Mathematical Modeling and Experimental Validation of Extracellular Vesicle-Mediated Tumor Suppressor MicroRNA Delivery and Propagation in Ovarian Cancer Cells.

Extracellular vesicle (EV)-mediated microRNA transfer and propagation from the donor cell to the recipient cell in the tumor microenvironment have significant implications, including the development of multidrug resistance (MDR). Although miRNA-encapsulated EV have been shown to have functional effects on recipient cells, the quantitative aspects of transfer kinetics and functional effects remain poorly understood. Intracellular events such as degradation of miRNA, loading of miRNA into EVs, cellular release of EVs, and their uptake by recipient cells govern the transfer and functional effect of encapsulated miRNA. Based on these rate-limiting steps, we developed a mathematical model using ordinary differential equations (model 1). We performed coculture experiments using ID8-VEGF ovarian cancer cells to demonstrate EV-mediated propagation of tumor suppressor miRNA Let7b administered with hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles. Using the experimental data and model fitting, we determined the rate constants for the kinetic events involved in the transfer from the donor cells to the recipient cells. In model 2, we performed Let7b transfection experiments in ID8-VEGF cells with HA-PEI nanoparticles to determine the concentration-effect relationship on HMGA2 mRNA levels. Lastly, in model 3, we combined model 1 and model 2 parameters to describe the kinetics and effect relationship of EV-Let7b in recipient cells to predict the minimum number of miRNA copies needed to show functional effects.

A phase 1 trial of lipid-encapsulated mRNA encoding a monoclonal antibody with neutralizing activity against Chikungunya virus.

Chikungunya virus (CHIKV) infection causes acute disease characterized by fever, rash and arthralgia, which progresses to severe and chronic arthritis in up to 50% of patients. Moreover, CHIKV infection can be fatal in infants or immunocompromised individuals and has no approved therapy or prevention. This phase 1, first-in-human, randomized, placebo-controlled, proof-of-concept trial conducted from January 2019 to June 2020 evaluated the safety and pharmacology of mRNA-1944, a lipid nanoparticle-encapsulated messenger RNA encoding the heavy and light chains of a CHIKV-specific monoclonal neutralizing antibody, CHKV-24 ( NCT03829384 ). The primary outcome was to evaluate the safety and tolerability of escalating doses of mRNA-1944 administered via intravenous infusion in healthy participants aged 18-50 years. The secondary objectives included determination of the pharmacokinetics of mRNA encoding for CHKV-24 immunoglobulin heavy and light chains and ionizable amino lipid component and the pharmacodynamics of mRNA-1944 as assessed by serum concentrations of mRNA encoding for CHKV-24 immunoglobulin G (IgG), plasma concentrations of ionizable amino lipid and serum concentrations of CHKV-24 IgG. Here we report the results of a prespecified interim analysis of 38 healthy participants who received intravenous single doses of mRNA-1944 or placebo at 0.1, 0.3 and 0.6 mg kg-1, or two weekly doses at 0.3 mg kg-1. At 12, 24 and 48 h after single infusions, dose-dependent levels of CHKV-24 IgG with neutralizing activity were observed at titers predicted to be therapeutically relevant concentrations (≥1 µg ml-1) across doses that persisted for ≥16 weeks at 0.3 and 0.6 mg kg-1 (mean t1/2 approximately 69 d). A second 0.3 mg kg-1 dose 1 week after the first increased CHKV-24 IgG levels 1.8-fold. Adverse effects were mild to moderate in severity, did not worsen with a second mRNA-1944 dose and none were serious. To our knowledge, mRNA-1944 is the first mRNA-encoded monoclonal antibody showing in vivo expression and detectable ex vivo neutralizing activity in a clinical trial and may offer a treatment option for CHIKV infection. Further evaluation of the potential therapeutic use of mRNA-1944 in clinical trials for the treatment of CHIKV infection is warranted.

Co-Silencing of Tissue Transglutaminase-2 and Interleukin-15 Genes in a Celiac Disease Mimetic Mouse Model Using a Nanoparticle-in-Microsphere Oral System.

