Bioactive Bone Substitute in a Rabbit Ulna Model: Preclinical Study.

BACKGROUND: Current therapies to effectively treat long-bone defects and extensive bone tissue loss remains limited. In this study, we created a new bone substitute by integrating advanced technologies such as structure patterning, controlled release of a bone growth factor and conjugation system for clinically effective bone regeneration. This novel bioactive bone substitute was evaluated for its safety and efficacy using a rabbit ulna model. METHODS: A three dimensional bone patterned cylindrical structure with 1.5 cm in length and 5 mm in diameter was printed using poly(L-lactic acid)(PLLA) as a weight-bearing support and space-filling scaffold. And a bone morphogenetic protein 2 (BMP2) was employed to enhance bone regeneration, and coated to a 3D PLLA using alginate catechol and collagen to prolong the release kinetics. This novel bone substitute (BS)was evaluated for its physico-chemical and biological properties in vitro, and histological analysis and radiographical analysis such as X-ray, CT and micro-CT image analysis were performed to evaluate new bone formation in vivo. RESULTS: The BS possesses an ideal shape and mechanically suitable proeperties for clinical use, with an easy-to-grab and break-resistant design at both ends, 80 ± 10 MPa of compression strength, and BMP2 release for two months. Histological analysis demonstrated the biocompability of BS with minimal inflammation and immune response, and X-ray, CT and micro-CT demonstrated effective new bone formation in rabbit ulna defect model. CONCLUSION: The preclinical study of a novel bioactive bone substitute has shown its safe and effective properties in an animal model suggesting its clinical potential.

rhBMP-2-Conjugated Three-Dimensional-Printed Poly(L-lactide) Scaffold is an Effective Bone Substitute.

BACKGROUND: Bone growth factors, particularly bone morphogenic protein-2 (BMP-2), are required for effective treatment of significant bone loss. Despite the extensive development of bone substitutes, much remains to be desired for wider application in clinical settings. The currently available bone substitutes cannot sustain prolonged BMP-2 release and are inconvenient to use. In this study, we developed a ready-to-use bone substitute by sequential conjugation of BMP to a three-dimensional (3D) poly(L-lactide) (PLLA) scaffold using novel molecular adhesive materials that reduced the operation time and sustained prolonged BMP release. METHODS: A 3D PLLA scaffold was printed and BMP-2 was conjugated with alginate-catechol and collagen. PLLA scaffolds were conjugated with different concentrations of BMP-2 and evaluated for bone regeneration in vitro and in vivo using a mouse calvarial model. The BMP-2 release kinetics were analyzed using ELISA. Histological analysis and micro-CT image analysis were performed to evaluate new bone formation. RESULTS: The 3D structure of the PLLA scaffold had a pore size of 400 µm and grid thickness of 187-230 µm. BMP-2 was released in an initial burst, followed by a sustained release for 14 days. Released BMP-2 maintained osteoinductivity in vitro and in vivo. Micro-computed tomography and histological findings demonstrate that the PLLA scaffold conjugated with 2 µg/ml of BMP-2 induced optimal bone regeneration. CONCLUSION: The 3D-printed PLLA scaffold conjugated with BMP-2 enhanced bone regeneration, demonstrating its potential as a novel bone substitute.

[Development and Effects of a Person-Centered Fall Prevention Program for Older Adults with Dementia in Long-Term Care Hospitals: For Older Adults with Dementia and Caregivers in Long-Term Care Hospitals].

PURPOSE: This study examined the effects of a person-centered fall prevention program for older adults with dementia in long-term care hospitals. METHODS: A nonequivalent control group pretest-posttest design was used. The study sample included 42 older adults with dementia (experimental group: 21, control group: 21) and 42 caregivers (experimental group: 21, control group: 21). The program comprised 48 sessions held over 12 weeks and included exercise intervention with resistance and balance, dance walking (45~60 min, three times/week), cognitive and emotional intervention (35~50 min, once per week), and person-centered fall prevention education (10 min, once per week). The program for caregivers consisted of six educational sessions (i.e., fall prevention competency enhancement and person-centered care strategy education, 80 min, once per week) for six weeks. Data were collected before participation and 12 weeks after program completion from February 18 to May 12, 2019. Data analysis was conducted using the chi-square test, t-test, and Mann-Whitney U test with SPSS/WIN 21.0. RESULTS: The experimental group of older adults with dementia showed significant improvement in physical and cognitive functions, and a decrease in depression, and behavioral and psychological symptoms, when compared with the control group. caregivers in the experimental group exhibited significant improvement in fall-related knowledge and person-centered care of older adults with dementia compared to the control group. CONCLUSION: The study findings indicate that this program was effective as a nursing intervention for fall prevention among older adults with dementia in long-term care hospitals.

