Soluble FcϵRII/CD23 in Patients with Autoimmune Diseases and Epstein-Barr Virus-Related Disorders: Analysis by ELISA for Soluble FcϵRII/CD23

The low-affinity Fc receptor for IgE (FcϵRII/CD23) and its soluble form (sCD23, IgE-binding factor) have multiple functions, and enhanced levels of these are associated with various immunological diseases. We established two sensitive ELISA systems using enzyme-conjugated mAb and biotinylated mAb. The detection limits of the ELISA systems were 0.03 and 1.0 ng/ml, which showed good correlation in the range 1.0-10 ng/ml. In the ELISA system using enzyme-conjugated mAb, the average sCD23 concentration in 303 normal healthy volunteers was 1.4 ± 0.3 ng/ml. In the ELISA system using biotinylated mAb, sCD23 levels in normal healthy volunteers showed almost the same values. In patients with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren syndrome, progressive systemic sclerosis, and mixed connective tissue disease, the sCD23 levels were significantly higher than those in normal individuals. Furthermore, in Epstein-Barr virus-related disorders after liver transplantation with immunosuppression, plasma levels of sCD23 rapidly Increased to more than 12 ng/ml when clinical symptoms were evident. In addition, the sCD23 values remained high, although elevated GOT levels gradually decreased to standard values and EBV hepatitis improved. These data suggest that sCD23 levels are a sensitive marker of autoimmune diseases and EBV-related disorders in addition to allergic disorders. The ELISA system for sCD23 may be an additional diagnostic tool in estimating the clinical courses of these diseases.     

Varying importance of soluble and membrane CD14 in endothelial detection of lipopolysaccharide

The endothelial response to LPS is critical in the recruitment of leukocytes, thereby allowing the host to survive Gram-negative infection. Herein, we investigated the roles of soluble CD14 (sCD14) and membrane CD14 (mCD14) in the endothelial response to low level LPS (0.1 ng/ml), intermediate level LPS (10 ng/ml), and high level LPS (1000 ng/ml). Removal of sCD14 from serum and sCD14-negative serum prevented low level LPS detection and subsequent response. Addition of recombinant sCD14 back into the endothelial system rescued the endothelial response. GPI-linked mCD14 removal from endothelium or endothelial treatment with a CD14 mAb prevented responses to low-level LPS even in the presence of sCD14. This demonstrates essential nonoverlapping roles for both mCD14 and sCD14 in the detection of low-level LPS. At intermediate levels of LPS, sCD14 was not required, but blocking mCD14 still prevented endothelial LPS detection and E-selectin expression, even in the presence of sCD14, suggesting that sCD14 cannot substitute for mCD14. At very high levels of LPS, the absence of mCD14 and sCD14 did not abrogate TLR4-dependent, E-selectin synthesis in response to LPS. The MyD88 independent pathway was detected in endothelium (presence of TRIF-related adaptor molecule TRAM). The MyD88-independent response (IFN-beta) in endothelium required mCD14 even at the highest LPS dose tested. Our results demonstrate an essential role for endothelial mCD14 that cannot be replaced by sCD14. Furthermore, we have provided evidence for a TRAM pathway in endothelium that is dependent on mCD14 even when other responses are no longer mCD14 dependent.     

