Circulating Endothelial Cells are Associated with Thromboembolic Events in Patients with Antiphospholipid Antibodies.

BACKGROUND: Endothelial damage has been described in antiphospholipid antibody (aPL)-positive patients. However, it is uncertain whether circulating endothelial cells (CECs)-which are released when endothelial injury occurs-can be a marker of patients at high risk for thrombosis. METHODS: Ninety-seven patients with aPL and/or systemic lupus erythematosus (SLE) were included. CECs were determined by an automated CellSearch system. We also assayed plasma levels of tissue factor-bearing extracellular vesicles (TF+/EVs) and soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) as markers of endothelial dysfunction/damage. RESULTS: Patients' mean age was 46.1 ± 13.9 years, 77 were women. Thirty-seven had SLE and 75 patients were suffering from antiphospholipid syndrome. Thirty-seven percent of patients presented a medical history of arterial thrombosis and 46% a history of venous thromboembolism (VTE). Thirteen patients had increased levels of CECs (>20/mL), with a mean CEC level of 48.3 ± 21.3 per mL. In univariate analysis, patients with obesity or medical history of myocardial infarction (MI), VTE, or nephropathy had a significant increased CEC level. In multivariate analysis, obesity (odds ratio [OR] = 6.07, 95% confidence interval [CI]: 1.42-25.94), VTE (OR = 7.59 [95% CI: 1.38-41.66]), and MI (OR = 5.5 [95% CI: 1.1-26.6)] were independently and significantly associated with elevated CECs. We also identified significant correlations between CECs and other markers of endothelial dysfunction: sTREM-1 and TF+/EVs. CONCLUSION: This study demonstrated that endothelial injury assessed by the levels of CECs was associated with thromboembolic events in patients with aPL and/or autoimmune diseases.

CellSearch technology applied to the detection and quantification of tumor cells in CSF of patients with lung cancer leptomeningeal metastasis.

OBJECTIVES: The diagnosis of solid cancer leptomeningeal metastasis (LM) relies on the cytology of cerebrospinal fluid (CSF) and/or imaging evidence of neuraxis, yet both lack sufficient sensitivity. The utility of the CellSearch, an FDA -approved technology, in assessing CSF tumor cell (CSFTC) was evaluated here in the diagnosis and treatment of patients with lung cancer-related LM. MATERIALS AND METHODS: In 18 patients with magnetic resonance imaging (MRI) confirmed LM due to lung cancer, 5 mL of CSF were collected in CellSave preservative tubes, which allow performing the assay within 96 h after sampling. Using a previously adapted CellSearch method, we detected, visualized and enumerated CSFTCs and compared the results with conventional cytology. In 3 patients, tumor cells were evaluated sequentially to explore the predictive role of CSFTCs enumeration in the treatment response monitoring. RESULTS: CSFTCs were disclosed in 14 of 18 MRI confirmed LM samples (median 785CSFTCs/5 mL CSF, range 1 to >20,000), yielding a sensitivity of 77.8%, compared with 44.4% for conventional cytology. CSFTC clusters were observed in 12 patients, similar to those previously described in blood as circulating tumor microemboli (CTM), and enumerated sequentially with reproducible results, which did not necessarily correlate with response to treatment. CONCLUSION: The CellSearch technology, applied to limited sample volumes and allowing delayed processing, could be of great interest in the diagnosis of LM in lung cancer patients.

Case Report: Detection and quantification of tumor cells in peripheral blood and ascitic fluid from a metastatic esophageal cancer patient using the CellSearch (®) technology.

Analysis of ascitic fluid should help to identify and characterize malignant cells in gastrointestinal cancer. However, despite a high specificity, the sensitivity of traditional ascitic fluid cytology remains insufficient, at around 60%. Since 2004 the CellSearch (®) technology has shown its advantages in the detection of circulating tumor cells (CTCs) in peripheral blood, which can perform an accurate diagnosis and molecular analysis at the same time. To our knowledge, no previous study has explored the potential utility of this technology for the detection and quantification of tumor cells in ascitic fluid samples. Herein we report a case of metastatic esophageal adenocarcinoma in a 70-year-old man presenting with dysphagia and a large amount of fluid in the peritoneal cavity. Analysis of a peripheral blood sample and ascites sample with the CellSearch (®) technology both revealed the presence of putative tumor cells that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. This study confirmed the hematogenous dissemination of esophageal cancer by the detection of circulating tumor cells in the peripheral blood, and is the first to demonstrate that tumor cells can be identified in ascitic fluid by using CellSearch (®) technology.