Fabrication and Characterization of Graphene Oxide-Coated Plate for Efficient Culture of Stem Cells.

BACKGROUND: For stem cell applications in regenerative medicine, it is very important to produce high-quality stem cells in large quantities in a short time period. Recently, many studies have shown big potential of graphene oxide as a biocompatible substance to enhance cell growth. We investigated if graphene oxide-coated culture plate can promote production efficiency of stem cells. METHODS: Three types of graphene oxide were used for this study. They are highly concentrated graphene oxide solution, single-layer graphene oxide solution, and ultra-highly concentrated single-layer graphene oxide solution with different single-layer ratios, and coated on cell culture plates using a spray coating method. Physiochemical and biological properties of graphene oxide-coated surface were analyzed by atomic force microscope (AFM), scanning electron microscope (SEM), cell counting kit, a live/dead assay kit, and confocal imaging. RESULTS: Graphene oxide was evenly coated on cell culture plates with a roughness of 6.4 ~ 38.2 nm, as measured by SEM and AFM. Young's Modulus value was up to 115.1 GPa, confirming that graphene oxide was strongly glued to the surface. The ex vivo stem cell expansion efficiency was enhanced as bone marrow-derived stem cell doubling time on the graphene oxide decreased compared to the control (no graphene oxide coating), from 64 to 58 h, and the growth rate increased up to 145%. We also observed faster attachment and higher affinity of stem cells to the graphene oxide compared to control by confocal microscope. CONCLUSION: This study demonstrated that graphene oxide dramatically enhanced the ex vivo expansion efficiency of stem cells. Spray coating enabled an ultra-thin coating of graphene oxide on cell culture plates. The results supported that utilization of graphene oxide on culture plates can be a promising mean for mass production of stem cells for commercial applications.

Collagen immobilization on ultra-thin nanofiber membrane to promote in vitro endothelial monolayer formation.

The endothelialization on the poly (ε-caprolactone) nanofiber has been limited due to its low hydrophilicity. The aim of this study was to immobilize collagen on an ultra-thin poly (ε-caprolactone) nanofiber membrane without altering the nanofiber structure and maintaining the endothelial cell homeostasis on it. We immobilized collagen on the poly (ε-caprolactone) nanofiber using hydrolysis by NaOH treatment and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo-N-hydroxysulfosuccinimide reaction as a cost-effective and stable approach. NaOH was first applied to render the poly (ε-caprolactone) nanofiber hydrophilic. Subsequently, collagen was immobilized on the surface of the poly (ε-caprolactone) nanofibers using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/sulfo-N-hydroxysulfosuccinimide. Scanning electron microscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and fluorescence microscopy were used to verify stable collagen immobilization on the surface of the poly (ε-caprolactone) nanofibers and the maintenance of the original structure of poly (ε-caprolactone) nanofibers. Furthermore, human endothelial cells were cultured on the collagen-immobilized poly (ε-caprolactone) nanofiber membrane and expressed tight junction proteins with the increase in transendothelial electrical resistance, which demonstrated the maintenance of the endothelial cell homeostasis on the collagen-immobilized-poly (ε-caprolactone) nanofiber membrane. Thus, we expected that this process would be promising for maintaining cell homeostasis on the ultra-thin poly (ε-caprolactone) nanofiber scaffolds.

Preparation and Characterization of Human Adipose Tissue-Derived Extracellular Matrix, Growth Factors, and Stem Cells: A Concise Review.

