Sequential growth factor exposure of human Ad-MSCs improves chondrogenic differentiation in an osteochondral biphasic implant.

Joint cartilage damage affects 10-12% of the world's population. Medical treatments improve the short-term quality of life of affected individuals but lack a long-term effect due to injury progression into fibrocartilage. The use of mesenchymal stem cells (MSCs) is one of the most promising strategies for tissue regeneration due to their ability to be isolated, expanded and differentiated into metabolically active chondrocytes to achieve long-term restoration. For this purpose, human adipose-derived MSCs (Ad-MSCs) were isolated from lipectomy and grown in xeno-free conditions. To establish the best differentiation potential towards a stable chondrocyte phenotype, isolated Ad-MSCs were sequentially exposed to five differentiation schemes of growth factors in previously designed three-dimensional biphasic scaffolds with incorporation of a decellularized cartilage matrix as a bioactive ingredient, silk fibroin and bone matrix, to generate a system capable of being loaded with pre-differentiated Ad-MSCs, to be used as a clinical implant in cartilage lesions for tissue regeneration. Chondrogenic and osteogenic markers were analyzed by reverse transcription-quantitative PCR and cartilage matrix generation by histology techniques at different time points over 40 days. All groups had an increased expression of chondrogenic markers; however, the use of fibroblast growth factor 2 (10 ng/ml) followed by a combination of insulin-like growth factor 1 (100 ng/ml)/TGFß1 (10 ng/ml) and a final step of exposure to TGFß1 alone (10 ng/ml) resulted in the most optimal chondrogenic signature towards chondrocyte differentiation and the lowest levels of osteogenic expression, while maintaining stable collagen matrix deposition until day 33. This encourages their possible use in osteochondral lesions, with appropriate properties for use in clinical patients.

Whole-Exome Sequencing, Proteome Landscape, and Immune Cell Migration Patterns in a Clinical Context of Menkes Disease.

Menkes disease (MD) is a rare and often lethal X-linked recessive syndrome, characterized by generalized alterations in copper transport and metabolism, linked to mutations in the ATPase copper transporting α (ATP7A) gene. Our objective was to identify genomic alterations and circulating proteomic profiles related to MD assessing their potential roles in the clinical features of the disease. We describe the case of a male patient of 8 months of age with silvery hair, tan skin color, hypotonia, alterations in neurodevelopment, presence of seizures, and low values of plasma ceruloplasmin. Trio-whole-exome sequencing (Trio-WES) analysis, plasma proteome screening, and blood cell migration assays were carried out. Trio-WES revealed a hemizygous change c.4190C > T (p.S1397F) in exon 22 of the ATP7A gene. Compared with his parents and with child controls, 11 plasma proteins were upregulated and 59 downregulated in the patient. According to their biological processes, 42 (71.2%) of downregulated proteins had a participation in cellular transport. The immune system process was represented by 35 (59.3%) downregulated proteins (p = 9.44 × 10-11). Additional studies are necessary to validate these findings as hallmarks of MD.

Fibroblast Growth Factor Type 2 (FGF2) Administration Attenuated the Clinical Manifestations of Preeclampsia in a Murine Model Induced by L-NAME.

Background: In preeclampsia, a hypertensive disorder of pregnancy, the poor remodeling of spiral arteries leads to placental hypoperfusion and ischemia, provoking generalized maternal endothelial dysfunction and, in severe cases, death. Endothelial and placental remodeling is important for correct pregnancy evolution and is mediated by cytokines and growth factors such as fibroblast growth factor type 2 (FGF2). In this study, we evaluated the effect of human recombinant FGF2 (rhFGF2) administration in a murine model of PE induced by NG-nitro-L-arginine methyl ester (L-NAME) to test if rhFGF2 administration can lessen the clinical manifestations of PE. Methods: Pregnant rats were administrated with 0.9% of NaCl (vehicle), L-NAME (60 mg/kg), FGF2 (666.6 ng/kg), L-NAME+FGF2 or L-NAME + hydralazine (10 mg/kg) from the 10th to 19th days of gestation. Blood pressure (BP), urine protein concentrations and anthropometric values both rat and fetuses were assessed. Histological evaluation of organs from rats delivered by cesarean section was carried out using hematoxylin and eosin staining. Results: A PE-like model was established, and it included phenotypes such as maternal hypertension, proteinuria, and fetal growth delay. Compared to the groups treated with L-NAME, the L-NAME + FGF2 group was similar to vehicle: the BP remained stable and the rats did not develop enhanced proteinuria. Both the fetuses and placentas from rats treated with L-NAME + FGF2 had similar values of weight and size compared with the vehicle. Conclusion: The intravenous administration of rhFGF2 showed beneficial and hypotensive effects, reducing the clinical manifestations of PE in the evaluated model.

