Cell-specific Antibodies Research Articles

MP61-03 A BIOFUNCTIONAL VASCULAR SCAFFOLD FOR REPLACING SMALL-DIAMETER BLOOD VESSELS

You have accessJournal of UrologyTransplantation & Vascular Surgery: Renal Transplantation & Vascular Surgery I1 Apr 2018MP61-03 A BIOFUNCTIONAL VASCULAR SCAFFOLD FOR REPLACING SMALL-DIAMETER BLOOD VESSELS Young Min Ju, Ickhee Kim, John Jackson, Anthony Atala, James Yoo, and Sang Jin Lee Young Min JuYoung Min Ju More articles by this author , Ickhee KimIckhee Kim More articles by this author , John JacksonJohn Jackson More articles by this author , Anthony AtalaAnthony Atala More articles by this author , James YooJames Yoo More articles by this author , and Sang Jin LeeSang Jin Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1982AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Disease of the renal vascular system has resulted in a clear clinical need for the development of functional vascular substitutes. We have previously fabricated a vascular scaffold by electrospinning of poly(e-caprolactone) (PCL) combined with collagen. In this study, we hypothesized that multiple bioconjugation of endothelial progenitor cells (EPC)/endothelial cell (EC) specific antibodies and anti-thrombogenic agents onto the vascular scaffold could facilitate in situ endothelialization while preventing blood clotting. METHODS Vascular scaffolds were fabricated by electrospinning of poly(e-caprolactone) (PCL) combined with collagen. Bioconjugation of endothelial progenitor cells (EPC)/endothelial cell (EC) specific antibodies and anti-thrombogenic agents were performed onto the vascular scaffold. The bioconjugated scaffolds were implanted in a sheep model of vascular defect. RESULTS The biofunctionalized scaffolds conjugated with EPC/EC-specific antibodies and heparin were able to achieve effective EPC/EC capturing and anti-thrombogenesis. In vivo experiment showed that these vascular scaffolds maintained a high degree of patency and structural integrity without eliciting a histologic inflammatory response over the course of 6-month period in sheep. Moreover, the matured EC coverage on the lumen and smooth muscle layer were observed at 6 months after implantation. CONCLUSIONS We demonstrate that the generation of the biofunctional vascular scaffold, along with existing tools for selection and directed immobilization of EPC/EC-specific antibodies and anti-thrombogenic agent, could provide the necessary components for successful tissue engineering of small diameter blood vessels in the renal vascular system. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e824 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Young Min Ju More articles by this author Ickhee Kim More articles by this author John Jackson More articles by this author Anthony Atala More articles by this author James Yoo More articles by this author Sang Jin Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The dynamic development of germ cells during chicken embryogenesis

Appropriate regulation of cell proliferation during embryogenesis is crucial for the maintenance of germness. An in-depth understanding of germ cell developmental processes may yield valuable information on germ cell biology and applied sciences. However, direct evidences about germ cell proliferation and cell cycling during avian embryonic development has not been well-studied. Thus, we explored chicken germ cell dynamics during embryonic development via flow cytometry employing a germ cell-specific anti-cVASA antibody (the chicken VASA homolog is termed CVH) and propidium iodide staining. The numbers of male germ cells increased significantly during early embryonic development, but proliferation was decreased significantly with accumulation at the G0/G1 phase after embryonic d 14 (E.14), indicating initiation of mitotic arrest in the testis. On the other hand, the number of female germ cells increased significantly throughout embryogenesis, and proliferating cells were continuously evident in the ovary to the time of hatching, although gradual accumulation of cells at the G2/M phase was also evident. 5-ethynyl-2΄-deoxyuridine (EdU) incorporation analysis revealed that populations of mitotically active germ cells existed in both sexes during late embryogenesis, indicating either the maintenance of stem cell populations, or asynchronous meiosis. Collectively, these results indicate that chicken germ cells exhibited conserved developmental processes that were clearly sexually dimorphic.

