Local Immunomodulation Research Articles

Comparative Evaluation of Lipid Profile, C-Reactive Protein and Paraoxonase-1 Activity in Dogs with Inflammatory Protein-Losing Enteropathy and Healthy Dogs.

Chronic inflammation alters lipoprotein metabolism and causes changes in the serum concentrations of lipids, C-reactive protein (CRP), and paraoxonase-1 activity (PON-1), an enzyme that may act as a local detoxifier, antioxidant, and immunomodulator in the gastrointestinal tract. Scarce information is available in dogs with protein-losing enteropathy secondary to chronic enteropathy (iPLE). The first aim was to describe and compare the lipid profiles, CRP concentrations and PON-1 activities in healthy dogs and in dogs with iPLE. The second aim was to evaluate correlations among clinicopathological, histologic data and lipid profiles in dogs with iPLE. Serum samples from 51 iPLE and 40 healthy dogs were used to study albumin, total protein, CRP, PON-1 activity, cholesterol, triglycerides and lipoprotein classes. Serum concentrations of albumin, total protein, cholesterol, PON-1 activity, and high-density and very-low-density lipoproteins were lower in iPLE dogs compared to healthy controls, while those of triglycerides, low-density lipoproteins, chylomicrons and CRP were higher. Significant correlations between the lipid profile and the existing chronic enteropathy activity index were not found. High-density and low-density lipoproteins correlated with CRP and PON-1. Triglycerides were significantly higher in dogs with both inflammation and lymphangiectasia. The results need to be confirmed in further studies.

Abstract B021: A preclinical murine model investigation; Focused ultrasound ablation of osteosarcoma induces local and systemic immunomodulation

Abstract B021: A preclinical murine model investigation; Focused ultrasound ablation of osteosarcoma induces local and systemic immunomodulation

Simvastatin-Loaded Polymeric Nanoparticles: Targeting Inflammatory Macrophages for Local Adipose Tissue Browning in Obesity Treatment.

Obesity is defined as chronic, low-grade inflammation within specific tissues. Given the escalating prevalence of obesity among individuals of all ages, obesity has reached epidemic proportions, posing an important public health challenge. Despite significant advancements in treating obesity, conventional approaches remain largely ineffective or involve severe side effects, thus underscoring the pressing need to explore and develop treatment approaches. Targeted and local immunomodulation using nanoparticles (NPs) can influence fat production and utilization processes. Statins, known for their anti-inflammatory properties, show the potential for mitigating obesity-related inflammation. A localized delivery option offers several advantages over oral and parenteral delivery methods. Here, we developed simvastatin (Sim) encapsulated within PLGA NPs (Sim-NP) for localized delivery of Sim to adipose tissues (ATs) for immunomodulation to treat obesity. In vitro experiments revealed the strong anti-inflammatory effects of Sim-NPs, which resulted in enhanced modulation of macrophage (MΦ) polarization and induction of AT browning. We then extended our investigation to an in vivo mouse model of high-fat-diet (HFD)-induced obesity. Sim-NP administration led to the controlled release of Sim within AT, directly impacting MΦ activity and inducing AT browning while inducing weight loss. Our findings demonstrated that Sim-NP administration effectively inhibited the progression of obesity-related inflammation, controlled white fat production, and enhanced AT modulation. These results highlight the potential of Sim-NP as a potent nanotherapy for treating obesity by modulating the immune system.

Prophylactic and therapeutic cancer vaccine with continuous localized immunomodulation

Selective in vivo immune cell manipulation offers a promising strategy for cancer vaccines. In this context, spatiotemporal control over recruitment of specific cells, and their direct exposure to appropriate immunoadjuvants and antigens are key to effective cancer vaccines. We present an implantable 3D-printed cancer vaccine platform called the ‘NanoLymph’ that enables spatiotemporally-controlled recruitment and manipulation of immune cells in a subcutaneous site. Leveraging two reservoirs each for continuous immunoadjuvant release or antigen presentation, the NanoLymph attracts dendritic cells (DCs) on site and exposes them to tumor-associated antigens. Upon local antigen-specific activation, DCs are mobilized to initiate a systemic immune response. NanoLymph releasing granulocyte-macrophage colony-stimulating factor and CpG-oligodeoxynucleotides with irradiated whole cell tumor lysate inhibited tumor growth of B16F10 murine melanoma in a prophylactic and therapeutic vaccine setting. Overall, this study presents the NanoLymph as a versatile cancer vaccine development platform with replenishable and controlled local release of antigens and immunoadjuvants.

