ß-catenin mRNA encapsulated in SM-102 lipid nanoparticles enhances bone formation in a murine tibia fracture repair model.

Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing. In this study, we aimed to develop an osteoanabolic therapy which activates the Wnt/ß-catenin pathway, a molecular driver of endochondral ossification. We hypothesize that using an mRNA-based therapeutic encoding ß-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes. To optimize a delivery platform built on recent advancements in liposomal technologies, two FDA-approved ionizable phospholipids, DLin-MC3-DMA (MC3) and SM-102, were used to fabricate unique ionizable lipid nanoparticle (LNP) formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model. Using firefly luciferase mRNA as a reporter gene to track and quantify transfection, SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection, minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs. The generated ß-cateninGOF mRNA encapsulated in SM-102 LNPs (SM-102-ß-cateninGOF mRNA) showed bioactivity in vitro through upregulation of downstream canonical Wnt genes, axin2 and runx2. When testing SM-102-ß-cateninGOF mRNA therapeutic in a murine tibia fracture model, histomorphometric analysis showed increased bone and decreased cartilage composition with the 45 µg concentration at 2 weeks post-fracture. µCT testing confirmed that SM-102-ß-cateninGOF mRNA promoted bone formation in vivo, revealing significantly more bone volume over total volume in the 45 µg group. Thus, we generated a novel mRNA-based therapeutic encoding a ß-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery.

Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity.

The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. The goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed B7-H3; whereas, levels were undetectable on normal dog tissues. Both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little anti-tumor activity was generated by B7-H3 CAR T cells; whereas, B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept.

Interventions impacting the accessibility of sexual reproductive health services for head porters in sub-Saharan Africa- A scoping review protocol.

Head porters working in markets in sub-Saharan Africa (SSA) are one of the world's most vulnerable and socioeconomically disadvantaged groups. They consist predominantly of uneducated women and girls seeking to escape poverty, early marriage, and other issues of domestic violence. Most female head porters are in their reproductive years and often lack access to sexual reproductive health services (SRHS) despite being at high risk for sexually transmitted infections (STIs), unplanned pregnancies, and gender-based violence. The low priority for women and girls' SRH in many SSA countries highlights the need to explore the factors influencing the accessibility of services for failure to do so restrains human development. An initial search of the literature was conducted and revealed no current scoping or systematic reviews on the accessibility to SRHS for female head porters in SSA. We outline a scoping review protocol, using the Joanna Briggs Institute methodology, to determine the interventions that influence the accessibility of SRHS for female head porters in SSA. The protocol is registered with Open Science Framework (https://osf.io/hjfkd). Findings will not only be valuable for female head porters but for all vulnerable female groups in SSA who experience high SRH risks and social disparities.

Mineral coated microparticles doped with fluoride and complexed with mRNA prolong transfection in fracture healing.

Introduction: Impaired fracture healing, specifically non-union, has been found to occur up to 14% in tibial shaft fractures. The current standard of care to treat non-union often requires additional surgeries which can result in long recovery times. Injectable-based therapies to accelerate fracture healing have the potential to mitigate the need for additional surgeries. Gene therapies have recently undergone significant advancements due to developments in nanotechnology, which improve mRNA stability while reducing immunogenicity. Methods: In this study, we tested the efficacy of mineral coated microparticles (MCM) and fluoride-doped MCM (FMCM) to effectively deliver firefly luciferase (FLuc) mRNA lipoplexes (LPX) to the fracture site. Here, adult mice underwent a tibia fracture and stabilization method and all treatments were locally injected into the fracture. Level of osteogenesis and amount of bone formation were assessed using gene expression and histomorphometry respectively. Localized and systemic inflammation were measured through gene expression, histopathology scoring and measuring C-reactive protein (CRP) in the serum. Lastly, daily IVIS images were taken to track and measure transfection over time. Results: MCM-LPX-FLuc and FMCM-LPX-FLuc were not found to cause any cytotoxic effects when tested in vitro. When measuring the osteogenic potential of each mineral composition, FMCM-LPX-FLuc trended higher in osteogenic markers through qRT-PCR than the other groups tested in a murine fracture and stabilization model. Despite FMCM-LPX-FLuc showing slightly elevated il-1ß and il-4 levels in the fracture callus, inflammation scoring of the fracture callus did not result in any differences. Additionally, an acute systemic inflammatory response was not observed in any of the samples tested. The concentration of MCM-LPX-FLuc and FMCM-LPX-FLuc that was used in the murine fracture model did not stimulate bone when analyzed through stereological principles. Transfection efficacy and kinetics of delivery platforms revealed that FMCM-LPX-FLuc prolongs the luciferase signal both in vitro and in vivo. Discussion: These data together reveal that FMCM-LPX-FLuc could serve as a promising mRNA delivery platform for fracture healing applications.

