Paired Peripheral Blood Samples Research Articles

CD8+ T cell profiles in patients with rheumatoid arthritis and their relationship to disease activity.

CD8+ T cells are abundant in rheumatoid arthritis (RA). However, their role in disease pathogenesis is poorly defined. This study was undertaken to investigate the relationship between disease activity and CD8+ T cell phenotypes, production of cytokines, and production of cytotoxic molecules in the peripheral blood (PB) and synovial fluid (SF) of patients with RA. CD8+ T cell phenotypes were determined in 96 patients with RA (44 with disease in remission, 34 with active disease, 18 with low disease activity) and in 64 sex- and age-matched healthy controls. Ten paired PB and SF samples from patients with active RA were analyzed. The expression of surface markers, cytokines, and proteolytic enzymes in CD8+ T cells was evaluated using flow cytometry. PB CD8+ T cells from RA patients with active disease exhibited an effector (CD27-CD62L-) phenotype (P = 0.005), with elevated expression of proinflammatory cytokines (tumor necrosis factor α [TNFα], interferon-γ [IFNγ], interleukin-6 [IL-6], IL-17A) when compared to healthy controls. In a state of remission, the same phenotype observed in patients with active disease persisted, including a significant increase in the frequency of CD69 (P < 0.001), but lower cytokine production was observed. SF CD8+ T cells from RA patients expressed more robust effector memory (CD27+CD62L-) and activated (CD69+) profiles compared to the T cell subsets in paired PB samples. Production of cytokines (IL-6, IL-17A, and IFNγ) by CD8+ T cells from RA PB was positively correlated within individual donors. Moreover, production of cytokines (TNFα, IFNγ, and IL-17A) by CD8+ T cells from RA PB positively correlated with the Disease Activity Score in 28 joints. The activation status and proinflammatory potential of CD8+ T cell subsets observed in the RA patients in this study strongly suggest that a phenotype of local and systemic cytotoxic effector T cells plays a role in this disease.

A study on FoxP3 and Tregs in paired samples of peripheral blood and synovium in rheumatoid arthritis.

There is an increasing evidence suggesting the role of fork head boxP3 (FoxP3) in the development and the regulation of CD4+CD25+ Treg cells. T-cell regulatory mechanisms in rheumatoid arthritis patients were evaluated by the contributing factors such as pro-inflammatory cytokines, circulating immune complexes, HLA DR expression, ligand binding biomarkers, FoxP3 expression in paired samples of peripheral blood (PB) and synovial fluid (SF). These cellular responses were further correlated with the humoral immune responses such as anti-cyclic citrullinated peptides IgG (CCP), circulating immune complex-c1q IgG (CIC), immunoglobulin G (IgG) and immunoglobulin M (IgM) of the rheumatoid arthritis factor (RAF). The results suggest a definitive role of Tregs in the homeostatic control because there is an increase in FoxP3 (37%) and HLA-DR (45%) expression in the synovial fluid as compared to PB. Furthermore, humoral responses as a downstream effector mechanism are positively correlated with the pathogenesis of rheumatoid arthritis (RA). A positive relationship exists between quantitative anti-CCP production and the expression of HLA-DR. The study relates an increased and pivotal role of B cell activation in the synovial fluid thereby permitting the need to ablate the targeted B cell immune responses.

Assessment of Central Nervous System Involvement in Pediatric Acute Lymphoblastic Leukemia Patients Using Next-Generation Sequencing Method

