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Preliminary work has shown that select triacylglycerols (TAGs) are upregulated in a preclinical model of MGD, suggesting that TAGs may be an important outcome variable in research involving human meibomian gland epithelial cells (HMGECs). The purpose of this study was to explore the HMGEC TAG lipidome in culture conditions known to influence differentiation. HMGECs were differentiated in DMEM/F12 with 10 ng/ml EGF, FBS (2% or 10%), and rosiglitazone (0, 20, or 50 μM) for two or five days. Following culture, lipids were extracted, processed, and directly infused into a Triple TOF 5600 mass spectrometer (SCIEX, Framingham, MA) with electrospray ionization. MS and MS/MS ALL spectra were acquired in the positive ion mode and performed with the SWATH technology. Only the TAGs that were present in all 48 samples were included in the analysis. Multiple regression techniques were utilized to assess the effects of each factor (FBS, rosiglitazone, and culture duration) on each expressed TAG. The HMGEC TAG lipidome consisted of 115 TAGs with 42-62 carbons and zero to 10 double bonds. Fatty acyl chains had 14 to 26 carbons and zero to five double bonds. C18:1 (oleic acid, 25/115, 21.7%) and C16:0 (palmitic acid, 16/115, 13.9%) were the most common fatty acids. FBS, rosiglitazone, and culture duration were significant predictors for 93 TAGs (80.9%) with R 2 values ranging from 0.20 to 0.77 (p < 0.05). FBS and rosiglitazone achieved significance (p < 0.05) for 80 (69.6%) and 67 TAGs (58.3%), respectively. Rosiglitazone demonstrated a selective upregulation of TAGs containing 16 or 18 carbons. Culture duration reached significance (p < 0.05) for only 36 TAGs (31.3%). When comparing the 10 most abundant C18:1-containing TAGs in meibum, FBS was a negative predictor for five TAGs (mean standardized coefficient [SC] = -0.58, p < 0.001), rosiglitazone was a positive predictor for six TAGs (mean SC = 0.41, p ≤ 0.03), and culture duration weakly influenced one TAG (SC = 0.27, p = 0.008). FBS and rosiglitazone, unlike culture duration, are powerful modulators of the TAG profile. Rosiglitazone induces changes that could be consistent with fatty acid synthesis, suggesting that quantifying the TAG lipidome could be an indirect measure of lipogenesis. Though both have been described as differentiating agents, FBS and rosiglitazone induce opposing effects on meibum-relevant TAGs. Culturing with rosiglitazone is associated with a TAG profile that is more consistent with the expected outcome of lipogenesis and with the profile observed in normal human meibum., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Abstract: Immune checkpoint inhibition has improved the clinical outcomes for numerous patients with cancer. However, the downside is a whole new spectrum of immune-related adverse events. We report a 68-year-old man with a history of nonsmall cell lung cancer presenting with a spontaneous corneal perforation in the right eye after 22 cycles of pembrolizumab. In addition, a chronic central nonhealing epithelial defect developed after performing a penetrating keratoplasty. Treatment with autologous serum drops resulted in complete healing of the corneal ulcer, where other conventional therapies had no effect. One month after reinitiating pembrolizumab therapy, our patient presented again with a corneal perforation in the fellow eye. This case describes relapsing sterile ulcerations associated with pembrolizumab use and presents an unexpected cure., Competing Interests: A. Sandrine has received speakers fees from BMS, Roche, Astra Zeneca, Merck, Pfizer, and Novartis. The remaining authors have no funding or conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Schistosomiasis is one of the most devastating parasitic disease in the world. Schistosoma spp. survive for decades within the vasculature of their human hosts. They have evolved a vast array of mechanisms to avoid the immune reaction of the host. Due to their sexual dimorphism, with the female worm lying within the gynecophoric canal of the male worm, it is the male that is exposed to the immediate environment and the soluble parts of the host's immune response. To understand how the worms are so successful in fending off the immune attacks of the host, comparative analyses of both worm sexes in human serum (with or without Praziquantel) were performed using scanning electron microscopy, transmission electron microscopy, and immunohistochemistry. Further, gene expression analyses of tegument-specific genes were performed. Following the incubation in human serum, males and females out of pairs show morphological changes such as an altered structure of the pits below the surface and an increased number of pits per area. In addition, female schistosomes presented a marked tuft-like repulsion of their opsonized surface. The observed resistance of females to Praziquantel seemed to depend on active proteins in the human serum. Moreover, different expression profiles of tegument-specific genes indicate different functions of female&#95;single and male&#95;single teguments in response to human serum. Our results indicate that female schistosomes developed different evasion strategies toward the host's immune system in comparison to males that might lead to more robustness and has to be taken into account for the development of new anti-schistosomal drugs.

Purpose: The aim of this study was to report a case of sterile corneal ulcer leading to perforation, which was treated effectively with autologous serum eye drops, topical regenerative agent (poly-carboxymethylglucose sulfate), steroids, and systemic immunosuppression in a patient with undiagnosed primary Sjögren's syndrome., Methods: A 74-year-old female presented with a month's history of gradually worsening blurry vision in her left eye. Ophthalmic examination revealed a central descemetocele with excessive corneal stromal melting and absence of signs of infection. A bandage contact lens was applied for tectonic support along with topical corticosteroid and antibiotic drops. Autoimmune screen disclosed a diagnosis of Sjögren's syndrome, and the patient was commenced on systemic immunosuppression. Forty-eight hours after presentation, the patient developed a localized corneal perforation, presenting with a flat anterior chamber., Results: Urgent amniotic membrane transplantation was arranged while topical dexamethasone, moxifloxacin, and autologous serum eye drops were administered. After 24 h of intensive topical treatment, a significant reforming of the anterior chamber and subsequent gradual regeneration of the corneal stroma were noted, thus postponing amniotic grafting. The patient remained under close monitoring, showing progressive clinical improvement. Regenerating agent eye drops (Cacicol20 ® ) were also applied over the next month, with careful and slow tapering of topical dexamethasone. Further improvement of corneal thickness was observed, and visual acuity increased to 20/80., Conclusion: This case report demonstrates the successful medical treatment of an autoimmune-related sterile corneal perforation without surgical intervention, highlighting the fact that early diagnosis and rigorous medical treatment with autologous serum and regenerating agent eye drops can effectively aid tissue regeneration and favorable visual rehabilitation.

Background: Despite a substantial body of research, no clear best practice guidelines exist for the assessment of hydration in athletes. Body water is stored in and shifted between different sites throughout the body complicating hydration assessment. This review seeks to highlight the unique strengths and limitations of various hydration assessment methods described in the literature as well as providing best practice guidelines., Main Body: There is a plethora of methods that range in validity and reliability, including complicated and invasive methods (i.e. neutron activation analysis and stable isotope dilution), to moderately invasive blood, urine and salivary variables, progressing to non-invasive metrics such as tear osmolality, body mass, bioimpedance analysis, and sensation of thirst. Any single assessment of hydration status is problematic. Instead, the recommended approach is to use a combination, which have complementary strengths, which increase accuracy and validity. If methods such as salivary variables, urine colour, vital signs and sensation of thirst are utilised in isolation, great care must be taken due to their lack of sensitivity, reliability and/or accuracy. Detailed assessments such as neutron activation and stable isotope dilution analysis are highly accurate but expensive, with significant time delays due to data analysis providing little potential for immediate action. While alternative variables such as hormonal and electrolyte concentration, bioimpedance and tear osmolality require further research to determine their validity and reliability before inclusion into any test battery., Conclusion: To improve best practice additional comprehensive research is required to further the scientific understanding of evaluating hydration status.

