Nerve growth factor promote VCAM-1-dependent monocyte adhesion and M2 polarization in osteosarcoma microenvironment: Implications for larotrectinib therapy.

Osteosarcoma is the most prevalent form of primary malignant bone tumor, primarily affecting children and adolescents. The nerve growth factors (NGF) referred to as neurotrophins have been associated with cancer-induced bone pain; however, the role of NGF in osteosarcoma has yet to be elucidated. In osteosarcoma samples from the Genomic Data Commons data portal, we detected higher levels of NGF and M2 macrophage markers, but not M1 macrophage markers. In cellular experiments, NGF-stimulated osteosarcoma conditional medium was shown to facilitate macrophage polarization from the M0 to the M2 phenotype. NGF also enhanced VCAM-1-dependent monocyte adhesion within the osteosarcoma microenvironment by down-regulating miR-513c-5p levels through the FAK and c-Src cascades. In in vivo xenograft models, the overexpression of NGF was shown to enhance tumor growth, while the oral administration of the TrK inhibitor larotrectinib markedly antagonized NGF-promoted M2 macrophage expression and tumor progression. These results suggest that larotrectinib could potentially be used as a therapeutic agent aimed at mitigating NGF-mediated osteosarcoma progression.

NAMPT enhances LOX expression and promotes metastasis in human chondrosarcoma cells by inhibiting miR-26b-5p synthesis.

Chondrosarcoma is a malignant bone tumor that emerges from abnormalities in cartilaginous tissue and is related with lung metastases. Nicotinamide phosphoribosyltransferase (NAMPT) is an adipocytokine reported to enhance tumor metastasis. Our results from clinical samples and the Gene Expression Omnibus data set reveal that NAMPT levels are markedly higher in chondrosarcoma patients than in normal individuals. NAMPT stimulation significantly increased lysyl oxidase (LOX) production in chondrosarcoma cells. Additionally, NAMPT increased LOX-dependent cell migration and invasion in chondrosarcoma by suppressing miR-26b-5p generation through the c-Src and Akt signaling pathways. Overexpression of NAMPT promoted chondrosarcoma metastasis to the lung in vivo. Furthermore, knockdown of LOX counteracted NAMPT-facilitated metastasis. Thus, the NAMPT/LOX axis presents a novel target for treating the metastasis of chondrosarcoma.

Targeting nerve growth factor-mediated osteosarcoma metastasis: mechanistic insights and therapeutic opportunities using larotrectinib.

Osteosarcoma (OS) therapy presents numerous challenges, due largely to a low survival rate following metastasis onset. Nerve growth factor (NGF) has been implicated in the metastasis and progression of various cancers; however, the mechanism by which NGF promotes metastasis in osteosarcoma has yet to be elucidated. This study investigated the influence of NGF on the migration and metastasis of osteosarcoma patients (88 cases) as well as the underlying molecular mechanisms, based on RNA-sequencing and gene expression data from a public database (TARGET-OS). In osteosarcoma patients, the expression of NGF was significantly higher than that of other growth factors. This observation was confirmed in bone tissue arrays from 91 osteosarcoma patients, in which the expression levels of NGF and matrix metallopeptidase-2 (MMP-2) protein were significantly higher than in normal bone, and strongly correlated with tumor stage. In summary, NGF is positively correlated with MMP-2 in human osteosarcoma tissue and NGF promotes osteosarcoma cell metastasis by upregulating MMP-2 expression. In cellular experiments using human osteosarcoma cells (143B and MG63), NGF upregulated MMP-2 expression and promoted wound healing, cell migration, and cell invasion. Pre-treatment with MEK and ERK inhibitors or siRNA attenuated the effects of NGF on cell migration and invasion. Stimulation with NGF was shown to promote phosphorylation along the MEK/ERK signaling pathway and decrease the expression of microRNA-92a-1-5p (miR-92a-1-5p). In in vivo experiments involving an orthotopic mouse model, the overexpression of NGF enhanced the effects of NGF on lung metastasis. Note that larotrectinib (a tropomyosin kinase receptor) strongly inhibited the effect of NGF on lung metastasis. In conclusion, it appears that NGF promotes MMP-2-dependent cell migration by inhibiting the effects of miR-92a-1-5p via the MEK/ERK signaling cascade. Larotrectinib emerged as a potential drug for the treatment of NGF-mediated metastasis in osteosarcoma.