Background: Successful outcome in childhood acute lymphoblastic leukemia (ALL) relies upon appropriate central nervous system (CNS)-directed therapy for treatment of subclinical or overt CNS leukemia. Patients with leukemia blasts detected in the cerebrospinal fluid (CSF) at diagnosis require intensified intrathecal therapy to avert a higher rate of relapse and poorer survival compared with patients without blasts in the CSF. Traditionally, CSF assessment is performed by morphological analysis of CSF smears with or without TdT assay or via flow cytometry; however, both of these methods are limited in their sensitivity. Moreover, these modalities rely on subjective operator judgment for identification of leukemic blasts, which can result in limited assay reproducibility. A precise definition of CNS involvement is necessary to avoid over- or under- treatment. The LymphoSIGHT™ platform is a next-generation sequencing-based method that is being used for minimal residual disease assessment in lymphoid malignancies (Faham et al., Blood 2012). The LymphoSIGHT™ platform has a sensitivity to detect one leukemic cell per million leukocytes in peripheral blood and/or bone marrow and has been shown to have prognostic value in adult ALL patients (Logan et al., Biol Blood Marrow Transplant 2014). In this pilot study, we assessed whether this sequencing method could be used to detect leukemic clonotypes in CSF samples from newly diagnosed pediatric ALL patients.Methods: Diagnostic bone marrow samples, and paired CSF and peripheral blood samples were obtained from two patients. Using universal primer sets, we amplified variable, diversity, and joining gene segments from immunoglobulin (Ig) heavy chain (IGH), Ig kappa chain (IGK), and T-cell receptor beta (TRB), delta (TRD) and gamma (TRG) loci from genomic DNA. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then quantitated in CSF (cell pellet and supernatant) and peripheral blood samples obtained at diagnosis and interim treatment time points. A quantitative and standardized measure of leukemic clonotype level per million leukocytes in each follow-up sample was determined.Results: Diagnostic bone marrow samples from the two pediatric ALL patients were used to identify the tumor-specific clonotypes. Three IGH tumor clonotyes, one IGK, and one TRD tumor clonotype were observed in the first patient. Three IGH tumor clonotypes were observed in the second patient. The level of tumor burden associated with each leukemic clonotype was then assessed in serial CSF samples from each of the two patients. In the first patient, both the CSF pellet and supernatant were positive by sequencing at diagnosis. Consistent with the sequencing results, this patient was determined to have CNS involvement at the time of diagnosis based on standard testing methods. The patient cleared at Day 22, and the CSF pellet and supernatant from this time point were also negative by sequencing. Serial samples from the second patient, who was shown to lack CNS involvement by standard testing methods, were also tested. Both the CSF cell pellet and supernatant were negative at the Day 0 and Day 22 time points for this patient, showing concordance with available clinical data (Table 1).Conclusions: This study demonstrates proof of principle that the LymphoSIGHT sequencing method can be used to assess CNS involvement in pediatric patients with ALL. Further studies are warranted to assess the diagnostic and prognostic value of the sequencing method for assessment of CNS disease. The study cohort has now been expanded to include an additional 35 pediatric patients with ALL, and data will be presented.Abstract 5343. Table 1Comparison of results in CSF samples using sequencing and traditional methods.PatientTime PointStandard analysis of CSF samplesSequencing analysis of CSF samplesWBC count/µLRBC count/µLCell numberPercent blastsLeukemia molecules/1M diploid genomes (cell pellet)Leukemia molecules/1M diploid genomes (supernatant)1Day 000857431,100366,8001Day 2200330002Day 000250002Day 220032000 DisclosuresWeng:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

OP0017 Gut-Derived IL-23R+CD3+CD4-CD8-CD56+T-BET+NKP44+ Cells Are Expanded in the Peripheral Blood, Synovial Fluid and Bone Marrow of Patients with Ankylosing Spondylitis and Produce IL-17 and IL-22

BackgroundChronic gut inflammation occurring in AS patients has been linked to active axial inflammation and the gut has been proposed as the main site of IL-23 production in AS patients....

AB0021 In Psoriatic Arthritis Synovial Tissue Harbors Expanded T-Cell Clones Which Are not Fully Represented in Synovial Fluid or Blood Samples

BackgroundT-cells play a key role in psoriatic arthritis (PsA). Nevertheless, the distribution and exact role of these cells remains unclear. Here, we study whether expanded T-cell clones are present in...

Interleukin‐17+CD8+ T Cells Are Enriched in the Joints of Patients With Psoriatic Arthritis and Correlate With Disease Activity and Joint Damage Progression

ObjectivePsoriatic arthritis (PsA) is associated with HLA class I genes, in contrast to the association with HLA class II in rheumatoid arthritis (RA). Since IL-17+ cells are considered important mediators of synovial inflammation, we sought to determine whether IL-17–producing CD8+ T cells may be found in the joints of patients with PsA and whether these cells might contribute to the disease process.MethodsMononuclear cells from paired samples of synovial fluid (SF) and peripheral blood (PB) from patients with PsA or patients with RA were stimulated ex vivo, and CD4− T cells were examined by flow cytometry for cytokine expression, cytotoxic markers, and frequencies of γ/δ or mucosal-associated invariant T cells. Clinical measures of arthritis activity (C-reactive protein [CRP] level, erythrocyte sedimentation rate [ESR], Disease Activity Score in 28 joints [DAS28]) and power Doppler ultrasound (PDUS) scores for the presence of active synovitis in the aspirated knee were recorded and assessed for correlations with immunologic markers.ResultsWithin the CD3+ T cell compartment, both IL-17+CD4− (predominantly CD8+) and IL-17+CD4+ T cells were significantly enhanced in the SF compared to the PB of patients with PsA (P = 0.0003 and P = 0.002, respectively; n = 21), whereas in patients with RA, only IL-17+CD4+ T cells were increased in the SF compared to the PB (P = 0.008; n = 14). The frequency of IL-17+CD4− T cells in PsA SF was positively correlated with the CRP level (r = 0.52, P = 0.01), ESR (r = 0.59, P = 0.004), and DAS28 (r = 0.52, P = 0.01), and was increased in patients with erosive disease (P < 0.05). In addition, the frequency of IL-17+CD4− T cells positively correlated with the PDUS score, a marker for active synovitis (r = 0.49, P = 0.04).ConclusionThese results show, for the first time, that the PsA joint, but not the RA joint, is enriched for IL-17+CD8+ T cells. Moreover, the findings reveal that the levels of this T cell subset are correlated with disease activity measures and the radiographic erosion status after 2 years, suggesting a previously unrecognized contribution of these cells to the pathogenesis of PsA.

