RESUMO
The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.
Assuntos
Opacidade da Córnea , Epitélio Corneano , Limbo da Córnea , Humanos , Limbo da Córnea/metabolismo , Córnea/metabolismo , Epitélio Corneano/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Diferenciação Celular/genética , Opacidade da Córnea/metabolismoRESUMO
The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.
Assuntos
Sítios Frágeis do Cromossomo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Endonucleases/metabolismo , Mitose , RecQ Helicases/metabolismo , Origem de Replicação , Sítios de Ligação , Proteína Quinase CDC2 , Instabilidade Cromossômica , Segregação de Cromossomos , Quinases Ciclina-Dependentes/metabolismo , DNA/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Células HEK293 , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Interferência de RNA , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , Fatores de Tempo , TransfecçãoRESUMO
PURPOSE: To compare the outcomes of deep anterior lamellar keratoplasty (DALK) using dehydrated versus standard organ culture-stored donor corneas for eyes with keratoconus. DESIGN: Prospective, randomized, single-center trial conducted in Italy. PARTICIPANTS: Adult patients (age ≥ 18 years) with keratoconus scheduled for elective DALK. METHODS: Patients undergoing successful type 1 bubble pneumatic dissection using a standard DALK technique were randomized during surgery to receive either dehydrated (n = 30) or standard organ culture-stored (n = 30) donor corneas. MAIN OUTCOME MEASURES: The primary study outcome was best spectacle-corrected visual acuity (BSCVA) 12 months after surgery. Secondary outcomes were refractive astigmatism (RA), endothelial cell density (ECD), and complication rates. RESULTS: Postoperative BSCVA did not significantly differ between groups at both time points: mean difference at 6 months was 0.030 logarithm of the minimum angle of resolution (logMAR; 95% confidence interval [CI], -0.53 to 0.10 logMAR; P = 0.471) and at 12 months was -0.013 logMAR (95% CI, -0.10 to 0.08 logMAR; P = 0.764). No significant differences between groups were observed in terms of postoperative RA and ECD at all time points. In the first 3 days after DALK, an epithelial defect was present in 10 patients (33%) in the organ culture cornea group and in 29 patients (97%) in the dehydrated cornea group. Complete re-epithelialization was achieved by day 7 in all patients (100%) in both groups. CONCLUSIONS: The study provides evidence that the use of dehydrated corneas is noninferior to the use of standard organ culture donor corneas for DALK. Corneal tissue dehydration represents a viable solution that can allow long-term cornea preservation and avoid wastage of unused corneas. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
Assuntos
Transplante de Córnea , Ceratocone , Técnicas de Cultura de Órgãos , Preservação de Órgãos , Doadores de Tecidos , Acuidade Visual , Humanos , Estudos Prospectivos , Masculino , Feminino , Adulto , Transplante de Córnea/métodos , Acuidade Visual/fisiologia , Ceratocone/cirurgia , Ceratocone/fisiopatologia , Preservação de Órgãos/métodos , Pessoa de Meia-Idade , Endotélio Corneano/patologia , Adulto Jovem , Córnea/cirurgia , Contagem de CélulasRESUMO
Olive leaves are a rich source of polyphenols with healthful properties and represent one of the most abundant waste products of olive oil production. The aims of this study were to explore the phenolic composition of olive leaves from the three main Tuscan cultivars (Leccino, Moraiolo and Frantoio) collected in Siena and Grosseto provinces and to investigate the possible use of these compounds as varietal and geographic origin markers. Discriminant factorial analysis (DFA) was used for distinguishing between different cultivars and locations. Apigenin and caffeoyl-secologanoside showed significant differences between cultivars. DFA showed that ligstroside, apigenin and luteolin have the most influence in determining the differences between sites, whereas total polyphenols, olacein and hydroxytyrosol acetate allowed for separation between leaves from the same province. The results of the present study indicate that concentrations of phenolic compounds, measured through high-resolution mass spectrometry, can be used as a marker for both the cultivar and of geographical origin of olive leaves, and possibly of olive-related products, as well as across small geographic scales (less than 50 km distance between sites).