Co-localization of lymphoid aggregates and lymphatic networks in nose- (NALT) and lacrimal duct-associated lymphoid tissue (LDALT) of mice

BackgroundThe lymphatic vascular pattern in the head of mice has rarely been studied, due to problems of sectioning and immunostaining of complex bony structures. Therefore, the association of head lymphoid tissues with the lymphatics has remained unknown although the mouse is the most often used species in immunology.ResultsHere, we studied the association of nasal and nasolacrimal duct lymphatics with lymphoid aggregates in 14-day-old and 2-month-old mice. We performed paraffin sectioning of whole, decalcified heads, and immunostaining with the lymphatic endothelial cell-specific antibodies Lyve-1 and Podoplanin. Most parts of the nasal mucous membrane do not contain any lymphatics. Only the region of the inferior turbinates contains lymphatic networks, which are connected to those of the palatine. Nose-associated lymphoid tissue (NALT) is restricted to the basal parts of the nose, which contain lymphatics. NALT is continued occipitally and can be found at both sides along the sphenoidal sinus, again in close association with lymphatic networks. Nasal lymphatics are connected to those of the ocular region via a lymphatic network along the nasolacrimal duct (NLD). By this means, lacrimal duct-associated lymphoid tissue (LDALT) has a dense supply with lymphatics.ConclusionsNALT and LDALT play a key role in the immune system of the mouse head, where they function as primary recognition sites for antigens. Using the dense lymphatic networks along the NLD described in this study, these antigens reach lymphatics near the palatine and are further drained to lymph nodes of the head and neck region.NALT and LDALT develop in immediate vicinity of lymphatic vessels. Therefore, we suggest a causative connection of lymphatic vessels and the development of lymphoid tissues.

A Whole Brain Staining, Embedding, and Clearing Pipeline for Adult Zebrafish to Visualize Cell Proliferation and Morphology in 3-Dimensions.

The field of macro-imaging has grown considerably with the appearance of innovative clearing methods and confocal microscopes with lasers capable of penetrating increasing tissue depths. The ability to visualize and model the growth of whole organs as they develop from birth, or with manipulation, disease or injury, provides new ways of thinking about development, tissue-wide signaling, and cell-to-cell interactions. The zebrafish (Danio rerio) has ascended from a predominantly developmental model to a leading adult model of tissue regeneration. The unmatched neurogenic and regenerative capacity of the mature central nervous system, in particular, has received much attention, however tools to interrogate the adult brain are sparse. At present there exists no straightforward methods of visualizing changes in the whole adult brain in 3-dimensions (3-D) to examine systemic patterns of cell proliferation or cell populations of interest under physiological, injury, or diseased conditions. The method presented here is the first of its kind to offer an efficient step-by-step pipeline from intraperitoneal injections of the proliferative marker, 5-ethynyl-2′-deoxyuridine (EdU), to whole brain labeling, to a final embedded and cleared brain sample suitable for 3-D imaging using optical projection tomography (OPT). Moreover, this method allows potential for imaging GFP-reporter lines and cell-specific antibodies in the presence or absence of EdU. The small size of the adult zebrafish brain, the highly consistent degree of EdU labeling, and the use of basic clearing agents, benzyl benzoate, and benzyl alcohol, makes this method highly tractable for most laboratories interested in understanding the vertebrate central nervous system in health and disease. Post-processing of OPT-imaged adult zebrafish brains injected with EdU illustrate that proliferative patterns in EdU can readily be observed and analyzed using IMARIS and/or FIJI/IMAGEJ software. This protocol will be a valuable tool to unlock new ways of understanding systemic patterns in cell proliferation in the healthy and injured brain, brain-wide cellular interactions, stem cell niche development, and changes in brain morphology.

