In response to the expanding landscape of the biotechnology industry and the increasing demand for comprehensive drug development as well as the conduct of preclinical and clinical trials, there is a growing need for employment of diverse animal models, including both small and large animals. The focus of this study was on refining ex vivo culture techniques for bioluminescence imaging following administration of intradermal injections in large animals. To examine the feasibility of our approach, varying concentrations of the rFluc protein were administered to rats and live imaging was employed to validate the corresponding levels of expression. Subsequently, following administration of rFluc to mini-pigs, ex vivo analyses were performed on sample tissues to assess the levels of protein expression across different concentrations. In particular, optimal culturing conditions that facilitated the sustained expression of the protein in samples post-euthanasia were identified. Moreover, by employing small animal imaging devices, we were able to capture clear images of the sample plates, which provided evidence of the successful application of our experimental techniques. The findings from this research represent a significant effort toward refining bioluminescence imaging methods tailored for use with large animal models—an imperative facet of contemporary drug development and biomedical research.

손 위생 제품이 다양화됨과 동시에 각 활용 방법에 따 라 그 효능을 평가하는 여러 시험 방법들이 보고되고 있 다. 하지만 평가 방법에 따라 각 제품의 항균 효능은 다 르게 나타나며, 이로 인해 제품의 실제적인 효능을 확인 하는 데에 어려움이 있을 수 있다. 손 위생 제품의 효능 평가방법 비교에 초점을 둔 연구는 매우 제한적이며, 특 히 돼지피부를 이용한 ex vivo에 대한 연구는 극히 드물 다. 이에 본 연구는 손 위생 제품 중 리브온 소독제와 워 시오프 세정제에 대해 각각의 항균 평가 방법을 종합적으 로 비교했고, ex vivo 시험에 영향을 미칠 수 있는 요인을 파악하여 연구 단계에서 효율적인 ex vivo 시험의 신뢰성 을 향상시키고자 하였다. in vitro 시험으로써 액체 현탁을 기반으로 하는 time-kill 시험을 진행했고, in vivo 시험은 최소 20명의 참여자를 대상으로 진행되었다. ex vivo 시험 은 규격화된 돼지 피부를 이용하여 in vivo 시험과 동일한 방법으로 진행하면서 소독제의 최적 처리량과 세정제 사 용 시 첨가되는 물의 양을 제안했다. 시험에 사용된 손 소 독제는 in vitro 시험에서 모두 5 log 이상의 세균 감소율 을 보인 반면, ex vivo와 in vivo에서는 훨씬 낮은 살균 활 성을 보였으며, 특히 알코올 함량이 낮은 손 소독제에서 는 1 log 미만의 살균 활성을 나타냈다. 반면에 손 세정 제의 in vitro 시험 결과, 대장균에 대해서는 1 log 이하의 낮은 항균력을 보였으나, ex vivo 와 in vivo 시험 결과에 서는 이보다 높은 항균력을 유사하게 나타냈다. 본 연구 에서는 ex vivo 와 in vivo 시험 방법이 리브온과 워시오 프 타입 제품의 두가지 다른 항균 메커니즘을 반영할 수 있음을 확인했다. 이로 인해 최적의 조건으로 설정된 ex vivo 시험은 빠르고 정확한 항균 평가법이 될 수 있음을 제시한다.

