressing Myeloblastic Leukemia K562 Cells.

Curcumin loaded in micelles of block copolymers of ω-methoxypoly(ethylene glycol) and N-(2-hydroxypropyl) methacrylamide modified with aliphatic dilactate (CD) or aromatic benzoyl group (CN) were previously reported to inhibit human ovarian carcinoma (OVCAR-3), human colorectal adenocarcinoma (Caco-2), and human lymphoblastic leukemia (Molt-4) cells. Myeloblastic leukemia cells (K562) are prone to drug resistance and differ in both cancer genotype and phenotype from the three mentioned cancer cells. In the present study, CD and CN micelles were prepared and their effects on K562 and normal cells were explored. The obtained CD and CN showed a narrow size distribution with diameters of 63 ± 3 and 50 ± 1 nm, respectively. The curcumin entrapment efficiency of CD and CN was similarly high, above 80% (84 ± 8% and 91 ± 3%). Both CD and CN showed suppression on WT1-expressing K562 and high cell-cycle arrest at the G2/M phase. However, CD showed significantly higher cytotoxicity to K562, with faster cellular uptake and internalization than CN. In addition, CD showed better compatibility with normal red blood cells and peripheral blood mononuclear cells than CN. The promising CD will be further investigated in rodents and possibly in clinical studies for leukemia treatment.

Reliable cell preparation protocol for Raman imaging to effectively differentiate normal leukocytes and leukemic blasts.

Leukemias are a remarkably diverse group of malignancies originating from abnormal progenitor cells in the bone marrow. Leukemia subtypes are classified according to the cell type that has undergone neoplastic transformation using demanding and time-consuming methods. Alternative is Raman imaging that can be used both for living and fixed cells. However, considering the diversity of leukemic cell types and normal leukocytes, and the availability of different sample preparation protocols, the main objective of this work was to verify them for leukemia and normal blood cell samples for Raman imaging. The effect of glutaraldehyde (GA) fixation in a concentration gradient (0.1 %, 0.5 %, and 2.5 % GA) on the molecular structure of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral blood mononuclear cells (PBMCs) was verified. Changes in the secondary structure of proteins within cells were indicated as the main effect of fixation, as shown by an increase in band intensity at 1041 cm-1, characteristic for in-plane δ(CH) deformation in phenylalanine (Phe). Different sensitivity of mononuclear and leukemic cells to fixation was observed. While the 0.1 % concentration of GA was too low to preserve the cell structure for an extended period of time, a GA concentration of 0.5 % seemed optimal for both normal and malignant cells. Chemical changes in PBMCs samples stored for 11 days were also investigated, which manifested in numerous modifications in the secondary structure of proteins and the content of nucleic acids. The impact of cell preculturing for 72 h after unbanking was verified, and there was no significant effect on the molecular structure of cells fixed with 0.5 % GA. In summary, the developed protocol for the preparation of samples for Raman imaging allows for the effective differentiation of fixed normal leukocytes from malignant T lymphoblasts.

Psychostimulant-induced aberrant DNA methylation in an in vitro model of human peripheral blood mononuclear cells.

BACKGROUND: Several reports have provided crucial evidence in animal models that epigenetic modifications, such as DNA methylation, may be involved in psychostimulant-induced stable changes at the cellular level in the brain. Epigenetic editors DNA methyltransferases (DNMTs) and ten-eleven translocation enzymes (TETs) coordinate expression of gene networks, which then manifest as long-term behavioural changes. However, the extent to which aberrant DNA methylation is involved in the mechanisms of substance use disorder in humans is unclear. We previously demonstrated that cocaine modifies gene transcription, via DNA methylation, throughout the brain and in peripheral blood cells in mice. RESULTS: We treated human peripheral blood mononuclear cells (PBMCs) from healthy male donors (n = 18) in vitro with psychostimulants (amphetamine, cocaine). After treatment, we assessed mRNA levels and enzymatic activities of TETs and DNMTs, conducted genome-wide DNA methylation assays and next-generation sequencing. We found that repeated exposure to psychostimulants decreased mRNA levels and enzymatic activity of TETs and 5-hydroxymethylation levels in PBMCs. These data were in line with observed hyper- and hypomethylation and mRNA expression of marker genes (IL-10, ATP2B4). Additionally, we evaluated whether the effects of cocaine on epigenetic editors (DNMTs and TETs) and cytokines interleukin-6 (IL-6) and IL-10 could be reversed by the DNMT inhibitor decitabine. Indeed, decitabine eliminated cocaine's effect on the activity of TETs and DNMTs and decreased cytokine levels, whereas cocaine increased IL-6 and decreased IL-10. CONCLUSIONS: Our data suggest that repeated psychostimulant exposure decreases TETs' enzymatic activity in PBMCs. Co-treatment with decitabine reversed TETs' levels and modulated immune response after repeated cocaine exposure. Further investigation is needed to clarify if TET could represent a putative biomarker of psychostimulant use and if DNMT inhibition could have therapeutic potential.