Maternal cafeteria diet exposure primes depression-like behavior in the offspring evoking lower brain volume related to changes in synaptic terminals and gliosis.

Maternal nutritional programming by caloric exposure during pregnancy and lactation results in long-term behavioral modification in the offspring. Here, we characterized the effect of maternal caloric exposure on synaptic and brain morphological organization and its effects on depression-like behavior susceptibility in rats' offspring. Female Wistar rats were exposed to chow or cafeteria (CAF) diet for 9 weeks (pre-pregnancy, pregnancy, and lactation) and then switched to chow diet after weaning. By postnatal day 60, the male Wistar rat offspring were tested for depressive-like behavior using operational conditioning, novelty suppressed feeding, sucrose preference, and open-field test. Brain macro and microstructural morphology were analyzed using magnetic resonance imaging deformation-based morphometry (DBM) and western blot, immunohistochemistry for NMDA and AMPA receptor, synaptophysin and myelin, respectively. We found that the offspring of mothers exposed to CAF diet displayed deficient motivation showing decrease in the operant conditioning, sucrose preference, and suppressed feeding test. Macrostructural DBM analysis showed reduction in the frontomesocorticolimbic circuit volume including the nucleus accumbens (NAc), hippocampus, and prefrontal cortex. Microstructural analysis revealed reduced synaptic terminals in hippocampus and NAc, whereas increased glial fibrillary acidic protein in hippocampus and lateral hypothalamus, as well as a decrease in the hippocampal cell number and myelin reduction in the dentate gyrus and hilus, respectively. Also, offspring exhibited increase of the GluR1 and GLUR2 subunits of AMPA receptor, whereas a decrease in the mGluR2 expression in hippocampus. Our findings reveal that maternal programming might prime depression-like behavior in the offspring by modulating macro and micro brain organization of the frontomesocorticolimbic circuit.

A Cellularized Biphasic Implant Based on a Bioactive Silk Fibroin Promotes Integration and Tissue Organization during Osteochondral Defect Repair in a Porcine Model.

In cartilage tissue engineering, biphasic scaffolds (BSs) have been designed not only to influence the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone, promoting the implant's integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a BS based on the assembly of a cartilage phase constituted by fibroin biofunctionalyzed with a bovine cartilage matrix, cellularized with differentiated autologous pre-chondrocytes and well attached to a bone phase (decellularized bovine bone) to promote cartilage regeneration in a model of joint damage in pigs. BSs were assembled by fibroin crystallization with methanol, and the mechanical features and histological architectures were evaluated. The scaffolds were cellularized and matured for 12 days, then implanted into an osteochondral defect in a porcine model (n = 4). Three treatments were applied per knee: Group I, monophasic cellular scaffold (single chondral phase); group II (BS), cellularized only in the chondral phase; and in order to study the influence of the cellularization of the bone phase, Group III was cellularized in chondral phases and a bone phase, with autologous osteoblasts being included. After 8 weeks of surgery, the integration and regeneration tissues were analyzed via a histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular BSs reached a Young's modulus of 805.01 kPa, similar to native cartilage. In vitro biological studies revealed the chondroinductive ability of the BSs, evidenced by an increase in sulfated glycosaminoglycans and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, in Group I, the defects were not reconstructed. In Groups II and III, a good integration of the implant with the surrounding tissue was observed. Defects in group II were fulfilled via hyaline cartilage and normal bone. Group III defects showed fibrous repair tissue. In conclusion, our findings demonstrated the efficacy of a biphasic and bioactive scaffold based on silk fibroin and cellularized only in the chondral phase, which entwined chondroinductive features and a biomechanical capability with an appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

A Bioactive Cartilage Graft of IGF1-Transduced Adipose Mesenchymal Stem Cells Embedded in an Alginate/Bovine Cartilage Matrix Tridimensional Scaffold.