Purpose: This study investigated the clinical manifestation and risk factors associated with remission in filamentary keratitis., Design: Retrospective, interventional, comparative case series., Methods: We retrospectively reviewed the medical records of 116 patients with filamentary keratitis diagnosed and treated between January 2012 and December 2018. We investigated the 5 causative factors including brain lesion, dry eye syndrome, autoimmune disease, ocular surgery or injury, and other conditions; treatment methods and duration; and remission status, and analyzed the risk factors associated with remission., Results: The mean age of the patients was 56.9 ± 19.1 years and the mean follow-up duration was 14.9 ± 22.8 months. The most common underlying condition associated with filamentary keratitis was identified as a brain lesion (36.2%), followed by dry eye syndrome (30.2%) and autoimmune disease (24.1%). A comparison of remission rates among the causative factors revealed that cases associated with brain lesions had significantly lower remission rates (33.3%) than those associated with other causative factors (>60%) (P = .001). After adjustment for sex, age, diabetes mellitus, and hypertension, the treatment failure rate in patients affected by brain lesions was 6.602-fold higher than that associated without brain lesion (P = .001). The treatment method-dependent differences in the remission rate were observed in brain lesion and dry eye syndrome (P = .041 and P = .005, respectively)., Conclusions: The most common condition leading to filamentary keratitis was a brain lesion, followed by dry eye syndrome and autoimmune disease. The treatment failure rate was statistically significantly low only in patients with filamentary keratitis associated with brain lesions., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pregnant women with obesity are at increased risk of parturition dysfunction; however, the biological mechanism has remained unknown. We hypothesized that molecules circulating in the serum of pregnant women with obesity may induce the aberrant expression of contraction-associated proteins (CAPs), leading to insufficient uterine contractions. This study aimed to investigate the effects of maternal serum on CAPs expression by human uterine smooth muscle cells (UtSMCs) and elucidate the influence of maternal obesity. Blood samples were collected from singleton pregnant women at 36-41 weeks of gestation before the onset of labor. UtSMCs were incubated in the serum, and the mRNA expressions of PTGFR, OXTR, GJA1, and PTGS2 were examined by RT-PCR. Progranulin (PGRN) is a circulating glycoprotein associated with insulin resistance characterized by the accumulation of visceral fat. The serum PGRN levels of the samples were measured by ELISA. After incubated with PGRN (100-1,000 ng/mL), mRNA expression of PTGFR, OXTR, and GJA1 and protein expression of CX43 were examined by RT-PCR and western blotting, respectively. The mRNA expressions of PTGFR, OXTR, and GJA1 showed significantly negative correlations with gestational weight gain (GWG). Serum PGRN levels showed a significantly positive correlation with GWG. High levels of PGRN suppressed the mRNA expression of GJA1 and the protein expression of CX43. The change in maternal serum induced by GWG suppressed the CAPs expression by UtSMCs. PGRN is one of the factors in the serum responsible for inhibiting the expression of CX43.

Aim: To compare the efficacy of cord blood and peripheral adult donor blood serum eyedrops, controlled for growth factor content, in the treatment of severe dry eye diseases (DED) resistant to conventional therapy., Methods: This was a multicentre randomised, double-masked, cross-over clinical trial. Sixty patients diagnosed as severe DED, associated to persistent corneal epithelial defects were randomised and equally assigned to group A (treated with cord blood serum (CBS)) or group B (treated with PBS), eyedrops administered eight times/day for 1 month. Primary outcome was the pretreatment and post-treatment change in corneal fluorescein staining. Secondary outcomes included the pretreatment and post-treatment change in Ocular Surface Disease Index (OSDI) questionnaire and Visual Analogue Score (VAS) of subjective symptoms, Schirmer I test, tear break-up time and conjunctival staining. Patients with relapse in signs or symptoms after further 2 months switched to the remaining group for one additional month. Data were statistically analysed (p<0.05)., Results: Corneal staining was more significantly reduced after the CBS treatment, both VAS and OSDI score reduction was observed in both groups, but group A reported significantly less grittiness and pain. Nineteen patients shifted in the crossover period, the within individual comparison confirmed a better recovery in the CBS treatment period. Reduction in epithelial damage was positively associated with epidermal growth factor, transforming growth factorα and platelet-derived growth factor content. Levels of interleukins (IL-13) were positively associated with symptom decrease., Conclusions: Overall, DED signs improved after both CBS and PBS treatments, with potential advantages of CBS for subjective symptoms and corneal damage reduction., Clinical Trial Registration: NCT03064984., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: The aim of this study was to compare the effects of local administrations of platelet-rich plasma (PRP) with autologous conditioned serum (ACS) on Achilles tendon healing in a rat model., Methods: In this study, 40 male Sprague-Dawley rats, aged 12 months and weighing 350 to 400 g were used. The rats were divided into three groups (n=10 in each group): a control group and two treatment groups (PRP vs ACS). A standardized procedure was applied for the complete rupture and repair of the Achilles tendon in each group. The PRP group received one dose of PRP on the operative area, and ACS group received ACS at 24, 48, and 72 hours after the surgery. The control group received no injection. Animals were sacrificed 30 days after the operation, and tendon healing in each group was assessed histopathologically based on Bonar's semi-quantitative score and Movin's semi-quantitative grading scale. For the biomechanical analyses, unoperated Achilles tendons of all rats in the control and ACS groups were also harvested, and pulling tests were applied to the specimen to measure the longitudinal axis strength. The highest force value among the data obtained was defined as the maximum strength value (Fmax)., Results: The mean Bonar's score was significantly lower in the PRP group (3.8±0.8) than in the ACS (4.8±0.45) and control groups (5.2±0.837) (p=0.0028). The mean Movin's score was significantly lower in the PRP group (7.80±1.49) than in the ACS (9.8±1) and control groups (11.2±2.4) (p=0.029). The ratio of type I collagen was significantly higher in the PRP group (60±6) than in the ACS (52±4.5) and control groups (42±9) (p=0.005). Biomechanical results obtained from operated sites were comparable in terms of Fmax among groups (PRP, 33.93±2.61; ACS, 35.24±3.26; control, 35.69±3.62) (p=0.674). Similarly, the results obtained from unoperated sites were comparable among groups (PRP, 47.71±1.21; ACS, 48.14±2; control, 49.14.69±1.88) (p=0.395)., Conclusion: In terms of histopathological results, PRP seems to be more effective than ACS for Achilles tendon healing in rats.

Myogenic differentiation mechanisms are generally assessed using a murine cell line placed in low concentrations of an animal-derived serum. To more closely approximate in vivo pathophysiological conditions, recent studies have combined the use of human muscle cells with human serum. Nevertheless, the in vitro studies of the effects of a human microenvironment on the differentiation process of human myoblasts require the identification of the culture conditions that would provide an optimal and reproducible differentiation process of human muscle cells. We assessed the differentiation variability resulting from the use of human myoblasts and serums from healthy subjects by measuring the myotube diameter, fusion index and surface covered by myotubes. We showed the preserved cell-dependent variability of the differentiation response of myoblasts cultured in human serums compared to FBS. We found that using a pool of serums reduced the serum-dependent variability of the myogenic response compared to individual serums. We validated our methodology by showing the atrophying effect of pooled serums from COPD patients on healthy human myotubes. By replacing animal-derived tissues with human myoblasts and serums, and by validating the sensitivity of cultured human muscle cells to a pathological microenvironment, this human cell culture model offers a valuable tool for studying the role of the microenvironment in chronic disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Significance: After epithelium-off crosslinking (CXL), epithelial closure time and post-operative pain are an important issue in terms of possible complications and patient comfort. We report a prospective randomized study about the use of autologous serum eye drops after CXL., Purpose: This study aims to evaluate the effect of autologous serum eye drops on epithelial healing and post-operative pain after CXL., Methods: Sixty patients diagnosed as having progressive keratoconus and treated with accelerated CXL (9 mW/cm for 10 minutes) randomly received 20% autologous serum eye drops (autologous serum group, n = 30) or artificial tears (control group, n = 30). Patients were evaluated every day after the surgery, and the day of epithelial closure was recorded. All patients were asked to report the maximum pain level using the Wong-Baker FACES Pain Rating Scale at the end of each day until the epithelial closure was completed. The change in topographic parameters and haze were recorded at 6 months., Results: The mean epithelial closure time was significantly lower in the autologous serum group than in the control group (2.37 ± 0.49 and 2.67 ± 0.47 days, respectively; P = .02). There was a statistically significant difference between the pain scores in the first and second days of surgery between the two groups (first-day autologous serum autologous serum group: 2.80 ± 0.66 and control group: 3.50 ± 0.82, P = .01; second-day autologous serum group: 1.73 ± 0.69 and control group: 2.20 ± 0.76, P = .02). Pre-operative and post-operative topographic parameters and haze at 6 months were similar between the two groups (P > .05 for all)., Conclusions: Use of autologous serum eye drops after CXL accelerates epithelial healing and reduces post-operative pain. Shortening the duration of epithelial closure would be beneficial in reducing possible complications and increasing patient comfort.