Acetaminophen overdose-induced acute liver injury can be alleviated by static magnetic field.

Acetaminophen (APAP), the most frequently used mild analgesic and antipyretic drug worldwide, is implicated in causing 46% of all acute liver failures in the USA and between 40% and 70% in Europe. The predominant pharmacological intervention approved for mitigating such overdose is the antioxidant N-acetylcysteine (NAC); however, its efficacy is limited in cases of advanced liver injury or when administered at a late stage. In the current study, we discovered that treatment with a moderate intensity static magnetic field (SMF) notably reduced the mortality rate in mice subjected to high-dose APAP from 40% to 0%, proving effective at both the initial liver injury stage and the subsequent recovery stage. During the early phase of liver injury, SMF markedly reduced APAP-induced oxidative stress, free radicals, and liver damage, resulting in a reduction in multiple oxidative stress markers and an increase in the antioxidant glutathione (GSH). During the later stage of liver recovery, application of vertically downward SMF increased DNA synthesis and hepatocyte proliferation. Moreover, the combination of NAC and SMF significantly mitigated liver damage induced by high-dose APAP and increased liver recovery, even 24 h post overdose, when the effectiveness of NAC alone substantially declines. Overall, this study provides a non-invasive non-pharmaceutical tool that offers dual benefits in the injury and repair stages following APAP overdose. Of note, this tool can work as an alternative to or in combination with NAC to prevent or minimize liver damage induced by APAP, and potentially other toxic overdoses.

NGF facilitates ICAM-1-dependent monocyte adhesion and M1 macrophage polarization in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder in which monocytes adhering to synovial tissue differentiate into the pro-inflammatory M1 macrophage phenotype. Nerve growth factors (NGF) referred to as neurotrophins have been associated with inflammatory events; however, researchers have yet to elucidate the role of NGF in RA. Our examination of clinical tissue samples and analysis of data sourced from the Gene Expression Omnibus dataset unveiled elevated expression levels of M1 macrophage markers in human RA synovial tissue samples compared to normal tissue, with no such distinction observed for M2 markers. Furthermore, immunofluorescence data depicted increased expression levels of NGF and M1 macrophages in RA mice in contrast to normal mice. It appears that NGF stimulation facilitates macrophage polarization from the M0 to the M1 phenotype. It also appears that NGF promotes ICAM-1 production in human RA synovial fibroblasts, which enhances monocyte adhesion through the TrkA, MEK/ERK, and AP-1 signaling cascades. Our findings indicate NGF/TrkA axis as a novel target for the treatment of RA.

Acupoint Thread Embedding Combined With Wenshen Bugu Decoction for the Treatment of Aromatase Inhibitor-Associated Musculoskeletal Symptom Among Postmenopausal Breast Cancer Patients: Study Protocol of a Randomized Controlled Trial.

BACKGROUND: Aromatase inhibitors (AIs) are recommended as the preferred therapy for postmenopausal women with hormone receptor-positive (HR+) breast cancer. As a result, aromatase inhibitor-associated musculoskeletal symptom (AIMSS) have become a major problem leading to therapy discontinuation and decreased quality of life in patients receiving adjuvant AIs treatment. Multiple therapies have been attempted, but have yielded limited clinical results. This study will be performed to determine whether acupoint thread embedding (ATE) combined with Wenshen Bugu Decoction can effectively treat AIMSS, so as to improve the AIs medication compliance of postmenopausal breast cancer patients. METHODS: This study will utilize a randomized, 2 parallel groups controlled trial design. A total of 128 eligible postmenopausal breast cancer women with AIMSS will be randomized to receive a 12-week treatment with Wenshen Bugu Decoction alone (control group) or in combination with ATE (treatment group) in a 1:1 ratio. The primary outcome will be the 12 week Brief Pain Inventory Worst Pain (BPI-WP) score. The secondary outcome measures will include response rate, Brief Pain Inventory-Short Form (BFI-SF), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Functional Assessment of Cancer Therapy-Endocrine Symptom (FACT-ES), Functional Assessment of Cancer Therapy-Breast (FACT-B), bone marrow density (BMD), blood markers of bone metabolite, Morisky medication adherence scale-8 (MMAS-8), credibility and expectancy, and survival outcomes. DISCUSSION: This trial may provide clinical evidence that ATE combined with Wenshen Bugu Decoction can be beneficial for treating AIMSS among postmenopausal breast cancer survivors. Our findings will be helpful to enhance the quality of life and reduce the occurrence of AIs withdrawal.

