Opposing Functions of Interferon Coordinate Adaptive and Innate Immune Responses to Cancer Immune Checkpoint Blockade.

Interferon-gamma (IFNG) augments immune function yet promotes T cell exhaustion through PDL1. How these opposing effects are integrated to impact immune checkpoint blockade (ICB) is unclear. We show that while inhibiting tumor IFNG signaling decreases interferon-stimulated genes (ISGs) in cancer cells, it increases ISGs in immune cells by enhancing IFNG produced by exhausted T cells (TEX). In tumors with favorable antigenicity, these TEX mediate rejection. In tumors with neoantigen or MHC-I loss, TEX instead utilize IFNG to drive maturation of innate immune cells, including a PD1+TRAIL+ ILC1 population. By disabling an inhibitory circuit impacting PD1 and TRAIL, blocking tumor IFNG signaling promotes innate immune killing. Thus, interferon signaling in cancer cells and immune cells oppose each other to establish a regulatory relationship that limits both adaptive and innate immune killing. In melanoma and lung cancer patients, perturbation of this relationship is associated with ICB response independent of tumor mutational burden.

CRISPR screen in regulatory T cells reveals modulators of Foxp3.

Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

Comparisons of Non-Oral Immune-Related Adverse Events Among Patients With Cancer With Different Oral Toxicity Profiles.

OBJECTIVES: Immune-related adverse events (irAEs) are common. Oral irAEs tend to cluster in patients who experience concurrent toxicities. We aimed to characterize the frequency and trajectory of non-oral irAEs in patients who developed oral irAEs, assess their relationship with non-oral irAEs, and compare those characteristics with patients without oral irAEs. METHODS: A retrospective chart review was conducted to identify patients who started ICIT between December 11, 2011, and September 15, 2019 (n = 4683) in the Mass General Brigham Registered Patient Data Registry. Demographic information, cancer diagnosis, ICIT regimen, treatment duration, and time and number of infusions to irAE onset were recorded. Non-oral irAEs were categorized into 13 groups. Patients with melanoma, pulmonary cancer, or head and neck cancer who had oral irAEs were then matched with those without oral irAEs to compare the prevalence of concomitant non-oral irAEs. RESULTS: Three hundred and fourteen patients with oral irAEs with a mean age of 65.9 ±â€…12.6 years (43.3% females) were included. Patients with multiple oral irAEs were more likely to have non-oral irAEs (OR: 2.7, 95% CI, 1.3-3.5), including cutaneous (OR: 1.7, 95% CI, 1.1-3.0), rheumatological (OR: 2.2, 95% CI, 1.1-4.2), thyroid (OR: 2.4, 95% CI, 1.2-4.9), and neurological irAEs (OR: 2.5, 95% CI, 1.0-6.3). Compared to matched patients with non-oral irAEs, patients with oral irAEs were more likely to have cutaneous (OR: 1.7, 95% CI, 1.0-2.8) and thyroid (OR: 2.86, 95% CI, 1.1-7.5) irAEs. The development of oral and non-oral irAEs is often coincidental. CONCLUSION: Patients who have non-oral irAEs should be monitored for development of oral irAEs for prompt management.

Mönckeberg's medial arteriosclerosis in the oral and maxillofacial region: A pilot study.

OBJECTIVE: To evaluate the prevalence of medial vascular calcifications in the oral and maxillofacial region and their association with systemic diseases. MATERIALS AND METHODS: The study included 211 consecutive patients with systemic diseases (January 2015-May 2016). Medical history and radiographic images were evaluated. Univariate analysis (t-test) was performed for continuous variables (age). The Chi square test was applied for the categorical variables (Mönckeberg medial arteriosclerosis [MMA], gender). RESULTS: There was a 6.2% prevalence of MMA. The mean age of patients with MMA was 65.46 ± 13.38. The prevalence of kidney disease in patients with MMA was significantly higher than in those without MMA (p < 0.001). This finding was maintained even after adjusting for other systemic diseases (OR = 31.84 [8.63-136.78]). CONCLUSION: A significant prevalence of MMA in kidney disease patients was observed in this pilot study.

HRD1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination.

The endoplasmic reticulum-associated protein degradation (ERAD) is responsible for recognizing and retro-translocating protein substrates, misfolded or not, from the ER for cytosolic proteasomal degradation. HMG-CoA Reductase (HMGCR) Degradation protein-HRD1-was initially identified as an E3 ligase critical for ERAD. However, its physiological functions remain largely undefined. Herein, we discovered that hepatic HRD1 expression is induced in the postprandial condition upon mouse refeeding. Mice with liver-specific HRD1 deletion failed to repress FGF21 production in serum and liver even in the refeeding condition and phenocopy the FGF21 gain-of-function mice showing growth retardation, female infertility, and diurnal circadian behavior disruption. HRD1-ERAD facilitates the degradation of the liver-specific ER-tethered transcription factor CREBH to downregulate FGF21 expression. HRD1-ERAD catalyzes polyubiquitin conjugation onto CREBH at lysine 294 for its proteasomal degradation, bridging a multi-organ crosstalk in regulating growth, circadian behavior, and female fertility through regulating the CREBH-FGF21 regulatory axis.

Oral lichen planus: comparative efficacy and treatment costs-a systematic review.

OBJECTIVE: To compare the reported efficacy and costs of available interventions used for the management of oral lichen planus (OLP). MATERIALS AND METHODS: A systematic literature search was performed from database inception until March 2021 in MEDLINE via PubMed and the Cochrane library following PRISMA guidelines. Only randomized controlled trials (RCT) comparing an active intervention with placebo or different active interventions for OLP management were considered. RESULTS: Seventy (70) RCTs were included. The majority of evidence suggested efficacy of topical steroids (dexamethasone, clobetasol, fluocinonide, triamcinolone), topical calcineurin inhibitors (tacrolimus, pimecrolimus, cyclosporine), topical retinoids, intra-lesional triamcinolone, aloe-vera gel, photodynamic therapy, and low-level laser therapies for OLP management. Based on the estimated cost per month and evidence for efficacy and side-effects, topical steroids (fluocinonide > dexamethasone > clobetasol > triamcinolone) appear to be more cost-effective than topical calcineurin inhibitors (tacrolimus > pimecrolimus > cyclosporine) followed by intra-lesional triamcinolone. CONCLUSION: Of common treatment regimens for OLP, topical steroids appear to be the most economical and efficacious option followed by topical calcineurin inhibitors. Large-scale multi-modality, prospective trials in which head-to-head comparisons interventions are compared are required to definitely assess the cost-effectiveness of OLP treatments.

Oral side effects of immune checkpoint inhibitor therapy (ICIT): An analysis of 4683 patients receiving ICIT for malignancies at Massachusetts General Hospital, Brigham & Women's Hospital, and the Dana-Farber Cancer Institute, 2011 to 2019.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are increasingly accepted as a treatment option for several cancers. Although various systemic immune-related adverse events (irAEs) have been characterized, the effect of ICIs on the oral cavity and contiguous structures is still poorly understood. METHODS: Electronic medical records of 4683 patients in the Mass General Brigham Registered Patient Data Registry who received ICI therapy (ICIT) between December 2011 and September 2019 were reviewed. Reports of oral conditions were categorized into oral mucosal disorders, xerostomia, and dysgeusia. After applying exclusion criteria, demographic characteristics and clinical features were summarized for the patients who had oral irAEs. RESULTS: In total, 317 patients developed oral conditions that were associated with ICIT (incidence, 6.8%; 317 of 4683 patients). These conditions included xerostomia (68.5%), oral mucosal disorders (33.4%), and dysgeusia (24.0%). In patients with oral irAEs, respiratory cancer (28.4%) was the most common primary cancer, followed by melanoma (26.2%), and head and neck cancer (14.8%). Oral mucosal disorders developed after the initiation of ICIT between 2 and 851 days (between 1 and 1332 days in patients with xerostomia and between 1 and 1455 days in patients with dysgeusia). Of all oral irAEs, 50.9% developed within 3 months, and 85.5% developed within 12 months. CONCLUSIONS: Oral side effects appear to be more common among patients who receive ICIT than has been previously reported. Concomitant cytotoxic regimens may exacerbate the risk of oral adverse events, perhaps representing the sum of the effects of different, but simultaneous or sequential, pathogenic mechanisms. Additional studies are warranted to better characterize oral irAEs and their biologic basis.

Endoplasmic reticulum-associated degradation and beyond: The multitasking roles for HRD1 in immune regulation and autoimmunity.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism against ER stress, wherein unfolded/misfolded proteins accumulated in the ER are transported to the cytosol for degradation by the ubiquitin-proteasome system. The ER resident E3 ubiquitin ligase HRD1 has been identified as a key ERAD factor that directly catalyzes ubiquitin conjugation onto the unfolded or misfolded proteins for proteasomal degradation. The abnormally increased HRD1 expression was discovered in rheumatoid synovial cells, providing the first evidence for HRD1 dysregulation involved in human inflammatory pathogenesis. Further studies shown that inflammatory cytokines involved in rheumatoid pathogenesis including IL-1ß, TNF-α, IL-17 and IL-26 induce HRD1 expression. Recent studies using mice with tissue-specific targeted deletion of HRD1 gene have revealed important functions of HRD1 in immune regulation and inflammatory diseases. HRD1 has been shown critical for dendritic cell expression of antigens to both CD4 and CD8 T cells. Both TCR and costimulatory receptor CD28 signaling induces HRD1 expression, which promotes T cell clonal expansion and IL-2 production. Together with the fact that HRD1 is required for maintaining the stability of regulatory T cell (Treg) stability, HRD1 appears to fine tone T cell immunity. In addition, HRD1 is involved in humoral immune response by regulating early B cell development and maintaining B cell survival upon recognition of specific antigen. HRD1 appears to target its substrates for ubiquitination through, either ERAD-dependent or -independent, at least two distinct molecular mechanisms in a cell or tissue specific manner to achieve its physiological functions. Dysregulation of HRD1 expression and/or it functions are involved in autoimmune inflammatory diseases in particular rheumatoid arthritis and lupus. Here, we review current findings on the mechanism of HRD1 protein in immune regulation and the involvement of HRD1 in the pathogenesis of autoimmune inflammatory diseases.

USP22 deficiency leads to myeloid leukemia upon oncogenic Kras activation through a PU.1-dependent mechanism.

Ras mutations are commonly observed in juvenile myelomonocytic leukemia (JMML) and chronic myelomonocytic leukemia (CMML). JMML and CMML transform into acute myeloid leukemia (AML) in about 10% and 50% of patients, respectively. However, how additional events cooperate with Ras to promote this transformation are largely unknown. We show that absence of the ubiquitin-specific peptidase 22 (USP22), a component of the Spt-Ada-GCN5-acetyltransferase chromatin-remodeling complex that is linked to cancer progression, unexpectedly promotes AML transformation in mice expressing oncogenic KrasG12D/+ USP22 deficiency in KrasG12D/+ mice resulted in shorter survival compared with control mice. This was due to a block in myeloid cell differentiation leading to the generation of AML. This effect was cell autonomous because mice transplanted with USP22-deficient KrasG12D/+ cells developed an aggressive disease and died rapidly. The transcriptome profile of USP22-deficient KrasG12D/+ progenitors resembled leukemic stem cells and was highly correlated with genes associated with poor prognosis in AML. We show that USP22 functions as a PU.1 deubiquitylase by positively regulating its protein stability and promoting the expression of PU.1 target genes. Reconstitution of PU.1 overexpression in USP22-deficient KrasG12D/+ progenitors rescued their differentiation. Our findings uncovered an unexpected role for USP22 in Ras-induced leukemogenesis and provide further insights into the function of USP22 in carcinogenesis.

JAK1-mediated Sirt1 phosphorylation functions as a negative feedback of the JAK1-STAT3 pathway.

The type III NAD-dependent histone deacetylase Sirt1 plays important roles in a variety of pathobiological functions through targeting either the acetylated histones or transcription factors. However, the molecular mechanisms underlying how the Sirt1 functions are regulated remain vague. Herein we identified that the Janus kinase 1 (JAK1) interacts with Sirt1 and catalyzes its phosphorylation at the tyrosine residues of 280 and 301, both of which are highly conserved and located in the histone deacetylase catalytic domain of Sirt1. IL-6 stimulation enhanced Sirt1 interaction with JAK1 and JAK1-mediated Sirt1 phosphorylation. Interestingly, JAK1-mediated Sirt1 phosphorylation did not alter Sirt1 deacetylase catalytic activity, but instead it is required for Sirt1 interaction with the downstream transcription factor STAT3. JAK1-mediated phosphorylation enhanced Sirt1 suppression of STAT3 acetylation and transcriptional activity. As a consequence, Sirt1 activation attenuates IL-6 activity in protecting cancer cells from chemotherapeutic drug-induced apoptosis. Our studies identify JAK1 as a previously unappreciated tyrosine kinase of Sirt1 and reveal a novel negative feedback of the JAK1-STAT3 pathway.

Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas.

Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity.

Effect of Adhesive Debonding on the Performance of Piezoelectric Sensors in Structural Health Monitoring Systems.

Piezoelectric (PZT) ceramic elements are often subjected to complex loads during in- service lifetime in structural health monitoring (SHM) systems, and debonding of both excitation actuators and receiving sensors have a negative effect on the monitoring signals. A first systematic investigation of debonding behaviors by considering actuators and sensors simultaneously was performed in this paper. The debonding areas of actuators were set in different percentage range from 0% to 70%, and sensors in 0%, 20%, 40% and 60%. The signal-based monitoring method was used to extract the characteristic parameters of both the amplitudes and phases of received signals. Experimental results revealed that as the debonding areas of the actuators increase, the normalized amplitude appears a quick decrease before 35% debonding area of actuators and then a slow rise until 60% of debonding reached. This may be explained that the 35% debonding turning point correspond to the coincidence of the excitation frequencies of peripheral actuators with the inherent frequency of the central piezoelectric sensor, and the 60% be the result of the maximum ability of piezoelectric sensor. The degrees of debonding of actuators and sensors also have significant influence on the phase angle offset, with large debonding of actuators increases the phase offset sharply. The research work may provide useful information for practical monitoring of SHM systems.

Human fallopian tube epithelium co-culture with murine ovarian follicles reveals crosstalk in the reproductive cycle.

STUDY QUESTION: Do interactions between human fallopian tube epithelium and murine follicles occur during an artificial reproductive cycle in a co-culture system in vitro? SUMMARY ANSWER: In a co-culture system, human fallopian tissues responded to the menstrual cycle mimetic by changes in morphology and levels of secreted factors, and increasing murine corpus luteum progesterone secretion. WHAT IS KNOWN ALREADY: The entire fallopian tube epithelium, including ciliated and secretory cells, can be regulated in the reproductive cycle. Currently, there are no in vitro culture models that can monitor fallopian tissues in real time in response to factors produced by the ovary. In addition, there are no reports on the impact of fallopian tissue on ovarian function during the menstrual cycle. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Human fallopian tissue (n = 24) was obtained by routine hysterectomies from women (aged 26-50 years, mean age = 43.6) who had not undergone exogenous hormonal treatment for at least 3 months prior to surgery. CD1 female mice were used for ovarian follicle isolation. The human fallopian epithelium layers were either co-cultured with five murine multilayer secondary follicles (150-180 µm follicles, encapsulated in one alginate gel bead) for 15 days or received stepwise steroid hormone additions for 13 days. The fallopian tissue morphology and cilia beating rate, as measured by an Andor Spinning Disk Confocal, were investigated. Oviduct-specific glycoprotein 1 (OVGP1), human insulin-like growth factor 1 (hIGF1), vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL8) as biological functional markers were measured either by ELISA or western blot to indicate dynamic changes in the fallopian epithelium during the reproductive cycle generated by mouse follicles or by stepwise steroid hormone induction. Three or four patients in each experiment were recruited for replicates. Data were presented as mean ± SD and further analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. MAIN RESULTS AND THE ROLE OF CHANCE: The cultured fallopian tube epithelium responded to exogenous steroid hormone stimulation, as demonstrated by enhanced cilia beating rate (~25% increase, P = 0.04) and an increase in OVGP1 secretion (P = 0.02) in response to 1 nM estradiol (E2) treatment when compared with 0.1 nM E2. Conversely, 10 nM progesterone plus 1 nM E2 suppressed cilia beating rate by ~30% (P = 0.008), while OVGP1 secretion was suppressed by 0.1 nM E2 plus 50 nM progesterone (P = 0.002 versus 1 nM E2 alone). Human fallopian tube epithelium was co-cultured with murine secondary follicles to mimic the human menstrual cycle. OVGP1 and VEGF-A secretion from fallopian tissue was similar with stepwise hormone treatment and when cultured with murine follicles. However, the secretion patterns of hIGF1 and IL8 differed in the luteal phase when comparing steroid treatment with follicle co-culture. In co-culture, hIGF1 secretion was suppressed in the luteal versus follicular phase (P = 0.005) but stepwise hormone treatment had no effect on hIGF1. In co-culture, IL8 secretion was also suppressed on luteal phase day 15 (P = 0.013) versus follicular phase day 7, but IL8 secretion increased continuously under high E2/progesterone treatment (P = 0.003 for D13 versus D3). In the co-culture system, the corpus luteum continuously produced progesterone in the presence of fallopian tube tissue until Day 18 while, without fallopian tissue, progesterone started to drop from Day 13. LIMITATIONS, REASONS FOR CAUTION: One limitation of this study is that murine follicles were used to mimic the human menstrual cycle. However, although secretion patterns of peptide hormones such as inhibins and activins differ in mice and humans, the co-culture system used here did reveal interactions between the tissues that govern reproductive function. WIDER IMPLICATIONS OF THE FINDINGS: In vitro co-culture models of fallopian reproductive tissues with ovarian follicles can provide an important tool for understanding fertility and for uncovering the mechanisms responsible for reduced fertility. In addition, the role of oviductal secretions and how they influence ovarian function, such as the production of progesterone during the menstrual cycle, can be uncovered using this model. LARGE-SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was funded by grants from the NIH (UH3TR001207), the American Cancer Society (RSG-12-230-01-TBG) and NIH (R01EB014806). The authors declare no competing financial interest.

The inorganic anatomy of the mammalian preimplantation embryo and the requirement of zinc during the first mitotic divisions.

BACKGROUND: Zinc is the most abundant transition metal in the mammalian oocyte, and dynamic fluxes in intracellular concentration are essential for regulating both meiotic progression and fertilization. Whether the defined pathways of zinc utilization in female meiosis directly translate to mitotic cells, including the mammalian preimplantation embryo, has not been studied previously. RESULTS: We determined that zinc is the most abundant transition metal in the preimplantation embryo, with levels an order of magnitude higher than those of iron or copper. Using a zinc-specific fluorescent probe, we demonstrated that labile zinc is distributed in vesicle-like structures in the cortex of cells at all stages of preimplantation embryo development. To test the importance of zinc during this period, we induced zinc insufficiency using the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). Incubation of embryos in media containing TPEN resulted in a developmental arrest that was specific to zinc chelation and associated with compromised mitotic parameters. The developmental arrest due to zinc insufficiency was associated with altered chromatin structure in the blastomere nuclei and decreased global transcription. CONCLUSIONS: These results demonstrate for the first time that the preimplantation embryo requires tight zinc regulation and homeostasis for the initial mitotic divisions of life.

Engineering the ovarian cycle using in vitro follicle culture.

STUDY QUESTION: Can cultured follicles model the ovarian cycle, including follicular- and luteal-phase hormone synthesis patterns and ovulation? SUMMARY ANSWER: Under gonadotrophin stimulation, murine follicles grown in an encapsulated three-dimensional system ovulate in vitro and murine and human follicle hormone synthesis mimics follicular and luteal phases expected in vivo. WHAT IS KNOWN ALREADY: Studies of the human ovary and follicle function are limited by the availability of human tissue and lack of in vitro models. We developed an encapsulated in vitro follicle growth (eIVFG) culture system, which preserves 3D follicular structure. Thus far, the alginate system has supported the culture of follicles from mice, dog, rhesus macaque, baboon and human. These studies have shown that cultured follicles synthesize steroid hormones similar to those observed during the follicular phase in vivo. STUDY DESIGN, SIZE, DURATION: Cultured murine follicles were treated with human chorionic gonadotrophin (hCG) and epidermal growth factor (EGF) and either assayed for luteinization or removed from alginate beads and assayed for ovulation. Human follicles were also cultured, treated with follicle-stimulating hormone (FSH), hCG and EGF to mimic gonadotrophin changes throughout the ovarian cycle, and culture medium was assayed for hormone production. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine and human follicles were cultured in alginate hydrogel and hormone production [17ß-estradiol, progesterone, inhibin A, inhibin B, activin A and anti-Müllerian hormone (AMH)] was quantified in medium by enzyme-linked immuno assay (ELISA). Human ovarian tissue was acquired from females between 6 and 34 years of age with a cancer diagnosis. These participants were undergoing ovarian tissue cryopreservation at National Physicians Cooperative sites as part of the Oncofertility Consortium. MAIN RESULTS AND THE ROLE OF CHANCE: When grown in this system, 96% of mouse follicles ovulated in response to hCG and released meiotically competent eggs. Ovulated follicles recapitulated transcriptional, morphologic and hormone synthesis patterns post-luteinizing hormone (LH/hCG). In addition to rodent follicles, individual human follicles secreted steroid and peptide hormones that mimicked the patterns of serum hormones observed during the menstrual cycle. LIMITATIONS, REASONS FOR CAUTION: This was a descriptive study of an in vitro model of ovulation and the ovarian hormone cycle. The ovulation studies were limited to murine tissue and further studies are needed to optimize conditions using other species. WIDER IMPLICATIONS OF THE FINDINGS: The eIVFG system reliably phenocopies the in vivo ovarian cycle and provides a new tool to study human follicle biology and the influence of cycling female hormones on other tissue systems in vitro. STUDY FUNDING/COMPETING INTERESTS: This work was supported by NIH U54 HD041857, NIH U54 HD076188, NIH UH2 E5022920, NIH UH3 TR001207 and F30 AG040916 (R.M.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Use of an organotypic mammalian in vitro follicle growth assay to facilitate female reproductive toxicity screening.

Screening of pharmaceutical, chemical and environmental compounds for their effects on reproductive health relies on in vivo studies. More robust and efficient methods to assess these effects are needed. Herein we adapted and validated an organotypic in vitro follicle growth (IVFG) assay to determine the impact of compounds on markers of ovarian function. We isolated mammalian follicles and cultured them in the presence of compounds with: (1) known fertotoxicity (i.e. toxicity to the reproductive system; cyclophosphamide and cisplatin); (2) no known fertotoxicity (nalbuphine); and (3) unknown fertotoxicity (Corexit EC 9500 A; CE, Nalco, Chicago, IL, USA). For each compound, we assayed follicle growth, hormone production and the ability of follicle-enclosed oocytes to resume meiosis and produce a mature egg. Cyclophosphamide and cisplatin caused dose-dependent disruption of follicle dynamics, whereas nalbuphine did not. The reproductive toxicity of CE, an oil dispersant used heavily during the 2010 Deepwater Horizon oil spill, has never been examined in a mammalian system. In the present study, CE compromised follicle morphology and functional parameters. Our findings demonstrate that this IVFG assay system can be used to distinguish fertotoxic from non-toxic compounds, providing an in vitro tool to assess the effects of chemical compounds on reproductive function and health.

Thalidomide for management of refractory oral mucosal diseases.

BACKGROUND: Thalidomide has anti-inflammatory properties and has been used off-label for multiple mucocutaneous disorders, but its application in managing refractory oral mucosal diseases is unclear. This study aimed to review the efficacy and safety of thalidomide in treating various oral mucosal disorders refractory to conventional therapies. METHODS: The medical records of patients who were prescribed thalidomide from 2002 through 2021 for oral mucosal disorders were reviewed. Data collected included demographic characteristics, oral mucosal disease diagnosis, treatment courses, and thalidomide dose, duration, response, and side effects. RESULTS: Thalidomide was prescribed for 28 patients with diagnoses of recurrent aphthous stomatitis (n = 14), inflammatory oral lichenoid lesions (n = 6), traumatic ulcerative granuloma with stroma eosinophilia (n = 5), chronic radiation-induced mucositis (n = 2), and orofacial granulomatosis (n = 1). Patients were treated for a median duration of 84 days (range 2-1,582). Clinical improvement was observed in 19 of 22 patients who completed at least 1 cycle of thalidomide (86.4%), with complete resolution in 12 patients (54.5%). Adverse events occurred in 75% of patients (n = 21), with 8 requiring thalidomide discontinuation. The most common adverse events included peripheral neuropathy (42.9%), drowsiness (28.6%), and constipation (21.4%). CONCLUSIONS: Thalidomide may be considered for the management of refractory oral mucosal disorders. Drug side effects are common and need monitoring closely during use.

Application of PRI-E-a combined learning method in oral and maxillofacial oncology education.

The traditional lecture-based learning (LBL) method is facing great challenges due to its low efficiency and single proceeding form. We designed a PRI-E learning mode that combined and modified problem-based, case-based, and evidence-based learning with a step-by-step approach. We evaluated the practical learning outcomes of using the PRI-E mode by comparing it with traditional lecture-based learning in oral and maxillofacial oncology education. "PRI-E" consists of the first letters of the English words Passion, Research, Innovation, and Education, and it means "the best Education". This prospective randomized controlled trial included 40 participants. We evenly divided the participants into the PRI-E (n = 20) and LBL group (n = 20) based on the entrance test scores. The same staff group designed and then taught the learning content with different group measures. The evaluation included the final test scores and questionnaire assessments. Without affecting the examination results, the PRI-E teaching method was more satisfactory and popular with participants in terms of ability development and classroom participation. Enacting the PRI-E teaching method required more time, but this did not affect its popularity among the participants. Compared with the LBL learning mode, the PRI-E learning mode was more organized and efficient in oral and maxillofacial oncology education without affecting academic performance. This model has a high degree of satisfaction, which is conducive to training students' comprehensive ability.

Pharmacokinetics, pharmacodynamics, and toxicity of a PD-1-targeted IL-15 in cynomolgus monkeys.

PF-07209960 is a novel bispecific fusion protein composed of an anti-PD-1 antibody and engineered IL-15 cytokine mutein with reduced binding affinity to its receptors. The pharmacokinetics (PK), pharmacodynamics (PD), and toxicity of PF-07209960 were evaluated following once every other week subcutaneous (SC) or intravenous (IV) administration to cynomolgus monkeys in a repeat-dose PKPD (0.01-0.3 mg/kg/dose) and GLP toxicity study (0.1-3 mg/kg/dose). PF-07209960 showed dose dependent pharmacokinetics with a terminal T1/2 of 8 and 13 hours following IV administration at 0.03 and 0.1 mg/kg, respectively. The clearance is faster than a typical IgG1 antibody. Slightly faster clearance was also observed following the second dose, likely due to increased target pool and formation of anti-drug antibodies (ADA). Despite a high incidence rate of ADA (92%) observed in GLP toxicity study, PD-1 receptor occupancy, IL-15 signaling (STAT5 phosphorylation) and T cell expansion were comparable following the first and second doses. Activation and proliferation of T cells were observed with largest increase in cell numbers found in gamma delta T cells, followed by CD4+ and CD8+ T cells, and then NK cells. Release of cytokines IL-6, IFNγ, and IL-10 were detected, which peaked at 72 hours postdose. There was PF-07209960-related mortality at ≥1 mg/kg. At scheduled necropsy, microscopic findings were generalized mononuclear infiltration in various tissues. Both the no observed adverse effect level (NOAEL) and the highest non severely toxic dose (HNSTD) were determined to be 0.3 mg/kg/dose, which corresponded to mean Cmax and AUC48 values of 1.15 µg/mL and 37.9 µg*h/mL, respectively.