Lysophosphatidylcholine (lysoPtdCho) is produced by the phospholipase A 2 -mediated hydrolysis of phosphatidylcholine and can stimulate proliferation and apoptosis of vascular smooth muscle cells. We examined the influence of fetal bovine serum (FBS) concentration in the culture medium on lysoPtdCho-mediated apoptosis and proliferation of human aortic smooth muscle cells (HASMCs) as well as on the activation of extracellular signal-regulated kinases (ERK)1/2. In the presence of 1% FBS, HASMC viability increased after lysoPtdCho treatment at 1 and 10 μM but decreased at 25 and 50 μM. However, lysoPtdCho increased HASMC viability in a dose-dependent manner in the presence of 10% FBS. The activity of caspase 3/7 in HASMCs was increased by 25 μM lysoPtdCho in the presence of 1% FBS, but not 10% FBS. Furthermore, lysoPtdCho at 1 and 10 μM triggered ERK1/2 phosphorylation in the presence of 1% FBS, but not at 10% FBS. Thus, lysoPtdCho-mediated HASMC apoptosis, proliferation, and ERK1/2 activation are dependent on the concentration of FBS.

Significance: There is a dearth of studies investigating the challenges encountered in dry eye practice. Profiling these barriers is crucial to improving dry eye diagnosis and patient care., Purpose: This study aimed to examine the diagnostic and treatment perspectives, and challenges in dry eye practice in Ghana., Methods: An anonymous paper-based or web survey regarding dry eye practice pattern, practice challenges, and access to diagnostic tools was distributed to 280 potential participants., Results: One hundred thirteen respondents completed the survey. Case history (92.5%), fluorescein tear breakup time (87.5%), and corneal fluorescein staining (72.5%) were the topmost procedures used for dry eye diagnosis. A preserved lubricant drop was the most commonly prescribed treatment of mild, moderate, and severe dry eye at the rates of 77.0, 83.2, and 77.0%, respectively. A few respondents prescribed cyclosporine (2.7%) or punctal plugs (5.3%) across all disease severities, and none used scleral lens, autologous serum tears, or thermal pulsation. Graduate professional training influenced the practice pattern of 82.3% of respondents, whereas continuing professional education influenced less than 1%. Approximately 70.1 and 92.8% of optometrists considered referring dry eye in children and cases that are unresponsive to treatment, respectively. Eighty-eight percent of practitioners indicated they experience a challenge in dry eye practice, with limited access to diagnostic tools (77.9%) and limited availability of effective dry eye medication on the Ghanaian market (50.4%) being the most frequent challenges. More than 85% of respondents had access to a fluorescein dye or slit-lamp biomicroscope; however, none had access to a phenol red thread, lissamine green dye, osmolarity technology, or meibography device., Conclusions: Practitioners' limited access to diagnostic tools/techniques and the limited effective dry eye treatments are major challenges encountered in dry eye practice in Ghana. Addressing these will improve dry eye practice and treatment outcomes in the country.

Purpose: To describe the safety and effectiveness of using autologous serum-based eye drops for the treatment of severe dry eye and persistent corneal epithelial defect., Methods: Literature searches of the PubMed and Cochrane Library databases were conducted most recently in March 2019. The searches identified 281 citations, which were reviewed in abstract form. Of these, 48 were selected for a full-text review, and 13 met the inclusion criteria and were assigned a quality-of-evidence rating by the panel methodologist. Eight of these studies were rated level II and 5 were rated level III; there were no level I studies., Results: This analysis included 10 studies of the use of autologous serum-based eye drops for severe dry eye disease and 4 studies of persistent epithelial defect. Several studies showed good effectiveness, with some improvement in symptoms, signs, or both. Eight of the studies reported improved symptoms for severe dry eye disease, and all noted improvement in at least 1 clinical sign. For persistent epithelial defects, all of the studies showed improvement, with 3 of the 4 demonstrating an improvement rate of more than 90%. Adverse events were rare., Conclusions: Although autologous serum-based tears may be effective in the treatment of severe dry eye and persistent epithelial defect, conclusions are limited owing to the absence of controlled trials., (Copyright © 2019 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Of all genetic mutations causing human disease, premature termination codons (PTCs) that result from splicing defaults, insertions, deletions, and point mutations comprise around 30%. From these mutations, around 11% are a substitution of a single nucleotide that change a codon into a premature termination codon. These types of mutations affect several million patients suffering from a large variety of genetic diseases, ranging from relatively common inheritable cancer syndromes to muscular dystrophy or very rare neuro-metabolic disorders. Over the past three decades, genetic and biochemical studies have revealed that certain antibiotics and other synthetic molecules can act as nonsense mutation readthrough-inducing drugs. These compounds bind a specific site on the rRNA and, as a result, the stop codon is misread and an amino acid (that may or may not differ from the wild-type amino acid) is inserted and translation occurs through the premature termination codon. This strategy has great therapeutic potential. Unfortunately, many readthrough agents are toxic and cannot be administered over the extended period usually required for the chronic treatment of genetic diseases. Furthermore, readthrough compounds only restore protein production in very few disease models and the readthrough levels are usually low, typically achieving no more than 5% of normal protein expression. Efforts have been made over the years to overcome these obstacles so that readthrough treatment can become clinically relevant. Here, we present the creation of a stable cell line system that constitutively expresses our dual-reporter vector harboring two cancer initiating nonsense mutations in the adenomatous polyposis coli (APC) gene. This system will be used as an improved screening method for isolation of new nonsense mutation readthrough inducers. Using these cell lines as well as colorectal cancer cell lines, we demonstrate that serum starvation enhances drug-induced readthrough activity, an observation which may prove beneficial in a therapeutic scenario that requires higher levels of the restored protein. KEY MESSAGES: Nonsense mutations affects millions of people worldwide. We have developed a nonsense mutation read-through screening tool. We find that serum starvation enhances antibiotic-induced nonsense mutation read-through. Our results suggest new strategies for enhancing nonsense mutation read-through that may have positive effects on a large number of patients.

Direct conversion is considered a promising approach to obtain tissue-specific cells for cell therapies; however, this strategy depends on exogenous gene expression that may cause undesired adverse effects such as tumorigenesis. By optimizing the Schwann cell induction system, which was originally developed for trans-differentiation of bone marrow mesenchymal stem cells into Schwann cells, we established a system to directly convert adult human skin fibroblasts into cells comparable to authentic human Schwann cells without gene introduction. Serial treatments with beta-mercaptoethanol, retinoic acid, and finally a cocktail of basic fibroblast growth factor, forskolin, platelet-derived growth factor-AA, and heregulin-β1 (EGF domain) converted fibroblasts into cells expressing authentic Schwann cell markers at an efficiency of approximately 75%. Genome-wide gene expression analysis suggested the conversion of fibroblasts into the Schwann cell-lineage. Transplantation of induced Schwann cells into severed peripheral nerve of rats facilitated axonal regeneration and robust functional recovery in sciatic function index comparable to those of authentic human Schwann cells. The contributions of induced Schwann cells to myelination of regenerated axons and re-formation of neuromuscular junctions were also demonstrated. Our data clearly demonstrated that cells comparable to functional Schwann cells feasible for the treatment of neural disease can be induced from adult human skin fibroblasts without gene introduction. This direct conversion system will be beneficial for clinical applications to peripheral and central nervous system injuries and demyelinating diseases., (© 2019 Wiley Periodicals, Inc.)