피부를 대상으로 한 살균을 목적으로 하는 외용소독제 의 경우 식품 취급자에 오염된 미생물의 사멸 또는 제거 를 목적으로 활용될 수 있으며, 최근 개인위생에 대한 관 심 증가에 따라 제품 소비 증가와 제품 다양화가 두드러 지게 나타나고 있다. 살균 효능은 소독제의 핵심 품질 평 가 요소로서 수행 절차 및 조건에 따라 상이한 결과가 나 타날 수 있기 때문에 시험법의 효율성과 정확성을 높이기 위한 연구가 필요하다. 이에 본 총설논문에서는 주요 제 형별(겔형, 액제형, 와이프형) 시험법 개발 현황을 파악하 고 시험법별 특장점 분석 결과와 최근 관련 연구를 통하 여 제시된 시사점을 기반으로 향후 효능 평가 체계의 발 전 방향을 제시하고자 하였다. 인체 대상 시험법의 경우 시험 유형에 따라 소독제를 시험 대상 피부 표면에 처리 하는 조건이 다양화되어 있어 시험법 간 동등성에 대한 평가를 통해 소독제 제품의 성분이나 특성에 따라 최적의 시험 유형을 파악하고 그에 대응되는 적절한 평가 체계 및 관련 규제의 표준화의 필요성을 시사하였다. 특히 와 이프형 소독제의 경우 처리 방식이 미생물 제거 및 살균 에 직접적으로 영향을 미침에도 불구하고 피부에 노출하 는 손 대상 처리를 위한 사용 패턴의 표준화 사례가 부족 하였다. 한편 [전처리 - 소독제 노출 - 미생물 회수] 등 각 시험 절차별로 결과에 영향을 미치는 주요 결정 요인을 발굴하는 연구의 지속 수행을 통해 기존 시험법을 개선하고 신규 시험법을 개발하고자 하는 노력이 요구된다. 최 근 활발하게 개발되고 있는 ex vivo 시험법은 인체 시험 의 제한적인 연구 재현성과 같은 한계를 극복하면서도 인 간 피부 환경을 구현하기 위한 기술의 적용을 통해 연구 결과의 신뢰도를 확보할 수 있을 것으로 판단된다. 한편 손 피부를 대상으로 한 균총 연구 등 소독제 처리 전후 미생물의 특성과 분포 분석 관련 연구가 최근 다수 보고 되고 있어 이를 활용한 미생물 군집 단위의 소독제 효능 평가 시험법의 확립이 기대된다. 본 연구를 통해 제시된 소독제 효능 시험법의 현황 기반 발전 전략은 보다 효과 적인 개인위생 관리 확립을 통해 손을 통해 교차 오염되 는 미생물에 의한 감염성 질병 발생을 최소화하여 공중보 건 및 식품 안전성 향상에 기여할 수 있다.

Spinach (Spinacia oleracea L.), a green leafy vegetable, is well known as a functional food due to its biological activities. Vascular calcification is associated with several disease conditions including atherosclerosis, diabetes, and chronic kidney disease (CKD), and is known to raise the risk of cardiovascular diseases related morbidity and mortality. However, there are no previous studies that have investigated the effects of fermented spinach exract (FSE) against aortic and its underlying mechanisms. Therefore, this study investigated the effects and action of possible mechanisms of FSE on inorganic phosphate (PI)-induced vascular calcification in ex vivo mouse aortic rings. PI increased vascular calcification through calcium deposition in ex vivo aortic rings. FSE inhibited calcium accumulation and osteogenic key marker, runt-related transcription factor 2 (Runx2), and bone Morphogenetic Protein 2 (BMP-2) protein expression in ex vivo aortic rings. And, FSE inhibited PI-induced extracellular signal-regulated kinase (ERK) and p38 phosphorylation in ex vivo aortic rings. These results show that FSE can prevent vascular calcification which may be a crucial way for the prevention and treatment of vascular disease association with vascular calcification.

Skin protects the body by mediating various immune responses against exogenous substances including bacteria, fungi, and viruses, in addition to its predominant role as a physical barrier. Despite the significant protection offered via various mechanisms, bacterial infection of the skin is one of the most common skin diseases in veterinary medicine. This study demonstrated the structural and immunological changes in the skin during infections with Pseudomonas aeruginosa and Staphylococcus pseudintermedius using skin explants from four healthy beagles. Skin structure was generally well preserved in uninfected controls, but defects in skin structure, including injury of keratinocytes and dermal–epidermal junctional disruption, were identified when skin explants were exposed to P. aeruginosa and S. pseudintermedius. On exposure to P. aeruginosa, marked linear cleft formation was noted along with acantholysis along the basal layer after 24 hours of culture. In addition to the defects in the skin structure, mRNA expression levels of the AMPs cBD103 and S100A8 were decreased, which was confirmed by immunohistochemical staining. Taken together, these results suggest that our ex vivo canine skin model is a research tool for investigating bacterial skin infections in dogs.

