Cells In Peripheral Blood Mononuclear Cells Research Articles

CXCL10/CXCR3 axis is associated with disease activity and the development of mucocutaneous lesions in patients with Beh\xe7et\u2019s disease

The objective of this study was to investigate CXC chemokines and its receptor in patients with Behcet’s disease (BD) and their associations with disease activity. Blood samples were collected from 109 BD patients and 36 age- and sex-matched healthy controls (HCs). Twenty-two follow-up blood samples were collected in BD patients. Serum CXC chemokines (CXCL1, CXCL8, CXCL9, CXCL10, CXCL12, CXCL13 and CXCL16) and cell surface marker expression (CD3, CD4 and CXCR3) in peripheral blood mononuclear cells (PBMCs) were assayed. Clinical features including disease activity were evaluated at the time of blood collection. CXCR3 expression in skin and intestinal lesions from BD patients and HCs was assessed via immunohistochemistry. Serum CXCL10 levels were correlated with disease activity in terms of Behçet’s Disease Current Activity Form (BDCAF) (p < 0.001). In follow-up BD patients, changes in serum CXCL10 levels tended to be correlated with those of BDCAF. The percentage of CXCR3 expression in CD3-positive cells in PBMCs was inversely correlated with serum CXCL10 levels in BD patients (p = 0.022). By immunohistochemistry, the number of CXCR3-positive mononuclear cells was higher in skin and intestinal lesions of BD patients than in those of HCs. These results suggest that the CXCL10/CXCR3 axis may contribute to the pathogenesis of BD.

CD4+ T helper cells and regulatory T cells in active lupus nephritis: an imbalance towards a predominant Th1 response?

The objective of this study was to evaluate the frequency of CD4+ T cell subsets in peripheral blood mononuclear cells (PBMC), urine and renal tissue from patients with lupus nephritis (LN). PBMC and urinary cells were collected from 17 patients with active LN, 20 disease controls (DC) with primary glomerulonephritis and 10 healthy controls (HC) and were analysed by flow cytometry with markers for T helper type 1 (Th1), Th2, Th17 and regulatory T cells (Treg ) cells. T cell subsets were assessed by immunohistochemistry from LN biopsy specimens from 12 LN patients. T cell subtypes in PBMC were re-evaluated at 6 months of therapy. CD4+ T cells were decreased in PBMC in LN compared with DC and HC (P = 0·0001). No differences were observed in urinary CD4+ T cell subsets between LN and DC. The frequency of urinary Th17 cells was higher in patients with non-proliferative than in proliferative LN (P = 0·041). CD3+ and T-box 21 ( Tbet+) cells were found in glomeruli and interstitium of LN patients, while forkhead box protein 3 (FoxP3), retinoid-related orphan receptor gamma (ROR-γ) and GATA binding protein 3 (GATA-3) were present only in glomeruli. Th1 cells in PBMC were correlated negatively with urinary Th1 cells (Rho=-0·531; P = 0·028) and with Tbet in renal interstitium (Rho=-0·782; P = 0·004). At 6 months, LN patients showed an increase in Th17 cells in PBMC. In conclusion, the inverse association between Th1 cells from PBMC and urinary/renal tissue indicate a role for Th1 in LN pathophysiology. Urinary Th17 cells were associated with less severe LN, and Th17 increased in PBMC during therapy. Urinary CD4+ T cells were not different between LN and DC.

Design and synthesis of novel nitrogen mustard-evodiamine hybrids with selective antiproliferative activity

A series of novel nitrogen mustard-evodiamine hybrids were synthesized and evaluated for their antitproliferative properties. The antiproliferative activities of 10a–d, 11a–d, and 12a–d against four different kinds of human cancer cell lines (PC-3, HepG2, THP-1 and HL-60) and human normal peripheral blood mononuclear cells (PBMC) were determined. The results showed that all the target hybrid compounds exhibited antiproliferative activities against tested human tumor cell lines to some extent and no antiproliferative activities (>200 μM) against human normal PBMC cells. The antiproliferative selectivity between tumorous and normal cells was very useful for further antitumor drug development. Among the target compounds, 12c showed the strongest cytotoxicity against two tumor cell lines (THP-1 and HL-60) with IC50 values of 4.05 μM and 0.50 μM, respectively, and selected for further mechanism study in HL-60 cells. The results showed that 12c could induce HL-60 cells apoptosis and arrest at G2 phase at low sub-micromolar concentrations via mitochondria-related pathways.