Cellular Microvesicles in the Blood of Patients with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex and largely unclear pathogenesis. Cellular phospholipid microvesicles released upon activation and/or death of a cell have been proposed to play a role in inflammatory autoimmune pathologies, including SLE. Here, circulating microvesicles of various cellular origins were marked with fluorescently labeled cell-specific antibodies and enumerated by flow cytometry in platelet-free plasma obtained from the heparinized blood of 29 SLE patients and 19 normal subjects. Significantly higher concentrations of endothelial-, monocyte-, and erythrocyte-derived microvesicles were found in the SLE patients compared to normal subjects with prevalence of microvesicles originating from endothelial cells. No significant difference was found for platelet-derived microvesicles. A correlation analysis of microvesicle counts with laboratory parameters and clinical features of SLE suggest differential implications of various cell-derived microvesicles in the pathogenesis of SLE. These data suggest that SLE is associated with functional alterations of endotheliocytes, monocytes, and erythrocytes followed by enhanced release of microvesicles that may contribute to inflammation and hypercoagulability.

Hypofractionated Irradiation Has Immune Stimulatory Potential and Induces a Timely Restricted Infiltration of Immune Cells in Colon Cancer Tumors.

In addition to locally controlling the tumor, hypofractionated radiotherapy (RT) particularly aims to activate immune cells in the RT-modified microenvironment. Therefore, we examined whether hypofractionated RT can activate dendritic cells (DCs), induce immune cell infiltration in tumors, and how the chronology of immune cell migration into tumors occurs to gain knowledge for future definition of radiation breaks and inclusion of immunotherapy. Colorectal cancer treatments offer only limited survival benefit, and immunobiological principles for additional therapies need to be explored with preclinical models. The impact of hypofractionated RT on CT26 colon cancer tumor cell death, migration of DCs toward supernatants (SN) of tumor cells, and activation of DCs by SN were analyzed. The subcutaneous tumor of a BALB/c-CT26 mouse model was locally irradiated with 2 × 5 Gy, the tumor volume was monitored, and the infiltration of immune cells in the tumor was determined by flow cytometry daily. Hypofractionated RT induced a mixture of apoptotic and necrotic CT26 cells, which is known to be in particular immunogenic. DCs that migrated toward SN of CT26 cells particularly upregulated the activation markers CD80 and CD86 when in contact with SN of irradiated tumor cells. After hypofractionated RT, the tumor outgrowth was significantly retarded and in the irradiated tumors an increased infiltration of macrophages (CD11bhigh/F4-80+) and DCs (MHC-II+), but only between day 5 and 10 after the first irradiation, takes place. While CD4+ T cells migrated into non-irradiated and irradiated tumors, CD8+ T cells were only found in tumors that had been irradiated and they were highly increased at day 8 after the first irradiation. Myeloid-derived suppressor cells and regulatory T cells show regular turnover in irradiated and non-irradiated tumors. Tumor cell-specific anti-IgM antibodies were enhanced in the serum of animals with irradiated tumors. We conclude that hypofractionated RT suffices to activate DCs and to induce infiltration of innate and adaptive immune cells into solid colorectal tumors. However, the presence of immune cells in the tumor which are beneficial for antitumor immune responses is timely restricted. These findings should be considered when innovative multimodal tumor treatment protocols of distinct RT with immune therapies are designed and clinically implemented.

Late onset cytopenias following haematopoietic stem cell transplant associated with viral infection and cell specific antibodies

This report describes a patient who received an allogeneic haematopoietic stem cell transplant and who, following a viral infection, developed late onset cytopenias associated with antibodies against red cells, platelets and granulocytes. Investigation of these cytopenias revealed the presence of lineage specific auto- and allo-antibodies, which were not present in either the donor or in the recipient prior to the viral infection. This case provides further evidence for the concept that viral challenges following HSCT can result in the production of cell specific antibodies that can have significant implications for patient management.