Background: Human adipose tissue is routinely discarded as medical waste. However, this tissue may have valuable clinical applications since methods have been devised to effectively isolate adipose-derived extracellular matrix (ECM), growth factors (GFs), and stem cells. In this review, we analyze the literature that devised these methods and then suggest an optimal method based on their characterization results. Methods: Methods that we analyze in this article include: extraction of adipose tissue, decellularization, confirmation of decellularization, identification of residual active ingredients (ECM, GFs, and cells), removal of immunogens, and comparing structural/physiological/biochemical characteristics of active ingredients. Results: Human adipose ECMs are composed of collagen type I-VII, laminin, fibronectin, elastin, and glycosaminoglycan (GAG). GFs immobilized in GAG include basic fibroblast growth factor (bFGF), transforming growth factor beta 1(TGF-b1), insulin like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), BMP4 (bone morphogenetic protein 4), nerve growth factor (NGF), hepatocyte growth factor (HGF), and epithermal growth factor (EGF). Stem cells in the stromal-vascular fraction display mesenchymal markers, self-renewal gene expression, and multi-differentiation potential. Conclusion: Depending on the preparation method, the volume, biological activity, and physical properties of ECM, GFs, and adipose tissue-derived cells can vary. Thus, the optimal preparation method is dependent on the intended application of the adipose tissue-derived products.

Full Thickness Skin Expansion ex vivo in a Newly Developed Reactor and Evaluation of Auto-Grafting Efficiency of the Expanded Skin Using Yucatan Pig Model.

BACKGROUND: Skin grafts are required in numerous clinical procedures, such as reconstruction after skin removal and correction of contracture or scarring after severe skin loss caused by burns, accidents, and trauma. The current standard for skin defect replacement procedures is the use of autologous skin grafts. However, donor-site tissue availability remains a major obstacle for the successful replacement of skin defects and often limits this option. The aim of this study is to effectively expand full thickness skin to clinically useful size using an automated skin reactor and evaluate auto grafting efficiency of the expanded skin using Yucatan female pigs. METHODS: We developed an automated bioreactor system with the functions of real-time monitoring and remote-control, optimization of grip, and induction of skin porosity for effective tissue expansion. We evaluated the morphological, ultra-structural, and mechanical properties of the expanded skin before and after expansion using histology, immunohistochemistry, and tensile testing. We further carried out in vivo grafting study using Yucatan pigs to investigate the feasibility of this method in clinical application. RESULTS: The results showed an average expansion rate of 180%. The histological findings indicated that external expansion stimulated cellular activity in the isolated skin and resulted in successful grafting to the transplanted site. Specifically, hyperplasia did not appear at the auto-grafted site, and grafted skin appeared similar to normal skin. Furthermore, mechanical stimuli resulted in an increase in COL1A2 expression in a suitable environment. CONCLUSIONS: These findings provided insight on the potential of this expansion system in promoting dermal extracellular matrix synthesis in vitro. Conclusively, this newly developed smart skin bioreactor enabled effective skin expansion ex vivo and successful grafting in vivo in a pig model.

Investigation of Exhaled Breath Samples from Patients with Alzheimer's Disease Using Gas Chromatography-Mass Spectrometry and an Exhaled Breath Sensor System.

Exhaled breath is a body secretion, and the sampling process of this is simple and cost effective. It can be non-invasively collected for diagnostic procedures. Variations in the chemical composition of exhaled breath resulting from gaseous exchange in the extensive capillary network of the body are proposed to be associated with pathophysiological changes. In light of the foreseeable potential of exhaled breath as a diagnostic specimen, we used gas chromatography and mass spectrometry (GC-MS) to study the chemical compounds present in exhaled breath samples from patients with Alzheimer's disease (AD), Parkinson's disease (PD), and from healthy individuals as a control group. In addition, we also designed and developed a chemical-based exhaled breath sensor system to examine the distribution pattern in the patient and control groups. The results of our study showed that several chemical compounds, such as 1-phenantherol and ethyl 3-cyano-2,3-bis (2,5,-dimethyl-3-thienyl)-acrylate, had a higher percentage area in the AD group than in the PD and control groups. These results may indicate an association of these chemical components in exhaled breath with the progression of disease. In addition, in-house fabricated exhaled breath sensor systems, containing several types of gas sensors, showed significant differences in terms of the normalized response of the sensitivity characteristics between the patient and control groups. A subsequent clustering analysis was able to distinguish between the AD patients, PD patients, and healthy individuals using principal component analysis, Sammon's mapping, and a combination of both methods, in particular when using the exhaled breath sensor array system A consisting of eight sensors. With this in mind, the exhaled breath sensor system could provide alternative option for diagnosis and be applied as a useful, effective tool for the screening and diagnosis of AD in the near future.

