Bioengineering of vascularized porcine flaps using perfusion-recellularization.

Large volume soft tissue defects greatly impact patient quality of life and function while suitable repair options remain a challenge in reconstructive surgery. Engineered flaps could represent a clinically translatable option that may circumvent issues related to donor site morbidity and tissue availability. Herein, we describe the regeneration of vascularized porcine flaps, specifically of the omentum and tensor fascia lata (TFL) flaps, using a tissue engineering perfusion-decellularization and recellularization approach. Flaps were decellularized using a low concentration sodium dodecyl sulfate (SDS) detergent perfusion to generate an acellular scaffold with retained extracellular matrix (ECM) components while removing underlying cellular and nuclear contents. A perfusion-recellularization strategy allowed for seeding of acellular flaps with a co-culture of human umbilical vein endothelial cell (HUVEC) and mesenchymal stromal cells (MSC) onto the decellularized omentum and TFL flaps. Our recellularization technique demonstrated evidence of intravascular cell attachment, as well as markers of endothelial and mesenchymal phenotype. Altogether, our findings support the potential of using bioengineered porcine flaps as a novel, clinically-translatable strategy for future application in reconstructive surgery.

Mice with a Pax2 missense variant display impaired glomerular repair.

PAX2 regulates kidney development, and its expression persists in parietal epithelial cells (PECs), potentially serving as a podocyte reserve. We hypothesized that mice with a Pax2 pathogenic missense variant (Pax2A220G/+) have impaired PEC-mediated podocyte regeneration. Embryonic wild-type mouse kidneys showed overlapping expression of PAX2/Wilms' tumor-1 (WT-1) until PEC and podocyte differentiation, reflecting a close lineage relationship. Embryonic and adult Pax2A220G/+ mice have reduced nephron number but demonstrated no glomerular disease under baseline conditions. Pax2A220G/+ mice compared with wild-type mice were more susceptible to glomerular disease after adriamycin (ADR)-induced podocyte injury, as demonstrated by worsened glomerular scarring, increased podocyte foot process effacement, and podocyte loss. There was a decrease in PAX2-expressing PECs in wild-type mice after adriamycin injury accompanied by the occurrence of PAX2/WT-1-coexpressing glomerular tuft cells. In contrast, Pax2A220G/+ mice showed no changes in the numbers of PAX2-expressing PECs after adriamycin injury, associated with fewer PAX2/WT-1-coexpressing glomerular tuft cells compared with injured wild-type mice. A subset of PAX2-expressing glomerular tuft cells after adriamycin injury was increased in Pax2A220G/+ mice, suggesting a pathological process given the worse outcomes observed in this group. Finally, Pax2A220G/+ mice have increased numbers of glomerular tuft cells expressing Ki-67 and cleaved caspase-3 compared with wild-type mice after adriamycin injury, consistent with maladaptive responses to podocyte loss. Collectively, our results suggest that decreased glomerular numbers in Pax2A220G/+ mice are likely compounded with the inability of their mutated PECs to regenerate podocyte loss, and together these two mechanisms drive the worsened focal segmental glomerular sclerosis phenotype in these mice.NEW & NOTEWORTHY Congenital anomalies of the kidney and urinary tract comprise some of the leading causes of kidney failure in children, but our previous study showed that one of its genetic causes, PAX2, is also associated with adult-onset focal segmental glomerular sclerosis. Using a clinically relevant model, our present study demonstrated that after podocyte injury, parietal epithelial cells expressing PAX2 are deployed into the glomerular tuft to assist in repair in wild-type mice, but this mechanism is impaired in Pax2A220G/+ mice.

Human-Induced Pluripotent Stem Cells in Plastic and Reconstructive Surgery.