Plasma endothelial microparticles reflect the extent of capillaroscopic alterations and correlate with the severity of skin involvement in systemic sclerosis

Introduction and aimEndothelial microparticles (EMPs) are membrane-coated vesicles shed from endothelial cells and are considered markers of the endothelial state. It has been shown that total numbers of circulating EMPs are increased in patients with systemic sclerosis (SSc), but their clinical correlations have not yet been investigated in detail. We aimed to assess possible relationships between circulating EMPs and clinical as well as laboratory features among SSc patients with special attention to possible association with alteration in microvascular morphology objectified on nailfold videocapillaroscopy and clinical signs of microvascular complications. Materials and methodsThe study included 47 SSc patients and 27 age- and sex-matched healthy controls. EMPs were identified with flow cytometry after staining platelet-poor plasma with combinations of fluorescent cell-specific monoclonal antibodies (anti-CD31, -51, -42b, -62E and Annexin V). The following types of EMPs were evaluated: total EMPs (CD31+/CD42b−), activated EMPs (CD62E+/AnnV−,) and apoptotic EMPs (CD62E+/AnnV+ or CD51+). Clinical evaluation of patients was obtained, including nailfold videocapillaroscopy. ResultsAll types of EMPs were significantly elevated in SSc patients as compared with healthy controls. We found significant inverse correlation between severity of skin involvement and values of total EMPs (r=−0.32; p=0.02) and their levels tended to be lower in SSc patients with digital ulcers when compared to those without ischaemic skin lesions (p=0.09). Total EMPs and activated EMPs showed correlations with the number of ramified capillaries (r=−0.40 and r=0.37, respectively, p<0.05 for both). Moreover, total EMPs inversely correlated with the severity of capillary loss (r=−0.35, p<0.05) and their levels were significantly lower in patients with late NVC pattern with respect to those with early microangiopathy (p<0.05). On the other hand, active NVC pattern was characterized by strongly elevated levels of activated EMPs when compared to an early vascular alteration (p<0.05). ConclusionsOur results suggest that quantity and phenotype of circulating EMPs might indicate on molecular vascular damage with endothelial dysfunction and to reflect progressive loss of capillaries consequencing in microvascular insufficiency in SSc patients.

Efficient Gene Transfer into Chicken Gonads by Combining Transposons with Polyethylenimine

&lt;p&gt;Transposon mediated transfection is a promising, safe, and convenient way to generate transgenic chicken compared with virus-mediated technology and the in vitro modification of primordial germ cells (PGCs). To establish a simple method for in vivo transfection of chicken PGCs, we applied four different transposon systems (PB, SB, Tol2, and ZB) to investigate the gene transfer efficiency of chicken gonads via direct injection of a mixture of transposon and transposase plasmids and transfection reagent (polyethylenimine, PEI) into the subgerminal cavity of Hamburger and Hamilton stage 2-3 chick embryos. We also compared the effect of the amount of plasmids injected on the gene transfer efficiency of chicken gonads. We found that over 70% of the gonads were green fluorescent protein (GFP)-positive across all four transposon groups, and that the proportion of GFP-positive gonads was not significantly different between different transposons. Some GFP positive cells in gonads were confirmed as germ cells by co-labeling with the germ cell specific antibody. We also found that the proportions of GFP-positive gonads decreased significantly with a decrease of plasmid dose from 100 ng to 20 or 50 ng. Here we revealed that a combination of transposons with PEI is a simple and efficient method for gene transfer into chicken gonads and able to transfect PGCs in vivo that could be used for the production of transgenic chickens.&lt;/p&gt;

Gene Expression Analysis of Neurons and Astrocytes Isolated by Laser Capture Microdissection from Frozen Human Brain Tissues.