Optimization of recombinant human platelet-derived growth factor-BB encapsulated in Poly (lactic-co-glycolic acid) microspheres for applications in wound healing.

Growth factors play multiple and critical roles in wound repair processes. Platelet-derived growth factor (PDGF) is a potent growth factor that is particularly important in the early inflammatory phase of wound healing. In order to extend the half-life of PDGF, polymeric encapsulation is used. In the current study, Poly (lactic-co-glycolic acid) (PLGA) microspheres containing recombinant human (rh) PDGF-BB were prepared to prolong the effectiveness of this growth factor. PLGA microspheres were optimized using a modified w/o/w-double-emulsion/solvent evaporation method by changing the processing conditions of stirring speed and emulsifier (polyvinyl alcohol) concentration. Microspheres prepared using the optimized method released rhPDGF-BB for up to three weeks. An in vitro migration assay showed a significant decrease in the wound area in cells treated with rhPDGF-BB microspheres compared to control cells. These findings demonstrate the potential of rhPDGF-BB encapsulated in microspheres to enhance wound healing.

Rapid expansion and auto-grafting efficiency of porcine full skin expanded by a skin bioreactor ex vivo.

Full skin auto-grafts are required for reconstruction of skin burns and trauma scars. However, currently available clinical approaches such as sheet skin graft, mesh skin grafts, artificial skin graft, and in vivo skin expansion have limitations due to their potential danger for secondary damage and scar formation at the donor site, and discomfort during skin expansion. We developed an advanced bioreactor system and evaluated its function in skin expansion using porcine full skin. The reactor was designed as a pneumatic cylinder type, was programmed to adjust the pressure and the operating time. The system was composed of culture chamber unit, environmental control unit, and monitoring unit. Skins were expanded at 200 kPa pneumatic force and the expanded skins were analyzed by immunohistochemistry and histology. Furthermore we carried out auto-grafting experiment of the expanded skins in vivo using Yucatan pigs and skins were harvested and histologically analyzed after 8 weeks. The results showed that the bioreactor expanded skins to 160% in 4 hours. Histological analysis of the expanded skins revealed that epidermal cells and dermal fibroblasts were viable and remained integrity. The results of auto-grafting experiment indicated that fibrosis and scars were not detected in the grafted skins. This study demonstrates that the newly developed skin bioreactor enabled to obtain large sized full skin rapidly and successful grating.

Genetic variation in the coagulation factor V gene and risk of femoral head osteonecrosis.

Osteonecrosis of the femoral head (ONFH) is characterized by the death of the cellular portion of the femoral head due a reduction or disruption in the blood supply. Certain studies have implicated coagulation disorders, including thrombophilia and hypofibrinolysis, in the pathogenesis of ONFH. The factor V (F5) Leiden mutation has been suggested to be a genetic risk factor for venous thromboembolism and osteonecrosis in Caucasian individuals, however, this association remains controversial in other populations. The present study aimed to identify polymorphisms of the F5 gene and performed a case­control study in a Korean population. The F5 gene was sequenced in 24 unrelated Korean individuals, and 16 polymorphisms were detected. A total of six polymorphisms were genotyped in 423 patients with ONFH and 348 control individuals. Analysis of the association between genotyped single nucleotide polymorphisms and haplotypes and with ONFH was performed. Comparison of the ONFH samples and the control individuals using logistic regression models revealed no statistically significant difference in the frequencies of the F5 polymorphisms and haplotypes. These findings suggested that F5 polymorphisms were not significant in the susceptibility to ONFH in the Korean population.

Improvement of Fat Transplantation: Fat Graft With Adipose-Derived Stem Cells and Oxygen-Generating Microspheres.

