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Children with T-ALL/LBL require prompt diagnosis and treatment. Flow cytometric analysis of T-lineage and immaturity markers usually leads to a straightforward diagnosis. However, rare cases of T-ALL expressing bright CD45 and lacking expression of immature markers can be a diagnostic conundrum and difficult to differentiate from mature T-cell lymphomas lacking surface CD3 expression or aberrantly expressing immature markers, which affects treatment decisions and prognosis. Here, we summarize the clinical, pathologic, and genetic features of 3 pediatric T-ALL cases with an entirely mature immunophenotype., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Stereotactic body radiation therapy (SBRT) versus surgery for operable early-stage non-small cell lung cancer (ES-NSCLC) remains highly debated. Herein, we used spatial proteomics to identify whether any molecular biomarker(s) associate with the efficacy of either modality, in efforts to optimize treatment selection between surgery and SBRT for this population., Methods and Materials: We evaluated biopsy tissue samples from 44 patients with ES-NSCLC treated with first-line SBRT (cohort 1) by GeoMx Digital Spatial Profiling (DSP) with a panel of 70 proteins in 5 spatial molecular compartments: tumor (panCK+), leukocyte (CD45+), lymphocyte (CD3+), macrophage (CD68+), and stroma (α-SMA+). To validate the findings in cohort 1, biopsy samples from 52 patients with ES-NSCLC who received SBRT (cohort 2) and 62 patients with ES-NSCLC who underwent surgery (cohort 3) were collected and analyzed by multiplex immunofluorescence (mIF)., Results: In cohort 1, higher CD44 expression in the lymphocyte compartment was associated with poorer recurrence-free survival (RFS) (DSP: P < .001; mIF: P < .001) and higher recurrence rate (DSP: P = .001; mIF: P = .004). mIF data from cohort 2 validated these findings (P < .05 for all). From cohort 3, higher lymphocyte CD44 predicted higher RFS after surgery (P = .003). Intermodality comparisons demonstrated that SBRT was associated with significantly higher RFS over surgery in CD44-low patients (P < .001), but surgery was superior to SBRT in CD44-high cases (P = .016)., Conclusions: Lymphocyte CD44 may not only be a predictor of SBRT efficacy in this population but also an important biomarker (pending validation by large prospective data) that could better sharpen selection for SBRT versus surgery in ES-NSCLC., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored., Methods: Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard.", Results: The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples., Conclusions: Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability., (© 2024 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Background: Circulating tumor cells (CTCs) shed into blood provide prognostic and/or predictive information. Previously, the authors established an assay to detect carcinoma cells from pleural fluid, termed effusion tumor cells (ETCs), by employing an immunofluorescence-based CTC-identification platform (RareCyte) on air-dried unstained ThinPrep (TP) slides. To facilitate clinical integration, they evaluated different slide processing and storage conditions, hypothesizing that alternative comparable conditions for ETC detection exist., Methods: The authors enumerated ETCs on RareCyte, using morphology and mean fluorescence intensity (MFI) cutoffs of >100 arbitrary units (a.u.) for epithelial cellular adhesion molecule (EpCAM) and <100 a.u. for CD45. They analyzed malignant pleural fluid from three patients under seven processing and/or staining conditions, three patients after short-term storage under three conditions, and seven samples following long-term storage at -80°C. MFI values of 4',6-diamidino-2-phenylindol, cytokeratin, CD45, and EpCAM were compared., Results: ETCs were detected in all conditions. Among the different processing conditions tested, the ethanol-fixed, unstained TP was most similar to the previously established air-dried, unstained TP protocol. All smears and Pap-stained TPs had significantly different marker MFIs from the established condition. After short-term storage, the established condition showed comparable results, but ethanol-fixed and Pap-stained slides showed significant differences. ETCs were detectable after long-term storage at -80°C in comparable numbers to freshly prepared slides, but most marker MFIs were significantly different., Conclusions: It is possible to detect ETCs under different processing and storage conditions, lending promise to the application of this method in broader settings. Because of decreased immunofluorescence-signature distinctions between cells, morphology may need to play a larger role., (© 2024 American Cancer Society.)

As humans age, their memory T cell compartment expands due to the lifelong exposure to antigens. This expansion is characterized by terminally differentiated CD8 + T cells (Temra), which possess NK cell-like phenotype and are associated with chronic inflammatory conditions. Temra cells are predominantly driven by the sporadic reactivation of cytomegalovirus (CMV), yet their epigenomic patterns and cellular heterogeneity remain understudied. To address this gap, we correlated their gene expression profiles with chromatin openness and conducted single-cell transcriptome analysis, comparing them to other CD8 + subsets and CMV-responses. We confirmed that Temra cells exhibit high expression of genes associated with cytotoxicity and lower expression of costimulatory and chemokine genes. The data revealed that CMV-responsive CD8 + T cells (Tcmv) were predominantly derived from a mixed population of Temra and memory cells (Tcm/em) and shared their transcriptomic profiles. Using ATAC-seq analysis, we identified 1449 differentially accessible chromatin regions between CD8 + Temra and Tcm/em cells, of which only 127 sites gained chromatin accessibility in Temra cells. We further identified 51 gene loci, including costimulatory CD27 , CD28 , and ICOS genes, whose chromatin accessibility correlated with their gene expression. The differential chromatin regions Tcm/em cells were enriched in motifs that bind multiple transcriptional activators, such as Jun/Fos, NFkappaB, and STAT, whereas the open regions in Temra cells mainly contained binding sites of T-box transcription factors. Our single-cell analysis of CD8 + CCR7 lo CD45RA hi sorted Temra population showed several subsets of Temra and NKT-like cells and CMC1 + Temra populations in older individuals that were shifted towards decreased cytotoxicity. Among CD8 + CCR7 lo CD45RA hi sorted cells, we found a decreased proportion of IL7R + Tcm/em-like and MAIT cells in individuals with high levels of CMV antibodies (CMV hi ). These results shed new light on the molecular and cellular heterogeneity of CD8 + Temra cells and their relationship to aging and CMV infection., Competing Interests: Author IF was employed by company Qiagen Aarhus A/S. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Türk, Filippov, Arnold, Zaugg, Tserel, Kisand and Peterson.)

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Breast cancer is the leading cause of cancer death in woman and tremendous efforts are undertaken to limit its dissemination and to provide effective treatment. Various histopathological parameters are routinely assessed in breast cancer biopsies to provide valuable diagnostic and prognostic information. MMP-11 and CD45 are tumor-associated antigens and potentially valuable biomarkers for grading aggressiveness and metastatic probability. This paper presents methods for quantitative and multiplexed imaging of MMP-11 and CD45 in breast cancer tissues and investigates their potential for improved cancer characterization and patient stratification. An immunohistochemistry-assisted laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method was successfully developed and optimized using lanthanide-tagged monoclonal antibodies as proxies to determine spatial distributions and concentrations of the two breast cancer biomarkers. The labeling degree of antibodies was determined via size exclusion-ICP-tandem mass spectrometry (SEC-ICP-MS/MS) employing online calibration via post-column isotope dilution analysis (IDA). The calibration of spatial distributions of labeled lanthanides in tissues was performed by ablating mold-prepared gelatin standards spiked with element standards. Knowledge of labeling degrees enabled the translation of lanthanide concentrations into biomarkers concentrations. The k-means clustering was used to select tissue areas for statistical analysis and mean concentrations were compared for sets of metastatic, non-metastatic and healthy samples. MMP-11 was expressed in stroma surrounding tumor areas, while CD45 was predominantly found inside tumor areas with high cell density. There was no significant correlation between CD45 and metastasis (P = 0.70); however, MMP-11 was significantly up-regulated (202%) in metastatic samples compared to non-metastatic (P = 0.0077) and healthy tissues (P = 0.0087)., (© The Author(s) 2022. Published by Oxford University Press.)