Correlation of the expression of miR-146a in peripheral blood mononuclear cells of patients with ankylosing spondylitis and inflammatory factors.

We investigated the expression of miR-146a in peripheral blood mononuclear cell (PBMC) of patients with ankylosing spondylitis (AS) and its correlation with inflammatory factors to explore the clinical significance. In total 45 patients with AS were selected at the Weifang People's Hospital from June, 2014 to January, 2016. At the same time, 30 healthy volunteers were also selected to serve as control group. Expression level of miR-146a in PBMC cells of patients in each group was detected by quantitative real-time-polymerase chain reaction (qRT-PCR). Levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in serum and the supernatant of culture medium of PBMC derived from each group were detected by enzyme-linked immunosorbent assay (ELISA). Correlations between expression level of miR-146a and serum inflammatory factors, and clinical indicators were analyzed. Clinical indicators included bath ankylosing spondylitis disease activity index (BASDAI), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and duration of morning stiffness. Expression level of miR-146a in PBMC of AS patients was significantly higher than that of healthy control (P<0.01); levels of TNF-α, IL-1β and IL-6 in serum and the supernatant of culture medium of PBMC derived from AS patients were significant compared to those of control group (P<0.01); expression of miR-146a in PBMC of patients with AS was positively correlated with the levels of TNF-α, IL-1β and IL-6 in serum (r=0.632, P<0.01; r=0.574, P<0.01; r=0.483, P<0.01). In addition, expression level of miR-146a in PBMC of patients with AS was positively correlated with BASDAI, ESR, CRP and duration of morning stiffness (r=0.551, P<0.01; r=0.738, P<0.01; r=0.685, P<0.01; r=0.497, P<0.01). Expression level of miR-146a in PBMC of AS patients was significantly increased and the expression level was positively correlated with the levels of TNF-α, IL-1β and IL-6 in serum (P<0.05). In addition, expression level of miR-146a in PBMC of AS patients was also positively correlated with BASDAI, ESR, CRP and duration of morning stiffness. Those results suggest that miR-146a may be involved in the pathogenesis of AS, and the expression level of miR-146a in PBMC cells may be helpful for diagnosis of AS and judgment of disease activity.

Rheumatoid arthritis fibroblast-like synoviocytes co-cultured with PBMC increased peripheral CD4+ CXCR5+ ICOS+ T cell numbers.

'Circulating' T follicular helper cells (Tfh), characterized by their surface phenotypes CD4+ chemokine receptor 5 (CXCR5)+ inducible co-stimulatory molecule (ICOS)+ , have been identified as the CD4+ T cell subset specialized in supporting the activation, expansion and differentiation of B cells. Fibroblast-like synoviocytes (FLS) are critical in promoting inflammation and cartilage destruction in rheumatoid arthritis (RA), and the interaction between FLS and T cells is considered to facilitate FLS activation and T cell recruitment. However, it remains unknown whether RA-FLS co-cultured with activated peripheral blood mononuclear cells (PBMC) has immunoregulatory effects on peripheral Tfh. In the present study, we co-cultured RA-FLS with or without anti-CD3/CD28-stimulated PBMC. The results showed that RA-FLS co-cultured with stimulated PBMC could increase the numbers of CD4+ CXCR5+ ICOS+ T cells of RA PBMC possibly via the production of interleukin (IL)-6, a critical cytokine involved in the differentiation of Tfh cells. We also observed increased reactive oxygen species (ROS) levels in the co-culture system of RA-FLS and PBMC. The percentage of CD4+ CXCR5+ ICOS+ T cells was decreased when ROS production was inhibited by N-acetyl-L-cysteine (NAC), a specific inhibitor which can decrease ROS production. In addition, we showed that the higher levels of tumour necrosis factor (TNF)-α and IL-1β in the co-culture system and the blocking of TNF receptor 2 (TNF-R2) and IL-1β receptor (IL-1βR) both decreased the numbers of CD4+ CXCR5+ ICOS+ T cells. Our study reveals a novel mechanistic insight into how the interaction of RA-FLS and PBMC participates in the RA pathogenesis, and also provides support for the biologicals application for RA.