Differential levels of soluble endoglin (CD105) in myeloid malignancies

Angiogenesis contributes to disease progression in solid and hematopoietic malignancies, and endoglin (CD105), a component of the transforming growth factor (TGF)-beta receptor complex, is a powerful marker of neovascularization. Elevated amounts of soluble CD105 (sCD105) have been recently identified in selected solid tumors but no data are available on sCD105 in hematopoietic malignancies. Therefore, levels of sCD105 were investigated in sera of patients with acute myeloid leukemia (AML) (n = 10) or chronic myeloproliferative disorders (CMD) (n = 28), and correlated with those of soluble TGF-beta(1) (sTGF-beta(1)). Dot blot assay detected higher amounts of sCD105 (P < 0.05) both in AML (4.34 +/- 2.62 OD/mm(2)) and in CMD (3.71 +/- 2.09 OD/mm(2)) patients than in healthy subjects (n = 14, 2.38 +/- 1.18 OD/mm(2)). Instead, enzyme-linked immunosorbent assay (ELISA) identified (P < 0.05) lower and higher levels of sTGF-beta(1) in AML (32,017 +/- 1,900 pg/ml) and CMD (60,700 +/- 19,200 pg/ml) patients, respectively, compared to healthy individuals (n = 11, 47,173 +/- 5,443 pg/ml). In essential thrombocythemia (ET) patients with thrombotic episodes, levels of sCD105 were lower (P < 0.05) compared to patients without thrombotic complications, and inversely correlated with those of sTGF-beta(1) (r = 0.94). Conversely, amounts of sCD105 directly correlated with levels of sTGF-beta(1) (r = 0.74) in ET patients without thrombotic events. Our results show that high levels of sCD105 are present in myeloid malignancies that are characterized by a high cellular proliferation rate, and suggest that an altered balance between sCD105 and sTGF-beta(1) might favor disease progression and clinical complications.     

系统性红斑狼疮患者血清中CD83与自身抗体的表达及其相互关系

目的：检测系统性红斑狼疮（systemic lupus erythematosus,SLE）患者血清中CD83（soluble CD 83,sCD 83）和多种自身抗体的表达水平,并探讨其相互关系。方法：ELISA检测患者可溶性CD 83和AnuA的表达,应用间接免疫荧光的方法检测抗cmDNA抗体,应用乳凝法检测血清中的DNP,采用胶体金标记和快速膜渗滤技术测定血清中的抗dsDNA抗体。结果：对照组患者血清中可溶性CD 83的表达为（0.26±0.10）ng/ml,实验组患者血清中可溶性CD 83的表达为（5.56±0.72）ng/ml。与对照组相比,实验组患者血清中可溶性CD 83的平均浓度明显升高。在抗dsDNA抗体阴性的51例系统性红斑狼疮患者中AnuA的阳性率明显高于抗DNP抗体和抗cmDNA抗体,同样在抗DNP抗体阴性的58例系统性红斑狼疮患者中AnuA的阳性率明显高于dsDNA抗体和抗cmDNA抗体。系统性红斑狼疮患者中可溶性CD83的水平（〈2.68 ng/ml）与各种自身抗体（抗dsDNA抗体、AnuA、抗DNP抗体和抗cmDNA抗体）水平的相关系数分别为（r=0.542,0.613,0.489和0.367）。具有高水平可溶性CD83的系统性红斑狼疮患者（≥2.68 ng/ml）,与各种自身抗体（抗dsDNA抗体,AnuA,抗DNP抗体和抗cmDNA抗体）水平的相关系数分别为（r=0.711,P〈0.05）、（r=0.845,P〈0.01）、（r=0.862,P〈0.01）和（r=0.724,P〈0.051）。结论：可溶性CD83通过活化DC细胞并激活补体系统,参与系统性红斑狼疮的发生发展,联合可溶性CD83和多种自身抗体的检测,能更明确系统性红斑狼疮患者病情的严重程度,有利于SLE的诊断和治疗。     

