Determining quantitative immunophenotypes and evaluating their implications

NASA Astrophysics Data System (ADS)

Redelman, Douglas; Hudig, Dorothy; Berner, Dave; Castell, Linda M.; Roberts, Don; Ensign, Wayne

2002-05-01

Quantitative immunophenotypes varied widely among > 100 healthy young males but were maintained at characteristic levels within individuals. The initial results (SPIE Proceedings 4260:226) that examined cell numbers and the quantitative expression of adhesion and lineage-specific molecules, e.g., CD2 and CD14, have now been confirmed and extended to include the quantitative expression of inducible molecules such as HLA-DR and perforin (Pf). Some properties, such as the ratio of T helper (Th) to T cytotoxic/suppressor (Tc/s) cells, are known to be genetically determined. Other properties, e.g., the T:B cell ratio, the amount of CD19 per B cell, etc., behaved similarly and may also be inherited traits. Since some patterns observed in these healthy individuals resembled those found in pathological situations we tested whether the patterns could be associated with the occurrence of disease. The current studies shows that there were associations between quantitative immunophenotypes and the subsequent incidence and severity of disease. For example, individuals with characteristically low levels of HLA-DR or B cells or reduced numbers of Pf+ Tc/s cells had more frequent and/or more severe upper respiratory infections. Quantitative immunophenotypes will be more widely measured if the necessary standards are available and if appropriate procedures are made more accessible.

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium

PubMed Central

Finak, Greg; Langweiler, Marc; Jaimes, Maria; Malek, Mehrnoush; Taghiyar, Jafar; Korin, Yael; Raddassi, Khadir; Devine, Lesley; Obermoser, Gerlinde; Pekalski, Marcin L.; Pontikos, Nikolas; Diaz, Alain; Heck, Susanne; Villanova, Federica; Terrazzini, Nadia; Kern, Florian; Qian, Yu; Stanton, Rick; Wang, Kui; Brandes, Aaron; Ramey, John; Aghaeepour, Nima; Mosmann, Tim; Scheuermann, Richard H.; Reed, Elaine; Palucka, Karolina; Pascual, Virginia; Blomberg, Bonnie B.; Nestle, Frank; Nussenblatt, Robert B.; Brinkman, Ryan Remy; Gottardo, Raphael; Maecker, Holden; McCoy, J Philip

2016-01-01

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping. PMID:26861911

[Comparative immunophenotypic characterization of human and monkey permanent lymphoid culture cells].

PubMed

Agrba, V Z; Lapin, B A; Medvedeva, N M; Ignatova, I E; Karal-Ogly, D D

2007-01-01

The aim of the study was to define the comparative immunophenotypic characteristics ofwidely spread lymphoid cell cultures, derived from Burkett's lymphoma named as Raji and P3HR-1 in comparison with analogous monkey cultures. It has been shown that P3HR-1 culture consists of similar type cells - activated B-lymphocytes CD23 with k phenotype, which demonstrates its monoclonality. Raji culture includes cells with markers of immature B-lymphocytes CD10 and CD24, as well as elements expressing CD10 antigens. T-cell markers were found in none of the cultures. In contrast to human cells, monkey lymphoid culture expressed both B- and T-cell markers. Moreover, in one of them, obtained from a green monkey, T-cells of suppressor type (CD8) prevailed. The immunophenotypic characteristics of primate lymphoid cell cultures, revealed by the study, are of great importance for their proper application to medical and biological studies.

Acute promyelocytic leukemia, hypogranular variant, with uncharacteristic staining with chloroacetate esterase.

PubMed

Dunphy, C H; Polski, J M; Johns, G; Evans, H L; Gardner, L J

2001-06-01

A diagnosis of the hypogranular variant of acute promyelocytic leukemia (APLv) may be difficult to establish based on cytomorphology alone. However, the great majority of cases have a classical immunophenotype by flow cytometric immunophenotyping (FCI) (CD13+, CD33+, dim CD64+, HLA-DR-, and CD34-) and a classical enzyme cytochemical (EC) staining pattern. [intensely staining with myeloperoxidase, Sudan Black B, and chloroacetate esterase (CAE) and negative with alpha'-naphthyl acetate and butyrate esterases]. Although the immunophenotype of APLv by FCI has varied in the literature (HLA-DR +/- and CD34 +/-), the EC staining pattern has remained constant. We report a case of APLv with characteristic cytomorphology, compatible FCI data (CD13+, CD33+, dim CD64+, HLA-DR +/-, and CD34-), chromosomal detection of t(15; 17), and molecular detection of the PML/RAR alpha fusion gene; however, staining of the leukemic cells with CAE was quite uncharacteristic. We describe our findings.

[Malignant peripheral nerve sheath tumor with perineural differentiation (malignant perineurinoma) of the cervix uteri].

PubMed

Dolzhikov, A A; Mukhina, T S

2014-01-01

The paper describes a case of a malignant peripheral nerve sheath tumor with perineural differentiation and at the rare site of the cervix uteri in a 57-year-old patient. The diagnosis was established on the basis of extensive immunohistochemical examination, by excluding the similar neoplasms and detecting an immunophenotype characteristic of perineural differentiation. There are data available in the literature on the morphological and immunophenotypical characteristics of this tumor.

Heterogeneity in acute undifferentiated leukemia.

PubMed

LeMaistre, A; Childs, C C; Hirsch-Ginsberg, C; Reuben, J; Cork, A; Trujillo, J M; Andersson, B; McCredie, K B; Freireich, E; Stass, S A

1988-01-01

From January 1985 to May 1987, we studied 256 adults with newly diagnosed acute leukemia. Acute undifferentiated leukemia (AUL) was diagnosed in 12 of the 256 (4.6%) cases when lineage could not be delineated by light microscopy and light cytochemistry. To further characterize the blasts, immunophenotyping, ultrastructural myeloperoxidase (UMPO), and ultrastructural platelet peroxidase parameters were examined in 10, 11, and 6 of the 12 cases, respectively. Five cases demonstrated UMPO and were reclassified as acute myeloblastic leukemia (AML). Of the six UMPO-negative cases, three had a myeloid and one had a mixed immunophenotype. One UMPO-negative patient with a myeloid immunophenotype was probed for the immunoglobulin heavy chain gene (JH) and the beta chain of the T-cell receptor gene (Tcr beta) with no evidence of rearrangement. Six cases were treated with standard acute lymphoblastic leukemia (ALL) chemotherapy and failed to achieve complete remission (CR). Various AML chemotherapeutic regimens produced CR in only 3 of the 12 cases. One case was treated with gamma interferon and the other 2 with high-dose Ara-C. Our findings indicate a myeloid lineage can be detected by UMPO (5/12) in some cases of AUL. A germline configuration with JH and Tcr beta in one case as well as a myeloid immunophenotype in 3 UMPO-negative cases raises the possibility that myeloid lineage commitment may occur in the absence of myeloid peroxidase (MPO) cytochemical positivity.

Immunophenotypic analysis of mast cells in mastocytosis: When and how to do it. Proposals of the Spanish Network on Mastocytosis (REMA).

PubMed

Escribano, Luis; Diaz-Agustin, Beatriz; López, Antonio; Núñez López, Rosa; García-Montero, Andrés; Almeida, Julia; Prados, Aranzazu; Angulo, Miguel; Herrero, Sonia; Orfao, Alberto

2004-03-01

Mastocytosis is a term used for a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow, liver, spleen, and lymph nodes, among others. In recent years, multiparameter flow cytometric studies have shown that pathologic MCs from patients with mastocytosis display unique aberrant immunophenotypic characteristics as compared with normal MCs. Among other features, pathologic MCs show aberrant expression of CD25 and CD2 antigens and abnormally high levels of the CD11c and CD35 complement receptors, the CD59 complement regulatory molecule, the CD63 lysosomal membrane antigen, and the CD69 early-activation antigen. In addition, MCs from mastocytosis express abnormally low levels of CD117 and unexpectedly high light scatter and autofluorescence characteristics. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this paper we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, information for their phenotypic characterization, and the criteria currently used for a correct interpretation of the immunophenotypic results obtained. Copyright 2004 Wiley-Liss, Inc.

Immunophenotypic characterization of bone marrow mast cells in mastocytosis and other mast cell disorders.

PubMed

Sánchez-Muñoz, Laura; Teodósio, Cristina; Morgado, José M; Escribano, Luis

2011-01-01

Mastocytosis is a term used to designate a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow (BM), liver, spleen, and lymph nodes, among others. Recent advances in our understanding of mast cell biology and disease resulted in the identification of important differences in the expression of mast cell surface antigens between normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts lead to the inclusion of this immunophenotypic abnormality in the World Health Organization diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients lacking histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this chapter, we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, for their phenotypic characterization, and the criteria currently used for correct interpretation of the immunophenotypic results obtained. Copyright © 2011 Elsevier Inc. All rights reserved.

Prognostic stratification improvement by integrating ID1/ID3/IGJ gene expression signature and immunophenotypic profile in adult patients with B-ALL.

PubMed

Cruz-Rodriguez, Nataly; Combita, Alba L; Enciso, Leonardo J; Raney, Lauren F; Pinzon, Paula L; Lozano, Olga C; Campos, Alba M; Peñaloza, Niyireth; Solano, Julio; Herrera, Maria V; Zabaleta, Jovanny; Quijano, Sandra

2017-02-28

Survival of adults with B-Acute Lymphoblastic Leukemia requires accurate risk stratification of patients in order to provide the appropriate therapy. Contemporary techniques, using clinical and cytogenetic variables are incomplete for prognosis prediction. To improve the classification of adult patients diagnosed with B-ALL into prognosis groups, two strategies were examined and combined: the expression of the ID1/ID3/IGJ gene signature by RT-PCR and the immunophenotypic profile of 19 markers proposed in the EuroFlow protocol by Flow Cytometry in bone marrow samples. Both techniques were correlated to stratify patients into prognostic groups. An inverse relationship between survival and expression of the three-genes signature was observed and an immunophenotypic profile associated with clinical outcome was identified. Markers CD10 and CD20 were correlated with simultaneous overexpression of ID1, ID3 and IGJ. Patients with simultaneous expression of the poor prognosis gene signature and overexpression of CD10 or CD20, had worse Event Free Survival and Overall Survival than patients who had either the poor prognosis gene expression signature or only CD20 or CD10 overexpressed. By utilizing the combined evaluation of these two immunophenotypic markers along with the poor prognosis gene expression signature, the risk stratification can be significantly strengthened. Further studies including a large number of patients are needed to confirm these findings.

Papillary syncytial metaplasia associated with endometrial breakdown exhibits an immunophenotype that overlaps with uterine serous carcinoma.

PubMed

McCluggage, W Glenn; McBride, Hilary A

2012-05-01

Uterine serous carcinoma (USC) is an aggressive variant of Type 2 endometrial carcinoma, which in most cases exhibits, at least focally, a papillary architecture. Occasionally, especially in small biopsy specimens, it may be difficult to distinguish between USC and a variety of metaplastic or reactive processes. In particular, papillary syncytial metaplasia (PSM), as a result of endometrial breakdown, may be confused with USC or its precursor serous endometrial intraepithelial carcinoma. In such cases, immunohistochemistry is often undertaken, the panel of markers usually including estrogen receptor (ER), p53, p16, and MIB1. The expected immunoprofile of USC is ER negative, p53 and p16 positive, and a high MIB1 proliferation index, although studies have shown that significant numbers of cases deviate from this immunophenotype. With regard to the aforementioned markers, PSM has not been studied extensively, but intuitively, the expected immunophenotype would be ER positive, p53 and p16 negative, and a low MIB1 proliferation index. After 2 index cases in which breaking down menstrual endometrium with florid PSM was misdiagnosed on an endometrial biopsy as USC or suspected USC, in part due to the observed immunophenotype, we studied the expression of ER, p53, p16, MIB1, and HMGA2 (a recently described useful marker of USC) in 10 further cases of PSM associated with endometrial breakdown. We illustrate that compared with a nonbreaking down endometrium, PSM is characterized by a decreased expression of ER and an increased expression of p53 (although still wild-type staining) and p16, the latter marker typically being diffusely positive. HMGA2 is negative, and there is a low MIB1 proliferation index. In cases of PSM, which are morphologically problematic, the immunophenotype may further heighten the suspicion of serous malignancy and potentially result in a misdiagnosis.

Multiparameter Flow Cytometry For Clinical Applications

NASA Astrophysics Data System (ADS)

Stewart, Carleton C.

1989-06-01

Flow Cytometry facilities are well established and provide immunophenotyping and DNA content measurement services. The application of immunophenotyping has been primarily in monitoring therapy and in providing further information to aid in the definitive diagnosis of immunological and neoplastic disease such as: immunodeficiency disease, auto immune disease, organ transplantation, and leukemia and lymphoma. DNA content measurements have been particularly important in determining the fraction of cycling cells and presence of aneuploid cells in neoplasia. This information has been useful in the management of patients with solid tumors.

[Bone marrow mononuclear cells from murine tibia after the space flight on biosatellite "Bion-M1"].

PubMed

Andreeva, E R; Goncharova, E A; Gornostaeva, A N; Grigor'eva, O V; Buravkova, L B

2014-01-01

Cellularity, viability and immunophenotype of mononuclear cells derived from the tibial marrow of C57bL/6 mice were measured after the 30-day "Bion-M1" space flight and subsequent 7-day recovery. Cell number in the flight group was significantly less than in the group of vivarium control. There was no difference in the parameter between the flight and control groups after the recovery. Viability of mononuclear cells was more than 95% in all examined groups. Flow cytometric analysis failed to show differences in bone marrow cell immunophenotype (CD45, CD34, CD90.1 (Thy1); however, the flight animals had more large-sized CD45+ mononuclears than the control groups of mice. These results indicate that spaceflight factors did not have significant damaging effects on the number or immunophenotype of murine bone marrow mononuclears. These observations are consistent with the previously made assumption of a moderate and reversible stress reaction of mammals to space flight.

Towards an Immunophenotype of Schizophrenia: Progress, Potential Mechanisms, and Future Directions

PubMed Central

Miller, Brian J; Goldsmith, David R

2017-01-01

The evidence to date, coupled with advances in immunology and genetics has afforded the field an unparalleled opportunity to investigate the hypothesis that a subset of patients with schizophrenia may manifest an immunophenotype, toward new potential diagnostics and therapeutics to reduce risk, alleviate symptoms, and improve quality of life in both at-risk populations and patients with established schizophrenia. In this paper, we will first summarize the findings on immune dysfunction in schizophrenia, including (1) genetic, prenatal, and premorbid immune risk factors and (2) immune markers across the clinical course of the disorder, including cytokines; C-reactive protein; immune cells; antibodies, autoantibodies and comorbid autoimmune disorders; complement; oxidative stress; imaging of neuroinflammation; infections; and clinical trials of anti-inflammatory agents and immunotherapy. We will then discuss a potential mechanistic framework toward increased understanding of a potential schizophrenia immunophenotype. We will then critically appraise the existing literature, and discuss suggestions for the future research agenda in this area that are needed to rigorously evaluate this hypothesis. PMID:27654215

Cell Encapsulating Biomaterial Regulates Mesenchymal Stromal/Stem Cell Differentiation and Macrophage Immunophenotype

PubMed Central

Cantu, David Antonio; Hematti, Peiman

2012-01-01

Bone marrow mesenchymal stromal/stem cell (MSC) encapsulation within a biomatrix could improve cellular delivery and extend survival and residence time over conventional intravenous administration. Although MSCs modulate monocyte/macrophage (Mø) immunophenotypic properties, little is known about how such interactions are influenced when MSCs are entrapped within a biomaterial. Furthermore, the impact of the cell-encapsulating matrix on MSC multipotency and on Møs, which infiltrate biomaterials, remains poorly understood. Here we elucidate this three-way interaction. The Mø immunophenotype and MSC differentiation were examined with regard to established and experimental collagen-based biomaterials for MSC entrapment. Tumor necrosis factor-α secretion was acutely inhibited at 4 days. MSCs cocultured with Møs demonstrated attenuated chondrocyte differentiation, whereas osteoblast differentiation was enhanced. Adipocyte differentiation was considerably enhanced for MSCs entrapped within the gelatin/polyethylene glycol-based matrix. A better understanding of the effect of cell encapsulation on differentiation potency and immunomodulation of MSCs is essential for MSC-based, biomaterial-enabled therapies. PMID:23197666

Human herpesviruses respiratory infections in patients with acute respiratory distress (ARDS).

PubMed

Bonizzoli, Manuela; Arvia, Rosaria; di Valvasone, Simona; Liotta, Francesco; Zakrzewska, Krystyna; Azzi, Alberta; Peris, Adriano

2016-08-01

Acute respiratory distress syndrome (ARDS) is today a leading cause of hospitalization in intensive care unit (ICU). ARDS and pneumonia are closely related to critically ill patients; however, the etiologic agent is not always identified. The presence of human herpes simplex virus 1, human cytomegalovirus and Epstein-Barr virus in respiratory samples of critically ill patients is increasingly reported even without canonical immunosuppression. The main aim of this study was to better understand the significance of herpesviruses finding in lower respiratory tract of ARDS patients hospitalized in ICU. The presence of this group of herpesviruses, in addition to the research of influenza viruses and other common respiratory viruses, was investigated in respiratory samples from 54 patients hospitalized in ICU, without a known microbiological causative agent. Moreover, the immunophenotype of each patient was analyzed. Herpesviruses DNA presence in the lower respiratory tract seemed not attributable to an impaired immunophenotype, whereas a significant correlation was observed between herpesviruses positivity and influenza virus infection. A higher ICU mortality was significantly related to the presence of herpesvirus infection in the lower respiratory tract as well as to impaired immunophenotype, as patients with poor outcome showed severe lymphopenia, affecting in particular T (CD3+) cells, since the first days of ICU hospitalization. In conclusion, these results indicate that herpesviruses lower respiratory tract infection, which occurs more frequently following influenza virus infection, can be a negative prognostic marker. An independent risk factor for ICU patients with ARDS is an impaired immunophenotype.

Morphologic Reproducibility, Genotyping, and Immunohistochemical Profiling Do Not Support a Category of Seromucinous Carcinoma of the Ovary.

PubMed

Rambau, Peter F; McIntyre, John B; Taylor, Jennifer; Lee, Sandra; Ogilvie, Travis; Sienko, Anna; Morris, Don; Duggan, Máire A; McCluggage, W Glenn; Köbel, Martin

2017-05-01

The 2014 World Health Organization Classification of Tumors of Female Reproductive Organs endorsed the new category of seromucinous carcinoma, a neoplasm that exhibits morphologic and immunophenotypic overlap with other histotypes of ovarian carcinoma. The goal of this study was to determine whether seromucinous carcinoma was a distinct histotype by assessing its diagnostic reproducibility and comparing its molecular composition to the 5 major histotypes of ovarian carcinoma. Thirty-two tumors diagnosed as seromucinous carcinomas from 2 centers were studied. Eighteen cases were randomly selected for a review set comprising a total of 50 ovarian carcinomas of various histotypes. Morphologic histotype was independently assessed by 4 pathologists. For the 32 seromucinous carcinomas, a histotype-specific immunophenotype was assigned using a diagnostic immunohistochemical panel. Histotype-specific genotype was assigned using a combination of immunohistochemistry and targeted next-generation sequencing for somatic mutations, including genes recurrently mutated in ovarian carcinomas. There was low to modest agreement between pathologists with the reference diagnosis of seromucinous carcinoma, ranging from 39% to 56% for the 4 observers. The immunophenotype was not unique but overlapped predominantly with endometrioid and to a lesser extent with mucinous and low-grade serous carcinoma. Genomic and immunohistochemical alterations were detected in a number of target genes, including KRAS (70%), PIK3CA (37%), PTEN (19%), and ARID1A (16%); no CTNNB1 mutations were identified. Nine cases (30%) harbored concurrent KRAS/PIK3CA mutations. An endometrioid genotype was assigned to 19 cases, a low-grade serous genotype to 9, and a mucinous genotype to 1 and 3 cases were uninformative. Integrating morphology, immunophenotype, and genotyping resulted in reclassifying the seromucinous carcinomas to endometrioid 23/32 (72%), low-grade serous 8/32 (25%), and mucinous 1/32 (3%). The morphologic diagnosis of seromucinous carcinomas is not very reliable and it does not exhibit a distinct immunophenotype or genotype. The molecular features overlap mostly with endometrioid and low-grade serous carcinomas. Our data suggest the category of seromucinous carcinoma be discontinued as ancillary molecular tests can assign cases to one of the major histotypes.

Immunophenotypic analysis of Waldenstrom's macroglobulinemia.

PubMed

San Miguel, J F; Vidriales, M B; Ocio, E; Mateo, G; Sánchez-Guijo, F; Sánchez, M L; Escribano, L; Bárez, A; Moro, M J; Hernández, J; Aguilera, C; Cuello, R; García-Frade, J; López, R; Portero, J; Orfao, A

2003-04-01

Immunophenotyping has become an essential tool for diagnosis of hematological malignancies. By contrast, for diagnosis of Waldenstrom's macroglobulinemia (WM) immunophenotyping is used only occasionally. From 150 patients with a IgM monoclonal gammopathy we have selected 60 cases with (1) morphological lymphoplasmocytoid bone marrow (BM) infiltration (>20%); (2) IgM paraprotein (>10g/L); and (3) absence of features of other lymphoma types. Immunophenotypic analysis was based on the use of the triple or quadruple monoclonal antibody (MoAb) combinations. To increase the sensitivity of the analysis of antigen expression, selected CD19(+)CD20(+) B cells were targeted. We have also explored the antigenic characteristics of both the plasma cell (PC) and mast cell (MC) compartments present in the BM from 15 WM patients. Clonal WM lymphocytes were characterized by the constant expression of pan-B markers (CD19, CD20, CD22, CD24) together with sIg, predominantly kappa (5:1, kappa:lambda ratio). A high proportion of cases (75%) were positive for FMC7 and CD25, but in contrast to hairy cell leukemia (HCL), these lymphocytes were always negative for CD103 and CD11c. CD10 antigen was also absent in all WM patients and less than one fifth of patients were positive for CD5 and CD23, while CD27, CD45RA, and BCL-2 were present in most malignant cells. In two cases, the coexistence of two different clones of B lymphocytes was identified, and in eight additional cases, intraclonal phenotypic heterogeneity was observed. As far as PCs are concerned, in most patients (85%) the number of PCs was within the normal range (median, 0.36%). The antigenic profile of these PCs differed from that observed in normal and myelomatous PC (CD38(++)CD19(++/-)CD56(-)CD45(++)CD20(+)). In three cases, PCs showed aberrant expression for CD5, CD22, or FMC7. Finally, the number of mast cells was significantly higher (0.058 +/- 0.13) as compared to normal BM (0.019 +/- 0.02) (P <.01), although they were immunophenotypically normal (CD117(+)CD2(-)CD25(-)). Copyright 2003 Elsevier Inc. All rights reserved.

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping.

PubMed

Tembhare, Prashant; Badrinath, Yajamanam; Ghogale, Sitaram; Patkar, Nikhil; Dhole, Nilesh; Dalavi, Pooja; Kunder, Nikesh; Kumar, Ashok; Gujral, Sumeet; Subramanian, P G

2016-03-01

Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B-cell acute lymphoblastic leukemia (B-ALL) and multiple myeloma (MM). Current methods of flow-cytometric (FC) DNA-ploidy evaluation are either technically difficult or limited to three- to four-color immunophenotyping and hence, challenging to evaluate DNA-ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six- to seven-color immunophenotyping and DNA-ploidy using a dye-FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra-assay variation for FCV was studied. Using this six-color immunophenotyping & FCV-protocol DNA-ploidy was determined in bone-marrow samples from 124 B-ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1-peak with FCV (mean-CV 4.1%) was slightly higher than PI (mean-CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra-assay variation was very low with CV of 0.005%. In B-ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near-hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near-hyperdiploidy in 8% and remaining 30% were diploid. FCV-based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of six-color immunophenotyping and DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions. © 2015 International Society for Advancement of Cytometry.

Primary central nervous system diffuse large B-cell lymphoma in the immunocompetent: Immunophenotypic subtypes and Epstein-Barr virus association.

PubMed

Mahadevan, Anita; Rao, Clementina Rama; Shanmugham, M; Shankar, Susarla Krishna

2015-01-01

Primary central nervous system diffuse large B-cell lymphoma (PCNSL DLBCL) in the immunocompetent is an uncommon tumor that has an activated B-cell immunophenotype resembling germinal center exit B cells. They also differ from primary central nervous diffuse large B-cell lymphomas in the immunocompromised as they show no association with the Epstein-Barr virus. To determine if immunophenotypic subtyping of PCNS DLBCL from Asian subcontinent are also different similar to its systemic counterpart is unclear, as there are only limited studies from Asia, and none from India. The immunohistochemical profile of 24 South Indian patients with primary central nervous system diffuse large B-cell lymphoma was studied using germinal center markers - CD10 and Bcl-6, and activation markers - MUM1 and CD138, which are markers for late/post germinal centre B cells. Insitu hybridization for EBV genome and LMP1 by immunohistochemistry was carried out in all cases to determine association with EBV. Centroblastic morphology and uniform activated B-cell phenotype with positivity for MUM1 was seen in 91.6% of tumors. Co-expression of Bcl-6 and MUM1 was evident in 50%, which is more frequent than in systemic diffuse large B-cell lymphomas. All cases were negative for Epstein-Barr virus using EBER in-situ hybridization and LMP1 immunohistochemistry. Primary diffuse large B-cell lymphoma in the immunocompetent is a distinct clinicopathological entity with centroblastic morphology, a uniform activated B-cell immunophenotype that is not associated with the Epstein-Barr virus regardless of geographic origin.

Overtraining and immune system: a prospective longitudinal study in endurance athletes.

PubMed

Gabriel, H H; Urhausen, A; Valet, G; Heidelbach, U; Kindermann, W

1998-07-01

A prospective longitudinal study investigated for 19 +/- 3) months whether immunophenotypes of peripheral leukocytes were altered in periods of severe training. Leukocyte membrane antigens (CD3, CD4, CD8, CD14, CD16, CD19, CD45, CD45RO, and CD56) of endurance athletes were immunophenotyped (dual-color flow cytometry) and list mode data analyzed by a self-learning classification system in a state of an overtraining syndrome (OT; N = 15) and several occasions without symptoms of staleness (NS; N = 70). Neither at physical rest nor after a short-term highly intensive cycle ergometer exercise session at 110% of the individual anaerobic threshold did cell counts of neutrophils, T, B, and natural killer cells differ between OT and NS. Eosinophils were lower during OT, activated T cells (CD3+HLA/DR+) showed slight increases (NS: 5.5 +/- 2.7; OT 7.3 +/- 2.4% CD3+ of cells; means +/- SD; P < 0.01) during OT without reaching pathological ranges. The cell-surface expression of CD45RO (P < 0.001) on T cells, but not cell concentrations of CD45RO+ T cells, were higher during OT. OT could be classified with high specificities (92%) and sensitivities (93%). It is concluded that OT does not lead to clinically relevant alterations of immunophenotypes in peripheral blood and especially that an immunosuppressive effect cannot be detected. Immunophenotyping may provide help with the diagnosis of OT in future, but the diagnostic approach presented here requires improvements before use in sports medicine practice is enabled.

Immunophenotyping does not improve predictivity of the local lymph node assay in mice.

PubMed

Strauss, Volker; Kolle, Susanne N; Honarvar, Naveed; Dammann, Martina; Groeters, Sibylle; Faulhammer, Frank; Landsiedel, Robert; van Ravenzwaay, Bennard

2015-04-01

The local lymph node assay (LLNA) is a regulatory accepted test for the identification of skin sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. However, there is evidence that LLNA is overestimating the sensitization potential of certain substance classes in particular those exerting skin irritation. Some reports describe the additional use of flow cytometry-based immunophenotyping to better discriminate irritants from sensitizing irritants in LLNA. In the present study, the 22 performance standards plus 8 surfactants were assessed using the radioactive LLNA method. In addition, lymph node cells were immunophenotyped to evaluate the specificity of the lymph node response using cell surface markers such as B220 or CD19, CD3, CD4, CD8, I-A(κ) and CD69 with the aim to allow a better discrimination above all between irritants and sensitizers, but also non-irritating sensitizers and non-sensitizers. However, the markers assessed in this study do not sufficiently differentiate between irritants and irritant sensitizers and therefore did not improve the predictive capacity of the LLNA. Copyright © 2014 John Wiley & Sons, Ltd.

A Method for Identification and Analysis of Non-Overlapping Myeloid Immunophenotypes in Humans

PubMed Central

Gustafson, Michael P.; Lin, Yi; Maas, Mary L.; Van Keulen, Virginia P.; Johnston, Patrick B.; Peikert, Tobias; Gastineau, Dennis A.; Dietz, Allan B.

2015-01-01

The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN-CD33+HLA-DR- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies. PMID:25799053

Developmental immunotoxicity (DIT): assays for evaluating effects of exogenous agents on development of the immune system.

PubMed

DeWitt, Jamie C; Peden-Adams, Margie M; Keil, Deborah E; Dietert, Rodney R

2012-02-01

Developmental immunotoxicity (DIT) occurs when exposure to environmental risk factors prior to adulthood, including chemical, biological, physical, or physiological factors, alters immune system development. DIT may elicit suppression, hyperactivation, or misregulation of immune responses and may present clinically as decreased resistance to pathogens, allergic and autoimmune diseases, and inflammatory diseases. Immunotoxicity testing guidelines established by the Environmental Protection Agency for adult animals (OPPTS 8703.7800) require functional tests and immunophenotyping that are suitable for detecting immunomodulation, especially immunosuppression. However, evaluating immune function in offspring that are not fully immunocompetent yields results that are challenging to interpret. Therefore, this unit will describe an optimum exposure scenario, reference two assays (immunophenotyping and histopathology) appropriate for detecting immunomodulation in weaning-age offspring, and reference four assays (immunophenotyping, histopathology, T cell-dependent antibody responses, and delayed-type hypersensitivity) appropriate for detecting immunomodulation in immunocompetent offspring. The protocol also will reference other assays (natural killer cell and cytotoxic T lymphocyte) with potential utility for assessing DIT. © 2012 by John Wiley & Sons, Inc.

Immunophenotypic analysis of hematopoiesis in patients suffering from Shwachman-Bodian-Diamond Syndrome.

PubMed

Mercuri, Angela; Cannata, Elisa; Perbellini, Omar; Cugno, Chiara; Balter, Rita; Zaccaron, Ada; Tridello, Gloria; Pizzolo, Giovanni; De Bortoli, Massimiliano; Krampera, Mauro; Cipolli, Marco; Cesaro, Simone

2015-10-01

Shwachman-Diamond syndrome is a rare disorder characterized by exocrine pancreatic insufficiency, skeletal abnormalities, and bone marrow failure, with high risk of leukemic evolution. The aim of the study was the immunophenotypic characterization of bone marrow cells from patients with Shwachman-Diamond syndrome to assess the maturation pathway of blood progenitor cells and to identify the presence of recurrent abnormalities. Bone marrow samples from nineteen patients and eleven controls were analyzed by multiparameter flow cytometry. We found a low frequency of CD34+ cells (P = 0.0179) and myeloid progenitors (P = 0.025), in the bone marrow of patients with Shwachman-Diamond syndrome as compared to the controls. A significant reduction in the percentage of granulocytes (P = 0.002) and an increase of monocytes (P < 0.001) were also evident in the bone marrow of patients. On the basis of these observations, future prospective assessments may be useful to verify the contribution of bone marrow immunophenotype in the early identification of the evolution toward aplasia or myelodysplasia. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Vortex-dislodged cells from bone marrow trephine biopsy yield satisfactory results for flow cytometric immunophenotyping.

PubMed

Bommannan, K; Sachdeva, M U S; Gupta, M; Bose, P; Kumar, N; Sharma, P; Naseem, S; Ahluwalia, J; Das, R; Varma, N

2016-10-01

A good bone marrow (BM) sample is essential in evaluating many hematologic disorders. An unsuccessful BM aspiration (BMA) procedure precludes a successful flow cytometric immunophenotyping (FCI) in most hematologic malignancies. Apart from FCI, most ancillary diagnostic techniques in hematology are less informative. We describe the feasibility of FCI in vortex-dislodged cell preparation obtained from unfixed trephine biopsy (TB) specimens. In pancytopenic patients and dry tap cases, routine diagnostic BMA and TB samples were complemented by additional trephine biopsies. These supplementary cores were immediately transferred into sterile tubes filled with phosphate-buffered saline, vortexed, and centrifuged. The cell pellet obtained was used for flow cytometric immunophenotyping. Of 7955 BMAs performed in 42 months, 34 dry tap cases were eligible for the study. Vortexing rendered a cell pellet in 94% of the cases (32 of 34), and FCI rendered a rapid diagnosis in 100% of the cases (32 of 32) where cell pellets were available. We describe an efficient procedure which could be effectively utilized in resource-limited centers and reduce the frequency of repeat BMA procedures. © 2016 John Wiley & Sons Ltd.

Immunophenotype of infiltrating cells in protocol renal allograft biopsies from tacrolimus-versus cyclosporine-treated patients.

PubMed

Serón, Daniel; O'Valle, Francisco; Moreso, Francesc; Gomà, Montse; Hueso, Miguel; Grinyó, Josep M; Garcia del Moral, Raimundo

2007-03-15

The prevalence of subclinical rejection is lower in patients receiving tacrolimus than in patients treated with cyclosporine. However, it is not known whether this difference is related to the modulation of a specific cell immunophenotype. We perform a two case-one control study in patients treated with tacrolimus (n=44) or cyclosporine (n=22) with a protocol biopsy performed at 4 to 6 months. Immunophenotype of infiltrating cells was evaluated with monoclonal antibodies directed against CD45 (all leukocytes), CD3 (T lymphocytes), CD68 (monocytes/macrophages), and CD20 (B lymphocytes) and expressed as interstitial positive cells/mm(2). The number of interstitial CD45 (290+/-209 vs. 696+/-560; P<0.01), CD3 (121+/-84 vs. 208+/-104; P<0.01), and CD68 (155+/-232 vs. 242+/-280; P<0.05) but not CD20 (137+/-119 vs. 197+/-154) positive cells was lower in tacrolimus-treated patients. T lymphocytes and macrophages interstitial infiltration was reduced in tacrolimus treated patients evaluated with protocol biopsies in comparison to cyclosporine-treated patients.

Does a p53 "Wild-type" Immunophenotype Exclude a Diagnosis of Endometrial Serous Carcinoma?

PubMed

Fadare, Oluwole; Roma, Andres A; Parkash, Vinita; Zheng, Wenxin; Walavalkar, Vighnesh

2018-01-01

An aberrant p53 immunophenotype may be identified in several histotypes of endometrial carcinoma, and is accordingly recognized to lack diagnostic specificity in and of itself. However, based on the high frequency with which p53 aberrations have historically been identified in endometrial serous carcinoma, a mutation-type immunophenotype is considered to be highly sensitive for the histotype. Using an illustrative case study and a review of the literature, we explore a relatively routine diagnostic question: whether the negative predictive value of a wild-type p53 immunophenotype for serous carcinoma is absolute, that is, whether a p53-wild type immunophenotype is absolutely incompatible with a diagnosis of serous carcinoma. The case is an advanced stage endometrial carcinoma that was reproducibly classified by pathologists from 3 institutions as serous carcinoma based on its morphologic features. By immunohistochemistry, the tumor was p53-wild type (DO-7 clone), diffusely positive for p16 (block positivity), and showed retained expression of PTEN, MSH2, MSH6, MLH1, and PMS2. Next generation sequencing showed that there indeed was an underlying mutation in TP53 (D393fs*78, R213*). The tumor was microsatellite stable, had a low mutational burden (4 mutations per MB), and displayed no mutations in the exonuclease domain of DNA polymerase epsilon (POLE) gene. Other genomic alterations included RB1 mutation (R46fs*19), amplifications in MYST3 and CRKL, and ARID1A deletion (splice site 5125-94_5138del108). A review of the recent literature identified 5 studies in which a total of 259 cases of serous carcinoma were whole-exome sequenced. The average TP53 mutational rate in endometrial serous carcinoma was only 75% (range, 60 to 88). A total of 12 (33%) of 36 immunohistochemical studies reported a p53-aberrant rate of <80% in endometrial serous carcinoma. We discuss in detail several potential explanations that may underlie the scenario of serous carcinoma-like morphology combined with p53-wild-type immunophenotype, including analytic limitations, a nonserous histotype displaying morphologic mimicry of serous carcinoma, and true biological phenomena (including the possibility of a TP53-independent pathway of endometrial serous carcinogenesis). Ultimately, our central thematic question is provisionally answered in the negative. At present, the available data would not support a categorical conclusion that a p53 alteration is a necessary and obligate component in the genesis and/or diagnosis of endometrial serous carcinoma. On the basis of their collective experience, the authors proffer some recommendations on the use of p53 immunohistochemistry in the histotyping of endometrial carcinomas.

Immunophenotypical characterization of canine mesenchymal stem cells from perivisceral and subcutaneous adipose tissue by a species-specific panel of antibodies.

PubMed

Ivanovska, Ana; Grolli, Stefano; Borghetti, Paolo; Ravanetti, Francesca; Conti, Virna; De Angelis, Elena; Macchi, Francesca; Ramoni, Roberto; Martelli, Paolo; Gazza, Ferdinando; Cacchioli, Antonio

2017-10-01

Immunophenotypical characterization of mesenchymal stem cells is fundamental for the design and execution of sound experimental and clinical studies. The scarce availability of species-specific antibodies for canine antigens has hampered the immunophenotypical characterization of canine mesenchymal stem cells (MSC). The aim of this study was to select a panel of species-specific direct antibodies readily useful for canine mesenchymal stem cells characterization. They were isolated from perivisceral and subcutaneous adipose tissue samples collected during regular surgeries from 8 dogs. Single color flow cytometric analysis of mesenchymal stem cells (P3) deriving from subcutaneous and perivisceral adipose tissue with a panel of 7 direct anti-canine antibodies revealed two largely homogenous cell populations with a similar pattern: CD29 + , CD44 + , CD73 + , CD90 + , CD34 - , CD45 - and MHC-II - with no statistically significant differences among them. Antibody reactivity was demonstrated on canine peripheral blood mononuclear cells. The similarities are reinforced by their in vitro cell morphology, trilineage differentiation ability and RT-PCR analysis (CD90 + , CD73 + , CD105 + , CD44 + , CD13 + , CD29 + , Oct-4 + gene and CD31 - and CD45 - expression). Our results report for the first time a comparison between the immunophenotypic profile of canine MSC deriving from perivisceral and subcutaneous adipose tissue. The substantial equivalence between the two populations has practical implication on clinical applications, giving the opportunity to choose the source depending on the patient needs. The results contribute to routine characterization of MSC populations grown in vitro, a mandatory process for the definition of solid and reproducible laboratory and therapeutic procedures. Copyright © 2017 Elsevier Ltd. All rights reserved.

Endometrial Serous Carcinoma With Clear-Cell Change: Frequency and Immunohistochemical Analysis.

PubMed

Hariri, Nosaibah; Qarmali, Morad; Fadare, Oluwole

2018-04-01

The diagnostic distinction between endometrial serous carcinoma (ESC) and endometrial clear-cell carcinoma (CCC) may occasionally be problematic, and one potentially contributing factor is the finding of clear cells in otherwise classic cases of ESC. This study aimed to define the frequency of this finding and comparatively assessed the immunophenotype of the clear cells. A review of 56 cases of ESC identified 8 (14.28%) with clear cells, representing 1% to 20% (median 7.5) of tumoral volume in these cases. In only 3 cases were clear cells discernible at low (×20) magnification. There was no significant difference in stage distribution or age between ESC patients with and without clear cells. The immunophenotypes of ESC-associated clear cells (group 1) were compared with foci of conventional ESC on another tissue block within the same case (group 2; n = 8) as well as a randomly selected cohort of CCC cases (group 3; n = 8). Groups 1 and 2 showed no significant differences regarding p53, ER, PR, Napsin-A, p504S, and hepatocyte nuclear factor 1β (HNF1β) expression, or regarding mitotic indices or Ki67 proliferation rate. In contrast, group 1 cases showed an immunophenotypic profile that was notably different from that of group 3 cases, with the former showing statistically significantly higher/more frequent expression of ER, PR, Ki67, and p53 and lower/less frequent expression of Napsin-A, p504S, and HNF1β. We conclude that clear-cell change is seen in 14% of ESCs and is discernible at low magnification in only 5%; these areas show an immunophenotype that is essentially identical to the associated background conventional ESC and are phenotypically dissimilar to CCC.

Reconstruction of the immune system after unrelated or partially matched T-cell-depleted bone marrow transplantation in children: immunophenotypic analysis and factors affecting the speed of recovery.

PubMed

Kook, H; Goldman, F; Padley, D; Giller, R; Rumelhart, S; Holida, M; Lee, N; Peters, C; Comito, M; Huling, D; Trigg, M

1996-08-01

We prospectively studied immune reconstitution in 102 children who underwent T-lymphocyte depleted bone marrow transplants using either closely matched unrelated donors or partially matched familial donors by assaying total lymphocyte counts (TLC), T-cell subsets, B cells, and natural killer cells. TLC, CD3+, and CD4+ T-cell counts remained depressed until 2 to 3 years posttransplant, whereas CD8+ T-cell counts normalized by 18 months, resulting in an inverted CD4:CD8 ratio until 12 months posttransplant. Although the percentage of NK cells was elevated early posttransplant, their absolute numbers remained normal. CD20+ B cells were depressed until 12 to 18 months posttransplant. Factors affecting immunophenotypic recovery were analyzed by nonparametric statistics. Younger patients tended to have higher TLC posttransplant. Higher marrow cell doses were not associated with hastened immunophenotypic recovery. Graft-versus-host disease (GVHD) and/or its treatment significantly delayed the immune reconstitution of CD3+, CD4+, and CD20+ cells. The presence of cytomegalovirus was associated with increased CD8+ counts and a decrease in the percentages of CD4+ and CD20+ cells.

EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

PubMed Central

Kalina, T; Flores-Montero, J; van der Velden, V H J; Martin-Ayuso, M; Böttcher, S; Ritgen, M; Almeida, J; Lhermitte, L; Asnafi, V; Mendonça, A; de Tute, R; Cullen, M; Sedek, L; Vidriales, M B; Pérez, J J; te Marvelde, J G; Mejstrikova, E; Hrusak, O; Szczepański, T; van Dongen, J J M; Orfao, A

2012-01-01

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database. PMID:22948490

Human immunotoxicologic markers of chemical exposures: preliminary validation studies.

PubMed

Wartenberg, D; Laskin, D; Kipen, H

1993-01-01

The circulating cells of the immune system are sensitive to environmental contaminants, and effects are often manifested as changes in the cell surface differentiation antigens of affected populations of cells, particularly lymphocytes. In this investigation, we explore the likelihood that variation in the expression of the surface markers of immune cells can be used as an index of exposure to toxic chemicals. We recruited 38 healthy New Jersey men to study pesticides effects: 19 orchard farmers (high exposure); 13 berry farmers (low exposure); and 6 hardware store owners (no exposure). Immunophenotyping was performed assaying the following cell surface antigens: CD2, CD4, CD8, CD14, CD20, CD26, CD29, CD45R, CD56, and PMN. Data were analyzed using univariate and multivariate methods. There were no significant differences among the groups with respect to routine medical histories, physical examinations, or routine laboratory parameters. No striking differences between groups were seen in univariate tests. Multivariate tests suggested some differences among groups and limited ability to correctly classify individuals based on immunophenotyping results. Immunophenotyping represents a fruitful area of research for improved exposure classification. Work is needed both on mechanistic understanding of the patterns observed and on the statistical interpretation of these patterns.

Uroplakin II Expression in Breast Carcinomas Showing Apocrine Differentiation: Putting Some Emphasis on Invasive Pleomorphic Lobular Carcinoma as a Potential Mimic of Urothelial Carcinoma at Metastatic Sites.

PubMed

Tajima, Shogo; Koda, Kenji

2016-01-01

Uroplakin II antibody is exclusively specific for urothelial carcinoma. Nonurothelial carcinoma has not been reported to be immunoreactive for uroplakin II. In the present study, we hypothesized that breast carcinoma showing apocrine differentiation, such as invasive pleomorphic lobular carcinoma (IPLC) and apocrine carcinoma (AC), stains positive for uroplakin II. We identified 6 cases of IPLC between 2000 and 2014 by searching a computerized pathological database. We randomly selected 10 cases of each classic invasive lobular carcinoma (cILC) and AC and five cases of apocrine metaplasia (AM) that coexisted in a surgically resected breast carcinoma specimen. Immunohistochemistry was performed for uroplakin II, GATA3, CK7, CK20, and other representative markers positive for urothelial carcinoma. All cases of IPLC, AC, and AM, except those of cILC, showed immunoreactivity for uroplakin II. Poorly differentiated urothelial carcinoma sometimes shows similar morphology to IPLC with the following immunophenotype: CK7+, CK20-, GATA3+, and uroplakin II+. In the present study, this immunophenotype was observed in all the cases of IPLC and AC. Therefore, when studying metastatic, poorly differentiated carcinoma showing the aforementioned immunophenotype, we should consider the possibility of it being IPLC in addition to metastatic urothelial carcinoma.

Multicolor-FICTION

PubMed Central

Martín-Subero, José Ignacio; Chudoba, Ilse; Harder, Lana; Gesk, Stefan; Grote, Werner; Novo, Francisco Javier; Calasanz, María José; Siebert, Reiner

2002-01-01

Phenotypic and genotypic analyses of cells are increasingly essential for understanding pathogenetic mechanisms as well as for diagnosing and classifying malignancies and other diseases. We report a novel multicolor approach based on the FICTION (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms) technique, which enables the simultaneous detection of morphological, immunophenotypic, and genetic characteristics of single cells. As prerequisite, multicolor interphase fluorescence in situ hybridization assays for B-cell non-Hodgkin’s lymphoma and anaplastic large-cell lymphoma have been developed. These assays allow the simultaneous detection of the most frequent primary chromosomal aberrations in these neoplasms, such as t(8;14), t(11;14), t(14;18), and t(3;14), and the various rearrangements of the ALK gene, respectively. To establish the multicolor FICTION technique, these assays were combined with the immunophenotypic detection of lineage- or tumor-specific antigens, namely CD20 and ALK, respectively. For evaluation of multicolor FICTION experiments, image acquisition was performed by automatic sequential capturing of multiple focal planes. Thus, three-dimensional information was obtained. The multicolor FICTION assays were applied to well-characterized lymphoma samples, proving the performance, validity, and diagnostic power of the technique. Future multicolor FICTION applications include the detection of preneoplastic lesions, early stage and minimal residual diseases, or micrometastases. PMID:12163366

2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia.

PubMed

Wood, Brent L; Arroz, Maria; Barnett, David; DiGiuseppe, Joseph; Greig, Bruce; Kussick, Steven J; Oldaker, Teri; Shenkin, Mark; Stone, Elizabeth; Wallace, Paul

2007-01-01

Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia. Copyright 2007 Clinical Cytometry Society.

Advantages of flow cytometry immunophenotyping for the diagnosis of central nervous system non-Hodgkin's lymphoma in AIDS patients.

PubMed

Subirá, D; Górgolas, M; Castañón, S; Serrano, C; Román, A; Rivas, F; Tomás, J F

2005-01-01

Neurological disorders are common in HIV-infected patients. Central nervous system (CNS) lymphoma should always be considered because it is an important cause of morbidity and mortality. To investigate the clinical utility of flow cytometry immunophenotyping (FCI) in diagnosing or discarding leptomeningeal involvement in HIV-infected patients and to compare its sensitivity with that of conventional cytological methods. Fifty-six cerebrospinal fluid (CSF) samples from 29 HIV-infected patients were independently evaluated by flow cytometry and cytology. The description of an aberrant immunophenotype was the criterion used to define the malignant nature of any CSF cell population. FCI and cytology gave concordant results for 48 of the 56 CSF samples studied: 37 were negative for malignancy and 11 had evidence of CNS lymphoma. Discordant results were obtained for eight CSF samples, and the accuracy of the FCI findings could be demonstrated for four CSF samples described as positive for malignancy according to the FCI criteria. A high level of agreement was found between the results obtained using the two methods, but FCI gave at least 25% higher sensitivity than conventional cytomorphological methods for the detection of malignant cells. This advantage suggests that, in case of negative flow cytometry results, disorders other than non-Hodgkin's lymphoma should be strongly considered.

Interim heterogeneity changes measured using entropy texture features on T2-weighted MRI at 3.0 T are associated with pathological response to neoadjuvant chemotherapy in primary breast cancer.

PubMed

Henderson, Shelley; Purdie, Colin; Michie, Caroline; Evans, Andrew; Lerski, Richard; Johnston, Marilyn; Vinnicombe, Sarah; Thompson, Alastair M

2017-11-01

To investigate whether interim changes in hetereogeneity (measured using entropy features) on MRI were associated with pathological residual cancer burden (RCB) at final surgery in patients receiving neoadjuvant chemotherapy (NAC) for primary breast cancer. This was a retrospective study of 88 consenting women (age: 30-79 years). Scanning was performed on a 3.0 T MRI scanner prior to NAC (baseline) and after 2-3 cycles of treatment (interim). Entropy was derived from the grey-level co-occurrence matrix, on slice-matched baseline/interim T2-weighted images. Response, assessed using RCB score on surgically resected specimens, was compared statistically with entropy/heterogeneity changes and ROC analysis performed. Association of pCR within each tumour immunophenotype was evaluated. Mean entropy percent differences between examinations, by response category, were: pCR: 32.8%, RCB-I: 10.5%, RCB-II: 9.7% and RCB-III: 3.0%. Association of ultimate pCR with coarse entropy changes between baseline/interim MRI across all lesions yielded 85.2% accuracy (area under ROC curve: 0.845). Excellent sensitivity/specificity was obtained for pCR prediction within each immunophenotype: ER+: 100%/100%; HER2+: 83.3%/95.7%, TNBC: 87.5%/80.0%. Lesion T2 heterogeneity changes are associated with response to NAC using RCB scores, particularly for pCR, and can be useful across all immunophenotypes with good diagnostic accuracy. • Texture analysis provides a means of measuring lesion heterogeneity on MRI images. • Heterogeneity changes between baseline/interim MRI can be linked with ultimate pathological response. • Heterogeneity changes give good diagnostic accuracy of pCR response across all immunophenotypes. • Percentage reduction in heterogeneity is associated with pCR with good accuracy and NPV.

Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples

DOE PAGES

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

2016-08-31

We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group ofmore » homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are available in the flowMatch package at www.bioconductor.org. It has been downloaded nearly 6,000 times since 2014.« less

FLOCK cluster analysis of plasma cell flow cytometry data predicts bone marrow involvement by plasma cell neoplasia.

PubMed

Dorfman, David M; LaPlante, Charlotte D; Li, Betty

2016-09-01

We analyzed plasma cell populations in bone marrow samples from 353 patients with possible bone marrow involvement by a plasma cell neoplasm, using FLOCK (FLOw Clustering without K), an unbiased, automated, computational approach to identify cell subsets in multidimensional flow cytometry data. FLOCK identified discrete plasma cell populations in the majority of bone marrow specimens found by standard histologic and immunophenotypic criteria to be involved by a plasma cell neoplasm (202/208 cases; 97%), including 34 cases that were negative by standard flow cytometric analysis that included clonality assessment. FLOCK identified discrete plasma cell populations in only a minority of cases negative for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria (38/145 cases; 26%). Interestingly, 55% of the cases negative by standard analysis, but containing a FLOCK-identified discrete plasma cell population, were positive for monoclonal gammopathy by serum protein electrophoresis and immunofixation. FLOCK-identified and quantitated plasma cell populations accounted for 3.05% of total cells on average in cases positive for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria, and 0.27% of total cells on average in cases negative for involvement by a plasma cell neoplasm by standard histologic and immunophenotypic criteria (p<0.0001; area under the curve by ROC analysis=0.96). The presence of a FLOCK-identified discrete plasma cell population was predictive of the presence of plasma cell neoplasia with a sensitivity of 97%, compared with only 81% for standard flow cytometric analysis, and had specificity of 74%, PPV of 84% and NPV of 95%. FLOCK analysis, which has been shown to provide useful diagnostic information for evaluating patients with suspected systemic mastocytosis, is able to identify neoplastic plasma cell populations analyzed by flow cytometry, and may be helpful in the diagnostic evaluation of bone marrow samples for involvement by plasma cell neoplasia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ovarian transitional cell carcinoma represents a poorly differentiated form of high-grade serous or endometrioid adenocarcinoma.

PubMed

Takeuchi, Tadahisa; Ohishi, Yoshihiro; Imamura, Hiroko; Aman, Murasaki; Shida, Kaai; Kobayashi, Hiroaki; Kato, Kiyoko; Oda, Yoshinao

2013-07-01

Ovarian transitional cell tumors include Brenner tumors (BTs) and transitional cell carcinoma (TCC; non-BTs) according to the most recent World Health Organization classification. However, it remains a matter of debate whether TCC represents a distinct entity or a morphologic variant of high-grade serous adenocarcinoma (HG-SC). The purpose of this study was to resolve the above question by clarifying the morphologic, immunohistochemical, and molecular features of TCC. We reviewed 488 cases of epithelial ovarian carcinomas and reclassified them on the basis of the most recent World Health Organization classification with the modifications proposed by Köbel and colleagues, and 35 cases of TCC were identified; 25 and 6 TCCs were admixed with HG-SC and endometrioid adenocarcinoma (EC), respectively, and the remaining 4 cases were pure TCC. TCC components were not observed in any clear cell carcinomas or mucinous adenocarcinomas. Only 2 cases of malignant BT were identified. In addition to TCCs, malignant BTs, and related adenocarcinomas, benign and borderline BTs were included in the following immunohistochemical and molecular analyses. Immunohistochemically, pure TCCs, TCCs admixed with HG-SC, and pure HG-SCs were characterized by frequent aberrant p53 expression (diffuse or null pattern) and WT1+/ER+/PR+/IMP2+ immunophenotype, whereas BTs, including benign, borderline, and malignant BTs, were characterized by lack of aberrant p53 expression and WT1-/ER-/PR-/IMP2- immunophenotype. In contrast to the BTs, pure ECs and TCCs admixed with EC showed an ER+/PR+ immunophenotype. Nearly all the tumors with a TP53 gene mutation by molecular analysis showed aberrant p53 staining patterns. In conclusion, TCC is not a distinct entity but a poorly differentiated form of serous or EC, as (1) most TCCs coexist with HG-SC (mostly) or EC (occasionally), and (2) the immunophenotype and molecular features are similar to those of HG-SC or EC but different from those of BTs.

Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group ofmore » homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are available in the flowMatch package at www.bioconductor.org. It has been downloaded nearly 6,000 times since 2014.« less

Distinct endotypes of steroid-resistant asthma characterized by IL-17Ahigh and IFN-γhigh immunophenotypes: Potential benefits of calcitriol

PubMed Central

Chambers, Emma S.; Nanzer, Alexandra M.; Pfeffer, Paul E.; Richards, David F.; Timms, Peter M.; Martineau, Adrian R.; Griffiths, Christopher J.; Corrigan, Christopher J.; Hawrylowicz, Catherine M.

2015-01-01

Background A small population of patients with severe asthma does not respond to glucocorticoids (steroid resistant [SR]). They have high morbidity, highlighting an urgent need for strategies to enhance glucocorticoid responsiveness. Objective We investigated the immunologic differences between steroid-sensitive (SS) and SR asthmatic patients and the effect on immunophenotype of oral calcitriol treatment because it has been previously shown to beneficially modulate the clinical response to glucocorticoids in patients with SR asthma. Methods CD8-depleted PBMCs were isolated from 12 patients with SS and 23 patients with SR asthma and cultured for 7 days with anti-CD3 and IL-2 with or without dexamethasone. Cytokine production was assessed in supernatants by using the Cytometric Bead Array. Patients with SR asthma were subsequently randomized to oral calcitriol or placebo therapy, and identical studies were repeated. Results Patients with SR asthma produced significantly increased IL-17A and IFN-γ levels compared with those in patients with SS asthma, although it was evident that cells from individual patients might overproduce one or the other of these cytokines. Production of IL-17A was inversely and production of IL-13 was positively associated with the clinical response to prednisolone. Oral calcitriol, compared with placebo, therapy of the patients with SR asthma significantly improved dexamethasone-induced IL-10 production in vitro while suppressing dexamethasone-induced IL-17A production. This effect mirrored the previously demonstrated improvement in clinical response to oral glucocorticoids in calcitriol-treated patients with SR asthma. Conclusions IL-17Ahigh and IFN-γhigh immunophenotypes exist in patients with SR asthma. These data identify immunologic pathways that likely underpin the beneficial clinical effects of calcitriol in patients with SR asthma by directing the SR cytokine profile toward a more SS immune phenotype, suggesting strategies for identifying vitamin D responder immunophenotypes. PMID:25772594

Mucinous cystadenocarcinoma of the breast with a basal-like immunophenotype.

PubMed

Deng, Yunte; Xue, Debin; Wang, Xiaoyan; Xu, Sanpeng; Ao, Qilin; Hu, Zhiyong; Wang, Guoping

2012-06-01

Mucinous cystadenocarcinoma (MCA) of the breast is extremely rare and was only recently described as a distinct variant of invasive ductal carcinoma of the breast. A case of MCA is reported in a 41-year-old woman. Mammographic and ultrasonographic examinations showed an irregularly shaped 10.0 × 8.0 × 5.5 cm lesion with patching calcification in the upper outer quadrant of the left breast. The gross examination revealed that the tumor has a well-circumscribed edge with a gelatinous cut surface and hemorrhage and necrosis were also noticed in the mass. Microscopically, the mass resembled mucinous cystic neoplasm of the ovary and pancreas closely, with cystic areas lined by columnar mucinous cells and associated with abundant extracellular and intracellular mucin, which is distinctively different from mucinous carcinoma with typically nests of low grade neoplastic cells floating in the mucin pool. The tumor cells were positive for CK7, CK20 and CDX2 were negative and displayed a typical immunophenotype of basal-like breast cancer (ER, PR, HER2 were negative, CK5/6 and EGFR were positive). Metastatic carcinoma was identified in three of 14 axillary lymph nodes. We describe here a very unusual case of breast MCA with basal-like immunophenotype. © 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.

MiT Family Translocation-Associated Renal Cell Carcinoma: A Contemporary Update With Emphasis on Morphologic, Immunophenotypic, and Molecular Mimics.

PubMed

Magers, Martin J; Udager, Aaron M; Mehra, Rohit

2015-10-01

Translocation-associated renal cell carcinoma (t-RCC) is a relatively uncommon subtype of renal cell carcinoma characterized by recurrent gene rearrangements involving the TFE3 or TFEB loci. TFE3 and TFEB are members of the microphthalmia transcription factor (MiT) family, which regulates differentiation in melanocytes and osteoclasts, and MiT family gene fusions activate unique molecular programs that can be detected immunohistochemically. Although the overall clinical behavior of t-RCC is variable, emerging molecular data suggest the possibility of targeted approaches to advanced disease. Thus, distinguishing t-RCC from its morphologic, immunophenotypic, and molecular mimics may have important clinical implications. The differential diagnosis for t-RCC includes a variety of common renal neoplasms, particularly those demonstrating clear cell and papillary features; in addition, because of immunophenotypic overlap and/or shared molecular abnormalities (ie, TFE3 gene rearrangement), a distinctive set of nonepithelial renal tumors may also warrant consideration. Directed ancillary testing is an essential aspect to the workup of t-RCC cases and may include a panel of immunohistochemical stains, such as PAX8, pancytokeratins, epithelial membrane antigen, carbonic anhydrase IX, HMB-45, and Melan-A. Dual-color, break-apart fluorescent in situ hybridization for TFE3 or TFEB gene rearrangement may be helpful in diagnostically challenging cases or when molecular confirmation is needed.

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

PubMed Central

Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation. PMID:28465691

Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions.

PubMed

Patrikoski, Mimmi; Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

2017-01-01

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes

PubMed Central

van Dongen, J J M; Lhermitte, L; Böttcher, S; Almeida, J; van der Velden, V H J; Flores-Montero, J; Rawstron, A; Asnafi, V; Lécrevisse, Q; Lucio, P; Mejstrikova, E; Szczepański, T; Kalina, T; de Tute, R; Brüggemann, M; Sedek, L; Cullen, M; Langerak, A W; Mendonça, A; Macintyre, E; Martin-Ayuso, M; Hrusak, O; Vidriales, M B; Orfao, A

2012-01-01

Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2–7 sequential design–evaluation–redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies. PMID:22552007

Regulators of apoptosis in cholangiocarcinoma.

PubMed

Jhala, Nirag C; Vickers, Selwyn M; Argani, Pedram; McDonald, Jay M

2005-04-01

Dysregulation of mediators of apoptosis is associated with carcinogenesis. For biliary duct cancers, p53 gene mutation is an important contributor to carcinogenesis. Mutations in the p53 gene affect transcription of the Fas gene, resulting in lack of Fas expression on cell membrane. It has been previously shown that cloned Fas-negative but not Fas-positive human cholangiocarcinoma cells are resistant to anti-Fas-mediated apoptosis and develop tumors in nude mice. In addition, interferon gamma induces Fas expression in Fas-negative cholangiocarcinoma cells and makes them susceptible to apoptosis. Therefore, it becomes important to characterize immunophenotypic expression of p53 and Fas in normal and neoplastic human tissues of the biliary tract to further understand the pathogenesis of the disease. To date, human studies to characterize differences in immunophenotypic expression of the Fas protein between intrahepatic and extrahepatic biliary duct cancers and in their precursor lesions have not been performed. To report the immunophenotypic expression of p53 and Fas expression in various stages in the development of bile duct cancers (intrahepatic and extrahepatic tumor location) and their association with tumor differentiation. Thirty bile duct cancer samples (13 intrahepatic and 17 extrahepatic) from 18 men and 12 women who ranged in age from 44 to 77 years (mean age, 65.6 years) were retrieved from the surgical pathology files. Hematoxylin-eosin-stained slides were evaluated for the type and grade of tumor and dysplastic changes in the biliary tract epithelium. Additional slides were immunohistochemically stained with p53 and anti-Fas mouse monoclonal antibody. The pattern of Fas distribution and percentage of cells positive for p53 and Fas expression were determined. The percentage of Fas-expressing cells is significantly (P = .01) more frequently noted in extrahepatic tumors compared with intrahepatic tumors. Furthermore, Fas expression decreased from dysplastic epithelium to cholangiocarcinoma (P = .01), and this decreasing trend continued from well to poorly differentiated tumors. Nuclear p53 expression was not identified in normal and dysplastic epithelium but was noted in 30% of carcinomas (P = .02). Fas expression is an early event in pathogenesis of bile duct cancers. Immunophenotypic expression of Fas is associated with well to moderately differentiated tumors but not with poor tumor differentiation.

Perivascular epithelioid cell neoplasms: pathology and pathogenesis.

PubMed

Folpe, Andrew L; Kwiatkowski, David J

2010-01-01

This review article summarizes our current understanding of the clinical, pathologic, immunohistochemical, and genetic aspects of perivascular epithelioid cell neoplasms, a rare group of related tumors defined by both morphologic and immunophenotypic criteria.

Cell and tissue microarray technologies for protein and nucleic acid expression profiling.

PubMed

Cardano, Marina; Diaferia, Giuseppe R; Falavigna, Maurizio; Spinelli, Chiara C; Sessa, Fausto; DeBlasio, Pasquale; Biunno, Ida

2013-02-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform.

[A rare tumor of the parapharyngeal space: myxoid chondrosarcoma].

PubMed

Bahri, I; Boudawara, T; Sellami, A; Khabir, A; Ghorgel, M; Drira, M; Daoud, J; Jlidi, R

2002-01-01

Extrasqueletal myxoid chondrosarcoma (EMC) is an uncommon soft tissue malignant tumor, locally aggressive with a high incidence of distant metastasis. It has distinctive clinical, immunophenotypic, cytogenetic and ultrastructural features. Most EMC are associated with the translocation t(9;22) (q22;q12). Their occurrence in the parapharyngeal space is extremely rare. Our objective is to discuss the difficulties of the histological diagnosis of EMC and to describe its immunophenotypic, cytogenetic features and clinical behavior. We report a case of a 67 years old woman who presented with a five months history of dysphagia. The oral examination found a mass displacing the posterior and left walls of the pharynx. Surgical resection of the tumor was undertaken. The pathologic examination concluded to the diagnosis of an EMC of the left parapharygeal space. Now, the patient is receiving an adjuvant radiotherapy.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

PubMed

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-08-17

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Comparative analysis of cytokeratin 15, TDAG51, cytokeratin 20 and androgen receptor in sclerosing adnexal neoplasms and variants of basal cell carcinoma.

PubMed

Evangelista, Mara Therese P; North, Jeffrey P

2015-11-01

Desmoplastic trichoepithelioma (DTE), morpheaform basal cell carcinoma (BCC) and microcystic adnexal carcinoma (MAC) are sclerosing adnexal neoplasms with overlapping histopathologic features. We compared cytokeratin 15, (CK15), T-cell death-associated gene 51 (TDAG51), cytokeratin 20 (CK20) and androgen receptor (AR) in differentiating these tumors and assessed their expression in BCC subtypes. Fifteen DTE, 15 infundibulocystic BCC, 18 micronodular BCC, 18 morpheaform BCC and 6 MAC were assessed for CK15, TDAG51, CK20 and AR expression. Quantitative CK15 staining was higher in DTE compared with BCC (p < 0.0001) and MAC (p = 0.02). Quantitative TDAG51 staining was higher in DTE than BCC (p < 0.0001). The CK20+AR- immunophenotype was 100% sensitive and specific in diagnosing DTE. The CK20-AR+ immunophenotype was 95.24% specific and 83.33% sensitive for BCC. The CK20-AR- immunophenotype was 83.33% sensitive and 90.91% specific for MAC. CK15, CK20 and AR were positive in 87, 53 and 67% of infundibulocystic BCC cases, respectively. Combination of CK20 and AR best differentiated these sclerosing adnexal neoplasms. Greater positivity for CK15 and TDAG51 generally favors benign lesions. Infundibulocystic BCC has higher CK20 and lower AR immunopositivity than other BCC variants and a high degree of CK15 and TDAG51 positivity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Current state of biology and diagnosis of clonal mast cell diseases in adults.

PubMed

Alvarez-Twose, I; Morgado, J M; Sánchez-Muñoz, L; García-Montero, A; Mollejo, M; Orfao, A; Escribano, L

2012-10-01

Mastocytosis comprises a heterogeneous group of disorders characterized by the presence of clonal mast cells (MC) in organs such as skin, bone marrow (BM), and gastrointestinal tract, among other tissues. The clonal nature of the disease can be established in most adult patients by the demonstration of activating KIT mutations in their BM MC. When highly sensitive techniques capable of identifying cells present at very low frequencies in a sample are applied, BM MC from virtually all systemic mastocytosis patients display unique immunophenotypical features, particularly the aberrant expression of CD25. By contrast, large, multifocal BM MC aggregates (the only World Health Organization major criterion for systemic mastocytosis) are absent in a significant proportion of patients fulfilling at least three minor criteria for systemic mastocytosis, particularly in subjects studied at early stages of the disease with very low MC burden. Moreover, recent molecular and immunophenotypical investigations of BM MC from patients with indolent systemic mastocytosis have revealed a close association of some biological features (e.g., multilineage involvement of hematopoiesis by the KIT mutation and an immature mast cell immunophenotype) with an increased risk for disease progression. These observations support the fact that, although the current consensus diagnostic criteria for systemic mastocytosis have been a major advance for the diagnosis and classification of the disease, rationale usage of the most sensitive diagnostic techniques available nowadays is needed to improve the diagnosis, refine the classification, and reach objective prognostic stratification of adult mastocytosis. © 2012 Blackwell Publishing Ltd.

Identification of immunophenotypic subtypes with different prognoses in extranodal natural killer/T-cell lymphoma, nasal type.

PubMed

Yu, Jian-Bo; Zuo, Zhuo; Zhang, Wen-Yan; Yang, Qun-Pei; Zhang, Ying-Chun; Tang, Yuan; Zhao, Sha; Mo, Xian-Ming; Liu, Wei-Ping

2014-11-01

To analyze the differentiation characteristics of extranodal natural killer/T-cell lymphoma, nasal type, one nude mouse model, cell lines SNK6 and SNT8, and 16 fresh human samples were analyzed by flow cytometry immunophenotyping and immunohistochemistry staining; and 115 archived cases were used for phenotypic detection and prognostic analysis. We found that CD25 was expressed by most tumor cells in all samples, and CD56(+)CD25(+) cells were the predominant population in the mouse model, the 2 cell lines, and 10 of the 16 fresh tumor samples; in the other 6 fresh tumor samples, the predominant cell population was of the CD16(+)CD25(+) phenotype, and only a minor population showed the CD56(+)CD25(+) phenotype. The phenotype detected by immunohistochemistry staining generally was consistent with the phenotype found by flow cytometry immunophenotyping. According to the expression of CD56 and CD16, 115 cases could be classified into 3 phenotypic subtypes: CD56(-)CD16(-), CD56(+)CD16(-), and CD56(dim/-)CD16(+). Patients with tumors of the CD56(dim/-)CD16(+) phenotype had a poorer prognosis than patients with tumors of the other phenotypes. Differentiation of extranodal natural killer/T-cell lymphoma, nasal type apparently resembles the normal natural killer cell developmental pattern, and these tumors can be classified into 3 phenotypic subtypes of different aggressiveness. Expression of CD56(dim/-)CD16(+) implies a poorer prognosis. Copyright © 2014 Elsevier Inc. All rights reserved.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

PubMed Central

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-01-01

We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

[Immunophenotype of lymphoblastic leukaemia in children in relation to clinical symptoms and laboratory tests, preceding its diagnosis].

PubMed

Zapolska, Beata; Krawczuk-Rybak, Maryna; Łuczyński, Włodzimierz; Zak, Janusz; Leszczyńska, Elzbieta

2004-01-01

The aim of study was to compare the clinical picture and results of laboratory tests according to the acute lymphoblastic leukaemia (ALL) immunophenotype. The observation was carried out on a group of 67 patients treated in the IIIrd Department of Paediatrics and Department of Children Oncology in the Medical Academy of Białystok from January 1994 to April 2001. This group consists of 4 children with pro-B acute lymphoblastic leukaemia, 52 children with pre-B cell ALL, 1 child with B-cell acute lymphoblastic leukaemia and 9 children with T-cell acute lymphoblastic leukaemia. Haemorrhagic diathesis. splenomegaly, enlargement of peripheral lymph nodes as well as higher values of white blood cells count, blasts count, haemoglobin concentration, haematocrit and LDH activity were observed more frequently in patients with T-cell leukaemia than in others.

Extranodal posttransplant plasmacytic hyperplasia with subsequent posttransplant plasmacytic malignancy: six-year interval case report and review of the literature.

PubMed

Dunphy, Cherie H; Galambos, Csaba; Polski, Jacek M; Evans, H Lance; Gardner, Laura J; Grosso, Leonard E; Montone, Kathleen T

2002-03-01

Posttransplant lymphoproliferative disorders (PTLDs) represent a morphologic, immunophenotypic, and genotypic spectrum of disease. Most recently, Knowles et al divided PTLDs into 3 distinct categories: (1) plasmacytic hyperplasia, (2) polymorphic B-cell hyperplasia and polymorphic B-cell lymphoma, and (3) immunoblastic lymphoma and multiple myeloma. Although one form of PTLD may progress to another form, only 1 previous case has been reported in which multiple myeloma developed 14 months after an original diagnosis of plasmacytic hyperplasia. The type of solid organ transplant was not specified in that case. We report a post--cardiac transplant plasmacytic hyperplasia developing 7 years posttransplant. Six years subsequent to the plasmacytic hyperplasia, the patient developed a posttransplant plasmacytic malignancy, supported by morphology, flow cytometric immunophenotyping, and genotypic studies. Since we have no data to support disseminated bony disease or an abnormal serum protein, we have not used the term "multiple myeloma" for this case.

Cell and Tissue Microarray Technologies for Protein and Nucleic Acid Expression Profiling

PubMed Central

Cardano, Marina; Diaferia, Giuseppe R.; Falavigna, Maurizio; Spinelli, Chiara C.; Sessa, Fausto; DeBlasio, Pasquale

2013-01-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform. PMID:23172795

Posttransplantation lymphoproliferative disease involving the pituitary gland.

PubMed

Meriden, Zina; Bullock, Grant C; Bagg, Adam; Bonatti, Hugo; Cousar, John B; Lopes, M Beatriz; Robbins, Mark K; Cathro, Helen P

2010-11-01

Posttransplantation lymphoproliferative disorders (PTLD) are heterogeneous lesions with variable morphology, immunophenotype, and molecular characteristics. Multiple distinct primary lesions can occur in PTLD, rarely with both B-cell and T-cell characteristics. Lesions can involve both grafted organs and other sites; however, PTLD involving the pituitary gland has not been previously reported. We describe a patient who developed Epstein-Barr virus-negative PTLD 13 years posttransplantation involving the terminal ileum and pituitary, which was simultaneously involved by a pituitary adenoma. Immunohistochemistry of the pituitary lesion showed expression of CD79a, CD3, and CD7 with clonal rearrangements of both T-cell receptor gamma chain (TRG@) and immunoglobulin heavy chain (IGH@) genes. The terminal ileal lesion was immunophenotypically and molecularly distinct. This is the first report of pituitary PTLD and illustrates the potentially complex nature of PTLD. Copyright © 2010 Elsevier Inc. All rights reserved.

Immunophenotype of nipple adenoma in a male patient.

PubMed

Fernandez-Flores, Angel; Suarez-Peñaranda, Jose-Manuel

2011-03-01

Adenoma of the nipple is rare in men. It must be distinguished from a breast carcinoma and from Paget disease. In this sense, immunohistochemistry can be of some help. In women, for instance, immunoexpression of c-erbB-2 favors a diagnosis of Paget disease, according to some studies. Nevertheless, we have not found any studies on HER2/neu status, estrogen receptors, or progesterone receptors in nipple adenoma of male patients. We present a case of an adenoma of the nipple in a 21-year-old man in which we carried out a wide immunohistochemical study. The lesion did not express estrogen receptors, progesterone receptors, or androgen receptors. The HercepTest was negative. Smooth muscle Actin and p63 were remarked in the basal layer of the tumoral tubules, supporting the benignancy of the lesion. This case of adenoma of the nipple in a male shows an immunophenotype that is similar to the ones reported in female patients.

Clinical utility of bone marrow flow cytometry in B-cell non-Hodgkin lymphomas (B-NHL).

PubMed

Perea, G; Altés, A; Bellido, M; Aventín, A; Bordes, R; Ayats, R; Remacha, A F; Espinosa, I; Briones, J; Sierra, J; Nomdedéu, J F

2004-09-01

To determine the efficacy of flow cytometry (FC) in the assessment of bone marrow (BM) in B-cell non-Hodgkin lymphoma (B-NHL). FC is a common practice, but is far from being validated. Morphological analysis and FC immunophenotyping were performed on 421 samples. T-cell lymphomas, Hodgkin's disease, chronic lymphocytic leukaemia and hairy cell leukaemia were not included in the study. Clonality was assessed by the standard kappa/lambda/CD19 test. Aberrant immunophenotypes present in the B-cell subpopulation were also investigated. A double-step procedure was employed in all cases to increase the sensitivity of the FC procedure. Of 380 evaluable samples, 188 corresponded to follicular lymphoma (FL), 58 to diffuse large B-cell lymphoma (DLBCL), 57 to mantle cell lymphoma (MCL), seven to Burkitt's lymphoma and the remaining 70 samples to other low-grade lymphomas. Morphological marrow infiltration was found in 148 cases, and flow immunophenotyping identified 138 cases with BM involvement. A concordance between the two methods was detected in 298 cases (79%). There was a discordance in 82 cases (21%): morphology positive/FC negative in 46 cases and morphology negative/FC positive in 36 (61% of all cases with discordance were from FL). There was no difference in outcome when patients with discordances were compared with patients without discordances. Most samples showed concordance between morphological and FC results. FC identified BM involvement in the absence of morphological infiltration. Morphology/FC discordance seems to have no influence on the outcome of FL patients. Copyright 2004 Blackwell Publishing Limited

Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion.

PubMed

Dessels, Carla; Durandt, Chrisna; Pepper, Michael S

2018-03-19

Pooled human platelet lysate (pHPL) has been used to expand adipose-derived stromal cells (ASCs) and can be formulated using fresh or expired buffy coats (BCs) which are then resuspended in either plasma or an additive solution. Not much is known about the effects that expired products and additive solutions have on ASC expansion, and the need for quality control and release criteria has been expressed. This pilot study compared proliferation, cell size, morphology and immunophenotype of ASCs expanded in the different pHPL alternatives versus foetal bovine serum (FBS). Quality control criteria were assessed prior to and during the manufacture of the pHPL alternatives. ASCs were then expanded in 1%, 2.5%, 5% or 10% of the different pHPL alternatives or in 10% FBS. Cell size, morphology, cell number and immunophenotype were measured using microscopy and flow cytometry. The majority of the pHPL alternatives were within the recommended ranges for the quality control criteria. ASCs expanded in the pHPL alternatives were smaller in size, displayed a tighter spindle-shaped morphology, increased cell growth and had a similar immunophenotype (with the exception of CD34 and CD36) when compared to ASCs expanded in FBS. Here we report on the effects that expired BC products and additive solutions have on ASC expansion. When taken together, our findings indicate that all of the pHPL alternatives can be considered to be suitable replacements for FBS for ASC expansion, and that expired BC products can be used as an alternative to fresh BC products.

Intraepithelial lymphocyte immunophenotype: a useful tool in the diagnosis of celiac disease.

PubMed

Saborido, Rebeca; Martinón, Nazareth; Regueiro, Alexandra; Crujeiras, Vanesa; Eiras, Pablo; Leis, Rosaura

2018-02-01

According to new ESPGHAN guidelines, gluten challenge is considered necessary when there is doubt about the initial diagnosis of celiac disease (CD). The main aim of this study was to quantify intraepithelial lymphocyte (IEL) immunophenotype on celiac patients on gluten-containing diet (GCD) compared to those on gluten-free diet (GFD). Another aim was to evaluate the clinical utility of IELs in the CD diagnosis, especially in selected patients on GFD where diagnostic uncertainty remains. IEL immunophenotype (TCRγδ and NK-like IELs) were studied by flow cytometry in 111 children with CD (81 children with CD on GCD and 30 celiac patients on GFD) and a control group (10 children). Duration of GFD was 5.4 ± 1.6 years. TCRγδ IELs in celiac patients receiving a GCD or GFD were significantly higher (p < 0.001) than in the control group. NK-like IELs in patients receiving a GCD or GFD were significantly lower than in the control group (p < 0.001). We observed a permanent decrease of NK-like IELs and an increment of TCRγδ IELs after following an adequate establishment and compliance of a long-term GFD in celiac patients. Recognition of IELs changes in the intestinal mucosa on celiac patients after long-term establishment of a GFD could constitute a useful tool for CD diagnosis in various situations: in which there is doubt about the initial diagnosis and repeat biopsy is necessary (avoiding the need of gluten challenges), and in those patients with symptoms/signs suggestive of CD who maintain a low gluten diet.

Characterization of Common Chromosomal Translocations and Their Frequencies in Acute Myeloid Leukemia Patients of Northwest Iran

PubMed Central

Amanollahi Kamaneh, Elnaz; Shams Asenjan, Karim; Movassaghpour Akbari, Aliakbar; Akbarzadeh Laleh, Parvin; Chavoshi, Hadi; Eivazi Ziaei, Jamal; Nikanfar, Alireza; Asvadi Kermani, Iraj; Esfahani, Ali

2016-01-01

Objective Detection of chromosomal translocations has an important role in diagnosis and treatment of hematological disorders. We aimed to evaluate the 46 new cases of de novo acute myeloid leukemia (AML) patients for common translocations and to assess the effect of geographic and ethnic differences on their frequencies. Materials and Methods In this descriptive study, reverse transcriptase-polymerase chain reaction (RT-PCR) was used on 46 fresh bone marrow or peripheral blood samples to detect translocations t (8; 21), t (15; 17), t (9; 11) and inv (16). Patients were classified using the French-American-British (FAB) criteria in to eight sub-groups (M0-M7). Immunophenotyping and biochemical test results of patients were compared with RT-PCR results. Results Our patients were relatively young with a mean age of 44 years. AML was relatively predominant in female patients (54.3%) and most of patients belonged to AML-M2. Translocation t (8; 21) had the highest frequency (13%) and t (15; 17) with 2.7% incidence was the second most frequent. CD19 as an immunophenotypic marker was at a relatively high frequency (50%) in cases with t (8; 21), and patients with this translocation had a specific immunophenotypic pattern of complete expression of CD45, CD38, CD34, CD33 and HLA-DR. Conclusion Similarities and differences of results in Iran with different parts of the world can be explained with ethnic and geographic factors in characterizations of AML. Recognition of these factors especially in other comprehensive studies may aid better diagnosis and management of this disease. PMID:27054117

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Qing-Feng; Wang, Wei-Hu; Wang, Shu-Lian

Purpose: To investigate, in a large cohort of patients, the immunophenotypic and clinical differences of nasal and extranasal extranodal nasal-type natural killer/T-cell lymphoma of the upper aerodigestive tract (UADT-NKTCL) and examine the relevance of the immunophenotype on the clinical behavior, prognosis, and treatment. Methods and Materials: A total of 231 patients with UADT-NKTCL were recruited. One hundred eighty-one patients had primary location in the nasal cavity (nasal UADT-NKTCL), and 50 patients had primary extranasal UADT-NKTCL. Results: Patients with extranasal UADT-NKTCL had more adverse clinical features, including advanced-stage disease, regional lymph node involvement, B symptoms, and poor performance status, than patientsmore » with nasal UADT-NKTCL. In addition, CD56 and granzyme B were less frequently expressed in extranasal UADT-NKTCL. The 5-year overall survival rate was 74.1% for the entire group and 76.0% for early-stage disease. The 5-year overall survival rate for extranasal UADT-NKTCL was similar or superior to that of nasal UADT-NKTCL for all disease stages (76.9% vs 73.4%, P=.465), stage I disease (75.9% vs 79.2%, P=.786), and stage II disease (83.3% vs 50.3%, P=.018). CD56 expression and a Ki-67 proliferation rate ≥50% predicted poorer survival for extranasal UADT-NKTCL but not for nasal UADT-NKTCL. Conclusions: Patients with nasal and extranasal UADT-NKTCL have significantly different clinical features, immunophenotypes, and prognosis. Extranasal UADT-NKTCL should be considered as a distinct subgroup apart from the most commonly diagnosed prototype of nasal UADT-NKTCL.« less

Phenotype in combination with genotype improves outcome prediction in acute myeloid leukemia: a report from Children’s Oncology Group protocol AAML0531

PubMed Central

Voigt, Andrew P.; Brodersen, Lisa Eidenschink; Alonzo, Todd A.; Gerbing, Robert B.; Menssen, Andrew J.; Wilson, Elisabeth R.; Kahwash, Samir; Raimondi, Susana C.; Hirsch, Betsy A.; Gamis, Alan S.; Meshinchi, Soheil; Wells, Denise A.; Loken, Michael R.