Articular cartilage injuries remain as a therapeutic challenge due to the limited regeneration potential of this tissue. Cartilage engineering grafts combining chondrogenic cells, scaffold materials, and microenvironmental factors are emerging as promissory alternatives. The design of an adequate scaffold resembling the physicochemical features of natural cartilage and able to support chondrogenesis in the implants is a crucial topic to solve. This study reports the development of an implant constructed with IGF1-transduced adipose-derived mesenchymal stem cells (immunophenotypes: CD105+, CD90+, CD73+, CD14-, and CD34-) embedded in a scaffold composed of a mix of alginate/milled bovine decellularized knee material which was cultivated in vitro for 28 days (3CI). Histological analyses demonstrated the distribution into isogenous groups of chondrocytes surrounded by a de novo dense extracellular matrix with balanced proportions of collagens II and I and high amounts of sulfated proteoglycans which also evidenced adequate cell proliferation and differentiation. This graft also shoved mechanical properties resembling the natural knee cartilage. A modified Bern/O'Driscoll scale showed that the 3CI implants had a significantly higher score than the 2CI implants lacking cells transduced with IGF1 (16/18 vs. 14/18), representing high-quality engineering cartilage suitable for in vivo tests. This study suggests that this graft resembles several features of typical hyaline cartilage and will be promissory for preclinical studies for cartilage regeneration.

Two novel mutations in the GAN gene causing giant axonal neuropathy.

BACKGROUND: Giant axonal neuropathy (GAN) is a rare neurodegenerative disease transmitted in an autosomal recessive mode. This disorder presents motor and sensitive symptoms with an onset in early childhood. Progressive neurodegeneration makes the patients wheelchair dependent by the end of the second decade of life. Affected individuals do not survive beyond the third decade of life. Molecular analysis has identified mutations in the gene GAN in patients with this disorder. This gene produces a protein called gigaxonin which is presumably involved in protein degradation via the ubiquitin-proteasome system. However, the underlying molecular mechanism is not clearly understood yet. METHODS: Here we present the first patient from Mexico with clinical data suggesting GAN. Sequencing of the GAN gene was carried out. Changes in the nucleotide sequence were investigated for their possible impact on protein function and structure using the publicly available prediction tools PolyPhen-2 and PANTHER. RESULTS: The patient is a compound heterozygous carrying two novel mutations in the GAN gene. The sequence analysis revealed two missense mutations in the Kelch repeats domain. In one allele, a C>T transition was found in exon 9 at the nucleotide position 55393 (g.55393C>T). In the other allele, a transversion G>T in exon 11 at the nucleotide position 67471 (g.67471G>T) was observed. Both of the bioinformatic tools predicted that these amino acid substitutions would have a negative impact on gigaxonin's function. CONCLUSION: This work provides useful information for health professionals and expands the spectrum of disease-causing mutations in the GAN gene and it is the first documented case in Mexican population.

Thymidylate synthase gene variants as predictors of clinical response and toxicity to fluoropyrimidine-based chemotherapy for colorectal cancer.

BACKGROUND: Fluoropyrimidines form the chemotherapy backbone of advanced and metastatic colorectal cancer (CRC). These drugs are frequently associated with toxicity events that result in dose adjustments and even suspension of the treatment. The thymidylate synthase (TYMS) gene is a potential marker of response and toxicity to fluoropyirimidines as this enzyme is the molecular target of these drugs. Our aim was to assess the association between variants of TYMS with response and toxicity to fluoropyrimidines in patients with CRC in independent retrospective and prospective studies. METHODS: Variants namely rs45445694, rs183205964, rs2853542 and rs151264360 of TYMS were genotyped in 105 CRC patients and were evaluated to define their association with clinical response and toxicity to fluoropyrimidines. Additionally, the relationship between genotypes and tumor gene expression was analyzed by quantitative polymerase chain reaction. RESULTS: The 2R/2R (rs45445694) was associated with clinical response (p=0.05, odds ratio (OR)=3.45) and severe toxicity (p=0.0014, OR=5.21, from pooled data). Expression analysis in tumor tissues suggested a correlation between the 2R/2R genotype and low TYMS expression. CONCLUSIONS: The allele 2R (rs45445694) predicts severe toxicity and objective response in advanced CRC patients. In addition, the alleles G(rs2853542) and 6bp-(rs151264360) are independent predictors of response failure to chemotherapy. This is the first study made on a Latin American population that points out TYMS gene variants have predictive values for response and toxicity in patients with CRC treated with fluoropyrimidine-based chemotherapy.

Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries.

Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated ß1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Análisis histopatológico del cartílago articular expuesto a metilprednisolona, ropivacaína y ácido hialurónico en un modelo animal de osteoartrosis / Histopathological analysis of the articular cartilage exposed to methylprednisolone, ropivacaine and hyaluronic acid in an animal model of osteoarthrosis

La osteoartrosis es un padecimiento del aparato locomotor con una prevalencia elevada y en crecimiento, paralela al envejecimiento de la población. La infiltración intraarticular de sustancias para aliviar la sintomatología de la osteoartrosis es una práctica común en el consultorio médico de los especialistas que atienden esta enfermedad. Aunque la sintomatología mejora con la infiltración de anestésicos locales, corticoesteroides y suplementos viscosantes, es aún incierto el efecto que estas sustancias tienen sobre la integridad del cartílago articular. Este estudio explora a nivel macroscópico e histológico el efecto de la infiltración de ropivacaína, metilprednisolona y ácido hialurónico sobre el cartílago articular en un modelo de osteoartrosis química en conejos (n=24). Nuestros resultados indican que en los grupos infiltrados con metilprednisolona (n=8) y ropivacaína (n=8) la estructura del cartílago articular presento alteraciones más severas con respecto a su grupo control, además de una disminución importante en la síntesis de matriz extracelular. En el grupo infiltrado con ácido hialurónico (n=8), las alteraciones macroscópicas e histológicas del cartílago articular mejoraron con respecto a su grupo control, presentando una estructura integra y síntesis de matriz extracelular normal.

Osteoarthritis is a musculoskeletal condition with a high prevalence, increasing with the aging of population. The intraarticular infiltration of substances to relieve the symptoms of osteoarthritis is a common practice in medical practice. Although symptoms improved with the infiltration of local anesthetics, corticosteroids and supplements, it is still uncertain what effect these substances have on the integrity of articular cartilage. This study explores the macroscopic and histological effects of infiltration of Ropivacaine, Methylprednisolone and Hyaluronic Acid on articular cartilage in a model of chemical osteoarthritis in rabbits (n=24). Our results indicate that in the infiltrated groups with Methylprednisolone (n=8) and Ropivacaine (n=8) the structure of articular cartilage present more severe alterations with respect to its control group and an important decrease in the synthesis of extracellular matrix. In-group infiltrated with hyaluronic acid (n=8), macroscopic and histological changes of articular cartilage improved with respect to its control group, presenting a normal structure and normal extracellular matrix synthesis.

Evaluation of in vivo pathogenicity of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis with different enzymatic profiles in a murine model of disseminated candidiasis.

Six isolates of the Candida parapsilosis complex with different enzymatic profiles were used to induce systemic infection in immunocompetent BALB/c mice. Fungal tissue burden was determined on days 2, 5, 10, and 15 post challenge. The highest fungal load irrespective of post-infection day was detected in the kidney, followed by the spleen, lung, and liver, with a tendency for the fungal burden to decrease by day 15 in all groups. Significant differences among the strains were not detected, suggesting that the three species of the "psilosis" group possess a similar pathogenic potential in disseminated candidiasis regardless of their enzymatic profiles.