Compressive properties and failure behavior of photocast hydroxyapatite gyroid scaffolds vary with porosity.

Hydroxyapatite is commonly used in tissue engineered scaffolds for bone regeneration due to its excellent bioactivity and slow degradation rate in the human body. A method of layer-wise, photopolymerized viscous extrusion, a type of additive manufacturing, was developed to fabricate hydroxyapatite gyroid scaffolds with 60%, 70%, and 80% porosities. This study uses this method to produce and evaluate calcium phosphate-based scaffolds. Gyroid topology was selected due to its interconnected porosity and superior, isotropic mechanical properties compared to typical rectilinear lattice structures. These 3D printed scaffolds were mechanically tested in compression and examined to determine the relationship between porosity, ultimate compressive strength, and fracture behavior. Compressive strength increased with decreasing porosity. Ultimate compressive strengths of the 60% and 70% porous gyroids are comparable to that of human cancellous bone, and higher than previously reported for hydroxyapatite rectilinear scaffolds. These gyroid scaffolds exhibited ultimate compressive strength increases between 1.5 and 6.5 times greater than expected, based on volume of material, as porosity is decreased. The Weibull moduli, a measure of failure predictability, were predictive of failure mode and found to be in the accepted range for engineering ceramics. The gyroid scaffolds were also found to be self-reinforcing such that initial failures due to minor manufacturing inconsistencies did not appear to be the primary cause of early failure of the scaffold. The porous gyroids exhibited scaffold failure characteristics that varied with porosity, ranging from monolithic failure to layer-by-layer failure, and demonstrated self-reinforcement in each porosity tested.

Immersive Reality Experience Technology for Reducing Social Isolation and Improving Social Connectedness and Well-being of Children and Young People Who Are Hospitalized: Open Trial.

BACKGROUND: Children and young people who are hospitalized can feel disconnected from their peers and families, which can, in turn, predispose them to psychological problems, including anxiety and depression. Immersive reality experience technology, recently developed by the New Zealand Patience Project Charitable Trust, may help to overcome these issues. Immersive reality experience technology uses immersive 360° live streaming and a virtual reality headset to enable children and young people who are hospitalized to connect through cameras located in either their school or home environment and via SMS text messaging with a designated buddy. OBJECTIVE: This trial aims to expand qualitative findings from a previous smaller proof of concept trial to ascertain the views of New Zealand children and young people who are hospitalized, their caregivers, and teachers regarding immersive reality experience technology and quantitatively evaluate the effectiveness of immersive reality experience technology in reducing social isolation and improving social connectedness and well-being using validated outcome measures. METHODS: An open trial of immersive reality experience technology was conducted between December 2019 and December 2020 for which 19 New Zealand children and young people aged 13 to 18 years who had been hospitalized at Starship Hospital-a specialist pediatric hospital in Auckland-for at least 2 weeks were recruited. All young people completed the Short Warwick-Edinburgh Mental Well-Being Scale, an abbreviated version of the Social Connectedness Scale, and the Social Inclusion Scale at baseline. Of the 19 participants, 10 (53%) used immersive reality experience technology as often as they wished over a 6-week period and completed postintervention measures. Semistructured interviews with a subset of the 10 young people, 4 caregivers, and 6 teachers were conducted immediately after the intervention. RESULTS: Participants reported improvements in social inclusion (mean change 3.9, SD 2.8; P=.06), social connectedness (mean change 14.2, SD 10.0; P=.002), and well-being (mean change 5.7, SD 4.0; P=.001). Key themes from interviews with participants, caregivers, and teachers were the importance of support for using immersive reality experience technology, connecting versus connectedness, choice and connection, and the value of setting it up and getting it right. Recommendations for improving connectedness via immersive reality experience and related technologies were also provided. CONCLUSIONS: Immersive reality experience technology can improve the social inclusion, social connectedness, and well-being of New Zealand children and young people who are hospitalized. With some technological modifications and simplified implementation, immersive reality experience and related technology could become part of standard care and support children and young people who are hospitalized in New Zealand and elsewhere to sustain family and peer cohesion, experience fewer psychological problems, and more easily return to normal life following the completion of treatment. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Network Registry ACTRN12619000252112; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=376837&isReview=true.

