Ruthenium-Cathepsin Inhibitor Conjugates for Green Light-Activated Photodynamic Therapy and Photochemotherapy.

Dysregulated cathepsin activity is linked to various human diseases including metabolic disorders, autoimmune conditions, and cancer. Given the overexpression of cathepsin in the tumor microenvironment, cathepsin inhibitors are promising pharmacological agents and drug delivery vehicles for cancer treatment. In this study, we describe the synthesis and photochemical and biological assessment of a dual-action agent based on ruthenium that is conjugated with a cathepsin inhibitor, designed for both photodynamic therapy (PDT) and photochemotherapy (PCT). The ruthenium-cathepsin inhibitor conjugate was synthesized through an oxime click reaction, combining a pan-cathepsin inhibitor based on E64d with the Ru(II) PCT/PDT fragment [Ru(dqpy)(dppn)], where dqpy = 2,6-di(quinoline-2-yl)pyridine and dppn = benzo[i]dipyrido[3,2-a:2',3'-c]phenazine. Photochemical investigations validated the conjugate's ability to release a triazole-containing cathepsin inhibitor for PCT and to generate singlet oxygen for PDT upon exposure to green light. Inhibition studies demonstrated the conjugate's potent and irreversible inactivation of purified and intracellular cysteine cathepsins. Two Ru(II) PCT/PDT agents based on the [Ru(dqpy)(dppn)] moiety were evaluated for photoinduced cytotoxicity in 4T1 murine triple-negative breast cancer cells, L929 fibroblasts, and M0, M1, and M2 macrophages. The cathepsin inhibitor conjugate displayed notable selectivity for inducing cell death under irradiation compared to dark conditions, mitigating toxicity in the dark observed with the triazole control complex [Ru(dqpy)(dppn)(MeTz)]2+ (MeTz = 1-methyl-1H-1,2,4-triazole). Notably, our lead complex is among a limited number of dual PCT/PDT agents activated with green light.

Metalloimmunotherapy with Rhodium and Ruthenium Complexes: Targeting Tumor-Associated Macrophages.

Tumor associated macrophages (TAMs) suppress the cancer immune response and are a key target for immunotherapy. The effects of ruthenium and rhodium complexes on TAMs have not been well characterized. To address this gap in the field, a panel of 22 dirhodium and ruthenium complexes were screened against three subtypes of macrophages, triple-negative breast cancer and normal breast tissue cells. Experiments were carried out in 2D and biomimetic 3D co-culture experiments with and without irradiation with blue light. Leads were identified with cell-type-specific toxicity toward macrophage subtypes, cancer cells, or both. Experiments with 3D spheroids revealed complexes that sensitized the tumor models to the chemotherapeutic doxorubicin. Cell surface exposure of calreticulin, a known facilitator of immunogenic cell death (ICD), was increased upon treatment, along with a concomitant reduction in the M2-subtype classifier arginase. Our findings lay a strong foundation for the future development of ruthenium- and rhodium-based chemotherapies targeting TAMs.

Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity.

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose-dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in chromosome 17 that was responsible for regulating the response. A/J and NOD segments of genes in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC-IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies.

The current state of robotic retromuscular repairs-a qualitative review of the literature.

BACKGROUND: The management of ventral incisional hernias (VIH) has undergone many iterations over the last 5 years due to evolution in surgical techniques and advancement in robotic surgery. Four general principles have emerged: mesh usage, retromuscular mesh placement, primary fascial closure, and usage of minimally invasive techniques when possible. The application of robotic retromuscular repairs in VIH allow these principles to be applied simultaneously. This qualitative review attempts to answer what robotic retromuscular repairs are described, which patients are selected for these techniques, and what are current outcomes. METHODS: Using the key words: "robotic retromuscular repair", "robotic Rives Stoppa", and "robotic transversus abdominis release", a PubMed search of articles written up to December 2019 was critically reviewed. RESULTS: 44 articles were encountered, 9 high-quality articles were analyzed for this manuscript. Level of evidence ranged from 2B to 2C. Robotic TAR patients had BMI of 33 kg/m2, defect sizes ranging from 7-14 cm wide to 12-19 cm long, longer OR times, no difference in surgical site events, and shorter length of stay (LOS). The techniques to perform robotic Rives Stoppa (RS) were heterogeneous; however, extended totally extraperitoneal (ETEP) approach is most described. Defect width for RS repairs ranged 4-7 cm and LOS was less than 1 day. Complication rates were low, there is no long-term data on hernia recurrence, and information on cost is limited. CONCLUSION: In short-term follow-up, robotic retromuscular repairs show promise that VIH can be repaired with intramuscular mesh, few complications, and shorter LOS. Data on hernia recurrence, long-term complications, and rigorous cost analysis are needed to demonstrate generalizability.

