Detection Of CD4 Research Articles

Abstract 2806: Progression of ductal carcinoma in situ (DCIS), is it in the immune microenvironment

Abstract Introduction: DCIS is a non-obligate precursor to invasive breast cancer (IBC). DCIS patients are treated similarly to breast cancer with surgery, often followed by radiotherapy and/or endocrine treatment. However, most DCIS lesions will never progress to IBC, indicating that overdiagnosis and overtreatment exists. DCIS lesions show variable amounts of immune cells, particularly in the periductal stroma. Immune escape might be a critical step for transition from DCIS to IBC. We aim to identify factors within the immune microenvironment of DCIS lesions that distinguish harmless from potentially hazardous DCIS. Methods: A case-control study is being conducted consisting of women with pure DCIS diagnosed between 1989-2005 with median follow-up of 12 years, treated with breast conserving surgery only. Cases are defined as women with DCIS developing subsequent ipsilateral breast cancer (iIBC), controls as women with DCIS without subsequent iIBC. Multispectral immunohistochemical imaging was performed on primary DCIS lesions, aiming at detection of CD20+ B-cells, CD8+ T-cells, CD3+ T-cells, CD3+Foxp3+ regulatory T-cells, and CD68+ macrophages. Density of immune cell subsets in cells/mm2, immune cell ratios and spatial relationships were calculated for 27 cases and 28 controls. These immune cell related factors were correlated to outcome and integrated with RNAseq data of pure microdissected DCIS. We performed gene set enrichment analysis on the correlation between DCIS gene expression and density of immune cell types with sample permutation (flexgsea R package). Results: Stromal lymphocyte, B-cell, CD8+ T-cell, regulatory T-cell and macrophage density did not significantly differ between cases and controls. Immune cell composition (CD8+ T-cell/lymphocyte, CD8+ T-cell/CD3+Foxp3+ regulatory T-cell and CD20+/lymphocyte ratio) and fraction of regulatory T-cells in close proximity of a CD8+ T-cell did not differ between cases and controls. We find a negative association between stromal B-cell density and DCIS gene expression of estrogen receptor (ESR1) targets. Higher stromal T-cell density was associated with proliferation and expression of genes characteristic for luminal B and basal-like subtypes. Furthermore, higher density of specific immune cell subsets within the DCIS compartment was associated with several immune and cancer pathways. Conclusion: A first set of analyzed DCIS cases (n=27) and controls (n=28) show no significant differences regarding immune cell density, composition and spatial relationships. Considering the entire group of DCIS patients (n=55), a negative association between stromal B-cell density and gene expression of ESR1 targets was found. Higher density of lymphocytes was associated with proliferation and expression of genes characteristic for luminal B and basal-like subtypes. The full set of 175 DCIS lesions will be presented at AACR Annual Meeting 2019. Citation Format: Mathilde M. Almekinders, Lindy Visser, Bram Thijssen, Rianne van der Linden, Charlotte van Rooijen, Petra Kristel, Annegien Broeks, Tycho Bismeijer, Lodewyk Wessels, Erik Hooijberg, Karin de Visser, Esther Lips, Jelle Wesseling, on behalf of the PRECISION team (PREvent ductal Carcinoma In Situ Invasive Overtreatment Now). Progression of ductal carcinoma in situ (DCIS), is it in the immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2806.

Abstract B055: Enhanced detection of T-cells targeting unique neoantigens and shared mutated oncogenes for personalized cancer immunotherapy

Abstract Adoptive cell transfer (ACT) of selected tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T-cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T-cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of tumor-neoantigen reactive TILs in epithelial cancers could be enhanced by enriching T-cells that express PD-1 and/or T-cell activation markers (CD134, CD137) followed by microwell culturing at limiting dilution cell concentrations to avoid overgrowth of non-reactive T-cells. Using this approach, in six patients with metastatic epithelial cancer including stomach, colon pancreatic and ovarian cancers, this method led to the detection of CD4 and CD8 T-cells targeting 19 neoantigens compared to only eight neoantigens recognized using our standard TIL fragment screening approach. In two patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of five neoantigen reactive-TCRs against five different mutations from one patient and a highly potent MHC-II-restricted KRASG12V reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated three MHC class-II-restricted TCRs targeting the TP53G245S “hot-spot” mutation. In conclusion, this approach provides a highly sensitive and specific platform to isolate clinically relevant neoantigen-reactive T-cells or their TCRs for cancer treatment. Citation Format: Rami Yossef, Eric Tran, Alena Gros, Drew Deniger, Gal Cafri, Steven A. Rosenberg. Enhanced detection of T-cells targeting unique neoantigens and shared mutated oncogenes for personalized cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B055.

