Sensitivity Of Peripheral Blood Mononuclear Cells Research Articles

The Combination of Panobinostat and Melphalan for the Treatment of Patients with Multiple Myeloma.

Histone deacetylase inhibitors show synergy with several genotoxic drugs. Herein, we investigated the biological impact of the combined treatment of panobinostat and melphalan in multiple myeloma (MM). DNA damage response (DDR) parameters and the expression of DDR-associated genes were analyzed in bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) from 26 newly diagnosed MM patients. PBMCs from 25 healthy controls (HC) were examined in parallel. Compared with the ex vivo melphalan-only treatment, combined treatment with panobinostat and melphalan significantly reduced the efficiency of nucleotide excision repair (NER) and double-strand-break repair (DSB/R), enhanced the accumulation of DNA lesions (monoadducts and DSBs), and increased the apoptosis rate only in patients' BMPCs (all p < 0.001); marginal changes were observed in PBMCs from the same patients or HC. Accordingly, panobinostat pre-treatment decreased the expression levels of critical NER (DDB2, XPC) and DSB/R (MRE11A, PRKDC/DNAPKc, RAD50, XRCC6/Ku70) genes only in patients' BMPCs; no significant changes were observed in PBMCs from patients or HC. Together, our findings demonstrate that panobinostat significantly increased the melphalan sensitivity of malignant BMPCs without increasing the melphalan sensitivity of PBMCs from the same patients, thus paving the way for combination therapies in MM with improved anti-myeloma efficacy and lower side effects.

Individual Lymphocyte Sensitivity to Steroids as a Reliable Biomarker for Clinical Outcome after Steroid Withdrawal in Japanese Renal Transplantation.

Recently, steroid reduction/withdrawal regimens have been attempted to minimize the side effects of steroids in renal transplantation. However, some recipients have experienced an increase/resumption of steroid administrations and acute graft rejection (AR). Therefore, we investigated the relationship between the individual lymphocyte sensitivity to steroids and the clinical outcome after steroid reduction/withdrawal. We cultured peripheral blood mononuclear cells (PBMCs) isolated from 24 recipients with concanavalin A (Con A) in the presence of methylprednisolone (MPSL) or cortisol (COR) for four days, and the 50% of PBMC proliferation (IC50) values and the PBMC sensitivity to steroids were calculated. Regarding the experience of steroid increase/resumption and incidence of AR within one year of steroid reduction/withdrawal, the IC50 values of these drugs before transplantation in the clinical event group were significantly higher than those in the event-free group. The cumulative incidence of steroid increase/resumption and AR in the PBMC high-sensitivity groups to these drugs before transplantation were significantly lower than those in the low-sensitivity groups. These observations suggested that an individual’s lymphocyte sensitivity to steroids could be a reliable biomarker to predict the clinical outcome after steroid reduction/withdrawal and to select the patients whose dose of steroids can be decreased and/or withdrawn after transplantation.

Increased Sensitivity of PBMCs Isolated from Patients with Rheumatoid Arthritis to DNA Damaging Agents Is Connected with Inefficient DNA Repair.

Rheumatoid arthritis (RA) is a systemic, inflammatory disease of the joints and surrounding tissues. RA manifests itself with severe joint pain, articular inflammation, and oxidative stress. RA is associated with certain types of cancer. We have assumed that RA patients’ increased susceptibility to cancer may be linked with genomic instability induced by impaired DNA repair and sensitivity to DNA damaging agents. The aim of this work was to analyze the sensitivity of peripheral blood mononuclear cells (PBMCs) isolated from RA patients to DNA damaging agents: tert-butyl hydroperoxide (TBH), bleomycin, ultraviolet (UV) radiation, and methyl methanesulfonate (MMS) and calculate the repair efficiency. TBH induce oxidative DNA lesions repaired mainly by base excision repair (BER). Bleomycin induced mainly DNA double-strand breaks repaired by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). We included 20 rheumatoid arthritis patients and 20 healthy controls and used an alkaline version of the comet assay with modification to measure sensitivity to DNA damaging agents and DNA repair efficiency. We found an increased number of DNA breaks and alkali-labile sites in the RA patients compared to those in the controls. Exposure to DNA damaging agents evoked the same increased damage in both groups, but we observed statistically higher PMBC sensitivity to TBH, MMS, bleomycin as well as UV. Examination of the repair kinetics of both groups revealed that the DNA lesions induced by TBH and bleomycin were more efficiently repaired in the controls than in the patients. These data suggest impaired DNA repair in RA patients, which may accelerate PBMC aging and/or lead to higher cancer incidence among RA patients.