Removal of Fc Glycans from [89Zr]Zr-DFO-Anti-CD8 Prevents Peripheral Depletion of CD8+ T Cells.

The N-linked biantennary glycans on the heavy chain of immunoglobulin G (IgG) antibodies (mAbs) are instrumental in the recognition of the Fc region by Fc-gamma receptors (FcγR). In the case of full-length mAb-based imaging tracers targeting immune cell populations, these Fc:FcγR interactions can potentially deplete effector cells responsible for tumor clearance. To bypass this problem, we hypothesize that the enzymatic removal of the Fc glycans will disrupt Fc:FcγR interactions and spare tracer-targeted immune cells from depletion during immunopositron emission tomography (immunoPET) imaging. Herein, we compared the in vitro and in vivo properties of 89Zr-radiolabeled CD8-specific murine mAb (anti-CD8wt, clone 2.43), a well-known depleting mAb, and its deglycosylated counterpart (anti-CD8degly). Deglycosylation was achieved via enzymatic treatment with the peptide: N-glycosidase F (PNGaseF). Both anti-CD8wt and anti-CD8degly mAbs were conjugated to p-SCN-Bn-desferrioxamine (DFO) and labeled with 89Zr. Bindings of both DFO-conjugated mAbs to FcγR and CD8+ splenocytes were compared. In vivo imaging and distribution studies were conducted to examine the specificity and pharmacokinetics of the radioimmunoconjugates in tumor-naive and CT26 colorectal tumor-bearing mice. Ex vivo analysis of CD8+ T cell population in spleens and tumors obtained postimaging were measured via flow cytometry and qRT-PCR. The removal of the Fc glycans from anti-CD8wt was confirmed via SDS-PAGE. A reduction in FcγR interaction was exhibited by DFO-anti-CD8degly, while its binding to CD8 remained unchanged. Tissue distribution showed similar pharmacokinetics of [89Zr]Zr-DFO-anti-CD8degly and the wt radioimmunoconjugate. In vivo blocking studies further demonstrated retained specificity of the deglycosylated radiotracer for CD8. From the imaging studies, no difference in accumulation in both spleens and tumors was observed between both radiotracers. Results from the flow cytometry analysis confirmed depletion of CD8+ T cells in spleens of mice administered with DFO-anti-CD8wt, whereas an increase in CD8+ T cells was shown with DFO-anti-CD8degly. No statistically significant difference in tumor infiltrating CD8+ T cells was observed in cohorts administered with the probes when compared to control unmodulated mice. CD8 mRNA levels from excised tumors showed increased transcripts of the antigen in mice administered with [89Zr]Zr-DFO-anti-CD8degly compared to mice imaged with [89Zr]Zr-DFO-anti-CD8wt. In conclusion, the removal of Fc glycans offers a straightforward approach to develop full length antibody-based imaging probes specifically for detecting CD8+ immune molecules with no consequential depletion of their target cell population in peripheral tissues.

An HER2 DNA vaccine with evolution-selected amino acid substitutions reveals a fundamental principle for cancer vaccine formulation in HER2 transgenic mice.

