CD45-positive Leukocytes Research Articles

(186) THE USE OF THE FLUORESCENT NUCLEAR STAIN DAPI TO IDENTIFY SEMINAL LEUKOCYTES

Abstract Introduction Emerging animal-based research highlights the requisite role of the immune system in maintaining tolerance to spermatozoa. Critically, however, there exists a paucity of studies investigating the role of the immune system in clinical male factor infertility. Current protocols investigating seminal fluid resident white blood cells (leukocytes) include peroxidase staining; however, further investigation of these cells is rarely performed due to the extremely low frequency of these cells. The AUA and ASRM have no guidelines regarding the workup or treatment of leukospermia. Objective We sought to develop a streamlined and simple assay to identify and better characterize rare leukocytes within spermatozoa-rich semen samples. Methods We identified 15 patients with “Too Numerous to Count” round cells from our seminal fluid cryobank. These semen samples were thawed and exposed to a formalin/saponin based buffer to fix and permeabilize cells. Cells were then stained with the fluorescent nuclear stain DAPI and fluorescently conjugated antibodies directed against CD45. Sample acquisition was performed on a BD LSRFortessa Flow Cytometer and data analyzed using FlowJo software. Results DAPI nuclear staining clearly separates DAPI dim spermatozoa (haploid cells) from DAPI bright leukocytes (diploid cells). Subsequent dual-channel CD45 antibody staining confirms the absence of CD45 on haploid spermatozoa (98% negative) and positivity of CD45 on diploid leukocytes (87.2% positive). Appropriate DAPI signal intensity of the leukocytes within the semen samples was confirmed by spiking semen samples with peripheral blood mononuclear cells. Overall, CD45 positive leukocytes represented 0.003% to 2.93% +/- 0.88% of the leukocytes found in the seminal plasma of infertile patients. Conclusions 50% of male factor infertility remains undefined. Preclinical evidence reveals a required role of the immune system in maintaining immunologic tolerance to “foreign” spermatozoa. Breaks in this system may perpetuate male factor infertility but clinical investigation of this paradigm remains to be pursued. Disclosure No.

(187) T LYMPHOCYTES IN HUMAN SEMEN: INSIGHTS INTO IMMUNE TOLERANCE OF SPERMATOGENESIS

Abstract Introduction Emerging preclinical work has demonstrated the requisite role of the adaptive immune system in maintaining tolerance toward “foreign” spermatozoa. As seen in animal models, breaks in this system directly causes male factor infertility. Depletion of tolerogenic T lymphocytes induces severe autoimmune orchitis, immune cell infiltration, and reduced sperm number and motility. Objective There exists no investigation of the adaptive regulatory immune system in the context of human male fertility. We interrogated our seminal fluid cryobank which consists of 1700 patient samples to study this highly relevant question in male fertility. Methods We identified 15 semen samples with increased round cells on semen analysis. Patient samples were thawed, fixed and permeabilized, and stained with the nuclear fluorescent stain DAPI. This differentiates DAPI dim spermatozoa [haploid] from DAPI bright leukocytes [diploid]. Cells were then stained with fluorescently conjugated antibodies (CD45, CD3, CD4, CD25 and Foxp3) to identify leukocytes and specifically, tolerogenic CD4 T Regulatory Cells (CD4 Tregs). Multiparameter flow cytometric analysis was performed (BD LSRFortessa) and data analyzed using GraphPad Prism Software. Results CD45 positive leukocytes as identified by flow cytometry ranged in frequency from 0.003% to 2.93% +/- 0.88% and correlated positively with WBC counts on semen analyses. We were able to reliably identify Foxp3+ CD4 Tregs in semen samples. Increased frequencies of CD4 Tregs were observed in patients with higher seminal fluid WBC counts. Interestingly, CD4 Treg frequencies were inversely correlated with sperm density and motility [p < 0.05 linear regression]. Conclusions We describe the first report of tolerogenic T lymphocytes studied in human semen. We reliably identified and documented increased frequencies of CD4 Tregs in patients with lower sperm density and motility. CD4 Tregs expand in inflammatory conditions to quell ongoing autoimmunity. Overall, we hypothesize that inadequate control of autoreactive anti-sperm lymphocytes by tolerogenic CD4 Tregs leads to poor sperm quality. Further mechanistic investigation of this paradigm remains highly warranted. Disclosure No.