A hallmark of plastic and reconstructive surgery is restoring form and function. Historically, tissue procured from healthy portions of a patient's body has been used to fill defects, but this is limited by tissue availability. Human-induced pluripotent stem cells (hiPSCs) are stem cells derived from the de-differentiation of mature somatic cells. hiPSCs are of particular interest in plastic surgery as they have the capacity to be re-differentiated into more mature cells, and cultured to grow tissues. This review aims to evaluate the applications of hiPSCs in the plastic surgery context, with a focus on recent advances and limitations. The use of hiPSCs and non-human iPSCs has been researched in the context of skin, nerve, vasculature, skeletal muscle, cartilage, and bone regeneration. hiPSCs offer a future for regenerated autologous skin grafts, flaps comprised of various tissue types, and whole functional units such as the face and limbs. Also, they can be used to model diseases affecting tissues of interest in plastic surgery, such as skin cancers, epidermolysis bullosa, and scleroderma. Tumorigenicity, immunogenicity and pragmatism still pose significant limitations. Further research is required to identify appropriate somatic origin and induction techniques to harness the epigenetic memory of hiPSCs or identify methods to manipulate epigenetic memory.

Tumor-specific cholinergic CD4+ T lymphocytes guide immunosurveillance of hepatocellular carcinoma.

Cholinergic nerves are involved in tumor progression and dissemination. In contrast to other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected. It remains unclear whether there is any form of cholinergic regulation of liver cancer. Here, we show that cholinergic T cells curtail the development of liver cancer by supporting antitumor immune responses. In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response and induction of two populations of CD4+ T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1+ T cells. Tumor antigens drove the clonal expansion of these cholinergic T cells in HCC. Genetic ablation of Chat in T cells led to an increased prevalence of preneoplastic cells and exacerbated liver cancer due to compromised antitumor immunity. Mechanistically, the cholinergic activity intrinsic in T cells constrained Ca2+-NFAT signaling induced by T cell antigen receptor engagement. Without this cholinergic modulation, hyperactivated CD25+ T regulatory cells and dysregulated PD-1+ T cells impaired HCC immunosurveillance. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology.

RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

Replication fork reversal is an important mechanism to protect the stability of stalled forks and thereby preserve genomic integrity. While multiple enzymes have been identified that can remodel forks, their regulation remains poorly understood. Here, we demonstrate that the ubiquitin ligase RFWD3, whose mutation causes Fanconi Anemia, promotes recruitment of the DNA translocase ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. Using electron microscopy, we show that RFWD3 stimulates fork remodeling in a ZRANB3-epistatic manner. Fork reversal is known to promote nascent DNA degradation in BRCA2-deficient cells. Consistent with a role for RFWD3 in fork reversal, inactivation of RFWD3 in these cells rescues fork degradation and collapse, analogous to ZRANB3 inactivation. RFWD3 loss impairs ZRANB3 localization to spontaneous nuclear foci induced by inhibition of the PCNA deubiquitinase USP1. We demonstrate that RFWD3 promotes PCNA ubiquitination and interaction with ZRANB3, providing a mechanism for RFWD3-dependent recruitment of ZRANB3. Together, these results uncover a new role for RFWD3 in regulating ZRANB3-dependent fork remodeling.

Light-Activated siRNA Endosomal Release (LASER) by Porphyrin Lipid Nanoparticles.

Lipid nanoparticles (LNPs) have achieved clinical success in delivering small interfering RNAs (siRNAs) for targeted gene therapy. However, endosomal escape of siRNA into the cytosol remains a fundamental challenge for LNPs. Herein, we report a strategy termed light-activated siRNA endosomal release (LASER) to address this challenge. We established a porphyrin-LNP by incorporating porphyrin-lipids into the clinically approved Onpattro formulation. The porphyrin-LNP maintained the physical properties of an LNP and generated reactive oxygen species (ROS) when irradiated with near-infrared (NIR) light. Using confocal microscopy, we revealed that porphyrin-lipids within the LNP translocate to endosomal membranes during endocytosis. The translocated porphyrin-lipids generated ROS under light irradiation and enabled LASER through endosomal membranes disruption as observed through GAL-9 recruitment and transmission electron microscopy (TEM). By establishing a quantitative confocal imaging method, we confirmed that porphyrin-LNPs can increase siRNA endosomal escape efficiency by up to 2-fold via LASER and further enhance luciferase target knockdown by 4-fold more in luciferase-transfected prostate cancer cells. Finally, we formulated porphyrin-LNPs encapsulated with gold nanoparticles (GNP) and visualized the LASER effect within prostate tumors via TEM, confirming the light-activated endosomal membrane disruption and subsequent GNP release into cytosols in vivo. Overall, porphyrin-LNPs and the LASER approach enhanced siRNA endosomal escape and significantly improved knockdown efficacy. We believe the versatility of this technology could be applied to various LNP-based RNA therapeutics.

