In Vivo and In Silico Studies of the Hepatoprotective Activity of Tert-Butylhydroquinone.

Tert-butylhydroquinone (TBHQ) is a synthetic food antioxidant with biological activities, but little is known about its pharmacological benefits in liver disease. Therefore, this work aimed to evaluate TBHQ during acute liver damage induced by CCl4 (24 h) or BDL (48 h) in Wistar rats. It was found that pretreatment with TBHQ prevents 50% of mortality induced by a lethal dose of CCl4 (4 g/kg, i.p.), and 80% of BDL+TBHQ rats survived, while only 50% of the BDL group survived. Serum markers of liver damage and macroscopic and microscopic (H&E staining) observations suggest that TBHQ protects from both hepatocellular necrosis caused by the sublethal dose of CCl4 (1.6 g/kg, i.p.), as well as necrosis/ductal proliferation caused by BDL. Additionally, online databases identified 49 potential protein targets for TBHQ. Finally, a biological target candidate (Keap1) was evaluated in a proof-of-concept in silico molecular docking assay, resulting in an interaction energy of -5.5491 kcal/mol, which was higher than RA839 and lower than monoethyl fumarate (compounds known to bind to Keap1). These findings suggest that TBHQ increases the survival of animals subjected to CCl4 intoxication or BDL, presumably by reducing hepatocellular damage, probably due to the interaction of TBHQ with Keap1.

Arthroscopic Matrix-Encapsulated Autologous Chondrocyte Implantation: A Pilot Multicenter Investigation in Latin America.

Objective. To evaluate minimum biosecurity parameters (MBP) for arthroscopic matrix-encapsulated autologous chondrocyte implantation (AMECI) based on patients' clinical outcomes, magnetic resonance imaging (MRI) T2-mapping, Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score, and International Cartilage Repair Society (ICRS) second-look arthroscopic evaluation, laying the basis for a future multicenter study. Design. Pilot clinical study. We analyzed the logistics to perform AMECI to treat focal chondral lesions in different hospitals following strict biosecurity parameters related to tissue and construct transportation, chondrocyte isolation, and cell expansion. Patient progress was analyzed with patient-reported outcome measures, MRI T2-mapping, MOCART, and ICRS arthroscopic second-look evaluation. Results. Thirty-five lesions in 30 patients treated in 7 different hospitals were evaluated. Cell viability before implantation was >90%. Cell viability in construct remnants was 87% ± 11% at 24 hours, 75% ± 17.1% at 48 hours, and 60% ± 8% at 72 hours after implantation. Mean final follow-up was 37 months (12-72 months). Patients showed statistically significant improvement in all clinical scores and MOCART evaluations. MRI T2-mapping evaluation showed significant decrease in relaxation time from 61.2 ± 14.3 to 42.9 ± 7.2 ms (P < 0.05). Arthroscopic second-look evaluation showed grade II "near normal" tissue in 83% of patients. Two treatment failures were documented. Conclusions. It was feasible to perform AMECI in 7 different institutions in a large metropolitan area following our biosecurity measures without any implant-related complication. Treated patients showed improvement in clinical, MRI T2-mapping, and MOCART scores, as well as a low failure rate and a favorable ICRS arthroscopic evaluation at a mid-term follow-up. Level of Evidence. 2b.

Co-culture of dedifferentiated and primary human chondrocytes obtained from cadaveric donor enhance the histological quality of repair tissue: an in-vivo animal study.

To compare the quality of the repair tissue in three-dimensional co-culture of human chondrocytes implanted in an in vivo model. Six cadaveric and five live human donors were included. Osteochondral biopsies from the donor knees were harvested for chondrocyte isolation. Fifty percent of cadaveric chondrocytes were expanded until passage-2 (P2) while the remaining cells were cryopreserved in passage-0 (P0). Fresh primary chondrocytes (P0f) obtained from live human donors were co-cultured. Three-dimensional constructs were prepared with a monolayer of passage-2 chondrocytes, collagen membrane (Geistlich Bio-Gide®), and pellet of non-co-cultured (P2) or co-cultured chondrocytes (P2 + P0c, P2 + P0f). Constructs were implanted in the subcutaneous tissue of athymic mice and left for 3 months growth. Safranin-O and Alcian blue staining were used to glycosaminoglycan content assessment. Aggrecan and type-II collagen were evaluated by immunohistochemistry. New-formed tissue quality was evaluated with an adaptation of the modified O'Driscoll score. Histological quality of non-co-cultured group was 4.37 (SD ±4.71), while co-cultured groups had a mean score of 8.71 (SD ±3.98) for the fresh primary chondrocytes and 9.57 (SD ±1.27) in the cryopreserved chondrocytes. In immunohistochemistry, Co-culture groups were strongly stained for type-II and aggrecan not seen in the non-co-cultured group. It is possible to isolate viable chondrocytes from cadaveric human donors in samples processed in the first 48-h of dead. There is non-significant difference between the numbers of chondrocytes isolated from live or cadaveric donors. Cryopreservation of cadaveric primary chondrocytes does not alter the capability to form cartilage like tissue. Co-culture of primary and passaged chondrocytes enhances the histological quality of new-formed tissue compared to non-co-cultured cells.

