BACKGROUND:Shifts in CD8+ T-cell subsets that are hallmarks of immunosenescence are observed in ageing and in conditions of chronic immune stimulation. Presently, there is limited documentation of such changes in lung cancer and other malignancies affecting the lungs.METHODS:Changes in CD8+ T-cell subsets, based on the expression of CD28 and CD57, were analysed in patients with various forms of cancer affecting the lungs, undergoing chemotherapy and in a control group over six months, using multi-colour flow cytometry.RESULTS:The differences between patients and controls, and the changes in the frequency of CD8+ T-cell subpopulations among lung cancer patients corresponded to those seen in immunosenescence: lower CD8-/CD8+ ratio, lower proportions of CD28+CD57- cells consisting of naïve and central memory cells, and higher proportions of senescent-enriched CD28-CD57+ cells among the lung cancer patients, with the stage IV lung cancer patients showing the most pronounced changes. Also observed was a tendency of chemotherapy to induce the formation of CD28+CD57+ cells, which, in line with the capacity of chemotherapy to induce the formation of senescent cells, might provide more evidence supporting CD28+CD57+ cells as senescent cells.CONCLUSION:Immunosenescence was present before the start of the treatment; it appeared to be pronounced in patients with advanced cases of malignancies affecting the lungs, and might not be averted by chemotherapy.

Background: During organismal aging, human T-cells shift towards less functional phenotypes, often called senescent cells. As these cells have not been well characterized, we aimed to relate surface markers of human T-cell senescence with characteristics of in vitro cellular aging and to further characterize these cells.Methods: We identified, by flow cytometry, subpopulations of CD8+ T-cells based on CD57 and CD28 expression, and tested them for some markers of cellular senescence, apoptosis, differentiation and homing.Results: Elderly persons presented significantly higher proportions not only of CD28-CD57+, but also of CD28+CD57+ cells. CD28+CD57+ cells had the highest expression of p16, p21, Bcl-2, CD95, CD45RO, CCR5 and PD-1, thereby arguing in favor of a senescent phenotype.Conclusion: Among CD8+ T-lymphocytes, CD28+ CD57+ cells represent a subset with some senescent features that are distinct from the CD28 -CD57+ cells. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Objective: To investigate early changes in leukocyte subsets and autonomic function as predictors of the development of poststroke infections.Methods: We assessed the time course of leukocyte subsets in the blood of 59 patients with acute ischemic stroke. We divided the patients into 2 groups: those who developed infections during the first 7 days after stroke onset and those who did not. We measured urinary norepinephrine and epinephrine concentrations and pulse rate variability indices within 24 hours of admission.Results: We found that the number of circulating natural killer (NK) cells within the first hours after stroke was higher in stroke patients who developed infections (mean 435 cells/mL; 95% confidence interval [CI] 321-588) than in stroke patients who did not develop infections (mean 236 cells/mL; 95% CI 186-300; p = 0.001). This was followed by a decrease in all lymphocyte subsets from admission to day 1, varying between 22% and 40%, which was not seen in patients without poststroke infection (mean increase varied between 2% and 23%; all p < 0.005). In the group that developed infections, pulse rate variability revealed a decreased high frequency component. These findings all remained significant after adjustment for age and stroke volume.Conclusions: High circulating NK cell count within the first hours after ischemic stroke onset followed by a drop in all lymphocyte subsets identified patients who developed infections and may be caused by a sympathovagal imbalance with sympathetic overweight. These findings need to be validated in larger studies.

Background: Changes in sub-populations of cytotoxic (CD8(+)) T-cells, which are observed in aging and in conditions of chronic immune stimulation, are not well-documented in cancer. Materials and Methods: Using flow cytometry, CD8(+) T-cell subsets were analyzed in patients with breast cancer undergoing DNA-damaging chemotherapy and in an older female control group during a six-month longitudinal study, to explore shifts in CD8(+) T-cells and the effect of DNA-damaging chemotherapy on different T-cell sub-populations. Results: As expected, there was a consistent decrease in absolute numbers of leukocytes, lymphocytes, T-cells and CD8(+) T-cells during chemotherapy in patients with cancer. Among the T-cells, there was a lower CD8(-)/CD8(+) ratio, persisting over the six months, in patients with cancer compared to controls. The proportion of CD28(-)CD57(+) cells also remained higher among patients with cancer throughout the sampling duration. The number of CD28(+)CD57(-) and CD28(-)CD5(-) cells decreased faster during DNA-damaging chemotherapy than CD28(+)CD57(+) and CD28(-)CD57(+) cells, while only CD28(-)CD57(-) cells showed a significant reconstitutive capacity after six months. Conclusion: Immunosenescence appeared to be pronounced in patients with breast cancer, with senescent CD8(+) T-cells playing a role. The normal condition was not restored after six months of chemotherapy.

