Fresh Cell Suspensions Research Articles

Tissue-resident memory T cells from a metastatic vaginal melanoma patient are tumor-responsive T cells and increase after anti-PD-1 treatment

BackgroundVaginal melanoma (VM) is a rare cancer and has a poor response to immune checkpoint blockade (ICB). CD8+Tissue Resident Memory (TRM) T cells proliferate in response to ICB and correlate...

Technical Updates to the Comet Assay In Vivo for Assessing DNA Damage in Zebrafish Embryos from Fresh and Frozen Cell Suspensions.

The Single-Cell Gel Electrophoresis, simply known as the Comet assay, is a sensitive and quick technique used to quantitate DNA damage, widely used to assess the effects of genotoxicants and mutagens in animal cells. Still, performing the assay on peripheral or cultured cells is far more expeditious and cost effective than solid tissue, especially from small biological model like the zebrafish embryo. The current work describes and validates a highly cost-effective protocol of the updated Comet assay designed for zebrafish embryos. Compared with the few previous applications of the Comet assay on this biological model, the present method successfully simplifies the process of cell harvesting and resuspending, producing a much higher yield of viable nucleoids with reduced basal DNA damage, even from a small number of embryos, and compatible with scoring with safe fluorescent dyes. Additionally, the protocol can be just as easily performed on freshly harvested cells of cells frozen in dimethyl sulfoxide (DMSO)-containing physiological buffer, without a significant increase of DNA damage, which is another highly relevant update, especially for researchers handling high numbers of samples.

FACS Analysis of Col1α Protein Levels in Primary Fibroblasts.

Chronic inflammatory diseases are often associated with organ fibrosis, a progressive condition in which excessive deposition of extracellular matrix (ECM), mainly composed of collagen I (Col I), is deposited by activated fibroblasts and severely impairs tissue architecture and function, eventually resulting in organ failure. Moreover, enhanced collagen deposition by activated fibroblasts and increased stiffness of the extracellular matrix were demonstrated to be associated with tumor progression and metastasis. In order to quantitatively analyze fibrotic activation of fibroblasts and collagen deposition, it is essential to assess collagen content. While various histological methods allow assessment of collagen in tissue sections (e.g., Masson trichrome and Sirius red), reliable measurement and quantification of collagen levels in vitro remain a challenge in the field. In this protocol, we utilize intracellular staining of Col1α and flow cytometry analysis to analyze collagen content in primary fibroblasts isolated from fresh single cell suspensions of metastases-bearing lungs.

Importance of Detection of Intracellular Myeloperoxidase, CD13, CD79a, CD22, CD3 and Terminal Deoxynucleotidyl Transferase By Flow Cytometry Diagnosis of Acute Leukemias