Assuntos
Olea , Fenóis , Folhas de Planta , Olea/química , Olea/classificação , Folhas de Planta/química , Fenóis/análise , Fenóis/química , Itália , Polifenóis/análise , Polifenóis/química , Biomarcadores , Geografia , Extratos Vegetais/químicaRESUMO
BACKGROUND: Rare primary malignant bone sarcomas (RPMBS) account for 5%-10% of primary high-grade bone tumors and represent a major treatment challenge. The outcome of patients with RPMBS enrolled in the EUROpean Bone Over 40 Sarcoma Study (EURO-B.O.S.S) is presented. METHODS: Inclusion criteria were as follows: age from 41 to 65 years and a diagnosis of high-grade spindle cell, pleomorphic, or vascular RPMBS. The chemotherapy regimen included doxorubicin 60 mg/m2 , ifosfamide 9 g/m2 , and cisplatin 90 mg/m2 ; postoperative methotrexate 8 g/m2 was added in case of a poor histologic response. Version 2.0 of the Common Terminology Criteria for Adverse Events, Kaplan-Meier curves, log-rank tests, and univariate Cox regression models were used. RESULTS: In total, 113 patients were evaluable for analysis. The median patient age was 52 years (range, 40-66 years), and 67 patients were men. Eighty-eight tumors were categorized as undifferentiated pleomorphic sarcomas (UPS), 20 were categorized as leiomyosarcomas, three were categorized as fibrosarcomas, and two were categorized as angiosarcomas. Eighty-three of 113 tumors were located in the extremities. Ninety-five of 113 patients presented with no evidence of metastases. After a median follow-up of 6.8 years (interquartile range [IQR], 3.5-9.8 years), the 5-year overall survival rate for patients with localized disease was 68.4% (IQR, 56.9%-77.5%), and it was 71.7% (IQR, 58.1%-81.6%) for patients with UPS and 54.9% (IQR, 29.5%-74.5%) for patients with leiomyosarcoma. Grade III-IV hematologic toxicity was reported in 81% patients; 23% had grade II-III neurotoxicity, and 37.5% had grade I-II nephrotoxicity. Five-year overall survival was significantly better for patients with localized disease, for patients who obtained surgical complete remission, and when the primary tumor was located in the extremities. CONCLUSIONS: The survival of patients who had RPMBS in the current series was similar to that of age-matched patients who had high-grade osteosarcoma treated according to the same protocol. An osteosarcoma-like chemotherapy may be proposed in patients who have RPMBS.
Assuntos
Neoplasias Ósseas , Leiomiossarcoma , Osteossarcoma , Sarcoma , Masculino , Humanos , Adulto , Pessoa de Meia-Idade , Idoso , Feminino , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sarcoma/patologia , Osteossarcoma/tratamento farmacológico , Terapia Combinada , Neoplasias Ósseas/patologia , Doxorrubicina , Ifosfamida , Leiomiossarcoma/tratamento farmacológicoRESUMO
Transparency of the human cornea is responsible for clear vision, which is maintained by a monolayer of non-proliferative human corneal endothelial cells (HCEnCs). Dysfunction of these cells can result in irreversible corneal blindness. It is important to identify key factors that limit the proliferation of HCEnCs and thus attempt to reverse them. Extracellular vesicles contain cargo which includes microRNAs (miRNAs) that can modulate a cellular function. In non small cell lung cancer, expression of miR-195-5p has been shown to inhibit proliferation; therefore, we aimed to investigate the inhibitory effect of miR-195-5p in inducing the proliferation of HCEnCs. Human corneal endothelial cell line (HCEC-12) and primary HCEnCs were cultured with miR-195-5p scramble, mimic or inhibitor. Corneal tissues from human cadaveric and FECD donors, and from pigs, mice and rabbits, were used for RT-PCR. miR-195-5p showed an abundance value of 11,363.31 a.u. When normalized against HCEnCs from cadaveric donors, FECD tissues showed a significant upregulation of miR-195-5p (p < 0.05) but was significantly downregulated in pig (p < 0.001), mouse (p < 0.01) and rabbit (p < 0.001) CEnCs, which have known proliferative capacity. Proliferation, cell doubling, and wound healing rates were significantly higher when miR-195-5p was inhibited. Inhibiting miR-195-5p showed a significant improvement in viability (HEC staining), decreased cell apoptosis (TdT-dNTP staining) and expression of ZO-1, NA+/K+-ATPase and Ki-67 markers. Expression of miR-195-5p is found in HCEnCs and FECD cells, which restricts the proliferation of these cells. However, inhibiting miR-195-5p can induce the proliferation of HCEnCs, which opens exciting directions for future research in prolonging FECD pathogenesis by increasing the proliferative capacity of HCEnCs using anti-miR therapy in vivo.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Humanos , Animais , Camundongos , Coelhos , Suínos , Células Endoteliais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proliferação de Células , CadáverRESUMO
Human epithelial stem cells (ESCs) are characterized by long-term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly-ectodermal dysplasia-clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ-secretase inhibitor; N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine T-butyl ester). All cells were able to differentiate in vitro but exhibited variable self-renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up- or down-regulated during their lifetime, thus allowing to identify druggable gene networks and off-the-shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells.