Spontaneous and activation-induced apoptosis of peripheral blood mononuclear cells in the pathogenesis of type 1 diabetes mellitus

Apoptosis is the leading mechanism of pancreatic β-cell destruction in type 1 diabetes mellitus (T1DM). Assessment of activation apoptosis in response to stimulation with mitogen or a specific antigen is considered more significant when studying apoptosis markers in peripheral blood mononuclear cells to clarify the role of programmed cell death in pathogenesis of various diseases, since the role of apoptosis in immune response is increased in activated cells. It has been established that the stimuli that activate resting Tlymphocytes initiate apoptotic death of activated T lymphocytes. Therefore, detection of spontaneous apoptosis only is not very informative. In addition, determination of cell sensitivity to apoptosis induction makes it possible to identify relations of pathological process to the enhancement or weakening of this sensitivity. The key point in the T1DM initiation is apoptosis resistance of activated autoreactive T lymphocytes, that migrate from bloodstream to the pancreas and take an active part in destruction of the pancreatic insular structures. Despite long studies of T1DM pathogenesis, the exact causes of resistance of effector T cell clones to apoptosis remain unclear. There are no facts that answer the question: to what extent is the ability of T lymphocytes of peripheral blood to enter into apoptosis associated with severity and duration of the disease. In this regard, the aim of the study was to evaluate the effectiveness of in vitro activation-induced apoptosis of T lymphocytes in the patients with T1DM, depending on the state of compensation and duration of the disease. The features of activationinduced apoptosis have been studied in cultures of peripheral blood mononuclear cells (PBMC) in the patients with T1DM. Phytohemagglutinin (PHA) and insulin were used as apoptosis inducers. Increased in vitro sensitivity of PBMC to activation-induced apoptosis was revealed in T1DM patients. The strongest apoptotic response to PHA was detected in cases of T1DM decompensation. Considering predominantly Tcells to undergo apoptosis in response to PHA stimulation, one may speak about high sensitivity of activated T lymphocytes to induced apoptosis in the patients with T1DM. The highest level of activation-induced apoptosis in response to insulin stimulation was revealed in the compensation phase of T1DM. We have found that the intensity of spontaneous and activation-induced apoptosis correlates with decompensation of the disease and the degree of β-cells secretory function disorder. In fact, strong direct correlation was observed between the percentage of hypodiploid cells and blood concentration of glucose, and the inverse correlation was shown between the number of apoptotic cells and serum levels of C-peptide. The data obtained are in accordance with the modern concept of T1DM immunopathogenesis, which includes a development of autoimmune diseases associated not only with enhanced apoptosis of target cells, but also with a defect in phagocytic clearance of apoptotic cells due to impaired efferocytosis, i.e., phagocytosis of apoptotic cells. Thus, the maximal increase in spontaneous and activation-induced apoptosis levels of peripheral blood lymphocytes during the DM-1 decompensation is explained not only by the hyperglycemia effects, but also by the secondary immune response to the so-called “late apoptotic” or “secondary necrotic” β-cells, due to their ineffective phagocytic clearance.

The Asthma-associated PER1-like domain-containing protein 1 (PERLD1) Haplotype Influences Soluble Glycosylphosphatidylinositol Anchor Protein (sGPI-AP) Levels in Serum and Immune Cell Proliferation

Post-glycosylphosphatidylinositol (GPI) attachment to proteins 3, also known as PGAP3 or PERLD1 (PER1-like domain-containing protein 1), participates in the lipid remodeling process of glycosylphosphatidylinositol (GPI) anchor proteins during post-translational modification. Functional defect in PERLD1 was previously hypothesized to influence this process in T-cells and their subsequent activation and proliferation. This current study aims to functionally characterize PERLD1 genetic variants and relate this with human immune cells proliferation rate upon stimulation. We first showed the association between a PERLD1 tag-single nucleotide polymorphism (tagSNP), rs2941504, and the development of asthma in our study population. This association remained significant after conditioning for the other asthma-associated SNP rs8076131 that is also located within the 17q12–21 region. Subsequent sequencing of 40 unrelated Singapore Chinese individuals identified 12 more common PERLD1 SNPs (minor allele frequency > 5%) that are in linkage disequilibrium (LD, r2 > 0.8) with rs2941504. Through in vitro studies, 7 of these SNPs were found to form a functional haplotype that influences alternative splicing of PERLD1 transcript. This result was validated in human peripheral blood mononuclear cell (PBMC), where the minor haplotype (Hap2) was shown to be associated with significantly increased PERLD1 truncated transcript. Additionally, Hap2 was found to be related to increased levels of several soluble GPI-anchored proteins (such as sCD55 and sCD59) in serum. Elevated sCD55 in the serum was demonstrated to reduce the proliferation rate of PBMCs upon phytohaemagglutinin (PHA) stimulation. Taken together, the current study has shown a functional PERLD1 haplotype, which modifies PBMC sensitivity upon stimulation and may contribute to the individual’s susceptibility to allergic asthma.