Background: The detection of Intracellular (IC) antigens by flow cytometry (FC) such as myeloperoxidase (MPO), cCD13, cCD79a, cCD22, cCD3 and Terminal deoxynucleotidyl Transferase (TdT) has become the useful tool in the differential diagnosis between acute myeloid leukemias (AML) and acute lymphoid leukemias (ALL). Through detection of myeloid antigens (MPO and cCD13), B cells precursors (cCD79a and cCD22) and precocity T-cells (cCD3) it has been possible to confirm the diagnosis of these acute leukemias. The detection of intracellular cell markers by FC usually requires previous permeabilization of fresh cell suspensions. TdT, also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells. TdT adds N-nucleotides to the V, D, and J exons of the TCR and BCR genes during antibody gene recombination, enabling the phenomenon of junctional diversity. In humans, terminal transferase is encoded by the DNTT gene. This antigen is expressed mostly in the nucleus cells from primary lymphoid organs, like the thymus and bone marrow. The TdT detection has also been shown to be useful in confirming the acute forms of B and T-lineage lymphoproliferative diseases by FC. The aim of this study was to demonstrate the importance of this cell markers' detection by FC in the differential diagnostic of acute leukemias. Methods: Bone marrow and/or peripheral blood leukemic cells from 50 cases of acute leukemia: 16 ALL and 36 AML. The cells were fixed and permeabilized in briefly exposed to Becton & Dickinson Lyse Solution at concentration of 10%, and subsequently labeled with monoclonal antibodies anti-MPO, TdT, CD3, CD13, CD22 and CD79a. Results: The MPO expression was observed in 35/36(97,22%) and cCD13 in all cases of AML and in none ALL patients. Three cases of MPO-positive ALL (FAB-L2) could be reclassified as M0-AML. These cases were CD34+;HLADR+;CD33-;CD13-;CD7+ and cCD13+. The intensity of TdT expression was observed in 15/16 (93.8%) of ALL and 5/36 (13.9%) of AML. The cCD22 and cCD79a were positive in 15/16 (93.8%) and all of pre-B ALL respectively and cCD3 was expressed in one case of Pre-T ALL that initial phenotype was CD34+/HLADR+/TdT+/CD7+ and sCD3-). Conclusions: These results indicate that monoclonal antibodies anti-MPO, cCD13, cCD79a, cCD22, cCD3 and TdT were excellent cell markers for the diagnosis and classification of acute leukemias and can be reliably detected by FC. This rapid and specific technique should be a valuable addition to routine immunophenotyping of acute leukemia. DisclosuresNo relevant conflicts of interest to declare.

What is the best protocol to cryopreserve immature mouse testicular cell suspensions?

From a clinical perspective, which parameters grant optimal cryopreservation of mouse testicular cell suspensions? We studied the effect of different cryopreservation rates, the addition of sugars, different vessels and the addition of an apoptotic inhibitor on the efficiency of testicular cell suspension cryopreservation. After thawing and warming, testicular cell suspensions were transplanted to recipient mice for further functional assay. After selecting the optimal cryopreservation procedure, a second experiment compared the transplantation efficiency between the selected freezing protocol and fresh testicular cell suspensions. Multiple- and single-step freezing did not differ significantly in terms of recovered viable cells (RVC) (33 ± 28% and 38 ± 25%). The addition of sucrose did not result in a higher RVC (33 ± 20%). Cells frozen in vials recovered better than those frozen in straws (52 ± 20% versus 33 ± 20%; P = 0.0049). The inclusion of an apoptosis inhibitor (z-VAD[Oe]-FMK) significantly increased the RVC after thawing (61 ± 18% versus 50 ± 17%; P = 0.0480). When comparing the optimal cryopreservation procedure with fresh testicular cell suspensions, a lower RVC (63 ± 11% versus 92 ± 4%; P < 0.0001) and number of donor-derived spermatogonial stem cell colonies per testis (34.04 ± 2.34 versus 16.78 ± 7.76; P = 0.0051) were observed. Upon freeze-thawing or vitrification-warming, and assessment of donor-derived spermatogenesis after transplantation, Dulbecco's modified Eagle's medium supplemented with 1.5M dimethyl-sulphoxide, 10% fetal calf serum and 60 µM of Z-VAD-(OMe)-FMK in vials at a freezing rate of -1°C/min was optimal.

Characterization of myeloid cell populations in human testes collected after sex reassignment surgery