Assuntos
Fenda Labial , Fissura Palatina , Displasia Ectodérmica , Fenda Labial/diagnóstico , Fissura Palatina/diagnóstico , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/genética , Humanos , Inibidores da Agregação Plaquetária , Células-TroncoRESUMO
BACKGROUND: According to retrospective osteosarcoma series, ABCB1/P-glycoprotein (Pgp) overexpression predicts for poor outcomes. A prospective trial to assess a risk-adapted treatment strategy using mifamurtide in Pgp+ patients was performed. METHODS: This was a phase 2, multicenter, uncontrolled trial including patients 40 years old or younger with nonmetastatic extremity high-grade osteosarcoma stratified according to Pgp expression. All patients received high-dose methotrexate, doxorubicin, and cisplatin (MAP) preoperatively. In Pgp+ patients, mifamurtide was added postoperatively and combined with MAP for a good histologic response (necrosis ≥ 90%; good responders [GRs]) or with high-dose ifosfamide (HDIFO) at 3 g/m2 /d on days 1 to 5 for a histologic response < 90% (poor responders [PRs]). Pgp- patients received MAP postoperatively. After an amendment, the cumulative dose of methotrexate was increased from 60 to 120 g/m2 (from 5 to 10 courses). The primary end point was event-free survival (EFS). A postamendment analysis was performed. RESULTS: In all, 279 patients were recruited, and 194 were included in the postamendment analysis: 70 (36%) were Pgp-, and 124 (64%) were Pgp+. The median follow-up was 51 months. For Pgp+ patients, 5-year EFS after definitive surgery (null hypothesis, 40%) was 69.8% (90% confidence interval [CI], 62.2%-76.2%): 59.8% in PRs and 83.7% in GRs. For Pgp- patients, the 5-year EFS rate was 66.4% (90% CI, 55.6%-75.1%). CONCLUSIONS: This study showed that adjuvant mifamurtide, combined with HDIFO for a poor response to induction chemotherapy, could improve EFS in Pgp+ patients. Overall, the outcomes compared favorably with previous series. Mifamurtide and HDIFO as salvage chemotherapy are worth further study.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/uso terapêutico , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Neoplasias Ósseas/cirurgia , Criança , Intervalo Livre de Doença , Extremidades/patologia , Humanos , Ifosfamida , Itália , Metotrexato , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Osteossarcoma/cirurgia , Estudos Prospectivos , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
The phase III clinical study of adjuvant liposomal muramyl tripeptide (MTP-PE) in resected high-grade osteosarcoma (OS) documented positive results that have been translated into regulatory approval, supporting initial promise for innate immune therapies in OS. There remains, however, no new approved treatment such as MTP-PE for either metastatic or recurrent OS. Whilst the addition of different agents, including liposomal MTP-PE, to surgery for metastatic or recurrent high-grade osteosarcoma has tried to improve response rates, a mechanistic hiatus exists in terms of a detailed understanding the therapeutic strategies required in advanced disease. Here we report a Bayesian designed multi-arm, multi-centre, open-label phase II study with randomisation in patients with metastatic and/or recurrent OS, designed to investigate how patients with OS might respond to liposomal MTP-PE, either given alone or in combination with ifosfamide. Despite the trial closing because of poor recruitment within the allocated funding period, with no objective responses in eight patients, we report the design and feasibility outcomes for patients registered into the trial. We demonstrate the feasibility of the Bayesian design, European collaboration, tissue collection with genomic analysis and serum cytokine characterisation. Further mechanistic investigation of liposomal MTP-PE alone and in combination with other agents remains warranted in metastatic OS.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Teorema de Bayes , Biomarcadores , Neoplasias Ósseas/patologia , Humanos , Lipossomos , Recidiva Local de Neoplasia/tratamento farmacológico , Osteossarcoma/patologia , FosfatidiletanolaminasRESUMO