Triiodothyronine induces a proinflammatory monocyte/macrophage profile and impedes cardiac regeneration

Neonatal mouse hearts can regenerate post-injury, unlike adult hearts that form fibrotic scars. The mechanism of thyroid hormone signaling in cardiac regeneration warrants further study. We found that triiodothyronine impairs cardiomyocyte proliferation and heart regeneration in neonatal mice after apical resection. Single-cell RNA-Sequencing on cardiac CD45-positive leukocytes revealed a pro-inflammatory phenotype in monocytes/macrophages after triiodothyronine treatment. Furthermore, we observed that cardiomyocyte proliferation was inhibited by medium from triiodothyronine-treated macrophages, while triiodothyronine itself had no direct effect on the cardiomyocytes in vitro. Our study unveils a novel role of triiodothyronine in mediating the inflammatory response that hinders heart regeneration.

Ferulic acid suppresses the inflammation and apoptosis in Kawasaki disease through activating the AMPK/mTOR/NF-κB pathway.

Kawasaki disease (KD) is a self-limiting and acute systemic vasculitis of unknown etiology, mainly affecting children. Ferulic acid (FA), a natural phenolic substance, has multiple pharmacological properties, including anti-inflammatory, anti-apoptosis, and anti-fibrosis, and so on. So far, the protective effects of FA on KD have not been explored. In this study, we established Candida albicans water soluble fraction (CAWS)-induced mouse coronary artery vasculitis of KD model and the tumor necrosis factor α (TNF-α)-induced human umbilical vein endothelial cells (HUVECs) injury model to investigate the anti-inflammatory and anti-apoptosis effects of FA on KD, and try to elucidate the underlying mechanism. Our in vivo results demonstrated that FA exerted anti-inflammatory effects on KD by inhibiting the infiltration of CD45-positive leukocytes and fibrosis around the coronary artery. Additionally, FA downregulated the levels of inflammatory and chemotactic cytokines, alleviated splenomegaly, and exhibited anti-apoptotic effects on KD by reducing TUNEL-positive cells, downregulating BAX expression, and upregulating BCL-2 expression. In addition, Our in vitro findings showed that FA could effectively inhibit TNF-α-induced HUVEC inflammation like NF-κB inhibitor QNZ by downregulating the expression of pro-inflammatory cytokines as well as attenuated TNF-α-induced HUVEC apoptosis by reducing apoptotic cell numbers and the BAX/BCL-2 ratio, which could be reversed by the AMPK inhibitor compound c (CC). The further mechanistic study demonstrated that FA could restrain vascular endothelial cell inflammation and apoptosis in KD through activating the AMPK/mTOR/NF-κB pathway. However, FA alone is hard to completely restore KD into normal condition. In conclusion, FA has potential protective effects on KD, suggesting its promising role as an adjuvant for KD therapy in the future.

The effect of fucoxanthin on the development of CCl<sub>4</sub>-induced liver fibrosis

Background. According to current concepts regarding hepatic fibrosis, myofibroblast differentiation from stellate cells, regulated by transforming growth factor-β (TGF-β), is a key step in its pathogenesis. Hence, inhibition of TGF-β-dependent activation of hepatic stellate cells has been suggested as a promising strategy for preventing the disease development.Aim. To explore whether the administration of fucoxanthin at a dose of 30 mg/kg is efficient in suppressing hepatic fibrosis.Materials and Methods. The experiments were carried out on 30 outbred ICR/CD1 mice which have been divided into three groups: intact animals, animals with untreated hepatic fibrosis which has been induced by intraperitoneal injections of CCl4 (2 μl/g, 6 weeks, twice per week), and animals which received fucoxanthin per os (30 mg/kg daily for 5 weeks) after inducing hepatic fibrosis as described above. Histological examination was performed by Sirius Red staining using the METAVIR fibrosis and activity score. Immunohistochemical analysis was performed by quantitation of α-SMA-positive myofibroblasts, CD45-positive leukocytes, and TIMP-1-positive regions. Further, we quantified TGF-β in liver homogenate as well as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the serum by means of enzyme-linked immunosorbent assay. An assessment of liver function was conducted by measuring serum alanine aminotransferase, aspartate aminotransferase, and albumin levels.Results. Fucoxanthin decreased the number of myofibroblasts and leukocytes, the volume of connective tissue and TIMP-1-positive regions, and the level of TGF-β in the liver homogenate, altogether indicative of ameliorated hepatic fibrosis. In accord, treatment with fucoxanthin reduced serum IL-1β, TNF-α, alanine aminotransferase and aspartate aminotransferase, and increased serum albumin.Conclusion. Treatment with fucoxanthin at a dose of 30 mg/kg has an antifibrotic effect and diminishes liver fibrosis.