Echinococcus multilocularis vesicular fluid inhibits activation and proliferation of natural killer cells.

Alveolar echinococcosis is a severe chronic helminthic disease that mimics slow-growing liver cancer. The immune evasion strategy of Echinococcus multilocularis Leuckart, 1863 remains poorly understood. The aim of this study was to investigate in vitro the impact of E. multilocularis vesicular fluid (Em-VF) on peripheral blood mononuclear cells (PBMC) and on natural killer (NK) cells. PBMC and NK cells were exposed to Em-VF (1 µg/ml) during six days. The effect of Em-VF was assessed on CD69, viability and proliferation, and on and transforming growth factor β (TGF-β), interferon γ (IFN-γ), interleukin 17 (IL-17) and interleukin 10, using flow cytometry and ELISA, respectively. Exposure to Em-VF had no bearing on PBMC's viability, proliferation and expression of CD69. In contrast, higher levels of IL-17 at day three and of TGF-β at day six were observed in PBMC supernatant after exposure to Em-VF (p < 0.05, Wilcoxon signed-rank test). Exposure to Em-VF induced a significant decrease of CD69 expression of NK cells at day three and a significant decrease of proliferation of NK cells at day six (p < 0.05, Wilcoxon signed-rank test). In contrast, NK cells viability and levels of cytokines did not vary significantly over Em-VF stimulation. Exposure to Em-VF had a significant bearing on activation and proliferation of NK cells. NK cells may play an important role in the immune response of the host against E. multilocularis.

Bone marrow IRF4 level in multiple myeloma: an indicator of peripheral blood Th17 and disease.

Interferon regulator factor 4 (IRF4) is characterized to be a member of interferon regulatory family, which is predominantly expressed in bone marrow plasma cells of patients with multiple myeloma (MM). Recent studies indicated IRF4 is critical for T-help cells (Th17) differentiation and interleukin-17 (IL-17) secretion. Here, a total of 58 MM patients were enrolled in this study, the proportions of Th17 cells and T regulatory (Treg) cells in peripheral blood mononuclear cells (PBMCs) were determined by flow cytometric analysis. Immunohistochemistry was employed to detect the IRF4 expression in bone marrow. Herein, we observed a significant increase of IRF4 in bone marrow accompanied with a notable up-regulation of Th17 cells in PBMC within MM patients compared with healthy donors. Furthermore, the proportions of Th17 cells and serum IL-17 levels were higher in patients with stage III than stage I & II MM patients, and those parameters were positively correlated with the expression of IRF4 in these cases. These results for the first time indicate that a crosstalk between IRF4 and Th17 cells is associated with MM prognosis, and IRF4 may be served an important target for MM immunotherapy.

Cytomegalovirus infection in HIV-infected and uninfected individuals is characterized by circulating regulatory T cells of unconstrained antigenic specificity.

Cytomegalovirus (CMV) infection is associated with immune-suppression in immune-compromised hosts and old adults. We previously showed that ex vivo CMV restimulation of peripheral blood mononuclear cells (PBMC) of CMV-seropositive volunteers expanded CD4+CD27-CD28- regulatory T cells (Tregs). Here we evaluate the phenotype and function of circulating CD4+CD27-CD28- T cells of CMV-seropositive adults. Compared with CMV-seronegative, CMV-seropositive adults had 10-fold higher CD4+CD27-CD28-% T cells in PBMC. Circulating CD4+CD27-CD28- T cells from both CMV-seropositive and seronegative donors expressed higher levels of TGFβ, granzyme B, CD39, CD147 and IL-35, and lower levels of CD127, compared with their parent circulating CD4+ T cells. However, only CMV-seropositive circulating CD4+CD27-CD28- had increased FOXP3 expression. CD4+CD27-CD28- sorted from the PBMC of CMV-seropositive donors expanded ex vivo in the presence of rhIL2 and inhibited ex vivo proliferation of autologous PBMC restimulated with CMV, varicella-zoster virus or C. albicans antigens. CD4+CD27-CD28- sorted from CMV-seronegative PBMC did not expand in the presence of rhIL2 and did not inhibit autologous PBMC proliferation. CD3+CD27-CD28- circulating T cells (≥80% CD8+) from CMV-seropositive HIV-infected donors also inhibited ex vivo proliferation of autologous PBMC restimulated with CMV or HIV. These data indicate that CMV-seropositive individuals have circulating Tregs that inhibit cell-mediated immune responses to CMV and other antigens and may be contribute to an immune-suppressive effect of CMV infection. Moreover, the phenotypic similarity between circulating CD4+CD27-CD28- Tregs with differentiated effector T cells suggests that the two T-cell subsets might evolve in parallel or in sequence from the same progenitor cells in response to CMV stimulation during reactivations.