PI3K inhibitor treatment ameliorates the glucocorticoid insensitivity of PBMCs in severe asthma

BackgroundGlucocorticoid (GC) insensitivity is an important feature of severe and fatal asthma. Oxidative stress can induce phosphoinositide-3-kinase (PI3K) activation, contributing to the development of GC insensitivity in chronic airway diseases. However, the underlying molecular mechanism of PI3K in the pathogenesis of severe asthma remains unknown.MethodsWe isolated peripheral blood mononuclear cells (PBMCs) from 34 participants (12 patients with mild/moderate asthma, 10 patients with severe asthma, and 12 control subjects). H2O2 was used to stimulate the human macrophage line U937 to mimic the oxidative stress status in severe asthma. The ability of candidate compounds, namely, azithromycin, PI3K inhibitors (BEZ235 and LY294002) and a p38 MAPK inhibitor (BIRB796), to ameliorate GC insensitivity in severe asthma was evaluated.ResultsPBMCs from patients with severe asthma exhibited dose-dependent and time-dependent GC insensitivity, which correlated with reduced activity of histone deacetylase 2 (HDAC2) (p < 0.05) and elevated expression of proinflammatory genes [nuclear factor-κB (NF-κB) and activator protein-1 (AP-1)] (p < 0.01) compared with these parameters in the control group. The PI3K inhibitors (BZE235 and LY294002) significantly restored the GC sensitivity of PBMCs from patients with severe asthma. In vitro, the PI3K inhibitors (BZE235 and LY294002) ameliorated GC insensitivity in H2O2/TNFα-induced IL-8 release from U937 cells by independently restoring the activity of HDAC2 or inhibiting the activation of transcription factors.ConclusionsThis study demonstrates that PI3K inhibitors ameliorate GC insensitivity in severe asthma by restoring HDAC2 activity and inhibiting the phosphorylation of nuclear signaling transcription factors.

PO-129 In vitro radiosensitivity and repair kinetics of PBMCs from prostate cancer patients and healthy donors evaluated by comet assay

IntroductionA high cellular radiosensitivity is connected with a risk for development of severe side effects after radiotherapy. In this study we have attempted to find a correlation between the initial radiosensitivity of in vitro irradiated peripheral blood mononuclear cells (PBMC) of prostate cancer patients and the adverse side effects of radiotherapy.Material and methodsPBMCs isolated from 7 prostate cancer patients, before the onset of radiotherapy and 3 healthy men were exposed in vitro to 2 Gy of x-ray radiation (Varian, 6 MeV). Following irradiation cell death was measured using the Annexin V/PI assay. DNA repair kinetics (0 and 24 hour after irradiation) was performed using the comet assay, and the results were expressed as% of tail DNA. Acute toxicity were graded according to the EORTC radiation morbidity scoring scale.Results and discussionsOur data show that the in vitro irradiation of PBMCs in prostate cancer patients initially caused a significantly higher DNA damage than in the control group of healthy donors. After a repair time of 24 hour, samples from all healthy donors showed no residual DNA damage (average 2.6%) in PBMCs. If all patients are observed for 0 hour and 24 hour after in vitro PBMCs irradiation then there is a significant reduction in the degree of DNA damage, from 35.5 to 30.4 tail% DNA. What is also important is that 4 patients had a very low level of DNA damage, which can be considered as normal cellular reaction to irradiation, while 3 patients had a very high level of damage. Also, in 5 out of 7 examined prostate cancer patients, there was an increase in the percentage of PBMCs in the early and late stages of apoptosis 24 hour after in vitro irradiation of their PBMCs. Most patients had mild side reactions to radiotherapy and were graded as grade 1 (per EORTC scale). Only in 1 of 7 patients observed side effects are classified as grade 2 and this is in correlation with a very high level of DNA damage (82.25% initial, versus 64.77% 24 hour after in vitro irradiation).ConclusionOur study was insufficient to reveal the relationship between the risk of developing side effects to radiotherapy and the sensitivity of PBMCs irradiated in vitro, measured by comet assay. A larger number of patients and further studies are necessary to confirm the potential application of the comet assay.

Fungal cell wall agents and bacterial lipopolysaccharide in organic dust as possible risk factors for pulmonary sarcoidosis.