IDH2 and TET2 mutations synergize to modulate T Follicular Helper cell functional interaction with the AITL microenvironment.

Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T cell lymphoma that originates from T follicular helper (Tfh) cells and exhibits a prominent tumor microenvironment (TME). IDH2 and TET2 mutations co-occur frequently in AITL, but their contribution to tumorigenesis is poorly understood. We developed an AITL mouse model that is driven by Idh2 and Tet2 mutations. Malignant Tfh cells display aberrant transcriptomic and epigenetic programs that impair TCR signaling. Neoplastic Tfh cells bearing combined Idh2 and Tet2 mutations show altered cross-talk with germinal center B cells that promotes B cell clonal expansion while decreasing Fas-FasL interaction and reducing B cell apoptosis. The plasma cell count and angiogenesis are also increased in the Idh2-mutated tumors, implying a major relationship between Idh2 mutation and the characteristic AITL TME. Our mouse model recapitulates several features of human IDH2-mutated AITL and provides a rationale for exploring therapeutic targeting of Tfh-TME cross-talk for AITL patients.

A noncoding single-nucleotide polymorphism at 8q24 drives IDH1-mutant glioma formation.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1R132H-driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Fabrication of succinate-alginate xerogel films for in vitro coupling of osteogenesis and neovascularization.

The osseointegration of metallic implants is reliant on a cascade of molecular interactions and the delivery of macromolecules to the implant environment that occurs before substantial bone formation. Early blood vessel formation is a requisite first step in the healing timeline for osteoid formation, where vascular development can be accelerated as a result of controlled hypoxic conditioning. In this study, alginate-derived xerogel films containing varied concentrations of disodium succinate salt which has been shown to induce pseudohypoxia (short-term hypoxic effects while maintaining an oxygenated environment) were developed. Xerogels were characterized for their morphology, succinate release over time and cellular response with osteoblast-mimicking Saos-2 and human umbilical vein endothelial cells (HUVEC). Scanning electron microscopy revealed a multiscale topography that may favour osseointegration and alamarBlue assays indicated no cytotoxic effects during in vitro proliferation of Saos-2 cells. pH measurements of eluted succinate reach 95 % of peak value after 7 h of immersion for all gels containing 10 mM of succinate or less, and 60 % within the first 40 min. In vitro exposure of HUVECs to succinate-conditioned media increased the net concentration of total proteins measured by bicinchoninic acid (BCA) assay and maintains stable vascular endothelial growth factor (VEGF) and extracellular platelet-derived growth factor (PDGF) for vessel formation through comparison of enzyme-linked immunosorbent assays (ELISAs) of the culture media and cell lysate. Tube formation assays also showed a sustained increase in tube diameter across the first 48 h of HUVEC culture when succinate concentrations of 1 and 10 µM in the xerogel. Overall, the succinate-alginate films serve as a prospective organic coating for bone-interfacing implant materials which may induce temporary pseudohypoxic conditions favourable for early angiogenesis and bone regeneration in vivo at succinate concentrations of 1 or 10 µM.

Loss of Epigenetic Regulation Disrupts Lineage Integrity, Induces Aberrant Alveogenesis, and Promotes Breast Cancer.

Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent "long-tail" breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 ("EpiDrivers"), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in ∼39% of human breast cancers, and ∼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology. SIGNIFICANCE: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711.

Cisplatin toxicity in the developing brain displays an absolute requirement for caspase-3.