Expression of Angiopoietin and VEGF in Cervical Cancer and its Clinical Significance.

ObjectiveThe aim of this study was to evaluate the expression of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) in cervical cancer and its clinical significance.MethodsImmunohistochemical assay was used to examine the expression of Ang-1/2 and VEGF in tumor tissue from 56 cervical squamous cell carcinoma patients treated with operation only (SCC-O group), as well as 51 subjects with cervical squamous cell carcinoma treated with neoadjuvant radiotherapy (SCC-RCO group, n=28) or neoadjuvant chemotherapy (SCC-CO group, n=23). Both microvessel density (MVD) and lymphatic vessel density (LVD) were examined in the three groups through detection of CD34 and D2-40 expression in respective tissue samples.ResultsWith the progression of cervical cancer, the positive expression scores of Ang-2 and VEGF were significantly increased (p<0.05). Compared with surgical intervention, neoadjuvant chemoradiotherapy significantly reduced the positive expression scores of Ang-1, Ang-2, and VEGF in cervical cancer tissues (p<0.05). The MVD values of the SCC-CO and SCC-RO groups were significantly reduced as compared to the SCC-O group (p<0.05). Similarly, the LVD values of the SCC-CO and SCC-RO groups were also significantly reduced when compared to those of the SCC-O group (p<0.05). However, LVD values of the SCC-CO and SCC-RO groups were not statistical different (p>0.05).ConclusionAng-1, Ang-2 and VEGF may play an important role in the development of cervical cancer. Mutual synergism of Ang-2 and VEGF demonstrated a close relationship with the generation of cervical blood and lymphatic vessels. Cervical cancer radiotherapy and chemotherapy could significantly inhibit the formation of blood vessels and lymphatic vessels in tumor tissue.

Abstract 2667: Patient-derived lymphoma cell lines and high-throughput screening enable identification of potential cytotoxic therapeutic agents for dogs and cats with lymphoma

Abstract Treatment success in lymphoma, the most common hematopoietic cancer seen in dogs and cats, continues to be elusive. The clinical arsenal is limited to a few conventional chemotherapeutic drug protocols that result in short-term responses. In this study, dog and cat patient-derived tumor cells were tested via high-throughput screening (HTS) against a pool of over 100 FDA-approved compounds for human cancer treatment as a means to identify potential treatment strategies that can be relatively easily adapted to the dog and cat, as well as over 500 protein kinase inhibitors as a means to identify potential oncogenic driver pathways. Cells from lymph node fine needle aspirates, pleural effusions, or peripheral blood mononuclear cells from over 20 lymphoma patients were placed in cell culture media with and without growth factors. Cells from two cases, ZS15 from a Boxer dog and MT16 from a domestic shorthair cat, have continued to expand for over 12 and 6 months, respectively. Characterization by flow cytometry for detection of CD3 and CD21 identified these patient-derived cells to be T-cell lymphocytes. HTS and follow-up dose response curve (DRC) assays were performed, identifying multiple drugs effective in inhibiting growth of MT16 and ZS15 cells at nanomolar concentrations in vitro; these included anthracenedione/anthracycline-class and actinomycin-class drugs, as well as mRNA synthesis, microtubule/spindle formation, proteasome, histone deacetylase, or insulin growth factor-1 receptor inhibitors, many of which are currently not used in veterinary species. Notably, ZS15 cell growth was inhibited by mammalian target of rapamycin (mTOR) inhibitors, corroborating previous exome sequencing data of Boxer T-cell lymphomas that identified recurrent driver mutations in the PTEN-mTOR pathway. In addition, many of these drugs showed much higher IC50s in HTS and DRC assays on primary canine and feline fibroblasts, used as surrogates for normal tissue, revealing the specificity of their action on the cancer cells. These results advocate use of HTS and DRC assays as a means for identification of oncogenic driving mechanisms and for identification of drugs novel to veterinary medicine that warrant further investigation as alternative treatments for lymphoma in companion animals, which in turn can serve as valuable and accessible models for human lymphomas. Citation Format: Garrick M. Moll, Vilma Yuzbasiyan-Gurkan. Patient-derived lymphoma cell lines and high-throughput screening enable identification of potential cytotoxic therapeutic agents for dogs and cats with lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2667.