BackgroundComposition of organic dust is very complex, involving particles of microbial, animal and plant origin. Several environmental exposure studies associate microbial cell wall agents in organic dust with various respiratory symptoms and diseases. The aim of the present study was to investigate the in vitro effects of the co-exposure of fungal cell wall agents (FCWAs) and bacterial lipopolysaccharide (LPS) on inflammatory immune responses of peripheral blood mononuclear cells (PBMCs) from patients with pulmonary sarcoidosis.MethodsPBMCs from 22 patients with pulmonary sarcoidosis and 20 healthy subjects were isolated and stimulated in vitro with FCWAs (soluble and particulate (1 → 3)-β-D-glucan, zymosan and chitosan) and/or LPS. Subsequently, cytokines were measured by ELISA and the mRNA expression of dectin-1, toll-like receptor 2 (TLR2), TLR4 and mannose receptor (MR) was analysed by real-time RT-PCR.ResultsPatients with sarcoidosis had a significantly higher secretion of inflammatory cytokines tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-10 and IL-12 (1.7-fold, 2.0-fold, 2.2-fold, and 2.8-fold, respectively; all p < 0.05) after in vitro co-stimulation of PBMCs with FCWAs and LPS. We showed that PBMCs from patients with sarcoidosis had a higher baseline mRNA expression of dectin-1, TLR2, TLR4 and MR (6-fold, 11-fold, 18-fold, and 4-fold, respectively). Furthermore, we found a reduced expression of dectin-1, TLR2 and TLR4 after stimulation with FCWAs and/or LPS, although the reduction was significantly weaker in patients than in healthy subjects.ConclusionsIn conclusion, co-stimulation with FCWAs and LPS of PBMC from patients with sarcoidosis caused a weaker reduction of dectin-1, TLR2, TLR4 receptors expression, which could increase the sensitivity of PBMCs, leading to excessive inflammatory cytokine responses and result in the development or progression of pulmonary sarcoidosis.

The State of Chromatin Condensation, the Expression of Genes Involved in DNA Damage Response and the DNA Repair Capacity Affect the Drug Sensitivity of PBMCs of Myeloma Patients Treated with Melphalan

Melphalan is an interstrand cross-link (ICL)-inducing agent and one of the most active chemotherapeuticdrugs in the treatment of multiple myeloma (MM). However, the molecular mechanisms contributing to differential response of MM patients to melphalan are poorly understood. Herein, we investigated the underlying mechanisms in processing and repair of melphalan-induced DNA lesions and their potent contribution to the outcome of anti-myeloma therapy.We studied peripheral blood mononuclear cells (PBMCs) from 15 newly diagnosed multiple myeloma (MM) patients (8M/7F; median age 61 years) responders (≥PR, n=9) and non-responders (< PR, n=6) to subsequent melphalan therapy. PBMCs from 25 healthy controls were also included in this study (HC; 14M/11F, median age 58 years). PBMCs were ex vivo treated with melphalan and the extent of the DNA damage formation/repair (monoadducts by using Southern blot; ICLs by using a quantitative PCR-based assay; double strand breaks (DSBs) using immunofluorescence quantification of γH2AX foci), the induction of the apoptotic pathway by using a photometric enzyme-immunoassay and the local chromatin condensation by using micrococcal nuclease digestion were examined. Finally, the expression of a focused panel of 84 genes involved in DNA damage response (DDR) pathways (ATM/ATR signaling, DNA repair pathways, cell cycle regulation, apoptosis) was also evaluated.Following ex vivo treatment of PBMCs with melphalan, in all individuals examined biphasic DNA repair kinetics were observed, including a fast first-phase of repair and a much slower second phase. Interestingly, the accumulation of monoadducts was inversely correlated with the first-phase repair capacity of the PBMCs, being significantly higher in HC than in responders and lowest in non-responders (all P<0.001). The second phase repair capacity showed no differences in all individuals analyzed. Also, although ICLs "unhooking" rates were similar in all individuals, accumulation of ICLs was significantly higher in HC compared to responders' PBMCs (P<0.01), due to higher levels of monoadducts that are precursors of ICLs left unrepaired in these cells, resulting in higher formation of ICLs. Minimal amounts of ICLs were observed in non-responders. Moreover, DSBs burden was significantly higher in HC than in responders, due to higher accumulation of ICL, which are precursors of DSBs and lower rates of DSB repair in these cells (P<0.01). Again, minimal amounts of DSBs were observed in non-responders.Interestingly, apoptosis rate of PBMCs was inversely correlated with the repair efficiency of both the first-phase monoadducts and the DSBs, with the apoptosis being significantly higher in HC compared to responders and lowest in non-responders (all P<0.05). In untreated PBMCs, we found an inverse correlation between the local chromatin condensation and the repair capacity. Therefore, we observed a progressive, significant increase in the looseness of the local chromatin structure, from HC to MM patients, with responders showing more condensed chromatin structure compared to non-responders (all P<0.05). Interestingly, by using a-amanitin, an inducer of chromatin condensation, PBMCs from non-responders showed the DNA repair capacity and the melphalan sensitivity similar to PBMCs from responders, suggesting that the state of the chromatin structure contributes to the response to melphalan therapy.Finally, microarray analyses of untreated PBMCs consistently point to an altered expression of several DNA damage response-related genes in MM patients compared to HC. Particularly, responders' PBMCs showed upregulation of 4 genes (ATR, CHEK2, XPA, XRCC1) and downregulation of 5 genes (ATM, MPG, UNG, CDKN1A, CDC25C) compared to non-responders (in all cases, fold changes between groups were >2, P<0.001), suggesting that changes in the molecular components of the DDR pathways correlate with the outcome of melphalan therapy.We conclude that the state of chromatin condensation, the expression of genes involved in DDR pathways and the repair capacity of monoadducts and DSBs affect the drug sensitivity of PBMCs and maybe used for the prediction of response of myeloma patients to melphalan therapy. DisclosuresTerpos:Celgene: Honoraria, Other: travel expenses; Novartis: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel expenses. Munshi:celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees. Dimopoulos:Janssen-Cilag: Honoraria; Novartis: Honoraria; Genesis: Honoraria; Amgen: Honoraria; Onyx: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