Osteochondral Regeneration Ability of Uncultured Bone Marrow Mononuclear Cells and Platelet-Rich Fibrin Scaffold.

Platelet-rich fibrin (PRF) and bone marrow mononuclear cells are potential scaffolds and cell sources for osteochondral regeneration. The main aim of this paper is to examine the effects of PRF scaffolds and autologous uncultured bone marrow mononuclear cells on osteochondral regeneration in rabbit knees. Three different types of PRF scaffolds were generated from peripheral blood (Ch-PRF and L-PRF) and bone marrow combined with uncultured bone marrow mononuclear cells (BMM-PRF). The histological characteristics of these scaffolds were assessed via hematoxylin-eosin staining, PicroSirius red staining, and immunohistochemical staining. Osteochondral defects with a diameter of 3 mm and depth of 3 mm were created on the trochlear groove of the rabbit's femur. Different PRF scaffolds were then applied to treat the defects. A group of rabbits with induced osteochondral defects that were not treated with any scaffold was used as a control. Osteochondral tissue regeneration was assessed after 2, 4, and 6 weeks by macroscopy (using the Internal Cartilage Repair Society score, X-ray) and microscopy (hematoxylin-eosin stain, safranin O stain, toluidine stain, and Wakitani histological scale, immunohistochemistry), in addition to gene expression analysis of osteochondral markers. Ch-PRF had a heterogeneous fibrin network structure and cellular population; L-PRF and BMM-PRF had a homogeneous structure with a uniform distribution of the fibrin network. Ch-PRF and L-PRF contained a population of CD45-positive leukocytes embedded in the fibrin network, while mononuclear cells in the BMM-PRF scaffold were positive for the pluripotent stem cell-specific antibody Oct-4. In comparison to the untreated group, the rabbits that were given the autologous graft displayed significantly improved healing of the articular cartilage tissue and of the subchondral bone. Regeneration was gradually observed after 2, 4, and 6 weeks of PRF scaffold treatment, which was particularly evident in the BMM-PRF group. The combination of biomaterials with autologous platelet-rich fibrin and uncultured bone marrow mononuclear cells promoted osteochondral regeneration in a rabbit model more than platelet-rich fibrin material alone. Our results indicate that autologous platelet-rich fibrin scaffolds combined with uncultured bone marrow mononuclear cells applied in healing osteochondral lesions may represent a suitable treatment in addition to stem cell and biomaterial therapy.

Perivascular adipose tissue promotes vascular dysfunction in murine lupus.

Patients with systemic lupus erythematosus (SLE) are at elevated risk for Q10 cardiovascular disease (CVD) due to accelerated atherosclerosis. Compared to heathy control subjects, lupus patients have higher volumes and densities of thoracic aortic perivascular adipose tissue (PVAT), which independently associates with vascular calcification, a marker of subclinical atherosclerosis. However, the biological and functional role of PVAT in SLE has not been directly investigated. Using mouse models of lupus, we studied the phenotype and function of PVAT, and the mechanisms linking PVAT and vascular dysfunction in lupus disease. Lupus mice were hypermetabolic and exhibited partial lipodystrophy, with sparing of thoracic aortic PVAT. Using wire myography, we found that mice with active lupus exhibited impaired endothelium-dependent relaxation of thoracic aorta, which was further exacerbated in the presence of thoracic aortic PVAT. Interestingly, PVAT from lupus mice exhibited phenotypic switching, as evidenced by "whitening" and hypertrophy of perivascular adipocytes along with immune cell infiltration, in association with adventitial hyperplasia. In addition, expression of UCP1, a brown/beige adipose marker, was dramatically decreased, while CD45-positive leukocyte infiltration was increased, in PVAT from lupus mice. Furthermore, PVAT from lupus mice exhibited a marked decrease in adipogenic gene expression, concomitant with increased pro-inflammatory adipocytokine and leukocyte marker expression. Taken together, these results suggest that dysfunctional, inflamed PVAT may contribute to vascular disease in lupus.