COVID-19's impact on the ocular surface has already been recognized, however the molecular mechanisms induced by the infection on the ocular surface are still unclear. The aim of this paper is to provide a first overview of the transcriptional perturbations caused by SARS-CoV-2 on the ocular surface by analyzing gene expression profile of corneoscleral ring samples from post-mortem SARS-CoV-2 positive donors (PD). The presence of SARS-CoV-2 on the ocular surface, in tears and corneal tissues has rarely been detected in infected individuals in both the presence and the absence of ocular manifestations. In this preliminary study, 6 human corneoscleral tissues of 3 PD and two tissues from a negative donor (CTRL) were obtained at the local eye bank. The presence of genomic and sub-genomic SARS-CoV-2 RNAs was assessed by qRT-PCR, while transcriptome analysis (RNA-sequencing) was performed by Illumina. Principal Component Analysis (PCA), search for differentially expressed genes (DEGs) and Gene Ontology (GO)-enrichment analysis were performed. Three samples from PD were found positive for SARS-CoV-2 genomic RNA, although the absence of sub-genomic RNAs indicated an inactive virus. PCA analysis grouped 3 different clusters, one including CTRL, and the other two including, respectively, PD with undetected SARS-CoV-2 (PD-SARS-neg) and PD with detected SARS-CoV-2 (PD-SARS-pos). The DEGs in common with the 2 PD clusters included several genes associable to the interferon pathway, such as ADAMTS4, RSAD2, MMP1, IL6, ISG15 and proinflammatory cytokines. Among the down-regulated genes we found AQP5. GO analysis revealed 77 GO terms over-represented in PD-SARS-neg vs. CTRL, and 17 GO terms in PD-SARS-pos vs. CTRL. The presence of SARS-CoV-2 RNA and RNA-sequencing reads in ocular surface tissues supports the possibility that the eye acts as an entry route. The modulation of early responsive genes, together with several ISGs suggests a potential protective responsiveness of the ocular tissues to SARS-CoV-2.
Assuntos
COVID-19 , Córnea/metabolismo , Humanos , RNA Viral , SARS-CoV-2 , TranscriptomaRESUMO
BACKGROUND: Chemotherapy negatively affects gonadal function, often resulting in premature ovarian failure (POF) due to ovarian reserve depletion. Mechanisms of gonadotoxicity, such as primordial follicle overactivation and "burnout", remain to be established. Ovarian tissue cryopreservation (OTC) before treatment plays an important role in safeguarding fertility. METHODS: This is a prospective observational study that aims to evaluate the feasibility of OTC after chemotherapeutic treatment initiation. Patients were divided into 2 groups depending on whether they received chemotherapy before the harvesting procedure (Group 1) or not (Group 2). The main outcomes of this study are serum anti-Mullerian hormone (AMH) levels and histological follicular counts on ovarian tissue biopsies. RESULTS: Between 2012 and 2020, 79 patients underwent OTC at our Hospital. Follicular counts from the ovarian biopsies of 30 post-pubertal patients and respective serum AMH levels were included in the analysis. AMH levels did not significantly differ between the 2 groups (P = 0.70) as well as the number of primordial follicles (P = 0.73). Ovarian biopsies of patients from Group 1 showed a higher number of primary follicles (P = 0.04) and atretic follicles (P = 0.05) with respect to Group 2. CONCLUSIONS: In conclusion, OTC appears to be feasible even after the start of chemotherapeutic treatment, since in treated patients, the main ovarian reserve indicators (number of primordial follicles and serum AMH levels) were not significantly reduced compared to untreated patients. The "burnout" theory of chemotherapeutic damage to the ovary seems to be supported by the higher number of primary follicles found in the ovaries of patients who received chemotherapy before OTC.