The testis has been described in animal models as a site of immune privilege, which protects spermatids against tissue damage during inflammation. Myeloid cells, including macrophages and dendritic cells (DC), are defined as key players in the testicular immune privilege in animal models. However, their distribution and frequency in human testis remain poorly described. To overcome the challenges related to tissue sampling, we obtained testicular tissue from men under hormonal therapy who elected to have sex reassignment surgery (SRS). We examined the distribution of myeloid cell populations in tissue sections using immunohistofluorescence and evaluated their relative frequencies in fresh testicular cell suspensions compared with matched blood using multi-parametric flow cytometry. We identified 4.9% of CD45+ leucocytes in testicular cell suspensions, of which 0.4% were B cells, demonstrating a low level of blood contamination. Myeloid cells (Lin−HLA-DR+) were located in the testicular interstitium and represented a median of 23.4% of testicular leucocytes, displaying higher HLA-DR expression compared to their counterparts in blood (p=0.001). The frequency of testicular myeloid cells was not linked with the duration of hormonal therapy. Resident macrophages (CD14+CD163+) constituted the most frequent myeloid cell subset and expressed high levels of CD163. Elevated proportion of myeloid DC (CD14−CD11c+) contrasted with the paucity of plasmacytoïd DC (CD14−CD123+) in testis. Myeloid-derived suppressor cells (Lin−HLA-DR−CD33hiCD11bhi) were not detected in the testis while constituting 0.5% of blood leucocytes. For the first time, we characterized myeloid cell subsets in human testes collected after SRS, providing a basis to assess their contribution to immune privilege.

Study of tumor-infiltrating T-cell reactivity to metastatic gastrointestinal cancers.

e14179 Background: A high density of tumor-infiltrating lymphocytes (TIL) found in gastrointestinal cancers has been associated with improved prognosis. Evidence supporting specific autologous tumor recognition by TIL remains sparse and limits the development of innovative immunotherapy approaches. Methods: Metastases from 15 GI cancer patients (13 colorectal, 1 gastric, 1 cholangiocarcinoma) processed into fragments and fresh tumor cell suspensions (FrTu) were used to grow TIL and cancer cell lines. TIL phenotype was assessed by flowcytometry and reactivity by coculture with available cryopreserved FrTu and cancer cell lines. Limiting dilution techniques were used to isolate reactive CD8+ TIL clones. T-cell receptors (TCR) were sequenced using PCR amplification. Results: TIL were expanded successfully in all 15 cases. TIL phenotype studied from 7 freshly resected colorectal cancer metastases revealed a higher percentage of immunosuppressive T cells (CD4+CD25HIFoxP3+) compared to adjacent normal tissue and blood (respectively 5.4 ± 1.0%, 1.3 ± 0.5%, and 1.0 ± 0.4% of CD3+ cells). Interferon-gamma release by TIL after their first outgrowth upon coculture with cryopreserved FrTu was significant in all 6 cases studied, with positive results ranging from 7% (3/42) to 89% (8/9) of patient’s distinct cultures. Two newly established cancer cell lines allowed extensive investigation of TIL reactivity. In a case of gastric cancer, 65% of the CD3+CD8+ TIL expanded from liver metastases expressed the same TCR. TIL clones expressing this TCR could recognize the autologous cancer cell line with a high degree of specificity, suggesting that within this tumor a productive immune response occurred. In a case of metastatic cholangiocarcinoma, two TIL clones with distinct TCR represented less than 1% of the CD3+CD8+ cells grown from an omental metastasis and the malignant ascites, but could nonetheless lyse the autologous tumor with specificity. Conclusions: Despite the immunosuppressive microenvironment of GI cancer metastases, CD8+ TIL that recognize autologous tumor with specificity can be identified and expanded in vitro. The adoptive transfer of TIL for the therapy of metastatic GI cancer patients is currently being studied.

Abstract 5199: Primary culture of human midgut carcinoid cell lines and isolation of a putative carcinoid stem cell population

