Peripheral Blood Mononuclear Cell Specimens Research Articles

Delayed HIV detection among infants exposed to postnatal antiretroviral prophylaxis during breastfeeding

The objective of this study is to determine whether detection of HIV infection was delayed in infants exposed to antiretroviral prophylaxis to prevent HIV transmission during breastfeeding. The Breastfeeding, Antiretrovirals and Nutrition (BAN) study was a randomized trial of 2369 mother-infant pairs conducted from 2004 to 2010. In addition to an intrapartum regimen, all mother-infant pairs were randomly assigned to three antiretroviral intervention arms during 28 weeks of breastfeeding: no further antiretroviral prophylaxis (control arm); infant-daily nevirapine (nevirapine arm); and maternal zidovudine, lamivudine and either nevirapine, nelfinavir or lopinavir-ritonavir (maternal arm). After breastfeeding cessation counselling and stopping the antiretroviral interventions by 28 weeks, 28 infant HIV infections occurred. To determine whether these infections occurred during the breastfeeding and antiretroviral intervention phase but had delayed detection on the antiretroviral arms, we performed ultrasensitive (droplet digital PCR) HIV testing on infants with stored peripheral blood mononuclear cell (PBMC) specimens at 24 weeks (n = 9). Of the nine infants, all three on the infant nevirapine arm had detectable HIV DNA at 24 weeks, compared with two of four on the maternal antiretroviral arm and one of two on the control arm. For infants with detectable HIV at 24 weeks, the median delay in detection between the ultrasensitive and standard assays was 18.3 weeks for the nevirapine arm, 15.4 weeks for the maternal arm and 9.4 weeks for the control arm. The prolonged inability to detect HIV with standard assays in the context of postnatal antiretroviral prophylaxis suggests that early antiretrovirals may restrict HIV replication sufficiently to lead to missed diagnosis among infected infants. Therefore, repeat virologic testing is warranted beyond the WHO-recommended point of testing at 6 weeks after breastfeeding cessation.

Abstract 3400: Mitochondrial profiling in AML patients treated with an Alvocidib containing regimen reveals MCL1 dependency in responder bone marrow

Abstract Background. Molecular profiles may provide insights into the likelihood of responding to traditional cytotoxic chemotherapy as well as important clues to the success of novel targeted agents. The anti-apoptotic BCL-2 family proteins have previously been shown to facilitate pro-survival and resistance to chemotherapy in AML, multiple myeloma, as well as in other types of cancer. Measuring the function of these proteins using BH3 profiling has proven to provide useful information in predicting AML treatment response with traditional ara-c based treatment. Here, we sought to determine if such an approach might detect response with the addition of Alvocidib to ara-c-based chemotherapy. Mitochrondrial apoptosis signaling was assessed in AML patients treated with Alvocidib, ara-c and mitoxantrone (FLAM). Methods. Pretreatment peripheral blood mononuclear cell and bone marrow specimens from 63 patients treated with FLAM were measured for mitochondrial outer membrane permeabilization following incubation with BH3 peptides as a surrogate for BCL-2 protein family function. Results. Analysis of the priming states (mitochondrial depolarization) in the patients who underwent complete remission (CR), compared with those who did not (NR), revealed that NOXA priming was significantly higher in CR bone marrow samples (median 44.5%) compared with NR samples (median 5.2%, p = 0.006). NOXA is known to interact most directly with MCL1, suggesting that these cancer cells may be MCL1 dependent. An additional multi-peptide algorithm using three BH3 members was also identified (involving BAD, PUMA, and BIM), which may also indirectly support MCL1 dependency. These BH3 priming profiles were both additive to known risk factors associated with clinical response to chemotherapy, including cytogenetic risk factors. Receiver operating characteristic (ROC) curve analysis of NOXA priming plus cytogenetics and MDS history indicated that these variables were highly predictive of response to FLAM (AUC-0.92, p = 0.0002). This correlation is distinct from our previous work which showed that the priming due to the BIM peptide predicts response to traditional ara-c based treatment. This work provides a basis for further studies into the MCL1 dependent action of Alvocidib, as well as a potential biomarker for identification of patients likely to respond to FLAM. Citation Format: Elisha J. Dettman, Steven L. Warner, Camille Doykan, Melissa Arn, Noel Blake, David J. Bearss, Michael Cardone, Bruce D. Smith. Mitochondrial profiling in AML patients treated with an Alvocidib containing regimen reveals MCL1 dependency in responder bone marrow. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3400. doi:10.1158/1538-7445.AM2015-3400

Abstract 4302: Long term monitoring of circulating regulatory T cells (Treg), myeloid derived suppressor cella (MDSC) and type I effector T cells in melanoma patients treated with neoadjuvant ipilimumab