Enhancement of endogenous immunity to tumor-associated self-antigens and neoantigens is the goal of preventive vaccination. Toward this goal, we compared the efficacy of the following HER2 DNA vaccine constructs: vaccines encoding wild-type HER2, hybrid HER2 vaccines consisting of human HER2 and rat Neu, HER2 vaccines with single residue substitutions and a novel human HER2 DNA vaccine, ph(es)E2TM. ph(es)E2TM was designed to contain five evolution-selected substitutions: M198V, Q398R, F425L, H473R and A622T that occur frequently in 12 primate HER2 sequences. These ph(es)E2TM substitutions score 0 to 1 in blocks substitutions matrix (BLOSUM), indicating minimal biochemical alterations. h(es)E2TM recombinant protein is recognized by a panel of anti-HER2 mAbs, demonstrating the preservation of HER2 protein structure. Compared to native human HER2, electrovaccination of HER2 transgenic mice with ph(es)E2TM induced a threefold increase in HER2-binding antibody (Ab) and elevated levels of IFNγ-producing T cells. ph(es)E2TM, but not pE2TM immune serum, recognized HER2 peptide p95 355LPESFDGDPASNTAP369, suggesting a broadening of epitope recognition induced by the minimally modified HER2 vaccine. ph(es)E2TM vaccination reduced tumor growth more effectively than wild-type HER2 or HER2 vaccines with more extensive modifications. The elevation of tumor immunity by ph(es)E2TM vaccination would create a favorable tumor microenvironment for neoantigen priming, further enhancing the protective immunity. The fundamental principle of exploiting evolution-selected amino acid substitutions is novel, effective and applicable to vaccine development in general.

Layer-by-layer films with bioreducible and nonbioreducible polycations for sequential DNA release.

Layer-by-layer (LbL) films containing cationic polyelectrolytes and anionic bioactive molecules such as DNA are promising biomaterials for controlled and localized gene delivery for a number of biomedical applications including cancer DNA vaccine delivery. Bioreducible LbL films made of disulfide-containing poly(amido amine)s (PAAs) and plasmid DNA can be degraded by redox-active membrane proteins through the thiol-disulfide exchange reaction to release DNA exclusively into the extracellular microenvironment adjacent to the film. In order to better understand the film degradation mechanism and nature of the released species, the bioreducible film degradation is studied by atomic force microscopy, fluorescence, and dynamic light scattering in solutions containing a reducing agent. The PAA/DNA LbL film undergoes fast bulk degradation with micrometer-sized pieces breaking off from the substrate. This bulk degradation behavior is arrested by periodic insertions of a nonbioreducible poly(ethylenimine) (PEI) layer. The LbL films containing PAA/DNA and PEI/DNA bilayers display sequential film disassembly and are capable of continuously releasing DNA nanoparticles over a prolonged time. Insertion of the PEI layer enables the bioreducible LbL films to transfect human embryonic kidney 293 cells. The data conclude that the PEI layer is effective as a barrier layer against interlayer diffusion during LbL film assembly and more importantly during film disassembly. Without the barrier layer, the high mobility of cleaved PAA fragments is responsible for bulk degradation of bioreducible LbL films, which may prevent their ultimate gene-delivery applications. This work establishes a direct link among film internal structure, disassembly mechanism, and transfection efficiency. It provides a simple method to design bioreducible LbL films for sequential and long-time DNA release.

Ru(II)-Photoactive Agents for Targeting ER Stress and Immunogenic Cell Death.

Immunotherapy has emerged as a promising avenue for cancer treatment by bolstering the immune system's ability to recognize and attack cancer cells. Photodynamic therapy shows potential in enhancing antitumor immunity, though the mechanisms behind its success are not fully understood. In this manuscript, we investigate two previously reported green light activated PCT/PDT agents where compound 2 - [Ru(tpy)(Me2bpy)( 3 )] 2+ , (tpy = 2,2':6',2''- terpyridine, Me2bpy = 6,6'-dimethyl-2,2'-bipyridine, 3 = pyridyl-BODIPY-I2,) - shows remarkable photoselectivity in assays containing both 2D cancer cells and 3D cocultures containing BALB/c macrophages and 4T1 murine breast cancer cells. Through flow cytometry and protein analysis, we found complex 2 displays superior evidence of induced endoplasmic reticulum (ER) stress markers and indicators of immunogenic cell death (ICD) compared to its ligand 3 , despite its weaker photoselectivity. Most importantly, these results were supported by in vivo studies where 2 produced anti-tumor immunity against the 4T1 tumor model in BALB/c mice. Complete tumor elimination was achieved in 2/8 mice, and these mice were both protected against a subsequent contralateral rechallenge and showed increased ex vivo peripheral tumor antigen-specific recall, suggesting memory T cells are induced by 2 . Signatures of M1 macrophage polarization were also evident in tumor tissue from the remaining 6/8 mice treated with 2 compared to untreated tumors. These findings demonstrate Ru(II) complexation plays a critical role in ER targeting which triggers ICD, highlighting the potential of Ru(II) agents as future in situ tumor vaccines.