CD14 is an acute-phase protein

The origin of soluble CD14 (sCD14) in the circulation is uncertain. To examine whether CD14 could be an acute-phase protein (APP), the levels of sCD14, IL-6, and C-reactive protein were determined by ELISA in serum and synovial fluid (SF) of patients with various arthropathies, and the regulation of CD14 synthesis was examined in liver cells. In patients with crystal-mediated or immunologically mediated arthritis (rheumatoid arthritis), serum levels of sCD14 were higher than or similar to those found in infection-mediated arthritis (reactive arthritis), precluding a relation with bacteria exposure. Levels of sCD14 were similar in SF and serum, and did not correlate with the number of SF leukocytes, excluding an important source from leukocyte membrane-bound CD14, by protease-mediated shedding. In contrast, serum levels of sCD14 in patients correlated with those of C-reactive protein, a classical APP, and IL-6, a cytokine known to regulate the synthesis of APP in the liver. Serum levels of sCD14 also correlated with disease activity in rheumatoid arthritis and reactive arthritis patients. IL-6 stimulated the production of CD14 by HepG2 hepatoma cells. By real-time PCR, the inducibility of CD14 by IL-6 was also observed at the mRNA level both in HepG2 cells and human primary hepatocytes. These in vitro results were confirmed by in vivo studies in IL-6(-/-) mice injected with turpentine, an experimental model of acute-phase response. Liver levels of CD14 mRNA increased in IL-6(+/+), but not in IL-6(-/-) mice. These results indicate that sCD14 can be considered as a type 2 APP.     

Enzyme-Linked Immunosorbent Assay for the Human Glial Fibrillary Acidic Protein Using a Mouse Monoclonal Antibody

Two enzyme-linked immunosorbent assays (ELISAs) have been developed for the quantification of soluble human glial fibrillary acidic protein (GFAP). The specificity of the assays for GFAP is ensured by the use of a monoclonal antibody directed against a GFAP-specific antigenic determinant. One ELISA is a four-layer system working in the concentration range 5-600 ng GFAP/ml. The other ELISA is a five-layer system and includes a biotin/avidin binding reaction. The latter assay has a working range of 0.5-60 ng GFAP/ml. The assays may be used for quantification of GFAP in CSFs, amniotic fluids, and extracts or homogenates of normal and pathological brain material. GFAP in serum could not be quantified because of unidentified interference. CSFs from 18 nonneurological subjects were found to contain 2-14 ng GFAP/ml (mean 4.1 ng/ml), whereas amniotic fluids from 50 normal pregnant women contained up to 24 ng GFAP/ml (mean 12.4 ng/ml). GFAP concentrations in CSFs from 32 multiple sclerosis patients were found not to be elevated compared to the control group.     

A soluble form of the human T cell differentiation antigen CD27 is released after triggering of the TCR/CD3 complex.

The human T cell Ag CD27 belongs to a recently defined family of cell surface receptors, including the nerve growth factor receptor, two distinct tumor necrosis factor receptors, and the B cell specific molecule CD40. On resting T cells, CD27 is a transmembrane homodimer with subunits of 50 to 55 kDa (p55). T cell activation via the TCR/CD3 complex causes a strong enhancement of p55 expression. Concomitantly, an alternative form of the CD27 molecule with a molecular mass of 28 to 32 kDa (p32) appears at the cell surface. With the use of ELISA, we here show that a soluble form of CD27 (sCD27) can be detected in the supernatant of T cells activated with anti-CD3 or combinations of anti-CD2 mAb. Moreover, sCD27 is found in both serum and urine from healthy donors. sCD27, purified from either culture supernatant or urine, has a molecular mass of 28 to 32 kDa and is, according to peptide mapping, structurally homologous to the p55 membrane form of CD27. Quantification of sCD27 levels may be used as a marker for T lymphocyte activation in vivo.     

Expression,regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases

Background

Inflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases.

Methods

sCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. In vitro studies were performed to investigate which factors regulate sCD14 release and mCD14 expression.

Results

sCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. In vitro, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition.

Conclusions

This study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.     

High concentration of soluble HLA-DR in the synovial fluid: generation and significance in "rheumatoid-like" inflammatory joint diseases