Celiac disease is a chronic inflammatory condition characterized by activation of the immune system in response to deamidation of gluten peptides brought about by tissue transglutaminase-2 (TG2). Overexpression of interleukin-15 (IL-15) in the intestinal epithelium and the lamina propria leads to the dysregulation of the immune system, leading to epithelial damage. The goal of this study was to develop an RNA interference therapeutic strategy for celiac disease using a combination of TG2 and IL-15 gene silencing in the inflamed intestine. TG2 and IL-15 silencing siRNA sequences, along with scrambled control, were encapsulated in a nanoparticle-in-microsphere oral system (NiMOS) and administered in a poly(I:C) mouse model of celiac disease. Single TG2 and IL-15 siRNA therapy and the combination showed effective gene silencing in vivo. Additionally, it was found that IL-15 gene silencing alone and combination in the NiMOS significantly reduced other proinflammatory cytokines. The tissue histopathology data also confirmed a reduction in immune cell infiltration and restoration of the mucosal architecture and barrier function in the intestine upon treatment. Overall, the results of this study show evidence that celiac disease can be potentially treated with an oral microsphere formulation using a combination of TG2 and IL-15 RNA interference therapeutic strategies.

Pharmacokinetics and Biodistribution Analysis of Small Interference RNA for Silencing Tissue Transglutaminase-2 in Celiac Disease After Oral Administration in Mice Using Gelatin-Based Multicompartmental Delivery Systems.

Background: RNA interference (RNAi) therapy has tremendous potential in treating diseases that are characterized by overexpression of genes. However, the biggest challenge to utilize the therapy is to engineer delivery systems that can efficiently transport small interfering RNA (siRNA) to appropriate target sites. Our objective in this study was to develop and evaluate multi-compartmental systems for the oral delivery of siRNA that targets the overexpressed TG2 gene (TG2-siRNA) in the small intestine for the treatment of celiac disease (CD). Materials and Methods: Two types of multicompartmental systems were developed and evaluated: (1) a solid-in-solid multicompartmental system featuring "nanoparticle in microsphere oral system (NiMOS)" where type B gelatin nanoparticles containing TG2-siRNA (TG2-NiMOS) were encapsulated within poly(ɛ-caprolactone) (PCL) based microspheres, and (2) a solid-in-liquid multicompartmental system, "Nanoparticle-in-Emulsion (NiE)" consisting of type-B gelatin nanoparticles containing TG2-siRNA encapsulated within safflower oil containing water-in-oil-in-water (W/O/W) multiple emulsion (TG2-NiE). Results: Evaluation of the biodistribution and pharmacokinetics (PK) after a single oral dose of siRNA containing multicompartmental systems to C57BL/6 mice showed that TG2-siRNA was delivered to the small intestine (duodenum, jejunum and ileum), and colon with minimal systemic exposure via both TG2-NiE and TG2-NiMOS systems. TG2-siRNA exposure (AUC0-t) in the duodenum, jejunum, ileum and colon was 56.4-, 34.3-, 85.5- and 35.5-fold greater for the TG2-NiMOS formulation, relative to the TG2-NiE formulation. Conclusion: The results of this study suggest that TG2-NiMOS formulation was more superior than TG2-NiE formulation in facilitating intestinal delivery of siRNA via the oral route of administration and can be potentially used in the treatment of CD.

Psychometric properties of the Malay version of motivation scales in drug treatment.

INTRODUCTION: In recognition of the role of motivation in drug use treatment, patient motivational screening instruments are needed for strategic planning and treatment. The aims of this study were to evaluate the reliability and validity of the Malay version of the Treatment Motivation Scale, and to compare the motivational levels of patients receiving substance abuse treatment with different modalities (inpatient vs. outpatient). The motivational scale consists of three scales: problem recognition, desire for help and treatment readiness. METHOD: A convenience sample of 102 patients was recruited from four Cure and Care Service Centres in Malaysia. RESULTS: Principal component analysis with varimax rotation supported two-factor solutions for each subscale: problem recognition, desire for help and treatment readiness, which accounted for 63.5%, 62.7% and 49.1% of the variances, respectively. The Cronbach's alpha coefficients were acceptable for the overall measures (24 items: ∝ = 0.89), the problem recognition scale (10 items; ∝ = 0.89), desire for help (6 items; ∝ = 0.64) and treatment readiness scale (8 items; ∝ = 0.60). The results also indicated significant motivational differences for different modalities, with inpatients having significantly higher motivational scores in each scale compared to outpatients. CONCLUSION: The present study pointed towards the favourable psychometric properties of a motivation for treatment scale, which can be a useful instrument for clinical applications of drug use changes and treatment.

Body dysmorphic disorder: beyond skin deep.