Piperine enhances carbohydrate/fat metabolism in skeletal muscle during acute exercise in mice

BackgroundExercise promotes energy metabolism (e.g., metabolism of glucose and lipids) in skeletal muscles; however, reactive oxygen species are also generated during exercise. Various spices have been reported to have beneficial effects in sports medicine. Here, we investigated the effects of piperine, an active compound in black pepper, to determine its effects on metabolism during acute endurance exercise.MethodsICR mice (n = 18) were divided into three groups: nonexercise (CON), exercise (EX), and exercise with piperine (5 mg/kg) treatment (EP). Mice were subjected to enforced exercise on a treadmill at a speed of 22 m/min for 1 h. To evaluate the inflammatory responses following exercise, fluorescence-activated cell sorting analysis was performed to monitor changes in CD4+ cells within the peripheral blood mononuclear cells (PBMCs) of mice. The expression levels of metabolic pathway components and redox-related factors were evaluated in the soleus muscle by reverse transcription polymerase chain reaction and western blotting.ResultsThere were no changes in the differentiation of immune cells in PBMCs in both the EX and EP groups compared with that in the CON group. Mice in the EX group exhibited a significant increase in the expression of metabolic pathway components and redox signal-related components compared with mice in the CON group. Moreover, mice in the EP group showed greater metabolic (GLUT4, MCT1, FAT/CD36, CPT1, CS) changes than mice in the EX group, and changes in the expression of redox signal components were lower in the EP group than those in the EX group.ConclusionOur findings demonstrate that piperine promoted beneficial metabolism during exercise by regulating carbohydrate/fat metabolism and redox signals. Therefore, piperine may be a candidate supplement for improvement of exercise ability.

Analysis of cysteine-X-cysteine motif chemokine ligands 9, 10, and 11, their receptor CXCR3, and their possible role on the recruitment of immune cells at the maternal-conceptus interface in pigs.

Chemokines play critical roles in the establishment and maintenance of pregnancy in animals. Cysteine-X-cysteine motif chemokine ligand 9 (CXCL9), CXCL10, and CXCL11 are involved in recruiting immune cells by binding to their shared receptor, CXC receptor 3 (CXCR3), in a variety of tissues. This study examined the expression and regulation of chemokines CXCL9, CXCL10, and CXCL11, their receptor CXCR3, and their role at the maternal-conceptus interface in pigs. The endometrium expressed CXCL9, CXCL10, CXCL11, and CXCR3 stage specifically during pregnancy, with the greatest abundance on Day 15 of pregnancy. It was noted that their expression was primarily localized to stromal cells, endothelial cells, or vascular smooth muscle cells in the endometrium. Interferon-γ increased the abundance of CXCL9, CXCL10, CXCL11 mRNAs, but not CXCR3, in endometrial explants. Furthermore, recombinant CXCL9 (rCXCL9), rCXCL10, and rCXCL11 proteins increased migration of cultured peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Recombinant CXCL9 and rCXCL10 caused migration of CD4+, CD8+, CD4+CD8+ T cells, and natural killer (NK) cells, and rCXCL11 increased migration of CD4+ T and NK cells in PBMCs. The present study demonstrated that interferon-γ-induced CXCL9, CXCL10, and CXCL11, and their receptor CXCR3 were expressed in the uterus in stage- and cell-type specific manners and increased the migration of T and NK cells, which showed the greatest endometrial infiltration on Day 15 of pregnancy. These results suggest that CXCL9, CXCL10, and CXCL11 may play an important role in the recruitment of immune cells into the endometrium during the implantation period inpigs.