Objective To investigate the distribution of CD11c + B220 + NK cells in peripheral lymphoid tissues and liver and the surface expression of plasmacytoid dendritic cell antigen-1 (PDCA-1) on CD11c + B220 + NK cells. Methods The spleen, lymph nodes and liver tissues of C57BL/6 mice were collected to prepare single-cell suspensions, and the proportion of CD11c + B220 + NK cells in the tissues and their surface expression of PDCA-1 were detected by multi-color flow cytometry. Results CD11c + B220 + NK cells were distributed widely in the spleen, lymph nodes and liver, with the highest proportion in the spleen (2.82±0.45)%. PDCA-1s were expressed in some of CD11c + B220 + NK cells in the tissues, particularly in the spleen tissues. Conclusion CD11c + B220 + NK cells are important subpopulation of NK cells in murine peripheral lymphoid tissues and liver. The expression of PDCA-1 on CD11c + B220 + NK cells is different in different tissues.

While the abundance and phenotype of tumor-infiltrating lymphocytes are linked with clinical survival, their spatial coordination and its clinical significance remain unclear. Here, we investigated the immune profile of intratumoral and peritumoral tissue of clear cell renal cell carcinoma patients (n = 64). We trained a cell classifier to detect lymphocytes from hematoxylin and eosin stained tissue slides. Using unsupervised classification, patients were further classified into immune cold, hot and excluded topographies reflecting lymphocyte abundance and localization. The immune topography distribution was further validated with The Cancer Genome Atlas digital image dataset. We showed association between PBRM1 mutation and immune cold topography, STAG1 mutation and immune hot topography and BAP1 mutation and immune excluded topography. With quantitative multiplex immunohistochemistry we analyzed the expression of 23 lymphocyte markers in intratumoral and peritumoral tissue regions. To study spatial interactions, we developed an algorithm quantifying the proportion of adjacent immune cell pairs and their immunophenotypes. Immune excluded tumors were associated with superior overall survival (HR 0.19, p = 0.02) and less extensive metastasis. Intratumoral T cells were characterized with pronounced expression of immunological activation and exhaustion markers such as granzyme B, PD1, and LAG3. Immune cell interaction occurred most frequently in the intratumoral region and correlated with CD45RO expression. Moreover, high proportion of peritumoral CD45RO+ T cells predicted poor overall survival. In summary, intratumoral and peritumoral tissue regions represent distinct immunospatial profiles and are associated with clinicopathologic characteristics., (© 2021. The Author(s).)

Advanced maternal age is a risk factor for female infertility, and placental dysfunction is considered one of the causes of pregnancy complications. We investigated the effects of advanced maternal aging on pregnancy outcomes and placental senescence. Female pregnant mice were separated into three groups: young (3 months old), middle (8-9 months old), and aged (11-13 months old). Although the body weights of young and middle dams gradually increased during pregnancy, the body weight of aged dams only increased slightly. The placental weight and resorption rate were significantly higher, and live fetal weights were reduced in a maternal age-dependent manner. Although mRNA expression of senescence regulatory factors (p16 and p21) increased in the spleen of aged dams, mRNA expression of p16 did not change and that of p21 was reduced in the placenta of aged dams. Using a cytokine array of proteins extracted from placental tissues, the expression of various types of senescence-associated secretory phenotype (SASP) factors was decreased in aged dams compared with young and middle dams. The aged maternal placenta showed reduced immune cell accumulation compared with the young placenta. Our present results suggest that models using pregnant mice older than 8 months are more suitable for verifying older human pregnancies. These findings suggest that general cellular senescence programs may not be included in the placenta and that placental functions, including SASP production and immune cell accumulation, gradually decrease in a maternal age-dependent manner, resulting in a higher rate of pregnancy complications.

Objectives: The treatment of pediatric acute lymphoblastic leukemias (ALL) has seen remarkable advances recently. However, relapse occurs in approximately 20% of cases which necessitates identifying additional high risk parameters for treatment intensification. The aim of this study is to assess the prognostic significance of CD45 antigen expression in pediatric ALL., Methods: We studied 363 pediatric patients with B cell precursor-ALL (BCP-ALL) (n = 313) and T-ALL (n = 50). The ratio of median fluorescence intensity of CD45 expressed in leukemic blasts and normal lymphocytes was calculated. The 75th percentile was taken as cut-off to categorise patients into CD45 high and CD45 low groups., Results: The 75th percentile was 0.141 in BCP-ALL and 0.548 in T-ALL. In BCP-ALL, there was a statistically significant association of age (≥10 years) (p = 0.027) and National Cancer Institute high risk group (p = 0.001) with high CD45 expression but not in T-ALL. Worse event-free survival (EFS) was seen with high CD45 expression in BCP-ALL (42.17% versus 60.83%, p = 0.0053). In T-ALL, there was no association between CD45 expression and EFS (CD45 high 40.40% versus low 67.35%, p = 0.414). The overall survival (OS) was 70% versus 60% (p = 0.38) in BCP-ALL and the OS was 82% versus 68% (p = 0.16) in T-ALL for CD45 low versus CD45 high groups, respectively., Conclusion: We conclude that high CD45 surface expression is associated with worse EFS in pediatric BCP-ALL., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: Cancer patient outcomes and selection for novel therapies are heavily influenced by the immune contexture of the tumor microenvironment. Esophageal cancer is associated with poor outcomes. In contrast to colorectal cancer, where the immunoscore is increasingly used in prognostic staging, little is known about the immune cell populations in esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (SCC), and their clinical significance., Methods: Tissue microarrays were constructed from resected tumor tissue of 72 EAC patients and 23 SCC patients. Immunohistochemical staining of CD3, CD8, CD56, CD68, CD45RO, CD69, IFN-γ, IL-10, IL-4, IL-17, TGF-β, FOXP3 and CD107a was performed. Positivity was examined in both the stromal and epithelial compartments. Statistical analysis was performed to identify differences in immune cell infiltration and functional phenotypes between cancer subtypes and tissue compartments., Results: This study identified that esophageal tumors are enriched with CD45RO+ and CD8+ cells and such positivity is significantly higher in SCC compared with EAC. Furthermore, the expression of CD45RO positively correlates with that of CD8 within the tumors of both patient cohorts, suggesting a dominance of memory cytotoxic T cells. This is supported by strong positivity of degranulation marker CD107a in the stromal compartment of EAC and SCC tumors. Cytokine staining revealed a mixed pro- and anti-inflammatory profile within EAC tumors., Conclusions: Esophageal tumors are enriched with memory cytotoxic T cells. Applying these measurements to a larger cohort will ascertain the clinical utility of assessing specific lymphocyte infiltrates in EAC and SCC tumors with regards to future immunotherapy use, patient prognosis and outcomes., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

With the advent of cancer immunology, mass cytometry has been increasingly employed to characterize the responses to cancer therapies and the tumor microenvironment (TME). One of its most notable applications is efficient multiplexing of samples into batches by dedicating a number of metal isotope channels to barcodes, enabling robust data acquisition and analysis. Barcoding is most effective when markers are present in all cells of interest. While CD45 has been shown to be a reliable marker for barcoding all immune cells in a given sample, a strategy to reliably barcode mouse cancer cells has not been demonstrated. To this end, we identified CD29 and CD98 as markers widely expressed by commonly used mouse cancer cell lines. We conjugated anti-CD29 and anti-CD98 antibodies to cadmium or indium metals and validated their utility in 10-plex barcoding of live cells. Finally, we established a potentially novel barcoding system incorporating the combination of CD29, CD98, and CD45 to multiplex 10 tumors from s.c. MC38 and KPC tumor models, while successfully recapitulating the known contrast in the PD1-PDL1 axis between the 2 models. The ability to barcode tumor cells along with immune cells empowers the interrogation of the tumor-immune interactions in mouse TME studies.

