Glucocorticoid Receptor ß Isoform Predominates in the Human Dysplastic Brain Region and Is Modulated by Age, Sex, and Antiseizure Medication.

The glucocorticoid receptor (GR) at the blood−brain barrier (BBB) is involved in the pathogenesis of drug-resistant epilepsy with focal cortical dysplasia (FCD); however, the roles of GR isoforms GRα and GRß in the dysplastic brain have not been revealed. We utilized dysplastic/epileptic and non-dysplastic brain tissue from patients who underwent resective epilepsy surgery to identify the GRα and GRß levels, subcellular localization, and cellular specificity. BBB endothelial cells isolated from the dysplastic brain tissue (EPI-ECs) were used to decipher the key BBB proteins related to drug regulation and BBB integrity compared to control and transfected GRß-overexpressed BBB endothelial cells. GRß was upregulated in dysplastic compared to non-dysplastic tissues, and an imbalance of the GRα/GRß ratio was significant in females vs. males and in patients > 45 years old. In EPI-ECs, the subcellular localization and expression patterns of GRß, Hsp90, CYP3A4, and CYP2C9 were consistent with GRß+ brain endothelial cells. Active matrix metalloproteinase levels and activity increased, whereas claudin-5 levels decreased in both EPI-ECs and GRß+ endothelial cells. In conclusion, the GRß has a major effect on dysplastic BBB functional proteins and is age and gender-dependent, suggesting a critical role of brain GRß in dysplasia as a potential biomarker and therapeutic target in epilepsy.

Precision in Oculofacial Surgery: Made-To-Specification Cast-Molded Implants in Orbital Reconstruction.

PURPOSE: To describe the utilization of customized made-to-specification porous polyethylene implants and to evaluate clinical characteristics and outcomes of patients who received these implants for unilateral orbital defects. METHODS: A retrospective review of 9 patients was performed. Three-dimensional surface models were generated from high-resolution computed tomography scans. Orbital constructs were modeled after the normal, contralateral orbits and mirrored across the vertical midline to generate the target orbital implant. Measured outcomes included globe position, extraocular motility, facial symmetry, and diplopia. RESULTS: Patients ranged 25-56 years old (mean: 37) and included 6 males and 3 females. Cases consisted of 6 orbital floor fractures due to trauma, 1 lateral wall defect after neurofibroma resection, 1 floor/medial wall defect after myxoma resection, and 1 superior orbital rim defect after intraosseous hemangioma resection. Seven patients had ≥1 prior repair. All patients had previous hard and soft tissue defects and varying degrees of restrictive globe motility. Patients exhibited improved ductions after implant placement and improved facial appearance and symmetry. Post-operatively, 1 patient was found to have a small orbital hematoma between the implant and orbital floor, resolving within weeks. CONCLUSIONS: Precision, personalized oculofacial surgery is the next wave in tailoring surgical care to the individual patient. Customizable implants are manufactured to specifically mold to an individual patient's unique bony architecture, which can lead to superior outcomes in reconstructing orbital and craniofacial bony defects. This technique is particularly useful in patients with prior unsuccessful repair.

Three-Dimensional Ultrasound Versus Computerized Tomography in Fat Graft Volumetric Analysis.

Studies evaluating fat grafting in mice have frequently used micro-computed tomography (micro-CT) as an accurate radiographic tool to measure longitudinal volume retention without killing the animal. Over the past decade, however, microultrasonography has emerged as an equally powerful preclinical imaging tool. Given their respective strengths in 3-dimensional reconstruction, there is no study to our knowledge that directly compares micro-CT with microultrasound in volumetric analysis. In this study, we compared the performance of micro-CT with microultrasound in the evaluation of adipose tissue graft volume in a murine model. Fifteen immunodeficient mice were given 200 µL of adipose tissue grafts. In vivo volumetric analysis of the grafts by micro-CT and microultrasound was conducted at discrete time points up to postoperative day 105. Three mice were killed at multiple time points, and explanted grafts were reimaged by CT and ultrasound, as mentioned previously. Analysis revealed that in vivo graft volumes measured by micro-CT do not differ significantly from those of microultrasound. Furthermore, both micro-CT and microultrasound were capable of accurately measuring fat grafts as in vivo volumes closely correlated with explanted volumes. Finally, ultrasound was found to yield improved soft tissue contrast compared with micro-CT. Therefore, either modality may be used, depending on experimental needs.