Role of Oxidative Stress in the Suppression of Immune Responses in Peripheral Blood Mononuclear Cells Exposed to Combustible Tobacco Product Preparation

Cigarette smoking is a major risk factor for several human diseases. Chronic inflammation, resulting from increased oxidative stress, has been suggested as a mechanism that contributes to the increased susceptibility of smokers to cancer and microbial infections. We have previously shown that whole-smoke conditioned medium (WS-CM) and total particulate matter (TPM) prepared from Kentucky 3R4F reference cigarettes [collectively called as combustible tobacco product preparations (TPPs)] potently suppressed agonist-stimulated cytokine secretion and target cell killing in peripheral blood mononuclear cells (PBMCs). Here we have investigated the role of oxidative stress from TPPs, which alters inflammatory responses in vitro. Particularly, we investigated the mechanisms of WS-CM-induced suppression of select cytokine secretions in Toll-like receptor (TLR) agonist-stimulated cells and target cell killing by effector cells in PBMCs. Pretreatment with N-acetyl cysteine (NAC), a precursor of reduced glutathione and an established anti-oxidant, protected against DNA damage and cytotoxicity caused by exposure to WS-CM. Similarly, secretion of tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 in response to TLR-4 stimulation was restored by pretreatment with NAC. Target cell killing, a functional measure of cytolytic cells in PBMCs, is suppressed by WS-CM. Pretreatment with NAC restored the target cell killing in WS-CM treated PBMCs. This was accompanied by higher perforin levels in the effector cell populations. Collectively, these data suggest that reducing oxidative stress caused by cigarette smoke components restores select immune responses in this ex vivo model.

RT-LAMP assay: an alternative approach for profiling of bovine heat shock protein 70 gene in PBMC cultured model.

The purpose of this study is to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) based assay for in vitro profiling of heat shock protein 70 (Hsp70) in bovine peripheral blood mononuclear cell (PBMC) culture model utilizing the absorbance level of magnesium pyrophosphate-a by-product of LAMP reaction. A set of bovine Hsp70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate by-product which signifies the degree of Hsp70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (P < 0.05) correlation between absorbance level and the fold change of Hsp70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine Hsp70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene.

CD4+ T cells in PBMC to predict the outcome of anti-PD-1 therapy.

11525 Background: Antibody blockade of programmed death 1 (PD-1), has led to durable responses and significant prolongation of overall survival in metastatic cancers including non-small cell lung cancer (NSCLC). However, in clinical trials, response rates were as low as 20 %, and approximately 50 % of the patients did not achieve benefits to prolong progression free survival. These results bring us a hypothesis that there are subgroups with distinct pre-existing anti-tumor immunity resulting in different responses to anti-PD-1 therapy. We reported that effector T cells, which are capable of mediating antitumor reactivity, are primed in LNs draining growing tumors and that these T cells exclusively belong to the T cells that down-regulated CD62L (CD62Llow) subpopulation. In the absence of purified tumor antigenic proteins or peptides on many tumors, the expression of the homing molecule CD62L on T cells may serve as a surrogate marker for identifying tumor-specific immune cells. Methods: We analyzed the peripheral blood mononuclear cells (PBMC) of 50 consecutive NSCLC patients who were planned to be treated with anti-PD-1 Ab, Nivolumab after obtaining written informed consent. The patients received Nivolumab at a dose of 3 mg per kilogram of body weight every 2 weeks. Tumor response was assessed with the use of the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, at week 8 and every 8 weeks thereafter. Results: The NSCLC patients who achieved partial response (PR) or stable disease (SD) had significantly (p = 4.1x10-7) more CD62Llow CD4+ T cells in PBMC than progressive disease (PD) patients. The percentages of CD62Llow in CD4+ T cells provided sensitivity 92.9 %, and specificity 96.7 % to predict the patients who had PD. Moreover, SD patients had significantly (p = 0.0067) less regulatory T cell subpopulation than PR patients, thus, it was possible to predict PR from SD. Conclusions: These results show that the major differences in pre-existing immunity among PR, SD, and PD patients to anti-PD-1 Ab existed in CD4+ T cell balance between primed effector and regulatory T cells. Further characterization of CD62Llow CD4+ T cells including mRNA microarray, checkpoint molecules, and chemokine receptors is going on.