Eosinophils Respond to Extracellular Matrix Treated Muscle Injuries but are Not Required for Macrophage Polarization.

The immune response to decellularized extracellular matrix (ECM) muscle injury is characterized by Th2 T cells, Tregs, M2-like macrophages, and an abundance of eosinophils. Eosinophils have previously been described as mediators of muscle regeneration but inhibit skin wound healing. In addition to response to wounding, a large number of eosinophils respond to biomaterial-treated muscle injury, specifically in response to decellularized ECM. ECM treatment of muscle wounds has been associated with positive outcomes in tissue regeneration, but the detailed mechanisms of action are still being evaluated. Here, this work investigates the role of these eosinophils in terms of their immunologic phenotype and subsequent effect on the local tissue microenvironment. These cells have a mixed phenotype showing both type-2 and regulatory gene upregulation and but are not required for macrophage polarization. Beyond the local tissue, ECM treatment is seen to induce a transient flux of eosinophils to the lungs but prevented a trauma-associated neutrophilia in the lungs of injured mice. This work believes this local and systemic immunomodulation contributes to the regenerative effects of the material and such distal tissue effects should be considered in therapeutic design and implementation.

P-306 GnRH agonists exert a positive influence on regulatory immune cells in the endometrium of women with adenomyosis

Abstract Study question How does treatment with GnRH-agonist (GnRH-a) prior to frozen embryo transfer (FET) in women with adenomyosis affect immune cell populations in endometrium and peripheral blood? Summary answer GnRH-a induces a decrease in monocytes and an increase in CD4+T-cells in peripheral blood and a decrease classical monocytes and T-regulatory cells in the endometrium What is known already Adenomyosis is associated with lower implantation and higher miscarriage rates. Observational studies have reported improved pregnancy outcomes in women with adenomyosis after assisted reproductive technologies (ART) and pre-treatment with GnRH-a prior to FET. However, the specific mechanisms of GnRHa action beyond central hormonal suppression remain unclear, but may be mediated through local and/or systemic immunomodulation. Study design, size, duration Prospective cohort study including infertile women with adenomyosis and GnRH-a treatment ≥3 months prior to FET. Pre- and post-treatment analysis of blood and endometrium samples were compared. Recruitment is ongoing with n = 30 women included thus far, 16 of whom have completed the study, while the remaining are still undergoing GnRH-a therapy. Participants/materials, setting, methods Adenomyosis was diagnosed sonographically if ≥ 2 criteria published by the MUSA group were met. Peripheral blood and endometrial samples were obtained before and after treatment with GnRH-a. Plasma cells and uterine Natural Killer (uNK) cells in endometrial biopsies were quantified with immunhistochemistry (IHC), and leukocyte populations in all samples were analysed using flow cytometry (FC). Pre- and post-treatment levels of immune cells and percentage of leukocyte populations were compared using Wilcoxon rank-sum tests (p < 0.05 significant) Main results and the role of chance Interim analysis of immune cell populations in peripheral blood and endometrium of the 16 women who already completed the study was performed. At baseline, IHC showed elevated plasma cell concentrations indicating chronic endometritis in 3/30 patients (10%), while uNK cells were elevated (>300/mm2) in 13/30 patients (43.3%). FC analysis of peripheral blood showed a significant decrease in percentage of monocytes (p = 0.01) and increase in percentage of T-cells (p = 0.03), specifically concentration of CD4+T-cells (p = 0.03), after treatment with GnRH-a as compared to pre-treatment levels. In the endometrium, following GnRH-a therapy, we observed a decrease in both the percentage of classical monocytes (p = 0.03) and T-regulatory cells (p = 0.03). All 16 participants who completed the study already underwent FET, resulting in ten pregnancies (62.5%), although four resulted in miscarriage, while the outcome of the remaining pregnancies is still pending. Limitations, reasons for caution These are preliminary results whose generalizability needs to be confirmed in a larger study population. No correction for multiple comparisons was performed and significant findings have to be interpreted with caution. Wider implications of the findings Elevated uNK on IHC cells seen at baseline may be associated with implantation failure, miscarriage. CD4+ and DN T-cells are important mediators of reproductive immune tolerance. In women with adenomyosis, GnRH-a may have a positive impact on these regulatory immune cells in peripheral blood as well as in the endometrium. Trial registration number Not applicable