BACKGROUND: Autologous fat transplantation has become increasingly popular in plastic surgery. However, high resorption rate limits the utility of this technique. To address this problem, this study examined fat transplantation with oxygen-generating microspheres and adipose-derived stem cells (ASCs) in a rat model. METHODS: The rats were assigned to four groups. Group 1 had fat transplantation only; group 2, fat transplantation with oxygen-generating microspheres; group 3, fat transplantation with ASCs; group 4, fat transplantation with oxygen-generating microspheres and ASCs. RESULTS: At postoperative 2 weeks, compared to the control group, weight and volume increased significantly in groups 3 and 4. The survival distance of fat cells from the margin of transplanted tissue was 247 µm in group 1, 379 µm group 2, 521 µm in group 3, and 669 µm in group 4. All of the experimental groups were significantly increased. Growth factors (fibroblast growth factor- 2 [FGF-2], insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor) analysis was performed through real-time polymerase chain reaction. Compared to the control group, the mean of the periods was statistically significant at FGF-2 in group 3 and FGF-2, insulin-like growth factor-1, and epidermal growth factor in group 4. CONCLUSIONS: In this study, fat transplantation was improved with oxygen-generating microspheres and ASCs. The oxygen-generating microspheres supply oxygen to adipocytes and ASCs where diffusion does not occur, increasing cell survival rate. Surviving ASCs become involved in the metabolic processes for adipocytes and induce angiogenesis. Therefore, fat transplantation result was improved. Excessive oxygen supply, however, reduces angiogenesis and may cause oxygen toxicity. So, further evaluation of oxygen-generating microspheres is necessary for application to tissues to determine appropriate oxygen concentration and a valid oxygen release period.

Pre-clinical efficacy and safety evaluation of human amniotic fluid-derived stem cell injection in a mouse model of urinary incontinence.

PURPOSE: Stem cell-based therapies represent new promises for the treatment of urinary incontinence. This study was performed to assess optimized cell passage number, cell dose, therapeutic efficacy, feasibility, toxicity, and cell trafficking for the first step of the pre-clinical evaluation of human amniotic fluid stem cell (hAFSC) therapy in a urinary incontinence animal model. MATERIALS AND METHODS: The proper cell passage number was analyzed with hAFSCs at passages 4, 6, and 8 at week 2. The cell dose optimization included 1×104, 1×105, and 1×106 cells at week 2. The in vivo cell toxicity was performed with 0.25×106, 0.5×106, and 1×106 cells at weeks 2 and 4. Cell tracking was performed with 1×106 cells at weeks 2 and 4. RESULTS: The selected optimal cell passage number was smaller than 6, and the optimal cell dose was 1×106 for the mouse model. In our pre-clinical study, hAFSC-injected animals showed normal values for several parameters. Moreover, the injected cells were found to be non-toxic and non-tumorigenic. Furthermore, the injected hAFSCs were rarely identified by in vivo cell trafficking in the target organs at week 2. CONCLUSION: This study demonstrates for the first time the pre-clinical efficacy and safety of hAFSC injection in the urinary incontinence animal model and provides a basis for future clinical applications.

Regulation Policy on Cell- and Tissue-Based Therapy Products in Korea.

Extensive investment on research and development of cell therapy products (CTPs) and tissue-engineered products (TEPs) has been made in Korea, and various commercial products are born in market. The Ministry of Food and Drug Safety (MFDS) in Korea regulates CTPs and TEPs as biological products under the authority of the Pharmaceutical Affairs Act. The Korean MFDS approved 16 CTPs and 4 stem CTPs and authorized 135 clinical trials, including 60 sponsor-investigator trials. Currently, the advent of stem cell technology and new biomaterials gives an impetus to develop more innovative CTPs and TEPs to treat intractable and serious diseases. This article deals about the regulatory process for approving CTPs and TEPs in Korea. Regulatory policies of the MFDS for supporting the development of novel products and ensuring the safety of the CTPs and TEPs are reviewed.

Non-invasive screening for Alzheimer's disease by sensing salivary sugar using Drosophila cells expressing gustatory receptor (Gr5a) immobilized on an extended gate ion-sensitive field-effect transistor (EG-ISFET) biosensor.