Value of HIV patients with regular follow-up as in-house internal controls of flow cytometry measurement of lymphocyte subsets.

BACKGROUND: Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. METHODS: Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells, and CD4+ absolute counts (ACs). RESULTS: Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08, and 0.18 for CD3%, CD4%, CD8%, and CD4 ACs, respectively. DISCUSSION: In-house follow-up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 International Clinical Cytometry Society.

Value of HIV patients with regular follow-up as in-house internal controls of flow cytometry measurement of lymphocyte subsets.

Background. Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. Methods. Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells and CD4+ absolute counts. Results. Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08 and 0.18 for CD3%, CD4% CD8% and CD4 absolute counts respectively. Discussion. In-house follow up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 Clinical Cytometry Society.

Detection and quantification of CSF malignant cells by the CellSearch technology in patients with melanoma leptomeningeal metastasis.

Melanoma is the most frequent solid tumor associated with leptomeningeal metastasis (LM). The usual diagnostic tools, that is, cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium-enhanced MRI of the entire neuraxis both lack effectiveness. The CellSearch Veridex technology for the detection of circulating tumor cells (CTC) in blood was designed for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer, which express EpCAM markers. We have previously adapted this technology to detect malignant cells in the CSF of breast cancer LM. Our objective here was to check if this technology would also allow the detection and the enumeration of CTC in the CSF of melanoma patients presenting with LM although melanoma does not express EpCAM markers. On the occasion of the intrathecal treatment of LM in 2 melanoma patients, 5 mL of CSF and 7.5 mL of blood were collected on CellSave Preservative Tubes and analyzed within 3 days after CSF sampling using a melanoma-dedicated kit. The CellSearch Veridex technology was then adapted to direct enrichment, enumeration, and visualization of melanoma cells in the CSF. CD146+, HMW-MAA+, CD34-, and CD45- cells with typical morphology could be observed and enumerated sequentially with reproducible results, corresponding to CSF melanoma cells (CSFMC). In contrast to the current gold standard cytomorphological analysis, this new approach allowed a precise quantification of CSFMC in all samples concomitantly analyzed. This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of melanoma patients with LM.

Development of a new method for identification and quantification in cerebrospinal fluid of malignant cells from breast carcinoma leptomeningeal metastasis.

BACKGROUND: The diagnosis of leptomeningeal metastasis (LM) in patients with solid tumors remains difficult. The usual diagnostic methods of cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium enhanced MRI of the entire neuraxis lack both specificity and sensitivity. The Veridex CellSearch® technology has been designed for the detection of circulating tumor cells (CTC) in blood from cancer patients and validated for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer. Our aim was to adapt this technology for the detection and the enumeration of tumor cells in the CSF of breast cancer patients presenting with LM. METHODS: On the occasion of a randomized phase III study evaluating the role of the intrathecal treatment in LM from breast cancer (DEPOSEIN, EudraCT N°: 2010-023134-23), the CellSearch® technology was adapted to direct enrichment, enumeration and visualization of tumor cells in 5 mL CSF samples, collected on CellSave® Preservative Tubes and analyzed within 3 days after CSF sampling. RESULTS: Sixteen CSF of 8 patients with primary breast cancer presenting with LM were studied. EpCAM+/cytokeratin + cells with typical morphology could be observed and enumerated sequentially with reproducible results in low or elevated numbers in 8 patients. CONCLUSION: This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of cancer patients with LM, especially to appreciate the efficacy of chemotherapy.

Serum sTREM-1 (soluble triggering receptor expressed on myeloid cells-1) associates negatively with embryo quality in infertility patients.