Liver Transplantation for Hepatic Trauma: A Study From the European Liver Transplant Registry.

BACKGROUND: Liver transplantation is the most extreme form of surgical management of patients with hepatic trauma, with very limited literature data supporting its use. The aim of this study was to assess the results of liver transplantation for hepatic trauma. METHODS: This retrospective analysis based on European Liver Transplant Registry comprised data of 73 recipients of liver transplantation for hepatic trauma performed in 37 centers in the period between 1987 and 2013. Mortality and graft loss rates at 90 days were set as primary and secondary outcome measures, respectively. RESULTS: Mortality and graft loss rates at 90 days were 42.5% and 46.6%, respectively. Regarding general variables, cross-clamping without extracorporeal veno-venous bypass was the only independent risk factor for both mortality (P = 0.031) and graft loss (P = 0.034). Regarding more detailed factors, grade of liver trauma exceeding IV increased the risk of mortality (P = 0.005) and graft loss (P = 0.018). Moreover, a tendency above the level of significance was observed for the negative impact of injury severity score (ISS) on mortality (P = 0.071). The optimal cut-off for ISS was 33, with sensitivity of 60.0%, specificity of 80.0%, positive predictive value of 75.0%, and negative predictive value of 66.7%. CONCLUSIONS: Liver transplantation seems to be justified in selected patients with otherwise fatal severe liver injuries, particularly in whom cross-clamping without extracorporeal bypass can be omitted. The ISS cutoff less than 33 may be useful in the selection process.

Elisidepsin Interacts Directly with Glycosylceramides in the Plasma Membrane of Tumor Cells to Induce Necrotic Cell Death.

Plasma membrane integrity is essential for cell life. Any major break on it immediately induces the death of the affected cell. Different molecules were described as disrupting this cell structure and thus showing antitumor activity. We have previously defined that elisidepsin (Irvalec®, PM02734) inserts and self-organizes in the plasma membrane of tumor cells, inducing a rapid loss of membrane integrity, cell permeabilization and necrotic death. Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane. Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane. Consequently, drug treatment did not induce the characteristic necrotic cell death. Furthermore, GM95, a mutant derivative from B16 mouse melanoma cells lacking ceramide glucosyltransferase (UGCG) activity and thus the synthesis of glycosylceramides, was also resistant to elisidepsin. Over-expression of UGCG gene in these deficient cells restored glycosylceramides synthesis, rendering them sensitive to elisidepsin, at a similar level than parental B16 cells. These results indicate that glycosylceramides act as membrane targets of elisidepsin, facilitating its insertion in the plasma membrane and the subsequent membrane permeabilization that leads to drug-induced cell death. They also indicate that cell membrane lipids are a plausible target for antineoplastic therapy.

Stellatolides, a new cyclodepsipeptide family from the sponge Ecionemia acervus: isolation, solid-phase total synthesis, and full structural assignment of stellatolide A.

The marine environment is a rich source of metabolites with potential therapeutic properties and applications for humans. Here we describe the first isolation, solid-phase total synthesis, and full structural assignment of a new class of cyclodepsipeptides from the Madagascan sponge Ecionemia acervus that shows in vitro cytotoxic activities at submicromolar concentrations. Seven structures belonging to a new family of compounds, given the general name stellatolides, were characterized. The sequence and stereochemistry of all the amino acids in these molecules were established by a combination of spectroscopic analysis, chemical degradation, and derivatization studies. Furthermore, the complete structure of stellatolide A was confirmed by an efficient solid-phase method for the first total synthesis and the full structural assignment of this molecule, including the asymmetric synthesis of the unique ß-hydroxy acid moiety (Z)-3-hydroxy-6,8-dimethylnon-4-enoic acid.

