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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_single and male_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.

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.

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.)

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.)

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: 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.)

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: 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.

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.

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.

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.

Background: To evaluate the efficacy of autologous serum (AS) eye drops for the symptomatic relief of severe dry eye syndrome (DES), as compared to conventional preservative-free artificial tears (PFAT)., Methods: This prospective double-blind randomized crossover study used the Ocular Surface Disease Index (OSDI), tear film break-up time (TBUT), Schirmer's Test, and OXFORD Scale at baseline and after each of two 1-month treatment periods to measure the effect of 20 % diluted AS eye drops vs. PFAT in 20 consecutive severe DES patients that were refractory to conventional treatment., Results: The study included 20 (18 female and two male) severe DES patients (40 eyes). Significantly higher TBUT (P < 0.001, Wilcoxon signed-rank test) and a greater decrease in OSDI score (55.18 % decrease in the AS treatment group vs. 19.50 % decrease in the PFAT treatment group) (P < 0.001, Student's paired samples t-test) were observed in the AS treatment group after 1 month of treatment. There wasn't a significant difference in Schirmer's test and OXFORD conjunctival and corneal vital dying grading scores between the two treatment groups after 1 month of treatment (P > 0.05 [Mann-Whitney U test])., Conclusions: AS eye drops were more effective than conventional eye drops for improving tear film stability and subjective comfort in patients with severe DES.

Purpose: To assess the efficacy of sodium hyaluronate as vehicle for diluting autologous serum., Methods: The concentration and temporal stability of EGF, TGF-β, PDGF-AB and albumin in fresh and frozen samples of autologous serum diluted with sodium hyaluronate and saline solution, as well as the pH, osmolarity and density was studied. In parallel, the clinic effects of autologous serum diluted to 20% with sodium hyaluronate were compared with another solution of autologous serum diluted to 20% with saline in a prospective, comparative, randomized and double-blind study in 26 patients (52 eyes) with Sjögren syndrome. Patients underwent a complete ophthalmic assessment including tear film evaluation and corneal and conjunctival impression cytology at the beginning of the study and 2 months later., Results: The growth factor (GF) concentration remained stable during 1 month at 4°C both in fresh and defrosted samples without any differences being found between both preparations. No differences were found related to osmolarity, pH and density between these preparations before and after frosting. Autologous serum diluted with sodium hyaluronate caused a significant improvement of the tear film stability, fluorescein and rose Bengal stain, break-up time, corneal and conjunctival squamous metaplasia as well as in the patient subjective perception., Conclusions: Sodium hyaluronate is an excellent vehicle for diluting autologous serum due to the gradual release of GF and increasing their duration and effect on the ocular surface. Preparations diluted with sodium hyaluronate are better tolerated by patients and require a lower number of drops administrations., (© 2013 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Background: In patients with ST-segment elevation myocardial infarction (STEMI) reperfused with primary coronary intervention (PCI), the dynamics of endothelial cell (EC) viability, apoptosis and necrosis and its relationship with the structural consequences on the left ventricle have not been addressed so far., Design: In 20 STEMI patients, we incubated human umbilical vein endothelial cells (HUVECs) with serum drawn before reperfusion and subsequently afterwards (24, 96 h, 30 days). Viability, apoptosis and necrosis percentages were evaluated by flow cytometry. Values were compared with 12 age- and sex-matched control subjects with normal coronary arteries. Cardiac magnetic resonance (CMR) was performed during the first week after infarction., Results: Serum from STEMI patients induced a progressive loss of EC viability, with a nadir of 67.7 ± 10.2% at 96 h (baseline: 75 ± 6% and controls: 80.2 ± 3.9%, P < 0.001 in both cases). This is due to an increase in apoptosis that peaked at 96 h after reperfusion (15.2 ± 7.1% vs. 11 ± 6 at baseline and 5.8 ± 1.6% in controls, P < 0.001 in both cases). However, no significant dynamic changes in EC necrosis were detected. Extensive myocardial oedema (> 30%, median of left ventricular mass) was the only CMR variable significantly associated with a higher percentage of EC apoptosis at 96 h (extensive vs. nonextensive oedema: 18.3 ± 6.8% vs. 12.1 ± 6.3%, P < 0.05)., Conclusions: Dynamic changes in EC viability occur in the setting of STEMI patients reperfused with PCI, these changes peak late after reperfusion, they are mainly the result of an increase of apoptosis and are associated with the presence of extensive myocardial oedema., (© 2013 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.)