S131 Peripheral Blood Mononuclear Cells From Children With Severe Asthma Exhibit An Impaired Corticosteroid Sensitivity, Which Also Correlates With Increasing Body Mass Index

Introduction Corticosteroid (CS) insensitivity contributes to the difficulty in managing children with severe asthma. 1 A better understanding of the molecular mechanisms driving this defective response could provide novel therapeutic options for these patients. Peripheral blood mononuclear cells (PBMCs) from adults with severe asthma have been used to demonstrate an impaired sensitivity to CS, enabling the delineation of potential underlying mechanisms. Whether CS insensitivity exists in PBMCs from severely asthmatic children, however, requires further validation. Objective To determine whether PBMCs from children with severe asthma have an impaired in vitro responsiveness to corticosteroids. Methods We conducted an observational feasibility study comparing the corticosteroid sensitivity of PBMCs from asthmatic children on British Thoracic Society treatment step 4–5 (n = 7) with healthy controls (n = 5). PBMCs from 5 ml of venous blood were plated in the presence of 100 ng/ml of lipopolysaccharide (LPS), and in the absence or presence of either 10 –8 M or 10 –6 M of dexamethasone (DEX). ELISA assays were used to determine the levels of TNF-α and IL-8, and the% suppression of these by DEX. Pearson product-moment correlation tests were conducted to determine the correlation between in vitro CS sensitivity and different clinical parameters. Results There was no difference in baseline or LPS-induced cytokine release from PBMCs between the two groups. The inhibition of TNF-α release by DEX was significantly diminished in children with asthma compared to healthy controls at 10 –8 M concentration ( p = 0 .018) but no differences were noticed at 10 –6 M concentration, or on LPS-induced IL-8 production. A significant inverse correlation between% inhibition of TNF-α and body mass index (BMI) at 10 –8 M (r=-0.84, p = 0 .02) and 10 –6 M DEX (r=-0.82, p = 0 .02) was found. Conclusions Our results show the existence of an impaired CS sensitivity in PBMCs from children with severe asthma, suggesting that these cells can be used for mechanistic investigations. Interestingly, we observed a negative correlation between CS sensitivity and BMI, a novel in vitro finding which supports the association between overweight/obese asthmatic children and a decreased clinical response to CS therapy. Together, these results merit further studies with a larger sample size. References Chung KF et al . ERJ 2014;43:343–373

Radiofrequency currents exert cytotoxic effects in NB69 human neuroblastoma cells but not in peripheral blood mononuclear cells

Recently, a number of electric and electrothermal therapies have been applied to the treatment of specific cancer types. However, the cellular and molecular mechanisms involved in the response to such therapies have not been well characterized yet. Capacitive-resistive electric transfer (CRET) therapy uses electric currents at frequencies within the 0.45–0.6 MHz range to induce hyperthermia in target tissues. Preliminary trials in cancer patients have shown consistent signs that CRET could slow down growth of tumor tissues in brain gliomas, without inducing detectable damage in the surrounding healthy tissue. Previous studies by our group have shown that subthermal treatment with 0.57-MHz electric currents can induce a cytostatic, not cytotoxic response in HepG2 human hepatocarcinoma cells; such effect being mediated by cell cycle alterations. In contrast, the study of the response of NB69 human neuroblastoma cells to the same electric treatment revealed consistent indications of cytotoxic effects. The present study extends the knowledge on the response of NB69 cells to the subthermal stimulus, comparing it to that of primary cultures of human peripheral blood mononuclear cells (PBMC) exposed to the same treatment. The results showed no sensitivity of PBMC to the 0.57 MHz subthermal currents and confirmed that the treatment exerts a cytotoxic action in NB69 cells. The data also revealed a previously undetected cytostatic response of the neuroblastoma cell line. CRET currents affected NB69 cell proliferation by significantly reducing the fraction of cells in the phase G2/M of the cell cycle at 12 h of exposure. These data provide new information on the mechanisms of response to CRET therapy, and are consistent with a cytotoxic and/or cytostatic action of the electric treatment, which would affect human cells of tumor origin but not normal cells with a low proliferation rate.

