Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Polyomavirus-associated Disseminated T-cell Lymphoma in a Colony of Zebra Finches (Taeniopygia guttata).

Four zebra finches in a closed research colony presented with variable clinical signs, including masses, skin lesions, shivering, and/or ruffled feathers. These birds were not responsive to treatment efforts; 3 died and one was euthanized. All 4 were submitted for necropsy to determine the cause of the clinical signs. Gross necropsy and histopathologic findings from all birds resulted in a diagnosis of round cell neoplasia in multiple organs, including the skin, liver, kidney, and reproductive tract, with intranuclear inclusion bodies in the neoplastic cells. In all 4 cases, immunohistochemical staining showed strong immunoreactivity for CD3 in 70% to 80% of the neoplastic round cells, with a relatively small subset that were immunopositive for Pax5. These findings supported a diagnosis of T-cell lymphoma. Frozen liver tissue from one case was submitted for next-generation sequencing (NGS), which revealed viral RNA with 100% sequence homology to canary polyomavirus strain 34639 that had originally been identified in a European goldfinch. Formalin-fixed paraffin-embedded scrolls from another case were also submitted for NGS, which revealed viral RNA with 97.2% sequence homology to canary polyomavirus strain 37273 that had originally been identified in a canary. To localize the virus in situ, RNAscope hybridization was performed using a probe designed to target the VP1 gene of the sequenced virus in frozen liver tissue. In all 4 cases, disseminated and robust hybridization signals were detected in neoplastic cells. These findings indicate that polyomaviruses have the potential to be oncogenic in zebra finches.

Toxicologic Pathology Forum: Considerations Regarding Determination of Adversity for Immunopathology Findings in Nonclinical Toxicology Studies with Immune-Modulating Therapeutics.

The evaluation of changes in the immune system serves to determine the efficacy and potential immunotoxicologic effects of new products under development. Toxicologic pathologists play critical roles in identifying immune system changes that drive the immunosafety determination. Standard pathology evaluations of therapies and chemicals remain similar; however, biopharmaceutical therapies have moved from simply affecting the immune system to being specifically developed to modify the immune system, which can impact interpretation. Recent explosive growth in immunomodulatory therapies presents a challenge to the toxicologic pathologist, toxicologist, and regulatory reviewer in terms of evaluating the clinical relevance and potential adversity of immune system changes. Beyond the recognition of such changes, there is an increasing expectation to evaluate, describe, and interpret how therapies affect complex immune system pathways for both immunomodulatory therapies and non-immunomodulatory drugs with off-target immunotoxic effects. In this opinion piece, considerations regarding immune system evaluation, the current landscape of immunomodulatory therapies, a brief description of immunotoxicologic (and immunopathologic) endpoints, the importance of integrating such immunosafety data, and relevance to adversity determination are discussed. Importantly, we describe how the current paradigm of determining adversity for immune system changes may be challenging or insufficient and propose a harmonized and flexible approach for assessing adversity.

Pretreatment with a novel Toll-like receptor 4 agonist attenuates renal ischemia-reperfusion injury.

Acute kidney injury (AKI) is common in surgical and critically ill patients. This study examined whether pretreatment with a novel Toll-like receptor 4 agonist attenuated ischemia-reperfusion injury (IRI)-induced AKI (IRI-AKI). We performed a blinded, randomized-controlled study in mice pretreated with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD), a synthetic Toll-like receptor 4 agonist. Two cohorts of male BALB/c mice received intravenous vehicle or PHAD (2, 20, or 200 µg) at 48 and 24 h before unilateral renal pedicle clamping and simultaneous contralateral nephrectomy. A separate cohort of mice received intravenous vehicle or 200 µg PHAD followed by bilateral IRI-AKI. Mice were monitored for evidence of kidney injury for 3 days postreperfusion. Kidney function was assessed by serum blood urea nitrogen and creatinine measurements. Kidney tubular injury was assessed by semiquantitative analysis of tubular morphology on periodic acid-Schiff (PAS)-stained kidney sections and by kidney mRNA quantification of injury [neutrophil gelatinase-associated lipocalin (Ngal), kidney injury molecule-1 (Kim-1), and heme oxygenase-1 (Ho-1)] and inflammation [interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (Tnf-α)] using quantitative RT-PCR. Immunohistochemistry was used to quantify proximal tubular cell injury and renal macrophages by quantifying the areas stained with Kim-1 and F4/80 antibodies, respectively, and TUNEL staining to detect the apoptotic nuclei. PHAD pretreatment yielded dose-dependent kidney function preservation after unilateral IRI-AKI. Histological injury, apoptosis, Kim-1 staining, and Ngal mRNA were lower in PHAD-treated mice and IL-1ß mRNA was higher in PHAD-treated mice. Similar pretreatment protection was noted with 200 mg PHAD after bilateral IRI-AKI, with significantly reduced Kim-1 immunostaining in the outer medulla of mice treated with PHAD after bilateral IRI-AKI. In conclusion, PHAD pretreatment leads to dose-dependent protection from renal injury after unilateral and bilateral IRI-AKI in mice.NEW & NOTEWORTHY Pretreatment with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide; a novel synthetic Toll-like receptor 4 agonist, preserves kidney function during ischemia-reperfusion injury-induced acute kidney injury.