2017-01-01

Diagnostic biomarkers can be used to determine relapse risk in acute myeloid leukemia, and certain genetic aberrancies have prognostic relevance. A diagnostic immunophenotypic expression profile, which quantifies the amounts of distinct gene products, not just their presence or absence, was established in order to improve outcome prediction for patients with acute myeloid leukemia. The immunophenotypic expression profile, which defines each patient’s leukemia as a location in 15-dimensional space, was generated for 769 patients enrolled in the Children’s Oncology Group AAML0531 protocol. Unsupervised hierarchical clustering grouped patients with similar immunophenotypic expression profiles into eleven patient cohorts, demonstrating high associations among phenotype, genotype, morphology, and outcome. Of 95 patients with inv(16), 79% segregated in Cluster A. Of 109 patients with t(8;21), 92% segregated in Clusters A and B. Of 152 patients with 11q23 alterations, 78% segregated in Clusters D, E, F, G, or H. For both inv(16) and 11q23 abnormalities, differential phenotypic expression identified patient groups with different survival characteristics (P<0.05). Clinical outcome analysis revealed that Cluster B (predominantly t(8;21)) was associated with favorable outcome (P<0.001) and Clusters E, G, H, and K were associated with adverse outcomes (P<0.05). Multivariable regression analysis revealed that Clusters E, G, H, and K were independently associated with worse survival (P range <0.001 to 0.008). The Children’s Oncology Group AAML0531 trial: clinicaltrials.gov Identifier: 00372593. PMID:28883080

Evaluation of Immunophenotypic and Molecular Biomarkers for Sézary Syndrome Using Standard Operating Procedures: A Multicenter Study of 59 Patients.

PubMed

Boonk, Stephanie E; Zoutman, Willem H; Marie-Cardine, Anne; van der Fits, Leslie; Out-Luiting, Jacoba J; Mitchell, Tracey J; Tosi, Isabella; Morris, Stephen L; Moriarty, Blaithin; Booken, Nina; Felcht, Moritz; Quaglino, Pietro; Ponti, Renata; Barberio, Emanuela; Ram-Wolff, Caroline; Jäntti, Kirsi; Ranki, Annamari; Bernengo, Maria Grazia; Klemke, Claus-Detlev; Bensussan, Armand; Michel, Laurence; Whittaker, Sean; Bagot, Martine; Tensen, Cornelis P; Willemze, Rein; Vermeer, Maarten H

2016-07-01

Differentiation between Sézary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sézary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sézary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sézary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sézary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4(+) T cells) and/or CD7 (≥40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sézary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sézary syndrome versus erythrodermic inflammatory dermatoses. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Immunophenotypic and Molecular Analysis of Human Dental Pulp Stem Cells Potential for Neurogenic Differentiation

PubMed Central

Fatima, Nikhat; Khan, Aleem A.; Vishwakarma, Sandeep K.

2017-01-01

Background: Growing evidence shows that dental pulp (DP) tissues could be a potential source of adult stem cells for the treatment of devastating neurological diseases and several other conditions. Aims: Exploration of the expression profile of several key molecular markers to evaluate the molecular dynamics in undifferentiated and differentiated DP-derived stem cells (DPSCs) in vitro. Settings and Design: The characteristics and multilineage differentiation ability of DPSCs were determined by cellular and molecular kinetics. DPSCs were further induced to form adherent (ADH) and non-ADH (NADH) neurospheres under serum-free condition which was further induced into neurogenic lineage cells and characterized for their molecular and cellular diversity at each stage. Statistical Analysis Used: Statistical analysis used one-way analysis of variance, Student's t-test, Livak method for relative quantification, and R programming. Results: Immunophenotypic analysis of DPSCs revealed >80% cells positive for mesenchymal markers CD90 and CD105, >70% positive for transferring receptor (CD71), and >30% for chemotactic factor (CXCR3). These cells showed mesodermal differentiation also and confirmed by specific staining and molecular analysis. Activation of neuronal lineage markers and neurogenic growth factors was observed during lineage differentiation of cells derived from NADH and ADH spheroids. Greater than 80% of cells were found to express β-tubulin III in both differentiation conditions. Conclusions: The present study reported a cascade of immunophenotypic and molecular markers to characterize neurogenic differentiation of DPSCs under serum-free condition. These findings trigger the future analyses for clinical applicability of DP-derived cells in regenerative applications. PMID:28566856

Correlation of karyotype and immunophenotype in childhood acute lymphoblastic leukemia; experience at the National Cancer Institute, Cairo University, Egypt.

PubMed

Hamouda, Faiza; El-Sissy, Azza H; Radwan, Ashraf K; Hussein, Hany; Gadallah, Farida H; Al-Sharkawy, Nahla; Sedhom, Eman; Ebeid, Emad; Salem, Shereen I

2007-06-01

To identify chromosomal pattern among the major immunophenotypic subgroups in Egyptian children with ALL, and its correlation with clinical presentation and disease free survival. Cytogenetic and immunophenotypic analysis were done for all patients. Patients received ALL-PNCI-III/98 chemotherapy protocol used at NCI, Cairo University. The frequency of pseudodiploidy and normal karyotype in the whole group was 42.9% and 33.3% respectively. The frequency of pseudodiploidy was 36.8% in CALLA positive early pre B, 30.7% in pre B cases, 71.4% in T cell cases and 100% in mature B cell cases. At 12 months, DFS was 50% for pseudodiploid group having pre B phenotype, compared to 16.6% for pseudodiploid group with CALLA positive early pre B ALL. Sixteen percent of the studied cases showed T cell phenotype, 71.4% of them showed pseudodiploid karyotype, all of them had high risk features. Hyperdiploidy was found in 31.5% of CALLA positive early pre B cases and was associated with favorable prognostic features and DFS of 66.6% at 12 months. Hyperdiploidy of >50 chromosome represented 62.5% of hyperdipoid cases, 80% of them were CALLA positive early pre B ALL carrying good risk features. Fifty percent of normal karyotypic patients showed pre B phenotype, while 42.8% showed CALLA positive early pre B ALL. Their age, TLC, DFS, were almost comparable. CALLA early pre B phenotype has a positive impact on chromosomal pattern having best outcome among patients with hyperdiploidy. The Pseudodiploid karyotype carries a better outcome with pre B phenotype.

Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS).

PubMed

Freeman, Christine M; Crudgington, Sean; Stolberg, Valerie R; Brown, Jeanette P; Sonstein, Joanne; Alexis, Neil E; Doerschuk, Claire M; Basta, Patricia V; Carretta, Elizabeth E; Couper, David J; Hastie, Annette T; Kaner, Robert J; O'Neal, Wanda K; Paine, Robert; Rennard, Stephen I; Shimbo, Daichi; Woodruff, Prescott G; Zeidler, Michelle; Curtis, Jeffrey L

2015-01-27

Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a "just-in-time" design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. This study was registered with ClinicalTrials.gov as NCT01969344 .

Prognosis of children with mixed phenotype acute leukemia treated on the basis of consistent immunophenotypic criteria

PubMed Central

Mejstrikova, Ester; Volejnikova, Jana; Fronkova, Eva; Zdrahalova, Katerina; Kalina, Tomas; Sterba, Jaroslav; Jabali, Yahia; Mihal, Vladimir; Blazek, Bohumir; Cerna, Zdena; Prochazkova, Daniela; Hak, Jiri; Zemanova, Zuzana; Jarosova, Marie; Oltova, Alexandra; Sedlacek, Petr; Schwarz, Jiri; Zuna, Jan; Trka, Jan; Stary, Jan; Hrusak, Ondrej

2010-01-01

Background Mixed phenotype acute leukemia (MPAL) represents a diagnostic and therapeutic dilemma. The European Group for the Immunological Classification of Leukemias (EGIL) scoring system unambiguously defines MPAL expressing aberrant lineage markers. Discussions surrounding it have focused on scoring details, and information is limited regarding its biological, clinical and prognostic significance. The recent World Health Organization classification is simpler and could replace the EGIL scoring system after transformation into unambiguous guidelines. Design and Methods Simple immunophenotypic criteria were used to classify all cases of childhood acute leukemia in order to provide therapy directed against acute lymphoblastic leukemia or acute myeloid leukemia. Prognosis, genotype and immunoglobulin/T-cell receptor gene rearrangement status were analyzed. Results The incidences of MPAL were 28/582 and 4/107 for children treated with acute lymphoblastic leukemia and acute myeloid leukemia regimens, respectively. In immunophenotypic principal component analysis, MPAL treated as T-cell acute lymphoblastic leukemia clustered between cases of non-mixed T-cell acute lymphoblastic leukemia and acute myeloid leukemia, while other MPAL cases were included in the respective non-mixed B-cell progenitor acute lymphoblastic leukemia or acute myeloid leukemia clusters. Analogously, immunoglobulin/T-cell receptor gene rearrangements followed the expected pattern in patients treated as having acute myeloid leukemia (non-rearranged, 4/4) or as having B-cell progenitor acute lymphoblastic leukemia (rearranged, 20/20), but were missing in 3/5 analyzed cases of MPAL treated as having T-cell acute lymphobastic leukemia. In patients who received acute lymphoblastic leukemia treatment, the 5-year event-free survival of the MPAL cases was worse than that of the non-mixed cases (53±10% and 76±2% at 5 years, respectively, P=0.0075), with a more pronounced difference among B lineage cases. The small numbers of MPAL cases treated as T-cell acute lymphoblastic leukemia or as acute myeloid leukemia hampered separate statistics. We compared prognosis of all subsets with the prognosis of previously published cohorts. Conclusions Simple immunophenotypic criteria are useful for therapy decisions in MPAL. In B lineage leukemia, MPAL confers poorer prognosis. However, our data do not justify a preferential use of current acute myeloid leukemia-based therapy in MPAL. PMID:20145275

Undifferentiated carcinoma of parotid gland.

PubMed Central

López, J I; Alfaro, J; Ballestin, C

1991-01-01

Two cases of undifferentiated carcinomas of the major salivary glands were studied using immunohistochemical techniques. Results showed that this entity was a high grade malignant neoplasm arising from the excretory duct. Despite the undifferentiated appearance multiple immunophenotypes were evident in both cases. PMID:2045506

Understanding Laboratory Tests

MedlinePlus

... it measures: Changes in the number and/or structure of chromosomes in a patient’s white blood cells or bone marrow cells How it is used: Diagnosis, deciding on appropriate treatment Immunophenotyping What it measures: Identifies cells based on the types of antigens present on the ...

[Cytomorphology of acute mixed leukemia].

PubMed

Sucić, Mirna; Batinić, Drago; Zadro, Renata; Mrsić, Sanja; Labar, Boris

2008-10-01

Biphenotypic acute leukemias (AL) with blasts expressing both myeloid and lymphoid antigens are grouped with undifferentiated AL and bilineal AL in the group of AL of ambiguous lineage. Not all AL with myeloid and lymphoid antigens (ALMy+Ly) are true biphenotypic AL. According to EGIL scoring system, true biphenotypic ALMy+Ly are those with a sum of antigens 2 or more points for both myeloid and lymphoid lineage or for B and T lineage. The aim of this study was to compare cytomorphology and immunophenotype of AL to better understand the relation of certain AL morphology, immunophenotype, cytogenetics and molecular biology of biphenotypic AL. The study included a group of 169 AL patients treated from 1985 till 1991, and a group of 102 AL patients treated from 1993 till 1996 at Zagreb University Hospital Center. Bone marrow and peripheral blood of the two groups of AL patients were analyzed according to Pappenheim (May-Grunwald-Giemsa), cytochemical and alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunocytochemical staining. Flow cytometry immunophenotyping of bone marrow was also done in both patient groups. In the group of 169 adult AL patients, 116 were cytomorphologically classified as acute myeloblastic leukemias (AML), 35 as acute lymphoblastic leukemias (ALL) and 18 as acute undifferentiated leukemias (ANLM). In 6 (3.4%) of 169 AL patients, blasts expressed both myeloid and lymphoid antigens. In the group of 102 AL patients there were 19 (18.6%) ALMy+Ly. In 64 patients cytomorphologically classified into AML subgroup out of 102 AL patients, there were 15 (14.7%/102; 23.4%/64) AML with lymphoid antigens (AMLLy+). In 35 patients cytomorphologically diagnosed as ALL and 3 as ANLM out of 102 AL, there were 4 (3.9%/102; 10.5%/38) ALL with myeloid antigens (ALLMy+). The incidence of mixed AL in 102 AL was more consistent with other studies, pointing to the necessity of myeloperoxidase (MPO), CD7 and TdT determination as part of standard immunophenotyping for better recognition of mixed AL. In both groups of 169 and 102 AL patients, the majority of AL cases were cytomorphologically classified as AML. In the group of 169 patients there were 5 AMLLy+ and in the group of 102 patients there were 15 AMLLy+. In one ANLM,My+ out of 169 AL and also one ANLM,My+ out of 102 AL, blasts were cytomorphologically undifferentiated; in 3 ALLMy+ of 102 AL blasts expressed lymphoid morphology. According to EGIL scoring system, among 15 AMLLy+ of 102 AL there were 4 true biphenotypic ALMy+Ly (1 M1, 2 M3, 1 M4), and in 4 ALMy+Ly with undifferentiated and lymphoid morphology there were 2 true biphenotypic AL (1 L2; 1 ANLM). In 3 ALLB+T out of 35 ALL, one was interlineal biphenotypic AL. These observations are consistent with other studies and WHO determinations indicating that the majority of true biphenotypic leukemias are associated with immature monoblastic or myeloid cytomorphology or with lymphoid or undifferentiated characteristics, but may also express any AML cytomorphology type. Thus, there is no direct correlation of leukemic cell cytomorphology and biphenotypic AL immunophenotype.

Authentication of Primordial Characteristics of the CLBL-1 Cell Line Prove the Integrity of a Canine B-Cell Lymphoma in a Murine In Vivo Model

PubMed Central

Reimann-Berg, Nicola; Walter, Ingrid; Fuchs-Baumgartinger, Andrea; Wagner, Siegfried; Kovacic, Boris; Essler, Sabine E.; Schwendenwein, Ilse; Nolte, Ingo; Saalmüller, Armin; Escobar, Hugo Murua

2012-01-01

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2−/−γc −/− mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45+, MHCII+, CD11a+ and CD79αcy+. PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies. PMID:22761949

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model.

PubMed

Rütgen, Barbara C; Willenbrock, Saskia; Reimann-Berg, Nicola; Walter, Ingrid; Fuchs-Baumgartinger, Andrea; Wagner, Siegfried; Kovacic, Boris; Essler, Sabine E; Schwendenwein, Ilse; Nolte, Ingo; Saalmüller, Armin; Murua Escobar, Hugo

2012-01-01

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-)γ(c) (-/-) mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+), MHCII(+), CD11a(+) and CD79αcy(+). PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.

Immature MEF2C-dysregulated T-cell leukemia patients have an early T-cell precursor acute lymphoblastic leukemia gene signature and typically have non-rearranged T-cell receptors

PubMed Central

Zuurbier, Linda; Gutierrez, Alejandro; Mullighan, Charles G.; Canté-Barrett, Kirsten; Gevaert, A. Olivier; de Rooi, Johan; Li, Yunlei; Smits, Willem K.; Buijs-Gladdines, Jessica G.C.A.M.; Sonneveld, Edwin; Look, A. Thomas; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

2014-01-01

Three distinct immature T-cell acute lymphoblastic leukemia entities have been described including cases that express an early T-cell precursor immunophenotype or expression profile, immature MEF2C-dysregulated T-cell acute lymphoblastic leukemia cluster cases based on gene expression analysis (immature cluster) and cases that retain non-rearranged TRG@ loci. Early T-cell precursor acute lymphoblastic leukemia cases exclusively overlap with immature cluster samples based on the expression of early T-cell precursor acute lymphoblastic leukemia signature genes, indicating that both are featuring a single disease entity. Patients lacking TRG@ rearrangements represent only 40% of immature cluster cases, but no further evidence was found to suggest that cases with absence of bi-allelic TRG@ deletions reflect a distinct and even more immature disease entity. Immature cluster/early T-cell precursor acute lymphoblastic leukemia cases are strongly enriched for genes expressed in hematopoietic stem cells as well as genes expressed in normal early thymocyte progenitor or double negative-2A T-cell subsets. Identification of early T-cell precursor acute lymphoblastic leukemia cases solely by defined immunophenotypic criteria strongly underestimates the number of cases that have a corresponding gene signature. However, early T-cell precursor acute lymphoblastic leukemia samples correlate best with a CD1 negative, CD4 and CD8 double negative immunophenotype with expression of CD34 and/or myeloid markers CD13 or CD33. Unlike various other studies, immature cluster/early T-cell precursor acute lymphoblastic leukemia patients treated on the COALL-97 protocol did not have an overall inferior outcome, and demonstrated equal sensitivity levels to most conventional therapeutic drugs compared to other pediatric T-cell acute lymphoblastic leukemia patients. PMID:23975177

Through the glass darkly: intraepithelial neoplasia, top-down differentiation and the road to pelvic serous cancer

PubMed Central

Crum, Christopher P; Herfs, Michael; Ning, Gang; Bijron, Jonathan G.; Howitt, Brooke E.; Jimenez, Cynthia A.; Hanamornroongruang, Suchanan; McKeon, Frank D.; Xian, Wa

2014-01-01

The origins of pelvic high grade serous cancer (HGSC) have become a subject of intense scrutiny in view of proposals to reduce the incidence of the disease via opportunistic salpingectomy in healthy women. Accumulated data implicates the fimbria as a site of origin and descriptive molecular pathology and experimental evidence strongly support a serous carcinogenic sequence in the fallopian tube. Both direct and indirect ("surrogate") precursors suggest the benign tube undergoes important biologic changes after menopause, acquiring abnormalities in gene expression that are shared with malignancy. However, the tube can be linked to only some HGSCs, recharging arguments that nearby peritoneum/ovarian surface epithelium (POSE) also hosts progenitors to this malignancy. A major sticking point is the difference in immunophenotype between POSE and Müllerian epithelium, essentially requiring mesothelial to Müllerian differentiation prior to or during malignant transformation to HGSC. However, there is emerging evidence that an embryonic or progenitor phenotype exists in the adult female genital tract with the capacity to differentiate, normally or during neoplastic transformation. Recently, a putative cell of origin to cervical cancer has been identified in the squamo-columnar (SC) junction, projecting a model whereby embryonic progenitors give rise to immuno-phenotypically distinct neoplastic progeny under stromal influences via "top down" differentiation. A similar pattern of differentiation is implied in the endometrium and the juxtaposition of disparate epithelial immuno-phenotypes (POSE and underlying Müllerian inclusions) recapitulates this in the ovary. While a sudden mesothelial-Mullerian transition remains to be proven, it would explain the rapid evolution, short asymptomatic interval, and absence of a defined epithelial starting point in many HGSCs. Resolving this question will be critical to both expectations from prophylactic salpingectomy and future approaches to pelvic serous cancer prevention. PMID:24030860

Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets.

PubMed

Boheler, Kenneth R; Gundry, Rebekah L

2017-01-01

Stem cells and their derivatives hold great promise to advance regenerative medicine. Critical to the progression of this field is the identification and utilization of antibody-accessible cell-surface proteins for immunophenotyping and cell sorting-techniques essential for assessment and isolation of defined cell populations with known functional and therapeutic properties. Beyond their utility for cell identification and selection, cell-surface proteins are also major targets for pharmacological intervention. Although comprehensive cell-surface protein maps are highly valuable, they have been difficult to define until recently. In this review, we discuss the application of a contemporary targeted chemoproteomic-based technique for defining the cell-surface proteomes of stem and progenitor cells. In applying this approach to pluripotent stem cells (PSCs), these studies have improved the biological understanding of these cells, led to the enhanced use and development of antibodies suitable for immunophenotyping and sorting, and contributed to the repurposing of existing drugs without the need for high-throughput screening. The utility of this latter approach was first demonstrated with human PSCs (hPSCs) through the identification of small molecules that are selectively toxic to hPSCs and have the potential for eliminating confounding and tumorigenic cells in hPSC-derived progeny destined for research and transplantation. Overall, the cutting-edge technologies reviewed here will accelerate the development of novel cell-surface protein targets for immunophenotyping, new reagents to improve the isolation of therapeutically qualified cells, and pharmacological studies to advance the treatment of intractable diseases amenable to cell-replacement therapies. Stem Cells Translational Medicine 2017;6:131-138. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

The effect of pro-inflammatory cytokines on immunophenotype, differentiation capacity and immunomodulatory functions of human mesenchymal stem cells.

PubMed

Pourgholaminejad, Arash; Aghdami, Nasser; Baharvand, Hossein; Moazzeni, Seyed Mohammad

2016-09-01

Mesenchymal stem cells (MSCs), as cells with potential clinical utilities, have demonstrated preferential incorporation into inflammation sites. Immunophenotype and immunomodulatory functions of MSCs could alter by inflamed-microenvironments due to the local pro-inflammatory cytokine milieu. A major cellular mediator with specific function in promoting inflammation and pathogenicity of autoimmunity are IL-17-producing T helper 17 (Th17) cells that polarize in inflamed sites in the presence of pro-inflammatory cytokines such as Interleukin-1β (IL-1β), IL-6 and IL-23. Since MSCs are promising candidate for cell-based therapeutic strategies in inflammatory and autoimmune diseases, Th17 cell polarizing factors may alter MSCs phenotype and function. In this study, human bone-marrow-derived MSCs (BM-MSC) and adipose tissue-derived MSCs (AD-MSC) were cultured with or without IL-1β, IL-6 and IL-23 as pro-inflammatory cytokines. The surface markers and their differentiation capacity were measured in cytokine-untreated and cytokine-treated MSCs. MSCs-mediated immunomodulation was analyzed by their regulatory effects on mixed lymphocyte reaction (MLR) and the level of IL-10, TGF-β, IL-4, IFN-γ and TNF-α production as immunomodulatory cytokines. Pro-inflammatory cytokines showed no effect on MSCs morphology, immunophenotype and co-stimulatory molecules except up-regulation of CD45. Adipogenic and osteogenic differentiation capacity increased in CD45+ MSCs. Moreover, cytokine-treated MSCs preserved the suppressive ability of allogeneic T cell proliferation and produced higher level of TGF-β and lower level of IL-4. We concluded pro-inflammatory cytokines up-regulate the efficacy of MSCs in cell-based therapy of degenerative, inflammatory and autoimmune disorders. Copyright © 2016. Published by Elsevier Ltd.

Acute leukemias of ambiguous lineage.

PubMed

Béné, Marie C; Porwit, Anna

2012-02-01

The 2008 edition of the WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues recognizes a special category called "leukemias of ambiguous lineage." The vast majority of these rare leukemias are classified as mixed phenotype acute leukemia (MPAL), although acute undifferentiated leukemias and natural killer lymphoblastic leukemias are also included. The major immunophenotypic markers used by the WHO 2008 to determine the lineage for these proliferations are myeloperoxidase, CD19, and cytoplasmic CD3. However, extensive immunophenotyping is necessary to confirm that the cells indeed belong to 2 different lineages or coexpress differentiation antigens of more than 1 lineage. Specific subsets of MPAL are defined by chromosomal anomalies such as the t(9;22) Philadelphia chromosome BCR-ABL1 or involvement of the MLL gene on chromosome 11q23. Other MPAL are divided into B/myeloid NOS, T/myeloid NOS, B/T NOS, and B/T/myeloid NOS. MPAL are usually of dire prognosis, respond variably to chemotherapy of acute lymphoblastic or acute myeloblastic type, and benefit most from rapid allogeneic hematopoietic stem cell transplantation.

Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines

PubMed Central

Korać, P; Jones, M; Dominis, M; Kušec, R; Mason, D Y; Banham, A H; Ventura, R A

2005-01-01

The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease. PMID:16311361

Childhood nodal marginal zone lymphoma with unusual clinicopathologic and cytogenetic features for the pediatric variant: a case report.

PubMed

Aqil, Barina; Merritt, Brian Y; Elghetany, M Tarek; Kamdar, Kala Y; Lu, Xinyan Y; Curry, Choladda V

2015-01-01

Nodal marginal zone lymphoma (NMZL) is a B-cell lymphoma that shares morphologic and immunophenotypic features with extranodal and splenic marginal zone lymphomas but lacks extranodal or splenic involvement at presentation. NMZL occurs mostly in adults with no sex predilection, at advanced stage (III or IV), with frequent relapses and a high incidence of tumoral genetic abnormalities including trisomies 3 and 18 and gain of 7q. Pediatric NMZL, however, is a rare but distinct variant of NMZL with characteristic features including male predominance, asymptomatic and localized (stage I) disease, low relapse rates with excellent outcomes, and a lower incidence of essentially similar genetic aberrations compared to adult NMZL. Here we describe a unique case of childhood NMZL with unusual clinicopathologic features for the pediatric variant including generalized lymphadenopathy, high-stage disease with persistence after therapy, unusual immunophenotype (CD5, CD23, and BCL6 positive), and unique chromosomal abnormalities including monosomy 20 and add(10)(p11.2).

Primary Intracranial Myoepithelial Neoplasm: A Potential Mimic of Meningioma.

PubMed

Choy, Bonnie; Pytel, Peter

2016-05-01

Myoepithelial neoplasms were originally described in the salivary glands but their spectrum has been expanding with reports in other locations, including soft tissue. Intracranial cases are exceptionally rare outside the sellar region where they are assumed to be arising from Rathke pouch rests. Two cases of pediatric intracranial myoepithelial neoplasm in the interhemispheric fissure and the right cerebral hemisphere are reported here. Imaging studies suggest that the second case was associated with cerebrospinal fluid dissemination. Both cases showed typical variation in morphology and immunophenotype between more epithelioid and more mesenchymal features. The differential diagnosis at this particular anatomic location includes meningioma, which can show some overlap in immunophenotype since both tumors express EMA as well as GLUT1. One case was positive for EWSR1 rearrangement by fluorescence in situ hybridization. One patient is disease free at last follow-up while the other succumbed to the disease within days illustrating the clinical spectrum of these tumors. © The Author(s) 2015.

Obesity Determines the Immunophenotypic Profile and Functional Characteristics of Human Mesenchymal Stem Cells From Adipose Tissue

PubMed Central

Pachón-Peña, Gisela; Serena, Carolina; Ejarque, Miriam; Petriz, Jordi; Duran, Xevi; Oliva-Olivera, W.; Simó, Rafael; Tinahones, Francisco J.

2016-01-01

Adipose tissue is a major source of mesenchymal stem cells (MSCs), which possess a variety of properties that make them ideal candidates for regenerative and immunomodulatory therapies. Here, we compared the immunophenotypic profile of human adipose-derived stem cells (hASCs) from lean and obese individuals, and explored its relationship with the apparent altered plasticity of hASCs. We also hypothesized that persistent hypoxia treatment of cultured hASCs may be necessary but not sufficient to drive significant changes in mature adipocytes. hASCs were obtained from subcutaneous adipose tissue of healthy, adult, female donors undergoing abdominal plastic surgery: lean (n = 8; body mass index [BMI]: 23 ± 1 kg/m2) and obese (n = 8; BMI: 35 ± 5 kg/m2). Cell surface marker expression, proliferation and migration capacity, and adipogenic differentiation potential of cultured hASCs at two different oxygen conditions were studied. Compared with lean-derived hASCs, obese-derived hASCs demonstrated increased proliferation and migration capacity but decreased lipid droplet accumulation, correlating with a higher expression of human leukocyte antigen (HLA)-II and cluster of differentiation (CD) 106 and lower expression of CD29. Of interest, adipogenic differentiation modified CD106, CD49b, HLA-ABC surface protein expression, which was dependent on the donor’s BMI. Additionally, low oxygen tension increased proliferation and migration of lean but not obese hASCs, which correlated with an altered CD36 and CD49b immunophenotypic profile. In summary, the differences observed in proliferation, migration, and differentiation capacity in obese hASCs occurred in parallel with changes in cell surface markers, both under basal conditions and during differentiation. Therefore, obesity is an important determinant of stem cell function independent of oxygen tension. Significance The obesity-related hypoxic environment may have latent effects on human adipose tissue-derived mesenchymal stem cells (hASCs) with potential consequences in mature cells. This study explores the immunophenotypic profile of hASCs obtained from lean and obese individuals and its potential relationship with the altered plasticity of hASCs observed in obesity. In this context, an altered pattern of cell surface marker expression in obese-derived hASCs in both undifferentiated and differentiated stages is demonstrated. Differences in proliferation, migration, and differentiation capacity of hASCs from obese adipose tissue correlated with alterations in cell surface expression. Remarkably, altered plasticity observed in obese-derived hASCs was maintained in the absence of hypoxia, suggesting that these cells might be obesity conditioned. PMID:26956208

Eosinophilia in a cat with acute leukemia

PubMed Central

Gilroy, Cornelia; Forzán, María; Drew, Anne; Vernau, William

2011-01-01

A 4-year-old castrated male domestic shorthaired cat with a history of vomiting and anorexia was diagnosed with leukemia with marked hepatic and splenic infiltration and concurrent eosinophilia with marked tissue infiltration. Despite thorough immunocytochemical and immunohistochemical immunophenotyping, the cell lineage of the leukemia was not identified. PMID:22379202

Cellular Consequences of Telomere Shortening in Histologically Normal Breast Tissues

DTIC Science & Technology

2010-09-01

quantitative PCR (10) or with a chemiluminescent-based slot blot assay that measures telomere DNA content, a proxy of telomere length (9, 11, 12). These...Subhawong AP, Subhawong T, Nassar H, et al. Most basal-like breast carcinomas demonstrate the same Rb-/p16+ immunophenotype as the HPV -related poorly

Immunophenotyping of acute leukaemias by flow cytometry: a review.

PubMed

Pamnani, R

2009-12-01

To provide an overview of the utility of flow cytometry for phenotyping of acute leukaemias and selection-of monoclonal antibodies. The literature review was obtained through internet, journals and chapters in the relevant books. Relevant articles and chapters on immunophenotyping of acute leukaemias were selected from respected international journals and books in the field of haematology and were reviewed. Complete articles relevant to the topic were selected and reviewed and the necessary information extracted for this review. Flow cytometry has been used extensively in recent years to characterise haemopoeitic malignancies and done routinely in the developed world. This technique has greatly improved the diagnosis and classification of haemopoeitic malignancies and has been recommended by World Health Organisation classification (WHO) of tumours of haemopoeitic and lymphoid tissue. Application of flow cytometry for the diagnosis of leukaemias has been recently introduced in Kenya and is currently being undertaken in research using limited but appropriate panels of monoclonal antibodies. It is hoped that findings of this research will inform the use of flow cytometry as an ancillary diagnostic technique in our resource-constrained set up.

Use of Monoclonal Antibodies for the Diagnosis of T-cell Malignancies: Applications and Limitations.

PubMed

Hastrup, N; Pallesen, G; Ralfikiaer, E

1990-01-01

Biopsy samples from 136 peripheral T-cell lymphomas have been examined and compared with benign inflammatory T-cell infiltrates in an attempt to establish whether immunohistological methods may help to improve the distinction between these conditions. The results confirm and extend previous reports and indicate that the aberrant T-cell phenotypes constitute the single most reliable criterion for the distinction between benign and malignant T-cell infiltrates. These phenotypes are expressed frequently in T-cell malignancies in. lymphoid organs and are also seen in a substantial number of biopsy samples from advanced cutaneous T-cell lymphomas (CTCL). In contrast, early CTCL do not express aberrant T-cell phenotypes and are indistinguishable from benign cutaneous conditions in terms of their immunophenotypic properties. It is concluded that immunophenotypic techniques form a valuable supplement to routine histological methods for the diagnosis of T-cell lymphomas in lymphoid organs. The methods may also help to improve the diagnosis of advanced CTCL, but are of no or only limited help for the recognition of the early stages.

Macrophages are related to goblet cell hyperplasia and induce MUC5B but not MUC5AC in human bronchus epithelial cells.

PubMed

Silva, Manuel A; Bercik, Premysl

2012-06-01

Airway goblet cell hyperplasia (GCH)--detectable by mucin staining--and abnormal macrophage infiltrate are pathological features present in many chronic respiratory disorders. However, it is unknown if both factors are associated. Using in-vivo and in-vitro models, we investigated whether macrophages are related with GCH and changes in mucin immunophenotypes. Lung sections from Sprague-Dawley rats treated for 48 h with one intra-tracheal dose of PBS or LPS (n=4-6 per group) were immunophenotyped for rat-goblet cells, immune, and proliferation markers. Human monocyte-derived macrophages (MDM) were pre-treated with or without LPS, immunophenotyped, and their supernatant, as well as cytokines at levels equivalent to supernatant were used to challenge primary culture of normal human bronchus epithelial cells (HBEC) in air-liquid interface, followed by MUC5B and MUC5AC mucin immunostaining. An association between increased bronchiolar goblet cells and terminal-bronchiolar proliferative epithelial cells confirmed the presence of GCH in our LPS rat model, which was related with augmented bronchiolar CD68 macrophage infiltration. The in-vitro experiments have shown that MUC5AC phenotype was inhibited when HBEC were challenged with supernatant from MDM pre-treated with or without LPS. In contrast, TNF-α and interleukin-1β at levels equivalent to supernatant from LPS-treated MDM increased MUC5AC. MUC5B was induced by LPS, supernatant from LPS-treated MDM, a mix of cytokines including TNF-α and TNF-α alone at levels present in supernatant from LPS-treated MDM. We demonstrated that macrophages are related with bronchiolar GCH, and that they induced MUC5B and inhibited MUC5AC in HBEC, suggesting a role for them in the pathogenesis of airway MUC5B-related GCH.

Immunophenotypic and genetic characteristics of diffuse large B-cell lymphoma in Taiwan.

PubMed

Chang, Sheng-Tsung; Chen, Shang-Wen; Ho, Chung-Han; Kuo, Chun-Chi; Sakata, Seiji; Takeuchi, Kengo; Chuang, Shih-Sung

2016-11-01

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma type. The immunophenotypic and genetic features of DLBCL in Taiwan have not been characterized. In this study, we performed immunohistochemical analysis and interphase fluorescence in situ hybridization (FISH) using tissue microarray sections to investigate a cohort of unselected DLBCL cases in a single institution in Taiwan from 1990 to 2010. Of the 153 cases investigated, CD10, bcl-6, and MUM1 were expressed in 16.3%, 71.2%, and 71.9% cases, respectively, with 27.5% (n = 42) of cases being classified as having a germinal center B-cell (GCB) origin by the Hans algorithm. By FISH analysis, 19.6%, 4.6%, 26.1%, and 3.9% cases showed rearrangement at IGH, BCL2, BCL6, and MYC loci, respectively, including three (2.0%) cases of double-hit lymphoma. As compared with the non-GCB tumors, GCB tumors more frequently expressed CD10 (p < 0.001) and bcl-6 (p = 0.001) with less frequent expression of MUM1 (p = 0.007). Moreover, GCB tumors more frequently exhibited rearrangement at the BCL2 (p = 0.024) and MYC (p = 0.038) loci than non-GCB tumors. However, there was no survival difference between these two groups. In this first series of DLBCL evaluation from Taiwan, we found that the relative frequency of GCB tumors among DLBCL was low in most East Asian countries. There is a wide range of BCL2 rearrangement rates, higher in the West and lower in East Asia. A larger and/or national study is warranted to better understand the immunophenotypic and molecular features of DLBCL in Taiwan and their respective impact on patient survival. Copyright © 2016. Published by Elsevier B.V.

Infectious mononucleosis mimicking lymphoma: distinguishing morphological and immunophenotypic features.

PubMed

Louissaint, Abner; Ferry, Judith A; Soupir, Chad P; Hasserjian, Robert P; Harris, Nancy L; Zukerberg, Lawrence R

2012-08-01

The diagnosis of infectious mononucleosis (acute Epstein-Barr virus (EBV) infection) is usually made on the basis of clinical and laboratory findings. However, an atypical clinical presentation occasionally results in a lymph node or tonsillar biopsy. The morphological features of EBV-infected lymphoid tissue can easily mimic lymphoma. Furthermore, the immunophenotype of the immunoblasts has not been well characterized. To assess the morphological spectrum of acute EBV infection and the utility of immunohistochemistry in diagnosing difficult cases that resemble lymphoma, we reviewed 18 cases of acute EBV infection submitted in consultation to our institution with an initial diagnosis of/or suspicion for lymphoma. Patients included nine male and nine female individuals with a median age of 18 years (range 9-69). Biopsies were obtained from lymph nodes (3/18) or Waldeyer's ring (15/18). Infectious mononucleosis was confirmed by monospot or serological assays in 72% of cases (13/18). All cases featured architectural distortion by a polymorphous infiltrate with an immunoblastic proliferation, sometimes forming sheets. Reed-Sternberg-like cells were present in 8/18 (44%) of the cases. Infiltrates were often accompanied by necrosis (10/18) and mucosal ulceration (6/15). The majority of immunoblasts in all cases were CD20+ B cells with a post-germinal center immunophenotype (strongly positive for MUM1/IRF4 (18/18), CD10- (18/18 negative) and BCL-6- (16/18 negative; 2/18 faint BCL-6 expression in <10% of immunoblasts)). Immunoblasts showed variable weak expression of BCL-2 and polyclonal expression of κ and λ immunoglobulin light chains in 81% cases. Reed-Sternberg-like cells in 8/8 cases were CD30+, CD15-, BOB.1+ and OCT-2+. In conclusion, an atypical lymphoid infiltrate with numerous MUM1+, CD10-, BCL-6- immunoblasts should raise the suspicion of a reactive process, such as infectious mononucleosis, and warrants additional consideration before a diagnosis of lymphoma is made.

Specific pathogen-free status alters immunophenotype in rhesus macaques: implications for the study of simian immunodeficiency virus.

PubMed

Santos, Rosemary V; Lin, Kuei-Chin; Mansfield, Keith; Wachtman, Lynn M

2011-10-01

The repertoire of viruses to which research primates are exposed, even in the absence of clinical disease, may contribute to experimental confounding. In this study we examined whether standard specific pathogen-free (SPF) rhesus macaques exposed to a wider spectrum of enzootic viruses and expanded SPF macaques derived to exclude a greater number of viral agents would display alterations in immune activation or immune cell populations. Given the impact of immunophenotype on human immunodeficiency virus (HIV) progression and the importance of the simian immunodeficiency virus (SIV) model for the study of HIV pathogenesis, we elected to additionally examine the impact of SPF status on the capacity of peripheral blood mononuclear cells (PBMCs) to support SIV replication. The expanded SPF group displayed significant immune alterations including increased serum interleukin (IL)-15 and a greater in vitro elaboration of GM-CSF, IL1ra, VEGF, IL-10, IL12/23, and MIP-1b. Consistent with reduced viral antigenic exposure in expanded SPF macaques, decreased CD4(+) and CD8(+) transitional and effector memory (T(EM)) cell populations were observed. Expanded SPF PBMC cultures also demonstrated an increased peak (192.61 ng/ml p27) and area under the curve in in vitro SIV production (1968.64 ng/ml p27) when compared to standard SPF macaques (99.32 ng/ml p27; p=0.03 and 915.17 ng/ml p27; p=0.03, respectively). In vitro SIV replication did not correlate with CD4(+) T(EM) cell counts but was highly correlated with serum IL-15 in the subset of animals examined. Findings suggest that an altered immunophenotype associated with the maintenance of primates under differing levels of bioexclusion has the potential to impact the outcome of SIV studies and models for which the measurement of immunologic endpoints is critical.

Eight color immunophenotyping of T-, B- and NK-cell subpopulations for characterization of chronic immunodeficiencies.

PubMed

A, Boldt; S, Borte; S, Fricke; K, Kentouche; F, Emmrich; M, Borte; F, Kahlenberg; U, Sack

2014-01-16

Background: The heterogeneity of primary and secondary immunodeficiencies demands for the development of a comprehensive flow cytometric screening system, based on reference values that support a standardized immunophenotypic characterization of most lymphocyte subpopulations. Methods: Peripheral blood samples from healthy adult volunteers (n=25) were collected and split into eight panel fractions (100µl each). Subsequently, pre-mixed 8-color antibody cocktails were incubated per specific panel of whole blood to detect and differentiate cell subsets of: (i) a general lymphocyte overviews, (ii) B-cell subpopulations, (iii) CD4+ subpopulations, (iv) CD8+ subpopulations, (v) regulatory T-cells, (vi) recent thymic emigrants, (vii) NK-cell subpopulations, (viii) NK-cell activation markers. All samples were lysed, washed and measured by flow cytometry. FACS DIVA software was used for data analysis and calculation of quadrant statistics (mean values, standard error of mean, percentile ranges). Results: Whole blood staining of lymphocytes provided the analysis of: (i) CD3+, 4+, 8+, 19+, 16/56+, and activated CD4/8 cells; (ii) immature, naïve, non-switched/switched, memory, (activated) CD21 low , transitional B-cells, plasmablasts/plasmacells; (iii and iv) naïve, central memory, effector, effector memory, TH1/TH2/TH17-like and CCR5+CD8-cells; (v) CD25+, regulatory T-cells (naïve/memory, HLA-DR+); (vi) α/β- and γ/δ-T-cells, recent thymic emigrants in CD4/CD8 cells; (vii) immature/mature CD56 bright , CD94/NKG2D+ NK-cells; and (viii) Nkp30, 44, 46 and CD57+NK-cells. Clinical examples and quadrant statistics are provided. Conclusion: The present study represents a practical approach to standardize the immunophenotyping of most T-, B- and NK-cell subpopulations. That allows differentiating, whether abnormalities or developmental shifts observed in lymphocyte subpopulations originates either from primary or secondary immunological disturbance. © 2014 Clinical Cytometry Society. Copyright © 2014 Clinical Cytometry Society.

Polymorphous low grade adenocarcinoma has a consistent p63+/p40- immunophenotype that helps distinguish it from adenoid cystic carcinoma and cellular pleomorphic adenoma.

PubMed

Rooper, Lisa; Sharma, Rajni; Bishop, Justin A

2015-03-01

Polymorphous low grade adenocarcinoma (PLGA) is a tumor of minor salivary glands that exhibits considerable morphologic overlap with adenoid cystic carcinoma and cellular pleomorphic adenoma, especially in small biopsy specimens. Unlike these other tumor types. PLGAs do not harbor a myoepithelial component, yet their frequent positivity for p63 diminishes the usefulness of this particular myoepithelial marker as a discriminating immunostain. p40 is an antibody that recognizes ΔNp63, a p63 isoform that is more specific for true myoepithelial differentiation. As such, p40 immunostaining could help distinguish PLGAs from adenoid cystic carcinomas and pleomorphic adenomas. In this study, p63 and p40 immunohistochemistry was performed on paraffin embedded, formalin fixed tissue from 11 PLGAs, 101 adenoid cystic carcinomas, and 31 pleomorphic adenomas. All 11 PLGAs (100 %) were positive for p63 but completely negative for p40. Among adenoid cystic carcinomas, 91 of 101 (90 %) were positive for p63 and 90/101 (89 %) were positive for p40. The single discordant p63+/p40- adenoid cystic carcinoma exhibited solid architecture and high grade features not typically seen in PLGA. Among pleomorphic adenomas, 21/31 (68 %) were positive for p63 and 13/31 (42 %) were positive for p40. For the pleomorphic adenomas, the discordant p63+/p40- staining pattern was seen only in the overtly mesenchymal chondromyxoid stroma. The cellular epithelial component of the pleomorphic adenomas demonstrated concordant p63+/p40+ or p63-/p40- immunophenotypes. PLGA consistently exhibits a p63+/p40- immunophenotype that can help distinguish it from adenoid cystic carcinoma and cellular pleomorphic adenoma, tumors that characteristically demonstrate concordant p63 and p40 immunostaining patterns. A p63/p40 immunohistochemical panel can provide a valuable tool for making the distinction between these morphologically similar but clinically divergent entities.

Mixed-phenotypic acute leukemia series from tertiary care center.

PubMed

Pawar, Ravikiran N; Banerjee, Sambhunath; Bramha, Subhajit; Krishnan, Shekhar; Bhattacharya, Arpita; Saha, Vaskar; Chakrapani, Anupam; Bhave, Saurabh; Chandy, Mammen; Nair, Reena; Parihar, Mayur; Arora, Neeraj; Mishra, D K

2017-01-01

Mixed-phenotype acute leukemias (MPALs) are a heterogeneous group of rare leukemias constituting approximately 2%-5% of all leukemias, in which assigning a single lineage of origin is not possible. They are diagnosed by either the presence of antigens of more than one lineage or by the presence of dual population of blasts belonging to two or more lineages. We highlight the clinicopathological, immunophenotype, and genetic data of a cohort (n = 14) of patients diagnosed and treated at our center. We retrospectively analyzed consecutive cases of MPAL diagnosed in our flow cytometry laboratory from May 2012 to August 2015. These cases were diagnosed based on immunophenotyping of peripheral blood/bone marrow aspirates and morphology/genetics wherever available as per the World Health Organization (WHO) 2008 guideline. Among 628 consecutive acute leukemia (AL) cases diagnosed and evaluated during this period, we identified 14 (2.2%) patients with MPAL fulfilling WHO 2008/EGIL criteria for immunological characterizing of AL criteria. Majority of these were males (n = 8, male:female ratio 1.3:1) and adults (n = 11, 78.5%). The median age of this cohort was 41 years (range 2-80). These cases were further classified as: B/myeloid (n = 9), T/myeloid (n = 4), and B/T MPAL (n = 1). Cytogenetics was available in 12 out of 14 cases, out of which, three cases had normal karyotype, three with t(9;22)(q34;q11), and two cases with complex karyotype. We also came across a rare case of B + T lymphoid MPAL who had mixed-lineage leukemia gene t(v; 11q23) rearrangement. MPAL is a complex entity with heterogeneous clinical, immunophenotypic, cytogenetic, and molecular features. Multiparametric flowcytometry by using comprehensive antibody panels is a valuable tool for diagnosis. Subsequent cytogenetic and molecular analysis for further prognostic stratification and treatment modalities are important.

Contribution of flow cytometry to the diagnosis of gastric lymphomas in endoscopic biopsy specimens.

PubMed

Almasri, N M; Zaer, F S; Iturraspe, J A; Braylan, R C

1997-07-01

Gastric lymphomas seem to have unique clinical, pathologic, and immunophenotypic features that set them apart from nodal lymphomas. Microscopic examination of endoscopic biopsy specimens is the most frequent procedure used to diagnose gastric tumors, but it is very difficult, and sometimes impossible, to recognize lymphomas in endoscopic samples by histologic or even immunohistologic methods. Because most gastric lymphomas are of B-cell origin, we used flow cytometry to assess B-cell clonality in gastric biopsy specimens containing dense lymphocytic infiltrates thought to represent lymphoma. We prepared viable cell suspensions from unfixed specimens obtained from 29 consecutive patients who had a previous microscopic diagnosis of suspicious gastric lymphoid infiltrates. We performed immunophenotypic studies with multicolor flow cytometry, and we assessed clonality by examination of immunoglobulin (Ig) light-chain expression analyzed exclusively on B cells identified by anti-CD20 or CD19 antibodies. The mean number of cells recovered was 1.04 x 10(6), from an average of 5.5 gastric biopsy fragments per patient. In 26 of the 29 patients, the number of cells was adequate for analysis. We detected B-cell monoclonality in 16 cases, including 5 in which the percentage of clonal B cells was less than 5%. Of the 16 cases, only 8 could be diagnosed as lymphomas on morphologic grounds alone; the remaining 8 patients had either suspicious lymphoid infiltrates or chronic gastritis. The three cases with an insufficient number of cells were considered non-neoplastic either on histologic grounds alone or in conjunction with Southern analysis of Ig genes. We conclude that flow cytometric immunophenotypic analysis of freshly prepared cell suspensions obtained from endoscopic biopsy specimens can be used to evaluate gastric lymphocytic infiltrates. Specifically, the analysis of surface Ig light-chain expression on B cells distinguishes between monoclonal (lymphoma) and polyclonal (nonlymphoma) infiltrates. The rapidity, ease, quantitative properties, and sensitivity of this technique make it a supplement to the morphologic assessment of gastric lymphoid infiltrates.

Differences of immunophenotypic markers and signaling molecules between adenocarcinomas of gastric cardia and distal stomach.

PubMed

Xue, Liying; Zhang, Xianghong; Li, Yuehong; Yang, Haiyan; Li, Xuemin; Mi, Jianmin; Wang, Hengshu; Wang, Junling; Yan, Xia

2011-04-01

During the past decades, the subsites of gastric carcinoma underwent significant changes. The incidence of the adenocarcinoma at distal stomach has been decreased, whereas cardiac adenocarcinoma remained increasing in many countries. The aim of this study was to investigate the differences between gastric cardiac and distal adenocarcinomas. We detected expressions of cytokeratins (cytokeratins 7, 14, 19, and 20) and mucins (mucins 1, 2, and 5AC) by immunohistochemistry and signaling molecules (p38, mitogen-activated protein kinase-interacting kinase 1 (MNK1), extracellular signal-regulated kinase, Jun N-terminal kinase, and phosphoinositide 3 kinase) by reverse transcription-polymerase chain reaction in both groups. The incidence of mucin 2 expression was lower in total (50.0%) and advanced-stage cases (52.0%) with cardiac adenocarcinomas than those in distal cases with total (70.2%) and advanced stage (71.4%), respectively. However, the staining for cytokeratin 14 was also significantly higher in total or advanced-stage tumors from the cardia. Our data showed no significant difference of cytokeratin 7/cytokeratin 20 pattern between 2 groups, but cytokeratin 20 expression was significantly higher in advanced-stage carcinomas of the cardia (58.7%) than in distal ones with advanced stage (38.3%). A multivariate analysis demonstrated different relationships between immunophenotypic markers and pathologic parameters in adenocarcinomas of the cardia and distal stomach. Moreover, significantly lower expressions of MNK1 and p38 in cardiac tumors were also detected. In summary, we found significant differences in patterns of immunophenotypic markers and expressions of signaling molecules between the 2 groups. It is indicated that adenocarcinoma of the cardia was different in histotype and histologic origin from distal adenocarcinoma. The cardiac adenocarcinoma might be a special subtype or an independent entity of gastric carcinoma in China. Copyright © 2011 Elsevier Inc. All rights reserved.

Evaluation of a Multicolor, Single-Tube Technique To Enumerate Lymphocyte Subpopulations▿

PubMed Central

Colombo, F.; Cattaneo, A.; Lopa, R.; Portararo, P.; Rebulla, P.; Porretti, L.

2008-01-01

To evaluate the fully automated FACSCanto software, we compared lymphocyte subpopulation counts obtained using three-color FACSCalibur-CELLQuest and six-color FACSCanto-FACSCanto software techniques. High correlations were observed between data obtained with these techniques. Our study indicated that FACSCanto clinical software is accurate and sensitive in single-platform lymphocyte immunophenotyping. PMID:18448621

Subpopulations of bovine T lymphocytes collected during foot-and-mouth disease virus infection are affected by freezing, but are subsequently stable in frozen samples

USDA-ARS?s Scientific Manuscript database

Immunophenotyping of peripheral-blood lymphocytes by flow cytometry is an important tool for infectious disease research. In many live-animal experiments and other longitudinal studies, the processing, prompt staining, and analysis of fresh samples is a logistical challenge and daily variation can c...

MHC class I target recognition, immunophenotypes and proteomic profiles of natural killer cells within the spleens of day-14 chick embryos

USDA-ARS?s Scientific Manuscript database

Chicken natural killer (NK) cells are not well defined, so little is known about the molecular interactions controlling their activity. At day 14 of embryonic development, chick spleens are a rich source of T-cellfree CD8aa+, CD3_ cells with natural killing activity. Cell-mediated cytotoxicity assay...

Treatment of dogs with lymphoma using a 12-week, maintenance-free combination chemotherapy protocol.

PubMed

Simon, D; Nolte, I; Eberle, N; Abbrederis, N; Killich, M; Hirschberger, J

2006-01-01

Treatment of lymphoma in dogs by long-term chemotherapy has favorable results. However, the efficacy of short-term, maintenance-free treatment protocols on remission and survival times in dogs has not been determined. That treatment using a 12-week chemotherapy protocol would be associated with satisfactory treatment outcome in dogs with lymphoma. 77 dogs with histologically or cytologically confirmed diagnosis of lymphoma. Prospective clinical trial in which dogs were treated with a 12-week chemotherapy protocol consisting of L-asparaginase, vincristine, cyclophosphamide, doxorubicin, and prednisolone. Complete remission rate was 76.3%. Multivariate logistic regression analysis revealed that clinical substage (P = .006) and immunophenotype (P = .003) had a significant influence on the likelihood of a dog achieving complete remission. Median duration of first complete remission was 243 days (range 19-1,191 days). The 6-month, 1-year, and 2-year remission rates were 68%, 28%, and 16%, respectively. In the multivariate analysis of patient variables, immunophenotype (P = .022) revealed a significant influence on first remission duration. Toxicosis was mild with the exception of 1 treatment-associated death. In this group of dogs the 12-week maintenance-free chemotherapy protocol was well tolerated and had satisfactory results.

Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

PubMed

Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

2016-06-02

After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro

PubMed Central

Kemény, Lajos V.; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B.

2016-01-01

After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells’ nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma–stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments. PMID:27271591

The role of multiparametric flow cytometry in the detection of minimal residual disease in acute leukaemia.

PubMed

Lee, Denise; Grigoriadis, George; Westerman, David

2015-12-01

Flow cytometry is the most accessible method for minimal residual disease (MRD) detection due to its availability in most haematological centres. Using a precise combination of different antibodies, immunophenotypic detection of MRD in acute leukaemia can be performed by identifying abnormal combinations or expressions of antigens on malignant cells at diagnosis, during and post treatment. These abnormal phenotypes, referred to as leukaemia-associated immunophenotypes (LAIPs) are either absent or expressed at low frequency in normal bone marrow (BM) cells and are used to monitor the behaviour and quantitate the amount of residual disease following treatment. In paediatric acute lymphoblastic leukaemia (ALL), the level of MRD by multiparametric flow cytometry (MPFC) during therapy is recognised as an important predictor of outcome. Although less extensively studied, adult ALL and adult and paediatric acute myeloid leukaemia (AML) have also demonstrated similar findings. The challenge now is incorporating this information for risk-stratification so that therapy can be tailored individually and ultimately improve outcome while also limiting treatment-related toxicity. In this review we will elaborate on the current and future role of MPFC in MRD in acute leukaemia while also addressing its limitations.

Brenner tumor of the ovary: a comparative immunohistochemical and ultrastructural study with transitional cell carcinoma of the bladder.

PubMed

Ordóñez, N G; Mackay, B

2000-01-01

Because of a fancied light microscopic resemblance to transitional epithelium (urothelium), Brenner tumor (BT) of the ovary is commonly described as a transitional cell neoplasm. An inability to detect a great deal of similarity between the two at the ultrastructural level prompted this electron microscopic study comparing 3 benign Brenner tumors with normal urothelium and 6 transitional cell carcinomas (TCC) of varying histologic grade from the urinary bladder. To complement the ultrastructural observations, the immunophenotype of 8 benign BTs was evaluated together with that of 12 TCCs of the bladder using antibodies to thrombomodulin (TM), cytokeratin 20, cytokeratin 7, and carcinoembryonic antigen (CEA), all of which have been shown to react with TCCs of urothelial origin. At the ultrastructural level, there was only limited evidence of a morphologic likeness between the epithelial cells of BTs and those of the benign or neoplastic urothelium. The immunophenotype of the two tumors also differed significantly in that there was no reactivity for TM or cytokeratin 20 in the BTs, while these markers were expressed in the TCCs. Both BTs and TCCs were positive for cytokeratin 7 and may express CEA.

A hormone map of human immune cells showing the presence of adrenocorticotropic hormone, triiodothyronine and endorphin in immunophenotyped white blood cells

PubMed Central

Pállinger, Éva; Csaba, György

2008-01-01

The amounts of adrenocorticotropic hormone (ACTH), endorphin and triiodothyronine (T3) in twenty-six blood samples from men and women who were healthy or had non-haematological diseases were determined by flow cytometry. Lymphocytes were immunophenotyped using monoclonal antibodies against cell surface antigens, and monocytes and granulocytes were separated by their size and granularity (using forward-scatter versus side-scatter dot plots). Each hormone was found in each cell type. The hormone content of lymphocytes was balanced, but the concentration of ACTH was significantly lower in activated T cells, that of endorphin was significantly lower in natural killer (NK) cells, and that of T3 was lower in both cell types compared with values for all lymphocytes. Monocytes and granulocytes contained very significantly more hormones than lymphocytes or monocytes. The concentration of endorphin was an order of magnitude higher in granulocytes than in monocytes or lymphocytes, reflecting the pain-relieving role of granulocytes during inflammation. Compared with monocytes, in granulocytes there was a higher concentration of ACTH and a lower concentration of T3, which suggests selective hormone production by these cells. PMID:18005034

Biology and Clinical Relevance of Acute Myeloid Leukemia Stem Cells.

PubMed

Reinisch, Andreas; Chan, Steven M; Thomas, Daniel; Majeti, Ravindra

2015-07-01

Evidence for the cancer stem cell model was first demonstrated in xenotransplanted blood and bone marrow samples from patients with acute myeloid leukemia (AML) almost two decades ago, supporting the concept that a rare clonal and mutated leukemic stem cell (LSC) population is sufficient to drive leukemic growth. The inability to eliminate LSCs with conventional therapies is thought to be the primary cause of disease relapse in AML patients, and as such, novel therapies with the ability to target this population are required to improve patient outcomes. An important step towards this goal is the identification of common immunophenotypic surface markers and biological properties that distinguish LSCs from normal hematopoietic stem and progenitor cells (HSPCs) across AML patients. This work has resulted in the development of a large number of potential LSC-selective therapies that target cell surface molecules, intracellular signaling pathways, and the bone marrow microenvironment. Here, we will review the basic biology, immunophenotypic detection, and clinical relevance of LSCs, as well as emerging biological and small-molecule strategies that either directly target LSCs or indirectly target these cells through modulation of their microenvironment. Copyright © 2015 Elsevier Inc. All rights reserved.

Humoral immunity and autism spectrum disorders.

PubMed

Fluegge, Keith

2017-05-01

Abnormal immune activation, particularly of a humoral nature, has consistently been described in the etiopathogenesis of autism spectrum disorders (ASD). In this journal, Mead and Ashwood (2015) reviewed immune abnormalities in autism and linked them to severity of classic autistic symptoms. However, there remains a lack of clarity as to how environmental risk factors in ASD may contribute to such immunophenotypes. The evidence presented herein highlights these immune deficits of a humoral nature in ASD. Moreover, aligned with prior research showing a link between chronic air pollution and suppression of humoral immunity, the author of this commentary has proposed that environmental exposure to pervasive air pollutants, particularly nitrous oxide (N 2 O), may target several anti-inflammatory biomarkers, including alpha 7 nicotinic acetylcholine receptor (α7nAChR) inhibition and stimulation of kappa opioid receptor (KOR) activity. Given that these physiological targets, in particular, may promote the oft-noted humoral immunophenotypes in ASD, including B cell survival and muted antibody responses, this correspondence supports an existing line of evidence that air pollution, and particularly exposure to environmental N 2 O, may be an important etiological risk factor in ASD. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Identification and immunophenotypic characterization of normal and pathological mast cells.

PubMed

Morgado, José Mário; Sánchez-Muñoz, Laura; Teodósio, Cristina; Escribano, Luís

2014-01-01

Mast cells (MCs) are secretory cells that are central players in human allergic disease and immune responses. With the exception of a few pathological situations, MCs are usually present at relatively low frequencies in most tissues. Since their first description, MCs in tissues were identified mostly using their morphological characteristics and their typical coloration when stained with aniline dyes. However, increasing availability of highly specific antibodies now permits the use of fluorescence-based flow cytometry as the method of choice for the quantification, characterization, and purification of cells in suspension. This technique allows for a rapid analysis of thousands of events and for the identification of cells present at frequencies as low as one event in 10(6) unwanted cells. This method also permits for simultaneous characterization of multiple antigens at a single-cell level, which is ideal in order to study rare populations of cells like MCs. Here we describe the basis of flow cytometry-based immunophenotyping applied to the study of MC. The protocol focuses on the study of human MCs present in body fluids (mainly bone marrow) but can easily be adapted to study MCs from other tissues and species.

[Primary breast lymphoma: a clinical, pathological and immunophenotypic study of eight cases].

PubMed

Ying, Jianming; Feng, Xiaoli; Liu, Xiuyun; Xie, Yongqiang; Sun, Yuntian

2002-12-01

To study the clinical, pathological and immunophenotypic characteristics of the primary breast lymphoma (PBL). Analyses of clinical history, preoperative findings, histological and immunohistochemical features of eight patients with PBL were performed. Malignant lymphoma was difficult to diagnose preoperatively. All patients were women. The age range was from 34 approximately 65 years (mean 46.4 years). The right breast was involved initially in three patients, the left in four. One patient presented bilateral involvement. Seven patients were assessed at stage IE, one with ipsolateral axillary lymph nodes involvement at stage IIE. According to the WHO classification, five patients were diagnosed as diffuse large B-cell lymphoma (4/5 centroblast, 1/5 immunoblast); the other three patients as MALT lymphoma, all with lymphoepithelial lesions. The paraffin-embedded tissues of all cases showed immunoreactivity for B-cell markers CD20, CD45RA. CD5 and CD10 were negative in all cases. Follow-up data were obtained in six patients, none recurred or died within 8 to 108 months after diagnosis. This study indicates that most PBL are diffuse large B-cell lymphoma and MALT lymphoma and have a better prognosis after comprehensive therapy.

Mucinous cystadenocarcinoma of the breast with amplification of the HER2-gene confirmed by FISH: The first case reported.

PubMed

Petersson, Fredrik; Pang, Brendan; Thamboo, Thomas P; Putti, Thomas Choudary

2010-06-01

We present the first case of a primary mucinous cystadenocarcinoma of the breast that, in addition to the characteristic immunophenotype (CK7(+), CK20(-), ER(-), PR(-), and cdx2(-)), showed a strong membranous HER2-protein expression and HER2-gene amplification documented by fluorescence in situ hybridization. Copyright 2010 Elsevier Inc. All rights reserved.

Pure Red Cell Aplasia After Chemotherapy for Hodgkin’s Lymphoma: In Vitro Evidence for T Cell Mediated Suppression of Erythropoiesis and Response to Sequential Cyclosporin and Erythropoietin

DTIC Science & Technology

1994-01-01

serologies revealed no evidence of recent or active parvovirus , hepatitis A, B, C, or Immunophenotyping studies on peripheral blood lym- Epstein-Barr...impair erythropoietin production in humans [27-291 and Norbert Frickhofer for the B 19 parvovirus studies and in mice [301. As was documented

Microanatomy of the cervical and anorectal squamocolumnar junctions: a proposed model for anatomical differences in HPV-related cancer risk

PubMed Central

Yang, Eric J.; Quick, Matthew C.; Hanamornroongruang, Suchanan; Lai, Keith; Doyle, Leona; McKeon, Frank D.; Xian, Wa; Crum, Christopher P.; Herfs, Michael

2015-01-01

Human papilloma virus (HPV) infection causes cancers and their precursors (high grade squamous intraepithelial lesions) near cervical and anal squamocolumnar junctions. Recently described cervical squamocolumnar junctions cells are putative residual embryonic cells near the cervical transformation zone. These cells appear multipotential and share an identical immunophenotype (strongly CK7-positive) with over 90% of high grade squamous intraepithelial lesions and cervical carcinomas. However, because the number of new cervical cancers discovered yearly world-wide is 17-fold that of anal cancer, we posed the hypothesis that this difference in cancer risk reflects differences in the transition zones at the two sites. The microanatomy of the normal anal transformation zone (n = 37) and topography and immunophenotype of anal squamous neoplasms (n = 97) were studied. A discrete anal transition zone was composed of multi-layered CK7-positive/p63-negative superficial columnar cells and an uninterrupted layer of CK7-negative/p63-positive basal cells. The CK7-negative/p63-positive basal cells were continuous with – and identical in appearance to - the basal cells of the mature squamous epithelium. This was in contrast to the cervical squamocolumnar junction, that harbored a single-layered CK7-positive/p63-negative squamocolumnar junction cell population. Of the 97 Anal intraepithelial neoplasia/squamous cell carcinomas evaluated, only 27% (26/97) appeared to originate near the anal transition zone and only 23% (22/97) were CK7-positive. This study thus reveals two fundamental differences between the anus and cervix: 1) the anal transition zone does not harbor a single monolayer of residual un-differentiated embryonic cells and 2) the dominant tumor immuno-phenotype is in keeping with an origin in metaplastic (CK7-negative) squamous rather than squamocolumnar junction (CK7-positive) epithelium. The implication is that at birth, the embryonic cells in the anal transition zone have already begun to differentiate, presenting a less vulnerable squamous metaplasia that - like vaginal and vulvar epithelium - is less prone to HPV directed carcinogenesis. This in turn underscores the link between cancer risk and a very small and discrete population of vulnerable squamocolumnar junction cells in the cervix. PMID:25975286

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes

PubMed Central

Sandes, Alex F.; Yamamoto, Mihoko; Matarraz, Sergio; Chauffaille, Maria de Lourdes L.F.; Quijano, Sandra; López, Antonio; Oguro, Tsutomu; Kimura, Eliza Y. S.; Orfao, Alberto

2012-01-01

Background Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes. Design and Methods We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated. Results Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n=5), CD42a (n=1) and CD61 (n=2), together with reactivity for CD34 (n=1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n=16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival. Conclusions Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders. PMID:22271903

Common Variable Immunodeficiency Non-Infectious Disease Endotypes Redefined Using Unbiased Network Clustering in Large Electronic Datasets.

PubMed

Farmer, Jocelyn R; Ong, Mei-Sing; Barmettler, Sara; Yonker, Lael M; Fuleihan, Ramsay; Sullivan, Kathleen E; Cunningham-Rundles, Charlotte; Walter, Jolan E

2017-01-01

Common variable immunodeficiency (CVID) is increasingly recognized for its association with autoimmune and inflammatory complications. Despite recent advances in immunophenotypic and genetic discovery, clinical care of CVID remains limited by our inability to accurately model risk for non-infectious disease development. Herein, we demonstrate the utility of unbiased network clustering as a novel method to analyze inter-relationships between non-infectious disease outcomes in CVID using databases at the United States Immunodeficiency Network (USIDNET), the centralized immunodeficiency registry of the United States, and Partners, a tertiary care network in Boston, MA, USA, with a shared electronic medical record amenable to natural language processing. Immunophenotypes were comparable in terms of native antibody deficiencies, low titer response to pneumococcus, and B cell maturation arrest. However, recorded non-infectious disease outcomes were more substantial in the Partners cohort across the spectrum of lymphoproliferation, cytopenias, autoimmunity, atopy, and malignancy. Using unbiased network clustering to analyze 34 non-infectious disease outcomes in the Partners cohort, we further identified unique patterns of lymphoproliferative (two clusters), autoimmune (two clusters), and atopic (one cluster) disease that were defined as CVID non-infectious endotypes according to discrete and non-overlapping immunophenotypes. Markers were both previously described {high serum IgE in the atopic cluster [odds ratio (OR) 6.5] and low class-switched memory B cells in the total lymphoproliferative cluster (OR 9.2)} and novel [low serum C3 in the total lymphoproliferative cluster (OR 5.1)]. Mortality risk in the Partners cohort was significantly associated with individual non-infectious disease outcomes as well as lymphoproliferative cluster 2, specifically (OR 5.9). In contrast, unbiased network clustering failed to associate known comorbidities in the adult USIDNET cohort. Together, these data suggest that unbiased network clustering can be used in CVID to redefine non-infectious disease inter-relationships; however, applicability may be limited to datasets well annotated through mechanisms such as natural language processing. The lymphoproliferative, autoimmune, and atopic Partners CVID endotypes herein described can be used moving forward to streamline genetic and biomarker discovery and to facilitate early screening and intervention in CVID patients at highest risk for autoimmune and inflammatory progression.

Immunopathogenesis in Autism: Regulatory T-Cells and Autoimmunity in Neurodevelopment

DTIC Science & Technology

2011-12-01

etiology of autism and related neurodevelopmental disorders is largely unknown. Myriad hypotheses have suggested that exogenous agents, such as...developmental exposure to PFOA of PFOS. However, autism risk cannot be determined from these data alone. Regulatory T cells, immunophenotyping...autoantibodies, CD3+, myelin basic protein, autism 1 JUL 2010 - 30 NOV 2011Final01-12-2011 W81XWH-10-1-0484 Immunopathogenesis in Autism : Regulatory T-Cells

The Role of Glucocorticoids and Neuroinflammation in Mediating the Effects of Stress on Drug Abuse

DTIC Science & Technology

2013-10-01

addiction. In the second year of this project, we made pivotal discoveries in how stress sensitizes the pro-inflammatory effects of methamphetamine ...immunophenotypic and functional characteristics. J Neurosci Methods 151, 121- 130. Krasnova et al., 2009. Methamphetamine toxicity and messengers of death...Tocharus et al., 2010. Melatonin attenuates methamphetamine -induced overexpression of pro- inflammatory cytokines in microglial cell lines. J Pineal Res

Difference of Morphology and Immunophenotype between Central and Peripheral Squamous Cell Carcinomas of the Lung

PubMed Central

Hayashi, Tomayoshi; Sano, Hisao; Egashira, Ryoko; Tabata, Kazuhiro; Tanaka, Tomonori; Kashima, Yukio; Nunomura, Sayuri

2013-01-01

Background. Recent agents, that is, pemetrexed and bevacizumab, have shown reproductive negative association between squamous histology. According to these agents' effectiveness, ruling out of the squamous histology is a significant issue for surgical pathologists. Several articles have proposed the distinction of peripheral type from central type of squamous cell carcinoma (SqCC) due to its similarity to adenocarcinoma, although little evidence to support the difference between these two types was published. In this study, we compared the clinicopathologic findings of central and peripheral pulmonary SqCCs. Material and Methods. 15 central and 35 peripheral types of SqCC from 2005 to 2010 were examined. Twelve morphological features were scored based on their intensity in the original H&E slides, and then, tissue microarray holding triplicated cores from 43 cases was immunohistochemically examined for cytokeratin (CK)7, CK14, TTF-1, Napsin A, p63, CK34βE12, CK5/6, and p53. Result. Most of the histological findings did not separate central and peripheral SqCCs; only the presence of emphysema, interstitial fibrosis, and entrapped pneumocytes inside the tumor showed statistic predominance in peripheral SqCC. This is the first immunophenotypic research in the central and peripheral types of SqCC. PMID:24069587

Effects of pre-analytical variables on flow cytometric diagnosis of canine lymphoma: A retrospective study (2009-2015).

PubMed

Comazzi, S; Cozzi, M; Bernardi, S; Zanella, D R; Aresu, L; Stefanello, D; Marconato, L; Martini, V

2018-02-01

Flow cytometry (FC) is increasingly being used for immunophenotyping and staging of canine lymphoma. The aim of this retrospective study was to assess pre-analytical variables that might influence the diagnostic utility of FC of lymph node (LN) fine needle aspirate (FNA) specimens from dogs with lymphoproliferative diseases. The study included 987 cases with LN FNA specimens sent for immunophenotyping that were submitted to a diagnostic laboratory in Italy from 2009 to 2015. Cases were grouped into 'diagnostic' and 'non-diagnostic'. Pre-analytical factors analysed by univariate and multivariate analyses were animal-related factors (breed, age, sex, size), operator-related factors (year, season, shipping method, submitting veterinarian) and sample-related factors (type of sample material, cellular concentration, cytological smears, artefacts). The submitting veterinarian, sample material, sample cellularity and artefacts affected the likelihood of having a diagnostic sample. The availability of specimens from different sites and of cytological smears increased the odds of obtaining a diagnostic result. Major artefacts affecting diagnostic utility included poor cellularity and the presence of dead cells. Flow cytometry on LN FNA samples yielded conclusive results in more than 90% of cases with adequate sample quality and sampling conditions. Copyright © 2018 Elsevier Ltd. All rights reserved.

The expression of MDM2/CDK4 gene product in the differential diagnosis of well differentiated liposarcoma and large deep-seated lipoma

PubMed Central

Pilotti, S; Torre, G Della; Mezzelani, A; Tamborini, E; Azzarelli, A; Sozzi, G; Pierotti, M A

2000-01-01

Ordinary lipomas are cytogenetically characterized by a variety of balanced rearrangements involving chromosome segment 12q13–15, whereas well differentiated liposarcomas (WDL) show supernumerary ring and giant marker chromosomes, known to contain amplified 12q sequences. The tight correlation between the presence of ring chromosomes and both amplification and overexpression of MDM2 and CDK4 genes suggests the exploration of the possibility that immunocytochemistry (ICC) might assist in the differential diagnosis of lipoma-like well differentiated liposarcomas (LL-WDL) and large deep-seated lipomas (LDSL). For this purpose, 21 cases of the former and 19 cases of the latter tumours were analysed by ICC and, according to the availability of material, by molecular and cytogenetic approaches. All lipomas displayed a null MDM2/CDK4 phenotype, whereas all LL-WDL showed MDM2/CDK4 or CDK4 phenotypes. Southern blot analysis performed on 16 suitable cases, complemented by fluorescence in situ hybridization and classical cytogenetic analysis in 11 cases, was consistent with, and further supported the immunophenotyping data. In conclusion, MDM2/CDK4 product-based immunophenotyping appears to represent a valuable method for the categorization of arguable LDSL. © 2000 Cancer Research Campaign PMID:10755400

Human natural killer cell development in secondary lymphoid tissues

PubMed Central

Freud, Aharon G.; Yu, Jianhua; Caligiuri, Michael A.

2014-01-01

For nearly a decade it has been appreciated that critical steps in human natural killer (NK) cell development likely occur outside of the bone marrow and potentially necessitate distinct microenvironments within extramedullary tissues. The latter include the liver and gravid uterus as well as secondary lymphoid tissues such as tonsils and lymph nodes. For as yet unknown reasons these tissues are naturally enriched with NK cell developmental intermediates (NKDI) that span a maturation continuum starting from an oligopotent CD34+CD45RA+ hematopoietic precursor cell to a cytolytic mature NK cell. Indeed despite the detection of NKDI within the aforementioned tissues, relatively little is known about how, why, and when these tissues may be most suited to support NK cell maturation and how this process fits in with other components of the human immune system. With the discovery of other innate lymphoid subsets whose immunophenotypes overlap with those of NKDI, there is also need to revisit and potentially re-characterize the basic immunophenotypes of the stages of the human NK cell developmental pathway in vivo. In this review, we provide an overview of human NK cell development in secondary lymphoid tissues and discuss the many questions that remain to be answered in this exciting field. PMID:24661538

Flow cytometric determination of quantitative immunophenotypes

NASA Astrophysics Data System (ADS)

Redelman, Douglas; Ensign, Wayne; Roberts, Don

2001-05-01

Immunofluorescent flow cytometric analysis of peripheral blood leucocytes is most commonly used to identify and enumerate cells defined by one or more clusters of differentiation (CD) antigens. Although less widely employed, quantitative tests that measure the amounts of CD antigens expressed per cell are used in some situations such as the characterization of lymphomas and leukocytes or the measurement of CD38 on CD3plu8pluT cells in HIV infected individuals. The CD antigens used to identify leukocyte populations are functionally important molecules and it is known that under- or over-expression of some CD antigens can affect cellular responses. For example, high or low expression of CD19 on B cells is associated with autoimmune conditions or depressed antibody responses, respectively. In the current studies, the quantitative expression of CD antigens on T cells, B cells and monocytes was determined in a group of age and sex-matched Marines at several times before and after training exercises. There was substantial variation among these individuals in the quantitative expression of CD antigens and in the number of cells in various populations. However, there was relatively little variation within individuals during the two months they were examined. Thus, the number of cells in leukocyte sub-populations and the amount of CD antigens expressed per cell appear to comprise a characteristic quantitative immunophenotype.

Surface-Micromachined Microfiltration Membranes for Efficient Isolation and Functional Immunophenotyping of Subpopulations of Immune Cells

PubMed Central

Oh, Boram; Lam, Raymond H. W.; Fan, Rong; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

2015-01-01

An accurate measurement of the immune status in patients with immune system disorders is critical in evaluating the stage of diseases and tailoring drug treatments. The functional cellular immunity test is a promising method to establish the diagnosis of immune dysfunctions. The conventional functional cellular immunity test involves measurements of the capacity of peripheral blood mononuclear cells to produce pro-inflammatory cytokines when stimulated ex vivo. However, this “bulk” assay measures the overall reactivity of a population of lymphocytes and monocytes, making it difficult to pinpoint the phenotype or real identity of the reactive immune cells involved. In this research, we develop a large surface micromachined polydimethylsiloxane (PDMS) microfiltration membrane (PMM) with high porosity, which is integrated in a microfluidic microfiltration platform. Using the PMM with functionalized microbeads conjugated with antibodies against specific cell surface proteins, we demonstrated rapid, efficient and high-throughput on-chip isolation, enrichment, and stimulation of subpopulations of immune cells from blood specimens. Furthermore, the PMM-integrated microfiltration platform, coupled with a no-wash homogeneous chemiluminescence assay (“AlphaLISA”), enables us to demonstrate rapid and sensitive on-chip immunophenotyping assays for subpopulations of immune cells isolated directly from minute quantities of blood samples. PMID:23335389

Alopecia universalis in a dog with testicular neoplasia.

PubMed

Outerbridge, Catherine A; White, Stephen D; Affolter, Verena K

2016-12-01

To describe a case of testicular neoplasia and alopecia universalis in a dog, and successful treatment of the latter with ciclosporin. Twelve-year-old intact male wirehaired fox terrier. Castration, skin biopsy for histopathology, lymphocyte immunophenotyping and clonality analysis of the canine T-cell receptor gamma locus (TCRγ) rearrangement. The dog presented with symmetrical generalized alopecia. Testicular enlargement was noted which on castration was determined to be caused by bilateral interstitial cell tumours, Sertoli cell tumours and a unilateral seminoma. During the four months after castration the alopecia became more severe and widespread. Histopathology of the skin showed moderate, multifocal, mural folliculitis, peribulbar mucinosis and lymphocytic bulbitis, and targeting of anagen hair follicles. Immunophenotyping of the infiltrate showed a population of well-differentiated, small CD3-positive T lymphocytes, some expressing CD4 and others CD8. Molecular analysis revealed a polyclonal lymphocytic infiltrate, substantiating the diagnosis of alopecia areata rather than lymphoma. Treatment with ciclosporin (4.6 mg/kg) and ketoconazole (4.6 mg/kg) resulted in complete hair regrowth. Ciclosporin treatment, in combination with ketoconazole, can be effective for treatment of alopecia universalis in the dog. Alopecia universalis may present with clinically noninflammatory, symmetrical, generalized alopecia, mimicking an endocrine alopecia, and skin biopsies are needed to confirm the diagnosis. © 2016 ESVD and ACVD.

Regulation of immunophenotype modulation of monocytes-macrophages from M1 into M2 by prostate cancer cell-culture supernatant via transcription factor STAT3.

PubMed

Solís-Martínez, R; Cancino-Marentes, M; Hernández-Flores, G; Ortiz-Lazareno, P; Mandujano-Álvarez, G; Cruz-Gálvez, C; Sierra-Díaz, E; Rodríguez-Padilla, C; Jave-Suárez, L F; Aguilar-Lemarroy, A; Bravo-Cuellar, A

2018-04-01

Transcription factor STAT3 has a prominent innate immunity effect on cancer progression. We determined the regulation of STAT3 in the immunophenotype modulation of macrophages from M1 into M2 induced by the cell-culture supernatant of the Prostate-Cancer line PC3. Monocytes-macrophages from healthy donors were cultured in the supernatant of PC3 cells, membrane proteins, and intracytoplasmic and phosphorylated STAT3 were measured using flow cytometry, while cytokines and growth factors were studied using luminescence. Cytotoxicity and nitric oxide were evaluated via colorimetric assays. The supernatant of PC3 prostate-tumor cells effectively induced macrophages toward an M2 profile, and the expression of phosphorylated STAT3 in the monocytes-macrophages notably increased, and mainly related to IL-10. In the group of monocytes-macrophages treated with a STAT3 inhibitor, the macrophages were induced toward an M1 phenotype. In this study, we showed that the secretion profile of PC3 prostate-cancer cells induces a change in macrophage phenotype from M1 into M2, and that the phenomenon is related to phosphorylation of transcription factor STAT3 and IL-10. Copyright © 2018. Published by Elsevier B.V.

Hodgkin's disease and CD30-positive anaplastic large cell lymphomas--a continuous spectrum of malignant disorders. A quantitative morphometric and immunohistologic study.

PubMed Central

Leoncini, L.; Del Vecchio, M. T.; Kraft, R.; Megha, T.; Barbini, P.; Cevenini, G.; Poggi, S.; Pileri, S.; Tosi, P.; Cottier, H.