Human placental organoids as a model to probe early gestation maternal immune dynamics.

During pregnancy, the human placenta establishes tolerance toward fetal allogeneic tissue, where specialized trophoblast subtypes play a complex role in local and peripheral immunomodulation. However, due to inadequate models to study the early gestation of the human placenta, each trophoblast subtype's role in modulating the maternal immune response has remained elusive. Here, we derived human placental organoids from early gestation trophoblast stem cells to (1) identify patterns of immunomodulatory protein expression by trophoblast subtype and (2) evaluate the effects of the placental organoid secretome on immune cell activation and regulation. We show that the three primary trophoblast phenotypes had distinct influences on immune cell phenotype and activation and that three-dimensional culture significantly alters trophoblast immunomodulation relative to traditional two-dimensional trophoblast culture.

Medicamentous Therapy of Malignant Eyelid Tumors

Although surgical therapy is often the first-line treatment for malignant eyelid tumors, pharmacological treatment approaches can also be included and pursued in the treatment plan. Narrative review with a selective literature search on PubMed and Google Scholar. Various pharmacological therapeutic principles are currently available. One option is the local application of agents within the tumor area. This can be achieved through cytostatically active drugs such as 5-fluorouracil for superficial basal cell carcinomas and precursors of squamous cell carcinomas, or through mitomycin C in specific cases of sebaceous gland carcinoma. Another form of pharmacological local therapy is local immunomodulation using Imiquimod for superficial basal cell carcinomas, actinic keratosis, and Bowen's disease. Furthermore, there are systemic pharmacological therapies like chemotherapies, for example in sebaceous cell carcinoma, or systemic immunomodulation using checkpoint inhibitors for example in basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, and melanoma. Additionally, targeted therapies offer yet another treatment modality that exploits the molecular biological characteristics of various tumor entities. Examples of this include Hedgehog inhibitors for basal cell carcinomas, EGFR inhibitors for squamous cell carcinomas, or BRAF inhibitors for melanomas. This review addresses these treatment options for malignant tumors of the eyelid and systematically organizes them for the reader. Even though the data on these eye tumors is still limited, the reported case studies using systemic therapies for malignant eyelid tumors demonstrate the potential of this treatment modality. However, the need for further research is high especially concerning the combination of different therapy principles for increasing the effectiveness of eyelid tumor therapy.

VEGFR-3 signaling in macrophages: friend or foe in disease?

Lymphatic vessels have been increasingly appreciated in the context of immunology not only as passive conduits for immune and cancer cell transport but also as key in local tissue immunomodulation. Targeting lymphatic vessel growth and potential immune regulation often takes advantage of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling to manipulate lymphatic biology. A receptor tyrosine kinase, VEGFR-3, is highly expressed on lymphatic endothelial cells, and its signaling is key in lymphatic growth, development, and survival and, as a result, often considered to be "lymphatic-specific" in adults. A subset of immune cells, notably of the monocyte-derived lineage, have been identified to express VEGFR-3 in tissues from the lung to the gut and in conditions as varied as cancer and chronic kidney disease. These VEGFR-3+ macrophages are highly chemotactic toward the VEGFR-3 ligands VEGF-C and VEGF-D. VEGFR-3 signaling has also been implicated in dictating the plasticity of these cells from pro-inflammatory to anti-inflammatory phenotypes. Conversely, expression may potentially be transient during monocyte differentiation with unknown effects. Macrophages play critically important and varied roles in the onset and resolution of inflammation, tissue remodeling, and vasculogenesis: targeting lymphatic vessel growth and immunomodulation by manipulating VEGFR-3 signaling may thus impact macrophage biology and their impact on disease pathogenesis. This mini review highlights the studies and pathologies in which VEGFR-3+ macrophages have been specifically identified, as well as the activity and polarization changes that macrophage VEGFR-3 signaling may elicit, and affords some conclusions as to the importance of macrophage VEGFR-3 signaling in disease.