Evaluation of the Immune Response within the Tumor Microenvironment in African American and non-Hispanic White Non-Small Cell Lung Cancer Patients.

BACKGROUND: African Americans have higher incidence and mortality from lung cancer than non-Hispanic Whites, but investigations into differences in immune response have been minimal. Therefore, we compared components of the tumor microenvironment among African Americans and non-Hispanic Whites diagnosed with non-small cell lung cancer (NSCLC) based on PD-L1 or tertiary lymphoid structure (TLS) status to identify differences of translational relevance. METHODS: Using a cohort of 280 NSCLC patients from the INHALE study (non-Hispanic White: n=155; African American: n=125), we evaluated PD-L1 tumor proportion score (<1% vs. ≥1%) and TLS status (presence/absence), comparing differences within the tumor microenvironment based on immune cell distribution and differential expression of genes. RESULTS: Tumors from African Americans had a higher proportion of plasma cell signatures within the tumor microenvironment than non-Hispanic Whites. In addition, gene expression patterns in African American PD-L1 positive samples suggest these tumors contained greater numbers of γδ T-cells and resting dendritic cells, along with fewer CD8+ T-cells after adjusting for age, sex, pack-years, stage, and histology. Investigation of differential expression of B-cell/plasma cell related genes between the two patient populations revealed that two immunoglobulin genes (IGKV2-29 and IGLL5) were associated with decreased mortality risk in African Americans. CONCLUSIONS: In the first known race-stratified analysis of tumor microenvironment components in lung cancer based on PD-L1 expression or TLS status, differences within the immune cell composition and transcriptomic signature were identified that may have therapeutic implications. IMPACT: Future investigation of racial variation within the tumor microenvironment may help direct the use of immunotherapy.

Interferon gamma immunoPET imaging to evaluate response to immune checkpoint inhibitors.

Introduction: We previously developed a 89Zr-labeled antibody-based immuno-positron emission tomography (immunoPET) tracer targeting interferon gamma (IFNγ), a cytokine produced predominantly by activated T and natural killer (NK) cells during pathogen clearance, anti-tumor immunity, and various inflammatory and autoimmune conditions. The current study investigated [89Zr]Zr-DFO-anti-IFNγ PET as a method to monitor response to immune checkpoint inhibitors (ICIs). Methods: BALB/c mice bearing CT26 colorectal tumors were treated with combined ICI (anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death 1 (PD-1)). The [89Zr]Zr-DFO-anti-IFNγ PET tracer, generated with antibody clone AN18, was administered on the day of the second ICI treatment, with PET imaging 72 hours later. Tumor mRNA was analyzed by quantitative reverse-transcribed PCR (qRT-PCR). Results: We detected significantly higher intratumoral localization of [89Zr]Zr-DFO-anti-IFNγ in ICI-treated mice compared to untreated controls, while uptake of an isotype control tracer remained similar between treated and untreated mice. Interestingly, [89Zr]Zr-DFO-anti-IFNγ uptake was also elevated relative to the isotype control in untreated mice, suggesting that the IFNγ-specific tracer might be able to detect underlying immune activity in situ in this immunogenic model. In an efficacy experiment, a significant inverse correlation between tracer uptake and tumor burden was also observed. Because antibodies to cytokines often exhibit neutralizing effects which might alter cellular communication within the tumor microenvironment, we also evaluated the impact of AN18 on downstream IFNγ signaling and ICI outcomes. Tumor transcript analysis using interferon regulatory factor 1 (IRF1) expression as a readout of IFNγ signaling suggested there may be a marginal disruption of this pathway. However, compared to a 250 µg dose known to neutralize IFNγ, which diminished ICI efficacy, a tracer-equivalent 50 µg dose did not reduce ICI response rates. Discussion: These results support the use of IFNγ PET as a method to monitor immune activity in situ after ICI, which may also extend to additional T cell-activating immunotherapies.