Body dysmorphic disorder (BDD) is a psychiatric illness that primarily affects adolescents and young adults of both sexes. Patients have a distorted self-image, which manifests as a preoccupation with slight or imagined defects in the face, nose, skin, hair or any part of the body that ultimately interferes with daily functioning. It is a relatively common yet long unrecognized problem. Patients often seek multiple physician assessments for their perceived defects and request cosmetic procedures. Early intervention can prevent a cycle of multiple surgeries, as the outcome is usually poor and may lead to exacerbation of symptoms, anger and litigation. BDD is a disabling, and even life-threatening, condition; it can lead to major depression and suicidal ideation. Selective serotonin reuptake inhibitors and cognitive behavioral therapy are the mainstay of treatment and are beneficial in most patients. A multidisciplinary approach is strongly recommended.

HLA-Cw*0409N is associated with HLA-A*2301 and HLA-B*4403-carrying haplotypes.

The associations of HLA-B*4402 and HLA-B*4403 with alleles of HLA-A and HLA-Cw were investigated in panels of HLA-B*4403 and HLA-B*4402 homozygous individuals and in selected individuals carrying HLA-Cw*04 and HLA-B*4403. Some of these individuals were genotyped and also carried (HLA-DRB1*0701, DQB1*02). Among the latter, we studied individuals carrying the conserved extended haplotype (CEH) [HLA-Cw*04, B*4403, FC31, DRB1*0701, DQB1*02]. Four different common (HLA-Cw*, B*44) haplotypes were identified that extended to the HLA-A locus: HLA-A*0201, Cw*0501, B*4402; HLA-A*2902, Cw*1601, B*4403; HLA-A*2301, Cw*0401, B*4403; and HLA-A*2301, Cw*0409N, B*4403. We identified eight unrelated examples of the allele HLA-Cw*0409N. HLA-A*2301 was associated with both HLA-Cw*0401 and HLA-Cw*0409N, suggesting that HLA-Cw*0409N may have arisen from a mutation in a CEH. We estimate that approximately 2 to 5 in 1000 Caucasian individuals carry the allele HLA-Cw*0409N, making it one of the most frequent null HLA alleles known to date. Our findings demonstrate the first example of three different HLA-Cw-determined subtypes of a common or CEH carrying a shared HLA-B allele, in this case HLA-B*4403.

A comparison of soft-tissue anchors in tendo achilles reattachment.

This prospective study evaluated four soft-tissue fixation modalities, used in seven different combinations, to reattach the tendo Achilles in 34 cadaveric specimens. Ultimate loads, elastic moduli, and modes of failure were evaluated by loading the specimen in a cantilevered fashion on an Instron. Mann-Whitney U tests were performed to compare the failure load data for statistical significance. Although the use of two Mitek SuperAnchors showed better load resistance than one anchor (p < .01), there was no significant improvement between using two or three anchors (one anchor 116 +/- 24 N, two anchors 234 +/- 21 N, three anchors 277 +/- 80 N). Two Bionx Bankart Tacks demonstrated no significant difference over using a single tack (one tack 178 +/- 57 N, two tacks 214 +/- 86 N). No statistical difference was observed between the screw and washer systems (screw with polyacetal resin washer 307 +/- 80 N, screw with metal washer 290 +/- 81 N). Both screw and washer systems did show greater stability when compared with a single Mitek SuperAnchor (p < .01) or a single Bionx Bankart Tack (p < .05). Similar analyses using the Mann-Whitney U tests were performed on the elastic modulus data. Analysis of the displacement data among all groups showed no statistical difference. Observations of the mode of failure exhibited 86% of Mitek SuperAnchor failed secondary to suture, and 70% of the Bionx Bankart Tack and 90% of the screw and washer systems failed because of the tendon shearing around the fixation. The comparisons of cost-effectiveness among the fixations showed the Synthes screw and polyacetal resin spiked washer to have the lowest cost to load ratio ($0.15/N).

Relative stability of tension band versus two-cortex screw fixation for treating fifth metatarsal base avulsion fractures.

This study assesses the strength of fixating avulsion fractures of the fifth metatarsal base with a 4.0-mm partially threaded cancellous screw crossing two cortices as compared to tension banding. Our data showed statistically significant fixation strength improvement over tension banding for avulsion fractures (p < 0.02) in both polystyrene foam models and fresh, nonpreserved frozen cadaveric samples. In cadavers, the screw fixations were able to withstand more than three times the load sustained by the tension band fixations. The study utilized the Instron 8500 tensiometer to apply physiologic loads to test the constructs until failure. The displacement and load data at failure show the limitations of both fixations. By increasing the load resistance while maintaining compression, the bicortical cancellous screw fixation created greater stability at the avulsion fracture of the fifth metatarsal base as compared to tension band stabilization.

