RESUMO
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/MSALL 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 R2 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.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Glândulas Tarsais/efeitos dos fármacos , Rosiglitazona/farmacologia , Triglicerídeos/metabolismo , Contagem de Células , Diferenciação Celular , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Lipidômica , Glândulas Tarsais/metabolismo , Soro/fisiologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Anti-Helmínticos/farmacologia , Proteínas de Helminto/metabolismo , Praziquantel/farmacologia , Schistosoma/efeitos dos fármacos , Soro/fisiologia , Animais , Resistência a Medicamentos , Feminino , Proteínas de Helminto/genética , Humanos , Evasão da Resposta Imune , Masculino , Schistosoma/metabolismo , Schistosoma/ultraestrutura , Fatores SexuaisRESUMO
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.
Assuntos
Desenvolvimento Muscular/efeitos dos fármacos , Mioblastos/citologia , Soro/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Pessoa de Meia-Idade , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Soro/metabolismo , Soroalbumina Bovina/farmacologiaRESUMO
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.
Assuntos
Academias e Institutos/organização & administração , Doenças da Córnea/terapia , Síndromes do Olho Seco/terapia , Soluções Oftálmicas/administração & dosagem , Oftalmologia/organização & administração , Soro , Avaliação da Tecnologia Biomédica/normas , Doenças da Córnea/patologia , Epitélio Corneano/patologia , Humanos , Soro/fisiologia , Resultado do Tratamento , Estados UnidosRESUMO
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.
Assuntos
Proteínas Contráteis/genética , Ganho de Peso na Gestação , Miócitos de Músculo Liso/metabolismo , Progranulinas/fisiologia , Útero/metabolismo , Adulto , Células Cultivadas , Proteínas Contráteis/metabolismo , Meios de Cultivo Condicionados/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Ganho de Peso na Gestação/genética , Ganho de Peso na Gestação/fisiologia , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Obesidade/genética , Obesidade/metabolismo , Obesidade/fisiopatologia , Parto/sangue , Parto/metabolismo , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/metabolismo , Complicações na Gravidez/fisiopatologia , Progranulinas/sangue , Progranulinas/farmacologia , Soro/fisiologia , Contração Uterina/genética , Contração Uterina/metabolismo , Útero/citologiaRESUMO
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.
Assuntos
Reagentes de Ligações Cruzadas , Ceratocone/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Riboflavina/uso terapêutico , Soro/fisiologia , Cicatrização/fisiologia , Adolescente , Adulto , Colágeno/metabolismo , Substância Própria/efeitos dos fármacos , Substância Própria/metabolismo , Desbridamento , Epitélio Corneano/fisiologia , Feminino , Humanos , Ceratocone/metabolismo , Masculino , Estudos Prospectivos , Raios Ultravioleta , Adulto JovemRESUMO
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.
Assuntos
Síndromes do Olho Seco/diagnóstico , Síndromes do Olho Seco/terapia , Padrões de Prática Médica/estatística & dados numéricos , Adulto , Ciclosporina/administração & dosagem , Técnicas de Diagnóstico Oftalmológico , Síndromes do Olho Seco/epidemiologia , Síndromes do Olho Seco/fisiopatologia , Feminino , Fluoresceína/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Gana/epidemiologia , Humanos , Imunossupressores/administração & dosagem , Lubrificantes Oftálmicos , Masculino , Pessoa de Meia-Idade , Optometristas/estatística & dados numéricos , Concentração Osmolar , Plug Lacrimal , Soro/fisiologia , Inquéritos e Questionários , Lágrimas/fisiologia , Resultado do Tratamento , Adulto JovemRESUMO
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-ß1 (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.
Assuntos
Diferenciação Celular/fisiologia , Fibroblastos/fisiologia , Traumatismos dos Nervos Periféricos/cirurgia , Recuperação de Função Fisiológica/fisiologia , Células de Schwann/fisiologia , Células de Schwann/transplante , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Locomoção/fisiologia , Masculino , Microscopia Eletrônica , Proteína P0 da Mielina/metabolismo , Traumatismos dos Nervos Periféricos/fisiopatologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/ultraestrutura , Soro/fisiologia , Pele/citologia , Fatores de Tempo , Tretinoína/farmacologiaRESUMO
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 â¼ 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.
Assuntos
Ilhas de CpG , Genes cdc/efeitos dos fármacos , Leucemia Mieloide Aguda , Oligodesoxirribonucleotídeos/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Evasão Tumoral/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Estabilidade de Medicamentos , Genes cdc/imunologia , Terapia Genética/métodos , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/uso terapêutico , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/genética , Soro/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Reatores Biológicos , Coleta de Amostras Sanguíneas/métodos , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/instrumentação , Terapia Baseada em Transplante de Células e Tecidos/métodos , Soro/fisiologia , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/fisiologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Meios de Cultura/farmacologia , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Dados Preliminares , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
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.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Regiões Promotoras Genéticas , Soro/fisiologia , Ácido Tióctico/farmacologia , Células Hep G2 , Histonas/metabolismo , Humanos , Ácido Tióctico/análogos & derivadosRESUMO
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.