Different cell types and multiple cellular connections characterize the human brain. Gene expression analysis using a specific population of cells is more accurate than conducting analysis of the whole tissue homogenate, particularly in the context of neurodegenerative diseases, where a specific subset of cells is affected by the different pathology. Due to the difficulty of obtaining homogenous cell populations, gene expression in specific cell-types (neurons, astrocytes, etc.) has been understudied. To leverage the use of archive resources of frozen human brains in studies of neurodegenerative diseases, we developed and calibrated a method to quantify cell-type specific—neuronal, astrocytes—expression profiles of genes implicated in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Archive human frozen brain tissues were used to prepare slides for rapid immunostaining using cell-specific antibodies. The immunoreactive-cells were isolated by Laser Capture Microdissection (LCM). The enrichment for a particular cell-type of interest was validated in post-analysis stage by the expression of cell-specific markers. We optimized the technique to preserve the RNA integrity, so that the RNA was suitable for downstream expression analyses. Following RNA extraction, the expression levels were determined digitally using nCounter Single Cell Gene Expression assay (NanoString Technologies®). The results demonstrated that using our optimized technique we successfully isolated single neurons and astrocytes from human frozen brain tissues and obtained RNA of a good quality that was suitable for mRNA expression analysis. We present here new advancements compared to previous reported methods, which improve the method's feasibility and its applicability for a variety of downstream molecular analyses. Our new developed method can be implemented in genetic and functional genomic research of neurodegenerative diseases and has the potential to significantly advance the field.

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming.

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

A Novel Universal Detection Agent for Time-Gated Luminescence Bioimaging.

Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.

Bio-nanocapsules displaying various immunoglobulins as an active targeting-based drug delivery system

The bio-nanocapsule (BNC) is an approximately 30-nm particle comprising the hepatitis B virus (HBV) envelope L protein and a lipid bilayer. The L protein harbors the HBV-derived infection machinery; therefore, BNC can encapsulate payloads such as drugs, nucleic acids, and proteins and deliver them into human hepatocytes specifically in vitro and in vivo. To diversify the possible functions of BNC, we generated ZZ-BNC by replacing the domain indispensable for the human hepatotrophic property of BNC (N-terminal region of L protein) with the tandem form of the IgG Fc-binding Z domain of Staphylococcus aureus protein A. Thus, the ZZ-BNC is an active targeting-based drug delivery system (DDS) nanocarrier that depends on the specificity of the IgGs displayed. However, the Z domain limits the animal species and subtypes of IgGs that can be displayed on ZZ-BNC. In this study, we introduced into BNC an Ig κ light chain-binding B1 domain of Finegoldia magna protein L (protein-L B1 domain) and an Ig Fc-binding C2 domain of Streptococcus species protein G (protein-G C2 domain) to produce LG-BNC. The LL-BNC was constructed in a similar way using a tandem form of the protein-L B1 domain. Both LG-BNC and LL-BNC could display rat IgGs, mouse IgG1, human IgG3, and human IgM, all of which not binding to ZZ-BNC, and accumulate in target cells in an antibody specificity-dependent manner. Thus, these BNCs could display a broad spectrum of Igs, significantly improving the prospects for BNCs as active targeting-based DDS nanocarriers. Statement of SignificanceWe previously reported that ZZ-BNC, bio-nanocapsule deploying the IgG-binding Z domain of protein A, could display cell-specific antibody in an oriented immobilization manner, and act as an active targeting-based DDS nanocarrier. Since the Z domain can only bind to limited types of Igs, we generated BNCs deploying other Ig-binding domains: LL-BNC harboring the tandem form of Ig-binding domain of protein L, and LG-BNC harboring the Ig binding domains of protein L and protein G sequentially. Both BNCs could display a broader spectrum of Igs than does the ZZ-BNC. When these BNCs displayed anti-CD11c IgG or anti-EGFR IgG, both of which cannot bind to Z domain, they could bind to and then enter their respective target cells.

Post-transplant lymphoproliferative disorders.

Post-transplant lymphoproliferative disorders (PTLDs) are a group of conditions that involve uncontrolled proliferation of lymphoid cells as a consequence of extrinsic immunosuppression after organ or haematopoietic stem cell transplant. PTLDs show some similarities to classic lymphomas in the non-immunosuppressed general population. The oncogenic Epstein-Barr virus (EBV) is a key pathogenic driver in many early-onset cases, through multiple mechanisms. The incidence of PTLD varies with the type of transplant; a clear distinction should therefore be made between the conditions after solid organ transplant and after haematopoietic stem cell transplant. Recipient EBV seronegativity and the intensity of immunosuppression are among key risk factors. Symptoms and signs depend on the localization of the lymphoid masses. Diagnosis requires histopathology, although imaging techniques can provide additional supportive evidence. Pre-emptive intervention based on monitoring EBV levels in blood has emerged as the preferred strategy for PTLD prevention. Treatment of established disease includes reduction of immunosuppression and/or administration of rituximab (a B cell-specific antibody against CD20), chemotherapy and EBV-specific cytotoxic T cells. Despite these strategies, the mortality and morbidity remains considerable. Patient outcome is influenced by the severity of presentation, treatment-related complications and risk of allograft loss. New innovative treatment options hold promise for changing the outlook in the future.