Predictors of At-Home Arterial Oxygen Desaturation Events in Ambulatory Surgical Patients.

OBJECTIVES: Little is known about the early recovery phase occurring at-home after anesthesia and surgery in ambulatory surgical patients. We studied quantitative oximetry and quality-of-life metrics in the first 48 hours after same-day orthopedic surgery examining the association between the recovery metrics and specific patient and procedural factors. METHODS: We used the STOP-Bang score to quantify patient risk for obstructive sleep apnea in 50 adult patients at 2 centers using continuous portable oximetry and patient journaling. Parametric statistical procedures were used to assess relationships among patient and procedural factors and desaturation events. RESULTS: Higher STOP-Bang scores were predictive of the number and duration of desaturation events below mild and severe thresholds for arterial oxygen saturation during their first 48 hours after discharge from ambulatory surgery. Older patients and patients with higher BMI in particular were at an increased risk of mild and severe arterial oxygen desaturation. Using a home CPAP reduced the number of desaturation events. Of interest, taking opiate analgesics decreased the number of desaturation events. CONCLUSIONS: Given the absence of systematic research of early ambulatory anesthesia/surgery recovery at home and concerns of postoperative respiratory events, our results have clear implications for patient safety. Our results imply that screening based on noninvasive STOP-Bang scores may allow for suggestions for recovery from ambulatory surgery, such as encouraging patients with high scores to use home CPAP and aggressive education regarding use of opiates.

Domestic stigmatisation: refocusing interventions for people living with HIV.

Domestic stigmatisation serves as an umbrella term for acts of enacted or felt stigma experienced in a person's domestic environment. This article reports on the term which transpired from a narrative inquiry in 2011 with people living with HIV (PLWH) who reported humiliation or segregation, experienced or perceived, within the domestic environment that rendered the individual disabled, diseased, unworthy, unhealthy, or deficient. A literature review about this form of stigma was conducted using the following inclusion criteria: 1) a peer-reviewed source; 2) published between 2011 and 2018; 3) access to full-text articles; 4) accessible in English; 5) reported from any country; and 6) using qualitative or mixed-method approaches. A total of 37 studies were included in the review - documenting 51 specific experiences of domestic stigmatisation (referred to as acts for the purpose of the review) across all studies. A matrix was developed detailing each study's' publication date, geographical context, participant gender (where possible) and the reported acts. A critical analysis is offered on the concept "domestic stigmatisation" and its relevance to domestic or family interventions. Deliberate attention to this concept can potentially refocus HIV stigma-reducing interventions to benefit families and promote coping strategies to reduce stigma-related stress associated with seropositive identities.

Combined delivery of FGF-2, TGF-ß1, and adipose-derived stem cells from an engineered periosteum to a critical-sized mouse femur defect.

Critical-sized long bone defects suffer from complications including impaired healing and non-union due to substandard healing and integration of devitalized bone allograft. Removal of the periosteum contributes to the limited healing of bone allografts. Restoring a periosteum on bone allografts may provide improved allograft healing and integration. This article reports a polysaccharide-based tissue engineered periosteum that delivers basic fibroblast growth factor (FGF-2), transforming growth factor-ß1 (TGF-ß1), and adipose-derived mesenchymal stem cells (ASCs) to a critical-sized mouse femur defect. The tissue engineered periosteum was evaluated for improving bone allograft healing and incorporation by locally delivering FGF-2, TGF-ß1, and supporting ASCs transplantation. ASCs were successfully delivered and longitudinally tracked at the defect site for at least 7 days post operation with delivered FGF-2 and TGF-ß1 showing a mitogenic effect on the ASCs. At 6 weeks post implantation, data showed a non-significant increase in normalized bone callus volume. However, union ratio analysis showed a significant inhibition in allograft incorporation, confirmed by histological analysis, due to loosening of the nanofiber coating from the allograft surface. Ultimately, this investigation shows our tissue engineered periosteum can deliver FGF-2, TGF-ß1, and ASCs to a mouse critical-sized femur defect and further optimization may yield improved bone allograft healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 900-911, 2017.

Docetaxel Accumulates in Lymphatic Circulation Following Subcutaneous Delivery Compared to Intravenous Delivery in Rats.