Generation of Human iPSCs by Episomal Reprogramming of Skin Fibroblasts and Peripheral Blood Mononuclear Cells.

Human-induced pluripotent stem cells (iPSCs) can be generated from patient-specific somatic cells by forced expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Sustained expression of the transgenes during reprogramming is crucial for the successful derivation of iPSCs. Integrating retroviruses have been used to achieve the required prolonged expression; however, issues of undesirable transgene expression in the iPSC-derived cell types post reprogramming can occur. Alternative non-integrating approaches to reprogram somatic cells into pluripotency have been established. Here, we describe a detailed method for generating human iPSCs from fibroblasts and peripheral blood mononuclear cells (PBMCs) using the non-integrating episomal plasmids. The delivery of the episomal plasmids into the somatic cells is achieved using a nucleofection technique, and reprogramming is performed in chemically defined media. This process takes approximately 30 days to establish the iPSC colonies. We also describe a method for growing iPSCs on vitronectin as well as procedures for the long-term expansion of iPSCs on human fibroblast feeder cells.

Ex Vivo Virotherapy with Myxoma Virus to Treat Cancer.

Myxoma virus (MYXV) is a member of the Poxviridae family and the genus Leporipoxvirus. In nature MYXV tropism is restricted to lagomorphs, and is specifically pathogenic only for European rabbits (Oryctolagus cuniculus), in which this virus causes the lethal systemic disease called myxomatosis. Importantly, although MYXV cannot cause any disease pathology in humans, mice, or any other domestic animals other than rabbit, this virus can productively infect and kill a variety of human and murine cancer cells, from either primary sources or cultured cancer cell lines. Therefore, in the last decade, MYXV has emerged as a novel oncolytic virus against hematologic malignancies and various solid cancers. One novel aspect of MYXV virotherapy is a new systemic virus delivery strategy to cancer sites in the recipient, by which adsorption of the virus to isolated leukocytes is conducted prior to reinfusion of the virus-infected cells back into the recipient, via a procedure called ex vivo virotherapy (EVV, or simply EV2). The EV2 delivery strategy thus exploits the inherent migratory properties of leukocytes to ferry MYXV to tissue sites bearing cancer cells that are accessible to leukocyte chemotaxis. Here, we describe EV2 procedures with MYXV to systemically deliver the virus to sites of disseminated and/or metastatic cancer in situ via infected leukocytes derived from either bone marrow or peripheral blood.

Elevated repulsive guidance molecule-a mRNA in peripheral blood mononuclear cells are associated with impaired leptomeningeal collaterals in patients with middle cerebral artery occlusions.