Myeloma stem cell concepts, heterogeneity and plasticity of multiple myeloma

We really enjoyed reading the recently published excellent review on myeloma concepts (Hajek et al, 2013). It was well timed for the presentation of myeloma data at the American Society of Hematology (ASH) 2013 meeting in New Orleans. Mainly Spanish data (Mateos et al 2013) is further translating the concept of heterogeneity and plasticity of multiple myeloma plasma cells into clinical practice. At the end of his comprehensive review Professor Hajek and his team recommended that ‘… To improve outcomes of future therapy, it is necessary to take into account the plasticity of MIC (myeloma-initiating cells) and altering dominance of genetically distinct subclones that occur as results of previous treatment.' We fully agree with this statement, but to make future therapy more efficient, even curative, we have to involve the patient's immune system. Myeloma evolution happens within and in very close interaction with the plasma cell microenvironment, particularly in close relationship with the compartments of the patient's immune system. In this regard, the question of whether the patient's immune system is the innocent bystander in this process is very much ignored in all current concepts or if it plays a key role in the initiation process of myeloma pathogenesis, remains unsolved and largely unexplored. In an attempt to obtain further insight into the potential role of the immune system in the long-term control of multiple myeloma, we have recently investigated the distribution and phenotype of different compartments of the immune system in paired peripheral blood and bone marrow samples from myeloma patients with long-term disease control, having received no treatment for more than 10 years, with that of age-matched healthy adults (Pessoa de Magalhães et al, 2013). Interestingly, the immune system capacity and repertoire of these myeloma patients was much stronger than that of the age-matched healthy adult group and other myeloma patients who failed to retain complete remission over time. Of note, in this study, long-term disease control was not restricted to patients with a complete response, because some reverted to a monoclonal gammopathy of undetermined significance (MGUS)-like profile after therapy, still not developing symptomatic disease for more than 10 years. A more detailed analysis of the different immune cell compartments showed increased numbers of cytotoxic T and natural killer (NK) cells and recovered dendritic cell counts, together with decreased percentages of regulatory T cells in the bone marrow; this was associated with recovered B-cell counts, including all B-cell compartments, in peripheral blood, and bone marrow as well as plasma cells. These results, together with the recurrent observation of the highly adverse prognostic impact of decreased normal plasma cell numbers in the bone marrow of myeloma patients, highlight the association of a preserved vs deteriorated immune system in long-term control vs malignant progression of monoclonal gammopathies (Pérez-Persona et al, 2007, 2010; Paiva et al, 2009, 2011, 2013). Therefore, the therapeutic plan for patients with multiple myeloma needs to include both sides on the battlefield (Kotoucek et al, 2012): myeloma plasma cells and myeloma stem cells by appreciating the features of plasticity and heterogeneity and, on the other hand, simultaneously rebuilding the patient's haematopoiesis in general, and immune system in particular (rather than to replace it with a donor haematopoiesis as a result of high treatment-related toxicity and a limited graft-versus-myeloma effect). The patient's immune system should then be able to take over the residual myeloma disease after successful chemoimmunotherapy. Pavel Kotouček and Alberto Orfao wrote the paper.