Chemotherapy and Abdominal Wall Closure Technique Increase the Probability of Postoperative Ventral Incisional Hernia in Patients With Colon Cancer.

BACKGROUND: Chemotherapy is associated with postoperative ventral incisional hernia (PVIH) after right hemicolectomy (RHC) for colon cancer, and abdominal wall closure technique may affect PVIH. We sought to identify clinical predictors of PVIH. METHODS: We retrospectively analyzed patients who underwent RHC for colon cancer from 2008-2018 and later developed PVIH. Time to PVIH was analyzed with Kaplan-Meier analysis, clinical predictors were identified with multivariable Cox proportional hazards modeling, and the probability of PVIH given chemotherapy and the suture technique was estimated with Bayesian analysis. RESULTS: We identified 399 patients (209 no adjuvant chemotherapy and 190 adjuvant chemotherapy), with an overall PVIH rate of 38%. The 5-year PVIH rate was 55% for adjuvant chemotherapy, compared with 38% for none (log-rank P < .05). Adjuvant chemotherapy (hazard ratio [HR] 1.65, 95% confidence interval [CI] 1.18-2.31, P < .01), age (HR .99, 95% CI .97-1.00, P < .01), body mass index (HR 1.02, 95% CI 1.00-1.04, P < .01), and neoadjuvant chemotherapy (HR 1.92, 95% CI 1.21-3.00, P < .01) were independently associated with PVIH. Postoperative ventral incisional hernia was more common overall in patients who received adjuvant chemotherapy (46% compared with 30%, P < .01). In patients who received adjuvant chemotherapy, the probability of PVIH for incision closure with #1 running looped polydioxanone was 42%, compared with 59% for incision closure with #0 single interrupted polyglactin 910. DISCUSSION: Exposure to chemotherapy increases the probability of PVIH after RHC, and non-short stitch incision closure further increases this probability, more so than age or body mass index. The suture technique deserves further study as a modifiable factor in this high-risk population.

COVID-19 compliance among urban trail users: Behavioral insights and environmental implications.

Public green spaces provide physical and mental respite, which have become essential and elevated services during the COVID-19 pandemic. As visitation to public parks and recreation areas increased during the pandemic, the challenge of maintaining visitor safety and protecting environmental resources was exacerbated. A key visitor safety practice during the COVID-19 onset was maintaining a physical distance of six feet (1.8 m) between groups. A novel data set documented and compared physical distancing compliance and off-trail behavior on multiple-use trails across multiple states and within select U.S. communities, attending to the impact of select environmental factors. Nearly 6000 observations revealed physical distancing compliance varied and the environmental factors of trail width, density, and signage influenced its variability. Similarly, off-trail movement was related to trail width and density. Clearly the environment matters as people negotiate the 'new normal' of physical distancing during physical activity and outdoor recreation participation. Given the ongoing COVID-19 pandemic and likelihood of future health crises, this project provides important information and insight for trail and other public green space management, monitoring, and modelling moving forward. Management implications: As both trail width and visitor density impacted physical distancing, a combination of trail design that accommodates distancing requirements and density management practices that provide sufficient trail user spacing is essential to retain safe and active trail use.Off-trail movement was influenced by both trail width and density, so ensuring safe off-trail spaces exist and using durable off-trail materials can minimize disturbance and protect visitors.Signage is inconsistently significant to influence trail-compliant distancing behavior, but optimizing its placement and content may improve effectiveness.Compliant trail behavior varied by trail width, visitor density, and trail location; therefore, site-specific information is necessary to understand possible visitor behavior and design/implement mitigation strategies.

Cases and context: Mask-related behaviors among U.S. trail visitors during the COVID-19 pandemic.