Cell-Based Soft Tissue Reconstruction in a Hydrogel Scaffold.

BACKGROUND: Renevia is a hyaluronin-gelatin crosslinked matrix scaffold that has been studied as an alternative to adipose transfer in soft tissue reconstruction. It is designed to emulate the native extracellular matrix environment by supporting stromal vascular fraction (SVF) cell attachment, survival, and proliferation, thus promoting cell-based volume restoration. However, the concentration of incorporated cells for a clinically relevant result has yet to be determined. METHODS: Five experimental groups of seven CD-1 nude immunodeficient mice were given 250 µL grafts of the following composition: 1 million human SVF cells per mL of Renevia scaffold, 6 million human SVF cells per mL scaffold, 12 million human SVF cells per mL scaffold, Renevia scaffold-alone or human adipose tissue-alone. Volumetric analysis was conducted at discrete time points over 16 weeks using 3-dimensional ultrasound, after which time the grafts were explanted for histologic analysis. RESULTS: At the conclusion of the study at week 16, the Renevia scaffold group incorporating the highest concentration of human SVF cells (12 million cells per mL scaffold) had significantly greater volume retention compared with the 2 lower concentrations, scaffold-alone and fat-alone groups. Histology of the 12 million scaffold group revealed abundant adipocyte formation within the scaffold, exceeding that observed in the 6 million, 1 million, and scaffold-alone groups. The 12 million group also demonstrated significantly increased vascularity per CD31 staining. CONCLUSIONS: Stromal vascular fraction cells coupled with Renevia hydrogel scaffold can enhance soft tissue volume reconstruction. In this study, we observed the greatest effect with 12 million cells per mL. From the perspective of volume retention, incorporation of higher concentrations of SVF cells with Renevia may be an alternative to conventional adipose tissue grafting.

Distinct uptake and elimination profiles for trastuzumab, human IgG, and biocytin-TMR in experimental HER2+ brain metastases of breast cancer.

BACKGROUND: The aim of this study is an improved understanding of drug distribution in brain metastases. Rather than single point snapshots, we analyzed the time course and route of drug/probe elimination (clearance), focusing on the intramural periarterial drainage (IPAD) pathway. METHODS: Mice with JIMT1-BR HER2+ experimental brain metastases were injected with biocytin-TMR and either trastuzumab or human IgG. Drugs/probes circulated for 5 min to 48 h, followed by perfusion. Brain sections were stained for human IgG, vascular basement membrane proteins laminin or collagen IV, and periarterial α-SMA. A machine learning algorithm was developed to identify metastases, metastatic microenvironment, and uninvolved brain in confocally scanned brain sections. Drug/probe intensity over time and total imaged drug exposure (iAUC) were calculated for 27,249 lesions and co-immunofluorescence with IPAD-vascular matrix analyzed in 11,668 metastases. RESULTS: In metastases, peak trastuzumab levels were 5-fold higher than human IgG but 4-fold less than biocytin-TMR. The elimination phase constituted 85-93% of total iAUC for all drugs/probes tested. For trastuzumab, total iAUC during uptake was similar to the small molecule drug probe biocytin-TMR, but slower trastuzumab elimination resulted in a 1.7-fold higher total iAUC. During elimination trastuzumab and IgG were preferentially enriched in the α-SMA+ periarterial vascular matrix, consistent with the IPAD clearance route; biocytin-TMR showed heterogeneous elimination pathways. CONCLUSIONS: Drug/probe elimination is an important component of drug development for brain metastases. We identified a prolonged elimination pathway for systemically administered antibodies through the periarterial vascular matrix that may contribute to the sustained presence and efficacy of large antibody therapeutics.

KMN-159, a novel EP4 receptor selective agonist, stimulates osteoblastic differentiation in cultured whole rat bone marrow.