Assuntos
Epitélio Corneano/fisiologia , Sangue Fetal/fisiologia , Ceratoplastia Penetrante , Soro/fisiologia , Adulto , Doenças da Córnea/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reepitelização , Acuidade Visual/fisiologia , Cicatrização/fisiologiaRESUMO
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).
Assuntos
Coagulação Sanguínea , Coleta de Amostras Sanguíneas , Hemólise , Controle de Qualidade , Testes Hematológicos , Humanos , Flebotomia/normas , Soro/fisiologiaRESUMO
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 µmol/L) on HPFs was lower than on HCFs (100 µmol/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.
Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histamina/farmacologia , Pterígio/patologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Túnica Conjuntiva/citologia , Difenidramina/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Nizatidina/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Soro/fisiologiaRESUMO
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.
Assuntos
Plaquetas/fisiologia , Movimento Celular , Proliferação de Células , Matriz Extracelular/metabolismo , Meniscos Tibiais/citologia , Plasma Rico em Plaquetas/fisiologia , Soro/fisiologia , Idoso , Células Cultivadas , Quimiotaxia , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Feminino , Humanos , Masculino , Meniscos Tibiais/metabolismo , Menisco , Pessoa de Meia-Idade , Proteoglicanas/metabolismoRESUMO
The pathogenic hallmark of systemic lupus erythematosus is the autoimmune response against self nuclear Ags, including dsDNA. The increased expression of the proinflammatory cytokine IL-1ß has been found in the cutaneous lesion and PBMCs from lupus patients, suggesting a potential involvement of this cytokine in the pathogenesis of lupus. IL-1ß is produced primarily by innate immune cells such as monocytes and can promote a Th17 cell response, which is increased in lupus. IL-1ß production requires cleaving pro-IL-ß into IL-1ß by the caspase-1-associated multiprotein complex called inflammasomes. In this study we show that self dsDNA induces IL-1ß production from human monocytes dependent on serum or purified IgG containing anti-dsDNA Abs by activating the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. Reactive oxygen species (ROS) and K(+) efflux were involved in this activation. Knocking down the NLRP3 or inhibiting caspase-1, ROS, and K(+) efflux decreased IL-1ß production. Supernatants from monocytes treated with a combination of self dsDNA and anti-dsDNA Ab(+) serum promoted IL-17 production from CD4(+) T cells in an IL-1ß-dependent manner. These findings provide new insights in lupus pathogenesis by demonstrating that self dsDNA together with its autoantibodies induces IL-1ß production from human monocytes by activating the NLRP3 inflammasome through inducing ROS synthesis and K(+) efflux, leading to the increased Th17 cell response.
Assuntos
Autoanticorpos/fisiologia , Proteínas de Transporte/metabolismo , DNA/fisiologia , Interleucina-1beta/biossíntese , Monócitos/imunologia , Monócitos/metabolismo , Adulto , Autoanticorpos/sangue , Proteínas de Transporte/sangue , Proteínas de Transporte/fisiologia , Caspase 1/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , DNA/sangue , DNA/imunologia , Humanos , Interleucina-1beta/sangue , Células Jurkat , Monócitos/citologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Soro/fisiologiaRESUMO
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-ß1 (TGF-ß1) 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-ß1 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.
Assuntos
Receptores ErbB/análise , Fibronectinas/análise , Falência Renal Crônica/sangue , Fator de Crescimento Derivado de Plaquetas/análise , Soro/química , Fator de Crescimento Transformador beta1/análise , Adulto , Idoso , Linhagem Celular , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/citologia , Feminino , Voluntários Saudáveis , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Soluções Oftálmicas , Soro/fisiologiaRESUMO
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.
Assuntos
Músculo Esquelético/citologia , Soro/fisiologia , Engenharia Tecidual , Animais , Sinalização do Cálcio , Bovinos , Técnicas de Cultura de Células , Linhagem Celular , Meios de Cultura , Glicólise , Metabolismo dos Lipídeos , Camundongos , Proteínas Musculares/metabolismo , Oxirredução , FenótipoRESUMO
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.
Assuntos
Apoptose/fisiologia , Células Endoteliais/fisiologia , Infarto do Miocárdio/fisiopatologia , Soro/fisiologia , Idoso , Idoso de 80 Anos ou mais , Técnicas de Imagem Cardíaca , Estudos de Casos e Controles , Sobrevivência Celular/fisiologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/terapia , Necrose/fisiopatologia , Intervenção Coronária PercutâneaRESUMO
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.