We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

γδ T cells are the unique T cell subgroup with their T cell receptors composed of γ chain and δ chain. Unlike αβ T cells, γδ T cells are non-MHC-restricted in recognizing tumor antigens, and therefore defined as innate immune cells. Activated γδ T cells can promote the anti-tumor function of adaptive immune cells. They are considered as a bridge between adaptive immunity and innate immunity. However, several other studies have shown that γδ T cells can also promote tumor progression by inhibiting anti-tumor response. Therefore, γδ T cells may have both anti-tumor and tumor-promoting effects. In order to clarify this contradiction, in this review, we summarized the functions of the main subsets of human γδ T cells in how they exhibit their respective anti-tumor or pro-tumor effects in cancer. Then, we reviewed recent γδ T cell-based anti-tumor immunotherapy. Finally, we summarized the existing problems and prospect of this immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Li, Zhang, Wu and Chen.)

The ability to accurately identify and quantify immune cell populations within adipose tissue is important in understanding the role of immune cells in metabolic disease risk. Flow cytometry is the gold standard method for immune cell quantification. However, quantification of immune cells from adipose tissue presents a number of challenges because of the complexities of working with an oily substance and the rapid deterioration of immune cell viability before analysis can be performed. Here we present a highly reproducible flow cytometry protocol for the quantification of immune cells in human adipose tissue, which overcomes these issues., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction/objective: Liquid-based cytology (LBC) is advantageous as multiple stained specimens can be prepared and used for additional assays such as immunocytochemical and molecular-pathological investigations. Two types of preservative-fixative solutions (fixatives) are used for nongynecologic specimens used in the BD SurePath-LBC (SP-LBC) method, and their components vary. However, few studies have evaluated the differences in antigen-retaining ability between these fixatives. Therefore, we investigated and compared the antigen-retaining ability of the fixatives in immunocytochemical staining (ICC) under long-term storage conditions., Materials and Methods: Sediments of cultured RAJI cells (derived from Burkitt's lymphoma) were added to each fixative (red and blue) and stored at room temperature for a specified period (1 h; 1 week; and 1, 3, and 6 months). The specimens were then prepared using the SP-LBC method and subjected to ICC. Positivity rate was calculated using the specimens fixed at room temperature for 1 h as a control. Antibodies against Ki67 expressed in the nucleus and against CD20 and leukocyte common antigen (LCA) expressed on the cell membrane were used., Results: For CD20 and LCA, the positivity rate increased with time in the red fixative compared with that in the control. In the blue fixative, the positivity rate was highest at 1 h and was maintained at a high level throughout the storage period. In contrast, the Ki67 positivity rate was highest at 1 h in both red and blue fixatives and markedly decreased with time. Therefore, although refrigerated (8°C) storage was used, no improvement was noted., Conclusions: Long-term storage is possible for cell membrane antigens at room temperature; however, it is unsuitable for intranuclear antigens. Therefore, we conclude that suitable fixative type and storage temperature differ based on antigen location. Further investigation is warranted., (© 2021 S. Karger AG, Basel.)

Several recent experimental studies have investigated the effects of caffeine and chlorogenic acid (CGA), representative ingredients of coffee, on nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). However, the results are conflicting, and their effects are yet to be clarified. In the present study, we examined the effects of caffeine and CGA on choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice, relatively new model mice of NASH. Seven-week-old male C57BL/6J mice were divided into the following groups: Control diet (control), CDAHFD (CDAHFD), CDAHFD supplemented with 0.05% ( w / w ) caffeine (caffeine), and CDAHFD supplemented with 0.1% ( w / w ) CGA (CGA). After seven weeks, the mice were killed and serum biochemical, histopathological, and molecular analyses were performed. Serum alanine aminotransferase (ALT) levels were significantly higher in the caffeine and CGA groups than in the CDAHFD group. On image analysis, the prevalence of Oil red O-positive areas (reflecting steatosis) was significantly higher in the caffeine group than in the CDAHFD group, and that of CD45R-positive areas (reflecting lymphocytic infiltration) in the hepatic lobule was significantly higher in the caffeine and CGA groups than in the CDAHFD group. Hepatic expression of interleukin (IL)-6 mRNA was higher in the caffeine and CGA groups than in the CDAHFD group, and the difference was statistically significant for the caffeine group. In conclusion, in the present study, caffeine and CGA significantly worsened the markers of liver cell injury, inflammation, and/or steatosis in NASH lesions in mice.

Recent efforts have focused on customizing orthobiologics, such as platelet-rich plasma (PRP) and bone marrow concentrate (BMC), to improve tissue repair. We hypothesized that oral losartan (a TGF-β1 blocker with anti-fibrotic properties) could decrease TGF-β1 levels in leukocyte-poor PRP (LP-PRP) and fibrocytes in BMC. Ten rabbits were randomized into two groups (N = 5/group): osteochondral defect + microfracture (control, group 1) and osteochondral defect + microfracture + losartan (losartan, group 2). For group 2, a dose of 10mg/kg/day of losartan was administrated orally for 12 weeks post-operatively. After 12 weeks, whole blood (WB) and bone marrow aspirate (BMA) samples were collected to process LP-PRP and BMC. TGF-β1 concentrations were measured in WB and LP-PRP with multiplex immunoassay. BMC cell populations were analyzed by flow cytometry with CD31, CD44, CD45, CD34, CD146 and CD90 antibodies. There was no significant difference in TGF-β1 levels between the losartan and control group in WB or LP-PRP. In BMC, the percentage of CD31+ cells (endothelial cells) in the losartan group was significantly higher than the control group ( p = 0.008), while the percentage of CD45+ cells (hematopoietic cells-fibrocytes) in the losartan group was significantly lower than the control group ( p = 0.03).

The isolation and analysis of circulating tumor cells (CTC) has the potential to provide minimally invasive diagnostic, prognostic and predictive information. Widespread clinical implementation of CTC analysis has been hampered by a lack of comparative investigation between different analytic methodologies in clinically relevant settings. The objective of this study was to evaluate four different CTC isolation techniques-those that rely on surface antigen expression (EpCAM or CD45 using DynaBeads® or EasySep™ systems) or the biophysical properties (RosetteSep™ or ScreenCell®) of CTCs. These were evaluated using cultured cells in order to calculate isolation efficiency at various levels including; inter-assay and inter-operator variability, protocol complexity and turn-around time. All four techniques were adequate at levels above 100 cells/mL which is commonly used for the evaluation of new isolation techniques. Only the RosetteSep™ and ScreenCell® techniques were found to provide adequate sensitivity at a level of 10 cells/mL. These techniques were then applied to the isolation and analysis of circulating tumor cells blood drawn from metastatic breast cancer patients where CTCs were detected in 54% (15/28) of MBC patients using the RosetteSep™ and 75% (6/8) with ScreenCell®. Overall, the ScreenCell® method had better sensitivity., Competing Interests: Funding for this study was provided in part from a Pfizer Investigator Initiated Research Grant (WS575460). This does not alter our adherence to PLOS ONE policies on sharing data and materials. We have no further relevant declarations (such as consultancies, patents, employment, product development) related to this funder or any other commercial entities.