Pharmacological inhibition of Na+/K+-ATPase induces neurovascular degeneration and glial cell alteration in the rat retina

Na+/K+-ATPase (NKA) plays an important role in ion homeostasis and neurotransmitter uptake. In the retina, multidirectional communications among neurons, glia, and blood vessels (that is, neuro-glio-vascular interaction) are crucial for maintaining tissue homeostasis. We investigated the role of NKA in the elements of neuro-glio-vascular unit in neonatal and adult rat retinas. Male Sprague-Dawley rats (1- and 8-week-old) were injected intravitreally with ouabain (20 nmol/eye), an inhibitor of NKA. Morphological changes in retinal neurons, glia, and blood vessels were examined. The intravitreal injection of ouabain decreased the number of cells in the ganglion cell layer, as well as the thicknesses of the inner plexiform and inner nuclear layers in neonatal and adult rats compared to age-matched controls. The ouabain-induced neuronal cell damage was partially prevented by D-(−)-2-amino-5-phosphonopentanoic acid, an antagonist of N-methyl-D-aspartic acid receptors. In the deep retinal vascular plexus of the ouabain-injected eyes, angiogenesis was delayed in neonatal rats, whereas capillary degeneration occurred in adult rats. The immunoreactivity of glutamine synthetase and vascular endothelial growth factor (VEGF) decreased in the retinas of neonatal and adult rats injected intravitreally with ouabain. The immunoreactivity of glial fibrillary acidic protein was enhanced in the retinas of ouabain-injected adult eyes. After the ouabain injection, CD45-positive leukocytes and Iba1-positive microglia increased in the inner retinal layer of neonatal rats, whereas they increased in the middle retinal layer of adult rats. These results suggest that the inhibition of NKA induces the degeneration of neuronal and vascular cells and alteration of glial cells in both neonatal and adult retinas. In addition to the direct effects of NKA inhibition, the disturbance of retinal glutamate metabolism and decreased VEGF expression may contribute to neurovascular degeneration. The activity of NKA is crucial for maintaining elements of neuro-glio-vascular unit in the retina.

Metformin Protects against NMDA-Induced Retinal Injury through the MEK/ERK Signaling Pathway in Rats.

Metformin, an anti-hyperglycemic drug of the biguanide class, exerts positive effects in several non-diabetes-related diseases. In this study, we aimed to examine the protective effects of metformin against N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinal damage in rats and determine the mechanisms of its protective effects. Male Sprague–Dawley rats (7 to 9 weeks old) were used in this study. Following intravitreal injection of NMDA (200 nmol/eye), the number of neuronal cells in the ganglion cell layer and parvalbumin-positive amacrine cells decreased, whereas the number of CD45-positive leukocytes and Iba1-positive microglia increased. Metformin attenuated these NMDA-induced responses. The neuroprotective effect of metformin was abolished by compound C, an inhibitor of AMP-activated protein kinase (AMPK). The AMPK activator, AICAR, exerted a neuroprotective effect in NMDA-induced retinal injury. The MEK1/2 inhibitor, U0126, reduced the neuroprotective effect of metformin. These results suggest that metformin protects against NMDA-induced retinal neurotoxicity through activation of the AMPK and MEK/extracellular signal-regulated kinase (ERK) signaling pathways. This neuroprotective effect could be partially attributable to the inhibitory effects on inflammatory responses.

Enzymatically Inactive Tissue-Type Plasminogen Activator Reverses Disease Progression in the Dextran Sulfate Sodium Mouse Model of Inflammatory Bowel Disease

Enzymatically inactive tissue-type plasminogen activator (EI-tPA) does not activate fibrinolysis, but interacts with the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1) in macrophages to block innate immune system responses mediated by toll-like receptors. Herein, we examined the ability of EI-tPA to treat colitis in mice, induced by dextran sulfate sodium. In two separate studies, designed to generate colitis of differing severity, a single dose of EI-tPA administered after inflammation established significantly improved disease parameters. EI-tPA-treated mice demonstrated improved weight gain. Stools improved in character and became hemoccult negative. Abdominal tenderness decreased. Colon shortening significantly decreased in EI-tPA-treated mice, suggesting attenuation of irreversible tissue damage and remodeling. Furthermore, histopathologic evidence of disease decreased in the distal 25% of the colon in EI-tPA-treated mice. EI-tPA did not decrease the number of CD45-positive leukocytes or F4/80-positive macrophage-like cells detected in extracts of colons from dextran sulfate sodium-treated mice as assessed by flow cytometry. However, multiple colon cell types expressed the NMDA-R, suggesting the ability of diverse cells, including CD3-positive cells, CD103-positive cells, Ly6G-positive cells, and epithelial cell adhesion molecule-positive epithelial cells to respond to EI-tPA. Mesenchymal cells that line intestinal crypts and provide barrier function expressed LRP1, thereby representing another potential target for EI-tPA. These results demonstrate that the NMDA-R/LRP1 receptor system may be a target for drug development in diseases characterized by tissue damage and chronic inflammation.