A modified surgical technique of fibrin glue-assisted double bipedicle conjunctival flaps for patients with ocular surface diseases.

This study aimed to describe and investigate the surgical outcome and complications of fibrin glue-assisted double bipedicle conjunctival flaps (CFs) (FADCOF), an alternative surgical technique that restores a stable ocular surface in patients with painful blinding ocular surface disease combined with a shortage of bulbar conjunctiva. Six eyes of six patients with painful blinding ocular surface disease were enrolled in this study. All patients had inadequate superior or inferior conjunctiva tissue to cover the whole corneal surface owing to previous surgeries or ocular surface diseases. These patients received FADCOF between 2009 and 2019. The main outcome included surgical success rate, visual analog scale (VAS) pain score, ocular inflammation score, and postoperative complications. Surgical success was defined as resolution of initial ocular complaints and restoration of a stable ocular surface with no flap melting, retraction, or dehiscence resulting in re-exposure of the corneal surface. All of the six eyes (100%) achieved surgical success. All patients reported significant improvement in subjective symptoms and complete resolution of ocular pain after the surgery (VAS pain score: 6.5 ± 0.5 preoperatively to 0.0 ± 0.0 at 1 month). Ocular inflammation score decreased significantly from a presurgical value of 1.83 ± 0.69 to 0.33 ± 0.47 1 month after the surgery. No postoperative complication was found during the long-term follow-up (range: 12-82 months). FADCOF is a reliable alternative for patients with painful blinding ocular surface diseases unsuitable for single total CF surgery. This surgical technique yields fast ocular surface stabilization, satisfactory recovery, and low complication rates.

Extending the utility of anterior corneal buttons through refrigeration and glycerol cryopreservation: utility rate and outcome analysis.

BACKGROUND/AIMS: To evaluate the utility rate, indication, outcome, and cost of refrigeration and glycerol cryopreservation for storing anterior corneal buttons during endothelial keratoplasty for subsequent use in tectonic lamellar patch grafting. METHOD: Anterior corneal buttons collected after precutting or prestripping during endothelial keratoplasty from January 2014 to December 2019 were preserved using the following protocol: (1) refrigeration for up to 4 weeks at 4°C in Optisol-GS and (2) glycerol cryopreservation for up to 2 years. The utility rate, outcome and cost of these cryopreserved anterior corneal buttons were retrospectively examined. RESULTS: During the 6-year study period, 26 anterior corneal buttons were refrigerated and 49 were cryopreserved for extended use. The utility rates for the refrigerated and cryopreserved anterior corneal buttons were 69.2% and 73.5%, respectively. Their average preservation periods were 0.53±0.05 and 12.76±0.94 months, respectively. Noninfection-related perforation was the leading indication for using the extendedly preserved anterior corneal buttons. The average postoperative follow-up periods were 10.03±2.91 and 14.35±2.17 months for refrigerated and cryopreserved anterior corneal buttons. Secondary keratoplasty was required by 7 of 18 (38.9%) and 6 of 36 (16.7%) patients receiving refrigerated and cryopreserved anterior corneal buttons, respectively. None of our patients developed graft infection from donor tissues. CONCLUSION: Cryopreservation can safely extend the utility of anterior corneal buttons. This method not only reduced the wastage of the limited donor tissue but also was cost-effective.

The ocular graft-versus-host disease: the path from current knowledge to future managements.