Impaired anti-inflammatory efficacy of n-butyrate in patients with IBD

The intestinal mucosa of patients with inflammatory bowel diseases (IBD) characteristically shows a high degree of inflammation when compared to healthy subjects. This appears to be attributable to an imbalance in local reactivity of inflammatory cells. In the present study, we tested the hypothesis that immune cells from patients with IBD are less sensitive to anti-inflammatory agents in the gut as exemplified by the short-chain fatty acid (SCFA) n-butyrate. Peripheral blood mononuclear cells (PBMC) of patients with IBD (22 Crohn`s Disease, CD; 9 Ulcerative Colitis, UC) and 20 healthy individuals were stimulated through TLR-4 and TLR-2 engagement, respectively, and the anti-inflammatory activity of n-butyrate (0·06-1 mM) on cytokine production (IL-1β, IL-10, IL-12/23p40, TNF-α) was assessed. Inhibition curves were generated, and effective doses (ED20-ED80) were determined. Hyperresponsiveness to TLR-2 activation reflected by increased IL-12/23p40 and TNF-α production was observed in patients with IBD. To inhibit the release of IL-12/23p40 from PBMC after activation via TLR2-agonists, higher concentrations of n-butyrate were required in patients with IBD , when compared to healthy subjects. With regard to TLR-4 activation, PBMC from patients with IBD and controls were equally responsive to the immunoregulatory effects of n-butyrate. Further analysis revealed that the impaired sensitivity of PBMC to the anti-inflammatory action of n-butyrate was independent from hyperreactivity of immunocompetent cells. Impaired sensitivity to the inhibitory action of n-butyrate in IBD may constitute a determinant in the pathogenesis of these inflammatory diseases.

Comparative Study of the Cellular Pharmacodynamics of Calcineurin Inhibitors between Patients with Chronic Renal Failure Awaiting Renal Transplantation and Cirrhosis Patients Awaiting Liver Transplantation

The in vitro response of peripheral blood mononuclear cells (PBMCs) to the suppressive effects of calcineurin inhibitors is known to correlate with the clinical efficacy of drugs used in renal transplantations. The present study was conducted to examine the differences of PBMC responses to calcineurin inhibitors between chronic renal failure (CRF) patients awaiting renal transplantation and cirrhosis patients awaiting liver transplantation. The study included 99 CRF patients awaiting renal transplantation and 27 cirrhosis patients awaiting liver transplantation. Twenty milliliters of venous blood was taken 1-7 days before transplantation. The in vitro drug concentrations giving 50% inhibition of PBMC blastogenesis stimulated with concanavalin A (IC(50)s) were calculated. The suppressive effects of tacrolimus against PBMC blastogenesis were more than 10-100 times stronger than those of cyclosporine. The median IC(50) value for cyclosporine against the CRF PBMCs was not significantly different from the median IC(50) value against the cirrhosis PBMCs. In contrast, tacrolimus sensitivity in cirrhosis PBMCs is approximately seven times higher than that in CRF PBMCs. The median IC(50) value for tacrolimus against cirrhosis PBMCs was significantly lower and therefore the effect was stronger in comparison to the CRF PBMCs (p < 0.001). These data suggest that the PBMCs of cirrhosis patients, in comparison to those of CRF patients, are highly sensitive to the suppressive effect of tacrolimus. However, PBMC sensitivity to cyclosporine was not significantly different between the CRF and cirrhosis patients. These observations raise the possibility that treatment with tacrolimus, rather than cyclosporine, may therefore be a better choice to reduce the risks of allograft rejection in liver transplantation.

Clinical Significance of the Cellular Pharmacodynamics of Tacrolimus in Living-Donor Liver Transplantation

Successful immunosuppressive therapy is critical for liver transplantation; however, a considerable number of patients experience fatal rejection or alternatively exhibit serious infection resulting from excessive immunosuppression. The in vitro tacrolimus response of peripheral blood mononuclear cells (PBMCs) before transplantation was compared to the clinical outcome up to 4 weeks after operation in 28 living-donor liver transplant recipients treated with tacrolimus. The tacrolimus IC(50) values against concanavalin A-induced PBMC blastogenesis in vitro were calculated. These recipients were classified into two groups with the mean tacrolimus IC(50) (0.18 ng/ml) as the cutoff point, after which the clinical outcome between the patient groups was compared. The allograft rejection incidence in the low-sensitivity group (IC(50) < 0.18 ng/ml; n = 16) was 6/12 (50.0%), which was significantly higher than the incidence of 2/16 (12.5%) in the high-sensitivity group (IC(50) > 0.18 ng/ml; n = 12) (p = 0.0297). In contrast, the infection incidence in the high-sensitivity group was 6/16 (37.5%), which was significantly higher than that of the low-sensitivity group (1/12; 8.3%) (p = 0.0401). These data suggest that patients exhibiting a low PBMC sensitivity to tacrolimus have a risk of rejection, whereas highly sensitive patients have a risk of infection in living-donor liver transplantations under tacrolimus therapy.