Background: Because of the lack of accurate values for applied forces in manual therapy, manual therapists relies on the magnitude of the individual’s perception during applying the force. However, excessive loading maneuvers carry risks for patients. Objective: To establish the relationship between the maximal force applied to swine skin with the specific region, sex, and baseline parameters of the subject. Design: Ex-vivo Study and laboratory Experimental research Methods: 3.5 kg of Korean pork sirloin that is a piece of swine was handled and it was set 3 dimensions; #A; #B; #C. Forty-seven participants who has no experience in physical therapy randomly carried out the experiment, indicated to push each place of the pressure spots with same posture and process under supervision from the instructor who has over 15 years of manual therapy, and we measured the pressure force in each time. Results: The biggest pressure force was recorded in spot #A, and #B was represented after #C. Pressure on #A showed certain statistic relation with height (r=.317, p<.05) and weight (r=.434, p<.01); pressure on #B showed certain relation which has statistical meaning with only height (r=.401, p<.01); pressure on #C emerged to have statistic relationship with height (r=.308, p< .05)and weight (r=.428, p<.01). The age aspect revealed relation with pressure on #A, #B and #C, but that was not statistically significant. Conclusions: It can be inferred that there is the most loss of pressure in the area where cartilage is like an island in the middle.

The hand held voltammetry systems searched diabetic assay using glucose sensor of fluorine nafion doped carbon nanotube electrode (FCNE). An inexpensive graphite carbon pencil was used as an Ag/AgCl reference and Pt counter electrode. Upon combining and using three electrode systems, optimum square wave (SW) stripping results were attained to 1.0-9.0 ug/L with 8 points. Statistic RSD precision was of 6.02 % with n=15 in 0.1 mg/L glucose. After a total of 200 second accumulation times, analytical detection limit of 0.8 ug/L was obtained. This developed technique was applied to urine samples from diabetic patients urine for fluid analysis, it was determined that the sensor can be used with a diagnostics in the ex vivo of live cells and non treated biological fluid.

Transplantation of stem cells, such as mesenchymal stem cells (MSCs), is a promising strategy for treating several types of intractable disorders. Mechanistically, it could not only replace damaged cells by direct contribution, but also establish an anti-inflammatory or immunomodulatory microenvironment. However, the cellular mechanisms underlying molecular and biological properties of stem cells during ex vivo expansion and also after transplantation in pathological environments remain largely elusive. We recently developed the cyanoacrylamide-based coumarin derivatives (Fluorescent real-time thiol tracer; FreSHtracer*) reversibly react with glutathione for monitoring of glutathione levels in living stem cells. These probes revealed that glutathione levels are heterogeneous among subcellular organelles and among individual cells and show dynamic changes and heterogeneity in repopulating stem cells depending on oxidative-stress or culture conditions. Importantly, a subpopulation of stem cells with high-glutathione levels exhibited increased self-renewal and migration activities in vitro and showed improved therapeutic efficiency in treating asthma. Furthermore, employing a novel combination of longitudinal intravital confocal fluorescence imaging and microcystoscopy in living animals, we investigated the distributions and properties of transplanted multipotent MSCs derived from human embryonic stem cells at single-cell resolution in real-time by performing confocal imaging of bladder tissues in a rat model of IC/BPS for up to 6 months post-transplantation. These novel real-time monitoring strategies demonstrate the novel molecular insight for maintaining stem cell functions and also enhance understanding of the in vivo behaviors of the engrafted stem cells, which is crucial to determine the efficacy and safety of stem cell-based therapies. This strategy may facilitate the translation of various stem cell-based approaches into clinical practice.

Mesenchymal stem cells (MSCs) are multipotent stem cells, which can be induced to differentiate into several cells. MSCs are also reported to possess immunomodulatory properties through secretion of inflammatory cytokines and generation of regulatory T (Treg) cells. Treg cells play an important role in allergic disorders, including atopic dermatitis. We examined the immunomodulatory effects of canine adipose tissue derived-MSCs (cAD-MSCs) in 3 groups: Group 1, untreated normal dog; Group 2, dogs with Dermatophagoides farinae ointment-induced atopic dermatitis; and Group 3, dogs with atopic dermatitis. Canine peripheral blood mononuclear cells (PBMCs) were collected from each group and co-cultured with cAD-MSCs. After co-culturing, PBMCs were separated and the expression of Treg cells was examined by flow cytometry. According to the results, the populations of Treg cells were increased in 3 ex vivo experimental groups, co-cultured with cAD-MSCs. These results would be important for the application of MSCs in clinical trials.

For ex-vivo diabetic control, the voltammetric diagnosis of glucose (GU) was conducted with a modified carbon nanotube paste electrode, using handheld analytical circuits. The optimum analytical conditions were attained within the 0.5-4.0 ug/L working range and at the 0.06 ug/L detection limit, which system was interfaced to the feedback circuits and was applied to human urine for diabetic-patient diagnosis. It can be used for ex-vivo flow control analysis, vascular flow detection and other medicinal assays. The equations of the patients’ urine are y=36.65x+12.13 and R²=0.987, those of the healthy person of y= 2.5x+10.9 and R²=0.928 (patients: 118 ug/L; healthy person: 12.34 ug/L).