BACKGROUND: Graft-versus-host disease (GVHD) is one of the major complications of allogenic haematopoietic stem cell transplantation (HSCT). The manifestation of ocular GVHD (oGVHD) is variable and may involve cornea, lacrimal gland, conjunctiva, eyelid, and/or nasolacrimal duct. We reviewed and summarized the current managements of oGVHD with specific focus on the emerging therapeutic advances. METHODS: PubMed, Web of Science, and Google Scholar were searched for relevant literatures published within 20 years. Keywords used included "Graft-Versus-Host Disease", "GVHD", "ocular", "ocular surface", "ocular GVHD", "oGVHD", "dry eye", "keratitis", etc. RESULTS: Current managements of oGVHD can be classified into topical immunosuppressants, local tear-preservatory treatments, local non-pharmacological/surgical interventions, and systemic treatments. Additionally, some innovative therapies with promising treatment effects have been proposed, including topical target therapies, epitheliotrophic and neurotrophic treatments, recombinant DNase eye drops, mesenchymal stromal cell injection, and more. CONCLUSIONS: Clinical managements of oGVHD are administered in a symptom-based, stepwise manner. The advances in innovative therapies may help improve clinical outcomes, and it is essential that physicians stay updated with these novel treatment options.

Toxic keratopathy related to antiseptics in nonocular surgery.

Antiseptics, especially those containing ethanol, are toxic to the ocular surface. Here, we report a 5-year-old girl with antiseptic-related eye injury following an uneventful bilateral tonsillectomy under general anesthesia. Before surgery, her eyes were protected and disinfection of perioral skin with ethanol-containing chlorhexidine followed. Whitening of the lower half of her right ocular surface was found after the surgery, and this indicated severe chemical burn. Prompt irrigation with normal saline, instillation of topical medication, and application of amniotic membrane containing device were performed, which led to a satisfactory result. Toxic eye injury could happen in head and neck surgeries under general anesthesia. Causes of ocular injury include improper eye protection, head positions leading to accumulation of excessive antiseptics, and improper usage of ethanol-containing antiseptics for skin preparation. The use of ethanol-free antiseptic solutions in the peri-ocular region and proper protection of eyes may reduce the risk of severe ocular surface injury in nonocular surgeries.

Spectral-domain optical coherence tomography for evaluating palisades of Vogt in ocular surface disorders with limbal involvement.

Spectral-domain optical coherence tomography (SD-OCT) has been used to observe the morphology of the palisades of Vogt (POV) with satisfactory resolutions. In this study, we used SD-OCT to examine the microstructure of the POV in ocular surface disorders with limbal involvement. We detect subclinical limbal pathologies based on five parameters, including (1) decreased epithelial thickness, (2) loss of the sharp stromal tip, (3) loss of the smooth epithelial-stromal interface, (4) dilated stromal vessels, and (5) decreased POV density. Eighteen eyes of 10 patients with advancing wavelike epitheliopathy (AWE) and 15 eyes of 9 patients with phlyctenular keratitis/ocular rosacea were recruited. SD-OCT could detect abnormal changes in the POV in 100% of the lesion sites. In presumed-healthy areas of the diseased eyes diagnosed by slit-lamp biomicroscopy, SD-OCT detected abnormal changes in the POV in 100% of the eyes in both groups. In patients with unilateral disease, abnormal changes in the POV were detected by SD-OCT in 50% and 100% of presumed-healthy eyes diagnosed by slit-lamp biomicroscopy in the AWE group and phlyctenular keratitis/ocular rosacea group, respectively. SD-OCT is powerful in detecting POV changes in ocular surface disorders and can provide useful information that cannot be provided by slit-lamp biomicroscopy.

Submicron spatial resolution optical coherence tomography for visualising the 3D structures of cells cultivated in complex culture systems.

Three-dimensional (3D) configuration of in vitro cultivated cells has been recognised as a valuable tool in developing stem cell and cancer cell therapy. However, currently available imaging approaches for live cells have drawbacks, including unsatisfactory resolution, lack of cross-sectional and 3D images, and poor penetration of multi-layered cell products, especially when cells are cultivated on semitransparent carriers. Herein, we report a prototype of a full-field optical coherence tomography (FF-OCT) system with isotropic submicron spatial resolution in en face and cross-sectional views that provides a label-free, non-invasive platform with high-resolution 3D imaging. We validated the imaging power of this prototype by examining (1) cultivated neuron cells (N2A cell line); (2) multilayered, cultivated limbal epithelial sheets (mCLESs); (3) neuron cells (N2A cell line) and mCLESs cultivated on a semitransparent amniotic membrane (stAM); and (4) directly adherent colonies of neuron-like cells (DACNs) covered by limbal epithelial cell sheets. Our FF-OCT exhibited a penetrance of up to 150 µm in a multilayered cell sheet and displayed the morphological differences of neurons and epithelial cells in complex coculture systems. This FF-OCT is expected to facilitate the visualisation of cultivated cell products in vitro and has a high potential for cell therapy and translational medicine research.