Development of Fe3O4/Ag core/shell-based multifunctional immunomagnetic nanoparticles for isolation and detection of CD34+ stem cells

ABSTRACTFe3O4/Ag core/shell nanoparticles functionalized with the free amino (NH2) functional groups (Fe3O4/Ag-NH2) were conjugated with fluorescent electron coupled dye (ECD)-antiCD34 antibody using the 1-ethyl-3-(3′-dimethyl-aminopropyl) carbodiimide (EDC) catalyst (ECD – Electron Coupled Dye or R Phycoerythrin-Texas Red is a fluorescent organic dye attached to the antibody). The characteristic fluorescence of ECD in the antibody was investigated and was used as a good indicator for estimating the percentage of the antibodies that were successfully conjugated with the nanoparticles. The conjugation efficiency was found to increase depending on the VNP:VAB ratio, where VNP and VAB are the volumes of the nanoparticle solution (concentration of 50 ppm) and the as-purchased antibody solution, respectively. The conjugation efficiency rapidly increased from approximately 18% to approximately 70% when VNP:VAB was increased from 2:1 to 100:1, and it gradually reached the saturated state at an efficiency of 95%, as the VNP:VAB was equal to 300:1. The bioactivity of the abovementioned conjugation product (denoted by Fe3O4/Ag-antiCD34) was evaluated in an experiment for the collection of stem cells from bone marrow samples.

Global Assessment of Dengue Virus-Specific CD4+ T Cell Responses in Dengue-Endemic Areas.

Dengue is a major public health problem worldwide. Assessment of adaptive immunity is important to understanding immunopathology and to define correlates of protection against dengue virus (DENV). To enable global assessment of CD4+ T cell responses, we mapped HLA-DRB1-restricted DENV-specific CD4+ T cell epitopes in individuals previously exposed to DENV in the general population of the dengue-endemic region of Managua, Nicaragua. HLA class II epitopes in the population of Managua were identified by an in vitro IFNγ ELISPOT assay. CD4+ T cells purified by magnetic bead negative selection were stimulated with HLA-matched epitope pools in the presence of autologous antigen-presenting cells, followed by pool deconvolution to identify specific epitopes. The epitopes identified in this study were combined with those previously identified in the DENV endemic region of Sri Lanka, to generate a "megapool" (MP) consisting of 180 peptides specifically designed to achieve balanced HLA and DENV serotype coverage. The DENV CD4MP180 was validated by intracellular cytokine staining assays. We detected responses directed against a total of 431 epitopes, representing all 4 DENV serotypes, restricted by 15 different HLA-DRB1 alleles. The responses were associated with a similar pattern of protein immunodominance, overall higher magnitude of responses, as compared to what was observed previously in the Sri Lanka region. Based on these epitope mapping studies, we designed a DENV CD4 MP180 with higher and more consistent coverage, which allowed the detection of CD4+ T cell DENV responses ex vivo in various cohorts of DENV exposed donors worldwide, including donors from Nicaragua, Brazil, Singapore, Sri Lanka, and U.S. domestic flavivirus-naïve subjects immunized with Tetravalent Dengue Live-Attenuated Vaccine (TV005). This broad reactivity reflects that the 21 HLA-DRB1 alleles analyzed in this and previous studies account for more than 80% of alleles present with a phenotypic frequency ≥5% worldwide, corresponding to 92% phenotypic coverage of the general population (i.e., 92% of individuals express at least one of these alleles). The DENV CD4 MP180 can be utilized to measure ex vivo responses to DENV irrespective of geographical location.

Programmed death 1 is highly expressed on CD8+ CD57+ T cells in patients with stable multiple sclerosis and inhibits their cytotoxic response to Epstein-Barr virus.

Growing evidence points to a deregulated response to Epstein-Barr virus (EBV) in the central nervous system of patients with multiple sclerosis (MS) as a possible cause of disease. We have investigated the response of a subpopulation of effector CD8+ T cells to EBV in 36 healthy donors and in 35 patients with MS in active and inactive disease. We have measured the expression of markers of degranulation, the release of cytokines, cytotoxicity and the regulation of effector functions by inhibitory receptors, such as programmed death 1 (PD-1) and human inhibitor receptor immunoglobulin-like transcript 2 (ILT2). We demonstrate that polyfunctional cytotoxic CD8+ CD57+ T cells are able to kill EBV-infected cells in healthy donors. In contrast, an anergic exhaustion-like phenotype of CD8+ CD57+ T cells with high expression of PD-1 was observed in inactive patients with MS compared with active patients with MS or healthy donors. Detection of CD8+ CD57+ T cells in meningeal inflammatory infiltrates from post-mortem MS tissue confirmed the association of this cell phenotype with the disease pathological process. The overall results suggest that ineffective immune control of EBV in patietns with MS during remission may be one factor preceding and enabling the reactivation of the virus in the central nervous system and may cause exacerbation of the disease.