Balanitis circinata as the sole clinical presentation of underlying sexually acquired reactive arthritis: A case report

A 35-year-old male was diagnosed with a case of HLA27-positive reactive arthritis post-evaluation. He presented with the only symptoms of recurrent genital ulcers on glans penis of 4 months duration. The patient was treated with nonsteroidal anti-inflammatory drugs, a short course of systemic steroids, and a local immunomodulator cream on the penile lesion. The patient responded well to the treatment and returned to his usual life activities with no occurrence so far.

Matrix-bound nanovesicle-associated IL-33 supports functional recovery after skeletal muscle injury by initiating a pro-regenerative macrophage phenotypic transition

Injuries to skeletal muscle are among the most common injuries in civilian and military populations, accounting for nearly 60% of extremity injuries. The standard of care for severe extremity injury has been focused upon limb salvage procedures and the utilization of tissue grafts or orthotics in conjunction with rehabilitation to avoid amputation. Nonetheless, many patients have persistent strength and functional deficits that permanently impact their quality of life. Preclinical and clinical studies have shown that partial restoration of functional skeletal muscle tissue following injury can be achieved by the implantation of a biologic scaffold composed of extracellular matrix (ECM). These favorable outcomes are mediated, at least in part, through local immunomodulation. The mechanisms underlying this immunomodulatory effect, however, are poorly understood. The present study investigates a potential mechanistic driver of the immunomodulatory effects; specifically, the effect of selected ECM components upon inflammation resolution and repair. Results show that the host response to skeletal muscle injury is profoundly altered and functional recovery decreased in il33−/− mice compared to age- and sex-matched wildtype counterparts by 14 days post-injury. Results also show that IL-33, contained within matrix-bound nanovesicles (MBV), supports skeletal muscle regeneration by regulating local macrophage activation toward a pro-remodeling phenotype via canonical and non-canonical pathways to improve functional recovery from injury compared to untreated il33−/− counterparts. Taken together, these data suggest that MBV and their associated IL-33 cargo represent a novel homeostatic signaling mechanism that contributes to skeletal muscle repair.

Dermatomycosis in cats (prevalence, diagnosis, course, treatment)

The article analyzes the literature on the distribution and diagnosis of cat dermatomycosis. Pathogenic fungi of the genera Trichophyton and Microsporum occupy a special place among the most common pathogens that provoke the development of dermatitis among small animals. Mycoses of small animals caused by dermatophytes are zoonoses, in which the potential for transmission of microorganisms from animals to humans and vice versa is high. Regarding the pathogenesis of fungal diseases in cats, it is believed that the infectious agent is arthrospores formed by fragmented hyphae. This pathology's mechanism of development is associated with spores' penetration into hair follicles. Mushroom components mainly cause hypersensitivity reactions with the development of a humoral and cell-mediated immune response. Issues of early diagnosis are also covered, which allows timely detection of sick animals and effective treatment. Diagnosis of fungal lesions is usually complex. In the practice of veterinary medicine, new methods of diagnosing dermatophytoses are increasingly used, particularly in detecting pathogenic fungi in dogs and cats using a PCR test. When examining cats suspected of having dermatomycosis, anamnestic data are taken into account (type of animal, sex, age, breed, housing and feeding conditions, general condition, behavioral reactions, presence of appetite, appearance), as well as clinical manifestations of skin lesions, namely the location of the pathological process, nature and its manifestations. Clinical signs of the disease in cat dermatomycosis are very diverse and are determined by the specific properties of the pathogens. Antifungal drugs of systemic and local action and immunomodulators play an essential role in the system of treatment and prevention measures.