Follow-up of a new arthroscopic technique for implantation of matrix-encapsulated autologous chondrocytes in the knee.

PURPOSE: The purpose of this study was to evaluate the clinical and sequential imaging follow-up results at a mean of 36 months after an arthroscopic technique for implantation of matrix-encapsulated autologous chondrocytes for the treatment of articular cartilage lesions on the femoral condyles. METHODS: Ten patients underwent arthroscopic implantation of autologous chondrocytes seeded onto a bioabsorbable scaffold. The patients were evaluated clinically using a visual analog scale (VAS) for pain and International Knee Documentation Committee (IKDC), Lysholm, and Tegner scores. Magnetic resonance imaging (MRI) T2-mapping and magnetic resonance observation of cartilage repair tissue (MOCART) evaluations were also performed. Second-look arthroscopic evaluation using the International Cartilage Repair Society (ICRS) grading classification was performed at 12 months. RESULTS: Compared with their preoperative values, at 36 months mean values ± standard deviation for the VAS scale for pain were 6.0 ± 1.5 to 0.3 ± 0.4. Improvement in clinical scores between preoperative values and 36-month follow-up values in subjective IKDC scores was 46.9 ± 18.5 to 77.2 ± 12.8; in Lysholm scores, it was 51.8 ± 25.1 to 87.9 ± 6.5, and in the Tegner activity scale it was 2.9 ± 1.7 to 5.9 ± 1.9. Mean T2 mapping and MOCART scores improved over time to 38.1 ± 4.4 ms and 72.5 ± 10, respectively. Mean ICRS score by second-look arthroscopy at 1 year was 10.4 ± 0.1. CONCLUSIONS: All clinical scores improved over time compared with the preoperative values. Clinical results are comparable with MRI T2 mapping and ICRS evaluations, suggesting that this arthroscopic technique for cell-based cartilage repair is efficacious and reproducible at a mean of 36 months of follow-up. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Ingeniería de tejidos y osteoartritis / Tissue engineering and osteoarthritis

Las lesiones de cartílago articular predisponen al desarrollo precoz de osteoartritis. La mayoría de las técnicas quirúrgicas actuales para el tratamiento de lesiones condrales dan lugar a la formación de fibrocartílago con propiedades bioquímicas y biomecánicas inferiores a las del cartílago articular. La ingeniería de tejidos puede ofrecer una alternativa moderna para el tratamiento de estas lesiones y de esta forma prevenir el desarrollo de osteoartritis en pacientes jóvenes activos. Existen diferentes alternativas en cuanto al tipo de células para implantar como tratamiento, ya sean el actual uso de condrocitos autólogos o células troncales mesenquimales. La otra variable es el tipo de andamio sobre el cual cultivar o sembrar la células para su implante: materiales sintéticos biocompatibles y bioabsorbibles, los derivados de fibrina o de colágeno de diferentes fuentes (bovina, porcina, de cola de rata, etc.), en forma de geles, esponjas, mallas, etc., y todas ellas con o sin la adición de factores de crecimiento. En la actualidad, el uso de condrocitos autólogos es una realidad, ya sea inyectados en suspensión bajo un parche de periostio o sembrados en colágeno. Casi todos los investigadores y las empresas de biotecnología están buscando técnicas para las que no se requieran dos intervenciones quirúrgicas, por lo cual muy probablemente habrá que pensar en células troncales y con sistemas de implantación artroscópicos (AU)

Articular cartilage lesions predispose to the development of early osteoarthritis. Most current surgical techniques give rise to the formation of fibrocartilage with biochemical and biomechanical properties inferior to those or articular cartilage. Tissue engineering could offer modern alternative to the treatment of these lesions and in this way, prevent the development of early osteoarthritis in young active patients. Different tissue engineering approaches rely on the current use of autologous chondrocytes, or the potential use of mesenchymal stem cells. Other variables rely on the type of scaffold to use such as synthetic biodegradable polymers, fibrin or collagen-derived scaffolds of different sources, bovine, porcine, rat tail, etc, in the form of gels, sponges, mesh, etc, and all of these with or without growth factors. The use of autologous chondrocytes is a reality at the present time, whether injected under a periosteum patch or seeded on collagen. However, most investigators and biotech companies are in search of onestep surgical procedures, for which reason stem cells have to be kept in mind, as well as systems that will allow arthroscopic implantation (AU)

Matrix-encapsulation cell-seeding technique to prevent cell detachment during arthroscopic implantation of matrix-induced autologous chondrocytes.