Immunosuppressant Drugs Mitigate Immune Responses Generated by Human Mesenchymal Stem Cells Transplanted into the Mouse Parenchyma

It has been widely accepted that mesenchymal stem cells (MSCs) can evade the immune surveillance of the recipient. However, emerging research cast doubt on whether MSCs are intrinsically immune-privileged. Previously, we observed that the transplantation of human MSCs (hMSCs) into the mouse parenchyma attracted a high infiltration of leukocytes into the injection tract. Thus, in order to reduce the immune responses generated by hMSCs, the aim of this study was to assess which immunosuppressant condition (dexamethasone only, tacrolimus only, or dexamethasone and tacrolimus together) would not only reduce the overall immune response but also enhance the persistence of MSCs engrafted into the caudate putamen of wild-type C57BL/6 mice. According to immunohistochemical analysis, compared to the hMSC only group, the administration of immunosuppressants (for all three conditions) reduced the infiltration of CD45-positive leukocytes and neutrophils at the site of injection. The highest hMSC persistence was detected from the group that received combinatorial administrations of dexamethasone and tacrolimus. Moreover, compared to the immunocompetent WT mouse, higher MSC engraftment was observed from the immunodeficient BALB/c mice. The results of this study support the use of immunosuppressants to tackle MSC-mediated immune responses and to possibly prolong the engraftment of transplanted MSCs.

Abstract WMP77: SUMO1 Plays an Important Role in Cardiac Dysfunction After Intracerebral Hemorrhage in Aged Mice

Background: The conjugation of the small ubiquitin-like modifier1 (SUMO1) plays an important role in numerous biological processes, including DNA repair and signal transduction. SUMO1 also reduces cardiac oxidative stress and hypertrophy as well as induces neuroprotective effects. Intracerebral hemorrhage (ICH) induces cardiac deficit in the absence of primary cardiac diseases in young adult wild type mice. In this study, we tested the hypothesis that SUMO1 plays a key role in regulating brain-heart interaction after ICH, and SUMO1 deficit leads to worse brain and heart deficit after ICH in aged mice. Methods: Aged (16-20 months) female Sumo1-deficient (SUMO1-/-) mice or wild-type (SUMO1+/+) mice were subjected to ICH by injecting collagenase IV into the basal ganglia. Cardiac function was measured by echocardiography before ICH induction and at 7 days after ICH. Modified neurological severity (mNSS) ,foot-fault and adhesive removal tests were performed at 1, 3, 6 days after ICH to investigate neurological function. Cognitive functional tests (odor and novel objective tests) were performed before sacrificing the mice at 10 days after ICH, and then histological and immunohistochemically staining were used to evaluate the mechanisms. Results: Compared to SUMO1+/+ mice, SUMO1-/- mice did not exhibit significant cardiac deficit before ICH in aged mice. Compared to SUMO1+/+ICH mice, SUMO1-/-ICH mice exhibit significantly (p<0.05) increased: 1) brain hemorrhage volume and induced worse neurological and cognitive deficits, 2) cardiac dysfunction identified by decreased cardiac contractile function measured by left ventricular ejection fraction (LVEF) and fractional shortening (FS); 3) cardiac hypertrophy and fibrosis; 4) ionized calcium binding adaptor molecule (IBA1) positive macrophages/microglia and CD45 positive leukocyte infiltration into both heart and brain tissue. Conclusions: Aged SUMO1 deficient mice subjected to ICH not only exhibit increased neurological and cognitive functional deficit, but also significantly increased cardiac dysfunction and inflammatory cell infiltration into heart. These data suggest that SUMO1 plays an important role in brain-heart interaction and SUMO1 impacts brain-cardiac dysfunction after ICH.