Aims: Serum is widely used for in vitro cell culture of eukaryotic cells. Although serum is well known to affect various biological activities in cancer cells, its effect in vasculogenic mimicry (VM) is not yet fully defined. Thus, this study investigated the role of serum in VM in human prostate cancer (PCa) PC-3 cells., Main Methods: Invasion assay and 3D culture VM tube formation assay are performed. VM-related molecules are checked by western blot and reverse transcriptase-polymerase chain reaction. Nuclear twist is detected by confocal after twist-FITC/DAPI double staining., Key Findings: Serum dramatically induced not only invasion but also VM. Serum increased the phosphorylation of erythropoietin-producing hepatocellular A2 (EphA2) without affecting EphA2 expression. Both the protein and mRNA expression levels of vascular endothelial cadherin (VE-cadherin) are up-regulated by serum. Twist expression was increased in the nucleus by serum. Serum activated AKT through phosphorylation, despite the unchanged AKT expression. Serum caused an increase in matrix metalloproteinase-2 (MMP-2) and laminin subunit 5 gamma-2 (LAMC2) protein expressions. Wortmannin, a phosphoinositide-3-kinase inhibitor, significantly decreased serum-induced invasion and VM., Significance: These results demonstrated that serum activates EphA2 and up-regulates twist/VE-cadherin, which in turn activate AKT that up-regulates MMP-2 and LAMC2, thereby inducing the invasion and VM of human PCa PC-3 cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) play a critical role in total body irradiation (TBI)-induced hematopoietic system injury. However, the mechanisms involved in ROS production in hematopoietic stem cells (HSCs) post TBI need to be further explored. In this study, we demonstrated that hematopoietic system injury in mice radiated with TBI was effectively alleviated when the blood circulation environment was changed via the injection of serum from non-radiated mice. Serum injection increased the survival of radiated mice and ameliorated TBI-induced hematopoietic system injury through attenuating myeloid skew, increasing HSC frequency, and promoting the reconstitution of radiated HSCs. Serum injection also decreased ROS levels in HSCs and regulated oxidative stress-related proteins. A serum proteome sequence array showed that proteins related to tissue injury and oxidative stress were regulated, and a serum-derived exosome microRNA sequence assay showed that the PI3K-Akt and Hippo signaling pathways were affected in radiated mice injected with serum from non-radiated mice. Furthermore, a significant increase in cell viability and a decrease in ROS were observed in radiated lineage - c-kit + cells treated with serum-derived exosomes. Similarly, an improvement in the impaired differentiation of HSCs was observed in radiated mice injected with serum-derived exosomes. Taken together, our observations suggest that serum from non-radiated mice alleviates HSC injury in radiated mice by improving the systemic environment after radiation, and exosomes contribute to this radioprotective effect as important serum active component., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: Dentin debris and organic components may affect the properties of intracanal irrigants. This study aimed to evaluate the effect of dentin powder (DP) and human serum (HS) on the antibacterial and antibiofilm activity of sodium hypochlorite (NaOCl) against Enterococcus faecalis., Design: DP from 100 to 6.25 mg/mL and HS from 10% to 0.3125% were interactively mixed and added into E. faecalis and 1% NaOCl solution. The live E. faecalis were counted after 1 min of contact. For biofilm testing, 7 days of E. faecalis biofilms were treated by 100 mg/mL DP and 10% HS alone or combination with 1% NaOCl solution for 1 min. Furthermore, after challenges, E. faecalis biofilms were stained with SYTO 9 and propidium iodide, and confocal laser scanning microscopy (CLSM) was used to determine the proportion of dead and live cells in the biofilm., Results: One hundred mg/mL DP or 10% HS alone showed the excellent inhibition of 1% NaOCl against planktonic E. faecalis, and the low concentration of DP and HS presented an additive inhibitory effect. The number of live bacteria in biofilms were significantly higher in the 1% NaOCl-treated group with DP or HS than without DP and HS (p <  0.05), and a higher percentage of dead bacteria was found in the challenge of NaOCl in the absence of DP and HS than in the presence of DP and HS., Conclusion: DP and HS generated the inhibition of antibacterial and antibiofilm activities of NaOCl, whereas the effect of HS was greater than DP., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

To understand the impact of fetal bovine serum (FBS) on metabolism and cellular architecture in addition to morphogenesis, we have identified FBS responsive proteome of Candida albicans. FBS induced 34% hyphae and 60% pseudohyphae in C. albicans at 30 °C while 98% hyphae at 37 °C. LC-MS/MS analysis revealed that 285 proteins modulated significantly in response to FBS at 30 °C and 37 °C. Out of which 152 were upregulated and 62 were downregulated at 30 °C while 18 were up and 53 were downregulated at 37 °C. Functional annotation suggests that FBS may inhibit glycolysis and fermentative pathway and enhance oxidative phosphorylation (OxPhos), TCA cycle, amino acid and fatty acid metabolism indicating a use of alternative energy source by C. albicans. OxPhos inhibition assay using sodium azide corroborated the correlation between inhibition of glycolysis and enhanced OxPhos with pseudohyphae formation. C. albicans induced hyphae in response to FBS irrespective of down regulation of Ras1,Asr1/Asr2, indicates the possible involvement of MAPK and cAMP-PKA independent pathway. The Cell wall of cells grown in presence of FBS at 30 °C was rich in mannan, Beta 1,3-glucan and chitin while membranes were rich in ergosterol compared to those grown at 37 °C., Significance of the Study: This is the first study suggesting a correlation between OxPhos and morphogenesis especially pseudohyphae formation in C. albicans. Our data also indicate that fetal bovine serum (FBS) induced morphogenesis is multifactorial and may involve MAPK and cAMP-PKA independent pathway. In addition to morphogenesis, our study provides an insight in to the modulation of metabolism and cellular architecture of C. albicans in response to FBS., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction and Objectives: Microvascular obstruction (MVO) exerts deleterious effects following acute myocardial infarction (AMI). We investigated coronary angiogenesis induced by coronary serum and the role of hypoxia-inducible factor-1A (HIF-1A) in MVO repair., Methods: Myocardial infarction was induced in swine by transitory 90-minute coronary occlusion. The pigs were divided into a control group and 4 AMI groups: no reperfusion, 1minute, 1 week and 1 month after reperfusion. Microvascular obstruction and microvessel density were quantified. The proangiogenic effect of coronary serum drawn from coronary sinus on endothelial cells was evaluated using an in vitro tubulogenesis assay. Circulating and myocardial HIF-1A levels and the effect of in vitro blockade of HIF-1A was assessed., Results: Compared with control myocardium, microvessel density decreased at 90-minute ischemia, and MVO first occurred at 1minute after reperfusion. Both peaked at 1 week and almost completely resolved at 1 month. Coronary serum exerted a neoangiogenic effect on coronary endothelial cells in vitro, peaking at ischemia and 1minute postreperfusion (32 ± 4 and 41 ± 9 tubes vs control: 3 ± 3 tubes; P < .01). Hypoxia-inducible factor-1A increased in serum during ischemia (5-minute ischemia: 273 ± 52 pg/mL vs control: 148 ± 48 pg/mL; P < .01) being present on microvessels of all AMI groups (no reperfusion: 67% ± 5% vs control: 15% ± 17%; P < .01). In vitro blockade of HIF-1A reduced the angiogenic response induced by serum., Conclusions: Coronary serum represents a potent neoangiogenic stimulus even before reperfusion; HIF-1A might be crucial. Coronary neoangiogenesis induced by coronary serum can contribute to understanding the pathophysiology of AMI., (Copyright © 2017 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Myeloproliferative neoplasms (MPN)-such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF)-are typically diseases of the elderly caused by acquired somatic mutations. However, it is largely unknown how the malignant clone interferes with normal hematopoiesis. In this study, we analyzed if serum of MPN patients comprises soluble factors that impact on hematopoietic stem and progenitor cells (HPCs)., Methods: CD34+ HPCs were cultured in medium supplemented with serum samples of PV, ET, or MF patients, or healthy controls. The impact on proliferation, maintenance of immature hematopoietic surface markers, and colony forming unit (CFU) potential was systematically analyzed. In addition, we compared serum of healthy young (<25 years) and elderly donors (>50 years) to determine how normal aging impacts on the hematopoiesis-supportive function of serum., Results: Serum from MF, PV and ET patients significantly increased proliferation as compared to controls. In addition, serum from MF and ET patients attenuated the loss of a primitive immunophenotype during in vitro culture. The CFU counts were significantly higher if HPCs were cultured with serum of MPN patients as compared to controls. Furthermore, serum of healthy young versus old donors did not evoke significant differences in proliferation or immunophenotype of HPCs, whereas the CFU frequency was significantly increased by serum from elderly patients., Conclusion: Our results indicate that serum derived from patients with MPN comprises activating feedback signals that stimulate the HPCs-and this stimulatory signal may result in a viscous circle that further accelerates development of the disease., Competing Interests: I (WW) am a PLOS ONE Editorial Board member. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Purpose: To describe the safety and efficacy of autologous serum tears (AST) in managing ocular surface disease resistant to conventional therapy in patients with systemic autoimmune disease(s)., Design: Retrospective, interventional case series., Methods: Records of patients from 2009 to 2015 with systemic autoimmune disease treated with AST (20%-50%) for chronic surface disease were analyzed. Standardized measures of subjective dry eye symptoms, objective dry eye staining of the cornea, and slit-lamp findings including punctate epithelial erosion (PEE), filamentary keratopathy (FK), and corneal epithelial defects (KED) were compared during first and last visit. We attempted to standardize outcomes by creating a scale from 1 to 4 for subjective and objective components: worsening (1), no improvement (2), partial improvement (3), and complete resolution (4)., Results: Fifty-one patients (101 eyes) were included. The mean age was 59.8 ± 13.2 years (72.5% female). Average use of AST was 14.3 ± 11.7 months. Complete objective improvement of initial slit-lamp findings was achieved in 30% and partial improvement in 55% of eyes. Presence of PEE, FK, and KED decreased from 92.1% to 52.5% (P < .001), from 22.8% to 9.9% (P = .02), and from 5% to 2% (P = .44) of the eyes, respectively. Full subjective improvement of symptoms was achieved in 34.6%, partial in 50.5%, and none in 14.9% of patients. No adverse side effects were noted during follow-up., Conclusions: AST are a safe and effective adjunct therapy in improving both objective signs and subjective symptoms of ocular surface disorders associated with systemic autoimmune disease(s)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Bone Marrow MSCs are an appealing source for several cell-based therapies. Many bioreactors, as the Quantum Cell Expansion System, have been developed to generate a large number of MSCs under Good Manufacturing Practice conditions by using Human Platelet Lysate (HPL). Previously we isolated in the human bone marrow a novel cell population, named Mesodermal Progenitor Cells (MPCs), which we identified as precursors of MSCs. MPCs could represent an important cell source for regenerative medicine applications. As HPL gives rise to a homogeneus MSC population, limiting the harvesting of other cell types, in this study we investigated the efficacy of pooled human AB serum (ABS) to provide clinically relevant numbers of both MSCs and MPCs for regenerative medicine applications by using the Quantum System., Methods: Bone marrow aspirates were obtained from healthy adult individuals undergoing routine total hip replacement surgery and used to generate primary cultures in the bioreactor. HPL and ABS were tested as supplements to culture medium. Morphological observations, cytofluorimetric analysis, lactate and glucose level assessment were performed., Results: ABS gave rise to both heterogeneous MSC and MPC population. About 95% of cells cultured in HPL showed a fibroblast-like morphology and typical mesenchymal surface markers, but MPCs were scarcely represented., Discussion: The use of ABS appeared to sustain a large scale MSC production, as well as the recovery of a subset of MPCs, and resulted a suitable alternative to HPL in the cell generation based on the Quantum System., (Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Purpose: To evaluate the role of umbilical cord serum (UCS) and autologous serum (AS) therapy in reepithelialization of corneal graft after keratoplasty in a randomized controlled trial., Methods: A total of 105 eyes with epithelial defect (ED) after keratoplasty (penetrating keratoplasty-67 and anterior lamellar keratoplasty-38) on the first postoperative day were included in the study. The eyes were randomized into three groups: UCS (n=35), AS (n=35), and artificial tears (AT) (n=35). All patients received standard postoperative medical therapy. The primary outcome measure was time to epithelialization, and secondary outcome measures were best-corrected visual acuity and graft clarity., Results: The ED healed completely in 103 eyes. The mean time for complete reepithelialization was 2.5±2.1, 3.1±2.2, and 4.5±1.4 days in UCS, AS, and AT groups, respectively. The mean percentage decrease in the size of the ED was significantly better in the UCS and AS groups as compared with the AT group (P=0.001). The rate of reepithelialization was comparable between the AS and UCS groups (P=0.3). On bivariate analysis, significant correlation was found between the mean size of postoperative ED, grade of the donor cornea (P=0.001), and the presence of preoperative ED (P=0.001). No complications were associated with the use of serum therapy., Conclusion: Most of the cases of postkeratoplasty corneal ED can be managed with AT only. The serum therapy (AS/UCS) helps in the faster reepithelialization of postkeratoplasty ED as compared with AT and may be considered as a treatment option for early epithelial healing.