BACKGROUND: The circulatory pathway for particles deposited outside of blood capillaries has not been well characterized for non-traditionally-delivered chemotherapeutics. MATERIALS AND METHODS: Blood and lymph pharmacokinetics of docetaxel (5 mg/kg) and carboplatin (14 and 28 mg/kg) following subcutaneous (s.c.) versus intravenous (i.v.) delivery were determined in a rodent model with catheterizations of both the thoracic lymphatic duct and jugular vein for prolonged synchronous blood and lymph sampling. RESULTS: Subcutaneous docetaxel demonstrates preferential lymphatic accumulation based on the area under the time-concentration curve (AUC0-24h) whereas i.v. docetaxel resulted in a greater plasma maximum concentration measured (Cmax). The apparent elimination half-life (t1/2) in lymph for docetaxel is greater following i.v. or s.c. delivery compared to t1/2 in blood. Carboplatin demonstrates a dose-dependent increase in plasma Cmax regardless of delivery route; the total carboplatin exposure over 24 h in lymph and plasma are comparable. CONCLUSION: Subcutaneous docetaxel achieves lymphatic accumulation greater than that of i.v. delivery.

Do Mesenchymal Stromal Cells Influence Microscopic Residual or Metastatic Osteosarcoma in a Murine Model?

BACKGROUND: Mesenchymal stromal cells (MSCs) have been shown in rodent models to promote primary and pulmonary metastatic sarcoma growth when injected in the presence of gross tumor. In theory, this would limit their use in a clinical setting after limb salvage treatment for osteosarcoma. Although concerning, these models do not translate to the clinical setting wherein MSCs could be used after primary tumor resection to aid in bone healing and incorporation of tumor endoprostheses. If we can determine whether the use of MSCs in this setting is safe, it might improve our ability to augment bone healing in patients undergoing limb salvage. QUESTIONS/PURPOSES: The purpose of this study was to determine (1) whether MSCs promote pulmonary metastatic disease progression in a murine osteosarcoma model; and/or (2) whether they affect local disease recurrence in the presence of microscopic residual osteosarcoma. METHODS: An orthotopic model of luciferase-expressing osteosarcoma was developed. At 10 days, resection of the primary tumor was performed. One hundred fourteen female C3H mice were inoculated with DLM8-luc osteosarcoma in the proximal tibia. Ninety-four mice developed orthotopic osteosarcoma with luciferase expression. Mice with bioluminescent evidence of a primary tumor received either a microscopically "clean" amputation at a time when residual microscopic metastatic disease was present in the lungs (pulmonary metastasis group; n = 65) or a "dirty" amputation (local recurrence group; n = 29). Mice were randomized to receive intravenous MSCs, MSCs at the surgical site, or no MSCs. Mice were monitored for development and progression of pulmonary metastasis and local recurrence by bioluminescence imaging and daily measurements at the surgical site. The number of pulmonary nodules, time to first evidence of metastasis, and size of recurrent tumor were compared using Kruskal-Wallis, analysis of variance, Welch's, t-tests, or Mann-Whitney tests as appropriate for the specific data sets with p < 0.05 considered significant. RESULTS: Mice receiving intravenous MSCs had a faster time to first detection of pulmonary metastasis (2.93 ± 1.90 days) compared with mice with local injection of MSCs (6.94 ± 6.78 days) or no MSCs (5.93 ± 4.55 days) (p = 0.022). MSC treatment did not influence whether mice developed local recurrence (p = 0.749) or size of recurrent tumors (p = 0.221). CONCLUSIONS: MSCs delivered to the surgical site did not promote local recurrence or size of recurrent tumors, but intravenous injection of MSCs did hasten onset of detection of pulmonary metastatic disease. Although local administration of MSCs into a surgical site does not appear to promote either pulmonary metastatic disease or local recurrence, large variation within groups and small numbers diminished statistical power such that a Type II error cannot be ruled out. CLINICAL RELEVANCE: If MSCs are to be used to augment bone healing in the postlimb salvage setting in patients with osteosarcoma, it will be important to understand their influence, if any, on pulmonary micrometastsis or residual microscopic local disease. Although murine models do not completely recapitulate the clinical scenario, these results suggest that intravenous delivery of MSCs may promote micrometastatic pulmonary disease. Local administration into a surgical wound, even in the presence of residual microscopic disease, may be safe, at least in this murine model, but further investigation is warranted before considering the use of MSCs for clinical use in patients with osteosarcoma.