Acute Leukemia in Horses.

Leukemia is broadly divided into acute and chronic lymphocytic and myeloid types based on the proportion of blasts, morphology of cells, and expression of specific antigens on neoplastic cells. Classifying leukemia in horses can be challenging if blasts predominate and since few antibodies to identify cell types are available. The objective of this study was to describe in detail the clinical and pathologic features of acute leukemia in horses. Twelve horses ranging from 0.2 to 25.9 years of age were diagnosed with acute leukemia. Six cases were classified as acute lymphocytic leukemia (ALL) based on predominance of blasts, lack of granulocytic or monocytic differentiation, and detection of CD3, CD20, and/or CD79a antigens by immunohistochemistry. Six other cases were classified as acute myeloid leukemia (AML) with myelomonocytic ( n = 4), basophilic ( n = 1), and eosinophilic ( n = 1) differentiation based on > 20% bone marrow blasts and partial leukocytic differentiation. Reactivity with antibodies to Iba-1/AIF-1, CD172a, and CD163 was determined for all cases of AML. Eleven horses had thrombocytopenia, 10 had neutropenia, 8 had anemia, all had blasts on blood films, and none had leukocytosis. Ten horses had increased serum acute phase proteins. Bone marrow cellularity ranged from 30% to 100%, and the proportion of blasts ranged from 80% to 100% and 30% to 60% in ALL and AML, respectively. Horses were severely ill at diagnosis and euthanized within days or weeks. Unique features of acute leukemia in horses compared to other species were variable lymphocyte antigen expression (ALL) and frequent inflammation (ALL and AML).

Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells

In centralized immune monitoring for a multi-center allergen immunotherapy trial, we observed frequent loss of CD4+ T cell integrity following staining of cultured PBMCs with our regulatory T cell flow cytometry panel. Samples were marked by a loss of total cellular events, altered scatter properties, and reduced CD3+CD4+ events. This occurred only in samples that were stained with Foxp3 and were therefore treated with Foxp3 fixation-permeabilization buffer. We identified granulocyte contamination in samples associated with a loss of integrity, and went on to test the impact of granulocyte depletion on day-old blood samples. Granulocyte depletion prevented loss of cell integrity and CD3+CD4+ events, and reduced variability in detection of Foxp3+ cells. Addition of purified neutrophils back to PBMCs altered scatter properties and detection of CD4+ T cells. Implementation of a granulocyte depletion step in our standard operating protocols has reduced assay failure due to loss of sample integrity from 31% to 0%. Routine incorporation of a granulocyte depletion step during PBMC isolation is recommended prior to downstream immune monitoring in blood with next-day processing.

Identification of the Immunological Profile in Some Egyptian Children with β- Thalassaemia Major Under Different Treatment Modalities in El Minia Region

Abstract Background: β-thalassaemia major is one of the chronic hemolytic anemias resulting from defect in β-globin chain. It requires frequent blood transfusion plus other treatment modalities. These treatment modalities may be associated with certain immunologic modulations. Objective: To assess the immunity status in children with β-thalassaemia major under different treatment regimens within El Minia, Egypt. Subjects and Methods: One hundred forty-four children were enrolled and classified into four groups. Thirty-six β-thalassaemia patients treated only with blood transfusion (group I). Thirty-six patients treated with transfusion and iron chelation (group II). Thirty-six patients treated with transfusion, iron chelation and subjected to splenectomy (group III). Group IV involved thirty-six apparently healthy age and sex matched children. CBC plus serum levels of ferritin, IgA, complement C3 and C4 were measured along with detection of CD3+, CD4+, CD8+, CD19+ and CD56+ lymphocyte percentages and absolute counts. Results: IgA levels were significantly higher in thalassaemia patients compared to controls (p<0.001) plus highly significant increase in IgA levels in splenectomized patients than non-splenectomized (p<0.001). Levels of C3 were significantly decreased in all patients compared with controls (p=0.001) with a highly significant decrease in C3 levels in splenectomized patients than non splenectomized ones (p<0.001) but no statistical difference between their C4 levels. Significant statistical differences were revealed regarding CD3+, CD4+ and CD8+ T lymphocyte percentages within thalassaemia groups when compared to each other’s and to controls. Splenectomized patients had higher significant levels regarding serum ferritin (p=0.02) along with CD3+ (p=0.05), CD4+ (p=0.05) and CD8+ (p=0.037) lymphocyte percentages compared to non-splenectomized. CD19+ lymphocyte percentages were significantly higher while CD56+ lymphocyte percentages were significantly lower in all patients compared with controls (p=0.02 and 0.05). Conclusion: Immune modulation occurs in thalassaemia patients with regional specific variations and is related to variations in treatment modalities.