Functional analysis of CD14+HLA-DR-/low myeloid-derived suppressor cells in patients with lung squamous cell carcinoma.

Immunomodulatory therapy is a potential effective treatment for advanced cancer that may provide an alternative to chemotherapy, which patients can experience adverse side effects to. Myeloid-derived suppressor cells (MDSCs) have been demonstrated to cause T-cell tolerance in rodents and humans; however, little is known about the role of MDSCs in squamous cell carcinoma. In the present study, the role of MDSCs in lung squamous cell carcinoma was investigated. Peripheral blood from 78 patients with lung squamous cell carcinoma and 30 healthy controls was examined for the presence and function of human MDSCs, denoted as monocyte differentiation antigen CD14-positive HLA class II histocompatibility antigen DR-negative/low (CD14+ HLA-DR-/low) cells by flow cytometry. The sorted T-cell surface glyoprotein CD3 (CD3)+ cells and CD14+HLA-DR-/low cells were subsequently co-cultured with a tumor cell line (NCI-H226). T-cell apoptosis was detected using annexin-V-fluorescein isothicyanate and 7-aminoactinomycin D. Interferon-γ (IFN-γ) levels were detected using an ELISA. The frequency of MDSCs in the peripheral blood mononuclear cells (PBMCs) from patients with lung squamous cell carcinoma was significantly higher compared with that of the healthy controls (P<0.05), whereas the frequency of T-cell surface glyoprotein CD4 (CD4)+ T cells and CD8+ T cells in PBMCs was significantly decreased (P<0.05). In an MDSC/CD8+ co-culture system, the proportion of CD8+ T-cell apoptosis significantly increased with the increase in ratio of MDSCs (P<0.05), while the proportion of tumor cell apoptosis significantly decreased (P<0.05). The concentration of IFN-γ significantly decreased with the increase in MDSCs (P<0.05). Therefore, MDSCs participate in the immune escape of lung squamous cell carcinoma, and may provide a possible therapeutic strategy for the treatment of this disease.

The Capability of Anti-ABO-Blood-Type Antibodies Production in Peripheral Blood Mononuclear Cells after ABO-Incompatible Liver Transplantation

Introduction: The anti-donor ABO antibody (Ab) titer doesn’t elevate in long-term followed-up ABO incompatible liver transplant (i-LTx) patients and antibody mediated rejection does not occur in the late graft liver according to our experience. To clarify the mechanism of unresponsiveness to ABO antigens, we examined the capability of the peripheral blood mononuclear cells (PBMCs) of long-term followed-up i-LTx patients to produce anti-donor ABO antibodies. Methods: The study subjects were the patients with blood-type B and O who had undergone i-LTx with preoperative rituximab induction (n = 5), without preoperative rituximab induction (n = 5), pediatric patients (under 15 years old at transplantation) without preoperative rituximab induction (n = 5), ABO-identical/compatible patients (n = 5), and healthy volunteers (n = 5). On day 0, 1 × 107 cells of PBMCs, obtained from the subjects, are engrafted into NOG mice by peritoneal injections. Mice, which chimerized with human PBMCs, were sensitized with A red blood cells on day 5, 7, and 9. On day 21, the sera were collected. Anti A Abs (IgM and IgG) were examined by flow cytometric analysis. Result: Anti A Ab titers of all the patients with i-LTx patients are hardly detected in the sera from the subjects. In chimeric mice, the production of anti A antibodies of pediatric recipients’ PBMCs was also suppressed. But, the ability of PBMCs to produce anti-A Abs still remained in the adult patients’ PBMCs regardless of perioperative rituximab induction. The production of anti A Ab was observed in identical/compatible patients and healthy volunteers (figure 1). Conclusion: The immune unresponsiveness to ABO antigens has existed in PBMCs from the i-LT pediatric patients. On the other hand, in the adult recipients, the ability to produce anti-donor ABO antibodies was maintained in PBMCs. This suggests that the timing of exposure to ABO antigens during evolution of humoral immune response is crucial to establish humoral immune tolerance.Figure

Increased CTLA-4+ T cells may contribute to impaired T helper type 1 immune responses in patients with chronic obstructive pulmonary disease.