In the search for its role in inflammatory joint diseases, soluble HLA-DR (sHLA-DR) was quantitated in 72 synovial fluids (SF) by a newly established immunoenzyme assay. Unlike other soluble receptors which accumulated only moderately (sCD25, sCD4) or negligibly (sHLA class I, sCD8) in the SF, SF sHLA-DR levels exceeded serum levels by up to 3 orders of magnitude and varied disease dependently from "control" values (traumatic synovitis and osteoarthritis: 9.9 +/- 6.1 ng/ml). Clear-cut different SF sHLA-DR values in HLA-DR-associated "rheumatoid-like" (136.5 +/- 130.0 ng/ml) vs HLA-B27-associated "spondylarthropathy-like" arthritic forms (28.4 +/- 29.1 ng/ml) were most significant comparing oligoarticular juvenile chronic arthritis type I (147.6 +/- 112.6 ng/ml) and type II (3.3 +/- 1.1 ng/ml), thus offering a new classification marker. Also ex vivo, large amounts of sHLA-DR were released spontaneously by SF mononuclear cells and found to be related to the T-cell activation state. SF sHLA-DR may be shed in large complexes or micelles, as it eluted mainly at >450 kDa on gel filtration. Western blotting revealed that the majority of SF sHLA-DR consisted of full-length alpha- and beta-chains. Minor fractions of smaller sized antigens seemed to be generated by proteolytic cleavage rather than by alternative splicing, since only minute amounts of HLA-DRB mRNA lacking the transmembrane exon could be amplified by RT-PCR. Distinct forms of high-dose sHLA-DR, able to provoke rather than to suppress T-cell responses, are discussed as contributing to some HLA-DR disease association.     

Control of lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis factor secretion in human peripheral blood monocytes.

We used flow cytometry to determine how LPS-binding protein (LBP) effects the binding of fluorescein-labeled LPS to human monocytes via receptor-dependent mechanisms. The addition of human, rabbit, mouse, or FCS strikingly increased the binding of LPS to monocytes compared with controls incubated in serum-free medium. This binding was totally prevented by preincubation of monocytes with MY4, an anti-CD14 mAb, or by enzymatic removal of CD14 from monocytes. Depletion of LBP from rabbit serum with anti-LBP antibodies also produced a similar suppression. Solutions of albumin did not support the enhanced binding observed in serum but the addition of purified rabbit LBP to albumin solutions resulted in binding similar to that observed in serum-containing medium. When type-specific anti-LPS mAb was added to human serum, LPS binding to monocytes occurred but was only partly inhibited by anti-CD14 mAb, suggesting that receptors other than CD14 (presumably Fc or complement receptors) were involved. Serum increased by 100- to 1000-fold the sensitivity of monocytes to the triggering by LPS resulting in TNF secretion. TNF secretion was inhibited by anti-CD14 mAb up to 100 ng/ml of LPS and by anti-LPS mAb up to 1 to 10 ng/ml. The inhibition of TNF secretion by anti-LPS mAb appeared to be the result of directing LPS to monocyte receptors other than CD14. In contrast, in medium containing normal as well as acute serum and in the absence of anti-LPS antibodies, the binding of LPS to monocytes and the triggering of TNF secretion appeared to be mediated mainly by interactions between CD14 and LBP-LPS complexes.     

Postoperative Serum Levels of sCD26 for Surveillance in Colorectal Cancer Patients

One of the main aims of the follow-up after curative resection of colorectal cancer is the early detection and treatment of tumor recurrence. We previously demonstrated decreased preoperative soluble CD26 (sCD26) levels in serum from colorectal cancer patients. We extended now the study to investigate if sCD26 levels in postoperative serum serve as marker of recurrence of the disease during surveillance. Soluble sCD26 was measured in pre- and postoperative serum samples of 43 patients with primary colorectal cancer. Carcinoembryonic antigen, carbohydrate antigen 19.9 and 72.4 levels were also measured during surveillance. The average follow-up period was 41.8±20.8 months. sCD26 levels during follow-up showed well-defined patterns in patients without disease (n = 28), and in patients with tumor persistence (n = 2), local recurrence (n = 3) or distant metastasis (n = 10). Disease-free patients showed stable levels between 460–850 ng/mL during follow-up, while high (over 850 ng/mL) and unstable sCD26 levels were found before recurrence was diagnosed. The mean maximum/minimum sCD26 ratios during surveillance were 1.52, 2.12 and 2.63 for patients with no recurrence, local recurrence and metastasis, respectively (p = 0.005). From the cut-off obtained from a receiver operator characteristics (ROC) curve built with the maximum/minimum sCD26 ratios and the upper and lower cut-offs of sCD26, we were able to discriminate patients with and without recurrent disease. We propose that the measurement of serum sCD26 during the follow-up of patients diagnosed of colorectal cancer could be valuable for the early detection of local and distant recurrence. A large, randomized, prospective trial should be performed to confirm our findings.     