Doping-induced assembly interface for noninvasive in vivo local and systemic immunomodulation.

Peripheral neural interfaces, potent in modulating local and systemic immune responses for disease treatment, face significant challenges due to the peripheral nerves' broad distribution in tissues like the fascia, periosteum, and skin. The incongruity between static electronic components and the dynamic, complex organization of the peripheral nervous system often leads to interface failure, stalling circuit research and clinical applications. To overcome these, we developed a self-assembling, tissue-adaptive electrode composed of a single-component cocktail nanosheet colloid, including dopants, conducting polymers, stabilizers, and an MXene catalyst. Delivered via a jet injector to designated nerve terminals, this assembly utilizes reactive oxygen species to catalytically dope poly (3,4-ethylenedioxythiophene), enhancing π-π interactions between nanosheets, and yielding a conductive, biodegradable interface. This interface effectively regulates local immune activity and promotes sensory and motor nerve functional restoration in nerve-injured mice, while engaging the vagal-adrenal axis in freely moving mice, eliciting catecholamine neurotransmitter release, and suppressing systemic cytokine storms. This innovative strategy specifically targets nerve substructures, bolstering local and systemic immune modulation, and paving the way for the development of self-adaptive dynamic neural interfaces.

Ablative and Immunostimulatory Effects of Histotripsy Ablation in a Murine Osteosarcoma Model.

Background: Osteosarcoma (OS) is the most frequently occurring malignant bone tumor in humans, primarily affecting children and adolescents. Significant advancements in treatment options for OS have not occurred in the last several decades, and the prognosis remains grim with only a 70% rate of 5-year survival. The objective of this study was to investigate the focused ultrasound technique of histotripsy as a novel, noninvasive treatment option for OS. Methods: We utilized a heterotopic OS murine model to establish the feasibility of ablating OS tumors with histotripsy in a preclinical setting. We investigated the local immune response within the tumor microenvironment (TME) via immune cell phenotyping and gene expression analysis. Findings: We established the feasibility of ablating heterotopic OS tumors with ablation characterized microscopically by loss of cellular architecture in targeted regions of tumors. We observed greater populations of macrophages and dendritic cells within treated tumors and the upregulation of immune activating genes 72 h after histotripsy ablation. Interpretation: This study was the first to investigate histotripsy ablation for OS in a preclinical murine model, with results suggesting local immunomodulation within the TME. Our results support the continued investigation of histotripsy as a novel noninvasive treatment option for OS patients to improve clinical outcomes and patient prognosis.

Local Immunomodulation of Human Periodontal Ligaments Stem Cells (PDLSC) in Periodontitis: A Systematic Review

Background: Human periodontal ligament stem cells (PDLSC) are multipotent cells, derived from periodontal ligament (PDL), that can attain the formation of new bone, cementum, and periodontal ligament. However, the quantity of stem cells found in PDL tissues is deficient. Therefore, in periodontitis, the limited PDLSCs make regeneration of the periodontium challenging and unpredictable. Objectives: To critically appraise the available experimental evidence of the local immunomodulation signaling systems and their surrounding functional molecules for repair and application in periodontal regeneration therapy. Methods: This systematic review was conducted using PRISMA 2020 guidelines and research papers were selected based on strict exclusion and inclusion criteria. PubMed, Cochrane Library, Google Scholar and Web of Science databases were used to collect all the research papers ranging from January 2012 to December 2022. Results: Nine out of 586 research papers that focused on PDLSCs during inflammatory conditions were selected based on the Cochrane Risk of Bias Assessment. Seven articles documented that non-coding microRNAs (ncRNAs) played critical roles in the modification of PDLSC multipotency in periodontitis. An article showed that the post-translational modifications are influenced by histone deacetylases (HDAC) indicating the involvement of novel epigenetic signaling systems. A natural product-derived functional molecule resveratrol is described to have anti-inflammatory properties through the nuclear factor kappa B (NF-kB) pathway. Conclusion: The results of our present systematic review clearly apprise that the ncRNAs signaling system and natural products derived anti-inflammatory functional molecules play a clinically significant therapeutic target in novel periodontitis therapy as preventive, diagnostic, therapeutic, and prognostic markers.