PD-L1 expression in basaloid squamous cell lung carcinoma: Relationship to PD-1+ and CD8+ tumor-infiltrating T cells and outcome

PD-1/PD-L1 inhibitors demonstrated durable clinical responses in patients with lung squamous cell carcinoma. However, the expression pattern of PD-L1 and the presence of CD8+ and PD-1+ tumor-infiltrating T cells in the basaloid variant of squamous cell carcinoma remain unknown. immunohistochemistry analysis of PD-L1 expression, with three recently validated monoclonal antibodies used in clinical trials (clones SP142, SP263, and 28-8), and detection of CD8+ and PD-1+ tumor-infiltrating T cells was performed on whole-tissue sections from 56 patients following surgery for basaloid squamous cell carcinoma. Data were correlated to clinicopathological parameters and outcome. Fair to poor concordance was observed between the SP142 vs SP263 clones, and SP142 vs 28-8 (κ range, 0.018–0.412), while the 28-8 and SP263 demonstrated a strong correlation in both the tumor cell and immune cell compartments (κ=0.883, and κ=0.721). Expression of PD-L1 correlated with a high content of CD8+ and PD-1+ tumor-infiltrating T cells when using SP142 (P=0.012; P=0.022), but not with SP263 or 28-8 (P=0.314; P=0.611). In the multivariate analysis, we found significantly better disease-free and overall survival rates for high PD-L1 expression with SP142, CD8+ and PD-1+ tumor-infiltrating T cells (P=0.003; P=0.007). No significant prognosis value was observed for SP263 and 28-8 clones, except a correlation between improved overall survival and SP263 in the univariate analysis (P=0.039), not confirmed in the multivariate model. In conclusion, we report that the expression of PD-L1 and the content of CD8+ and PD-1+ tumor-infiltrating T cells is an independent indicator of better outcome in basaloid squamous cell carcinoma patients, although the observed effect is dependent on the PD-L1 immunohistochemistry assay.

Abstract B081: Spatially-resolved, multiplexed (up to 800 plex) digital characterization of protein and mRNA abundance in FFPE tissue sections: Application to immuno-oncology

Abstract The Tumor Microenvironment (TME) has emerged as a key compartment that determines the overall effectiveness of cancer immunotherapy. Hence, it is very important to determine the abundance and location of key immune-regulators in the TME. Historically, standard immunohistochemistry (IHC) and immunofluorescence have been used to assess spatial heterogeneity of proteins and nucleic-acids in tissue slices. However, these techniques are inherently limited in utility because it has been difficult to quantify the abundance of multiple protein/nucleic-acids across a wide dynamic range. Here, we report the development and validation of a spatially-resolved protein and RNA detection platform with the potential to simultaneously quantify up to 800 targets with greater than 5 log10 of dynamic range from a single formalin-fixed paraffin-embedded (FFPE) slide. We demonstrate validation of this technology by characterization of a panel of immune proteins expressed in colorectal cancer samples, and we also demonstrate spatially resolved detection of RNA. In situ high-plex digital molecular profiling is enabled by the use of UV-photocleavable small indexing DNA-oligo tags that can be delivered to the target within the tissue via direct attachment to RNA binding probes or conjugation to primary antibodies and are quantified with the standard nCounter technology. A slide-mounted FFPE tissue section is bound with a multiplexed cocktail of oligo-labeled primary antibody or mRNA hybridization probes, and a microfluidic flow cell is attached to the slide. Low-plex (3 or 4 color fluorescence) visible wavelength probes are utilized to generate an overall view of the FFPE tissue slice morphology (e.g., nuclear staining probes, select antibody pairs such as anti-CD3 and anti-CD8). Using the visible wavelength morphology as a guide, regions of interest (ROI) in the tumor are identified (e.g., areas with tumor infiltrating leukocytes) and then sequentially illuminated with UV light to release the indexing-oligos off all the high-plex molecular profiling reagents. Using this approach and standard microscope instrumentation, the limits of detection enable near single cell resolution. Following each UV illumination cycle, the photocleaved indexing-oligos are released into the buffer-layer above the tissue slice, collected via microcapillary aspiration, and stored in an individual well of a microtiter-plate. The contents of each well can then be referenced back to the exact region of tumor that was illuminated by UV light. Oligos are then hybridized to the nCounter fluorescently labeled optical barcodes to permit ex-situ digital counting of as many as 800 different analytes localized within a single ROI in the tumor. As demonstration of the technology, simultaneous multiplexed detection of CD3, CD8, CD45R0, CD4, CD45, PD-1, PD-L1, Vista, TIM-3, B7-H3, Ki67 (plus additional key IO-targets) will be quantified from colorectal tumor biopsies using oligo-conjugated primary antibodies. Furthermore, we will demonstrate detection of key IO associated immune RNA targets using direct hybridization of oligo-labeled probes. The ability to measure DNA, RNA, and protein at up-to 800-plex from single slices of FFPE tissue may enable the discovery of key immune biomarkers in tumors and accelerate the development immunotherapy and their associated companion diagnostics. Citation Format: Dwayne Dunaway, Jaemyeong Jung, Chris Merritt, Isaac Sprague, Philippa Webster, Sarah Warren, Joseph Beechem. Spatially-resolved, multiplexed (up to 800 plex) digital characterization of protein and mRNA abundance in FFPE tissue sections: Application to immuno-oncology [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B081.