PROBLEM: Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is a useful biomarker of infection and inflammation. METHOD OF STUDY: We studied serum and follicular fluid sTREM-1 in infertile patients (N = 110) utilizing enzyme-linked immunosorbent assay. RESULTS: Serum and follicular sTREM-1 were in good correlation (Pearson's correlation 0.56, P < 0.0001) with higher values in follicular fluid (140.4 ± 34.4 and 115.6 ± 35.1 pg/mL, t-test, P < 0.0001). Endometriosis associated with lower follicular and serum sTREM-1 compared with male factor infertility patients (age-adjusted r = -25.7 pg/mL, P = 0.018; r = -22.1 pg/mL, P = 0.030). No associations between follicular or serum sTREM-1 and clinical parameters were found, except higher serum sTREM-1 associated with lower embryo quality in all patients (adjusted r = -0.3%, P = 0.033), with a cutoff value between 111.5 and 113.3 pg/mL (OR = 0.38, P = 0.048; OR = 0.34, P = 0.028) predicting that more than 39% of embryos would be with good quality. CONCLUSION: Serum sTREM-1 could represent a prognostic marker for female fecundity, probably indicating impaired inflammatory reaction of immune system.

Follicular proinflammatory cytokines and chemokines as markers of IVF success.

Cytokines are key modulators of the immune system and also contribute to regulation of the ovarian cycle. In this study, Bender MedSystems FlowCytomix technology was used to analyze follicular cytokines (proinflammatory: IL-1ß, IL-6, IL-18, IFN-γ, IFN-α, TNF-α, IL-12, and IL-23;, and anti-inflammatory: G-CSF), chemokines (MIP-1α, MIP-1ß, MCP-1, RANTES, and IL-8), and other biomarkers (sAPO-1/Fas, CD44(v6)) in 153 women undergoing in vitro fertilization (IVF). Cytokine origin was studied by mRNA analysis of granulosa cells. Higher follicular MIP-1α and CD44(v6) were found to correlate with polycystic ovary syndrome, IL-23, INF-γ, and TNF-α with endometriosis, higher CD44(v6) but lower IL-ß and INF-α correlated with tubal factor infertility, and lower levels of IL-18 and CD44(v6) characterized unexplained infertility. IL-12 positively correlated with oocyte fertilization and embryo development, while increased IL-18, IL-8, and MIP-1ß were associated with successful IVF-induced pregnancy.

Mutual benefits of B-ALL and HLDA/HCDM HLDA 9th Barcelona 2010.

The B-cell panel of the ninth HLDA was applied in a multicentre fashion to cryopreserved cells from 46 patients with acute lymphoblastic leukemia. The reagents were aliquoted and shipped to volunteer participants from the French Groupe d'Etude Immunologique des Leucémies (GEIL). All samples were tested in flow cytometry, and the results collected as of the strength of labeling of the leukemic clone as negative, weak or strong. Among the 64 antibodies tested, the strongest and most frequent staining was observed for CD305 (LAIR), CD229 (Ly9), CD200 (OX-2) and, to a lesser extent, CD361 (EVI2b). Details of the observations, and information about the molecules tested are provided in the manuscript as well as a summary table.

Position statement: Assessment strategy for implementation of the Immunology curriculum of the European Board of UEMS Medical Biopathology.

The assessment strategy for implementation of the Union of European Medical Specialists' (UEMS) Immunology curriculum is based on a combination of formative and summative assessments. The strategy comprises a combination of workplace-based assessments and knowledge-based assessments designed to ensure acquisition of key learning outcomes as defined in the curriculum. The purpose of this paper is to explicitly link the assessment strategy to the curriculum.

Local lymphocytic and epithelial activation in a case of autoimmune oophoritis.

OBJECTIVE: To further define the immunological tissular modifications in premature ovarian failure (POF). METHOD: The patient was followed up for premature ovarian failure and mild endometriosis associated with serum antiovarian antibodies. A laparoscopic ovarian biopsy was decided on to analyze the tissue and support the onset of immunosuppressive therapy. Immunohistochemistry was performed using monoclonal antibodies directed against T cell membrane markers, as well as activation molecules, to define the composition of the cellular infiltrate and the consequences on ovarian tissue. RESULT(S): A dense infiltration of activated T lymphocytes was observed in close contact with follicular epithelium expressing HLA-DR and CD40. CONCLUSION(S): This observation supports the role of cellular immunity in ovarian autoimmunity with features very similar to those reported in murine models and other human autoimmune endocrine pathologies.