Lipocalin-2 is an essential component of the innate immune response to Acinetobacter baumannii infection.

Acinetobacter baumannii is an opportunistic pathogen and an emerging global health threat. Within healthcare settings, major presentations of A. baumannii include bloodstream infections and ventilator-associated pneumonia. The increased prevalence of ventilated patients during the COVID-19 pandemic has led to a rise in secondary bacterial pneumonia caused by multidrug resistant (MDR) A. baumannii. Additionally, due to its MDR status and the lack of antimicrobial drugs in the development pipeline, the World Health Organization has designated carbapenem-resistant A. baumannii to be its priority critical pathogen for the development of novel therapeutics. To better inform the design of new treatment options, a comprehensive understanding of how the host contains A. baumannii infection is required. Here, we investigate the innate immune response to A. baumannii by assessing the impact of infection on host gene expression using NanoString technology. The transcriptional profile observed in the A. baumannii infected host is characteristic of Gram-negative bacteremia and reveals expression patterns consistent with the induction of nutritional immunity, a process by which the host exploits the availability of essential nutrient metals to curtail bacterial proliferation. The gene encoding for lipocalin-2 (Lcn2), a siderophore sequestering protein, was the most highly upregulated during A. baumannii bacteremia, of the targets assessed, and corresponds to robust LCN2 expression in tissues. Lcn2-/- mice exhibited distinct organ-specific gene expression changes including increased transcription of genes involved in metal sequestration, such as S100A8 and S100A9, suggesting a potential compensatory mechanism to perturbed metal homeostasis. In vitro, LCN2 inhibits the iron-dependent growth of A. baumannii and induces iron-regulated gene expression. To elucidate the role of LCN2 in infection, WT and Lcn2-/- mice were infected with A. baumannii using both bacteremia and pneumonia models. LCN2 was not required to control bacterial growth during bacteremia but was protective against mortality. In contrast, during pneumonia Lcn2-/- mice had increased bacterial burdens in all organs evaluated, suggesting that LCN2 plays an important role in inhibiting the survival and dissemination of A. baumannii. The control of A. baumannii infection by LCN2 is likely multifactorial, and our results suggest that impairment of iron acquisition by the pathogen is a contributing factor. Modulation of LCN2 expression or modifying the structure of LCN2 to expand upon its ability to sequester siderophores may thus represent feasible avenues for therapeutic development against this pathogen.

A Single Nucleotide Polymorphism in SH2B3/LNK Promotes Hypertension Development and Renal Damage.

BACKGROUND: SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism in SH2B3 (rs3184504) results in substitution of tryptophan (Trp) for arginine (Arg) at amino acid 262 and is a top association signal for hypertension in human genome-wide association studies. Whether this variant is causal for hypertension, and if so, the mechanism by which it impacts pathogenesis is unknown. METHODS: We used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of this SH2B3 polymorphism. Mice underwent angiotensin II (Ang II) infusion to evaluate differences in blood pressure (BP) elevation and end-organ damage including albuminuria and renal fibrosis. Cytokine production and Stat4 phosphorylation was also assessed in Arg/Arg and Trp/Trp T cells. RESULTS: Trp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8+ T cells from Ang II-infused Trp/Trp mice produce significantly more interferon gamma (IFNg) compared to Arg/Arg controls. Interleukin-12 (IL-12)-induced IFNg production is greater in Trp/Trp compared to Arg/Arg CD8+ T cells. In addition, IL-12 enhances Stat4 phosphorylation to a greater degree in Trp/Trp compared to Arg/Arg CD8+ T cells, suggesting that Trp-encoding SH2B3 exhibits less negative regulation of IL-12 signaling to promote IFNg production. Finally, we demonstrated that a multi-SNP model genetically predicting increased SH2B3 expression in lymphocytes is inversely associated with hypertension and hypertensive chronic kidney disease in humans.. CONCLUSIONS: Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

Confirmation of Pathogen 'Burnout' in Mouse Colonies with Previous Evidence of Infection with Parvovirus and Rotavirus.