Assuntos
Antineoplásicos , Reserva Ovariana , Hormônio Antimülleriano , Antineoplásicos/efeitos adversos , Feminino , Humanos , Folículo Ovariano , Ovário/patologia , Estudos ProspectivosRESUMO
PURPOSE: To compare the effects on adhesive and structural properties of newer preservation conditions to those obtained with an established, standardized protocol (dimethyl sulfoxide at -180 °C). In attempt to simplify and enhance the safety of the procedure, we tested dextran-based freezing medium and a dry condition (no medium) at temperatures of -80 °C. METHODS: Five patches of human amniotic membrane were obtained from three different donors. For each donor, five preservation condition were tested: dimethyl sulfoxide at -180 °C, dimethyl sulfoxide at -80 °C, dextran-based medium at -180 °C, dextran-based medium at -80 °C and dry freezing at -80 °C (no medium). At the end of four months storage period, adhesive properties and structure were analyzed. RESULTS: None of the newer preservation protocols showed differences in adhesive and structural properties of the tissues. The stromal layer always kept its adhesiveness, while both structure and basement membrane were not altered by any the preservation protocol. CONCLUSIONS: Switching from liquid nitrogen cryopreservation to -80 °C would reduce manipulation, simplify the procedure, making it also cheaper. The use of dextran-based freezing medium or no medium at all (dry condition) would avoid the potential toxicity of the dimethyl sulfoxide-based freezing media.
Assuntos
Crioprotetores , Dimetil Sulfóxido , Humanos , Crioprotetores/farmacologia , Âmnio , Dextranos , Criopreservação/métodosRESUMO
Phthalocyanines (Pcs) are promising candidates for photodynamic therapy (PDT) due to their absorption in the phototherapeutic window. However, the highly aromatic Pc core leads to undesired aggregation and decreased reactive oxygen species (ROS) production. Therefore, short PEG chain functionalized A3B type asymmetric Pc photosensitizers (PSs) were designed in order to decrease aggregation and increase the aqueous solubility. Here we report the synthesis, characterization, optical properties, cellular localization, and cytotoxicity of three novel Pc-based agents (LC31, MLC31, and DMLC31Pt). The stepwise functionalization of the peripheral moieties has a strong effect on the distribution coefficient (logP), cellular uptake, and localization, as well as photocytotoxicity. Additional experiments have revealed that the presence of the malonic ester moiety in the reported agent series is indispensable in order to induce photocytotoxicity. The best-performing agent, MLC31, showed mitochondrial targeting and an impressive phototoxic index (p.i.) of 748 in the cisplatin-resistant A2780/CP70 cell line, after a low-dose irradiation of 6.95 J/cm2. This is the result of a high photocytotoxicity (IC50 = 157 nM) upon irradiation with near-infrared (NIR) light, and virtually no toxicity in the dark (IC50 = 117 µM). Photocytotoxicity was subsequently determined under hypoxic conditions. Additionally, a preliminarily pathway investigation of the mitochondrial membrane potential (MMP) disruption and induction of apoptosis by MLC31 was carried out. Our results underline how agent design involving both hydrophilic and lipophilic peripheral groups may serve as an effective way to improve the PDT efficiency of highly aromatic PSs for NIR light-mediated cancer therapy.