A119: Deep Sequencing Analysis of the T Regulatory and T Effector Repertoire in Juvenile Idiopathic Arthritis

Background/Purpose:T regulatory (Treg) function has been shown to be impaired in juvenile idiopathic arthritis (JIA) as synovial fluid (SF) Tregs are unable to suppress T effector cells (Teffs) found in the joint. The cause of this breach in immunologic tolerance is not fully understood, meriting the further study of Tregs and Teffs in JIA. The T cell receptor (TCR) endows T lymphocytes with antigen specificity and mediates interactions between T cells and their environment. Yet, little is known about the comparative specificities, clonality, and diversity of Tregs and Teffs in JIA. Further, the Treg repertoire has not been studied in adult or pediatric inflammatory arthritis. Therefore, we endeavored to characterize these TCR repertoires in JIA through deep sequencing technology.Methods:Mononuclear cells were isolated by Ficoll gradient centrifugation from paired peripheral blood (PB) and SF samples. Tregs (CD4+CD25+CD127low) and Teffs (CD4+CD25−) were sorted by flow cytometry. The TCRβ chain was amplified by multiplex PCR with genomic DNA serving as the template and then deep sequenced (ImmunoSEQ™). Sequences were aligned to the reference International ImMunoGeneTics information system. Further analysis utilizing Immunoglobulin Analysis Tool software was done to assess clonality, diversity, and variable (V), diversity (D), and joining (J) usage. Statistical analysis was completed with paired/unpaired Wilcoxon signed‐rank and Kruskal‐Wallis tests.Results:Three girls and 2 boys with oligoarticular (n = 2), extended oligoarticular (n = 2), and psoriatic (n = 1) arthritis were studied. Patients were similar in age (7–12 yrs) and ANA status (negative n = 4). Treatment consisted of NSAIDs (n = 2), methotrexate (n = 1), leflunomide (n = 1), and leflunomide with etanercept (n = 1). Tregs, measured as % of CD4+ cells, were enriched in the SF compared to PB (mean ± SEM: SF Tregs 13.2 ± 1.8; PB Tregs 5.1 ± 0.6). SF Teff repertoire was less diverse (p = 0.008) and more clonal (p = 0.008) than PB Teffs. In SF Teffs, a single complementarity determining region 3 (CDR3) was shared across all patients, though different V and J genes were used to create this CDR3, suggesting antigen selection. SF Tregs had the largest clonal expansions compared to PB Tregs (p = 0.016), PB Teffs (p = 0.008) and SF Teffs (p = 0.032). The degree of SF Treg clonality correlated with the active joint count. V gene usage, particularly Vβ29, in unique clonotypes was skewed in SF Tregs compared to PB Teffs, PB Tregs, and SF Teffs (p = 0.022); Vβ29 gene overuse was most prominent in patients with more active joints.Conclusion:We report on the first deep sequencing analysis of T cells in JIA. An antigen‐driven process was suggested by the clonality and shared CDR3 clonotypes in SF Teffs. This study is the first to demonstrate abnormalities in the Treg repertoire in inflammatory arthritis. The SF Treg repertoire had the most pronounced clonality and skewing of V gene usage, and these abnormalities correlated with disease severity. These results suggest that Treg insufficiency in JIA may be partially due to an aberrant Treg repertoire in the arthritic joint. Studies of additional patients with functional correlates are needed to augment these findings.

Evaluation of reliability on STR typing at leukemic patients used for forensic purposes

Over the past decades, main advances in the field of molecular biology, coupled with benefits in genomic technologies, have led to detailed molecular investigations in the genetic diversity generated by researchers. Short tandem repeat (STR) loci are polymorphic loci found throughout all eukaryotic genome. DNA profiling identification, parental testing and kinship analysis by analysis of STR loci have been widely used in forensic sciences since 1993. Malignant tissues may sometimes be the source of biological material for forensic analysis, including identification of individuals or paternity testing. There are a number of studies on microsatellite instability in different types of tumors by comparing the STR profiles of malignant and healthy tissues on the same individuals. Defects in DNA repair pathways (non-repair or mis-repair) and metabolism lead to an accumulation of microsatellite alterations in genomic DNA of various cancer types that result genomic instabilities on forensic analyses. Common forms of genomic instability are loss of heterozygosity (LOH) and microsatellite instability (MSI). In this study, the applicability of autosomal STR markers, which are routinely used in forensic analysis, were investigated in order to detect genotypes in blood samples collected from leukemic patients to estimate the reliability of the results when malignant tissues are used as a source of forensic individual identification. Specimens were collected from 90 acute and 10 chronic leukemia volunteers with oral swabs as well as their paired peripheral blood samples from the Oncology Centre of the Department of Hematology at Istanbul University, during the years 2010-2011. Specimens were tested and compared with 16 somatic STR loci (CSFIPO, THO1, TPOX, vWA, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11 and FGA) widely used in forensic identification and kinship. Only two STR instabilities were encountered among 100 specimens. An MSI in the FGA loci and a LOH in the D2S1338 loci were determined in two individuals separately. Our results demonstrate that the use of the biological samples from leukemia patients in forensic identification and kinship testing is questionable, especially if known microsatellite instability is available. Genetic instabilities may alter the STR polymorphism, leading to potential errors on forensic identification of individuals. Therefore, typing of autosomal STRs from leukemia patients should be performed with both healthy and malignant tissue samples of individual as references.