Pathogen monitoring and colony health management are critical components of any rodent research program. From an operational perspective, rodent facilities are protected from unwanted infectious agents by facility-specific bioexclusion criteria, sanitation of the physical environment, and personal protective equipment. Another important preventative measure is the use of room health levels to provide traffic patterns for animal care and research staff as they move between rooms of differing health status. For mice, our institution uses a tiered room level system with 6 defined categories, ranging from level 1 (strictest entry criteria) to 6 (least stringent entry criteria). Level 6 is defined as rooms with mice that have tested positive for mouse parvovirus (MPV) or mouse rotavirus (MRV) or both on sentinel serology at any point in time in the past and no decontamination. Because many of our mouse rooms had historically been positive for MPV and/or MRV and because of the high financial and logistic challenges of using repeated test-and-cull for elimination, we had tolerated the potential presence of MPV and MRV and had developed management practices that would promote 'burnout' (that is, elimination of infectious agents due to absence of susceptible hosts) of these pathogens. Analysis of sentinel data showed that we had 28 rooms in 4 facilities for which excluded pathogens had not been identified in 3 y or more. We therefore developed a hybrid testing strategy involving both PCR analysis and serology and implemented it in sentinels and in select colony mice to determine whether the rooms had undergone successful burnout and were free of MPV and MRV. All test results obtained during the assessment were negative for both viruses, and the rooms were subsequently upgraded to level 5 (free from excluded pathogens and allowing two-way movement in and out of housing room). All upgraded rooms have remained negative on subsequent quarterly routine sentinel serology for over 3 y. Our testing strategy for confirming pathogen burnout may be a useful and cost-efficient model for other academic rodent research programs that face a similar situation.

Symmetric Dimethylarginine Is a Sensitive Biomarker of Glomerular Injury in Rats.

Glomerular filtration rate is the gold-standard method for assessment of renal function but is rarely performed in routine toxicity studies. Standard serum biomarkers of renal function are insensitive and become elevated only with significant loss of organ function. Symmetric dimethylarginine (SDMA) is a ubiquitous analyte that is freely filtered by the glomerulus and can be detected in serum. It has shown utility for the detection of renal injury in dogs and cats in clinical veterinary practice, but the potential utility of SDMA to detect renal injury in preclinical species or toxicity studies has not been thoroughly investigated. We utilized a well-characterized glomerular toxicant, puromycin aminonucleoside, to induce podocyte injury and subsequent proteinuria in young male Sprague-Dawley rats. At the end of 1 or 2 weeks, blood, urine, and kidney tissue were collected for analysis. One week following a single 50 mg/kg dose, urea nitrogen, creatinine, and albumin mean values were within historical control ranges, while SDMA was increased. Glomerular changes in these animals included periodic acid-Schiff positive globules within podocytes, podocyte hypertrophy by light microscopy, and podocyte degeneration with effacement of foot processes by electron microscopy (EM). Taken together, our data indicate that SDMA may be a useful biomarker for early detection of glomerular toxicities in rats.

Deep Learning Approaches and Applications in Toxicologic Histopathology: Current Status and Future Perspectives.

Whole slide imaging enables the use of a wide array of digital image analysis tools that are revolutionizing pathology. Recent advances in digital pathology and deep convolutional neural networks have created an enormous opportunity to improve workflow efficiency, provide more quantitative, objective, and consistent assessments of pathology datasets, and develop decision support systems. Such innovations are already making their way into clinical practice. However, the progress of machine learning - in particular, deep learning (DL) - has been rather slower in nonclinical toxicology studies. Histopathology data from toxicology studies are critical during the drug development process that is required by regulatory bodies to assess drug-related toxicity in laboratory animals and its impact on human safety in clinical trials. Due to the high volume of slides routinely evaluated, low-throughput, or narrowly performing DL methods that may work well in small-scale diagnostic studies or for the identification of a single abnormality are tedious and impractical for toxicologic pathology. Furthermore, regulatory requirements around good laboratory practice are a major hurdle for the adoption of DL in toxicologic pathology. This paper reviews the major DL concepts, emerging applications, and examples of DL in toxicologic pathology image analysis. We end with a discussion of specific challenges and directions for future research.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Rabbit.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and non-proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the laboratory rabbit used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Hypoxia inducible factor signaling in breast tumors controls spontaneous tumor dissemination in a site-specific manner.