CXCL12 loaded-dermal filler captures CXCR4 expressing melanoma circulating tumor cells

Development of distant metastasis relies on interactions between cancer and stromal cells. CXCL12, also known as stromal-derived factor 1α (SDF-1α), is a major chemokine constitutively secreted in bone marrow, lymph nodes, liver and lung, playing a critical role in the migration and seeding of neoplastic cells. CXCL12 activates the CXCR4 receptor that is overexpressed in several human cancer cells. Recent evidence reveals that tumors induce pre-metastatic niches in target organ producing tumor-derived factors. Pre-metastatic niches represent a tumor growth-favoring microenvironment in absence of cancer cells. A commercially available dermal filler, hyaluronic acid (HA) -based gel, loaded with CXCL12 (CLG) reproduced a “fake” pre-metastatic niche. In vitro, B16-hCXCR4-GFP, human cxcr4 expressing murine melanoma cells efficiently migrated toward CLG. In vivo, CLGs and empty gels (EGs) were subcutaneously injected into C57BL/6 mice and 5 days later B16-hCXCR4-GFP cells were intravenously inoculated. CLGs were able to recruit a significantly higher number of B16-hCXCR4-GFP cells as compared to EGs, with reduced lung metastasis in mice carrying CLG. CLG were infiltrated by higher number of CD45-positive leukocytes, mainly neutrophils CD11b+Ly6G+ cells, myeloid CD11b+Ly6G- and macrophages F4/80. CLG recovered cells recapitulated the features of B16-hCXCR4-GFP (epithelial, melanin rich, MELAN A/ S100/ c-Kit/CXCR4 pos; α-SMA neg). Thus a HA-based dermal filler loaded with CXCL12 can attract and trap CXCR4+tumor cells. The CLG trapped cells can be recovered and biologically characterized. As a corollary, a reduction in CXCR4 dependent lung metastasis was detected.

1747-P: 3D Imaging and Quantification of Pancreatic Immune Cell Infiltration during Emergence and Progression of Spontaneous Autoimmune Diabetes in Nonobese Diabetic (NOD) Mice

The non-obese diabetic (NOD) mouse develops diabetes as the result of spontaneous autoimmune insulitis and is widely used as a translational polygenic model of type 1 diabetes. Disease progression in the NOD mouse model is slow, involving a long prodromal phase of pancreatic islet infiltration before autoimmune β-cell destruction and manifestation of diabetic hyperglycemia, making the NOD mouse ideal for probing the effectiveness of potential disease prevention therapies. Improved understanding of the spatial dynamics of immune cell infiltration of the pancreas during the emergence and natural progression of autoimmune diabetes would permit further resolution of drug treatment effects in this model. The present study therefore aimed to image the spontaneous development and progression of autoimmune diabetes dynamically and at 3-dimensional cellular resolution in the NOD mouse. Female NOD mice were terminated before diabetes onset, at 14 and 18 weeks of age, and pancreata were isolated and divided into two alternating samples consisting of either the head and tail. One half was stained for insulin and CD45 using iDISCO whole-mount immunohistochemistry and subsequently scanned using a light sheet microscope. Islet infiltration was quantified using an in-house developed algorithm. The other half of each pancreas was simultaneously evaluated by flow cytometry to discriminate and quantify immune cells of lymphoid and myeloid lineage. Infiltrating CD45-positive leukocytes progressively increased in abundance over time, however, with highly variable distribution in the pancreas. Flow cytometry confirmed extensive leukocyte infiltration and revealed predominant changes in T-cell profiles. In conclusion, 3D imaging allows for visualization and unbiased quantitative analysis of the unsynchronized spreading of insulitis into different parts of the pancreas in NOD mice. Disclosure B.B. Boland: Employee; Self; Gubra, MedImmune. A. Manresa-Arraut: None. R.V. Grônlund: None. U. Roostalu: Employee; Self; Gubra. J. Hecksher-Sørensen: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. N. Vrang: Board Member; Self; Gubra. Stock/Shareholder; Self; Gubra. J. Jelsing: Stock/Shareholder; Self; Gubra. L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S.

Isolation of mouse CD45 positive leukocytes from tissues

Abstract Immune cell function is often tissue-specific, therefore isolating cells from their tissue microenvironment is necessary to better understand their role in health and disease. This can be challenging in the presence of non-immune, tissue-derived cells and cellular debris from tissue dissociation. These factors reduce the leukocyte start frequency, which can prolong the isolation process and limit the identification of small but critical leukocyte subsets. To overcome these obstacles, we developed a simple and rapid selection method to enrich for CD45+ leukocytes from mouse tissues. Starting with a single cell suspension, cells are labelled with an antibody complex that links CD45+ cells to magnetic particles and following magnetic separation, the isolated CD45+ cells are ready for use. Using healthy mouse lung tissue as an example, CD45+ leukocytes were enriched from 63.5 ± 9.4% to 97.1 ± 1.2% purity, and the recovery of viable CD45+ cells was 37.7 ± 14.5% (mean ± SD, n=37). Preliminary testing on tumors from a mouse 4T1 breast tumor model resulted in efficient enrichment of tumor-infiltrating leukocytes. Importantly, the composition of immune subsets from both healthy lung tissues and tumor samples was maintained following CD45 selection. To demonstrate functionality, T cells within the CD45 selected population from spleen were able to upregulate CD25 and CD69, and proliferate upon stimulation. In as little as 20 minutes, the EasySep™ Mouse CD45 Isolation Kit allows researchers to easily enrich for functional leukocytes from tissue samples. Examining immune cells directly from healthy and tumor tissues will enhance our understanding of how these cells function and aid in developing new approaches to combat disease progression.