A5.16 miRNA expression profile in Bulgarian patients with rheumatoid arthritis compared to patients with osteoarthritis and healthy controls in regard to their use as biomarkers in the clinical practice

Background and ObjectivesThe pathogenesis of rheumatoid arthritis (RA) is still unknown. Recent data suggest that micro-ribonucleic acids (microRNAs) and their targets are involved in the inflammatory process and may perpetuate...

A8.10 Human memory t cells from the bone marrow are resting and maintain long-lasting memory

ObjectivesMemory T cells are essential components of immunological memory, but little is known about their maintenance. Recirculating memory T cells of blood and secondary lymphoid organs slowly decay in numbers....

Elevated Fetal Adipsin/Acylation-Stimulating Protein (ASP) in Obese Pregnancy: Novel Placental Secretion via Hofbauer Cells

Obesity in pregnancy is associated with increased risks of obesity in the offspring. We investigated the relationship between obesity in pregnancy and circulating maternal and fetal levels of adipose tissue-derived factors adipsin and acylation stimulating protein (ASP) in lean and obese mothers. Paired peripheral and cord blood samples were taken. Paired fat and placenta tissue were taken for explant culture. Media were assayed for secreted adipsin and ASP. Clinical parameters assayed included fasting insulin, glucose, and adipsin. The study was conducted at a university hospital maternity unit. Patients included 35 lean [body mass index (BMI) 19-25 kg/m(2), mean age 32 years and 39 obese (BMI) > 30 kg/m(2), mean age 32.49 years] pregnant Caucasian women, delivered by cesarean section at term. Identification of placental macrophages [Hofbauer cells (HBCs)], as a source of adipsin and ASP was determined. HBCs secreted both adipsin and ASP. Cord levels of adipsin (1663.78 ± 52.76 pg/mL) and ASP (354.48 ± 17.17 ng/mL) were significantly elevated in the offspring of obese mothers compared with their lean controls [1354.66 ± 33.87 pg/mL and 302.63 ± 14.98 ng/mL, respectively (P < .05 for both)]. Placentae from obese mothers released significantly more adipsin and ASP than placentae from lean mothers [546.0 ± 44 pg/mL · g vs 284.56 ± 43 pg/mL · g and 5485.75 ± 163.32 ng/mL · g vs 2399.16 ± 181.83 ng/mL · g, respectively (P < .05 for both)]. Circulating fetal adipsin and ASP positively correlated with maternal BMI (r = 0.611, P < .0001, and r = 0.391, P < .05, respectively). Fetal adipsin correlated positively with maternal (r = 0.482, P < .01) and fetal homeostasis model assessment of insulin resistance (r = 0.465, P < .01). We demonstrate novel secretion of adipsin and ASP by placental HBCs.

Characteristics of differentiated CD8+ and CD4+ T cells present in the human brain

Immune surveillance of the central nervous system (CNS) by T cells is important to keep CNS-trophic viruses in a latent state, yet our knowledge of the characteristics of CNS-populating T cells is incomplete. We performed a comprehensive, multi-color flow-cytometric analysis of isolated T cells from paired corpus callosum (CC) and peripheral blood (PB) samples of 20 brain donors. Compared to PB, CC T cells, which were mostly located in the perivascular space and sporadically in the parenchyma, were enriched for cells expressing CD8. Both CD4(+) and CD8(+) T cells in the CC had a late-differentiated phenotype, as indicated by lack of expression of CD27 and CD28. The CC contained high numbers of T cells expressing chemokine receptor CX3CR1 and CXCR3 that allow for homing to inflamed endothelium and tissue, but hardly cells expressing the lymph node-homing receptor CCR7. Despite the late-differentiated phenotype, CC T cells had high expression of the IL-7 receptor α-chain CD127 and did not contain the neurotoxic cytolytic enzymes perforin, granzyme A, and granzyme B. We postulate that CNS T cells make up a population of tissue-adapted differentiated cells, which use CX3CR1 and CXCR3 to home into the perivascular space, use IL-7 for maintenance, and lack immediate cytolytic activity, thereby preventing immunopathology in response to low or non-specific stimuli. The presence of these cells in this tightly regulated environment likely enables a fast response to local threats. Our results will enable future detailed exploration of T-cell subsets in the brain involved in neurological diseases.