Abstract Background: Research in carcinoid and neuroendocrine tumors (NETs) of midgut origin is limited by a lack of cell lines. The existing foregut NET lines, BON and H727, are phenotypically distinct from midgut NETs highlighting the importance of establishing midgut derived NET cell lines. Therapeutic options are limited for midgut NETs. NET cell lines are difficult to culture for long periods (thus few cell lines). We are developing methods to improve primary and prolonged NET cell culture in an attempt to identify novel therapeutic approaches. Along these lines, we are conducting studies to identify a putative cancer stem cell population in human NET specimens to provide new insights regarding potential targets in NETs. Methods: Sterile human midgut NET surgical specimens were digested to a single cell suspension and fibroblast depleted with magnetic beads. The NET cells were maintained on collagen I coated plates for primary culture. A portion of the primary cell culture was used for FACS by the Aldefluor assay to isolate potential cancer stem cells (CSCs). Sorted cells were grown in serum-free media on low attachment plates to assess sphere formation and preserved for RNA isolation and subsequent gene array. Subcutaneous xenografts in nude and NOD/SCID gamma mice using fresh tumor implants and NET cell suspensions were performed as potential means to further propagate NET cells. Results: The NET markers, somatostatin receptor 2 and chromogranin A, were detectable in cultures from a midgut carcinoid liver metastasis at passage 3 and 5, as well as in one mouse xenograft. While retention of NET markers from xenograft implants is promising, tumor growth remains limited and slow, with tumors in 5/59 mice over 4-10 months. We are now studying tumor xenografts in more immunodeficient NOD/SCID gamma mice with current patient samples to potentially enhance xenograft growth. FACS sorting identified an ALDH+ population of NET cells, which formed spheres (a characteristic of CSCs) more frequently than ALDH(−) negative cells. A FACS sorted midgut sample plated at 10k cells/well in sphere forming media produced spheres as early as 8 days in the ALDH+ population, while the ALDH- cells did not form spheres (sham sorted cells formed rare spheres), which was also observed in other samples where viable cells were obtained. Additionally, the ALDH+ cells from this population were cutlured on plastic and remained viable for 4 weeks, while the ALDH- population was not viable in culture. RNA isolation for gene array is underway for the ALDH+ population to characterize these cells in midgut carcinoids. Conclusions: We have developed a system allowing for NET primary culture for up to 4 weeks. We have also identified a putative CSC population in NETs. These techniques are the first step in obtaining cells for in vitro studies and in identifying novel therapeutic targets for NETs. Supported by the The Raymond and Beverly Sackler Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2011-5199

EARLY AND RELIABLE DIAGNOSIS OF NON-HODGKIN LYMPHOMA in CHILDHOOD AND ADOLESCENCE: Contribution of Cytomorphology and Flow Cytometric Immunophenotyping

Non-Hodgkin lymphoma (NHL) represents one of the most rapidly growing malignancies in childhood and adolescence. About 80% of patients now are cured with adequate treatment. Serious complications at presentation due to tumor lysis syndrome or local tumor effects are commonly observed. Thus, a rapid diagnosis with the least invasive procedure enabling the initiation of early and specific therapy is necessary to diminish early fatality or persistent impairment. In 56 centrally registered patients with NHL, cytomorphologic analyses (FAB criteria) of May-Grünwald-Giemsa–stained touch imprints or malignant effusions and flow cytometric immunophenotyping (EGIL criteria) of fresh cell suspensions with a standardized panel of monoclonal antibodies were performed. The authors identified 23 patients with Burkitt lymphoma by the combination of FAB L3 morphology and a mature B-cell phenotype and 22 patients with lymphoblastic lymphoma by FAB L1/L2 morphology and a T-/B-cell precursor phenotype. They also found 11 patients with large cell lymphomas, 3 of them with anaplastic large cell lymphoma (T-cell phenotype; NPM/ALK-positive). In the remaining 8 patients diffuse large B-cell lymphoma was suspected by the combined use of cytologic and immunophenotypic findings (mature B-cell phenotype). In all cases with available solid tumor material (n = 42/56) the preliminary diagnosis was confirmed by histopathology. Burkitt lymphoma, lymphoblastic lymphoma, and, in a few cases, some large cell lymphomas could be classified reliably by cytomorphology and immunophenotyping of freshly obtained tumor cell material, enabling an early start of specific lymphoma treatment.

Prediction of in vitro intrinsic clearance from hepatocytes: comparison of suspensions and monolayer cultures.