Hypoxia is a common feature in tumors and induces signaling that promotes tumor cell survival, invasion, and metastasis, but the impact of hypoxia inducible factor (HIF) signaling in the primary tumor on dissemination to bone in particular remains unclear. To better understand the contributions of hypoxia inducible factor 1 alpha (HIF1α), HIF2α, and general HIF pathway activation in metastasis, we employ a PyMT-driven spontaneous murine mammary carcinoma model with mammary specific deletion of Hif1α, Hif2α, or von Hippel-Lindau factor (Vhl) using the Cre-lox system. Here we show that Hif1α or Hif2α deletion in the primary tumor decreases metastatic tumor burden in the bone marrow, while Vhl deletion increases bone tumor burden, as hypothesized. Unexpectedly, Hif1α deletion increases metastatic tumor burden in the lung, while deletion of Hif2α or Vhl does not affect pulmonary metastasis. Mice with Hif1α deleted tumors also exhibit reduced bone volume as measured by micro computed tomography, suggesting that disruption of the osteogenic niche may be involved in the preference for lung dissemination observed in this group. Thus, we reveal that HIF signaling in breast tumors controls tumor dissemination in a site-specific manner.

Top-Down Fabricated microPlates for Prolonged, Intra-articular Matrix Metalloproteinase 13 siRNA Nanocarrier Delivery to Reduce Post-traumatic Osteoarthritis.

Post-traumatic osteoarthritis (PTOA) associated with joint injury triggers a degenerative cycle of matrix destruction and inflammatory signaling, leading to pain and loss of function. Here, prolonged RNA interference (RNAi) of matrix metalloproteinase 13 (MMP13) is tested as a PTOA disease modifying therapy. MMP13 is upregulated in PTOA and degrades the key cartilage structural protein type II collagen. Short interfering RNA (siRNA) loaded nanoparticles (siNPs) were encapsulated in shape-defined poly(lactic-co-glycolic acid) (PLGA) based microPlates (µPLs) to formulate siNP-µPLs that maintained siNPs in the joint significantly longer than delivery of free siNPs. Treatment with siNP-µPLs against MMP13 (siMMP13-µPLs) in a mechanical load-induced mouse model of PTOA maintained potent (65-75%) MMP13 gene expression knockdown and reduced MMP13 protein production in joint tissues throughout a 28-day study. MMP13 silencing reduced PTOA articular cartilage degradation/fibrillation, meniscal deterioration, synovial hyperplasia, osteophytes, and pro-inflammatory gene expression, supporting the therapeutic potential of long-lasting siMMP13-µPL therapy for PTOA.

Amelioration of post-traumatic osteoarthritis via nanoparticle depots delivering small interfering RNA to damaged cartilage.

The progression of osteoarthritis is associated with inflammation triggered by the enzymatic degradation of extracellular matrix in injured cartilage. Here we show that a locally injected depot of nanoparticles functionalized with an antibody targeting type II collagen and carrying small interfering RNA targeting the matrix metalloproteinase 13 gene (Mmp13), which breaks down type II collagen, substantially reduced the expression of MMP13 and protected cartilage integrity and overall joint structure in acute and severe mouse models of post-traumatic osteoarthritis. MMP13 inhibition suppressed clusters of genes associated with tissue restructuring, angiogenesis, innate immune responses and proteolysis. We also show that intra-articular injections of the nanoparticles led to greater reductions in disease progression than either a single injection or weekly injections of the steroid methylprednisolone. Sustained drug retention by targeting collagen in the damaged extracellular matrix of osteoarthritic cartilage may also be an effective strategy for the treatment of osteoarthritis with other disease-modifying drugs.

Shape-Defined microPlates for the Sustained Intra-articular Release of Dexamethasone in the Management of Overload-Induced Osteoarthritis.