Coating cortical bone allografts with periosteum-mimetic scaffolds made of chitosan, trimethyl chitosan, and heparin.

Bone allografts have very limited healing leading to high rates of failure from non-union, fracture, and infection. The limited healing of bone allografts is due in large part to devitalization and removal of the periosteum, which removes osteogenic cells and osteoinductive signals. Here we report techniques for directly coating cortical bone with tissue scaffolds, and evaluate the scaffolds' capacity to support osteoprogenitor cells. Three types of coatings are investigated: N,N,N-trimethyl chitosan-heparin polyelectrolyte multilayers, freeze-dried porous chitosan foam coatings, and electrospun chitosan nanofibers. The freeze-dried and electrospun scaffolds are also further modified with polyelectrolyte multilayers. All of the scaffolds are durable to subsequent aqueous processing, and are cytocompatible with adipose-derived stem cells. Alkaline phosphatase and receptor activator of nuclear factor kappa-B ligand expression at days 7 and 21 suggest that these scaffolds support an osteoprogenitor phenotype. These scaffolds could serve as periosteum mimics, deliver osteoprogenitor cells, and improve bone allograft healing.

Transcription factor Ets1 cooperates with estrogen receptor α to stimulate estradiol-dependent growth in breast cancer cells and tumors.

The purpose of this study was to explore the role of transcription factor Ets1 in estrogen receptor α (ERα)-positive breast cancer progression. We expressed human Ets1 or empty vector in four human ERα-positive breast cancer cell lines and observed increased colony formation. Further examination of cellular responses in stable Ets1-expressing MCF7 clones displayed increased proliferation, migration, and invasion. Ets1-expressing MCF7 tumors grown in the mammary fat pads of nude mice exhibited increased rates of tumor growth (7.36±2.47 mm(3)/day) compared to control MCF7 tumors (2.52±1.70 mm(3)/day), but maintained their dependence on estradiol for tumor growth. Proliferation marker Ki-67 staining was not different between control and Ets1-expressing tumors, but Ets1-expressing tumors exhibited large necrotic centers and elevated apoptotic staining. Ets1 was shown to cooperate with ERα and the p160 nuclear receptor coactivator (NCOA/SRC) family to increase activation of a consensus estrogen response element luciferase reporter construct. Ets1-expressing MCF7 cells also exhibited elevated expression of the ERα target genes, progesterone receptor and trefoil factor 1. Using GST-pulldown assays, Ets1 formed stable complexes containing both ERα and p160 nuclear receptor coactivators. Taken together, these data suggest that the Ets1-dependent estradiol sensitization of breast cancer cells and tumors may be partially due to the ability of Ets1 to cooperate with ERα and nuclear receptor coactivators to stimulate transcriptional activity of estrogen-dependent genes.

Speed of human biological form and motion processing.

Recent work suggests that biological motion processing can begin within ~110 ms of stimulus onset, as indexed by the P1 component of the event-related potential (ERP). Here, we investigated whether modulation of the P1 component reflects configural processing alone, rather than the processing of both configuration and motion cues. A three-stimulus oddball task was employed to evaluate bottom-up processing of biological motion. Intact point-light walkers (PLWs) or scrambled PLWs served as distractor stimuli, whereas point-light displays of tool motion served as standard and target stimuli. In a second experiment, the same design was used, but the dynamic stimuli were replaced with static point-light displays. The first experiment revealed that dynamic PLWs elicited a larger P1 as compared to scrambled PLWs. A similar P1 increase was also observed for static PLWs in the second experiment, indicating that these stimuli were more salient than static, scrambled PLWs. These findings suggest that the visual system can rapidly extract global form information from static PLWs and that the observed P1 effect for dynamic PLWs is not dependent on the presence of motion cues. Finally, we found that the N1 component was sensitive to dynamic, but not static, PLWs, suggesting that this component reflects the processing of both form and motion information. The sensitivity of P1 to static PLWs has implications for dynamic form models of biological motion processing that posit temporal integration of configural cues present in individual frames of PLW animations.

Orthotopic model of canine osteosarcoma in athymic rats for evaluation of stereotactic radiotherapy.