The challenge of simultaneously providing outdoor recreation opportunities while protecting the public from SARS-CoV-2 virus and COVID-19 transmission, as well as future pandemics, remains foremost on managers' minds. Safe spaces and cultures are paramount for managers and visitors alike. Recommended protective measures against COVID-19 included physically distancing 1.8 m (six-feet) between parties and mask-wearing when distancing is not possible. Adoption of these protective measures is relatively unknown but essential to inform recreation management and planning through future health crises. Such adoption is likely influenced by both the pandemic context and site context, particularly related to visitor density. An observational study assessed mask-wearing behaviors among trail walkers on multiple trails in the United States from November 2020 through May 2021. Trained observers identified if walking groups were prepared to mask or had masks correctly worn as well as if encounters were compliant with the 1.8 m recommendations. Data collected across seven U.S. states enabled comparisons of mask-related behaviors across sites as well as considerations to: the influence of the pandemic context in terms of cases and vaccination rates, mask mandates, and trail density. Results from nearly 3000 encounters revealed significant variance in visible masks, low compliance of mask-wearing in encounters less than 1.8 m, significant influence of both COVID-19 cases and vaccination rates on mask wearing at half the sites, and no impact of state-level mask mandates when controlling for cases and vaccinations. Integrating public health data can inform predictions of compliant behaviors, or lack thereof, and needs exist to advance a safety culture.

Host genetic background regulates the capacity for anti-tumor antibody-dependent phagocytosis.

Background: Antitumor antibody, or targeted immunotherapy, has revolutionized cancer treatment and markedly improved patient outcomes. A prime example is the monoclonal antibody (mAb) trastuzumab, which targets human epidermal growth factor receptor 2 (HER2). However, like many targeted immunotherapies, only a subset of patients benefit from trastuzumab long-term. In addition to tumor-intrinsic factors, we hypothesize that host genetics may influence subsequent immune activation. Methods: To model the human population, we produced F1 crosses of genetically heterogeneous Diversity Outbred (DO) mice with BALB/c mice (DOCF1). Distinct DOCF1 mice were orthotopically implanted with the BALB/c-syngeneic TUBO mammary tumor line, which expresses the HER2 ortholog rat neu. Treatment with anti-neu mAb clone 7.16.4 began once tumors reached ∼200 mm 3 . Genetic linkage and quantitative trait locus (QTL) effects analyses in R/qtl2 identified loci associated with tumor growth rates. Locus validation was performed with BALB/c F1 crosses with recombinant-inbred Collaborative Cross (CC) strains selected for therapy-associated driver genetics (CCxCF1). The respective roles of natural killer (NK) cells and macrophages were investigated by selective depletion in vivo. Ex vivo macrophage antibody-dependent phagocytosis (ADCP) assays were evaluated by confocal microscopy using 7.16.4-opsonized E2Crimson-expressing TUBO tumor cells. Results: We observed a divergent response to anti-tumor antibody therapy in DOCF1 mice. Genetic linkage analysis detected a locus on chromosome 10 that correlates to a robust response to therapy, which was validated in CCxCF1 models. Single-cell RNA sequencing of tumors from responder and non-responder models identified key differences in tumor immune infiltrate composition, particularly within macrophage (Mφ) subsets. This is further supported by ex vivo analysis showing Mφ ADCP capacity correlates to in vivo treatment outcomes in both DOCF1 and CCxCF1 models. Conclusions: Host genetics play a key regulatory role in targeted immunotherapy outcomes, and putative causal genes are identified in murine chromosome 10 which may govern Mφ function during ADCP.

Predicting physical distancing on recreational trails during COVID-19.

The emergence of Coronavirus 19 led to societal and behavioral changes, including intensified use of many public parks and trails for mental respite and leisure time physical activity. As visitors sought stress-relief in the great outdoors, they also encountered stressful situations as they navigated risk exposure. Recommendations to physically distance between parties was a key component to reduce risk, but compliance is unknown in the outdoor arena. This observational study of more than 10 000 trail user encounters documented distancing and enabled predictive analysis that revealed wider trails, smaller groups and signage led to greater distancing compliance. Managers and planners can integrate these findings immediately and in consideration of future trail designs to minimize risk exposure. Management implications: Select site features increase odds of distancing compliance and can inform management decisions and designs immediately and in addressing future use surges: wider trails, unpaved surfaces, and COVID-19 signage.As distancing compliance waned with time but signage increased compliance, innovative and dynamic signs may sustain compliance and multi-media communications should be considered.Both activity size and group type influence distancing so considering group size recommendations and activity separation are in order.