Endometrial human chorionic gonadotropin (hCG) expression is a marker for adequate secretory transformation of the endometrium.

Successful embryo implantation into the endometrium depends on embryonic characteristics and proper endometrial development. Reproductive medicine often focuses on embryo quality, whereas reliable diagnostic tests for endometrial receptivity are still needed. We previously found that human chorionic gonadotropin (hCG), one of the earliest proteins secreted by the embryo, was also expressed by the luteal phase endometrium around the implantation window. Here, we tested our hypothesis of endometrial hCG as an implantation marker. Endometrial biopsies and serum samples were taken from patients undergoing routine infertility diagnostics. Correlations of immunohistochemically detected endometrial hCG expression with adequate endometrial secretory transformation, the infiltration of CD45-positive leukocytes, clinical diagnostic parameters, and endometrial thickness were analyzed. A highly significant correlation between the endometrial score, as a measurement for regular secretory transformation, and the intensity of hCG staining was found. The invasion of CD45-positive leukocytes increased with progressing endometrial secretory transformation and rising endometrial hCG expression. In addition, serum progesterone concentrations correlated with hCG expression by the endometrial glands. Our results suggest endometrial hCG as a possible diagnostic parameter characterizing the endometrial secretory transformation and, thus, possibly also its implantation capability.

Presence of lytic Epstein-Barr virus infection in nasopharyngeal carcinoma.

Chromogenic Epstein-Barr virus-encoded RNA (EBER) in situ hybridization (EBER-ISH) is the gold standard to detect Epstein-Barr virus (EBV) but it is difficult to use in conjunction with immunohistochemistry (IHC). In this study, our purpose was to validate the sensitivity and specificity of RNAscope in detection of EBV infection in nasal epithelium and its stroma. Fluorescence-based RNAscope EBER-ISH, BRLF1-ISH, and lineage marker-IHC were performed on archived formalin-fixed paraffin-embedded tissues from normal nasal cavity (n = 5), nasopharynx (n = 8), and nasopharyngeal carcinoma (NPC) specimens (n = 10). The EBERs were detected in 10 of 10 NPC samples but was absent in all normal tissues from the nasal cavity and nasopharynx. The EBERs were exclusively located in pan-cytokeratin (pan-CK)-positive tumor epithelial cells but not in CD45-positive leukocytes and vimentin-positive stromal fibroblasts. The level of EBER expression varied in tumor cells within patient and between patients as well. Additionally, 5 of 10 patients had positive BRLF-ISH. We developed a simple and reproducible method to simultaneously detect mRNA and protein in formalin-fixed paraffin-embedded tissues of NPC. As a single staining, traditional EBER continues to be useful; however, for interpretation of the phenotype of EBV-infected cells, RNAscope is superior. Significantly, we showed that lytic EBV infection took place in NPC tumors.

A Novel Mouse Model of Traumatic Optic Neuropathy Using External Ultrasound Energy to Achieve Focal, Indirect Optic Nerve Injury

Traumatic optic neuropathy (TON) is a devastating cause of permanent visual loss following blunt injury to the head. Animal models for TON exist, but most fail to recapitulate the clinical scenario of closed head indirect trauma to the nerve and subsequent neurodegeneration. Thus, we developed a clinically-relevant animal model for TON using a novel ultrasonic pulse injury modality (sonication-induced TON; SI-TON). To trigger TON, a microtip probe sonifier was placed on the supraorbital ridge directly above the entrance of the optic nerve into the bony canal. An ultrasonic pulse was then delivered to the optic nerve. After injury, the number of RGCs in the retina as well as visual function measured by PERG steadily decreased over a two-week period. In the optic nerve, pro-inflammatory markers were upregulated within 6 hours following injury. Immunohistochemistry showed activation of microglia and infiltration of CD45-positive leukocytes in the optic nerve and initiation of a gliotic response. The SI-TON model is capable of delivering a non-contact concussive injury to the optic nerve and induce TON in mice. Thus, our data indicate that the SI-TON model reliably recapitulates the pathophysiology and progressive neurodegeneration seen in the human manifestation.