Decorin is down‐regulated in multiple myeloma and MGUS bone marrow plasma and inhibits HGF‐induced myeloma plasma cell viability and migration

Decorin is a stromal-produced small leucine-rich proteoglycan known to attenuate tumour pro-survival, migration, proliferation and angiogenic signalling pathways. Recent studies have shown that decorin interacts with the hepatocyte growth factor (HGF) receptor c-Met, a potential key pathway in multiple myeloma (MM). Decorin levels in paired peripheral blood and bone marrow plasma samples from healthy volunteers (HV) (n=23), and patients with monoclonal gammopathy of undetermined significance (MGUS) (n=41) and MM (n=19) were determined by ELISA. Further, the ability of decorin to inhibit HGF-induced effects on MM cell lines were analysed in vitro using cell viability and Transwell migration assays. We found that decorin concentrations were significantly higher (P<0.05) in bone marrow (BM) plasma from HVs (median 35.2ng/mL; range, 15.3-99.1) compared with MGUS (median 22.5ng/mL; range, 11.1-59.5) and patients with MM (median 21.5ng/mL; range, 10.6-35.9). Decorin levels were higher in BM plasma than in peripheral blood in all groups, with a BM/PB ratio of 3.9, 3.4 and 2.5 for HV, MGUS and MM, respectively. A positive correlation (Spearman's ρ=0.51, P<0.05) was found between simultaneously measured levels of HGF and decorin in BM plasma in HVs, but not in MGUS or MM samples. Functionally, decorin inhibited HGF-induced migration and viability of INA-6 and ANBL-6 MM cell lines, independent of c-Met down-regulation. Our results show that decorin is down-regulated in MGUS and MM bone marrow plasma and that it inhibits HGF-induced viability and migration of myeloma cell lines in vitro.

Single Cell Analysis of Lymph Node Tissue from HIV-1 Infected Patients Reveals that the Majority of CD4+ T-cells Contain One HIV-1 DNA Molecule

Genetic recombination contributes to the diversity of human immunodeficiency virus (HIV-1). Productive HIV-1 recombination is, however, dependent on both the number of HIV-1 genomes per infected cell and the genetic relationship between these viral genomes. A detailed analysis of the number of proviruses and their genetic relationship in infected cells isolated from peripheral blood and tissue compartments is therefore important for understanding HIV-1 recombination, genetic diversity and the dynamics of HIV-1 infection. To address these issues, we used a previously developed single-cell sequencing technique to quantify and genetically characterize individual HIV-1 DNA molecules from single cells in lymph node tissue and peripheral blood. Analysis of memory and naïve CD4+ T cells from paired lymph node and peripheral blood samples from five untreated chronically infected patients revealed that the majority of these HIV-1-infected cells (>90%) contain only one copy of HIV-1 DNA, implying a limited potential for productive recombination in virus produced by these cells in these two compartments. Phylogenetic analysis revealed genetic similarity of HIV-1 DNA in memory and naïve CD4+ T-cells from lymph node, peripheral blood and HIV-1 RNA from plasma, implying exchange of virus and/or infected cells between these compartments in untreated chronic infection.

Predominance of activated, clonally expanded T helper type 17 cells within the CD4+ T cell population in psoriatic lesions

Recent evidence points to the T helper type 17 (Th17) subset as key in the pathogenesis of psoriasis, but cells of this type in lesions remain to be fully characterized. Here we isolated, enumerated, functionally tested and clonotyped the CD4(+) Th cell population ex vivo from lesional biopsies and paired peripheral blood samples from psoriasis patients. Th17 cells were over-represented dramatically in lesions from all patients, representing 49-93% of CD4(+) Th cells compared with 3-18% in blood. Most lesional Th17 cells produced interleukin (IL)-17A ex vivo without further stimulation and expressed the CD45RO(+) phenotype characteristic of activated or memory cells. There was no increase in 'natural' [CD25(hi) forkhead box protein 3 (FoxP3(+))] regulatory T cells in lesions versus peripheral blood, but there was enrichment of 'induced' IL-10(+) regulatory T cell numbers in biopsies from some patients. The lesional Th17 cells exhibited a bias in T cell receptor Vβ chain usage, suggestive of specific expansion by antigen. The therapeutic challenge is to overcome the dominance of overwhelming numbers of such antigen-specific Th17 cells in psoriatic lesions.