Notch1 is associated with the differentiation of human bone marrow‑derived mesenchymal stem cells to cardiomyocytes.

Notch signaling is involved in the early process of differentiation to determine the fate of stem cells. However, the precise role of Notch in human bone marrow-derived mesenchymal stem cells (hBMSCs) remains unclear. The present study aimed to investigate the involvement of Notch signalling during the course of hBMSC differentiation into cardiomyocytes using hBMSCs, with multilineage differentiation ability, isolated and purified from human bone marrow. Flow cytometric analysis revealed that CD29, CD44 and CD90 were highly expressed on the surface of cells in their fifth passage, whereas detection of CD34, CD45, CD54 and HLA-DR was negative. Visualization of morphological changes, western blotting, immunocytochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) demonstrated that hBMSCs differentiate into cardiomyocytes through treatment with 5-azacytidine (5-aza). Transmission electron microscopy revealed ultramicroscopic details of differentiated hBMSCs. Western blotting and immunocytochemistry demonstrated increased protein expression levels of α-actin and cardiac troponin T expression, and RT-qPCR revealed increased mRNA expression of Notch1 early in the process of differentiation (days 1, 4 and 7), and increased mRNA expression levels of the transcription factors GATA binding protein-4 and NK2 homeobox 5 at day 28 day. In conclusion, differentiation of hBMSCs into cardiomyocytes was induced in vitro by 5-aza, and was associated with upregulation of Notch1, GATA binding protein-4 and Nkx2.5 expression. Overexpression of the Notch1 signaling pathway may represent a potential mechanism underlying the differentiation of hBMSCs.

Primary cutaneous B‐cell lymphoma other than marginal zone: clinicopathologic analysis of 161 cases: Comparison with current classification and definition of prognostic markers

Categorization of primary cutaneous B‐cell lymphomas (PCBCL) other than marginal zone (MZL) represents a diagnostic challenge with relevant prognostic implications. The 2008 WHO lymphoma classification recognizes only primary cutaneous follicular center cell lymphoma (PCFCCL) and primary cutaneous diffuse large B‐cell lymphoma, leg type (PCDLBCL‐LT), whereas the previous 2005 WHO/EORTC classification also included an intermediate form, namely PCDLBCL, other. We conducted a retrospective, multicentric, consensus‐based revision of the clinicopathologic characteristics of 161 cases of PCBCL other than MZL. Upon the histologic features that are listed in the WHO classification, 96 cases were classified as PCFCCL and 25 as PCDLBCL‐LT; 40 further cases did not fit in the former subgroups in terms of cytology and/or architecture, thus were classified as PCDLBCL, not otherwise specified (PCDLBCL‐NOS). We assigned all the cases a histogenetic profile, based on the immunohistochemical detection of CD10, BCL6, and MUM1, and a “double hit score” upon positivity for BCL2 and MYC. PCDLBCL‐NOS had a clinical presentation more similar to PCFCCL, whereas the histology was more consistent with the picture of a diffuse large B‐cell lymphoma, as predominantly composed of centroblasts but with intermixed a reactive infiltrate of small lymphocytes. Its behavior was intermediate between the other two forms, particularly when considering only cases with a “non‐germinal B‐cell” profile, whereas “germinal center” cases resembled PCFCCL. Our data confirmed the aggressive behavior of PCDLBC‐LT, which often coexpressed MYC and BCL2. The impact of single factors on 5‐year survival was documented, particularly histogenetic profile in PCDLBCL and BCL2 translocation in PCFCCL. Our study confirms that a further group—PCDLBCL‐NOS—exists, which can be recognized through a careful combination of histopathologic criteria coupled with adequate clinical information.