Immunomodulators have been used in recent years to reactivate host anti-tumor immunity in several hematological malignancies. This report describes the effect of activating natural killer T (NKT) cells by α-Galactosylceramide (α-GalCer) in the 5T33MM model of multiple myeloma (MM). NKT cells are T lymphocytes, co-expressing T and NK receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain. We followed iNKT numbers during the development of the disease in both 5T33MM mice and MM patients and found that their numbers dropped dramatically at the end stage of the disease, leading to a loss of total IFN-γ secretion. We furthermore observed that α-GalCer treatment significantly increased the survival of 5T33MM diseased mice. Taken together, our data demonstrate for the first time the possibility of using a preclinical murine MM model to study the effects of α-GalCer and show promising results of α-GalCer treatment in a low tumor burden setting.

As a rule, myelodysplastic syndromes (MDS) are accompanied by peripheral cytopenia in one or more cell lineages [1]. Platelet function defects (PFDs) are frequently seen in MDS, but usually only cause mild bleeding symptoms [2,3]. We report for the fi rst time a case of MDS that manifested as a clinically severe and initially unexplained PFD with completely normal blood counts. Th e diagnosis of MDS was made retrospectively, after the patient had gradually progressed toward refractory thrombocytopenia with minimal dysplastic features, and fi nally acute myeloid leukemia (AML). Th is is a case of a 56-year-old man who presented with a 6-year history of recurrent rectal blood loss for which he was admitted to our hospital on several occasions. Multiple colonoscopies and gastroscopies could fi nd no clear cause aside from the presence of internal hemorrhoids, for which he was surgically treated. Th is intervention, however, had very little impact on his symptoms. Th e patient reported a recently developed bleeding trend, presenting as recurrent epistaxis, for which cauterization was needed on two occasions, as well as a tendency to develop cutaneous purpura. Th ere was no family history of a bleeding disorder. In between hemorrhagic episodes, complete blood counts and clotting studies, including von Willebrand factor antigen and activity tests, were unremarkable. Kidney and functional liver tests as well as serum protein electrophoresis revealed no abnormalities. Th ere were no signs of increased fi brinolysis (D-dimers 476 μ g/L). Platelet light transmission aggregometry was disturbed with all tested agonists: the aggregation response to adenosine diphosphate, collagen and ristocetin was 35%, 47% and 60%, respectively; there was a strikingly absent response to arachidonic acid. A possible impact of medication was excluded by repeated analysis under controlled circumstances. Due to the lack of any bleeding diathesis in the patient ’ s history, a genetic defect was unlikely. Any plausible explanation for the fi ndings could not be found at that point, and hence the patient was treated symptomatically with frequent erythrocyte transfusions and tranexamic acid, having very little impact on the bleeding frequency. Six years after the first symptoms appeared, the patient developed an isolated thrombocytopenia: hemoglobin concentration 13.4 g/dL, red cell count 4.6 10 12 /L, mean corpuscular volume 90.7 fL, platelet count 95 10 9 /L, white cell count 11.6 10 9 /L. A blood film showed the presence of giant platelets. Hemorrhagic incidents became more common: over a period of 2 years the patient was hospitalized five times because of massive rectorrhagia for which transfusion of red blood cells was frequently mandatory. Endoscopic and colonoscopic investigations were again unremarkable except for the sporadic detection of small arteriovascular malformations. These lesions were difficult to observe because of persistent bleeding and blood clots in the gastrointestinal tract. Both angiography and Meckel scan were negative. Thrombocytopenia and the appearance of large platelets prompted the diagnosis of idiopathic thrombocytopenic purpura (ITP). A bone marrow aspirate revealed an increased number of megakaryocytes, lacking any other significant abnormalities. This finding supported the diagnosis of ITP. The patient was treated with corticosteroids, inducing a modest but only temporary response of platelet count and bleeding. Tranexamic acid was able to stop the blood loss for a brief period, but the patient quickly relapsed despite increasing the dosage. Ultimately, donor platelets were administered at a regular interval, with a good impact on the symptoms. Th is patient ’ s case was put up for reevaluation when cytogenetic analysis of the bone marrow revealed the presence of trisomy 21 as the sole karyotypic abnormality, present in 15 of 20 analyzed metaphases. Th is very rare acquired chromosomal aberration has sporadically been