The Novel Methods for Analysis of Exosomes Released from Endothelial Cells and Endothelial Progenitor Cells.

Exosomes (EXs) are cell-derived vesicles that mediate cell-cell communication and could serve as biomarkers. Here we described novel methods for purification and phenotyping of EXs released from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads and fluorescence quantum dots (Q-dots®) techniques. EXs from the culture medium of ECs and EPCs were isolated and detected with cell-specific antibody conjugated microbeads and second antibody conjugated Q-dots by using nanoparticle tracking analysis (NTA) system. The sensitivities of the cell origin markers for ECs (CD105, CD144) and EPCs (CD34, KDR) were evaluated. The sensitivity and specificity were determined by using positive and negative markers for EXs (CD63), platelets (CD41), erythrocytes (CD235a), and microvesicles (Annexin V). Moreover, the methods were further validated in particle-free plasma and patient samples. Results showed that anti-CD105/anti-CD144 and anti-CD34/anti-KDR had the highest sensitivity and specificity for isolating and detecting EC-EXs and EPC-EXs, respectively. The methods had the overall recovery rate of over 70% and were able to detect the dynamical changes of circulating EC-EXs and EPC-EXs in acute ischemic stroke. In conclusion, we have developed sensitive and specific microbeads/Q-dots fluorescence NTA methods for EC-EX and EPC-EX isolation and detection, which will facilitate the functional study and biomarker discovery.

Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments.

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix.

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the β-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description ofhow to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists' discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Correlated responses of respiratory disease and immune capacity traits of Landrace pigs selected for Mycoplasmal pneumonia of swine (MPS) lesion

Five generations of Landrace pigs selected for average daily gain, backfat thickness, Mycoplasmal pneumonia of swine (MPS) lesion score, and plasma cortisol levels, was executed to decrease the MPS lesion score. Genetic parameters and correlated genetic responses for respiratory disease and peripheral blood immune traits were estimated in 1395 Landrace pigs. We estimated the negative genetic correlation of MPS lesion score with phagocytic activity (PA) at 7 weeks of age (-0.67). The breeding values of PA at 7 weeks of age and 105 kg body weight and the correlated selection response of the ratio of granular leukocytes to lymphocytes at 105 kg body weight were significantly increased, and sheep red blood cell-specific antibody production (AP) was significantly decreased in a selection-dependent manner. Increasing of natural immunological indicators (e.g. PA) and decreasing of humoral immunological indicator (e.g. AP) were observed due to genetically decreasing MPS lesion score.

Rapid and automated flow cytometry crossmatch for optimizing inter-assay and inter-operator consistency