The effect of human platelet lysate on corneal nerve regeneration.

AIM: This study aimed to test whether human platelet lysate (HPL) has neurotrophic ability for corneal nerve regeneration. METHODS: We measured the neurotrophic factors in human peripheral serum (HPS) and two commercially available HPLs, UltraGRO and PLTMax. In vitro, we compared the growth rates, neuronal differentiation and immunostaining of neuron markers in mouse neuroblastoma cell line (Neuro-2a) and primary culture of mouse trigeminal ganglion cells that were cultivated in different concentrations of fetal bovine serum, HPS and HPL. In vivo, we created corneal wounds on Sprague Dawley rats with a rotating burr and evaluated the effects of topical HPL on wound healing and corneal nerve regeneration by in vivo confocal microscopy and corneal aesthesiometry. RESULTS: HPLs had significantly higher concentrations of various neurotrophic factors compared with HPS (p<0.05). In Neuro-2a cells, 3% HPL was better at promoting neuronal growth and differentiation compared with HPS at the same concentration. HPL was also found to have superior neurotrophic effects compared with HPS in primary cultures of mouse trigeminal ganglion cells. In vivo, HPL-treated eyes had better corneal epithelial wound healing rate, nerve regeneration length and corneal touch threshold compared with eyes treated with artificial tears (p<0.05). CONCLUSION: HPL has significantly higher concentrations of neurotrophic factors compared with HPS. It showed not only in vitro but also in vivo corneal neurotrophic abilities. Our results suggest that HPL may have a potential role in the treatment of diseases related to corneal nerve damage or degeneration.

Epithelial basement membrane dystrophy after femtosecond laser-assisted LASIK successfully treated with in vivo confocal microscopy-assisted photorefractive keratectomy.

Photorefractive keratectomy (PRK) was performed to treat corneal epithelial basement membrane dystrophy (EBMD) found after femtosecond laser-assisted laser in situ keratomileusis (FS-LASIK) in a 29-year-old man with previous silent cornea. The patient was diagnosed with post-FS-LASIK EBMD by slitlamp examination and in vivo confocal microscopy (IVCM). Initial treatment with topical lubricants and alcohol soaking was unsuccessful, and the patient continued to have blurred vision and discomfort. The patient underwent a PRK procedure, and the symptoms resolved dramatically; residual refractive errors were also corrected. IVCM revealed a characteristic presentation of EBMD before PRK and normal corneal epithelium after treatment. Patients with asymptomatic EBMD might suffer from recurrent corneal erosions after FS-LASIK. Surface ablation including PRK might lead to successful treatment outcomes, and IVCM might provide helpful information before and after treatment.

Use of white light in vivo confocal microscopy for the detection of spatial changes in the corneal nerves in cases of early-stage Acanthamoeba keratitis with radial keratoneuritis.

Purpose: Radial keratoneuritis (RK) is a common feature of Acanthamoeba keratitis (AK). In vivo confocal microscopy (IVCM) is noninvasive and provides real-time images for the diagnosis of corneal diseases by allowing the visualization of corneal structures and morphologies of living organisms at the cellular level. Images of AK with RK obtained using commercial white light IVCM devices have not been frequently evaluated. In the present study, a white light IVCM device was used to evaluate the corneal findings and describe spatial changes in the corneal nerves at different depths in cases of early-stage AK with RK. Methods: In this retrospective, observational study, white light IVCM images focused on RK were evaluated for Acanthamoeba cysts/trophozoites, corneal deposits, and altered corneal nerves, with special emphasis on three-dimensional spatial changes in the corneal nerves at different depths. Results: Seventeen eyes of 17 patients exhibiting early-stage AK with RK were included in the study. Acanthamoeba cysts/trophozoites were observed in the corneal epithelium of 13 eyes and stroma of 7 eyes. Alterations in the corneal nerve morphology and density were observed from the basal epithelial layer to the stromal layer in 12 eyes. Acanthamoeba trophozoites were attached to the corneal stromal nerves in five eyes. Conclusion: These findings suggest that white light IVCM can identify consistent corneal findings, particularly spatial changes in the corneal nerves, in cases of early-stage AK with RK.