Purpose: Presently, our clinic is the only centre in Scandinavia that offers patients with corneal surface pathology including limbal stem cell deficiency (LSCD) transplantation of ex vivo expanded limbal epithelial cells (LECs). We here present clinical data of the first nine patients with LSCD who were transplanted with autologous LECs expanded in medium completely free of any animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), and with autologous human serum as the only growth supplement., Methods: We conducted a noncomparative retrospective study of patients with LSCD at our centre between 2009 and 2011. The diagnosis was based on history and clinical signs. A biopsy was taken from healthy limbus, and the epithelium was expanded on amniotic membrane (AM) in medium containing autologous serum and subsequently transplanted to the affected eye., Results: Successful outcome was defined as relief of pain and photophobia and/or improved best corrected visual acuity (BCVA) and/or reestablishment of a stable corneal epithelium and regression of corneal vascularization. Five of the nine transplanted patients (55.6%) had an improvement in either subjective symptoms or objective findings (11- to 28-month follow-up)., Conclusions: Our clinical study shows that patients with LSCD can be treated successfully with transplantation of LECs expanded ex vivo in a medium with autologous serum as the only growth supplement. The use of this novel culture system, which is devoid of animal-derived products and non-human/recombinant growth factors (including Cholera Toxin), reduces the risks of inter-species disease transmission and host immune responses to xenogenic proteins, both obvious advantages for the patient., (© 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.)

Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB's role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB's role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway's role in LD dynamics and the VLDL pathway., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Pulmonary artery endothelial cells (PAEC) in an intact vessel are continually exposed to serum, but unless injured, do not proliferate, constrained by confluence. In contrast, pulmonary artery smooth muscle cells (PASMC) attain, and maintain, confluence in the presence of minimal serum, protected from serum's stimulatory effects except when the endothelial barrier becomes more permeable. We hypothesized therefore, that confluent PASMC may be less constrained by contact inhibition in the presence of serum than PAEC and tested this idea by exposing confluent non-transformed human PAEC and PASMC to media containing increasing concentrations of fetal bovine serum (FBS) and determining cell growth over 7 days. PAEC that had attained confluence in low serum did not proliferate even when exposed to 5% serum, the highest concentration tested. In contrast, PASMC that attained confluence in low serum did proliferate once serum levels were increased, an effect that was dose dependent. Consistent with this observation, PASMC had more BrdU incorporation and a greater percentage of cells in S phase in 5% compared to 0.2% FBS, whereas no such difference was seen in PAEC. These results suggest that confluent human PAEC are resistant to the stimulatory effects of serum, whereas confluent PASMC can proliferate when serum levels are increased, an effect mediated in part by differences in phosphoinositide 3-kinase activation. This observation may be relevant to understanding the PASMC hyperplasia observed in humans and animals with pulmonary hypertension in which changes in endothelial permeability due to hypoxia or injury expose the underlying smooth muscle to serum.

Purpose: Pterygium is characterized as invasive, proliferative fibrovascular altered conjunctival tissue. The extensive vascular network is likely to significantly contribute to the progression of the disease. In the present study, we investigated the effects of reduced serum (to mimic a suppressed blood supply) on cell signaling events and the functional role of the calcium store in cultured, pterygial-derived fibroblasts., Methods: Pure fibroblast cultures were established from cell outgrowths of pterygial tissue. Growth and migration of pterygial-derived fibroblast was evaluated using a patch growth assay, MTS assay, and a scratch wound assay. Intracellular calcium levels were determined using Fura-2 detection in response to ligand stimulation using a 96-well plate format., Results: A progressive increase in serum concentration resulted in promotion of pterygial cell growth detected using the MTS assay. A significant increase in intracellular calcium level was observed in response to histamine (10 and 100 μM), ATP (10 and 100 μM), acetylcholine (10 and 100 μM) and epidermal growth factor (10 ng/mL) in serum-maintained cells. However, no significant changes were observed when cells were maintained in serum-free medium. Thapsigargin (1 μM), a Ca-ATPase inhibitor, induced a significantly greater increase in intracellular calcium level in the serum-maintained group relative to serum-starved cells. In both cases, elevation of intracellular calcium was reduced when calcium-free bathing medium was used. Preincubation of cells with 1 μM thapsigargin ablated ligand-induced calcium responses. Disruption of calcium signaling through thapsigargin treatment significantly perturbed cell growth and migration., Conclusions: Receptor-induced calcium signaling activity is suppressed in pterygial-derived fibroblasts in response to serum deprivation. This correlates with reduced growth rates and a depleted endoplasmic reticulum calcium store. The store plays a key role in cell growth and migration of pterygial-derived fibroblasts. Therefore, the strategic reduction of the vascular network in pterygium will affect the calcium store level and in turn affect functional responses associated with pterygia.