Event Abstract Back to Event Senescene associated cells in human CD8+ T-lymphocytes and their homing characteristics Oscar O. Onyema1*, Rose Njemini1, Louis N. Forti1, Ivan Bautmans1, Joeri L. Aerts2, Marc De Waele3 and Tony Mets1, 4 1 Vrije Universiteit Brussel, Gerontology Department, Belgium 2 Vrije Universiteit Brussel, Immunology and Physiology Department, Belgium 3 Universitair Ziekenhuis Brussel, Hematology Department, Belgium 4 Universitair Ziekenhuis Brussel, Geriatrics Department, Belgium The expression of CD57 and the absence of CD28 on T-lymphocytes have been used as "senescence" markers. The association of these markers with characteristics of senescence that emerge during in vitro cellular aging is, however, not clear. Using flow cytometry, CD8+ T-cells were analyzed in old and young human subjects for their expression of CD57 and CD28. The subpopulations were tested for various markers of cellular senescence, apoptosis, homing and differentiation, and for their proliferation capacity. Significantly higher proportions of CD28+CD57+ and CD28-CD57+ cells were found in old subjects. CD28+CD57+ cells lacked proliferation. They also had the highest intensity of expression of the senescence markers: p16 and p21, the pro-survival protein: Bcl-2, and the apoptosis and survival regulator: CD95, and the highest frequency of Bcl-2 and CD95 expressing cells, favouring a likely role of these proteins in promoting senescence in CD28+CD57+ cells despite their significantly higher proportion of cells positive for the apoptosis indicator, Annexin-V, compared with other subpopulations. The frequency of CD45RO and CCR5 expressing cells was also highest in CD28+CD57+ cells. CD45RO, CCR5 and CD95 are highly expressed by cell populations believed to be dominated by senescent cells. CD28-CD57+ cells also showed appreciable senescence behaviour; however, their heterogeneity emphasized in this study, and our observation of a subpopulation of CXCR2+ cells among them indicates the possibility of a subpopulation of CD28-CD57+ cells that might represent a more pronounced senescent phenotype. Although there are evidences from our study that different cell phenotypes might represent senescent cells from different T cell homing environments shown by their level of expression of CD62L and CCR7, we conclude that among CD8+ T-lymphocytes, characteristics that correspond to a senescent cell type are most prominent in CD28+CD57+ cells, thus providing further evidence for the existence of senescent cells in vivo and their possible importance in aging. Acknowledgements The study was supported by a scientific grant from the "Wetenschappelijk Fonds Willy Gepts" from the Universitair Ziekenhuis Brussel. References 1. Acosta JC, O'Loghlen A, Banito A, Guijarro MV, Augert A, Raguz S, Fumagalli M, Da Costa M, Brown C, Popov N, Takatsu Y, Melamed J, d'Adda di Fagagna F, Bernard D, Hernando E , Gil J (2008). Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell. 133, 1006-1018. 2. Appay V, van Lier RA, Sallusto F , Roederer M (2008). Phenotype and function of human T lymphocyte subsets: consensus and issues. Cytometry A. 73, 975-983. 3. Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ, Crotty LE, Casazza JP, Kuruppu J, Migueles SA, Connors M, Roederer M, Douek DC , Koup RA (2003). Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells. Blood. 101, 2711-2720. 4. Campisi J , d'Adda di Fagagna F (2007). Cellular senescence: when bad things happen to good cells. Nature reviews. Molecular cell biology. 8, 729-740. 5. Champagne P, Ogg GS, King AS, Knabenhans C, Ellefsen K, Nobile M, Appay V, Rizzardi GP, Fleury S, Lipp M, Forster R, Rowland-Jones S, Sekaly RP, McMichael AJ , Pantaleo G (2001). Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature. 410, 106-111. 6. Jeyapalan JC, Ferreira M, Sedivy JM , Herbig U (2007). Accumulation of senescent cells in mitotic tissue of aging primates. Mechanisms of ageing and development. 128, 36-44. Keywords: cellular senescence, p16, p21, CD28, CD57, CXCR2 Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Onyema OO, Njemini R, Forti LN, Bautmans I, Aerts JL, De Waele M and Mets T (2013). Senescene associated cells in human CD8+ T-lymphocytes and their homing characteristics. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00973 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Mr. Oscar O Onyema, Vrije Universiteit Brussel, Gerontology Department, Brussels, 1090, Belgium, oonyema@vub.ac.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Oscar O Onyema Rose Njemini Louis N Forti Ivan Bautmans Joeri L Aerts Marc De Waele Tony Mets Google Oscar O Onyema Rose Njemini Louis N Forti Ivan Bautmans Joeri L Aerts Marc De Waele Tony Mets Google Scholar Oscar O Onyema Rose Njemini Louis N Forti Ivan Bautmans Joeri L Aerts Marc De Waele Tony Mets PubMed Oscar O Onyema Rose Njemini Louis N Forti Ivan Bautmans Joeri L Aerts Marc De Waele Tony Mets Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