Immunoglobulin G4+ clones identified by next-generation sequencing dominate the B cell receptor repertoire in immunoglobulin G4 associated cholangitis

Immunoglobulin G4 (IgG4)-associated cholangitis (IAC) is a manifestation of the recently discovered idiopathic IgG4-related disease. The majority of patients have elevated serum IgG4 levels and/or IgG4-positive B-cell and plasma cell infiltrates in the affected tissue. We hypothesized that clonally expanded, class-switched IgG4-positive B cells and plasma cells could be causal to these poorly understood phenomena. In a prospective cohort of six consecutive IAC patients, six healthy controls, and six disease controls, we used a novel next-generation sequencing approach to screen the B-cell receptor (BCR) repertoires, in blood as well as in affected tissue, for IgG4+ clones. A full repertoire analysis of the BCR heavy chain was performed using GS-FLX/454 and customized bioinformatics algorithms (>10,000 sequences/sample; clones with a frequency ≥0.5% were considered dominant). We found that the most dominant clones within the IgG+ BCRheavy repertoire of the peripheral blood at baseline were IgG4+ only in IAC patients. In all IAC patients, but none of the controls, IgG4+ BCR clones were among the 10 most dominant BCR clones of any immunoglobulin isotype (IgA, IgD, IgM, and IgG) in blood. The BCR repertoires of the duodenal papilla comprised the same dominant IgG4+ clones as the paired peripheral blood samples. In all IAC patients, after 4 and 8 weeks of corticosteroid therapy the contribution of these IgG4+ clones to the IgG+ repertoire as well as to total BCR repertoire was marginalized, mirroring sharp declines in serum IgG4 titers and regression of clinical symptoms. The novel finding of highly abundant IgG4+ BCR clones in blood and tissue of patients with active IAC, which disappear upon corticosteroid treatment, suggests that specific B cell responses are pivotal to the pathogenesis of IAC. (HEPATOLOGY 2013 ).

Abstract 1458: Efficient isolation of circulating tumor cells in small cell lung cancer patients using size- and geometry-controlled microcavity array system.

Abstract Background: We have developed a microcavity array (MCA) system integrated with a miniaturized device for the isolation of circulating tumor cells (CTC) without relying on EpCAM expression. The MCA system allows the filtration of tumor cells from whole blood on the basis of differences in the size between tumor and blood cells. The previous study has shown that the MCA system is potentially superior to the conventional epithelial antigen-based method for detecting CTCs in non-small cell lung cancer. Recently, we further optimized the shape and porosity of MCA to efficiently isolate smaller tumor cells as in small cell lung cancer (SCLC). Here we report the results of clinical study in SCLC patients comparing MCA system to CellSearch system and exploratory study of novel rectangular-shaped MCA system. Methods: The MCA is made of nickel by electroforming and circular MCA is fabricated with 8-9 μm diameter pores. Meanwhile, novel rectangular-shaped MCA is fabricated with 5-9 μm-wide and 30-60 μm-long pores. NCI-H69 (12 μm in diameter) SCLC cells were used for spike-in experiments. CTCs were defined as cells with round to oval morphology, a visible nucleus, positive for cytokeratin and negative for CD45. For the clinical evaluation, paired peripheral blood samples were collected from 18 SCLC patients to compare the performance of the CellSearch system and the MCA system for CTC detection. Results: The recovery rate of spiked NCI-H69 cells with the rectangular-shaped MCA (80 ± 2%) was significantly higher than that with the circular MCA (67 ± 4%) (p=0.01, t-test). In addition, the carryover of leukocytes on the rectangular-shaped MCA was 7-fold lower than that on the circular MCA in the experiment using healthy donor blood. The fluctuation of flow resistance during filtration with the rectangular-shaped MCA (&amp;lt;1.5 kPa) was smaller than that with the circular MCA (2 kPa); the rectangular-shaped MCA enabled recovery of small tumor cells with high efficiency. In clinical study, compared to the CellSearch, the circular and rectangular-shaped MCA system identified significantly more SCLC patients (9 of 18 vs. 18 of 18 vs. 17 of 18, respectively) as CTC positive. No significant difference was observed in the CTC counts between the rectangular-shaped MCA (median 16, range 0-545 cells/7.5 mL) and the circular MCA (median 23, range 2-2329 cells/7.5 mL) (p=0.77, Wilcoxon test). In contrast, the number of captured leukocytes on the rectangular-shaped MCA was significantly lower than that on the circular MCA (p&amp;lt;0.0001, Wilcoxon test), suggesting that implementing the rectangular-shaped MCA diminishes a considerable number of carryover leukocytes. Conclusion: The MCA system has a potential as a tool for the efficient recovery of CTCs in small cell type tumors with high purity, while offering the additional advantages in cost, portability, and capacity to perform more detailed analyses of CTCs. Citation Format: Masahito Hosokawa, Hirotsugu Kenmotsu, Yasuhiro Koh, Tomoko Yoshino, Tsuyoshi Tanaka, Tateaki Naito, Toshiaki Takahashi, Haruyasu Murakami, Yukiko Nakamura, Asuka Tsuya, Takehito Shukuya, Akira Ono, Hiroaki Akamatsu, Reiko Watanabe, Sachiyo Ono, Hisashige Kanbara, Tadashi Matsunaga, Nobuyuki Yamamoto. Efficient isolation of circulating tumor cells in small cell lung cancer patients using size- and geometry-controlled microcavity array system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1458. doi:10.1158/1538-7445.AM2013-1458