OBJECTIVE: To develop an orthotopic model of canine osteosarcoma in athymic rats as a model for evaluating the effects of stereotactic radiotherapy (SRT) on osteosarcoma cells. ANIMALS: 26 athymic nude rats. PROCEDURES: 3 experiments were performed. In the first 2 experiments, rats were injected with 1 × 10(6) Abrams canine osteosarcoma cells into the proximal aspect of the tibia (n = 12) or distal aspect of the femur (6). Tumor engraftment and progression were monitored weekly via radiography, luciferase imaging, and measurement of urine pyridinoline concentration for 5 weeks and histologic evaluation after euthanasia. In the third experiment, 8 rats underwent canine osteosarcoma cell injection into the distal aspect of the femur and SRT was administered to the affected area in three 12-Gy fractions delivered on consecutive days (total radiation dose, 36 Gy). Percentage tumor necrosis and urinary pyridinoline concentrations were used to assess local tumor control. The short-term effect of SRT on skin was also evaluated. RESULTS: Tumors developed in 10 of 12 tibial sites and all 14 femoral sites. Administration of SRT to rats with femoral osteosarcoma was feasible and successful. Mean tumor necrosis of 95% was achieved histologically, and minimal adverse skin effects were observed. CONCLUSIONS AND CLINICAL RELEVANCE: The orthotopic model of canine osteosarcoma in rats developed in this study was suitable for evaluating the effects of local tumor control and can be used in future studies to evaluate optimization of SRT duration, dose, and fractionation schemes. The model could also allow evaluation of other treatments in combination with SRT, such as chemotherapy or bisphosphonate, radioprotectant, or parathyroid hormone treatment.

A possible role of multidrug resistance-associated protein 2 (Mrp2) in hepatic excretion of PCB126, an environmental contaminant: PBPK/PD modeling.

3,3',4,4',5'-Pentachlorobiphenyl (PCB126) is a carcinogenic environmental pollutant and its toxicity is mediated through binding with aryl hydrocarbon receptor (AhR). Earlier, we found that PCB126 treated F344 rats had 110-400 times higher PCB126 concentration in the liver than in the fat. Protein binding was suspected to be a major factor for the high liver concentration of PCB126 despite its high lipophilicity. In this research, we conducted a combined pharmacokinetic/pharmacodynamic study in male F344 rats. In addition to blood and tissue pharmacokinetics, we use the development of hepatic preneoplastic foci (glutathione-S-transferase placental form [GSTP]) as a pharmacodynamic endpoint. Experimental data were utilized for building a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model. PBPK/PD modeling was consistent with the experimental PK and PD data. Salient features of this model include: (1) bindings between PCB126 and hepatic proteins, particularly the multidrug resistance-associated protein (Mrp2), a protein transporter; (2) Mrp2-mediated excretion; and (3) a relationship between area under the curve of PCB126 in the livers and % volume of GSTP foci. Mrp2 involvement in PCB126 pharmacokinetics is supported by computational chemistry calculation using a three-dimensional quantitative structure-activity relationship model of Mrp2 developed by S. Hirono et al. (2005, Pharm. Res. 22, 260-269). This work, for the first time, provided a plausible role of a versatile hepatic transporter for drugs, Mrp2, in the disposition of an important environmental pollutant, PCB126.

Hepatic sequestration of chlordecone and hexafluoroacetone evaluated by pharmacokinetic modeling.

Chlordecone (CD) and mirex (M) differ by a single carbonyl group in CD in place of two chlorines in M. Although both compounds are lipophilic, their tissue distributions differ markedly: CD concentrations are highest in liver; M concentrations are highest in fat. We used tissue time course data in rats from our laboratory for CD and M and literature data from monkeys to develop PBPK models to study differences in liver and fat partitioning. The PK model for M had partitioning in tissue without specific hepatic binding. The CD model had partitioning similar to M, and also included liver binding: the maximal binding (B(max)) and binding affinity constant (Kd) required to describe the rat data were 370 nmol/g liver and 100 nM, respectively. To see if other ketones with electron withdrawing constituents at the alpha carbon were also preferentially distributed to liver, we developed a PBPK description for tissue distribution of hexafluoroacetone (HFA). Compared to acetone, HFA is known to be preferentially sequestered in liver and more slowly excreted unchanged from the body. Acetone is more equally distributed to tissues. HFA distribution was evaluated with a PBPK model that included hepatic binding. B(max) and Kd were 1.58 micromol/g liver and 301 microM. In summary, liver sequestration of CD and HFA most likely represents relatively high-affinity but reversible binding of activated carbonyls in these compounds (activated by the presence of electron withdrawing substituents on the alpha-carbons) with glutathione and glutathione transferases, that are present at much higher concentrations in liver than in other tissues. Strong, but reversible hemithioketal formation with active sulfhydryls may also be associated with the toxic responses to CD and HFA.