1990-01-01

The authors have examined cellular areas of lymphoma tissue in 28 cases of Hodgkin's disease (HD) or anaplastic large cell lymphoma (ALCL, 'Ki-1 cell lymphoma') to evaluate the boundaries between the two entities. Methods applied included conventional histology; test point analysis; semiautomated morphometry of nuclear profile features of Reed-Sternberg and other atypical large cells (RSALCs); and immunohistochemistry of these elements on all paraffin sections and, in 15 cases, on frozen sections. Mean nuclear profile morphotypes of RSALCs per case varied independently of immunophenotype and histologic diagnosis. Conversely, immunohistochemistry demonstrated significant, although not consistent, preferential positivities of these CD30+ elements for CD15 in HD, and for epithelial membrane antigen (EMA) and CD43 in ALCLs. In the latter, RSALCs also exhibited a tendency for CD45 and CD45RO positivity and for the expression of T-cell-associated antigens. However, there were considerable overlaps. This continuous spectrum of RSALC nuclear profile morphotypes and immunophenotypes, ranging from HD over questionable cases, intermediate between HD and ALCL, to ALCLs, was paralleled by differences in the reactive component of lymphomas. Lymphocytes and granulocytes were significantly deficient in ALCLs. Images Figure 1 PMID:2173409

CD200 is a useful diagnostic marker for identifying atypical chronic lymphocytic leukemia by flow cytometry.

PubMed

Ting, Y S; Smith, S A B C; Brown, D A; Dodds, A J; Fay, K C; Ma, D D F; Milliken, S; Moore, J J; Sewell, W A

2018-05-27

Immunophenotyping by flow cytometry is routinely employed in distinguishing between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Inclusion of CD200 has been reported to contribute to more reliable differentiation between CLL and MCL. We investigated the value of CD200 in assessment of atypical CLL cases. CD200 expression on mature B cell neoplasms was studied by eight-color flow cytometry in combination with a conventional panel of flow cytometry markers. The study included 70 control samples, 63 samples with CLL or atypical CLL phenotype, 6 MCL samples, and 40 samples of other mature B cell neoplasms. All CLL samples were positive for CD200, whereas MCL samples were dim or negative for CD200. Of the CLL samples, 7 were atypical by conventional flow cytometry, with Matutes scores ≤3. These cases were tested for evidence of a t(11;14) translocation, characteristic of MCL, and all were negative, consistent with their classification as atypical CLL. All these atypical CLL samples were strongly positive for CD200. CD200 proved to be a useful marker for differentiation between CLL and MCL by flow cytometry. In particular, CD200 was useful in distinguishing CLL samples with atypical immunophenotypes from MCL. © 2018 John Wiley & Sons Ltd.

The impact of ex vivo clinical grade activation protocols on human T-cell phenotype and function for the generation of genetically modified cells for adoptive cell transfer therapy.

PubMed

Tumeh, Paul C; Koya, Richard C; Chodon, Thinle; Graham, Nicholas A; Graeber, Thomas G; Comin-Anduix, Begoña; Ribas, Antoni

2010-10-01

Optimized conditions for the ex vivo activation, genetic manipulation, and expansion of human lymphocytes for adoptive cell therapy may lead to protocols that maximize their in vivo function. We analyzed the effects of 4 clinical grade activation and expansion protocols over 3 weeks on cell proliferative rate, immunophenotype, cell metabolism, and transduction efficiency of human peripheral blood mononuclear cells (PBMCs). Peak lentiviral transduction efficiency was early (days 2 to 4), at a time when cells showed a larger size, maximal uptake of metabolic substrates, and the highest level of proximal T-cell receptor signaling engagement. Anti-CD2/3/28 activation beads induced greater proliferation rate and skewed PBMCs early on to a CD4 phenotype when compared with the cells cultured in OKT3. Multicolor surface phenotyping demonstrated that changes in T-cell surface markers that define T-cell functional phenotypes were dependent on the time spent in culture as opposed to the particular activation protocol. In conclusion, ex vivo activation of human PBMCs for adoptive cell therapy demonstrate defined immunophenotypic and functional signatures over time, with cells early on showing larger sizes, higher transduction efficiency, maximal metabolic activity, and zeta-chain-associated protein-70 activation.

Classification and clinical behavior of blastic plasmacytoid dendritic cell neoplasms according to their maturation-associated immunophenotypic profile

PubMed Central

Martín-Martín, Lourdes; López, Antonio; Vidriales, Belén; Caballero, María Dolores; Rodrigues, António Silva; Ferreira, Silvia Inês; Lima, Margarida; Almeida, Sérgio; Valverde, Berta; Martínez, Pilar; Ferrer, Ana; Candeias, Jorge; Ruíz-Cabello, Francisco; Buadesa, Josefa Marco; Sempere, Amparo; Villamor, Neus

2015-01-01

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of leukemia/lymphoma, whose diagnosis can be difficult to achieve due to its clinical and biological heterogeneity, as well as its overlapping features with other hematologic malignancies. In this study we investigated whether the association between the maturational stage of tumor cells and the clinico-biological and prognostic features of the disease, based on the analysis of 46 BPDCN cases classified into three maturation-associated subgroups on immunophenotypic grounds. Our results show that blasts from cases with an immature plasmacytoid dendritic cell (pDC) phenotype exhibit an uncommon CD56− phenotype, coexisting with CD34+ non-pDC tumor cells, typically in the absence of extramedullary (e.g. skin) disease at presentation. Conversely, patients with a more mature blast cell phenotype more frequently displayed skin/extramedullary involvement and spread into secondary lymphoid tissues. Despite the dismal outcome, acute lymphoblastic leukemia-type therapy (with central nervous system prophylaxis) and/or allogeneic stem cell transplantation appeared to be the only effective therapies. Overall, our findings indicate that the maturational profile of pDC blasts in BPDCN is highly heterogeneous and translates into a wide clinical spectrum -from acute leukemia to mature lymphoma-like behavior-, which may also lead to variable diagnosis and treatment. PMID:26056082

Role of flow-cytometric immunophenotyping in prediction of BCR/ABL1 gene rearrangement in adult B-cell acute lymphoblastic leukemia.

PubMed

Corrente, Francesco; Bellesi, Silvia; Metafuni, Elisabetta; Puggioni, Pier Luigi; Marietti, Sara; Ciminello, Angela Maria; Za, Tommaso; Sorà, Federica; Fianchi, Luana; Sica, Simona; De Stefano, Valerio; Chiusolo, Patrizia

2018-05-01

We performed a retrospective analysis of 88 adult patients with B-ALL diagnosed in our center by a flow-cytometric assessment. Immunophenotypic expression of leukemic cells was explored by simultaneous evaluation of positivity, percentage of expressing cells and median fluorescence intensity (MFI). BCR/ABL1 fusion transcripts were assessed by RT-PCR analysis and were identified in 36 patients (40.9%). CD10 and CD34 were positive in the totality of BCR/ABL1-positive cases. Patients with gene rearrangement had a greater frequency of CD66c, CD13 and CD33 positivity compared with BCR/ABL1-negative cases. Moreover, BCR/ABL1-positive cases exhibited a greater median percentage and MFI values of CD13, CD33, CD66c, CD10, CD34 and CD25 expressions, but a lower median percentage and MFI values of CD38 and CD22 expressions than patients without gene rearrangement. Multivariate logistic regression analysis showed that CD10, CD38 and CD13 expressions were independent predictors for the presence of BCR/ABL1 rearrangement. Predictive probabilities of molecular occurrence based on these markers are proposed. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

The immunophenotype of mast cells and its utility in the diagnostic work-up of systemic mastocytosis.

PubMed

Teodosio, Cristina; Mayado, Andrea; Sánchez-Muñoz, Laura; Morgado, José M; Jara-Acevedo, María; Álvarez-Twose, Ivan; García-Montero, Andrés C; Matito, Almudena; Caldas, Caldas; Escribano, Luis; Orfao, Alberto

2015-01-01

SM comprises a heterogeneous group of disorders, characterized by an abnormal accumulation of clonal MCs in 1 or more tissues, frequently involving the skin and BM. Despite the fact that most adult patients (>90%) carry the same genetic lesion (D816V KIT mutation), the disease presents with multiple variants with very distinct clinical and biologic features, a diverse prognosis, and different therapeutic requirements. Recent advances in the standardization of the study of BM MC by MFC allowed reproducible identification and characterization of normal/reactive MCs and their precursors, as well as the establishment of the normal MC maturational profiles. Analysis of large groups of patients versus normal/reactive samples has highlighted the existence of aberrant MC phenotypes in SM, which are essential for the diagnosis of the disease. In turn, 3 clearly distinct and altered maturation-associated immunophenotypic profiles have been reported recently in SM, which provide criteria for the distinction between ISM patients with MC-restricted and multilineage KIT mutation; thus, immunphenotyping also contributes to prognostic stratification of ISM, particularly when analysis of the KIT mutation on highly purified BM cells is not routinely available in the diagnostic work-up of the disease. © Society for Leukocyte Biology.

Multi-color CD34⁺ progenitor-focused flow cytometric assay in evaluation of myelodysplastic syndromes in patients with post cancer therapy cytopenia.

PubMed

Tang, Guilin; Jorgensen, L Jeffrey; Zhou, Yi; Hu, Ying; Kersh, Marian; Garcia-Manero, Guillermo; Medeiros, L Jeffrey; Wang, Sa A

2012-08-01

Bone marrow assessment for myelodysplastic syndrome (MDS) in a patient who develops cytopenia(s) following cancer therapy is challenging. With recent advances in multi-color flow cytometry immunophenotypic analysis, a CD34(+) progenitor-focused 7-color assay was developed and tested in this clinical setting. This assay was first performed in 73 MDS patients and 53 non-MDS patients (developmental set). A number of immunophenotypic changes were differentially observed in these two groups. Based on the sensitivity, specificity and reproducibility, a core panel of markers was selected for final assessment that included increased total CD34(+) myeloblasts; decreased stage I hematogones; altered CD45/side scatter; altered expression of CD13, CD33, CD34, CD38, CD117, and CD123; aberrant expression of lymphoid or mature myelomonocytic antigens on CD34(+) myeloblasts; and several marked alterations in maturing myelomonocytic cells. The data were translated into a simplified scoring system which was then used in 120 patients with cytopenia(s) secondary to cancer therapy over a 2-year period (validation set). With a median follow-up of 11 months, this assay demonstrated 89% sensitivity, 94% specificity, and 92% accuracy in establishing or excluding a diagnosis of MDS. Copyright © 2012 Elsevier Ltd. All rights reserved.

Immunophenotyping of HPV Types 16 and 18 among Sudanese Patients with Oral Lesions

PubMed Central

Ginawi, Ibrahim A. M.; Mahgoub, Ebtihag A.; Ahmed, Hussain G.

2012-01-01

Objective The aim of this study was to screen patients with oral lesions for the presence of Human Papilloma Virus (HPV) types 16 and 18. Methods Sixty patients aged between 11-80 years with a mean age of 46 years were examined using immunohistological techniques. All samples were retrieved from RICK during the period from August 2009 to August 2010. Out of 60 patients, 50 had Oral Squamous Cell Carcinomas (OSCCs) and the remaining ten had benign oral lesions, included as internal control. Results Of the 50 patients with OSCCs, 10 (20%) showed positive immunohistochemical results for HPV types 16 and 18 of which 50% were detected among males and 50% were demonstrated among females. The ten positive findings were Immunophenotyped as follows: five were positive with HPV type 16, four with type 18 and one was positive for HPV types 16 and18. All patients with benign oral lesions were negative for HPV immunohistochemistry. Conclusion The study suggests the role of HPV 16 and 18 in the etiology of oral cancers in different parts of Sudan. However, the use of molecular techniques such as PCR are needed to confirm the results of immunohistochemistry in the role of the HPV in developing of OSCC in Sudan. PMID:22811767

Immunohistochemical/histochemical double staining method in the study of the columnar metaplasia of the oesophagus.

PubMed

Cabibi, D; Giannone, A G; Mascarella, C; Guarnotta, C; Castiglia, M; Pantuso, G; Fiorentino, E

2014-03-05

Intestinal metaplasia in Barrett's oesophagus (BO) represents an important risk factor for oesophageal adenocarcinoma. Instead, few and controversial data are reported about the progression risk of columnar-lined oesophagus without intestinal metaplasia (CLO), posing an issue about its clinical management. The aim was to evaluate if some immunophenotypic changes were present in CLO independently of the presence of the goblet cells. We studied a series of oesophageal biopsies from patients with endoscopic finding of columnar metaplasia, by performing some immunohistochemical stainings (CK7, p53, AuroraA) combined with histochemistry (Alcian-blue and Alcian/PAS), with the aim of simultaneously assess the histochemical features in cells that shows an aberrant expression of such antigens. We evidenced a cytoplasmic expression of CK7 and a nuclear expression of Aurora A and p53,  both in goblet cells of BO and in non-goblet cells of CLO, some of which showing mild dysplasia. These findings suggest that some immunophenotypic changes are present in CLO and they can precede the appearance of the goblet cells or can be present independently of them, confirming the conception of BO as the condition characterized by any extention of columnar epithelium. This is the first study in which a combined immunohistochemical/histochemical method has been applied to Barrett pathology.

The impact of ex vivo clinical grade activation protocols on human T cell phenotype and function for the generation of genetically modified cells for adoptive cell transfer therapy

PubMed Central

Tumeh, Paul C.; Koya, Richard C.; Chodon, Thinle; Graham, Nicholas A.; Graeber, Thomas G.; Comin-Anduix, Begoña; Ribas, Antoni

2011-01-01

Optimized conditions for the ex vivo activation, genetic manipulation, and expansion of human lymphocytes for adoptive cell therapy (ACT) may lead to protocols that maximize their in vivo function. We analyzed the effects of four clinical grade activation and expansion protocols over three weeks on cell proliferative rate, immunophenotype, cell metabolism, and transduction efficiency of human peripheral blood mononuclear cells (PBMCs). Peak lentiviral transduction efficiency was early (days 2 to 4), at a time when cells demonstrated a larger size, maximal uptake of metabolic substrates, and the highest level of proximal TCR signaling engagement. Anti-CD2/3/28 activation beads induced greater proliferation rate and skewed PBMCs early on to a CD4 phenotype when compared to the cells cultured in OKT3. Multicolor surface phenotyping demonstrated that changes in T cell surface markers that define T cell functional phenotypes were dependent on the time spent in culture as opposed to the particular activation protocol. In conclusion, ex vivo activation of human PBMCs for ACT demonstrate defined immunophenotypic and functional signatures over time, with cells early on showing larger sizes, higher transduction efficiency, maximal metabolic activity and ZAP-70 activation. PMID:20842061

Evaluation of Plasma Platelet Microparticles in Thrombotic Thrombocytopenic Purpura.

PubMed

Tahmasbi, Leila; Karimi, Mehran; Kafiabadi, Sedigheh Amini; Nikougoftar, Mahin; Haghpanah, Sezaneh; Ranjbaran, Reza; Moghadam, Mohamad

2017-01-01

Platelet microparticles (PMPs) have a procoagulant activity about 50-100 times greater than active platelets due to high expression of negatively charged phospholipids on their surfaces. In this study, we evaluated microparticle immunophenotyping and also plasma PMPs level in patients with Thrombotic Thrombocytopenic Purpura (TTP) in Southern Iran. We had two study groups: 15 TTP patients and 15 healthy control group and PMPs from platelet concentrate (PC) at the 5 th day of storage. Microparticles were prepared in two steps, by low and high centrifugation followed by size confirmation via 'Dynamic Light Scattering (DLS)' Zetasizer. Immunophenotyping of PMPs was done via flow cytometry, using a FACS Calibur flow cytometer (BD, USA). PMPs counts were obtained using Partec-cyflow and Polysciences Microbeads (1 micron in diameter). Results were analyzed using FlowJo 7.6 (Treestar, USA) and Partec FlowMax software. Our results showed that the majority of microparticles in TTP patients and normal individuals were PMPs and also demonstrated that the plasma PMPs level in TTP patients was higher than the normal control group ( P -value<0.001). It seems that elevated PMPs level in TTP patients could be related to thrombotic events. Nevertheless, more studies are needed to confirm these results. © 2017 by the Association of Clinical Scientists, Inc.

Expression of aberrant CD markers in acute leukemia: a study of 100 cases with immunophenotyping by multiparameter flowcytometry.

PubMed

Sarma, Anupam; Hazarika, Munlima; Das, Debabrata; Kumar Rai, Avdhesh; Sharma, Jagannath Dev; Bhuyan, Chidananda; Kataki, Amal Chandra

2015-01-01

Acute leukemia is a heterogenous disease having diverse phenotypes. Immunophenotyping by flowcytometry is essential for diagnosis of myeloid and lymphoid subtypes. Aberrant phenotype incidence is controversial and dissimilar results have been reported by different groups. Purpose of the study was to determine the incidence of aberrant phenotypes in North East Indian patients with acute leukemia. We analysed a total of 100 cases (AML = 36, ALL = 61, MPAL = 3) by multiparametric flow cytometry using an acute panel of monoclonal antibodies (MoAbs). The MoAbs were selected to identify differentiation-associated antigens of both myeloid and lymphoid lineages. Aberrant phenotypes were found in 21 (58.3%) cases of AML, 36 (59.2%) cases of B-ALL and 6 (66.7%) cases of T-ALL. CD7 was the most frequent lymphoid associated antigen found in 33% of AML cases while CD117 was the myeloid antigen most frequently detected in ALL (54%) cases. Aberrant expression of CD 117 is highly significant by Fischer's exact test (P< 0.0001). We conclude that aberrant phenotypes are present in a great majority of acute leukemia patients of North East India. Future studies will be directed to correlate of these markers with prognosis and therapeutic response.

Human immunophenotyping via low-variance, low-bias, interpretive regression modeling of small, wide data sets: Application to aging and immune response to influenza vaccination.

PubMed

Holmes, Tyson H; He, Xiao-Song

2016-10-01

Small, wide data sets are commonplace in human immunophenotyping research. As defined here, a small, wide data set is constructed by sampling a small to modest quantity n,1

Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

PubMed Central

2014-01-01

Introduction Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank. Methods The BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA. Results The harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied. Conclusions The BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, the BM-MSCs and AT-MSCs showed fastest ‘‘in vitro’’ differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes. PMID:24559797

CD3-CD4+ lymphoid variant of hypereosinophilic syndrome: nodal and extranodal histopathological and immunophenotypic features of a peripheral indolent clonal T-cell lymphoproliferative disorder.

PubMed

Lefèvre, Guillaume; Copin, Marie-Christine; Roumier, Christophe; Aubert, Hélène; Avenel-Audran, Martine; Grardel, Nathalie; Poulain, Stéphanie; Staumont-Sallé, Delphine; Seneschal, Julien; Salles, Gilles; Ghomari, Kamel; Terriou, Louis; Leclech, Christian; Morati-Hafsaoui, Chafika; Morschhauser, Franck; Lambotte, Olivier; Ackerman, Félix; Trauet, Jacques; Geffroy, Sandrine; Dumezy, Florent; Capron, Monique; Roche-Lestienne, Catherine; Taieb, Alain; Hatron, Pierre-Yves; Dubucquoi, Sylvain; Hachulla, Eric; Prin, Lionel; Labalette, Myriam; Launay, David; Preudhomme, Claude; Kahn, Jean-Emmanuel

2015-08-01

The CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome is characterized by hypereosinophilia and clonal circulating CD3(-)CD4(+) T cells. Peripheral T-cell lymphoma has been described during this disease course, and we observed in our cohort of 23 patients 2 cases of angio-immunoblastic T-cell lymphoma. We focus here on histopathological (n=12 patients) and immunophenotypic (n=15) characteristics of CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome. Atypical CD4(+) T cells lymphoid infiltrates were found in 10 of 12 CD3(-)CD4(+) L-HES patients, in lymph nodes (n=4 of 4 patients), in skin (n=9 of 9) and other extra-nodal tissues (gut, lacrymal gland, synovium). Lymph nodes displayed infiltrates limited to the interfollicular areas or even an effacement of nodal architecture, associated with proliferation of arborizing high endothelial venules and increased follicular dendritic cell meshwork. Analysis of 2 fresh skin samples confirmed the presence of CD3(-)CD4(+) T cells. Clonal T cells were detected in at least one tissue in 8 patients, including lymph nodes (n=4 of 4): the same clonal T cells were detected in blood and in at least one biopsy, with a maximum delay of 23 years between samples. In the majority of cases, circulating CD3(-)CD4(+) T cells were CD2(hi) (n=9 of 14), CD5(hi) (n=12 of 14), and CD7(-)(n=4 of 14) or CD7(low) (n=10 of 14). Angio-immunoblastic T-cell lymphoma can also present with CD3(-)CD4(+) T cells; despite other common histopathological and immunophenotypic features, CD10 expression and follicular helper T-cell markers were not detected in lymphoid variant of hypereosinophilic syndrome patients, except in both patients who developed angio-immunoblastic T-cell lymphoma, and only at T-cell lymphoma diagnosis. Taken together, persistence of tissular clonal T cells and histopathological features define CD3(-)CD4(+) lymphoid variant of hypereosinophilic syndrome as a peripheral indolent clonal T-cell lymphoproliferative disorder, which should not be confused with angio-immunoblastic T-cell lymphoma. Copyright© Ferrata Storti Foundation.

CD3−CD4+ lymphoid variant of hypereosinophilic syndrome: nodal and extranodal histopathological and immunophenotypic features of a peripheral indolent clonal T-cell lymphoproliferative disorder

PubMed Central

Lefèvre, Guillaume; Copin, Marie-Christine; Roumier, Christophe; Aubert, Hélène; Avenel-Audran, Martine; Grardel, Nathalie; Poulain, Stéphanie; Staumont-Sallé, Delphine; Seneschal, Julien; Salles, Gilles; Ghomari, Kamel; Terriou, Louis; Leclech, Christian; Morati-Hafsaoui, Chafika; Morschhauser, Franck; Lambotte, Olivier; Ackerman, Félix; Trauet, Jacques; Geffroy, Sandrine; Dumezy, Florent; Capron, Monique; Roche-Lestienne, Catherine; Taieb, Alain; Hatron, Pierre-Yves; Dubucquoi, Sylvain; Hachulla, Eric; Prin, Lionel; Labalette, Myriam; Launay, David; Preudhomme, Claude; Kahn, Jean-Emmanuel

2015-01-01

The CD3−CD4+ lymphoid variant of hypereosinophilic syndrome is characterized by hypereosinophilia and clonal circulating CD3−CD4+ T cells. Peripheral T-cell lymphoma has been described during this disease course, and we observed in our cohort of 23 patients 2 cases of angio-immunoblastic T-cell lymphoma. We focus here on histopathological (n=12 patients) and immunophenotypic (n=15) characteristics of CD3−CD4+ lymphoid variant of hypereosinophilic syndrome. Atypical CD4+ T cells lymphoid infiltrates were found in 10 of 12 CD3−CD4+ L-HES patients, in lymph nodes (n=4 of 4 patients), in skin (n=9 of 9) and other extra-nodal tissues (gut, lacrymal gland, synovium). Lymph nodes displayed infiltrates limited to the interfollicular areas or even an effacement of nodal architecture, associated with proliferation of arborizing high endothelial venules and increased follicular dendritic cell meshwork. Analysis of 2 fresh skin samples confirmed the presence of CD3−CD4+ T cells. Clonal T cells were detected in at least one tissue in 8 patients, including lymph nodes (n=4 of 4): the same clonal T cells were detected in blood and in at least one biopsy, with a maximum delay of 23 years between samples. In the majority of cases, circulating CD3−CD4+ T cells were CD2hi (n=9 of 14), CD5hi (n=12 of 14), and CD7−(n=4 of 14) or CD7low (n=10 of 14). Angio-immunoblastic T-cell lymphoma can also present with CD3−CD4+ T cells; despite other common histopathological and immunophenotypic features, CD10 expression and follicular helper T-cell markers were not detected in lymphoid variant of hypereosinophilic syndrome patients, except in both patients who developed angio-immunoblastic T-cell lymphoma, and only at T-cell lymphoma diagnosis. Taken together, persistence of tissular clonal T cells and histopathological features define CD3−CD4+ lymphoid variant of hypereosinophilic syndrome as a peripheral indolent clonal T-cell lymphoproliferative disorder, which should not be confused with angio-immunoblastic T-cell lymphoma. PMID:25682606

Bone marrow mononuclears from murine tibia after spaceflight on biosatellite

NASA Astrophysics Data System (ADS)

Andreeva, Elena; Roe, Maria; Buravkova, Ludmila; Andrianova, Irina; Goncharova, Elena; Gornostaeva, Alexandra

Elucidation of the space flight effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this is provided by project "BION -M1". The purpose of this study was to evaluate the effects of a 30-day flight on biosatellite "BION - M1" and the subsequent 7-day recovery on the quantity, viability, immunophenotype of mononuclears from murine tibia bone marrow. Also the in vitro characterization of functional capacity of multipotent mesenchymal stromal cells (MSCs) was scheduled. Under the project, the S57black/6 mice were divided into groups: spaceflight/vivarium control, recovery after spaceflight/ vivarium control to recovery. Bone marrow mononuclears were isolated from the tibia and immunophenotyped using antibodies against CD45, CD34, CD90 on a flow cytometer Epics XL (Beckman Coulter). A part of the each pool was frozen for subsequent estimation of hematopoietic colony-forming units (CFU), the rest was used for the evaluation of fibroblast CFU (CFUf) number, MSC proliferative activity and osteogenic potency. The cell number in the flight group was significantly lower than in the vivarium control group. There were no differences in this parameter between flight and control groups after 7 days of recovery. The mononuclears viability was more than 95 percent in all examined groups. Flow cytometric analysis showed no differences in the bone marrow cell immunophenotype (CD45, CD34, CD90.1 (Thy1)), but the flight animals had more large-sized CD45+mononuclears, than the control groups of mice. There was no difference in the CFUf number between groups. After 7 days in vitro the MSC number in flight group was twice higher than in vivarium group, after 10 days - 4 times higher. These data may indicate a higher proliferative activity of MSCs after spaceflight. MSCs showed the same and high alkaline phosphatase activity, both in flight and in the control groups, suggesting no effect of spaceflight factors on early osteogenic potency of stromal cells. These results indicate that spaceflight factors had no significant damaging effects on the murine bone marrow mononuclears. These observations are consistent with previously made assumption of moderate and reversible stress reaction of mammals on spaceflight conditions. This work was supported by Program of Basic Research of IMBP RAS

Analysis of tumour-infiltrating lymphocytes reveals two new biologically different subgroups of breast ductal carcinoma in situ.

PubMed

Beguinot, Marie; Dauplat, Marie-Melanie; Kwiatkowski, Fabrice; Lebouedec, Guillaume; Tixier, Lucie; Pomel, Christophe; Penault-Llorca, Frederique; Radosevic-Robin, Nina

2018-02-03

Tumour-infiltrating lymphocytes (TILs) have been demonstrated to significantly influence prognosis and response to therapy of invasive breast cancer (IBC). Thus, it has been suggested that TIL density or/and immunophenotype could serve as biomarkers for selection of IBC patients for immunotherapy. However, much less is known about significance of TILs in breast ductal carcinoma in situ (DCIS). We retrospectively investigated TIL density and immunophenotype in 96 pure DCIS and 35 microinvasive carcinomas (miCa). TIL density was assessed on H&E-stained breast biopsy sections as the percentage of tumour stromal area occupied by TILs, and classified into 4 grades: 0 (0%-9%), 1 (10-29%), 2 (30-49%) and 3 (50%-100%). TIL immunophenotype was assessed by immunohistochemistry for CD8, CD4, FoxP3, CD38 or CD20. Compared to pure DCIS, miCa contained significantly more cases with TIL density grade 3 (p = 0.028). Concordantly, CD8+, CD4+ and CD38+ cells were more numerous in miCa than in pure DCIS. In the pure DCIS subgroup with TIL density grades 2 and 3, all TIL subpopulations were more numerous than in the pure DCIS with TIL density grades 0 and 1, however the ratio between T-lymphocytes (CD8+ and CD4+) and B-lymphocytes (CD20+) was significantly lower (p = 0.029). On the other side, this ratio was significantly higher in miCa, in comparison with pure DCIS having TIL density grades 2 and 3 (p = 0.017). By cluster analysis of tumour cell pathobiological features we demonstrated similarity between miCa and the pure DCIS with TIL density grades 2 and 3. The only significant difference between those two categories was in the ratio of T- to B-TILs, higher in miCa. Results indicate that TIL density level can distinguish 2 biologically different DCIS subgroups, one of which (DCIS with ≥30% TILs, the TIL-rich DCIS) is like miCa. Similarity of TIL-rich pure DCIS and miCa as well as the role of B-lymphocytes in DCIS invasiveness are worth further investigating with regards to the potential development of immunotherapy-based prevention of DCIS progression.

Childhood acute leukemias are frequent in Mexico City: descriptive epidemiology.

PubMed

Pérez-Saldivar, María Luisa; Fajardo-Gutiérrez, Arturo; Bernáldez-Ríos, Roberto; Martínez-Avalos, Armando; Medina-Sanson, Aurora; Espinosa-Hernández, Laura; Flores-Chapa, José de Diego; Amador-Sánchez, Raquel; Peñaloza-González, José Gabriel; Alvarez-Rodríguez, Francisco Javier; Bolea-Murga, Victoria; Flores-Lujano, Janet; Rodríguez-Zepeda, María Del Carmen; Rivera-Luna, Roberto; Dorantes-Acosta, Elisa María; Jiménez-Hernández, Elva; Alvarado-Ibarra, Martha; Velázquez-Aviña, Martha Margarita; Torres-Nava, José Refugio; Duarte-Rodríguez, David Aldebarán; Paredes-Aguilera, Rogelio; Del Campo-Martínez, María de Los Ángeles; Cárdenas-Cardos, Rocío; Alamilla-Galicia, Paola Hillary; Bekker-Méndez, Vilma Carolina; Ortega-Alvarez, Manuel Carlos; Mejia-Arangure, Juan Manuel

2011-08-17

Worldwide, acute leukemia is the most common type of childhood cancer. It is particularly common in the Hispanic populations residing in the United States, Costa Rica, and Mexico City. The objective of this study was to determine the incidence of acute leukemia in children who were diagnosed and treated in public hospitals in Mexico City. Included in this study were those children, under 15 years of age and residents of Mexico City, who were diagnosed in 2006 and 2007 with leukemia, as determined by using the International Classification of Childhood Cancer. The average annual incidence rates (AAIR), and the standardized average annual incidence rates (SAAIR) per million children were calculated. We calculated crude, age- and sex-specific incidence rates and adjusted for age by the direct method with the world population as standard. We determined if there were a correlation between the incidence of acute leukemias in the various boroughs of Mexico City and either the number of agricultural hectares, the average number of persons per household, or the municipal human development index for Mexico (used as a reference of socio-economic level). Although a total of 610 new cases of leukemia were registered during 2006-2007, only 228 fit the criteria for inclusion in this study. The overall SAAIR was 57.6 per million children (95% CI, 46.9-68.3); acute lymphoblastic leukemia (ALL) was the most frequent type of leukemia, constituting 85.1% of the cases (SAAIR: 49.5 per million), followed by acute myeloblastic leukemia at 12.3% (SAAIR: 6.9 per million), and chronic myeloid leukemia at 1.7% (SAAIR: 0.9 per million). The 1-4 years age group had the highest SAAIR for ALL (77.7 per million). For cases of ALL, 73.2% had precursor B-cell immunophenotype (SAAIR: 35.8 per million) and 12.4% had T-cell immunophenotype (SAAIR 6.3 per million). The peak ages for ALL were 2-6 years and 8-10 years. More than half the children (58.8%) were classified as high risk. There was a positive correlation between the average number of persons per household and the incidence of the pre-B immunophenotype (Pearson's r, 0.789; P = 0.02). The frequency of ALL in Mexico City is among the highest in the world, similar to those found for Hispanics in the United States and in Costa Rica.

Human Immunophenotyping via Low-Variance, Low-Bias, Interpretive Regression Modeling of Small, Wide Data Sets: Application to Aging and Immune Response to Influenza Vaccination

PubMed Central

Holmes, Tyson H.; He, Xiao-Song

2016-01-01

Small, wide data sets are commonplace in human immunophenotyping research. As defined here, a small, wide data set is constructed by sampling a small to modest quantity n, 1 < n < 50, of human participants for the purpose of estimating many parameters p, such that n < p < 1,000. We offer a set of prescriptions that are designed to facilitate low-variance (i.e. stable), low-bias, interpretive regression modeling of small, wide data sets. These prescriptions are distinctive in their especially heavy emphasis on minimizing use of out-of-sample information for conducting statistical inference. That allows the working immunologist to proceed without being encumbered by imposed and often untestable statistical assumptions. Problems of unmeasured confounders, confidence-interval coverage, feature selection, and shrinkage/denoising are defined clearly and treated in detail. We propose an extension of an existing nonparametric technique for improved small-sample confidence-interval tail coverage from the univariate case (single immune feature) to the multivariate (many, possibly correlated immune features). An important role for derived features in the immunological interpretation of regression analyses is stressed. Areas of further research are discussed. Presented principles and methods are illustrated through application to a small, wide data set of adults spanning a wide range in ages and multiple immunophenotypes that were assayed before and after immunization with inactivated influenza vaccine (IIV). Our regression modeling prescriptions identify some potentially important topics for future immunological research. 1) Immunologists may wish to distinguish age-related differences in immune features from changes in immune features caused by aging. 2) A form of the bootstrap that employs linear extrapolation may prove to be an invaluable analytic tool because it allows the working immunologist to obtain accurate estimates of the stability of immune parameter estimates with a bare minimum of imposed assumptions. 3) Liberal inclusion of immune features in phenotyping panels can facilitate accurate separation of biological signal of interest from noise. In addition, through a combination of denoising and potentially improved confidence interval coverage, we identify some candidate immune correlates (frequency of cell subset and concentration of cytokine) with B cell response as measured by quantity of IIV-specific IgA antibody-secreting cells and quantity of IIV-specific IgG antibody-secreting cells. PMID:27196789

Augmenting Trastuzumab Therapy against Breast Cancer through Selective Activation of NK Cells

DTIC Science & Technology

2014-12-01

purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines including MCF7 (A and E...purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A...and Whiteside, T.L. 2007. A novel multiparametric flow cytometry -based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons

Acute Myeloid Leukemia with MYC Rearrangement and JAK2 V617F Mutation

PubMed Central

Ohanian, Maro; Bueso-Ramos, Carlos; Ok, Chi Young; Lin, Pei; Patel, Keyur; Alattar, Mona Lisa; Khoury, Joseph D.; Rozovski, Uri; Estrov, Zeev; Huh, Yang O.; Cortes, Jorge; Abruzzo, Lynne V.

2016-01-01

Little is known about MYC dysregulation in myeloid malignancies, and we can find no published studies that have evaluated MYC protein expression in primary cases of myelodysplastic syndromes (MDS) or acute myeloid leukemias (AML). We describe the clinical, morphologic, immunophenotypic, cytogenetic, and molecular genetic findings in two MDS/AML cases that contained both MYC rearrangement and JAK2-V617F mutation. We demonstrate MYC protein expression by immunohistochemistry in both patients. PMID:26382622

Is dermatitis palmaris sicca an irritant contact dermatitis?

PubMed

Chen, Fu-Juan; Liu, Zhen; Zhou, Ying; Chen, Yong-Hua; Fan, Yi-Ming

2013-01-01

Dermatitis palmaris sicca (DPS) is a common dry-fissured palmar dermatitis in Asian women. It may be an irritant contact dermatitis, but the immunophenotype of the cells in its infiltrate is unknown. The aim of this study was to evaluate the role of inflammatory cells in the pathogenesis of DPS. Patch testing was done in 68 patients with DPS, 87 subjects with hand eczema, and 31 healthy subjects. Immunophenotyping of cutaneous inflammatory cells was performed in 8 patients with DPS, 10 subjects with hand eczema, and 8 healthy individuals. Positive patch rates were higher in patients with DPS and those with hand eczema compared with healthy controls, but strong positive (++ or +++) reactions in DPS were fewer compared with hand eczema. Density of CD3, CD4, CD8, and CD68 cells in skin lesions of DPS and hand eczema was significantly higher than that in normal skin. Sparse CD20 cells were present only in hand eczema. Compared with hand eczema, the number of CD3, CD8, CD68, and dermal CD1a cells decreased, but epidermal CD1a cells and CD4/CD8 ratio increased in DPS. The absolute lack of CD20 cells and relative scarcity of dermal CD8 and CD1a cells in skin lesions might be insufficient to induce contact hypersensitivity, so DPS may be an irritant but not allergic contact dermatitis.

Heparin concentration is critical for cell culture with human platelet lysate.

PubMed

Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang

2013-09-01

Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Cervical cancer cell supernatants induce a phenotypic switch from U937-derived macrophage-activated M1 state into M2-like suppressor phenotype with change in Toll-like receptor profile.

PubMed

Sánchez-Reyes, Karina; Bravo-Cuellar, Alejandro; Hernández-Flores, Georgina; Lerma-Díaz, José Manuel; Jave-Suárez, Luis Felipe; Gómez-Lomelí, Paulina; de Celis, Ruth; Aguilar-Lemarroy, Adriana; Domínguez-Rodríguez, Jorge Ramiro; Ortiz-Lazareno, Pablo Cesar

2014-01-01

Cervical cancer (CC) is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV) is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated) and M2 (alternatively activated). Macrophage polarization exerts profound effects on the Toll-like receptor (TLR) profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages.

Benign and Malignant Brenner Tumors Show an Absence of TERT Promoter Mutations That Are Commonly Present in Urothelial Carcinoma.

PubMed

Khani, Francesca; Diolombi, Mairo L; Khattar, Pallavi; Huang, Weihua; Fallon, John T; Epstein, Jonathan I; Zhong, Minghao

2016-09-01

Brenner tumors are uncommon ovarian neoplasms, which have morphologic and immunophenotypical features of transitional cell (urothelial) differentiation. The origin of Brenner tumors is perplexing, but they are believed to arise from transitional cell metaplasia occurring within the ovary and/or fallopian tube, although it is controversial whether this metaplasia is truly along transitional cell lines. Recently, TERT promoter mutations have been identified in urothelial carcinoma (UC) with high frequency (approximately 70%), and the current literature suggests a potential diagnostic and/or prognostic role of these mutations in UC. Molecular evidence supporting that Brenner tumors represent neoplasms exhibiting transitional cell differentiation is scant. To explore this further, we investigated a series of 19 Brenner tumors of the ovary (15 benign and 4 malignant) for the presence of TERT promoter mutations after genomic DNA extraction from formalin-fixed paraffin-embedded tissue blocks and standard polymerase chain reaction sequencing. TERT promoter mutations were not identified in any of the cases (0/19). The absence of TERT promoter mutations in Brenner tumors suggests that despite the morphologic and some immunophenotypical resemblance to non-neoplastic and neoplastic transitional epithelium, Brenner tumors may exhibit a molecularly distinct pathogenesis. The findings also may portend diagnostic utility in rare cases wherein it is difficult to distinguish a primary malignant Brenner tumor of the ovary from metastatic UC.

Phenotypic and Genetic Characterization of Circulating Tumor Cells by Combining Immunomagnetic Selection and FICTION Techniques

PubMed Central

Campos, María; Prior, Celia; Warleta, Fernando; Zudaire, Isabel; Ruíz-Mora, Jesús; Catena, Raúl; Calvo, Alfonso; Gaforio, José J.

2008-01-01

The presence of circulating tumor cells (CTCs) in breast cancer patients has been proven to have clinical relevance. Cytogenetic characterization of these cells could have crucial relevance for targeted cancer therapies. We developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) from healthy donors was spiked with a predetermined number of human breast cancer cells. Nucleated cells were separated by double density gradient centrifugation of blood samples. Tumor cells (TCs) were immunomagnetically isolated with an anti-cytokeratin antibody and placed onto slides for FICTION analysis. For immunophenotyping and genetic characterization of TCs, a mixture of primary monoclonal anti-pancytokeratin antibodies was used, followed by fluorescent secondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multicolor probe. Our results show that TCs can be efficiently isolated from peripheral blood and characterized by FICTION. Because genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclines and herceptin therapies, respectively, this novel methodology could be useful for a better classification of patients according to the genetic alterations of CTCs and for the application of targeted therapies. (J Histochem Cytochem 56:667–675, 2008) PMID:18413646

Comprehensive characterization of mesenchymal stromal cells from patients with Fanconi anaemia.

PubMed

Mantelli, Melissa; Avanzini, Maria Antonia; Rosti, Vittorio; Ingo, Daniela M; Conforti, Antonella; Novara, Francesca; Arrigo, Giulia; Boni, Marina; Zappatore, Rita; Lenta, Elisa; Moretta, Antonia; Acquafredda, Gloria; de Silvestri, Annalisa; Cirillo, Valentina; Cicchetti, Elisa; Algeri, Mattia; Strocchio, Luisa; Vinti, Luciana; Starc, Nadia; Biagini, Simone; Sirleto, Pietro; Bernasconi, Paolo; Zuffardi, Orsetta; Maserati, Emanuela; Maccario, Rita; Zecca, Marco; Locatelli, Franco; Bernardo, Maria Ester

2015-09-01

Fanconi anaemia (FA) is an inherited disorder characterized by pancytopenia, congenital malformations and a predisposition to develop malignancies. Alterations in the haematopoietic microenvironment of FA patients have been reported, but little is known regarding the components of their bone marrow (BM) stroma. We characterized mesenchymal stromal cells (MSCs) isolated from BM of 18 FA patients both before and after allogeneic haematopoietic stem cell transplantation (HSCT). Morphology, fibroblast colony-forming unit (CFU-F) ability, proliferative capacity, immunophenotype, differentiation potential, ability to support long-term haematopoiesis and immunomodulatory properties of FA-MSCs were analysed and compared with those of MSCs expanded from 15 age-matched healthy donors (HD-MSCs). FA-MSCs were genetically characterized through conventional karyotyping, diepoxybutane-test and array-comparative genomic hybridization. FA-MSCs generated before and after HSCT were compared. Morphology, immunophenotype, differentiation potential, ability in vitro to inhibit mitogen-induced T-cell proliferation and to support long-term haematopoiesis did not differ between FA-MSCs and HD-MSCs. CFU-F ability and proliferative capacity of FA-MSCs isolated after HSCT were significantly lower than those of HD-MSCs. FA-MSCs reached senescence significantly earlier than HD-MSCs and showed spontaneous chromosome fragility. Our findings indicate that FA-MSCs are defective in their ability to survive in vitro and display spontaneous chromosome breakages; whether these defects are involved in pathophysiology of BM failure syndromes deserves further investigation. © 2015 John Wiley & Sons Ltd.

IL-17 genetic and immunophenotypic evaluation in chronic graft-versus-host disease.

PubMed

Resende, Renata Gonçalves; Correia-Silva, Jeane de Fátima; Silva, Tarcília Aparecida; Salomão, Ulisses Eliezer; Marques-Silva, Luciano; Vieira, Érica Leandro Marciano; Dutra, Walderez Ornelas; Gomez, Ricardo Santiago

2014-01-01

Although interleukin-17 (IL-17) is a recently discovered cytokine associated with several autoimmune diseases, its role in the pathogenesis of chronic graft-versus-host disease (cGVHD) was not established yet. The objective of this study was to investigate the association of IL17A and IL17F genes polymorphisms and IL-17A and IL-17F levels with cGVHD. IL-17A expression was also investigated in CD4(+) T cells of patients with systemic cGVHD. For Part I of the study, fifty-eight allo-HSCT recipients and donors were prospectively studied. Blood samples were obtained to determine IL17A and IL17F genes polymorphisms. Cytokines levels in blood and saliva were assessed by ELISA at days +35 and +100 after HSCT. In Part II, for the immunophenotypic evaluation, eight patients with systemic cGVHD were selected and the expression of IL-17A was evaluated. We found association between recipient AA genotype with systemic cGVHD. No association was observed between IL-17A levels and cGVHD. Lower IL-17A levels in the blood were associated with AA genotype. In flow cytometry analysis, decreased expression of IL-17A was observed in patients with cGVHD after stimulation. In conclusion, IL-17A may have an important role in the development of systemic cGVHD.

Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells.

PubMed

Kim, Dae Seong; Ko, Young Jong; Lee, Myoung Woo; Park, Hyun Jin; Park, Yoo Jin; Kim, Dong-Ik; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

2016-11-01

Culture of mesenchymal stem cells (MSCs) under ambient conditions does not replicate the low oxygen environment of normal physiological or pathological states and can result in cellular impairment during culture. To overcome these limitations, we explored the effect of hypoxia (1 % O 2 ) on the biological characteristics of MSCs over the course of different culture periods. The following biological characteristics were examined in human bone marrow-derived MSCs cultured under hypoxia for 8 weeks: proliferation rate, morphology, cell size, senescence, immunophenotypic characteristics, and the expression levels of stemness-associated factors and cytokine and chemokine genes. MSCs cultured under hypoxia for approximately 2 weeks showed increased proliferation and viability. During long-term culture, hypoxia delayed phenotypic changes in MSCs, such as increased cell volume, altered morphology, and the expression of senescence-associated-β-gal, without altering their characteristic immunophenotypic characteristics. Furthermore, hypoxia increased the expression of stemness and chemokine-related genes, including OCT4 and CXCR7, and did not decrease the expression of KLF4, C-MYC, CCL2, CXCL9, CXCL10, and CXCR4 compared with levels in cells cultured under normoxia. In conclusion, low oxygen tension improved the biological characteristics of MSCs during ex vivo expansion. These data suggest that hypoxic culture could be a useful method for increasing the efficacy of MSC cell therapies.

Sunset yellow FCF, a permitted food dye, alters functional responses of splenocytes at non-cytotoxic dose.

PubMed

Yadav, Ashish; Kumar, Arvind; Tripathi, Anurag; Das, Mukul

2013-03-13

Sunset yellow FCF (SY), a permitted food color, is extensively used in various food preparations and quite often exceeds the permissible levels (100-200 mg/kg). Several toxicity studies on SY are reported, however immunomodulatory properties have not been explored yet. To investigate the immunotoxic properties of SY, splenocytes were isolated, cultured and subjected to mitogen stimulated proliferation assay (lipopolysaccharide, LPS or concanavalin A, Con A), mixed lymphocyte reaction (MLR) assay, immunophenotypic analysis of cell surface receptor expression and assay for cytokines release in the culture supernatants were performed in the presence of SY. Since SY did not exhibit any cytotoxicity up to 250 μg/ml, this dose was used for further studies. It was observed that SY (250 μg/ml) significantly (p<0.05) suppressed the mitogen induced proliferation of splenocytes and MLR response. Further, immunophenotypic analysis revealed that SY alters the relative expression of CD3e/CD4/CD8 in T cells and CD19 in B-cells. Consistent with the suppression of T-cell and B-cell responses and altered surface receptor expression, SY also lowered the expression of IL2, IL4, IL6, IL-17, IFN-γ and TNF-α cytokines. These results suggest that non-cytotoxic dose of SY may have immunomodulatory effects. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Chronic alcohol consumption is associated with changes in the distribution, immunophenotype, and the inflammatory cytokine secretion profile of circulating dendritic cells.

PubMed

Laso, Francisco Javier; Vaquero, José Miguel; Almeida, Julia; Marcos, Miguel; Orfao, Alberto

2007-05-01

Alcoholism is frequently associated with altered immune responses, limited information being available on its effects on dendritic cells (DC). In the present study we analyze the effects of chronic alcoholism on circulating DC. For the first time we studied the numerical distribution of DC in peripheral blood (PB), their immunophenotype, and their ex vivo pattern of spontaneous cytokine secretion, in chronic alcoholic patients without liver disease (AWLD group; n=17) and active ethanol (EtOH) intake, as well as in subjects with alcohol liver cirrhosis (ALC group; n=21). A significantly decreased HLADR expression and an increased reactivity for CD123 was observed on PB DC from AWLD patients; additionally, increased secretion of interleukin (IL) 1beta, IL6, IL12, and tumor necrosis factor-alpha (TNFalpha) by DC was also noted in this group. Conversely, patients with ALC and at least 1 year of alcohol withdrawal (ALCAW group) showed a decreased number of total circulating DC, whereas ALC patients with active EtOH intake (ALCET group) had an abnormally low production of IL1beta and TNFalpha by PB DC. Chronic alcoholism in the absence of liver disease is associated with an increased secretion of inflammatory cytokines by PB DC, whereas ALCAW and ALCET patients show decreased numbers of circulating DC and reduced secretion of these cytokines, respectively.

ALK-negative anaplastic large cell lymphoma with urinary bladder involvement diagnosed in urine cytology: A case report and literature review.

PubMed

Lobo, João; Henrique, Rui; Monteiro, Paula; Lobo, Cláudia

2017-04-01

Anaplastic large cell lymphoma is an aggressive T-cell neoplasm. It rarely involves the urinary bladder, with just twelve cases reported thus far and only one being ALK-negative. Immunophenotyping (particularly for ALK) is mandatory, both for prognostic and therapeutic reasons. Herein, we report the case of a patient with an ALK-negative anaplastic large cell lymphoma involving the bladder which was diagnosed and fully characterized by immunocytochemistry in urine cytology. The patient underwent a cystoscopy and the urine sample disclosed tumor diathesis background and aggregates of atypical cells, with evidence of multinucleation and mitotic figures. Immunocytochemistry revealed strong membrane/Golgi positivity for CD30 and negativity for ALK. The patient was submitted to transurethral resection for therapeutic purposes, which confirmed the diagnosis. To the best of our knowledge, this represents only the third case of anaplastic large cell lymphoma with bladder involvement diagnosed in urine cytology and the very first with diagnostic findings allowing for immunophenotyping of the disease in a bladder wash. The present report reinforces the role of urine cytology as a suitable method for establishing an earlier diagnosis and characterization of the disease, avoiding submitting patients to invasive procedures like transurethral resections. Diagn. Cytopathol. 2017;45:354-358. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues.

PubMed

Carrion, Ricardo; Brasky, Kathleen; Mansfield, Keith; Johnson, Curtis; Gonzales, Monica; Ticer, Anysha; Lukashevich, Igor; Tardif, Suzette; Patterson, Jean

2007-06-01

Lassa virus causes thousands of deaths annually in western Africa and is considered a potential biological weapon. In an attempt to develop a small nonhuman primate model of Lassa fever, common marmosets were subcutaneously inoculated with Lassa virus strain Josiah. This inoculation resulted in a systemic disease with clinical and morphological features mirroring those in fatal human Lassa infection: fever, weight loss, high viremia and viral RNA load in tissues, elevated liver enzymes, and severe morbidity between days 15 and 20. The most prominent histopathology findings included multifocal hepatic necrosis with mild inflammation and hepatocyte proliferation, lymphoid depletion, and interstitial nephritis. Cellular aggregates in regions of hepatocellular necrosis were largely composed of HAM56-positive macrophages, devoid of CD3-positive and CD20-positive cells, and characterized by marked reductions in the intensity of HLA-DP, DQ, DR staining. A marked reduction in the major histocompatibility complex class II expression was also observed in the lymph nodes. Immunophenotypic alterations in spleen included reductions in overall numbers of CD20-positive and CD3-positive cells and the disruption of lymphoid follicular architecture. These findings identify the common marmoset as an appropriate model of human Lassa fever and present the first experimental evidence that replication of Lassa virus in tissues is associated with alterations that would be expected to impair adaptive immunity.

Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression.

PubMed

Irioda, Ana Carolina; Cassilha, Rafael; Zocche, Larissa; Francisco, Julio Cesar; Cunha, Ricardo Correa; Ferreira, Priscila Elias; Guarita-Souza, Luiz Cesar; Ferreira, Reginaldo Justino; Mogharbel, Bassam Felipe; Garikipati, Venkata Naga Srikanth; Souza, Daiany; Beltrame, Mirian Perlingeiro; de Carvalho, Katherine Athayde Teixeira

2016-01-01

Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d), cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

Orthotopic xenografts of RCC retain histological, immunophenotypic and genetic features of tumors in patients

PubMed Central

Grisanzio, Chiara; Seeley, Apryle; Chang, Michelle; Collins, Michael; Di Napoli, Arianna; Cheng, Su-Chun; Percy, Andrew; Beroukhim, Rameen; Signoretti, Sabina

2013-01-01

Renal cell carcinoma (RCC) is an aggressive malignancy with limited responsiveness to existing treatments. In vivo models of human cancer, including RCC, are critical for developing more effective therapies. Unfortunately, current RCC models do not accurately represent relevant properties of the human disease. The goal of this study was to develop clinically relevant animal models of RCC for preclinical investigations. We transplanted intact human tumor tissue fragments orthotopically in immunodeficient mice. The xenografts were validated by comparing the morphologic, phenotypic, and genetic characteristics of the kidney tumor tissues before and after implantation. Twenty kidney tumors were transplanted into mice. Successful tumor growth was detected in 19 cases (95%). The histopathologic and immunophenotypic features of the xenografts and those of the original tumors largely overlapped in all the cases. Evaluation of genetic alterations in a subset of 10 cases demonstrated that the grafts largely retained the genetic features of the pre-implantation RCC tissues. Indeed, primary tumors and corresponding grafts displayed identical VHL mutations. Moreover, an identical pattern of DNA copy amplification or loss was observed in 6 of 10 cases (60%). In summary, orthotopic engrafting of RCC tissue fragments can be successfully used to generate animal models that closely resemble RCC in patients. These models will be invaluable for in vivo preclinical drug testing, and for deeper understanding of kidney carcinogenesis. PMID:21710693

Correlation of Cell Surface Biomarker Expression Levels with Adhesion Contact Angle Measured by Lateral Microscopy.

PubMed

Walz, Jenna A; Mace, Charles R

2018-06-05

Immunophenotyping is typically achieved using flow cytometry, but any influence a biomarker may have on adhesion or surface recognition cannot be determined concurrently. In this manuscript, we demonstrate the utility of lateral microscopy for correlating cell surface biomarker expression levels with quantitative descriptions of cell morphology. With our imaging system, we observed single cells from two T cell lines and two B cell lines adhere to antibody-coated substrates and quantified this adhesion using contact angle measurements. We found that SUP-T1 and CEM CD4+ cells, both of which express similar levels of CD4, experienced average changes in contact angle that were not statistically different from one another on surfaces coated in anti-CD4. However, MAVER-1 and BJAB K20 cells, both of which express different levels of CD20, underwent average changes in contact angle that were significantly different from one another on surfaces coated in anti-CD20. Our results indicate that changes in cell contact angles on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Our lateral microscopy approach offers a more reproducible and quantitative alternative to evaluate adhesion compared to commonly used wash assays and can be extended to many additional immunophenotyping applications to identify cells of interest within heterogeneous populations.

Dendritic Cells Promote Macrophage Infiltration and Comprise a Substantial Proportion of Obesity-Associated Increases in CD11c+ Cells in Adipose Tissue and Liver

PubMed Central

Stefanovic-Racic, Maja; Yang, Xiao; Turner, Michael S.; Mantell, Benjamin S.; Stolz, Donna B.; Sumpter, Tina L.; Sipula, Ian J.; Dedousis, Nikolaos; Scott, Donald K.; Morel, Penelope A.; Thomson, Angus W.; O’Doherty, Robert M.

2012-01-01

Obesity-associated increases in adipose tissue (AT) CD11c+ cells suggest that dendritic cells (DC), which are involved in the tissue recruitment and activation of macrophages, may play a role in determining AT and liver immunophenotype in obesity. This study addressed this hypothesis. With the use of flow cytometry, electron microscopy, and loss-and-gain of function approaches, the contribution of DC to the pattern of immune cell alterations and recruitment in obesity was assessed. In AT and liver there was a substantial, high-fat diet (HFD)–induced increase in DC. In AT, these increases were associated with crown-like structures, whereas in liver the increase in DC constituted an early and reversible response to diet. Notably, mice lacking DC had reduced AT and liver macrophages, whereas DC replacement in DC-null mice increased liver and AT macrophage populations. Furthermore, delivery of bone marrow–derived DC to lean wild-type mice increased AT and liver macrophage infiltration. Finally, mice lacking DC were resistant to the weight gain and metabolic abnormalities of an HFD. Together, these data demonstrate that DC are elevated in obesity, promote macrophage infiltration of AT and liver, contribute to the determination of tissue immunophenotype, and play a role in systemic metabolic responses to an HFD. PMID:22851575

Intestinal double-positive CD4+CD8+ T cells of neonatal rhesus macaques are proliferating, activated memory cells and primary targets for SIVMAC251 infection

PubMed Central

Wang, Xiaolei; Das, Arpita; Lackner, Andrew A.; Veazey, Ronald S.

2008-01-01

Peripheral blood and thymic double-positive (DP) CD4+CD8+ T cells from neonates have been described earlier, but the function and immunophenotypic characteristics of other tissue-derived DP T cells are not clearly understood. Here, we demonstrate the functional and immunophenotypic characteristics of DP cells in 6 different tissues, including thymus from normal neonatal rhesus macaques (Macaca mulatta) between 0 and 21 days of age. In general, intestinal DP T cells of neonates have higher percentages of memory markers (CD28+CD95+CD45RAlowCD62Llow) and proliferation compared with single-positive (SP) CD4+ and CD8+ T cells. In addition, percentages of DP T cells increase and CD62L expression decreases as animals mature, suggesting that DP cells mature and proliferate with maturity and/or antigen exposure. Consistent with this, intestinal DP T cells in neonates express higher levels of CCR5 and are the primary targets in simian immunodeficiency virus (SIV) infection. Finally, DP T cells produce higher levels of cytokine in response to mitogen stimulation compared with SP CD4+ or CD8+ T cells. Collectively, these findings demonstrate that intestinal DP T cells of neonates are proliferating, activated memory cells and are likely involved in regulating immune responses, in contrast to immature DP T cells in the thymus. PMID:18820133

Flow cytometric discrimination of seven lineage markers by using two fluorochromes

PubMed Central

Boin, Francesco; Giardino Torchia, Maria Letizia; Borrello, Ivan; Noonan, Kimberly A.; Neil, Matthew; Soloski, Mark J.

2017-01-01

Flow cytometry is the primary immunological technique used to analyze multiple parameters on complex cell populations. We present a staining method that identifies major human mononuclear lymphoid and myeloid populations (CD4+ and CD8+ T cells, γδ T cells, B cells, NK cells and monocytes), using only two fluorochromes and a minimal number of cells. Our approach increases the number of markers recordable on most flow cytometers allowing for a deeper and more comprehensive immunophenotyping. PMID:29190813

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology.

PubMed

Pavon, Lorena Favaro; Sibov, Tatiana Tais; Caminada de Toledo, Silvia Regina; Mara de Oliveira, Daniela; Cabral, Francisco Romero; Gabriel de Souza, Jean; Boufleur, Pamela; Marti, Luciana C; Malheiros, Jackeline Moraes; Ferreira da Cruz, Edgar; Paiva, Fernando F; Malheiros, Suzana M F; de Paiva Neto, Manoel A; Tannús, Alberto; Mascarenhas de Oliveira, Sérgio; Silva, Nasjla Saba; Cappellano, Andrea Maria; Petrilli, Antonio Sérgio; Chudzinski-Tavassi, Ana Marisa; Cavalheiro, Sérgio

2018-04-24

Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients ( n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

Immunophenotypic analysis of the Kaposi sarcoma herpesvirus (KSHV; HHV-8)-infected B cells in HIV+ multicentric Castleman disease (MCD).

PubMed

Chadburn, A; Hyjek, E M; Tam, W; Liu, Y; Rengifo, T; Cesarman, E; Knowles, D M

2008-11-01

Kaposi sarcoma herpesvirus (KSHV) is aetiologically related to Kaposi sarcoma, classical and extracavitary primary effusion lymphoma (PEL; EC-PEL) and multicentric Castleman disease (MCD), entities preferentially occurring in HIV-infected individuals. Characterization of HIV-associated PELs/EC-PELs suggests that the KSHV-infected malignant cells originate from a pre-terminal stage of B-cell differentiation. However, only limited phenotypic studies have been performed on HIV+ MCD, including for PR domain containing 1 with zinc finger domain/B lymphocyte-induced maturation protein 1 (PRDM1/BLIMP1), a key regulator of terminal B-cell differentiation. The aim was to characterize KSHV-infected cells in 17 cases of HIV+ MCD. Double immunohistochemistry and immunohistochemistry-in situ hybridization were used to characterize the KSHV-infected cells in MCD; the results were compared with the phenotypic profiles of 39 PELs/EC-PELs and seven PEL cell lines. Whereas the immunophenotype of KSHV-infected cells in MCD and malignant KSHV+ PEL cells was similar (PAX5, Bcl-6-; PRDM1/BLIMP1, IRF4/MUM1+; Ki67+), the MCD KSHV-infected cells differed, as they expressed OCT2, cytoplasmic lambda immunoglobulin; variably expressed CD27; lacked CD138; and were Epstein-Barr virus negative. Although both PEL and MCD originate from KSHV-infected pre-terminally differentiated B cells, these findings, with previously reported genetic studies, indicate HIV+ MCD may arise from extrafollicular B cells, whereas PELs may originate from cells that have traversed the germinal centre.

The clinical characteristics and the features of immunophenotype of peripheral lymphocytes of adult onset chronic active Epstein-Barr virus disease at a Tertiary Care Hospital in Beijing.

PubMed

Luo, Ling; Wang, Huanling; Fan, Hongwei; Xie, Jing; Qiu, Zhifeng; Li, Taisheng

2018-03-01

Chronic active Epstein-Barr virus (CAEBV) infection is a rare disease with high mortality. Most of CAEBV patients have been reported from Japan and are pediatric cases.The goal was to describe the clinical characteristics and the immunophenotypic features of peripheral lymphocytes in adult onset CAEBV patients.We retrospectively reviewed and analyzed all adult onset CAEBV cases admitted to Peking Union Medical College Hospital (PUMCH) between 2012 and 2016. Demographic, clinical, laboratory data, and the immunophentyping data of peripheral lymphocytes were collected.There were 28 adult onset CAEBV patients. The median age was 45 (range, 20-81). Most of the patients presented with fever; splenomegaly; lymphadenopathy and hepatitis. Unlike pediatric cases reported, the manifestations of cardiovascular diseases in our patients were pulmonary arterial hypertension, decreased cardiac function and aorta vasculitis. Prevalence of interstitial pneumonitis in our patients were comparatively higher and prevalence of hypersensitivity to mosquito bites were comparatively lower than that reported by Japan. In this study, CAEBV patients had decreased B cell, NK cell, CD4 cell and CD8 cell counts. The prevalence of low level of B cells, NK cells, CD4 cells was relatively higher than reported ever.Chinese adult onset CAEBV patients have different clinical characteristics and are featured by an immunosuppression status as demonstrated by decreased B cell, NK cell, CD4 cell and CD8 cell.

Comparison of quantitative flow cytometric data provided by panels with lower and increased color number

NASA Astrophysics Data System (ADS)

Bocsi, József; Mittag, Anja; Pierzchalski, Arkadiusz; Baumgartner, Adolf; Dähnert, Ingo; Tárnok, Attila

2012-03-01

To date the flow cytometry (FCM) industry is booming with new generations of commercial clinical instruments. Long-term clinical studies have the dilemma that moving to new instruments being capable of more complex cell-analysis makes it difficult to compare new data with those obtained on older instruments with less complex analysis panels. Since 15 years we conduct follow-up studies on children with congenital heart diseases. In this period we moved from 2- to 3- and now to 10-color FCM immunophenotyping panels. Questions arise how to compare and transfer data from lower to higher level of complexity. Two comparable antibody panels for leukocyte immunophenotyping (12-tube 2-colors, and 9-tube 4-colors) were measured on a BD FACScalibur FCM (calibration: Spherotech beads) in 19 blood samples from children with congenital heart disease. This increase of colors was accompanied by moving antibodies that were in the 2-color panel either FITC or PE labeled to red dyes such as PerCP or APC. Algorithms were developed for bridging data for quantitative characterization of antigen expression (mean fluorescence intensity) and frequency of different cell subpopulations in combination with rainbow bead standard data. This approach worked for the most relevant antibodies (CD3, CD4, CD8 etc.) well, but rendered substantial uncertainty for activation markers (CD69 etc.). Our techniques are particularly well suited to the analysis in long-term studies and have the potential to compare older and recent results in a standardized way.

CD30 expression in follicular lymphoma.

PubMed

Gardner, L J; Polski, J M; Evans, H L; Perkins, S L; Dunphy, C H

2001-08-01

CD30(+) anaplastic large cell lymphomas were originally described as being of T-cell, null cell, and B-cell origin. CD30, however, is not a specific marker of anaplastic large cell lymphoma and has been found to be expressed in reactive as well as neoplastic populations as a probable activation marker. In addition, CD30(+) cells have also been described in both diffuse large B-cell and follicular lymphomas (FLs), resembling the pattern seen in reactive tonsils and lymph nodes. We report an index case of FL with CD30 expression, which on initial touch preparations and flow cytometric immunophenotyping revealed a prominent population of CD30(+) cells with marked cellular pleomorphism (anaplasia) in a background of typical FL. Immunohistochemistry of the paraffin section for CD30 in our index case confirmed unequivocal CD30(+) pleomorphic cells in the malignant nodules in occasional clusters. This case prompted a study of additional cases of FL for pattern of immunoreactivity with CD30 on paraffin sections. Twenty-two additional cases of FL (grades 1-3) were retrieved for CD30 immunoperoxidase staining as in the index case. This study demonstrated 32% of the additional cases of FL had definitive CD30(+), large, pleomorphic malignant cells by paraffin immunohistochemistry. In 2 cases (9%), the pattern of immunoreactivity with CD30 showed clustering and variable staining of large cells, as our index case. This study underscores the morphologic and immunophenotypic spectrum of FL that includes CD30 staining and cellular pleomorphism.

Systematic donor blood qualification by flow cytometry would have been able to avoid CLL-type MBL transmission after unrelated hematopoietic stem cell transplantation.

PubMed

Ferrand, Christophe; Garnache-Ottou, Francine; Collonge-Rame, Marie Agnès; Larosa, Fabrice; Blanc, Michel; Behar, Catherine; Giannoli, Catherine; Garnier, Frédérico; Tiberghien, Pierre; Deconinck, Eric; Rohrlich, Pierre Simon

2012-03-01

The current screening for eligibility of unrelated volunteer marrow donors comprises a complete clinical check-up, a blood CBC and serum protein immunoelectrophoresis. This allows to eliminate acute leukemias, myeloproliferative and myelodysplastic disorders, myelomas and MGUS. To date, the risk of transmission of chronic lymphocytic leukemia (CLL) disease is only evaluated by the clinical evaluation and CBC. We report here the case of a CLL-type MBL disease occurring in a 12-year-old boy after unrelated BMT. Deep biological investigations, as Immunophenotyping, cytogenetic and molecular biology allow us to determine the donor origin of the CLL clone. In 2010, 14.2% donor (105/737) for unrelated hematopoietic stem cell transplantation were over 45y. It is currently estimated (USA) that 1 in 210 men and women will be diagnosed with CLL during their lifetime. Given the long asymptomatic phase of CLL, this raises the case for a detection strategy analog to that used for MGUS and myeloma through serum protein electrophoresis. This case-report, to our knowledge, of a CLL-type MBL unrelated donor-to-recipient transmission through BMT raises ethical and practical questions, such as the proper information about disease transmission risk. The cost-effectiveness of a systematic peripheral blood Immunophenotyping in donors elder than 40y at time of stem cell donation should be evaluated. © 2012 John Wiley & Sons A/S.

Human herpesvirus 8-associated lymphoma mimicking cutaneous anaplastic large T-cell lymphoma in a patient with human immunodeficiency virus infection.

PubMed

Li, Meng-Fang; Hsiao, Cheng-Hsiang; Chen, Yi-Lin; Huang, Wen-Ya; Lee, Yi-Hsuan; Huang, Hsien-Neng; Lien, Huang-Chun

2012-02-01

Primary effusion lymphoma, a human herpesvirus 8 (HHV8)-associated lymphoma, is uncommon, and it is usually seen in human immunodeficiency virus (HIV)-infected patients. It presents as a body cavity-based lymphomatous effusion, but several cases of the so-called solid primary effusion lymphoma presenting as solid tumors without associated lymphomatous effusion have been reported. They have similar clinical, histopathological and immunophenotypical features. Most of them have a B-cell genotype. This suggests the solid variant may represent a clinicopathological spectrum of primary effusion lymphoma. We report a case of HHV8-associated lymphoma histopathologically and immunophenotypically mimicking cutaneous anaplastic large cell lymphoma. The patient was a 31-year-old HIV-seropositive man presenting with skin nodules over his right thigh. Biopsy of the nodules showed anaplastic large cells infiltrating the dermis. These malignant cells strongly expressed CD3, CD30 and CD43. Cutaneous anaplastic large T-cell lymphoma was initially diagnosed, but further tests, including immunoreactivity for HHV8 protein and clonal rearrangements of immunoglobulin genes, confirmed the diagnosis of HHV8-associated B-cell lymphoma with aberrant T-cell marker expression. This case provides an example of solid primary effusion lymphoma mimicking cutaneous anaplastic large T-cell lymphoma and highlights the importance of HHV8 immunohistochemistry and molecular tests in the diagnosis of HHV8-associated lymphoma with a cutaneous presentation. Copyright © 2011 John Wiley & Sons A/S.

Immunophenotyping of rheumatoid arthritis reveals a linkage between HLA-DRB1 genotype, CXCR4 expression on memory CD4(+) T cells, and disease activity.

PubMed

Nagafuchi, Yasuo; Shoda, Hirofumi; Sumitomo, Shuji; Nakachi, Shinichiro; Kato, Rika; Tsuchida, Yumi; Tsuchiya, Haruka; Sakurai, Keiichi; Hanata, Norio; Tateishi, Shoko; Kanda, Hiroko; Ishigaki, Kazuyoshi; Okada, Yukinori; Suzuki, Akari; Kochi, Yuta; Fujio, Keishi; Yamamoto, Kazuhiko

2016-07-07

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that leads to destructive arthritis. Although the HLA class II locus is the strongest genetic risk factor for rheumatoid arthritis, the relationship between HLA class II alleles and lymphocyte activation remains unclear. We performed immunophenotyping of peripheral blood mononuclear cells on 91 HLA-DRB1-genotyped RA patients and 110 healthy donors. The frequency of memory CXCR4(+)CD4(+) T cells, and not Th1 and Th17 cells, was significantly associated with disease severity by multiple linear regression analysis. RA patients with one or more susceptible HLA-DR haplotypes (shared epitope: SE) displayed a significantly higher frequency of memory CXCR4(+)CD4(+) T cells. Moreover, the frequency of memory CXCR4(+)CD4(+) T cells significantly correlated with the expression level of HLA-DR on B cells, which was elevated in RA patients with SE. In vitro analysis and transcriptomic pathway analysis suggested that the interaction between HLA-DR and T cell receptors is an important regulator of memory CXCR4(+)CD4(+) T cells. Clinically, a higher frequency of memory CXCR4(+)CD4(+) T cells predicted a better response to CTLA4-Ig. Memory CXCR4(+)CD4(+) T cells may serve as a powerful biomarker for unraveling the linkage between HLA-DRB1 genotype and disease activity in RA.

Anti-TNF and thiopurine therapy in pregnant IBD patients does not significantly alter a panel of B-cell and T-cell subsets in 1-year-old infants.

PubMed

Kattah, Michael G; Milush, Jeffrey M; Burt, Trevor; McCabe, Robert P; Whang, Michael I; Ma, Averil; Mahadevan, Uma

2018-04-03

Infants exposed to combination therapy with anti-tumor necrosis factor (anti-TNF) agents and thiopurines may exhibit increased infections at 1 year of age compared to unexposed infants. We hypothesized that this increased risk of infection is due to abnormal development of the newborn immune system. We immunophenotyped B-cell and T-cell subsets using multiparameter flow cytometry in 1-year-old infants whose mothers were exposed to therapeutic agents for IBD. We analyzed samples from infants exposed to infliximab (IFX) or adalimumab (ADA) monotherapy (IFX/ADA, n = 11), certolizumab pegol (CZP) monotherapy (CZP, n = 4), IFX or ADA plus thiopurine combination therapy (IFX/ADA + IM, n = 4), and CZP plus thiopurine combination therapy (CZP + IM, n = 2). Percentages of B cells, CD4 + T helper cells, T regulatory cells (T regs ), and CD8 + cytotoxic T cells, were similar among the groups. Infants exposed to combination therapy (IFX/ADA + IM) exhibited trends toward fewer CD27 + B cells, switched memory B cells, plasmablasts, interferon gamma (IFNγ)-producing CD4 + and CD8 + T cells, and CCR5 + CD4 + T cells, but these did not reach statistical significance. Multiparameter immunophenotyping of major B-cell and T-cell subsets suggests that the adaptive newborn immune system develops largely unaltered after exposure to combination therapy as compared to anti-TNF monotherapy.

Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis.

PubMed

Kadić, Elma; Moniz, Raymond J; Huo, Ying; Chi, An; Kariv, Ilona

2017-02-02

Comprehensive understanding of cellular immune subsets involved in regulation of tumor progression is central to the development of cancer immunotherapies. Single cell immunophenotyping has historically been accomplished by flow cytometry (FC) analysis, enabling the analysis of up to 18 markers. Recent advancements in mass cytometry (MC) have facilitated detection of over 50 markers, utilizing high resolving power of mass spectrometry (MS). This study examined an analytical and operational feasibility of MC for an in-depth immunophenotyping analysis of the tumor microenvironment, using the commercial CyTOF™ instrument, and further interrogated challenges in managing the integrity of tumor specimens. Initial longitudinal studies with frozen peripheral blood mononuclear cells (PBMCs) showed minimal MC inter-assay variability over nine independent runs. In addition, detection of common leukocyte lineage markers using MC and FC detection confirmed that these methodologies are comparable in cell subset identification. An advanced multiparametric MC analysis of 39 total markers enabled a comprehensive evaluation of cell surface marker expression in fresh and cryopreserved tumor samples. This comparative analysis revealed significant reduction of expression levels of multiple markers upon cryopreservation. Most notably myeloid derived suppressor cells (MDSC), defined by co-expression of CD66b + and CD15 + , HLA-DR dim and CD14 - phenotype, were undetectable in frozen samples. These results suggest that optimization and evaluation of cryopreservation protocols is necessary for accurate biomarker discovery in frozen tumor specimens.

Utility of peripheral blood immunophenotyping by flow cytometry in the diagnosis of pediatric acute leukemia.

PubMed

Metrock, Laura K; Summers, Ryan J; Park, Sunita; Gillespie, Scott; Castellino, Sharon; Lew, Glen; Keller, Frank G

2017-10-01

Childhood acute leukemia is traditionally diagnosed from a bone marrow aspirate (BMA). New-onset acute leukemia patients do not always have visible circulating blasts in the peripheral blood (PB) at diagnosis. While the role of bone marrow flow cytometry for the diagnosis of acute leukemia is well established, the utility of PB flow cytometry (PBFC) is unknown. We performed a single-institution retrospective analysis to compare PBFC versus BMA in establishing or excluding a diagnosis of childhood acute leukemia. We retrospectively identified 485 PBFC samples with concurrent BMA from 2008 to 2013. Results of four-color flow cytometry for immunophenotypic characterization of leukemic versus nonclonal disease were characterized. Sensitivity and specificity were calculated among patients without a known diagnosis or prior therapy. Among 485 samples eligible for analysis, 120 had negative PBFC and BMA, 359 had positive PBFC and BMA, 3 had negative PBFC and positive BMA, and 3 had positive PBFC and negative BMA. There were small but significant differences in sensitivity (100 vs. 93.8%; P = 0.002) and positive predictive value (100 vs. 93.8%; P = 0.002) favoring BMA over PBFC among those demonstrating absence of circulating morphologic blasts. PBFC has high sensitivity and specificity for the diagnosis of childhood acute leukemia. The predictive value of PBFC remains high for patients without visible circulating blasts and may enhance the diagnostic process for determining the indications for marrow testing. © 2017 Wiley Periodicals, Inc.

Serum of myeloproliferative neoplasms stimulates hematopoietic stem and progenitor cells.

PubMed

Lubberich, Richard K; Walenda, Thomas; Goecke, Tamme W; Strathmann, Klaus; Isfort, Susanne; Brümmendorf, Tim H; Koschmieder, Steffen; Wagner, Wolfgang

2018-01-01

Myeloproliferative neoplasms (MPN)-such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF)-are typically diseases of the elderly caused by acquired somatic mutations. However, it is largely unknown how the malignant clone interferes with normal hematopoiesis. In this study, we analyzed if serum of MPN patients comprises soluble factors that impact on hematopoietic stem and progenitor cells (HPCs). CD34+ HPCs were cultured in medium supplemented with serum samples of PV, ET, or MF patients, or healthy controls. The impact on proliferation, maintenance of immature hematopoietic surface markers, and colony forming unit (CFU) potential was systematically analyzed. In addition, we compared serum of healthy young (<25 years) and elderly donors (>50 years) to determine how normal aging impacts on the hematopoiesis-supportive function of serum. Serum from MF, PV and ET patients significantly increased proliferation as compared to controls. In addition, serum from MF and ET patients attenuated the loss of a primitive immunophenotype during in vitro culture. The CFU counts were significantly higher if HPCs were cultured with serum of MPN patients as compared to controls. Furthermore, serum of healthy young versus old donors did not evoke significant differences in proliferation or immunophenotype of HPCs, whereas the CFU frequency was significantly increased by serum from elderly patients. Our results indicate that serum derived from patients with MPN comprises activating feedback signals that stimulate the HPCs-and this stimulatory signal may result in a viscous circle that further accelerates development of the disease.

Immaturity associated antigens are lost during induction for T cell lymphoblastic leukemia: implications for minimal residual disease detection

PubMed Central

Roshal, Mikhail; Fromm, Jonathan R; Winter, Stuart; Dunsmore, Kimberly; Wood, Brent

2011-01-01

Background Induction chemotherapy for acute leukemia often leads to antigenic shifts in residual abnormal blast populations. Studies in precursor B cell ALL (B-ALL) and AML have demonstrated that chemotherapy commonly results in the loss of antigens associated with immaturity, limiting their utility for minimal residual disease (MRD) detection. Little information is available about the stability of these antigens in precursor T cell ALL (T-ALL) though it is presumed that CD99 and TdT are highly informative based on limited studies. Methods In a longitudinal investigation, we explored patterns of lineage specific and immaturity associated antigens in T-ALL in a large cohort of patients treated under the multicenter Children's Oncology Group (COG) protocol. All samples were analyzed using multicolor flow cytometry in a standardized fashion at a single institution. Results We report that markers of immaturity particularly, TdT and CD99 dramatically decline on leukemic blasts during therapy. CD34 and CD10 expression is confined to a minority of pre-treatment samples and is also not stable. In contrast, lineage associated markers including CD2, CD3, CD4, CD5, CD7 and CD8 failed to show significant trends. Conclusions Our study strongly argues for expansion of immunophenotyping panels for T-ALL MRD to decrease reliance on immature antigens. This study represents the first demonstration of consistent immunophenotypic shifts in T-ALL. PMID:20155852

Diversity and divergence of the glioma-infiltrating T-cell receptor repertoire

PubMed Central

Sims, Jennifer S.; Grinshpun, Boris; Feng, Yaping; Ung, Timothy H.; Neira, Justin A.; Samanamud, Jorge L.; Canoll, Peter; Shen, Yufeng; Sims, Peter A.; Bruce, Jeffrey N.

2016-01-01

Although immune signaling has emerged as a defining feature of the glioma microenvironment, how the underlying structure of the glioma-infiltrating T-cell population differs from that of the blood from which it originates has been difficult to measure directly in patients. High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. We have defined immunophenotypes of whole repertoires based on TCRseq of the α- and β-chains from glioma tissue, nonneoplastic brain tissue, and peripheral blood from patients. Using information theory, we partitioned the diversity of these TCR repertoires into that from the distribution of VJ cassette combinations and diversity due to VJ-independent factors, such as selection due to antigen binding. Tumor-infiltrating lymphocytes (TILs) possessed higher VJ-independent diversity than nonneoplastic tissue, stratifying patients according to tumor grade. We found that the VJ-independent components of tumor-associated repertoires diverge more from their corresponding peripheral repertoires than T-cell populations in nonneoplastic brain tissue, particularly for low-grade gliomas. Finally, we identified a “signature” set of TCRs whose use in peripheral blood is associated with patients exhibiting low TIL divergence and is depleted in patients with highly divergent TIL repertoires. This signature is detectable in peripheral blood, and therefore accessible noninvasively. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to antiglioma vaccines and immunotherapy. PMID:27261081

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

PubMed

Marín-Llera, Jessica Cristina; Chimal-Monroy, Jesús

2018-05-01

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues. © 2018 International Federation for Cell Biology.

The clinical characteristics and the features of immunophenotype of peripheral lymphocytes of adult onset chronic active Epstein-Barr virus disease at a Tertiary Care Hospital in Beijing

PubMed Central

Luo, Ling; Wang, Huanling; Fan, Hongwei; Xie, Jing; Qiu, Zhifeng; Li, Taisheng

2018-01-01

Abstract Chronic active Epstein-Barr virus (CAEBV) infection is a rare disease with high mortality. Most of CAEBV patients have been reported from Japan and are pediatric cases. The goal was to describe the clinical characteristics and the immunophenotypic features of peripheral lymphocytes in adult onset CAEBV patients. We retrospectively reviewed and analyzed all adult onset CAEBV cases admitted to Peking Union Medical College Hospital (PUMCH) between 2012 and 2016. Demographic, clinical, laboratory data, and the immunophentyping data of peripheral lymphocytes were collected. There were 28 adult onset CAEBV patients. The median age was 45 (range, 20–81). Most of the patients presented with fever; splenomegaly; lymphadenopathy and hepatitis. Unlike pediatric cases reported, the manifestations of cardiovascular diseases in our patients were pulmonary arterial hypertension, decreased cardiac function and aorta vasculitis. Prevalence of interstitial pneumonitis in our patients were comparatively higher and prevalence of hypersensitivity to mosquito bites were comparatively lower than that reported by Japan. In this study, CAEBV patients had decreased B cell, NK cell, CD4 cell and CD8 cell counts. The prevalence of low level of B cells, NK cells, CD4 cells was relatively higher than reported ever. Chinese adult onset CAEBV patients have different clinical characteristics and are featured by an immunosuppression status as demonstrated by decreased B cell, NK cell, CD4 cell and CD8 cell. PMID:29489682

Intestinal metaplasia in Barrett's oesophagus may be an epiphenomenon rather than a preneoplastic condition, and CDX2-positive cardiac-type epithelium is associated with minute Barrett's tumour.