BACKGROUND: In the event of middle cerebral artery occlusion (MCAO), leptomeningeal collaterals (LMCs) play a crucial role in determining the survival of brain tissue distal to occlusion. Previous findings indicated that genes controlling arteriogenesis can impact the extent of LMCs. Therefore, probe for potential genetic parameters correlating of arteriogenesis may be clinically useful in predicting LMCs status in MCAO. During the screening process, we focused on repulsive guidance molecule a (RGMa), which has been reported to play a negative role in angiogenesis after stroke by decreasing the proliferation, migration, and tube formation of endothelial cells (ECs) in vivo and in vitro. Indeed, endothelial function plays a main role in arteriogenesis and is essential in determining the LMCs status. Therefore, in present study, we aimed to testify the hypothesis that RGMa might be associated with LMCs status in MCAO. METHODS: We prospectively enrolled patients with acute M1 MCA +/- intracranial internal carotid artery (ICA) occlusions (n=96) and healthy controls (n=33). Status of LMCs was evaluated according to computed tomographic angiography (CTA) on admission. Baseline RGMa mRNA expression was quantified by using quantitative real-time PCR. RESULTS: Patients with poor LMCs showed significantly higher RGMa mRNA levels than patients with good LMCs status (P=0.001) as well as healthy controls (P=0.002), respectively; whereas good LMCs group showed similar baseline RGMa levels than controls (P=1.000). RGMa mRNA level and baseline NIHSS score were independent predictors for impaired LMCs. CONCLUSIONS: In MCAO patients, elevated PBMCs RGMa mRNA levels were associated with impaired LMCs status, indicating that measurement of RGMa mRNA expression in the early phase of stroke, together with other clinical approaches, was logically expected to be useful for predicting LMCs status. Moreover, a role for RGMa in leptomeningeal arteriogenesis following ischemic stroke can be hypothesized.

Effect of porous tantalum on the biological response of human peripheral mononuclear cells exposed to Porphyromonas gingivalis.

AIM: To evaluate biological behavior of human peripheral mononuclear cells (PBMC) in contact with porous tantalum (PT) and Porphyromonas gingivalis (Pg). METHODS: Pg was incubated for 8 hours. The groups formed were: PBMC (control), PBMC + PT, PBMC + Pg and PBMC + PT + Pg. Cell viability was evaluated using MTT assay. The morphology and adhesion of PBMC to PT was evaluated using scanning electron microscopy. Expression of interleukin (IL)-10, transforming growth factor (TGF)-ß, matrix metallopeptidase (MMP)-9 and receptor activator of nuclear factor-κΒ ligand (RANKL) was determined by enzyme-linked immunosorbent assay. RESULTS: MTT assay revealed that PT did not interfere in the mitochondrial activity of PBMC (P > .05). Scanning electron microscopy showed the adherence of PBMC to PT. IL-10 levels in PBMC + PT were similar to PBMC and lower than PBMC + Pg. TGF-ß levels in PBMC + PT were higher than PBMC and PBMC + Pg. MMP-9 levels in PBMC + PT were similar to PBMC and lower than PBMC + Pg and PBMC + PT + Pg. RANKL levels in PBMC + PT were lower than in PBMC. CONCLUSION: PT did not affect PBMC viability, allowed cell adhesion, reduced expression of RANKL and enhanced TGF-ß in comparison with the control group.

Increased protein intake affects pro-opiomelanocortin (POMC) processing, immune function and IGF signaling in peripheral blood mononuclear cells of home-dwelling old subjects using a genome-wide gene expression approach.

BACKGROUND: Adequate protein intake among older adults is associated with better health outcomes such as immune function and metabolic regulation of skeletal muscle, but conflicting results make it difficult to define the optimal intake. To further understand the impact of protein intake on metabolic processes, the aim of the study was to explore genome-wide gene expression changes in peripheral blood mononuclear cells (PBMCs) in home-dwelling old subjects after increased protein intake for 12 weeks. METHOD: In a parallel double-blind randomized controlled intervention study, subjects (≥ 70 years) received a protein-enriched milk (2 × 20 g protein/day, n = 14, mean (±SD) age 76.9 ± 4.9 years) or an isocaloric carbohydrate drink (n = 17, mean (±SD) age 77.7 ± 4.8 years) for breakfast and evening meal for 12 weeks. PBMCs were isolated before and after the intervention. Microarray analysis was performed using Illumina technology. Serum levels of gut peptides and insulin growth factor (IGF)-1 were also measured. RESULTS: In total 758 gene transcripts were regulated after increased protein intake, and 649 gene transcripts were regulated after intake of carbohydrates (p < 0.05). Forty-two of these genes were overlapping. After adjusting for multiple testing, 27 of the 758 gene transcripts were regulated (FDR, q-value < 0.25) after protein intake. Of these 25 were upregulated and two downregulated. In particular, genes and signaling pathways involved in pro-opiomelanocortin (POMC) processing, immune function, and IGF signaling were significantly altered. CONCLUSIONS: PBMCs can be used to study gene expression changes after long-term protein intake, as many signaling pathways were regulated after increased protein intake. The functional significance of these findings needs to be further investigated. TRIAL REGISTRATION: ClinicalTrials.gov, ID no. NCT02218333. The study was registered on August 18, 2014.