Relationship between plasma leptin or soluble cleaved leptin-receptor concentrations and glucocorticoid sensitivity of peripheral blood mononuclear cells in patients with nephrotic syndrome

Leptin is a small peptide hormone which centrally regulates weight. Leptin receptor (OB-R) is expressed in hematopoietic cells, the central nervous, and immune systems. OB-R bears a homology similar to members of the class Ι cytokine family, and therefore, leptin appears to modulate immune responses. The aim of this study was to examine the effect of plasma leptin, soluble OB-R (sOB-R), and the free leptin index (FLI), the ratio between leptin and sOB-R levels, on the sensitivities of peripheral blood mononuclear cells (PBMCs) to prednisolone and cyclosporine in 16 healthy subjects and seven nephrotic syndrome (NS) patients. The NS patients had significantly higher serum sOB-R and lower FLI, compared with the healthy subjects (respectively, P = 0.0026, P = 0.0383). Whereas, the NS patients had significantly lower PBMC sensitivity to prednisolone ( P = 0.0049). PBMCs sensitivity to cyclosporine was not significantly different between the healthy subjects and the NS patients. In addition, when the data from all subjects were analyzed, there was a significantly positive correlation between plasma sOB-R concentrations and the IC 50 values of prednisolone ( P = 0.0478). In contrast, plasma leptin concentrations and FLIs did not correlate significantly with the prednisolone and cyclosporine IC 50 values. From these observations it can be suggested that plasma leptin has little effect on PBMC sensitivity to immunosuppressive drugs in NS patients. Molecular background(s) for the influence of sOB-R on the PBMC sensitivity to glucocorticoids remain(s) to be elucidated.

A rapid and efficient strategy to generate antigen-specific human monoclonal antibody by in vitro immunization and the phage display method

An in vitro immunization (IVI) protocol was developed for inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs). After antigen sensitization of PBMCs by IVI, B cells producing antigen-specific antibody can be propagated within a week. Here, we attempted to establish a rapid, efficient strategy to obtain antigen-specific antibody by the phage display method using in vitro immunized PBMCs. Heavy and light chain variable region genes were easily amplified from these PBMCs immunized with mite extract (ME). After generating a combinatorial phage library (1.6 × 10 5 members), 4 antigen-specific clones were selected by 5 panning rounds using biotinylated antigen and streptavidin magnetic beads. Next, we combined variable region genes of these selected clones with human IgG constant region genes and produced human IgG-type antibody. Direct and competitive enzyme-linked immunosorbent assays demonstrated that the mAb 1C11 clone bound specifically to ME. We thus established a rapid, efficient method to obtain antigen-specific human antibody genes and produce human monoclonal IgG antibody using the phage antibody library generated from in vitro immunized PBMCs.

The Role of Immune Response to Staphylococcus aureus Superantigens and Disease Severity in Relation to the Sensitivity to Tacrolimus in Atopic Dermatitis

Fukushima H, Hirano T, Shibayama N, et al. Int Arch Allergy Immunol. 2006;141:281–289 PURPOSE OF THE STUDY. To examine the prevalence and role of Staphylococcus aureus superantigens on the pathophysiology and immunosuppressive drug sensitivity in patients with atopic dermatitis (AD). STUDY POPULATION. Twenty-nine patients with AD and 13 healthy control patients were included in the study. METHODS. Twenty-nine patients with AD were classified into 2 groups on the basis of their clinical AD scores: a low-score group (n = 14), corresponding to patients with mild-to-moderate AD, and a high-score group (n = 15), corresponding to patients with severe AD. Plasma antistaphylococcal enterotoxin B or toxic-shock syndrome toxin-1 (TSST-1) immunoglobulin E (IgE) levels were measured for the patients and healthy subjects by enzyme-linked immunosorbent assay. Also, individual drug sensitivity was estimated by determining the drug concentrations that would provide 50% inhibition (IC50) of peripheral blood mononuclear cell (PBMC) proliferation in vitro. RESULTS. The levels of plasma anti-staphylococcal enterotoxin B or TSST-1 IgE in the patients with severe AD were significantly higher than those in the patients with mild-to-moderate AD (P &amp;lt; .05 and P &amp;lt; .01, respectively). When stimulated with concanavalin A in vitro, PBMCs in the patients with severe AD exhibited low sensitivity to the suppressive efficacy of tacrolimus (FK506) as compared with the patients with mild-to-moderate AD (P &amp;lt; .01). Furthermore, there was a significant correlation between the IC50 values of FK506 and plasma anti–TSST-1 IgE levels (P &amp;lt; .01). CONCLUSIONS. PBMCs in patients with severe AD exhibited lower sensitivity to FK506 and had higher plasma levels of anti–TSST-1 IgE as compared with the patients with mild AD. S aureus superantigens seem to be one of the causes of decreased PBMC sensitivity to FK506; therefore, an alternative treatment would be useful on the basis of individual drug-sensitivity data and anti–TSST-1 IgE levels. REVIEWER COMMENTS. Patients with AD commonly develop superinfections that require antimicrobial treatment. This study suggests that untreated superinfection may contribute to the ineffectiveness of topical medications that are commonly used as routine skin care for patients with AD. Attentiveness to the presence of concomitant skin infection is necessary for comprehensive skin care for patients with AD.

Cooperative effects of corticosteroids and catecholamines upon immune deviation of the type-1/type-2 cytokine balance in favor of type-2 expression in human peripheral blood mononuclear cells

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high levels of catecholamines (CT) and corticosteroids (CS). Although both CS and CT individually can inhibit the production of T-helper 1 (TH1, type-1 like) cytokines and simultaneously promote the production of T-helper 2 (TH2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination CT and CS in immune responses that may be more physiologically relevant. We therefore investigated the combined effects of in vitro CT and CS upon the type-1/type-2 cytokine balance of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of superimposed acute and chronic stress. Results demonstrated a significant decrease in type-1 cytokine production (IFN-γ) and a significant increase in type-2 cytokine production (IL-4, IL-10) in our CS + CT incubated cultures when compared to either CT or CS agents alone. Furthermore, variable enhancement of type-1/type-2 immune deviation occurred depending upon when the CT was added. The data suggest that CS can increase the sensitivity of PBMC to the immunomodulatory effects of CT and establishes an in vitro model to study the combined effects of in vivo type-1/type-2 cytokine alterations observed in acute and chronic stress.

The effect of methylprednisolone on proliferation of PBMCs obtained from steroid‐sensitive and steroid‐resistant rheumatoid arthritis patients

Objective: Glucocorticoids (GCs) are among the most frequently used drugs for the treatment of rheumatoid arthritis (RA). Unfortunately, up to 30% of patients with RA fail to respond to the treatment. We investigated the hypothesis that patients with RA who did not respond to GC treatment have steroid‐resistant peripheral blood mononuclear cells (PBMCs).Methods: Forty‐four patients with RA were enrolled in the study. PBMCs were isolated from blood samples. The effect of methylprednisolone (MP) on the proliferation of stimulated cells was measured. After taking the blood samples, 10 days of MP therapy (20 mg i.v.) was started, in order to classify the patients into either a GC‐sensitive (RA/GCS) or a GC‐resistant (RA/GCR) group.Results: A quarter of our patients did not show any improvement after short‐term GC therapy and were assigned to the RA/GCR group. The inhibition of PBMC proliferation after MP treatment was significantly lower in the RA/GCR as compared to the RA/GCS group.Conclusion: Based on the close relationship between clinically observed GC resistance and a diminished response of PBMCs to MP treatment, we conclude that measurement of the steroid sensitivity of PBMCs may be a useful tool in predicting the therapeutic effect of GC in patients with RA.

P-Glycoprotein Function in Peripheral Blood Mononuclear Cells of Myasthenia Gravis Patients Treated with Tacrolimus

Tacrolimus hydrate (FK506) reduces the symptoms of myasthenia gravis (MG) due to its immunosuppressive properties. A drug efflux pump P-glycoprotein (P-gp) actively transports FK506 out of target cells, thereby reducing their efficacy. We investigated the influence of FK506 therapy on the P-gp function of peripheral-blood mononuclear cells (PBMCs) in MG patients. Six MG patients treated with FK506 (MG(FK+)), four MG patients treated without FK506 administration (MG(FK-)), and 18 healthy subjects were included in this study. P-gp function was estimated by transporter activity that was inferred from a decrease in fluorescent P-gp substrate Rhodamine 123 (Rh123) and its inhibition by cyclosporine A (CsA). The P-gp efflux function in MG (FK+) patients assessed by the Kolmogorov-Smirnov (KS) statistic D was lower than in the healthy subjects (p=0.0084). However, PBMC sensitivity to FK506 in MG (FK+) patients was significantly higher compared to that of the healthy subjects (p=0.02). There was a significant correlation between the Rh123 efflux activity and PBMC sensitivity to FK506 in vitro (p=0.011). The data raise the possibility that FK506 treatment attenuated P-gp function in the PBMCs of the MG patients.