Expression of functional toll-like receptors by B-chronic lymphocytic leukemia cells.

This study reports that B-chronic lymphocytic leukemia (B-CLL) cells display the same pattern of toll-like receptors (TLRs) proteins expression as normal B-cells, yet with overexpression of TLR9. Furthermore, TLR7 and TLR9 appear to be functional and liable to respond to specific ligands, respectively imidazoquinolines and CpG-ODN thus potentially opening new therapeutic approaches.

Modulation of the triggering receptor expressed on myeloid cells-1 pathway during pneumonia in rats.

BACKGROUND: Triggering receptor expressed on myeloid cells-1 (TREM-1) is a cell-surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. The aim of the present study was to investigate the effect of the modulation of the TREM-1 pathway during experimental pneumonia in rats. METHODS: Adult male Wistar rats were intratracheally inoculated with Pseudomonas aeruginosa (PAO1 strain) and randomly treated or not treated with an analogue synthetic peptide derived from the extracellular moiety of TREM-1 (LP17). RESULTS: P. aeruginosa induced a severe pneumonia associated with signs of severe sepsis within the first 24 h. In septic rats, LP17 improved hemodynamic status, attenuated the development of lactic acidosis and hypoxemia, modulated lung and systemic inflammatory responses and coagulation activation, reduced lung histological damage, and improved survival. CONCLUSIONS: The modulation of the TREM-1 pathway by the use of such synthetic peptides as LP17 appears beneficial during P. aeruginosa pneumonia in rats in attenuating lung and systemic inflammatory responses.

Corticosteroids in patients with antiovarian antibodies undergoing in vitro fertilization: a prospective pilot study.

OBJECTIVE: Antiovarian autoantibodies (AOA) have been associated with reproductive failure, especially in in vitro fertilization (IVF) patients. Thus, the success rate of IVF might be improved by the use of corticosteroids. However, therapeutic trials with these drugs have yielded conflicting results, particularly because of heterogeneous inclusion criteria. Among women with previous IVF failure, we selected those who presented with a positive serum AOA assay, and analysed the efficacy of corticosteroids in improving the IVF outcome in these patients. METHODS: One hundred patients with serum AOA detected by ELISA and at least two previously failed IVF attempts were selected. These patients underwent a further IVF cycle with 0.5 mg/kg prednisolone, started on the first day of the treatment cycle. In patients who became pregnant, corticosteroids were administered until the end of the first trimester of pregnancy and then progressively discontinued. AOA were assessed before and after oocyte retrieval. Clinical data of the corticosteroid-treated cycle were compared with data from the preceding IVF cycle for each patient. RESULTS: No adverse effects resulting from corticosteroids were observed. Post oocyte retrieval antiovarian IgG were significantly lower in corticosteroid-treated attempts when compared with the preceding cycles. Twenty-six pregnancies resulted in the birth of 30 healthy children. The pregnancy rate, implantation rate, and live birth rate were 38.8%, 17.8%, and 26.5% respectively in prednisolone-treated cycles. CONCLUSION: This study confirms the usefulness of corticosteroids in improving the success rate in a subset of patients with previous IVF failure and significant serum AOA levels.

Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock.

The triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. To investigate the effect of the modulation of the TREM-1 pathway during experimental murine sepsis, we used analogue synthetic peptides derived from the extracellular moiety of TREM-1. The TREM-1 ligand was expressed on both peritoneal and peripheral neutrophils during experimental peritonitis in mice. The TREM-1 peptides inhibited the recognition by TREM-1 of its ligand and protected endotoxinic mice from death. In septic rats, the TREM-1 peptides improved the hemodynamic status, attenuated the development of lactic acidosis, modulated the production of such proinflammatory cytokines as tumor necrosis factor alpha and interleukin-1beta, and improved survival. The protective effect of these peptides on arterial pressure could partly be explained by a decreased production of nitric oxide. These data suggest that in vivo modulation of TREM-1 might be a suitable therapeutic tool for the treatment of sepsis.