Due to the time-dependent loss of cytochrome P450 (P450)-mediated metabolism in freshly isolated hepatocytes, several types of culture systems have been developed to extend their lifespan. The aim of this study was to evaluate the ability of monolayer cultures of rat hepatocytes to determine the in vitro CL(int) compared with suspensions of freshly isolated hepatocytes. Seven compounds were incubated in rat hepatocyte suspensions and monolayer cultures, and in vitro CL(int) was obtained via metabolite formation (12 pathways) or substrate depletion approaches. Only two compounds (tolbutamide and 7-ethoxycoumarin) gave comparable (within 2-fold) in vitro CL(int) in both suspensions and monolayer cultures. Although the overall rank order of compounds was the same in both models (covering a range of 3-4 orders of magnitude), the prediction of in vitro CL(int) for high-turnover compounds (seven pathways) was lower for monolayer cultures compared with suspensions, probably due to an uptake rate limitation leading to increases in K(M). In general, there was an average 50% loss of the P450 activity in monolayers based on a decrease in V(max) relative to suspensions. However, monolayer cultures gave a higher estimation of in vitro CL(int) for the low-turnover compound S-warfarin compared with fresh cell suspensions due to a decrease in the K(M) of the four individual metabolites. The use of hepatocyte monolayer cultures may offer the potential advantage of extending the lower end of the usable clearance range (below 0.1 microl/min/10(6) cells) for predicting in vivo CL(int).

Neural progenitor cells do not differentiate prematurely in presenilin-1 null mutant mice

Mice with a null mutation of the presenilin-1 (PS1−/−) gene die during late intrauterine life or shortly after birth and exhibit defects in cortical development. A previous report suggested that neurons differentiate prematurely in PS1−/− brain [M. Handler, X. Yang, J. Shen, Presenilin-1 regulates neuronal differentiation during neurogenesis, Development 127 (2000) 2593–2606]. Here we reexamined the issue of whether premature neuronal differentiation occurs in PS1−/− brain using fresh cell suspensions from embryonic E11.5 and E13.5 telencephalon where individual cell phenotypes can be easily determined with cell type specific markers. Immunostaining with seven neuronal specific markers (MAP2, β-III tubulin, GABA, reelin, GluR2/3, calbindin, and calretinin) failed to reveal any evidence of premature neuronal differentiation in PS1−/− telencephalon. We also determined the fraction of cells expressing the neural progenitor marker nestin and found no evidence for premature depletion of neural progenitor cells in PS1−/− telencephalon. Moreover, based on MAP2 staining of tissue sections from E12.5 embryos the topography of newly generated neurons also appeared to be undisturbed in the telencephalon of PS1−/− embryos. These studies thus argue that premature neuronal differentiation is unlikely to be a core pathophysiological feature underlying the aberrant cortical development that occurs in PS1−/− brain.

CORRECTION TO "TISSUE DISTRIBUTION, STABILITY, AND PHARMACOKINETICS OF APO2 LIGAND/TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND IN HUMAN COLON CARCINOMA COLO205 TUMOR-BEARING MICE"

Due to the time-dependent loss of cytochrome P450 (P450)-mediated metabolism in freshly isolated hepatocytes, several types of culture systems have been developed to extend their lifespan. The aim of this study was to evaluate the ability of monolayer cultures of rat hepatocytes to determine the in vitro CL(int) compared with suspensions of freshly isolated hepatocytes. Seven compounds were incubated in rat hepatocyte suspensions and monolayer cultures, and in vitro CL(int) was obtained via metabolite formation (12 pathways) or substrate depletion approaches. Only two compounds (tolbutamide and 7-ethoxycoumarin) gave comparable (within 2-fold) in vitro CL(int) in both suspensions and monolayer cultures. Although the overall rank order of compounds was the same in both models (covering a range of 3-4 orders of magnitude), the prediction of in vitro CL(int) for high-turnover compounds (seven pathways) was lower for monolayer cultures compared with suspensions, probably due to an uptake rate limitation leading to increases in K(M). In general, there was an average 50% loss of the P450 activity in monolayers based on a decrease in V(max) relative to suspensions. However, monolayer cultures gave a higher estimation of in vitro CL(int) for the low-turnover compound S-warfarin compared with fresh cell suspensions due to a decrease in the K(M) of the four individual metabolites. The use of hepatocyte monolayer cultures may offer the potential advantage of extending the lower end of the usable clearance range (below 0.1 microl/min/10(6) cells) for predicting in vivo CL(int).