Background: Stem/progenitor cells have been identified in human milk. However, characterization and percentages of cell subsets in human milk using hematopoietic stem and progenitor cell markers according to the differential expression of CD45, i.e., as CD45 dim/+ (mainly hematopoietic stem/progenitor cells) and CD45 - (mainly non-hematopoietic stem/progenitor cells), have not been assessed to date., Research Aim: To characterize stem/progenitor-like cell phenotypes in human milk and to report the percentages of these cells at two different lactation stages compared to peripheral blood., Methods: Human milk samples paired with peripheral blood samples ( N = 10) were analyzed by flow cytometry using CD45, CD34, CD133, CD38, and lineage-negative markers. The percentage of cell subsets was analyzed in colostrum (Day 3 postpartum) and transitional milk (Day 5/6 postpartum) and compared with the peripheral blood counterpart., Results: The percentage of CD45 - CD34 + cells was predominant in both colostrum and transitional milk. The percentage of CD45 +/high CD133 + cells was high in colostrum while the percentage of CD45 - CD133 + cells was high in transitional milk. Furthermore, the median percentages of the CD45 - CD34 + , CD45 - CD133 + , and CD45 dim CD133 + cell subsets were higher in colostrum than its peripheral blood counterpart (0.11% vs. 0.002%; 0.17% vs. 0.0005%; 0.09% vs. 0.05%, p = .04, respectively); also CD45 - CD34 - CD133 + and CD45 dim CD34 - CD133 + cell subsets were higher in colostrum than peripheral blood (1.32% vs. 0.0% and 2.4% vs. 0.06%, p = .04), respectively)., Conclusion: Early human milk is an abundant reservoir of hematopoietic stem/progenitor-like cells in the CD45 +/high population and non-hematopoietic stem/progenitor-like cells in the CD45 - population.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease. The current lack of an effective vaccine to simultaneously protect against the four serotypes of DENV in seronegative individuals is a major unmet medical need. Further, the immunological basis for protective immunity in the setting of DENV infection or vaccination is not fully understood. Our team has developed a live attenuated tetravalent dengue virus vaccine that provides complete protection in a human model of dengue virus challenge. The goal of this study was to define, in the context of protective human vaccination, the quality of vaccine-induced DENV-specific CD8 + and CD4 + T cells and the temporal dynamics associated with their formation and maintenance. Multifunctional, DENV-specific CD8 + and CD4 + T cells developed 8-14 days after vaccination and were maintained for at least 6 months. Virus-specific CD8 T + cells were a mixture of effector memory T cells (T EM ) and effector memory T cells re-expressing CD45RA (T EMRA ), with T EM cells predominating until day 21 post-vaccination and T EMRA cells thereafter. The majority of virus-specific CD4 + T cells were T EM with a small fraction being T EMRA . The frequency of virus-specific CD8 + and CD4 + T cells were further skewed to the T EMRA phenotype following either a second dose of the tetravalent vaccine or challenge with a single serotype of DENV. Collectively, our study has defined the phenotypic profile of antiviral CD8 + and CD4 + T cells associated with protective immunity to DENV infection and the kinetics of their formation and maintenance., (Copyright © 2020 Graham, Eisenhauer, Diehl, Pierce, Whitehead, Durbin, Kirkpatrick, Sette, Weiskopf, Boyson and Botten.)

The non-polymorphic nature of CD1 proteins creates a situation in which T cells with invariant T cell receptors (TCRs), like CD1d-specific NKT cells, are present in all humans. CD1b is an abundant protein on human dendritic cells that presents M. tuberculosis ( Mtb ) lipid antigens to T cells. Analysis of T cell clones suggested that semi-invariant TCRs exist in the CD1b system, but their prevalence in humans is not known. Here we used CD1b tetramers loaded with mycolic acid or glucose monomycolate to study polyclonal T cells from 150 Peruvian subjects. We found that CD1b tetramers loaded with mycolic acid or glucose monomycolate antigens stained TRAV1-2 + GEM T cells or TRBV4-1 + LDN5-like T cells in the majority of subjects tested, at rates ~10-fold lower than NKT cells. Thus, GEM T cells and LDN5-like T cells are a normal part of the human immune system. Unlike prior studies measuring MHC- or CD1b-mediated activation, this large-scale tetramer study found no significant differences in rates of CD1b tetramer-mycobacterial lipid staining of T cells among subjects with Mtb exposure, latent Mtb infection or active tuberculosis (TB) disease. In all subjects, including "uninfected" subjects, CD1b tetramer + T cells expressed memory markers at high levels. However, among controls with lower mycobacterial antigen exposure in Boston, we found significantly lower frequencies of T cells staining with CD1b tetramers loaded with mycobacterial lipids. These data link CD1b-specific T cell detection to mycobacterial exposure, but not TB disease status, which potentially explains differences in outcomes among CD1-based clinical studies, which used control subjects with low Mtb exposure., (Copyright © 2020 Lopez, Iwany, Suliman, Reijneveld, Ocampo, Jimenez, Calderon, Lecca, Murray, Moody and Van Rhijn.)

Background Aims: In 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards., Methods: Twelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures., Results: Excellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells., Conclusions: The current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary, or financial interest in the products or companies described in this article., (Copyright © 2019 International Society for Cell and Gene Therapy. All rights reserved.)

Gliomas are the most common malignant brain tumors in the pediatric population. Even though great efforts have been made to understand their distinctive molecular characteristics, there has not been any improvements in the median survival in decades. In children, high-grade glial tumors have a median survival of 9-15 months. It has recently been demonstrated that pediatric high-grade gliomas (pHGG) are biologically and molecularly different from the adult counterparts, which could explain why conventional treatments universally fail. The development of an in vivo pHGG model harboring the specific genetic alterations encountered in pediatric gliomas is imperative in order to study the molecular basis that drives the progression and aggressiveness of these tumors. It would also enable harnessing these results for the development of novel therapeutic approaches. Our lab has implemented a method to induce brain tumors using transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mouse brain. One of the main advantages of this method is that tumors are induced by altering the genome of the host cells, allowing us to recapitulate the salient features of the human disease. In this chapter we describe a method to isolate two cell populations from tumors generated in situ in mice, i.e., one population enriched in tumor cells and another population enriched in CD45+ cells. We also present methodologies as to how tumor infiltrating immune cells can be phenotypically characterized using flow cytometry., (© 2020 Elsevier Inc. All rights reserved.)

Background: Naringenin is a biologically active analgesic, anti-inflammatory, and antioxidant flavonoid. Naringenin targets in inflammation-induced articular pain remain poorly explored., Methods: The present study investigated the cellular and molecular mechanisms involved in the analgesic/anti-inflammatory effects of naringenin in zymosan-induced arthritis. Mice were pre-treated orally with naringenin (16.7-150 mg/kg), followed by intra-articular injection of zymosan. Articular mechanical hyperalgesia and oedema, leucocyte recruitment to synovial cavity, histopathology, expression/production of pro- and anti-inflammatory mediators and NFκB activation, inflammasome component expression, and oxidative stress were evaluated., Results: Naringenin inhibited articular pain and oedema in a dose-dependent manner. The dose of 50 mg/kg inhibited leucocyte recruitment, histopathological alterations, NFκB activation, and NFκB-dependent pro-inflammatory cytokines (TNF-α, IL-1β, and IL-33), and preproET-1 mRNA expression, but increased anti-inflammatory IL-10. Naringenin also inhibited inflammasome upregulation (reduced Nlrp3, ASC, caspase-1, and pro-IL-1β mRNA expression) and oxidative stress (reduced gp91 phox mRNA expression and superoxide anion production, increased GSH levels, induced Nrf2 protein in CD45 + hematopoietic recruited cells, and induced Nrf2 and HO-1 mRNA expression)., Conclusions: Naringenin presents analgesic and anti-inflammatory effects in zymosan-induced arthritis by targeting its main physiopathological mechanisms. These data highlight this flavonoid as an interesting therapeutic compound to treat joint inflammation, deserving additional pre-clinical and clinical studies.

