Porcine cells express more than one functional ligand for the human lymphocyte activating receptor NKG2D

Abstract: Background: Xenotransplantation could ameliorate the severe shortage of donor organs. The initial results of transplantation from genetically‐modified pig donors to primate recipients suggest that hyperacute rejection can be overcome, but thrombotic microangiopathy and the human anti‐pig cellular immune response remain as significant impediments to successful clinical xenotransplantation. NKG2D is an activating immunoreceptor found on human natural killer (HuNK) cells, CD8⁺ and γδ T cells. Signaling through NKG2D mediates cytotoxicity and cytokine secretion by NK cells and co‐stimulation of T cells. Methods: Chinese hamster ovary P (CHOP) cells were transfected with human NKG2D and used in cell–cell binding studies with porcine epithelial, and endothelial cell lines. Soluble recombinant NKG2D‐Fc was used to stain various porcine cells and tissues to indicate ligand expression. Porcine cells were used as targets in cytotoxicity assays with the HuNK cell lines NKL and YT, with and without enzymatic removal of pULBP1 and antibody blockade of NKG2D signaling. Results and conclusions: In this study, we demonstrate the expression of ligands for human NKG2D on porcine cell lines of endothelial and epithelial origin, islet cell clusters and rejecting kidney. HuNK cells were activated to kill pig cells expressing NKG2D ligands, and cytotoxicity was inhibited by antibody blockade of NKG2D. A previous study identified pULBP1 as the principal ligand for human NKG2D on pig aortic endothelial cells. In the current study, renal epithelial and intestinal endothelial cells each expressed high surface levels of pULBP1, but binding of soluble recombinant NKG2D and NKG2D‐dependent cytotoxicity against these cells persisted after the enzymatic removal of pULBP1, strongly suggesting the presence of at least one additional functional ligand for human NKG2D in these cell types.     

Three‐dimensional printing in orthopaedic surgery: review of current and future applications

Three‐dimensional (3D) printing is a rapidly evolving technology with the potential for significant contributions to surgical practice. There are many current applications for 3D printing technology with future applications being explored. This technology has applications in preoperative planning, education, custom manufacturing (implants, prosthetics and surgical guides) and exciting potential for biological applications. This article reviews the current and future applications of 3D technology in orthopaedic surgery.     

Computer-aided surgery using 3D rendering of maxillofacial pathology and trauma

Background Technological advances in the field of robotics and computer‐assisted surgery continue to revolutionize medicine and surgery. The specialty of maxillofacial surgery has benefited from this advancement. One of the areas of the specialty where this advancement has made significant contributions is in the management of complex soft tissue and bony pathological conditions. Equally significant benefits have also been seen in the reconstruction of traumatic facial defects. Methods Reviews of the literature and retrospective cases from the University of Florida College of Medicine at Jacksonville were undertaken. Results An update on the current use of three‐dimensional (3D) imaging of maxillofacial pathology and a report of a case example are presented. Conclusion The use of computer‐aided rendering of 3D images of the disease process or defects allows for the surgeon to manipulate the information in the preoperative setting to aid in the planning of the surgery. This preoperative planning is, in effect, a simulation of the planned surgery. This paper reviews one of the commonly used methods of integrating computer‐aided surgery in the daily practice of maxillofacial surgery. The advantages as well as the drawbacks of this technique are reviewed. Copyright © 2007 John Wiley & Sons, Ltd.     

Applications of three‐dimensional printing technology in urological practice

A rapid expansion in the medical applications of three‐dimensional (3D)‐printing technology has been seen in recent years. This technology is capable of manufacturing low‐cost and customisable surgical devices, 3D models for use in preoperative planning and surgical education, and fabricated biomaterials. While several studies have suggested 3D printers may be a useful and cost‐effective tool in urological practice, few studies are available that clearly demonstrate the clinical benefit of 3D‐printed materials. Nevertheless, 3D‐printing technology continues to advance rapidly and promises to play an increasingly larger role in the field of urology. Herein, we review the current urological applications of 3D printing and discuss the potential impact of 3D‐printing technology on the future of urological practice.     