Neuroprotection by intravenous transplantation of bone marrow mononuclear cells from 5-fluorouracil pre-treated rats in a model of ischemic stroke

Our previous studies showed that bone marrow mononuclear cells (BMMNCs) from 5-fluorouracil (5-FU) pre-treated rats (named BMRMNCs) had a better therapeutic efficacy in ischemia/reperfusion rats as compared to BMMNCs from untreated rats. This study was undertaken to further explore the potential mechanisms underlying the neuroprotective effects of BMRMNCs in the same model. Rats were intravenously pre-treated with 5-FU, and BMRMNCs were collected 7 days later and subjected to flow cytometry for detection of CD34, CD45 and CD90. Middle cerebral artery occlusion (MCAO) was induced in rats, and BMMNCs and BMRMNCs were independently transplanted via the tail vein at 24 h after MCAO. NISSL staining was performed 14 days after cell transplantation and the viable cells in the hippocampus were counted. Stromal cell-derived factor 1 (SDF-1) mRNA expression was detected in the penumbra at 7 and 14 days after treatment. The contents of pro-inflammatory cytokines and growth factors as well as microvessel density (MVD) were determined at 14 days. Results showed more BMRMNCs were positive for CD34, CD45 and CD90. After transplantation, more viable cells were observed in the hippocampus of BMRMNCs treated rats. In addition, BMRMNCs transplantation significantly increased MVD, reduced pro-inflammatory cytokines and raised growth factors in the penumbra. However, the SDF-1 mRNA expression was comparable between BMRMNCs group and BMMNCs group. Our results indicate that BMRMNCs are likely to more effectively improve the local microenvironment to increase viable cells and elevate angiogenesis, exerting neuroprotective effects on cerebral ischemia in rats.

Comparison of three different flow cytometers in the clinical assessment of lymphocyte subsets

Objective To evaluate the consistency and accuracy among 3 brands of flow cytometers(BriCyte E6, BD FACSCanto Ⅱ and Beckman Coulter FC 500) in the detection of lymphocyte subsets. Methods According to the methodology, the BriCyte E6 was compared with 2 flow cytometers commonly used in clinical detection. Seventy-three cases (40 male and 33 female) of anticoagulation peripheral blood specimens were collected in the clinical laborartory department of Xinhua Hospital in July 2015 and the percentage (%) and absolute number (#) of the lymphocyte subsets were detected by 3 different flow cytometers within samples collected 4 h. Results There were good consistency among the 3 flow cytometers (R2>0.95, R2 from 0.969 5 to 0.992 4) in the detection of lymphocyte subsets percentage, so did in the detection of absolute number (R2>0.95, R2 from 0.969 1 to 0.993 3). As to the precision evaluation, in the detectionof CD8%, T#, CD4+ T# and CD8+ T#, BriCyte E6 achieved a low CV% compared with FACSCanto Ⅱ and FC 500 (Friedman statistics are 16.720, 11.840, 15.760 and 15.430, P=0.000 2, 0.027, 0.000 4, 0.000 4, respectively). In the detection of T%, CD4%, NK%, B%, NK#, B#, there was no significant difference among the 3 flow cytometers (Friedman statistics are 4.242, 3.916, 0.852, 2.595, 1.835 and 0.578, P=0.119 9, 0.141 2, 0.653 2, 0.273 3, 0.399 6, 0.749 0, respectively). Conclusions The 3 flow cytometers have a good consistency in the detection of lymphocyte subsets. BriCyte E6 may be an alternative or complement of existing flow cytometers.(Chin J Lab Med, 2016, 39: 361-365) Key words: Flow cytometry; Comparative study; Lymphocyte subsets; Diagnostic equipment

The immunotoxic effects of short term chronic exposure to Titanium Dioxide Nanoparticles on spleen of adult albino rats and the role of after toxic effect follow up