Downregulation of the preadipocyte marker Delta-like 1 homologue (Dlk1), an inhibitor of adipogenesis, has been suggested to be a prerequisite for adipogenic differentiation to occur, and low Dlk1 levels are often used to verify adipogenesis. Mouse preadipocytic cell lines such as 3T3-L1, as well as primary derived preadipocytes, are important models to study adipogenic differentiation and obesity. However, in vitro adipogenic differentiation of primary derived preadipocytes remains incomplete, and identification of factors that will improve the adipogenic differentiation process is thus of high value. In this study we show that horse serum fails to improve adipogenic differentiation of mouse preadipocytes (both 3T3-L1 cells and primary derived mouse preadipocytes) as otherwise reported for bone marrow derived adipogenic precursors. Unexpectedly, while Dlk1 levels were indeed decreased using horse serum, this did not correlate with a high degree of adipogenic differentiation. In conclusion, our novel results thus reveal that horse serum clearly is insufficient for adipogenic differentiation of mouse preadipocytes and that low levels of Dlk1 alone are a poor marker of mouse in vitro adipogenesis. We would also like to emphasize that it is very important for the field of cellular differentiation that researchers thoroughly investigate the effect of individual reagents in their protocols. Such data will increase understanding of the limitations and possibilities of individual systems., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O(3) per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H(2)O(2)) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Unlabelled: Chitosan is a biocompatible polysaccharide composed of glucosamine and N-acetylglucosamine. The polymer has a unique behavior of fluctuating between soluble chains at pH 6 and insoluble microparticles at pH 7. The purpose of this study was to test the hypothesis that chitosan structure, solubility state, and serum influence the rate of cell uptake. Chitosans with 80% and 95% degree of deacetylation (medium and low viscosity) were tagged with rhodamine and analyzed for particle size, media solubility, and uptake by HEK293 epithelial cells using live confocal microscopy and flow cytometry. In media pH 7.4 with or without 10% serum, chitosans fully precipitated into 0.5 to 1.4 µm diameter microparticles with a slight negative charge. During 24 h of culture in serum-free medium, chitosan particles remained extracellular. In cultures with serum, particles were taken up into intracellular vesicles in a serum dose-dependent manner. Opsonization of chitosan with serum, or replacement of serum by epidermal growth factor (EGF) failed to mediate serum-free chitosan particle uptake. Serum stimulated cells to acidify the media, partly by lactate generation. Media acidified to pH 6.5 by 7 mM lactate maintained 50% of chitosan in the soluble fraction, and led to minor uniform serum-free uptake in small vesicles., Conclusion: Media acidification mediates minor in vitro uptake of non-biofouled soluble chitosan chains, while serum-biofouled insoluble chitosan microparticles require sustained serum exposure to generate energy required for macropinocytosis.