Spheroid‐based 3D cell cultures identify salinomycin as a promising drug for the treatment of chondrosarcoma

Chondrosarcoma (CS) is a cartilage malignancy of adulthood that is treated by surgery alone, since chemotherapy is considered ineffective. Unfortunately, a large proportion of patients with CS develop lung metastases, and several die of the disease. In this study, we compared 3D‐spheroid cultures and conventional cell monolayer models in order to identify the best way to select anticancer agents that could be effective for the systemic control of CS. Using SW1353 cells, we developed a three‐dimensional (3D) in vitro culture model to mimic in vivo features of CS microenvironment and evaluated the efficacy of different drugs to modulate CS cell proliferation and survival in 2D versus 3D‐cultures. Doxorubicin (DXR) and cisplatin, that are widely employed in sarcomas, were less effective on 3D‐CS spheroids when compared to standard monolayer models, whereas treatment with the ionophore salinomycin (SAL) had a strong cytotoxic effect both on 2D and 3D‐cultures. Furthermore, as demonstrated by the reduced viability and the enhanced DXR nuclear localization, SAL enhanced DXR cytotoxicity in 3D‐CS spheroids also at sub‐lethal doses. SAL activity on 3D‐CS spheroids was mediated by a significant induction of apoptosis via caspase activation. This study demonstrates that preclinical tests significantly differ in monolayer and 3D cultures of CS cells. Using this approach, SAL, alone or, at sub‐lethal concentrations, in combination with DXR, represents a promising agent for the systemic treatment of CS. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2305–2312, 2018.

     

Matrix Stiffness in Three‐Dimensional Systems Effects on the Behavior of C3A Cells

The purpose of this study was to evaluate in vitro how the modulation of stiffness in a three‐dimensional (3D) system independently influenced the behaviors of hepatocytes. Cells of a human hepatocyte cell line, C3A, which have been used in a clinically tested bioartificial liver support system, were conducted as cell models. Using a 3D system of “mechanically tunable” alginate hydrogels, matrix stiffness was modeled by corresponding to values in normal and fibrotic livers. Through observing the cellular morphology, viability, functional protein analysis, and gene expression, the effect of the 3D matrix stiffness on C3A cells was investigated. When cultured in stiff hydrogels (12 Kpa), C3A cells adopt a growth arrested and dedifferentiated phenotype, whereas in soft hydrogels (1 Kpa), they remain differentiated phenotype. The behavior of C3A cells can be modulated via independent tuning of mechanical stimuli in the 3D alginate hydrogels, which is different from that in the two‐dimensional (2D) systems. The results indicate the importance of matrix stiffness choice for liver tissue engineering.     

Expression of green fluorescent protein in human foreskin fibroblasts for use in 2D and 3D culture models

The availability of fibroblasts that express green fluorescent protein (GFP) would be of interest for the monitoring of cell growth, migration, contraction, and other processes within the fibroblast‐populated collagen matrix and other culture systems. A plasmid lentiviral vector‐GFP (pLV‐GFP) was utilized for gene delivery to produce primary human foreskin fibroblasts (HFFs) that stably express GFP. Cell morphology, cell migration, and collagen contraction were compared between nontransduced HFFs and transduced GFP‐HFFs; no differences were observed. Immunocytochemical staining showed no differences in cell morphology between nontransduced and GFP‐HFFs in both two‐dimensional and three‐dimensional culture systems. Furthermore, there was no significant difference in cellular population growth within the collagen matrix populated with nontransduced vs. GFP‐HFFs. Within the limits of our assays, we conclude that transduction of GFP into HFFs did not alter the observed properties of HFFs compared with nontransduced fibroblasts. The GFP‐HFFs may represent a new tool for the convenient monitoring of living primary fibroblast processes in two‐dimensional or three‐dimensional culture.     

The Potential Role of Dedicated 3D Breast CT as a Diagnostic Tool: Review and Early Clinical Examples