Desmoid tumors (DTs) are local aggressive neoplasms, whose therapeutic approach has remained so far unsolved and in many instances controversial. Nowadays, immunotherapy appears to play a leading role in the treatment of various tumor types. Characterization of the tumor immune microenvironment (TME) and immune checkpoints can possibly help identify new immunotherapeutic targets for DTs. We performed immunohistochemistry (IHC) on 33 formalin-fixed paraffin-embedded (FFPE) tissue sections from DT samples to characterize the TME and the immune checkpoint expression profile. We stained for CD3, CD4, CD8, CD20, FoxP3, CD45RO, CD56, CD68, NKp46, granzyme B, CD27, CD70, PD1 and PD-L1. We investigated the expression of the markers in the tumoral stroma, as well as at the periphery of the tumor. We found that most of the tumors showed organization of lymphocytes into lymphoid aggregates at the periphery of the tumor, strongly resembling tertiary lymphoid organs (TLOs). The tumor expressed a significant number of memory T cells, both at the periphery and in the tumoral stroma. In the lymphoid aggregates, we also recognized a significant proportion of regulatory T cells. The immune checkpoint ligand PD-L1 was negative on the tumor cells in almost all samples. On the other hand, PD1 was partially expressed in lymphocytes at the periphery of the tumor. To conclude, we are the first to show that DTs display a strong immune infiltration at the tumor margins, with formation of lymphoid aggregates. Moreover, we demonstrated that there is no PD-L1-driven immune suppression present in the tumor cells.

Objective: To explore the expression of CD45 in newly diagnosed multiple myeloma (MM) and its relationship with clinical efficacy and prognosis. Methods: This study retrospectively analyzed expression and distribution of CD45 in 130 cases of newly diagnosed MM, comparing clinical efficacy and prognosis in CD45(+)/CD45(-) groups. Results: ①The CD45(+) group was 33 cases (25.38%) , and CD45(-) group was 97 cases (74.62%) . ②The objective remission rate (ORR) of CD45(+) and CD45(-)group was 33.33% and 64.95%, respectively. The difference was statistically significant ( P =0.002) . For patients in Bortezomib regimen, the ORR of CD45(+) and CD45(-) group was 35.71% and 66.25%, respectively. The difference was statistically significant ( P =0.005) . ③The median progress free survival (PFS) of CD45(+) group and CD45(-) group was 29.8 (95% CI 10.0-59.0) months vs 34.5 (95% CI 6.0-69.0) months ( χ (2)=14.59, P <0.001) and the median overall survival (OS) was 32.5 (95% CI 10.0-68.0) months vs 37.6 (95% CI 6.0-78.0) months ( χ (2)=11.42, P =0.001) , respectively. Among the patients in bortezomib regimen, The median PFS and median OS of CD45 (+) group and CD45(-) group were 30.3 (95% CI 10.0-59.0) months vs 36.3 (95% CI 6.0-69.0) months ( χ (2)=14.75, P =0.001) and 34.0 (95% CI 10.0-68.0) months vs 39.5 (95% CI 6.0-78.0) months ( χ (2)=10.62, P =0.001) . ④Cox risk regression model analysis showed that serum creatinine≥176.8 μmol/L ( HR =5.078, 95% CI 1.744-14.723, P =0.001) , CD45 positive ( HR =14.504, 95% CI 0.168-0.42, P =0.001) , LDH≥220 IU/L ( HR =1.308, 95% CI 1.16-2.417, P =0.015) were independent risk prognostic factors. Conclusion: CD45 expression is a risk prognostic factor of MM patients. Bortezomib did not improve the poor prognosis of CD45(+) MM patients.

Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45 + leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints. NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-μm filter can replace density-mediated mononuclear cell separation which usually results in 50-70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.

Background: Impaired T cell immune function exists in end-stage renal disease (ESRD) patients. Dialysis treatment may lead to changes in T cell subsets. In the present study, we aimed to identify alterations of T cell phenotypes in ESRD patients, especially in those receiving peritoneal dialysis (PD), and analyze the potential associated factors., Methods: In the present study, 110 PD patients and 110 age/gender-matched hemodialysis (HD) patients who met the inclusion criteria were studied. Pre-dialysis blood samples were obtained and analyzed by flow cytometry to detect the expression of CD45RO and CCR7. Univariate and multivariate regression analyses were used to determine the factors associated with the alteration of T cell phenotypes., Results: In all dialysis patients, age was associated with the frequencies of both CD4+ and CD8+ naïve T cells, effector memory (EM) T cells and effector memory RA (EMRA) T cells but not central memory (CM) T cells. Dialysis modality was also associated with T cell subsets. Compared with HD patients, PD patients showed an increase in both CD4+ and CD8+ CM T cells and a reduction in both CD4+ and CD8+ EM and EMRA T cells. However, the number of CD4+ naïve T cells was lower and the number of CD8+ naïve T cells was higher in PD patients than those in HD patients. In PD patients, further multivariate analysis revealed that the frequency of CD4+ naïve T cells was positively associated with nPCR, while the frequency of CD8+ naïve T cells was negatively associated with age., Conclusion: In dialysis patients, the dialysis modality and age influence T cell subsets. There is a progression from naïve to effector T cells in HD patients compared with PD patients. In PD patients, different factors may influence the frequencies of CD4+ and CD8+ naïve T cells.

Mass cytometry (CyTOF) is a critical cell profiling tool in acquiring multiparameter proteome data at the single-cell level. A major challenge in CyTOF analysis is sample-to-sample variance arising from the pipetting process, staining variation, and instrument sensitivity. To reduce such variations, cell barcoding strategies that enable the combination of individual samples prior to antibody staining and data acquisition on CyTOF are often utilized. The most prevalent barcoding strategy is based on a binary scheme that cross-examines the existence or nonexistence of certain mass signals; however, it is limited by low barcoding efficiency and high cost, especially for large sample size. Herein, we present a novel barcoding method for CyTOF application based on mass ratiometry. Different mass tags with specific fixed ratios are used to label CD45 antibody to achieve sample barcoding. The presented method exponentially increases the number of possible barcoded samples with the same amount of mass tags compared with conventional methods. It also reduces the overall time for the labeling process to 40 min and avoids the need for expensive commercial barcoding buffer reagents. Moreover, unlike the conventional barcoding process, this strategy does not pre-permeabilize cells before the barcoding procedure, which offers additional benefits in preserving surface biomarker signals.