Purpose: Patients' perceptions of the effectiveness of a treatment, or perceived treatment effectiveness (PTE), play an important role in medicine. This study aimed to evaluate patients' PTE in dry eye disease (DED) and investigate factors contributing to these patients' perceptions., Methods: This cross-sectional study included 66 patients with DED. At enrollment, all patients had comprehensive ophthalmic assessment. In addition, to evaluate the patient's PTE, they were asked to use a 10-point scale ranging from "strongly disagree (score 1)" to "strongly agree (score 10)" to score their views on whether their DED treatments had been effective. Changes in clinical parameters of DED over time during their care were also evaluated retrospectively and correlated with the patients' PTE., Results: The mean age of patients was 55.7 years; 79% were women. Regarding patients' PTE, 36.4% strongly (score 10) and 53.0% moderately (scores 6-9) believed that their DED treatment had been effective. However, 10.6% thought that their treatment had not been effective (scores 1-5). Less favorable PTE for the DED treatment was significantly associated with a younger age (P < 0.001), current use of antidepressant medications (P = 0.01), and a higher Ocular Surface Disease Index score (P = 0.01) at enrollment., Conclusions: A majority of patients with DED have positive perceptions regarding the effectiveness of their treatments. Less favorable perceptions are associated with more severe ocular symptoms and nonocular parameters such as younger age and current antidepressant use. In DED management, assessing patients' PTE should be considered as an important part of clinical practice.

Purpose: To investigate the morphological changes of corneal epithelium and subbasal nerves by in vivo confocal microscopy in patients with ocular surface disease (OSD) treated with cord blood serum (CBS) eye drops., Methods: Twenty patients with OSD (mean age 61.1 ± 12.6 years) were included in this prospective 1-arm study and treated with CBS eye drops for 2 months. Corneal sensitivity, Schirmer test score, breakup time, subjective symptoms [Ocular Surface Disease Index (OSDI) and Visual Analogue Scale (VAS)], and corneal staining were evaluated before (T0) and after (T1) treatment. In vivo confocal microscopy analyzed giant epithelial cells, subbasal nerve number and tortuosity, neuromas, beading, and dendritic cells (DCs) in the central cornea., Results: OSDI, Visual Analogue Scale, and Oxford grading values significantly decreased at T1 versus T0 (respectively, 44.1 ± 18.9 vs. 74.2 ± 13.9; 3.7 ± 1.5 vs. 8.9 ± 0.9; and 2.4 ± 1.1 vs. 3.3 ± 1.3; P < 0.0001), whereas corneal sensitivity, Schirmer test score, and breakup time significantly increased (respectively, 49.5 ± 2.6 vs. 47.9 ± 2.9; 3.2 ± 2.0 vs. 2.4 ± 2.2; 4.6 ± 3.1 vs. 3.8 ± 2.1; P < 0.0001). Corneal nerve morphology improved at T1 versus T0 with a higher total nerve number (3.4 ± 1.6 vs. 2.5 ± 1.6 per frame) and lower tortuosity (3.0 ± 0.7 vs. 3.5 ± 0.6) (P < 0.01). The number of patients presenting with giant epithelial cells, beading, and neuromas decreased at T1. DC density did not change after treatment. The detection of neuromas and higher DC density at T0 were associated with greater OSDI reduction at T1 (P < 0.001)., Conclusions: CBS eye drops significantly improved corneal nerve morphology and subjective symptoms in patients with severe OSD. The presence of neuromas and higher dendritic cell density at baseline were associated with greater reduction of discomfort symptoms after treatment.

Purpose: To investigate the effects of human breast milk on corneal epithelial wound healing., Methods: The effects of human breast milk on epithelial healing is compared with autologous serum and artificial tears on 24 female Bal-b/C mice. A central corneal epithelial defect was created using a 2 mm trephine. Four groups were formed. By a random pick-up, topical human breast milk 4 × 1 was given to Group 1, topical mouse autologous serum 4 × 1 was applied to Group 2, and preservative-free artificial tears 4 × 1 was applied to Group 3.Group 4 was evaluated as control. Biomicroscopical examination was performed on days 1, 2, and 3. Mice were sacrificed on the third day. Histopathological and electron microscopic examinations were performed as well., Results: The fastest and best healing group was Group 1, followed by Group 2. Re-epithelization was not complete even at the end of the second day in groups 3 and 4., Conclusions: The rich content of human breast milk may be an alternative to epithelial healers and artificial tears.