An in vitro interval before transplantation of mesencephalic reaggregates does not compromise survival or functionality

Grafts of primary ventral mesencephalic tissue and cell suspensions to the denervated striatum are currently utilized as a treatment strategy for Parkinson's disease. Survival rates of grafted dopamine (DA) neurons are extremely poor (5–20%) and is even poorer in grafts to the aged striatum. Short pretreatment of grafted cells with various survival-promoting agents has elicited 2- to 3-fold improvements in these survival rates. However, the duration of pretreatment is limited by the necessity of implanting the embryonic cells within a critical period after tissue harvest, potentially limiting the beneficial effects of these interventions. This study details the use of a modified mesencephalic reaggregate culture system combined with striatal-derived trophic factor support to provide an extended ex vivo cell culture interval before grafting. Mesencephalic cell suspension grafts implanted immediately following dissociation were compared to grafts of an equivalent number of cells reaggregated in the presence of striatal oligodendrocyte-type-2 astrocyte (SO2A) conditioned medium for 3 or 7 days. All grafts were placed in the denervated striatum of young adult male Fischer 344 rats. Rotational assessment of amphetamine-induced rotations indicates that aggregates maintained for 3 days in culture present statistically similar functional recovery profiles as compared to cell suspension grafts. Grafts of mesencephalic reaggregates maintained in vitro for 7 days did not display significant improvements in functional recovery. Immunohistochemical analysis for tyrosine hydroxylase immunoreactive (THir) neurons conducted at 10 weeks post-grafting revealed equivalent survival rates of THir neurons in grafts of fresh cell suspensions and aggregates held in culture for 3 days. Grafts of reaggregates held in culture for 7 days possessed significantly fewer THir neurons, about 25% of the cell suspension or 3-day aggregate grafts. This ex vivo reaggregate system allows for extended pretreatment (3 days) of mesencephalic cells with survival-promoting agents and immunological screening of tissue before transplantation.

Angiotensin-(1–7) stimulates water transport in rat inner medullary collecting duct: evidence for involvement of vasopressin V2 receptors

The peptide angiotensin-(1-7) [Ang-(1-7)] is known to enhance water transport in rat inner medullary collecting duct (IMCD). The aim of this study was to determine the mechanism of the Ang-(1-7) effect on osmotic water permeability (Pf). Pf was measured in the normal rat IMCD perfused in vitro in presence of agonists [Ang-(1-7), arginine vasopressin (AVP) and Ang-(3-8)], and antagonists of the angiotensin and the vasopressin cascade. Ang-(1-7), but not Ang-(3-8), increased Pf significantly. The effect of Ang-(1-7) on Pf was abolished by its selective antagonist, A-779, added before or after Ang-(1-7). Prostaglandin E2 and the protein kinase A inhibitor H8 also blocked the Ang-(1-7) effect. Blockade of vasopressin V1 receptors by antagonists did not change the Ang-(1-7) effect, but pre-treatment with a V2 antagonist abolished the effect of Ang-(1-7) on Pf. Similarly, pre-treatment with A-779 inhibited AVP's effect on Pf. Forskolin-stimulated Pf was blocked both by A-779 and by the V2 antagonist. Finally, Ang-(1-7) increased cAMP levels in fresh IMCD cell suspensions whilst the forskolin-stimulated cAMP synthesis was decreased by A-779 and the V2 antagonist. These data provide evidence that Ang-(1-7) interacts via its receptor with the AVP V2 system through a mechanism involving adenylate-cyclase activation.