Body fluids are often used as specimens for medical diagnosis. With the advent of advanced analytical techniques in biotechnology, the diagnostic potential of saliva has been the focus of many studies. We recently reported the presence of excess salivary sugars, in patients with Alzheimer's disease (AD). In the present study, we developed a highly sensitive, cell-based biosensor to detect trehalose levels in patient saliva. The developed biosensor relies on the overexpression of sugar sensitive gustatory receptors (Gr5a) in Drosophila cells to detect the salivary trehalose. The cell-based biosensor was built on the foundation of an improved extended gate ion-sensitive field-effect transistor (EG-ISFET). Using an EG-ISFET, instead of a traditional ion-sensitive field-effect transistor (ISFET), resulted in an increase in the sensitivity and reliability of detection. The biosensor was designed with the gate terminals segregated from the conventional ISFET device. This design allows the construction of an independent reference and sensing region for simultaneous and accurate measurements of samples from controls and patients respectively. To investigate the efficacy of the cell-based biosensor for AD screening, we collected 20 saliva samples from each of the following groups: participants diagnosed with AD, participants diagnosed with Parkinson's disease (PD), and a control group composed of healthy individuals. We then studied the response generated from the interaction of the salivary trehalose of the saliva samples and the Gr5a in the immobilized cells on an EG-ISFET sensor. The cell-based biosensor significantly distinguished salivary sugar, trehalose of the AD group from the PD and control groups. Based on these findings, we propose that salivary trehalose, might be a potential biomarker for AD and could be detected using our cell-based EG-ISFET biosensor. The cell-based EG-ISFET biosensor provides a sensitive and direct approach for salivary sugar detection and may be used in the future as a screening method for AD.

Applicability and safety of in vitro skin expansion using a skin bioreactor: a clinical trial.

BACKGROUND: Tissue expansion is an effective and valuable technique for the reconstruction of large skin lesions and scars. This study aimed to evaluate the applicability and safety of a newly designed skin expanding bioreactor system for maximizing the graft area and minimizing the donor site area. METHODS: A computer-controlled biaxial skin bioreactor system was used to expand skin in two directions while the culture media was changed daily. The aim was to achieve an expansion speed that enabled the skin to reach twice its original area in two weeks or less. Skin expansion and subsequent grafting were performed for 10 patients, and each patient was followed for 6 months postoperatively for clinical evaluation. Scar evaluation was performed through visual assessment and by using photos. RESULTS: The average skin expansion rate was 10.54%±6.25%; take rate, 88.89%±11.39%; and contraction rate, 4.2%±2.28% after 6 months. Evaluation of the donor and recipient sites by medical specialists resulted in an average score of 3.5 (out of a potential maximum of 5) at 3 months, and 3.9 at 6 months. The average score for patient satisfaction of the donor site was 6.2 (out of a potential maximum of 10), and an average score of 5.2 was noted for the recipient site. Histological examination performed before and after the skin expansion revealed an increase in porosity of the dermal layer. CONCLUSIONS: This study confirmed the safety and applicability of the in vitro skin bioreactor, and further studies are needed to develop methods for increasing the skin expansion rate.

Combined injection of three different lineages of early-differentiating human amniotic fluid-derived cells restores urethral sphincter function in urinary incontinence.

OBJECTIVE: To investigate whether a triple combination of early-differentiated cells derived from human amniotic fluid stem cells (hAFSCs) would show synergistic effects in urethral sphincter regeneration. MATERIALS AND METHODS: We early-differentiated hAFSCs into muscle, neuron and endothelial progenitor cells and then injected them into the urethral sphincter region of pudendal neurectomized ICR mice, as single-cell, double-cell or triple-cell combinations. Urodynamic studies and histological, immunohistochemical and molecular analyses were performed. RESULTS: Urodynamic study showed significantly improved leak point pressure in the triple-cell-combination group compared with the single-cell- or double-cell-combination groups. These functional results were confirmed by histological and immunohistochemical analyses, as evidenced by the formation of new striated muscle fibres and neuromuscular junctions at the cell injection site. Molecular analysis showed higher target marker expression in the retrieved urethral tissue of the triple-cell-combination group. The injection of early-differentiated hAFSCs suppressed in vivo host CD8 lymphocyte aggregations and did not form teratoma. The nanoparticle-labelled early-differentiated hAFSCs could be tracked in vivo with optical imaging for up to 14 days after injection. CONCLUSION: Our novel concept of triple-combined early-differentiated cell therapy for the damaged sphincter may provide a viable option for incontinence treatment.

Comparative analysis of whole-blood interferon-γ and flow cytometry assays for detecting post-treatment immune responses in patients with active tuberculosis.