The most failures in the process of laboratory analysis occur at the pre-analytical stage. The percentage of samples of blood serum with hemolysis and percentage of samples of EDTA of whole blood with clots are largely applied as indices of quality of venous blood sampling. The analysis of data from 28 laboratories established no relationship between percentage ofsamples with hemolysis and percentage of samples with clots. Prior to implementation of more large-scaled studies, the level less than 1% of samples with hemolysis can be accepted as a minimal level of quality of blood sampling and the level less than 0.4% of samples of total blood with clots can be accepted as a minimal level of quality ofpreparation of samples after blood drawing from vein for analysis using hematological analyzers. both indices can be applied for estimating efficiency of measures concerning amelioration of quality of procedure of blood drawing from vein. By all appearances, the ideal sample for control of quality of blood drawing in the nearest future can become sample of blood drew from vein into vial with citrate for analysis using modern coagulometric analyzer. This sample can become a source for calculation of several indices of quality of venous blood drawing (hemolysis, clots, vial filling and violation of ration sample-anticoagulant).

Purpose: To evaluate the effect of 10% human serum (HS), 5% platelet-rich plasma (PRP), and 5% autologous conditioned plasma (ACP) on migration, proliferation, and extracellular matrix (ECM) synthesis of human meniscus cells., Methods: Cell migration and proliferation on stimulation with HS, PRP, and ACP were assessed by chemotaxis assays and measurement of genomic DNA content. Meniscus cells were cultivated in pellets stimulated with 10% HS, 5% PRP, or 5% ACP. Meniscal ECM formation was evaluated by histochemical staining of collagen type I, type II, and proteoglycans and by analysis of fibrochondrocyte marker gene expression., Results: Human meniscus cells were significantly attracted by all 3 blood-derived products (10% HS and 5% ACP: P = .0001, 5% PRP: P = .0002). Cell proliferation at day 9 was significantly increased on stimulation with 10% HS (P = .0001) and 5% PRP (P = .0002) compared with 5% ACP and controls. Meniscus cell pellet cultures showed the formation of a well-structured meniscal ECM with deposition of collagen type I, type II, and proteoglycans on stimulation with 10% HS, whereas 5% PRP or 5% ACP resulted in the formation of an inhomogeneous and more fibrous ECM. Stimulation with 10% HS and 5% ACP showed a significant induction of fibrochondrocyte marker genes such as aggrecan (HS: P = .0002, ACP: P = .0147), cartilage oligomeric matrix protein (HS: P = .0002, ACP: P = .0005), and biglycan (HS: P = .0002, ACP: P = .0003), whereas PRP showed no inducing effect., Conclusions: Among all tested blood-derived products, only stimulation with HS showed the formation of a meniscal ECM as well as positive cell proliferating and migrating effects in vitro. Regarding a potential biological repair of nonvascular meniscus lesions, our results may point toward the use of HS as a beneficial augment in regenerative meniscus repair approaches., Clinical Relevance: Our findings may suggest that HS might be a beneficial augment for meniscus repair., (Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Cell synchronization is an approach to obtain cell populations of the same stage, which is a prerequisite to studying the regulation of cell cycle progression in vivo. Serum starvation and thymidine double blocking (TdR) are two important practices in studying cell cycle synchronization. However, their effects on canine cancer cells as well as the regulatory mechanisms by these two methods are poorly understood. In this study, we determined the optimum conditions of serum starvation and TdR and their effects on cell cycle synchronization. We further explored the involvement of PI3K/Akt signaling pathway in the cell cycle synchronization by investigating the expression of three key genes (p27, p53 and bcl-2). Serum starvation resulted in a reversible cell cycle arrest and synchronously progress through G0/G1. The highest percentage of CHMm cells (87.47%) in G0/G1 stage was obtained after 42 h incubation with 0.5% fetal bovine serum (FBS). TdR double blocking could arrest 98.9% of CHMm cells in G1/S phase (0 h of release), and could arrest 93.74% of CHMm cells in S phase after 4h of release. We also found that the p27, p53, bcl-2 genes were most highly expressed in G0/G1 phase. Our current work revealed that serum starvation and TdR methods could achieve sufficient synchronization of CHMm cells. Moreover, the expression of p27, p53 and bcl-2 genes was related to cyclical movements and apoptosis. Our results will provide a new insight into cell cycle regulation and reprogramming of canine cancer cells induced by serum starvation and TdR blocking., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Targeting oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) in acute myeloid leukemia (AML) can reduce blast survival and tumor immune evasion. Decoy oligodeoxynucleotides (dODNs), which comprise STAT3-specific DNA sequences are competitive inhibition of STAT3 transcriptional activity. To deliver STAT3dODN specifically to myeloid cells, we linked STAT3dODN to the Toll-like receptor 9 (TLR9) ligand, cytosine guanine dinucleotide (CpG). The CpG-STAT3dODN conjugates are quickly internalized by human and mouse TLR9(+)immune cells (dendritic cells, B cells) and the majority of patients' derived AML blasts, including leukemia stem/progenitor cells. Following uptake, CpG-STAT3dODNs are released from endosomes, and bind and sequester cytoplasmic STAT3, thereby inhibiting downstream gene expression in target cells. STAT3 inhibition in patients' AML cells limits their immunosuppressive potential by reduced arginase expression, thereby partly restoring T-cell proliferation. Partly chemically modified CpG-STAT3dODNs have >60 hours serum half-life which allows for IV administration to leukemia-bearing mice (50% effective dose ∼ 2.5 mg/kg). Repeated administration of CpG-STAT3dODN resulted in regression of human MV4-11 AML in mice. The antitumor efficacy of this strategy is further enhanced in immunocompetent mice by combining direct leukemia-specific cytotoxicity with immunogenic effects of STAT3 blocking/TLR9 triggering. CpG-STAT3dODN effectively reducedCbfb/MYH11/MplAML burden in various organs and eliminated leukemia stem/progenitor cells, mainly through CD8/CD4 T-cell-mediated immune responses. In contrast, small-molecule Janus kinase 2/STAT3 inhibitor failed to reproduce therapeutic effects of cell-selective CpG-STAT3dODN strategy. These results demonstrate therapeutic potential of CpG-STAT3dODN inhibitors with broad implications for treatment of AML and potentially other hematologic malignancies., (© 2016 by The American Society of Hematology.)

The hormone-like polypeptide, fibroblast growth factor 21 (FGF21), is a major modulator of lipid and glucose metabolism and an exploratory treatment strategy for obesity related metabolic disorders. The costs of recombinant FGF21 and mode of delivery by injection are important constraints to its wide therapeutic use. The stimulation of endogenous FGF21 production through diet is being explored as an alternative approach. To that end, we examined the mechanism(s) by which serum manipulation and lipoic acid (a dietary activator of FGF21) induce FGF21 in human hepatocellular carcinoma HepG2 cells. Serum withdrawal markedly induced FGF21 mRNA levels (88 fold) and FGF21 secreted in the media (19 fold). Lipoic acid induced FGF21 mRNA 7 fold above DMSO-treated control cells and FGF21 secretion 3 fold. These effects were several-fold greater than those of PPARα agonist, Wy14643, which failed to induce FGF21 above and beyond the induction seen with serum withdrawal. The use of transcription inhibitor, actinomycin D, revealed that de novo mRNA synthesis drives FGF21 secretion in response to serum starvation. Four previously unrecognized loci in FGF21 promoter were nucleosome depleted and enriched in acetylated histone H3 revealing their role as transcriptional enhancers and putative transcription factor binding sites. FGF21 did not accumulate to a significant degree in induced HepG2 cells, which secreted FGF21 time dependently in media. We conclude that lipoic acid cell signaling connects with the transcriptional upregulation of FGF21 and it may prove to be a safe and affordable means to stimulate FGF21 production., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose . It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs). Methods . Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs); ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R) antagonist (Diphenhydramine Hydrochloride) and histamine receptor-2 (H2R) antagonist (Nizatidine) were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results . The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10  μ mol/L) on HPFs was lower than on HCFs (100  μ mol/L), and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion . Histamine may play an important role in the proliferation of HPFs and act through H1R.