CD64指数、sCD14、sCD163与胸腔镜肺癌根治术后并发重症肺部感染的关系研究

摘要 目的：探讨CD64指数、可溶性白细胞分化抗原14（sCD14）、可溶性白细胞分化抗原163（sCD163）与胸腔镜肺癌根治术后并发重症肺部感染的关系。方法：选择2020年1月至2023年1月南通市肿瘤医院收治的行胸腔镜肺癌根治术治疗的非小细胞肺癌（NSCLC）患者共415例。根据术后是否并发重症肺部感染将患者分为感染组（21例）和未感染组（394例）。检测外周血CD64指数和血清sCD14、sCD163水平。多因素Logistic回归分析影响胸腔镜肺癌根治术后并发重症肺部感染的因素。受试者工作特征（ROC）曲线分析CD64指数、sCD14、sCD163预测胸腔镜肺癌根治术后并发重症肺部感染的价值。结果：感染组外周血CD64指数、血清sCD14、sCD163水平高于未感染组（P＜0.05）。多因素Logistic回归分析显示高CD64指数、sCD14、sCD163、降钙素原（PCT）是胸腔镜肺癌根治术后并发重症肺部感染的危险因素（P＜0.05）,高呼气峰值流量（PEF）是保护因素（P＜0.05）。CD64指数、sCD14、sCD163、PCT预测胸腔镜肺癌根治术后并发重症肺部感染的曲线下面积为0.834、0.815、0.842、0.784,联合预测曲线下面积为0.928,高于单独预测。结论：血清CD64指数、sCD14、sCD163、PCT水平升高是胸腔镜肺癌根治术后并发重症肺部感染的危险因素。联合CD64指数、sCD14、sCD163、PCT可较好地预测术后重症肺部感染的风险。     

CD14 gene promoter polymorphism in different clinical forms of tuberculosis

Mycobacterium tuberculosis interacts with monocyte-macrophages through cell surface molecules including CD14. A soluble form of CD14 (sCD14) exists in human serum, and higher amounts of it are found in tuberculosis. A polymorphism on CD14 gene promoter was associated with increased sCD14 levels in some diseases. To evaluate whether this polymorphism associates with tuberculosis, its clinical forms, and increased sCD14, genotype/allele frequencies in tuberculosis patients were compared with the controls. Results confirmed increased levels of sCD14 in patients with tuberculosis, and those with miliary tuberculosis had the highest levels. sCD14 decreased to normal levels after anti-tuberculosis treatment. No association was found between the CD14 polymorphism and tuberculosis or sCD14 levels. Results suggest that sCD14 may be involved in anti-tuberculosis immune response, but its increase is a consequence of infection rather than a predisposed genetic trait. Measuring sCD14 in tuberculosis may help monitor anti-tuberculosis treatment.     

Analysis of Neurotrophin/Receptor Interactions with a gD-Flag-Modified Quantitative Kinase Receptor Activation (gD.KIRA) Enzyme-Linked Immunosorbent Assay