Comparison of select cancer biomarkers in human circulating and bulk tumor cells using magnetic nanoparticles and a miniaturized micro-NMR system

Circulating tumor cells (CTC) harvested from peripheral blood have received significant interest as sources for serial sampling to gauge treatment efficacy. Nanotechnology and microfluidic based approaches are emerging to facilitate such analyses. While of considerable clinical importance, there is little information on how similar or different CTCs are from their shedding bulk tumors. In this clinical study, paired tumor fine needle aspirate and peripheral blood samples were obtained from cancer patients during image-guided biopsy. Using targeted magnetic nanoparticles and a point-of-care micro-NMR system, we compared selected biomarkers (EpCAM, EGFR, HER-2 and vimentin) in both CTC and fine needle biopsies of solid epithelial cancers. We show a weak correlation between each paired sample, suggesting that use of CTC as “liquid biopsies” and proxies to metastatic solid lesions could be misleading. From the Clinical EditorIn this clinical study, paired tumor fine needle aspirate and peripheral blood samples were obtained from patients with solid epithelial cancers during image-guided biopsy. Using targeted magnetic nanoparticles and a point-of-care micro-NMR system, the authors compared selected biomarkers in both circulating tumor cells (CTC) and fine needle biopsies, demonstrating a weak correlation between each paired sample, suggesting that use of CTC could be misleading in this context.

Local renin-angiotensin system in the thyroid – myth or fact?

Beside the basic control system associated with the hypothalamus-hypophysis-thyroid axis and thyrotropin, a growing evidence suggests that thyroid function is influenced by a vast spectrum of locally produced factors. Renin-angiotensin system (RAS) is considered as one of the most powerful and effective regulatory systems in humans. In the “classical” cascade of RAS, angiotensinogen is enzymatically conversed to the most active element of RAS – angiotensin (Ang) II. Ang II exerts its effects via two main receptors: AT1R and AT2R. The understanding of RAS biological function was significantly changed by the discovery of new enzymatic cascades leading to the production of molecules with different than Ang II functional properties – for instance Ang (1-7), molecule reacting with its specific receptor – Mas. Beside the growing complexity of RAS-structure, diverse expression patterns of RAS components in various tissues formed a basis for a concept of “local RAS” in many organs including brain and immune system. It was shown that many organ-specific effects of hormonal factors, including the influence of thyroid hormones on cardiovascular system, were dependent on the modulation of local RAS components. However, very little is known about the role of RAS in the control of thyroid function. In our immunohistochemical analysis of operative specimens we observed for the first time expression of RAS components in human thyroid. Interestingly, although our recently published findings suggest a direct regulatory influence of thyroid function on the phenotype and activity of immune cells in humans, no studies regarding the influence of human RAS on the course of such common inflammatory process as thyroiditis are available. Flow Cytometry analyses performed in our laboratory on thyroid fine needle aspiration biopsy material allowed us to assess quantitatively and phenotypically the immune cell populations of local thyroid blood. The comparison of results obtained with paired peripheral blood and local thyroid blood samples suggested differences in the structure of antigen presenting cell populations, e.g. monocytes, known to be the main source of RAS components in the immune system. Interestingly, RAS positive immune cells have been shown to correlate with clinical course of various inflammatory and malignant diseases. In conclusion, better knowledge about local RAS in the thyroid seems to be of great importance for the understanding of both: thyroid physiology and pathological processes associated with inflammatory and malignant disorders of this gland.