Purpose: To evaluate the effect of autologous serum on the rate of epithelial healing and clinical results after laser epithelial keratectomy (LASEK) for correction of myopia., Materials and Methods: Thirty eyes of 15 patients received autologous serum drops (Study Group) while 30 eyes of 15 patients received conventional artificial tears (Control Group) after LASEK. LASEK was performed with 25-second application of 18% alcohol. Laser ablation was performed with the ESIRIS excimer laser (SCHWIND, Kleinostheim, Germany). Patients were seen daily until epithelial closure, and at 1, 3, 6, and 12 months. Time to epithelial healing was the main outcome measure. Uncorrected visual acuity (UCVA), manifest refraction, and haze were recorded., Results: Preoperative myopic spherical equivalent refraction was -2.98 ± 1.13 diopters (D) in the study group and -2.65 ± 1.01 D in the control group (p = 0.264). The mean time to epithelial healing was about 1 day shorter in the eyes receiving autologous serum than the eyes receiving conventional treatment (2.78 ± 0.40 days versus 3.73 ± 0.58 days, respectively) (p = 0.001). All eyes achieved 20/25 or better UCVA at 6 months. Over 90% of eyes were within ±0.50 D of emmetropia at 12 months in both groups. No significant difference was noted in the incidence of haze., Conclusions: Autologous serum eye drops seem to accelerate epithelial healing after LASEK, which may shorten the duration of early postoperative discomfort by about 1 day.

Purpose: The purpose of this study is to assess the stability of the growth factors (GF) in autologous serum eyedrops under different storage conditions., Methods: The concentration of epidermal growth factor (EGF), transforming growth factor-β (TGF-β1), platelet-derived growth factor AB (PDGF-AB), and albumin was measured in fresh and defrosted samples of autologous serum under different storage conditions. The fresh and defrosted samples were cooled at 4 °C, and they were studied immediately after preparation, or after defrosting, and after 1, 2, 3, and 4 weeks. The concentration of GF was also assessed after 1, 3, 6, and 9 months at -20 °C. We also investigated how the different storage conditions influence the biological effects of autologous serum on conjunctival and corneal cell cultures., Results: The concentration of EGF, TGF-β1, PDGF-AB, and albumin remained stable over the 4 weeks at 4 °C, both in fresh and in defrosted samples. Likewise, no statistically significant differences were found between the GF concentration in fresh samples and after 1, 3, 6, and 9 months of freezing at -20 °C. Moreover, no differences were found on the cell proliferation and differentiation between cultured cells with fresh or defrosted samples after 4 weeks at 4 °C or after 1, 3, 6, or 9 months at -20 °C., Conclusions: Long-term storage of autologous serum eyedrops at -20 °C does not affect the concentration of GF, simplifies clinical logistics, and reduces the frequency of blood extractions from the patients.

Purpose: To investigate the composition and biological activity of serum epitheliotrophic factors from chronic renal failure (CRF) patients vs. healthy controls., Methods: Twenty, 50 and 100 % autologous serum eyedrops (ASEs) were prepared from 16 CRF patients and 16 normal subjects. Serum epithelial growth factor (EGF), platelet-derived growth factor-AB (PDGF-AB), transforming growth factor-β1 (TGF-β1) and fibronectin levels were quantified using enzyme-linked immunosorbent assay kits. A 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assay was used to compare the proliferative effects of serum from CRF patients and healthy donors in a human corneal epithelial cell (HCEC) culture model. Migration assays were conducted via manual scraping of HCECs for migratory potential of ASEs. Morphologic changes were studied with transmission electron microscopy (TEM)., Results: EGF, PDGF-AB and TGF-β1 levels in ASEs from healthy donors were significantly higher than in serum of CRF patients. Cellular proliferation was similar in the CRF patient and normal control groups. ASEs from the normal group had a significantly higher effect on cell migration. ASEs in both groups facilitated better proliferation and migration than the negative control. Furthermore, we observed an enhancement after incubation with diluted serum vs. undiluted serum. In TEM analysis, HCECs incubated with CRF patients' 50 % ASEs showed some loss of microvilli without alterations of cytoplasmic organelles., Conclusions: Epitheliotrophic factors concentrations and biologic activities from CRF patient sera differed from healthy controls. ASEs in CRF patients are also helpful in the corneal healing process, especially when applied at a 50 % concentration.

A facile photolithographic platform for the design of cell-guiding polymeric substrates is introduced. Specific areas of the substrate are photo-deactivated for the subsequent growth of bioresistant polymer brushes, creating zones for cell proliferation, and protein adhesion., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Skeletal muscle phenotype is regulated by a complex interaction between genetic, hormonal, and electrical inputs. However, because of the interrelatedness of these factors in vivo it is difficult to determine the importance of one over the other. Over the last 5 years, we have engineered skeletal muscles in the European Union (EU) and the United States (US) using the same clone of C2C12 cells. Strikingly, the dynamics of contraction of the muscles was dramatically different. Therefore, in this study we sought to determine whether the hormonal milieu (source of fetal bovine serum (FBS)) could alter engineered muscle phenotype. In muscles engineered in serum of US origin time-to-peak tension (2.2-fold), half relaxation (2.6-fold), and fatigue resistance (improved 25%) all showed indications of a shift towards a slower phenotype. Even though there was a dramatic shift in the rate of contraction, myosin heavy chain expression was the same. The contraction speed was instead related to a shift in calcium release/sensitivity proteins (DHPR = 3.1-fold lower, slow CSQ = 3.4-fold higher, and slow TnT = 2.4-fold higher) and calcium uptake proteins (slow SERCA = 1.7-fold higher and parvalbumin = 41-fold lower). These shifts in calcium dynamics were accompanied by a partial shift in metabolic enzymes, but could not be explained by purported regulators of muscle phenotype. These data suggest that hormonal differences in serum of USDA and EU origin cause a shift in calcium handling resulting in a dramatic change in engineered muscle function., (© 2014 Wiley Periodicals, Inc.)

New therapeutic strategies are required for critical size bone defects, because the gold standard of transplanting autologous bone from an unharmed area of the body often leads to several severe side effects and disadvantages for the patient. For years, tissue engineering approaches have been seeking a stable, axially vascularized transplantable bone replacement suitable for transplantation into the recipient bed with pre-existing insufficient conditions. For this reason, the arteriovenous loop model was developed and various bone substitutes have been vascularized. However, it has not been possible thus far to engineer a primary stable and axially vascularized transplantable bone substitute. For that purpose, a primary stable silica-embedded nanohydroxyapatite (HA) bone substitute in combination with blood, bone marrow, expanded, or directly retransplanted mesenchymal stem cells, recombinant human bone morphogenetic protein 2 (rhBMP-2), and different carrier materials (fibrin, cell culture medium, autologous serum) was tested subcutaneously for 4 or 12 weeks in the sheep model. Autologous serum lead to an early matrix change during degradation of the bone substitute and formation of new bone tissue. The best results were achieved in the group combining mesenchymal stem cells expanded with 60 μg/mL rhBMP-2 in autologous serum. Better ingrowth of fibrovascular tissue could be detected in the autologous serum group compared with the control (fibrin). Osteoclastic activity indicating an active bone remodeling process was observed after 4 weeks, particularly in the group with autologous serum and after 12 weeks in every experimental group. This study clearly demonstrates the positive effects of autologous serum in combination with mesenchymal stem cells and rhBMP-2 on bone formation in a primary stable silica-embedded nano-HA bone grafting material in the sheep model. In further experiments, the results will be transferred to the sheep arteriovenous loop model in order to engineer an axially vascularized primary stable bone replacement in clinically relevant size for free transplantation.