PubMed

Watanabe, Gen; Ajioka, Yoichi; Takeuchi, Manabu; Annenkov, Alexey; Kato, Takashi; Watanabe, Kaori; Tani, Yusuke; Ikegami, Kikuo; Yokota, Yoko; Fukuda, Mutsumi

2015-01-01

Although intestinal-type epithelium in Barrett's oesophagus has been traditionally recognized as having a distinct malignant potential, whether this also holds true for cardiac-type epithelium remains controversial. The aim of this study was to identify a type of epithelium that is highly associated with Barrett's tumour. We analysed tumours and the corresponding background mucosa with special regard to tumour size in 40 cases of superficial Barrett's tumour by using immunohistochemical staining for CDX2, CD10, MUC2, MUC5AC, and MUC6. Intestinal metaplasia in tumour-adjacent mucosa was not associated with tumour size, but was significantly correlated with the extent of Barrett's oesophagus (P < 0.001). The majority (69.2%, 9/13) of small tumours (≤10 mm) had no intestinal metaplasia in adjacent non-neoplastic mucosae. Minute (≤5 mm) tumours were significantly associated with a gastric immunophenotype (P < 0.001). Purely gastric-immunophenotype tumour cells expressed CDX2, and cardiac-type epithelium adjacent to small tumours also showed low-level CDX2 expression. Our data suggest that intestinal metaplasia in Barrett's oesophagus is an epiphenomenon rather than a preneoplastic condition, and that CDX2-positive cardiac-type epithelium is highly associated with minute Barrett's tumour. Further prospective studies are needed to evaluate the risk of malignancy of cardiac-type epithelium with regard to sub-morphological intestinalization. © 2014 The Authors. Histopathology published by John Wiley & Sons Ltd.

Bio-electrospraying of human mesenchymal stem cells: An alternative for tissue engineering

PubMed Central

Braghirolli, D. I.; Zamboni, F.; Chagastelles, P. C.; Moura, D. J.; Saffi, J.; Henriques, J. A. P.; Pilger, D. A.; Pranke, P.

2013-01-01

Bio-electrospraying (BES) is a technique used for the processing of cells and can be applied to tissue engineering. The association of BES with scaffold production techniques has been shown to be an interesting strategy for the production of biomaterials with cells homogeneously distributed in the entire structure. Various studies have evaluated the effects of BES on different cell types. However, until the present moment, no studies have evaluated the impact of BES time on mesenchymal stem cells (MSC). Therefore, the aim of this work was to standardise the different parameters of BES (voltage, flow rate, and distance of the needle from the collecting plate) in relation to cell viability and then to evaluate the impact of BES time in relation to viability, proliferation, DNA damage, maintenance of plasticity and the immunophenotypic profile of MSC. Using 15 kV voltage, 0.46 ml/h flow rate and 4 cm distance, it was possible to form a stable and continuous jet of BES without causing a significant reduction in cell viability. Time periods between 15 and 60 min of BES did not cause alterations of viability, proliferation, plasticity, and immunophenotypic profile of the MSC. Time periods above 30 min of BES resulted in DNA damage; however, the DNA was able to repair itself within five hours. These results indicate that bio-electrospraying is an adequate technique for processing MSC which can be safely applied to tissue engineering and regenerative medicine. PMID:24404063

Proposed minimal panel of antibodies for cost-effectiveness and accuracy in acute leukemias immunophenotyping: Prospective study at a tertiary care center.

PubMed

Singh, Neha; Pati, Hara Prasad P; Tyagi, Seema; Deka, Roopam; Sharma, Rahul; Saxena, Renu

2016-07-01

Flowcytometry has an essential role in the diagnosis and classification of acute leukemias. However, there exists a great degree of inter-laboratory variability on issues like panel selection, antibody combinations, gating strategies, fluorochromes, and clonal selection. The primary aim of this study was to derive a minimal panel of antibodies and evaluate its diagnostic usefulness in acute leukemias by flowcytometry by using the detailed immune-phenotype of different lineage-specific or non-specific markers. This prospective observational study involved 400 newly diagnosed cases of acute leukemias. Bone marrow aspirate samples were subjected to morphological evaluation, cytogenetics and flow cytometric immunophenotyping. A minimal panel of eight antibodies comprising of CD45/CD34/CD19/MPO/cytoCD3/CD64/CD117/CD79a was derived by applying different permutations and combinations with a diagnostic yield of 97.5%. The minimal panel was further validated by testing in an independent cohort of patients with similar demographic characteristics, where it showed a high diagnostic yield of 98% in comparison with the screening panels proposed by other recently published studies. It may be concluded that the diagnostic performance of the eight antibody panel is better than most other panels used across the different laboratories in terms of yield, number of antibodies used and the scientific approach used to derive and validate the results and so henceforth may be applied in any setting with limited resources for better diagnostic accuracy.

Patient-derived tumor xenografts of lung squamous cell carcinoma alter long non-coding RNA profile but not responsiveness to cisplatin.

PubMed

Lu, Dapeng; Luo, Peng; Zhang, Ju; Ye, Yuanyuan; Wang, Qi; Li, Ming; Zhou, Hangcheng; Xie, Mingran; Wang, Baolong

2018-06-01

Lung squamous cell carcinoma (LSCC), the second most common type of lung cancer, has received limited attention. Patient-derived tumor xenografts (PDTXs) are useful preclinical models to reproduce the diverse heterogeneity of cancer, but it is important to identify potential variations during their establishment. A total of 18 PDTXs were established from 37 the surgical specimens and 16 were serially passaged to third generation. Second- and third-generation xenografts had a faster growth rate in mice. The tumor implantation success rate was associated with poorer differentiation, larger tumor volume and higher expression of Ki-67. The xenografts largely retained histological and key immunophenotypic features (including p53, p63, cytokeratin5/6, and E-cadherin). However, increased Ki-67 expression was identified in partial xenografts. Long non-coding RNA (lncRNA) and mRNA expression in third-generation xenografts differed from that of matched primary tumors. Gene Ontology and pathway analysis showed that mRNAs involved in cell cycle, and metabolism regulation were generally upregulated in xenografts, while those associated with immune responses were typically downregulated. Furthermore, the responses of xenografts to cisplatin were consistent with clinical outcome. In the present study, PDTXs of SCC were successfully established, and closely resembled their original tumor regarding their immunophenotype and response to cisplatin. Overall, PDTXS of LSCC altered the lncRNA profile and increased the proliferative activity of cancer cells, whilst retaining responsiveness to cisplatin.

Comprehensive Astronaut Immune Assessment Following a Short-Duration Space Flight

NASA Technical Reports Server (NTRS)

Crucian, Brian; Stowe, Raymond; Yetman, Deborah; Pierson, Duane; Sams, Clarence

2006-01-01

Immune system dysregulation has been demonstrated to occur during spaceflight and has the potential to cause serious health risks to crewmembers participating in exploration class missions. As a part of an ongoing NASA flight experiment assessing viral immunity (DSO-500), a generalized immune assessment was performed on 3 crewmembers who participated in the recent STS-114 Space Shuttle mission. The following assays were performed: (1) comprehensive immunophenotype analysis; (2) T cell function/intracellular cytokine profiles; (4) secreted Th1/Th2 cytokine profiles via cytometric bead array. Immunophenotype analysis included a leukocyte differential, lymphocyte subsets, T cell subsets, cytotoxic/effector CD8+ T cells, memory/naive T cell subsets and constitutively activated T cells. Study timepoints were L-180, L-65, L-10, R+0, R+3 and R+14. Detailed data are presented in the poster text. As expected from a limited number of human subjects, data tended to vary with respect to most parameters. Specific post-flight alterations were as follows (subject number in parentheses): Granulocytosis (2/3), reduced NK cells (3/3), elevated CD4/CD8 ratio (3/3), general CD8+ phenotype shift to a less differentiated phenotype (3/3), elevated levels of memory CD4+ T cells (3/3), loss of L-selectin on T cell subsets (3/3), increased levels of activated T cells (2/3), reduced IL-2 producing T cell subsets (3/3), levels of IFNg producing T cells were unchanged. CD8+ T cell expression of the CD69 activation markers following whole blood stimulation with SEA+SEB were dramatically reduced postflight (3/3), whereas other T cell function assessments were largely unchanged. Cytometric bead array assessment of secreted T cell cytokines was performed, following whole blood stimulation with either CD3/CD28 antibodies or PMA+ionomycin for 48 hours. Specific cytokines assessed were IFNg, TNFa, IL-2, IL-4, IL-5, IL-10. Following CD3/CD28 stimulation, all three crewmembers had a mission-associated reduction in the levels of secreted IFNg. One crewmember had a post-flight inversion in the IFNg/IL-10 ratio postflight, which trended back to baseline by R+14. Detailed cytokine data are presented in the poster text. This testing regimen was designed to correlate immunophenotype changes (thought to correspond to specific in-vivo immune responses or pathogenesis), against altered leukocyte function and cytokine profiles. In-flight studies are required to determine if post-flight alterations are reflective of the in-flight condition, or are a response to landing and readaptation.

Extensive characterization of the immunophenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage− cells

PubMed Central

Almeida, J; Bueno, C; Alguero, M C; Sanchez, M L; Cañizo, M C; Fernandez, M E; Vaquero, J M; Laso, F J; Escribano, L; San Miguel, J F; Orfao, A

1999-01-01

Dendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II+/lineage− were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16 ± 0.06% of the PB nucleated cells and 55.9 ± 11.9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12 ± 0.04% of PB nucleated cells and 44.53 ± 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor α-chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1β (97 ± 5% of positive cells), IL-6 (96 ± 1.1% of positive cells), IL-12 (81.5 ± 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-α) (84 ± 22.1% of positive cells) as well as chemokines such as IL-8 (99 ± 1% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production. PMID:10594557

[An analysis of immunophenotyping of peripheral lymphocytes in adult patients with infectious mononucleosis and chronic active Epstein-Barr virus infection].

PubMed

Xie, J; Wang, H L; Qiu, Z F; Li, T S

2016-06-01

To determine the immunophenotypic features of peripheral lymphocytes in adult patients with Epstein-Barr virus(EBV)-associated infectious mononucleosis(IM) and chronic active EBV infection (CAEBV). Eighteen IM patients, 12 CAEBV patients and 18 healthy donors were included. Lymphocyte subsets including CD3(-)CD19(+) B cells, CD3(-)CD16/56(+) NK cells, CD4(+) and CD8(+) T cells in peripheral blood were measured by flow cytometry. The expression of activation markers (HLA-DR and CD38) on CD8(+) T cells and CD28 expression on T cells were also determined. Kruskal-Wallis H and Mann-Whitney U tests were used to compare variables among groups. IM patients had dramatically increased CD8(+) T cell counts than healthy donors (5.22×10(9)/L vs 0.54×10(9)/L, P<0.001). B cell counts moderately reduced in patients with IM than in healthy donors. No difference was found in absolute CD4(+) T cell and NK cell counts between IM and healthy donors. The levels of HLA-DR and CD38 on CD8(+) T cells significantly increased in IM patients compared with those in healthy controls. The intensity of CD28 on CD8(+) T cells significantly decreased, which was not seen on CD4(+) T cells. The median cell counts of B, NK, CD4(+) T and CD8(+) T subsets in CAEBV patients were 0.02×10(9)/L, 0.06×10(9)/L, 0.26×10(9)/L and 0.21×10(9)/L respectively, which were significantly lower than those in healthy donors (0.22×10(9)/L, 0.38×10(9)/L, 0.78×10(9)/L, 0.54×10(9)/L)and IM patients (0.12×10(9)/L, 0.40×10(9)/L, 0.91×10(9)/L, 5.22×10(9)/L). The positive rates of HLA-DR and CD38 on CD8(+) T cells in CAEBV patients were higher than those in healthy controls, but lower than those in IM patients. The immunophenotypic pattern in adult patients with IM is characterized by a dramatic increase of extensively activated CD8(+) T cells, a moderate reduction of CD19(+) B cells and no significant change of CD4(+) T cells and CD16/56(+) NK cells. CAEBV is featured by an immunosuppression status as demonstrated by significantly decreased B, NK, CD4(+) T and CD8(+) T subsets.

Childhood acute leukemias are frequent in Mexico City: descriptive epidemiology

PubMed Central

2011-01-01

Background Worldwide, acute leukemia is the most common type of childhood cancer. It is particularly common in the Hispanic populations residing in the United States, Costa Rica, and Mexico City. The objective of this study was to determine the incidence of acute leukemia in children who were diagnosed and treated in public hospitals in Mexico City. Methods Included in this study were those children, under 15 years of age and residents of Mexico City, who were diagnosed in 2006 and 2007 with leukemia, as determined by using the International Classification of Childhood Cancer. The average annual incidence rates (AAIR), and the standardized average annual incidence rates (SAAIR) per million children were calculated. We calculated crude, age- and sex-specific incidence rates and adjusted for age by the direct method with the world population as standard. We determined if there were a correlation between the incidence of acute leukemias in the various boroughs of Mexico City and either the number of agricultural hectares, the average number of persons per household, or the municipal human development index for Mexico (used as a reference of socio-economic level). Results Although a total of 610 new cases of leukemia were registered during 2006-2007, only 228 fit the criteria for inclusion in this study. The overall SAAIR was 57.6 per million children (95% CI, 46.9-68.3); acute lymphoblastic leukemia (ALL) was the most frequent type of leukemia, constituting 85.1% of the cases (SAAIR: 49.5 per million), followed by acute myeloblastic leukemia at 12.3% (SAAIR: 6.9 per million), and chronic myeloid leukemia at 1.7% (SAAIR: 0.9 per million). The 1-4 years age group had the highest SAAIR for ALL (77.7 per million). For cases of ALL, 73.2% had precursor B-cell immunophenotype (SAAIR: 35.8 per million) and 12.4% had T-cell immunophenotype (SAAIR 6.3 per million). The peak ages for ALL were 2-6 years and 8-10 years. More than half the children (58.8%) were classified as high risk. There was a positive correlation between the average number of persons per household and the incidence of the pre-B immunophenotype (Pearson's r, 0.789; P = 0.02). Conclusions The frequency of ALL in Mexico City is among the highest in the world, similar to those found for Hispanics in the United States and in Costa Rica. PMID:21846410

Next-Generation Molecular Histology Using Highly Multiplexed Ion Beam Imaging (MIBI) of Breast Cancer Tissue Specimens for Enhanced Clinical Guidance

DTIC Science & Technology

2015-07-01

SLC7A5, NRDG1, HTF9C, CEACAM5). Gene-expression assays using qRT-PCR, array hybridization, and RNA sequence assays have also been developed. The...and RNA sequence assays have also been developed. The OncotypeDX, for example, uses a panel of 21 genes (16 analytical, 5 controls: Ki67, STK15...Provide a brief list of keywords (limit to 20 words). Breast Cancer Diagnosis Pathology Immunophenotype Multiplex Morphology RNA In Situ

Cutaneous Pseudolymphomas.

PubMed

Romero-Pérez, D; Blanes Martínez, M; Encabo-Durán, B

2016-10-01

The term cutaneous pseudolymphoma refers to benign reactive lymphoid proliferations in the skin that simulate cutaneous lymphomas. It is a purely descriptive term that encompasses various reactive conditions with a varied etiology, pathogenesis, clinical presentation, histology, and behavior. We present a review of the different types of cutaneous pseudolymphoma. To reach a correct diagnosis, it is necessary to contrast clinical, histologic, immunophenotypic, and molecular findings. Even with these data, in some cases only the clinical course will confirm the diagnosis, making follow-up essential. Copyright © 2016 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.

Flow Cytometry in Diagnosis of Myelomatous Pleural Effusion: A Case Report.

PubMed

Arora, Parul; Gupta, Sanjeev Kumar; Mallik, Nabhajit; Mittal, Reena; Sharma, Om Dutt; Kumar, Lalit

2016-06-01

Plasma cell myeloma is a multifocal plasma cell neoplasm associated with increased monoclonal protein in serum and/or urine. Pleural effusions in patients with myeloma are uncommon (6 %). However, effusions due to direct infiltration of the pleura by plasma cells (myelomatous pleural effusion) are extremely rare (<1 %) and usually seen with IgA myeloma. The diagnosis of such cases requires pleural fluid cytology, electrophoresis or pleural biopsy. We present a case of myelomatous pleural effusion diagnosed using flow cytometry immunophenotyping in addition to the pleural fluid cytology. A 45 year old female was diagnosed as plasma cell myeloma (IgG kappa) in 2007. She received multiple lines of therapy during the course of her treatment including thalidomide, dexamethasone, lenalidomide, bortezomib, and doxorubicin based regimens. However, the patient had progressive extramedullary disease and developed pleural effusion in 2014. Cytological examination of the pleural fluid showed degenerative changes. Few preserved areas showed mononuclear cells including morphologically abnormal plasma cells. Immunophenotyping of these cells by flow cytometry revealed a pattern indicating neoplastic plasma cells. There was expression of CD38, CD138, and CD56, with absence of CD19, CD10 and CD45. This confirmed the diagnosis of myelomatous pleural effusion. Subsequently, the patient was offered a dexamethasone, cyclophosphamide, etoposide and cisplatin based regimen but, she declined further treatment and succumbed to her disease 3 months later. Myelomatous pleural effusion is a rare complication of plasma cell myeloma. Flow cytometry can be used as an adjunctive technique in its diagnosis particularly in cases with equivocal cytology and electrophoresis findings.

Minimally differentiated erythroleukaemia (AML M6 'variant'): a rare subset of AML distinct from AML M6. Groupe Français d'Hématologie Cellulaire.

PubMed

Garand, R; Duchayne, E; Blanchard, D; Robillard, N; Kuhlein, E; Fenneteau, O; Salomon-Nguyen, F; Grange, M J; Rousselot, P; Demur, C

1995-08-01

We describe eight cases of erythroleukaemia distinct from FAB-AML M6, which demonstrate minimal erythroid differentiation not associated with a myeloblastic component. Three infants (including a Down's syndrome) and two adults presented with a de novo leukaemia. One case was preceded by an untreated refractory anaemia with excess of blasts and one by polycythaemia vera. One case presented with an inaugural blast crisis of chronic myeloid leukaemia. In four patients the leukaemic cells showed a proerythroblast-like morphology. The four other were initially classified as undifferentiated AL (two cases) or AML MO (two cases) because of the immature aspect of the cells, their lack of myeloperoxidase activity and the absence of B, T lymphoid and myeloid (My) marker expressions apart from the CD33 antigen. Immunophenotyping in three cases showed an immature erythroblast profile (glycophorins A and B+, spectrin+). In the five others the erythroid nature was recognized by the expression of ABH blood group system on fresh cells (four cases) and glycophorin A on cells after 3 d in vitro culture with erythropoietin (EPO) + IL3 (two cases). Moreover, an erythroid colony growth of leukaemic origin was observed in three patients. In conclusion, the study of erythroid marker expression is of particular importance when immunophenotyping leukaemic cells with a proerythroblast-like morphology or an undifferentiated aspect and a HLA DR-, CD36++, B-, T-, My- (CD33 +/-) phenotype. We propose the term AML M6 'variant' for this rare type of AML.

Haematological profile of patients with mixed-phenotype acute leukaemia from a tertiary care centre of north India

PubMed Central

Sharma, Manupriya; Sachdeva, Man Updesh Singh; Bose, Parveen; Varma, Neelam; Varma, Subhash; Marwaha, R.K.; Malhotra, Pankaj

2017-01-01

Background & objectives: Mixed-phenotype acute leukaemia (MPAL) is a rare neoplasm with no definite treatment protocols and a distinctly poor outcome. Advancement in polychromatic flow cytometry has made its identification easier. This prospective study was designed to identify cases of MPAL and study their clinical presentation and haematological profile in a tertiary care hospital in north India. Methods: Ethylenediaminetetraacetic acid (EDTA)-anticoagulated bone marrow aspirate samples of patients diagnosed as acute leukaemia (AL) on the basis of morphology were utilized for immunophenotyping. A comprehensive panel of fluorochrome-labelled monoclonal antibodies targeting myeloid, B-cell, T-cell and immaturity markers was utilized. The patients diagnosed to have MPAL, on the basis of the World Health Organization 2008 classification, were selected for further analyses. Results: There were 15 (2.99%) patients with MPAL of the total 501 cases of AL. Seven were children, all males and mean age of 5.08±3.88 yr. Eight were adults, male:female=6:2 and mean age of 21.43±5.74 yr. Eight were diagnosed as B/myeloid and seven were T/myeloid. No association was observed between age and immunophenotype of MPAL. On morphology, 11 were diagnosed as AML and four as ALL, and no specific morphology of blasts was predictive of a MPAL. Interpretation & conclusions: MPAL appeared to be a rare neoplasm (2.99% of AL cases). A comprehensive primary panel of monoclonal antibodies should be used to identify this neoplasm known to have a poor outcome. PMID:28639598

Analysis of peroxidase-negative acute unclassifiable leukemias by monoclonal antibodies. 1. Acute myelogenous leukemia and acute myelomonocytic leukemia.

PubMed

Imamura, N; Tanaka, R; Kajihara, H; Kuramoto, A

1988-11-01

In this study, pretreatment peripheral and/or bone marrow blasts from 12 patients with acute unclassifiable leukemia (AUL) expressing the myeloid-related cell-surface antigen (CD 11) were isolated for further analysis. Despite a lack of myeloperoxidase (MPO) activity, 1 patient's blasts contained cytoplasmic Auer rods. The circulating blasts from another patient expressed MPO while maintaining the same surface phenotype during 20 months of clinical follow-up. In addition, the blasts from 3 cases demonstrated both myelomonocytic and monocyte-specific surface antigens, whereas the remaining 9 cases completely lacked any monocyte-specific antigen detectable by monoclonal antibodies, Mo2, My4 and Leu M3 (CD 14). The first case eventually was diagnosed as acute myelomonocytic leukemia and the second as acute myelogenous leukemia by means of immunophenotypic analysis using flow cytometry (FACS IV). In addition, the presence of MPO protein was identified in the cytoplasm of blast cells from 5 patients with AUL by means of a cytoplasmic immunofluorescence test using a monoclonal antibody (MA1). Our study indicates that non-T, non-B AUL expressing OKM1 (CD 11) antigens include acute leukemias which are unequivocally identifiable as being of either myeloid or myelomonocytic origin. However, further investigations, including immunophenotypic and cytoplasmic analysis, ultrastructural cytochemistry and gene analysis with molecular probes (tests applicable to normal myeloid cells), are necessary in order to determine the actual origin of blasts and to recognize the differentiation stages of the various types of leukemic cells from patients with undifferentiated forms of leukemia.

CD5-expressing B-cell lymphomas/leukemias: relatively high frequency of CD5+ B-cell lymphomas with an overall poor prognosis in Nagasaki Japan.

PubMed

Kamihira, S; Hirakata, Y; Atogami, S; Sohda, H; Tsuruda, K; Yamada, Y; Tomonaga, M

1996-06-01

To characterize CD5+ B-cell neoplasms in Japan, where chronic lymphocytic leukemia (CLL) is rare and of different subtypes in comparison with Western countries, we collected 58 cases of CD5+ B-cell lymphomas/leukemias and analyzed their clinicopathologic features. According to the French-American-British (FAB) and standard histologic classification, the cases corresponded to small lymphocytic lymphoma (SLL, group I; n = 22, consisting of CLL, n = 10, CLL/PL, n = 3, and CLLmixed, n = 7); intermediate differentiated lymphoma/mantle cell lymphoma (IDL/MCL, group II, n = 18); and others with CD5-positive lymphomas (group III, n = 18). The CD5+ B-cell lymphomas showed morphologic and prognostic variability among the three groups. The clinical and immunophenotypic features were remarkably consistent in leukemic disease being seen in 73% of all cases, splenomegaly in 63%, and intense CD19, CD20, surface membrane immunogobulin M (SmIgM) or SmIgM and SmIgD, light-chain expression, and no CD10 expression. The median survival time of groups I, II, and III was 7.8, 3.3, and 0.8 years, respectively. These findings suggest that CD5 antigens may serve as valid markers for the prognosis and clinical features of B-cell lymphomas and that CD5+ B-cell lymphomas with an overall poor prognosis occurs at a relatively high frequency in Japan. This also suggests that a combination of immunophenotypic and morphologic features is of value for characterizing CD5+ B-cell neoplasms.

Fluorescence immunophenotyping and interphase cytogenetics (FICTION) detects BCL6 abnormalities, including gene amplification, in most cases of nodular lymphocyte-predominant Hodgkin lymphoma.

PubMed

Bakhirev, Alexei G; Vasef, Mohammad A; Zhang, Qian-Yun; Reichard, Kaaren K; Czuchlewski, David R

2014-04-01

BCL6 translocations are a frequent finding in B-cell lymphomas of diverse subtypes, including some cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). However, reliable analysis of BCL6 rearrangements using fluorescence in situ hybridization is difficult in NLPHL because of the relative paucity of neoplastic cells. Combined immunofluorescence microscopy and fluorescence in situ hybridization, or fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION), permits targeted analysis of neoplastic cells. To better define the spectrum of BCL6 abnormalities in NLPHL using FICTION analysis. We performed an optimized FICTION analysis of 24 lymph nodes, including 11 NLPHL, 5 follicular hyperplasia with prominent progressive transformation of germinal centers, and 8 follicular hyperplasia without progressive transformation of germinal centers. BCL6 rearrangement was identified in 5 of 11 cases of NLPHL (46%). In addition, BCL6 gene amplification, with large clusters of BCL6 signals in the absence of chromosome 3 aneuploidy, was detected in 3 of 11 cases of NLPHL (27%). One NLPHL showed extra copies of BCL6 present in conjunction with multiple copies of chromosome 3. Altogether, we detected BCL6 abnormalities in 9 of 11 cases of NLPHL (82%). None of the progressive transformation of germinal centers or follicular hyperplasia cases showed BCL6 abnormalities by FICTION. To our knowledge, this is the first report of BCL6 gene amplification in NLPHL. Our optimized protocol for FICTION permits detection of cytogenetic abnormalities in most NLPHL cases and may represent a useful ancillary diagnostic technique.

Cryopreservation of human whole blood allows immunophenotyping by flow cytometry up to 30days after cell isolation.

PubMed

Paredes, R Madelaine; Tadaki, Douglas K; Sooter, Amanda; Gamboni, Fabia; Sheppard, Forest

2018-01-01

Immunophenotyping of whole blood (WB) by flow cytometry (FC) is used clinically to assess a patient's immune status and also in biomedical research. Current protocols recommend storage of immunolabeled samples at 4°C with FC analysis to be completed within seven days. This data acquisition window can be extended to up to one year post-labeling, but this requires cryopreservation of the samples at ultra-low temperatures (≤-80°C or in liquid nitrogen). In this study we optimized a standardized cryopreservation protocol to enable preservation of immunolabeled, human WB samples at -20°C for FC and tested its effectiveness after 0, 5, 15 or 30days. Analysis of stored samples shows that this protocol effectively preserves immunolabeled WB samples and that the duration of storage has no effect on morphology, viability or frequency of WB cell subpopulations, and that the intensity of fluorescent signal from labeled extracellular markers is fully preserved for at least 15days, and up to 30days for some markers. We demonstrate that using this protocol, we are able to differentiate resting versus activated WB cells as demonstrated by detection of significantly increased expression of CD11b by myeloid cells in WB samples pretreated with LPS (100μg/mL for 12h). Finally, we show that this method allows for labeling and detection of the intracellular cytokine (IL-8) up to 30days following cryopreservation from myeloid cells, in previously labeled and cryopreserved WB samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of an Atypical Enzootic Bovine Leukosis in Japan by Using a Novel Classification of Bovine Leukemia Based on Immunophenotypic Analysis

PubMed Central

Nishimori, Asami; Okagawa, Tomohiro; Maekawa, Naoya; Goto, Shinya; Ikebuchi, Ryoyo; Nakahara, Ayako; Chiba, Yuzumi; Ikeda, Masaho; Murata, Shiro; Ohashi, Kazuhiko

2017-01-01

ABSTRACT Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLV-associated lymphoma occurs predominantly in adult cattle of >3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection. PMID:28659325

Growth on poly(L-lactic acid) porous scaffold preserves CD73 and CD90 immunophenotype markers of rat bone marrow mesenchymal stromal cells.

PubMed

Zamparelli, Alessandra; Zini, Nicoletta; Cattini, Luca; Spaletta, Giulia; Dallatana, Davide; Bassi, Elena; Barbaro, Fulvio; Iafisco, Michele; Mosca, Salvatore; Parrilli, Annapaola; Fini, Milena; Giardino, Roberto; Sandri, Monica; Sprio, Simone; Tampieri, Anna; Maraldi, Nadir M; Toni, Roberto

2014-10-01

Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(L-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different subpopulations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.

Cell Growth Characteristics, Differentiation Frequency, and Immunophenotype of Adult Ear Mesenchymal Stem Cells

PubMed Central

Staszkiewicz, Jaroslaw; Frazier, Trivia P.; Rowan, Brian G.; Bunnell, Bruce A.; Chiu, Ernest S.; Gimble, Jeffrey M.

2010-01-01

Ear mesenchymal stem cells (EMSCs) represent a readily accessible population of stem-like cells that are adherent, clonogenic, and have the ability to self-renew. Previously, we have demonstrated that they can be induced to differentiate into adipocyte, osteocyte, chondrocyte, and myocyte lineages. The purpose of the current study was to characterize the growth kinetics of the cells and to determine their ability to form colonies of fibroblasts, adipocytes, osteocytes, and chondrocytes. In addition, the immunophenotypes of freshly isolated and culture-expanded cells were evaluated. From 1 g of tissue, we were able to isolate an average of 7.8 × 106 cells exhibiting a cell cycle length of ∼2–3 days. Colony-forming unit (CFU) assays indicated high proliferation potential, and confirmed previously observed multipotentiality of the cells. Fluorescence-activated cell sorting (FACS) showed that EMSCs were negative for hematopoietic markers (CD4, CD45), proving that they did not derive from circulating hematopoietic cells. The FACS analyses also showed high expression of stem cell antigen-1 (Sca-1) with only a minor population of cells expressing CD117, thus identifying Sca-1 as the more robust stem cell biomarker. Additionally, flow cytometry data revealed that the expression patterns of hematopoietic, stromal, and stem cell markers were maintained in the passaged EMSCs, consistent with the persistence of an undifferentiated state. This study indicates that EMSCs provide an alternative model for in vitro analyses of adult mesenchymal stem cells (MSCs). Further studies will be necessary to determine their utility for tissue engineering and regenerative medical applications. PMID:19400629

Correlation between heat shock proteins, adiponectin, and T lymphocyte cytokine expression in type 2 diabetics.

PubMed

Mahmoud, Fadia F; Haines, David; Dashti, Ali A; El-Shazly, Sherief; Al-Najjar, Fawzia

2018-05-11

Type 2 diabetes mellitus (T2DM) features insulin resistance, hyperglycemia, dyslipidemia, overproduction of inflammatory cytokines, and systemic oxidative stress. Here, heat shock proteins Hsp70 and Hsp 90, adiponectin, and heme oxygenase-1 (HO-1, Hsp32) are profiled in peripheral blood mononuclear cells (PBMC) and serum from 25 T2DM patients and 25 healthy control subjects. Cells cultured with phorbol 12-myristate 13-acetate/ionomycin were evaluated by three-color flow cytometry for immunophenotypic biomarkers. Plasma HO-1, Hsp, and adiponectin levels were assayed by enzyme-linked immunosorbent assay (ELISA). Relative to healthy controls, T2DM patients exhibited significantly elevated plasma Hsp70, and representation of T helper immunophenotypes activated to express inflammatory cytokines, including CD4+ IFN-γ+, CD4+ TNF-α+, CD4+ IL-6+, CD4+ IL-1β+ T cells, significantly lower representation of CD4+ IL-10+ T cells, plasma adiponectin and cell-associated HO-1 expression-with no significant differences in plasma Hsp90 between T2DM and healthy controls. Plasma HO-1 and adiponectin in T2DM patients inversely correlated with TNF-α and showed inverse correlation between serum LDL and plasma HO-1. Moreover, TNF-α and Hsp90 in T2DM patients correlated positively with fasting blood glucose (FBG). These results demonstrate correlation between potentially pathogenic T cells, HO-1, and adiponectin, additionally revealing a T helper (Th)1-related character of T2DM immunopathogenesis, suggesting potential for novel T cell-related management strategies for T2DM and related co-morbidities.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology

PubMed Central

Pavon, Lorena Favaro; Sibov, Tatiana Tais; Caminada de Toledo, Silvia Regina; Mara de Oliveira, Daniela; Cabral, Francisco Romero; Gabriel de Souza, Jean; Boufleur, Pamela; Marti, Luciana C.; Malheiros, Jackeline Moraes; Ferreira da Cruz, Edgar; Paiva, Fernando F.; Malheiros, Suzana M.F.; de Paiva Neto, Manoel A.; Tannús, Alberto; Mascarenhas de Oliveira, Sérgio; Silva, Nasjla Saba; Cappellano, Andrea Maria; Petrilli, Antonio Sérgio; Chudzinski-Tavassi, Ana Marisa; Cavalheiro, Sérgio

2018-01-01

Background Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model. PMID:29774098

Immunophenotypic Alterations in Resident Immune Cells and Myocardial Fibrosis in the Aging Rhesus Macaque (Macaca mulatta) Heart

PubMed Central

Macri, Sheila C.; Bailey, Charles C.; de Oca, Nicole Monts; Silva, Nilsa A.; Rosene, Douglas L.; Mansfield, Keith G.; Miller, Andrew D.

2012-01-01

The rhesus macaque (Macaca mulatta) is used extensively in translational biomedical research and drug development studies and is an important model of aging. Macaques often develop myocardial fibrosis with age which can result in the loss of normal cardiac architecture with the expansion of the extracellular matrix and deposition of collagen. The etiology and pathogenesis of this pernicious process is poorly understood. Cardiac fibrosis was assessed using histologic and immunohistochemical techniques in cardiac tissue sections from 34 rhesus macaques. Overall left ventricular and left ventricular mid-myocardial interstitial/perivascular fibrosis were positively correlated with age (r=0.6522, p<0.0001 and r=0.4704, p=0.005, respectively). When divided into young (mean=2.8 years), middle-aged (mean=17.5 years), and advanced age (mean=29.2 years) groups, immunophenotypic characterization of antigen presenting cells revealed differential expression of CD163 and DC-SIGN between the young and middle-aged groups compared to the advanced age group (p<0.0001). HAM-56 expression decreased significantly in the advanced age cohort (p=0.0021). The expression of CD8, CD163, and DCSIGN correlated positively with age (r=0.3999, p= 0.0191; r=0.5676, p=0.0005; r=0.5245, p=0.0014 respectively). These results show the importance of myocardial fibrosis as a common age-related pathology and additionally, alterations in T cell, macrophage, and dendritic cell phenotype in rhesus macaque myocardium are associated with age but unassociated with the fibrosis. PMID:22328408

Inflammatory cells in minor salivary glands of patients with chronic hepatitis C: immunophenotype, pattern of distribution, and comparison with liver samples.

PubMed

Caldeira, Patrícia Carlos; Oliveira e Silva, Karla Rachel; Vidigal, Paula Vieira Teixeira; Grossmann, Soraya de Mattos Camargo; do Carmo, Maria Auxiliadora Vieira

2014-05-01

To characterize the immunophenotype and the distribution of the inflammatory infiltrate (INF) in salivary glands (SG) of patients with chronic hepatitis C, comparing with laboratorial data (genotype, viral load, METAVIR, and HCV RNA in SG), and liver. INF was classified as diffuse or focal. Immunohistochemistry for CD3, CD20, CD8, CD4, CD57, CD68, and S100 was performed in 61 SG and 59 livers. Diffuse INF was more common in SG than in liver. CD3(+), CD20(+), and CD8(+) were the most frequent cells in both tissues, with few CD57(+), CD68(+), and S100(+) cells. CD4(+) cells were common in liver, but rare in SG. Liver presented higher indexes for all markers, except S100(+) (p<0.05). Higher CD3(+), CD20(+), and CD8(+) (p<0.05) were observed in SG with focal infiltrate than with diffuse infiltrate. In liver, CD20(+) and CD3(+) were higher in focal infiltrate, and CD68(+) in diffuse infiltrate (p<0.05). Comparisons with laboratorial data did not show statistical significance. The INF in SG was mainly composed by T and B lymphocytes, mostly cytotoxic T cells. The glandular INF can present differences in composition according to its distribution. A more intense inflammation was observed in liver, but similar cell types were identified in SG, except for CD4(+). Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Immunophenotyping of the cerebrospinal fluid as a prognostic factor at diagnosis of acute lymphoblastic leukemia in children and adolescents.

PubMed

Cancela, Camila Silva Peres; Murao, Mitiko; Assumpção, Juliana Godoy; Souza, Marcelo Eduardo de Lima; de Macedo, Antonio Vaz; Viana, Marcos Borato; De Oliveira, Benigna Maria

2017-03-01

This study aimed at evaluating the use of immunophenotyping (IMP) in the identification of blast cells in the cerebrospinal fluid (CSF) of children and adolescents with acute lymphoblastic leukemia (ALL). Sixty-seven patients aged 18 years or younger were included. Fifty-five CSF samples were analyzed at initial diagnosis and 17 at the time of relapse. A cytological analysis (CA) was performed in all 72 samples, while IMP was done in 63. Blasts were identified in only three samples by CA, whereas all three samples were found negative by IMP, one of which had no isolation of nucleated cells after centrifugation. Among the samples analyzed by IMP, 11 showed a positive blast count, two of which had been inconclusive using CA. No equivalence was found between CA and IMP results (p = 0.55). CSF IMP positivity was not associated with other risk factors for ALL relapse. Among the 55 patients included at the time of diagnosis of ALL, eight relapsed during follow-up. Considering the cases of central nervous system (CNS) relapse, one of the patients belonged to the CSF IMP-positive group (11%) at diagnosis, and the other two cases, to the IMP-negative (5%) group. Detection of CSF blast cells using IMP was associated with a worse overall (p < 0.0001) and event-free survival (p < 0.0001). These results show that CSF IMP may be a useful additional method to conventional CA in the diagnosis of CNS involvement in ALL, and for the identification of high-risk subgroups that would benefit from an intensified therapy.

AIDS-related lymphoma. Histopathology, immunophenotype, and association with Epstein-Barr virus as demonstrated by in situ nucleic acid hybridization.

PubMed Central

Hamilton-Dutoit, S. J.; Pallesen, G.; Franzmann, M. B.; Karkov, J.; Black, F.; Skinhøj, P.; Pedersen, C.

1991-01-01

To investigate the range of pathology shown by acquired immune deficiency syndrome (AIDS)-related lymphomas arising in an epidemiologically well-defined group of patients, all cases of lymphoma recognized in Danish human immunodeficiency virus (HIV)-infected individuals up to the end of 1988 were studied. Twenty-seven cases (26 high-grade non-Hodgkin's lymphoma [NHL], 1 Hodgkin's disease) were found, to give a cumulative incidence rate of 8% among Danish AIDS patients. Morphologically most NHL patients were classified into two groups: 1) high-grade tumors with a predominant population of immunoblasts, either monomorphic or more often polymorphic with plasmacytic differentiation; 2) Burkitt-type. Of 26 NHLs, 22 had a B-cell paraffin-section immunophenotype and 4 were non-B, non-T. Epstein-Barr virus (EBV) DNA was demonstrated in tumor cells of 12 of 24 cases (50%) using in situ nucleic acid hybridization with a 35S-labeled probe in paraffin sections. Epstein-Barr virus DNA was found in 65% of group 1 and 20% of group 2 tumors. This study suggests the existence of two main groups of AIDS-related lymphoma with different pathogeneses. First there are immunoblast-rich lesions, which usually are associated with EBV and morphologically resemble lymphomas described in immunosuppressed organ-transplantation patients. Second there are Burkitt-type tumors in which EBV sequences are less common and that may be pathogenetically analogous to sporadic Burkitt's lymphoma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1846263

Protection against renal ischemia-reperfusion injury through hormesis? Dietary intervention versus cold exposure.

PubMed

Shushimita, Shushimita; Grefhorst, Aldo; Steenbergen, Jacobie; de Bruin, Ron W F; Ijzermans, Jan N M; Themmen, Axel P N; Dor, Frank J M F

2016-01-01

Dietary restriction (DR) and fasting (FA) induce robust protection against the detrimental effects of renal ischemia-reperfusion injury (I/RI). Several mechanisms of protection have been proposed, such as hormesis. Hormesis is defined as a life-supporting beneficial effect resulting from the cellular responses to single or multiple rounds of (mild) stress. The cold exposure (CE) model is a stress model similar to DR, and has been shown to have hormetic effects and has proved to increase longevity. CE is considered to be the most robust method to increase metabolism through activation of brown adipocytes. BAT has been considered important in etiology of obesity and its metabolic consequences. Since DR, FA, and CE models are proposed to work through hormesis, we investigated physiology of adipose tissue and effect on BAT in these models and compared them to ad libitum (AL) fed mice. We also studied the differential effect of these stress models on immunological changes, and effect of CE on renal I/RI. We show similar physiological changes in adiposity in male C57Bl/6 mice due to DR, FA and CE, but the CE mice were not protected against renal I/RI. The immunophenotypic changes observed in the CE mice were similar to the AL animals, in contrast to FA mice, that showed major immunophenotypic changes in the B and T cell development stages in primary and secondary lymphoid organs. Our findings thus demonstrate that DR, FA and CE are hormetic stress models. DR and FA protect against renal I/IR, whereas CE could not. Copyright © 2015 Elsevier Inc. All rights reserved.

Fifteen years of external quality assessment in leukemia/lymphoma immunophenotyping in The Netherlands and Belgium: A way forward.

PubMed

Preijers, Frank W M B; van der Velden, Vincent H J; Preijers, Tim; Brooimans, Rik A; Marijt, Erik; Homburg, Christa; van Montfort, Kees; Gratama, Jan W

2016-05-01

In 1985, external quality assurance was initiated in the Netherlands to reduce the between-laboratory variability of leukemia/lymphoma immunophenotyping and to improve diagnostic conclusions. This program consisted of regular distributions of test samples followed by biannual plenary participant meetings in which results were presented and discussed. A scoring system was developed in which the quality of results was rated by systematically reviewing the pre-analytical, analytical, and post-analytical assay stages using three scores, i.e., correct (A), minor fault (B), and major fault (C). Here, we report on 90 consecutive samples distributed to 40-61 participating laboratories between 1998 and 2012. Most samples contained >20% aberrant cells, mainly selected from mature lymphoid malignancies (B or T cell) and acute leukemias (myeloid or lymphoblastic). In 2002, minimally required monoclonal antibody (mAb) panels were introduced, whilst methodological guidelines for all three assay stages were implemented. Retrospectively, we divided the study into subsequent periods of 4 ("initial"), 4 ("learning"), and 7 years ("consolidation") to detect "learning effects." Uni- and multivariate models showed that analytical performance declined since 2002, but that post-analytical performance improved during the entire period. These results emphasized the need to improve technical aspects of the assay, and reflected improved interpretational skills of the participants. A strong effect of participant affiliation in all three assay stages was observed: laboratories in academic and large peripheral hospitals performed significantly better than those in small hospitals. © 2015 International Clinical Cytometry Society. © 2015 International Clinical Cytometry Society.

Identifying Stem-like Cells Using Mitochondrial Membrane Potential | Center for Cancer Research

Cancer.gov

Therapies that are based on living cells promise to improve treatments for metastatic cancer and for many degenerative diseases. Lasting treatment of these maladies may require the durable persistence of cells. Long-term engraftment of cells – for months or years – and the generation of large numbers of progeny are characteristics of stem cells. Most approaches to isolate viable hematopoetic stem cells and therapeutically active T cells are based on immunophenotyping using highly multicolored flow cytometry. However, these methods do not directly measure the metabolic features of cells, which are known to be important in predicting cell fate.

[Clinical characteristics of human recombination activating gene 1 mutations in 8 immunodeficiency patients with diverse phenotypes].

PubMed

Yu, G; Wang, W J; Liu, D R; Tao, Z F; Hui, X Y; Hou, J; Sun, J Q; Wang, X C

2018-03-02

Objective: To investigate the clinical characteristics of 8 immunodeficiency cases caused by human recombination activating gene 1 (RAG1) mutations, and to explore the relationship among genotypes, clinical manifestations and immunophenotypes. Methods: Clinical data were collected and analyzed from patients with RAG1 mutations who visited the Department of Clinical Immunology, Children's Hospital of Fudan University between October 2013 and June 2017. The data included clinical manifestations, immunophenotypes and genotypes. Results: A total of 8 patients were diagnosed with RAG1 deficiency (6 boys and 2 girls). The minimum age of onset was 2 months, and the maximum age was 4 months. The minimum age of diagnosis was 2 months, and the maximum age was 13 years. Four patients had a family history of infant death due to severe infections. Two cases were born to the same consanguineous parents. All cases had recurrent infections, including involvement of respiratory tract (8 cases), digestive tract (6 cases), urinary tract (1 case), and central nervous system (1 case). The pathogens of infection included bacteria, viruses and fungi. Rotavirus was found in 3 cases, cytomegalovirus (CMV) in 5 cases, bacillus Calmette-Guérin adverse reaction in 2 cases (1 of whom had a positive acid-fast smear from lymph node puncture fluid), fungal infection in 3 cases. One case had multiple nodular space-occupying lesions in lungs and abdominal cavity complicated with multiple bone destruction. The peripheral blood lymphocyte counts of all patients ranged between 0.1 ×10(9)/L and 3.3×10(9)/L (median, 0.65×10(9)/L). Eosinophilia was found in 3 cases (range, (0.48-1.69) ×10(9)/L). The patients were classified according to immunophenotype as severe combined immunodeficiency phenotype (4 cases), leaky severe combined immunodeficiency (2 cases), Omenn syndrome (1 case) and combined immunodeficiency (1 case) . Decreased serum IgG levels were found in 3 cases, increased serum IgM levels in 3 cases, increased serum IgE levels in 5 cases. RAG1 homozygous mutations were detected in 5 cases and RAG1 compound heterozygous mutations in 3 cases. Two novel mutations and six previously reported mutations were identified. Three cases were successfully treated with hematopoietic stem cell transplantation. Four cases died due to infections, and the 13 year-old patient was still under follow-up in the outpatient clinic. Conclusions: Different RAG1 gene mutations can lead to diverse clinical presentations and immune phenotypes. Clinicians should pay attention to the family history of infant death with severe infection. In that situation, immunological evaluation and gene detection should be performed as early as possible.

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro.

PubMed

Griffiths, Sarah; Baraniak, Priya R; Copland, Ian B; Nerem, Robert M; McDevitt, Todd C

2013-12-01

Multipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown. MSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to "rescue" the proliferative capacity of MSCs. hPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS. hPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

PubMed

de Soure, António M; Fernandes-Platzgummer, Ana; Moreira, Francisco; Lilaia, Carla; Liu, Shi-Hwei; Ku, Chen-Peng; Huang, Yi-Feng; Milligan, William; Cabral, Joaquim M S; da Silva, Cláudia L

2017-05-01

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGRO TM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO TM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO TM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Hepatocyte growth factor enhances the inflammation-alleviating effect of umbilical cord-derived mesenchymal stromal cells in a bronchiolitis obliterans model.

PubMed

Cao, Xiao-Pei; Han, Dong-Mei; Zhao, Li; Guo, Zi-Kuan; Xiao, Feng-Jun; Zhang, Yi-Kun; Zhang, Xiao-Yan; Wang, Li-Sheng; Wang, Heng-Xiang; Wang, Hua

2016-03-01

Specific and effective therapy for prevention or reversal of bronchiolitis obliterans (BO) is lacking. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF) gene modified mesenchymal stromal cells (MSCs) on BO. A mouse model of experimental BO was established by subcutaneously transplanting the tracheas from C57BL/6 mice into Balb/C recipients, which were then administered saline, Ad-HGF-modified human umbilical cord-MSCs (MSCs-HGF) or Ad-Null-modified MSCs (MSCs-Null). The therapeutic effects of MSCs-Null and MSCs-HGF were evaluated by using fluorescence-activated cell sorting (FACS) for lymphocyte immunophenotype of spleen, enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (rt-PCR) for cytokine expression, and histopathological analysis for the transplanted trachea. The histopathologic recovery of allograft tracheas was improved significantly after MSCs-Null and MSCs-HGF treatment and the beneficial effects were particularly observed in MSCs-HGF-treated mice. Furthermore, the allo-transplantation-induced immunophenotype disorders of the spleen, including regulatory T (Treg), T helper (Th)1, Th2 and Th17, were attenuated in both cell-treated groups. MSCs-HGF treatment reduced expression and secretion of inflammation cytokines interferon-gamma (IFN-γ), and increased expression of anti-inflammatory cytokine interleukin (IL)-4 and IL-10. It also decreased the expression level of the profibrosis factor transforming growth factor (TGF)-β. Treatment of BO with HGF gene modified MSCs results in reduction of local inflammation and promotion in recovery of allograft trachea histopathology. These findings might provide an effective therapeutic strategy for BO. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Renal cell carcinoma, unclassified with medullary phenotype: poorly differentiated adenocarcinomas overlapping with renal medullary carcinoma.

PubMed

Sirohi, Deepika; Smith, Steven C; Ohe, Chisato; Colombo, Piergiuseppe; Divatia, Mukul; Dragoescu, Ema; Rao, Priya; Hirsch, Michelle S; Chen, Ying-Bei; Mehra, Rohit; Amin, Mahul B

2017-09-01

Renal medullary carcinoma (RMC) is a highly aggressive renal cell carcinoma arising in the collecting system and requiring careful correlation with status of sickle cell trait. A panel of international experts has recently proposed provisional diagnostic terminology, renal cell carcinoma, unclassified, with medullary phenotype, based on encountering an extraordinarily rare tumor with RMC morphology and immunophenotype in an individual proven not to have a hemoglobinopathy. Herein, we extend this observation to a cohort of 5 such tumors, morphologically similar to RMC, lacking SMARCB1 expression by immunohistochemistry, but each without evidence of a hemoglobinopathy. The tumors arose in 4 men and 1 woman with a mean age of 44 years, occurring in 3 left and 2 right kidneys. Clinically, aggression was apparent with involvement of perinephric adipose tissue in all 5 cases, nodal metastasis in 4 of 5 cases, and death of disease in 4 of 5 cases within 3-27 months. Histologic sections showed poorly differentiated adenocarcinoma, often with solid and nested growth patterns, as well as infiltrative glandular, tubulopapillary, cribriform, or reticular growth. Rhabdoid and sarcomatoid cytomorphology was seen in a subset. All tumors showed PAX8 nuclear positivity and SMARCB1 loss, with OCT3/4 expression in 4 of 5 cases. In summary, this first series of renal cell carcinoma, unclassified, with medullary phenotype documents tumors with morphologic, immunophenotypic, and prognostic features of RMC occurring in individuals without sickle cell trait. Although greater biologic and molecular understanding is needed, the available evidence points to these cases representing a sporadic counterpart to sickle cell trait-associated RMC. Copyright © 2017 Elsevier Inc. All rights reserved.

Cellular immune response of pigeons in the conditions of endotoxin fever and pyrogenic tolerance.

PubMed

Dudek, K; Bednarek, D

2011-01-01

The aim of this study was to investigate changes in selected parameters of cellular immune response in the conditions of endotoxin fever and pyrogenic tolerance in pigeons. On the first day of observation the experimental birds (n = 18) were intravenously injected with Escherichia coli LPS at a dose of 10 microg/kg b.w., while the control animals (n = 6) received apyrogenic physiological saline also in the form of injection. On the second and the third day of the experiment LPS was injected additionally at 24 h intervals. Four and a half hours after the saline and pyrogen administration blood samples were collected from the control and experimental pigeons. The following immunological assays were performed: WBC, leucogram and immunophenotyping of lymphocyte subsets in peripheral blood, i.e. CD 3+ (T lymphocytes), CD 4+ (T helper lymphocytes) and CD 8+ (T suppressor/cytotoxic lymphocytes) cells. In the conditions of endotoxin fever (i.e. after the first LPS injection) leucopenia, monocytopenia, heterophilia and eosinophilia were observed. Additionally, the immunophenotyping of peripheral blood lymphocytes indicated an increase in percentage of CD 3+, CD 4+ and CD 8+ cells in response to the single injection of LPS. In contrast, the consecutive injections of LPS, which created a pyrogenic tolerance effect, caused a decrease in WBC value, heteropenia, eosinopenia and lymphocytosis. Moreover, during this state an increase in percentage of CD 3+ and CD 8+ cells was demonstrated in contrast to the percentage of CD 4+ lymphocytes. The general tendencies in cellular immune response of the affected pigeons in the conditions of endotoxin fever and pyrogenic tolerance aim at activation of defence mechanisms against LPS for its prompt elimination from the animal's organism.

IgG4 Expression in Primary Cutaneous Marginal Zone Lymphoma: A Multicenter Study.

PubMed

De Souza, Aieska; Ferry, Judith A; Burghart, Daniel R; Tinguely, Marianne; Goyal, Amrita; Duncan, Lyn M; Kutzner, Heinz; Kempf, Werner

2017-02-01

Primary cutaneous marginal zone lymphoma (PCMZL) is the second most common B-cell lymphoma of the skin. A recent study has demonstrated a strikingly high prevalence of immunoglobulin (Ig)G4 expression in PCMZL with plasmacytic differentiation. The objective was to investigate the incidence of IgG4 expression in PCMZL, and its correlation with clinical and immunophenotypic features. Multicenter study that utilized immunohistochemistry and in-situ hybridization to evaluate the expression of IgG4, Ig light (κ and λ), and heavy chains (IgM, IgG), and the ratio of T (CD3+) and B (CD20+) cells in biopsy specimens from 30 patients with PCMZL and to correlate these findings with the clinical features. IgG4 expression was observed in 4 out of 30 patients (13%) with PCMZL. Patients with IgG4-positive lymphomas were 57 to 77 years of age (mean, 69) at biopsy. The lesions were solitary in 2 patients with IgG4-positive lymphomas, and were most commonly located on the trunk. Patients with IgG4-negative lymphomas experienced earlier disease onset at an average age of 53 years. The majority of the IgG4-negative cases presented with localized disease, on the trunk and upper extremities. There was no significant difference in the IgG4-positive versus negative cases for the following parameters: Ig κ or λ restriction, B-cell or T-cell predominance, and site of the lesions. IgG4 expression was observed in a minority of PCMZL patients. We did not identify significant clinical or immunophenotypic differences between IgG4 positive and negative cases.

Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions.

PubMed

Castiglia, Sara; Mareschi, Katia; Labanca, Luciana; Lucania, Graziella; Leone, Marco; Sanavio, Fiorella; Castello, Laura; Rustichelli, Deborah; Signorino, Elena; Gunetti, Monica; Bergallo, Massimiliano; Bordiga, Anna Maria; Ferrero, Ivana; Fagioli, Franca

2014-06-01

Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). Bone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed. The MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed. We demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice-compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

TFE3 Translocation Associated Perivascular Epithelioid Cell Neoplasm (PEComa) of the Gynecologic Tract: Morphology, Immunophenotype, Differential Diagnosis

PubMed Central

Schoolmeester, J. Kenneth; Dao, Linda N.; Sukov, William R.; Park, Kay J.; Murali, Rajmohan; Hameed, Meera R.; Soslow, Robert A.

2016-01-01

TFE3 translocation associated PEComa is a distinct form of perivascular epithelioid cell neoplasm, the features of which are poorly defined owing to their general infrequency and limited prior reports with confirmed rearrangement or fusion totaling nine cases. Recent investigation has found a lack of TSC gene mutation in these tumors compared to their nonrearranged counterparts which underscores the importance of recognizing the translocated variant due to hypothetical ineffectiveness of targeted mTOR inhibitor therapy. Six cases were identified and TFE3 rearrangement was confirmed by FISH. Patient age ranged 46 to 66 years (median 50) and none had a history of tuberous sclerosis complex. Three cases arose in the uterine corpus, one in the vagina, and one pelvic tumor and one pulmonary tumor were likely a recurrence/metastasis from a probable uterine primary. Five cases had purely clear cell epithelioid morphology that showed a spectrum of atypia while one case had a mixture of clear cell epithelioid and spindle cells. A mostly consistent immunophenotype was observed in the purely clear cell epithelioid cases: each demonstrated diffuse TFE3, HMB45, CathepsinK labeling, either focal or no melanA staining and variably weak reactivity to smooth muscle markers. The mixed clear cell epithelioid and spindle cell case had a similar pattern in its epithelioid component, but strong muscle marker positivity in its spindle cell component. Follow up ranged 1 to 57 months. Three cases demonstrated aggressive behavior and three cases had no evidence of recurrence. Both GYN-specific and traditional sets of criteria for malignancy were evaluated. The GYN model showed improved inclusion and specificity in comparison to the traditional model. PMID:25517951

TFE3 translocation-associated perivascular epithelioid cell neoplasm (PEComa) of the gynecologic tract: morphology, immunophenotype, differential diagnosis.

PubMed

Schoolmeester, J Kenneth; Dao, Linda N; Sukov, William R; Wang, Lu; Park, Kay J; Murali, Rajmohan; Hameed, Meera R; Soslow, Robert A

2015-03-01

TFE3 translocation-associated PEComa is a distinct form of perivascular epithelioid cell neoplasm, the features of which are poorly defined owing to their general infrequency and limited prior reports with confirmed rearrangement or fusion. Recent investigation has found a lack of TSC gene mutation in these tumors compared with their nonrearranged counterparts, which underscores the importance of recognizing the translocated variant because of hypothetical ineffectiveness of targeted mTOR inhibitor therapy. Six cases were identified, and TFE3 rearrangement was confirmed by fluorescence in situ hybridization. Patient age ranged from 46 to 66 years (median 50 y), and none had a history of a tuberous sclerosis complex. Three cases arose in the uterine corpus, 1 in the vagina, 1 pelvic tumor, and 1 pulmonary tumor that was likely a recurrence/metastasis from a probable uterine primary. Five cases had clear cell epithelioid morphology that showed a spectrum of atypia, while 1 case had a mixture of clear cell epithelioid and spindle cells. A mostly consistent immunophenotype was observed in the clear cell epithelioid cases: each demonstrated diffuse TFE3, HMB45, cathepsinK labeling, either focal or no melanA staining, and variably weak reactivity to smooth muscle markers. The mixed clear cell epithelioid and spindle cell case had a similar expression pattern in its epithelioid component but strong muscle marker positivity in its spindle cell component. Follow-up ranged from 1 to 57 months. Three cases demonstrated aggressive behavior, and 3 cases had no evidence of recurrence. Both GYN-specific and traditional sets of criteria for malignancy were evaluated. The GYN model showed improved inclusion and specificity in comparison to the traditional model.

A comparative immunohistochemical and immunophenotypical study on lymphocytes expression in patients affected by oral lichen planus.

PubMed

Lorenzini, Guido; Viviano, Massimo; Chisci, Elettra; Chisci, Glauco; Picciotti, Maria

2013-09-01

Oral lichen planus (OLP) is an immune-mediated mucocutaneous disease of uncertain aetiology. OLP has many manifestations: reticular, erosive, atrophic, plaque like, papular, bullous, with unique etiopathogenetic working. The purpose of this study is to find a link between different clinical types of lichen and the alterations of lymphocytes on peripheral blood and oral mucosa. A total of 21 patients were enrolled in this study. The mean age of patients was 53.82 years, between 31 and 78 years. OLP Diagnosis was afterwards confirmed by histopathology. Selected patients underwent to clinical evaluation, lesion characterization, incisional biopsy, samples histological analysis, peripheral blood collection. Blood specimens were submitted to cell count determination with differential, characterization of populations and circulating lymphocyte subpopulations using monoclonal antibodies in flow cytometry. Referring to the clinical presentation of lesions, patients were divided in two groups: red lesions (RL) and white lesions (WL) and compared with an age-matched control group. The results of the immunophenotypic study showed correlation between WL and the expression of CD19 lymphocytes (r = 0.693, P = 0.0005). The results of immunohistochemical study performed on histological specimens showed a significant correlation between RL group and expression of all lymphocyte tested (CD3 r = 0.722 P = 0.0002, CD4 r = 0.579 P = 0.0060, CD56 r = 0.513 P = 0.0173, CD8 r = 0.548 P = 0.0102). We assume there is the responsibility of the expression of lymphocytes, not only type but also as quantity, in determining RL or WL manifestation of OLP. Circulating lymphocytes may have a role, too. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Monoclonal antibody fluorescence for routine lymphocyte subpopulation analysis with the Abbott CELL-DYN Sapphire haematology analyser.

PubMed

Molero, T; Lemes, A; DE LA Iglesia, S; Scott, C S

2007-12-01

Using previously described procedures, this study quantified T-cell, T-cell subset, B-cell and NK-cell populations with the CD-Sapphire haematology analyser in a series of patients with mild to moderate lymphocytosis. Lymphocyte counts ranged from 6.0 to 14.9 x 10(9)/l, with 86/97 being <10.0 x 10(9)/l. Immunophenotyping (CD3/CD19/HLA-DR, CD4/CD8 and CD16/CD56 combinations) was performed using EDTA-anticoagulated blood, automated CD-Sapphire analysis and subsequent software processing. Of 35 samples from younger (<12 years) patients, 22 (63%) had nonspecific lymphocyte changes, 4 (11%) showed specific increases in nonreactive T-Helper or T-Suppressor cells, and five showed a reactive T-cell lymphocytosis. The remaining four were classified as 'Transient/Persistent NK-associated (NKa) Expansion' (n = 3) and specific B-cell lymphocytosis (n = 1). For older patients (n = 59), 15 (25%) had an increase (>1.5 x 10(9)/l) in B-cells, and seven investigated for surface immunoglobulin expression were all found to be clonal. The remaining samples were categorized as 'Transient/Persistent NK-associated (NKa) Expansion' (13/59), Reactive Lymphocytosis (5/59), 'Reactive Lymphocytosis or Transient/Persistent NKa Expansion' (8/59), specific T-Helper cell (n = 8) or T-Suppressor cell (n = 3) lymphocytosis, and 'Lymphocytosis of Undetermined Significance' (n = 7). This study has demonstrated the feasibility of applying limited immunophenotyping protocols to the investigation of patients with abnormal lymphocyte counts in routine haematology. By using commercially purchased liquid monoclonal reagents to determine lymphocyte subpopulation profiles, haematology laboratories can provide more definitive information of potential clinical importance.

Cutaneous squamous and neuroendocrine carcinoma: genetically and immunohistochemically different from Merkel cell carcinoma

PubMed Central

Pulitzer, Melissa P; Brannon, A Rose; Berger, Michael F; Louis, Peter; Scott, Sasinya N; Jungbluth, Achim A; Coit, Daniel G; Brownell, Isaac; Busam, Klaus J

2016-01-01

Cutaneous neuroendocrine (Merkel cell) carcinoma most often arises de novo in the background of a clonally integrated virus, the Merkel cell polyomavirus, and is notable for positive expression of retinoblastoma 1 (RB1) protein and low expression of p53 compared with the rare Merkel cell polyomavirus-negative Merkel cell carcinomas. Combined squamous and Merkel cell tumors are consistently negative for Merkel cell polyomavirus. Little is known about their immunophenotypic or molecular profile. Herein, we studied 10 combined cutaneous squamous cell and neuroendocrine carcinomas for immunohistochemical expression of p53, retinoblastoma 1 protein, neurofilament, p63, and cytokeratin 20 (CK20). We compared mutation profiles of five combined Merkel cell carcinomas and seven ‘pure’ Merkel cell carcinomas using targeted next-generation sequencing. Combined tumors were from the head, trunk, and leg of Caucasian males and one female aged 52–89. All cases were highly p53- and p63-positive and neurofilament-negative in the squamous component, whereas RB1-negative in both components. Eight out of 10 were p53-positive, 3/10 p63-positive, and 3/10 focally neurofilament-positive in the neuroendocrine component. Six out of 10 were CK20-positive in any part. By next-generation sequencing, combined tumors were highly mutated, with an average of 48 mutations per megabase compared with pure tumors, which showed 1.25 mutations per megabase. RB1 and p53 mutations were identified in all five combined tumors. Combined tumors represent an immunophenotypically and genetically distinct variant of primary cutaneous neuroendocrine carcinomas, notable for a highly mutated genetic profile, significant p53 expression and/or mutation, absent RB1 expression in the context of increased RB1 mutation, and minimal neurofilament expression. PMID:26022453

Assessment of bone marrow lymphocytic status during tyrosine kinase inhibitor therapy and its relation to therapy response in chronic myeloid leukaemia.

PubMed

El Missiry, Mohamed; Adnan Awad, Shady; Rajala, Hanna L; Al-Samadi, Ahmed; Ekblom, Marja; Markevän, Berit; Åstrand-Grundström, Ingbritt; Wold, Maren; Svedahl, Ellen Rabben; Juhl, Birgitte Ravn; Bjerrum, Ole Weis; Haulin, Inger; Porkka, Kimmo; Olsson-Strömberg, Ulla; Hjorth-Hansen, Henrik; Mustjoki, Satu

2016-05-01

Tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukaemia have been reported to induce immunomodulatory effects. We aimed to assess peripheral blood (PB) and bone marrow (BM) lymphocyte status at the diagnosis and during different TKI therapies and correlate it with treatment responses. BM and PB samples were acquired from 105 first-line TKI-treated patients. Relative number of BM lymphocytes was evaluated from MGG-stained BM aspirates, and immunophenotypic analyses were performed with multicolour flow cytometry. Early 3-month expansion of BM lymphocytes was found during all different TKIs (imatinib n = 71, 20 %; dasatinib n = 25, 21 %; nilotinib n = 9, 22 %; healthy controls n = 14, 12 %, p < 0.0001). Increased PB lymphocyte count was only observed during dasatinib therapy. The BM lymphocyte expansion was associated with early molecular response; patients with 3-month BCR-ABL1 <10 % showed higher lymphocyte counts than patients with BCR-ABL1 >10 % (23 vs. 17 %, p < 0.05). Detailed phenotypic analysis showed that BM lymphocyte expansion consisted of various lymphocyte subclasses, but especially the proportion of CD19+ B cells and CD3negCD16/56+ NK cells increased from diagnostic values. During dasatinib treatment, the lymphocyte balance in both BM and PB was shifted more to cytotoxic direction (increased CD8+CD57+ and CD8+HLA-DR+ cells, and low T regulatory cells), whereas no major immunophenotypic differences were observed between imatinib and nilotinib patients. Early BM lymphocytosis occurs with all current first-line TKIs and is associated with better treatment responses. PB and BM immunoprofile during dasatinib treatment markedly differs from both imatinib- and nilotinib-treated patients.

Periodontal-ligament-derived stem cells exhibit the capacity for long-term survival, self-renewal, and regeneration of multiple tissue types in vivo.

PubMed

Menicanin, Danijela; Mrozik, Krzysztof Marek; Wada, Naohisa; Marino, Victor; Shi, Songtao; Bartold, P Mark; Gronthos, Stan

2014-05-01

Primary periodontal ligament stem cells (PDLSCs) are known to possess multidifferentiation potential and exhibit an immunophenotype similar to that described for bone-marrow-derived mesenchymal stem cells. In the present study, bromo-deoxyuridine (BrdU)-labeled ovine PDLSCs implanted into immunodeficient mice survived after 8 weeks post-transplantation and exhibited the capacity to form bone/cementum-like mineralized tissue, ligament structures similar to Sharpey's fibers with an associated vasculature. To evaluate self-renewal potential, PDLSCs were recovered from harvested primary transplants 8 weeks post-transplantation that exhibit an immunophenotype and multipotential capacity comparable to primary PDLSCs. The re-derived PDLSCs isolated from primary transplants were implanted into secondary ectopic xenogeneic transplants. Histomorphological analysis demonstrated that four out of six donor re-derived PDLSC populations displayed a capacity to survive and form fibrous ligament structures and mineralized tissues associated with vasculature in vivo, although at diminished levels in comparison to primary PDLSCs. Further, the capacity for long-term survival and the potential role of PDLSCs in dental tissue regeneration were determined using an ovine preclinical periodontal defect model. Autologous ex vivo-expanded PDLSCs that were prelabeled with BrdU were seeded onto Gelfoam(®) scaffolds and then transplanted into fenestration defects surgically created in the periodontium of the second premolars. Histological assessment at 8 weeks post-implantation revealed surviving BrdU-positive PDLSCs associated with regenerated periodontium-related tissues, including cementum and bone-like structures. This is the first report to demonstrate the self-renewal capacity of PDLSCs using serial xenogeneic transplants and provides evidence of the long-term survival and tissue contribution of autologous PDLSCs in a preclinical periodontal defect model.

Periodontal-Ligament-Derived Stem Cells Exhibit the Capacity for Long-Term Survival, Self-Renewal, and Regeneration of Multiple Tissue Types in Vivo

PubMed Central

Menicanin, Danijela; Mrozik, Krzysztof Marek; Wada, Naohisa; Marino, Victor; Shi, Songtao; Bartold, P. Mark

2014-01-01

Primary periodontal ligament stem cells (PDLSCs) are known to possess multidifferentiation potential and exhibit an immunophenotype similar to that described for bone-marrow-derived mesenchymal stem cells. In the present study, bromo-deoxyuridine (BrdU)–labeled ovine PDLSCs implanted into immunodeficient mice survived after 8 weeks post-transplantation and exhibited the capacity to form bone/cementum-like mineralized tissue, ligament structures similar to Sharpey's fibers with an associated vasculature. To evaluate self-renewal potential, PDLSCs were recovered from harvested primary transplants 8 weeks post-transplantation that exhibit an immunophenotype and multipotential capacity comparable to primary PDLSCs. The re-derived PDLSCs isolated from primary transplants were implanted into secondary ectopic xenogeneic transplants. Histomorphological analysis demonstrated that four out of six donor re-derived PDLSC populations displayed a capacity to survive and form fibrous ligament structures and mineralized tissues associated with vasculature in vivo, although at diminished levels in comparison to primary PDLSCs. Further, the capacity for long-term survival and the potential role of PDLSCs in dental tissue regeneration were determined using an ovine preclinical periodontal defect model. Autologous ex vivo–expanded PDLSCs that were prelabeled with BrdU were seeded onto Gelfoam® scaffolds and then transplanted into fenestration defects surgically created in the periodontium of the second premolars. Histological assessment at 8 weeks post-implantation revealed surviving BrdU-positive PDLSCs associated with regenerated periodontium-related tissues, including cementum and bone-like structures. This is the first report to demonstrate the self-renewal capacity of PDLSCs using serial xenogeneic transplants and provides evidence of the long-term survival and tissue contribution of autologous PDLSCs in a preclinical periodontal defect model. PMID:24351050

Rapid and simple immunophenotypic characterization of lymphocytes using a new test.

PubMed

Bellido, M; Rubiol, E; Ubeda, J; Estivill, C; López, O; Manteiga, R; Nomdedéu, J F

1998-08-01

In this paper, we report our experience of lymphocyte phenotyping of a series of 108 consecutive samples using a simple flow cytometry test (Lymphogram). The kit consists of a combination of 5 different markers conjugated with three fluorochromes (CD8-FITC, CD19-FITC, CD56-PE, CD3-PE, CD4-PECy5) in the same tube. This allows identification of different T-cells, NK subpopulations and B lymphocytes. The samples were divided into three groups: samples with absolute lymphocytosis (> 5 x 10(9)/L) (n = 50), samples with relative lymphocytosis (> 50%) (n = 24) and other categories for which a lymphocyte immunophenotype was required (T-cell lymphoma and estimation of blood involvement in chronic lymphoproliferative disorders (CLPD) (n = 34). When CD19+ cells exceeded the normal range or there was a suspicion of CLPD without B-cell lymphopenia, clonality was investigated by means of light chain restriction analysis. In the first group, 29 samples were abnormal (10 CLPD, 3 polyclonal B-cell lymphocytosis, 13 inversions of the CD4/CD8 ratio and 3 cases with CD4 lymphocytosis) and 21 samples were regarded as normal. In the second group 7 samples showed abnormalities (2 CLPD, 3 inverted CD4/CD8 ratios and 2 with a relative increase in CD4 cells). In one sample from the third group B-cell clonality without lymphocytosis was detected whereas in 18 samples a polyclonal pattern was observed. The presence of B-cell lymphopenia precluded further clonality study in 13 samples. Lymphogram associated with clonality analysis is a rapid, easy and cheap method of assessing lymphocyte phenotypes in the majority of clinically relevant situations.

The effect of Me2SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality.

PubMed

Morris, Timothy J; Picken, Andrew; Sharp, Duncan M C; Slater, Nigel K H; Hewitt, Christopher J; Coopman, Karen

2016-12-01

With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like Me 2 SO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to Me 2 SO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to Me 2 SO at 37 °C post-thaw, membrane integrity dropped to ∼70% and only ∼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to Me 2 SO before, and after, cryopreservation. Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Identification of IgH gene rearrangement and immunophenotype in an animal model of Epstein-Barr virus-associated lymphomas.

PubMed

Zhang, Yang; Peng, Xueqin; Tang, Yunlian; Gan, Xiaoning; Wang, Chengkun; Xie, Lu; Xie, Xiaoli; Gan, Runliang; Wu, Yimou

2016-10-01

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma. Because the susceptible hosts of EB virus are limited to human and cotton-top tamarins (Saguinus oedipus), there have been no appropriate animal models until the lymphoma model induced by EBV in human peripheral blood lymphocyte (hu-PBL)/SCID chimeric mice was reported. However, it is still controversial whether the EBV-associated lymphoma induced in hu-PBL/SCID mice is a monoclonal tumor. In this study, we transplanted normal human peripheral blood lymphocytes (hu-PBL) from six donors infected with EBV into SCID mice to construct hu-PBL/SCID chimeric mice. The induced tumors were found in the mediastinum or abdominal cavity of SCID mice. Microscopic observation exhibited tumor cells that were large and had a plasmablastic, centroblastic or immunoblastic-like appearance. Immunophenotyping assays showed the induced tumors were LCA-positive, CD20/CD79a-positive (markers of B cells), and CD3/CD45RO-negative (markers of T cells). A human-specific Alu sequence could be amplified by Alu-PCR. This confirmed that induced tumors were B-cell lymphomas originating from the transplanted human lymphocytes rather than mouse cells. EBER in situ hybridization detected positive signals in the nuclei of the tumor cells. Expression of EBV-encoded LMP1, EBNA-1, and EBNA-2 in the tumors was significantly positive. PCR-based capillary electrophoresis analysis of IgH gene rearrangement revealed a monoclonal peak and single amplification product in all six cases of induced tumors. This indicated that EBV can induce monoclonal proliferation of human B lymphocytes and promotes the development of lymphoma. J. Med. Virol. 88:1804-1813, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Donor-Matched Comparison of Chondrogenic Potential of Equine Bone Marrow- and Synovial Fluid-Derived Mesenchymal Stem Cells: Implications for Cartilage Tissue Regeneration

PubMed Central

Zayed, Mohammed; Caniglia, Christopher; Misk, Nabil; Dhar, Madhu S.

2017-01-01

Mesenchymal stem cells (MSCs) have been demonstrated to be useful for cartilage tissue regeneration. Bone marrow (BM) and synovial fluid (SF) are promising sources for MSCs to be used in cartilage regeneration. In order to improve the clinical outcomes, it is recommended that prior to clinical use, the cellular properties and, specifically, their chondrogenic potential must be investigated. The purpose of this study is to compare and better understand the in vitro chondrogenic potential of equine bone marrow-derived mesenchymal stem cells (BMMSCs) and synovial fluid-derived mesenchymal stem cells (SFMSCs) populated from the same equine donor. BM- and SF-derived MSCs cultures were generated from five equine donors, and the MSCs were evaluated in vitro for their morphology, proliferation, trilineage differentiation, and immunophenotyping. Differences in their chondrogenic potentials were further evaluated quantitatively using glycosaminoglycan (GAG) content and via immunofluorescence of chondrogenic differentiation protein markers, SRY-type HMG box9, Aggrecan, and collagen II. The BMMSCs and SFMSCs were similar in cellular morphology, viability, and immunophenotype, but, varied in their chondrogenic potential, and expression of the key chondrogenic proteins. The SFMSCs exhibited a significant increase in GAG content compared to the BMMSCs (P < 0.0001) in three donors, suggesting increased levels of chondrogenesis. The expression of the key chondrogenic proteins correlated positively with the GAG content, suggesting that the differentiation process is dependent on the expression of the target proteins in these three donors. Our findings suggest that even though SFMSCs were hypothesized to be more chondrogenic relative to BMMSCs, there was considerable donor-to-donor variation in the primary cultures of MSCs which can significantly affect their downstream application. PMID:28149840

Evaluation of optimal DNA staining for triggering by scanning fluorescence microscopy (SFM)

NASA Astrophysics Data System (ADS)

Mittag, Anja; Marecka, Monika; Pierzchalski, Arkadiusz; Malkusch, Wolf; Bocsi, József; Tárnok, Attila

2009-02-01

In imaging and flow cytometry, DNA staining is a common trigger signal for cell identification. Selection of the proper DNA dye is restricted by the hardware configuration of the instrument. The Zeiss Imaging Solutions GmbH (München, Germany) introduced a new automated scanning fluorescence microscope - SFM (Axio Imager.Z1) which combines fluorescence imaging with cytometric parameters measurement. The aim of the study was to select optimal DNA dyes as trigger signal in leukocyte detection and subsequent cytometric analysis of double-labeled leukocytes by SFM. Seven DNA dyes (DAPI, Hoechst 33258, Hoechst 33342, POPO-3, PI, 7-AAD, and TOPRO-3) were tested and found to be suitable for the implemented filtersets (fs) of the SFM (fs: 49, fs: 44, fs: 20). EDTA blood was stained after erythrocyte lysis with DNA dye. Cells were transferred on microscopic slides and embedded in fluorescent mounting medium. Quality of DNA fluorescence signal as well as spillover signals were analyzed by SFM. CD45-APC and CD3-PE as well as CD4-FITC and CD8-APC were selected for immunophenotyping and used in combination with Hoechst. Within the tested DNA dyes DAPI showed relatively low spillover and the best CV value. Due to the low spillover of UV DNA dyes a triple staining of Hoechst and APC and PE (or APC and FITC, respectively) could be analyzed without difficulty. These results were confirmed by FCM measurements. DNA fluorescence is applicable for identifying and triggering leukocytes in SFM analyses. Although some DNA dyes exhibit strong spillover in other fluorescence channels, it was possible to immunophenotype leukocytes. DAPI seems to be best suitable for use in the SFM system and will be used in protocol setups as primary parameter.

A diffusely enlarged pancreas: the (un)usual suspect.

PubMed

Magalhães-Costa, Pedro; Brito, Maria José; Pinto-Marques, Pedro

2016-12-01

An 81-years-old female presented with obstructive jaundice and a non-specific clinical picture of nausea and appetite loss. Labs demonstrated a conjugated hyperbilirrubinemia (7.7 mg/dL), increased aspartate aminotransferase and alanine aminotransferase (10xULN and 8xULN, respectively), increased lactate dehydrogenase (10xULN) and serum lipase (3xULN). CA 19.9 was 342 U/mL (Ref value < 37 U/mL). There was no evidence of peripheral lymphadenopathy or hepatosplenomegaly. Imaging (Figure 1A and 1B) revealed a marked homogeneous enlargement of the pancreas (without any well-defined mass), dilation of the extra and intra-hepatic bile ducts and ascites. Endoscopic ultrasound (Figure 1C and 1D) identified an enlarged homogeneous hypoechoic pancreas, without any well-defined lesion, no dilation of the main pancreatic duct, no peripancreatic or celiac enlarged lymph nodes. A fine-needle biopsy was performed yielding, on cytological examination and cell-block technique (Figure 2A and 2B), numerous medium/large sized atypical lymphoid cells that displayed a B-cell lineage immunophenotype (Figure 2A-2F). Even though, further characterization (by flow cytometric immunophenotyping) could not be obtained, a final diagnosis of primary pancreatic lymphoma (PPL) was assumed. Primary pancreatic lymphoma is a remarkably rare tumor of the pancreas, representing approximately 0.5% of all pancreatic neoplasms and <2% of all lymphomas (1,2). A correct diagnosis is crucial because therapeutic management differs from other pancreatic malignancies (pancreatic ductal adenocarcinoma, neuroendocrine tumor and metastases) (2,3). Two morphologic patterns of PPL are recognized: a focal form (occurring in the pancreatic head in 80% of cases) and a rarer diffuse/infiltrative pattern, as depicted herein, emulating an acute/autoimmune pancreatitis (1).

Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development.