KMN-159 is the lead compound from a series of novel difluorolactam prostanoid EP4 receptor agonists aimed at inducing local bone formation while avoiding the inherent side effects of systemic EP4 activation. KMN-159 is a potent, selective small molecule possessing pharmacokinetic properties amenable to local administration. Unfractionated rat bone marrow cells (BMCs) were treated once at plating with escalating doses of KMN-159 (1 pM to 10 µM). The resulting elevated alkaline phosphatase (ALP) levels measured 9 days post-dose are consistent with increased osteoblastic differentiation and exposure to KMN-159 at low nanomolar concentrations for as little as 30 min was sufficient to induce complete osteoblast differentiation of the BMCs from both sexes and regardless of age. ALP induction was blocked by an EP4 receptor antagonist but not by EP1 or EP2 receptor antagonists and was not induced by EP2 or EP3 receptor agonists. Addition of BMCs to plates coated with KMN-159 24 days earlier resulted in ALP activation, highlighting the chemical stability of the compound. The expression of phenotype markers such as ALP, type I collagen, and osteocalcin was significantly elevated throughout the osteoblastic differentiation timecourse initiated by KMN-159 stimulation. An increased number of tartrate-resistant acid phosphatase-positive cells was observed KMN-159 or PGE2 treated BMCs but only in the presence of exogenous receptor activator of nuclear factor kappa-Β ligand (RANKL). No change in the number of adipocytes was observed. KMN-159 also increased bone healing in a rat calvarial defect model with a healing rate equivalent to recombinant human bone morphogenetic protein-2. Our studies show that KMN-159 is able to stimulate osteoblastic differentiation with a very short time of exposure, supporting its potential as a therapeutic candidate for augmenting bone mass.

Fat Grafting into Younger Recipients Improves Volume Retention in an Animal Model.

BACKGROUND: Soft-tissue deficits associated with various craniofacial anomalies can be addressed by fat grafting, although outcomes remain unpredictable. Furthermore, consensus does not exist for timing of these procedures. Whereas some advocate approaching soft-tissue reconstruction after the underlying skeletal foundation has been corrected, other studies have suggested that earlier grafting may exploit a younger recipient niche that is more conducive to fat graft survival. As there is a dearth of research investigating effects of recipient age on fat graft volume retention, this study compared the effectiveness of fat grafting in younger versus older animals through a longitudinal, in vivo analysis. METHODS: Human lipoaspirate from three healthy female donors was grafted subcutaneously over the calvaria of immunocompromised mice. Volume retention over 8 weeks was evaluated using micro-computed tomography at three experimental ages: 3 weeks, 6 months, and 1 year. Histologic examination was performed on explanted grafts to evaluate graft health and vascularity. Recipient-site vascularity was also evaluated by confocal microscopy. RESULTS: The greatest retention of fat graft volume was noted in the youngest group compared with both older groups (p < 0.05) at 6 and 8 weeks after grafting. Histologic and immunohistochemical analyses revealed that improved retention in younger groups was associated with greater fat graft integrity and more robust vascularization. CONCLUSION: The authors' study provides evidence that grafting fat into a younger recipient site correlates with improved volume retention over time, suggesting that beginning soft-tissue reconstruction with fat grafting in patients at an earlier age may be preferable to late correction.

Utilizing Confocal Microscopy to Characterize Human and Mouse Adipose Tissue.