Cisplatin is a member of a widely utilized class of chemotherapeutic agent that initiates DNA damage response, cell cycle arrest, and p53-dependent apoptotic cell death in concert with DNA­platinum adduct formation. While normal programmed cell death (PCD) can occur in the developing neuroepithelium in the absence of caspase-3 within certain genetic backgrounds, we observed an absolute dependency upon this executioner caspase with respect to cisplatin-induced PCD in the developing central nervous system (CNS). We therefore examined the nature of this genotoxic injury in the CNS in vivo, in which cisplatin treatment causes widespread cellular injury consistent with hallmarks of apoptosis which are averted upon caspase-3 inhibition. Examination of cisplatin-mediated injury as a function of time revealed the presence of an alternative, delayed form of necroptosis-like cell death which manifests in Casp3-/- neuroepithelia for several days following the normal pattern of apoptosis. Together, these findings suggest a coordinated regulation of these disparate PCD pathways in response to genotoxic stress in vivo and highlight the unique and critical role which caspase-3 plays among executioner caspases in coordinating apoptotic versus necroptotic responsiveness of the developing CNS to genotoxic injury.

Ionic liquid treatment for efficient sample preparation of hydrated bone for scanning electron microscopy.

This study presents a new protocol for preparing bone samples for scanning electron microscopy (SEM) using a room temperature ionic liquid (RTIL) treatment method. RTIL-based solutions can be adopted as an alternative to lengthy and laborious traditional means of preparation for SEM due to their unique low-vapour pressure and conductive properties. Applied to biological samples, RTILs can be used quickly and efficiently to observe hydrated, unfixed structures in typical SEM systems. This first-time feasibility study of the optimization of this protocol for bone was explored through various SEM modalities using two distinct ionic liquids, 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMI][BF4]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMI][BF4]), at varying concentrations of 5, 10, and 25 % v/v in aqueous solution through an addition-based method. Based on qualitative observations in the SEM, a 60-second solution addition treatment of 10 % v/v [BMI][BF4] performed the best in imaging hydrated, unfixed bone samples, resulting in minimal charge buildup and no solution pooling on the surface. The treatment was applied effectively to a variety of bone samples, notably flat and polished, as well as highly topographical bone fracture surfaces of both healthy and osteoporotic human bone samples. In comparison to conventionally dehydrated bone, the RTIL treatment better preserved the natural bone structure, resulting in minimal microcracking in observed structures.

The Protexin complex counters resection on stalled forks to promote homologous recombination and crosslink repair.

Protection of stalled replication forks is critical to genomic stability. Using genetic and proteomic analyses, we discovered the Protexin complex containing the ssDNA binding protein SCAI and the DNA polymerase REV3. Protexin is required specifically for protecting forks stalled by nucleotide depletion, fork barriers, fragile sites, and DNA inter-strand crosslinks (ICLs), where it promotes homologous recombination and repair. Protexin loss leads to ssDNA accumulation and profound genomic instability in response to ICLs. Protexin interacts with RNA POL2, and both oppose EXO1's resection of DNA on forks remodeled by the FANCM translocase activity. This pathway acts independently of BRCA/RAD51-mediated fork stabilization, and cells with BRCA2 mutations were dependent on SCAI for survival. These data suggest that Protexin and its associated factors establish a new fork protection pathway that counteracts fork resection in part through a REV3 polymerase-dependent resynthesis mechanism of excised DNA, particularly at ICL stalled forks.

Therapeutic inhibition of USP9x-mediated Notch signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a breast cancer subtype that lacks targeted treatment options. The activation of the Notch developmental signaling pathway, which is a feature of TNBC, results in the secretion of proinflammatory cytokines and the recruitment of protumoral macrophages to the tumor microenvironment. While the Notch pathway is an obvious therapeutic target, its activity is ubiquitous, and predictably, anti-Notch therapies are burdened with significant on-target side effects. Previously, we discovered that, under conditions of cellular stress commonly found in the tumor microenvironment, the deubiquitinase USP9x forms a multiprotein complex with the pseudokinase tribbles homolog 3 (TRB3) that together activate the Notch pathway. Herein, we provide preclinical studies that support the potential of therapeutic USP9x inhibition to deactivate Notch. Using a murine TNBC model, we show that USP9x knockdown abrogates Notch activation, reducing the production of the proinflammatory cytokines, C-C motif chemokine ligand 2 (CCL2) and interleukin-1 beta (IL-1ß). Concomitant with these molecular changes, a reduction in tumor inflammation, the augmentation of antitumor immune response, and the suppression of tumor growth were observed. The pharmacological inhibition of USP9x using G9, a partially selective, small-molecule USP9x inhibitor, reduced Notch activity, remodeled the tumor immune landscape, and reduced tumor growth without associated toxicity. Proving the role of Notch, the ectopic expression of the activated Notch1 intracellular domain rescued G9-induced effects. This work supports the potential of USP9x inhibition to target Notch in metabolically vulnerable tissues like TNBC, while sparing normal Notch-dependent tissues.