PURPOSE: The goal of this study is to evaluate the efficiency of obtaining a large number of viable cells within a construct that will not be detached by high fluid flow during arthroscopic implantation. METHODS: Arthroscopic osteochondral biopsy specimens were obtained from the medial femoral trochlea of 8 horses. Chondrocytes were isolated by collagenase digestion and expanded in M199 media until confluency. After 10 to 12 days, cultures were trypsinized and cells resuspended in culture media. Then, 5 x 10(6) cells x mL(-1) were seeded on a culture dish and the same amount in a flask. Once extracellular matrix was formed, a polyglycolic/polylactic acid disk was placed in the culture dish. Cells obtained from the culture flasks (2 x 10(7) cells) were seeded onto the polymer and encapsulated by lifting the monolayer of cells and matrix from the bottom of the dish with surgical forceps. On days 1, 3, 5, 7, and 9, viability was evaluated by calcein fluorescence. Fiber cell attachment was evaluated before implantation by environmental scanning electron microscopy. Six horses were implanted with naive cell-polymer constructs, and two horses were implanted with adenoviral vector with green fluorescent protein (AdGFP)-transduced cells. Biopsy specimens of repair tissue were evaluated at 8 weeks in 6 horses and at 4 weeks in the 2 horses implanted with AdGFP-transduced cells by second-look arthroscopy and biopsy, histochemistry, and confocal laser scanning microscopy via MitoTracker Red 580 (Invitrogen [Molecular Probes], Gibco, Carlsbad, CA) to assess cell viability. RESULTS: Viability and attachment of cells to polymer were confirmed by calcein fluorescence microscopy and environmental scanning electron microscopy. Consistency of the construct was ideal for implantation between 7 and 9 days. Repair tissue with AdGFP chondrocytes after 4 weeks showed fluorescent cells also positive to MitoTracker probe by confocal laser scanning microscopy. Repair tissue after 8 weeks showed very cellular new tissue formation with good attachment to subchondral bone and adjacent cartilage. CONCLUSIONS: The matrix-encapsulation cell-seeding technique allowed us to maintain a sufficient number of viable cells within the polymer construct despite the high-pressure fluid flow that occurred during arthroscopic implantation when we used a pump for direct visualization. CLINICAL RELEVANCE: Arthroscopic implantation of cell-polymer constructs via a fluid pump can be performed without the risk of cell loss with the use of a simple cell-seeding technique.

[Tissue engineering and osteoarthritis]. / Ingeniería de tejidos y osteoartritis.

Articular cartilage lesions predispose to the development of early osteoarthritis. Most current surgical techniques give rise to the formation of fibrocartilage with biochemical and biomechanical properties inferior to those or articular cartilage. Tissue engineering could offer a modern alternative to the treatment of these lesions and in this way, prevent the development of early osteoarthritis in young active patients. Different tissue engineering approaches rely on the current use of autologous chondrocytes, or the potential use of mesenchymal stem cells. Other variables rely on the type of scaffold to use such as synthetic biodegradable polymers, fibrin or collagen-derived scaffolds of different sources, bovine, porcine, rat tail, etc, in the form of gels, sponges, mesh, etc, and all of these with or without growth factors. The use of autologous chondrocytes is a reality at the present time, whether injected under a periosteum patch or seeded on collagen. However, most investigators and biotech companies are in search of onestep surgical procedures, for which reason stem cells have to be kept in mind, as well as systems that will allow arthroscopic implantation.

Mycophenolate mofetil can be used as monotherapy late after liver transplantation.

We report our experience with calcineurin inhibitor (CNI) withdrawal and MMF monotherapy in 50 adult liver transplant (OLT) recipients with CNI-related toxicity. Thirty-four patients had chronic renal dysfunction (CRD) associated with arterial hypertension, 11 had only CRD and other five patients had hypertension. The mean time between OLT and introduction of MMF was 81 months. After the introduction of MMF, CNI was progressively reduced and withdrawn if possible. At the end of the follow up (mean time: 18 months) CNI was withdrawn in 39 patients (78%), and there was a significant decrease from baseline in serum creatinine (1.81-1.49 mg/dL; p < 0.0001), BUN (76.6-52.8 mg/dL; p < 0.0001) and uric acid (9-7.5 mg/dL; p < 0.0001) levels, and an increase in creatinine clearance (44.7-55.1 mL/min; p < 0.0001). Excluding patients who developed graft rejection and two patients who died, CRD improved in 32 of 40 patients (80%), and arterial hypertension improved in 22 of 29 patients (76%). Five patients (10%) developed acute rejection, and one patient (2%) chronic rejection. Twenty-six patients (52%) experienced side-effects, with asthenia, herpes virus infection, and diarrhea being the most common. Only eight patients (16%) required MMF dose reduction. In conclusion, MMF monotherapy late after OLT improves CRD and hypertension in most patients, is safe and well tolerated.