Significant advances in our understanding of human obesity, endocrinology, and metabolism have been made possible by murine comparative models, in which anatomically analogous fat depots are utilized; however, current research has questioned how truly analogous these depots are. In this study, we assess the validity of the analogy from the perspective of cellular architecture. Whole tissue mounting, confocal microscopy, and image reconstruction software were used to characterize the three-dimensional structure of the inguinal fat pad in mice, gluteofemoral fat in humans, and subcutaneous adipose tissue of the human abdominal wall. Abdominal and gluteofemoral adipose tissue specimens from 12 human patients and bilateral inguinal fat pads from 12 mice were stained for adipocytes, blood vessels, and a putative marker for adipose-derived multipotent progenitor cells, cluster of differentiation 34 (CD34). Samples were whole-mounted and imaged with laser scanning confocal microscopy. Expectedly, human adipocytes were larger and demonstrated greater size heterogeneity. Mouse fat displayed significantly higher vascular density compared with human fat when normalized to adipocyte count. There was no significant difference in the concentration of CD34-positive (CD34+) stromal cells from either species. However, the mean distance between CD34+ stromal cells and blood vessels was significantly greater in human fat. Finally, mouse inguinal fat contained larger numbers of brown adipocytes than did human gluteofemoral or human abdominal fat. Overall, the basic architecture of human adipose tissue differs significantly from that of mice. Insofar as human gluteofemoral fat differs from human abdominal adipose tissue, it was closer to mouse inguinal fat, being its comparative developmental analog. These differences likely confer variance in functional properties between the two sources and thus must be considered when designing murine models of human disease.

Deferoxamine Preconditioning of Irradiated Tissue Improves Perfusion and Fat Graft Retention.

BACKGROUND: Radiation therapy is a mainstay in the treatment of many malignancies, but collateral damage to surrounding tissue, with resultant hypovascularity, fibrosis, and atrophy, can be difficult to reconstruct. Fat grafting has been shown to improve the quality of irradiated skin, but volume retention of the graft is significantly decreased. Deferoxamine is a U.S. Food and Drug Administration-approved iron-chelating medication for acute iron intoxication and chronic iron overload that has also been shown to increase angiogenesis. The present study evaluates the effects of deferoxamine treatment on irradiated skin and subsequent fat graft volume retention. METHODS: Mice underwent irradiation to the scalp followed by treatment with deferoxamine or saline and perfusion and were analyzed using laser Doppler analysis. Human fat grafts were then placed beneath the scalp and retention was also followed up to 8 weeks radiographically. Finally, histologic evaluation of overlying skin was performed to evaluate the effects of deferoxamine preconditioning. RESULTS: Treatment with deferoxamine resulted in significantly increased perfusion, as demonstrated by laser Doppler analysis and CD31 immunofluorescent staining (p < 0.05). Increased dermal thickness and collagen content secondary to irradiation, however, were not affected by deferoxamine (p > 0.05). Importantly, fat graft volume retention was significantly increased when the irradiated recipient site was preconditioned with deferoxamine (p < 0.05). CONCLUSIONS: The authors' results demonstrated increased perfusion with deferoxamine treatment, which was also associated with improved fat graft volume retention. Preconditioning with deferoxamine may thus enhance fat graft outcomes for soft-tissue reconstruction following radiation therapy.

A Review of Cell-Based Strategies for Soft Tissue Reconstruction.

Soft tissue reconstruction to restore volume to damaged or deficient tissue beneath the skin remains a challenging endeavor. Current techniques are centered around autologous fat transfer, or the use of synthetic substitutes, however, a great deal of scientific inquiry has been made into both the molecular mechanisms involved in, and limitations of, de novo adipogenesis, that is, the formation of new adipose tissue from precursor cells. To best comprehend these mechanisms, an understanding of defined markers for adipogenic differentiation, and knowledge of both commercially available and primary cell lines that enable in vitro and in vivo studies is necessary. We review the growth factors, proteins, cytokines, drugs, and molecular pathways that have shown promise in enhancing adipogenesis and vasculogenesis, in addition to the multitude of scaffolds that act as delivery vehicles to support these processes. While progress continues on these fronts, equally important is how researchers are optimizing clinically employed strategies such as autologous fat transfer through cell-based intervention, and the potential to augment this approach through isolation of preferentially adipogenic or angiogenic precursor subpopulations, which exists on the horizon. This review will highlight the novel molecular and synthetic modifications currently being studied for inducing adipose tissue regeneration on a cellular level, which will expand our arsenal of techniques for approaching soft tissue reconstruction.

Health-Related Quality-of-Life Instruments for Pediatric Patients with Diverse Facial Deformities: A Systematic Literature Review.