BACKGROUND: Intracellular cytokine flow cytometry (ICCFC) has been explored to detect tuberculosis (TB) infections; however, there are little data regarding its use to examine the dynamic responses of Mycobacterium tuberculosis (MTB)-specific T-cells after anti-tuberculous therapy. The aim of this study was to analyze both dynamic changes in functional MTB antigen-specific T-cell subsets and interferon-gamma (IFN-γ) levels using ICCFC and the QuantiFERON-TB Gold In-Tube (QFT-IT) test, respectively, following anti-tuberculous treatment in patients with active TB. METHODS: Twenty-six patients with active TB were enrolled in the study, and QFT-IT and ICCFC were performed simultaneously both before and after treatment. IFN-γ levels (QFT-IT test) and the numbers of IFN-γ- or tumor necrosis factor-alpha (TNF-α)-expressing T-cells (ICCFC assay) were examined after stimulation with MTB antigen. RESULTS: There was no significant reduction in the mean IFN-γ concentrations measured by the QFT-IT test after anti-tuberculous treatment (P = 0.314). ICCFC analysis showed that the numbers of IFN-γ(+) /CD4(-) T-cells, and CD4(+) T-cells producing TNF-α, either alone or in combination with IFN-γ, were significantly reduced after anti-tuberculous treatment. The IFN-γ(+) /TNF-α(+) /CD4(+) T-cell subset showed the greatest difference between untreated and treated patients with active TB (area under the curve = 0.734, P = 0.004). CONCLUSIONS: Unlike the QFT-IT test, ICCFC provides diverse immunological information about dynamic changes in the number of MTB antigen-specific T-cells following anti-tuberculous therapy. Thus, analysis of MTB antigen-stimulated T-cell responses using ICCFC might have a role to play in monitoring treatment responses in patients with active TB.

MYOD mediates skeletal myogenic differentiation of human amniotic fluid stem cells and regeneration of muscle injury.

INTRODUCTION: Human amniotic fluid stem (hAFS) cells have been shown to differentiate into multiple lineages, including myoblasts. However, molecular mechanisms underlying the myogenic differentiation of hAFS cells and their regenerative potential for muscle injury remain to be elucidated. METHODS: In order to induce myogenic differentiation of hAFS cells, lentiviruses for MYOD were constructed and transduced into hAFS cells. Formation of myotube-like cells was analyzed by immunocytochemistry, and expression of molecular markers for myoblasts was analyzed by reverse transcription polymerase chain reaction and Western blotting. For in vivo muscle regeneration, MYOD transduced hAFS cells were injected into left tibialis anterior (TA) muscles injured with cardiotoxin, and muscle regeneration was analyzed using hematoxylin and eosin, immunocytochemistry and formation of neuro-muscular junction. RESULTS: MYOD expression in hAFS cells successfully induced differentiation into multinucleated myotube-like cells. Consistently, significant expression of myogenic marker genes, such as MYOG, DES, DMD and MYH, was induced by MYOD. Analysis of pre-myogenic factors showed that expression of PAX3, MEOX1 and EYA2 was significantly increased by MYOD. MYOD was phosphorylated and localized in the nucleus. These results suggest that in hAFS cells, MYOD is phosphorylated and localized in the nucleus, thus inducing expression of myogenic factors, resulting in myogenic differentiation of hAFS cells. To test regenerative potential of MYOD-transduced hAFS cells, we transplanted them into injured muscles of immunodeficient BALB/cSlc-nu mice. The results showed a substantial increase in the volume of TA muscle injected with MYOD-hAFS cells. In addition, TA muscle tissue injected with MYOD-hAFS cells has more numbers of neuro-muscular junctions compared to controls, indicating functional restoration of muscle injury by MYOD-hAFS cells. CONCLUSIONS: Collectively, our data suggest that transduction of hAFS cells with MYOD lentiviruses induces skeletal myogenic differentiation in vitro and morphological and functional regeneration of injured muscle in vivo.

RET fusion genes in Korean non-small cell lung cancer.

Recently, rearranged during transfection (RET) fusions have been identified in approximately 1% of non-small cell lung cancer (NSCLC). To know the prevalence of RET fusion genes in Korean NSCLCs, we examined the RET fusion genes in 156 surgically resected NSCLCs using a reverse transcriptase polymerase chain reaction. Two KIF5B-RET fusions and one CCDC6-RET fusion were identified. All three patients were females and never smokers with adenocarcinomas. RET fusion genes were mutually exclusive from EGFR, KRAS mutations and EML4-ALK fusion. RET fusion genes occur 1.9% (3 of 156) of surgically treated NSCLC patients in Koreans.