PubMed

Endale, Mehari; Ahlfeld, Shawn; Bao, Erik; Chen, Xiaoting; Green, Jenna; Bess, Zach; Weirauch, Matthew T; Xu, Yan; Perl, Anne Karina

2017-05-15

Many studies have investigated the source and role of epithelial progenitors during lung development; such information is limited for fibroblast populations and their complex role in the developing lung. In this study, we characterized the spatial location, mRNA expression and Immunophenotyping of PDGFRα + fibroblasts during sacculation and alveolarization. Confocal microscopy identified spatial association of PDGFRα expressing fibroblasts with proximal epithelial cells of the branching bronchioles and the dilating acinar tubules at E16.5; with distal terminal saccules at E18.5; and with alveolar epithelial cells at PN7 and PN28. Immunohistochemistry for alpha smooth muscle actin revealed that PDGFRα + fibroblasts contribute to proximal peribronchiolar smooth muscle at E16.5 and to transient distal alveolar myofibroblasts at PN7. Time series RNA-Seq analyses of PDGFRα + fibroblasts identified differentially expressed genes that, based on gene expression similarity were clustered into 7 major gene expression profile patterns. The presence of myofibroblast and smooth muscle precursors at E16.5 and PN7 was reflected by a two-peak gene expression profile on these days and gene ontology enrichment in muscle contraction. Additional molecular and functional differences between peribronchiolar smooth muscle cells at E16.5 and transient intraseptal myofibroblasts at PN7 were suggested by a single peak in gene expression at PN7 with functional enrichment in cell projection and muscle cell differentiation. Immunophenotyping of subsets of PDGFRα + fibroblasts by flow cytometry confirmed the predicted increase in proliferation at E16.5 and PN7, and identified subsets of CD29 + myofibroblasts and CD34 + lipofibroblasts. These data can be further mined to develop novel hypotheses and valuable understanding of the molecular and cellular basis of alveolarization. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development☆

PubMed Central

Endale, Mehari; Ahlfeld, Shawn; Bao, Erik; Chen, Xiaoting; Green, Jenna; Bess, Zach; Weirauch, Matthew T.; Xu, Yan; Perl, Anne Karina

2017-01-01

Many studies have investigated the source and role of epithelial progenitors during lung development; such information is limited for fibroblast populations and their complex role in the developing lung. In this study, we characterized the spatial location, mRNA expression and Immunophenotyping of PDGFRα+ fibroblasts during sacculation and alveolarization. Confocal microscopy identified spatial association of PDGFRα expressing fibroblasts with proximal epithelial cells of the branching bronchioles and the dilating acinar tubules at E16.5; with distal terminal saccules at E18.5; and with alveolar epithelial cells at PN7 and PN28. Immunohistochemistry for alpha smooth muscle actin revealed that PDGFRα+ fibroblasts contribute to proximal peribronchiolar smooth muscle at E16.5 and to transient distal alveolar myofibroblasts at PN7. Time series RNA-Seq analyses of PDGFRα+ fibroblasts identified differentially expressed genes that, based on gene expression similarity were clustered into 7 major gene expression profile patterns. The presence of myofibroblast and smooth muscle precursors at E16.5 and PN7 was reflected by a two-peak gene expression profile on these days and gene ontology enrichment in muscle contraction. Additional molecular and functional differences between peribronchiolar smooth muscle cells at E16.5 and transient intraseptal myofibroblasts at PN7 were suggested by a single peak in gene expression at PN7 with functional enrichment in cell projection and muscle cell differentiation. Immunophenotyping of subsets of PDGFRα+ fibroblasts by flow cytometry confirmed the predicted increase in proliferation at E16.5 and PN7, and identified subsets of CD29+ myofibroblasts and CD34+ lipofibroblasts. These data can be further mined to develop novel hypotheses and valuable understanding of the molecular and cellular basis of alveolarization. PMID:28408205

Spindle cell oncocytoma of the pituitary gland with follicle-like component: organotypic differentiation to support its origin from folliculo-stellate cells.

PubMed

Vajtai, Istvan; Beck, Jürgen; Kappeler, Andreas; Hewer, Ekkehard

2011-08-01

Spindle cell oncocytoma (SCO) is a rare, non-adenomatous tumor originating from the anterior pituitary gland. Composed of fusiform, mitochondrion-rich cells sharing several immunophenotypic and ultrastructural properties with folliculo-stellate cells (FSC), SCO has been proposed to represent a neoplastic counterpart of the latter. To date, however, SCO has failed to meet one criterion commonly used in histological-based taxonomy and diagnostics; that of recapitulating any of FSCs' morphologically defined developmental or physiological states. We describe a unique example of SCO wherein a conventional fascicular texture was seen coexisting with and organically merging into follicle-like arrangements. The sellar tumor of 2.7 × 2.6 × 2.5 cm was transphenoidally resected from a 55-year old female. Preoperative magnetic resonance imaging indicated an isointense, contrast enhancing mass with suprasellar extension. Histology showed multiple rudimentary to well-formed, follicle-like cavities on a classical spindle cell background; while all the participating cells exhibited an SCO immunophenotype, including positivity for S100 protein, vimentin, EMA, Bcl-2, and TTF-1, as well as staining with the antimitochondrial antibody 113-1. Conversely no expression of GFAP, follicular-epithelial cytokeratin, carcinoembryonic antigen, or anterior pituitary hormones was detected. Ultrastructurally, tumor cells facing follicular lumina displayed organelles of epithelial specialization, in particular surface microvilli and apical tight junctions. This constellation is felt to be reminiscent of FSCs' metaplastic transition to follicular epithelium, as observed during embryonic development and physiological renewal of the hormone-secreting parenchyma. Such finding is apt to being read as a supporting argument for SCO's descent from the FSC lineage.

HGF Gene Modification in Mesenchymal Stem Cells Reduces Radiation-Induced Intestinal Injury by Modulating Immunity.

PubMed

Wang, Hua; Sun, Rui-Ting; Li, Yang; Yang, Yue-Feng; Xiao, Feng-Jun; Zhang, Yi-Kun; Wang, Shao-Xia; Sun, Hui-Yan; Zhang, Qun-Wei; Wu, Chu-Tse; Wang, Li-Sheng

2015-01-01

Effective therapeutic strategies to address intestinal complications after radiation exposure are currently lacking. Mesenchymal stem cells (MSCs), which display the ability to repair the injured intestine, have been considered as delivery vehicles for repair genes. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF)-gene-modified MSCs on radiation-induced intestinal injury (RIII). Female 6- to 8-week-old mice were radiated locally at the abdomen with a single 13-Gy dose of radiation and then treated with saline control, Ad-HGF or Ad-Null-modified MSCs therapy. The transient engraftment of human MSCs was detected via real-time PCR and immunostaining. The therapeutic effects of non- and HGF-modified MSCs were evaluated via FACS to determine the lymphocyte immunophenotypes; via ELISA to measure cytokine expression; via immunostaining to determine tight junction protein expression; via PCNA staining to examine intestinal epithelial cell proliferation; and via TUNEL staining to detect intestinal epithelial cell apoptosis. The histopathological recovery of the radiation-injured intestine was significantly enhanced following non- or HGF-modified MSCs treatment. Importantly, the radiation-induced immunophenotypic disorders of the mesenteric lymph nodes and Peyer's patches were attenuated in both MSCs-treated groups. Treatment with HGF-modified MSCs reduced the expression and secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ), increased the expression of the anti-inflammatory cytokine IL-10 and the tight junction protein ZO-1, and promoted the proliferation and reduced the apoptosis of intestinal epithelial cells. Treatment of RIII with HGF-gene-modified MSCs reduces local inflammation and promotes the recovery of small intestinal histopathology in a mouse model. These findings might provide an effective therapeutic strategy for RIII.

A prospective study of the immunophenotype and temporal changes in the histologic lesions of canine demodicosis.

PubMed

Caswell, J L; Yager, J A; Parker, W M; Moore, P F

1997-07-01

Mural folliculitis is a consistent histologic lesion of canine demodicosis. The objective of this study was to describe the immunophenotype and to evaluate temporal changes in histologic lesions of demodicosis during the course of therapy. Five dogs with demodicosis were examined and biopsied biweekly for up to 14 weeks; three dogs were evaluated once only. Lymphocyte subsets infiltrating the lesions were quantified using immunohistochemistry to detect CD3, CD21, CD4, and CD8 antigens. Lymphocyte subsets in blood were analyzed from four dogs using flow cytometry. Mural folliculitis was always present during clinically active disease. In contrast, following resolution of clinical lesions, perifolliculitis and/or perifollicular granulomas were present but mural folliculitis was absent. Most lymphocytes infiltrating the follicular epithelium in lesions of mural folliculitis were CD3+ and CD8+; the ratio of CD4+ :CD8+ cells in this epithelium was 0.032. In contrast, the perifollicular dermis contained approximately equal numbers of CD4+ cells and CD8+ cells, with slightly fewer CD21+B cells. In peripheral blood, the ratio of CD4+:CD8+ lymphocytes was reduced and the percentage of CD8+ cells was increased in three of four dogs. These results indicate that mural folliculitis is a consistent lesion of clinically active canine demodicosis and is characterized by infiltration of the follicular epithelium by CD3+ CD8+ T lymphocytes. These lymphocytes are cytotoxic T cells, which may mediate the injury to the follicular epithelium in demodicosis. Alternatively, CD8+ T cells may play a role in resistance to Demodex canis infection or may represent a deleterious immune response in dogs that develop demodicosis.

Clinical applicability and prognostic significance of molecular response assessed by fluorescent-PCR of immunoglobulin genes in multiple myeloma. Results from a GEM/PETHEMA study.

PubMed

Martinez-Lopez, Joaquin; Fernández-Redondo, Elena; García-Sánz, Ramón; Montalbán, María Angeles; Martínez-Sánchez, Pilar; Pavia, Bruno; Mateos, María Victoria; Rosiñol, Laura; Martín, Marisa; Ayala, Rosa; Martínez, Rafael; Blanchard, María Jesus; Alegre, Adrian; Besalduch, Joan; Bargay, Joan; Hernandez, Miguel T; Sarasquete, María Eugenia; Sanchez-Godoy, Pedro; Fernández, Manuela; Blade, Joan; San Miguel, Jesús F; Lahuerta, Juan Jose

2013-12-01

Minimal residual disease monitoring is becoming increasingly important in multiple myeloma (MM), but multiparameter flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) techniques are not routinely available. This study investigated the prognostic influence of achieving molecular response assessed by fluorescent-PCR (F-PCR) in 130 newly diagnosed MM patients from Grupo Español Multidisciplinar de Melanoma (GEM)2000/GEM05 trials (NCT00560053, NCT00443235, NCT00464217) who achieved almost very good partial response after induction therapy. As a reference, we used the results observed with simultaneous MFC. F-PCR at diagnosis was performed on DNA using three different multiplex PCRs: IGH D-J, IGK V-J and KDE rearrangements. The applicability of F-PCR was 91·5%. After induction therapy, 64 patients achieved molecular response and 66 non-molecular response; median progression-free survival (PFS) was 61 versus 36 months, respectively (P = 0·001). Median overall survival (OS) was not reached (NR) in molecular response patients (5-year survival: 75%) versus 66 months in the non-molecular response group (P = 0·03). The corresponding PFS and OS values for patients with immunophenotypic versus non-immunophenotypic response were 67 versus 42 months (P = 0·005) and NR (5-year survival: 95%) versus 69 months (P = 0·004), respectively. F-PCR analysis is a rapid, affordable, and easily performable technique that, in some circumstances, may be a valid approach for minimal residual disease investigations in MM. © 2013 John Wiley & Sons Ltd.

Cutaneous squamous and neuroendocrine carcinoma: genetically and immunohistochemically different from Merkel cell carcinoma.

PubMed

Pulitzer, Melissa P; Brannon, A Rose; Berger, Michael F; Louis, Peter; Scott, Sasinya N; Jungbluth, Achim A; Coit, Daniel G; Brownell, Isaac; Busam, Klaus J

2015-08-01

Cutaneous neuroendocrine (Merkel cell) carcinoma most often arises de novo in the background of a clonally integrated virus, the Merkel cell polyomavirus, and is notable for positive expression of retinoblastoma 1 (RB1) protein and low expression of p53 compared with the rare Merkel cell polyomavirus-negative Merkel cell carcinomas. Combined squamous and Merkel cell tumors are consistently negative for Merkel cell polyomavirus. Little is known about their immunophenotypic or molecular profile. Herein, we studied 10 combined cutaneous squamous cell and neuroendocrine carcinomas for immunohistochemical expression of p53, retinoblastoma 1 protein, neurofilament, p63, and cytokeratin 20 (CK20). We compared mutation profiles of five combined Merkel cell carcinomas and seven 'pure' Merkel cell carcinomas using targeted next-generation sequencing. Combined tumors were from the head, trunk, and leg of Caucasian males and one female aged 52-89. All cases were highly p53- and p63-positive and neurofilament-negative in the squamous component, whereas RB1-negative in both components. Eight out of 10 were p53-positive, 3/10 p63-positive, and 3/10 focally neurofilament-positive in the neuroendocrine component. Six out of 10 were CK20-positive in any part. By next-generation sequencing, combined tumors were highly mutated, with an average of 48 mutations per megabase compared with pure tumors, which showed 1.25 mutations per megabase. RB1 and p53 mutations were identified in all five combined tumors. Combined tumors represent an immunophenotypically and genetically distinct variant of primary cutaneous neuroendocrine carcinomas, notable for a highly mutated genetic profile, significant p53 expression and/or mutation, absent RB1 expression in the context of increased RB1 mutation, and minimal neurofilament expression.

A morphological and immunophenotypic map of the immune response in Merkel cell carcinoma.

PubMed

Walsh, Noreen M; Fleming, Kirsten E; Hanly, John G; Dakin Hache, Kelly; Doucette, Steve; Ferrara, Gerardo; Cerroni, Lorenzo

2016-06-01

The susceptibility of Merkel cell carcinoma to the host immune response has prompted a search for effective immunotherapy. CD8-positive T lymphocytes are considered key effectors of this response, but the cellular infiltrates also harbor tumor-protective agents. By developing a comprehensive morphological and immunophenotypic map of tumor-infiltrating lymphocytes (TILS) in Merkel cell carcinoma, we aimed to establish a useful template for future studies. Twenty-two cases (mean age, 79years [range, 52-95]; male-female ratio, 10:12) were studied. TILS were categorized as brisk (7), nonbrisk (9), and absent(6). Merkel cell polyomavirus (MCPyV)-positive (16) and -negative (6) cases were included, as were those with pure (18) and combined (4) morphologies. One MCPyV+ case had undergone spontaneous regression. Immunohistochemical markers included CD3, CD4, CD8, CD20, CD68, FoxP3, PD-1, and CD123. Statistical analysis used Fisher exact tests and Spearman correlations. There was a significant correlation between brisk TILs and MCPyV+ status (P=.025). CD8+ T lymphocytes predominated, were present in significantly higher proportions in brisk infiltrates (P=.003), and showed a significant predilection for the intratumoral environment (P=.003). Immune inhibitors including T regulatory cells (FOXP3+) and PD-1+ "exhausted" immunocytes were present in lower proportions. Our findings support (1) the link between a brisk immune response and MCPyV positivity, (2) the supremacy of CD8+ cells in effecting immunity, and (3) the incorporation of immune inhibitors within the global infiltrate. Efforts to therapeutically arm the "effectors" and disarm the "detractors" are well focused. These will likely have the greatest impact on MCPyV-positive cases. Copyright © 2016 Elsevier Inc. All rights reserved.

Malignant and Tuberculous Pleural Effusions: Immunophenotypic Cellular Characterization

PubMed Central

de Aguiar, Lucia Maria Zanatta; Antonangelo, Leila; Vargas, Francisco S.; Zerbini, Maria Cláudia Nogueira; Sales, Maria Mirtes; Uip, David E.; Saldiva, Paulo Hilário Nascimento

2008-01-01

INTRODUCTION AND OBJECTIVES Tuberculosis and cancer are the main causes of pleural effusion. Pleural involvement is associated with migration of immune cells to the pleural cavity. We sought to characterize the immunophenotype of leukocytes in the pleural effusion and peripheral blood of patients with tuberculosis or malignancy. METHODS Thirty patients with tuberculosis (14) or malignancy (16) were studied. A control group included 20 healthy blood donors. RESULTS Malignant phycoerythrin pleural effusions showed higher percentages of CD3, CD4, CD3CD45RO, and CD20CD25 lymphocytes and lower percentages of CD3CD25 and CD20HLA-DR when compared to PB lymphocytes. Compared to PB, tuberculous effusions had a higher percentage of lymphocytes that co-expressed CD3, CD4, CD3CD45RO, CD3TCRαβ, CD3CD28, and CD20 and a lower percentage of CD14, CD8 and CD3TCRγδ-positive lymphocytes. Malignant effusions presented higher expression of CD14 whereas tuberculous effusions had higher expression of CD3 and CD3CD95L. Peripheral blood cells from tuberculosis patients showed higher expression of CD14, CD20CD25 and CD3CD95L. Compared with the control cells, tuberculosis and cancer peripheral blood cells presented a lower percentage of CD3CD4 and CD3CD28-positive cells as well as a higher percentage of CD3CD8, CD3CD25 and CD3CD80-positive cells. CONCLUSIONS Tuberculous and malignant peripheral blood is enriched with lymphocytes with a helper/inducer T cell phenotype, which are mainly of memory cells. CD14-positive cells were more frequently found in malignant effusions, while CD3-positive cells expressing Fas ligand were more frequently found in tuberculous effusions. PMID:18925324

Effects Of Hypoxia in Long-Term In Vitro Expansion of Human Bone Marrow Derived Mesenchymal Stem Cells.

PubMed

Pezzi, Annelise; Amorin, Bruna; Laureano, Álvaro; Valim, Vanessa; Dahmer, Alice; Zambonato, Bruna; Sehn, Filipe; Wilke, Ianaê; Bruschi, Lia; Silva, Maria Aparecida Lima da; Filippi-Chiela, Eduardo; Silla, Lucia

2017-10-01

Mesenchymal stem cells (MSC) are considered multipotent stromal, non-hematopoietic cells with properties of self-renovation and differentiation. Optimal conditions for culture of MSC have been under investigation. The oxygen tension used for cultivation has been studied and appears to play an important role in biological behavior of mesenchymal cells. The aim is characterize MSC in hypoxia and normoxia conditions comparing their morphological and functional characteristics. Bone marrow-derived mesenchymal stem cells obtained from 15 healthy donors and cultured. MSC obtained from each donor were separated into two cultivation conditions normoxia (21% O 2 ) and hypoxia (three donors at 1%, three donors at 2%, five donors at 3%, and four donors at 4% O 2 ) up to second passage. MSC were evaluated for proliferation, differentiation, immunophenotyping, size and cell complexity, oxidative stress, mitochondrial activity, and autophagy. Culture conditions applied did not seem to affect immunophenotypic features and cellular plasticity. However, cells subjected to hypoxia showed smaller size and greater cellular complexity, besides lower proliferation (P < 0.002). Furthermore, cells cultured in low O 2 tension had lower mitochondrial activity (P < 0.03) and a reduced tendency to autophagy, although oxidative stress did not vary among groups (P < 0.39). Oxygen tension seems to be a key regulator of cellular adaptation in vitro, and metabolic effects underlying this variable remain undescribed. Heterogeneity or even lack of results on the impact of oxygen concentration used for expanding MSC highlights the need for further research, in order to optimize conditions of cultivation and expansion and achieve greater safety and therapeutic efficacy. J. Cell. Biochem. 118: 3072-3079, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Reduced Activated T Lymphocytes (CD4+CD25+) and Plasma Levels of Cytokines in Parkinson's Disease.

PubMed

Rocha, Natalia Pessoa; Assis, Frankcinéia; Scalzo, Paula Luciana; Vieira, Érica Leandro Marciano; Barbosa, Izabela Guimarães; de Souza, Mariana Soares; Christo, Paulo Pereira; Reis, Helton José; Teixeira, Antonio Lucio

2018-02-01

Parkinson's disease (PD) is the second most common neurodegenerative disease. The cause of neurodegeneration in PD is not completely understood, and evidence has shown that inflammatory/immune changes may be involved in PD pathophysiology. Herein, we aimed to determine the profile of the peripheral immune system in patients with PD in comparison with controls. Forty patients with PD and 25 age- and gender-matched controls were enrolled in this study. From these, 23 PD patients and 21 controls were included in the immunophenotyping analyses. Peripheral blood was drawn on the same day of the clinical assessment and submitted to plasma separation for enzyme-linked immunosorbent assay or cytometric bead array. Immunophenotyping analyses of the peripheral blood were performed by flow cytometry. We found that patients with PD presented peripheral immune changes evidenced by decreased percentage of T lymphocytes (CD3+ cells), especially activated T lymphocytes (CD4+CD25+ cells), when compared with controls. In line with these results, we also found decreased plasma levels of the cytokines IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A in the PD group. In vitro experiments demonstrated that the production of cytokines by peripheral blood mononuclear cells harvested from healthy young donors was reduced after exposure to the anti-parkinsonian drugs levodopa and pramipexole. Our data corroborate the hypothesis that immunological mechanisms are involved in PD. It is not clear whether the differences that we have found are due to adaptive mechanisms or to changes associated with PD, including pharmacological treatment, or even directly related to the disease pathophysiology. Future studies are needed in this regard.

Splenic morphological changes are accompanied by altered baseline immunity in a mouse model of sickle-cell disease.

PubMed

Szczepanek, Steven M; McNamara, Jeffrey T; Secor, Eric R; Natarajan, Prabitha; Guernsey, Linda A; Miller, Lauren A; Ballesteros, Enrique; Jellison, Evan; Thrall, Roger S; Andemariam, Biree

2012-11-01

Although functional asplenia from infarctions may be a major contributor to increased infectious mortality in sickle-cell disease (SCD), this relationship has not been fully defined. We used the transgenic Berkeley SCD mouse to define blood and splenic immunophenotypic differences in this model compared with C57BL/6 and hemizygous controls. In the serum of SCD mice, we found increased IgG2a and suppressed IgM, IgG2b, and IgA levels. Serum IL-6 levels in SCD mice were elevated, whereas IL-1α, CXCL10, and CCL5 levels were decreased. The blood of SCD mice had higher white blood cell counts, with an increased percentage of lymphocytes and decreases in other leukocytes. Immunophenotyping of lymphocytes revealed higher percentages of CD8(+) and T-regulatory cells and lower percentages of B cells. SCD mouse spleens exhibited histological disorganization, with reduction of defined lymphoid follicles and expansion of red pulp, a greater than fourfold increase in splenic mononuclear cells, marked expansion of the nucleated red blood cell fraction, and B-cell and CD8(+) T-cell lymphopenia. Within the splenic B-cell population, there was a significant decrease in B-1a B cells, with a corresponding decrease in IgA secreting plasma cells in the gut. Confocal microscopy of spleens demonstrated complete disruption of the normal lymphofollicular structure in the white pulp of SCD mice without distinct B, T, and marginal zones. Our findings suggest that altered SCD splenic morphological characteristics result in an impaired systemic immune response. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Non-invasive in vivo molecular imaging of intra-articularly transplanted immortalized bone marrow stem cells for osteoarthritis treatment.

PubMed

Peng, Bou-Yue; Chiou, Chi-Sheng; Dubey, Navneet Kumar; Yu, Sung-Hsun; Deng, Yue-Hua; Tsai, Feng-Chou; Chiang, Han-Sun; Shieh, Ying-Hua; Chen, Wei-Hong; Deng, Win-Ping

2017-11-14

Pathophysiology of osteoarthritis (OA) is characterized by progressive loss of articular cartilage in the knee-joints. To impart regenerative ability in lowly metabolizing chondrocytes, the bone marrow stem cells (BMSCs) has recently been recognized as a superior alternative treatment for OA. However, study of primary BMSCs-mediated chondrogenesis is difficult due to progressive cellular aging and replicative senescence. To obtain a therapeutic cell population for OA, BMSCs were immortalized by human papilloma virus (HPV)-16 E6/E7 along with mCherry luciferase (mCL), a gene marker for non-invasive imaging, and designated as iBMSCs-mCL. Next, their cell morphology, population doubling time (PDT) and colony forming ability (CFU) were evaluated. Furthermore, pluripotency and immunophenotypic markers were investigated. To deduce therapeutic ability, iBMSCs-mCL were intra-articularly injected into right knee of anterior cruciate ligament transaction (ACLT)-OA mice model and tracked through non-invasive bioluminescence imaging. Cell morphology of iBMSCs-mCL was similar to parental BMSCs. PDT and CFU ability of iBMSCs-mCLs were significantly increased. Pluripotency and immunophenotypic markers were highly expressed in iBMSC-mCL. Long-term survival and tri-lineage differentiation particularly chondrogenic potential of iBMSCs-mCL were also demonstrated in vitro and then in vivo which was monitored through non-invasive imaging. Intensive bioluminescent signals in iBMSCs-mCL administered knee-joint indicated a marked in vivo survival and proliferation of iBMSCs-mCL. Immunohistochemical staining for type II collagen (IHC of Col II) and alcian blue & safranin o staining of proteoglycans also corroborated cartilage regeneration by iBMSCs-mCL. Conclusively, iBMSCs-mCL maintains stemness and in vivo cartilage regeneration potential suggesting a promising avenue for development of OA therapeutics.

[Solitary fibrous tumors and hemangiopericytomas of the meninges: Immunophenotype and histoprognosis in a series of 17 cases].

PubMed

Savary, Caroline; Rousselet, Marie-Christine; Michalak, Sophie; Fournier, Henri-Dominique; Taris, Michaël; Loussouarn, Delphine; Rousseau, Audrey

2016-08-01

The 2007 World Health Organization (WHO) classification of tumors of the central nervous system distinguishes meningeal hemangiopericytomas (HPC) from solitary fibrous tumors (TFS). In the WHO classification of tumors of soft tissue and bone, those neoplasms are no longer separate entities since the discovery in 2013 of a common oncogenic event, i.e. the NAB2-STAT6 gene fusion. A shared histopronostic grading system, called "Marseille grading system", was recently proposed, based on hypercellularity, mitotic count and necrosis. We evaluated the immunophenotype and histoprognosis in a retrospective cohort of intracranial HPC and TFS. Fifteen initial tumors and 2 recurrences were evaluated by immunohistochemistry for STAT6, CD34, EMA, progesterone receptors and Ki67. The pronostic value of the WHO and the Marseille grading systems was tested on 12 patients with clinical follow-up. Initial tumors were 11 HPC and 4 SFT. STAT6 and CD34 were expressed in 16/17 tumors, EMA and progesterone receptors in 2 and 5 cases, respectively. The Ki67 labelling index was 6.25% in HPC and 3% in SFT. Half of the tumors recurred between 2 years and 9 years after initial diagnosis (mean time 5 years). No statistical difference in the risk of recurrence was associated with either grade (WHO or Marseille), in this small cohort. The diagnosis of HPC and TFS is facilitated by the almost constant immuno-expression of STAT6, and this justifies their common classification. The high rate of recurrence implies a very long-term follow-up because the current grading systems do not accurately predict the individual risk. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Two-Photon Microscopy Imaging of thy1GFP-M Transgenic Mice: A Novel Animal Model to Investigate Brain Dendritic Cell Subsets In Vivo

PubMed Central

Laperchia, Claudia; Allegra Mascaro, Anna L.; Sacconi, Leonardo; Andrioli, Anna; Mattè, Alessandro; De Franceschi, Lucia; Grassi-Zucconi, Gigliola; Bentivoglio, Marina; Buffelli, Mario; Pavone, Francesco S.

2013-01-01

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for in vivo brain studies with two-photon fluorescence (TPF) microscopy. Mice of the thy1GFP-M line have been engineered for selective expression of green fluorescent protein (GFP) in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells in vivo and characterized in brain sections their immunophenotype. With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity) of dendritic cells (DCs), and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of thy1GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs in situ. The results indicate that the thy1GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in thy1GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions. PMID:23409142

Rapid Flow cytometric prenatal diagnosis of primary immunodeficiency (PID) disorders.

PubMed

Mishra, Anju; Gupta, Maya; Dalvi, Aparna; Ghosh, Kanjaksha; Madkaikar, Manisha

2014-04-01

Primary Immunodeficiency diseases (PID) are a heterogeneous group of inherited disorders of immune system. Immunophenotypic evaluation of PIDs using flowcytometry provides important clues for diagnosis of these disorders, though confirmation requires identification of underlying molecular defects. Prenatal diagnosis (PND) forms an important component of management in families affected with severe PID. However, molecular diagnostic facilities for each of these diseases are not available and may not be possible to perform in all cases. In such scenario we opted for phenotypic prenatal diagnosis by cordocentesis for families with index case having immunophenotypically well characterized PID. Normal reference ranges of lymphocyte subsets, CD 18/CD11 integrins on leukocytes, MHC class II expression and oxidative burst activity of fetal neutrophils at 18 weeks of gestation were previously established on 30 cord blood samples. PND was performed in 13 families with PIDs. Maternal contamination was ruled out by VNTR analysis. Out of 13 fetuses, nine were found to be unaffected (three cases with leukocyte adhesion deficiency (LAD-I), four cases with severe combined immunodeficiency diseases (SCID), one with X-linked agammaglobulinemia (XLA), and one with chronic granulomatous disease (CGD)] and three were found to be affected (one with T-B+NK-SCID, one with MHC class II deficiency and one with LAD-I). Diagnosis was confirmed by testing the cord blood samples after delivery and further follow-up of the children. In one family diagnosis could not be offered due to maternal contamination. No procedure related complications were observed. Flowcytometry offers rapid and sensitive method for prenatal diagnosis and genetic counseling for selected phenotypically well characterized PID in cases where molecular diagnostic facilities are not available.

A retrospective review of acute myeloid leukaemia in 35 dogs diagnosed by a combination of morphologic findings, flow cytometric immunophenotyping and cytochemical staining results (2007-2015).

PubMed

Davis, L L; Hume, K R; Stokol, T

2018-06-01

Acute myeloid leukaemia (AML) is an uncommon, rapidly progressive neoplasm in dogs. The aim of this retrospective study was to characterize the clinical presentation, haematologic findings, diagnostic imaging results, treatment and survival time of a contemporary cohort of dogs with AML. Diagnosis was based on >20% blasts in bone marrow or blood identified as myeloid based on morphologic findings, flow cytometric immunophenotyping and cytochemical staining. Medical records of 35 dogs diagnosed with AML from 2007 to 2015 were included. Most dogs presented with inappetence (66%) and lethargy (57%) and physical examination findings of peripheral lymphadenopathy (74%) and tachypnea (62%). Common haematologic findings were quantifiable circulating blasts (85%; median blast count 35 700/μL; range: 300-276 500/μL), anaemia (median haematocrit 34%; range: 11%-52%) and thrombocytopenia (median 57 000/μL; range: 9000-252 000/μL). Bicytopenia and pancytopenia were each found in 44% of dogs. Follow-up information was available for 34 dogs. The overall median survival time from diagnosis was 19 days (range: 1-121 days). Clinical progression in some dogs was not as rapid as previously reported. Haematologic responses to various chemotherapeutics were documented in 3 dogs, with associated survival times of 62, 103 and 121 days. Dogs treated with prednisone or a combination of chemotherapy and prednisone had improved survival compared to dogs that received symptomatic care only (P < .0001). Our results show canine AML has an overlapping clinical presentation with lymphoma. The prognosis for canine AML remains extremely guarded. Further studies are needed to optimize therapeutic regimens for dogs with AML. © 2017 John Wiley & Sons Ltd.

A novel population of local pericyte precursor cells in tumor stroma that require Notch signaling for differentiation.

PubMed

Patenaude, Alexandre; Woerher, Stefan; Umlandt, Patricia; Wong, Fred; Ibrahim, Rawa; Kyle, Alastair; Unger, Sandy; Fuller, Megan; Parker, Jeremy; Minchinton, Andrew; Eaves, Connie J; Karsan, Aly

2015-09-01

Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1(+) precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146(+)/CD45(-)/CD31(-/lo) pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1(hi)/CD146(-)/CD45(-)/CD31(-). The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1(-/lo)/CD146(-)/CD45(-)/CD31(-) cells. Our results suggest that a local Sca1(hi)/CD146(-)/CD45(-)/CD31(-) pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes. Copyright © 2015 Elsevier Inc. All rights reserved.

[Impaired endometrial receptivity in primary infertility in women with undifferentiated connective tissue dysplasia and hereditary thrombophilia].

PubMed

Zanozin, A S; Demura, T A; Kolosovsky, D Yu; Faizullina, N M; Kogan, E A

The concurrence of undifferentiated connective tissue dysplasia (uCTD) and hereditary thrombophilia (HT) often accompanies female infertility, in the pathogenesis of which impaired endometrial receptivity plays an important role. to investigate endometrial morphological and immunophenotypic features in patients with primary infertility in the presence of uCTD and HT. The pipelle endometrial biopsy specimens taken in the implantation window were examined in 81 patients, including 13 women with a clinical diagnosis of uCTD, 40 with HT, 19 with uCTD concurrent with HT, and in a control group of 9 heathy surrogate mothers. Morphological, immunohistochemical, and morphometric examinations were done to study the paraffin-embedded endometrial biopsy sections stained with hematoxylin and eosin, pikrofuksin by van Gieson, and with toluidine blue. Immunohistochemical tests were carried out using primary antibodies against ER, PgR, LIF, PAI-1, VEGF, Collagen I, Collagen III, fibronectin, laminin, MMP-2, and MMP-9. The uCTD, HT, and uCTD + HT groups were found to have signs of decreased endometrial receptivity as dramatically lower counts of mature pinopodes, slower endometrial maturation, reduced expression of the receptivity marker LIF, and deviations of the stromal progesterone-estrogen index from the normal value. Sclerotic foci with type III collagen accumulation were detected in the endometrial stroma. uCTD and HT and especially their concurrence are commonly a concomitant disease and risk factors for infertility in women due to impaired endometrial receptivity. In uCTD, connective tissue remodeling processes are substantially retarded, which ultimately leads to increased processes of endometrial stromal sclerosis, reduced endometrial receptivity, and infertility. The most pronounced morphological and immunophenotypical changes have been ascertained to develop in the uCTD + NT group. The findings may be used to predict and devise new infertility treatments in patients with uCTD + NT.

T-cell chronic lymphocytic leukemia in a double yellow-headed Amazon parrot (Amazona ochrocephala oratrix).

PubMed

Osofsky, Anna; Hawkins, Michelle G; Foreman, Oded; Kent, Michael S; Vernau, William; Lowenstine, Linda J

2011-12-01

An adult, male double yellow-headed Amazon parrot (Amazona ochrocephala oratrix) was diagnosed with chronic lymphocytic leukemia based on results of a complete blood cell count and cytologic examination of a bone marrow aspirate. Treatment with oral chlorambucil was attempted, but no response was evident after 40 days. The bird was euthanatized, and the diagnosis of chronic lymphocytic leukemia was confirmed on gross and microscopic examination of tissues. Neoplastic lymphocytes were found in the bone marrow, liver, kidney, testes, and blood vessels. Based on CD3-positive immunocytochemical and immunohistochemical immunophenotyping, the chronic lymphocytic leukemia was determined to be of T-cell origin.

Promyelocytic blast crisis of chronic myelogenous leukaemia with translocations (9;22) and (15;17).

PubMed

Scolnik, M P; Palacios, M F; Acevedo, S H; Castuma, M V; Larripa, I B; Palumbo, A; Moiraghi, E B; Sasot, A M; Huberman, A B

1998-09-01

The promyelocytic blast crisis is a rare form of transformation during the evolution of chronic myeloid leukaemia (CML). We report a case of promyelocytic blast crisis with t(15;17) in addition to t(9;22). The morphology and immunophenotype of the blasts were similar to those seen in acute promyelocytic leukaemia (APL). The t(15;17) was confirmed by FISH. The patient had evidence of coagulopathy with clinical and laboratory findings of disseminated intravascular coagulation (DIC). This report highlights the importance of correlating the results of multiple diagnostic methods in order to establish a correct diagnosis of the promyelocytic blast crisis of CML.

Flow cytometric analysis of normal and neoplastic mast cells: role in diagnosis and follow-up of mast cell disease.

PubMed

Escribano, Luis; Garcia Montero, Andres C; Núñez, Rosa; Orfao, Alberto

2006-08-01

Human mast cells (MCs) are directly derived from human pluripotent CD34+ stem and progenitor hematopoietic cells with stem cell factor being a critical growth factor supporting human MC proliferation, differentiation, and survival. Because of the advantages that flow cytometry offers (it allows rapid, objective, and sensitive multiparameter analysis of high numbers of cells from a sample, with information being provided on the basis of a single cell), it has become the method of choice in the past decade for immunophenotypic identification, enumeration, and characterization of human MCs in bone marrow and other tissue specimens.

Multidimensional data analysis in immunophenotyping.

PubMed

Loken, M R

2001-05-01

The complexity of cell populations requires careful selection of reagents to detect cells of interest and distinguish them from other types. Additional reagents are frequently used to provide independent criteria for cell identification. Two or three monoclonal antibodies in combination with forward and right-angle light scatter generate a data set that is difficult to visualize because the data must be represented in four- or five-dimensional space. The separation between cell populations provided by the multiple characteristics is best visualized by multidimensional analysis using all parameters simultaneously to identify populations within the resulting hyperspace. Groups of cells are distinguished based on a combination of characteristics not apparent in any usual two-dimensional representation of the data.

[Role of cytology in hematopathological diagnostics].

PubMed

Bode, B; Tinguely, M

2012-07-01

The role of cytology has so far been underrecognized in the diagnostic process of hematopathological questions. This article presents an algorithm which allows a stepwise work-up of cytology specimens obtained by minimally invasive ultrasound-guided fine needle aspiration in patients with unexplained lymph node swelling. Moreover, it is shown how the selective separation of cytology specimens allows the application of immunophenotypic analysis including flow cytometry and immunohistochemistry as well as molecular analyses, such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) strategies. With the integrative procedure presented, cytology offers an excellent cost-effective tool for the diagnostic approach of patients with suspected hematopathological malignancies allowing a high diagnostic accuracy, ideal for initial diagnosis or follow-up.

Adult systemic Epstein-Barr virus-positive T-cell lymphoproliferative disease: A case report.

PubMed

Wang, Youping; Liu, Xinyue; Chen, Yan

2015-09-01

Systemic Epstein-Barr virus (EBV)-positive T-cell lymphoproliferative disease (EBV + T-LPD) occurs mainly in Asia and South America and is extremely rare in adults. The disease is characterized by a clonal proliferation of EBV-infected T cells with a cytotoxic immunophenotype and is associated with a poor clinical outcome and can be life-threatening. The majority of the patients have evidence of systemic disease, often with lymph node, liver and spleen involvement. The present study describes a case of adult systemic EBV + T-LPD with high fever, systemic lymphadenopathy, hepatosplenomegaly, nose-pharynx neoplasm, pancytopenia, EB virus infection and proliferative bone marrow, with the aim of improving the understanding of the condition.

[Insulitis in type 1 diabetes].

PubMed

In't Veld, P; Klöppel, G

2016-05-01

Insulitis is considered to be the key morphological lesion of type 1 diabetes mellitus (T1DM) for which the diagnostic criteria were recently defined. From the immunophenotype of the lymphocytic infiltration, its frequency and extent during the course of T1DM and the presence of autoantibodies against beta cell proteins, it has been deduced that T1DM is a chronic autoimmune disease leading to gradual destruction of the insulin-producing cells of the islets of Langerhans in the pancreas, profound insulin deficiency and chronic hyperglycemia. This review article presents the morphological findings that support this hypothesis and addresses questions that need to be answered in order to further clarify the pathogenesis and to develop specific treatment options.

Novel Confocal Microscopic and Flow Cytometric Based Assays to Visualize and Detect the (Beta)2-Adrenergic Receptor in Human Lymphocyte and Mononuclear Cell Populations

NASA Technical Reports Server (NTRS)

Salicru, A. N.; Crucian, B. E.; Nelman, M. A.; Sams, C. F.; Actor, J. K.; Marshall, G. D.

2006-01-01

The data show that immunophenotyping of leukocyte populations with (beta)2AR is possible with the commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the 2AR. The FC assay has applications for measuring alterations in total (beta)2AR in human leukocyte populations as changes in fluorescence. In addition, CM confirms that both surface and intracellular compartments stain positively for the (beta)2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.

[Solid cystic papillary tumor of the pancreas].

PubMed

Bahri, I; Njim, L; Khabir, A; Mahmoudi, H; Ghorbel, A; Zakhama, A; Jlidi, R

2001-11-01

Solid cystic papillary tumors of the pancreas are rare; they occur most commonly in young women. Despite their characteristic microscopic appearance, their immunophenotype is not specific. Their prognosis is excellent after complete surgical resection. The study aim was to report two cases in female patients who were 15 and 20 years old; the first tumor was discovered fortuitously and the second girl presented with abdominal pain and vomiting. Both tumors were encapsulated and located in the tail of the pancreas. The histological study showed the papillary architecture mixed with solid areas. Immunohistochemical staining was positive only for vimentin in one case and positive for cytokeratin, chromogranin, synaptophysin, neuron specific enolase, vimentin and protein S100 in the second case.

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.

PubMed

Ornatsky, Olga I; Kinach, Robert; Bandura, Dmitry R; Lou, Xudong; Tanner, Scott D; Baranov, Vladimir I; Nitz, Mark; Winnik, Mitchell A

2008-01-01

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.

Pathology of nodal marginal zone lymphomas.

PubMed

Pileri, Stefano; Ponzoni, Maurilio

Nodal marginal zone B cell lymphomas (NMZLs) are a rare group of lymphoid disorders part of the spectrum of marginal zone B-cell lymphomas, which encompass splenic marginal one B-cell lymphoma (SMZL) and extra nodal marginal zone of B-cell lymphoma (EMZL), often of MALT-type. Two clinicopathological forms of NMZL are recognized: adult-type and pediatric-type, respectively. NMZLs show overlapping features with other types of MZ, but distinctive features as well. In this review, we will focus on the salient distinguishing features of NMZL mostly under morphological/immunophenotypical/molecular perspectives in views of the recent acquisitions and forthcoming updated 2016 WHO classification of lymphoid malignancies. Copyright © 2016 Elsevier Ltd. All rights reserved.

MRI phenotypes with high neurodegeneration are associated with peripheral blood B-cell changes.

PubMed

Comabella, Manuel; Cantó, Ester; Nurtdinov, Ramil; Río, Jordi; Villar, Luisa M; Picón, Carmen; Castilló, Joaquín; Fissolo, Nicolás; Aymerich, Xavier; Auger, Cristina; Rovira, Alex; Montalban, Xavier

2016-01-15

Little is known about the mechanisms leading to neurodegeneration in multiple sclerosis (MS) and the role of peripheral blood cells in this neurodegenerative component. We aimed to correlate brain radiological phenotypes defined by high and low neurodegeneration with gene expression profiling of peripheral blood mononuclear cells (PBMC) from MS patients. Magnetic resonance imaging (MRI) scans from 64 patients with relapsing-remitting MS (RRMS) were classified into radiological phenotypes characterized by low (N = 27) and high (N = 37) neurodegeneration according to the number of contrast-enhancing lesions, the relative volume of non-enhancing black holes on T1-weighted images, and the brain parenchymal fraction. Gene expression profiling was determined in PBMC using microarrays, and validation of selected genes was performed by polymerase chain reaction (PCR). B-cell immunophenotyping was conducted by flow cytometry. Microarray analysis revealed the B-cell specific genes FCRL1, FCRL2, FCRL5 (Fc receptor-like 1, 2 and 5 respectively), and CD22 as the top differentially expressed genes between patients with high and low neurodegeneration. Levels for these genes were significantly down-regulated in PBMC from patients with MRI phenotypes characterized by high neurodegeneration and microarray findings were validated by PCR. In patients with high neurodegeneration, immunophenotyping showed a significant increase in the expression of the B-cell activation markers CD80 in naïve B cells (CD45+/CD19+/CD27-/IgD+), unswitched memory B cells (CD45+/CD19+/CD27+/IgD+), and switched memory B cells (CD45+/CD19+/CD27+/IgD-), and CD86 in naïve and switched memory B cells. These results suggest that RRMS patients with radiological phenotypes showing high neurodegeneration have changes in B cells characterized by down-regulation of B-cell-specific genes and increased activation status. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells*

PubMed Central

Kanderova, Veronika; Kuzilkova, Daniela; Stuchly, Jan; Vaskova, Martina; Brdicka, Tomas; Fiser, Karel; Hrusak, Ondrej; Lund-Johansen, Fridtjof

2016-01-01

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients. To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p < 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation. In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study. PMID:26785729

TCRαβ+/CD4+ Large Granular Lymphocytosis

PubMed Central

Lima, Margarida; Almeida, Julia; dos Anjos Teixeira, Maria; Alguero, Maria del Carmen; Santos, Ana Helena; Balanzategui, Ana; Queirós, Maria Luís; Bárcena, Paloma; Izarra, Antonio; Fonseca, Sónia; Bueno, Clara; Justiça, Benvindo; Gonzalez, Marcos; San Miguel, Jesús F.; Orfao, Alberto

2003-01-01

Large granular lymphocyte (LGL) leukemia is a well-recognized disease of mature T-CD8+ or less frequently natural killer cells; in contrast, monoclonal expansions of CD4+ T-LGL have only been sporadically reported in the literature. In the present article we have explored throughout a period of 56 months the incidence of monoclonal expansions of CD4+ T-LGL in a population of 2.2 million inhabitants and analyzed the immunophenotype and the pattern of cytokine production of clonal CD4+ T cells of a series of 34 consecutive cases. Like CD8+ T-LGL leukemias, CD4+ T-LGL leukemia patients have an indolent disease; however, in contrast to CD8+ T-LGL leukemias, they do not show cytopenias and autoimmune phenomena and they frequently have associated neoplasias, which is usually determining the clinical course of the disease. Monoclonal CD4+ T-LGLshowed expression of TCRαβ, variable levels of CD8 (CD8−/+dim) and a homogeneous typical cytotoxic (granzyme B+, CD56+, CD57+, CD11b+/−) and activated/memory T cell (CD2+bright, CD7−/+dim, CD11a+bright, CD28−, CD62L− HLA-DR+) immunophenotype. In addition, they exhibited a Th1 pattern of cytokine production [interferon-γ++, tumor necrosis factor-α++, interleukin (IL-2)−/+, IL-4−, IL-10−, IL-13−]. Phenotypic analysis of the TCR-Vβ repertoire revealed large monoclonal TCR-Vβ expansions; only a restricted number of TCR-Vβ families were represented in the 34 cases analyzed. These findings suggest that monoclonal TCRαβ+/CD4+/NKa+/CD8−/+dim T-LGL represent a subgroup of monoclonal LGL lymphoproliferative disorders different from both CD8+ T-LGL and natural killer cell-type LGL leukemias. Longer follow-up periods are necessary to determine the exact significance of this clonal disorder. PMID:12875995

Air Pollution and Lymphocyte Phenotype Proportions in Cord Blood

PubMed Central

Hertz-Picciotto, Irva; Herr, Caroline E.W.; Yap, Poh-Sin; Dostál, Miroslav; Shumway, Robert H.; Ashwood, Paul; Lipsett, Michael; Joad, Jesse P.; Pinkerton, Kent E.; Šrám, Radim J.

2005-01-01

Effects of air pollution on morbidity and mortality may be mediated by alterations in immune competence. In this study we examined short-term associations of air pollution exposures with lymphocyte immunophenotypes in cord blood among 1,397 deliveries in two districts of the Czech Republic. We measured fine particulate matter < 2.5 μm in diameter (PM2.5) and 12 polycyclic aromatic hydrocarbons (PAHs) in 24-hr samples collected by versatile air pollution samplers. Cord blood samples were analyzed using a FACSort flow cytometer to determine phenotypes of CD3+ T-lymphocytes and their subsets CD4+ and CD8+, CD19+ B-lymphocytes, and natural killer cells. The mothers were interviewed regarding sociodemographic and lifestyle factors, and medical records were abstracted for obstetric, labor and delivery characteristics. During the period 1994 to 1998, the mean daily ambient concentration of PM2.5 was 24.8 μg/m3 and that of PAHs was 63.5 ng/m3. In multiple linear regression models adjusted for temperature, season, and other covariates, average PAH or PM2.5 levels during the 14 days before birth were associated with decreases in T-lymphocyte phenotype fractions (i.e., CD3+ CD4+, and CD8+), and a clear increase in the B-lymphocyte (CD19+) fraction. For a 100-ng/m3 increase in PAHs, which represented approximately two standard deviations, the percentage decrease was −3.3% [95% confidence interval (CI), −5.6 to −1.0%] for CD3+, −3.1% (95% CI, −4.9 to −1.3%) for CD4+, and −1.0% (95% CI, −1.8 to −0.2%) for CD8+ cells. The corresponding increase in the CD19+ cell proportion was 1.7% (95% CI, 0.4 to 3.0%). Associations were similar but slightly weaker for PM2.5. Ambient air pollution may influence the relative distribution of lymphocyte immunophenotypes of the fetus. PMID:16203253

Evaluation of bone marrow specimens with acute myelogenous leukemia for CD34, CD15, CD117, and myeloperoxidase.

PubMed

Dunphy, C H; Polski, J M; Evans, H L; Gardner, L J

2001-08-01

Immunophenotyping of bone marrow (BM) specimens with acute myelogenous leukemia (AML) may be performed by flow cytometric (FC) or immunohistochemical (IH) techniques. Some markers (CD34, CD15, and CD117) are available for both techniques. Myeloperoxidase (MPO) analysis may be performed by enzyme cytochemical (EC) or IH techniques. To determine the reliability of these markers and MPO by these techniques, we designed a study to compare the results of analyses of these markers and MPO by FC (CD34, CD15, and CD117), EC (MPO), and IH (CD34, CD15, CD117, and MPO) techniques. Twenty-nine AMLs formed the basis of the study. These AMLs all had been immunophenotyped previously by FC analysis; 27 also had had EC analysis performed. Of the AMLs, 29 had BM core biopsies and 26 had BM clots that could be evaluated. The paraffin blocks of the 29 BM core biopsies and 26 BM clots were stained for CD34, CD117, MPO, and CD15. These results were compared with results by FC analysis (CD34, CD15, and CD117) and EC analysis (MPO). Immunodetection of CD34 expression in AML had a similar sensitivity by FC and IH techniques. Immunodetection of CD15 and CD117 had a higher sensitivity by FC analysis than by IH analysis. Detection of MPO by IH analysis was more sensitive than by EC analysis. There was no correlation of French-American-British (FAB) subtype of AML with CD34 or CD117 expression. Expression of CD15 was associated with AMLs with a monocytic component. Myeloperoxidase reactivity by IH analysis was observed in AMLs originally FAB subtyped as M0. CD34 can be equally detected by FC and IH techniques. CD15 and CD117 are better detected by FC analysis and MPO is better detected by IH analysis.

[Clinicopathological study of non-Langerhans cell histiocytosis in central nervous system].

PubMed

Zhang, Tingting; Fu, Yongjuan; Lu, Dehong; Li, Cunjiang; Piao, Yueshan

2015-09-01

To explore the clinicopathological features and imaging characteristics of non-Langerhans cell histiocytosis in central nerve system, thus to facilitate the diagnosis and differential diagnosis. A total of ten cases were enrolled in the study, with seven cases of Rosai-Dorfman disease (RDD) and three cases of xanthoma disseminatum (XD). Data on the clinicopathological features, imaging, immunophenotype and prognosis were collected and analyzed. Seven patients with RDD, 5 males and 2 females with the mean age of 46.7 years old, all presented as dural-based or intraparenchymal hypo- to isointense lesions on T1 and T2 with post-contrast enhancement. The polymorphous admixture of histiocytes, lymphocytes and plasma cells was observed in a fibrous stroma, with emperipolesis of some histiocytes. The immunohistostaining of CD11c, CD68, MAC387 and S-100 was positive in the histiocytes, while the staining of CD1α was negative. Five patients recovered after the operation, while one patient died of the disease. All the 3 XD patients were female, with the median age of 20.7 years old. All XD patients presented as multiple intraparenchymal hypointense lesions on T1 and hyperintense lesions on T2 with post-contrast enhancement. The infiltration of foam-like histiocytes, a few Touton giant cells, lymphocytes and eosnophils was observed in all XD patients. The immunohistostaining of CD68 and CD11c was positive in the histiocytes and that of MAC387 partly positive, while the staining of S-100 and CD1α was negative. One XD patient survived well, while another one died of the disease. The diagnosis of RDD and XD should be based on their typical morphology and immunophenotype and should be differentiated from Langerhans cell histiocytosis and other non-Langerhans cell histiocytosis. Non-Langerhans cell histiocytosis in central nerve system often presents untypical clinical presentation and imaging features, thus the communication and cooperation between clinician and pathologist is needed.

A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation.

PubMed

Yagita, M; Huang, C L; Umehara, H; Matsuo, Y; Tabata, R; Miyake, M; Konaka, Y; Takatsuki, K

2000-05-01

We present the establishment of a natural killer (NK) leukemia cell line, designated KHYG-1, from the blood of a patient with aggressive NK leukemia, which both possessed the same p53 point mutation. The immunophenotype of the primary leukemia cells was CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16+, CD56+, CD57+ and HLA-DR+. A new cell line (KHYG-1) was established by culturing peripheral leukemia cells with 100 units of recombinant interleukin (IL)-2. The KHYG-1 cells showed LGL morphology with a large nucleus, coarse chromatin, conspicuous nucleoli, and abundant basophilic cytoplasm with many azurophilic granules. The immunophenotype of KHYG-1 cells was CD1-, CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16-, CD25-, CD33+, CD34-, CD56+, CD57-, CD122+, CD132+, and TdT-. Southern blot analysis of these cells revealed a normal germline configuration for the beta, delta, and gamma chains of the T cell receptor and the immunoglobulin heavy-chain genes. Moreover, the KHYG-1 cells displayed NK cell activity and IL-2-dependent proliferation in vitro, suggesting that they are of NK cell origin. Epstein-Barr virus (EBV) DNA was not detected in KHYG-1 cells by Southern blot analysis with a terminal repeat probe from an EBV genome. A point mutation in exon 7 of the p53 gene was detected in the KHYG-1 cells by PCR/SSCP analysis, and direct sequencing revealed the conversion of C to T at nucleotide 877 in codon 248. The primary leukemia cells also carried the same point mutation. Although the precise role of the p53 point mutation in leukemogenesis remains to be clarified, the establishment of an NK leukemia cell line with a p53 point mutation could be valuable in the study of leukemogenesis.

Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats

PubMed Central

Wang, Sa A.; Pozdnyakova, Olga; Jorgensen, Jeffrey L.; Medeiros, L. Jeffrey; Stachurski, Dariusz; Anderson, Mary; Raza, Azra; Woda, Bruce A.

2009-01-01

Background The presence of paroxysmal nocturnal hemoglobinuria clones in the setting of aplastic anemia or myelodysplastic syndrome has been shown to have prognostic and therapeutic implications. However, the status of paroxysmal nocturnal hemoglobinuria clones in various categories of myelodysplastic syndrome and in other bone marrow disorders is not well-studied. Design and Methods By using multiparameter flow cytometry immunophenotypic analysis with antibodies specific for four glycosylphosphatidylinositol-anchored proteins (CD55, CD59, CD16, CD66b) and performing an aerolysin lysis confirmatory test in representative cases, we assessed the paroxysmal nocturnal hemoglobinuria-phenotype granulocytes in 110 patients with myelodysplastic syndrome, 15 with myelodysplastic/myeloproliferative disease, 5 with idiopathic myelofibrosis and 6 with acute myeloid leukemia. Results Paroxysmal nocturnal hemoglobinuria-phenotype granulocytes were detected in nine patients with low grade myelodysplastic syndrome who showed clinicopathological features of bone marrow failure, similar to aplastic anemia. All paroxysmal nocturnal hemoglobinuria-positive cases demonstrated loss of the four glycosylphosphatidylinositol-anchored proteins, with CD16−CD66b− clones being larger than those of CD55−CD59− (p<0.05). Altered glycosylphosphatidylinositol-anchored protein expression secondary to granulocytic hypogranulation, immaturity, and/or immunophenotypic abnormalities was present in a substantial number of cases and diagnostically challenging. Conclusions These results show that routine screening for paroxysmal nocturnal hemoglobinuria clones in patients with an intrinsic bone marrow disease who show no clinical evidence of hemolysis has an appreciable yield in patients with low grade myelodysplastic syndromes. The recognition of diagnostic caveats and pitfalls associated with the underlying intrinsic bone marrow disease is essential in interpreting paroxysmal nocturnal hemoglobinuria testing correctly. In our experience, the CD16/CD66b antibody combination is superior to CD55/CD59 in screening for subclinical paroxysmal nocturnal hemoglobinuria because it detects a large clone size and is less subject to analytical interference. PMID:19001281

Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats.

PubMed

Wang, Sa A; Pozdnyakova, Olga; Jorgensen, Jeffrey L; Medeiros, L Jeffrey; Stachurski, Dariusz; Anderson, Mary; Raza, Azra; Woda, Bruce A

2009-01-01

The presence of paroxysmal nocturnal hemoglobinuria clones in the setting of aplastic anemia or myelodysplastic syndrome has been shown to have prognostic and therapeutic implications. However, the status of paroxysmal nocturnal hemoglobinuria clones in various categories of myelodysplastic syndrome and in other bone marrow disorders is not well-studied. By using multiparameter flow cytometry immunophenotypic analysis with antibodies specific for four glycosylphosphatidylinositol-anchored proteins (CD55, CD59, CD16, CD66b) and performing an aerolysin lysis confirmatory test in representative cases, we assessed the paroxysmal nocturnal hemoglobinuria-phenotype granulocytes in 110 patients with myelodysplastic syndrome, 15 with myelodysplastic/myeloproliferative disease, 5 with idiopathic myelofibrosis and 6 with acute myeloid leukemia. Paroxysmal nocturnal hemoglobinuria-phenotype granulocytes were detected in nine patients with low grade myelodysplastic syndrome who showed clinicopathological features of bone marrow failure, similar to aplastic anemia. All paroxysmal nocturnal hemoglobinuria-positive cases demonstrated loss of the four glycosylphosphatidylinositol-anchored proteins, with CD16(-)CD66b(-) clones being larger than those of CD55(-)CD59(-) (p<0.05). Altered glycosylphosphatidylinositol-anchored protein expression secondary to granulocytic hypogranulation, immaturity, and/or immunophenotypic abnormalities was present in a substantial number of cases and diagnostically challenging. These results show that routine screening for paroxysmal nocturnal hemoglobinuria clones in patients with an intrinsic bone marrow disease who show no clinical evidence of hemolysis has an appreciable yield in patients with low grade myelodysplastic syndromes. The recognition of diagnostic caveats and pitfalls associated with the underlying intrinsic bone marrow disease is essential in interpreting paroxysmal nocturnal hemoglobinuria testing correctly. In our experience, the CD16/CD66b antibody combination is superior to CD55/CD59 in screening for subclinical paroxysmal nocturnal hemoglobinuria because it detects a large clone size and is less subject to analytical interference.

Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour, both term and preterm.

PubMed

Zhang, Jianhong; Shynlova, Oksana; Sabra, Sally; Bang, Annie; Briollais, Laurent; Lye, Stephen J

2017-10-01

The onset of labour in rodents and in humans is associated with physiological inflammation which is manifested by infiltration of activated maternal peripheral leukocytes (mPLs) into uterine tissues. Here, we used flow cytometry to immunophenotype mPLs throughout gestation and labour, both term and preterm. Peripheral blood was collected from non-pregnant women and pregnant women in the 1st, 2nd and 3rd trimesters. Samples were also collected from women in active labour at term (TL) or preterm (PTL) and compared with women term not-in-labour (TNIL) and preterm not-in-labour (PTNIL). Different leukocyte populations were identified by surface markers such as CD45, CD14, CD15, CD3, CD4, CD8, CD19 and CD56. Their activation status was measured by the expression levels of CD11b, CD44, CD55, CD181 and CD192 proteins. Of all circulating CD45+ leukocytes, we detected significant increases in CD15+ granulocytes (i) in pregnant women versus non-pregnant; (ii) in TL women versus TNIL and versus pregnant women in the 1st/2nd/3rd trimester; (iii) in PTL women versus PTNIL. TL was characterized by (iv) increased expressions of CD11b, CD55 and CD192 on granulocytes; (v) increased mean fluorescent intensity (MFI) of CD55 and CD192 on monocytes; (vi) increased CD44 MFI on CD3+ lymphocytes as compared to late gestation. In summary, we have identified sub-populations of mPLs that are specifically activated in association with gestation (granulocytes) or with the onset of labour (granulocytes, monocytes and lymphocytes). Additionally, beta regression analysis created a set of reference values to rank this association between immune markers of pregnancy and to identify activation status with potential prognostic and diagnostic capability. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Germ cell tumour growth patterns originating from clear cell carcinomas of the ovary and endometrium: a comparative immunohistochemical study favouring their origin from somatic stem cells.

PubMed

Nogales, Francisco F; Prat, Jaime; Schuldt, Maolly; Cruz-Viruel, Nelly; Kaur, Baljeet; D'Angelo, Emanuela; Matias-Guiu, Xavier; Vidal, August; McCluggage, W Glenn; Oosterhuis, J Wolter

2018-03-01

To report a series of 11 ovarian and one endometrial neoplasm in elderly patients with mixed clear cell tumour and germ cell tumour (GCT) components, to compare their immunohistochemical profiles and demonstrate a putative stem cell population. The clear cell tumours included 11 clear cell carcinomas (CCC) and one borderline clear cell tumour, while the GCT always included glandular yolk sac tumour (YST). In four cases, there were also foci of teratoma with immature neuroepithelial and endodermal tissues and undifferentiated areas showing true embryoids. To distinguish between the clear cell and YST components, the following antibodies were used: HNF1-β, napsin-A, cytokeratin 7 (CK7), PAX8, EMA, AFP, SALL4, villin, glypican-3 (GPC-3), GATA3, HepPar-1, OCT4, CDX2, CD30 and SOX2. HNF1-β, CK7, EMA and GPC-3 were often expressed in both components. Other markers had higher specificity for each cellular lineage; napsin-A and PAX8 were expressed only in CCC, while SALL4, villin, AFP and HepPar-1 were positive in the glandular YST component but negative in the clear cell component. OCT4 expression occurred in six of 10 cases and consistently in teratoma (four of four). There is considerable immunophenotypical overlap between the two components in these mixed neoplasms, and a panel of markers should be used to facilitate the distinction. We propose that OCT4-expressing somatic cancer cells differentiate into GCT and represent spontaneously induced pluripotent stem cells, possibly conditioned by age-related epigenetic factors. These neoplasms have features of prepubertal type GCT showing lack of 12p gain, preponderance of YST and coexistence with immature neuroectoderm. However, there may also be undifferentiated stem cell areas with embryoid bodies, of the type seen in postpubertal testicular GCT, but lacking a complete embryonal carcinoma immunophenotype. © 2017 John Wiley & Sons Ltd.

Aberrant lymphoid antigen expression in acute myeloid leukemia in Saudi Arabia.

PubMed

El-Sissy, Azza H; El-Mashari, May A; Bassuni, Wafaa Y; El-Swaayed, Aziza F

2006-09-01

Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification and is considered particularly useful for identifying aberrant lineage association of acute leukemia, biphenotypic and bilineal acute leukemia, as well as monitoring minimal residual disease. Some immunophenotypes correlate with cytogenetic abnormalities and prognosis. Is to determine aberrant lymphoid antigen expression in Saudi acute myeloid leukemia (AML), correlate them with FAB subtypes, evaluate early surface markers CD7 and CD56, and to investigate the role of cytoplasmic CD79a (a B cell marker that is assigned a high score of 2.0 in the WHO classification). Thirty four newly diagnosed AML cases were included in this study, 47% showed aberrant lymphoid antigen expression. CD9 was the most frequently expressed lymphoid antigen (29.4%) followed by CD7 & CD19 (11.8%), CD4 (8.8%) and CD22 (2.9%). CD9 was expressed in 3/6 (50%) of M3 cases, CD7 was expressed in 11.8% and was mostly confined to FAB M1 and M2 and associated with immature antigens CD34, HLA-DR and TdT. CD56 was expressed in 7/34 (20.6%) cases, three of these cases (42.9%) belonged to the monocytic group. CD56 was also detected in 2 cases with 11q23 rearrangement. CD56 was expressed in 2/7 (28.6%) M2 cases, and was associated with t (8;21) (q22;q22) together with CD19. Co-expression of CD56 and CD7 was detected in 2.9% of the cases. CD79a was expressed in one case together with CD19, diagnosed as acute biphenotypic leukemia, and was associated with t(8;21) (q22;q22). Minimal residual disease in AML is very difficult to trace, detection of aberrant expression of lymphoid antigens will make it easier. The high score given to CD79a by EGIL is questionable based on cytogenetic classification.

Acute lymphoblastic leukemia in children and adolescents: prognostic factors and analysis of survival

PubMed Central

Lustosa de Sousa, Daniel Willian; de Almeida Ferreira, Francisco Valdeci; Cavalcante Félix, Francisco Helder; de Oliveira Lopes, Marcos Vinicios

2015-01-01

Objective To describe the clinical and laboratory features of children and adolescents with acute lymphoblastic leukemia treated at three referral centers in Ceará and evaluate prognostic factors for survival, including age, gender, presenting white blood cell count, immunophenotype, DNA index and early response to treatment. Methods Seventy-six under 19-year-old patients with newly diagnosed acute lymphoblastic leukemia treated with the Grupo Brasileiro de Tratamento de Leucemia da Infância – acute lymphoblastic leukemia-93 and -99 protocols between September 2007 and December 2009 were analyzed. The diagnosis was based on cytological, immunophenotypic and cytogenetic criteria. Associations between variables, prognostic factors and response to treatment were analyzed using the chi-square test and Fisher's exact test. Overall and event-free survival were estimated by Kaplan–Meier analysis and compared using the log-rank test. A Cox proportional hazards model was used to identify independent prognostic factors. Results The average age at diagnosis was 6.3 ± 0.5 years and males were predominant (65%). The most frequently observed clinical features were hepatomegaly, splenomegaly and lymphadenopathy. Central nervous system involvement and mediastinal enlargement occurred in 6.6% and 11.8%, respectively. B-acute lymphoblastic leukemia was more common (89.5%) than T-acute lymphoblastic leukemia. A DNA index >1.16 was found in 19% of patients and was associated with favorable prognosis. On Day 8 of induction therapy, 95% of the patients had lymphoblast counts <1000/μL and white blood cell counts <5.0 × 109/L. The remission induction rate was 95%, the induction mortality rate was 2.6% and overall survival was 72%. Conclusion The prognostic factors identified are compatible with the literature. The 5-year overall and event-free survival rates were lower than those reported for developed countries. As shown by the multivariate analysis, age and baseline white blood cell count were independent prognostic factors. PMID:26190424

Clinical, immunophenotypic, and molecular characteristics of well-differentiated systemic mastocytosis.

PubMed

Álvarez-Twose, Iván; Jara-Acevedo, María; Morgado, José Mário; García-Montero, Andrés; Sánchez-Muñoz, Laura; Teodósio, Cristina; Matito, Almudena; Mayado, Andrea; Caldas, Carolina; Mollejo, Manuela; Orfao, Alberto; Escribano, Luis

2016-01-01

Well-differentiated systemic mastocytosis (WDSM) is a rare variant of systemic mastocytosis (SM) characterized by bone marrow (BM) infiltration by mature-appearing mast cells (MCs) often lacking exon 17 KIT mutations. Because of its rarity, the clinical and biological features of WDSM remain poorly defined. We sought to determine the clinical, biological, and molecular features of a cohort of 33 patients with mastocytosis in the skin in association with BM infiltration by well-differentiated MCs and to establish potential diagnostic criteria for WDSM. Thirty-three patients with mastocytosis in the skin plus BM aggregates of round, fully granulated MCs lacking strong CD25 and CD2 expression in association with clonal MC features were studied. Our cohort of patients showed female predominance (female/male ratio, 4:1) and childhood onset of the disease (91%) with frequent familial aggregation (39%). Skin involvement was heterogeneous, including maculopapular (82%), nodular (6%), and diffuse cutaneous (12%) mastocytosis. KIT mutations were detected in only 10 (30%) of 33 patients, including the KIT D816V (n = 5), K509I (n = 3), N819Y (n = 1), and I817V (n = 1) mutations. BM MCs displayed a unique immunophenotypic pattern consisting of increased light scatter features, overexpression of cytoplasmic carboxypeptidase, and aberrant expression of CD30, together with absent (79%) or low (21%) positivity for CD25, CD2, or both. Despite only 9 (27%) of 33 patients fulfilling the World Health Organization criteria for SM, our findings allowed us to establish the systemic nature of the disease, which fit with the definition of WDSM. WDSM represents a rare clinically and molecularly heterogeneous variant of SM that requires unique diagnostic criteria to avoid a misdiagnosis of cutaneous mastocytosis per current World Health Organization criteria. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Enteropathy-associated T-cell lymphoma--a clinicopathologic and array comparative genomic hybridization study.

PubMed

Ko, Young Hyeh; Karnan, Sivasundaram; Kim, Kyeong Mee; Park, Cheol Keun; Kang, Eun Suk; Kim, Young Ho; Kang, Won Ki; Kim, Seok Jin; Kim, Won Seog; Lee, Woo Yong; Chun, Ho Kyung; Seto, Masao

2010-09-01

According to the new World Health Organization classification system, there are 2 types of enteropathy-associated T-cell lymphoma. Type 1 is associated with celiac disease and accounts for the majority of cases in Western countries, whereas type 2 is not associated with celiac disease. To characterize enteropathy-associated T-cell lymphoma types in Korea, we carried out clinicopathologic and immunophenotypic analyses of 8 Koreans with enteropathy-associated T-cell lymphoma and investigated genomic profile using array comparative genomic hybridization. The tumors involved the small intestine in 5 patients and the colorectum in 3 patients. Two patients carried an HLA DQB10302 allele that corresponds to HLA DQ8. None of the patients had gluten-sensitive malabsorption syndrome. Intraepithelial lymphocytosis was observed in all patients. The sizes of the tumor cells were small or small-to-medium in 7 cases and medium-to-large in 1 case. The immunophenotypes of the tumor cells were CD4-CD8+CD56+ in 4 cases, CD4-CD8+CD56- in 1 case, CD4-CD8-CD56+ in 2 cases, and CD4-CD8-CD56- in 1 case. Array comparative genomic hybridization analysis showed that chromosome 9q33-q34.1 gain was present in 4 (80%) of the 5 cases examined. Other recurrent genomic alterations were gain of 6p21.1-21.31 (3/5, 60%), gain of 19q (2/5), and the loss of 3p12.1-p12.2 (2/5) and 3q26.31 (2/5). These results suggest that the most prevalent type of enteropathy-associated T-cell lymphoma in this geographic region is type 2, and the genetic changes associated with it are similar to those in Western countries. Copyright 2010 Elsevier Inc. All rights reserved.

Different immunophenotypical apoptotic profiles characterise megakaryocytes of essential thrombocythaemia and primary myelofibrosis.

PubMed

Florena, A M; Tripodo, C; Di Bernardo, A; Iannitto, E; Guarnotta, C; Porcasi, R; Ingrao, S; Abbadessa, V; Franco, V

2009-04-01

Essential thrombocythaemia (ET) and primary myelofibrosis (PMF) share some clinical and pathological features, but show different biological behaviour and prognosis. The latest contributions to understanding the nature of these disorders have focused on bone marrow microenvironment remodelling and proliferative stress, recognising megakaryocytes (MKCs) as "key-cells". The aim of this study was to investigate the apoptotic profile of ET and PMF MKCs in order to further characterise the biology of these disorders. Bone marrow biopsy samples from 30 patients with ET, and 30 patients with PMF, were immunophenotypically studied for the expression of pro-apoptotic (Fas, Fas-L, Bax, Bad) and anti-apoptotic (Bcl-2, Bcl-XL, hTERT (human telomerase reverse transcriptase)) molecules and the "executioner" molecule caspase-3. The fraction of MKCs undergoing apoptosis was assessed by deoxynucleotidyl transferase-mediated dUTP nick-end labelling. Only the mitochondrial pathway seemed to be involved in MKC apoptosis. The anti-apoptotic molecule Bcl-XL was predominantly found in ET MKCs (50.5% of ET MKCs versus 35% of PMF MKCs; p = 0.036), while pro-apoptotic molecules Bax and Bad showed a prevalent expression in PMF MKCs (30.5% of ET MKCs versus 55% of PMF MKCs; 41% of ET MKCs versus 52% of PMF MKCs; p = 0.001 and p = 0.068, respectively). A significant fraction of PMF MKCs were committed to apoptosis according to caspase-3 expression and TUNEL, while only few ET cells were committed to apoptosis. hTERT was significantly more expressed in PMF (32% of ET MKCs versus 46% of PMF MKCs; p = 0.022), in agreement with the proliferative nature of this disease. It was found that ET and PMF MKCs, which barely differ in terms of morphology and aggregation, are characterised by markedly different apoptotic profiles. The rather high apoptotic fraction of PMF was able to support the fibrotic nature of this process, while the anti-apoptotic profile of ET cells fits well with their "steady" maturative state.

Mixed and Ambiguous Endometrial Carcinomas: A Heterogenous Group of Tumors With Different Clinicopathologic and Molecular Genetic Features.

PubMed

Espinosa, Iñigo; D'Angelo, Emanuela; Palacios, José; Prat, Jaime

2016-07-01

Besides endometrioid, serous, and clear cell carcinomas, there are endometrial carcinomas exhibiting mixed and ambiguous morphologic features. We have analyzed the immunophenotype (p53, p16, β-catenin, ER, HNF-1B, MLH1, and Ki-67) and mutational status (PTEN, KRAS, PIK3CA, and POLE) of 7 mixed carcinomas and 13 ambiguous carcinomas, all of them classified initially as mixed carcinomas. Only 2 of the 7 (28%) mixed carcinomas showed different immunophenotypes in different components. All but 2 tumors (5/7, 71%) overexpressed p53 and p16 and were negative for ER. Both carcinomas (2/7, 28%) showed a prominent micropapillary component that resembled an ovarian low-grade serous carcinoma and merged with villoglandular endometrioid carcinoma. The ambiguous carcinomas exhibited glandular architecture, high nuclear grade, and overlapping features of endometrioid and serous carcinomas. All tumors overexpressed p53 and p16, and the majority of cases (12/13, 92%) were negative for ER. KRAS mutations were identified in 3 of 7 (42%) mixed carcinomas, including the 2 cases with a "low-grade" serous-like component. PIK3CA mutations occurred in 2 (2/13, 15%) ambiguous carcinomas and PTEN mutations in 1 (1/7, 14%) mixed and 1 (1/13, 8%) ambiguous carcinoma. POLE exonuclease domain mutations were encountered in a case of mixed undifferentiated and well-differentiated (dedifferentiated) carcinoma. Two of the 7 (29%) mixed endometrial carcinomas and 5 of the 13 (38%) ambiguous carcinomas had extended beyond the pelvis (stages III and IV). Two of the 7 (29%) patients with mixed endometrial carcinoma and 6 of 12 (50%) patients with ambiguous endometrial carcinoma were alive with disease or had died of tumor. Our results show that, biologically, many so-called mixed carcinomas represent serous carcinomas with ambiguous morphology. Our series include 2 true mixed endometrial carcinomas with a "low-grade serous"-like component, microcystic, elongated, or fragmented features, KRAS mutations, and aggressive behavior.

Endocervical Adenocarcinoma With Morphologic Features of Both Usual and Gastric Types: Clinicopathologic and Immunohistochemical Analyses and High-risk HPV Detection by In Situ Hybridization.

PubMed

Wada, Tomoko; Ohishi, Yoshihiro; Kaku, Tsunehisa; Aman, Murasaki; Imamura, Hiroko; Yasutake, Nobuko; Sonoda, Kenzo; Kato, Kiyoko; Oda, Yoshinao

2017-05-01

The fourth edition of the World Health Organization classification set up new entities of endocervical adenocarcinoma (ECA), namely the "usual type" and "gastric type." These 2 types are considered to be distinct histogenetically because of their differing immunophenotypes, human papillomavirus (HPV) status, and prognoses. Usual-type ECAs (U-ECAs) are virtually always associated with high-risk human papillomavirus (HR-HPV) infection. Gastric-type ECAs (G-ECAs) are believed not to be associated with HR-HPV infection. Morphologically, U-ECA cells are characterized by mucin-poor and eosinophilic cytoplasm, resembling endometrioid carcinoma (a pseudoendometrioid feature). G-ECA cells are characterized by abundant clear or pale, mucinous cytoplasm and distinct cell borders. However, in routine practice we noticed that some ECAs contain morphologically usual type-like components and gastric type-like components in a single tumor; we have named these "G+U" ECAs. The histogenesis of such tumors has not been investigated. We conducted the present study to clarify the clinicopathologic and immunohistochemical features and HPV status of G+U ECAs, and to determine whether G+U ECAs are genuine G-ECAs mimicking U-ECAs or genuine U-ECAs with gastric type-like morphology. We retrospectively analyzed a series of 70 consecutive cases of ECA diagnosed as mucinous ECA, endocervical type, and we reclassified them on the basis of the latest World Health Organization classification. We identified 48 (69%) pure U-ECAs, 9 pure G-ECAs, and 13 G+U ECAs. Ten of the 13 G+U ECAs (77%) showed no HR-HPV infection by in situ hybridization (HPV-unrelated G+U ECAs) and showed frequent HIK1083 expression and aberrant p53 expression in both usual type-like and gastric type-like components. The other 3 G+U ECAs showed HR-HPV infection (HPV-related G+U EACs) and frequent p16+/p53-/HIK1083- immunophenotype in both usual type-like and gastric type-like components. The U-ECAs were characterized by HR-HPV infection detected by in situ hybridization and frequent p16+/p53-/HIK1083- immunophenotype, similar to that of the HPV-related G+U ECAs. In contrast, the pure G-ECAs were characterized by the absence of HPV infection and frequent HIK1083 expression and aberrant p53 expression, similar to that of HPV-unrelated G+U ECAs. G+U ECAs thus represent a heterogenous group composed of genuine G-ECAs and genuine U-ECAs. Most of the G+U ECAs we examined were genuine HPV-unrelated G-ECAs with usual type-like components showing mucin-poor, eosinophilic cytoplasm (pseudoendometrioid morphology). A small population of G+U ECAs was genuine HPV-related U-ECAs with gastric type-like components showing mucin-rich, voluminous cytoplasm. Thus, both types of ECAs can occasionally display patterns of differentiation suggesting a component of the other type but true mixed tumors do not appear to exist. Ancillary techniques (immunohistochemical analysis of p16, p53, and HPV DNA detection assays) should be used to assure proper classification of tumors with mixed morphologic features.

[Systemic lupus erythematosus and anaemia].

PubMed

Falcão, S; Barros, R; Mateus, M; Nero, P; de Matos, A Alves; Pimentão, J Bravo; Ribeiro, I; Weigert, A; Branco, J C

2007-01-01

The authors report the case of a 48-years-old Caucasian women, with a previous diagnosis of systemic lupus erythematosus characterized by asthenia, fever, skin rash, alopecia, Raynaud's phenomenon, arthritis, pericardial effusion, interstitial pulmonary involvement, diffuse proliferative glomerulonephritis with crescents and anemia. The presence of severe anemia refractory to high doses of glucocorticoids (1 mg/ /Kg/day), iron therapy and blood transfusions, associated with a low reticulocyte count determined the execution of a bone marrow aspiration, biopsy and immunophenotyping, which were compatible with the diagnosis of Myelodysplastic Syndrome. The treatment with erythropoietin (5.000U 3x/week) and cyclophosphamide pulses (1 gr/m(2) month) induced complete regression of morphologic bone marrow changes and anemia. The main causes of anemia in lupus patients are discussed.

Disseminated Nonleukemic Myeloid Sarcoma of the Spleen With Involvement of the Liver in an Infant.

PubMed

Rao, Yueli; Wu, Yuanyuan; Dong, Ao; Zhu, Kun; Li, Wei; Cai, Shenyang; Yang, Min; Yan, Jie

2017-05-01

Nonleukemic myeloid sarcoma (MS) is a rare tumor that can occur in several locations without myeloid leukemia. We reported a first case of nonleukemic MS of the spleen involving the liver in a 5-month-old boy presenting with hematochezia, petechial hemorrhage, fever, and hepatosplenomegaly. Bone marrow trephine biopsy and immunophenotypic flow cytometry revealed no evidence of myeloid leukemia. The patient underwent liver biopsy and splenectomy. Clinicopathology and immunohistochemistry suggested a disseminated nonleukemic MS. The patient died of respiratory failure on the seventh postoperative day. Early diagnosis of a disseminated nonleukemic MS may be quite important for patient survival and it should be considered one of the differential diagnoses of hepatosplenomegaly with atypical clinical features.

Acute B cell lymphoblastic leukaemia in a 12-week-old greyhound.

PubMed

Adams, J; Mellanby, R J; Villiers, E; Baines, S; Woodger, N

2004-11-01

A 12-week-old greyhound had a two-day history of lethargy, inappetence and shifting lameness. Clinical examination revealed pyrexia and hepatosplenomegaly. Haematological examination showed anaemia, thrombocytopenla, neutropenla and large numbers of atypical mononuclear leucocytes. A diagnosis of acute B cell lymphoblastic leukaemia was made following flow cytometric immunophenotyping of the leucocytes. The owner declined further evaluation and the dog was treated symptomatically with antibiotics. After a brief improvement, the dog's condition deteriorated and it was euthanased four days after initial presentation. The case was unusual because acute lymphoid leukaemia in the dog is most frequently reported in mature animals. This is in contrast to humans, where acute leukaemia is one of the most common childhood cancers.

Oral plasmablastic lymphoma as the first manifestation of AIDS*

PubMed Central

do Vale, Daniela Assis; Rogado, Carolina Martelli; de Carvalho, Danielle Lima Corrêa; Trierveiler, Marilia; Ortega, Karem López

2017-01-01

Plasmablastic lymphoma is a non-Hodgkin lymphoma characterized by its plasmacytic differentiation and predilection for the oral cavity. It is among the lymphomas most commonly associated with AIDS. This report details a case of a HIV-positive patient with a 1-month history of an exophytic mass in the gingival area of the upper left quadrant. The diagnosis of plasmablastic lymphoma was made based on its histopathological and immunophenotypical features. She was treated with chemotherapy followed by autologous hematopoietic stem cell transplantation. Despite complete resolution of the lesion, the patient died of cardiorespiratory arrest. This case illustrates plasmablastic lymphoma as the first clinical manifestation of AIDS, highlighting the importance of differentiating between a potentially malignant lesion and other pathologic processes. PMID:29267464

Primary breast leiomyosarcoma and synchronous homolateral lung cancer: a case report

PubMed Central

Meroni, Stefano; Voulaz, Emanuele; Alloisio, Marco; De Sanctis, Rita; Bossi, Paola; Cariboni, Umberto; De Simone, Matilde; Cioffi, Ugo

2017-01-01

Radiological and histological features of breast leiomyosarcoma can mimic a wide variety of other breast lesions, such as mesenchymal tumors, breast lymphomas, poorly differentiated carcinomas and metaplastic breast carcinomas. The authors present the case of a 62-year-old woman with a primary breast leiomyosarcoma with synchronous ipsilateral lung adenocarcinoma. The latter was an incidental finding during pre-surgical staging examinations. Clinicopathological, immunophenotypic and imaging features cancer are described. A brief review of the literature on imaging findings and management of breast leiomyosarcoma is presented. The authors discuss the differential diagnoses in breast imaging and of the extra-mammary incidental findings. Surgical resection remains the cornerstone of treatment, while radiation therapy and chemotherapy remain to be defined on a single-patient basis. PMID:29312765

Pure Erythroleukemia (Variant Acute Myeloid Leukemia-vAML-M6) with Deletion of Chromosome 20, Mainly Presenting as Late Erythroblasts, a Unique Case Report with Review of Literature.