BACKGROUND: Treatment for patients with diverse craniofacial conditions is complex and long-term. Craniofacial conditions profoundly influence health-related quality of life, and patient- and parent-reported outcomes provide a critical and complementary perspective on the multidisciplinary treatment of patients. However, little is known regarding the health-related quality of life among children with diverse craniofacial conditions. The purpose of this study was to systematically review the literature regarding patient- and parent-reported outcomes measures for patients with diverse craniofacial conditions. METHODS: Articles from the PubMed, PsychINFO, CINAHL, Embase/MEDLINE, Scopus, and Web of Science databases that used patient- and/or parent-reported outcome instruments in patients with diverse craniofacial conditions were reviewed. Diagnoses included were cleft lip and/or palate, craniosynostosis, microtia, craniofacial microsomia, facial vascular malformations, and congenital nevi across pediatric populations (0 to 22 years of age). RESULTS: Six hundred ninety articles were identified, and 155 were selected for inclusion. One hundred twenty different health-related quality-of-life tools were used to analyze factors such as physical, psychological, or social function. Of these, the 10 most common psychometrically tested tools were identified in 59 studies. Five tools had both parent and patient versions. Two tools were developed and validated for patients with diverse craniofacial conditions, but neither was developed for nonadolescent children. CONCLUSIONS: Many parent- and patient-reported instruments are used to measure varying health-related quality of life factors in this population, but no tool exists that was developed and psychometrically tested in different facial deformities that measures comprehensive health-related quality of life issues across all pediatric ages. This study will guide the development of new tools to measure the parent and patient health-related quality-of-life perspective in patients with diverse craniofacial conditions.

Interleukin-6, tissue factor and von Willebrand factor in acute decompensated heart failure: relationship to treatment and prognosis.

Arterial thrombotic and thromboembolic complications are increased in congestive heart failure (CHF), and are a particular problem in acute decompensated heart failure, which carries a poor prognosis. As interleukin-6 (IL-6) has been shown to induce the potent procoagulant tissue factor (TF) in experimental models, we hypothesized that the pro-inflammatory IL-6 may be one mechanism contributing to thrombosis in heart failure, mediated via endothelial expression of TF on activated/damaged cells [indicated by plasma von Willebrand factor (vWF)]. Seventy-seven patients (67% men, New York Heart Association class III-IV, 87%) with acute CHF were recruited, and were compared with 53 chronic stable CHF patients in sinus rhythm (66% men, New York Heart Association class III-IV, 2%) and 37 healthy controls (68% men). Acute CHF patients in sinus rhythm had elevated baseline levels of IL-6 (P < 0.0001), TF (P = 0.041) and vWF (P < 0.0001) (all measured by enzyme-linked immunosorbent assay) compared with both chronic CHF and healthy control groups. A correlation exists in acute CHF between baseline TF and IL-6 (Spearman r = 0.64, P < 0.0001). After 3 months treatment, with control or alleviation of heart failure symptoms in 40 patients, there was a fall in levels of IL-6 (P < 0.0001) and vWF (P < 0.0001), but levels still remained significantly higher than healthy controls. Patients who died at 6 months follow-up also had higher baseline levels of IL-6 (P = 0.008), TF (P = 0.037) and vWF (P = 0.039) when compared with those who remained alive. Elevated IL-6 may contribute to the thrombotic and thromboembolic complications in acute heart failure, in a process mediated via increased TF and vWF. Improvement of symptoms and plasma markers after treatment of acute CHF and prediction of prognosis by the markers may be useful in the clinical setting.

Human chorionic gonadotropin interactions with immobilized anti-hCG studied by quartz-crystal microbalance with dissipation monitoring.

Previous studies have found that commercial human pregnancy tests are often too insensitive to function to their advertised >99% accuracy. Improper orientation of proteins used for recognition of ligands in sensors can often prevent the binding site from being available to the ligand, thereby decreasing sensor sensitivity. We have developed a simple method for the immobilization of anti-human chorionic gonadotropin on a sensor surface that maximizes its sensitivity by ensuring ligand binding sites are exposed and densely packed. This surface also has an improved regenerative capacity over previously reported human chorionic gonadotropin sensors, retaining 99% of initial sensitivity after six regeneration cycles with 8M urea.