A rapid, sensitive, and high-capacity assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a “kinase receptor activation” or KIRA-ELISA, utilizes two separate microtiter plates, one for cell culture and ligand stimulation, and the other for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of neurotrophin-induced trkA, trkB, or trkC activation. It utilizes a trkA, trkB, or trkC receptor fused with a 26-amino-acid polypeptide flag derived from HSV glycoprotein D (gD.trkA, B, or C, respectively) on the amino-terminus, stably transfected into CHO cells. Stimulated receptors were solubilized with Triton X-100 buffer and then captured in ELISA wells coated with gD-specific mAb. The degree of receptor autophosphorylation was quantified by anti-phosphotyrosine ELISA. Reproducible standard curves were generated with an EC₅₀of approximately 16 ng/ml NGF for gD.trkA KIRA, 11 ng/ml for NT4/5 and 20 ng/ml for BDNF in gD.trkB KIRA, and 9.4 ng/ml for NT3 in gD.trkC KIRA. When the gD.trkA KIRA assay was used to quantify serum NGF or NT3 following administration to rats, the assay agreed well with currently existing ELISA assays. When the gD.trkA KIRA assay was used to test several NGF variants, as well as NGF stability samples, the capacity of the assay to quantify ligand bioactivity compared well with the more widely used radioreceptor binding and PC 12 cell survival assays. The gD.trk KIRA assays show great potential as rapid bioassays, capable of quantitative, consistent, and stability indicating analyses.     

Cutting edge: human B cell function is regulated by interaction with soluble CD14: opposite effects on IgG1 and IgE production

The mechanism(s) controlling activation of naive B cells, their proliferation, Ag receptor affinity maturation, isotype switching, and their fate as memory or plasma cells is not fully elucidated. Here we show that between 24 and 60% of CD19+ cells in PBMC bind soluble CD14 (sCD14). Tonsillar B cells also bind sCD14, but preferentially the CD38-ve/low cells. Interaction of sCD14 with B cells resulted in higher levels of IgG1 and marked inhibition of IgE production by activated tonsillar B cells and Ag-stimulated PBMC. We found that sCD14 interfered with CD40 signaling in B cells, inhibited IL-6 production by activated B cells, and increased the kinetics and magnitude of CD40 ligand expression on T cells. Together with the previously reported effects on T cells, these findings define sCD14 as a novel soluble regulatory factor capable of modulating cellular and humoral immune responses by interacting directly with T and B cells.     

Elevated Soluble CD163 in Gestational Diabetes Mellitus: Secretion from Human Placenta and Adipose Tissue

Recently soluble CD163 (sCD163), a cleaved form of the macrophage receptor CD163, was identified as a macrophage-specific risk-predictor for developing Type 2 Diabetes. Here, we investigate circulating levels of sCD163 in gestational diabetes mellitus (GDM). Furthermore, given the role of the placenta in the pathogenesis of GDM, we assessed placental contribution to sCD163 secretion. Paired maternal (venous) and umbilical vein blood samples from GDM (n = 18) and Body Mass Index (BMI) matched control women (n = 20) delivered by caesarean section at 39–40 week gestation were assessed for circulating levels of sCD163, Tumour necrosis factor alpha (TNF-α) and Interleukin 6 (IL-6). Media from explant culture of maternal subcutaneous fat and corresponding placental tissues were assayed for these same molecules. CD163 positive cell numbers were determined in placental and adipose tissues of GDM and control women. We found significantly elevated circulating sCD163 levels in GDM mothers (688.4±46.9 ng/ml vs. 505.6±38.6 ng/ml) and their offspring (418.2±26.6 ng/ml vs. 336.3±24.4 ng/ml [p<0.05 for both]) as compared to controls, together with elevated circulating TNF-α and IL-6 levels. Moreover, both GDM placentae (268.1±10.8 ng/ml/mg vs. 187.6±20.6 ng/ml/mg) and adipose explants (41.1±2.7 ng/ml/mg vs. 26.6±2.4 ng/ml/mg) released significantly more sCD163 than controls. Lastly, significantly more CD163 positive cells were observed in GDM placentae (25.7±1.1 vs. 22.1±1.2) and adipose tissue (19.1±1.1 vs 12.7±0.9) compared to controls. We describe elevated sCD163 levels in GDM and identify human placenta as a novel source of sCD163 suggesting that placental tissues might contribute to the increased levels of circulating sCD163 in GDM pregnancies.     