Abstract Background: Patients with regionally advanced melanoma were treated with neoadjuvant ipilimumab in a previously reported study (Tarhini. PLOS One 2014) where significant changes in circulating Treg, MDSC and peptide specific type I CD4+ and CD8+ T cells were observed early on-treatment (6 weeks) that correlated with clinical outcome. Methods: Patients were treated with ipilimumab (10 mg/kg IV every 3 weeks x 2 doses) bracketing surgery. Blood (serum / peripheral blood mononuclear cells) specimens were collected at baseline and during treatment for up to 9 months. We conducted longer term monitoring in patients with available specimens at 3 (n = 28), 6 (n = 22) and 9 (n = 13) months utilizing multicolor flow cytometry. We compared the frequencies of circulating suppressive Treg and MDSC on-study to baseline levels, as well as frequencies of CD4+ and CD8+ T cells specific to shared tumor-associated antigens (Gp-100, MART-1, NY-ESO-1) utilizing overlapping peptide libraries (15-mer peptides overlapping by 4). The prognostic value of cell frequencies in relation to the probability of the event of progression at 9 months was tested. Results: Baseline levels of Treg (CD4+/CD25hi+/CD39+) were significantly associated with 9 months progression free survival (PFS) (p = 0.04). A significant increase in Treg (CD4+CD25hi+Foxp3+ and CD4+/CD25hi+/CD39+) frequencies reported previously at 6 weeks after initiation of ipilimumab appears to have reversed starting 3 months as no significant changes were seen at this or any of the later time points compared to baseline. However, in monitoring the time dependent change over time in circulating Treg there was a trend towards an association with PFS (p = 0.09). Unlike what we had observed and reported at 6 weeks, no significant decrease in MDSC levels was seen at 3 months or later time points compared to baseline. We detected evidence of spontaneous in vivo cross presentation resulting in type I (interferon-γ producing), fully activated (CD69+) CD4+ and CD8+ antigen-specific T-cell immunity against cancer-testis (NY-ESO-1) as well as melanocytic lineage (MART-1, gp100) antigens in the absence of therapeutic vaccination. These responses were significantly potentiated at 6 weeks and persisted at 3, 6 and 9 months following the initiation of ipilimumab. Conclusions: Neoadjuvant ipilimumab significantly modulates the levels of cellular mediators of immune suppression (Treg and MDSC) early on-treatment with less significant changes seen on long term follow up. Significant potentiation of the type I effector T cell response is seen early and persists on long term monitoring. These biomarkers warrant further investigation in relation to the mechanism of action of ipilimumab and for their potential prognostic and therapeutic predictive value. (Supported by NIH award P50CA121973 and BMS) Citation Format: Janet Retseck, Amanda Gillespie-Twardy, Alexis Nasr, Hui-Min Lin, Yan Lin, John Kirkwood, Lisa H. Butterfield, Haris Zahoor, Cindy Sander, Ahmad A. Tarhini. Long term monitoring of circulating regulatory T cells (Treg), myeloid derived suppressor cella (MDSC) and type I effector T cells in melanoma patients treated with neoadjuvant ipilimumab. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4302. doi:10.1158/1538-7445.AM2015-4302

Dysregulated miR-103 and miR-143 expression in peripheral blood mononuclear cells from induced prediabetes and type 2 diabetes rats

The progression from normal glucose tolerance (NGT) to type 2 diabetes (T2D) occurs through an intermediate state of glucose intolerance known as pre-diabetes. This transition is usually a gradual phenomenon that occurs over 5–10years. Among the routinely practiced T2D screening criteria, like, FPG, IFG, IGT or HbA1c, still the issue of a preferable one is debated. The newly emerged microRNAs are created much hope to act as a class of suitable diabetes gene signature detectable at an early stage of the disease development. Although T2D related miRNA fluctuations are reported from the main insulin target organs, sampling of these organs for the sake of screening due to its invasive nature is not practicable. Peripheral blood mononuclear cells (PBMCs) are in constant touch with all body organs hence may exhibit the trace of miRNA changes which take place in insulin target organs.In this study we have evaluated miR-103 and miR-143 expression in three groups of rats namely; normal control, high fat diet (HFD) which is corresponding to prediabetes state, and high fat diet/streptozotocin (STZ) induced T2D.Quantitative real time PCR was used for profiling the selected miRNA expression at various time intervals of the three defined groups of rats. In prediabetes and overt diabetes stages, miR-103 showed significantly elevated expression in PBMC specimens compared to the normal healthy control group. Overexpression pattern of mir-143 was statistically significant in T2D compared to non-diabetic controls. However in HFD (prediabetic) rats also we observed an increasing pattern of miR-143 compared to the normal controls but it was not statistically significant.

Abstract 907: Bcl-xL dependence predicts response to alvocidib in chronic lymphocytic leukemia patients

Abstract Introduction. Chronic lymphocytic leukemia (CLL) patients may benefit from personalized strategies targeting specific therapies to individuals with favorable molecular profiles. Many therapeutics exhibit response in a subset of patients, and factors such as age or cytogenetics are insufficient to predict treatment success with high accuracy. We assessed mitochondrial functionality in apoptosis signaling for identification of CLL patients likely to exhibit clinical response to treatment with alvocidib. Effects of this agent have been shown to involve pathways of apoptosis, providing the rationale for our study. Patients and Methods. Relapsed/refractory patients comprising OSU0055 and Sanofi EFC6663 clinical trials were analyzed. Pretreatment peripheral blood mononuclear cell (PBMC) specimens were divided into training and validation sets for response-correlative assessment. Blinded to outcomes, we analyzed specimens by flow cytometry-based BH3 profiling, indirectly measuring induction of mitochondrial outer membrane permeabilization in response to treatment with BH3-only peptides (Bim, Noxa, Bad, Bmf, Hrk) as surrogates for Bcl-2 family functions. Findings were correlated with disease characteristics and treatment outcome in the proof-of-principal set and tested for confirmation in the validation set. Results. Response data were available for 62 patients; training and test sets comprised 30 and 32 patients, respectively. Regression analyses indicated a correlation between clinical response (ordered 3 categories; PD, SD, PR) and priming in training set with Bim(0.1) (p=.014) and Hrk (p=.0098), that later validated in the test set for both markers (p=.0051 and p=.015, respectively). In total (n=62), Bim regression displayed a p=.0027 and Hrk regression a p=.00046. When analyzed as PD/SD vs PR, the combined cohort yielded Bim p=.04, and Hrk p=.0039 by logistic regression. Using area under the receiver operating curve (AUC) to quantify the accuracy of outcome prediction, Bim AUC = 0.73 (CI[.60-.85]; p=.0004) and Hrk AUC = .73 (CI[.61-.86]; p=.0002). Hrk benefitted from adjustment for trisomy12 status (AUC=0.83; CI[.71-.95]; p<.0001). Analysis of BH3 profiling and tumor lysis syndrome (TLS) indicated correlation between TLS and Bad priming (p=.012 log regression; AUC=.75; CI[.60-.89]; p=.0007) that benefitted from inclusion of ECOG status and patient age (AUC=.85; CI[.73-.97]; p<.0001). Conclusion. Bim and Hrk are significantly associated with response and, thus, engaging Bcl-xL dependence may be a component of response to alvocidib in relapsed CLL patients. Interestingly, TLS is predicted by a distinct BH3 profiling peptide readout, Bad, consistent with alvocidib-associated TLS driven by Bcl-2-dependency. Taken together, these biomarkers may predict patient response to investigational CDK inhibitors in CLL and concurrently identify patients at risk for treatment-related toxicity. Citation Format: William E. Pierceall, Steven L. Warner, Ryan J. Lena, Camille Doykan, Noel Blake, Michael Elashoff, Daniel D. Von Hoff, David J. Bearss, Michael H. Cardone, Michael Grever, Mark C. Lanasa, John C. Byrd, Amy J. Johnson. Bcl-xL dependence predicts response to alvocidib in chronic lymphocytic leukemia patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 907. doi:10.1158/1538-7445.AM2014-907

The Center for HIV/AIDS Vaccine Immunology (CHAVI) multi-site quality assurance program for cryopreserved Human Peripheral Blood Mononuclear Cells

The Center for HIV/AIDS Vaccine Immunology (CHAVI) consortium was established to determine the host and virus factors associated with HIV transmission, infection and containment of virus replication, with the goal of advancing the development of an HIV protective vaccine. Studies to meet this goal required the use of cryopreserved Peripheral Blood Mononuclear Cell (PBMC) specimens, and therefore it was imperative that a quality assurance (QA) oversight program be developed to monitor PBMC samples obtained from study participants at multiple international sites. Nine site-affiliated laboratories in Africa and the USA collected and processed PBMCs, and cryopreserved PBMC were shipped to CHAVI repositories in Africa and the USA for long-term storage. A three-stage program was designed, based on Good Clinical Laboratory Practices (GCLP), to monitor PBMC integrity at each step of this process. The first stage evaluated the integrity of fresh PBMCs for initial viability, overall yield, and processing time at the site-affiliated laboratories (Stage 1); for the second stage, the repositories determined post-thaw viability and cell recovery of cryopreserved PBMC, received from the site-affiliated laboratories (Stage 2); the third stage assessed the long-term specimen storage at each repository (Stage 3). Overall, the CHAVI PBMC QA oversight program results highlight the relative importance of each of these stages to the ultimate goal of preserving specimen integrity from peripheral blood collection to long-term repository storage.

Molecular epidemiology of different hepatitis C genotypes in serum and peripheral blood mononuclear cells in jahrom city of iran.

Background:The Hepatitis C Virus (HCV) is considered essentially hepatotropic, yet the virus compartments have also been found in important extra hepatic sites. Detection of HCV RNA in extra hepatic reservoirs such as peripheral blood mononuclear cells (PBMCs) is important for determining disease progression and treatment effectiveness.Objectives:The present study aimed to determine different HCV genotypes in patients' plasma and PBMC specimens, in Jahrom city of Iran.Patients and Methods:Blood samples of 137 patients with established HCV were collected at the Honari clinic. These patients were anti-HCV and plasma HCV RNA positive. After plasma RNA extraction and obtaining a pellet of approximately 3-5 × 106 PBMCs, Real-time PCR was performed, using specific-genotype primers. Finally, data analysis was done by the Statistical Package for Social Sciences (SPSS) software.Results:Subtype 3 was the most common genotype in plasma (57.7%) and PBMCs (51.1%). Subtype 1a was detected in 36.5% and 30.7% of plasma samples and PBMCs, respectively whereas subtype 4 was not detected in any of the cases. There was a genotype difference between plasma and PBMCs of 12.4% of patients. In four patients no genotype was detected in their plasma but genotype 3 was detected in the PBMCs.Conclusions:It is suggested that determination of the target genotype by plasma subtyping for choosing the proper antiviral therapy is essential but may result in therapy failure. HCV genotyping in PBMC samples, along with plasma specimens, might be more beneficial. Therefore determining the HCV genotype in PBMCs, before beginning the therapy is useful due to the possibility of occult infection detection.

Altered miR-143 and miR-150 expressions in peripheral blood mononuclear cells for diagnosis of non-small cell lung cancer

Background Sensitive and specific biomarkers for identifying early stage of non-small cell lung cancer (NSCLC) are urgently needed to improve the therapeutic outcome and reduce the mortality. Small non-coding microRNAs (miRNAs) are key components of cancer development and are considered as potential biomarkers for cancer diagnosis and for monitoring treatment. The aim of this study was to determine whether aberrant miRNA expression can be used as a marker in peripheral blood mononuclear cells (PBMC) for the diagnosis of NSCLC. Methods The levels of two mature miRNAs (miR-143 and miR-150) were detected by probe-based stem-loop quantitative reverse-transcriptase PCR (RT-qPCR) in PBMC of 64 patients with NSCLC and 26 healthy individuals, and the relationship between miR-143 and miR-150 levels and clinical and pathological factors was explored. Results All endogenous miRNAs were present in peripheral blood in a remarkably stable form and detected by RT-qPCR. MiR-143 expression in the PBMC specimens was significantly lower in NSCLC patients than in healthy individuals (P <0.0001). MiR-150 expression in the PBMC specimens was not significantly different between NSCLC patients and healthy individuals (P=0.260). MiR-150 expression was significantly higher in lung adenocarcinoma patients than in healthy individuals (P=0.001). There was a very strong difference in the expression level of miR-150 between lung adenocarcinoma patients and lung squamous cell carcinoma patients (P <0.0001). In receiver operating characteristic curve (ROC) analysis, low expression of miR-143 showed the area under the ROC (AUC) of 0.885 for distinguishing cancer patients from healthy subjects. High expression of miR-150 had an AUC of 0.834 for distinguishing lung adenocarcinoma patients from healthy subjects. High expression of miR-150 had an AUC of 0.951 for distinguishing lung adenocarcinoma from lung squamous cell carcinoma. The miR-150 level was significantly associated with distant metastasis (P=0.014). Conclusions It is indicated that there is a potential for using miR-143 as a novel diagnostic biomarker for NSCLC. Moreover, miR-150 can be a highly accurate marker for differentiating adenocarcinoma from squamous cell carcinoma.

BH3 Profiling Discriminates Response to Cytarabine-Based Treatment of Acute Myelogenous Leukemia

As acute myelogenous leukemia (AML) patient response to cytarabine-based standard-of-care treatment is variable, stratification into subgroups by biomarker-predicted response may lead to improved clinical outcomes. Here, we assess cell mitochondrial depolarization to proapoptotic signaling BH3-only peptides as a surrogate for the function of Bcl-2 family proteins to address clinical response to cytarabine-based therapy in patients with AML (N = 62). Peripheral blood mononuclear cell (PBMC) or bone marrow aspirate specimens were obtained from newly diagnosed patients with AML, viably preserved, and assayed by flow cytometry following BH3 profile assay with individual BH3 peptides. Mann-Whitney analysis indicates biomarker correlation with response to induction therapy: Notably, BIM priming was highly significant (P = 2 × 10(-6)) with a compelling sensitivity/specificity profile [area under curve (AUC) = 0.83; 95% confidence interval (CI), 0.73-0.94; P = 2 × 10(-10)]. Multivariate analysis indicates improved profiles for BIM readout + patient age (AUC = 0.89; 95% CI, 0.81-0.97) and BIM + patient age + cytogenetic status (AUC = 0.91; 95% CI, 0.83-0.98). When patients were stratified by cytogenetic status, BIM readout was significant for both intermediate (P = 0.0017; AUC = 0.88; 95% CI, 0.71-1.04) and unfavorable (P = 0.023; AUC = 0.79; 95% CI, 0.58-1.00) risk groups, demonstrating predictive power independent of cytogenetics. Additional analyses of secondary clinical endpoints displayed correlation between overall survival (P = 0.037) and event-free survival (P = 0.044) when patients were stratified into tertiles by BIM peptide response. Taken together, these results highlight the potential utility of BH3 profiling in personalized diagnostics of AML by offering actionable information for patient management decisions.

Mcl-1 Dependence Predicts Response To Vorinostat and Gemtuzumab Ozogamicin In Acute Myeloid Leukemia

IntroductionThe outcome of older adults with acute myeloid leukemia (AML) using standard curative-intent chemotherapeutics remains dismal because of increasing risks of treatment-related mortality and therapeutic resistance associated with advancing patient age, accumulation of medical comorbidities, and changing disease biology. As a result, current expert guidelines, e.g. by the National Comprehensive Cancer Network (NCCN), recommend that these patients should receive investigational therapies. Still, investigational therapeutics typically exhibit response in only a subset of patients, and factors such as age or cytogenetics are often insufficient to predict treatment success with high accuracy. Here, we utilized BH3 profiling, a method for assessing mitochondrial functionality in apoptosis signaling, as a biomarker strategy for identification of patients likely to exhibit clinical response to an investigational regimen of a histone deacetylase (HDAC) inhibitor, vorinostat, in combination with gemtuzumab ozogamicin (GO), an immunoconjugate consisting of an anti-CD33 antibody and a toxic calicheamicin moiety. Cytotoxic effects of these agents have been shown to involve mitochondrial pathways of apoptosis, providing the scientific rationale for our study. Patients and MethodsThirty-one adults aged 60 years or older received treatment with vorinostat and GO for newly diagnosed AML (NCT00673153). For 26 of these patients, pretreatment peripheral blood mononuclear cell (PBMC) or bone marrow (BM) aspirate specimens were available. Blinded to outcomes, we analyzed these specimens by flow cytometry-based BH3 profiling. This assay indirectly measures induction of mitochondrial outer membrane permeabilization in response to treatment with BH3-only peptides, including activators (e.g. BIM) and sensitizers (e.g. NOXA, PUMA, BAD, HRK, BMF) as a surrogate for the function of Bcl-2 family proteins. Findings from these profiling studies were then correlated with disease characteristics and treatment outcome. ResultsBH3 profiling was technically successful in 23 of 26 tested specimens. Log regression analyses indicated a correlation between priming with NOXA, a selective modulator of the anti-apoptotic protein Mcl-1, and response to induction therapy (i.e. achievement of complete remission [CR]/CRp vs. resistance; p=.027). Using the area under the receiver operating curve (AUC) to quantify the accuracy of outcome prediction, the % priming achieved with NOXA yielded an AUC of 0.83 (95% CI: 0.65-1.00; p=.00042). After adjustment for age, the AUC associated with NOXA priming increased further (AUC=0.88 [0.75-1.00]). The % priming with NOXA was also statistically significantly associated with the duration of overall survival (OS; p=.027), with an AUC of 0.87 (0.64-1.00; p=.0017). Patients in the highest tertile of NOXA priming exhibited a median OS of 453.5 days vs. the other patients median OS of 132 days (p=.12 Mantel-Cox). ConclusionOur data show that the degree of priming indicated by NOXA identifies Mcl-1 dependence that is associated with response and outcome of vorinostat/GO in older adults with AML. This finding would be compatible with a pivotal role of Bcl-2 family protein-mediated mitochondrial depolarization for the clinical efficacy of GO and vorinostat, consistent with previous in vitro studies. Importantly, Mcl-1 -dependence as indicated by BH3 profiling of NOXA response may predict response to investigational therapeutic regimens for AML and, if integrated as predictive biomarker, may improve patient outcomes through identifying subsets of patients likely to respond to treatment with HDAC inhibitors and immunoconjugates including calicheamicins when given in combination. Disclosures:Pierceall:Eutropics: Employment, Equity Ownership. Lena:Eutropics: Employment, Equity Ownership. Blake:Noel Blake: Employment, Equity Ownership. Elaschoff:Eutropics: Consultancy. Cardone:Eutropics: Employment, Equity Ownership.