CD28-, CD57+ and KLRG1+ are cell surface markers that have been used to describe senescent T-lymphocytes in humans. However, the relationship among these phenotypes during aging, and their relationship with the concept of in vitro cellular aging have not been well established. Using five-colour flow cytometry, we analyzed peripheral blood T-lymphocytes for their expression of CD28, CD57 and KLRG1 in 11 young (Y) and 11 old (O) apparently healthy human subjects. The proportions of CD28- and CD57+ cells were significantly higher among the T-cell populations of O compared to Y subjects; the proportion of KLRG1+ cells was significantly higher only among CD8+ cells. Populations that were more frequent in the elderly participants were characterised as CD28+ CD57+, CD28- CD57+ or CD28- CD57-. The expression of p16 and p21, considered as markers for in vitro senescence, was higher in CD28+ CD57+ cells than in other subpopulations in both age groups. The expression of p21 was age-related, which was not the case for p16. Thus, although both p16 and p21 are involved in T-cell senescence, they appear to behave differently. CMV infection and shifts in subpopulations are unlikely as explanations of the observed differences. Their higher levels of p16 and p21 expression, coupled with their higher prevalence in the elderly participants make CD28+ CD57+ cells the subpopulation of T-cells most closely corresponding to the concept of senescent cells.

The World Health Organization introduced flow cytometry as an additional criterion for diagnosis of myelodysplastic syndromes (MDS). Aberrant antigen expression on bone marrow (BM) blasts may identify "low-grade MDS." This study aimed to examine differences in antigen expression on CD34+ BM cells between patients with MDS and those with secondary cytopenia. BM aspirates of 175 patients with cytopenia were classified as MDS or secondary cytopenia. Expression of stem cell antigens (CD34, CD133), myeloid antigens (CD13, CD33), B-cell antigens (CD19, CD10), growth factor receptors (CD117, CD123), and chemokine receptor (CD184) was examined. Thirty-two normal adults and 49 patients with CD34+ acute myeloid leukemia (AML) were also examined. High percentage of CD34+ cells, CD117 and CD123 overexpression, and abnormal CD45 expression on these cells are the best markers for MDS. These phenotypic aberrancies correlate with number of blasts and degree of dysplasia, and were similar to those in CD34+ AML, thus reflecting the relationship between these disorders.

Young children with persistent wheezing pose a diagnostic and therapeutical challenge to the pediatrician.

Article Reference values for new red blood cell and platelet parameters on the Abbott Diagnostics Cell-Dyn Sapphire was published on May 1, 2012 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 50, issue 5).

Human leukocyte Ag-G, a tolerogenic molecule that acts on cells of both innate and adaptive immunity, plays an important role in tumor progression, transplantation, placentation, as well as the protection of the allogeneic fetus from the maternal immune system. We investigated HLA-G mRNA and protein expression in human embryonic stem cells (hESC) derived from the inner cell mass (ICM) of blastocysts. hESC self-renew indefinitely in culture while maintaining pluripotency, providing an unlimited source of cells for therapy. HLA-G mRNA was present in early and late passage hESC, as assessed by real time RT-PCR. Protein expression was demonstrated by flow cytometry, immunocytochemistry, and ELISA on an hESC extract. Binding of HLA-G with its ILT2 receptor demonstrated the functional active status. To verify this finding in a physiologically relevant setting, HLA-G protein expression was investigated during preimplantation development. We demonstrated HLA-G protein expression in oocytes, cleavage stage embryos, and blastocysts, where we find it in trophectoderms but also in ICM cells. During blastocyst development, a downregulation of HLA-G in the ICM cells was present. This data might be important for cell therapy and transplantation because undifferentiated hESC can contaminate the transplant of differentiated stem cells and develop into malignant cancer cells.