LMAP-07 POTENTIAL MECHANISM OF IMMUNE ESCAPE RELATED TO TUMOR-ASSOCIATED MACROPHAGE IN GLIOBLASTOMA AFTER COMBINED THERAPY WITH RADIOTHERAPY AND IMMUNE CHECKPOINT BLOCKER

Abstract Immune checkpoint blockers (ICBs) have revolutionized the treatment of cancer. Radiotherapy (RT) has been demonstrated to increase the survival benefit of ICBs in some cancers through its local and systemic immunomodulation, although its efficacy in glioblastoma (GBM) is very limited. With a thorough investigation of the immune responses to single and dual treatment utilizing single-cell RNA-seq analyses on the synergy of RT and anti-PD-L1 antibody (aPD-L1), we studied the potential mechanism of immune escape in GBM even when RT and ICB are combined. Our findings demonstrated a statistically significant longer survival of mice treated with RT and aPD-L1 combined than either single modality of treatment in an orthotopic syngeneic GBM model achieving a 70% cure rate. In the tumor tissues collected from the mouse brain that failed dual treatment, i.e., immune escaped, immune cell profiling still demonstrated much lower T regulatory cells and higher CD8+ cytotoxic T cells infiltrated to tumor, and elevated CD8+/CD4+ and CD8+/Treg ratios in the dual therapy group compared to single therapies, as expected with synergistic effects of dual treatment. RT with aPD-L1 markedly reduced CD8+ and CD4+ T exhaustion cells and elevated T-memory cells in comparison to single modality groups. Nevertheless, compared to the RT alone or no treatment control group, dual therapy failed to decrease the proportion of Arginase1-positive glioma-associated macrophages (GAM) in macrophage/monocytes in tumor microenvironment, which were considerably increased if treated with aPD-L1 only. Moreover, the combination of RT with aPD-L1 did not alter immunosuppressive gene expression in the macrophage/monocyte groups. In conclusion, our research demonstrated that RT and aPD-L1 together may elicit a potent and durable antitumor immune response in the GBM mouse model through regulating lymphocytes but not macrophages. Our findings point to the urgent need for research into GAM-modulating drug and their potential to prevent immunological escape.

Localized immunomodulation technologies to enable cellular and organoid transplantation

Localized immunomodulation technologies are rapidly emerging as a new modality with the potential to revolutionize transplantation of cells and organs. In the past decade, cell-based immunomodulation therapies saw clinical success in the treatment of cancer and autoimmune diseases. In this review, we describe recent advances in engineering solutions for the development of localized immunomodulation techniques focusing on cellular and organoid transplantation. We begin by describing cell transplantation and highlighting notable clinical successes, particularly in the areas of stem cell therapy, chimeric antigen receptor (CAR)-T cell therapy, and islet transplantation. Next, we detail recent preclinical studies centered on genome editing and biomaterials to enhance localized immunomodulation. We close by discussing future opportunities to improve clinical and commercial success using these approaches to facilitate long-term immunomodulation technologies.

Advances in cutaneous squamous cell carcinoma.

Human malignancies arise predominantly in tissues of epithelial origin, where the stepwise transformation from healthy epithelium to premalignant dysplasia to invasive neoplasia involves sequential dysregulation of biological networks that govern essential functions of epithelial homeostasis. Cutaneous squamous cell carcinoma (cSCC) is a prototype epithelial malignancy, often with a high tumour mutational burden. A plethora of risk genes, dominated by UV-induced sun damage, drive disease progression in conjunction with stromal interactions and local immunomodulation, enabling continuous tumour growth. Recent studies have identified subpopulations of SCC cells that specifically interact with the tumour microenvironment. These advances, along with increased knowledge of the impact of germline genetics and somatic mutations on cSCC development, have led to a greater appreciation of the complexity of skin cancer pathogenesis and have enabled progress in neoadjuvant immunotherapy, which has improved pathological complete response rates. Although measures for the prevention and therapeutic management of cSCC are associated with clinical benefit, the prognosis remains poor for advanced disease. Elucidating how the genetic mechanisms that drive cSCC interact with the tumour microenvironment is a current focus in efforts to understand, prevent and treat cSCC.