Background : Vitamin D has regulatory effects on different cells of the immune system and low levels are associated with several immune-mediated diseases. Aim : To investigate the association between neonatal 25-hydroxy vitamin D (25-OHD) level and the expression of lymphocyte activation markers (HLA-DR, CD69, CD25, CD45RA) on T-lymphocyte subpopulations and its impact in neonatal infection. Methods : 25-OHD level was measured in the cord blood of 56 neonates and their mothers using an enzyme immune-assay method. Based on the 25-OHD level, infants were categorised into four groups: severe deficiency ( n =  7), moderate deficiency ( n =  21), mild deficiency ( n =  15) and normal 25-OHD level ( n =  13). Mothers were classified into deficient ( n =  18), insufficient ( n =  21) and normal levels ( n =  17). T-lymphocyte subpopulations and lymphocyte activation markers were investigated using flow cytometry. Results : There was a positive correlation between maternal and cord blood 25-OHD levels ( r  = 0.503, p  = 0.001). The group with severe 25-OHD deficiency had the significantly lowest level of total lymphocytes, CD3+ T lymphocytes, CD4+ T-helper and CD8+ T-cytotoxic lymphocytes and CD4+CD45RA+ naïve T-cells compared with the other groups. The frequencies of CD8+CD25+, CD4+CD25+ and CD4+HLA-DR+ activated T-lymphocytes were significantly lower in the severe, moderate and mild deficiency groups than in the normal group. Seven of 43 (16.27%) infants with 25-OHD deficiency were admitted with sepsis to the neonatal intensive care unit and there were no cases of sepsis in the normal 25-OHD group. Conclusion : Vitamin D deficiency is associated with a reduction of lymphocyte subsets and altered T-lymphocyte activation which are considered to be risk factors for neonatal infection.

The most prominent histologic lesion in antibody-mediated rejection is microvascular inflammation (MVI); however, its recognition and scoring can be challenging and poorly reproducible between pathologists. We developed a dual immunohistochemical (IHC)-stain (anti-CD34/anti-CD45 for endothelium/leukocytes) as ancillary tool to improve on the semi-quantitative Banff scores and allow quantification of MVI. We examined the relationship between CD34-CD45 IHC-based quantitative MVI score (the inflamed peritubular capillary ratio, iptcr) and renal-graft failure or donor-specific antibodies (DSA) strength at the time of biopsy. Quantitative iptcr score was significantly associated with renal graft failure (hazard ratio 1.81, per 1 SD-unit [0.13 points] of iptcr-increase; P = 0.026) and predicted the presence and strength of DSA (ordinal odds ratio: 2.42; P = 0.005; 75 biopsies/60 kidney transplant recipients; 30 HLA- and/or ABO-incompatible). Next, we assessed inter-pathologist agreement for ptc score and ptc extent (focal/diffuse) using CD34-CD45 IHC as compared to conventional stain. Compared to conventional stain, CD34-CD45 IHC significantly increased inter-pathologist agreement on ptc score severity and extent (κ-coefficient from 0.52-0.80 and 0.46-0.68, respectively, P < 0.001). Our findings show that CD34-CD45 IHC improves reproducibility of MVI scoring and facilitates MVI quantification and introduction of a dual anti-CD34/CD45 has the potential to improve recognition of MVI ahead of DSA results., (© 2018 Steunstichting ESOT.)

Diffuse large B-cell lymphoma (DLBCL) is characterized by medium- to large-sized neoplastic cells that express a wide range of B-cell markers including CD19, CD20, CD22, and CD79a. Also, as this is a hematopoietic malignancy, there is expression of the leukocyte common antigen CD45. Lack of CD20 expression occurs in a specific rare heterogeneous subgroup of DLBCL including primary effusion lymphoma, plasmablastic lymphoma, ALK-positive large B-cell lymphoma, and large B-cell lymphoma arising in HHV8+ multicentric Castleman disease. In this article, we report a rare case of CD20- and CD45-negative Epstein-Barr virus-positive DLBCL in which the entities listed above were ruled out, thereby posing a significant diagnostic challenge. Arriving at the correct diagnosis of Epstein-Barr virus-positive DLBCL was supported by immunoreactivity for the B-cell transcription factor Oct-2 and the pan-B cell marker CD79a.

Objective: We aimed to evaluate the sensitivity of fine needle aspiration (FNA) for the diagnosis of Hodgkin's lymphoma (HL) in HIV-infected patients., Study Design: An electronic search was conducted to retrospectively identify patients diagnosed with HL who underwent FNA followed by confirmatory biopsy. FNAs were categorized as negative, atypical/suspicious/positive, or nondiagnostic. Diagnostic sensitivity in HIV+ and HIV- patients was statistically compared via Fisher's exact test, with a p value <0.05 considered significant., Results: Thirty-six patients meeting inclusion criteria were identified (24 HIV- and 12 HIV+). Average age was 36.0 ± 11.5 and 36.5 ± 7.4 years (means ± SD) in HIV- and HIV+ patients, respectively. The male-to-female ratio was 1.4:1 in HIV- patients versus 3:1 in HIV+ patients. Among these 36 patients, a total of 42 FNAs were performed. Overall sensitivity of FNA was 66.7% (95% confidence interval: 52.4-80.9%). When stratified by HIV status, a statistically significant difference in FNA sensitivity was detected, as sen-sitivity was 84.6% (70.8-98.4%) in HIV- patients versus only 37.5% (13.8-61.2%) in HIV+ patients (p =0.003)., Conclusion: The diagnostic sensitivity of FNA biopsy was significantly attenuated in the HIV+ cohort. In HIV-infected patients presenting with lymphadenopathy, increased clinical suspicion of HL is critical to avoid misdiagnosis., (© 2019 S. Karger AG, Basel.)

Background: Pityriasis rosea is a common papulosquamous disorder. However, its etiology and pathogenesis remain unclear., Objective: We investigate the types of inflammatory cells infiltrating the lesional skin of pityriasis rosea and demonstrate whether T-cell-mediated immunity is involved in the pathogenesis of this condition or not., Methods: The biopsies were taken from the lesional skin of 35 cases of patients diagnosed with pityriasis rosea. The specimens were prepared in paraffin sections, then submitted to routine immunohistochemistry procedures using monoclonal antibodies directed against CD3, CD4, CD8, CD20 and CD45RO and horseradish peroxidase-labeled goat anti-human antibodies. The positive sections were determined by the ratio and staining intensity of positive inflammatory cells., Results: The mean score of positive CD3, CD4, CD8, and CD45RO staining was respectively 3.74±3.88, 5.67±4.40, 2.94±3.42 and 7.68±4.33 in these pityriasis rosea patients (P<0.001). The percentage of positive staining was 54.29% (19/35), 69.7% (23/33), 40% (14/35) and 79.41% (27/34) (P<0.05). However, the staining of CD20 was negative in all samples. The mean score of CD3 staining in patients with time for remission ≤60 days (4.90±4.21) was higher than that in patients with time for remission >60 days (2.00±2.5) (P<0.05), whereas no statistical difference in the mean score of CD4, CD8 and CD45RO staining was observed. study liMitations: The sample size and the selected monoclonal antibody are limited, so the results reflect only part of the cellular immunity in the pathogenesis of pityriasis rosea., Conclusion: Our findings support a predominantly T-cell mediated immunity in the development of pityriasis rosea.