Regarding therapies for treatment of corneal wounds, ex vivo corneal culture is the most effective for minimizing expensive animal studies. Eighteen porcine enucleated eyes were soaked in 0.2% povidone iodine solution for disinfection prior to cornea excision. Subsequently, corneas were excised from whole eyes and filled with an agar/medium mixture. Corneas were transferred into culture dishes, after which culture medium was added until the limbus was covered. Cultures were then placed onto a plate rocker to mimic blinking action, followed by incubation at 37°C and 5% CO2. Corneas were harvested on Days 0, 3, and 7 after incubation, and optical coherence tomography (OCT) was performed on Day 7. Two eyes from each group were fixed in 2% glutaraldehyde/4% paraformaldehyde for low-vacuum scanning electron microscopy (LV-SEM), and four eyes from each group were fixed in 10% neutral-buffered formalin for histological analysis. OCT results showed that central corneal thickness significantly increased by Day 7 compared to Day 0 (P<0.05). Using LV-SEM, gaps between endothelial cells were detected on Day 7 of ex vivo culture. In the histological evaluation, four to five stratified squamous cell layers, wing cells, and basal cells in the epithelium as well as flat-shaped keratocytes in the stroma were found on Day 0. By Day 7, stratified squamous cells and basal cells had decreased in number, and slightly round-shaped keratocytes were observed; however, the number of keratocytes was similar to that on Day 0. In this short-term ex vivo culture, epithelium and endothelium were sensitive to culture, whereas stroma and keratocytes were well maintained. An additional deswelling method will be needed to obtain more successful results in porcine corneal ex vivo culture.

For ex-vivo diabetic control, the voltammetric diagnosis of glucose (GU) was conducted with a modified carbon nanotube paste electrode, using handheld analytical circuits. The optimum analytical conditions were attained within the 0.5-4.0 ug/L working range and at the 0.06 ug/L detection limit, which system was interfaced to the feedback circuits and was applied to human urine for diabetic-patient diagnosis. It can be used for ex-vivo flow control analysis, vascular flow detection, and other medicinal assays.

Teratocytes (TCs) are the cells derived from the embryonic serosal membrane of some parasitic hymenopteran insects. As a parasitic factor, TCs are multifunctional in host regulation by inducing nutritional, immune, and developmental alterations. However, little is understood about their genetic constituents. This study reveals a comprehensive view of the genes expressed by TCs through a transcriptome analysis based on RNAseq technology. More than 6.29 Gb sequences were used to assemble 34,686 contigs (>200 bp) and annotated into different functional categories. The TC transcriptome profile was clearly distinct from those of hemocytes and the fat body. The TC transcriptome contained components of insulin signaling and biosynthesis of juvenile hormone and 20-hydroxyecdysone. TCs also expressed various groups of digestive enzymes, supporting its nutritional role for the growing parasitoid larvae in parasitism. Furthermore, this transcriptome analysis annotated two kinds of immunosuppressive serine protease inhibitors (serpins) and Rho GTPase-activating proteins (RhoGAPs). To determine the biological functions of these factors, we devised ex vivo RNA interference (RNAi) by conducting knockdown of gene expression in in vitro cultured TCs followed by injection of the treated TCs to test insects. Ex vivo RNAi revealed that some serpins and RhoGAPs expressed in TCs inhibited host cellular immunity. This study reports a transcriptome of the unique TC animal cell, and its immunosuppressive genetic factors using ex vivo RNAi technology.

Platycodon grandiflorum have been used as a traditional remedy and food source. This study was performed to investigate the immunomodulating effects of Platycodon grandiflorum in mouse, using ex vivo experiments. Six to seven-week old mice were fed ad libitum on a chow diet, and water extract of Platycodon grandiflorum was orally administrated at two different concentractions (50 and 500 ㎎/㎏ B.W./day) every other day for four weeks. In ex vivo experiments, the highest proliferation of splenocytes and levels of cytokine (IL-1β, IL-6, TNF-α) production were observed in 500 ㎎/㎏ BW/day supplementation group for all three cytokines stimulated by LPS. In conclusion, this study suggests that Platycodon grandiflorum extracts may enhance the immune function by regulating the splenocytes proliferation and cytokine production capacity by activating macrophages in mice.