Electron Microscopy Imaging Applications of Room Temperature Ionic Liquids in the Biological Field: A Review.

For biological imaging using electron microscopy (EM), the use of room-temperature ionic liquids (RTILs) has been proposed as an alternative to traditional lengthy preparation methods. With their low vapor pressures and conductivity, RTILs can be applied onto hard-to-image soft and/or wet samples without dehydration - allowing for a more representative, hydrated state of material and opening the possibility for visualization of in situ physiological processes using conventional EM systems. However, RTILs have yet to be utilized to their full potential by microscopists and microbiologists alike. To this end, this review aims to provide a comprehensive summary of biological applications of RTILs for EM to bridge the RTIL, in situ microscopy, and biological communities. We outline future research avenues for the use of RTILs for the EM observation of biological samples, notably i) RTIL selection and optimization, ii) applications for live cell processes and iii) electron beam and ionic liquid interaction studies.

Necroptotic-Apoptotic Regulation in an Endothelin-1 Model of Cerebral Ischemia.

The primary forms of cell death seen in ischemic stroke are of two major types: a necrotic/necroptotic form, and an apoptotic form that is frequently seen in penumbral regions of injury. Typically apoptotic versus necroptotic programmed cell death is described as competitive in nature, where necroptosis is often described as playing a backup role to apoptosis. In the present study, we examined the relationship between these two forms of cell death in a murine endothelin-1 model of ischemia-reperfusion injury in wildtype and caspase-3 null mice with and without addition of the pharmacologic RIPK1 phosphorylation inhibitor necrostatin-1. Analyses of ischemic brain injury were performed via both cellular and volumetric assessments, electron microscopy, TUNEL staining, activated caspase-3 and caspase-7 staining, as well as CD11b and F4/80 staining. Inhibition of caspase-3 or RIPK1 phosphorylation demonstrates significant neural protective effects which are non-additive and exhibit significant overlap in protected regions. Interestingly, morphologic analysis of the cortex demonstrates reduced apoptosis following RIPK1 inhibition. Consistent with this, RIPK1 inhibition reduces the levels of both caspase-3 and caspase-7 activation. Additionally, this protection appears independent of secondary inflammatory mediators. Together, these observations demonstrate that the necroptotic protein RIPK1 modifies caspase-3/-7 activity, ultimately resulting in decreased neuronal apoptosis. These findings thus modify the traditional exclusionary view of apoptotic/necroptotic signaling, revealing a new form of interaction between these dominant forms of cell death.

Hypoxia-inducible factor 1 alpha limits dendritic cell stimulation of CD8 T cell immunity.

Dendritic cells are sentinels of the immune system and represent a key cell in the activation of the adaptive immune response. Hypoxia-inducible factor 1 alpha (HIF-1α)-a crucial oxygen sensor stabilized during hypoxic conditions-has been shown to have both activating and inhibitory effects in immune cells in a context- and cell-dependent manner. Previous studies have demonstrated that in some immune cell types, HIF-1α serves a pro-inflammatory role. Genetic deletion of HIF-1α in macrophages has been reported to reduce their pro-inflammatory function. In contrast, loss of HIF-1α enhanced the pro-inflammatory activity of dendritic cells in a bacterial infection model. In this study, we aimed to further clarify the effects of HIF-1α in dendritic cells. Constitutive expression of HIF-1α resulted in diminished immunostimulatory capacity of dendritic cells in vivo, while conditional deletion of HIF-1α in dendritic cells enhanced their ability to induce a cytotoxic T cell response. HIF-1α-expressing dendritic cells demonstrated increased production of inhibitory mediators including IL-10, iNOS and VEGF, which correlated with their reduced capacity to drive effector CD8+ T cell function. Altogether, these data reveal that HIF-1α can promote the anti-inflammatory functions of dendritic cells and provides insight into dysfunctional immune responses in the context of HIF-1α activation.