Explants of lymphoid tissue provide a rare opportunity to assess the organization of the immune system in a living, dynamic environment. Traditionally, ex vivo immunostaining is conducted in fixed tissue sections, while live tissues are analyzed using genetically engineered fluorescent reporters or adoptively transferred, pre-labelled cell populations. Here, we validated a protocol for immunostaining and imaging in live, thick slices of lymph node tissue, thus providing a spatial "map" of the lymph node while maintaining the viability and functionality of the slices. Using anti-B220/CD45R (B cell) as a prototype antibody, the procedure for immunostaining was tested for sufficient signal to noise with respect to staining time, temperature, and wash time, and the specificity was verified in comparison to isotype controls. Immunostaining signal in live tissue slices was detectable to atleast 120 μm deep for both whole antibodies and F(ab') 2 fragments using the staining procedure. This procedure revealed the expected changes in B cell organization in lymph nodes from immunized mice. Cell surface staining with most antibodies did not induce cytokine secretion, and cytokine secretion in response to T cell stimulation was unaffected by immunostaining. Staining with known a mitogenic antibody (anti-CD3) simultaneously labelled the cells and activated the tissue, confirming that reagents for live immunostaining must be selected judiciously. As a proof of concept, this method was used to reveal the dynamic distribution of CD69, a T cell activation marker, in lymph node slices before and after ex vivo stimulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tumour-induced immunosuppression plays a role in the development and progression of cancer. Of interest is the interaction between programmed death-1 and programmed death ligand-1 (PD-L1) which can be targeted through immune checkpoint blockade; however, there are limited data surrounding the composition of the immune milieu in prostate cancer. We preliminarily assessed 21 radical prostatectomies in therapy-naïve patients for immune markers and PD-L1 expression. The immune infiltrates were higher in adenocarcinoma than benign prostate (lymphocytes p<0.001, macrophages p=0.010) with 5% of cases being PD-L1 high (≥5% expression). Increased peritumoural CD68 and CD163 expression correlated with lower grade group (GG) (p=0.024 and p=0.014, respectively) with a trend towards increased CD68 expression in lower stage cases (p=0.086). There was also increased CD45 expression in lower GGs (p=0.063). We found the immune infiltrate in acinar prostate cancer to be extremely heterogeneous with an overall immunophenotype unlikely to respond to immune checkpoint blockade., Competing Interests: Competing interests: MRD has received compensation from Hoffman – La Roche and Astra Zeneca for participating on advisory boards and honoraria from Astra Zeneca. BX has received honoraria from Merck., (© Author(s) (or their employer(s)) 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

Chronic psychological stress is associated with accelerated biological aging, immune dysfunction, and premature morbidity and mortality. Changes in the relative proportions of T cell subpopulations are thought to be a characteristic of immunological aging; however, understanding of whether these changes are associated with chronic psychological stress is incomplete. This study investigated associations between chronic caregiving stress and distributions of T cell phenotypes in a sample of high stress mothers of children with Autism Spectrum Disorder (caregivers; n = 91) and low stress mothers of neurotypical children (controls; n = 88). Immune markers assessed were naïve (CD45RA + CD62L+), central memory (CD45RA-CD62L+), and effector memory (CD45RA-CD62L-) CD4+ and CD8+ T cells. We also examined the ratio of effector to naïve (E:N) CD4+ and CD8+ T cells. In models adjusted for age, body mass index, race/ethnicity, and antidepressant use, caregivers displayed higher percentages of effector memory CD8+ and CD4+ T cells as well as lower percentages of naïve CD8+ T cells and central memory CD8+ and CD4+ T cells compared to controls. Caregivers also displayed significantly higher E:N ratios for both CD4+ and CD8+ T cells. These findings were also independent of cytomegalovirus infection status. Furthermore, higher parental stress, across both groups, was related to several immune parameters. These findings provide preliminary evidence that chronic parental caregiving stress is associated with changes in relative proportions of T cell subpopulations that are consistent with accelerated immunological aging., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC) in recent years. The current study was designed to sort the minipig (mp) perivascular stem cells (PSCs) and investigate the osteogenic potential. Purification of human PSCs was achieved via fluorescence‑activated cell sorting (FACS) from human liposuction samples [cluster of differentiation (CD)45‑CD34‑CD146+ perithelial cells and CD45‑CD34+CD146‑ adventitial cells]. Subsequently, PSCs were isolated from mp adipose tissue samples (n=9), characterized and, using purified mpPSCs (obtained by FACS, which is used in human PSC purification), the mpPSC osteogenic and adipogenic potential was evaluated by Alizarin Red S and Oil Red O staining in vitro, respectively. The cell morphometry was observed following cell isolation and culture, and hematoxylin and eosin staining was performed to identify the fat tissue structure and vascular distribution. Osteogenic and adipogenic differentiation‑associated gene expression levels were analyzed by reverse transcription‑quantitative polymerase chain reaction. The results demonstrated that the same antigens used for human PSC identification and isolation were working in mp tissue (CD45, CD146 and CD34). The two cell groups: CD45‑CD34‑CD146+ pericytes and CD45‑CD34+CD146‑ adventitial cells were successfully isolated from the subcutaneous fat in the posterior neck of mps, mpPSCs accounted for 8.6% of the stromal vascular fraction (SVF) with 1.4% pericytes and 7.2% adventitial cells. mpPSCs demonstrated characteristics of MSCs, including cell surface marker expression, colony forming unit‑fibroblast inclusion, and the stronger osteogenic and adipogenic differentiation potential than that of the non‑selected vascular stromal cells. The mRNA expression levels of osteocalcin, collagen, type I, α1 and peroxisome proliferator‑activated receptor‑γ in the mpPSCs group were significantly higher than those of the unsorted pSVF group (P<0.05). Thus, the current study successfully isolated and cultured CD146+ and CD34+ cell populations from mp tissues, characterized the cells' PSC‑like phenotype and identified their distinctly osteogenic and adipogenic potential.

Craniofacial defect is a critical problem in dental clinic, which has a tremendous impact on patients' quality of life. Mesenchymal stem cell-based therapy has emerged as a promising approach for tissue defect repair. However, reduced survival after mesenchymal stem cells (MSCs) transplantation remains as a major problem in this area, which hampers the outcome of regeneration. Recently, the mechanism to mobilize endogenous MSCs for tissue regeneration has received increasing attentions, as it does not require exogenous cell transplantation. The primary goal of this study was to confirm the role of intravenous substance P in mobilizing endogenous CD45 - CD11b - CD29 + MSCs in critical-sized bone defect animals and to investigate the effects of substance P on calvarial bone repair. Flow cytometry analyses revealed that intravenous substance P promoted the mobilization of endogenous CD45 - CD11b - CD29 + MSCs after bone defect. In addition, Micro-CT showed that intravenous substance P improved the outcomes of calvarial bone repair. Furthermore, we discovered that systemic injection of substance P attenuated inflammation and enhanced the survival of the local-transplanted GFP + MSCs. Our findings suggested that substance P together with its mobilized CD45 - CD11b - CD29 + MSCs helped improve calvarial defect repair through regulating inflammatory conditions and promoting the survival of local-transplanted cells.

Background: Evidence on the changes in the absolute counts of monocyte subpopulations in sepsis is missing., Methods: Firstly, absolute counts of circulating CD14 pos /HLA-DR pos /CD45 pos monocytes were measured by flow cytometry in 70 patients with Gram-negative sepsis and in 10 healthy volunteers. In the second phase, immunophenotyping was performed and the absolute count of circulating inflammatory monocytes and of circulating CD14 dim /CD16 pos /CD45 pos patrolling monocytes were measured in another 55 patients and 10 healthy volunteers. Measurements were repeated on days 3, 7, and 10. Results were correlated with survival after 28 days., Results: Greater numbers of CD14 pos /HLA-DR pos /CD45 pos monocytes were found on day 1 in survivors compared to nonsurvivors (p = 0.030). Receiver operating characteristic (ROC) analysis showed that a cutoff higher than 337 cells/mm 3 on day 1 could discriminate between survivors and nonsurvivors with a positive predictive value (PPV) of 91.1%. Logistic regression including Sequential Organ Failure Assessment (SOFA) score and Acute Physiology and Chronic Health Evaluation (APACHE) II score showed that an absolute count greater than 337 cells/mm 3 was independently associated with unfavorable outcome (odds ratio (OR) 0.19, p = 0.050). The absolute counts of inflammatory and of CD14 dim /CD16 pos /CD45 pos monocytes were greater in patients than healthy controls during the entire 10 days of follow-up. The absolute counts on day 3 of CD14 dim /CD16 pos /CD45 pos monocytes were greater in survivors than nonsurvivors (p = 0.027). ROC analysis revealed that the cutoff at 27 cells/mm 3 could discriminate between survivors and nonsurvivors with PPV of 94.1%. Logistic regression including age, SOFA score, and APACHE II score showed that an absolute count greater than 27 cells/mm 3 was independently associated with unfavorable outcome (OR 0.06, p = 0.033). Logistic regression analysis showed that intra-abdominal infection on day 1 was predictive of low CD14 dim / CD16 pos /CD45 pos count on day 3., Conclusion: Circulating counts of inflammatory and patrolling monocytes are greatly increased in Gram-negative sepsis. Absolute counts of CD14 pos /HLA-DR pos /CD45 pos monocytes on day 1 and CD14 dim /CD16 pos /CD45 pos monocytes on day 3 are independently associated with final outcome., Trial Registration: ClinicalTrials.gov, NCT01223690 . Registered retrospectively on 18 October 2010.