Impaired T helper type 1 (Th1) function is implicated in the susceptibility of patients with chronic obstructive pulmonary disease (COPD) to respiratory infections, which are common causes of acute exacerbations of COPD (AECOPD). To understand the underlying mechanisms, we assessed regulatory T (Treg) cells and the expression of an inhibitory T-cell receptor, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). Cryopreserved peripheral blood mononuclear cells (PBMC) from patients with AECOPD (n = 17), patients with stable COPD (sCOPD; n = 24) and age-matched healthy non-smoking controls (n = 26) were cultured for 24 hr with brefeldin-A or monensin to detect intracellular or surface CTLA-4 (respectively) by flow cytometry. T cells in PBMC from AECOPD (n = 9), sCOPD (n = 14) and controls (n = 12) were stimulated with anti-CD3 with and without anti-CTLA-4 blocking antibodies and cytokines were quantified by ELISA. Frequencies of circulating T cells expressing intracellular CTLA-4 were higher in sCOPD (P = 0·01), whereas patients with AECOPD had more T cells expressing surface CTLA-4 than healthy controls (P = 0·03). Increased frequencies of surface CTLA-4+ CD4+ T cells and CTLA-4+ Treg cells paralleled increases in plasma soluble tumour necrosis factor receptor-1 levels (r = 0·32, P = 0·01 and r = 0·29, P = 0·02, respectively) in all subjects. Interferon-γ responses to anti-CD3 stimulation were inversely proportional to frequencies of CD4+ T cells expressing intracellular CTLA-4 (r = -0·43, P = 0·01). Moreover, CTLA-4 blockade increased the induction of interferon-γ, tumour necrosis factor-α and interleukin-6 in PBMC stimulated with anti-CD3. Overall, chronic inflammation may expand sub-populations of T cells expressing CTLA-4 in COPD patients and therefore impair T-cell function. CTLA-4 blockade may restore Th1 function in patients with COPD and so aid the clearance of bacterial pathogens responsible for AECOPD.

Anti-inflammatory drugs decrease infection of brain endothelial cells with EHV-1 in vitro.

Equine herpesvirus-associated myeloencephalopathy is the result of endothelial cell infection of the spinal cord vasculature with equine herpesvirus-1 (EHV-1) during cell-associated viraemia. Endothelial cell infection requires contact between infected peripheral blood mononuclear and endothelial cells. Inflammation generated during viraemia likely upregulates adhesion molecule expression on both cell types increasing contact and facilitating endothelial cell infection. Evaluating the role of anti-inflammatory drugs in decreasing endothelial cell infection with EHV-1. In vitro assay, crossover design, multiple drug testing. In vitro modified infectious centre assay using immortalised carotid artery endothelial cells or primary brain endothelial cells with plaque counts per well as outcome. Cells were either anti-inflammatory drug treated or left untreated. Significant reduction of plaque count when cells were treated compared with untreated cells. No dose-dependent effect when drug concentrations were increased to 10× dose. Treatment of both peripheral blood mononuclear cells (PBMC) and endothelial cells (EC) is required for significant plaque count reduction. In vitro study. Anti-inflammatory drugs decrease infection of endothelial cells likely by reducing contact between EHV-1 infected PBMC and endothelial cells in vitro. The role of adhesion molecules in this process needs further investigation. In vitro results suggest anti-inflammatory drug therapy during EHV-1 infection and viraemia in horses could be clinically relevant.

Glucose Modifies the Immune Interaction Between Mononuclear and Human Colon Carcinoma Cells

Background: Glucose is one of the principal energy suppliers for normal cell development. Impaired glucose metabolism may lead to serious health and immune impediments including carcinogenesis. The question posed in the present work was if glucose, at several concentrations, affects cytokine production by normal human peripheral blood mononuclear cells (PBMC) and if glucose is able to modulate the immune balance between PBMC and colon cancer cells. Methods: PBMC and human colon cancer cells from HT-29 and RKO lines were separately incubated with glucose at concentrations of 1.25, 2.5 and 5 mg/ml and the capacity for production of TNFα, IL-1?, IL-6, IFNγ, IL-1ra IL- 10, and IL-2 was examined. In another set of experiments the effect of glucose on the secretion of these cytokines by PBMC co-incubated with carcinoma cells was evaluated. Results: Unstimulated PBMC incubated with glucose showed a slight to moderate inhibition of IFNγ and IL-10 secretion, whereas PBMC stimulated with LPS were not affected. Addition of glucose to PBMC stimulated by HT- 29 colon carcinoma cells showed a slight spurring of TNFα production and a marked inhibition of IL-1β, IFNγ and IL-10, an effect significantly less pronounced when glucose was added to co-cultures of PBMC with RKO cells. Conclusions: Glucose exerted an inhibitory effect on inflammatory cytokine production by PBMC stimulated by HT-29 carcinoma cells except for TNFα and IL-6. The results indicate that glucose alters the immune dialog between mononuclear and cancer cells, a phenomenon that may present an additional link between hyperglycemia and carcinogenesis.