Intoxicación crónica con el fruto maduro de karwinskia humboldtiana en ratas wistar: daño renal / Chronic intoxication with ripe fruit of karwinskia humboldtiana in wistar rats: renal damage

Karwinskia humboldtiana (Kh) es un arbusto venenoso responsable de numerosos casos de intoxicación accidental en humanos. En la literatura se ha descrito a la intoxicación crónica con Kh como uno polineuropatía sin describir si existen o no alteraciones en órganos distintos al SNC y SNP como lo es el riñón. El objetivo de este estudio fue evaluar la morfología renal en un modelo de intoxicación crónica con Kh. Se utilizaron 32 ratas Wistar, se dividieron en cuatro grupos (n=8) en donde 5 ratas de cada grupo fueron intoxicadas y 3 fueron control no intoxicadas. A las ratas intoxicadas se les administraron por vía oral 3,5 g/kg del fruto seco y molido de Kh fraccionados en 5 dosis de 1,5; 0,5; 0,5; 0,5 y 0,5 g/kg los días 0, 3, 7, 10 y 14 respectivamente. Las ratas control solo recibieron agua. Cada grupo fue sacrificado a diferentes tiempos según la evolución de la parálisis. Se obtuvieron muestras de riñón, se procesaron hasta obtener bloques de parafina y resinas epóxicas, se obtuvieron cortes y se tiñeron y contrastaron para su observación al microscopio de luz y electrónico de transmisión (MET) respectivamente. A microscopia de luz identificamos congestión vascular, necrosis de los túbulos contorneados y fibrosis de la cápsula de renal, en la etapa de parálisis se realizo un conteo de los glomérulos afectados en las muestras tratadas con Kh, a MET además de los hallazgos previamente descritos se identificó la presencia de abundantes depósitos de matriz extracelular en la membrana basal de la cápsula renal y en la barrera de filtración de todos los grupos intoxicados, siendo más evidentes en el grupo de recuperación, lo que demuestra que la intoxicación crónica con Kh es una intoxicación sistémica y no exclusiva del SNC y SNP.

Karwinskia humboldtiana (Kh) is a poisonous shrub causing a number a accidental intoxications in humans. In previous studies, damage has been reported to Peripheral and Central Nervous System. Main intoxication sign is the presence of paralysis. However, no studies have been documented about damage to other organs like the kidney. The objective of this research is to evaluate kidney histology during chronic intoxication. Thirty two (32) Wistar rats were divided into 4 groups (n=8). For each group, 5 rats were intoxicated with Kh and 3 received water only as a control. Intoxicated rats received 3.5 g/Kg body weight of dry powder of Kh fruit, fractionated in 5 doses as follows 1.5, 0.5, 0.5, 0.5, 05 on days 0, 3, 7, 10 and 14 respectively. Control rats received water only. Each group was euthanized at different times during paralysis evolution. Samples of kidney were obtained and processed by routine technique until paraffin embedding for light microscopy studies, and in epoxy resins for transmission electron microscopy. Sections were obtained and stained with H&E, Masson's trichrome, and treated for PAS with diastase reaction to demonstrate basal membranes. At the light microscopic level we observed blood vessel congestion, tubular necrosis and fibrosis of renal capsule. Both at Light microscopy and electron microscopy, it was identified a thickening of the filtration barrier and of renal capsule, in all intoxicated animals, especially in the recovery group. These findings demonstrate that Kh causes a systemic intoxication and not only of the nervous system, as has been considered up to now.

[Diagnosis and treatment of Pompe disease]. / Diagnóstico y tratamiento en la enfermedad de Pompe.

Pompe disease is a rare, progressive and often fatal neuromuscular disorder. It is caused by a deficiency of the lysosomal alpha-glucosidase. Among glycogen storage disorders, it is one of the most common. Its clinical manifestations can start at any moment of life, with a very variable symptomatology. In this article, we show an extended revision of the literature in regards to the main medical aspects of Pompe disease: etiology, psychopathology, epidemiology, clinical variants, pathological diagnosis, and enzyme replacement therapy. With this information, we created a diagnostic and therapeutic guide, which is addressed to specialists and to first-level physicians, in order to let them identify both the classic and the late forms of this disease. We describe as well the best, timely, multidisciplinary treatment in use. Also, we show some suggestions to the proper functioning of health institutions, and routes to diagnosis. We conclude that Pompe disease may be properly diagnosed and treated if health care professionals follow the internationally approved recommendations.