Neutrophils, in cooperation with serum, are vital gatekeepers of a host's microbiome and frontline defenders against invading microbes. Yet because human neutrophils are not amenable to many biological techniques, the mechanisms governing their immunological functions remain poorly understood. We here combine state-of-the-art single-cell experiments with flow cytometry to examine how temperature-dependent heat treatment of serum affects human neutrophil interactions with "target" particles of the fungal model zymosan. Assessing separately both the chemotactic as well as the phagocytic neutrophil responses to zymosan, we find that serum heat treatment modulates these responses in a differential manner. Whereas serum treatment at 52°C impairs almost all chemotactic activity and reduces cell-target adhesion, neutrophils still readily engulf target particles that are maneuvered into contact with the cell surface under the same conditions. Higher serum-treatment temperatures gradually suppress phagocytosis even after enforced cell-target contact. Using fluorescent staining, we correlate the observed cell behavior with the amounts of C3b and IgG deposited on the zymosan surface in sera treated at the respective temperatures. This comparison not only affirms the critical role of complement in chemotactic and adhesive neutrophil interactions with fungal surfaces, but also unmasks an important participation of IgGs in the phagocytosis of yeast-like fungal particles. In summary, this study presents new insight into fundamental immune mechanisms, including the chemotactic recruitment of immune cells, the adhesive capacity of cell-surface receptors, the role of IgGs in fungal recognition, and the opsonin-dependent phagocytosis morphology of human neutrophils. Moreover, we show how, by fine-tuning the heat treatment of serum, one can selectively study chemotaxis or phagocytosis under otherwise identical conditions. These results not only refine our understanding of a widely used laboratory method, they also establish a basis for new applications of this method.

Purpose: We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders., Methods: The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR., Results: We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP., Conclusions: PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-β1 (TGF-β1), which is presumed to be reduced systemically with advanced age. The aim of this study was to investigate whether human serum from elderly donors would have an inhibiting effect on the expression of collagen and collagen-related genes as well as on cell proliferative capacity in tendon cells from young individuals. There was no difference in systemic TGF-β1 levels in serum obtained from young and elderly donors, and we found no difference in collagen expression when cells were subjected to human serum from elderly versus young donors. In addition, tendon cell proliferation was similar when culture medium was supplemented with serum of different donor age. These findings suggest that factors such as the cell intrinsic capacity or the tissue-specific environment rather than systemic circulating factors are important for functional capacity throughout life in human tendon cells., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Pygeum africanum (Tadenan) is a popular phytotherapeutic agent used in the treatment of symptomatic benign prostatic hyperplasia. The active compounds of the drug have not been identified, and determining the plasma concentration of the drug is, therefore, not possible. Because there are conflicting results on the efficacy of this drug, we aimed to investigate its effect on prostate cell growth in vitro using human serum collected before and after Pygeum africanum intake. We used primary and organotypic cultures of human prostatic stromal myofibroblast cell line WPMY and prostatic epithelial cell line PNT2. We also used fresh benign prostatic tissue. The serum of a treated man induced decreases in the proliferation of primary cells, organotypic cells and WPMY cells but not PNT2 cells. We also analysed the effect of treated serum on the gene expression profile of WPMY cells. The transcriptome analysis revealed an upregulation of genes involved in multiple tumour suppression pathways and a downregulation of genes involved in inflammation and oxidative-stress pathways. The oral intake of Pygeum africanum resulted in serum levels of active substances that were sufficient to inhibit the proliferation of cultured myofibroblasts prostatic cells. This inhibition was associated with changes in the transcriptome.

Purpose: To compare the effects of topical human amniotic fluid (HAF), topical human serum (HS), and topical artificial tears in a mouse model of dry eye., Methods: Thirty C57BL/6 mice were divided into 3 treatment groups: HAF, HS, and preservative-free artificial tears. Dry eye was induced by an injection of botulinum toxin B (BTX-B) into the lacrimal gland. Tear production and ocular surface fluorescein staining were evaluated in each mouse at 6 time points during a 4-week period. Goblet cell density was assessed in stained histological sections. Apoptotic keratocytes were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling test assay., Results: A significant decrease in tear production was observed 3 days after BTX-B injection in all groups. At week 1, the HAF and HS groups had improved tear production compared with the control group (P < 0.001 and P = 0.003, respectively). HAF had a significantly improved fluorescein staining score compared with the HS (P = 0.043) and control (P = 0.007) groups at week 2. Goblet cell density was significantly decreased in the control group compared with the HAF and HS groups (P < 0.001). No difference in the amount of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive keratocytes was observed among the groups., Conclusion: HAF was superior to HS and artificial tears for improving corneal staining within 2 weeks of therapy in this induced mouse model of keratoconjunctivitis sicca. Clinical studies are needed to ascertain the benefits of these therapies in patients with ocular surface disorders associated with dry eye.