PubMed

Rasool, Javid; Geelani, Sajad; Khursheed; Yasir; Lone, Mohd Suhail; Shaban, Mohd

2014-03-01

Acute erythroleukemia is characterized by a predominant immature erythroid population and accounts for approximately 2-5 % of all cases of acute leukemia. Two subtypes are recognized based on the presence or absence of a significant myeloid component: erythroleukemia and pure erythroid leukemia. Erythroleukemia is predominantly a disease of adults, while pure erythroid leukemia can be seen in any age including children. Here is a case of pure erythroleukemia presenting mainly as late erythroblasts which was diagnosed on bone marrow examination, cytochemistry and was confirmed on immunophenotyping. Possibly this is the only case so for demonstrating deletion of long arm of chromosome 20 in pure erythroleukemia.

Anaphylaxis as a clinical manifestation of clonal mast cell disorders.

PubMed

Matito, A; Alvarez-Twose, I; Morgado, J M; Sánchez-Muñoz, L; Orfao, A; Escribano, L

2014-08-01

Clonal mast cell disorders comprise a heterogeneous group of disorders characterized by the presence of gain of function KIT mutations and a constitutively altered activation-associated mast cell immunophenotype frequently associated with clinical manifestations related to the release of mast cells mediators. These disorders do not always fulfil the World Health Organization (WHO)-proposed criteria for mastocytosis, particularly when low-sensitive diagnostic approaches are performed. Anaphylaxis is a frequent presentation of clonal mast cell disorders, particularly in mastocytosis patients without typical skin lesions. The presence of cardiovascular symptoms, e.g., hypotension, occurring after a hymenoptera sting or spontaneously in the absence of cutaneous manifestations such as urticaria is characteristic and differs from the presentation of anaphylaxis in the general population without mastocytosis.

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry

PubMed Central

Ornatsky, Olga I.; Kinach, Robert; Bandura, Dmitry R.; Lou, Xudong; Tanner, Scott D.; Baranov, Vladimir I.; Nitz, Mark; Winnik, Mitchell A.

2008-01-01

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping. PMID:19122859

Extramedullary (EMP) relapse in unusual locations in multiple myeloma: Is there an association with precedent thalidomide administration and a correlation of special biological features with treatment and outcome?

PubMed

Katodritou, E; Gastari, V; Verrou, E; Hadjiaggelidou, C; Varthaliti, M; Georgiadou, S; Laschos, K; Xirou, P; Yiannaki, E; Constantinou, N; Markala, D; Zervas, K

2009-08-01

Extramedullary relapse constitutes an uncommon manifestation of multiple myeloma (MM), characterized by highly malignant histology, special biological features, resistance to treatment and poor outcome. Its incidence has been increased during the last years, probably due to the introduction of novel strategies in the management of MM, including intensified treatment and immunomodulatory drugs. Here we report nine cases of extramedullary relapse of MM, presented in unusual locations, seven of which had previously been treated with thalidomide-containing regimens (TCR). Our aim was to explore the morphological, immunophenotypical, molecular and laboratory characteristics accompanying EMP-relapse and seek possible correlations with treatment and clinical outcome.

Acute Leukemia with a Translocation T(4;11)(q21;q23): a Distinct Clinicopathological Entity: Report of a Case with Cytogenetic Clonal Evolution and Review of 146 Cases of the Literature.

PubMed

Léglise, M C; Rivière, D; Brière, J

1990-01-01

We present a cytogenetic clonal evolution that correlates morphological and immunological shifts in a case of a patient with a t(4;11) (q21;q23) acute leukemia. We take this opportunity to review 146 cases reported so far, with special reference to morphology, immunophenotyping, cytogenetics, clinical characteristics and evolution. Particular features are underlined, and prognosis, leukemic stem cell origin, chromosomal breakpoints and genes involved are discussed. A relationship between this type of leukemia and exposure to carcinogens is suggested by a high rate of secondary leukemia in adults and a high frequency in newborns and infants.

Validation of Procedures for Monitoring Crewmember Immune Function

NASA Technical Reports Server (NTRS)

Pierson, Duane; Crucian, Brian; Mehta, Satish; Stowe, Raymond; Uchakin, Peter; Quiriarte, Heather; Sams, Clarence

2010-01-01

The objective of this Supplemental Medical Objective (SMO) is to determine the status of the immune system, physiological stress and latent viral reactivation (a clinical outcome that can be measured) during both short and long-duration spaceflight. In addition, this study will develop and validate an immune monitoring strategy consistent with operational flight requirements and constraints. Pre-mission, in-flight and post-flight blood and saliva samples will be obtained from participating crewmembers. Assays included peripheral immunophenotype, T cell function, cytokine profiles, viral-specific immunity, latent viral reactivation (EBV, CMV, VZV), and stress hormone measurements. To date, 18 short duration (now completed) and 8 long-duration crewmembers have completed the study. The long-duration phase of this study is ongoing. For this presentation, the final data set for the short duration subjects will be discussed.

Immune System Dysregulation, Viral Reactivation and Stress During Short-Duration Spaceflight

NASA Technical Reports Server (NTRS)

Pierson, Duane; Sams, Clarence; Crucian, Brian; Mehta, Satish; Stowe, Raymond; Uchakin, Peter; Quiriarte, Heather

2010-01-01

The objective of this NASA Short-Duration Bioastronautics Investigation (SDBI) was to assess spaceflight-associated immune dysregulation. Many previous studies have investigated this phenomenon post-flight, and found altered distribution and function of the peripheral leukocyte populations. Alterations in cytokine production profiles have also been reported. Unfortunately, post-flight data may be altered by the stress associated with high-G re-entry and readaptation to unit gravity following deconditioning. Therefore, the current study collected blood and saliva samples from crewmembers immediately before landing, and returned those samples to Earth for terrestrial analysis. Assays include peripheral comprehensive immunophenotype, T cell function, cytokine profiles, viral-specific immunity, latent viral reactivation (EBV, CMV, VZV), and stress hormone measurements. A total of 18 short duration crewmembers completed the study and the final data will be presented.

T-cell receptor gene rearrangement in Epstein-Barr virus infectious mononucleosis.

PubMed

Marbello, L; Riva, M; Veronese, S; Nosari, A M; Ravano, E; Colosimo, A; Paris, L; Morra, E

2012-09-01

This report describes the case of a previously healthy young man who presented with fever, pharyngitis, cervical lymphadenopathy, lymphocytosis, and severe thrombocytopenia. Serological tests for Epstein-Barr virus were diagnostic of a primary Epstein-Barr virus infectious mononucleosis but severe thrombocytopenia aroused the suspicion of a lymphoproliferative disease. T-cell receptor gene analysis performed on peripheral and bone marrow blood revealed a T-cell receptor γ-chain rearrangement without the evidence of malignancy using standard histologic and immunophenotype studies. Signs and symptoms of the infectious disease, blood count, and T-cell receptor gene rearrangement resolved with observation without the evidence of emergence of a lymphoproliferative disease. In the contest of a suspected lymphoproliferative disease, molecular results should be integrated with all available data for an appropriate diagnosis.

A morphological study of penile chancroid lesions in human immunodeficiency virus (HIV)-positive and -negative African men with a hypothesis concerning the role of chancroid in HIV transmission.

PubMed

Magro, C M; Crowson, A N; Alfa, M; Nath, A; Ronald, A; Ndinya-Achola, J O; Nasio, J

1996-10-01

Chancroid, the most common cause of genital ulceration in Africa, is known to be associated epidemiologically with heterosexual transmission of human immunodeficiency virus (HIV). The pathophysiological mechanisms by which chancroid might facilitate the spread of HIV are obscure. To investigate the role of chancroid in HIV transmission, the authors studied the histological features of biopsies from 11 men with penile chancroid lesions including five who were serologically positive for HIV. The histomorphologic and immunophenotypic nature of the inflammatory infiltrates suggests that there is a significant role for cell-mediated immunity in the host response to Hemophilus ducreyi infection. This response may be critical to the role of chancroid in HIV transmission.

Update on Mastocytosis (Part 1): Pathophysiology, Clinical Features, and Diagnosis.

PubMed

Azaña, J M; Torrelo, A; Matito, A

2016-01-01

Mastocytosis is a term used to describe a heterogeneous group of disorders characterized by clonal proliferation of mast cells in various organs. The organ most often affected is the skin. Mastocytosis is a relatively rare disorder that affects both sexes equally. It can occur at any age, although it tends to appear in the first decade of life, or later, between the second and fifth decades. Our understanding of the pathophysiology of mastocytosis has improved greatly in recent years, with the discovery that somatic c-kit mutations and aberrant immunophenotypic features have an important role. The clinical manifestations of mastocytosis are diverse, and skin lesions are the key to diagnosis in most patients. Copyright © 2015 Elsevier España, S.L.U. and AEDV. All rights reserved.

Gene expression of stem cells at different stages of ontological human development.

PubMed

Allegra, Adolfo; Altomare, Roberta; Curcio, Patrizia; Santoro, Alessandra; Lo Monte, Attilio I; Mazzola, Sergio; Marino, Angelo

2013-10-01

To compare multipotent mesenchymal stem cells (MSCs) obtained from chorionic villi (CV), amniotic fluid (AF) and placenta, with regard to their phenotype and gene expression, in order to understand if MSCs derived from different extra-embryonic tissues, at different stages of human ontological development, present distinct stemness characteristics. MSCs obtained from 30 samples of CV, 30 of AF and 10 placentas (obtained from elective caesarean sections) were compared. MSCs at second confluence cultures were characterized by immunophenotypic analysis with flow cytometry using FACS CANTO II. The expression of the genes Oct-4 (Octamer-binding transcription factor 4, also known as POU5F1), Sox-2 (SRY box-containing factor 2), Nanog, Rex-1 (Zfp-42) and Pax-6 (Paired Box Protein-6), was analyzed. Real-time quantitative PCR was performed by ABI Prism 7700, after RNA isolation and retro-transcription in cDNA. Statistical analysis was performed using non-parametric test Kruskal-Wallis (XLSTAT 2011) and confirmed by REST software, to estimate fold changes between samples. Each gene was defined differentially expressed if p-value was <0.05. Cells from all samples were negative for haematopoietic antigens CD45, CD34, CD117 and CD33 and positive for the typical MSCs antigens CD13, CD73 and CD90. Nevertheless, MSCs from AF and placentas showed different fluorescence intensity, reflecting the heterogeneity of these tissues. The gene expression of OCT-4, SOX-2, NANOG was not significantly different among the three groups. In AF, REX-1 and PAX-6 showed a higher expression in comparison to CV. MSCs of different extra-embryonic tissues showed no differences in immunophenotype when collected from second confluence cultures. The expression of OCT-4, NANOG and SOX-2 was not significantly different, demonstrating that all fetal sources are suitable for obtaining MSCs. These results open new possibilities for the clinical use of MSCs derived from easily accessible sources, in order to develop new protocols for clinical and experimental research. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Histopathological and immunophenotypical criteria for the diagnosis of Sézary syndrome in differentiation from other erythrodermic skin diseases: a European Organisation for Research and Treatment of Cancer (EORTC) Cutaneous Lymphoma Task Force Study of 97 cases.

PubMed

Klemke, C D; Booken, N; Weiss, C; Nicolay, J P; Goerdt, S; Felcht, M; Géraud, C; Kempf, W; Assaf, C; Ortonne, N; Battistella, M; Bagot, M; Knobler, R; Quaglino, P; Arheiliger, B; Santucci, M; Jansen, P; Vermeer, M H; Willemze, R

2015-07-01

Patients with erythrodermic disease are a diagnostic challenge regarding the clinical and histological differential diagnosis. To evaluate histopathological and immunohistochemical diagnostic markers for Sézary syndrome. Ninety-seven erythrodermic cases [Sézary syndrome (SS), n = 57; erythrodermic inflammatory dermatoses (EIDs), n = 40] were collected by the EORTC Cutaneous Lymphoma Task Force histopathology group. Evaluation criteria were (i) epidermal and dermal changes; (ii) morphology of the infiltrate; (iii) immunohistochemical analysis of marker loss (CD2, CD3, CD4, CD5 and CD7); (iv) bystander infiltrate by staining for CD8, FOXP3 and CD25; and (v) expression of Ki-67, CD30, PD-1 and MUM-1. The workshop panel made a correct diagnosis of SS in 51% of cases (cutaneous T-cell lymphoma 81%) and of EID in 80% without clinical or laboratory data. Histology revealed a significantly increased degree of epidermotropism (P < 0.001) and more intraepidermal atypical lymphocytes (P = 0.0014) in SS biopsies compared with EID. Pautrier microabscesses were seen only in SS (23%) and not in EID (P = 0.0012). SS showed significantly more dermal cerebriform and blastic lymphocytes than EID. Immunohistochemistry revealed a significant loss of CD7 expression (< 50%) in 33 of 51 (65%) cases of SS compared with two of 35 (6%) EID (P < 0.001). The lymphocytic infiltrate in SS skin samples was found significantly to express PD-1 (P = 0.0053), MUM-1 (P = 0.0017) and Ki-67 (P < 0.001), and showed less infiltration of CD8(+) lymphocytes (P < 0.001). A multivariate analysis identified CD7 loss, increased numbers of small cerebriform lymphocytes, low numbers of CD8(+) lymphocytes and increased proliferation (Ki-67(+) lymphocytes) as the strongest indicators for the diagnosis of SS. A number of different histological and immunophenotypical criteria are required to differentiate between SS and EIDs. © 2015 British Association of Dermatologists.

Renal cell carcinoma with t(6:11) (p21;q12). A case report highlighting distinctive immunohistologic features of this rare tumor.

PubMed

Arneja, Sarabjeet Kaur; Gujar, Neeraj

2015-01-01

Renal cell carcinoma (RCC) with t(6:11) (p21;q12) are extremely rare, fewer than 30 cases have been reported in literature. These tumors are characterized by specific chromosomal translocation involving TFEB, as against the more commonly known TFE3 (Xp11.2) translocation associated RCCs. The distinctive immnohistologic features are helpful in enabling a diagnosis of this rare tumor, otherwise diagnosed by fluorescence in situ hybridization assay, specific for detecting TFEB gene rearrangement. Herein, we report a case of this rare tumor in a 11 years old boy, with the objective of highlighting distinctive light microscopic and immuno-phenotypic features of this rare sub-type of translocation associated renal cell carcinoma, otherwise diagnosed by fluorescence in situ hybridization technique. Morphologically tumor showed distinctive biphasic population of cells, large epitheloid cells with voluminous eosinophillic cytoplasm and smaller cells with much lesser amount of cytoplasm and small rounded nuclei. The smaller cells at places clustered around hyaline pink material forming "pseudorosettes". population. Immunohistochemically both types of tumor cells showed negativity for pan CK (cytokeratin), EMA (epitheleal membrane antigen) and TFE3 (transcription factor E3). HMB 45 (human melanoma black 45) and Melan- A /MART 1 (melanoma antigen recognized by T cells) were moderate to strongly expressed. On review of literature, most RCCs with t(6;11) translocation have been reported to be negative for pan cytokeratins and EMA. Published literature also shows that the most distinctive immunohistochemical feature of t(6;11) translocation RCC is nuclear staining for TFEB protein. Immunostains for TFE3 have always been negative in the reported cases. It is noteworthy that immunoreactivity for melanocytic markers HMB45 and Melan A and immunonegativity for epithelial markers pan CK and EMA may lead to misdiagnosis of angiomyolipoma to the unwary. Knowledge of distinctive morphological and immuno-histochemical features of this tumor can help in establishing a diagnosis of this rare subset of translocation associated RCC on routine hematoxylin and eosin (H and E) staining and immunophenotyping. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

An early thymic precursor phenotype predicts outcome exclusively in HOXA-overexpressing adult T-cell acute lymphoblastic leukemia: a Group for Research in Adult Acute Lymphoblastic Leukemia study.

PubMed

Bond, Jonathan; Marchand, Tony; Touzart, Aurore; Cieslak, Agata; Trinquand, Amélie; Sutton, Laurent; Radford-Weiss, Isabelle; Lhermitte, Ludovic; Spicuglia, Salvatore; Dombret, Hervé; Macintyre, Elizabeth; Ifrah, Norbert; Hamel, Jean-François; Asnafi, Vahid

2016-06-01

Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acute lymphoblastic leukemias, but it is unclear whether these constitute a homogeneous clinical entity, and the biological consequences of HOXA overexpression have not been systematically examined. We characterized the biology and outcome of 55 HOXA-positive cases among 209 patients with adult T-cell acute lymphoblastic leukemia uniformly treated during the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies. HOXA-positive patients had markedly higher rates of an early thymic precursor-like immunophenotype (40.8% versus 14.5%, P=0.0004), chemoresistance (59.3% versus 40.8%, P=0.026) and positivity for minimal residual disease (48.5% versus 23.5%, P=0.01) than the HOXA-negative group. These differences were due to particularly high frequencies of chemoresistant early thymic precursor-like acute lymphoblastic leukemia in HOXA-positive cases harboring fusion oncoproteins that transactivate HOXA Strikingly, the presence of an early thymic precursor-like immunophenotype was associated with marked outcome differences within the HOXA-positive group (5-year overall survival 31.2% in HOXA-positive early thymic precursor versus 66.7% in HOXA-positive non-early thymic precursor, P=0.03), but not in HOXA-negative cases (5-year overall survival 74.2% in HOXA-negative early thymic precursor versus 57.2% in HOXA-negative non-early thymic precursor, P=0.44). Multivariate analysis further revealed that HOXA positivity independently affected event-free survival (P=0.053) and relapse risk (P=0.039) of chemoresistant T-cell acute lymphoblastic leukemia. These results show that the underlying mechanism of HOXA deregulation dictates the clinico-biological phenotype, and that the negative prognosis of early thymic precursor acute lymphoblastic leukemia is exclusive to HOXA-positive patients, suggesting that early treatment intensification is currently suboptimal for therapeutic rescue of HOXA-positive chemoresistant adult early thymic precursor acute lymphoblastic leukemia. The GRAALL-2003 and -2005 studies were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively. Copyright© Ferrata Storti Foundation.

Improvement of cardiac function by placenta-derived mesenchymal stem cells does not require permanent engraftment and is independent of the insulin signaling pathway.

PubMed

Passipieri, Juliana A; Kasai-Brunswick, Tais H; Suhett, Grazielle; Martins, Andreza B; Brasil, Guilherme V; Campos, Dilza B; Rocha, Nazareth N; Ramos, Isalira P; Mello, Debora B; Rodrigues, Deivid C; Christie, Beatriz B; Silva-Mendes, Bernardo J; Balduíno, Alex; Sá, Renato M; Lopes, Laudelino M; Goldenberg, Regina C; Campos de Carvalho, Antonio C; Carvalho, Adriana B

2014-08-21

The objective of this work was to evaluate the efficacy of placenta-derived mesenchymal stem cell (MSC) therapy in a mouse model of myocardial infarction (MI). Since MSCs can be obtained from two different regions of the human term placenta (chorionic plate or villi), cells obtained from both these regions were compared so that the best candidate for cell therapy could be selected. For the in vitro studies, chorionic plate MSCs (cp-MSCs) and chorionic villi MSCs (cv-MSCs) were extensively characterized for their genetic stability, clonogenic and differentiation potential, gene expression, and immunophenotype. For the in vivo studies, C57Bl/6 mice were submitted to MI and, after 21 days, received weekly intramyocardial injections of cp-MSCs for 3 weeks. Cells were also stably transduced with a viral construct expressing luciferase, under the control of the murine stem cell virus (MSCV) promoter, and were used in a bioluminescence assay. The expression of genes associated with the insulin signaling pathway was analyzed in the cardiac tissue from cp-MSCs and placebo groups. Morphology, differentiation, immunophenotype, and proliferation were quite similar between these cells. However, cp-MSCs had a greater clonogenic potential and higher expression of genes related to cell cycle progression and genome stability. Therefore, we considered that the chorionic plate was preferable to the chorionic villi for the isolation of MSCs. Sixty days after MI, cell-treated mice had a significant increase in ejection fraction and a reduction in end-systolic volume. This improvement was not caused by a reduction in infarct size. In addition, tracking of cp-MSCs transduced with luciferase revealed that cells remained in the heart for 4 days after the first injection but that the survival period was reduced after the second and third injections. Quantitative reverse transcription-polymerase chain reaction revealed similar expression of genes involved in the insulin signaling pathway when comparing cell-treated and placebo groups. Improvement of cardiac function by cp-MSCs did not require permanent engraftment and was not mediated by the insulin signaling pathway.

Histological and immunohistochemical characterization of the inflammatory and glial cells in the central nervous system of goat fetuses and adult male goats naturally infected with Neospora caninum.

PubMed

Costa, Rafael Carneiro; Orlando, Débora Ribeiro; Abreu, Camila Costa; Nakagaki, Karen Yumi Ribeiro; Mesquita, Leonardo Pereira; Nascimento, Lismara Castro; Silva, Aline Costa; Maiorka, Paulo César; Peconick, Ana Paula; Raymundo, Djeison Lutier; Varaschin, Mary Suzan

2014-12-14

Neospora caninum is an apicomplexan protozoan that is considered one of the main agents responsible for abortion in ruminants. The lesions found in the central nervous system (CNS) of aborted fetuses show multifocal necrosis, gliosis, and perivascular cuffs of mononuclear cells, but the inflammatory and glial cells have not been immunophenotypically characterized. The lesions in the CNS of infected adult animals have rarely been described. Therefore, in this study, we characterized the lesions, the immunophenotypes of the inflammatory and glial cells and the expression of MHC-II and PCNA in the CNS of goats infected with N. caninum. The CNS of eight aborted fetuses and six adult male goats naturally infected with N. caninum were analyzed with lectin histochemistry (RCA1) and immunohistochemistry (with anti-CD3, -CD79α, -GFAP, -MHC-II, and -PCNA antibodies). All animals were the offspring of dams naturally infected with N. caninum. The microscopic lesions in the CNS of the aborted fetuses consisted of perivascular cuffs composed mainly of macrophages (RCA1(+)), rare T lymphocytes (CD3(+)), and rare B lymphocytes (CD79α(+)). Multifocal necrosis surrounded by astrocytes (GFAP(+)), gliosis composed predominantly of monocytic-lineage cells (macrophages and microglia, RCA1(+)), and the cysts of N. caninum, related (or not) to the lesions were present. Similar lesions were found in four of the six male goats, and multinucleate giant cells related to focal gliosis were also found in three adult goats. Anti-GFAP immunostaining showed astrocytes characterizing areas of glial scarring. Cysts of N. caninum were found in three adult male goats. The presence of N. caninum was evaluated with histopathology, immunohistochemistry, and PCR. Immunohistochemistry demonstrated anti-PCNA labeling of macrophages and microglia in the perivascular cuffs and the expression of MHC-II by microglia and endothelial cells in the CNS of the aborted fetuses and adult male goats. Macrophages and microglia were the predominant inflammatory cells in the CNS of aborted fetuses and healthy adult male goats infected with N. caninum. Activated astrocytes were mainly associated with inflamed areas, suggesting that astrocytes were involved in the resolution of the lesions.

[Clinical and laboratory features of pediatric acute myeloid leukemia with inversion of chromosome 16].

PubMed

He, Ya-xiang; Xue, Yong-quan; Wang, Hong-ying; Yang, Nai-chao; Shao, Xue-jun; Xu, Jun; Ji, Zheng-hua; Huang, Yi-ping; Ding, Yun-fang; Hu, Shao-yan

2012-08-01

To evaluate the clinical and laboratory features of pediatric inv(16) acute myeloid leukemia (AML) retrospectively. Dual color fluorescence in situ hybridization (D-FISH) using a LSI CBFβ inv(16) break apart probe labeled by Spectrum red and Spectrum green was performed in 15 acute myeloid leukemia cases, including 13 cases with or without abnormal eosinophils but with positive core binding factor β (CBFβ)-MYH11 fusion transcript detected by RT-PCR, and 2 cases with trisomy 8 (+8). The results were compared with the morphology, immunophenotype, karyotype and RT-PCR. Morphologically, 12 cases were diagnosed as M(4)EO, 2 as M(4), and 1 as M(2a). Immunophenotypically, all 13 AML cases with inv(16) showed positive expression of CD(13) and CD(33), but without the expression of any lymphoid lineage antigens. Karyotyping analysis with G-banding detected inv(16) in 10 AML cases, including 9 M(4)EO cases and 1 M(2a), but only 5 positive cases were detected using R-banding technique. Among them, 2 cases had simultaneous +8 and trisomy22 (+22), one had +22 only in addition to inv(16). D-FISH revealed a CBFβ-MYH11 rearrangement in 13 cases of AML with positive RT-PCR results, and the mean positive rate of cell detection was 55.15% (range 37.0% - 86.0%). The complete remission rate (CR) and median survival period in this series of inv(16) AML were 81.5%and 11 months, respectively, of whom, 8 cases were still in CR. Relapse and karyotypic evolution were seen in case 5 with +8, +22 in addition to inv(16). AML with inv(16) is a special subtype. Most cases belong to M(4)EO. Its prognosis is good in general, but it seems to be an unfavorable feature for AML with inv(16) and +8, +22 simultaneously, especially with karyotypic evolution. For detection of inv(16), G-banding technique is evidently superior to R-banding technique. D-FISH combined with RT-PCR are more sensitive and reliable than chromosome banding analysis.

Markers of endothelial dysfunction and leucocyte activation in Saudi and non-Saudi haplotypes of sickle cell disease.

PubMed

Al Najjar, Salwa; Adam, Soheir; Ahmed, Nessar; Qari, Mohamed

2017-01-01

Sickle cell disease (SCD) is an autosomal recessive inherited hemoglobinopathy, characterized by chronic hemolysis and recurrent vaso-occlusive crisis (VOC). This study investigates changes in leucocyte subsets and the relationship between cell adhesion molecule expression and disease manifestations in patients during steady state and acute VOC. We compared soluble E-selectin and P-selectin levels in 84 SCD patients, in steady state and during VOC to 84 healthy controls. Using immunophenotyping, we also compared lymphocyte subsets in these three groups. Further, we compared E-selectin and P-selectin levels in patients of Saudi ethnicity to non-Saudi patients, in all three groups. Lymphocyte subsets showed high percentages of total T lymphocytes, T helper and suppressor lymphocytes, B lymphocytes as well as NK cells in patients with SCD during steady state, while B lymphocytes and NK cells were significantly higher during acute VOC crisis. High levels of both soluble E-selectin (sE-selectin) and soluble P-selectin (sP-selectin) markers were demonstrated in the serum of patients with SCD during both steady state and acute VOC. Levels of selectins were significantly higher in acute VOC. The immunophenotypic expression of L-selectin, on leucocytes, was high in SCD both during steady state and during acute VOC in comparison to normal control subjects. There was no significant difference in all three study groups between Saudi and non-Saudi patients. These findings suggest that patients with SCD have increased expression of adhesion molecules: E-selectin and P-selectin, which play an important role in the pathogenesis of VOC. Despite the distinct phenotype of Saudi patients with SCD, there was no significant difference in levels of soluble E-selectin and soluble P-selectin between Saudi and non-Saudi patients in all three groups. While sickle cell disease is a well-recognized state of chronic inflammation, the role of specific adhesion molecules is steadily unraveling. Studies are underway to investigate the potential role of selectin antagonists, for prevention and reversal of acute vascular occlusions in SCD patients.

Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

PubMed

Kalina, Tomas; Flores-Montero, Juan; Lecrevisse, Quentin; Pedreira, Carlos E; van der Velden, Vincent H J; Novakova, Michaela; Mejstrikova, Ester; Hrusak, Ondrej; Böttcher, Sebastian; Karsch, Dennis; Sędek, Łukasz; Trinquand, Amelie; Boeckx, Nancy; Caetano, Joana; Asnafi, Vahid; Lucio, Paulo; Lima, Margarida; Helena Santos, Ana; Bonaccorso, Paola; van der Sluijs-Gelling, Alita J; Langerak, Anton W; Martin-Ayuso, Marta; Szczepański, Tomasz; van Dongen, Jacques J M; Orfao, Alberto

2015-02-01

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

Trisomy/tetrasomy 13 in seven cases of acute leukemia.

PubMed

Sreekantaiah, C; Baer, M R; Morgan, S; Isaacs, J D; Miller, K B; Sandberg, A A

1990-11-01

We report the clinical presentation and the morphologic, histochemical, and immunophenotypic characteristics of seven patients with acute leukemia who had trisomy/tetrasomy 13 as the sole cytogenetic abnormality in their leukemia. Five patients had trisomy 13 at diagnosis of acute leukemia. All five of these patients had undifferentiated leukemias. The sixth patient, who had French-American-British (FAB) type M2 acute nonlymphocytic leukemia (ANLL), and the seventh patient with biphenotypic acute leukemia developed the trisomic clone as a new abnormality late in the course of their disease. A review of the literature revealed 28 previously reported hematologic malignancies with trisomy 13 or tetrasomy 13q as a solitary cytogenetic abnormality. Trisomy 13 appears to represent another rare but nonrandom cytogenetic abnormality in acute leukemia. In our series trisomy 13 is largely associated with acute leukemia with little myeloid or lymphoid differentiation.

Identification of CD56 and CD57 by flow cytometry in Ewing's sarcoma or primitive neuroectodermal tumor.

PubMed

Gardner, L J; Polski, J M; Fallon, R; Dunphy, C H

1998-07-01

CD56 and CD57 are commonly considered as natural killer and neuroectodermal markers, but their expression has been identified in a wide spectrum of neoplasms including some cases of Ewing's sarcoma (ES) and primitive neuroectodermal tumor (PNET). We report two cases of small, round blue cell tumor (SRBCT), in which flow cytometry immunophenotyping (FCI) detected strong expression of CD56 and CD57 (one case). Immunohistochemical staining with Leu-19 and Leu-7 confirmed the FI results. Although CD56 and CD57 expression is consistent with ES/PNET, it can be potentially misleading if results of FCI are interpreted in the absence of other findings. These cases suggest the utility of FCI in undifferentiated SRBCT. The literature on CD56 and CD57 expression in ES/PNET is reviewed and discussed.

Case report: Concomitant Chronic Lymphocytic Leukaemia and Cytogenetically Normal de novo Acute Leukaemia in a Patient.

PubMed

Kajtár, Béla; Rajnics, Péter; Egyed, Miklós; Alizadeh, Hussain

2015-01-01

The simultaneous occurrence of acute myeloid leukaemia with untreated chronic lymphocytic leukemia is extremely rare. We report a case of a 74-year-old man who was evaluated for macrocytic anaemia. Based on the morphology and immunophenotyping analysis of peripheral blood, a diagnosis of chronic lymphocytic leukemia was established. Subsequently, the bone marrow examination revealed the presence of two distinct, coexisting CLL and AML clones. Cytogenetic and molecular genetic analysis detected deletion 13q14.3 and unmutated immunoglobulin variable heavy-chain in the CLL clone, only. The AML and CLL clones did not share clonality, and the AML did not involve the peripheral blood. A diagnosis of cytogenetically normal de novo AML occurring concurrently with untreated CLL has not been reported previously in English literature. © 2015 by the Association of Clinical Scientists, Inc.

Histologic Mimics of Basal Cell Carcinoma.

PubMed

Stanoszek, Lauren M; Wang, Grace Y; Harms, Paul W

2017-11-01

- Basal cell carcinoma (BCC) is the most common human malignant neoplasm and is a frequently encountered diagnosis in dermatopathology. Although BCC may be locally destructive, it rarely metastasizes. Many diagnostic entities display morphologic and immunophenotypic overlap with BCC, including nonneoplastic processes, such as follicular induction over dermatofibroma; benign follicular tumors, such as trichoblastoma, trichoepithelioma, or basaloid follicular hamartoma; and malignant tumors, such as sebaceous carcinoma or Merkel cell carcinoma. Thus, misdiagnosis has significant potential to result in overtreatment or undertreatment. - To review key features distinguishing BCC from histologic mimics, including current evidence regarding immunohistochemical markers useful for that distinction. - Review of pertinent literature on BCC immunohistochemistry and differential diagnosis. - In most cases, BCC can be reliably diagnosed by histopathologic features. Immunohistochemistry may provide useful ancillary data in certain cases. Awareness of potential mimics is critical to avoid misdiagnosis and resulting inappropriate management.

[Primary lymphoma of the central nervous system: 20 years' experience in a referral hospital].

PubMed

Calderón-Garcidueñas, A L; Pacheco-Calleros, J; Castelán-Maldonado, E; Nocedal-Rustrián, F C

Primary central nervous system lymphomas (PCNSL) are rare neoplasms. AIM. To study the clinical aspects and the immuno-phenotype of all cases of PCNSL in a 20 years lapse in a referral hospital in Northeastern Mexico. From January 1986 to December 2005 all PCNSL histologically confirmed were studied. The primary lymphomas were 1% of malignant central nervous system neoplasms. 21 cases were studied (ages from 9-70 years) with male predominance (2:1). 24% patients had immuno-suppression. The more frequent clinical data were: papilledema (71%), headache (62%), paresis (48%) and seizures (33%). 33% of patients died during the first six months after diagnosis. The T lymphomas were 19% of cases and corresponded to small cell type. PCNSL are still a diagnostic challenge. Multicenter studies are required in order to determine the best treatment protocol.

A bursal pentapeptide (BPP-I), a novel bursal-derived peptide, exhibits antiproliferation of tumor cell and immunomodulator activity.

PubMed

Feng, Xiu L; Liu, Qing T; Cao, Rui B; Zhou, Bin; Wang, Fang Q; Deng, Wen L; Qiu, Ya F; Zhang, Yu; Ishag, Hassan; Ma, Zhi Y; Zheng, Qi S; Chen, Pu Y

2012-06-01

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds. Here, we isolated a novel bursal pentapeptide I (BPP-I), LGPGP, from BF. BPP-I could play inhibition effect on MCF-7 but not on CEF or Vero cell proliferation in vitro, and enhance antitumor factor p53 protein expression. Also, BPP-I stimulated antibody production in a dose-dependent manner in hybridoma cell. Furthermore, BPP-I could induce various immune responses in mice immunization experiments, including increase antibody production and cytokines IL-4 and IFN-γ level, and induce T-cell immunophenotyping. These results suggest that BPP-I is a potential immunomodulator of antitumor and immunity. The study could provide some novel insights on the probable candidate reagent for the antitumor and immune improvement.

Is alkaline phosphatase the smoking gun for highly refractory primitive leukemic cells?

PubMed Central

Rico, Laura G.; Juncà, Jordi; Ward, Mike D.; Bradford, Jolene; Petriz, Jordi

2016-01-01

With the aim to detect candidate malignant primitive progenitor populations, we modified an original alkaline phosphatase (ALP) stem cell detection method based on the identification of alkaline phosphatase fluorescent cells in combination with flow cytometry immunophenotyping. Over a period of one year, we have been using this technique to study its activity in patients with leukemia and lymphoma, showing that changes in the alkaline phosphatase levels can be used to detect rare populations of highly refractory malignant cells. By screening different blood cancers, we have observed that this activity is not always restricted to CD34+ leukemic cells, and can be overexpressed in CD34 negative leukemia. We have verified that this method gives accurate and reproducible measurements and our preliminary results suggest that CD34+/ALPhigh cells appear to sustain leukemogenesis over time. PMID:27732563

Flow cytometry: basic principles and applications.

PubMed

Adan, Aysun; Alizada, Günel; Kiraz, Yağmur; Baran, Yusuf; Nalbant, Ayten

2017-03-01

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.

Inv(7)(q22q36) in refactory anemia with excess blasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rayburn, J.; Stegeman, D.; Berger, C.

1994-09-01

Morphological review of bone marrow from an 89 year-old male revealed an immature cell population with increased blasts (25% CD34 positive). However, the morphology was not sufficiently clear to discriminate lymphoid from myeloid precursors. Immunophenotypically, there was evidence for both lymphoid and myeloid derivation with dual expression of CD5 and CD20, aberrant expression of CD19 versus CD20, and an increased CD13 population. Twenty percent (20%) of the cells were TdT positive. Cytogenetically, an inversion of chromosome 7, inv(7)(q22q36), was observed in 9 of 20 cells. This abnormality has been reported only once previously, in association with refractory anemia with excessmore » blasts (RAEB). The patient, to date, has not developed an acute leukemic process, but remains in a myelodysplastic state, defined as RAEB.« less

Blastic plasmacytoid dendritic cell neoplasm: report of two pediatric cases.

PubMed

Dharmani, Preeti Ashok; Mittal, Neha Manish; Subramanian, P G; Galani, Komal; Badrinath, Yajamanam; Amare, Pratibha; Gujral, Sumeet

2015-01-01

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of acute leukemia that typically follows a highly aggressive clinical course in adults, whereas experience in children with this disease is very limited. We report cases of two children in whom bone marrow showed infiltration by large atypical monocytoid 'blast-like' cells which on immunophenotyping expressed CD4, CD56, HLA-DR and CD33 while were negative for CD34 other T-cell, B-cell and myeloid markers. The differential diagnoses considered were AML, T/NK-cell leukemia and acute undifferentiated leukemia. Additional markers CD303/BDCA-2 and CD123 which are recently validated plasmacytoid dendritic cell markers were done which helped us clinch the diagnosis of this rare neoplasm. An accurate diagnosis of BPDCN is essential in order to provide prompt treatment. Due to its rarity and only recent recognition as a distinct clinicopathological entity, no standardized therapeutic approach has been established for BPDCN.

Massive periosteal reaction a presenting feature of acute megakaryocytic leukemia.

PubMed

Ueda, Takahiro; Ito, Yasuhiko; Maeda, Miho; Fukunaga, Yoshitaka

2007-12-01

Acute megakaryoblastic leukemia (AML M7) is a biologically heterogeneous form of acute myeloid leukemia accounting for 14.6% of cases. In many instances in the past, AML M7 has been classified as undifferentiated leukemia, myelodysplasia, myelofibrosis or some other disease because of its complex clinical presentation or the difficulty of obtaining and interpreting bone marrow samples. However, with currently available morphological, cytochemical, cytogenetic and immunophenotypic methods, AML M7 can now be reliably diagnosed. Although the radiographic spectrum of bony changes in leukemia have been well characterized, skeletal X-ray abnormalities in the setting of AML M7 in pediatric patients have been described in few reports that were associated with bone marrow fibrosis. Here we report on a 14-month-old girl who presented with a massive periosteal reaction of the extremities and clavicles associated with myelofibrosis, a presenting feature of AML M7. The bone changes were very unusual in this case.

CD4+ acute undifferentiated leukaemia, probably early monoblastic type, developing in a patient with myelodysplastic syndrome and bone marrow fibrosis.

PubMed

Guenova, M; Taskov, H; Zechev, J

1997-07-01

We report here a patient who presented with pancytopenia, hypercellular bone marrow and three-lineage dysplasia associated with an increase of reticulin fibres. After a 5-month period, anaemia and thrombocytopenia progressed very rapidly and the white blood count increased showing 45% blasts with monocytoid morphology, but cytochemically undifferentiated in nature. The immunophenotype revealed an unusual expression of CD4, CD36 and HLA-DR in the absence of any other myeloid or lymphoid lineage-associated markers. The patient died unexpectedly during the course of chemotherapy. The occurrence of CD4, CD36 and HLA-DR on the blast cells cannot determine the lineage of differentiation with certainty but provides some evidence that the leukaemic cells were probably derived from a very early monocytic progenitor with maturation arrest. These cells had apparently complex interactions with pathologic megakaryocytes and cytokine production.

[Acute hybrid leukemia. Review of the literature and presentation of a case].

PubMed

Guzzini, F; Angelopoulos, N; Banfi, L; Coppetti, D; Ceppi, M; Camerone, G

1990-03-01

In the last years, the development of immunophenotypic and molecular analyses allowed to recognize several cases of hybrid acute leukemia (AL), whose blast cell display both lymphoid and myeloid features. Hybrid, or mixed-lineage, AL seems to have distinct clinical manifestations and hematological findings, and is mainly characterized by resistance to chemotherapy and poor prognosis. We report on a patient with AL, which showed a very rapid switch from the lymphoblastic phenotype exhibited at presentation to a myelomonoblastic one, appeared at first relapse, and lastly progressed to an undifferentiated leukemia in the terminal phase. Together with this morphologic and cytochemical evolution, leukemic cells expressed, besides the primary early-B antigens, new immunological markers related to T-lymphocytic and myeloid lineages. Based on this observation and current understanding of the ontogenesis of hematologic malignancies, we discuss biological mechanisms which are likely to underlie hybrid leukemia.

Umbilical cord fibroblasts: Could they be considered as mesenchymal stem cells?

PubMed Central

Zeddou, Mustapha; Relic, Biserka; Malaise, Michel G

2014-01-01

In cell therapy protocols, many tissues were proposed as a source of mesenchymal stem cells (MSC) isolation. So far, bone marrow (BM) has been presented as the main source of MSC despite the invasive isolation procedure related to this source. During the last years, the umbilical cord (UC) matrix was cited in different studies as a reliable source from which long term ex vivo proliferating fibroblasts were isolated but with contradictory data about their immunophenotype, gene expression profile, and differentiation potential. Hence, an interesting question emerged: Are cells isolated from cord matrix (UC-MSC) different from other MSCs? In this review, we will summarize different studies that isolated and characterized UC-MSC. Considering BM-MSC as gold standard, we will discuss if UC-MSC fulfill different criteria that define MSC, and what remain to be done in this issue. PMID:25126385

Gamma delta T-cell large granular lymphocyte lymphoma in a dog.

PubMed

Ortiz, Ana Liza; Carvalho, Sofia; Leo, Chiara; Riondato, Fulvio; Archer, Joy; Cian, Francesco

2015-09-01

A 2-year and 6-month-old female neutered Labrador Retriever with Horner syndrome, megaesophagus, and a mediastinal mass was referred to the Queen Mother Hospital for Animals of the Royal Veterinary College. A large granular lymphocyte (LGL) lymphoma was diagnosed on cytology; flow cytometric analysis revealed a γδ T-cell phenotype (CD3+, CD5+, CD45+, TCRγδ+, CD4-, CD8-, CD34-, CD21-). Chemotherapy was started with a combination of lomustine, vincristine, procarbazine, and prednisolone, followed by bleyomicin. Euthanasia was elected by the owners, due to progressive deterioration and lack of quality of life, 28 days after diagnosis. This is the first cytologic and immunophenotypic characterization of a canine γδ T-cell lymphoma with LGL morphology and probably of mediastinal origin. The role of chemotherapy in delaying the disease progression remains unknown. © 2015 American Society for Veterinary Clinical Pathology.

Radiosensitive orbital metastasis as presentation of occult colonic adenocarcinoma.

PubMed

Ludmir, Ethan B; McCall, Shannon J; Czito, Brian G; Palta, Manisha

2014-09-19

An 82-year-old man presented with progressive right frontal headaches. The patient's history was significant for benign polyps on surveillance colonoscopy 2 years prior, without high-grade dysplasia or carcinoma. MRI revealed an enhancing lesion arising within the superomedial aspect of the right orbit. Lesion biopsy demonstrated histological appearance and immunophenotype suggestive of colonic adenocarcinoma. Staging positron emission tomography/CT showed visceral metastases and diffuse activity in the posterior rectosigmoid, consistent with metastatic colon cancer. Treatment of the orbital lesion with external beam radiotherapy to 30 Gy resulted in significant palliation of the patient's headaches. The patient expired 2 months following treatment completion due to disease progression. Orbital metastasis as the initial presentation of an occult colorectal primary lesion is exceedingly rare, and occurred in this patient despite surveillance colonoscopy. Radiotherapy remains an efficacious modality for treatment of orbital metastases. 2014 BMJ Publishing Group Ltd.

Mucinous cystadenocarcinoma of the breast: the challenge of diagnosing a rare entity

PubMed Central

Koufopoulos, Nektarios; Goudeli, Christina; Syrios, John; Filopoulos, Evangelos; Khaldi, Lubna

2017-01-01

Mucinous cystadenocarcinoma is an extremely rare variant of primary breast tumor which is histologically similar to mucinous cystadenocarcinoma of the ovary and pancreas. Herein we report a case of a 63 years old woman diagnosed with diverse histological types of non-synchronous rare primary breast tumors, a medullary carcinoma of the right breast and a mucinous cystadenocarcinoma of the left breast. Macroscopically the neoplasm appeared multilocular filled with mucoid material. Under light microscopy the cystic areas were lined by columnar cells with abundant intracellular and extracellular mucin. Solid areas were composed of tall columnar cells with intracellular mucin. Moderate to marked atypia was noticed and tumor cells stained positive for cytokeratin 7 and negative for cytokeratin 20. Moreover tumor cells displayed a basal like immunophenotype expressed as followed: ER negative, PR negative, HER-2 negative, cytokeratin (CK5/6) positive and EGFR positive. PMID:29081926

Mucinous cystadenocarcinoma of the breast: the challenge of diagnosing a rare entity.

PubMed

Koufopoulos, Nektarios; Goudeli, Christina; Syrios, John; Filopoulos, Evangelos; Khaldi, Lubna

2017-10-03

Mucinous cystadenocarcinoma is an extremely rare variant of primary breast tumor which is histologically similar to mucinous cystadenocarcinoma of the ovary and pancreas. Herein we report a case of a 63 years old woman diagnosed with diverse histological types of non-synchronous rare primary breast tumors, a medullary carcinoma of the right breast and a mucinous cystadenocarcinoma of the left breast. Macroscopically the neoplasm appeared multilocular filled with mucoid material. Under light microscopy the cystic areas were lined by columnar cells with abundant intracellular and extracellular mucin. Solid areas were composed of tall columnar cells with intracellular mucin. Moderate to marked atypia was noticed and tumor cells stained positive for cytokeratin 7 and negative for cytokeratin 20. Moreover tumor cells displayed a basal like immunophenotype expressed as followed: ER negative, PR negative, HER-2 negative, cytokeratin (CK5/6) positive and EGFR positive.

Clinicopathologic features and management of blastoid variant of mantle cell lymphoma.

PubMed

Shrestha, Rajesh; Bhatt, Vijaya Raj; Guru Murthy, Guru Subramanian; Armitage, James O

2015-01-01

The blastoid variant of mantle cell lymphoma (MCL), which accounts for less than one-third of MCL, may arise de novo or as a transformation from the classical form of MCL. Blastoid variant, which predominantly involves men in their sixth decade, has frequent extranodal involvement (40-60%), stage IV disease (up to 85%) and central nervous system (CNS) involvement. Diagnosis relies on morphological features and is challenging. Immunophenotyping may display CD23 and CD10 positivity and CD5 negativity in a subset. Genetic analysis demonstrates an increased number of complex genetic alterations. Blastoid variant responds poorly to conventional chemotherapy and has a short duration of response. Although the optimal therapy remains to be established, CNS prophylaxis and the use of aggressive immunochemotherapy followed by autologous stem cell transplant may prolong the remission rate and survival. Further studies are crucial to expand our understanding of this disease entity and improve the clinical outcome.

Germinoma with an extensive rhabdoid cell component centered at the corpus callosum.

PubMed

Tajima, Shogo; Koda, Kenji

2017-03-01

Intracranial germinomas comprise 0.5-2.0 % of all central nervous system (CNS) tumors and 50-60 % of CNS germ cell tumors. They most frequently originate in the pineal gland and the suprasellar region. The corpus callosum is an extremely uncommon location for germinoma formation. Herein, we report about a 20-year-old man with a germinoma centered at the corpus callosum and that extended to both cerebral hemispheres. In addition to its location, this case is unique in that the amount of tumor cells with rhabdoid morphology exceeded that of tumor cells with typical morphology. The rhabdoid cell component showed an immunophenotype compatible with germinoma. While the presence of rhabdoid cells is generally regarded as a sign of aggressive behavior, the patient has been doing well for at least 4 years since undergoing radiation therapy and chemotherapy. The cellular composition of germinoma might not critically affect prognosis with adequate treatment.

Multiplexed immunophenotyping of human antigen-presenting cells in whole blood by polychromatic flow cytometry

PubMed Central

Fung, Erik; Esposito, Laura; Todd, John A.; Wicker, Linda S.

2010-01-01

We describe two modular protocols for immunostaining and multiparameter flow cytometric analysis of major human antigen-presenting cells (dendritic cells, monocytes, B lymphocytes) in minimally manipulated whole blood. Simultaneous detection of up to eight colors is enabled by careful selection and testing of cell-subset-defining monoclonal antibodies (anchor markers) in the appropriate fluorochrome combinations, to demonstrate the quantification of surface expression levels of molecules involved in chemotaxis (e.g. CX3CR1, CCR2), adhesion (e.g. CD11b, CD62L), antigen presentation (e.g. CD83, CD86, CD209) and immune regulation (e.g. CD101) on circulating antigen-presenting cells. Each immunostaining reaction requires as little as 50–100 μl of peripheral whole blood, no density-gradient separation, and the entire procedure from preparation of reagents to flow cytometry can be completed in <5 h. PMID:20134434

Juvenile xanthogranuloma of the corneal limbus: report of two cases and review of the literature.

PubMed

Mocan, Mehmet C; Bozkurt, Banu; Orhan, Diclehan; Kuzey, Gamze; Irkec, Murat

2008-07-01

To report the clinical and histopathologic findings of limbal juvenile xanthogranuloma lesions in 2 pediatric cases. Case report and literature review. Two cases (a 7-month-old girl and a 7-year-old boy), both of whom developed a yellowish corneoscleral limbal mass not associated with other ocular or systemic findings, were evaluated. Both cases underwent simple excision of the lesion followed by local steroid treatment. Histopathologic examination in both cases revealed foamy histiocytes within an inflammatory infiltrate. One of the lesions also harbored multinucleate giant cells. Immunohistochemical staining was positive for CD68 and negative for S-100 and CD1a in both cases. Juvenile xanthogranuloma may rarely present with an isolated lesion occurring only at the corneoscleral limbus. The diagnosis in the early stages of this disorder may be established with immunophenotyping and characteristic histopathologic features even without the presence of the multinucleate giant cells.

Anaplastic large cell lymphoma of scrotal skin.

PubMed

Mehdi, Itrat; Praseeda, Indira; Al-Bahrani, Bassim Jaffer; Satayapal, Namrata; Monem, Assam Abdel; Al Kharusi, Suad

2011-11-01

Cutaneous T cell lymphoma (CTCL) is an uncommon diverse group of lympho-proliferative disorders involving the skin. They vary considerably in clinical presentation, microscopic features and immunophenotyping. The diagnosis is challenging, zealous, and often not easy. CD30+ve anaplastic large cell lymphoma is extremely rare. Its clinical spectrum varies from a solitary unifocal skin lesion of excellent prognosis to a multi focal systemic disease having a poor out come. The diagnosis is quite cumbersome, and often difficult. The differential diagnosis include from benign skin lesions to secondary cutaneous involvement by lymphoma. A correct diagnosis is integral with a complete metastatic/staging work up to avoid over treatment. The treatment options depend on extent of disease involvement and include surgical excision, surveillance, local radiotherapy, and systemic chemotherapy. The prognosis is good with unifocal local disease. We present here a very rare case of CD30+ ALCL of scrotal skin, in a middle aged male patient.

Endometrial metastasis of colorectal cancer with coincident endometrial adenocarcinoma.

PubMed

Colling, Richard; Lopes, Tito; Das, Nagiindra; Mathew, Joe

2010-11-05

Metastasis to the uterine corpus is uncommon and secondary colorectal tumours of the endometrium are rare. We describe a uterine tumour with components of both primary endometrial and metastatic colorectal carcinomata. In this case, a 72-year-old obese woman presented with a 2-week history of postmenopausal bleeding per vaginum and weight loss. She had an abdominoperineal resection 3 years previously for a Dukes stage B rectal carcinoma. A transvaginal ultrasonography showed a thickened endometrium. Histology immunophenotyping showed a CK7+, CK20+, CA125- and CEA+ colorectal metastasis (a profile consistent with her previous cancer) associated with a primary CK7+, CK20-, CA125+ and CEA- endometroid endometrial adenocarcinoma. We conclude this represents endometrial metastasis of colorectal carcinoma with coincident primary endometrial adenocarcinoma. We speculate as to whether the endometrial carcinoma arose de novo or was induced by the colorectal metastasis, or whether the primary endometrial tumour provided a fertile site for the colorectal metastasis.

[Immunomorphologic features of epithelial-stromal relationships at hyperplasia and endometrial carcinoma].

PubMed

Bantysh, B B; Paukov, v S; Kogan, E A

2012-01-01

The results of a immunomorphologic comprehensive study of epithelial-stromal relationships in the uterus hyperplasia and endometrial cancer suggest that the suppressor gene of cancer (PTEN) plays a key role in the process of neoplastic transformation of endometrial hyperplasia and adenocarcinoma development. For the first time the existence of two highly differentiated endometrial adenocarcinoma immunophenotype were detected The first one is a PTEN-negative endometrial aedenocarcinoma, characterized by an almost complete inhibition of tumor suppressor gene PTEN in the epithelium of the glands and stromal cell of the tumor The second type is a PTEN-positive endometrial adenocarcinoma, in which epithelial and stromal tumor suppressor gene PTEN activity has retained Based on these results we have formulated a hypothesis about the different types of endometrial hyperplasia morphogenesis and its possible transfer to cervical cancer associated with features of tumor suppressor gene PTEN.

A case of hairy cell leukemia variant.

PubMed

Găman, Amelia Maria; Dobrea, Camelia Marioara; Găman, Mihnea Alexandru

2015-01-01

Hairy cell leukemia variant (HCLv) is a rare B-cell chronic lymphoproliferative disorder with features of the classic HCL but presenting some particularities, a poor response to conventional therapy of classic HCL and a more aggressive course of disease with shorter survival than classic HCL. We present a case of a 52-year-old man hospitalized in July 2012 in the Clinic of Hematology of Craiova, Romania, having splenomegaly, leukocytosis with lymphocytosis, anemia and thrombocytopenia, without monocytopenia, which exposed, in the peripheral blood and bone marrow cells, intermediate morphology between hairy cells and prolymphocytes and immunophenotype of mature B-cell phenotype CD19, CD20, CD22, CD11c, CD103, low positive for CD25 and negative for CD3, diagnosed with HCL variant, with no response to conventional chemotherapy and interferon-alpha, an aggressive course of disease and a survival of less than a year from diagnosis.

[Plasmablastic lymphoma].

PubMed

Fernández-Álvarez, Rubén; Sancho, Juan-Manuel; Ribera, Josep-María

2016-11-04

Plasmablastic lymphoma (PBL) is a rare and aggressive subtype of non-Hodgkin lymphoma that commonly occurs in human immunodeficiency virus (HIV)-positive individuals, and affects oral sites. Occasionally, it has been described in HIV-negative patients and involving non-oral sites. Pathologically, PBL is a high-grade B-cell lymphoma that displays the immunophenotype of a terminally differentiated B-lymphocyte with loss of B-cell markers (CD20) and expression of plasma-cell antigens. Epstein-Barr virus infection and MYC rearrangements are frequently observed. Treatment of PBL is challenging because of the lack of established treatment and poor outcomes, with median survival times shorter than one year. In this review, we discuss the clinical and epidemiologic spectrum of PBL as well as its distinct pathological features. Finally, we summarize the currently available approaches for the treatment of patients with PBL. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

The role of flow cytometry in companion animal diagnostic medicine.

PubMed

Tarrant, Jacqueline M

2005-11-01

Flow cytometry is a powerful tool for characterising the composition of complex cell populations. The accuracy and precision of this technology for describing and enumerating cells exceeds traditional methods. The number of diagnostic veterinary laboratories with access to a dedicated machine is increasing, and there is the potential to offer a clinical flow cytometry service. The improved availability of monoclonal antibodies (mAb) to cell markers expressed by the leukocytes of companion animals, permits the implementation of comprehensive mAb panels suitable for diagnosis of lympho- and myeloproliferative disease. Reticulated erythrocyte and platelet quantification, antiglobulin assays for immune-mediated cytopenias, lymphocyte subset analysis, and immunophenotyping of lymphoma and leukemia, have been validated for companion animal samples on the flow cytometer. It is now timely to consider the role of flow cytometry in diagnostic practice, and the requirement for quality assurance and standardization of testing procedures.

CD4 expression on EL4 cells as an epiphenomenon of retroviral transduction and selection.

PubMed

Logan, Grant J; Spinoulas, Afroditi; Alexander, Stephen I; Smythe, Jason A; Alexander, Ian E

2004-04-01

The EL4 murine tumour cell line, isolated from a chemically induced lymphoma over 50 years ago, has been extensively exploited in immunological research. The conclusions drawn from many of these studies have been based on the presumption that EL4 cells maintain a stable phenotype during experimental manipulation. To the contrary, we have observed 100-fold greater expression of cell surface CD4 (CD4(high)) on a subpopulation of EL4 cells following retroviral transduction and G418 selection when compared with unmodified populations. Although the mechanism responsible for this effect remains to be elucidated, the unexpected expression of CD4, a molecule that functions as both a coreceptor with the T-cell receptor and ligand for the pro-inflammatory cytokine IL-16, has the potential to influence experimental outcomes. Upregulation of CD4 should be excluded when EL4 cells are utilized in experiments requiring a consistent immuno-phenotype.

Late ovarian relapse of TEL/AML1 positive ALL confirming that TEL deletion is a secondary event in leukemogenesis.

PubMed

Ly-Sunnaram, Beatrice; Henry, Catherine; Gandemer, Virginie; Mee, Franseza Le; Burtin, Florence; Blayau, Martine; Cayuela, Jean-Michel; Oster, Magalie; Clech, Philippe; Rambeau, Marc; Marie, Celine; Pampin, Cecilia; Edan, Christine; Gall, Edouard Le; Goasguen, Jean E

2005-09-01

We describe here a late extramedullary ovarian relapse in an 18-year-old female who was diagnosed with hypotetraploid cell acute lymphoblastic leukaemia (cALL) at the age of 6. At both occurrences of the disease cells were analyzed by morphology, immunophenotyping, cytogenetics and molecular methods. TEL/AML1 was detected by RT-PCR and FISH analysis in both events. We demonstrated, using detection of IGH/TCR rearrangements and TEL/AML1 breakpoints sequencing that the cells were clonally related. Moreover, interphasic FISH using TEL and AML1 probes showed the loss of a second TEL at the time of relapse. This observation confirms that TEL/AML1 alone is not sufficient to trigger ALL and that TEL deletion is a secondary event in leukemogenesis. To our knowledge, it is the first complete description of extramedullary ALL relapse combining all methodologies.

Cells Isolated from Human Periapical Cysts Express Mesenchymal Stem Cell-like Properties

PubMed Central

Marrelli, Massimo; Paduano, Francesco; Tatullo, Marco

2013-01-01

We provide a detailed description of mesenchymal stem cells (MSCs) isolated from human periapical cysts, which we have termed hPCy-MSCs. These cells have a fibroblast-like shape and adhere to tissue culture plastic surfaces. hPCy-MSCs possess high proliferative potential and self-renewal capacity properties. We characterised the immunophenotype of hPCy-MSCs (CD73+, CD90+, CD105+, CD13+, CD29+, CD44+, CD45-, STRO-1+, CD146+) by flow cytometry and immunofluorescence. hPCy-MSCs possess the potential to differentiate into osteoblast- and adipocyte-like cells in vitro. Multi-potentiality was evaluated with culture-specific staining and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis for osteo/odontogenic and adipogenic markers. This is the first report to indicate that human periapical cysts contain cells with MSC-like properties. Taken together, our findings indicate that human periapical cysts could be a rich source of MSCs. PMID:24250252

Cells isolated from human periapical cysts express mesenchymal stem cell-like properties.

PubMed

Marrelli, Massimo; Paduano, Francesco; Tatullo, Marco

2013-01-01

We provide a detailed description of mesenchymal stem cells (MSCs) isolated from human periapical cysts, which we have termed hPCy-MSCs. These cells have a fibroblast-like shape and adhere to tissue culture plastic surfaces. hPCy-MSCs possess high proliferative potential and self-renewal capacity properties. We characterised the immunophenotype of hPCy-MSCs (CD73(+), CD90(+), CD105(+), CD13(+), CD29(+), CD44(+), CD45(-), STRO-1(+), CD146(+)) by flow cytometry and immunofluorescence. hPCy-MSCs possess the potential to differentiate into osteoblast- and adipocyte-like cells in vitro. Multi-potentiality was evaluated with culture-specific staining and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis for osteo/odontogenic and adipogenic markers. This is the first report to indicate that human periapical cysts contain cells with MSC-like properties. Taken together, our findings indicate that human periapical cysts could be a rich source of MSCs.

Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

PubMed

Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

2018-01-01

The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Circulating tumor cell isolation: the assets of filtration methods with polycarbonate track-etched filters

PubMed Central

Dolfus, Claire; Piton, Nicolas; Toure, Emmanuel

2015-01-01

Circulating tumor cells (CTCs) arise from primary or secondary tumors and enter the bloodstream by active or passive intravasation. Given the low number of CTCs, enrichment is necessary for detection. Filtration methods are based on selection of CTCs by size using a filter with 6.5 to 8 µm pores. After coloration, collected CTCs are evaluated according to morphological criteria. Immunophenotyping and fluorescence in situ hybridization techniques may be used. Selected CTCs can also be cultivated in vitro to provide more material. Analysis of genomic mutations is difficult because it requires adapted techniques due to limited DNA materials. Filtration-selected CTCs have shown prognostic value in many studies but multicentric validating trials are mandatory to strengthen this assessment. Other clinical applications are promising such as follow-up, therapy response prediction and diagnosis. Microfluidic emerging systems could optimize filtration-selected CTCs by increasing selection accuracy. PMID:26543334

Isolation and Characterization of Canine Amniotic Membrane-Derived Multipotent Stem Cells

PubMed Central

Kim, Hyung-Sik; Kang, Kyung-Sun

2012-01-01

Recent studies have shown that amniotic membrane tissue is a rich source of stem cells in humans. In clinical applications, the amniotic membrane tissue had therapeutic effects on wound healing and corneal surface reconstruction. Here, we successfully isolated and identified multipotent stem cells (MSCs) from canine amniotic membrane tissue. We cultured the canine amniotic membrane-derived multipotent stem cells (cAM-MSCs) in low glucose DMEM medium. cAM-MSCs have a fibroblast-like shape and adhere to tissue culture plastic. We characterized the immunophenotype of cAM-MSCs by flow cytometry and measured cell proliferation by the cumulative population doubling level (CPDL). We performed differentiation studies for the detection of trilineage multipotent ability, under the appropriate culture conditions. Taken together, our results show that cAM-MSCs could be a rich source of stem cells in dogs. Furthermore, cAM-MSCs may be useful as a cell therapy application for veterinary regenerative medicine. PMID:23024756

Locally infiltrative ameloblastic fibroma in a rhesus macaque (Macaca mulatta) with characterizations of its proliferating activity and biological behavior

PubMed Central

Liu, David X.; Doyle, Lara A.; Bouljihad, Mostafa T.; Didier, Peter J.; Gilbert, Margaret H.; Wang, Xiaolei; Pahar, Bapi; Bohm, Rudolf P.; Veazey, Ronald S.; Lackner, Andrew A.

2014-01-01

An 8-year-old male rhesus macaque (Macaca mulatta) presented with unilateral enlargement of the left mandible. Radiographs revealed a marked expansion of the left mandible with a multilocular radiolucent mass with abundant osteolysis. The mass was grossly firm, fleshy, and gelatinous on the cut surface. Histologically, the mass was locally infiltrative and composed of neoplastic epithelial and mesenchymal components that stained positive for cytokeratin and vimentin, respectively. Occasional densely spherical condensations of fibroblasts resembling the cap stage of odontogenesis were present in the mesenchyma. Immunohistochemical staining with Ki-67, S-100, and CD34 indicated that both epithelial and mesenchymal components of the neoplasm had low proliferation. Alcian blue, periodic acid–Schiff, and trichrome stains showed an immature stromal component with no collagen formation. Based on the clinical, histologic, and immunophenotypic features, the tumor was identified as a locally infiltrative ameloblastic fibroma. PMID:22529141

Pagetoid reticulosis (epitheliotropic cutaneous T-cell lymphoma) in an adult alpaca (Vicugna pacos).

PubMed

Hasbach, Andrea E; Stern, Adam W

2016-07-01

A 9-year-old, intact female alpaca (Vicugna pacos) was presented for a second opinion with a 1-year history of nonpruritic, multifocal scaling and crusted cutaneous lesions, mainly involving skin on the face, axillae, and ventral abdomen. Clinical abnormalities were limited to the skin, and the alpaca was otherwise healthy. The initial veterinarian had examined the alpaca, found no evidence of ectoparasites with laboratory testing, and had tried several trial therapies including oral antibiotics, ivermectin, and topical use of betadine solution. At the time of presentation, the lesions had neither improved nor worsened with any attempted therapy, and multiple skin biopsies were collected. Histopathology and immunohistochemical staining findings were consistent with the pagetoid reticulosis type of cutaneous epitheliotropic T-cell lymphoma. Our report describes the clinical, histopathologic, and immunophenotypic features of pagetoid reticulosis epitheliotropic cutaneous T-cell lymphoma in an alpaca. © 2016 The Author(s).

Isolation of circulating plasma cells from blood of patients diagnosed with clonal plasma cell disorders using cell selection microfluidics.

PubMed

Kamande, Joyce W; Lindell, Maria A M; Witek, Małgorzata A; Voorhees, Peter M; Soper, Steven A

2018-02-19

Blood samples from patients with plasma cell disorders were analysed for the presence of circulating plasma cells (CPCs) using a microfluidic device modified with monoclonal anti-CD138 antibodies. CPCs were immuno-phenotyped using a CD38/CD56/CD45 panel and identified in 78% of patients with monoclonal gammopathy of undetermined significance (MGUS), all patients with smouldering and symptomatic multiple myeloma (MM), and none in the controls. The burden of CPCs was higher in patients with symptomatic MM compared with MGUS and smouldering MM (p < 0.05). FISH analysis revealed the presence of chromosome 13 deletions in CPCs that correlated with bone marrow results. Point mutations in KRAS were identified, including different mutations from sub-clones derived from the same patient. The microfluidic assay represents a highly sensitive method for enumerating CPCs and allows for the cytogenetic and molecular characterization of CPCs.

A "live" biopsy in a small-cell lung cancer patient by detection of circulating tumor cells.

PubMed

Bevilacqua, Simona; Gallo, Marianna; Franco, Renato; Rossi, Antonio; De Luca, Antonella; Rocco, Gaetano; Botti, Gerardo; Gridelli, Cesare; Normanno, Nicola

2009-07-01

A 71-year-old patient with a pulmonary lesion was diagnosed with a low-grade neuroendocrine tumor following examination of a fine needle aspiration biopsy. Analysis of a peripheral blood sample with the CellSearch system revealed the presence of putative circulating tumor cells (CTC) that were positive for EpCAM and cytokeratin (CK) expression. Since EpCAM is not usually expressed in neuroendocrine tumors, we performed a biopsy of liver metastases. Morphological and immunophenotypical characterization revealed that the patient had an EpCAM and CK positive small-cell lung cancer (SCLC). By using the CellSearch apparatus, EpCAM/CK positive CTC were detected in peripheral blood samples from 3 out of 4 additional SCLC patients. This study is the first to demonstrate that CTC can be identified in SCLC patients by using the CellSearch system.

The PAX2-null immunophenotype defines multiple lineages with common expression signatures in benign and neoplastic oviductal epithelium

PubMed Central

Ning, Gang; Bijron, Jonathan G.; Yamamoto, Yusuke; Wang, Xia; Howitt, Brooke E.; Herfs, Michael; Yang, Eric; Hong, Yue; Cornille, Maxence; Wu, Lingyan; Hanamornroongruang, Suchanan; McKeon, Frank D.; Crum, Christopher P.; Xian, Wa

2014-01-01

The oviducts contain high grade serous cancer (HGSC) precursors (serous tubal intraepithelial neoplasia or STINs), which are γ-H2AXp- and TP53 mutation-positive. Although they express wild type p53, secretory cell outgrowths (SCOUTs) are associated with older age and serous cancer; moreover both STINs and SCOUTs share a loss of PAX2 expression (PAX2n). We evaluated PAX2 expression in proliferating adult and embryonic oviductal cells, normal mucosa, SCOUTs, Walthard cell nests (WCNs), STINs and HGSCs, and the expression of genes chosen empirically or from SCOUT expression arrays. Clones generated in vitro from embryonic gynecologic tract and adult fallopian tube were Krt7p/PAX2n/EZH2p and underwent ciliated (PAX2n/EZH2n/FOXJ1p) and basal (Krt7n/EZH2n/Krt5p) differentiation. Similarly non-ciliated cells in normal mucosa were PAX2p but became PAX2n in multilayered epithelium undergoing ciliated or basal (Walthard cell nests or WCN) cell differentiation. PAX2n SCOUTs fell into two groups; Type I were secretory or secretory/ciliated with a “tubal” phenotype and were ALDH1n and β-cateninmem (membraneous only). Type II displayed a columnar to pseudostratified (endometrioid) phenotype, with an EZH2p, ALDH1p, β-cateninnc (nuclear and cytoplasmic), stathminp, LEF1p, RCN1p and RUNX2p expression signature. STINs and HGSCs shared the Type I immunophenotype of PAX2n, ALDH1n, β-cateninmem, but highly expressed EZH2p, LEF1p, RCN1p, and stathminp. This study, for the first time, links PAX2n with proliferating fetal and adult oviductal cells undergoing basal and ciliated differentiation and shows that this expression state is maintained in SCOUTs, STINs and HGSCs. All three entities can demonstrate a consistent perturbation of genes involved in potential tumor suppressor gene silencing (EZH2), transcriptional regulation (LEF1), regulation of differentiation (RUNX2), calcium binding (RCN1) and oncogenesis (stathmin). This shared expression signature between benign and neoplastic entities links normal progenitor cell expansion to abnormal and neoplastic outgrowth in the oviduct and exposes a common pathway that could be a target for early prevention. PMID:25130537

[Primary central nervous system diffuse large B cell lymphoma: a clinicopathologic and molecular study].

PubMed

Ma, Z P; Ainiwaer, Babayi; Liu, Z Y; Shi, X L; Cui, W L; Zhang, W; Li, X X

2016-11-08

Objective: To investigate clinicopathologic characteristics, immunophenotype and EB virus-related molecular genetic alterations in primary central nervous system diffuse large B cell lymphoma (DLBCL) along with correlation with clinical prognosis. Methods: A total of 30 cases of primary central nervous system DLBCL were retrospectively studied by retrieving clinical data, histological evaluation and immunophenotyping by EnVision two steps methods. The expression of EBER mRNA was detected by in situ hybridization and bcl-2, bcl-6 and C-MYC gene abnormalities were analyzed by interphase fluorescence in situ hybridization. Results: The cases included 18 males and 12 females (sex ratio of 1.5∶1.0) with an age ranging from 24 to 78 years (average age of 52 years, the median age of 53 years). The single primary clinical presentation was focal neurologic deficits. Tumor locations were supratentorial (21 cases), subtentorial (7 cases), involving both locations in 2 cases. Diffuse growth pattern was observed with large lymphoid cells mostly resembling centroblasts with abundant basophilic cytoplasm with oval to round, vesicular nuclei containing fine chromatin. An angiocentric and angiodestructive growth pattern was also present. Other features included perivascular space invasion. Immunohistochemical staining using a panel of CD10, bcl-6 and MUM1, six cases were germinal center-like (GCB) and 24 cases were non-germinal central-like (non-GCB). The positive rates of bcl-2, bcl-6 and C-MYC were 53.3% (16/30), 80.0% (24/30) and 20.0% (6/30), respectively. Genetic alterations were detected by FISH and the gene arrangement rates of bcl-2, bcl-6 and C-MYC were 3.3% (1/30), 16.7% (5/30) and 3.3% (1/30), respectively. There were 19 cases in stage 0-1 disease and 11 cases had stage 2-3 disease. Postoperative follow-up for average 13.6 months showed the median survival of 10 months, one-year survival of 46.7% and 16 patients died within a year. Conclusions: The clinical prognosis of primary central nerve system DLBCL depends on age, clinical performence status score, IPI score, immune classification and treatment. Patients typically progress rapidly with the high mortality within one year of diagnosis. Surgical resection combined with high-dose methotrexate or cytarabine chemotherapy offer the best treatment option.

Overexpression of syndecan-1, MUC-1, and putative stem cell markers in breast cancer leptomeningeal metastasis: a cerebrospinal fluid flow cytometry study.

PubMed

Cordone, Iole; Masi, Serena; Summa, Valentina; Carosi, Mariantonia; Vidiri, Antonello; Fabi, Alessandra; Pasquale, Alessia; Conti, Laura; Rosito, Immacolata; Carapella, Carmine Maria; Villani, Veronica; Pace, Andrea

2017-04-11

Cancer is a mosaic of tumor cell subpopulations, where only a minority is responsible for disease recurrence and cancer invasiveness. We focused on one of the most aggressive circulating tumor cells (CTCs) which, from the primitive tumor, spreads to the central nervous system (CNS), evaluating the expression of prognostic and putative cancer stem cell markers in breast cancer (BC) leptomeningeal metastasis (LM). Flow cytometry immunophenotypic analysis of cerebrospinal fluid (CSF) samples (4.5 ml) was performed in 13 consecutive cases of BCLM. Syndecan-1 (CD138), MUC-1 (CD227) CD45, CD34, and the putative cancer stem cell markers CD15, CD24, CD44, and CD133 surface expression were evaluated on CSF floating tumor cells. The tumor-associated leukocyte population was also characterized. Despite a low absolute cell number (8 cell/μl, range 1-86), the flow cytometry characterization was successfully conducted in all the samples. Syndecan-1 and MUC-1 overexpression was documented on BC cells in all the samples analyzed; CD44, CD24, CD15, and CD133 in 77%, 75%, 70%, and 45% of cases, respectively. A strong syndecan-1 and MUC-1 expression was also documented by immunohistochemistry on primary breast cancer tissues, performed in four patients. The CSF tumor population was flanked by T lymphocytes, with a different immunophenotype between the CSF and peripheral blood samples (P ≤ 0.02). Flow cytometry can be successfully employed for solid tumor LM characterization even in CSF samples with low cell count. This in vivo study documents that CSF floating BC cells overexpress prognostic and putative cancer stem cell biomarkers related to tumor invasiveness, potentially representing a molecular target for circulating tumor cell detection and LM treatment monitoring, as well as a primary target for innovative treatment strategies. The T lymphocyte infiltration, documented in all CSF samples, suggests a possible involvement of the CNS lymphatic system in both lymphoid and cancer cell migration into and out of the meninges, supporting the extension of a new form of cellular immunotherapy to LM. Due to the small number of cases, validation on large cohorts of patients are warranted to confirm these findings and to evaluate the impact and value of these results for diagnosis and management of LM.

Equine bone marrow-derived mesenchymal stromal cells are heterogeneous in MHC class II expression and capable of inciting an immune response in vitro

PubMed Central

2014-01-01

Introduction The horse is a valuable species to assess the effect of allogeneic mesenchymal stromal cells (MSCs) in regenerative treatments. No studies to date have examined recipient response to major histocompatibility complex (MHC)-mismatched equine MSCs. The purposes of this study were to immunophenotype MSCs from horses of known MHC haplotype and to compare the immunogenicity of MSCs with differing MHC class II expression. Methods MSCs and peripheral blood leukocytes (PBLs) were obtained from Thoroughbred horses (n = 10) of known MHC haplotype (ELA-A2, -A3, and -A9 homozygotes). MSCs were cultured through P8; cells from each passage (P2 to P8) were cryopreserved until used. Immunophenotyping of MHC class I and II, CD44, CD29, CD90, LFA-1, and CD45RB was performed by using flow cytometry. Tri-lineage differentiation assays were performed to confirm MSC multipotency. Recombinant equine IFN-γ was used to stimulate MHC class II negative MSCs in culture, after which expression of MHC class II was re-examined. To assess the ability of MHC class II negative or positive MSCs to stimulate an immune response, modified one-way mixed leukocyte reactions (MLRs) were performed by using MHC-matched and mismatched responder PBLs and stimulator PBLs or MSCs. Proliferation of gated CFSE-labeled CD3+ responder T cells was evaluated via CFSE attenuation by using flow cytometry and reported as the number of cells in the proliferating T-cell gate. Results MSCs varied widely in MHC class II expression despite being homogenous in terms of “stemness” marker expression and ability to undergo trilineage differentiation. Stimulation of MHC class II negative MSCs with IFN-γ resulted in markedly increased expression of MHC class II. MLR results revealed that MHC-mismatched MHC class II-positive MSCs caused significantly increased responder T-cell proliferation compared with MHC-mismatched MHC class II-negative and MHC-matched MSCs, and equivalent to that of the positive control of MHC-mismatched leukocytes. Conclusions The results of this study suggest that MSCs should be confirmed as MHC class II negative before allogeneic application. Additionally, it must be considered that even MHC class II-negative MSCs could upregulate MHC class II expression if implanted into an area of active inflammation, as demonstrated with in vitro stimulation with IFN-γ. PMID:24461709

High-grade endometrial carcinoma: serous and grade 3 endometrioid carcinomas have different immunophenotypes and outcomes.

PubMed

Alkushi, Abdulmohsen; Köbel, Martin; Kalloger, Steve E; Gilks, C Blake

2010-07-01

High-grade endometrial carcinomas are a heterogeneous group of tumors and include grade 3 endometrioid (EC-3), serous (SC), and clear cell carcinomas (CCC). There are conflicting data about the prognosis of these subtypes of high-grade endometrial carcinoma; this may be a result of lack of reproducibility in classifying tumor cell type. The purpose of this study was to examine differences in immunophenotype and outcome in a series of high-grade endometrial carcinomas, focusing on the comparison of EC-3 versus SC. We selected 180 endometrial carcinomas of SC, EC, or CCC type. No mixed carcinomas were included in the study. We chose the following immunohistochemical markers, estrogen receptor (ER), insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), p16, p53, progesterone receptor (PR), and phosphatase and tensin homolog (PTEN) as being significantly differentially expressed in endometrial carcinoma subtypes. The tumors were stratified into 4 groups on the basis of their cell type and grade: EC grade 1 or 2, EC-3, SC, and CCC. Univariate survival analysis revealed significant differences in outcome between the 4 groups (P<0.0001), with significantly longer disease-specific survival for grade 1 or 2 EC versus EC-3 (P=0.0001), and EC-3 versus SC (P=0.0003). p16, PTEN, and IMP3 expression was observed more frequently in SC compared with EC-3 (P<0.0001, P=0.021, and P=0.031, respectively). These 3 markers showed the highest sensitivity and specificity in distinguishing between EC-3 and SC, with receiver operating characteristics area under the curve of 0.85, 0.69, and 0.71, respectively. ER and p53 approached but did not reach significance for differential expression in EC-3 versus SC (P=0.055 and P=0.068, respectively). A combination of p16 and PTEN predicts EC-3 versus SC with a sensitivity of 90.0% and specificity of 96.8%. p16 and PTEN can aid in distinguishing between EC-3 and SC of the endometrium, and are superior to ER, PR, and p53 for this purpose. EC-3 carcinomas have a significantly better prognosis than SC carcinomas of the endometrium.

The Genetic Architecture of the Human Immune System: A Bioresource for Autoimmunity and Disease Pathogenesis

PubMed Central

Roederer, Mario; Quaye, Lydia; Mangino, Massimo; Beddall, Margaret H.; Mahnke, Yolanda; Chattopadhyay, Pratip; Tosi, Isabella; Napolitano, Luca; Barberio, Manuela Terranova; Menni, Cristina; Villanova, Federica; Di Meglio, Paola; Spector, Tim D.; Nestle, Frank O.

2015-01-01

Summary Despite recent discoveries of genetic variants associated with autoimmunity and infection, genetic control of the human immune system during homeostasis is poorly understood. We undertook a comprehensive immunophenotyping approach, analysing 78,000 immune traits in 669 female twins. From the top 151 heritable traits (up to 96% heritable), we used replicated GWAS to obtain 297 SNP associations at 11 genetic loci explaining up to 36% of the variation of 19 traits. We found multiple associations with canonical traits of all major immune cell subsets, and uncovered insights into genetic control for regulatory T cells. This dataset also revealed traits associated with loci known to confer autoimmune susceptibility, providing mechanistic hypotheses linking immune traits with the etiology of disease. Our data establish a bioresource that links genetic control elements associated with normal immune traits to common autoimmune and infectious diseases, providing a shortcut to identifying potential mechanisms of immune-related diseases. PMID:25772697

[Mesenchymal stem cells: definitions, culture and potential applications].

PubMed

Ceron, Willy; Lozada-Requena, Iván; Ventocilla, Kiomi; Jara, Sandra; Pinto, Milagros; Cabello, Marco; Aguilar, José L

2016-01-01

In recent years, mesenchymal stem cells (MSC) have become very important due to their high plasticity and their ability to release paracrine factors able to interact with various cell types, tissues and organs. The use of MSC in regenerative medicine became of vital importance, since they do not express histocompatibility MHC molecules class II nor costimulant molecules, and low expression of MHC class I, will not be rejected by individuals of same species, they could be used in an autologous, and eventually, allogeneic manner. However, it is important to scientifically demonstrate many properties, including immunomodulatory ones. Having several sources of obtaining, it should be standardized the best one to ensure the purity and quality of these cells. Finally, it is important when working with these cells, that characteristics of cell culture, immunophenotyping and differentiation capacity are fully demonstrated. MSC have been applied in several clinical uses. Among them, their ability to improve, and even heal chronic ulcers, as diabetic, has attracted attention for its potential therapeutic impact.