Changes in the extracellular matrix surrounding human chronic wounds revealed by 2-photon imaging.

Chronic wounds are a growing problem worldwide with no effective therapeutic treatments available. Our objective was to understand the composition of the dermal tissue surrounding venous leg ulcers and diabetic foot ulcers (DFU). We used novel 2-photon imaging techniques alongside classical histology to examine biopsies from the edges of two common types of chronic wound, venous leg ulcers and DFU. Compared to normal intact skin, we found that collagen levels are significantly reduced throughout the dermis of venous leg ulcer biopsies and DFU, with a reduction in both fibril thickness and abundance. Both wound types showed a significant reduction in elastin in the upper dermis, but in DFU, the loss was throughout the dermis. Loss of extracellular matrix correlated with high levels of CD68- and CD18-positive leukocytes. 2-photon imaging of the extracellular matrix in the intact tissue surrounding a chronic wound with a hand-held device may provide a useful clinical indicator on the healing progression or deterioration of these wounds.

Abstract 1627: Enhancement of anti-cancer immunity by OMX, a novel oxygen carrier immunotherapeutic that ameliorates the hypoxic tumor microenvironment

Abstract Hypoxia is a hallmark of cancer and a driver of tumor progression and poor patient outcomes. By generating an immunosuppressive tumor microenvironment that limits cytotoxic T lymphocyte (CTL) infiltration and activation, hypoxia limits the effectiveness of cancer immunotherapy and thus promotes tumor cell evasion of the host immune response. Omniox has developed a first-in-class anti-cancer immunotherapeutic, OMX, specifically designed to reverse tumor hypoxia to enhance cancer immunotherapy efficacy. In preclinical models, we have demonstrated that OMX accumulates in rodent subcutaneous and orthotopic tumors, as well as spontaneous canine melanomas and brain tumors, resulting in significant tumor hypoxia reduction.Here, using multiple subcutaneous syngeneic mouse tumor models (MC38, CT26, 4T1), we assessed OMX effects on intratumoral CTLs and immunosuppressive regulatory T cells (Treg), as well as the anti-tumor potential of OMX as a single agent and in combination with established immunotherapies. Using quantitative immunohistochemistry, we confirmed reports that hypoxic tumor areas are devoid of CTLs. Accordingly, by flow cytometry we observed a negative correlation between tumor hypoxia and CTL infiltration. While OMX single agent treatment did not affect the overall CD45-positive leukocyte population, Treg cells were selectively depleted and the CTL:Treg ratio was substantially increased, suggesting that OMX induced a shift towards immunosensitization. Consistent with this finding, we observed OMX single agent anti-tumor efficacy in MC38 colon tumors. Impressively, anti-tumor effects of OMX single agent were equivalent to that of a single treatment of the checkpoint inhibitor anti-CTLA4. We next assessed whether OMX would enhance the efficacy of checkpoint inhibitors when used in combination. In CT26 colon tumors, OMX exhibited combination anti-tumor activity with anti-CTLA4, giving rise to faster cures and a greater number of complete and durable responders compared to anti-CTLA4 alone. Of note, this enhanced response was observed for both early-stage and late-stage CT26 tumors. In 4T1 breast tumors, known to be insensitive to checkpoint inhibitors, treatment of early-stage (~60mm3) tumors with combination OMX and anti-PD1 resulted in a 27% response rate, compared to a 0% response rate to anti-PD1 alone. Taken together, our data suggest that OMX, by delivering oxygen to hypoxic tumor areas, induces a microenvironmental change from an immunosuppressive to an immunopermissive state. Given that OMX is well-tolerated in both small and large animals, and that its mechanism of action is upstream of numerous major immunosuppressive pathways, OMX holds great clinical potential to synergize with multiple immunotherapeutic agents to enhance tumor control by restoring anti-cancer immune responses in cancer patients. Citation Format: Kevin G. Leong, Yuqiong Pan, Jonathan A. Winger, Stephen P. Cary, Natacha Le Moan, Ana Krtolica. Enhancement of anti-cancer immunity by OMX, a novel oxygen carrier immunotherapeutic that ameliorates the hypoxic tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1627. doi:10.1158/1538-7445.AM2017-1627