Changes in the expression of α1B-adrenoceptor in peripheral mononuclear cells correlates with blood pressure and plasmatic homocysteine.

Human peripheral mononuclear cells (HPMC) have been suggested as a practical surrogate for myocardial or vascular cells. Present work analyses if changes in the expression of α1-adrenoceptors (ARs) in HPMC are related to the hypertensive state and its clinical consequences. Quantitative RT-PCR was employed to evaluate the mRNA levels of the three α1-ARs (α1A, α1B, α1D) in HPMC isolated from normotensive and hypertensive patients, and also in tissues from two animal models of hypertension: spontaneously hypertensive rats (SHR) and hypertension induced by chronic treatment with L-NAME. In patients, 24-h ambulatory blood pressure and serum biochemical profile were also recorded. We found that α1B-AR expression was higher in HPMC from hypertensive patients and correlated with blood pressure and plasmatic homocysteine. A rise in the α1B-AR expression in kidneys, but not in heart from hypertensive animal models was also found. α1D-AR did not change in HPMC, not in rat heart or kidney, but a significant correlation with plasmatic aldosterone was found. In conclusion, we have proved that α1-ARs mRNA expression in HPMC correlates with clinical variables and could be used as a potential biomarker in hypertensive patients.

EFFECTS OF COMBRETUM HEREROENSE AND CANTHIUMMUNDIANUM water EXTRACTS ON PRODUCTION AND EXPRESSION OF INTERLEUKIN-4.

BACKGROUND: Combretum hereroense and Canthium mundianum are two plants commonly used by traditional healers in the Northern region of Limpopo, South Africa for the treatment of diarrhea and inflammation. In the present study, the effects of their water extracts on the production and expression of interleukin-4 by peripheral blood mononuclear cells (PBMC'S) from HIV positive and negative individuals was evaluated. MATERIALS AND METHODS: Blood samples were collected from both HIV positive and HIV negative volunteers and were used for the purification of Peripheral blood mononuclear cells (PBMC). The PBMCs were cultured together with the water extracts after activation with phytohemagglutinin (PHA) for three days. Solid-phase sandwich ELISA (MABTECH) kit was used to detect IL-4 on un-stimulated and stimulated PBMC'S with phytohemaglutinin (PHA) and plant extracts, followed by the isolation of RNA using RNAesy Qiagen mini kit from the cells. Reverse transcriptase real time PCR was used to evaluate IL-4 gene expression by the cells. RESULTS: Combretum hereroense showed higher production of IL-4 at three different concentrations and a significant expression of mRNA with 4-fold amplification increase at 300µg/ml and 2-fold amplification increase at 20µg/ml. Canthium mundianum also showed increased production of IL-4 at 300µg/ml, but inhibited its production at 20µg/ml. Both extracts showed no expression at 50µg/ml. The response of the PBMCs from HIV negative individuals was more pronounced than that of HIV positive individuals who mostly increased production of IL4 at smaller concentrations unlike their HIV negative counterparts. Although in vitro studies do not necessarily predict in vivo outcomes, the plant extracts modulated the immune system by enhancing the production and expression of IL-4 in both HIV- and HIV+ individuals at different concentrations. CONCLUSIONS: For the first time we have shown that the immunomodulatory effect of medicinal plants may depend on the clinical status of the individual. The present study revealed that the effect of the water extracts from the two plants on IL-4 expression and production is dependent on the microbiological state of the individual and is dose dependent. Further studies are needed to identify the active components in the extracts and also characterize the patients further for a better understanding of the mechanisms of action of these extracts.

High CD6 and low chemokine receptor expression on peripheral blood lymphocytes correlates with MRI gadolinium enhancement in MS.