The MHC influences NK and NKT cell functions associated with immune abnormalities and lifespan.

The lifespans of H-2 congenic mice differ significantly. The B10.AKM (H-2m) strain has a median survival time (MST) of 15 months, whereas the B10.BR (H-2k) strain has an MST of 24 months. It was previously shown that B10.AKM mice at 13-15 months of age have immunological function comparable to those of B10.BR mice at 22-26 months of age. These functions include: a low proliferative response, reduced levels of intracellular calcium release [Ca2+]i, and an increase in the frequency of memory helper T-cells (CD4+ CD44hiCD45RBlo). In this report similar deficiencies were demonstrated in B10.AKM mice at 2-4 months of age and show that activated spleen NK1.1+CD4+ T (NKT) cells from young B10.AKM mice produce a significantly higher level of IL-4 but a lower level of IFN-gamma as compared to NKT cells from B10.BR mice of the same age. Also, the cytotoxic activity of natural killer (NK) cells from spleens of young (2-4 months) as well as adult (12-16 months) B10.AKM mice is significantly lower (P < 0.01) than that of NK cells from B10.BR mice. These findings suggest that the NKT activity in young B10.AKM mice is a factor for the early onset of immune dysfunction leading to a shorter lifespan.

A YAC-based physical map of the mouse genome.

A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

Irreversible inhibition of epidermal growth factor receptor tyrosine kinase with in vivo activity by N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785).

It has been shown previously that 4-anilino quinazolines compete with the ability of ATP to bind the epidermal growth factor receptor (EGF-R), inhibit EGF-stimulated autophosphorylation of tyrosine residues in EGF-R, and block EGF-mediated growth. Since millimolar concentrations of ATP in cells could reduce the efficacy of 4-anilino quinazolines in cells and the activity of these compounds would not be sustained once they were removed from the body, we reasoned that irreversible inhibitors of EGF-R might improve the activity of this series of compounds in animals. Molecular modeling of the EGF-R kinase domain was used to design irreversible inhibitors. We herein describe one such inhibitor: N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]2-butynamide, known as CL-387,785. This compound covalently bound to EGF-R. It also specifically inhibited kinase activity of the protein (IC50 = 370+/-120 pM), blocked EGF-stimulated autophosphorylation of the receptor in cells (ic50 approximately 5 nM), inhibited cell proliferation (IC50 = 31-125 nM) primarily in a cytostatic manner in cell lines that overexpress EGF-R or c-erbB-2, and profoundly blocked the growth of a tumor that overexpresses EGF-R in nude mice (when given orally at 80 mg/kg/day for 10 days, daily). We conclude that CL-387,785 is useful for studying the interaction of small molecules with EGF-R and may have clinical utility.

A comprehensive genetic map of the mouse genome.

The availability of dense genetic linkage maps of mammalian genomes makes feasible a wide range of studies, including positional cloning of monogenic traits, genetic dissection of polygenic traits, construction of genome-wide physical maps, rapid marker-assisted construction of congenic strains, and evolutionary comparisons. We have been engaged for the past five years in a concerted effort to produce a dense genetic map of the laboratory mouse. Here we present the final report of this project. The map contains 7,377 genetic markers, consisting of 6,580 highly informative simple sequence length polymorphisms integrated with 797 restriction fragment length polymorphisms in mouse genes. The average spacing between markers is about 0.2 centimorgans or 400 kilobases.

c-Ha-ras gene mutation and activation precede pathological changes in DMBA-induced in vivo carcinogenesis.

We have previously reported a stage-specific and sequential overexpression of the c-Ha-ras and c-erbB genes in 7, 12-dimethylbenzanthracene (DMBA)-induced in vivo carcinogenesis in hamster buccal pouch epithelium (HBPE). In this investigation, the immunoreactive protein product of the c-Ha-ras gene (p21 protein) was identified in HBPE cells, specifically in treated tissues and cultured cells established after 3 weeks of DMBA treatment. Microscopic examination did not show any histopathological changes in these tissues. The p21 protein was detected in a few selective cells, which were dispersed away from the more densely populated basal layer. The overexpression of the c-Ha-ras gene was accompanied by a point mutation of A----T in codon 61 (CAA), inducing an amino acid substitution from the wild-type glutamine to leucine in the peptide. The concurrent molecular modifications preceded any detectable histopathological changes. The cellular morphology and orientation in treated HBPE at this early stage was indistinguishable from the control tissue. Yet the genetic alterations, such as the point mutation and overexpression of the gene, were evident at the predysplastic stage. Amplification and overexpression of the second proto-oncogene, c-erbB, and its product, epidermal growth factor receptor (EGFR), were detected in HBPE cells at the later stages of extensive cell proliferation and invasion. By using double antibodies and two immunoreporter systems, we demonstrated overexpression of both c-Ha-ras and c-erbB genes in the same HBPE cells during this chemically induced in vivo carcinogenesis.