Laboratory tests for pulmonary sarcoidosis (percentage lymphocytes and CD4/CD8 ratio in bronchoalveolar lavage fluid and serum angiotensin-converting enzyme activity) lack sensitivity and specificity. In a retrospective study of 153 subjects under suspicion of pulmonary sarcoidosis (36 cases and 117 patients with other diseases [control patients]), we defined likelihood ratios (LRs) for rationally selected result intervals of these tests, which improve clinical interpretation as compared with dichotomous interpretation based on a single cutoff value. By using logistic regression analysis, we further integrated the 3 individual tests into a unified algorithm that could rule out diagnosis in 57 (48.7%) of the 177 control subjects and confirm diagnosis in 12 (33%) of the 36 pulmonary sarcoidosis cases. We conclude that use of LRs improves interpretation of laboratory tests for pulmonary sarcoidosis. In addition, we present a prediction algorithm based on the combination of laboratory tests that helps clinicians confirm or exclude diagnosis in almost half of the study population.

The enumeration and identification of blood cells in body fluids offers important information for the diagnosis and treatment of various medical conditions. Manual microscopic methods (hemacytometer total cell count and cytocentrifuged differential count) have inherent analytic and economic disadvantages but are still considered the "gold standard" methods. We evaluated the analytic and clinical performance of the Cell-Dyn Sapphire hematology analyzer (Abbott Diagnostics Division, Santa Clara, CA) for automated blood cell counting and leukocyte differential counting in cerebrospinal fluid, serous fluid (peritoneal and pleural fluid), and continuous ambulatory peritoneal dialysis fluid, and we compared the performance with the respective manual methods. In the present article, we describe its applicability for the distinct body fluids, and we highlight limitations and caveats.

OBJECTIVE The metabolic outcome of islet cell transplants in type 1 diabetic patients is variable. This retrospective analysis examines whether differences in recipient characteristics at the time of transplantation are correlated with inadequate graft function. RESEARCH DESIGN AND METHODS Thirty nonuremic C-peptide–negative type 1 diabetic patients had received an intraportal islet cell graft of comparable size under an ATG-tacrolimus–mycophenolate mofetil regimen. Baseline patient characteristics were compared with outcome parameters during the first 6 posttransplant months (i.e., plasma C-peptide, glycemic variability, and gain of insulin independence). Correlations in univariate analysis were further examined in a multivariate model. RESULTS Patients that did not become insulin independent exhibited significantly higher counts of B-cells as well as a T-cell autoreactivity against insulinoma-associated protein 2 (IA2) and/or GAD. In one of them, a liver biopsy during posttransplant year 2 showed B-cell accumulations near insulin-positive β-cell aggregates. Higher baseline total lymphocytes and T-cell autoreactivity were also correlated with lower plasma C-peptide levels and higher glycemic variability. CONCLUSIONS Higher total and B-cell counts and presence of T-cell autoreactivity at baseline are independently associated with lower graft function in type 1 diabetic patients receiving intraportal islet cells under ATG-tacrolimus–mycophenolate mofetil therapy. Prospective studies are needed to assess whether control of these characteristics can help increase the function of islet cell grafts during the first year posttransplantation.

Validation of clinical laboratory reports is the final step before transmission to the clinician, whereby test results are checked for intrinsic consistency and compatibility with the information available on the patient. Validation can be carried out manually by laboratory professionals or assisted by an automated system, which is currently the case in many laboratories (1, 2). The primary aim of validation is to prevent reporting of analytically erroneous test results. Furthermore, it can deliver a valuable contribution to the medical interpretation of deviant results, thereby reinforcing the advising role towards clinicians, and conceding to recent proposals of medical consultancy as a key competency of laboratory medicine (3, 4). We evaluated the performance of the automated validation system VALAB (Validation Assistee pour LABoratoire, Instrumentation Laboratory, Barcelona, Spain) and of our laboratory professionals in validating routine haematological test results. All laboratory reports (ns591) of 1 day containing haematological test results and other patient information (such as, age, sex, urgency of request, ambulatory or hospitalised, medical specialty of the prescribing physician, previous results) were submitted for validation to VALAB and to a group of laboratory professionals (ns7). VALAB and the laboratory professionals had to decide independently which reports needed a more detailed revision and/or some type of action (such as retesting or a call to the clinician) and should, for that reason, not be validated immediately. All decisions were registered. After individual decisions, the laboratory professionals obtained a consensus on which reports should be retained (inconsistent reports) and which reports