Immunohistochemical detection of immunoglobulin light chain expression in B-cell non-Hodgkin lymphomas using formalin-fixed, paraffin-embedded tissues and a heat-induced epitope retrieval technique.

Definitive diagnosis of B-cell non-Hodgkin lymphomas often requires demonstration of B-cell monoclonality. Immunohistochemical detection of monotypic immunoglobulin light chain expression, and thereby B-cell monoclonality, may be accomplished readily using fresh cell suspensions or frozen tissue sections. However, immunohistochemical detection of immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues is more difficult; with few exceptions, techniques suitable for formalin-fixed, paraffin-embedded tissues are not widely available. This report describes and validates a method for detecting immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues using a heat-induced epitope retrieval technique. This method was evaluated in a series of 113 cases of B-cell non-Hodgkin lymphoma, including 73 cases with correlative flow cytometric immunophenotyping data. Monotypic light chain expression was demonstrated in 91 (81%) of 113 cases, including several small core biopsy specimens with extremely limited tissue. Compared with the reference method (flow cytometric immunophenotyping), the specificity of the assay was 100%. Interobserver reproducibility was excellent, with 87% concordance between two independent observers categorizing cases as indeterminate, suggestive or diagnostic of kappa or lambda light chain restriction (Cohen kappa statistic: 0.81). In summary, the described method permits demonstration of immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues in approximately 80% of cases of B-cell non-Hodgkin lymphoma with a high degree of specificity and excellent interobserver reproducibility. The assay is sufficiently robust for diagnostic use in small biopsies in which fresh tissue is unavailable.

Epidermal CD8+ T cells reactive with group A streptococcal antigens in chronic plaque psoriasis.

Chronic plaque psoriasis is a T cell mediated disease associated with group A streptococci (GAS). We have previously shown the presence of a psoriasis-specific dermal Th1 subset that recognizes GAS antigens. To assess whether GAS-reactive T cells are also present in lesional epidermis, fresh cell suspensions or T cell lines isolated from lesional epidermis of 33 psoriasis patients were stained for intracellular interferon-gamma after stimulation with GAS antigens. The patients were typed by PCR for HLA-DR7 and HLA-Cw6 expression. A subset of GAS-reactive CD8+ T cells (2.4% +/- 2.4) was found in 14/21 (67%) fresh cell suspensions. A smaller subset of GAS-reactive CD4+ T cells (0.9% +/- 0.9) was found in 13/21 (62%) fresh cell suspensions, which was expanded in the T cell lines. There was a significant inverse correlation between the proportions of GAS-reactive CD4+ and CD8+ T cells in the fresh suspensions (r = -0.48, P = 0.0277). The presence of GAS-reactive CD4+ or CD8+ T cells did not correlate with HLA-DR7 or HLA-Cw6 expression, respectively. This study has demonstrated GAS-reactive CD8+, and to a lesser extent CD4+, T cell subsets in psoriatic epidermis and provides further evidence that GAS antigens may play a role in the pathogenesis of chronic plaque psoriasis.

MHC-class I expression on prostate carcinoma and modulation by IFN-gamma

In this study, we investigated MHC-class I expression on both prostate cancer and normal prostate and compared those data with the number of CD8+ lymphocyte. Secondary, we investigated effect of IFN-gamma to the MHC-class I expression on prostate carcinoma. Twenty cryo-preserved benign prostate samples (Seven normal prostates and five benign prostatic hypertrophy samples.) and fifteen prostate carcinoma samples were used for immunohistochemistry of CD8 and MHC-class I. Eleven fresh single cell suspensions of prostate carcinoma were used for IFN-gamma study. After 24 hours IFN-gamma stimulation, MHC-I expression was measured by FACs analysis. Interestingly, significant correlation was observed between MHC-class I expression and the CD8+ lymphocyte infiltrate (r = 0.705, P < 0.0001). After 24 hrs IFN-gamma stimulation, MHC-class I expression was up-regulated in all samples (p < 0.05). Reduced expression of MHC-class I was thought as one of the factor which is related to the reduced degree of TILs (tumor infiltratig lymphocytes) in prostate cancer. IFN-gamma which is secreted mainly from CTL (cytotoxic T lymphocyte) might increase the degree of TIL through up-regulated MHC-class I.