Cardioprotective effect of cromakalim (potassium channel opener) in isoproterenol induced myocardial infarction in rats.

Animals pretreated with cromakalim (1 mg/kg,po) along with isoproterenol (85 mg/kg,sc) showed less myocardial degenerative changes on histopathological examinations when compared with those treated with isoproterenol alone. Cromakalim's beneficial effects on myocardium were in dose-dependent manner. Administration of cromakalim (po) lowered significantly the serum LDH and SGOT and depleted intracytoplasmic glycogen as demonstrated by periodic schiff staining procedure. Increase in blood clotting time was highly significant (P less than 0.001). The results suggest cardioprotective effect of cromakalim in isoproterenol induced myocardial infarction.

Role of ultraviolet radiation in the induction of melanocytic tumors in hairless mice following 7,12-dimethylbenz(a)anthracene application and ultraviolet irradiation.

We examined the role of UVR (UV radiation) (UVA, 320-400 nm; UVB, 290-320 nm; and the combination of UVA and UVB) as a promoter in the induction of cutaneous melanoma. One hundred and seventy hairless mice (Skh-hr2), 6-8 weeks old, were treated in 8 groups: group I, DMBA [7,12-dimethylbenz(a)anthracene] plus UVA; group II, DMBA plus UVA plus UVB; group III, DMBA plus UVB; group IV, DMBA; group V, UVA; group VI, UVA plus UVB; group VII, UVB; group VIII, control. DMBA (0.5% solution) was applied once to promote the formation of dermal melanocytic nevus-like lesions while UVR treatments were conducted 3 times/week for 30 weeks. The mice were examined periodically for the development of multiple pigmented lesions, papillomas, squamous cell carcinomas, melanomas, and lymphomas. Treatment with DMBA plus UVA, DMBA plus UVB, and DMBA plus UVA plus UVB stimulated the development of multiple pigmented nevus-like lesions (85-100%) in mice of groups I, II, III, and IV. Upon necroscopy, 27-33% of animals in groups I, II, and III receiving UVR treatments developed clinically and histologically characterized melanomas. Treatment with DMBA alone did not produce melanomas. DMBA-treated animals in groups I, II, and III which received UVR treatments also developed lymphomas (21-50%). Animals treated with DMBA alone or those that received UVB or the combination of UVB plus UVA (without DMBA) developed only papillomas and squamous cell carcinomas (25-47%). Skin tumors were analyzed for the presence of point mutations in the ras gene. Polymerase chain reaction amplification of DNA and selective oligonucleotide hybridization revealed mutations in the 61st codon of the N-ras gene in the precursor nevus-like lesions and melanoma samples studied. This study suggests that UVR (both UVA and UVB) plays a role as a promoter in the stimulation of melanoma and lymphoma development in hairless mice.

Comparison of cellular protooncogene activation and transformation-related activity of human melanocytes and metastatic melanoma.

Two cell lines (NH and HM1), established from patients with metastatic melanomas, were evaluated for the presence of activated cellular protooncogenes. Northern blot analysis demonstrated increased expression of the c-myc gene (from 9 to 14 times) in NH and HM1 cell lines by densitometric comparison with human melanocyte cell lines. Analysis of the expression of 13 additional cellular protooncogenes revealed either no detectable levels (c-fms, c-abl, v-src, c-erb A1, c-erb B, v-mos, TGF beta, and c-myb) or unaltered expression levels (cH-ras, N-ras, c-fos, and c-sis) in normal human melanocytes and metastatic melanomas. Elevated expression of the c-myc gene was also detected in two long-term cultured melanoma cell lines (RPMI 7951 and SKMEL-30). Analysis of c-myc expression by in situ hybridization in HM1 cells showed that expression was not localized to a sub-population of cycling cells and all cells were overexpressing c-myc mRNA. Differences in relative abundance of c-myc transcripts suggests a relationship with the ability of DNA from these cell lines to efficiently transform NIH 3T3 cells and form colonies on soft agar.