La enfermedad de Pompe es un trastorno neuromuscular raro, progresivo, de curso rápido, debilitante y frecuentemente letal. Es causada por la deficiencia de la enzima lisosomal alfa-glucosidasa. Se considera uno de los trastornos por almacenamiento de glucógeno más frecuentes, cuyas manifestaciones pueden iniciar en cualquier momento de la vida con sintomatología muy variable. Se presenta una revisión extensa de la literatura acerca de los principales aspectos médicos sobre la enfermedad de Pompe: etiología y fisiopatología, epidemiología, variantes clínicas, diagnóstico patológico y terapia de reemplazo enzimático. Con esta información se generó una guía diagnóstico-terapéutica dirigida a los especialistas y a los médicos de primer nivel de atención médica, con la finalidad de permitirles identificar los casos de las formas clásica y tardía del padecimiento, para proveer un adecuado tratamiento oportuno y multidisciplinario. Asimismo, se emiten recomendaciones para el funcionamiento de las instituciones de salud y se ofrecen rutas diagnósticas de fácil aplicación. Se concluye que la enfermedad de Pompe puede diagnosticarse y tratarse adecuadamente si se siguen los estándares emitidos en el ámbito internacional.

Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer.

INTRODUCTION: Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-ß1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. METHODS: Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-ß1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. RESULTS: Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. CONCLUSION: Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the development of cell-based therapies for cartilage repair.

Las células TC-1 implantadas invaden a las fibras musculares esqueléticas adyacentes en un modelo murino de cáncer / Implanted TC-1 cells invade neighboring skeletal muscle fibers in a cancer murine model

La línea celular TC-1 se ha utilizado en la evaluación de la inmunoterapia antitumoral. No existen reportes sobre el efecto de las células TC-1 en tejidos adyacentes cuando se implantan en ratones C57BL/6. El objetivo de este trabajo fue evaluar la interacción entre las células TC-1 implantadas y las fibras musculares adyacentes. Se emplearon 8 ratones con células TC-1 implantadas y 3 ratones sin células. Se colectó el sitio del implante de las células tumorales a 10 días, las muestras se procesaron para microscopia de luz y electrónica de transmisión. Se realizaron tinciones con HyE y tricrómico de Masson, histoquímica con PAS, e inmunohistoquímica para identificar citoqueratinas, actina específica de músculo y metaloproteinasa de la matriz-9 (MMP-9). También se comparó el diámetro de las fibras musculares en ambos grupos de estudio. En el análisis histológico se observaron masas de células TC-1 que infiltran el tejido muscular y separan a las fibras entre sí. Las fibras musculares mostraron variaciones en la intensidad de la tinción y disminución de su diámetro. Se observaron masas de células tumorales TC-1 que invaden la fibra hacia el interior y logran atravesar la lámina externa y el sarcolema que las rodea. Se observó positividad a MMP-9 en el citoplasma de las células TC-1, y en el espacio entre las células tumorales y las fibras musculares. En el análisis ultraestructural se observó fragmentación y variaciones en el grosor de la lámina externa y vesículas subsarcolemales en el sitio en donde las células TC-1 invaden las fibras.Ahí, las células TC1 emiten proyecciones de membrana similares a pseudópodos....

TC-1 cells implanted in C57BL/6 mice are a model for evaluation of anti-tumor immunotherapy. To date there are no reports on the effect of implanted TC-1 cells upon neighboring striated muscle cells. The objective of this work was to evaluate the morphology of the interaction established among the implanted cells and the striated muscle cells. The study was carried out as follows: 8 adult C57BL/6 mice received 5x104 cells IP. As a control, 3 mice received no cells. 10 days after cells injection, no signs of tumor are present yet, and the site of cells injection was collected for morphological studies. Samples were processed for light and transmission electron microscopy. Histological sections were stained with H & E, Masson trichromic method, PAS histochemistry and immunohistochemistry for cytocheratins AE1/AE3, muscle specific actin and for matrix metalloproteinase-9. Cross section diameter of muscle sections was compared among experimental and control groups. The histological evaluation showed groups of tumor cells, infiltrating the spaces among muscle fibers. Muscle fibers showed variations in the cross section diameter as well as in the staining pattern. TC-1 cells were seen very close to muscle cells, invading the lamina externa and sarcolema to finally form groups of cells located within the sarcoplasm. This finding was demonstrated by the specific immunolabel for each kind of cell. Reactivity for metalloproteinase-9 was observed within the tumor cells and in the space mediating between the tumor cells and the muscle fiber. At the ultrastructural level, variations of the thickness of lamina externa were observed, as well as interruptions of this structure. Sarcolema also showed fragmentation, and close to these sites a number of subsarcolemmal vesicles were seen. In the vicinity of the muscle fiber, TC-1 cells formed membrane projections directed towards muscle membrane...