Clinical Significance of Soluble CD26 in Malignant Pleural Mesothelioma

There is no established single diagnostic marker for malignant pleural mesothelioma (MPM). CD26 is a 110 kDa, multifunctional, membrane-bound glycoprotein that has dipeptidyl peptidase IV (DPPIV) enzyme activity. The aim of this study was to evaluate the clinical significance of soluble CD26 (sCD26) in patients with MPM. The study included 80 MPM patients, 79 subjects with past asbestos exposure (SPE), and 134 patients with other benign pleural diseases (OPD) that were included as a control group. sCD26 levels and DPPIV activity in serum and/or pleural fluid were determined using an ELISA kit. Serum sCD26 levels and DPPIV enzyme activity in patients with MPM were significantly decreased compared with those in the SPE group (P = 0.000). The level of serum sCD26 was significantly decreased in patients with advanced stages of MPM compared with those with earlier stages (P = 0.047). The median OS of patients with MPM who had higher DPPIV enzyme activity was significantly longer than that of those with lower DPPIV enzyme activity (P = 0.032). The sCD26 levels in the pleural fluid of MPM patients with an epithelioid subtype were significantly increased compared with the OPD cohort (P = 0.012). Moreover, DPPIV enzyme activity in the pleural fluid of patients with MPM with an epithelioid subtype were significantly increased compared with those in the OPD cohort (P = 0.009). Patients with MPM who had lower specific DPPIV activity, determined as DPPIV/sCD26, showed significantly prolonged survival compared with those with higher specific DPPIV activity (P = 0.028). Serum sCD26 and DPPIV enzyme activity appear to be useful biomarkers for differentiating patients with MPM from SPE. The sCD26 levels or DPPIV enzyme activity in pleural fluid appear to be biomarkers in patients with an epithelioid subtype of MPM. DPPIV activity in serum or pleural fluid appears to be predictive for the prognosis of patients with MPM.     

Soluble CD14 Levels Reflect Liver Inflammation in Patients with Nonalcoholic Steatohepatitis

Background & Aims

Liver inflammation is a risk factor for the progression of nonalcoholic fatty liver disease (NAFLD). However, the diagnosis of liver inflammation is very difficult and invasive liver biopsy is still the only method to reliably detect liver inflammation. We previously reported that overexpression of CD14 in Kupffer cells may trigger the progression to nonalcoholic steatohepatitis (NASH) via liver inflammation following hyper-reactivity to low-dose lipopolysaccharide. Therefore, the aim of this study was to investigate the relationship between soluble type of CD14 (sCD14) and histological features in patients with NAFLD.

Methods

Our cohort consisted of 113 patients with liver biopsy-confirmed NAFLD and 21 age-matched healthy controls. Serum sCD14 levels were measured by an enzyme-linked immunosorbent assay.

Results

Serum sCD14 levels were significantly associated with diagnosis of NASH and the area under the receiver operator characteristic curve (AUROC) to distinguish between not NASH and NASH was 0.802. Moreover, serum sCD14 levels were significantly associated with the disease activity based on NAFLD activity score and hepatic CD14 mRNA expression, which is correlated with membrane CD14 (mCD14) expression, in patients with NAFLD. In multiple regression analysis, the serum sCD14 levels were independently associated with liver inflammation. The AUROC to distinguish between mild and severe liver inflammation in patients with NAFLD was 0.752.

Conclusions

We found that serum sCD14 levels increased significantly with increasing liver inflammation grade in patients with NAFLD, reflecting increased hepatic CD14 expression. Serum sCD14 is a promising tool to predict the worsening of liver inflammation, and may offer a potential biomarker for evaluation of therapeutic effects in NAFLD.     

Development of a Quantitative Bead Capture Assay for Soluble IL-7 Receptor Alpha in Human Plasma

Background

IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Rα (CD127) and common γ (CD132) chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127) is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults.

Methodology/Principal Findings

We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay''s range was 3.2–1000 ng/mL. The concentration of plasma sCD127 was 164±104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year.

Conclusions/Significance

This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases.