Accelerating diabetic wound healing by ROS-scavenging lipid nanoparticle-mRNA formulation.

Current treatment options for diabetic wounds face challenges due to low efficacy, as well as potential side effects and the necessity for repetitive treatments. To address these issues, we report a formulation utilizing trisulfide-derived lipid nanoparticle (TS LNP)-mRNA therapy to accelerate diabetic wound healing by repairing and reprogramming the microenvironment of the wounds. A library of reactive oxygen species (ROS)-responsive TS LNPs was designed and developed to encapsulate interleukin-4 (IL4) mRNA. TS2-IL4 LNP-mRNA effectively scavenges excess ROS at the wound site and induces the expression of IL4 in macrophages, promoting the polarization from the proinflammatory M1 to the anti-inflammatory M2 phenotype at the wound site. In a diabetic wound model of db/db mice, treatment with this formulation significantly accelerates wound healing by enhancing the formation of an intact epidermis, angiogenesis, and myofibroblasts. Overall, this TS LNP-mRNA platform not only provides a safe, effective, and convenient therapeutic strategy for diabetic wound healing but also holds great potential for clinical translation in both acute and chronic wound care.

The elucidation of species-specific receptor pharmacology: a case study using subtype selective para- and meta-carborane estrogen receptor agonists.

Estrogen receptors are essential pharmacological targets for treating hormonal disorders and estrogen-dependent malignancies. Selective activation of estrogen receptor (ER) ß is hypothesized to provide therapeutic benefit with reduced risk of unwanted estrogenic side-effects associated with ERα activity. However, activating ERß without activating α is challenging due to the high sequence and structural homology between the receptor subtypes. We assessed the impact of structural modifications to the parent compound OSU-ERß-12 on receptor subtype binding selectivity using cell-free binding assays. Functional selectivity was evaluated by transactivation in HEK-293 cells overexpressing human or murine estrogen receptors. In vivo selectivity was examined through the uterotrophic effects of the analogs after oral administration in estrogen-naïve female mice. Furthermore, we evaluated the in vivo pharmacokinetics of the analogs following single dose IV and oral administration. Regarding selectivity, a single compound exhibited greater functional selectivity than OSU-ERß-12 for human ERß. However, like others in the meta-carborane series, its poor in vivo pharmacokinetics limit its suitability for further development. Surprisingly, and at odds with their pharmacokinetic and in vitro human activity data, most analogs potently induced uterotrophic effects in estrogen-naïve female mice. Further investigation of activity in HEK293 cells expressing murine estrogen receptors revealed species-specific differences in the ER-subtype selectivity of these analogs. Our findings highlight species-specific receptor pharmacology and the challenges it poses to characterizing developmental therapeutics in preclinical species. Significance Statement This study investigates para- and meta-substituted carborane analogs targeting estrogen receptors, revealing the greater selectivity of carborane analogs for human ERß compared to the mouse homolog. These findings shed light on the intricacies of using preclinical species in drug development to predict human pharmacology. The report also provides insights for the refinement and optimization of carborane analogs as potential therapeutic agents for estrogen-related disease states.

STING Agonist-Derived LNP-mRNA Vaccine Enhances Protective Immunity Against SARS-CoV-2.

Lipid nanoparticle (LNP)-mediated delivery of messenger RNA (mRNA) COVID-19 vaccines has provided large-scale immune protection to the public. To elicit a robust immune response against SARS-CoV-2 infections, antigens produced by mRNAs encoding SARS-CoV-2 Spike glycoprotein need to be efficiently delivered and presented to antigen-presenting cells such as dendritic cells (DCs). As concurrent innate immune stimulation can facilitate the antigen presentation process, a library of non-nucleotide STING agonist-derived amino lipids (SALs) was synthesized and formulated into LNPs for mRNA delivery. SAL12 lipid nanoparticles (SAL12-LNPs) were identified as most potent in delivering mRNAs encoding the Spike glycoprotein (S) of SARS-CoV-2 while activating the STING pathway in DCs. Two doses of SAL12 S-LNPs by intramuscular immunization elicited potent neutralizing antibodies against SARS-CoV-2 in mice.

Ectopic expression of pericentric HSATII RNA results in nuclear RNA accumulation, MeCP2 recruitment, and cell division defects.

Within the pericentric regions of human chromosomes reside large arrays of tandemly repeated satellite sequences. Expression of the human pericentric satellite HSATII is prevented by extensive heterochromatin silencing in normal cells, yet in many cancer cells, HSATII RNA is aberrantly expressed and accumulates in large nuclear foci in cis. Expression and aggregation of HSATII RNA in cancer cells is concomitant with recruitment of key chromatin regulatory proteins including methyl-CpG binding protein 2 (MeCP2). While HSATII expression has been observed in a wide variety of cancer cell lines and tissues, the effect of its expression is unknown. We tested the effect of stable expression of HSATII RNA within cells that do not normally express HSATII. Ectopic HSATII expression in HeLa and primary fibroblast cells leads to focal accumulation of HSATII RNA in cis and triggers the accumulation of MeCP2 onto nuclear HSATII RNA bodies. Further, long-term expression of HSATII RNA leads to cell division defects including lagging chromosomes, chromatin bridges, and other chromatin defects. Thus, expression of HSATII RNA in normal cells phenocopies its nuclear accumulation in cancer cells and allows for the characterization of the cellular events triggered by aberrant expression of pericentric satellite RNA.

Clinical predictors and risk factors for vaginal mesh extrusion.

PURPOSES: Our study aims to enhance the accuracy of the clinical diagnosis in patients with vaginal mesh extrusion following transvaginal mesh placement for pelvic organ prolapse using significant clinical parameters and risk factors. METHODS: All patients who underwent vaginal mesh removal were retrospectively reviewed from January 2000 to May 2014. Eligible patients were divided into two groups according to the presence of vaginal mesh extrusion. RESULTS: A total of 862 patients, 798 were included. 357 (44.7%) had evidence of vaginal mesh extrusion, and 441 (55.3%) had no evidence of vaginal mesh extrusion. The mean age of the vaginal mesh extrusion group was slightly higher than in the group without vaginal mesh extrusion (58.7 ± 11.2 vs. 56.4 ± 11.5, respectively; p = 0.002). From multivariate analysis, the significant clinical correlations for vaginal mesh extrusion were vaginal bleeding [60 (16.9) vs. 14 (3.2%), p < 0.001], hispareunia [48 (13.5) vs. 15 (3.4%), OR = 4.163, p < 0.001], and vaginal discharge [45 (12.6) vs. 18 (4.1%), p = 0.001]. The risk factors were multiple mesh implantations [218 (67.06) vs. 175 (39.68%), p < 0.001] and menopause [314 (88) vs. 364 (82.7%), p = 0.145]. Demographic data, including BMI, sexual activity, vaginal atrophy, both local and systemic hormonal use, smoking status, and hysterectomy status, were not significantly different, as well as the clinical symptoms including dyspareunia, vaginal infection, and symptomatic vaginal bulge. CONCLUSIONS: Vaginal bleeding, hispareunia, and vaginal discharge were the most significant clinical predictors for raising suspicion of vaginal mesh extrusion. Multiple mesh implantations were a significant risk factor for extrusion.

Changing Default Fluoroscopy Equipment Settings Decreases Entrance Skin Dose in Patients.

PURPOSE: Proper fluoroscopic education and protocols may reduce the patient radiation dose but few prospective studies in urology have been performed. Using optically stimulated luminescent dosimeters we tested whether fluoroscopy time and/or entrance skin dose would decrease after educational and radiation reduction protocols. MATERIALS AND METHODS: At default manufacturer settings fluoroscopy time and entrance skin dose were prospectively measured using optically stimulated luminescent dosimeters in patients undergoing ureteroscopy, retrograde pyelogram/stent or percutaneous nephrolithotomy with access for stone disease. A validated radiation safety competency test was administered to urology faculty and residents before and after web based, hands-on fluoroscopy training. Default fluoroscopy settings were changed from continuous to intermittent pulse rate and from standard to half-dose output. Fluoroscopy time and entrance skin dose were then measured again. RESULTS: The cohorts of 44 pre-protocol and 50 post-protocol patients with stones were similarly matched. The change in mean fluoroscopy time and entrance skin dose from pre-protocol to post-protocol was -0.6 minutes and -11.6 mGy (33%) for percutaneous nephrolithotomy (p = 0.62 and <0.001), 0.5 minutes and -0.1 mGy (34%) for ureteroscopy (p = 0.42 and 0.31), and 0.1 minute and -0.1 mGy (29%) for retrograde pyelogram/stent (p = 0.85 and 0.49, respectively). Urologist post-training test scores increased 30% from pretraining scores (p = 0.1). CONCLUSIONS: Radiation safety training protocols improved clinical knowledge but did not significantly alter fluoroscopy time. Changing equipment default settings to intermittent pulse rate (12 frames per second) and half-dose lowered the entrance skin dose by 30% across all endourology patients but most significantly during percutaneous nephrolithotomy. To limit patient radiation exposure fluoroscopy default settings should be decreased before all endourology procedures and image equipment manufacturers should consider lowering standard default renal settings.

Transurethral radiofrequency collagen denaturation for the treatment of women with urinary incontinence.

BACKGROUND: Transurethral radiofrequency collagen denaturation is a relatively novel, minimally invasive device-based intervention used to treat individuals with urinary incontinence (UI). No systematic review of the evidence supporting its use has been published to date. OBJECTIVES: To evaluate the efficacy of transurethral radiofrequency collagen denaturation, compared with other interventions, in the treatment of women with UI.Review authors sought to compare the following.• Transurethral radiofrequency collagen denaturation versus no treatment/sham treatment.• Transurethral radiofrequency collagen denaturation versus conservative physical treatment.• Transurethral radiofrequency collagen denaturation versus mechanical devices (pessaries for UI).• Transurethral radiofrequency collagen denaturation versus drug treatment.• Transurethral radiofrequency collagen denaturation versus injectable treatment for UI.• Transurethral radiofrequency collagen denaturation versus other surgery for UI. SEARCH METHODS: We conducted a systematic search of the Cochrane Incontinence Group Specialised Register (searched 19 December 2014), EMBASE and EMBASE Classic (January 1947 to 2014 Week 50), Google Scholar and three trials registries in December 2014, along with reference checking. We sought to identify unpublished studies by handsearching abstracts of major gynaecology and urology meetings, and by contacting experts in the field and the device manufacturer. SELECTION CRITERIA: Randomised and quasi-randomised trials of transurethral radiofrequency collagen denaturation versus no treatment/sham treatment, conservative physical treatment, mechanical devices, drug treatment, injectable treatment for UI or other surgery for UI in women were eligible. DATA COLLECTION AND ANALYSIS: We screened search results and selected eligible studies for inclusion. We assessed risk of bias and analysed dichotomous variables as risk ratios (RRs) with 95% confidence intervals (CIs) and continuous variables as mean differences (MDs) with 95% CIs. We rated the quality of evidence using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) approach. MAIN RESULTS: We included in the analysis one small sham-controlled randomised trial of 173 women performed in the United States. Participants enrolled in this study had been diagnosed with stress UI and were randomly assigned to transurethral radiofrequency collagen denaturation (treatment) or a sham surgery using a non-functioning catheter (no treatment). Mean age of participants in the 12-month multi-centre trial was 50 years (range 22 to 76 years).Of three patient-important primary outcomes selected for this systematic review, the number of women reporting UI symptoms after intervention was not reported. No serious adverse events were reported for the transurethral radiofrequency collagen denaturation arm or the sham treatment arm during the 12-month trial. Owing to high risk of bias and imprecision, we downgraded the quality of evidence for this outcome to low. The effect of transurethral radiofrequency collagen denaturation on the number of women with an incontinence quality of life (I-QOL) score improvement ≥ 10 points at 12 months was as follows: RR 1.11, 95% CI 0.77 to 1.62; participants = 142, but the confidence interval was wide. For this outcome, the quality of evidence was also low as the result of high risk of bias and imprecision.We found no evidence on the number of women undergoing repeat continence surgery. The risk of other adverse events (pain/dysuria (RR 5.73, 95% CI 0.75 to 43.70; participants = 173); new detrusor overactivity (RR 1.36, 95% CI 0.63 to 2.93; participants = 173); and urinary tract infection (RR 0.95, 95% CI 0.24 to 3.86; participants = 173) could not be established reliably as the trial was small. Evidence was insufficient for assessment of whether use of transurethral radiofrequency collagen denaturation was associated with an increased rate of urinary retention, haematuria and hesitancy compared with sham treatment in 173 participants. The GRADE quality of evidence for all other adverse events with available evidence was low as the result of high risk of bias and imprecision.We found no evidence to inform comparisons of transurethral radiofrequency collagen denaturation with conservative physical treatment, mechanical devices, drug treatment, injectable treatment for UI or other surgery for UI. AUTHORS' CONCLUSIONS: It is not known whether transurethral radiofrequency collagen denaturation, as compared with sham treatment, improves patient-reported symptoms of UI. Evidence is insufficient to show whether the procedure improves disease-specific quality of life. Evidence is also insufficient to show whether the procedure causes serious adverse events or other adverse events in comparison with sham treatment, and no evidence was found for comparison with any other method of treatment for UI.

Engineering LNPs with polysarcosine lipids for mRNA delivery.

Since the approval of the lipid nanoparticles (LNP)-mRNA vaccines against the SARS-CoV-2 virus, there has been an increased interest in the delivery of mRNA through LNPs. However, current LNP formulations contain PEG lipids, which can stimulate the generation of anti-PEG antibodies. The presence of these antibodies can potentially cause adverse reactions and reduce therapeutic efficacy after administration. Given the widespread deployment of the COVID-19 vaccines, the increased exposure to PEG may necessitate the evaluation of alternative LNP formulations without PEG components. In this study, we investigated a series of polysarcosine (pSar) lipids as alternatives to the PEG lipids to determine whether pSar lipids could still provide the functionality of the PEG lipids in the ALC-0315 and SM-102 LNP systems. We found that complete replacement of the PEG lipid with a pSar lipid can increase or maintain mRNA delivery efficiency and exhibit similar safety profiles in vivo.

LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing.

Adipose stem cells (ASCs) have attracted considerable attention as potential therapeutic agents due to their ability to promote tissue regeneration. However, their limited tissue repair capability has posed a challenge in achieving optimal therapeutic outcomes. Herein, we conceive a series of lipid nanoparticles to reprogram ASCs with durable protein secretion capacity for enhanced tissue engineering and regeneration. In vitro studies identify that the isomannide-derived lipid nanoparticles (DIM1T LNP) efficiently deliver RNAs to ASCs. Co-delivery of self-amplifying RNA (saRNA) and E3 mRNA complex (the combination of saRNA and E3 mRNA is named SEC) using DIM1T LNP modulates host immune responses against saRNAs and facilitates the durable production of proteins of interest in ASCs. The DIM1T LNP-SEC engineered ASCs (DS-ASCs) prolong expression of hepatocyte growth factor (HGF) and C-X-C motif chemokine ligand 12 (CXCL12), which show superior wound healing efficacy over their wild-type and DIM1T LNP-mRNA counterparts in the diabetic cutaneous wound model. Overall, this work suggests LNPs as an effective platform to engineer ASCs with enhanced protein generation ability, expediting the development of ASCs-based cell therapies.

Advancements of in vitro transcribed mRNA (IVT mRNA) to enable translation into the clinics.

The accelerated progress and approval of two mRNA-based vaccines to address the SARS-CoV-2 virus were unprecedented. This record-setting feat was made possible through the solid foundation of research on in vitro transcribed mRNA (IVT mRNA) which could be utilized as a therapeutic modality. Through decades of thorough research to overcome barriers to implementation, mRNA-based vaccines or therapeutics offer many advantages to rapidly address a broad range of applications including infectious diseases, cancers, and gene editing. Here, we describe the advances that have supported the adoption of IVT mRNA in the clinics, including optimization of the IVT mRNA structural components, synthesis, and lastly concluding with different classes of IVT RNA. Continuing interest in driving IVT mRNA technology will enable a safer and more efficacious therapeutic modality to address emerging and existing diseases.

Close the cancer-immunity cycle by integrating lipid nanoparticle-mRNA formulations and dendritic cell therapy.

Effective cancer immunotherapy is usually blocked by immunosuppressive factors in the tumour microenvironment, resulting in tumour promotion, metastasis and recurrence. Here we combine lipid nanoparticle-mRNA formulations and dendritic cell therapy (named CATCH) to boost the cancer-immunity cycle via progressive steps to overcome the immunosuppressive tumour microenvironment. Multiple types of sugar-alcohol-derived lipid nanoparticles are conceived to modulate the cancer-immunity cycle. First, one type of lipid nanoparticle containing CD40 ligand mRNA induces robust immunogenic cell death in tumoural tissues, leading to the release of tumour-associated antigens and the expression of CD40 ligand. Next, dendritic cells engineered by another type of lipid nanoparticle encapsulating CD40 mRNA are adoptively transferred, which are then activated by the CD40 ligand molecules in tumoural tissues. This promotes the secretion of multiple cytokines and chemokines, and the upregulation of co-stimulatory molecules on dendritic cells, which are crucial for reprogramming the tumour microenvironment and priming the T-cell responses. After dendritic cells present tumour-associated antigens to T cells, all the above stepwise events contribute to boosting a potent tumour-specific T-cell immunity that eradicates established tumours, suppresses distal lesions and prevents tumour rechallenge.

A systematic review of the quality of evidence of ablative therapy for small renal masses.

PURPOSE: We critically assessed the methodological and reporting quality of published studies of ablative techniques for small renal masses. MATERIALS AND METHODS: We performed a systematic PubMed® and EMBASE® literature search from January 1966 to March 2010 to identify all full text, original research publications on ablative therapy for renal masses. Six reviewers working independently in 3 teams performed duplicate data abstraction using Strengthening the Reporting of Observational Studies in Epidemiology criteria, which were pilot tested in a separate sample. RESULTS: A total of 117 original research publications published in a 15-year period (1995 to 2009) met eligibility criteria. No randomized, controlled trials were identified. All studies were observational and 88.9% had 1 arm with no comparison group. Median sample size was 18 patients (IQR 5.5, 40.0) and 53.8% of studies included 20 or fewer patients. Median followup was 14.0 months (IQR 8.0, 23.8) and only 19.7% of studies had an average followup of greater than 24 months. Of the studies 20.5% mentioned the number of operators involved and only 6.0% provided information on their experience level. Of the studies 66.7% addressed the recurrence rate. Disease specific and overall survival was reported in only 15.4% and 16.2% of studies, respectively. CONCLUSIONS: The published literature on the therapeutic efficacy of ablative therapy for renal masses is largely limited to uncontrolled, 1-arm observational studies. In the absence of higher quality evidence ablative therapy outside research studies should be limited to select patients who are not candidates for surgical intervention.

Delivery of CRISPR-Cas9 system for screening and editing RNA binding proteins in cancer.

RNA-binding proteins (RBPs) play an important role in RNA metabolism, regulating the stability, localization, and functional dynamics of RNAs. Alternation in the RBP-RNA network has profound implications in cellular physiology, and is related to the development and spread of cancer in certain cases. To regulate the expression of specific genes and their biological activities, various strategies have been applied to target RBPs for cancer treatments, including small-molecule inhibitors, small-interfering RNA, peptides, and aptamers. Recently, the deployment of the CRISPR-Cas9 technology has provided a new platform for RBP screening and regulation. This review summarizes the delivery systems of the CRISPR-Cas9 system and their role in RBP-based cancer therapeutics, including identification of novel RBPs and regulation of cancer-associated RBPs. The efficient delivery of the CRISPR-Cas9 system is important to the profound understanding and clinical transition of RBPs as cancer therapeutic targets.

Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy.

Cytokines are important immunotherapeutics with approved drugs for the treatment of human cancers. However, systemic administration of cytokines often fails to achieve adequate concentrations to immune cells in tumors due to dose-limiting toxicity. Thus, developing localized therapy that directly delivers immune-stimulatory cytokines to tumors may improve the therapeutic efficacy. In this study, we generated novel lipid nanoparticles (LNPs) encapsulated with mRNAs encoding cytokines including IL-12, IL-27 and GM-CSF, and tested their anti-tumor activity. We first synthesized ionizable lipid materials containing di-amino groups with various head groups (DALs). The novel DAL4-LNP effectively delivered different mRNAs in vitro to tumor cells and in vivo to tumors. Intratumoral injection of DAL4-LNP loaded with IL-12 mRNA was most potent in inhibiting B16F10 melanoma tumor growth compared to IL-27 or GM-CSF mRNAs in monotherapy. Furthermore, intratumoral injection of dual DAL4-LNP-IL-12 mRNA and IL-27 mRNA showed a synergistic effect in suppressing tumor growth without causing systematic toxicity. Most importantly, intratumoral delivery of IL-12 and IL-27 mRNAs induced robust infiltration of immune effector cells, including IFN-γ and TNF-α producing NK and CD8+ T cells into tumors. Thus, intratumoral administration of DAL-LNP loaded with IL-12 and IL-27 mRNA provides a new treatment strategy for cancer.

Nanomaterials-Mediated Co-Stimulation of Toll-Like Receptors and CD40 for Antitumor Immunity.

Toll-like receptors (TLRs) and CD40-related signaling pathways represent critical bridges between innate and adaptive immune responses. Here, an immunotherapy regimen that enables co-stimulation of TLR7/8- and CD40-mediated pathways is developed. TLR7/8 agonist resiquimod (R848) derived amino lipids, RAL1 and RAL2, are synthesized and formulated into RAL-derived lipid nanoparticles (RAL-LNPs). The RAL2-LNPs show efficient CD40 mRNA delivery to DCs both in vitro (90.8 ± 2.7%) and in vivo (61.3 ± 16.4%). When combined with agonistic anti-CD40 antibody, this approach can produce effective antitumor activities in mouse melanoma tumor models, thereby suppressing tumor growth, prolonging mouse survival, and establishing antitumor memory immunity. Overall, RAL2-LNPs provide a novel platform toward cancer immunotherapy by integrating innate and adaptive immunity.

Harnessing lipid nanoparticles for efficient CRISPR delivery.

The CRISPR-Cas system has revolutionized the biomedical research field with its simple and flexible genome editing method. In October 2020, Emmanuelle Charpentier and Jennifer A. Doudna were awarded the 2020 Nobel Prize in chemistry in recognition of their outstanding contributions to the discovery of CRISPR-Cas9 genetic scissors, which allow scientists to alter DNA sequences with high precision. Recently, the first phase I clinical trials in cancer patients affirmed the safety and feasibility of ex vivo CRISPR-edited T cells. However, specific and effective CRISPR delivery in vivo remains challenging due to the multiple extracellular and intracellular barriers. Here, we discuss the recent advances in novel lipid nanomaterials for CRISPR delivery and describe relevant examples of potential therapeutics in cancers, genetic disorders, and infectious diseases.

Construction of Messenger RNA (mRNA) Probes Delivered By Lipid Nanoparticles to Visualize Intracellular Protein Expression and Localization at Organelles.

Organelles are specialized compartments, where various proteins reside and play crucial roles to maintain essential cellular structures and functions in mammalian cells. A comprehensive understanding of protein expressions and subsequent localizations at each organelle is of great benefit to the development of organelle-based therapies. Herein, a set of single or dual organelle labeling messenger RNAs (SOLAR or DOLAR) is designed as novel imaging probes, which encode fluorescent proteins with various organelle localization signals. These mRNA probes enable to visualize the protein localizations at different organelles and investigate their trafficking from ribosomal machinery to specific organelles. According to the in vitro results, SOLAR probes show organelle targeting capabilities consistent with the design. Moreover, DOLAR probes with different linkers display distinct targeting properties depending on different organelle localization signals. Additionally, these mRNA probes also exhibit organelle labeling ability in vivo when delivered by lipid nanoparticles (LNPs). Therefore, these mRNA-based probes provide a unique tool to study cell organelles and may facilitate the design of organelle-based therapies.

Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy.

Antibodies targeting costimulatory receptors of T cells have been developed for the activation of T cell immunity in cancer immunotherapy. However, costimulatory molecule expression is often lacking in tumor-infiltrating immune cells, which can impede antibody-mediated immunotherapy. Here, we hypothesize that delivery of costimulatory receptor mRNA to tumor-infiltrating T cells will enhance the antitumor effects of antibodies. We first design a library of biomimetic nanoparticles and find that phospholipid nanoparticles (PL1) effectively deliver costimulatory receptor mRNA (CD137 or OX40) to T cells. Then, we demonstrate that the combination of PL1-OX40 mRNA and anti-OX40 antibody exhibits significantly improved antitumor activity compared to anti-OX40 antibody alone in multiple tumor models. This treatment regimen results in a 60% complete response rate in the A20 tumor model, with these mice being resistant to rechallenge by A20 tumor cells. Additionally, the combination of PL1-OX40 mRNA and anti-OX40 antibody significantly boosts the antitumor immune response to anti-PD-1 + anti-CTLA-4 antibodies in the B16F10 tumor model. This study supports the concept of delivering mRNA encoding costimulatory receptors in combination with the corresponding agonistic antibody as a strategy to enhance cancer immunotherapy.

The Role of Chronic Mesh Infection in Delayed-Onset Vaginal Mesh Complications or Recurrent Urinary Tract Infections: Results From Explanted Mesh Cultures.

OBJECTIVES: Vaginal mesh complications necessitating excision are increasingly prevalent. We aim to study whether subclinical chronically infected mesh contributes to the development of delayed-onset mesh complications or recurrent urinary tract infections (UTIs). METHODS: Women undergoing mesh removal from August 2013 through May 2014 were identified by surgical code for vaginal mesh removal. Only women undergoing removal of anti-incontinence mesh were included. Exclusion criteria included any women undergoing simultaneous prolapse mesh removal. We abstracted preoperative and postoperative information from the medical record and compared mesh culture results from patients with and without mesh extrusion, de novo recurrent UTIs, and delayed-onset pain. RESULTS: One hundred seven women with only anti-incontinence mesh removed were included in the analysis. Onset of complications after mesh placement was within the first 6 months in 70 (65%) of 107 and delayed (≥6 months) in 37 (35%) of 107. A positive culture from the explanted mesh was obtained from 82 (77%) of 107 patients, and 40 (37%) of 107 were positive with potential pathogens. There were no significant differences in culture results when comparing patients with delayed-onset versus immediate pain, extrusion with no extrusion, and de novo recurrent UTIs with no infections. CONCLUSIONS: In this large cohort of patients with mesh removed for a diverse array of complications, cultures of the explanted vaginal mesh demonstrate frequent low-density bacterial colonization. We found no differences in culture results from women with delayed-onset pain versus acute pain, vaginal mesh extrusions versus no extrusions, or recurrent UTIs using standard culture methods. Chronic prosthetic infections in other areas of medicine are associated with bacterial biofilms, which are resistant to typical culture techniques. Further studies using culture-independent methods are needed to investigate the potential role of chronic bacterial infections in delayed vaginal mesh complications.

Cigarette smoke condensate-induced transformation of normal human breast epithelial cells in vitro.

In the present study, we showed that a single-dose treatment of normal breast epithelial cell line, MCF10A, for 72 h with cigarette smoke condensate (CSC) resulted in a transformed phenotype. The anchorage-dependent growth of these cells was decreased due to increased cell cycle arrest in S-G2/M phase; however, the surviving cells developed resistance due to an increased Bcl-xL to Bax ratio. Levels of PCNA and gadd45 proteins--involved in DNA repair in response to genomic damage--were increased, suggesting that the cells were responding to CSC-induced genomic damage. The transformation of MCF10A cells was determined by their colony-forming efficiency in soft-agar in an anchorage-independent manner. CSC-treated MCF10A cells efficiently formed colonies in soft-agar. We then re-established cell lines from the soft-agar colonies and further examined the persistence of their transforming characteristics. The re-established cell lines, when plated after 17 passages without CSC treatment, still formed colonies in the soft-agar. An increased staining of neuropilin-1 (NRP-1) further showed a transformation characteristic of MCF10A cells treated with CSC. In summary, our results suggest that CSC is capable of transforming the MCF10A cells in vitro, supporting the role of cigarette smoking and increased risk for breast cancer.