Anti-angiogenic effects of interleukin-12 delivered by a novel hyperthermia induced gene construct.

PURPOSE: Interleukin-12 (IL-12) is a pro-inflammatory cytokine possessing anti-cancer and anti-angiogenic properties. This study quantitatively assessed the anti-angiogenic effect of IL-12 delivered using an adenoviral vector with murine IL-12 placed under control of a heat shock promoter. This approach limits systemic toxicity by restricting IL-12 delivery locally to the tumour. The kinetics of the downstream cytokines interferon-gamma (IFN-gamma) and interferon inducible protein-10 (IP-10) and other molecules affecting angiogenesis, vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) were also studied. MATERIALS AND METHODS: 4T1 tumours were grown in Balb/C mice and the AdhspmIL-12 construct was injected intra-tumourally. The tumours were heated after 24 h using a water bath. At various time points post-heating the tumours were collected and quantitatively assessed for cytokine production and vascularity. RESULTS: A significant reduction was seen in the tumour vasculature of the treated group vs. the control group mice. Systemic effects of IL-12 were limited to generalized immunostimulation. No hepatoxicity was noted. CONCLUSIONS: This study suggests that IL-12 can be effectively delivered using a gene-based approach with a heat shock promoter. This results in quantitatively measurable anti-angiogenesis and general immunostimulation. The complex inter-play of other pro- and anti-angiogenic factors (IFN-gamma, IP-10, VEGF and PAI-1) was also studied.

Regional induction of CYP1A1 in rat liver following treatment with mixtures of PCB 126 and PCB 153.

Liver enzyme induction has been shown previously to be regional with clear borders between induced and uninduced regions in vivo, and cells either fully induced or not induced in vitro. The current study examined this phenomenon in vivo by evaluating enzyme induction after exposure to PCB 126 and PCB 153 in female Fisher 344 (F344) and male Sprague-Dawley (SD) rats. IHC revealed a regional induction of CYP1A1 after exposure to PCB 126, apparent in the centrilobular region at lower doses and progressing to panlobular with higher doses. PCB 153 exposure induced CYP2B1/2 in the centrilobular region, which spread to the midzonal region as the dose increased, but never became panlobular even at the highest dosage tested. In rats treated with PCB 126 in combination with high doses of PCB 153, induction of CYP1A1 occurred preferentially in the periportal region, a reversal from the pattern seen with PCB 126 alone. This CYP1A1 induction pattern reversal is a unique example of complex biological interactions between coplanar (PCB 126) and noncoplanar (PCB 153) halogenated aromatic hydrocarbons.

Induction of CYP1A1 in primary rat hepatocytes by 3,3',4,4',5-pentachlorobiphenyl: evidence for a switch circuit element.

In vivo induction of CYP1A1 in hepatocytes by aryl hydrocarbon receptor agonists is heterogeneous. Using immunohistochemistry, cells appear to be either induced or not induced as if the response of an individual cell is better represented as a switch. We have examined induction of CYP1A1 in vitro in primary rat hepatocytes to distinguish the responses of populations of cells and responses of individual cells. Cells were treated with various concentrations of the aryl hydrocarbon receptor agonist, 3,3',4,4',5-pentachlorobiphenyl. Concentration-response and time-course responses were determined for the population of cells by Western blotting for CYP1A1 protein and by real-time RT-PCR for CYP1A1 mRNA. Individual cell responses were visualized by immunocytochemistry (ICC) for protein and by in situ hybridization (ISH) for mRNA. CYP1A1 mRNA was quantified by frequency distribution analysis of grains observed on the ISH slides. Population responses showed time- and concentration-related increases in induction. Single cell responses appeared as all-or-none in the field, with cells appearing to be induced and others appearing to be not induced. Even at the highest concentrations (2.5 x 10(-7) M), some hepatocytes remained unresponsive. Distribution frequencies of single cell induction were more consistent with a switch with variable levels of induction in cells depending on treatment concentration. Combined with the reports from in vivo studies, our results support a switch with rheostat behavior for individual hepatocytes. Mechanistic studies in liver cell lines that are confirmed to exhibit switch-like induction of single cells will be necessary to assess the molecular pathways of this circuit element.