Diagnosis with an ex-vivo gold sensor was done using a modified fluorine-doping sensor, and cyclic voltammetry (CV) redox potentials of 0.4 V anodic and -0.2 V cathodic were obtained. Both peak currents were optimized using square-wave (SW) stripping voltammetry, and an analytical working range of 10-80 ug/L SW was attained. The precision of the 10-mg/L Au was 0.765 (n=8) RSD under the optimum conditions, and the analytical detection limit approached 0.006 ug/L (S/N=3) with only a 60 sec accumulation time. The developed method was used to examine the mouse droppings for medicinal diagnosis.

The T-cell receptor (TCR) engages with an antigen and initiates a signaling cascade that leads to the activation of transcription factors. Roquin, a protein encoded by the RC- 3H1 gene and characterized as an immune regulator, was recently identified as a novel RING-type ubiquitin ligase family member, but the mechanisms by which Roquin regulates T-cell responses are unclear. We used the EL-4 murine lymphoma cell line to elucidate the role of Roquin in vitro. Roquin-overexpressing EL-4 cells became hyper-responsive after anti-CD3/CD28 stimulation in vitro and were a major source of the cytokines IL-2 and TNF-α. Upon activation, these cells showed particularly enhanced production of IL-2 and TNF-α. To clarify the important role played by Roquin in T-cell responses ex vivo, we generated T-cell-specific Roquin transgenic (Tg) mice. Roquin-Tg CD4+ T-cells showed enhanced production of IL-2 and TNF-α in response to TCR stimulation with anti-CD28 co-stimulation. Further studies are necessary to investigate the role of Roquin in the regulation of primary T-cell activation, survival, and differentiation.

In vivo nicotine is associated with Alzheimer's, Parkinson's and lung cancer. Diagnostic assays of these diseases depend on very low analytical detection limits. In this study, a sensitive analytical method was examined using a voltammetric graphite pencil electrode (GPE) and a modified carbon nanotube paste electrode (CNE). The optimum analytical conditions for both electrodes were compared using square wave anodic stripping voltammetry (SW) and cyclic voltammetry (CV) obtaining 400 sec accumulation time and oxidation peak. Under optimum parameters, the stripping working range of GPE was 5.0-40.0μg/L, CNE: 0.1-0.8 and 5-50μg/L. Quantification limits were 5.0μg/L for GPE and 0.1μg/L for CNE, while detection limits were 0.6μg/L for GPE and 0.07μg/L for CNE. A standard deviation of 10.0μg/L was observed for 0.064 GPE and 0.095 CNE (n = 12) using 400 sec accumulation time. The results obtained can be applied to non.treated urine and ex vivo biological diagnostics.

Several types of white blood cells, such as T cells, B cells, and macrophages, are involved in the immune response. In particular, the processes of T-cell activation play a crucial role in an adaptive immune response, whereby the T-cell receptor (TCR) engages with an antigen and signals a cascade that leads to the activation of transcription factors (AP-1, NF-κB, and NFAT) that are critically involved in cytokine production. Roquin, encoded by the RC3H1 gene and characterized as an immune regulator, was recently identified as a novel RING-type ubiquitin ligase family member, but the mechanisms by which Roquin proteins regulate T-cell responses are unclear. To elucidate the role of Roquin in vitro, murine lymphoma EL-4 cells were used. Roquin overexpressing Tcells became hyper-responsive upon anti-CD3/CD28 stimulation in vitro and were a major source of cytokines such as IL-2, TNF-α, IL-6, and IL-10. Upon activation, these cells showed preferentially enhanced production of IL-2 and TNF-α, but not IFN-γ. To clarify the important role of Roquin in the T-cell response ex vivo, we generated T-cell-specific Roquin-transgenic (Tg) mice having a higher expression of Roquin in T cells as compared to wild-type mice. Using Roquin-Tg mice, we studied whether immune responses are affected ex vivo. Roquin-Tg CD4+ T cells showed enhanced production of IL-2 or TNF-α to TCR stimulation with anti-CD28 costimulation via up-regulation of CD28. T-cell proliferation also increased in Roquin-Tg CD4+ T cells after anti-CD3/CD28 treatment. Further studies on the role of Roquin in the regulation of primary T-cell activation, survival, and differentiation may be anticipated.