E3 ligase RFWD3 is a novel modulator of stalled fork stability in BRCA2-deficient cells.

BRCA1/2 help maintain genomic integrity by stabilizing stalled forks. Here, we identify the E3 ligase RFWD3 as an essential modulator of stalled fork stability in BRCA2-deficient cells and show that codepletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity upon replication stress. Stalled forks in BRCA2-deficient cells accumulate phosphorylated and ubiquitinated replication protein A (ubq-pRPA), the latter of which is mediated by RFWD3. Generation of this intermediate requires SMARCAL1, suggesting that it depends on stalled fork reversal. We show that in BRCA2-deficient cells, rescuing fork degradation might not be sufficient to ensure fork repair. Depleting MRE11 in BRCA2-deficient cells does block fork degradation, but it does not prevent fork collapse and cell sensitivity in the presence of replication stress. No such ubq-pRPA intermediate is formed in BRCA1-deficient cells, and our results suggest that BRCA1 may function upstream of BRCA2 in the stalled fork repair pathway. Collectively, our data uncover a novel mechanism by which RFWD3 destabilizes forks in BRCA2-deficient cells.

Tumor cell expression of B7-H4 correlates with higher frequencies of tumor-infiltrating APCs and higher CXCL17 expression in human epithelial ovarian cancer.

B7-H4, an immune suppressive member of the B7 family, is highly expressed in a wide variety of human malignancies making it an attractive immunotherapeutic target. However, the association between B7-H4 expression in the tumor microenvironment and the immune infiltrate has not been comprehensively examined. To evaluate the immune tumor microenvironment, we analyzed epithelial ovarian tumors from 28 patients using flow cytometry, immunohistochemistry, functional, and genomic analyses. We determined B7-H4 expression patterns and compared the immune infiltrates of tumors with high and low surface expression of B7-H4. Frequencies and phenotypes of tumor and immune cells were determined using multiple flow cytometry panels. Immunohistochemistry was used to analyze cellular infiltration and location. Publicly available datasets were interrogated to determine intratumoral cytokine and chemokine expression. We found that B7-H4 was predominantly expressed by tumor cells in the epithelial ovarian tumor microenvironment. Surface expression of B7-H4 on tumor cells was correlated with higher levels of infiltrating mature antigen-presenting cells. Further, expression of CXCL17, a monocyte and dendritic cell chemoattractant, correlated strongly with B7-H4 expression. T cells expressed activation markers, but T cells expressing a combination of markers associated with T cell activation/exhaustion phenotype were not prevalent. Overall, our data suggest that B7-H4 is associated with a pro-inflammatory tumor microenvironment.

Endothelin-1-mediated cerebral ischemia in mice: early cellular events and the role of caspase-3.

Over the past 30 years a number of animal models of cerebral ischemic injury have been developed. Middle cerebral artery occlusion (MCAO) in particular reproduces both ischemic and reperfusion elements and is widely utilized as a model of ischemic stroke in rodents. However substantial variability exists in this model even in clonal inbred mice due to stochastic elements of the cerebral vasculature. Models such as MCAO thus exhibit significant irreducible variabilities with respect to their zone of injury as well as inducing a sizable volume of injury to the cerebrum with damage to sub-cortical structures, conditions not typically seen for the majority of human clinical strokes. An alternative model utilizes endothelin-1 application focally to cerebral vasculature, resulting in an ischemic reperfusion injury which more closely mimics that seen in human clinical stroke. In order to further define this model we demonstrate that intra-cortical administration of ET-1 results in a highly reproducible pattern of tissue injury which is limited to the cerebral cortex, characterizing the early cellular and molecular events which occur during the first 24 h post-injury. In addition we demonstrate that caspase-3 is both necessary and sufficient to regulate a majority of cortical cell death observed during this period. The enhanced survival effects seen upon genetic deletion of caspase-3 appear to arise as a result of direct modification of cell autonomous PCD signaling as opposed to secondary effectors such as granulocyte infiltration or microglia activation. Taken together these findings detail the early mechanistic features regulating endothelin-1-mediated ischemic injury.