Hepatic progenitor cells (HPCs) play an important regenerative role in acute and chronic liver pathologies. Liver disease research often necessitates the grading of disease severity, and pathologists' reports are the current gold-standard for assessment. However, it is often impractical to recruit pathologists in large cohort studies. In this study we utilise PerkinElmer's "InForm" software package to semi-automate the scoring of patient liver biopsies, and compare outputs to a pathologist's assessment. We examined a cohort of eleven acute hepatitis samples and three non-alcoholic fatty liver disease (NAFLD) samples, stained with HPC markers (GCTM-5 and Pan Cytokeratin), an inflammatory marker (CD45), Sirius Red to detect collagen and haematoxylin/eosin for general histology. InForm was configured to identify presumptive HPCs, CD45 +ve inflammatory cells, areas of necrosis, fat and collagen deposition (p < 0.0001). Hepatitis samples were then evaluated both by a pathologist using the Ishak-Knodell scoring system, and by InForm through customised algorithms. Necroinflammation as evaluated by a pathologist, correlated with InForm outputs (r 2  = 0.8192, p < 0.05). This study demonstrates that the InForm software package provides a useful tool for liver disease research, allowing rapid, and objective quantification of the presumptive HPCs and identifies histological features that assist with assessing liver disease severity, and potentially can facilitate diagnosis.

Development of miniaturized devices for the rapid and sensitive detection of analyte is crucial for various applications across healthcare, pharmaceutical, environmental, and other industries. Here, we report on the detection of unlabeled analyte by using fluorescently labeled, antibody-conjugated microtubules in a kinesin-1 gliding motility assay. The detection principle is based on the formation of fluorescent supramolecular assemblies of microtubule bundles and spools in the presence of multivalent analytes. We demonstrate the rapid, label-free detection of CD45+ microvesicles derived from leukemia cells. Moreover, we employ our platform for the label-free detection of multivalent proteins at subnanomolar concentrations, as well as for profiling the cross-reactivity between commercially available secondary antibodies. As the detection principle is based on the molecular recognition between antigen and antibody, our method can find general application where it identifies any analyte, including clinically relevant microvesicles and proteins.

Background: This study assesses how the metastatic immune landscape is impacting the response to treatment and the outcome of colorectal cancer (CRC) patients., Methods: Complete curative resection of metastases (n = 441) was performed for two patient cohorts (n = 153). Immune densities were quantified in the center and invasive margin of all metastases. Immunoscore and T and B cell (TB) score were analyzed in relation to radiological and pathological responses and patient's disease-free (DFS) and overall survival (OS) using multivariable Cox proportional hazards models. All statistical tests were two-sided., Results: The spatial distribution of immune cells within metastases was nonuniform. Patients, as well as metastases of the same patient, had variable immune infiltrates and response to therapy. A beneficial response was statistically significantly associated with increased immune densities. Among all metastases, Immunoscore (I) and TB score evaluated in the least immune-infiltrated metastases were the strongest predictors for DFS and OS (five-year follow-up, Immunoscore: I 3-4: DFS rate = 27.9%, 95% CI = 15.2 to 51.3; vs I 0-1-2: DFS rate = 12.3%, 95% CI = 4.9 to 30.6; HR = 0.45, 95% CI = 0.28 to 0.70, P = .02; I 3-4: OS rate = 64.6%, 95% CI = 46.6 to 89.6; vs I 0-1-2: OS rate = 32.5%, 95% CI = 17.2 to 61.4; HR = 0.32, 95% CI = 0.15 to 0.66, P = .001, C-index = 65.9%; five-year follow-up, TB score: TB 3-4: DFS rate = 25.7%, 95% CI = 14.2 to 46.6; vs TB 0-1-2: DFS rate = 5.0%, 95% CI = 0.8 to 32.4; HR = 0.36, 95% CI = 0.22 to 0.57, P < .001; TB 3-4: OS rate = 63.7%, 95% CI = 46.4 to 87.5; vs TB 0-1-2: OS rate: 21.4%, 95% CI = 9.2 to 49.8; HR = 0.25, 95% CI = 0.12 to 0.51, P < .001, C-index = 67.8%). High TB score and Immunoscore patients had a median survival of 70.5 months, while low patients survived only 25.1 to 38.3 months. Nonresponding patients with high-immune infiltrates had prolonged DFS (HR = 0.28, 95% CI = 0.15 to 0.52, P = .001) and OS (HR = 0.25, 95% CI = 0.1 to 0.62, P = .001). The immune parameters remained the only statistically significant prognostic factor associated with DFS and OS in multivariable analysis (P < .001), while response to treatment was not., Conclusions: Response to treatment and prolonged survival of metastatic CRC patients were statistically significantly associated with high-immune densities quantified into the least immune-infiltrated metastasis., (© The Author 2017. Published by Oxford University Press.)

In-depth knowledge of the mechanisms induced by early postischemic cardiac endogenous mesenchymal stem cells (MSCs) in the acutely ischemic heart could advance our understanding of cardiac regeneration. Herein, we aimed to identify, isolate, and initially characterize the origin, kinetics and fate of cardiac MSCs. This was facilitated by in vivo genetic cell fate mapping through green fluorescent protein (GFP) expression under the control of vimentin induction after acute myocardial infarction (MI). Following permanent ligation of the left anterior descending coronary artery in CreER+ mTom/mGFP+ mice, vimentin/GFP+ cells revealed ischemia-responsive activation, survival, and local enrichment inside the peri-infarction border zone. Fluorescence-activated cell sorting (FACS)-isolated vimentin/GFP+ cells could be strongly expanded in vitro with clonogenic precursor formation and revealed MSC-typical cell morphology. Flow-cytometric analyses demonstrated an increase in cardiac vimentin/GFP+ cells in the ischemic heart, from a 0.6% cardiac mononuclear cell (MNC) fraction at 24 h to 1.6% at 72 h following MI. Sca-1+CD45- cells within the vimentin/GFP+ subtype of this MNC fraction increased from 35.2% at 24 h to 74.6% at 72 h after MI. The cardiac postischemic vimentin/GFP+ MNC subtype showed multipotent adipogenic, chondrogenic, and osteogenic differentiation potential, which is distinctive for MSCs. In conclusion, we demonstrated a seemingly proliferative first response of vimentin- induced cardiac endogenous MSCs in the acutely ischemic heart. Genetically, GFP-targeted in vivo cell tracking, isolation, and in vitro expansion of this cardiac MSC subtype could help to clarify their reparative status in inflammation, fibrogenesis, cell turnover, tissue homeostasis, and myocardial regeneration., (© 2019 S. Karger AG, Basel.)