Mammography is the gold standard in routine screening for the detection of breast cancer in the general population. However, limitations in sensitivity, particularly in dense breasts, has motivated the development of alternative imaging techniques such as digital breast tomosynthesis, whole breast ultrasound, breast‐specific gamma imaging, and more recently dedicated breast computed tomography or “breast CT”. Virtually all diagnostic work‐ups of asymptomatic nonpalpable findings arise from screening mammography. In most cases, diagnostic mammography and ultrasound are sufficient for diagnosis, with magnetic resonance imaging (MRI) playing an occasional role. Digital breast tomosynthesis, a limited‐angle tomographic technique, is increasingly being used for screening. Dedicated breast CT has full three‐dimensional (3D) capability with near‐isotropic resolution, which could potentially improve diagnostic accuracy. In current dedicated breast CT clinical prototypes, 300–500 low‐dose projections are acquired in a circular trajectory around the breast using a flat panel detector, followed by image reconstruction to provide the 3D breast volume. The average glandular dose to the breast from breast CT can range from as little as a two‐view screening mammogram to approximately that of a diagnostic mammography examination. Breast CT displays 3D images of the internal structures of the breast; therefore, evaluation of suspicious features like microcalcifications, masses, and asymmetries can be made in multiple anatomical planes from a single scan. The potential role of breast CT for diagnostic imaging is illustrated here through clinical examples such as imaging soft tissue abnormalities and microcalcifications. The potential for breast CT to serve as an imaging tool for extent of disease evaluation and for monitoring neo‐adjuvant chemotherapy response is also illustrated.     

A Novel Possible Strategy Based on Self‐Assembly Approach to Achieve Complete Periodontal Regeneration

Limitations of current regeneration modalities underscore the importance of restoring the three‐dimensional (3D) microenvironment of periodontal development, which is able to elicit the intrinsic capacity of mesenchymal stem cells to proceed to engage in a redevelopment‐like program. With increased attention for the potential therapeutic applications of periodontal ligament stem cells (PDLSCs) in periodontal regeneration, it has been proposed that bone marrow mesenchymal stem cells (BMMSCs) are very likely another cell source of physiological repair of periodontal tissues. With this in mind, enlightened from the research targeting the fabrication of laminar structures such as liver and kidney with heterotypic stratification of cell sheets, we proposed a novel possible strategy based on self‐assembly approach, which is akin to the physiological phenomenon that occurs during organogenesis, to enhance complete reconstruction of functional complex periodontium‐organ systems. We assumed that in this strategy, using the intrinsic capacity of monodispersed cells to self‐assemble into a microtissue such as a 3D spheroid, bilayered cell pellet constructs comprising calcified bone‐forming cell pellets (i.e., BMMSCs) and cementum/PDL‐forming cell pellets (i.e., PDLSCs) would be fabricated in vitro in a tissue‐mimicking way and then implanted into periodontal defects. We hypothesize that this novel strategy might open new options to reconstruct extended periodontal defects and then achieve the ultimate goal of predictable and complete regeneration of the periodontium.     

Usefulness of Intraoperative Real‐Time 3D Transesophageal Echocardiography in Cardiac Surgery

Abstract Background: Recent advances in three‐dimensional (3D) echocardiography allow to obtain real‐time 3D transesophageal (RT3DTEE) images intraoperatively. Methods: Preoperative transthoral echocardiography (TTE) revealed: hypertrophic ventricular septum (TTE:19.3 mm), systolic anterior motion (SAM) not causing obstruction and malcoaptation of the anterior mitral valve leaflet (AMVL), and posterior mitral valve leaflet (PMVL) with severe mitral regurgitation. Results: Intraoperative TEE with a x7‐2t MATRIX‐array transducer (Philips, Andover, MA, USA) with a transducer frequency of x7–2 t mHz, connected to a iE33 (Philips), shows us that the main mechanism and site of regurgitation was an AMVL cleft. We also measured a 24.3‐mm thickness of the ventricular septum and analyzing the 3D full volume acquisition revealed that there was no SAM. Conclusion: Intraoperative RT3DTEE permitted comprehensive 3D viewing of the mitral valve revealing the mechanism of mitral valve regurgitation, SAM, and the exact width of the hypertrophic ventricular septum.     

Comparison of 2 versus 3 dimensional fracture mapping strategies for 3 dimensional computerized tomography reconstructions of scapula neck and body fractures

Fracture mapping has been used in the understanding of injury patterns in different bones. To our knowledge, there are no applications of this technique using three‐dimensional (3D) morphologic fracture characteristics. Previously, scapula fractures were mapped by transferring information from 3D computed tomography to a two‐dimensional (2D) template. Cole et al. determined that 3D Computerized Tomography (CT) scans were more reliable compared to plain radiographs in terms of scapular angulation, translation, and glenopolar angle measurements. Thus, we hypothesized that if there is a difference between fracture lines drawn in 3D and in 2D, then the 3D mapping would yield more accurate fracture patterns. We completed a retrospective, comparative study (evidence level III) utilizing CT imaging from a single center scapular registry. We studied ten patients with scapula fractures in whom bilateral CT scans were obtained. Fractures were mapped both two and three‐dimensionally, and we measured deviations between the fracture lines that were drawn with each approach. The measured deviations ranged from 10.4 mm to 28.0 mm when comparing 2D versus 3D techniques, with the mean deviation being 4.0 mm and 10.4 mm, respectively. Half of the 2D renderings possessed hidden fracture lines that were later revealed on 3D imaging. Three‐dimensional renderings were more accurate when compared to 2D fracture mapping methods. This more accurate technique will allow for better understanding of 3D morphology and provide a basis for future fracture mapping in any bone. Accurate mapping is important because surgical approach, reduction, fixation, and implant design and selection are based on fracture patterns. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:265–271, 2018.     