Assuntos
Neoplasias Ovarianas , Fotoquimioterapia , Linhagem Celular Tumoral , Feminino , Humanos , Mitocôndrias , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologiaRESUMO
Ovarian endometriosis may increase the risk of malignancy. Several studies have suggested atypical endometriosis as the direct precursor of endometriosis-associated ovarian cancer. We performed an advanced, systematic search of the online medical databases PubMed and Medline. The search revealed n = 40 studies eligible for inclusion in this systematic review. Of these, n = 39 were finally included. The results from included studies are characterized by high heterogeneity, but some consistency has been found for altered expression in phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway, ARID1a, estrogen and progesterone receptors, transcriptional, nuclear, and growth factors in atypical endometriosis. Although many targets have been proposed as biomarkers for the presence of atypical endometriosis, none of them has such strong evidence to justify their systematic use in clinical practice, and they all need expensive molecular analyses. Further well-designed studies are needed to validate the evidence on available biomarkers and to investigate novel serum markers for atypical endometriosis.
Assuntos
Endometriose , Neoplasias Ovarianas , Lesões Pré-Cancerosas , Biomarcadores/análise , Carcinoma Epitelial do Ovário , Endometriose/patologia , Feminino , Humanos , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases , Lesões Pré-Cancerosas/patologiaRESUMO
Currently, endothelial keratoplasty is the gold standard for the surgical treatment of Fuchs endothelial corneal dystrophy (FECD). Despite the remarkable success in terms of surgical outcomes, a shortage of corneal donor tissue poses a limitation to performing endothelial keratoplasty in many parts of the world. Cell therapy is a potential alternative strategy to keratoplasty and is currently under investigation. Considering that corneas with FECD may contain relatively healthy endothelial cells, samples obtained by descemetorhexis of eyes undergoing EK for FECD can be used for ex vivo expansion of endothelial cells as an autologous cell culture. In this study, we established corneal endothelial cell cultures derived from 40 patients that underwent endothelial keratoplasty for advanced FECD. Several parameters were evaluated including patient characteristics such as age, gender, and endothelial cell density as well as various processing and cell culture protocols based on different combinations of shipping temperatures, stabilization periods and treatment methods for corneal endothelial cell dissociation. FECD cultures were classified into three groups as: (i) no cells, (ii) cell cultures with endothelial-like morphology or (iii) cell cultures with fibroblast-like features. Our data seem to suggest that some factors can influence FECD cell culture characteristics including young age, high paracentral endothelial cell density, low shipping temperature and short stabilization period prior to cell isolation. Treatment with type 1 collagenase for cell isolation can delay endothelial-to-mesenchymal transition, but does not increase proliferative capacity. Although heterologous corneal endothelial cultures from healthy donors have shown encouraging outcomes, the feasibility of autologous cell therapy as a potential treatment for FECD remains challenging. Low initial cell concentration as well as endothelial to mesenchymal transition are the main obstacles to the application of FECD cultures in the clinical setting.
Assuntos
Lâmina Limitante Posterior/cirurgia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Endotélio Corneano/citologia , Distrofia Endotelial de Fuchs/cirurgia , Idoso , Biomarcadores/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Separação Celular , Endotélio Corneano/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE: To report the impact of establishing and maintaining a high intracameral pressure (ICP) of 200 mmHg on UT-DSAEK graft preparation using an artificial anterior chamber pressuriser (ACP) control unit (Moria SA, Antony, France). METHOD: Retrospective laboratory and clinical study. Four paired donor corneas were mounted on an artificial anterior chamber and subjected to 70 mmHg ("low") and 200 mmHg ("high") ICP using an ACP system. The central corneal thinning rate was measured after 5 min using AS-OCT and the endothelial cell viability was analysed using trypan blue and live/dead staining following 70 mmHg and 200 mmHg ICP. Visual outcomes and complications in a clinical case series of nine patients with bullous keratopathy who underwent UT-DSAEK using 200 mmHg ICP during graft preparation are reported. RESULTS: Laboratory outcomes showed 2 ± 1% and 2 ± 2% dead cells following 70 mmHg and 200 mmHg ICP respectively. Percentage viability in the 70 mmHg group (52.94 ± 5.88%) was not found to be significantly different (p = 0.7) compared to the 200 mmHg group (59.14 ± 10.43%). The mean corneal thinning rate after applying 200 mmHg ICP was 27 ± 13 µm/min centrally (7.2%/min). In the clinical case series, two cases were combined with cataract surgery. Re-bubbling rate was 11%. At the last follow-up (259 ± 109 days), graft thickness was 83 ± 22 µm centrally, endothelial cell density was 1175 ± 566 cell/mm2 and the BCVA of 0.08 ± 0.12 logMAR was recorded with no episodes of rejection. CONCLUSION: ACP control unit for UT-DSAEK graft preparation helps in consistently obtaining UT-DSAEK grafts without compromising endothelial cell viability.