Anti-CD10 immunoperoxidase staining of paraffin-embedded acute leukemias: Comparison with flow cytometric immunophenotyping

CD10 is common in B-precursor acute lymphoblastic leukemia (ALL) but is rare in acute myeloid leukemia (AML). However, until recently, analysis for CD10 has generally required fresh or frozen tissue. 56C6 is a monoclonal antibody that is now commercially available for the detection of CD10 in routinely processed paraffin-embedded tissue. Immunoperoxidase stains for CD10 on paraffin-embedded bone marrow core biopsy specimens (B5-fixed, decalcified) and marrow aspirate clots (formalin-fixed) were compared with flow cytometric immunophenotyping for CD10 on fresh cell suspensions in 20 cases of AML and in 30 cases of ALL. CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 98% (49 of 50) of acute leukemias. The results matched in 100% (20 of 20) of AML. Five percent (1 of 20) of AMLs expressed CD10. Two of the AMLs with monocytoid differentiation were interpreted as negative for CD10 by flow cytometry, although these had nonspecific dim immunofluorescence for multiple markers, including CD10, and these cases were negative by immunohistochemistry. CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 97% (29 of 30) of ALL. Eighty-four percent (21 of 25) of B-precursor ALL and 40% (2/5) of T-lineage ALL expressed CD10 by immunohistochemistry. In 1 case of B-precursor ALL, CD10 was dimly positive in 24% of the blasts by flow cytometry but negative by immunohistochemistry. We conclude that immunohistochemical staining of paraffin-embedded tissue, either B5- or formalin-fixed, is an effective method for the detection of CD10 in acute leukemia. This technique is useful in distinguishing AML from ALL. HUM PATHOL 31:1051-1054.

Historical review of lymphomas.

Historical review of lymphomas.

GFAP mRNA positive glia acutely isolated from rat hippocampus predominantly show complex current patterns

Electrophysiologically complex glial cells have been widely identified from different regions of the central nervous system and constitute a dominant glial type in juvenile mice or rats. As these cells express several types of ion channels and neurotransmitter channels that were thought to be only present in neurons, this glial cell type has attracted considerable attention. However, the actual classification of these electrophysiologically complex glial cells remains unclear. They have been speculated to be an immature astrocyte because, although these cells show positive staining for the predominantly astrocytic marker S 100β, it has not been possible to show staining for the commonly accepted mature astrocytic marker, glial fibrillary acidic protein (GFAP). To address the question of whether these cells might express GFAP at the transcript level, we combined patch-clamp electrophysiological recording with single cell RT-PCR for GFAP mRNA in glial cells acutely isolated from 4 to 12 postnatal day rats. In fresh cell suspensions from the CA1 region, complex glial cells were found to be the dominant cell type (65% total cells). We found that the majority of these electrophysiologically complex cells (74%) were positive for GFAP mRNA. We also showed that the complex cells responded to AMPA and GABA application, and these were also GFAP mRNA positive. We also fixed and stained the preparations for GFAP without electrophysiological recording to better preserve GFAP immunoreactively. In agreement with other studies, only 1.5% of these presumed electrophysiologically complex cells, based on morphology, showed immunoreactivity for GFAP. The expression of GFAP at the transcript level indicates GFAP (−)/GFAP mRNA (+) glial cells have an astrocytic identity. As single cell RT-PCR is able to detect both GFAP (−)/GFAP mRNA (+) and GFAP (+)/GFAP mRNA (+) astrocytic subtypes, the present study also suggests it is a feasible approach for astrocytic lineage studies.