Computerized 3D morphological analysis of glenoid orientation

An accurate preoperative measurement of glenoid orientation is crucial for evaluating pathologies and successful total shoulder arthroplasty. Existing methods may be labor‐intensive, observer‐dependent, and sensitive to the misalignment between the scapula plane and CT scanning direction. In this study, we proposed a computation framework and performed an automated analysis of the glenoid orientation based on 3D surface data. Three‐dimensional models of 12 scapulae were analyzed. The glenoid cavity and external anatomical features were automatically extracted from these 3D models. Glenoid version was calculated using the scapula plane and the fulcrum axis alternatively. Glenoid inclination was measured both relative to transverse axis of the scapula and the medial pole‐inferior tip axis. The mean (±SD) of the fulcrum‐based glenoid version was ?0.55° (±4.17°), while the scapular‐plane‐based glenoid version was ?5.05° (±3.50°). The mean (±SD) of glenoid inclinations based on the medial pole and inferior tip was 12.75° (±5.03°) while the mean (±SD) of the glenoid inclination based on the medial pole and glenoid center was 4.63° (±4.86°). Our computational framework was able to extract the reproducible morphological measures free of inter‐ and intra‐ observer variability. For the first time in 3D, we showed that the fulcrum axis was practically perpendicular to the glenoid plane normal (radial line), and thus extended the fulcrum‐based glenoid version for quantifying 3D glenoid orientation. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:692–698, 2016.

     

Expression and Regulation of the Vitamin D Receptor in the Zebrafish,Danio rerio

Vitamin D and vitamin D metabolites such as 25‐hydroxyvitamin D and 1α,25‐dihydroxyvitamin D [1α,25(OH)₂D₃] circulate in the serum of fish. The receptor for 1α,25(OH)₂D₃ (VDR) has previously been cloned from fish intestine, and ligand binding assays have shown the presence of the VDR in the gills, intestine, and liver of fish. Using immunohistochemical methods with specific antibodies against the VDR, we now report that the VDR is widely expressed in tissues of the adult male and female zebrafish, Danio rerio, specifically in epithelial cells of gills, tubular cells of the kidney, and absorptive cells in the intestine. Additionally, the VDR is expressed in the skin, the olfactory organ, the retina, brain, and spinal cord. Sertoli cells of the testis, oocytes, acinar cells of the pancreas, hepatocytes, and bile duct epithelial cells express substantial amounts of the receptor. Osteoblast‐like cells and chondrocytes also express VDR. Preimmune serum and antiserum preadsorbed with Danio VDR protein fails to detect VDR in the same tissues. The VDR is also present in the developing eye, brain, and otic vesicle of 48‐ and 96‐h postfertilization zebrafish embryos. Parenteral administration of 1α,25(OH)₂D₃ increases concentrations of VDR in intestinal epithelial cells but not in epithelial cells of the gills. Lithocholic acid, however, does not alter concentrations of VDR after parenteral administration. The data suggest that VDR is widely distributed in tissues of the zebrafish, D. rerio, and is likely to play important roles in epithelial transport, bone, and endocrine function. Furthermore, concentrations of the receptor seem to be regulated by its ligand, 1α,25‐dihydroxyvitamin D but not by lithocholic acid. Zebrafish may serve as a useful model in which to assess the function of the VDR in diverse tissues.     

Exponential stability analysis for 2D discrete switched systems with state delays

This paper is concerned with the delay‐dependent exponential stability analysis of two‐dimensional (2D) discrete switched systems with state delays described by the Roesser model; the delays under consideration are varying. By constructing an appropriate Lyapunov‐Krasovskii functional and using the average dwell time approach, new delay‐dependent sufficient conditions for the exponential stability of the system under study are proposed. In order to obtain less conservative conditions, the delay partitioning method is adopted as well as the free‐weighting matrix technique. The proposed conditions are formulated in the format of linear matrix inequality. The effectiveness and the reduced conservatism of the developed results are shown by illustrative examples.     