Effects of Lavandula angustifolia essential oil on Interleukin 23 and Brain-Derived neurotrophic factor gene expression in peripheral blood mononuclear cells of multiple sclerosis patients

Introduction: We aimed to determine the effect of lavandula angustifolia essential oil (LEO) on IL-23 and brain-derived neurotrophic factor (BDNF) gene expressions in peripheral blood mononuclear cells (PBMCs) of relapse-remitting MS (RRMS) patients. Methods : LEO was prepared using the hydrodistilation method on the plants aerial parts. 8 female RRMS patients and 8 healthy sex and age matched controls were entered into this study. PBMC cells were separated using Ficoll method and were treated with a concentration of 225 µg/ml LEO which and then the mRNAs were used for determining the effects of LEO on IL-23 and BDNF gene expressions using Quantitative Real Time PCR technique. Moreover in order to determine the anti-inflammatory effects of LEO, we measured the gene expression of IL-6 and IL-23 in stimulated healthy PBMC cells treated with LEO. Results: Results showed that there is no significant difference between PBMC of patients compared to healthy controls in case of IL-23 gene expression. Moreover, LEO has no significant effect on gene expression of IL-23 in PBMC of neither patients nor control. Also the results showed that BDNF gene expression is reduced to 41% compared to healthy controls and LEO can increase the BDNF gene expression by 81% in patients PBMCs. Moreover we observed that LEO can significantly reduce the LPS stimulated IL-6 gene expression in healthy PBMCs but had no significant effect on IL-23 gene expression. Conclusion : The present study demonstrated that L.angustifolia essential oil may have a protective effect against neuron damage via increasing the gene expression of BDNF in PBMCs from RRMS patients. However, further studies are necessary to confirm our results.

Research Article Anthocyanins-loaded Eudragit® L100 nanoparticles: in vitro cytotoxic and genotoxic analysis

Anthocyanins are flavonoids compounds that have a great therapeutic potential acting as antioxidants, anti-inflammatory and anti-carcinogenic agents. They are consumed through food, pharmaceutical and cosmetics industries as a natural alternative to synthetic dyes. However, anthocyanins are unstable depending on pH, temperature, light and oxygen variations, which led to the production of its encapsulation in nanoparticles as an alternative to increase its stability and protect them from chemical degradation for human consumption. Due to the fact that many of the beneficial properties of nanoparticles could also be harmful to human health, and the need of prior toxicological evaluation of any medicinal product, this research aims to evaluate the cytotoxic and genotoxic effects of anthocyanins-loaded Eudragit® L100 nanoparticles (AN). A range of AN doses were tested with a preliminary MTT assay, which allowed selecting five concentrations for comet and micronucleus assays: 0.2, 2, 20, 125 and 250 µg/mL, respectively, on human peripheral blood mononuclear cells (PBMC) (cells without liver metabolizing enzymes) and human hepatoma cell line HepG2 (with liver metabolizing enzymes). Results showed absence of cytotoxic effect in MTT test on both cell types after 24 h of exposure. However, the nuclear division index in PBMC cells assessed by micronucleus test indicates a decrease in the cell division, after 28 h of exposure. Genotoxicity analysis showed that AN did not produce significant genotoxic effects detected in comet assay, in either cell type. However, the micronucleus test on HepG2 cells showed that at concentration of 2 µg/mL and higher, AN produced clastogenic/aneugenic effects. Under our experimental conditions and limitations, the observed cytotoxic and genotoxic effects, not previously reported in the literature, may be due to anthocyanin metabolization and related to its concentrations and time of exposure