Oligosaccharides from Palm Kernel Cake Enhances Adherence Inhibition and Intracellular Clearance of Salmonella enterica Serovar Enteritidis In Vitro.

Salmonella enterica serovar (ser.) Enteritidis (S. Enteritidis) is a foodborne pathogen often associated with contaminated poultry products. This study evaluated the anti-adherence and intracellular clearance capability of oligosaccharides extracted from palm kernel cake (PKC), a by-product of the palm oil industry, and compared its efficacy with commercial prebiotics- fructooligosaccharide (FOS) and mannanoligosaccharide (MOS)-against S. Enteritidis in vitro. Based on the degree of polymerization (DP), PKC oligosaccharides were further divided into 'Small' (DP ≤ 6) and 'Big' (DP > 6) fractions. Results showed that the Small and Big PKC fractions were able to reduce (p < 0.05) S. Enteritidis adherence to Cancer coli-2 (Caco-2) cells at 0.1 mg/ mL while MOS and FOS showed significant reduction at 1.0 mg/mL and 10.0 mg/mL, respectively. In terms of S. Enteritidis clearance, oligosaccharide-treated macrophages showed better S. Enteritidis clearance over time at 50 µg/mL for Small, Big and MOS, while FOS required a concentration of 500 µg/mL for a similar effect. This data highlights that oligosaccharides from PKC, particularly those of lower DP, were more effective than MOS and FOS at reducing S. Enteritidis adherence and enhancing S. Enteritidis clearance in a cell culture model.

Tear cytokine profiling in patients with superior limbic keratoconjunctivitis who underwent medical treatment or in conjunction with surgical management.

PURPOSE: To determine the correlation between superior limbic keratoconjunctivitis (SLK) and selected tear cytokines and to evaluate the efficacy of these cytokines in monitoring the response of patients with SLK to either medical treatment alone or in combination with conjunctival resection. DESIGN: A cohort study. METHODS: Twenty-five eyes of 13 patients with SLK were assigned to either medically responsive or surgical treatment groups depending on their responses 1 month after initial medical treatment. Treatment efficacy was assessed by improvements in clinical grading and decreases in the levels of tear cytokines. RESULTS: Fourteen eyes were improved by medical treatment alone, whereas satisfactory outcomes were achieved for the remaining 11 eyes after surgical management. The overall grading prior to medical treatment was lower in medically responsive group compared with surgical treatment group (p=0.0139). Among the examined tear cytokines, monocyte chemoattractant protein (MCP)-1 was positively associated with the severity of clinical grading (p=0.0251). While both treatments significantly decreased the levels of MCP-1 and interleukin-6, surgical treatment also decreased the levels of interferon-γ and tumour necrosis factor-α. Notably, overall cytokine levels after surgical treatment were lower than those after medical treatment alone. CONCLUSIONS: Since an association of tear MCP-1 level with the clinical grading and treatment response of SLK was observed in this study, tear MCP-1 may be a potential indicator of SLK disease severity. According to the degree to which the tear cytokine levels were decreased, surgical treatment appears to be an effective treatment modality for patients with SLK who are refractory to medical treatment alone.

Clinical features and outcomes of Acanthamoeba keratitis in a tertiary hospital over 20- year period.

PURPOSE: To analyze the outcomes of Acanthamoeba keratitis (AK) in terms of different clinical presentations in a tertiary hospital in Taiwan over a 20- year period. METHODS: This is a retrospective case series. Patients with AK diagnosed at the National Taiwan University Hospital between January 1996 and December 2015 were identified. A diagnosis of AK was made on the basis of positive Acanthamoeba smear/cultures or pathological identification of Acanthamoeba cysts on keratoplasty specimens. Patient demographics, clinical presentations, treatment courses, and final visual outcomes were collected and analyzed. Visual acuity, postoperative complications and graft survivals were measured as outcomes. RESULTS: Of the 62 patients with AK in our study, 64.5% were medically treated while 35.5% underwent surgical treatment. In those with ring infiltrate, 52.4% patients could be successfully treated with medications. In eyes receiving penetrating keratoplasty, postoperative complications were more common in therapeutic penetrating keratoplasty (TPK) than those in optical penetrating keratoplasty (OPK) group (82.4% versus 40%, p = 0.04). The graft size was larger in TPK than that in OPK group (8.56 versus 7.83 mm, p = 0.002). Furthermore, post-operative glaucoma, which was the major complication, was found to be associated with larger graft size (p = 0.02) and dilated pupil/iris atrophy (p = 0.01). CONCLUSION: Even in advanced cases with ring infiltrate, eradication of infection with anti-amoebic drugs is possible. In those requiring keratoplasty, the surgical timing should be made meticulously considering graft size and signs of dilated pupil/iris atrophy in terms of post-operative glaucoma.