Modulation of natural killer (NK) receptors on NK (CD3-/CD56+), T (CD3+/CD56-) and NKT-like (CD3+/CD56+) cells after heart transplantation.

BACKGROUND: After undergoing heart transplantation and the subsequent compulsive immunosuppressive treatments, patients are at risk of rejection episodes, infectious complications or cancer development. Thus, it is probable that the various subsets of peripheral cytotoxic lymphocytes are modulated in such patients. This area of study can now be investigated by examining the numerous recently described natural killer (NK)-cell-related surface receptors. METHODS: A prospective cohort of 60 heart transplant recipients and 60 controls was studied. The partitioning of lymphocyte subsets, especially NK (CD3-/CD56+), T (CD3+/CD56-) and NKT-like (CD3+/CD56+) cells, was compared in both groups using multi-parametric flow cytometry. Moreover, expression of a series of seven NK-related receptors was compared on the three subsets defined by CD56 expression. RESULTS: A significant increase in NK-cell levels was observed in transplanted patients, as compared with controls, whereas T and NKT-like cells were in similar proportions in both groups. Two NK-related receptors showed significantly different levels of expression in heart transplant recipients: the cytotoxic effector, CD244, which was in a significantly increased proportion on T and NKT-like cells; and the activating receptor, CD161, which was expressed significantly less on NK and NKT-like cells, but more on T cells. CONCLUSIONS: These findings indicate that cytotoxic NK-related cells, increased in proportion, also display increased levels of activity-associated markers in heart transplant recipients. Viral infection or the immunosuppressive regimen could be responsible for the modulation of regulatory receptors on NK and NKT-like cells in heart transplant recipients.

Measurement of immunoglobulins G, A, and M levels in B-lymphocytes culture.

Nephelometric immunoassays were developed for human IgG, IgA, and IgM quantitation in B-lymphocytes culture media. They allowed measurement of immunoglobulin (Ig) levels over a broad range of concentrations with good accuracy and precision. The kinetics of Ig production in B-lymphocyte cultures was followed and the mean amount of each Ig was determined in six different samples after three days of culture. The nephelometric immunoassays reported here could be used to study, in vitro, the influence of various molecules (inhibitory or amplifying effect) on B-lymphocytes' functional capacities.

Surface and soluble triggering receptor expressed on myeloid cells-1: expression patterns in murine sepsis.

OBJECTIVE: To examine the expression patterns of the triggering receptor expressed on myeloid cells (TREM)-1 during experimental septic shock. DESIGN: Animal study. SETTING: Animal research laboratory. SUBJECTS: Male BALB/c mice, 7-9 wks of age. INTERVENTIONS: Septic shock was induced by cecal ligation and puncture in eight mice. Eight additional animals were sham-operated and served as a control group. All animals were resuscitated by fluid infusion and administered antibiotics. Kill was performed under anesthesia 12, 24, or 48 hrs later. MEASUREMENTS AND MAIN RESULTS: Surface expression of TREM-1 was analyzed using flow cytometry on peripheral blood cells, peritoneal macrophages and neutrophils, splenic macrophages, and Kupffer cells. Gene expression was also studied in these same cells using reverse transcription-polymerase chain reaction. Tumor necrosis factor-alpha, interleukin-1beta, and soluble TREM-1 concentrations were determined in plasma and peritoneal lavage fluid. Sepsis strongly induced TREM-1 gene expression, which translated into an up-regulation of TREM-1 surface expression on neutrophils and monocytes/macrophages both at the focus on infection as well as distally. Moreover, sepsis induced the release of significant levels of soluble TREM-1. Plasma soluble TREM-1 concentrations negatively correlated with tumor necrosis factor-alpha and interleukin-1beta levels at 12 hrs. CONCLUSIONS: These results provide new information as to the regulation of TREM-1 during sepsis. Considering that both cell-surface and soluble TREM-1 were strongly up-regulated during infection, this study may add support to the putative usefulness of TREM-1 as a diagnostic tool.