Elevated Klotho Promoter Methylation Is Associated with Severity of Chronic Kidney Disease

Klotho (KL) expression is down-regulated in the renal tissues of chronic kidney disease (CKD) animal models and patients with end-stage renal disease. The putative role of KL promoter hypermethylation in the progression of CKD remains unclear. The present study aimed to determine renal and peripheral blood mononuclear cells (PBMC) levels of KL promoter methylation and analyze their relationship with clinical and histological severity in patients with CKD. Using bisulfite pyrosequencing, renal and PBMC levels of KL promoter methylation were quantified in 47 patients with CKD. 47 nephrectomy specimens of patients with renal cell carcinoma and 48 PBMC specimens of healthy volunteers were used as renal tissue and PBMC controls, respectively. Renal expression of KL protein was assayed by immunohistochemistry staining. Receiver operating characteristic (ROC) curve was used to identify the optimal cut-off value of PBMC KL promoter methylation level for renal KL promoter hypermethylation. Higher levels of KL promoter methylation were observed in renal tissue and PBMC in patients with CKD compared with controls (8.79±3.24 vs. 5.17±1.11%, P<0.001; 7.20±2.79 vs. 3.27±0.79%, P<0.001). In these patients, renal KL methylation level correlated inversely with renal KL immunostaining intensity (ρ=-0.794, P<0.001). Estimated glomerular filtration rate correlated inversely with renal and PBMC levels of KL promoter methylation (r=-0.829, P<0.001; r=-0.645, P<0.001), while tubulointerstistial fibrosis score correlated positively (ρ=0.826, P<0.001; ρ=0.755, P<0.001). PBMC KL promoter methylation level correlated positively with renal KL promoter methylation level in patients with CKD (r=0.787, P<0.001). In ROC curve, the area under curve was 0.964 (P<0.001) and the optimal cut-off value was 5.83% with a sensitivity of 93.8% and specificity of 86.7% to predict renal KL promoter hypermethylation. The degree of KL promoter methylation is associated with clinical and histological severity of CKD. PBMC KL promoter methylation level may act as a potential biomarker of renal KL promoter hypermethylation.

TLR2-Activated B Cells are Phenotypically Similar to the Abnormal Circulating B Cells Seen Preceding the Diagnosis of AIDS-Related NHL Diagnosis

AIDS-related non-Hodgkin lymphoma (AIDS-NHL) is a common AIDS-defining cancer. Prior studies suggest that chronic B-cell activation precedes AIDS-NHL diagnosis. Activation of B cells by multiple factors, including Toll-like receptor (TLR) signaling, leads to the expression of activation-induced cytidine deaminase (AID), a DNA mutating molecule that can contribute to oncogene translocations/mutations, leading to NHL. The goal of this study was to determine whether surface markers expressed on activated and/or germinal center B cells, and AID expression, were elevated on circulating B cells preceding AIDS-NHL and to determine if TLR signaling contributes to this activated B-cell phenotype. Stored viable peripheral blood mononuclear cell specimens, obtained before AIDS-NHL diagnosis, were assessed by multicolor flow cytometry. Additionally, B cells isolated from peripheral blood mononuclear cell were exposed to TLR ligands in vitro, after which B-cell phenotype was assessed by flow cytometry. An elevated fraction of B cells expressing CD10, CD71, or CD86 was seen in those who went on to develop AIDS-NHL. AID expression was detected in some who developed AIDS-NHL, but not in HIV+ or HIV- controls. TLR2-stimulated purified B cells exhibited the activated B-cell phenotype observed in HIV+ subjects before AIDS-NHL diagnosis. These results indicate that an elevated fraction of B cells display an activated/germinal center phenotype in those HIV+ subjects who go on to develop AIDS-NHL and suggest that TLR2-mediated activation may play a role in HIV infection-associated B-cell activation, potentially contributing to the genesis of AIDS-NHL.

Occult Hepatitis C Virus Infection in Candidates for Liver Transplant With Cryptogenic Cirrhosis

BackgroundOccult hepatitis C virus (HCV) infection is a new entity described by the presence of HCV-RNA in liver biopsy and/or peripheral blood mononuclear cell (PBMC) specimens, and undetectable levels or absence of HCV-RNA and in the absence or presence of anti HCV antibodies in plasma by current laboratory methods.ObjectivesTo evaluate the detection of HCV-RNA in PBMC specimens of the liver transplant candidates with cryptogenic cirrhosis by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR).Patients and MethodsFrom November 2007 to March 2013, 45 patients from Liver Transplant Center of Imam Khomeini Hospital, Tehran, were enrolled in this cross sectional study. PBMC specimens were separated from the peripheral blood sample. After extraction of RNA from plasma and PBMC specimens, HCV-RNA status was tested by RT-nested PCR. The 5′-untranslated region (5′-UTR) genotyping of HCV-RNA amplified from PBMC specimens was performed by a standard methodology with the INNO-LiPATM HCV II kit. The PCR products of 5′-UTR were sequenced after cloning into the pJET1.2 / blunt cloning vector.ResultsOf 45 patients, 4 (8.9% [95% CI: 4.4-15.6]) had detectable genomic HCV-RNA in their PBMC specimens. HCV genotypes were determined in the PBMCs of these subjects showed that 2 (50.0%) subjects with occult HCV infection had HCV subtype 3a, and 2 (50.0%) had HCV subtype 1b.ConclusionsThis study found that 8.9 % of the Iranian candidates for liver transplant with cryptogenic cirrhosis had occult HCV infection. Therefore, designing prospective studies focusing on the diagnosis of occult HCV infection in these subjects prior to liver transplantation could be valuable.

Decreased Level of Olfactory Receptors in Blood Cells Following Traumatic Brain Injury and Potential Association with Tauopathy

Traumatic brain injury (TBI) is a leading cause of death and disability among children and young adults in the United States. In this study, we explored whether changes in the gene expression profile of peripheral blood mononuclear cells (PBMC) may provide a clinically assessable "window" into the brain, reflecting molecular alterations following TBI that might contribute to the onset and progression of TBI clinical complications. We identified three olfactory receptor (OR) TBI biomarkers that are aberrantly down-regulated in PBMC specimens from TBI subjects. Down-regulation of these OR biomarkers in PBMC was correlated with the severity of brain injury and TBI-specific symptoms. A two- biomarker panel comprised of OR11H1 and OR4M1 provided the best criterion for segregating the TBI and control cases with 90% accuracy, 83.3% sensitivity, and 100% specificity. We found that the OR biomarkers are ectopically expressed in multiple brain regions, including the entorhinal-hippocampus system known to play an important role in memory formation and consolidation. Activation of OR4M1 led to attenuation of abnormal tau phosphorylation, possibly through JNK signaling pathway. Our results suggested that addition of the two-OR biomarker model to current diagnostic criteria may lead to improved TBI detection for clinical trials, and decreased expression of OR TBI biomarkers might be associated with TBI-induced tauopathy. Future studies exploring the physiological relevance of OR TBI biomarkers in the normal brain and in the brain following TBI will provide a better understanding of the biological mechanisms underlying TBI and insights into novel therapeutic targets for TBI.

BH3 profiling as a predictive biomarker for response to cytarabine-based treatment of acute myelogenous leukemia.

7022 Background: Acute myelogenous leukemia (AML) is the most frequently diagnosed of the leukemias with approximately 13,000 new cases per year in the U.S. Although intense therapeutic discovery efforts have been directed towards AML, cytarabine-based treatment remains unchanged as the standard-of-care (SOC). As patient response to SOC is variable, segregating patients based on biomarker-driven predicted response may lead to improved clinical outcomes. One personalized diagnostic approach that may provide actionable information in patient management is BH3 profiling, a surrogate functional biomarker assay that assesses cell mitochondrial response to pro-apoptotic signaling. Methods: Blinded to outcomes, we profiled an AML cohort (n=63) treated with cytarabine-based therapy using a panel of BH3 peptides with response as a primary endpoint. Peripheral blood mononuclear cell (PBMC) or bone marrow aspirate (BM) specimens were obtained from newly diagnosed AML patients and viably preserved. Specimens were assayed by flow cytometry following cell permeabilization and incubation with potentiometric JC-1 mitochondrial dye and individual BH3 peptides. Results: Mann-Whitney analysis indicates BH3 profiling biomarkers are correlated with response to induction therapy. Among BH3 peptides, BIM was highly significant (p=2X10-6; CI[0.73,0.94]) with a notable sensitivity/specificity profile (AUC=0.84; p=2X10-10). Multivariate analysis indicates improved profiles for BIM + patient age (AUC=0.89; CI[0.81,0.97])and BIM + patient age +cytogenetic status (AUC=0.91; CI[0.83,0.98]). When patients were stratified by cytogenetic status, BIM was significant for both intermediate (p=0.0017; AUC=0.88; CI[0.71,1.04]) and unfavorable (p=0.016; AUC=0.80; CI[0.60,1.00]), demonstrating that the predictive power of the assay is independent of cytogenetics. Conclusions: Here, BH3 profiling predicts patient response to cytarabine-based treatment regimens with accuracies of 90%. Thus, patients may potentially benefit from being given alternate therapies while at the same time spared the toxicities, cost, and time lost associated with a therapy less likely to exhibit response.

Interferon-stimulated genes are associated with peginterferon/ribavirin treatment response regardless of IL28B alleles in hepatitis C virus/HIV-coinfected patients

The interaction between interferon-stimulated genes (ISGs) expression, IL28B genotypes and hepatitis C treatment outcomes has been mainly evaluated in the liver tissue from hepatitis C virus (HCV)-monoinfected patients but with controversial results. Herein, we examined whether more easily accessible peripheral blood mononuclear cells (PBMCs) could be used for this purpose in HIV-HCV coinfected patients, a population in whom HCV-induced liver disease progression occurs more rapidly and treatment response is lower. Gene expression profiles were examined using the human whole genome Agilent microarray platform in PBMCs collected from HIV/HCV-coinfected patients who had completed a course of peginterferon/ribavirin therapy with validated outcomes. Patients were split out according to the achievement of sustained virological response (SVR) and IL28B rs12979860 genotypes. The GeneSpringGX software was used to select genes differentially expressed in the different groups. Nineteen HIV/HCV-coinfected individuals receiving antiretroviral therapy and having undetectable plasma HIV-RNA were examined. Global gene expression profiles showed 42 genes differentially expressed according to treatment outcome and 56 according to IL28B genotype. Common genes were not found and functions differed for genes belonging to either group. Whereas at least 26 out of 37 repressed genes (70.3%) in SVR patients were ISGs, none of the 56 differentially expressed genes in carriers of distinct IL28B variants were ISGs (P < 0.0001). Baseline expression of ISGs in PBMCs from HCV/HIV-coinfected patients influence the response to peginterferon/ribavirin therapy, regardless of IL28B genotypes. PBMC specimens can reliably be used for evaluating ISGs expression in clinical practice.

Longitudinal Change in Telomere Length and the Chronic Stress Response in a Randomized Pilot Biobehavioral Clinical Study: Implications for Cancer Prevention

Shortened telomere length is associated with increased cancer incidence and mortality. Populations experiencing chronic stress have accelerated telomere shortening. In this exploratory study, we examined associations between longitudinal changes in patient reported outcomes (PRO) of psychologic distress and peripheral blood mononuclear cell (PBMC) telomere length to test the hypothesis that modulation of the chronic stress response would also modulate telomere dynamics. Archived PBMC specimens (N = 22) were analyzed from a completed and reported randomized, longitudinal trial that showed a psychosocial telephone counseling intervention improved quality of life (QOL) and modulated stress-associated biomarkers in cervical cancer survivors. PROs and biospecimens were collected at baseline and 4 months postenrollment. Telomere length of archived PBMCs was evaluated using the flow-FISH assay. Longitudinal changes in psychologic distress, measured by the Brief Symptom Inventory-18, were significantly associated with increased telomere length within the CD14(+) (monocyte) population (r = -0.46, P = 0.043); a similar trend was observed for the CD14(-) population. Longitudinal changes in telomere length of the CD14(-) subset, primarily T lymphocytes, were associated with longitudinal increases in the naive T-cell population (r = 0.49, P = 0.052). Alterations in the chronic stress response were associated with modulation of telomere length in PBMCs, with evidence for mobilization of "younger" cells from progenitor populations. These data provide preliminary support for the (i) capacity to modulate the chronic stress response and the associated accelerated telomere shortening, (ii) inclusion of telomere length in the biobehavioral paradigm, and (iii) potential link between the chronic stress response and biologic mechanisms responsible for genomic integrity and carcinogenesis.

Chronic immune activation is a distinguishing feature of liver and PBMC gene signatures from HCV/HIV coinfected patients and may contribute to hepatic fibrogenesis

Hepatitis C virus/human immunodeficiency virus (HCV/HIV) coinfected patients demonstrate accelerated progression to severe liver injury in comparison to HCV monoinfected patients, although the underlying mechanisms are unclear owing to infection of separate tissue compartments with two distinct viral pathogens. Microarray analysis of paired liver biopsy and peripheral blood mononuclear cell (PBMC) specimens from HCV/HIV coinfected and HCV monoinfected patients identified a gene expression signature associated with increased inflammation and immune activation that was present only in liver and PBMC samples from coinfected patients. We also identified in these samples liver- and PBMC-specific signatures enriched with fibrogenic/hepatic stellate activation and proinflammatory genes, respectively. Finally, Bayesian networks were constructed by assimilating these data with existing data from liver and PBMC samples from other cohorts, augmenting enrichment of biologically important pathways and further indicating that chronic immune activation in HCV/HIV coinfection may exacerbate liver disease progression in coinfected patients.

A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.

Expression of CD4+ forkhead box P3 (FOXP3)+ regulatory T cells in inflammatory bowel disease

Forkhead box P3 (FOXP3) plays an important role in the development and function of CD4(+) regulatory T (Treg) cells. In this study the percentage of CD4(+) FOXP3(+) Treg cells in peripheral blood mononuclear cells (PBMC) and the frequency of Treg cells in the colonic mucosa of patients with inflammatory bowel disease (IBD) were investigated. The percentage of CD4(+) FOXP3(+) Treg cells in PBMC was analyzed by flow cytometry. Immunohistochemistry was used to examine the FOXP3(+) cells in the inflamed mucosa. Real-time polymerase chain reaction and Western blot were used to detect the expressions of FOXP3 mRNA and protein in PBMC and mucosal biopsy specimens of IBD patients, respectively. Together with the decrease of percentage of Treg cells in PBMC, we found that the frequency of Treg cells increased significantly in inflamed mucosa of active or inactive Crohn's disease (CD) and ulcerative colitis (UC). The expressions of FOXP3 mRNA and protein increased in inflamed mucosa when compared with those in healthy controls, especially the FOXP3 mRNA in patients with active CD or UC. Interestingly, the expression of FOXP3 protein in active UC was higher than that in active CD. There was a decrease of CD4(+) FOXP3(+) Treg cells in peripheral blood and an accumulation of Treg cells in inflamed mucosa. These data suggested that the suppressive function of Treg cells may be partially inhibited and this could be an important factor in the recurrence of disease, especially in UC.