The Neural Differentiation Potential of Limbal Stem Cells: A Model for Studying the Multipotency of Limbal Stem Cells.

PURPOSE: To investigate the multipotency, especially the neural differentiation potential, of limbal stem cells (LSCs) using a coculture system and to provide a relevant literature review. METHODS: Limbal tissue was harvested from adult New Zealand white rabbits and treated with collagenase A. Small pieces of the resulting limbal epithelial sheets were cocultured with a neuroblastoma cell line (Neuro-2A) in transwells. Morphological observation of the growing epithelial sheets was accomplished by microscopy, and marker expression was detected by immunocytochemistry for ßIII-tubulin and microtubule-associated protein 2. A literature review of associated studies was performed. RESULTS: In the coculture group, directly adherent colonies of neuron-like (DACN) cells were observed among the growing limbal epithelial sheets from day 3. The DACN cells exhibited neuron-like morphology. The control group comprising limbal cell sheets cultured alone showed a very small number of DACN cells at the end of the culture period (day 14). Immunocytochemical staining revealed that the DACN cells were positive for ßIII-tubulin and microtubule-associated protein 2, confirming the neuronal phenotype of the neuron-like cells. By contrast, the DACN cells in the control group produced negative results. In previous reports, LSCs and niches exhibited neural potential, but most differentiated neural cells were observed as floating spheres, in contrast to the DACN cells observed in the present study. CONCLUSIONS: We developed a coculture system of LSCs and Neuro-2A neuroblastoma cells and obtained DACN cells with neural differentiation potential. Our findings confirm the neural potential of LSCs, consistent with previous reports, but in a form other than floating spheres.

Anti-Diabetic Effect of a Shihunine-Rich Extract of Dendrobium loddigesii on 3T3-L1 Cells and db/db Mice by Up-Regulating AMPK-GLUT4-PPARα.

The stems of Dendrobium loddigesii, a Chinese herb, are often used to treat diabetes and its polar extract is rich in shihunine, a water-soluble Orchidaceae alkaloid, but little is known about the anti-diabetes effects and mechanism of shihunine. This study investigated the anti-diabetic effect of a shihunine-rich extract of D. loddigesii (DLS) based on 3T3-L1 cells and db/db mice. The underlying mechanisms were primarily explored using Western blot analysis and immunohistochemical staining. The 3T3-L1 cell experiments showed that DLS can reduce the intracellular accumulation of oil droplets as well as triglycerides (p < 0.001) and promote the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2deoxyglucose (2-NBDG) uptake of 3T3-L1 cells (p < 0.001). The animal experiments confirmed that after 8 weeks of DLS treatment, the body weight, fasting blood sugar, and serum lipid levels of mice were significantly lowered, and the oral glucose tolerance test and serum insulin level were significantly improved compared to the no-treatment diabetes mellitus group. Further histomorphology observation led to the conclusion that the quantities of islet cells were significantly increased and the increase in adipose cell size was significantly suppressed. The immunohistochemical test of pancreatic tissue revealed that DLS inhibited the expression of cleaved cysteine aspartic acid-specific protease 3 (cleaved caspase-3). Western blot experiments showed that DLS had agonistic effects on adenosine monophosphate (AMP)-activated protein kinase phosphorylation (p-AMPK) and increased the expression levels of peroxisome proliferator-activated receptor α (PPARα) and glucose transporter 4 (GLUT4) in liver or adipose tissues. These data suggest that the shihunine-rich extract of D. loddigesii is an anti-diabetic fraction of D. loddigesii. Under our experimental condition, DLS at a dose of 50 mg/kg has good anti-diabetic efficacy.