Osteoarthritis (OA) is treated with the intra-articular injection of steroids such as dexamethasone (DEX) to provide short-term pain management. However, DEX treatment suffers from rapid joint clearance. Here, 20 × 10 µm, shape-defined poly(d,l-lactide-co-glycolide)acid microPlates (µPLs) are created and intra-articularly deposited for the sustained release of DEX. Under confined conditions, DEX release is projected to persist for several months, with only ∼20% released in the first month. In a highly rigorous murine knee overload injury model (post-traumatic osteoarthritis), a single intra-articular injection of Cy5-µPLs is detected in the cartilage surface, infrapatellar fat pad/synovium, joint capsule, and posterior joint space up to 30 days. One intra-articular injection of DEX-µPL (1 mg kg-1) decreased the expression of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6, and matrix metalloproteinase (MMP)-13 by approximately half compared to free DEX at 4 weeks post-treatment. DEX-µPL also reduced load-induced histological changes in the articular cartilage and synovial tissues relative to saline or free DEX. In sum, the µPLs provide sustained drug release along with the capability to precisely control particle geometry and mechanical properties, yielding long-lasting benefits in overload-induced OA. This work motivates further study and development of particles that provide combined pharmacological and mechanical benefits.

Effect of pore size and spacing on neovascularization of a biodegradble shape memory polymer perivascular wrap.

Neointimal hyperplasia (NH) is a main source of failures in arteriovenous fistulas and vascular grafts. Several studies have demonstrated the promise of perivascular wraps to reduce NH via promotion of adventitial neovascularization and providing mechanical support. Limited clinical success thus far may be due to inappropriate material selection (e.g., nondegradable, too stiff) and geometric design (e.g., pore size and spacing, diameter). The influence of pore size and spacing on implant neovascularization is investigated here for a new biodegradable, thermoresponsive shape memory polymer (SMP) perivascular wrap. Following an initial pilot, 21 mice were each implanted with six scaffolds: four candidate SMP macroporous designs (a-d), a nonporous SMP control (e), and microporous GORETEX (f). Mice were sacrificed after 4 (N = 5), 14 (N = 8), and 28 (N = 8) days. There was a statistically significant increase in neovascularization score between all macroporous groups compared to nonporous SMP (p < .023) and microporous GORETEX (p < .007) controls at Day 28. Wider-spaced, smaller-sized pore designs (223 µm-spaced, 640 µm-diameter Design c) induced the most robust angiogenic response, with greater microvessel number (p < .0114) and area (p < .0055) than nonporous SMPs and GORETEX at Day 28. This design also produced significantly greater microvessel density than nonporous SMPs (p = 0.0028) and a smaller-spaced, larger-sized pore (155 µm-spaced, 1,180 µm-sized Design b) design (p = .0013). Strong neovascularization is expected to reduce NH, motivating further investigation of this SMP wrap with controlled pore spacing and size in more advanced arteriovenous models.

Effects of Acrylic Tunnel Enrichment on Anxiety-Like Behavior, Neurogenesis, and Physiology of C57BL/6J Mice.

Environmental enrichment for mice lags behind the standard enrichment offered to other laboratory rodents due to concerns about environmental variability and, in specific contexts, aggression. Our objective in this study was to evaluate concerns that the introduction of structural enrichment in the form of a single red acrylic mouse tunnel into murine housing may confound study findings. We measured effects on anxiety-like behaviors (elevated zero maze and open field activity), hippocampal neurogenesis, body weight gain, and physiologic markers of stress (adrenal gland weight, plasma corticosterone concentration, and neutrophil:lymphocyte ratio). Male and female C57BL/6J mice were randomly assigned to one of 2 groups: a standard-housed control group with enrichment consisting of paper nesting material, or an enriched group that received a single acrylic tunnel in addition to nesting material. All results fell within biologically normal ranges regardless of treatment, and variability (standard deviation) was not significantly different between groups for any measure. Mice in the enriched group showed modest differences during open field testing suggestive of decreased anxiety, traveling farther and depositing fewer fecal boli than standard-housed mice. Male mice in the tunnel-enriched group gained more body weight than standard-housed male mice. No significant effects by treatment were found in neurogenic or physiologic parameters. These results indicate that provision of simple structural enrichment is unlikely to have confounding effects on murine anxiety-like behaviors, neurogenesis, body weight gain, or physiologic parameters. We therefore recommend the inclusion of simple structural enrichment, such as an acrylic tunnel, to the standard environmental enrichment of social housing and nesting material for mice.

A Genetic Mouse Model Recapitulates Immune Checkpoint Inhibitor-Associated Myocarditis and Supports a Mechanism-Based Therapeutic Intervention.