CT exam and 2D and 3D reconstructions in facial tumors diagnosis

This study presents the CT imaging of a group of maxillofacial tumors that include benign tumors and primary and secondary malignant processes. MATERIAL AND METHOD: All patients were CT explored with and without intravenous contrast, using specific head and neck protocols. RESULTS: We studied the topography of the tumors, their impact on neighborhood regions and post-treatment results. The diagnostic of all patients had histological confirmation. In maxillofacial tumors and tumor-like diseases, 3D imaging was significantly superior to 2D axial imaging in showing the tumor shape and spatial relationships between tumors and surrounding structures. Two dimensional imaging and MPR imaging were excellent to reveal internal structure and pathological changes of tumors. 2D imaging and MPR imaging also achieved better results in showing tumors extended to soft tissues.     

3D细胞培养和类器官在骨髓源性间充质干细胞成骨分化中的研究进展

类器官（organoids）来源于自体的组织及干细胞,是通过体外3D培养后形成的细胞团块,这种团块具有原始组织及器官的三维结构,并保留了相对应的功能和遗传特征。由于其具有模拟特定机体器官的发育和疾病发生发展的潜能,这一模型在多种药物的筛选和分子机制研究中拥有更多的优势。近年来,已有实验表明骨髓源性间充质干细胞（BMSCs）通过3D培养及成骨分化可以形成骨的类器官,并可以植入机体发挥特定的作用。这种骨类器官模型的构建,不仅可以为骨质疏松等相关疾病研究提供更多方法,还在骨组织移植及修复等组织工程学中发挥重要作用。本文就BMSCs成骨分化的类器官相关3D模型研究进展作一综述,为BMSCs成骨分化的类器官的基础和临床研究提供更多理论依据和思路。     

ITIM‐dependent negative signaling pathways for the control of cell‐mediated xenogeneic immune responses

Xenotransplantation is an innovative field of research with the potential to provide us with an alternative source of organs to face the severe shortage of human organ donors. For several reasons, pigs have been chosen as the most suitable source of organs and tissues for transplantation in humans. However, porcine xenografts undergo cellular immune responses representing a major barrier to their acceptance and normal functioning. Innate and adaptive xenogeneic immunity is mediated by both the recognition of xenogeneic tissue antigens and the lack of inhibition due to molecular cross‐species incompatibilities of regulatory pathways. Therefore, the delivery of immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐dependent and related negative signals to control innate (NK cells, macrophages) and adaptive T and B cells might overcome cell‐mediated xenogeneic immunity. The proof of this concept has already been achieved in vitro by the transgenic overexpression of human ligands of several inhibitory receptors in porcine cells resulting in their resistance against xenoreactivity. Consequently, several transgenic pigs expressing tissue‐specific human ligands of inhibitory coreceptors (HLA‐E, CD47) or soluble competitors of costimulation (belatacept) have already been generated. The development of these robust and innovative approaches to modulate human anti‐pig cellular immune responses, complementary to conventional immunosuppression, will help to achieve long‐term xenograft survival. In this review, we will focus on the current strategies to enhance negative signaling pathways for the regulation of undesirable cell‐mediated xenoreactive immune responses.     

维生素D受体检测方法的研究进展

维生素D不仅能够调节钙磷代谢,影响骨的生长,还能调节机体的免疫活性和细胞生长与分化。维生素D的功能是通过激活细胞核内的维生素D受体（vitamin D receptor, VDR)来实现的,因此,VDR是靶细胞响应维生素D反应的决定因素。VDR高表达的组织,如肠、肾脏、骨和甲状旁腺等,是维生素D的主要靶组织。过去40年中,为了测定VDR在机体各个组织中的分布,建立了多种检测方法。然而,这些测定方法的应用也产生了一些具有争议的结果,这可能是由检测方法的特异性不同导致的。近年来,本研究团队建立了一套特异性强、灵敏性高的免疫测定方法,并将其成功应用于测定组织中VDR。本文将对这些检测方法的的优点和各自的局限性进行讨论,并指出测定方法的选择是成功鉴定靶组织中VDR的关键。