The role of C-X-C chemokine receptor type 4 (CXCR4) in umbilical mesenchymal stem cells (UMSCs) as therapy for liver disease is ill understood. The aim of the study was to evaluate rat UMSCs (rUMSCs) on CXCR4 expression and homing to injured liver tissue. rUMSCs were isolated from umbilical cords of pregnant rats. Acute liver failure (ALF) models were developed using D-galactosamine. CXCR4 expression induction by serum from rats with ALF (LFS), cytokines, growth factors, and LPS was analyzed. CXCR4 expression was analyzed by RT-PCR, western blot, and flow cytometry. rUMSCs were labeled with carboxyfluorescein and pretreated with LFS to induce CXCR4 expression and were transplanted into ALF rats. Animals were sacrificed 48 h and 1 week after transplantation. Liver-homing rUMSCs were observed under fluorescence microscopy. rUMSCs were successfully isolated, expressing CD90 and CD106, but not CD34 and CD45. mRNA and protein expressions of CXCR4 were strongly up-regulated by LFS and by the mixture of cytokines, stem cell factor, and LPS (CM). Expression of cell surface CXCR4 on rUMSCs in groups treated with LFS (42.37 ± 1.60 %) and CM (40.17 ± 1.78 %) was higher than that in the untreated control group (9.67 ± 1.06 %) (both P < 0.001). At 48 h after transplantation, more rUMSCs pretreated with LFS appeared in the portal area, and migrated to the liver parenchyma after 1 week. LFS strongly induced the surface expression of CXCR4 on rUMSCs. Increasing CXCR4 expression on rUMSCs may enhance their homing ability to injured liver tissue, and may eventually be used for treating liver diseases.

Background and Objective: As important components of bone marrow microenvironment, bone marrow endothelial cells (BMECs) have important roles in regulating haematopoietic functions of the bone marrow. In preliminary study, we found the humoral inhibitor in liver cirrhosis (LC) could lead to ultrastructure alterations of the bone marrow endothelium. The present study aimed to investigate functional changes occurred in BMECs during LC., Methods: A multi-step approach combining microarray microRNA (miRNA) and mRNA expression profiling and bioinformatics analysis was used to generate a specific miRNA-mRNA regulatory network in BMECs treated with supplemented medium with 20% pooled sera from 26 patients with LC or 10 healthy volunteers as a control group for 48h., Results: A total of 372 differentially expressed miRNAs and 1872 differentially expressed mRNAs were identified by miRNA and mRNA microarray, respectively. Twenty-one dysregulated miRNAs and their 23 dysregulated target mRNAs confirmed by experiment (31 miRNA-mRNA interactions) were screened based on databases miRecords and miRTarBase. Bioinformatics analysis revealed that the functions of these miRNA-mRNA interactions were involved in the positive regulation of apoptosis, negative regulation of cell proliferation, cell cycle arrest and so on. The results of flow cytometry analysis showed that the LC serum treated-BMECs had a higher apoptosis percentage (17.57±1.84%) compared to the control group (10.2±1.55%). (P<0.05) CONCLUSIONS: Thirty-one miRNA-mRNA pairs combined with 21 dysregulated miRNAs and their 23 target mRNAs identified by the multi-step approach are involved in BMECs treated with LC serum. The results indicated that LC serum-induced apoptosis in BMECs were regulated by a complicated miRNA-mRNA regulatory network., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Background: The majority of sera from patients with primary membranous nephropathy have autoantibodies against the M-type phospholipase A2 receptor (PLA2R) which is expressed on human podocytes. The rabbit variant of PLA2R attaches to collagen type IV via the fibronectin type II domain, which is also present in the human variant of PLA2R., Design: To assess whether the human PLA2R variant is also involved in attachment to collagen type IV, we conducted a cell adhesion assay on a collagen-coated surface using PLA2R-transfected and mock-transfected human embryonic kidney (HEK) cells. To test the hypothesis that sera from patients containing anti-PLA2R antibodies interfere with the adhesion of podocytes to collagen, we performed cell adhesion assays on a collagen type IV-coated surface using positive and negative serum samples from patients and cultured human podocytes in vitro expressing PLA2R., Results: The HEK cell adhesion assay confirmed an enhanced attachment of PLA2R-transfected cells to collagen type IV. We confirmed diminished podocyte adhesion in the presence of serum with anti-PLA2R antibodies. The concentration of anti-PLA2R antibodies correlated with proteinuria and to the degree of diminished adhesion of podocytes., Conclusions: We demonstrated that serum of patients containing autoantibodies directed to PLA2R interferes with the ability of podocytes to attach to collagen type IV in vitro, providing evidence of a serum soluble pathogenic factor interfering with podocyte adhesion in membranous nephropathy., (© 2014 Stichting European Society for Clinical Investigation Journal Foundation.)

Purpose: Autologous serum eye drops are considered to be safe and efficient for the treatment of dry eyes associated with Sjögren syndrome (SS). The purpose of this study was to compare the clinical efficacies of autologous serum eye drops in the management of primary and secondary SS., Methods: Patients with primary (n = 20; 35 eyes) and secondary (n = 14; 27 eyes) SS dry eye were included. Serum concentrations of proinflammatory cytokines [tumor necrosis factor α, interleukin (IL)-1β, IL-6, and IL-8] were measured by a multiplex immunobead assay. The ocular symptom scores, ocular staining grades, and tear break-up time were evaluated before and after 4 weeks of 50% autologous serum eye drop application., Results: At enrollment, patients with secondary SS had higher serum proinflammatory cytokine levels (tumor necrosis factor α, IL-1β, IL-6, and IL-8) than patients with primary SS (P < 0.01). After 4 weeks of autologous serum eye drop treatment, patients with primary SS had significantly improved ocular symptoms (P < 0.01), ocular surface staining grades (P < 0.01), and tear break-up time (P < 0.05). However, patients with secondary SS had no improvement (P > 0.05)., Conclusions: Our results suggest that autologous serum eye drops might not be effective for the treatment of secondary SS because of elevated serum proinflammatory cytokine levels.

Purpose: The aim of this study was to compare the effect on corneal wound healing of 3 differently manufactured blood derivatives [autologous serum (AS), platelet-rich plasma, and serum derived from plasma rich in growth factors (s-PRGF)]., Methods: Scratch wound-healing assays were performed on rabbit primary corneal epithelial cultures and human corneal epithelial cells. Additionally, mechanical debridement of rabbit corneal epithelium was performed. Wound-healing progression was assessed by measuring the denuded areas remaining over time after treatment with each of the 3 blood derivatives or a control treatment., Results: In vitro data show statistically significant differences in the healing process with all the derivatives compared with the control, but 2 of them (AS and s-PRGF) induced markedly faster wound healing. In contrast, although the mean time required to complete in vivo reepithelization was similar to that of AS and s-PRGF treatment, only wounds treated with s-PRGF were significantly smaller in size from 2.5 days onward with respect to the control treatment., Conclusions: All 3 blood derivatives studied are promoters of corneal reepithelization. However, the corneal wound-healing progresses differently with each derivative, being faster in vitro under AS and s-PRGF treatment and producing in vivo the greatest decrease in wound size under s-PRGF treatment. These findings highlight that the manufacturing process of the blood derivatives may modulate the efficacy of the final product.

Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

Purpose: We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells., Methods: Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression., Results: Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells., Conclusions: Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Objective: In multifocal motor neuropathy (MMN), the destruction of the blood-nerve barrier (BNB) has been considered to be the key step in the disease process. The purpose of the present study was to ascertain whether sera from patients with MMN can open the BNB, and which component of patient sera is the most important for this disruption., Methods: We evaluated the effects of sera from patients with MMN, patients with amyotrophic lateral sclerosis, and control subjects on the expression of tight junction proteins and vascular cell adhesion molecule-1 (VCAM-1), and on the transendothelial electrical resistance (TEER) in human peripheral nerve microvascular endothelial cells (PnMECs)., Results: The sera from patients with MMN decreased the claudin-5 protein expression and the TEER in PnMECs. However, this effect was reversed after application of an anti-vascular endothelial growth factor (anti-VEGF) neutralising antibody. The VEGF secreted by PnMECs was significantly increased after exposure to the sera from patients with MMN. The sera from patients with MMN also increased the VCAM-1 protein expression by upregulating the nuclear factor kappa-B (NF-κB) signalling. The immunoglobulin G purified from MMN sera decreased the expression of claudin-5 and increased the VCAM-1 expression in PnMECs., Conclusions: The sera from MMN patients may disrupt the BNB function via the autocrine secretion of VEGF in PnMECs, or the exposure to autoantibodies against PnMECs that are contained in the MMN sera. Autoantibodies against PnMECs in MMN sera may activate the BNB by upregulating the VCAM-1 expression, thereby allowing for the entry of a large number of circulating inflammatory cells into the peripheral nervous system.