Immune checkpoint inhibitors (ICI) targeting CTLA4 or PD-1/PD-L1 have transformed cancer therapy but are associated with immune-related adverse events, including myocarditis. Here, we report a robust preclinical mouse model of ICI-associated myocarditis in which monoallelic loss of Ctla4 in the context of complete genetic absence of Pdcd1 leads to premature death in approximately half of mice. Premature death results from myocardial infiltration by T cells and macrophages and severe ECG abnormalities, closely recapitulating the clinical and pathologic hallmarks of ICI-associated myocarditis observed in patients. Using this model, we show that Ctla4 and Pdcd1 functionally interact in a gene dosage-dependent manner, providing a mechanism by which myocarditis arises with increased frequency in the setting of combination ICI therapy. We demonstrate that intervention with CTLA4-Ig (abatacept) is sufficient to ameliorate disease progression and additionally provide a case series of patients in which abatacept mitigates the fulminant course of ICI myocarditis. SIGNIFICANCE: We provide a preclinical model of ICI-associated myocarditis which recapitulates this clinical syndrome. Using this model, we demonstrate that CTLA4 and PD-1 (ICI targets) functionally interact for myocarditis development and that intervention with CTLA4-Ig (abatacept) attenuates myocarditis, providing mechanistic rationale and preclinical support for therapeutic clinical studies.See related commentary by Young and Bluestone, p. 537.This article is highlighted in the In This Issue feature, p. 521.

Retraction: Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

[This retracts the article DOI: 10.18632/oncotarget.6427.].

Severe Periocular Edema after Intraarterial Carboplatin Chemotherapy for Retinoblastoma in a Rabbit (Oryctolagus cuniculus) Model.

Endovascular microcatheter-based intraarterial (ophthalmic artery) chemotherapy is becoming widely used for the clinical treatment of intraocular retinoblastoma due to its apparent increased efficacy compared with traditional intravenous chemotherapy; however local ocular complications are not uncommon. Carboplatin is a chemotherapeutic agent used in both intravenous and intraarterial chemotherapy. We used rabbits to assess pharmacokinetics and ocular and systemic toxicity after intraarterial carboplatin infusion. Subsequent to unilateral intraarterial administration of carboplatin, severe unilateral or bilateral periocular edema occurred in 6 adult male New Zealand white rabbits. Time to onset varied from less than 4 h after administration (n = 3, 50 mg) to approximately 24 h afterward (n = 3, 25 mg). After becoming symptomatic, 5 of the 6 animals were promptly euthanized, and the remaining animal (25 mg treatment) was medically managed for 4 d before being euthanized due to intractable edema-related lagophthalmos. Globes and orbits from all 6 euthanized rabbits were harvested en bloc; whole-mount sections were prepared for histologic evaluation, which revealed drug-induced vasogenic edema in confined spaces as the main underlying pathogenesis. Transient and self-limiting periocular edema is a common side effect of intraarterial chemotherapy but is thought to occur predominantly with melphalan monotherapy or combination therapy using melphalan, carboplatin, and topotecan. The severity of this adverse consequence in rabbits was unexpected, and its use in the study was subsequently discontinued. Although the definitive cause for this vasotoxicity and striking clinical presentation is unknown, we suspect species-specific anatomic features and sensitivity might have contributed to amplified complications after intraarterial carboplatin chemotherapy of the eye. Due to the adverse effects of intraarterial carboplatin chemotherapy that we observed in 2 experimental cohorts of rabbits, we recommend caution regarding its use in this species.

Society of Toxicologic Pathology Digital Pathology and Image Analysis Special Interest Group Article*: Opinion on the Application of Artificial Intelligence and Machine Learning to Digital Toxicologic Pathology.

Toxicologic pathology is transitioning from analog to digital methods. This transition seems inevitable due to a host of ongoing social and medical technological forces. Of these, artificial intelligence (AI) and in particular machine learning (ML) are globally disruptive, rapidly growing sectors of technology whose impact on the long-established field of histopathology is quickly being realized. The development of increasing numbers of algorithms, peering ever deeper into the histopathological space, has demonstrated to the scientific community that AI pathology platforms are now poised to truly impact the future of precision and personalized medicine. However, as with all great technological advances, there are implementation and adoption challenges. This review aims to define common and relevant AI and ML terminology, describe data generation and interpretation, outline current and potential future business cases, discuss validation and regulatory hurdles, and most importantly, propose how overcoming the challenges of this burgeoning technology may shape toxicologic pathology for years to come, enabling pathologists to contribute even more effectively to answering scientific questions and solving global health issues. [Box: see text].