Induction of proapoptotic antibodies to triple-negative breast cancer by vaccination with TRAIL death receptor DR5 DNA.

TNF-related apoptosis-inducing ligand receptor 2 [TRAIL-R2 or death receptor 5 (DR5)] is expressed at elevated levels in a broad range of solid tumors to mediate apoptotic signals from TRAIL or agonist antibodies. We tested the hypothesis that DR5 DNA vaccination will induce proapoptotic antibody to trigger apoptosis of tumor cells. BALB/c mice were electrovaccinated with DNA-encoding wild-type human DR5 (phDR5) or its derivatives. Resulting immune serum or purified immune IgG induced apoptosis in triple-negative breast cancer (TNBC) cells, which were also TRAIL sensitive. The proapoptotic activity of immune serum at dilutions of 0.5-2% was comparable to that of 1-2 µg/ml of TRAIL. Apoptotic activity of immune serum was enhanced by antibody crosslinking. Apoptotic cell death induced by anti-DR5 antibody was shown by the cleavage of PARP and caspase-3. In contrast, immune serum had no effect on the proliferation of activated human T cells, which expressed low levels of DR5. In vivo, hDR5 reactive immune serum prevented growth of SUM159 TNBC cells in severe combined immune-deficient mice. DR5-specific IFN-γ-secreting T cells were also induced by DNA vaccination. Furthermore, the feasibility to overcome immune tolerance to self DR5 was shown by the induction of mouse DR5-binding antibody after electrovaccination of BALB/c mice with pmDR5ectm-Td1 encoding a fusion protein of mouse DR5 and an immunogenic fragment of tetanus toxin. These findings support DR5 as a promising vaccine target for controlling TNBC and other DR5-positive cancers.

Using the Collaborative Cross and Diversity Outbred Mice in Immunology.

The Collaborative Cross (CC) and the Diversity Outbred (DO) stock mouse panels are the most powerful murine genetics tools available to the genetics community. Together, they combine the strength of inbred animal models with the diversity of outbred populations. Using the 63 CC strains or a panel of DO mice, each derived from the same 8 parental mouse strains, researchers can map genetic contributions to exceptionally complex immunological and infectious disease traits that would require far greater powering if performed by genome-wide association studies (GWAS) in human populations. These tools allow genes to be studied in heterozygous and homozygous states and provide a platform to study epistasis between interacting loci. Most importantly, once a quantitative phenotype is investigated and quantitative trait loci are identified, confirmatory genetic studies can be performed, which is often problematic using the GWAS approach. In addition, novel stable mouse models for immune phenotypes are often derived from studies utilizing the DO and CC mice that can serve as stronger model systems than existing ones in the field. The CC/DO systems have contributed to the fields of cancer immunology, autoimmunity, vaccinology, infectious disease, allergy, tissue rejection, and tolerance but have thus far been greatly underutilized. In this article, we present a recent review of the field and point out key areas of immunology that are ripe for further investigation and awaiting new CC/DO research projects. We also highlight some of the strong computational tools that have been developed for analyzing CC/DO genetic and phenotypic data. Additionally, we have formed a centralized community on the CyVerse infrastructure where immunogeneticists can utilize those software tools, collaborate with groups across the world, and expand the use of the CC and DO systems for investigating immunogenetic phenomena. © 2022 Wiley Periodicals LLC.

Evaluation of an ImmunoPET Tracer for IL-12 in a Preclinical Model of Inflammatory Immune Responses.