Benzo[f]azino[2,1-a]phthalazinium cations: novel DNA intercalating chromophores with antiproliferative activity.

New azaquinolizinium-type cations have been obtained from isochromane. The synthesis was completed over seven steps and included as the key feature an intramolecular Westphal condensation. This first example of the intramolecular process allowed the preparation of benzo[f]pyrido[2,1-a]phthalazinium and benzo[f]quino[2,1-a]phthalazinium salts, which were evaluated as DNA intercalators, DNA topoisomerase I inhibitors, and antiproliferative compounds. Both cationic systems behave as DNA intercalators and exhibit antiproliferative activity. The pentacyclic benzo[f]quino[2,1-a]phthalazinium cations also have an inhibitory effect on the catalytic activity of DNA topoisomerase I, without trapping of cleavage complexes. Structural characterization using density functional theory indicates that the fused ring systems are slightly nonplanar, and additional molecular modeling studies suggest a preferred orientation for the intercalating chromophores within a typical CpG or TpG intercalation site.

Synthesis of natural ecteinascidins (ET-729, ET-745, ET-759B, ET-736, ET-637, ET-594) from cyanosafracin B.

The semisynthetic process initially described for the synthesis of 1 (ET-743) has been extended to the preparation of other natural ecteinascidins. For the synthesis of 2 (ET-729) a demethylation of a N-Me intermediate was carried out by a selective oxidation with MCPBA. Other natural ecteinascidins (ET-745, ET-759B, ET-736, ET-637, ET-594) were accessible from key intermediate 25. The described methodologies allow for the preparation of a wide variety of ecteinascidins by procedures that can be easily scaled up.

Prevalencia de infertilidad en un sector urbano-rural / Prevalence of infertility in a urban-rural area

Para contribuir a mejorar nuestros conocimientos sobre la infertilidad se diseñó este estudio cuyo objetivo principal fue determinar su prevalencia en un sector de salud urbano-rural. Se realizó una encuesta en un sector del municipio Nuevitas, de la provincia CamagÜey de mujeres en edad reproductiva (15 a 49 años), seleccionadas al azar. Se aplicó un cuestionario diseñado por la OMS para investigaciones de este tipo, con ligeras modificaciones. Se compararon los resultados con otra encuesta sobre infertilidad realizada en el país. Se halló que el 13,4 por ciento de las mujeres casadas o en unión conyugal eran infértiles (11,8 por ciento del total de las mujeres encuestadas), lo que representa una prevalencia moderada del trastorno, bastante similar al 12,1 por ciento informado en el municipio Plaza en Ciudad de La Habana. El 0.7 por ciento de las mujeres casadas tenía una infertilidad primaria y el 12,7 por ciento una infertilidad secundaria. La gran mayoría de las mujeres fértiles y presumiblemente fértiles era menor de 35 años, mientras que la mayoría de las infértiles era mayor de 30 años

Prevalencia de infertilidad en un sector urbano-rural / Prevalence of infertility in a urban-rural area

Para contribuir a mejorar nuestros conocimientos sobre la infertilidad se diseñó este estudio cuyo objetivo principal fue determinar su prevalencia en un sector de salud urbano-rural. Se realizó una encuesta en un sector del municipio Nuevitas, de la provincia CamagÜey de mujeres en edad reproductiva (15 a 49 años), seleccionadas al azar. Se aplicó un cuestionario diseñado por la OMS para investigaciones de este tipo, con ligeras modificaciones. Se compararon los resultados con otra encuesta sobre infertilidad realizada en el país. Se halló que el 13,4 por ciento de las mujeres casadas o en unión conyugal eran infértiles (11,8 por ciento del total de las mujeres encuestadas), lo que representa una prevalencia moderada del trastorno, bastante similar al 12,1 por ciento informado en el municipio Plaza en Ciudad de La Habana. El 0.7 por ciento de las mujeres casadas tenía una infertilidad primaria y el 12,7 por ciento una infertilidad secundaria. La gran mayoría de las mujeres fértiles y presumiblemente fértiles era menor de 35 años, mientras que la mayoría de las infértiles era mayor de 30 años (AU)