Endometrial gene expression of DEFB4B, LYZ, and SLPI in biopsy samples of mares resistant or susceptible to endometritis

Endometritis is one of the main causes of subfertility in mares and therefore one of the most common reasons for economic losses in the horse breeding industry. Multiparous older mares are more susceptible to the development of persistent endometritis after mating, artificial insemination, or foaling since aging is associated with an altered systemic immune response and anatomical defects that interfere with physiological uterine clearance (Katila et.al. Animals. 2022; 12:779). In this context, their inability to recover from endometritis can lead to degenerative changes in the endometrium (e.g., glandular degenerative changes and associated peri-glandular and/or diffuse stromal fibrosis) (Canisso et.al. International Journal of Molecular Sciences 2020; 21:1432). This study aimed to evaluate possible variations in endometrial gene expression of the genes Defensin Beta 4B (DEFB4B), Lysozyme (LYZ), and Secretory Leukocyte Peptidase Inhibitor (SLPI) between resistant and susceptible mares. These three genes have already been identified as involved in the equine innate immune response and in the chronic uterine inflammatory response (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). Endometrial biopsy samples were collected from 19 mares. Mares were divided into two groups concerning their age and reproductive history: the "resistant group" (RES) (N=4) consisted of maiden mares 3.00±0.00 years old with endometrial biopsy grade I according to Kenney and Doig classification (1986); the "susceptible group" (SUS) (N=15)was composed of multiparous mares 17.33±3.28 years old, with endometrial biopsy of grade IIA, IIB or III. Total RNA was extracted from each sample and the corresponding cDNAs were prepared by retro-transcription using dedicated commercial kits. The samples were then amplified by qRT-PCR procedure as described by Marth and colleagues (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). The housekeeping gene GADPH was amplified in each sample as internal control and for data normalization. To minimize possible experimental errors, for each sample three technical replicates were performed and the quality of the extracted RNA was evaluated by spectrophotometry and gel electrophoresis. The results showed a statistically significant difference in the expression of the genes LYZ (p=0.0003) and DEFB4B (p=0.0273) which were upregulated in SUS versus RES mares. This suggests that lower gene expression found in young mares are attributable to a more balanced local immunomodulation, with consequent reduction of the inflammatory pathways and slower progression towards the deposition of fibrotic tissue compared to older mares. No differences in the expression of the SLPI gene were found between the two groups (p=0.8854). In conclusion, despite these promising results, larger studies are needed before LYZ and DEFB4B genes could be used as biomarkers to identify the mares susceptible to endometritis.

Sustained release hydrogel for durable locoregional chemoimmunotherapy for BRAF-mutated melanoma

The wide prevalence of BRAF mutations in diagnosed melanomas drove the clinical advancement of BRAF inhibitors in combination with immune checkpoint blockade for treatment of advanced disease. However, deficits in therapeutic potencies and safety profiles motivate the development of more effective strategies that improve the combination therapy's therapeutic index. Herein, we demonstrate the benefits of a locoregional chemoimmunotherapy delivery system, a novel thermosensitive hydrogel comprised of gelatin and Pluronic® F127 components already widely used in humans in both commercial and clinical products, for the co-delivery of a small molecule BRAF inhibitor with immune checkpoint blockade antibody for the treatment of BRAF-mutated melanoma. In vivo evaluation of administration route and immune checkpoint target effects revealed intratumoral administration of antagonistic programmed cell death protein 1 antibody (aPD-1) lead to potent antitumor therapy in combination with BRAF inhibitor vemurafenib. The thermosensitive F127-g-Gelatin hydrogel that was evaluated in multiple murine models of BRAF-mutated melanoma that facilitated prolonged local drug release within the tumor (>1 week) substantially improved local immunomodulation, tumor control, rates of tumor response, and animal survival. Thermosensitive F127-g-Gelatin hydrogels thus improve upon the clinical benefits of vemurafenib and aPD-1 in a locoregional chemoimmunotherapy approach for the treatment of BRAF-mutated melanoma.