Intraepithelial lymphocytes, scores, mimickers and challenges in diagnosing gluten-sensitive enteropathy (celiac disease)

PubMed Central

Sergi, Consolato; Shen, Fan; Bouma, Gerd

2017-01-01

The upper digestive tract is routinely scoped for several causes of malabsorption, and the number of duodenal biopsy specimens has increased notably in the last 10 years. Gluten-sensitive enteropathy (GSE) is an autoimmune disease, which shows an increasing prevalence worldwide and requires a joint clinico-pathological approach. The classical histopathology of GSE with partial or total villous blunting is well recognized, but the classification of GSE is not straightforward. Moreover, several mimickers of GSE with intraepithelial lymphocytosis have been identified in the last 20 years, with drug interactions and medical comorbidities adding to the conundrum. In this review, we report on the normal duodenal mucosa, the clinical presentation and laboratory diagnosis of GSE, the duodenal intraepithelial lymphocytes and immunophenotype of GSE-associated lymphocytes, the GSE mimickers, the differences “across oceans” among guidelines in diagnosing GSE, and the use of a synoptic report for reporting duodenal biopsies in both children and adults in the 21st century. PMID:28216964

Prognostic value of baseline absolute lymphocyte concentration and neutrophil/lymphocyte ratio in dogs with newly diagnosed multi-centric lymphoma.

PubMed

Mutz, M; Boudreaux, B; Kearney, M; Stroda, K; Gaunt, S; Shiomitsu, K

2015-12-01

Canine multi-centric B-cell lymphoma shares similarities with diffuse large B-cell (Non-Hodgkin's) lymphoma (NHL) in people. In people with NHL, lymphopenia at diagnosis and first relapse and neutrophil/lymphocyte ratio (N:L) > 3.5 are negative prognostic factors for survival. The objective of this study was to determine if lymphocyte concentration at diagnosis and first relapse and N:L were prognostic for survival in dogs with newly diagnosed multi-centric lymphoma. Medical records of 77 dogs with multi-centric lymphoma treated with a CHOP-based chemotherapy protocol were retrospectively evaluated. Absolute lymphocyte concentration and N:L ratio at presentation of dogs pre-treated with steroids was not significantly different from dogs who had not received steroids. On multivariate analysis, only immunophenotype remained significant for progression-free survival (PFS), whereas no variables remained significant for ST. A prospective study of these haematologic variables is warranted to assess their true significance. © 2013 John Wiley & Sons Ltd.

Retrospective analysis for treatment of naïve canine multicentric lymphoma with a 15-week, maintenance-free CHOP protocol.

PubMed

Curran, K; Thamm, D H

2016-08-01

Standard of care treatment of dogs with multicentric lymphoma includes combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP); however, owners may be hesitant to commit the resources necessary to complete a lengthy, multi-drug protocol. One hundred thirty-four client-owned dogs with multicentric lymphoma were treated with a 15-week CHOP chemotherapy protocol. The overall response rate was 98% with 104 dogs experiencing a complete response (CR). The median progression-free survival (PFS) time for all dogs was 176 days, and the median disease-specific overall survival time was 311 days. Prognostic factors identified on multivariate analysis as significant for PFS included substage, immunophenotype, hospitalization for adverse events, need for dose reduction, presence of neutrophilia at diagnosis, presence of anemia and experiencing a CR as best response to therapy. In conclusion, this protocol may be a viable alternative to CHOP protocols using a larger number of treatments. © 2015 John Wiley & Sons Ltd.

Immunological Identification of Trypanosoma cruzi Lineages in Human Infection Along the Endemic Area

PubMed Central

Risso, Marikena G.; Sartor, Paula A.; Burgos, Juan M.; Briceño, Luis; Rodríguez, Eva M.; Guhl, Felipe; Chavez, Omar Triana; Espinoza, Berta; Monteón, Victor M.; Russomando, Graciela; Schijman, Alejandro G.; Bottasso, Oscar A.; Leguizamón, Maria Susana

2011-01-01

Genotyping studies show a polarized geographic distribution of Trypanosoma cruzi lineages in humans. Here, we assessed their distribution along Latin America through an immunological approach we designated Western blot (WB) assay with Trypomastigote small-surface antigen (TSSA) I and TSSA II (TSSA-WB). These antigens are expressed by T. cruzi I (TCI; now TcI) and T. cruzi II (TCII; reclassified as TcII to TcVI) parasites. TSSA-WB showed good concordance with genotyping tests. An unexpected frequency of TSSA II recognition was observed in Colombia, Venezuela, and Mexico (northern region of Latin America). In Argentina and Paraguay (southern region), immunophenotyping confirmed the already reported TCII (TcII to TcVI) dominance. The lineage distribution between these regions showed significant difference but not among countries within them (except for Colombia and Venezuela). TSSA-WB shows TCII emergence in the northern region where TCI was reported as dominant or even as the unique T. cruzi lineage infecting humans. PMID:21212206

The promising potential of menstrual stem cells for antenatal diagnosis and cell therapy.

PubMed

Khoury, Maroun; Alcayaga-Miranda, Francisca; Illanes, Sebastián E; Figueroa, Fernando E

2014-01-01

Menstrual-derived stem cells (MenSCs) are a new source of mesenchymal stem cells isolated from the menstrual fluid. Currently, there is a growing interest in their clinical potential due to fact that they are multipotent, highly proliferative, and easy to obtain in a non-invasive manner. Sampling can be repeated periodically in a simplified and reproducible manner devoid of complications that no existing cell source can match. MenSCs are also free of ethical dilemmas, and display novel properties with regard to presently known adult derived stem cells. This review details their distinctive biological properties regarding immunophenotype and function, proliferation rate, differentiation potential, and paracrine effects mediated by secreted factors. Their possible role in antenatal diagnosis is also discussed. While more insight on their immunomodulatory and diagnostic properties is needed, the impact of clinical and epidemiological factors, such as age, use of contraceptives, or hormonal status still requires further investigations to properly assess their current and future use in clinical application and diagnosis.

USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation.

PubMed

Torre, Sabrina; Polyak, Maria J; Langlais, David; Fodil, Nassima; Kennedy, James M; Radovanovic, Irena; Berghout, Joanne; Leiva-Torres, Gabriel A; Krawczyk, Connie M; Ilangumaran, Subburaj; Mossman, Karen; Liang, Chen; Knobeloch, Klaus-Peter; Healy, Luke M; Antel, Jack; Arbour, Nathalie; Prat, Alexandre; Majewski, Jacek; Lathrop, Mark; Vidal, Silvia M; Gros, Philippe

2017-01-01

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15 L749R ) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15 L749R -associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.

Endometrial stromal tumors: the new WHO classification.

PubMed

Conklin, Christopher M J; Longacre, Teri A

2014-11-01

Endometrial stromal tumors are rare uterine mesenchymal neoplasms that have intrigued pathologists for years, not only because they commonly pose diagnostic dilemmas, but also because the classification and pathogenesis of these tumors has been widely debated. The current World Health Organization recognizes 4 categories of endometrial stromal tumor: endometrial stromal nodule (ESN), low-grade endometrial stromal sarcoma (LG-ESS), high-grade endometrial stromal sarcoma (HG-ESS), and undifferentiated uterine sarcoma (UUS). uterine sarcoma. These categories are defined by the presence of distinct translocations as well as tumor morphology and prognosis. Specifically, the JAZF1-SUZ12 (formerly JAZF1-JJAZ1) fusion identifies a large proportion of ESN and LG-ESSs, whereas the YWHAE-FAM22 translocation identifies HG-ESSs. The latter tumors appear to have a prognosis intermediate between LG-ESS and UUS, which exhibits no specific translocation pattern. This review (1) presents the clinicopathologic features of endometrial stromal tumors; (2) discusses their immunophenotype; and (3) highlights the recent advances in molecular genetics which explain their pathogenesis and lend support for a new classification system.

Establishment of a novel feline leukemia virus (FeLV)-negative B-cell cell line from a cat with B-cell lymphoma.

PubMed

Mochizuki, Hiroyuki; Takahashi, Masashi; Nishigaki, Kazuo; Ide, Tetsuya; Goto-Koshino, Yuko; Watanabe, Shinya; Sato, Hirofumi; Sato, Masahiko; Kotera, Yukiko; Fujino, Yasuhito; Ohno, Koichi; Uchida, Kazuyuki; Tsujimoto, Hajime

2011-04-15

We established a novel feline B-cell line, MS4, from the neoplastic pleural effusion of a cat with cutaneous B-cell lymphoma. Immunophenotype staining of the MS4 cells was positive for CD20, CD79α, and IgA and negative for CD3, CD4, CD5, CD8α, CD18, CD21, CD22, IgM, IgG, Ig light chain, and MHC class II. PCR analysis for immunoglobulin heavy chain gene rearrangements revealed a monoclonal rearrangement, whereas no clonal rearrangement of the T-cell receptor γ gene was detected. Southern blotting with an exogenous feline leukemia virus (FeLV) U3 probe revealed no integration of exogenous FeLV provirus. The MS4 cell line is the first FeLV-negative feline B-cell lymphoma cell line, and may be used to investigate the pathogenesis of spontaneously occurring feline lymphoma and the development of new therapies. Copyright © 2011 Elsevier B.V. All rights reserved.

A Case of Early Ocular Manifestation of Maculopathy in a 37 year Old Male Patient of CLL.

PubMed

Mehdi, Syed Riaz; Tandon, Nishi; Khan, Sufia Ahmad; Ahmad, Sharique

2014-09-01

B cell chronic lymphocytic leukemia (CLL) also called chronic lymphocytic leukemia is a disease of elderly, but in rare cases it can occur in young adults. Majority of patients present with no specific symptoms, and CLL is suspected on the basis of high total and relative lymphocyte counts, discovered on routine blood examination. This patient was referred from medicine outpatient in our institute, Era's Lucknow Medical College & Hospital for routine complete blood count. His total leukocyte count came out to be alarmingly high. The general blood picture and bone marrow examinations were consistent with CLL. On immunophenotyping CD5 and CD23 were positive. His cervical lymph nodes biopsy was reported as a case of CLL/small lymphocytic lymphoma. On his complaint of hazy vision his funduscopy revealed maculopathy. This makes our case a unique and rare one, as maculopathy in a young patient of CLL has not yet been reported from India.

Gastrointestinal stromal tumors: the histology report.

PubMed

Dei Tos, Angelo P; Laurino, Licia; Bearzi, Italo; Messerini, Luca; Farinati, Fabio

2011-03-01

Gastrointestinal stromal tumors (GISTs) represent a mesenchymal neoplasm occurring primarily in the gastrointestinal tract, and showing differentiation toward the interstitial cell of Cajal. Its incidence is approximately 15 case/100,000/year. Stomach and small bowel are the most frequently affected anatomic sites. GIST represents a morphological, immunophenotypical and molecular distinct entity, the recognition of which has profound therapeutic implications. In fact, they have shown an exquisite sensitivity to treatment with the tyrosine kinase inhibitor imatinib. Diagnosis relies upon morphology along with immunodetection of KIT and/or DOG1. When dealing with KIT negative cases, molecular analysis of KIT/PDGFRA genes may help in confirming diagnosis. Molecular evaluation of both genes are in any case recommended as mutational status provides key predictive information. Pathologists also play a key role in providing an estimation of the risk of biological aggressiveness, which is currently based on anatomic location of the tumor, size, and mitotic activity. Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd.. All rights reserved.

The genetic architecture of the human immune system: a bioresource for autoimmunity and disease pathogenesis.

PubMed

Roederer, Mario; Quaye, Lydia; Mangino, Massimo; Beddall, Margaret H; Mahnke, Yolanda; Chattopadhyay, Pratip; Tosi, Isabella; Napolitano, Luca; Terranova Barberio, Manuela; Menni, Cristina; Villanova, Federica; Di Meglio, Paola; Spector, Tim D; Nestle, Frank O

2015-04-09

Despite recent discoveries of genetic variants associated with autoimmunity and infection, genetic control of the human immune system during homeostasis is poorly understood. We undertook a comprehensive immunophenotyping approach, analyzing 78,000 immune traits in 669 female twins. From the top 151 heritable traits (up to 96% heritable), we used replicated GWAS to obtain 297 SNP associations at 11 genetic loci, explaining up to 36% of the variation of 19 traits. We found multiple associations with canonical traits of all major immune cell subsets and uncovered insights into genetic control for regulatory T cells. This data set also revealed traits associated with loci known to confer autoimmune susceptibility, providing mechanistic hypotheses linking immune traits with the etiology of disease. Our data establish a bioresource that links genetic control elements associated with normal immune traits to common autoimmune and infectious diseases, providing a shortcut to identifying potential mechanisms of immune-related diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Integration of lyoplate based flow cytometry and computational analysis for standardized immunological biomarker discovery.

PubMed

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R; Nestle, Frank O

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.

Integration of Lyoplate Based Flow Cytometry and Computational Analysis for Standardized Immunological Biomarker Discovery

PubMed Central

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A. Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R.; Nestle, Frank O.

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases. PMID:23843942

Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors.

PubMed

Patin, Etienne; Hasan, Milena; Bergstedt, Jacob; Rouilly, Vincent; Libri, Valentina; Urrutia, Alejandra; Alanio, Cécile; Scepanovic, Petar; Hammer, Christian; Jönsson, Friederike; Beitz, Benoît; Quach, Hélène; Lim, Yoong Wearn; Hunkapiller, Julie; Zepeda, Magge; Green, Cherie; Piasecka, Barbara; Leloup, Claire; Rogge, Lars; Huetz, François; Peguillet, Isabelle; Lantz, Olivier; Fontes, Magnus; Di Santo, James P; Thomas, Stéphanie; Fellay, Jacques; Duffy, Darragh; Quintana-Murci, Lluís; Albert, Matthew L

2018-03-01

The quantification and characterization of circulating immune cells provide key indicators of human health and disease. To identify the relative effects of environmental and genetic factors on variation in the parameters of innate and adaptive immune cells in homeostatic conditions, we combined standardized flow cytometry of blood leukocytes and genome-wide DNA genotyping of 1,000 healthy, unrelated people of Western European ancestry. We found that smoking, together with age, sex and latent infection with cytomegalovirus, were the main non-genetic factors that affected variation in parameters of human immune cells. Genome-wide association studies of 166 immunophenotypes identified 15 loci that showed enrichment for disease-associated variants. Finally, we demonstrated that the parameters of innate cells were more strongly controlled by genetic variation than were those of adaptive cells, which were driven by mainly environmental exposure. Our data establish a resource that will generate new hypotheses in immunology and highlight the role of innate immunity in susceptibility to common autoimmune diseases.

High prevalence of atypical hyperplasia in the endometrium of patients with epithelial ovarian cancer.

PubMed

Mingels, Marjanka J J M; Masadah, Rina; Geels, Yvette P; Otte-Höller, Irene; de Kievit, Ineke M; van der Laak, Jeroen A W M; van Ham, Maaike A P C; Bulten, Johan; Massuger, Leon F A G

2014-08-01

The aim of the present study is to determine the prevalence of endometrial premalignancies in women diagnosed with epithelial ovarian cancer (EOC). Endometrial and ovarian specimens of 186 patients with EOC were retrospectively selected using the nationwide pathology network and registry, and sections were comprehensively reviewed: 136 (73%) serous, 19 (10%) endometrioid, 15 (8%) mucinous, seven (4%) clear cell, and nine (5%) undifferentiated. Immunohistochemical phenotypes were compared for patients with serous EOC with concurrent endometrial pathology. In 31%, endometrial (pre)malignancy was found: carcinoma in 3%, endometrial intraepithelial carcinoma (EIC) in 4%, and atypical hyperplasia in 24%. Atypical hyperplasia was found in 47% of endometrioid EOCs but in 7% to 33% of other subtypes. Body mass index was higher concurrent to atypical hyperplasia (P=.001). Serous EOC and EIC immunophenotypes were comparable, whereas atypical hyperplasia was expressed differently. Apart from synchronous endometrial carcinoma, endometrial premalignancies should be taken into account when determining optimal treatment for women diagnosed with EOC. Copyright© by the American Society for Clinical Pathology.

Functional characterization of mouse spinal cord infiltrating CD8+ lymphocytes

PubMed Central

Deb, Chandra; Howe, Charles L

2011-01-01

Understanding the immunopathogenesis of neuroimmunological diseases of the CNS requires a robust method for isolating and characterizing the immune effector cells that infiltrate the spinal cord in animal models. We have developed a simple and rapid isolation method that produces high yields of spinal cord infiltrating leukocytes from a single demyelinated spinal cord and which maintains high surface expression of key immunophenotyping antigens. Using this method and the Theiler’s virus model of chronic demyelination, we report the presence of spinal cord infiltrating acute effector CD8+ lymphocytes that are CD45hiCD44loCD62L− and a population of spinal cord infiltrating target effector memory CD8+ lymphocytes that are CD45hiCD44hiCD62L−. These cells respond robustly to ex vivo stimulation by producing interferon γ but do not exhibit specificity for Theiler’s virus in a cytotoxicity assay. We conclude that target-derived lymphocytes in a mouse model of chronic spinal cord demyelination may have unique functional specificities. PMID:19596449

Microglial brain region-dependent diversity and selective regional sensitivities to ageing

PubMed Central

Grabert, Kathleen; Michoel, Tom; Karavolos, Michail H; Clohisey, Sara; Baillie, J Kenneth; Stevens, Mark P; Freeman, Tom C; Summers, Kim M; McColl, Barry W

2015-01-01

Microglia play critical roles in neural development, homeostasis and neuroinflammation and are increasingly implicated in age-related neurological dysfunction. Neurodegeneration often occurs in disease-specific spatially-restricted patterns, the origins of which are unknown. We performed the first genome-wide analysis of microglia from discrete brain regions across the adult lifespan of the mouse and reveal that microglia have distinct region-dependent transcriptional identities and age in a regionally variable manner. In the young adult brain, differences in bioenergetic and immunoregulatory pathways were the major sources of heterogeneity and suggested that cerebellar and hippocampal microglia exist in a more immune vigilant state. Immune function correlated with regional transcriptional patterns. Augmentation of the distinct cerebellar immunophenotype and a contrasting loss in distinction of the hippocampal phenotype among forebrain regions were key features during ageing. Microglial diversity may enable regionally localised homeostatic functions but could also underlie region-specific sensitivities to microglial dysregulation and involvement in age-related neurodegeneration. PMID:26780511

A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues.

PubMed Central

Casey, T. T.; Cousar, J. B.; Collins, R. D.

1988-01-01

Routine fixation and paraffin embedding destroys many hematopoietic and lymphoid differentiation antigens detected by flow cytometry or frozen section immunohistochemistry. On the other hand, morphologic evaluation is difficult in flow cytometric or frozen section studies. A simplified three-step plastic embedding system using acetone-fixed tissues embedded in glycol-methacrylate (GMA) resin has been found to provide both excellent morphologic and antigenic preservation. With our system, a wide variety of antigens are detected in plastic sections without trypsinization or prolonged embedding procedures; pan-B (CD19, CD22), pan-T (CD7, CD5, CD3, CD2), T-subset (CD4, CD8, CD1, CD25) markers as well as surface immunoglobulin and markers for myeloid and mononuclear-phagocyte cells are preserved. In summary, modifications of plastic embedding techniques used in this study simplify the procedure, apparently achieve excellent antigenic preservation, and facilitate evaluation of morphologic details in relation to immunocytochemical markers. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:3282442

Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia.

PubMed

Tothova, Zuzana; Krill-Burger, John M; Popova, Katerina D; Landers, Catherine C; Sievers, Quinlan L; Yudovich, David; Belizaire, Roger; Aster, Jon C; Morgan, Elizabeth A; Tsherniak, Aviad; Ebert, Benjamin L

2017-10-05

Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34 + human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents. Copyright © 2017 Elsevier Inc. All rights reserved.

Principles and applications of flow cytometry and cell sorting in companion animal medicine.

PubMed

Wilkerson, Melinda J

2012-01-01

Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.

Hairy-cell leukemia: a rare blood disorder in Asia.

PubMed

Josephine, F P; Nissapatorn, V

2006-01-01

We report a 68-year-old Indian man who was referred to the Hematology Unit for investigation for thrombocytopenia, an incidental finding during a pre-operative screening for prostatectomy. Physical examination was unremarkable. There was no splenomegaly, hepatomegaly or lymphadenopathy. Complete blood counts showed normal hemoglobin and total white cell count with moderate thrombocytopenia. Hairy-cell leukemia was diagnosed based on peripheral blood film, bone-marrow aspirate and trephine biopsy findings, supported by immunophenotyping results by flow cytometry. The purpose of this report is to create awareness of this uncommon presentation and to emphasize that a single-lineage cytopenia or absence of splenomegaly does not exclude the diagnosis of hairy-cell leukemia. Careful attention to morphological detail is important for early diagnosis, especially when low percentages of "hairy" cells are present in the peripheral blood and bone marrow. Early diagnosis is important to ensure that patients obtain maximum benefit from the newer therapeutic agents that have greatly improved the prognosis in this rare disorder.

Cardiac presentation of ALK positive anaplastic large cell lymphoma.

PubMed

Lim, Z Y; Grace, R; Salisbury, J R; Creamer, D; Jayaprakasam, A; Ho, A Y L; Devereux, S; Mufti, G J; Pagliuca, A

2005-12-01

Cardiac involvement as an initial presentation of malignant lymphoma is a rare occurrence. We report the case of an immunocompetent 29-year-old male who presented with syncope and arrythmias secondary to a ventricular cardiac mass. Transcutaneous cardiac biopsy was non-diagnostic, therefore an open cardiac biopsy was performed from which a provisional diagnosis of a cardiac inflammatory pseudotumour was made. Six months after presentation, he developed several subcutaneous lesions with systemic symptoms. Histological and immunophenotypic review of the initial cardiac biopsy revealed features consistent with a diagnosis of CD30, ALK1 positive anaplastic large cell lymphoma (ALCL). Despite intensive treatment with combination chemotherapy, there was significant progression of disease, and he died 11 months after diagnosis. The overall prognosis of cardiac lymphoma remains poor, which may be due to the often late presentation of the tumour. To our knowledge, this is the first reported case of a cardiac ALK positive ALCL. Although rare, cardiac presentation of ALCL should be added to the list of differential diagnoses of cardiac lymphomas.

CHARACTERIZATION OF AN EQUINE MACROPHAGE CELL LINE: APPLICATION TO STUDIES OF EIAV INFECTION

PubMed Central

Fidalgo-Carvalho, Isabel; Craigo, Jodi K.; Barnes, Shannon; Costa-Ramos, Carolina; Montelaro, Ronald C.

2009-01-01

EIAV is a monocyte/macrophage tropic virus. To date, even though EIAV has been under investigation for numerous years, very few details have been elucidated about EIAV/macrophage interactions. This is largely due to the absence of an equine macrophage cell line that would support viral replication. Herein we describe the spontaneous immortalization and generation of a clonal equine macrophage-like (EML) cell line with the functional and immunophenotype characteristics of differentiated equine monocyte derived macrophage(s) (eMDM(s)). These cells possess strong non-specific esterase (NSE) activity, are able to phagocytose fluorescent bioparticles, and produce nitrites in response to LPS. The EML-3C cell line expresses the EIAV receptor for cellular entry (ELR1) and supports replication of the virulent EIAVPV biological clone. Thus, EML-3C cells provide a useful cell line possessing equine macrophage related properties for the growth and study of EIAV infection as well as of other equine macrophage tropic viruses. PMID:19038510

Variant hairy cell leukemia following papillary urothelial neoplasm of bladder.

PubMed

Beyan, Cengiz; Kaptan, Kürsat

2014-03-01

A 65 years old man was admitted with multiple lymphadenopathy, weight loss, night sweats and fatigue for 2 months. He had been treated for bladder cancer 2 years ago. Leukocyte count was 37.9 x10(9)/l. Peripheral blood smear had 91% lymphocytes. Lymphocytes had large nuclei with prominent nucleoli, heterogeneous appearance, and large cytoplasm with hairy projections. Flow cytometric immunophenotyping revealed CD20, CD22, CD24, CD45 and HLA-DR positivity. Atypical lymphocytes were stained with tartrate resistant acid phosphatase. Increased metabolic activity was detected in multiple lymph nodes, bone marrow and extremely enlarged spleen with positron emission tomography-computed tomography. Excisional biopsy of the left axillary lymph node revealed infiltration with diffuse B-cell leukemia/lymphoma. Immunohistochemistry showed CD20 positive atypical cells with weak expression of CD11c. The patient was diagnosed as a case of variant hairy cell leukemia and cladribine was administered. A probable second primary malignancy should be kept in mind in cases with a defined malignancy in the presence of unusual symptoms.

Systems immunology reveals markers of susceptibility to West Nile virus infection.

PubMed

Qian, Feng; Goel, Gautam; Meng, Hailong; Wang, Xiaomei; You, Fuping; Devine, Lesley; Raddassi, Khadir; Garcia, Melissa N; Murray, Kristy O; Bolen, Christopher R; Gaujoux, Renaud; Shen-Orr, Shai S; Hafler, David; Fikrig, Erol; Xavier, Ramnik; Kleinstein, Steven H; Montgomery, Ruth R

2015-01-01

West Nile virus (WNV) infection is usually asymptomatic but can cause severe neurological disease and death, particularly in older patients, and how individual variations in immunity contribute to disease severity is not yet defined. Animal studies identified a role for several immunity-related genes that determine the severity of infection. We have integrated systems-level transcriptional and functional data sets from stratified cohorts of subjects with a history of WNV infection to define whether these markers can distinguish susceptibility in a human population. Transcriptional profiles combined with immunophenotyping of primary cells identified a predictive signature of susceptibility that was detectable years after acute infection (67% accuracy), with the most prominent alteration being decreased IL1B induction following ex vivo infection of macrophages with WNV. Deconvolution analysis also determined a significant role for CXCL10 expression in myeloid dendritic cells. This systems analysis identified markers of pathogenic mechanisms and offers insights into potential therapeutic strategies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Cell Surface Markers Expression in Postmenopausal Women and Relation to Obesity and Bone Status.

PubMed

Horváthová, Mira; Ilavská, Silvia; Štefíková, Kornélia; Szabová, Michaela; Krivošíková, Zora; Jahnová, Eva; Tulinská, Jana; Spustová, Viera; Gajdoš, Martin

2017-07-11

The age-related changes and hormonal deprivation in postmenopausal women are associated with the immune response alteration. The excessive fat accumulation, local and systemic inflammation may lead to dysregulation in immune function and relevant health problems, including obesity and osteoporosis. We analyzed the expression of cell surface markers in the venous blood specimens, stained with fluorophores-conjugated monoclonal antibodies and analysed by multicolour flow cytometry. The significant changes of cytotoxic, naive, and memory T-lymphocytes, plasmacytoid dendritic cells (DCs) were in postmenopausal women versus fertile women. Body mass index (BMI) affected markedly the cell surface expression of CD265/RANK. Osteoporosis is linked to reduced percentage of plasmacytoid DCs, and elevated natural Treg cells ( p < 0.05). The confounding factors such as women age, BMI, bone mineral density (BMD), waist size and tissue fat affect the expression of RANK on myeloid DCs and CD40L on T-lymphocytes that might be the immunophenotypic modulators after menopause.

The Cell Surface Markers Expression in Postmenopausal Women and Relation to Obesity and Bone Status

PubMed Central

Horváthová, Mira; Ilavská, Silvia; Štefíková, Kornélia; Szabová, Michaela; Krivošíková, Zora; Jahnová, Eva; Tulinská, Jana; Spustová, Viera; Gajdoš, Martin

2017-01-01

The age-related changes and hormonal deprivation in postmenopausal women are associated with the immune response alteration. The excessive fat accumulation, local and systemic inflammation may lead to dysregulation in immune function and relevant health problems, including obesity and osteoporosis. We analyzed the expression of cell surface markers in the venous blood specimens, stained with fluorophores-conjugated monoclonal antibodies and analysed by multicolour flow cytometry. The significant changes of cytotoxic, naive, and memory T-lymphocytes, plasmacytoid dendritic cells (DCs) were in postmenopausal women versus fertile women. Body mass index (BMI) affected markedly the cell surface expression of CD265/RANK. Osteoporosis is linked to reduced percentage of plasmacytoid DCs, and elevated natural Treg cells (p < 0.05). The confounding factors such as women age, BMI, bone mineral density (BMD), waist size and tissue fat affect the expression of RANK on myeloid DCs and CD40L on T-lymphocytes that might be the immunophenotypic modulators after menopause. PMID:28696349

Change in the immunophenotype of a somatotroph adenoma resulting in gigantism.

PubMed

Thawani, Jayesh P; Bailey, Robert L; Burns, Carrie M; Lee, John Y K

2014-01-01

Examining the pathologic progression of a pituitary adenoma from the point of a prepubescent child to an adult with gigantism affords us an opportunity to consider why patients may develop secretory or functioning tumors and raises questions about whether therapeutic interventions and surveillance strategies could be made to avoid irreversible phenotypic changes. A patient underwent a sublabial transsphenoidal resection for a clinically non-functioning macroadenoma in 1999. He underwent radiation treatment and was transiently given growth hormone (GH) supplementation as an adolescent. His growth rapidly traversed several percentiles and he was found to have elevated GH levels. The patient became symptomatic and was taken for a second neurosurgical procedure. Pathology and immunohistochemical staining demonstrated a significantly higher proportion of somatotroph cells and dense granularity; he was diagnosed with a functional somatotroph adenoma. While it is likely that the described observations reflect the manifestations of a functional somatotroph adenoma in development, it is possible that pubertal growth, GH supplementation, its removal, or radiation therapy contributed to the described endocrine and pathologic changes.

Pilot study of erlotinib in patients with acute myeloid leukemia.

PubMed

Sayar, Hamid; Czader, Magdalena; Amin, Chirag; Cangany, Mary; Konig, Heiko; Cripe, Larry D

2015-02-01

We conducted a pilot study to investigate clinical efficacy of tyrosine kinase inhibitor erlotinib in the treatment of acute myeloid leukemia (AML). A total of 11 patients with de novo AML were treated, including 2 with relapsed and/or refractory disease and 9 older patients with previously untreated AML. Patients with high baseline leukocyte count were excluded. Erlotinib was given orally at 150 mg per day continuously in 28-day cycles. The treatment was tolerated well, and no toxicities were observed. An initial reduction in circulating blasts, followed by disease progression, was observed in 2 patients. Nine other patients did not demonstrate any response in blood or bone marrow. Baseline and post-cycle 1 flow-cytometry were performed on bone marrow blasts to investigate signs of differentiation. No immunophenotypic changes suggestive of differentiation were observed. This pilot study did not demonstrate response to standard doses of erlotinib in patients with AML. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biology and relevance of human acute myeloid leukemia stem cells.

PubMed

Thomas, Daniel; Majeti, Ravindra

2017-03-23

Evidence of human acute myeloid leukemia stem cells (AML LSCs) was first reported nearly 2 decades ago through the identification of rare subpopulations of engrafting cells in xenotransplantation assays. These AML LSCs were shown to reside at the apex of a cellular hierarchy that initiates and maintains the disease, exhibiting properties of self-renewal, cell cycle quiescence, and chemoresistance. This cancer stem cell model offers an explanation for chemotherapy resistance and disease relapse and implies that approaches to treatment must eradicate LSCs for cure. More recently, a number of studies have both refined and expanded our understanding of LSCs and intrapatient heterogeneity in AML using improved xenotransplant models, genome-scale analyses, and experimental manipulation of primary patient cells. Here, we review these studies with a focus on the immunophenotype, biological properties, epigenetics, genetics, and clinical associations of human AML LSCs and discuss critical questions that need to be addressed in future research. © 2017 by The American Society of Hematology.

Microflow1, a sheathless fiber-optic flow cytometry biomedical platform: demonstration onboard the international space station.

PubMed

Dubeau-Laramée, Geneviève; Rivière, Christophe; Jean, Isabelle; Mermut, Ozzy; Cohen, Luchino Y

2014-04-01

A fiber-optic based flow cytometry platform was designed to build a portable and robust instrument for space applications. At the core of the Microflow1 is a unique fiber-optic flow cell fitted to a fluidic system and fiber coupled to the source and detection channels. A Microflow1 engineering unit was first tested and benchmarked against a commercial flow cytometer as a reference in a standard laboratory environment. Testing in parabolic flight campaigns was performed to establish Microflow1's performance in weightlessness, before operating the new platform on the International Space Station. Microflow1 had comparable performances to commercial systems, and operated remarkably and robustly in weightlessness (microgravity). Microflow1 supported immunophenotyping as well as microbead-based multiplexed cytokine assays in the space environment and independently of gravity levels. Results presented here provide evidence that this fiber-optic cytometer technology is inherently compatible with the space environment with negligible compromise to analytical performance. © 2013 International Society for Advancement of Cytometry.

Hematologic and lymphocyte immunophenotypic reference values for normal rhesus monkey (Macaca mulatta) umbilical cord blood; gravidity may play a role in study design.

PubMed

Rogers, Linda B; Kaack, M Bernice; Henson, Michael C; Rasmussen, Terri; Henson, Elizabeth; Veazey, Ronald S; Krogstad, Donald J; Davison, Billie B

2005-06-01

Hematology and flow cytometry reference values for rhesus umbilical cord blood (UCB) were established in 17 healthy infant rhesus monkeys delivered by elective cesarean section 10 days preterm. The infants were born to age matched, singly caged primigravid or secundigravid dams. The hematology and flow cytometry values were determined by automated cell counter and by FACS. No significant differences were observed with respect to infant gender. With respect to gravida, the primigravid infants had a significantly higher percentage (P= 0.05) of CD20(+) B lymphocytes in UCB. These results provide useful reference values for future studies of maternal - fetal disease transmission, vaccine and drug evaluation in non-human primate pregnancy, as well as fetal programming and immune modulation, gene therapy and the use of UCB as a source of stem cells for research and transplantation. Importantly, our results suggest that maternal gravidity may be an important variable to consider.

The etiologic role of infectious antigens in sarcoidosis pathogenesis

PubMed Central

Celada, Lindsay J.; Hawkins, Charlene; Drake, Wonder P.

2015-01-01

Sarcoidosis is a granulomatous disease of unknown etiology, characterized by a Th1 immunophenotype, most commonly involving the lung, skin, lymph node and eyes. Molecular and immunologic studies continue to strengthen the association of sarcoidosis with infectious antigens, particularly those derived from Propionibacterium and Mycobacterium species. Independent studies report the presence of microbial nucleic acids and proteins within sarcoidosis specimens. Complementary immunologic studies also support the role of infectious agents in sarcoidosis pathogenesis. Th-1 immune responses directed against mycobacterial virulence factors have been detected within sarcoidosis diagnostic bronchoalveolar lavage (BAL). Th1 and Th17 immune responses against propionibacteria have also been reported. More recently, case reports and clinical trials from Japanese, European and American investigators have emerged regarding the efficacy of antimicrobials against Propionibacterium and Mycobacterium species on pulmonary and cutaneous sarcoidosis. While these clinical investigations are not conclusive, they support increasing efforts to identify novel therapeutics, such as antimicrobials, that will impact the observed increase in sarcoidosis morbidity and mortality. PMID:26593133

An update on classification, genetics, and clinical approach to mixed phenotype acute leukemia (MPAL).

PubMed

Khan, Maliha; Siddiqi, Rabbia; Naqvi, Kiran

2018-06-01

Mixed phenotype acute leukemia (MPAL) is an uncommon diagnosis, representing only about 2-5% of acute leukemia cases. The blast cells of MPAL express multilineage immunophenotypic markers and may have a shared B/T/myeloid phenotype. Due to historical ambiguity in the diagnosis of MPAL, the genetics and clinical features of this disease remain poorly characterized. Based on the 2008 and 2016 World Health Organization classifications, myeloid lineage is best determined by presence of myeloperoxidase, while B and T lymphoid lineages are demonstrated by CD19 and cytoplasmic CD3 expression. MPAL typically carries a worse prognosis than either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL). Given the rarity of MPAL, there is a lack of prospective trial data to guide therapy; treatment generally relies on ALL-like regimens followed by consolidation chemotherapy or hematopoietic stem cell transplant (HSCT). Here, we review the updated classification, biology, clinical features, and treatment approach to MPAL.

Present and future of personalized medicine in CLL.

PubMed

Montserrat, Emili; Bauman, Tycho; Delgado, Julio

2016-03-01

Medicine has been 'personalized' (i.e. centred in persons) since its foundation. Recently, however, the term 'personalized medicine' (or, better, 'precision medicine') has been introduced to define 'a form of medicine that uses information about a person's genes, proteins, and environment to prevent, diagnose, and treat disease'. This concept has gained momentum thanks to next-generation-sequencing (NGS) techniques that allow identification of molecular characteristics unique to the patient and to the tumour. It is hoped that NGS will not only contribute to a better understanding of chronic lymphocytic leukaemia (CLL), but will identify disease subsets that could benefit from specific treatment interventions. Recent advances in diagnosis (e.g. high-resolution immunophenotyping, markers of genetic abnormalities), prognosis (e.g. biomarkers), response predictors [e.g. del(17p)/TP53 mutations even at subclonal level], treatment (e.g. BCR signalling inhibitors, BCL2 antagonists, CAR-T cells) and methods to evaluate minimal residual disease constitute good examples of tools facilitating 'personalized' management of patients with CLL. Copyright © 2016. Published by Elsevier Ltd.

Multinode acoustic focusing for parallel flow cytometry

PubMed Central

Piyasena, Menake E.; Suthanthiraraj, Pearlson P. Austin; Applegate, Robert W.; Goumas, Andrew M.; Woods, Travis A.; López, Gabriel P.; Graves, Steven W.

2012-01-01

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 microns, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multi-node acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 microns in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of CD4+ cellular immunophenotyping assay. This approach will have significant impact towards the creation of high throughput flow cytometers for rare cell detection applications (e.g. circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry. PMID:22239072

Comprehensive T-cell immunophenotyping and next-generation sequencing of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas.

PubMed

Poropatich, Kate; Fontanarosa, Joel; Swaminathan, Suchitra; Dittmann, Dave; Chen, Siqi; Samant, Sandeep; Zhang, Bin

2017-11-01

The success of programmed cell death 1 (PD-1) inhibition in achieving a clinical response in a subset of head and neck squamous cell carcinoma (HNSCC) patients emphasizes the need to better understand the immunobiology of HNSCC. Immunophenotyping was performed for 30 HCSCC patients [16 human papillomavirus (HPV)-positive; 14 HPV-negative] on matched tissue from the primary tumour site, locally metastatic cervical lymph nodes (LNs), uninvolved local cervical LNs, and peripheral blood. CD4 + and CD8 + T-cell lymphocytes obtained from tissue were analysed for expression levels of the inhibitory receptors PD-1, TIM-3 and CTLA-4. Next-generation sequencing of the T-cell receptor (TCR) β chain was performed on patients (n = 9) to determine receptor repertoire diversity and for clonality analysis. HPV-negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8 + T cells expressing CTLA-4 in tumour tissue (P = 0.0013) and in peripheral blood (P = 0.0344) than HPV-positive patients, as well as higher expression levels of TIM-3 + PD-1 + CD8 + T cells (P = 0.0072) than controls. For all patients, PD-1 expression on CD8 + T cells - particularly in HPV-negative HNSCC cases - strongly correlated (r = 0.63, P = 0.013) with tumour size at the primary site. The top CD8 + TCR clones from tumour tissue significantly overlapped with circulating peripheral blood TCR clones (r = 0.946), and HPV-positive patients had frequently expanded TCR clones that were more hydrophobic - and potentially more immunogenic - than those from HPV-negative patients. Collectively, our findings demonstrate, for the first time, that high-stage HPV-negative HNSCC patients with primary tumours at different sites in the head and neck have elevated peripheral CTLA-4 + CD8 + T-cell levels, that tumour-familiar CD8 + T cells are detectable in peripheral blood from HNSCC patients, and that TCRs from HPV-positive HNSCC patients potentially recognize distinctly immunogenic cognate antigens. However, our findings are preliminary, and need to be further confirmed in a larger patient cohort; also, how these factors affect patient response to immunotherapy needs to be determined. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tanzanian malignant lymphomas: WHO classification, presentation, ploidy, proliferation and HIV/EBV association

PubMed Central

2010-01-01

Background In Tanzania, the International Working Formulation [WF] rather than the WHO Classification is still being used in diagnosing malignant lymphomas (ML) and the biological characterization including the HIV/EBV association is sketchy, thus restraining comparison, prognostication and application of established therapeutic protocols. Methods Archival, diagnostic ML biopsies (N = 336), available sera (N = 35) screened by ELISA for HIV antibodies and corresponding clinical/histological reports at Muhimbili National Hospital (MNH) in Tanzania between 1996 and 2006 were retrieved and evaluated. A fraction (N = 174) were analyzed by histopathology and immunohistochemistry (IHC). Selected biopsies were characterized by flow-cytometry (FC) for DNA ploidy (N = 60) and some by in-situ hybridization (ISH) for EBV-encoded RNA (EBER, N = 37). Results A third (38.8%, 109/281) of the ML patients with available clinical information had extranodal disease presentation. A total of 158 out of 174 biopsies selected for immunophenotyping were confirmed to be ML which were mostly (84. 8%, 134/158) non-Hodgkin lymphoma (NHL). Most (83.6%, 112/134) of NHL were B-cell lymphomas (BCL) (CD20+), of which 50.9%, (57/112) were diffuse large B-cell (DLBCL). Out of the 158 confirmed MLs, 22 (13.9%) were T-cell [CD3+] lymphomas (TCL) and 24 (15.2%) were Hodgkin lymphomas (HL) [CD30+]. Furthermore, out of the 60 FC analyzed ML cases, 27 (M:F ratio 2:1) were DLBCL, a slight majority (55.6%, 15/27) with activated B-cell like (ABC) and 45% (12/27) with germinal center B-cell like (GCB) immunophenotype. Overall, 40% (24/60) ML were aneuploid mostly (63.0%, 17/27) the DLBCL and TCL (54.5%, 6/11). DNA index (DI) of FC-analyzed ML ranged from 1.103-2.407 (median = 1.51) and most (75.0%) aneuploid cases showed high (>40%) cell proliferation by Ki-67 reactivity. The majority (51.4%, 19/37) of EBER ISH analyzed lymphoma biopsies were positive. Of the serologically tested MLs, 40.0% (14/35) were HIV positive, mostly with high (≥40.0%) Ki-67 reactivity. Conclusions According to the 2001 WHO Classification, most subtypes are represented in Tanzanian ML. Extranodal presentation was common among MNH lymphoma patients who also showed high aneuploidy, tumor proliferation (KI-67) and EBER positivity. DLBCL was frequent and phenotype heterogeneity appeared similar to observations in Western countries suggesting applicability of established intervention approaches. HIV was apparently associated with high ML cell proliferation but extended studies are needed to clarify this. PMID:20591198

Heterogeneity of macrophages in injured trigeminal nerves: cytokine/chemokine expressing vs. phagocytic macrophages.

PubMed

Lee, SeungHwan; Zhang, Ji

2012-08-01

Macrophages are important immune effector cells in both innate and adaptive immune responses. Injury to peripheral nerves triggers activation of resident macrophages and infiltration of haematogenous macrophages, which they play critical roles in Wallerian degeneration and neuropathic pain. As macrophages are able to change their phenotypes in response to environment cues, we attempt to identify distinct phenotypes of macrophages in injured nerves and to understand the potential contribution of each macrophage subpopulation to the genesis of neuropathic pain associated with nerve injury. Rat mental nerves (terminal branches of trigeminal nerve) were loosely ligated. Sensitivity to mechanical stimuli at the lower lip area was monitored using calibrated von Frey Hairs. We examined the expression pattern of Iba-1, MAC1 and ED1 which allow us to reveal the immunophenotypes of macrophages at different time points post-injury. Functional status of each macrophage subpopulation was further investigated by colocalization with cytokines/chemokines, myelin basic protein and MHC II antigen, which reflect respectively secretory, phagocytic and antigen presentation properties of activated macrophages. Following nerve injury, a burst of Iba-1(+) macrophages was found in injured mental nerves. Among them, we detected two major immunophenotypes: MAC1(+) cytokines/chemokines secreting macrophages and ED1(+) phagocytic macrophages. Small, round shaped MAC1(+) macrophages were distributed essentially around the lesion site and existed only at early time points. Large, irregular and foamy ED1(+) macrophages were found among damaged nerve fibers and they persisted for at least 3 months post-injury. Although ED1(+) macrophages did not secrete inflammatory mediators, they were able to express neurotransmitter CGRP and MHC II at later time points. In parallel, we observed that mechanical allodynia developed after the nerve ligation was at its lowest level within 1 month. Although slightly increased afterwards, the head escape threshold maintained significantly lower than before injury until 3 months. We suggest that MAC1(+) macrophages contribute to the initiation of neuropathic pain by releasing cytokines/chemokines, and ED1(+) macrophages may contribute in maintaining the hypersensitivity under other mechanisms. Our results highlighted the heterogeneity and the plasticity of macrophages in response to the injury and provided further information on their potential involvement in neuropathic pain. Exploring the full spectrum of macrophage phenotypes in injured nerve is necessary. Individual macrophage population may be selectively targeted by cell-specific intervention for an effective treatment of neuropathic pain. Copyright © 2012 Elsevier Inc. All rights reserved.

A case of mistaken identity: CD11c-eYFP(+) cells in the normal mouse brain parenchyma and neural retina display the phenotype of microglia, not dendritic cells.

PubMed

Dando, Samantha J; Naranjo Golborne, Cecilia; Chinnery, Holly R; Ruitenberg, Marc J; McMenamin, Paul G

2016-08-01

Under steady-state conditions the central nervous system (CNS) is traditionally thought to be devoid of antigen presenting cells; however, putative dendritic cells (DCs) expressing enhanced yellow fluorescent protein (eYFP) are present in the retina and brain parenchyma of CD11c-eYFP mice. We previously showed that these mice carry the Crb1(rd8) mutation, which causes retinal dystrophic lesions; therefore we hypothesized that the presence of CD11c-eYFP(+) cells within the CNS may be due to pathology associated with the Crb1(rd8) mutation. We generated CD11c-eYFP Crb1(wt/wt) mice and compared the distribution and immunophenotype of CD11c-eYFP(+) cells in CD11c-eYFP mice with and without the Crb1(rd8) mutation. The number and distribution of CD11c-eYFP(+) cells in the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice. CD11c-eYFP(+) cells were distributed throughout the inner retina, and clustered in brain regions that receive input from the external environment or lack a blood-brain barrier. CD11c-eYFP(+) cells within the retina and cerebral cortex of CD11c-eYFP Crb1(wt/wt) mice expressed CD11b, F4/80, CD115 and Iba-1, but not DC or antigen presentation markers, whereas CD11c-eYFP(+) cells within the choroid plexus and pia mater expressed CD11c, I-A/I-E, CD80, CD86, CD103, DEC205, CD8α and CD135. The immunophenotype of CD11c-eYFP(+) cells and microglia within the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice; however, CD11c and I-A/I-E expression was significantly increased in CD11c-eYFP Crb1(rd8/rd8) mice. This study demonstrates that the overwhelming majority of CNS CD11c-eYFP(+) cells do not display the phenotype of DCs or their precursors and are most likely a subpopulation of microglia. GLIA 2016. GLIA 2016;64:1331-1349. © 2016 Wiley Periodicals, Inc.

Differential expression of cell cycle and WNT pathway-related genes accounts for differences in the growth and differentiation potential of Wharton's jelly and bone marrow-derived mesenchymal stem cells.

PubMed

Batsali, Aristea K; Pontikoglou, Charalampos; Koutroulakis, Dimitrios; Pavlaki, Konstantia I; Damianaki, Athina; Mavroudi, Irene; Alpantaki, Kalliopi; Kouvidi, Elisavet; Kontakis, George; Papadaki, Helen A

2017-04-26

In view of the current interest in exploring the clinical use of mesenchymal stem cells (MSCs) from different sources, we performed a side-by-side comparison of the biological properties of MSCs isolated from the Wharton's jelly (WJ), the most abundant MSC source in umbilical cord, with bone marrow (BM)-MSCs, the most extensively studied MSC population. MSCs were isolated and expanded from BM aspirates of hematologically healthy donors (n = 18) and from the WJ of full-term neonates (n = 18). We evaluated, in parallel experiments, the MSC immunophenotypic, survival and senescence characteristics as well as their proliferative potential and cell cycle distribution. We also assessed the expression of genes associated with the WNT- and cell cycle-signaling pathway and we performed karyotypic analysis through passages to evaluate the MSC genomic stability. The hematopoiesis-supporting capacity of MSCs from both sources was investigated by evaluating the clonogenic cells in the non-adherent fraction of MSC co-cultures with BM or umbilical cord blood-derived CD34 + cells and by measuring the hematopoietic cytokines levels in MSC culture supernatants. Finally, we evaluated the ability of MSCs to differentiate into adipocytes and osteocytes and the effect of the WNT-associated molecules WISP-1 and sFRP4 on the differentiation potential of WJ-MSCs. Both ex vivo-expanded MSC populations showed similar morphologic, immunophenotypic, survival and senescence characteristics and acquired genomic alterations at low frequency during passages. WJ-MSCs exhibited higher proliferative potential, possibly due to upregulation of genes that stimulate cell proliferation along with downregulation of genes related to cell cycle inhibition. WJ-MSCs displayed inferior lineage priming and differentiation capacity toward osteocytes and adipocytes, compared to BM-MSCs. This finding was associated with differential expression of molecules related to WNT signaling, including WISP1 and sFRP4, the respective role of which in the differentiation potential of WJ-MSCs was specifically investigated. Interestingly, treatment of WJ-MSCs with recombinant human WISP1 or sFRP4 resulted in induction of osteogenesis and adipogenesis, respectively. WJ-MSCs exhibited inferior hematopoiesis-supporting potential probably due to reduced production of stromal cell-Derived Factor-1α, compared to BM-MSCs. Overall, these data are anticipated to contribute to the better characterization of WJ-MSCs and BM-MSCs for potential clinical applications.

Results of therapy for acute lymphoblastic leukemia in black and white children.

PubMed

Pui, Ching-Hon; Sandlund, John T; Pei, Deqing; Rivera, Gaston K; Howard, Scott C; Ribeiro, Raul C; Rubnitz, Jeffrey E; Razzouk, Bassem I; Hudson, Melissa M; Cheng, Cheng; Raimondi, Susana C; Behm, Frederick G; Downing, James R; Relling, Mary V; Evans, William E

2003-10-15

Treatment results for acute lymphoblastic leukemia (ALL) clearly have improved over the past decade, but black children have not fared as well as white children in large national trials. To compare the clinical outcomes of therapy for black and white children with ALL treated at a single institution. A retrospective analysis of 412 children and adolescents (68 black, 338 white, and 6 other race) with newly diagnosed ALL who were treated consecutively at a pediatric cancer center in Memphis, Tenn. Patients were enrolled from December 1991 to July 1998 in successive Total Therapy studies regardless of race, ethnicity, or ability to pay and received risk-directed therapy according to stringent criteria. All patients received the same intensive, remission-induction therapy followed by 120 weeks of risk-assigned postremission therapy that included reinduction treatment, pulses of high-dose methotrexate, and early intensification of intrathecal chemotherapy. Event-free and overall survival rates for black and white children were estimated by the method of Kaplan and Meier and compared with the Mantel-Haenszel test and by Cox proportional hazards regression analysis, adjusting for known prognostic factors. The 68 black children were significantly more likely than the 338 white children to have higher-risk prognostic features, including an initial leukocyte count greater than 100 x 10(3)/ microL, a T-cell immunophenotype, and the t(1;19) chromosomal translocation with E2A-PBX1 fusion, and were less likely to have hyperdiploid blast cells, a favorable prognostic factor in childhood ALL. However, the clinical outcomes for these 2 cohorts were not significantly different: 5-year event-free and overall survival rates were 80.7% (95% confidence interval [CI], 70.3%-91.1%) and 86.2% (95% CI, 77.2%-95.2%) for black children vs 79.4% (95% CI, 74.7%-84.1%) and 85.0% (95% CI, 80.9%-89.1%) for white children. Ten-year results also were comparable, but the CIs were wide because of the small numbers of patients who had been followed up for 10 years or more. The lack of a racial effect on the long-term outcome of therapy was still apparent in a multivariate Cox regression analysis, adjusting for sex, age, presenting leukocyte count, leukemic cell DNA index, immunophenotype, and central nervous system status. With equal access to effective antileukemic therapy, black and white children with ALL can expect the same high rate of cure.

Growing B Lymphocytes in a Three-Dimensional Culture System

NASA Technical Reports Server (NTRS)

Wu, J. H. David; Bottaro, Andrea

2010-01-01

A three-dimensional (3D) culture system for growing long-lived B lymphocytes has been invented. The capabilities afforded by the system can be expected to expand the range of options for immunological research and related activities, including testing of immunogenicity of vaccine candidates in vitro, generation of human monoclonal antibodies, and immunotherapy. Mature lymphocytes, which are the effectors of adaptive immune responses in vertebrates, are extremely susceptible to apoptotic death, and depend on continuous reception of survival-inducing stimulation (in the forms of cytokines, cell-to-cell contacts, and antigen receptor signaling) from the microenvironment. For this reason, efforts to develop systems for long-term culture of functional, non-transformed and non-activated mature lymphocytes have been unsuccessful until now. The bone-marrow microenvironment supports the growth and differentiation of many hematopoietic lineages, in addition to B-lymphocytes. Primary bone-marrow cell cultures designed to promote the development of specific cell types in vitro are highly desirable experimental systems, amenable to manipulation under controlled conditions. However, the dynamic and complex network of stromal cells and insoluble matrix proteins is disrupted in prior plate- and flask-based culture systems, wherein the microenvironments have a predominantly two-dimensional (2D) character. In 2D bone-marrow cultures, normal B-lymphoid cells become progressively skewed toward precursor B-cell populations that do not retain a normal immunophenotype, and such mature B-lymphocytes as those harvested from the spleen or lymph nodes do not survive beyond several days ex vivo in the absence of mitogenic stimulation. The present 3D culture system is a bioreactor that contains highly porous artificial scaffolding that supports the long-term culture of bone marrow, spleen, and lymph-node samples. In this system, unlike in 2D culture systems, B-cell subpopulations developing within 3D cultures that have been modified to foster lymphopoiesis retain an immunophenotype that closely recapitulates cells in fresh bone marrow harvests. The 3D culture system has been found to be capable of supporting long-lived (8 weeks) populations of B and T lymphocytes from peripheral lymphoid organs, in the absence of activation signals, to an extent not achievable by conventional culture techniques. Interestingly, it has been found that 3D-culture B cells display a phenotype that has characteristics of both B1a and B2 cells. These promising preliminary observations suggest that the 3D culture system could be used with success in the study of peripheral-B-lymphocyte biology and in the development of biotechnological techniques and processes.

Biomarker evaluation of face transplant rejection: association of donor T cells with target cell injury.

PubMed

Lian, Christine Guo; Bueno, Ericka M; Granter, Scott R; Laga, Alvaro C; Saavedra, Arturo P; Lin, William M; Susa, Joseph S; Zhan, Qian; Chandraker, Anil K; Tullius, Stefan G; Pomahac, Bohdan; Murphy, George F

2014-06-01

This series of 113 sequential biopsies of full facial transplants provides findings of potential translational significance as well as biological insights that could prompt reexamination of conventional paradigms of effector pathways in skin allograft rejection. Serial biopsies before, during, and after rejection episodes were evaluated for clinicopathological assessment that in selected cases included specific biomarkers for donor-versus-recipient T cells. Histologic evidence of rejection included lymphocyte-associated injury to epidermal rete ridges, follicular infundibula, and dermal microvessels. Surprisingly, during active rejection, immune cells spatially associated with target cell injury consisted abundantly or predominantly of lymphocytes of donor origin with an immunophenotype typical of the resident memory T-cell subset. Current dogma assumes that skin allograft rejection is mediated by recipient T cells that attack epidermal targets, and the association of donor T cells with sites of target cell injury raises questions regarding the potential complexity of immune cell interactions in the rejection process. A more histopathologically refined and immune-based biomarker approach to assessment of rejection of facial transplants is now indicated.

A novel method for detection of phosphorylation in single cells by surface enhanced Raman scattering (SERS) using composite organic-inorganic nanoparticles (COINs).

PubMed

Shachaf, Catherine M; Elchuri, Sailaja V; Koh, Ai Leen; Zhu, Jing; Nguyen, Lienchi N; Mitchell, Dennis J; Zhang, Jingwu; Swartz, Kenneth B; Sun, Lei; Chan, Selena; Sinclair, Robert; Nolan, Garry P

2009-01-01

Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using "Composite Organic-Inorganic Nanoparticles" (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.

A Novel Method for Detection of Phosphorylation in Single Cells by Surface Enhanced Raman Scattering (SERS) using Composite Organic-Inorganic Nanoparticles (COINs)

PubMed Central

Shachaf, Catherine M.; Elchuri, Sailaja V.; Koh, Ai Leen; Zhu, Jing; Nguyen, Lienchi N.; Mitchell, Dennis J.; Zhang, Jingwu; Swartz, Kenneth B.; Sun, Lei; Chan, Selena; Sinclair, Robert; Nolan, Garry P.

2009-01-01

Background Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities. Methodology/Principal Findings To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using “Composite Organic-Inorganic Nanoparticles” (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry. Conclusions/Significance Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells. PMID:19367337

CD19-positive acute myeloblastic leukemia with trisomy 21 as a sole acquired karyotypic abnormality

PubMed Central

Wang, Hua-feng; Cheng, Yi-zhi; Wang, Huan-ping; Chen, Zhi-mei; Lou, Ji-yu; Jin, Jie

2009-01-01

We report that a 63-year-old Chinese female had acute myeloblastic leukemia (AML) in which trisomy 21 (+21) was found as the sole acquired karyotypic abnormality. The blasts were positive for myeloperoxidase, and the immunophenotype was positive for cluster of differentiation 19 (CD19), CD33, CD34, and human leukocyte antigens (HLA)-DR. The chromosomal analysis of bone marrow showed 47,XX,+21[2]/46,XX[18]. Fluorescent in situ hybridization (FISH) showed that three copies of AML1 were situated in separate chromosomes, and that t(8;21) was negative. The patient did not have any features of Down syndrome. A diagnosis of CD19-positive AML-M5 was established with trisomy 21 as a sole acquired karyotypic abnormality. The patient did not respond well to chemotherapy and died three months after the diagnosis. This is the first reported case of CD19-positive AML with trisomy 21 as the sole cytogenetic abnormality. The possible prognostic significance of the finding in AML with +21 as the sole acquired karyotypic abnormality was discussed. PMID:19882758

Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Myoung Woo; Moon, Young Joon; Yang, Mal Sook

2007-06-29

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types,more » including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.« less

Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia with "normal" FISH: correlations with clinicobiologic parameters.

PubMed

Rigolin, Gian Matteo; Cibien, Francesca; Martinelli, Sara; Formigaro, Luca; Rizzotto, Lara; Tammiso, Elisa; Saccenti, Elena; Bardi, Antonella; Cavazzini, Francesco; Ciccone, Maria; Nichele, Ilaria; Pizzolo, Giovanni; Zaja, Francesco; Fanin, Renato; Galieni, Piero; Dalsass, Alessia; Mestichelli, Francesca; Testa, Nicoletta; Negrini, Massimo; Cuneo, Antonio

2012-03-08

It is unclear whether karyotype aberrations that occur in regions uncovered by the standard fluorescence in situ hybridization (FISH) panel have prognostic relevance in chronic lymphocytic leukemia (CLL). We evaluated the significance of karyotypic aberrations in a learning cohort (LC; n = 64) and a validation cohort (VC; n = 84) of patients with chronic lymphocytic leukemia with "normal" FISH. An abnormal karyotype was found in 21.5% and 35.7% of cases in the LC and VC, respectively, and was associated with a lower immunophenotypic score (P = .030 in the LC, P = .035 in the VC), advanced stage (P = .040 in the VC), and need for treatment (P = .002 in the LC, P = < .0001 in the VC). The abnormal karyotype correlated with shorter time to first treatment and shorter survival in both the LC and the VC, representing the strongest prognostic parameter. In patients with chronic lymphocytic leukemia with normal FISH, karyotypic aberrations by conventional cytogenetics with novel mitogens identify a subset of cases with adverse prognostic features.

AHR prevents human IL-1R1hi ILC3 differentiation to natural killer cells

PubMed Central

Hughes, Tiffany; Briercheck, Edward L.; Freud, Aharon G.; Trotta, Rossana; McClory, Susan; Scoville, Steven D.; Keller, Karen; Deng, Youcai; Cole, Jordan; Harrison, Nicholas; Mao, Charlene; Zhang, Jianying; Benson, Don M.; Yu, Jianhua; Caligiuri, Michael A.

2014-01-01

SUMMARY Accumulating evidence indicates that human natural killer (NK) cells develop in secondary lymphoid tissue (SLT) through a so-called “stage 3” developmental intermediate minimally characterized by a CD34-CD117+CD94- immunophenotype that lacks mature NK cell function. This stage 3 population is heterogeneous, potentially composed of functionally distinct innate lymphoid cell (ILC) types that includes interleukin-1 receptor (IL-1R1) positive, IL-22-producing ILC3s. Whether human ILC3s are developmentally related to NK cells is a subject of ongoing investigation. Here we show that antagonism of the aryl hydrocarbon receptor (AHR) or silencing of AHR gene expression promotes differentiation of tonsillar IL-22-producing IL-1R1hi human ILC3s to CD56brightCD94+ IFN-gamma-producing cytolytic mature NK cells expressing eomesodermin (EOMES) and T-Box Protein 21 (TBX21 or TBET). Hence, AHR is a transcription factor that prevents human IL-1R1hi ILC3s from differentiating into NK cells. PMID:24953655

Factors predicting transformation of asymptomatic IgM monoclonal gammopathy.

PubMed

Greco, Antonino; Tedeschi, Alessandra; Varettoni, Marzia; Nichelatti, Michele; Paris, Laura; Ricci, Francesca; Vismara, Eleonora; Morra, Enrica

2011-02-01

We evaluated the risk of transformation of asymptomatic immunoglobulin (Ig) M monoclonal gammopathy (aIgM MG) into symptomatic lymphoproliferative disease in 287 patients all analyzed for bone marrow histopathology and immunophenotyping. This series included 201 patients with IgM MG of undetermined significance (IgM MGUS) and 86 with smoldering Waldenström's macroglobulinemia (sWM). After a median of 50 months (range, 12-322 months), 32 cases of aIgM-MG (11.1%) evolved into symptomatic malignant lymphoproliferative disease, as follows: symptomatic WM (n=26), non-Hodgkin lymphoma (n=6). The cumulative transformation percentage at 5 and 10 years was 8% and 19.5%, respectively. The parameters significantly correlated with evolution were, at univariate analysis, BM lymphoplasmacytic infiltration, high erythrocyte sedimentation rate, serum MC, serum IgM size, and serum IgA size. Among patients with aIgM-MG, those at high risk of evolution were patients with sWM, a distinct entity with serum IgM monoclonal protein≥3 g/dL and/or ≥10% bone marrow lymphoplasmacytic infiltration.

Liquid-based cytology and cell block immunocytochemistry in veterinary medicine: comparison with standard cytology for the evaluation of canine lymphoid samples.

PubMed

Fernandes, N C C A; Guerra, J M; Réssio, R A; Wasques, D G; Etlinger-Colonelli, D; Lorente, S; Nogueira, E; Dagli, M L Z

2016-08-01

Liquid-based Cytology (LBC) consists of immediate wet cell fixation with automated slide preparation. We applied LBC, cell block (CB) and immunocytochemistry to diagnose canine lymphoma and compare results with conventional cytology. Samples from enlarged lymph nodes of 18 dogs were collected and fixed in preservative solution for automated slide preparation (LBC), CB inclusion and immunophenotyping. Two CB techniques were tested: fixed sediment method (FSM) and agar method (AM). Anti-CD79a, anti-Pax5, anti-CD3 and anti-Ki67 were used in immunocytochemistry. LBC smears showed better nuclear and nucleolar definition, without cell superposition, but presented smaller cell size and worse cytoplasmic definition. FSM showed consistent cellular groups and were employed for immunocytochemistry, while AM CBs presented sparse groups of lymphocytes, with compromised analysis. Anti-Pax-5 allowed B-cell identification, both in reactive and neoplastic lymph nodes. Our preliminary report suggests that LBC and FSM together may be promising tools to improve lymphoma diagnosis through fine-needle aspiration. © 2015 John Wiley & Sons Ltd.

Circulating B cells in type 1 diabetics exhibit fewer maturation-associated phenotypes.

PubMed

Hanley, Patrick; Sutter, Jennifer A; Goodman, Noah G; Du, Yangzhu; Sekiguchi, Debora R; Meng, Wenzhao; Rickels, Michael R; Naji, Ali; Luning Prak, Eline T

2017-10-01

Although autoantibodies have been used for decades as diagnostic and prognostic markers in type 1 diabetes (T1D), further analysis of developmental abnormalities in B cells could reveal tolerance checkpoint defects that could improve individualized therapy. To evaluate B cell developmental progression in T1D, immunophenotyping was used to classify circulating B cells into transitional, mature naïve, mature activated, and resting memory subsets. Then each subset was analyzed for the expression of additional maturation-associated markers. While the frequencies of B cell subsets did not differ significantly between patients and controls, some T1D subjects exhibited reduced proportions of B cells that expressed transmembrane activator and CAML interactor (TACI) and Fas receptor (FasR). Furthermore, some T1D subjects had B cell subsets with lower frequencies of class switching. These results suggest circulating B cells exhibit variable maturation phenotypes in T1D. These phenotypic variations may correlate with differences in B cell selection in individual T1D patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Technical Advances in the Measurement of Residual Disease in Acute Myeloid Leukemia

PubMed Central

Roloff, Gregory W.; Lai, Catherine; Dillon, Laura W.

2017-01-01

Outcomes for those diagnosed with acute myeloid leukemia (AML) remain poor. It has been widely established that persistent residual leukemic burden, often referred to as measurable or minimal residual disease (MRD), after induction therapy or at the time of hematopoietic stem cell transplant (HSCT) is highly predictive for adverse clinical outcomes and can be used to identify patients likely to experience clinically evident relapse. As a result of inherent genetic and molecular heterogeneity in AML, there is no uniform method or protocol for MRD measurement to encompass all cases. Several techniques focusing on identifying recurrent molecular and cytogenetic aberrations or leukemia-associated immunophenotypes have been described, each with their own strengths and weaknesses. Modern technologies enabling the digital quantification and tracking of individual DNA or RNA molecules, next-generation sequencing (NGS) platforms, and high-resolution imaging capabilities are among several new avenues under development to supplement or replace the current standard of flow cytometry. In this review, we outline emerging modalities positioned to enhance MRD detection and discuss factors surrounding their integration into clinical practice. PMID:28925935

Assessment of ixekizumab, an interleukin-17A monoclonal antibody, for potential effects on reproduction and development, including immune system function, in cynomolgus monkeys.

PubMed

Clarke, D O; Hilbish, K G; Waters, D G; Newcomb, D L; Chellman, G J

2015-12-01

The reproductive and developmental toxicity of ixekizumab, a selective inhibitor of interleukin-17A (IL-17A), was assessed in the following studies in cynomolgus monkeys: fertility (3-month dosing), embryo-fetal development (EFD; dosing from gestation day (GD) 20 through 139), and pre-postnatal development (PPND; dosing from GD 20 through parturition). Because IL-17A has functional roles in innate and humoral immunity, immune system modulation was evaluated in the EFD and PPND studies; immunological evaluations in infants comprised peripheral blood immunophenotyping, Natural Killer cell cytolytic activity, and T-cell-dependent antibody (IgG and IgM) primary and secondary responses to antigen challenge. Ixekizumab exposure was sustained during the dosing periods in most adult monkeys. Fetal exposure at Cesarean section (GD 140-142; EFD study) was 18-25% of maternal exposure and ixekizumab was present in infants for up to 29 weeks postpartum. There were no adverse effects attributed to ixekizumab in any study. Importantly, immune system development and maturation were unaffected. Copyright © 2015 Elsevier Inc. All rights reserved.

[Clinico-pathologic Characteristics of Adult Patients with Atypical Infectious Mononucleosis].

PubMed

Yu, Ya-Ping; Song, Ping; An, Zhi-Ming; Zhou, Xiao-Gang; Li, Feng; Wang, Li-Ping; Mei, Jian-Gang; Zhai, Yong-Ping

2016-12-01

To investigate the clinicopathologic characteristics of adult patients with atypical infectious mononucleosis(IM). From January 2003 to December 2013, a total of 5 cases of atypical IM misdiagnosed as lymphoma were selected, and the clinico-pathological characteristics and efficacy of treatment were analyzed. Biopsy of lymph node or tonsil was performed to evaluate the possibility of lymphoma. Peripheral blood EBV antibody and EBV-DNA were examined by ELISA and real-time fluorescence quantitative PCR, respectively. All the cases were considered as lymphoma on the basis of morphological features in initial evaluation before relapse. These features included a florid immunoblastic proliferation, distortion of the underlying nodal or tonsillar architecture and the presence of necrosis. The immunophenotypic features, EBV encoded RNA (EBER) in situ hybridization and the gene rearrangement of immunoglobulin or T cell receptor may be helpful for the distinction of atypical IM from lymphoma. IM as EBV-related lymphoproliferative process shows marked clinical and histological diversity. Atypical case of IM may mimic many different type of lymphoma in clinical and pathologic features, and the misdiagnosis should be avoided by using molecular and pathological examination.

Histopathologic and immunohistochemical features of Hashimoto thyroiditis.

PubMed

Amani, H Kazem

2011-01-01

Intrathyroid lymphoid tissue is accrued in Hashimoto thyroiditis (HT). Histologically, this acquired lymphoid tissue bears a close resemblance to mucosa-associated lymphoid tissue (MALT) and can evolve to lymphoma. To demonstrate the morphological, and immunohistochemical profiles of Hashimoto thyroiditis and to ascertain the importance of light chain restriction in distinguishing HT with extensive lymphoplasmacytoid infiltrate from MALT lymphoma. We studied histopathologically and immunohistochemically (CD20, CD3, Igk, Igl and cytokeratin) 30 cases of HT for evaluation of the lymphoid infiltrate and the presence of lymphoepithelial lesions (LELs). Distinguishing between early thyroid lymphoma and HT was evaluated by light chain restriction. These findings were compared with two cases of primary thyroid lymphoma. The histopathological findings were characteristic of HT. Immunohistochemistry confirmed inconspicuous, rare B-cell LELs as well as a prominent T-lymphocyte population. Testing for light chain restriction showed polyclonal population of plasma cells. The cases of MALT lymphoma had distinct destructive lymphoepithelial lesions, B-cell immunophenotyping and showed kappa light chain restriction in the plasmacytoid population. Hashimoto thyroiditis differs both histopathologically and immunohistochemically from thyroid lymphoma. In suspicious cases, immunohistochemistry could be helpful in reaching a definitive diagnosis.

Enhanced Ex Vivo Expansion of Human Hematopoietic Progenitors on Native and Spin Coated Acellular Matrices Prepared from Bone Marrow Stromal Cells

PubMed Central

Wasnik, Samiksha; Kantipudi, Suma; Kirkland, Mark A.; Pande, Gopal

2016-01-01

The extracellular microenvironment in bone marrow (BM) is known to regulate the growth and differentiation of hematopoietic stem and progenitor cells (HSPC). We have developed cell-free matrices from a BM stromal cell line (HS-5), which can be used as substrates either in native form or as tissue engineered coatings, for the enhanced ex vivo expansion of umbilical cord blood (UCB) derived HSPC. The physicochemical properties (surface roughness, thickness, and uniformity) of native and spin coated acellular matrices (ACM) were studied using scanning and atomic force microscopy (SEM and AFM). Lineage-specific expansion of HSPC, grown on these substrates, was evaluated by immunophenotypic (flow cytometry) and functional (colony forming) assays. Our results show that the most efficient expansion of lineage-specific HSPC occurred on spin coated ACM. Our method provides an improved protocol for ex vivo HSPC expansion and it offers a system to study the in vivo roles of specific molecules in the hematopoietic niche that influence HSPC expansion. PMID:26981135

Genomic analyses identify recurrent MEF2D fusions in acute lymphoblastic leukaemia

PubMed Central

Gu, Zhaohui; Churchman, Michelle; Roberts, Kathryn; Li, Yongjin; Liu, Yu; Harvey, Richard C.; McCastlain, Kelly; Reshmi, Shalini C.; Payne-Turner, Debbie; Iacobucci, Ilaria; Shao, Ying; Chen, I-Ming; Valentine, Marcus; Pei, Deqing; Mungall, Karen L.; Mungall, Andrew J.; Ma, Yussanne; Moore, Richard; Marra, Marco; Stonerock, Eileen; Gastier-Foster, Julie M.; Devidas, Meenakshi; Dai, Yunfeng; Wood, Brent; Borowitz, Michael; Larsen, Eric E.; Maloney, Kelly; Mattano Jr, Leonard A.; Angiolillo, Anne; Salzer, Wanda L.; Burke, Michael J.; Gianni, Francesca; Spinelli, Orietta; Radich, Jerald P.; Minden, Mark D.; Moorman, Anthony V.; Patel, Bella; Fielding, Adele K.; Rowe, Jacob M.; Luger, Selina M.; Bhatia, Ravi; Aldoss, Ibrahim; Forman, Stephen J.; Kohlschmidt, Jessica; Mrózek, Krzysztof; Marcucci, Guido; Bloomfield, Clara D.; Stock, Wendy; Kornblau, Steven; Kantarjian, Hagop M.; Konopleva, Marina; Paietta, Elisabeth; Willman, Cheryl L.; L. Loh, Mignon; P. Hunger, Stephen; Mullighan, Charles G.

2016-01-01

Chromosomal rearrangements are initiating events in acute lymphoblastic leukaemia (ALL). Here using RNA sequencing of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2D-BCL9. Examination of an extended cohort of 1,164 B-ALL cases identified 30 cases with MEF2D rearrangements, which include an additional fusion partner, FOXJ2; thus, MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitor treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukaemia, for which new therapeutic approaches should be considered. PMID:27824051

Effects of low-doses of Bacillus spp. from permafrost on differentiation of bone marrow cells.

PubMed

Kalyonova, L F; Novikova, M A; Kostolomova, E G

2015-01-01

The effects of a new microorganism species (Bacillus spp., strain M3) isolated from permafrost specimens from Central Yakutia (Mamontova Mountain) on the bone marrow hemopoiesis were studied on laboratory mice. Analysis of the count and immunophenotype of bone marrow cells indicated that even in low doses (1000-5000 microbial cells) these microorganisms modulated hemopoiesis and lymphopoiesis activity. The percentage of early hemopoietic precursors (CD117(+)CD34(-)) increased, intensity of lymphocyte precursor proliferation and differentiation (CD25(+)CD44(-)) decreased, and the percentage of lymphocytes released from the bone marrow (CD25(+)CD44(+)) increased on day 21 after injection of the bacteria. These changes in activity of hemopoiesis were associated with changes in the level of regulatory T lymphocytes (reduced expression of TCRαβ) and were most likely compensatory. The possibility of modulating hemopoiesis activity in the bone marrow by low doses of one microorganism strain isolated from the permafrost could be useful for evaluating the effects of other low dose bacteria on the bone marrow hemopoiesis.

Laser Scanning Cytometry

PubMed Central

Pozarowski, Piotr; Holden, Elena; Darzynkiewicz, Zbigniew

2013-01-01

Summary The laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of analytical capabilities. Multilaser-excited fluorescence emitted from individual cells is measured at several wavelength ranges, rapidly (up to 5000 cells/min), with high sensitivity and accuracy. The following applications of LSC are reviewed: (1) identification of cells that differ in degree of chromatin condensation (e.g., mitotic or apoptotic cells or lymphocytes vs granulocytes vs monocytes); (2) detection of translocation between cytoplasm vs nucleus or nucleoplasm vs nucleolus of regulatory molecules such as NF- κB, p53, or Bax; (3) semiautomatic scoring of micronuclei in mutagenicity assays; (4) analysis of fluorescence in situ hybridization; (5) enumeration and morphometry of nucleoli; (6) analysis of phenotype of progeny of individual cells in clonogenicity assay; (7) cell immunophenotyping; (8) visual examination, imaging, or sequential analysis of the cells measured earlier upon their relocation, using different probes; (9) in situ enzyme kinetics and other time-resolved processes; (10) analysis of tissue section architecture; (11) application for hypocellular samples (needle aspirate, spinal fluid, etc.); (12) other clinical applications. Advantages and limitations of LSC are discussed and compared with flow cytometry. PMID:16719355

Nephrogenic diabetes insipidus in initial stage of acute lymphoblastic leukemia and relapse after haploidentical hematopoietic stem-cell transplantation: A case report.

PubMed

Li, Dezhi; Liu, Qian; Feng, Zhifang; Zhang, Qi; Feng, Saran

2018-06-01

Nephrogenic diabetes insipidus (NDI) rarely presents in the initial stage of acute lymphoblastic leukemia (ALL) and relapse due to renal infiltration is also rare. A 19-year-old man presented with weakness, polydipsia, and polyuria for 1 month. NDI was diagnosed with insignificant response to a water deprivation test after stimulation with vasopressin injection. Bone marrow examination combined with immunophenotypic analysis, cerebrospinal cytology, and abdominal ultrasonography confirmed the diagnoses of precursor B cell ALL with renal infiltration. The patient accepted standardized combination chemotherapy and ultimately had sustained remission, and his polydipsia and polyuria disappeared after 3 days of treatment. The ALL relapsed 1 year later and he received haploidentical stem cell transplantation (haplo-SCT) from his father. One year later, he again developed NDI, with bilateral renal enlargement because of extramedullary relapse, leading to subsequent death. This case demonstrates unusual early renal involvement in ALL presenting with initial NDI. Interestingly, the NDI returned with the relapse of renal infiltration 1 year after haplo-SCT. This case suggests that NDI was probably secondary to renal leukemic infiltration.

Epstein-Barr virus-positive cytotoxic T-cell lymphoma followed by chronic active Epstein-Barr virus infection-associated T/NK-cell lymphoproliferative disorder: a case report.

PubMed

Kato, Seiichi; Miyata, Tomoko; Takata, Katsuyoshi; Shimada, Satoko; Ito, Yoshinori; Tomita, Akihiro; Elsayed, Ahmed Ali; Takahashi, Emiko; Asano, Naoko; Kinoshita, Tomohiro; Kimura, Hiroshi; Nakamura, Shigeo

2013-12-01

A 30-year-old female patient presented with intestinal Epstein-Barr virus (EBV)-positive cytotoxic T-cell lymphoma (EBV+ CTL), which was surgically resected. Fourteen years later, she returned to our hospital with hypersensitivity to mosquito bites and was diagnosed with chronic active EBV infection-associated T/NK-cell lymphoproliferative disorder (CAEBV/TNK-LPD). She developed systemic EBV+ CTL at age 47 years during the 2.5-year clinical course of CAEBV/TNK-LPD, despite multiagent chemotherapy and allogeneic stem cell transplantation. Afterward, she had a rapidly deteriorating clinical course and died at age 48 years. The immunophenotype of the EBV+ CTL was consistently a CD3, CD8, and cytotoxic molecule-positive type with the same clonality in polymerase chain reaction analysis of T-cell receptor-γ chain gene rearrangement. This is the first reported case of EBV+ CTL preceding the clinical presentation of CAEBV/TNK-LPD. The present case was unique in suggesting a close relationship between EBV+ CTL and chronic active EBV infection. © 2013.

B-cell acute lymphoblastic leukemia with mature phenotype and MLL rearrangement: report of five new cases and review of the literature.

PubMed

Sajaroff, Elisa Olga; Mansini, Adrian; Rubio, Patricia; Alonso, Cristina Noemí; Gallego, Marta S; Coccé, Mariela C; Eandi-Eberle, Silvia; Bernasconi, Andrea Raquel; Ampatzidou, Maria; Paterakis, George; Papadhimitriou, Stefanos I; Petrikkos, Loizos; Papadakis, Vassilios; Polychronopoulou, Sophia; Rossi, Jorge G; Felice, Maria Sara

2016-10-01

The association between mature-B phenotype and MLL abnormalities in acute lymphoblastic leukemia (ALL) is a very unusual finding; only 14 pediatric cases have been reported so far. We describe the clinical and biological characteristics and outcome of five pediatric cases of newly diagnosed B lineage ALL with MLL abnormalities and mature immunophenotype based on light chain restriction and surface Ig expression. Blasts showed variable expression of CD10/CD34/TdT. MLL abnormalities with no MYC involvement were detected in all patients by G-banding, FISH, and/or RT-PCR. Three patients were treated according to Interfant protocol, one to ALLIC-09, and one received B-NHL-BFM-2004. All patients achieved complete remission and three of them relapsed. Despite the small cohort size, it could be postulated that B lineage ALL with MLL abnormalities and mature phenotype is a distinct entity that differs both from the typical Pro B ALL observed in infants and mature B-ALL with high MYC expression.

Change in the immunophenotype of a somatotroph adenoma resulting in gigantism

PubMed Central

Thawani, Jayesh P.; Bailey, Robert L.; Burns, Carrie M.; Lee, John Y. K.

2014-01-01

Background: Examining the pathologic progression of a pituitary adenoma from the point of a prepubescent child to an adult with gigantism affords us an opportunity to consider why patients may develop secretory or functioning tumors and raises questions about whether therapeutic interventions and surveillance strategies could be made to avoid irreversible phenotypic changes. Case Description: A patient underwent a sublabial transsphenoidal resection for a clinically non-functioning macroadenoma in 1999. He underwent radiation treatment and was transiently given growth hormone (GH) supplementation as an adolescent. His growth rapidly traversed several percentiles and he was found to have elevated GH levels. The patient became symptomatic and was taken for a second neurosurgical procedure. Pathology and immunohistochemical staining demonstrated a significantly higher proportion of somatotroph cells and dense granularity; he was diagnosed with a functional somatotroph adenoma. Conclusions: While it is likely that the described observations reflect the manifestations of a functional somatotroph adenoma in development, it is possible that pubertal growth, GH supplementation, its removal, or radiation therapy contributed to the described endocrine and pathologic changes. PMID:25396071

Experimental murine fascioliasis derives early immune suppression with increased levels of TGF-β and IL-4.

PubMed

Chung, Joon-Yong; Bae, Young-An; Yun, Doo-Hee; Yang, Hyun-Jong; Kong, Yoon

2012-12-01

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19(+) B cells was observed as early as week 1 post-infection while CD4(+)/CD8(+) T cells were down-regulated. Accumulation of Mac1(+) cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-α mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1β expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-β were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-β and IL-4 during the early stages of infection.

Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods.

PubMed

Øbro, Nina F; Ryder, Lars P; Madsen, Hans O; Andersen, Mette K; Lausen, Birgitte; Hasle, Henrik; Schmiegelow, Kjeld; Marquart, Hanne V

2012-01-01

Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.

On-chip immune cell activation and subsequent time-resolved magnetic bead-based cytokine detection.

PubMed

Kongsuphol, Patthara; Liu, Yunxiao; Ramadan, Qasem

2016-10-01

Cytokine profiling and immunophenotyping offer great potential for understanding many disease mechanisms, personalized diagnosis, and immunotherapy. Here, we demonstrate a time-resolved detection of cytokine from a single cell cluster using an in situ magnetic immune assay. An array of triple-layered microfluidic chambers was fabricated to enable simultaneous cell culture under perfusion flow and detection of the induced cytokines at multiple time-points. Each culture chamber comprises three fluidic compartments which are dedicated to, cell culture, perfusion and immunoassay. The three compartments are separated by porous membranes, which allow the diffusion of fresh nutrient from the perfusion compartment into the cell culture compartment and cytokines secretion from the cell culture compartment into the immune assay compartment. This structure hence enables capturing the released cytokines without disturbing the cell culture and without minimizing benefit gain from perfusion. Functionalized magnetic beads were used as a solid phase carrier for cytokine capturing and quantification. The cytokines released from differential stimuli were quantified in situ in non-differentiated U937 monocytes and differentiated macrophages.

Smoldering Multiple Myeloma

PubMed Central

Gao, Minjie; Yang, Guang; Kong, Yuanyuan; Wu, Xiaosong; Shi, Jumei

2015-01-01

Smoldering multiple myeloma (SMM) is an asymptomatic precursor stage of multiple myeloma (MM) characterized by clonal bone marrow plasma cells (BMPC) ≥ 10% and/or M protein level ≥ 30 g/L in the absence of end organ damage. It represents an intermediate stage between monoclonal gammopathy of undetermined significance (MGUS) and symptomatic MM. The risk of progression to symptomatic MM is not uniform, and several parameters have been reported to predict the risk of progression. These include the level of M protein and the percentage of BMPC, the proportion of immunophenotypically aberrant plasma cells, and the presence of immunoparesis, free light-chain (FLC) ratio, peripheral blood plasma cells (PBPC), pattern of serum M protein evolution, abnormal magnetic resonance imaging (MRI), cytogenetic abnormalities, IgA isotype, and Bence Jones proteinuria. So far treatment is still not recommended for SMM, because several trials suggested that patients with SMM do not benefit from early treatment. However, the Mateos et al. trial showed a survival benefit after early treatment with lenalidomide plus dexamethasone in patients with high-risk SMM. This trial has prompted a reevaluation of early treatment in an asymptomatic patient population. PMID:26000300

Preparation of myeloid derived suppressor cells (MDSC) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (AutoMACS).

PubMed

Nelson, Nadine; Szekeres, Karoly; Cooper, Denise; Ghansah, Tomar

2012-06-18

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to naïve counterparts. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo. Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to naïve MDSC. MDSC percentages are significantly less in lymphoid compartments of naïve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1(+) leukocytes from naïve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1(+) leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1(+) leukocytes from spleens of naïve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas naïve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from naïve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1(+) leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1(+) enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (naïve vs. tumor- bearing) in in vivo and in vitro assays.

Spiral Ganglion Stem Cells Can Be Propagated and Differentiated Into Neurons and Glia

PubMed Central

Zecha, Veronika; Wagenblast, Jens; Arnhold, Stefan; Edge, Albert S. B.; Stöver, Timo

2014-01-01

Abstract The spiral ganglion is an essential functional component of the peripheral auditory system. Most types of hearing loss are associated with spiral ganglion cell degeneration which is irreversible due to the inner ear's lack of regenerative capacity. Recent studies revealed the existence of stem cells in the postnatal spiral ganglion, which gives rise to the hope that these cells might be useful for regenerative inner ear therapies. Here, we provide an in-depth analysis of sphere-forming stem cells isolated from the spiral ganglion of postnatal mice. We show that spiral ganglion spheres have characteristics similar to neurospheres isolated from the brain. Importantly, spiral ganglion sphere cells maintain their major stem cell characteristics after repeated propagation, which enables the culture of spheres for an extended period of time. In this work, we also demonstrate that differentiated sphere-derived cell populations not only adopt the immunophenotype of mature spiral ganglion cells but also develop distinct ultrastructural features of neurons and glial cells. Thus, our work provides further evidence that self-renewing spiral ganglion stem cells might serve as a promising source for the regeneration of lost auditory neurons. PMID:24940560

Aggressive natural killer-cell leukemia with jaundice and spontaneous splenic rupture: a case report and review of the literature.

PubMed

Gao, Li-min; Liu, Wei-ping; Yang, Qun-pei; Li, Hui-fang; Chen, Jun-jie; Tang, Yuan; Zou, Yan; Liao, Dian-Ying; Liu, Yan-mei; Zhao, Sha

2013-03-11

Aggressive natural killer cell leukemia/lymphoma (ANKL) is a rare aggressive form of NK-cell neoplasm. We report an uncommon case of 36-year-old male who showed jaundice and spontaneous splenic rupture. The diagnosis was established by the biopsy of liver and spleen. The monomorphous medium-size neoplastic cells infiltrated into portal areas and sinus of liver as well as the cords and sinus of the spleen. Necrosis, mitotic figures and significant apoptosis could be seen easily. These neoplastic cells demonstrated a typical immunophenotype of CD3ε+, CD56+, CD16+, Granzyme B+, TIA-1+. T-cell receptor γ (TCR-γ) gene rearrangement analysis showed germline configuration and the result of in situ hybridization for Epstein-Barr virus-encoded RNA (EBER-ISH) was positive. The patient has undergone an aggressive clinical course and died of multi-organ function failure 14 days later after admission. To the best of our knowledge, this is the first case of ANKL with spontaneous splenic rupture, and we should pay more attention to recognize it. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2048154883890867.

Acute B lymphoblastic leukaemia-propagating cells are present at high frequency in diverse lymphoblast populations

PubMed Central

Rehe, Klaus; Wilson, Kerrie; Bomken, Simon; Williamson, Daniel; Irving, Julie; den Boer, Monique L; Stanulla, Martin; Schrappe, Martin; Hall, Andrew G; Heidenreich, Olaf; Vormoor, Josef

2013-01-01

Leukaemia-propagating cells are more frequent in high-risk acute B lymphoblastic leukaemia than in many malignancies that follow a hierarchical cancer stem cell model. It is unclear whether this characteristic can be more universally applied to patients from non-‘high-risk’ sub-groups and across a broad range of cellular immunophenotypes. Here, we demonstrate in a wide range of primary patient samples and patient samples previously passaged through mice that leukaemia-propagating cells are found in all populations defined by high or low expression of the lymphoid differentiation markers CD10, CD20 or CD34. The frequency of leukaemia-propagating cells and their engraftment kinetics do not differ between these populations. Transcriptomic analysis of CD34high and CD34low blasts establishes their difference and their similarity to comparable normal progenitors at different stages of B-cell development. However, consistent with the functional similarity of these populations, expression signatures characteristic of leukaemia propagating cells in acute myeloid leukaemia fail to distinguish between the different populations. Together, these findings suggest that there is no stem cell hierarchy in acute B lymphoblastic leukaemia. PMID:23229821

Trisomy 19 as the sole chromosomal anomaly in hematologic neoplasms.

PubMed

Johansson, B; Billström, R; Mauritzson, N; Mitelman, F

1994-05-01

Trisomy 19 was found as the sole chromosomal aberration in three hematologic malignancies: one chronic myelomonocytic leukemia and two cases of of immunophenotypically immature acute myeloid leukemia (AML). A compilation of previously published hematologic neoplasms with +19 as the only change reveals that this anomaly is strongly associated with myeloid malignancies; 25 of 31 cases have been myelodysplastic syndromes (MDS) or AML. Eight of the 11 MDS cases have been either refractory anemia (RA) or RA with excess of blasts, and four of the 14 AML cases have had preleukemic myelodysplastic cases phase, with the +19 accruing during the time of leukemic transformation. The AML cases have, in general, been either or early maturation arrest, i.e. undifferentiated or AML-M1/M2, or of myelomonocytic-monoblastic origin, i.e., AML-M4/M5. None of the MDS or AML cases with +19 had had a previous history of radio- or chemotherapy. We conclude that trisomy 19, as the sole anomaly, is a characteristic abnormality in de novo myeloid malignancies. No clinical features seem to characterize patients with +19 AML and MDS and the prognostic impact of the aberration remains to be elucidated.

Rectal lymphoma in 11 dogs: a retrospective study.

PubMed

Van den Steen, N; Berlato, D; Polton, G; Dobson, J; Stewart, J; Maglennon, G; Hayes, A M; Murphy, S

2012-10-01

To retrospectively evaluate the clinical behaviour and immunophenotype of lymphoma of the rectum in dogs. Eleven dogs diagnosed with lymphoma of the rectum on histopathology were retrospectively reviewed. Immunohistochemistry with CD3 and CD79a antibodies was performed at diagnosis or retrospectively. Treatment protocol varied with six dogs undergoing surgery and adjuvant chemotherapy, two received chemotherapy after only incisional biopsy, one had surgical resection only, one was treated symptomatically and one dog was not treated. Chemotherapy treatment consisted of either a -low-dose COP (cyclophosphamide - prednisolone - vincristine) protocol (four dogs) or a six-week CHOP-based (cyclophosphamide - vincristine - -prednisolone - anthracycline) protocol (four dogs). Dogs that received chemotherapy lived significantly longer than dogs that did not receive chemotherapy (2352 versus 70 days). Median survival time was not reached, and there was an overall mean survival time of 1697 days. Immunohistochemistry was performed in 10 of 11 samples, and was consistent with B-cell -lymphoma in all cases. Canine lymphoma of the rectum is associated with a favourable prognosis. Immunohistochemical evaluation of these lesions was consistent with B-cell lymphoma in all cases in which it was examined. © 2012 British Small Animal Veterinary Association.

Evaluation of a low density DNA microarray for small B-cell non-Hodgkin lymphoma differential diagnosis.

PubMed

Gillet, Jean-Pierre; Molina, Thierry Jo; Jamart, Jacques; Gaulard, Philippe; Leroy, Karen; Briere, Josette; Theate, Ivan; Thieblemont, Catherine; Bosly, Andre; Herin, Michel; Hamels, Jacques; Remacle, Jose

2009-03-01

Lymphomas are classified according to the World Health Organisation (WHO) classification which defines subtypes on the basis of clinical, morphological, immunophenotypic, molecular and cytogenetic criteria. Differential diagnosis of the subtypes is sometimes difficult, especially for small B-cell lymphoma (SBCL). Standardisation of molecular genetic assays using multiple gene expression analysis by microarrays could be a useful complement to the current diagnosis. The aim of the present study was to develop a low density DNA microarray for the analysis of 107 genes associated with B-cell non-Hodgkin lymphoma and to evaluate its performance in the diagnosis of SBCL. A predictive tool based on Fisher discriminant analysis using a training set of 40 patients including four different subtypes (follicular lymphoma n = 15, mantle cell lymphoma n = 7, B-cell chronic lymphocytic leukemia n = 6 and splenic marginal zone lymphoma n = 12) was designed. A short additional preliminary analysis to gauge the accuracy of this signature was then performed on an external set of nine patients. Using this model, eight of nine of those samples were classified successfully. This pilot study demonstrates that such a microarray tool may be a promising diagnostic approach for small B-cell non-Hodgkin lymphoma.

An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.

PubMed

Pinto, Alexander R; Paolicelli, Rosa; Salimova, Ekaterina; Gospocic, Janko; Slonimsky, Esfir; Bilbao-Cortes, Daniel; Godwin, James W; Rosenthal, Nadia A

2012-01-01

Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP(+) population within the adult Cx(3)cr1(GFP/+) knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45(+)CD11b(+)GFP(+)) are distinct from mononuclear CD45(+)CD11b(+)GFP(+) cells sorted from the spleen and brain of adult Cx(3)cr1(GFP/+) mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis.

Behçet's disease-like syndrome secondary to microbial infection: a case report and review of the literature.

PubMed

Zhang, Lingshu; Xu, Yuan; Peng, Yun; Yan, Bing; Liu, Yi

2015-01-01

Behçet's disease (BD)-like syndrome is an extremely rare situation occurred after Mycobacterium tuberculosis infection and virus infection. We reported a 45-year-old woman who visited our hospital complaining of swollen left ankle, painful genital ulcer, redness in the left eye and skin rash on lower limbs. The patient had a history of pleural tuberculosis and had received anti-tuberculous therapy for one year. Her left cervical lymph node sample demonstrated tubercle bacilli DNA fragmentation. The diagnosis of tuberculous lymphadenitis and Behçet's disease (BD)-like syndrome were made. This patient's symptoms remitted following treatment with anti-tuberculous therapy. This case indicates that some microbial infection can trigger the onset of BD-like syndrome in genetically susceptible subjects. However, treatment strategy of BD-like syndrome secondary to infection is totally different from primary BD. The aim of this case report is to present our experience of the different clinical signs and treatment of BD-like syndrome to expedite its early diagnosis in future. Combination of clinical, radiological, immunophenotypic, pathological, and genetic data contribute to improving the rate of diagnosis.

CD4-Negative Variant of Cutaneous Blastic Plasmacytoid Dendritic Cell Neoplasm With a Novel PBRM1 Mutation in an 11-Year-Old Girl.

PubMed

Yigit, Nuri; Suarez, Luisa Fernanda; Roth, Lisa Giulino; Orazi, Attilio; Tam, Wayne

2017-05-01

We report a rare case of CD4- cutaneous blastic plasmacytoid dendritic cell neoplasm (BPDCN) with a novel PBRM1 mutation. An 11-year-old girl presented with an enlarged mass on her left arm and underwent an incisional biopsy. Histopathologic examination and immunohistochemistry studies showed a monotonous proliferation of blasts that were CD4-, CD56+, and CD123+. There was no evidence of leukemic dissemination. Next-generation sequencing detected PBRM1 and CIC gene abnormalities. We confirmed and validated a novel PBRM1 mutation by conventional polymerase chain reaction and Sanger sequencing. CD4- variant of BPDCN may be mistaken for myeloid sarcoma or extramedullary lymphoblastic leukemia/lymphoma because of their overlapping morphologic and immunophenotypic features; thus, a careful clinicopathologic evaluation is essential to reach the correct diagnosis. PBRM1 mutation seems to be a driver event in this case. Our study underscores the importance of alterations in chromatin remodeling in the pathogenesis of BPDCN. © American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Fibroblastic connective tissue nevus.

PubMed

Velez, Moises J; Billings, Steven D; Weaver, Joshua A

2016-01-01

Fibroblastic connective tissue nevus (FCTN) is a newly recognized, benign cutaneous mesenchymal lesion of fibroblasts/myofibroblastic lineage, which expands the classification of connective tissue nevi. We present three cases of FCTN and discuss significant clinical, morphologic and immunophenotypic overlap with dermatomyofibroma. Our cases were from young women, aged 32, 24 and 10, and presented as 1.2 and 1 cm nodules on the posterior neck and right upper flank, respectively while presenting as a linear plaque of the right posterior thigh in the latter case. The lesions showed a poorly circumscribed proliferation of hypercellular spindle cells arranged in short to longer intersecting fascicles entrapping adnexal structures. Superficial adipose tissue was also entrapped in one case. The spindle cells had fibroblastic features with pale eosinophilic cytoplasmic extensions and inconspicuous nucleoli. The spindle cells were positive for CD34 in two cases. One case was negative for CD34, smooth muscle actin (SMA), desmin and S100. The overall features were consistent with a diagnosis of FCTN. In two cases, we further elucidated the fibroblastic differentiation of the spindle cells in FCTN with electron microscopy, which has not been previously described. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Surface topography of hairy cell leukemia cells compared to other leukemias as seen by scanning electron microscopy.

PubMed

Polliack, Aaron; Tadmor, Tamar

2011-06-01

This short review deals with the ultrastructural surface architecture of hairy cell leukemia (HCL) compared to other leukemic cells, as seen by scanning electron microscopy (SEM). The development of improved techniques for preparing blood cells for SEM in the 1970s readily enabled these features to be visualized more accurately. This review returns us to the earlier history of SEM, when the surface topography of normal and neoplastic cells was visualized and reported for the first time, in an era before the emergence and use of monoclonal antibodies and flow cytometry, now used routinely to define cells by their immunophenotype. Surface microvilli are characteristic for normal and leukemic lymphoid cells, myelo-monocytic cells lack microvilli and show surface ruffles, while leukemic plasma and myeloma cells and megakaryocytes display large surface blebs. HCL cell surfaces are complex and typically 'hybrid' in nature, displaying both lymphoid and monocytic features with florid ruffles of varying sizes interspersed with clumps of short microvilli cytoplasm. The surface features of other leukemic cells and photomicrographs of immuno-SEM labeling of cells employing antibodies and colloidal gold, reported more than 20 years ago, are shown.

Role of Synchrotron infra red microspectroscopy in studying epidermotropism of cutaneous T-cell lymphoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

El Bedewi, A.; El Anany, G; El Mofty, M

2010-01-01

The molecular mechanisms of epidermotropism in mycosis fungoides (MF) are not well understood to date. The aim of this study was to differentiate between epidermal and dermal lymphocytes within the skin of MF patients. This study was done on 10 MF patients with a mean age of 50 years diagnosed clinically in the Department of Dermatology, Cairo University, Egypt. A 6 mm biopsy was taken from each patient in order to confirm the diagnosis. Skin biopsies were cut, put on low e-slides and then stained with H&E. Further examination with Synchrotron infrared (IR) microspectroscopy was done in National Synchrotron Lightmore » Source - Brookhaven National Laboratory, New York, USA. Immunophenotyping using antibodies CD3, CD4, CD8, CD20 and CD30 was also done. Statistical analysis was done by Student's t-test and cluster analysis. Both epidermal and dermal lymphocytes were clustered separately. Also, Amide I and RNA and DNA within the lymphocytes were significantly different between the epidermis and the dermis. The biochemical analysis of protein, RNA and DNA with Synchrotron IR microspectroscopy is a promising tool for studying epidermotropism in cutaneous T-cell lymphoma.« less

Identification of early B cell precursors (stage 1 and 2 hematogones) in the peripheral blood.

PubMed

Kurzer, Jason H; Weinberg, Olga K

2018-05-25

Differentiating malignant B-lymphoblasts from early benign B cell precursors (hematogones) is a vital component of the diagnosis of B-lymphoblastic leukaemia. It has been previously reported that only late-stage B cell precursors circulate in the peripheral blood. Consequently, flow cytometric detection of cells with immunophenotypic findings similar to earlier stage precursors in the peripheral blood justifiably raises concern for involvement by B-lymphoblastic leukaemia. We report here, however, that benign early B cell precursors can indeed be detected in the peripheral blood, thus complicating the interpretation of flow cytometric findings derived from these sample types. A retrospective search of our collective databases identified 13 cases containing circulating early stage B cell precursors. The patients ranged in age from 15 days to 85 years old. All positive cases demonstrated that the earlier B cell precursors were associated with later stage precursors, a finding that could help differentiate these cells from B-lymphoblastic leukaemia. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Basaloid large cell lung carcinoma presenting as cutaneous metastasis at the colostomy site after abdominoperineal resection for rectal carcinoma.

PubMed

Sabater-Marco, Vicente; García-García, José Angel; Roig-Vila, José Vicente

2013-08-01

The occurrence of a tumor at the colostomy site after abdominoperineal resection for rectal carcinoma is rare and it may be related to a previously resected carcinoma or another primary tumor. We report a 61-year-old man who developed an ulcerated skin nodule at her colostomy site 6 years after resection of a rectal adenocarcinoma. Histopathologically, the skin nodule was composed of atypical large and pleomorphic cells with high mitotic rate and they were arranged in nests and within lymphatic channels in the dermis. The neoplastic cells were immunoreactive for cytokeratin (CK) AE1/3, CK7, CK34ßE12, epithelial membrane antigen and vimentin while detection of human papillomavirus and Epstein-Barr virus DNA was negative. A diagnosis of basaloid large cell carcinoma of pulmonary origin was suggested and it was confirmed by computed tomography-guided fine needle aspiration of a right subpleural mass. A metastatic tumor at the colostomy site is an exceptional finding and may be the first manifestation of lung cancer, especially if it consist of pleomorphic large cells with high mitotic rate and basaloid immunophenotype. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Keratocyst of the buccal mucosa: is it odontogenic?

PubMed

Ide, Fumio; Kikuchi, Kentaro; Miyazaki, Yuji; Mishima, Kenji; Saito, Ichiro; Kusama, Kaoru

2010-11-01

Odontogenic keratocyst (OKC) of the buccal mucosa, the diagnosis of which is based on subjective histologic evaluation, is a controversial entity of questionable existence. This report describes 2 rare cases of parakeratinized cyst arising from the buccal mucosa. Case 1 was a 60-year-old man with a 3-cm cyst and case 2 was a 16-year-old boy with a microcyst incidentally discovered on histology. Both lesions were essentially identical in histologic appearance and immunophenotype to intraosseous and gingival OKC, but they were clearly different from orthokeratinized odontogenic cysts and buccal mucosal epidermoid cysts. Step sections failed to reveal any kind of odontogenic tissue or skin adnexa in the cyst wall. These microscopic characteristics reflexively lead to the diagnosis of OKC, if the extragingival occurrence in the buccal mucosa cannot be considered. An alternative nonodontogenic origin includes a keratocyst of the skin, ie, an unusual mucosal presentation of cutaneous keratocyst. Because its true nature, either odontogenic or epidermal, cannot be conclusively proven at this time, we propose a more descriptive and noncommittal term, "mucosal keratocyst," for a particular cyst in a buccal location that is morphologically indistinguishable from OKC. Copyright © 2010 Mosby, Inc. All rights reserved.

SSAT/AHPBA Joint Symposium on Evaluation and Treatment of Benign Liver Lesions

PubMed Central

Chun, Yun Shin; House, Michael G.; Kaur, Harmeet; Loyer, Evelyne M.; Paradis, Valérie; Vauthey, Jean-Nicolas

2013-01-01

Background Benign liver lesions are common incidental radiologic findings. Methods Experts convened in 2011 at a Society for Surgery of the Alimentary Tract/ Americas Hepato-Pancreato-Biliary Association joint symposium to discuss the evaluation and treatment of benign liver lesions. Results Most benign liver lesions can be accurately diagnosed with high-quality imaging, including ultrasonography, multiphase computed tomography, and magnetic resonance imaging, particularly with hepatocyte-specific contrast agents. Percutaneous biopsy is reserved for lesions that cannot be characterized radiographically, and its accuracy is improved with immunophenotypic markers. Hepatic cysts are the most commonly diagnosed benign liver lesions; these must be distinguished from malignant cystic lesions, which are rare. Among the solid benign liver lesions, hemangiomas and focal nodular hyperplasia seldom require treatment. In contrast, hepatocellular adenomas are associated with a risk for complications. A new classification system for hepatocellular adenomas based on genetic and phenotypic features can help guide patient care. In patients who are symptomatic or at risk for complications, multidisciplinary evaluation and treatment based on clinicopathologic, radiographic, and molecular analysis is needed. Conclusions Most benign liver lesions can be accurately diagnosed radiographically and do not require treatment. Treatment is necessary for patients with symptoms or at risk for complications. PMID:23377783

Frequency of paroxysmal nocturnal hemoglobinuria in patients attended in Belém, Pará, Brazil

PubMed Central

de Brito Junior, Lacy Cardoso; Cardoso, Maria do Socorro de Oliveira; Rocha, Euzamar Gaby; Anijar, Herika; Cunha, Mariana; Saraiva, João Carlos Pina

2011-01-01

Background Paroxysmal nocturnal hemoglobinuria is a hematological disease with complex physiopathology. It is genetically characterized by a somatic mutation in the PIG-A gene (phosphatidylinositol glycan anchor biosynthesis, class A), in which the best known antigens are DAF (decay accelerating factor or CD55) and MIRL (membrane inhibitor of reactive lysis or CD59). Objective To determine the frequency of paroxysmal nocturnal hemoglobinuria in patients attended at the HEMOPA foundation from November 2008 to July 2009. Method Thirty patients, with ages ranging from two to 79 years old and suspected of having paroxysmal nocturnal hemoglobinuria were examined. All patients were immunophenotyped by flow cytometry for the CD5, CD59, CD16 and CD45 antigens. Results Paroxysmal nocturnal hemoglobinuria was identified in nine of the thirty patients investigated. Another 3 cases had inconclusive results with CD59-negative labeling only for neutrophils. The highest frequency of paroxysmal nocturnal hemoglobinuria patients (7/9) and inconclusive cases (2/3) were between 19 years old and 48 years old, with a median of 28 years. Conclusion These results show the importance of flow cytometry to identify cases in which patients are deficient in only one antigen (CD59). PMID:23284241

Immune Abnormalities in Autism Spectrum Disorder-Could They Hold Promise for Causative Treatment?

PubMed

Gładysz, Dominika; Krzywdzińska, Amanda; Hozyasz, Kamil K

2018-01-06

Autism spectrum disorders (ASD) are characterized by impairments in language and communication development, social behavior, and the occurrence of stereotypic patterns of behavior and interests. Despite substantial speculation about causes of ASD, its exact etiology remains unknown. Recent studies highlight a link between immune dysfunction and behavioral traits. Various immune anomalies, including humoral and cellular immunity along with abnormalities at the molecular level, have been reported. There is evidence of altered immune function both in cerebrospinal fluid and peripheral blood. Several studies hypothesize a role for neuroinflammation in ASD and are supported by brain tissue and cerebrospinal fluid analysis, as well as evidence of microglial activation. It has been shown that immune abnormalities occur in a substantial number of individuals with ASD. Identifying subgroups with immune system dysregulation and linking specific cellular immunophenotypes to different symptoms would be key to defining a group of patients with immune abnormalities as a major etiology underlying behavioral symptoms. These determinations would provide the opportunity to investigate causative treatments for a defined patient group that may specifically benefit from such an approach. This review summarizes recent insights into immune system dysfunction in individuals with ASD and discusses the potential implications for future therapies.

Use of Pig as a Model for Mesenchymal Stem Cell Therapies for Bone Regeneration.

PubMed

Rubessa, Marcello; Polkoff, Kathryn; Bionaz, Massimo; Monaco, Elisa; Milner, Derek J; Holllister, Scott J; Goldwasser, Michael S; Wheeler, Matthew B

2017-10-02

Bone is a plastic tissue with a large healing capability. However, extensive bone loss due to disease or trauma requires extreme therapy such as bone grafting or tissue-engineering applications. Presently, bone grafting is the gold standard for bone repair, but presents serious limitations including donor site morbidity, rejection, and limited tissue regeneration. The use of stem cells appears to be a means to overcome such limitations. Bone marrow mesenchymal stem cells (BMSC) have been the choice thus far for stem cell therapy for bone regeneration. However, adipose-derived stem cells (ASC) have similar immunophenotype, morphology, multilineage potential, and transcriptome compared to BMSC, and both types have demonstrated extensive osteogenic capacity both in vitro and in vivo in several species. The use of scaffolds in combination with stem cells and growth factors provides a valuable tool for guided bone regeneration, especially for complex anatomic defects. Before translation to human medicine, regenerative strategies must be developed in animal models to improve effectiveness and efficiency. The pig presents as a useful model due to similar macro- and microanatomy and favorable logistics of use. This review examines data that provides strong support for the clinical translation of the pig model for bone regeneration.

Peripheral T cell lymphomas: an immunological study of seven unusual cases.

PubMed

Raziuddin, S; Latif, A B; Arif, S; Ahad, A; Zaidi, A Z

1988-05-01

A multiparameter study of malignant lymph node cells and peripheral blood lymphocytes of seven patients with peripheral T cell lymphoma is presented. The results of monoclonal marker studies showed three cases of helper-suppressor T cell lymphoma (OKT4+, OKT8+), one case of suppressor T cell lymphoma (OKT8+), and three cases of helper T cell lymphoma (OKT4+). Immunophenotypic heterogeneity of neoplastic T cells with expression of pan-T antigens, OKT3+, and OKT11+ (erythrocyte rosetting+) was observed in most patients. Six of the seven cases tested showed Ia and DR antigens. No relationship was detected between patterns of reactivity with T cell reagents and histological types. When tested, the in-vitro malignant T cells of five patients proliferated in response to concanavalin A (Con A), but had poor response to phytohaemagglutinin. The interleukin 2 receptors showed maximum expression on Con A-activated T cells of five patients, and phytohaemagglutinin-activated T cells of one patient. The neoplastic T cells (OKT4+, OKT8+) of one patient studied had suppressor activity for IgG and IgA, and helper activity for IgM synthesis on pokeweed mitogen-induced normal B cell differentiations.

Reticulocyte analysis using flow cytometry.

PubMed

Corberand, J X

1996-12-01

Automation of the reticulocyte count by means of flow cytometry has considerably improved the quality of this investigation. This article deals firstly with the reasons for the poor performance of the microscopic technique and with the physiological principles underlying identification and classification of reticulocytes using RNA labeling. It then outlines the automated methods currently on the market, which can be classified in three categories: a) "general-purpose" cytofluorometers, which in clinical laboratories usually deal with lymphocyte immunophenotyping; b) the only commercially available cytofluorometer dedicated to the reticulocyte count; this automat has the advantage of requiring no human intervention as it merely needs to be fed with samples; c) hematology analyzers with specific modules for automatic counting of reticulocytes previously incubated with a non-fluorescent dye. Of the various fluorescent markers available, thiazole orange, DEQTC iodide and auramine are most often used for this basic hematology test. The quality of the count, the availability of new reticulocyte indices (maturation index, percentage of young reticulocytes) and rapidity of the count give this test renewed value in the practical approach to the diagnosis of anemia, and also open new perspectives in the surveillance of aplastic anemia after chemotherapy or bone marrow grafting.

Identification of a novel fusion gene, IRF2BP2-RARA, in acute promyelocytic leukemia.

PubMed

Yin, C Cameron; Jain, Nitin; Mehrotra, Meenakshi; Zhagn, Jianhua; Protopopov, Alexei; Zuo, Zhuang; Pemmaraju, Naveen; DiNardo, Courtney; Hirsch-Ginsberg, Cheryl; Wang, Sa A; Medeiros, L Jeffrey; Chin, Lynda; Patel, Keyur P; Ravandi, Farhad; Futreal, Andrew; Bueso-Ramos, Carlos E

2015-01-01

Acute promyelocytic leukemia (APL) is characterized by the fusion of retinoic acid receptor alpha (RARA) with promyelocytic leukemia (PML) or, rarely, other gene partners. This report presents a patient with APL with a novel fusion between RARA and the interferon regulatory factor 2 binding protein 2 (IRF2BP2) genes. A bone marrow examination in a 19-year-old woman who presented with ecchymoses and epistaxis showed morphologic and immunophenotypic features consistent with APL. PML oncogenic domain antibody was positive. Results of fluorescence in situ hybridization, conventional cytogenetics, reverse transcription-polymerase chain reaction (RT-PCR), and oligonucleotide microarray for PML-RARA and common APL variant translocations were negative. Next-generation RNA-sequencing analysis followed by RT-PCR and direct sequencing revealed distinct breakpoints within IRF2BP2 exon 2 and RARA intron 2. The patient received all-trans retinoic acid, arsenic, and gemtuzumab ozogamicin, and achieved complete remission. However, the disease relapsed 10 months later, 2 months after consolidation therapy. This is the first report showing involvement of IRF2BP2 in APL, and it expands the list of novel RARA partners identified in APL. Copyright © 2015 by the National Comprehensive Cancer Network.

Pediatric nodal marginal zone lymphoma may develop in the adult population.

PubMed

Gitelson, Elena; Al-Saleem, Tahseen; Robu, Valentin; Millenson, Michael M; Smith, Mitchell R

2010-01-01

Pediatric nodal marginal zone lymphoma (NMZL) is described as a separate variant of NMZL in the most recent WHO classification of tumors of hematologic and lymphoid tissues. It has distinctive morphology and clinical presentation and stands out as an indolent disease with remarkably better overall prognosis compared to classic NMZL. Here we report two adult patients with NMZL with clinical and morphologic features consistent with pediatric NMZL (pNMZL) and review available literature describing the clinical and histologic presentation of pNMZL. Two men, ages 44 and 18 years, each presented with localized cervical lymphadenopathy, both demonstrated florid proliferation of the marginal zone and disruption of reactive germinal centers, progressive transformation of germinal centers-like morphologic features typical for pNMZL and clonal disease with immunophenotype consistent with NMZL. This is the first report of pNMZL in a middle-aged person. Distinct histologic features and characteristic benign clinical course will help to distinguish this rare variant from other NMZL in the adults. Clinically, recognition is important to understand the true incidence of this rare form in the adult population and to avoid unnecessary overtreatment of this indolent form.

Evaluation of the immunological cellular response of Cebus apella exposed to the carcinogen N-methyl-N-nitrosourea and treated with CANOVA®.

PubMed

Feio, Danielle Cristinne Azevedo; Muniz, José Augusto Pereira Carneiro; Montenegro, Raquel Carvalho; Burbano, Rommel Rodriguez; De Brito Junior, Lacy Cardoso; De Lima, Patrícia Danielle Lima

2014-01-01

The immune response modifier Canova® is a homeopathic remedy indicated for patients with depressed immune system, since this drug appears to increase adaptive immunity and induce an immune response against multiple and severe pathological conditions, including cancer. We evaluated the pattern of immune cellular response in non-human primates of the species Cebus apella exposed to N-methyl-N-nitrosourea (MNU) with and without Canova®. Twelve animals were divided into four groups, with three animals each: negative control and three experimental groups, MNU-alone (35 days); MNU (35 days)-plus-Canova® (3 days) and Canova®-alone (3 days). The animals received MNU orally and Canova® by three intravenous injections. Evaluation of the cellular immune response was performed by immunophenotyping of T-lymphocytes (CD4(+), CD8(+)), B-lymphocytes and natural killer cells. Analysis was also performed of the cell cycle. Our results suggest an increase of T-lymphocytes (CD4(+)CD3(+)) only in the Canova® group, while in the MNU-plus-Canova® group only B-lymphocytes increased. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Immune Response to Marburg Virus Angola Infection in Nonhuman Primates.

PubMed

Fernando, Lisa; Qiu, Xiangguo; Melito, P Leno; Williams, Kinola J N; Feldmann, Friederike; Feldmann, Heinz; Jones, Steven M; Alimonti, Judie B

2015-10-01

The 2005 outbreak of Marburg virus (MARV) infection in Angola was the most lethal MARV infection outbreak in history, with a case-fatality rate (90%) similar to that for Zaire ebolavirus (EBOV) infection. However, very little is known about the pathogenicity of MARV Angola, as few studies have been conducted to date. Therefore, the immune response was examined in MARV Angola-infected nonhuman primates. Cynomolgus macaques were infected with MARV Angola and monitored for survival. The effect of MARV Angola on the immune system was examined by immunophenotyping whole-blood and by analyzing cytokine and chemokine levels in plasma and spleen specimens, using flow cytometry. The prominent clinical findings were rapid onset of disease and death (mean time after infection, 6.7 days), fever, depression, anorexia, petechial rash, and lymphopenia. Specifically, T, B, and natural killer cells were severely depleted in the blood by day 6. The typical cytokine storm was present, with levels of interferon γ, tumor necrosis factor, interleukin 6, and CCL2 rising in the blood early during infection. MARV Angola displayed the same virulence and disease pathology as EBOV. MARV Angola appears to cause a more rapid onset and severe outcome of infection than other MARV strains. © Crown copyright 2015.

Barcoding of live human PBMC for multiplexed mass cytometry*

PubMed Central

Mei, Henrik E.; Leipold, Michael D.; Schulz, Axel Ronald; Chester, Cariad; Maecker, Holden T.

2014-01-01

Mass cytometry is developing as a means of multiparametric single cell analysis. Here, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a CyTOF® instrument. Using six different anti-CD45 antibody (Ab) conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and reduces wet work and antibody consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45-barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and should be applicable to fluorescence flow cytometry as well. PMID:25609839

Reference ranges of lymphocyte subsets balanced for age and gender from a population of healthy adults in Chongqing District of China.

PubMed

Zhang, Kejun; Wang, Feng; Zhang, Mingxu; Cao, Xinglu; Yang, Shaojun; Jia, Shuangrong; Wang, Lixin; Luo, Jie; Deng, Shaoli; Chen, Ming

2016-11-01

The enumeration of lymphocyte subsets plays an essential role in the monitoring of immunological disorders. Immunophenotyping values have been found to be influenced by race, age, gender, and environmental conditions. Therefore, it is important to establish reference ranges for healthy adults from the local population for clinical decision-making. The current study aimed to establish a normal reference range for peripheral blood lymphocyte subsets in healthy adults from the Chongqing District of China by using single-platform flow cytometry. Age- and gender-specific reference ranges were established in 268 healthy adult males and females between 21 and 60 years of age. The CD8+ cell counts decreased with age, CD4+ cell percentages and counts increased with age, and total T cell percentages were higher in the female population. Our results are similar to those reported from other parts of China but different from some results reported from other countries; this further stresses the need to establish local reference ranges by region. Our results will help in the management of patients with human immunodeficiency virus and other immunological disorders in Chongqing District. © 2015 International Clinical Cytometry Society. © 2015 International Clinical Cytometry Society.

Highly multiparametric analysis by mass cytometry.

PubMed

Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Nitz, Mark; Winnik, Mitchell A; Tanner, Scott

2010-09-30

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays. Copyright © 2010 Elsevier B.V. All rights reserved.

T lymphocyte subpopulations diverge in commercially raised chickens

PubMed Central

Bridle, Byram W.; Julian, Richard; Shewen, Patricia E.; Vaillancourt, Jean-Pierre

2006-01-01

Abstract To evaluate immunocompetence in commercially raised chickens, we immunophenotyped Dekalb Delta and H&N White Leghorn (WLH) hybrids, 20 chickens in each of 3 age groups (9 wk [juvenile], 25 wk [young adult], and 79 or 80 wk [adult]), for circulating CD3+, CD4+, CD8+, TCR1+, TCR2+, and TCR3+ lymphocytes. The proportion of CD3+ T cells, including CD4+ and CD8+ subsets, was increased in the hybrids as compared with published values for laboratory-raised outbred WLH chickens. The proportion of the TCR2+ (Vβ1) T cell subpopulation was also increased. An age-related decrease in the proportion of TCR1+ (γδ) T cells was noted in both hybrids. Further, a remarkably low CD4:CD8 ratio was evident in all age groups of both hybrids, indicating decreased immunocompetence. Overall, these experiments provide age-related proportions of various peripheral-blood T lymphocyte subpopulations in commercially raised Dekalb Delta and H&N chickens that diverge from the proportions in laboratory-raised outbred WLH chickens and suggest reduced immunocompetence. Such a decline in immunocompetence, including humoral immune capacity, could be attributed to genetic selection for production traits, environmental factors associated with commercial operations, and intense immunization. PMID:16850940

Tumor Associated Mesenchymal Stromal Cells Show Higher Immunosuppressive and Angiogenic Properties Compared to Adipose Derived MSCs.

PubMed

Langroudi, Ladan; Hassan, Zuhair Muhammad; Soleimani, Masoud; Hashemi, Seyed Mahmoud

2015-12-01

Differentiation, migratory properties and availability of Mesenchymal Stromal Cells (MSC) have become an important part of biomedical research. However, the functional heterogeneity of cells derived from different tissues has hampered providing definitive phenotypic markers for these cells. To characterize and compare the phenotype and cytokines of adipose derived MSCs (AD-MSCs) and tumoral-MSCs (T-MSCs) isolated from mammary tumors of BALB/c mice. Immunophenotyping and in vitro differentiation tests were used for MSC characterization. Cytokine and enzyme profiles were assessed using ELISA and Real-time PCR, respectively. T-MSCs expressed significantly higher levels of HLA-DR (p=0.04). Higher levels of PGE2 and COX-2 enzyme were also observed in T-MSCs (p=0.07 and p=0.00, respectively). Additionally, T-MSCs expressed higher levels of iNOS and MMP9 (p=0.01 and p=0.01, respectively). T-MSCs were also able to induce higher levels of proliferation and migration of HUVEC endothelial cells in wound scratch assay compared to AD-MSCs (p=0.015). Functional differences showed by the surface markers of MSCs, cytokine and enzyme production indicate the effect of different microenvironments on MSCs phenotype and function.

A case of primary mucosa-associated lymphoid tissue lymphoma of the vagina.

PubMed

Yoshinaga, Kousuke; Akahira, Jun-Ichi; Niikura, Hitoshi; Ito, Kiyoshi; Moriya, Takuya; Murakami, Takashi; Kameoka, Jun-Ichi; Ichinohasama, Ryo; Okamura, Kunihiro; Yaegashi, Nobuo

2004-09-01

We report the first case of primary mucosa-associated lymphoid tissue (MALT) lymphoma of the vagina, the diagnosis of which is supported by genetic and immunophenotypic studies. A 65-year-old, para 2 woman presented to our hospital in July 1997 with a history of prolonged vaginal discharge. Although cytologic examination suggested possible malignancy, a biopsy of the vaginal wall was diagnosed as chronic inflammation. In June 2000, she underwent gynecologic examination because of anuria. Excisional biopsy revealed subepithelial infiltration of atypical lymphoid cells that stained for CD20, CD79a, and BCL-2; stained weakly for IgM; and did not stain for CD3, CD5, CD7, CD10, CD56, CD23, and IgD, suggesting marginal zone B-cell lineage. Monoclonality was detected by Southern blot analysis, and this patient was finally diagnosed as having primary MALT lymphoma of the vagina. She received 3 cycles of chemotherapy (THP-COP) and concurrent radiation to the whole pelvis. The patient is alive and well 40 months after treatment. Because the vagina is one of the mucosa-associated tissues, MALT lymphoma, though rare, must be included in the differential diagnosis of the vaginal neoplasms.

Acute erythremic myelosis (true erythroleukaemia): a variant of AML FAB-M6.

PubMed

Hasserjian, R P; Howard, J; Wood, A; Henry, K; Bain, B

2001-03-01

Classic erythroleukaemia (acute myeloid leukaemia M6, or M6 AML) is defined as an excess of myeloblasts in an erythroid predominant background. Leukaemia variants in which the primitive blast cells are demonstrably erythroid are extremely rare and poorly characterised. Variably referred to as "true erythroleukaemia" or "acute erythremic myelosis", they are often included within the M6 AML category even though they do not meet strict criteria for this type of AML. Two cases of acute erythroid neoplasia are presented with clinical, morphological, immunophenotypic, and cytogenetic analysis. Both patients presented with profound anaemia, one in a setting of long standing myelodysplasia. Bone marrow examination revealed a predominant population of highly dysplastic erythroid cells in both cases. In one case, the liver was infiltrated by neoplastic erythroid cells. Both patients died within four months of diagnosis. This report illustrates that cases of acute leukaemia occur in which the dominant neoplastic cell is a primitive erythroid cell without an accompanying increase in myeloblasts. This does not preclude the neoplastic clone originating in a multipotent haemopoietic stem cell, as suggested by cases arising in patients with myelodysplasia. Acute erythremic myelosis should be recognised as a distinct variant of M6 AML.

Immune functions in the Fisher rat fed beluga whale (Delphinapterus leucas) blubber from the contaminated St. Lawrence estuary

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lapierre, P.; Guise, S. De; Muir, D.C.G.

1999-02-01

In order to assess the immunotoxic potential of food naturally contaminated with PCBs and other organohalogens, Fisher rats were fed a diet in which the lipids originated from the blubber of either a highly polluted St. Lawrence beluga or a relatively uncontaminated Arctic beluga. After a period of 2 months, different immune functions were evaluated, including lymphoblastic transformation, natural killer cell activity, plaque-forming cells, phagocytosis, oxidative burst, and immunophenotyping. For all assays, rats fed at St. Lawrence beluga blubber diet or a mixture of Arctic and St. Lawrence beluga blubber diet were not different from control rats fed a dietmore » containing Arctic beluga blubber. These results are inconsistent with the well-known immunosuppressive effects of organochlorines in numerous species and with the lesions suggestive of organochlorine-related immunosuppression that are observed in St. Lawrence belugas. The lack of observable immunotoxic effects in rats fed contaminated beluga blubber might be explained by antagonistic effects in the organohalogen mixture, by a response specific to the rat, by a strain-related lack of sensitivity to organochlorines, or by insufficient dose due to the shortness of the exposure period or the route of exposure.« less

Desmoplastic ganglioglioma of the spinal cord in a western European hedgehog (Erinaceus europaeus).

PubMed

Ulrich, Reiner; Stan, Alexandru C; Fehr, Michael; Mallig, Carolin; Puff, Christina

2010-11-01

Gangliogliomas are composed of neoplastic glial and neuronal cells and are extremely rare tumors of the central nervous system of domestic animals. The present report describes the clinical presentation and the pathomorphological and immunophenotypical characteristics of a desmoplastic ganglioglioma in the spinal cord of a 3-year-old male western European hedgehog (Erinaceus europaeus). Clinically, the hedgehog exhibited a skin wound and therapy-resistant paresis of the left hind limb. Necropsy showed dilatation of the urinary bladder. Histologic examination of the thoracic spinal cord revealed a focally extensive infiltrative mass, which consisted of multiple nodules of smaller bipolar or oligopolar glial cells and variably sized polygonal, ganglionic, neuron-like cells embedded in variable amounts of microcystic neuropilic matrix. An area of spindle-shaped cells arranged in interwoven fascicles and surrounded by a prominent network of reticulin fibers was interpreted as desmoplastic leptomeningeal stroma. Immunohistochemistry revealed a moderate number of glial fibrillary acidic protein and S-100-positive cells and processes. In addition, the ganglionic neuron-like cells expressed neurofilament, microtubule-associated protein-2, and neuron-specific enolase. In summary, this spinal cord tumor was composed of astroglial and neuronal cellular elements, justifying the diagnosis of a desmoplastic ganglioglioma.

Squamous cell carcinoma of the uterine cervix associated with osteoclast-like giant cells: A case report and review of the literature.

PubMed

Yu, Guohua; Lin, Chunhua; Wang, Wei; Han, Yekun; Qu, Guimei; Zhang, Tingguo

2014-10-01

Squamous cell carcinoma is a common malignant tumor of the uterine cervix. The present study reports the case of squamous cell carcinoma of the uterine cervix with osteoclast-like giant cells (OGCs) in an 84-year-old female who had suffered from irregular vaginal bleeding for one month. Colposcopy was performed and a cauliflower-like mass was identified in the front lip of the uterine cervix. Biopsy was then performed, and the tumor was found to be composed of epithelial cell nests, ranging in size. The neoplastic cells exhibited unclear boundaries and eosinophilic cytoplasm. Additionally, the nuclei were atypical and mitosis was observed. Among the epithelial nests, there were numerous OGCs with abundant eosinophilic cytoplasm, as well as multinucleation with bland nuclei. By immunohistochemical staining, the epithelial cells were positive for cytokeratin, while negative for CD68 and vimentin. By contrast, the immunophenotype of the OGCs was the exact opposite. Based on the histological characters, a diagnosis of squamous cell carcinoma of the uterine cervix associated with OGCs was made. Considering the age of the patient, radiotherapy was administered. The patient succumbed to brain metastasis of the tumor after eight months of follow-up.

Amniotic fluid stem cells prevent follicle atresia and rescue fertility of mice with premature ovarian failure induced by chemotherapy.

PubMed

Xiao, Guan-Yu; Liu, I-Hsuan; Cheng, Chun-Chun; Chang, Chia-Chun; Lee, Yen-Hua; Cheng, Winston Teng-Kuei; Wu, Shinn-Chih

2014-01-01

Chemotherapy used to treat cancer may cause irreversible premature ovarian failure (POF). Of late, amniotic fluid stem cells (AFSCs) provide a novel source for regenerative medicine because of their primitive stage, low immunogenicity, and easy accessibility. In this study, we isolated AFSCs from transgenic mice that ubiquitously express enhanced green fluorescence protein (EGFP). These AFSCs exhibited morphologies, immunophenotypes, and mesoderm trilineage differentiation potentials similar to mesenchymal stem cells (MSCs). Further, AFSCs proliferated faster than MSCs and expressed OCT4, a marker for pluripotency. To investigate their potential in recovering fertility in POF model, AFSCs were transplanted into the ovaries of mice with POF six weeks post induction using chemotherapeutic drugs, busulfan and cyclophosphamide. AFSCs could rescue the reproductive ability of mice with POF by preventing follicle atresia and sustaining the healthy follicles. Notably, the transplanted AFSCs did not differentiate into granulosa and germline cells in vivo. After one month, the decreased numbers of transplanted AFSCs accompanied with the reduced beneficial effects indicated that the therapeutic efficacy were directly from AFSCs. These findings demonstrated the therapeutic effects of AFSCs and suggested the promise of AFSCs for treating infertility and POF caused by chemotherapy.

[The combination of chronic myeloid leukemia and multiple myeloma in one patient].

PubMed

Romanenko, N A; Bessmel'tsev, S S; Udal'eva, V Iu; Zenina, M N; Martynkevich, I S; Rugal', V I; Abdulkadyrov, K M

2013-01-01

The article describes the clinical observation of a patient with simultaneous course of lymphoid and myeloid neoplasms. The patient developed two diseases--chronic myeloid leukemia (CML) and multiple myeloma (MM), which were confirmed by corroborated hemogram, myelogram, immunophenotyping of bone marrow cells, biopsy, immunohistochemical, cytogenetic, biochemical and radiological studies. Target therapy of CML with tyrosine kinase inhibitors (imatinib at the standard dose of 400 mg per day) has provided a complete cytogenetic remission at 6 months and major molecular response at 18 months of treatment. Administration of 2 courses of programmed treatment "BD" > (bortezomib + dexamethasone) resulted in a very good partial response, which was maintained through a year and a half. However, against the background of programmed treatment there were developed complications as polyneuropathy of grade 2, which was treated with thioctacide, milgamy, and anemia of grade 2, successfully treated with epoetin beta. Subsequently, the patient was administered continuously with imatinib 400 mg that kept the major molecular response. Relapsed MM was revealed in 20 months and confirmed by a full clinical and hematological examination. The absence of organ dysfunction allowed choosing a supervisory tactics for the patient.

Diagnostic value of medical thoracoscopy in malignant pleural effusion induced by non-Hodgkin's lymphoma

PubMed Central

Wang, Zhen; Wu, Yan-Bing; Xu, Li-Li; Jin, Mu-Lan; Diao, Xiao-Li; Wang, Xiao-Juan; Tong, Zhao-Hui; Shi, Huan-Zhong

2017-01-01

Malignant pleural effusion (MPE) appears in up to 20% of patients with non-Hodgkin's lymphoma (NHL). The present study aimed to assess the efficacy of medical thoracoscopy (MT) in the diagnosis of patients with MPE induced by NHL. Between July 2005 and June 2014, 833 patients with pleural effusions of unknown etiology underwent MT in Beijing Chaoyang Hospital (Beijing, China), where diagnostic thoracocentesis or/and blind pleural biopsy had failed to yield an answer. Demographic, radiographic, thoracoscopic, histological and immunophenotyping data of 10 NHL patients with MPE were then retrospectively analyzed. Under medical thoracoscopy, pleural nodules (in n=6 patients), hyperemia (n=5), plaque-like lesions (n=4), pleural thickening (n=3), cellulose (n=3), ulcer (n=2), adhesion (n=2), and scattered hemorrhagic spots (n=1) were observed on the surface of parietal pleura. Histopathological and immunohistochemical analysis of pleural biopsy samples led to a correct diagnosis of B-cell NHL in 7 patients and T-lymphoblastic NHL in 2 patients. Data from the present study demonstrated that pleural biopsy through MT achieved a definite diagnosis of NHL in 9 out of 10 (90%) patients with MPE induced by NHL. Therefore, MT is a useful method for diagnosing MPE induced by NHL. PMID:29344253

Diagnostic value of medical thoracoscopy in malignant pleural effusion induced by non-Hodgkin's lymphoma.

PubMed

Wang, Zhen; Wu, Yan-Bing; Xu, Li-Li; Jin, Mu-Lan; Diao, Xiao-Li; Wang, Xiao-Juan; Tong, Zhao-Hui; Shi, Huan-Zhong

2017-12-01

Malignant pleural effusion (MPE) appears in up to 20% of patients with non-Hodgkin's lymphoma (NHL). The present study aimed to assess the efficacy of medical thoracoscopy (MT) in the diagnosis of patients with MPE induced by NHL. Between July 2005 and June 2014, 833 patients with pleural effusions of unknown etiology underwent MT in Beijing Chaoyang Hospital (Beijing, China), where diagnostic thoracocentesis or/and blind pleural biopsy had failed to yield an answer. Demographic, radiographic, thoracoscopic, histological and immunophenotyping data of 10 NHL patients with MPE were then retrospectively analyzed. Under medical thoracoscopy, pleural nodules (in n=6 patients), hyperemia (n=5), plaque-like lesions (n=4), pleural thickening (n=3), cellulose (n=3), ulcer (n=2), adhesion (n=2), and scattered hemorrhagic spots (n=1) were observed on the surface of parietal pleura. Histopathological and immunohistochemical analysis of pleural biopsy samples led to a correct diagnosis of B-cell NHL in 7 patients and T-lymphoblastic NHL in 2 patients. Data from the present study demonstrated that pleural biopsy through MT achieved a definite diagnosis of NHL in 9 out of 10 (90%) patients with MPE induced by NHL. Therefore, MT is a useful method for diagnosing MPE induced by NHL.

Chromosomal rearrangement of the PAX-5 locus in lymphoplasmacytic lymphoma with t(9;14)(p13;q32).

PubMed

Iida, S; Rao, P H; Ueda, R; Chaganti, R S; Dalla-Favera, R

1999-06-01

B-cell non-Hodgkin's lymphoma (NHL) consists of heterogeneous subtypes based on histologic, immunophenotypic, and clinical findings. Recent advances in molecular biology have provided us new insights into the pathogenesis of this neoplasm at the genetic level, such as the deregulation of the protooncogenes adjoining the immunoglobulin gene (Ig) loci, which is a specific event in mature B-cell tumors. Moreover, involvement of certain protooncogenes corresponds to certain subtypes of NHL. Recently, we found that t(9;14)(p13;q32) chromosomal translocation associated with lymphoplasmacytic lymphoma (LPL) juxtaposes PAX-5 gene encoding for an essential transcription factor (BSAP: B-cell specific activator protein) for B-cell proliferation and differentiation to the Ig heavy chain gene (IgH) locus. This results in deregulated expression of the PAX-5 mRNA. We also developed a diagnostic FISH (fluorescence in situ hybridization) procedure which is able to detect 80% of the widely scattering 9p13 breakpoints involved in this translocation. Thus, an understanding of the PAX-5 gene's physiological role in B-cell development and the pathological role in tumorigenesis may lead to the optimal clinical treatment strategy for LPL and LPL-derived diffuse large cell lymphoma (DLCL).

Liver Metastases From Renal Oncocytoma With Vascular Extension.

PubMed

Cacciamani, Giovanni; Cima, Luca; Ficial, Miriam; Novella, Giovanni; Siracusano, Salvatore; Tedeschi, Umberto; Balzarro, Matteo; Montin, Umberto; Cerruto, Maria A; De Marco, Vincenzo; Porcaro, Antonio B; Hes, Ondrej; DʼErrico, Antonia; Martignoni, Guido; Ghimenton, Claudio; Zaza, Gianluigi; Artibani, Walter; Brunelli, Matteo; Eccher, Albino

2017-07-04

The 2016 World Health Organization Renal Tumor Classification defines renal oncocytoma (RO) as a benign epithelial tumor; however, malignant histopathologic features have been documented. Rare cases with metastases have been reported. We describe the case of a 62-year-old woman who was referred to the Urology Clinic for a routine work-up. Magnetic resonance imaging and computerized tomography showed a 7-cm mass in the middle and lower portions of the left kidney and 2 suspected liver metastases. The patient underwent surgery. Microscopically both renal and liver lesions presented solid, solid-nested, and microcystic architecture, composed predominantly of large eosinophilic cells without any worrisome pattern except the vascular extension. The cells were positive for S100A1, CD117, and PAX-8 and negative for CAIX, CK7, and AMACR. Fluorescence in situ hybridization showed a disomic profile for the chromosomes 1, 2, 6, 7, 10, 17. No mutation of coding sequence of the SDHB, SDHC, SDHD, VHL, and BHD genes and no loss of heterozygosity at 3p were found. The final diagnosis was "RO" according to the 2016 World Health Organization Renal Tumor Classification with "liver metastases." This report provides a wide clinical-pathologic, immunophenotypical and molecular documentation of a RO with liver metastases.

A Reliable and Reproducible Model for Assessing the Effect of Different Concentrations of α-Solanine on Rat Bone Marrow Mesenchymal Stem Cells.

PubMed

Ordóñez-Vásquez, Adriana; Jaramillo-Gómez, Lorenza; Duran-Correa, Camilo; Escamilla-García, Erandi; De la Garza-Ramos, Myriam Angélica; Suárez-Obando, Fernando

2017-01-01

Αlpha-solanine ( α -solanine) is a glycoalkaloid present in potato (Solanum tuberosum) . It has been of particular interest because of its toxicity and potential teratogenic effects that include abnormalities of the central nervous system, such as exencephaly, encephalocele, and anophthalmia. Various types of cell culture have been used as experimental models to determine the effect of α -solanine on cell physiology. The morphological changes in the mesenchymal stem cell upon exposure to α -solanine have not been established. This study aimed to describe a reliable and reproducible model for assessing the structural changes induced by exposure of mouse bone marrow mesenchymal stem cells (MSCs) to different concentrations of α -solanine for 24 h. The results demonstrate that nonlethal concentrations of α -solanine (2-6  μ M) changed the morphology of the cells, including an increase in the number of nucleoli, suggesting elevated protein synthesis, and the formation of spicules. In addition, treatment with α -solanine reduced the number of adherent cells and the formation of colonies in culture. Immunophenotypic characterization and staining of MSCs are proposed as a reproducible method that allows description of cells exposed to the glycoalkaloid, α -solanine.

A Reliable and Reproducible Model for Assessing the Effect of Different Concentrations of α-Solanine on Rat Bone Marrow Mesenchymal Stem Cells

PubMed Central

Ordóñez-Vásquez, Adriana; Jaramillo-Gómez, Lorenza; Duran-Correa, Camilo

2017-01-01

Αlpha-solanine (α-solanine) is a glycoalkaloid present in potato (Solanum tuberosum). It has been of particular interest because of its toxicity and potential teratogenic effects that include abnormalities of the central nervous system, such as exencephaly, encephalocele, and anophthalmia. Various types of cell culture have been used as experimental models to determine the effect of α-solanine on cell physiology. The morphological changes in the mesenchymal stem cell upon exposure to α-solanine have not been established. This study aimed to describe a reliable and reproducible model for assessing the structural changes induced by exposure of mouse bone marrow mesenchymal stem cells (MSCs) to different concentrations of α-solanine for 24 h. The results demonstrate that nonlethal concentrations of α-solanine (2–6 μM) changed the morphology of the cells, including an increase in the number of nucleoli, suggesting elevated protein synthesis, and the formation of spicules. In addition, treatment with α-solanine reduced the number of adherent cells and the formation of colonies in culture. Immunophenotypic characterization and staining of MSCs are proposed as a reproducible method that allows description of cells exposed to the glycoalkaloid, α-solanine. PMID:29201465

Immunocytochemical markers in acute leukaemias diagnosis.

PubMed

Gluzman, D F; Nadgornaya, V A; Sklyarenko, L M; Ivanovskaya, T S; Poludnenko, L Yu; Ukrainskaya, N I

2010-09-01

The study included 1742 patients with acute myeloblastic leukaemias (AML) and acute lymphoblastic leukaemias (ALL), Kyiv city residents and patients from 20 regions of Ukraine. Bone marrow and blood smears were sent at diagnosis to Reference Center. The analysis was based on May-Grünvald-Giemza (MGG) stain and cytochemical reactions (MPO, acNSE, CAE, AP, PAS). Immunocytochemical techniques (APAAP, LSAB) and broad panel of monoclonal antibodies (MoAbs) against lineage specific and differentiation antigens of leukocytes were employed for immunophenotyping of leukemic blast cells directly in blood and bone marrow smears. Different types of AML were defined by the expression of the cell surface and cytoplasmic antigens. Immunocytochemical study was required especially in diagnosing of AML with minimal differentiation, acute megakaryoblastic leukaemia, acute erythroid leukaemia and acute leukaemias of ambiguous lineage. Acute lymphoblastic leukaemias was broadly classified into B-lineage and T-lineage ALL. According to the degree of B-lymphoid differentiation of the blast cells four subtypes of B-lineage ALL were established. T-lineage ALL observed in patients were also divided into four subtypes. Immunocytochemical examination was required to diagnose AL of ambiguous lineage with no clear evidence of lineage differentiation (acute undifferentiated leukaemia) or those with blasts that express markers of more than one lineage (mixed phenotype acute leukaemias).

Relapsed Acute Promyelocytic Leukemia Lacks "Classic" Leukemic Promyelocyte Morphology and Can Create Diagnostic Challenges.

PubMed

Dayton, Vanessa J; McKenna, Robert W; Yohe, Sophia L; Dolan, Michelle M; Courville, Elizabeth; Alvarez, Harold; Linden, Michael A

2017-01-01

Although current therapies for acute promyelocytic leukemia (APL), such as all- trans retinoic acid and arsenic trioxide, usually result in remission, some patients relapse. Early recognition of relapse is critical for prompt intervention. In this study, we systematically reviewed morphologic, immunophenotypic, and cytogenetic findings in paired diagnostic and relapsed APL cases and describe and quantify the changes in blast morphology at relapse. By electronic database search, we identified eight paired diagnostic and relapsed APL cases for which peripheral blood or bone marrow smears were available for review. For two cases, diagnostic material was available for relapse after hematopoietic cell transplantation. Neoplastic hypergranular or microgranular promyelocytes with indented or bivalve nuclei predominated at diagnosis in all patients. Most patients had undifferentiated blasts at relapse and/or hypergranular blast equivalents with round to oval nuclei. Classic acute promyelocytic leukemia cells with bivalve nuclei and bundles of cytoplasmic Auer rods were easily identifiable in fewer than half of cases at diagnosis and rare to absent in all relapsed cases. Morphologic features of relapsed APL overlap with other types of acute myeloid leukemia, creating diagnostic challenges, especially if no history is available when relapsing patients seek treatment for care. © American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Paradoxical aging in HIV: immune senescence of B Cells is most prominent in young age.

PubMed

Rinaldi, Stefano; Pallikkuth, Suresh; George, Varghese K; de Armas, Lesley R; Pahwa, Rajendra; Sanchez, Celeste M; Pallin, Maria Fernanda; Pan, Li; Cotugno, Nicola; Dickinson, Gordon; Rodriguez, Allan; Fischl, Margaret; Alcaide, Maria; Gonzalez, Louis; Palma, Paolo; Pahwa, Savita

2017-04-01

Combination antiretroviral therapies (cART)can lead to normal life expectancy in HIV-infected persons, and people aged >50 yrs represent the fastest growing HIV group. Although HIV and aging are independently associated with impaired humoral immunity, immune status in people aging with HIV is relatively unexplored. In this study influenza vaccination was used to probe age associated perturbations in the B cell compartment of HIV-negative "healthy controls" (HC) and virologically controlled HIV-infected participants on cART (HIV) (n=124), grouped by age as young (<40 yrs), middle-aged (40-59yrs) or old ( > 60 yrs). H1N1 antibody response at d21 post-vaccination correlated inversely with age in both HC and HIV. Immunophenotyping of cryopreserved PBMC demonstrated increased frequencies of double negative B cells and decreased plasmablasts in old compared to young HC. Remarkably, young HIV were different from young HC but similar to old HC in B cell phenotype, influenza specific spontaneous (d7) or memory (d21) antibody secreting cells. We conclude that B cell immune senescence is a prominent phenomenon in young HIV in comparison to young HC, but distinctions between old HIV and old HC are less evident though both groups manifest age-associated B cell dysfunction.

Trisomy 13 in a patient with common acute lymphoblastic leukemia: description of a case and review of the literature.

PubMed

Spirito, Francesca R; Mancini, Marco; Derme, Valentina; Cimino, Giuseppe; Testi, Anna Maria; Tafuri, Agostino; Vitale, Antonella; Foà, Robin

2003-07-01

Trisomy 13 occurring as a single cytogenetic abnormality has been associated with undifferentiated or biphenotypic acute leukemias and with an adverse prognostic outcome. We describe for the first time a case of B-cell common acute lymphoblastic leukemia (ALL) with trisomy 13 at diagnosis in an 18-year-old boy. The leukemic cells did not express myelocytic or T-cell associated antigens and no molecular abnormalities were detected. Following treatment, according to the GIMEMA ALL 0496 protocol, the patient achieved a brief (2 months) complete remission. At relapse, cytogenetic analysis showed karyotypic evolution that included two novel subclones carrying a del(6q), a del(7q), and an add(17q) in association with trisomy 13. In addition, immunophenotypic analysis revealed the coexpression of the CD33 and CD7 antigens on common ALL blasts, in accordance with other reported cases that displayed a predominant biphenotypic leukemia profile. The patient failed to obtain a second remission and died soon after due to infective complications. This report indicates that trisomy 13 can be found also in B-lineage ALL and underlines that this cytogenetic abnormality may identify a subgroup of male patients with clonal evolution potential and an adverse clinical outcome.

Acute myeloid leukemia mimicking primary testicular neoplasm. Presentation of a case with review of literature.

PubMed

McIlwain, Laura; Sokol, Lubomir; Moscinski, Lynn C; Saba, Hussain I

2003-04-01

We describe a new unique case of acute myeloid leukemia (AML) in a 21-yr-old male presenting with abdominal pain, bilateral testicular masses and gynecomastia. Further work-up with computed tomography of the chest, abdomen and pelvis revealed massive retroperitoneal, peripancreatic and mediastinal lymphadenopathy, suggesting primary testicular neoplasm. The patient was subjected to right orchiectomy that showed infiltration of testicular tissue with malignant cells, originally misinterpreted as undifferentiated carcinoma. Immunohistochemistry studies, however, showed these cells to be strongly positive for myeloperoxidase and CD45, indicating a myeloid cell origin. Bone marrow (BM) aspirate and biopsy demonstrated replacement of marrow with immature myeloid cells. Both the morphology and immunophenotype of the blast cells were consistent with AML type M4 (acute myelo-monocytic leukemia), using French-American-British (FAB) classification. The patient received standard induction chemotherapy with cytosine arabinoside (ARA-C) and daunorubicin followed with two cycles of consolidation therapy with high dose ARA-C, which resulted in remission of BM disease and resolution of lymphadenopathy and left testicular masses. After the second cycle of consolidation therapy, the patient developed sepsis that was complicated by refractory disseminated intravascular coagulopathy. He expired with a clinical picture of multiple organ failure. The unique features of this case are presented and the related literature is reviewed.

[A case of de novo acute basophilic leukaemia: diagnostic criteria and review of the literature].

PubMed

Staal-Viliare, A; Latger-Cannard, V; Rault, J P; Didion, J; Grégoire, M J; Bologna, S; Witz, B; Jonveaux, P; Lecompte, T; Rio, Y

2006-01-01

We report a case of a de novo acute basophilic leukaemia, revealed by an infectious pneumopathy in a 73 year old man. The full blood count revealed an hyperleucocytosis associated with an unregenerative normocytic normochrom anaemia and a thrombocytopenia. The blood and bone marrow smears showed a mixture of undifferentiated blast cells and basophiloblasts (high nucleo-cytoplasmic ratio, coarse basophilic cytoplasmic granules), along with basophilic precursors and basophilic polymorphonuclears. All the blasts were MPO negative but positive for the toluidine blue metachromatic coloration, which is considered as consistent with basophilic lineage. Immunophenotypic studies showed myeloid blasts, without maturity marker, CD 117 negative and CD203 cytoplasmic positive, the latter known to be highly representative of the basophilic lineage. This very clear-cut phenotype, associated with the morphology of cells, were arguments to ascertain the basophilic lineage of the blasts without the need of electron microscopic study. Cytogenetic and RNA analysis revealed the presence of a Philadelphia chromosome and of a BCR-ABL transcript with the unusual junction e6a2. Thus, imatinib was added to the conventional chimiotherapy and the patient is currently in complete remission. This clinical prompted allows us to review the literature on acute basophilic leukaemia and to state on the different diagnostic criteria of this rare disorder.

[Primary diffuse large B-cell lymphoma of the uterine cervix--a case report].

PubMed

Okudaira, Taeko; Nagasaki, Akitoshi; Miyagi, Takashi; Nakazato, Tetsuro; Taira, Naoya; Kudaka, Wataru; Maehama, Toshiyuki; Takasu, Nobuyuki

2008-08-01

Primary malignant lymphoma of the female genital tract is an extremely rare clinical entity. We report a case of primary non-Hodgkin lymphoma of the uterine cervix. A 68-year-old woman presented with abnormal genital bleeding in May 2002. A coloposcopic examination revealed a mass in the uterine cervix. Magnetic resonance imaging showed a bulky cervical tumor(7.5 x 8 cm)invading the right parametrium and adjacent levator ani muscle. Involvement of pelvic lymph nodes was also observed. The uterine lesion exhibited homogenous hypointensity on T1 weight image and isointense to hyperintense on T2-weight image. No other lesions were detected by the whole-body computed tomography, gallium scintigraphy, and bone marrow examination. Although cytology of the smear from the uterine cervix was nondiagnostic, the histologic examination of the punch biopsy material showed a diffuse proliferation of atypical lymphoid cells. Immunophenotypic studies revealed tumor cells were positive for CD19, CD20, CD30, and k-chain. A diagnosis of diffuse large B-cell lymphoma of the uterine cervix, clinical stage IIE was made. The patient was treated with 6 cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone(CHOP)chemotherapy followed by the involved field irradiation. She remains alive and free of disease more than 5 years after the diagnosis.

Immunological Prediction of Cytomegalovirus (CMV) Replication Risk in Solid Organ Transplantation Recipients: Approaches for Regulating the Targeted Anti-CMV Prevention Strategies

PubMed Central

2017-01-01

The current cytomegalovirus (CMV) prevention strategies in solid organ transplantation (SOT) recipients have contributed towards overcoming the detrimental effects caused by CMV lytic infection, and improving the long-term success rate of graft survival. Although the quantification of CMV in peripheral blood is the standard method, and an excellent end-point for diagnosing CMV replication and modulating the anti-CMV prevention strategies in SOT recipients, a novel biomarker mimicking the CMV control mechanism is required. CMV-specific immune monitoring can be employed as a basic tool predicting CMV infection or disease after SOT, since uncontrolled CMV replication mostly originates from the impairment of immune responses against CMV under immunosuppressive conditions in SOT recipients. Several studies conducted during the past few decades have indicated the possibility of measuring the CMV-specific cell-mediated immune response in clinical situations. Among several analytical assays, the most advancing standardized tool is the QuantiFERON®-CMV assay. The T-Track® CMV kit that uses the standardized enzyme-linked immunospot assay is also widely employed. In addition to these assays, immunophenotyping and intracellular cytokine analysis using flow cytometry (with fluorescence-labeled monoclonal antibodies or peptide-major histocompatibility complex multimers) needs to be adequately standardized and validated for potential clinical applications. PMID:29027383

Monitoring of benzene-induced hematotoxicity in mice by serial leukocyte counting using a microcavity array.

PubMed

Hosokawa, Masahito; Asami, Marie; Yoshino, Tomoko; Tsujimura, Noriyuki; Takahashi, Masayuki; Nakasono, Satoshi; Tanaka, Tsuyoshi; Matsunaga, Tadashi

2013-02-15

Monitoring of hematotoxicity, which requires serial blood collection, is difficult to carry out in small animals due to a lack of non-invasive, individual animal-appropriate techniques that enable enumeration of leukocyte subsets from limited amounts of whole blood. In this study, a microfluidic device equipped with a microcavity array that enables highly efficient separation of leukocytes from submicroliters of whole blood was applied for hematotoxicity monitoring in mice. The microcavity array can specifically separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Mouse leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered almost 100% of mouse leukocytes in 0.1 μL of whole blood without the effect of serial blood collection such as changes in body weight and total leukocyte count. We assessed benzene-associated hematotoxicity in mice using this system. Mice were administered with benzene once daily and the depression of leukocyte numbers induced in individual mice was successfully monitored from tail vein blood collected every other day for 2 weeks. Serial monitoring of the leukocyte number in individual mice will contribute to the understanding of hematotoxicity and reduction of the number of animal experiment trials. Copyright © 2012 Elsevier B.V. All rights reserved.

Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration.

PubMed

Koenig, Sarah; Probst, Irmelin; Becker, Heinz; Krause, Petra

2006-12-01

Oval cells constitute a heterogeneous population of proliferating progenitors found in rat livers following carcinogenic treatment (2-acetylaminofluorene and 70% hepatectomy). The aim of this study was to investigate the cellular pattern of various differentiation and cell type markers in this model of liver regeneration. Immunophenotypic characterisation revealed at least two subtypes emerging from the portal field. First, a population of oval cells formed duct-like structures and expressed bile duct (CD49f) as well as hepatocytic markers (alpha-foetoprotein, CD26). Second, a population of non-ductular oval cells was detected between and distally from the ductules expressing the neural marker nestin and the haematopoietic marker Thy1. Following oval cell isolation, a subset of the nestin-positive cells was shown to co-express hepatocytic and epithelial markers (albumin, CD26, pancytokeratin) and could be clearly distinguished from anti-desmin reactive hepatic stellate cells. The gene expression profiles (RT-PCR) of isolated oval cells and oval cell liver tissue were found to be similar to foetal liver (ED14). The present results suggest that the two oval cell populations are organised in a zonal hierarchy with a marker gradient from the inner (displaying hepatocytic and biliary markers) to the outer zone (showing hepatocytic and extrahepatic progenitor markers) of the proliferating progeny clusters.

Clearance of apoptotic photoreceptors: elimination of apoptotic debris into the subretinal space and macrophage-mediated phagocytosis via phosphatidylserine receptor and integrin alphavbeta3.

PubMed

Hisatomi, Toshio; Sakamoto, Taiji; Sonoda, Koh-Hei; Tsutsumi, Chikako; Qiao, Hong; Enaida, Hiroshi; Yamanaka, Ichiro; Kubota, Toshiaki; Ishibashi, Tatsuro; Kura, Shinobu; Susin, Santos A; Kroemer, Guido

2003-06-01

The effective phagocytotic clearance of apoptotic debris is fundamental to the maintenance of neural tissues during apoptosis. Retinal photoreceptors undergo apoptosis after retinal detachment. Although their induction phase of apoptosis has been well discussed, their phagocytotic process remains quite unclear. We herein demonstrate that apoptotic photoreceptors are selectively eliminated from their physiological localization, the outer nuclear layer, to the subretinal space, and then phagocytosed by monocyte-derived macrophages. This could be shown by an ultrastructural and immunophenotypic analysis. Moreover, in chimera mice expressing transgenic green fluorescent protein in bone marrow-derived cells, the local infiltration of macrophages could be detected after retinal detachment-induced photoreceptor apoptosis. The local injection of an antibody blocking the phosphatidylserine receptor (PSR) or a peptide (GRGDSP)-blocking integrin alphavbeta3 revealed that phagocytotic clearance involves the PSR as well as integrin alphavbeta3 in vivo. Importantly, the level of blockade obtained with these reagents was different. Although anti-PSR increased the frequency of apoptotic cells that fail to bind to macrophages, GRGDSP prevented the engulfment (but not the recognition) of apoptotic photoreceptor cells by macrophages. To our knowledge, this is the first report describing the mechanisms through which apoptotic photoreceptors are selectively eliminated via a directional process in the subretinal space.

Primary diffuse large B cell lymphoma arising from a leiomyoma of the uterine corpus.

PubMed

Zhao, Lianhua; Ma, Qiang; Wang, Qiushi; Zeng, Ying; Luo, Qingya; Xiao, Hualiang

2016-01-20

Primary diffuse large B cell lymphoma (DLBCL) of the uterus is rare, and primary DLBCL arising from a uterine leiomyoma (collision tumor) has not been reported in the literature. We describe the clinical, histological, immunohistochemical, and molecular features of primary DLBCL arising from a leiomyoma in the uterine corpus. A 73-year-old female patient had a uterine mass for 23 years. An ultrasound scan revealed marked enlargement of the uterus, measuring 18.2 × 13 × 16.3 cm, with a 17.6 × 10.9 × 11.6 cm hypoechoic mass in the uterine corpus. The tumors consisted of medium- to large-sized cells exhibiting a diffuse pattern of growth with a well-circumscribed leiomyoma. The neoplastic cells strongly expressed CD79α, CD20 and PAX5. Molecular analyses indicated clonal B-cell receptor gene rearrangement. To the best of our knowledge, no previous cases of primary DLBCL arising from a leiomyoma have been reported. It is necessary to differentiate a diagnosis of primary DLBCL arising from a leiomyoma from that of leiomyoma with florid reactive lymphocytic infiltration (lymphoma-like lesion). Careful analysis of clinical, histological, immunophenotypic, and genetic features is required to establish the correct diagnosis.

Multiparameter immune profiling of operational tolerance in liver transplantation.

PubMed

Martínez-Llordella, M; Puig-Pey, I; Orlando, G; Ramoni, M; Tisone, G; Rimola, A; Lerut, J; Latinne, D; Margarit, C; Bilbao, I; Brouard, S; Hernández-Fuentes, M; Soulillou, J-P; Sánchez-Fueyo, A

2007-02-01

Immunosuppressive drugs can be completely withdrawn in up to 20% of liver transplant recipients, commonly referred to as 'operationally' tolerant. Immune characterization of these patients, however, has not been performed in detail, and we lack tests capable of identifying tolerant patients among recipients receiving maintenance immunosuppression. In the current study we have analyzed a variety of biological traits in peripheral blood of operationally tolerant liver recipients in an attempt to define a multiparameter 'fingerprint' of tolerance. Thus, we have performed peripheral blood gene expression profiling and extensive blood cell immunophenotyping on 16 operationally tolerant liver recipients, 16 recipients requiring on-going immunosuppressive therapy, and 10 healthy individuals. Microarray profiling identified a gene expression signature that could discriminate tolerant recipients from immunosuppression-dependent patients with high accuracy. This signature included genes encoding for gammadelta T-cell and NK receptors, and for proteins involved in cell proliferation arrest. In addition, tolerant recipients exhibited significantly greater numbers of circulating potentially regulatory T-cell subsets (CD4+ CD25+ T-cells and Vdelta1+ T cells) than either non-tolerant patients or healthy individuals. Our data provide novel mechanistic insight on liver allograft operational tolerance, and constitute a first step in the search for a non-invasive diagnostic signature capable of predicting tolerance before undergoing drug weaning.

Characterization of skin blister fluids from children with Epstein-Barr virus-associated lymphoproliferative disease.

PubMed

Wada, Taizo; Toma, Tomoko; Miyazawa, Hanae; Koizumi, Eiko; Shirahashi, Tetsujiro; Matsuda, Yusuke; Yachie, Akihiro

2018-04-01

Epstein-Barr virus (EBV)-associated T- or natural killer (NK)-cell lymphoproliferative disease (LPD) is a heterogeneous group of disorders characterized by chronic proliferation of EBV-infected lymphocytes. Patients may present with severe skin manifestations, including hypersensitivity to mosquito bites (HMB) and hydroa vacciniforme (HV)-like eruption, which are characterized by blister formation and necrotic ulceration. Skin biopsy specimens show inflammatory reactions comprising EBV-infected lymphocytes. However, blister fluids have not been fully assessed in patients with this disease. Blister fluids were collected from three patients with EBV-associated LPD: two with HMB and one with HV. Immunophenotyping of blister lymphocytes and measurement of tumor necrosis factor (TNF)-α in blister fluids were performed. The patients with HMB and HV exhibited markedly increased percentages of NK and γδ T cells, respectively, in both peripheral blood and blister fluids. These NK and γδ T cells strongly expressed the activation marker human leukocyte antigen-DR and were considered to be cellular targets of EBV infections. TNF-α was highly elevated in all blister fluids. Severe local skin reactions of EBV-associated LPD may be associated with infiltrating EBV-infected lymphocytes and a high TNF-α concentration in blister fluids. © 2018 Japanese Dermatological Association.

Epstein–Barr virus-positive T/NK-cell lymphoproliferative disorders

PubMed Central

Cai, Qingqing; Chen, Kailin; Young, Ken H

2015-01-01

Epstein–Barr virus, a ubiquitous human herpesvirus, can induce both lytic and latent infections that result in a variety of human diseases, including lymphoproliferative disorders. The oncogenic potential of Epstein–Barr virus is related to its ability to infect and transform B lymphocytes into continuously proliferating lymphoblastoid cells. However, Epstein–Barr virus has also been implicated in the development of T/natural killer cell lymphoproliferative diseases. Epstein–Barr virus encodes a series of products that mimic several growth, transcription and anti-apoptotic factors, thus usurping control of pathways that regulate diverse homeostatic cellular functions and the microenvironment. However, the exact mechanism by which Epstein–Barr virus promotes oncogenesis and inflammatory lesion development remains unclear. Epstein–Barr virus-associated T/natural killer cell lymphoproliferative diseases often have overlapping clinical symptoms as well as histologic and immunophenotypic features because both lymphoid cell types derive from a common precursor. Accurate classification of Epstein–Barr virus-associated T/natural killer cell lymphoproliferative diseases is a prerequisite for appropriate clinical management. Currently, the treatment of most T/natural killer cell lymphoproliferative diseases is less than satisfactory. Novel and targeted therapies are strongly required to satisfy clinical demands. This review describes our current knowledge of the genetics, oncogenesis, biology, diagnosis and treatment of Epstein–Barr virus-associated T/natural killer cell lymphoproliferative diseases. PMID:25613730