Assuntos
Doenças da Córnea , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Câmara Anterior/cirurgia , Doenças da Córnea/diagnóstico , Doenças da Córnea/cirurgia , Endotélio Corneano , Humanos , Estudos Retrospectivos , Doadores de TecidosRESUMO
STUDY OBJECTIVE: To assess the postoperative likelihood of conception in patients with endometriomas managed by either CO2 laser vaporization or cystectomy. DESIGN: A retrospective study with prospective recording of data. SETTING: University hospital. PATIENTS: One hundred and forty-two patients with symptomatic endometriomas. INTERVENTIONS: Patients underwent a standardized laparoscopic stripping technique (Group 1) or cyst vaporization with CO2 fiber laser (Group 2). Patients wishing to become pregnant were allowed to attempt a spontaneous conception after surgery. If spontaneous conception failed, patients were referred for in vitro fertilization (IVF). MEASUREMENTS AND MAIN RESULTS: The primary objective was to compare pregnancy rates between the 2 groups. The secondary objective was the identification of independent predictors of pregnancy. Thirty-nine women in Group 1 (53.4%) and 39 women in Group 2 (56.5%) desired to conceive after surgery. Three patients (7.7%) in Group 1 became pregnant following donor-IVF and were excluded. Pregnancies were recorded in 72.2% of patients treated with cystectomy and in 74.3% of those managed with CO2 fiber laser (pâ¯=â¯.83). Twenty patients (55.6%) in Group 1 and 14 patients (35.9%) in Group 2 conceived spontaneously (pâ¯=â¯.08). Among patients who failed spontaneous conception, 21 patients (28%) achieved pregnancy through IVF (Group 1: nâ¯=â¯6, 16.7%; Group 2: nâ¯=â¯15, 38.5%; pâ¯=â¯.08). Twenty patients (26.7%) never became pregnant. Age at the time of surgery (odds ratio (OR)â¯=â¯0.86; 95% Confidence intervals (CI): 0.78-0.96, pâ¯=â¯.002) and duration of infertility (OR=0.80; 95% CI: 0.69-0.92, pâ¯=â¯.006) were identified as independent indicators for pregnancy. CONCLUSION: CO2 laser-treated endometrioma is associated with pregnancy rates equal to those observed after cystectomy and favorable IVF outcomes. The one step CO2 fiber laser technique may represent a viable alternative to cystectomy.
Assuntos
Cistectomia , Endometriose/cirurgia , Lasers de Gás/uso terapêutico , Doenças Ovarianas/cirurgia , Taxa de Gravidez , Adulto , Dióxido de Carbono , Feminino , Fertilização in vitro/estatística & dados numéricos , Humanos , Laparoscopia , Gravidez , Estudos RetrospectivosRESUMO
BACKGROUND: Selective lamellar corneal transplantation (keratoplasty) has overtaken full thickness penetrating keratoplasty as the graft choice for endothelial failure. Even more recently eye bank prepared tissues are becoming increasing popular as a way to reduce the risks of tissue loss and stress during endothelial keratoplasty preparation in the surgical theatre. This study compares costs between surgeon and eye bank prepared tissues for Descemet's stripping automated endothelial keratoplasty (DSAEK) and Descemet's membrane endothelial keratoplasty (DMEK). METHODS: Retrospective study conducted at the Royal Liverpool University Hospital including endothelial keratoplasties with a minimum of 6 months follow-up time. Cost analysis included surgical expenses, tissue acquisition fees, cost of patient's ward admission and out-patient expenses, including cost of re-bubbling procedures, costs of visits, anterior segment imaging and optometrist visits within the first 6 months follow-up. RESULTS: Ninety-eight eyes of 98 patients were included in the study of which 42 underwent DSAEK surgery and 56 DMEK surgery. Cost analysis of surgical expenses in the DSAEK group showed a significant difference between using surgeon prepared and eye bank prepared tissue (£3866 ± 296 and £4389 ± 360, respectively; p < 0.01) and the same was found in the DMEK group (£3682 ± 167 and £4162 ± 167 for surgeon prepared and eye bank prepared tissues, respectively; p < 0.01). Cost of out-patient visits did not differ significantly in either group. CONCLUSIONS: At the Royal Liverpool University Hospital, eye bank prepared tissues had higher surgical expenses compared to those prepared by the surgeon, while the post-operative care expenses were similar between the two groups.