Correlation between gadolinium-enhancing [Gd(+)] lesions on MRI and expression of CD6 molecules and a group of chemokine receptors on peripheral blood (PB) and cerebrospinal fluid (CSF) immune cells was measured in multiple sclerosis (MS) patients. Twenty remitting-relapsing MS patients with (n=10) and without (n=10) Gd(+) lesions entered the study. mRNA and surface expression of CD6 and CCR1, CCR2, CCR3 and CCR5 was measured by immunostaining and flow cytometry. Expression of mRNA and surface staining for CD6 in PB T lymphocytes was increased in Gd(+) compared to Gd(-) patients (p<0.01; p<0.05, respectively). CD6 mRNA correlated with the number and size of Gd(+) lesions (r=0.67, and r=0.65 respectively). mRNA and surface expression for CCR1, CCR2, and CCR3 in PB cells was lower in Gd(+) compared to Gd(-) MS patients (p<0.05, p<0.05). The frequency of cells co-expressing CD6 with CCR1 and CCR5 was low in PB T lymphocytes and high in CSF (p<0.05, p<0.05). These results suggest that Gd(+) correlates with increased expression of CD6 and decreased expression of chemokine receptors on PB T lymphocytes. Co-expression of CD6 with CCR1 and CCR5 predisposes cells for transmigration into CSF.

Marine n-3 Fatty Acids and Gene Expression in Peripheral Blood Mononuclear Cells.

Intake of marine n-3 fatty acids has been shown to have beneficial effects on cardiovascular disease. Gene expression analyses in peripheral blood mononuclear cells (PBMCs) are used to understand the underlying mechanisms of action of marine n-3 fatty acids. The aim of this review was to summarize the effects mediated by marine n-3 fatty acids on gene expression in PBMCs. A systematic literature search was conducted in PubMed in May 2014 and 14 papers were included. Targeted gene expression studies were reported in 9 papers and focused on genes involved in lipid metabolism and inflammation. Whole genome transcriptome analyses were conducted in 5 papers, and processes and pathways related to atherosclerotic plaque formation such as inflammation, oxidative stress response, cell cycle, cell adhesion, and apoptosis were modulated after fish oil supplementation. PBMC gene expression profiling has a potential to clarify further the molecular effects of fish oil consumption on human health.

Determinants of Risk Infection During Therapy with Anti TNF-Alpha Blocking Agents in Rheumatoid Arthritis.

The use of TNF-alpha antagonists (infliximab, etanercept, adalimumab) has changed the course of many rheumatic diseases including rheumatoid arthritis (RA). Since their approval, some questions regarding their safety including infections have been observed. The aim of the study was to evaluate the changes in cytokines levels and cells subsets in patients with RA during anti TNF blocking agents treatment and the possible effect on infections' development. We evaluated in 89 RA patients [39 treated with etanercept (ETN), 29 with adalimumab (ADA) and 21 with infliximab (IFN)] at baseline and after 6 months the following parameters: procalcitonin, ESR, CRP, cytokines as TNF, IL-6, IL-10, IL-8 and the TNF/IL-10 ratio, and peripheral mononuclear cells as CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD3- /CD16+/56+, CD14+HLADR+, CD20+, CD19+/CD38+. Peripheral mononuclear cells were detected by flow cytometric system Cytomics FC500 and cytokines circulating levels by a quantitative sandwich enzyme immunoassay technique (Human IL-8 Instant ELISAe Bioscience, Human IL-6 Instant ELISA e Bioscience, Human IL-10 Instant ELISAe Bioscience and Human TNF-a Quantikine immunoassay RD system). A lower reduction of CD14+HLADR+ in ADA group 54.6±10.4% vs ETA 48.4±15.7% vs INF 40.7±16.5%, p<0.039 was found. No differences in all three groups on peripheral mononuclear cells CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, CD 20+, CD19+/CD38+, CD3-/CD16+/56+, and cytokine circulating levels were found. The number of infections at 6 months was: 10.3% in ADA group, 12.8% in ETN group and 19.04% in IFN group. A correlation was found between the reduction in CD14+HLADR+ cells and IFN treatment. Our data showed that the level of CD14+HLADR+ cells was reduced during therapy with IFN. ADA and ETN don't reduce lymphocyte populations and their subsets such as CD14+HLADR+ cells that play an important role host defence.