The immune cytokine interleukin-12 (IL-12) is involved in cancer initiation and progression, autoimmunity, as well as graft versus host disease. The ability to monitor IL-12 via imaging may provide insight into various immune processes, including levels of antitumor immunity, inflammation, and infection due to its functions in immune signaling. Here, we report the development and preclinical evaluation of an antibody-based IL-12-specific positron emission tomography (PET) tracer. To mimic localized infection and stimulate IL-12 production, BALB/c mice were administered lipopolysaccharide (LPS) intramuscularly. [89Zr]Zr-DFO-αIL12 tracer was given one hour post LPS administration and PET images were taken after 5, 24, 48, and 72 hours. We observed significantly higher uptake in LPS-treated mice as compared to controls. Biodistribution of the tracer was evaluated in a separate cohort of mice, where tracer uptake was elevated in muscle, spleen, lymph nodes, and intestines after LPS administration. To evaluate the utility of [89Zr]Zr-DFO-αIL12 as an indicator of antigen presenting cell activation after cancer immunotherapy, we compared PET imaging with and without intratumoral delivery of oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (Adv/GM-CSF), which we have shown promotes anti-tumor immunity. BALB/c mice were inoculated orthotopically with the mouse mammary carcinoma line TUBO. Once TUBO tumors reached a volume of ~50 mm3, mice were treated with either three intratumoral injections of 108 PFU Adv/GM-CSF or vehicle control, given every other day. Upon the last dose, [89Zr]Zr-DFO-αIL12 was injected intravenously and 72 hours later all mice were imaged via PET. Tumor-specific uptake of [89Zr]Zr-DFO-αIL12 was higher in Adv/GM-CSF treated mice versus controls. Tissues were harvested after imaging, and elevated levels of macrophages and CD8+ Tc cells were detected in Adv/GM-CSF treated tumors by immunohistochemistry. We validated that IL-12 expression was induced after Adv/GM-CSF by qRT-PCR. Importantly, expression of genes activated by IL-12 (IFNγ, TNFα, and IL-18) were unaffected after IL-12 imaging relative to mice receiving an IgG control tracer, suggesting the tracer antibody does not significantly disrupt signaling. Our results indicate that targeting soluble cytokines such as IL-12 by PET imaging with antibody tracers may serve as a noninvasive method to evaluate the function of the immune milieu in situ.

Protocolized Training of Advanced Practice Providers for Robotic Surgery Improves the Quality of Intraoperative Assistance.

BACKGROUND: The expansion of robotic surgery requires identifying factors of competent robotic bedside assisting. Surgical trainees desire more robotic console time, and we hypothesized that protocolized robotic surgery bedside training could equip Advanced Practice Providers (APPs) to meet this growing need. No standardized precedent exists for training APPs. METHODS: We designed a pilot study consisting of didactic and clinical skills. APPs completed didactic tests followed by proctored clinical skills checklists intraoperatively. Operating surgeons scored trainees with 10-point Likert scale (< 5 not confident, > 5 = confident). APPs scoring > 5 advanced to a solo practicum. Competence was defined as: didactic test score > 75th percentile, completing < 5 checklists, scoring > 5 on the practicum. The probability of passing the practicum was calculated with Bayes theorem. RESULTS: Of 10 APP trainees, 5 passed on initial attempt. After individualized development plans, 4 passed retesting. Differences in trainee factors were not statistically significant, but the probability of passing the practicum was < 50% if more than four checklists were needed. CONCLUSIONS: Clinical experience, not didactic knowledge, determines the probability of intraoperative competence. Increasing clinical proctoring did not result in higher probability of competence. Early identification of APPs needing individualized improvement increases the proportion of competent APPs.

A diversity outbred F1 mouse model identifies host-intrinsic genetic regulators of response to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICI) have improved outcomes for a variety of malignancies; however, many patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent host genetic background influences response. To investigate this, we utilized the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are an outbred stock generated by crossbreeding eight inbred founder strains, and CC mice are recombinant inbred mice generated from the same eight founders. We generated 207 DOB6F1 mice representing 48 DO dams and demonstrated that these mice reliably accept the C57BL/6-syngeneic B16F0 tumor and that host genetic background influences response to ICI. Genetic linkage analysis from 142 mice identified multiple regions including one within chromosome 13 that associated with therapeutic response. We utilized 6 CC strains bearing the positive (NZO) or negative (C57BL/6) driver genotype in this locus. We found that 2/3 of predicted responder CCB6F1 crosses show reproducible ICI response. The chromosome 13 locus contains the murine prolactin family, which is a known immunomodulating cytokine associated with various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Prolactin augmented ICI response against B16F0, with increased CD8 infiltration and 5/8 mice exhibiting slowed tumor growth relative to controls. This study highlights the role of host genetics in ICI response and supports the use of F1 crosses in the DO and CC mouse populations as powerful cancer immunotherapy models.