Assuntos
Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Cirurgiões , Custos e Análise de Custo , Bancos de Olhos , Humanos , Estudos RetrospectivosRESUMO
The aim of this study is to set up a standardized and reproducible method to determine the potency (= stem cell content) of human conjunctival cell cultures by means of immunofluorescence-based analyses. This will help the development of new Advanced Therapy Medicinal Products (ATMPs) to use in future cell therapy clinical studies when fewer cells are available to perform the quality controls. To achieve this purpose, a reference standard was investigated and the expression levels of ΔNp63α (considered as a marker of conjunctival stem cells) was correlated to cell size. The limbal hTERT cells were used as reference standard to define the expression value of ΔNp63α. The mean intensity value of limbal hTERT cells ranging between 15 and 20 µm in diameter was used to distinguish between ΔNp63α bright and not bright cells. As ΔNp63α bright expression was mainly seen in the smaller cell size group (10-15 µm), we defined as conjunctival stem cells (= potency) those cells which were bright and with sizes between 10 and 15 µm. Assays on cells from clonal analyses were used to validate the method, as they do allow to observe a decrease in potency (Holoclones > Meroclones > Paraclones). The stem cell content of conjunctival grafts was found to be 11.3% ± 5.0 compared to 21.9% ± 0.6, 9.0% ± 8.1 and 0% from Holoclones, Meroclones and Paraclones, respectively. This new method, here named as Standardized Method for Potency Quantification, will allow to detect the potency in conjunctival cell cultures, thus obtaining a quality control assay responding to the GMP standards required for ATMP release.
Assuntos
Técnicas de Cultura de Células , Túnica Conjuntiva , Terapia Baseada em Transplante de Células e Tecidos , Células Epiteliais , Imunofluorescência , Humanos , Limbo da Córnea , Células-TroncoRESUMO
Cancer is one of the main causes of death worldwide. Chemotherapy, despite its severe side effects, is to date one of the leading strategies against cancer. Metal-based drugs present several potential advantages when compared to organic compounds and they have gained trust from the scientific community after the approval on the market of the drug cisplatin. Recently, we reported the ruthenium complex ([Ru(DIP)2 (sq)](PF6 ) (where DIP is 4,7-diphenyl-1,10-phenantroline and sq is semiquinonate) with a remarkable potential as chemotherapeutic agent against cancer, both in vitro and in vivo. In this work, we analyse a structurally similar compound, namely [Ru(DIP)2 (mal)](PF6 ), carrying the flavour-enhancing agent approved by the FDA, maltol (mal). To possess an FDA approved ligand is crucial for a complex, whose mechanism of action might include ligand exchange. Herein, we describe the synthesis and characterisation of [Ru(DIP)2 (mal)](PF6 ), its stability in solutions and under conditions that resemble the physiological ones, and its in-depth biological investigation. Cytotoxicity tests on different cell lines in 2D model and on HeLa MultiCellular Tumour Spheroids (MCTS) demonstrated that our compound has higher activity than cisplatin, inspiring further tests. [Ru(DIP)2 (mal)](PF6 ) was efficiently internalised by HeLa cells through a passive transport mechanism and severely affected the mitochondrial metabolism.