Background Flow cytometry crossmatch (FCXM) is a high sensitive tool to identify donor cell specific antibodies in recipient’s serum. However, the procedure is time consuming and fraught with inter-assay and inter-operator variabilities. Aim To optimize instrumentation for automated tagging (AT) in FCXM that will both quicken the procedure and exclude inter-operator variability. Methods Serum, spleen, or peripheral blood mononuclear cells were isolated from human subjects. AT was performed using a combination of Precision-XS and EL406 instruments (BioTek Instruments, Inc.) and compared to manual tagging (MT) (both in 96 well plates). FITC-goat α-human IgG channel shift (CS) of >40 for T cells and >50 for B cells was considered positive. Results AT of two cell specimens took 90 min vs. 100 min by MT. AT did not cause cell loss. Automated Tagging vs. Manual Tagging: AT and MT of specimens (N = 6) provided similar CS results (T cell-(P = 0.8) and B cell-FCXM (P = 0.9); (Fig 1A)) and median channel intensity results for T cell FCXM (%CV: negative control (NC) = 8 vs. 11; positive control (PC) = 10 vs. 14) and B cell FCXM (%CV: NC = 6 vs. 17; PC = 10 vs. 9). Intra-assay variability: When AT was performed in duplicates in a plate (N = 3), no intra-assay variability was noted in CS results (T cell FCXM: P = 0.1; B cell FCXM: P = 0.1 (Fig 1B)) or in median channel intensity results [T cell FCXM (%CV: NC = 1 vs. 5; PC = 4 vs. 2) and B cell FCXM (%CV: NC = 2 vs. 1; PC = 5 vs. 4).] Inter-assay variability: When AT was performed in two different plates (N = 3), no inter-assay variability was noted in CS results (T cell FCXM: P = 0.8; B cell FCXM: P = 0.9 (Fig 1C)) or in median channel intensity results [T cell FCXM (%CV: NC = 8; and PC = 6) and B cell FCXM (%CV: NC = 11; PC = 5)]. Inter-operator variability: Inter-operator variability was not noted when FCXM were performed by AT (Fig 1D). Conclusions Combination of Precision-XS- and EL406-instruments helps making a FCXM faster and automatous, and provides reliable results with inter-assay and inter-operator consistency. Download : Download full-size image

Abstract 1589: Investigation of circulating tumor cells from head and neck cancer patients undergoing radiation therapy: A pilot study

Abstract Background: Circulating tumor cell (CTC) enumeration provides prognostic information in patients with metastatic breast, prostate, colorectal and lung cancer. However, the effect of radiation therapy (RT) on CTCs in patients with primary head and neck malignancies has not been explored. The purpose of this exploratory study is to examine how CTCs, as measured by a novel cell capture technique, respond to RT in patients with head and neck cancer. Methods: A total of a 16 patients undergoing definitive radiation for primary, non-metastatic head and neck cancer were enrolled in this pilot study. Peripheral blood was collected at 5 time points, including at baseline prior to starting RT, at the first week, mid-point and final week during treatment, and at least 4 weeks after completion of RT. Quantification of CTCs was performed with a novel device that used a biomimetic combination of three cancer cell-specific antibodies (aEpCAM, aHer-2, and aEGFR) immobilized through poly(amidoamine) dendrimers and E-selectin, which respectively induce concurrent stationary binding and dynamic cell rolling of the tumor cells. Results: CTCs were successfully detected in all patients before the start of radiation (100%), including patients with HPV/p16 positive or negative tumors. The novel CTC device yielded up to a 4-fold enhancement in capture efficiency relative to the aEpCAM-functionalized surface. The average CTC count in patients before RT was 386 CTCs per mL (range, 18 to 1134 CTCs per mL), significantly higher than the average CTC count of 90 CTCs per mL after the completion of radiation (range, 5 to 393 CTCs per mL). All but one patient demonstrated a decrease in measured CTCs at sequential time points during the course of RT. The patient with an elevation in CTCs during RT had residual metastatic carcinoma detected after left level II selective cervical neck dissection, which was performed 12 weeks post-RT due to persistent radiographic lymph nodal abnormality. At a median follow-up of 1.5 months, no patient has had a local or distant failure. Conclusions: We have demonstrated that our novel technology can affectively capture CTCs in head and neck cancer patients. Importantly, our pilot data shows CTCs decrease over the course of RT, suggesting CTCs can be a predicative biomarker for radiotherapy response. Further study will investigate associations between CTC kinetics and clinical outcomes. Citation Format: Michael J. Eblan, Ja Hye Myung, Joseph M. Caster, Bhishamjit S. Chera, Seungpyo Hong, Andrew Z. Wang. Investigation of circulating tumor cells from head and neck cancer patients undergoing radiation therapy: A pilot study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1589. doi:10.1158/1538-7445.AM2015-1589