Objective: The usage of Titanium Dioxide NanoParticles (TiO2NPs) on a large scale of applications was a reason of a variety of many health problems. The aim of the current study was to evaluate the immune toxic effects of short term administration of TiO2NPs on spleen. Material and Methods: Forty Adult male Rats were equally divided into four groups as follows: Group I: negative control, Group II: positive control, Group III: received TiO2NPs (1200 mg/kg) orally daily for 12 weeks. Group IV: follow up group received TiO2NPs by the same dose, route and for the same duration as TiO2NPs group and then they were left untreated for another 8 weeks. Total leukocytes, differential leukocytic counts, Interleukins IL-2 and IL-10 levels were measured, the spleen sections were examined immunohistochemically for the detection of CD4+ and iNOS expressing cells. Histopathological alterations in the spleen were also evaluated. Moreover, DNA damage was evaluated by comet assay. The results: TiO2NPs exposure for 12 weeks resulted in significant decreased in the total and deferential leukocytic counts, serum interleukins IL-2 as well as IL-10. It caused marked decrease in CD4+T-lymphocytes and increase in iNOS expressing cells indicating oxidative stress in spleen tissues. It also caused histopathological disruption of spleen architecture and produced DNA damage in splenocytes. Discontinuation of TiO2NPs administration for 8 weeks resulted in significant improvement of leukocytes, interleukins, increase in CD4+ T-lymphocytes and decrease in iNOS expressing cells in spleen tissues. Moreover, there was moderate improvement in histopathological alterations and DNA damage. Conclusion:TiO2NPs consumption have immunotoxic effects which may resulted from genetic damage and oxidative stress in the spleen of adult male albino rats which could be improved by its discontinuation for a period of time and it was recommended to increase the period of discontinuation as complete improvement may occur

Development and characterization of monoclonal antibodies against goose CD3ɛ extracellular domain and their application in detection of CD3+ T lymphocytes

CD3 is one of the most important cell surface markers of T lymphocytes, and has an important function in signal transmission during antigen recognition. In this study, monoclonal antibodies against goose CD3ɛ extracellular domain were developed and characterized for the first time. The purified recombinant goose CD3ɛ extracellular domain protein and pcDNA3.1-GoCD3ɛex were used as immunogens to develop mAbs. Furthermore, recombinant protein was used to screen for mAbs. The characteristics of mAbs were identified through IFA, I-ELISA, Western blot, FCM, and LSCM analyzes. Results showed that mAbs 3C11 and 5A3 may be good candidates for use in detecting goose T lymphocytes and in immunoassaying and exploring the function of the CD3ɛ molecule.

Effect of highly active anti-retroviral therapy on reducing HIV/AIDS related death in Hebei, 1989-2013

To investigate the effect of highly active anti-retroviral therapy (HAART) on reducing HIV/AIDS related death. The analysis was conducted by using the data of 4,148 HIV/AIDS cases reported in Hebei province from 1989 to 2013. Regular follow-up, CD4 detection, registration of death were carried out for them. Free HAART has been provided to people living with HIV/AIDS who met the treatment requirement since 2003. Of 4,148 HIV/AIDS cases, 12,451.48 person years were observed, 968 cases died due to all registered death causes. The death density was 7.77/100 person years. The death density was 2.87/100 person years for the HIV/AIDS cases receiving HAART, and 16.58/100 for the HIV/AIDS cases receiving no HAART. In 1,894 AIDS cases, a total of 4,774.48 person years were observed from onset to death, 581 cases died due to all registered death causes, and the death density was 121.69/100 person years. The death density was 4.77/100 person years for the cases receiving HAART, and 125.92/100 person years for the cases receiving no HAART. In the cases with CD less than 200/mm3, the death density was 22.9/100 person years for those receiving no HAART and 5.3/100 person years for those receiving HAART. The annual analysis found that the death rate due to all registered death causes declined as the increase of HAART coverage in people living with HIV/AIDS. The expanding of HAART coverage in people infected with HIV can reduce death rate among them. Further expanding of HAART can effectively reduce the death among people living with HIV/AIDS.

Significance of CD34 expression in thymic tumor

Objective To investigate the expression of CD34 and its clinical significances in different types of thymic tumor. Methods The expressions of CD34 in 61 thymic tumor tissues were detected by SP immunohistochemical method, then, the MVD was calculated. The patients included 7 cases of type A, 9 cases of type AB, 16 cases of type B1, 10 cases of type B2, 10 cases of type B3 and 9 cases of type C according to 2004 WHO Classification of thymic tumor. Results CD34 expression level was significantly increased from type A to type C. The MVDs were 3.78±2.12, 5.72±2.79, 7.51±3.34, 10.89±5.10, 12.31±4.08 and 16.51±6.24 in A, AB, B1, B2, B3 and C type of thymic tumor (F=2.048, P=0.047). The expression level in B2+B3+C type was significantly higher than A+AB+B1 type (t=6.034, P<0.05). Conclusions The expression of CD34 in different types of thymic tumor suggests that it may take some action in tumor-associated angiogenic function. Detection of CD34 may be useful to evaluate malignancy of thymic tumor. Key words: Thymic tumor; CD34; Immunohistochemistry