Evaluación del efecto del tratamiento con ácido lipoico administrado a la rata Wistar intoxicada con el fruto de Karwinskia humboldtiana / Evaluation of the effect of treatment with lipoic acid administered to the Wistar rat intoxicated with the fruit of Karwinskia humboldtiana

La ingesta accidental de fruto de Karwinskia humboldtiana ocasiona una parálisis flácida, simétrica, progresiva y ascendente, similar al síndrome de Guillain-Barré. Evoluciona en el transcurso de 3 a 12 meses hasta su recuperación total, pero los casos graves terminan en la muerte por insuficiencia respiratoria. No existe un tratamiento específico. La lesión histopatológica descrita en nervio periférico de pacientes, y animales de experimentación corresponde a una desmielinización segmentaria acompañada de degeneración Walleriana. Una de las toxinas extraídas a partir de la semilla, la T-514, ocasiona un incremento de radicales libres in vitro. Los radicales libres se han relacionado con la desmielinización que se presenta en otros tipos de neuropatías como en la diabética. Ya que la lesión ultraestructural que se presenta en los modelos animales de diabetes es similar a la que se observa en la intoxicación experimental con fruto de K. humboldtiana, se decidió administrar un potente agente antioxidante, el ácido a-lipoico en un modelo de intoxicación crónica por fruto de K. humboldtiana. Sin embargo, no se observó mejoría sobre las manifestaciones clínicas evaluadas en los animales o sobre las lesiones histopatológicas presentes en el nervio periférico. Estos resultados sugieren que los radicales libres no son el mecanismo principal de lesión sobre el nervio periférico en la polineuropatía causada por K. humboldtiana.

The accidental ingestion of Karwinskia humboldtiana causes a flaccid, symmetrical, progressive and ascending paralysis, similar to Guillain-Barre syndrome. It evolves over the course of 3 to 12 months until full recovery, but severe cases end in death due to respiratory failure. There is no specific treatment. The histopathological lesions described in peripheral nerve of patients and in experimental animals, corresponds to segmental demyelination accompanied by Wallerian degeneration. One of the toxins extracted from the seed, T-514, causes an increase of free radicals in vitro. Free radicals have been associated to demyelination that occurs in other types of neuropathy such as diabetic neuropathy. Since the ultrastructural damage that occurs in animal models of diabetes is similar to that observed in experimental poisoning with the fruit of K. humboldtiana, we decided to administer a powerful antioxidant, a-lipoic acid, in a model of chronic poisoning due of K. humboldtiana. However, no improvement was observed on the clinical manifestations evaluated in animals or in the histopathological lesions in the peripheral nerve. These results suggest that free radicals are not the primary mechanism of injury on the peripheral nerve caused by K. humboldtiana.

Human bone morphogenetic protein 2-transduced mesenchymal stem cells improve bone regeneration in a model of mandible distraction surgery.

BACKGROUND: Bone morphogenetic proteins (BMPs) are actively involved in ossification, and BMP-2 participates throughout the entire process. Gene therapy for bone regeneration using adenovirus-expressing BMPs has been successful in small mammals, but it has not been satisfactory in large mammals. METHODS: We generated a 3-component implant (3C graft) comprising autologous mesenchymal stem cells (MSCs), ex vivo transduced with an adenovirus vector-expressing BMP-2 and embedded in a demineralized human bone matrix (DBM). RESULTS: In vitro studies demonstrated vector-induced osteogenesis; osteoblast population and mineralization of the extracellular matrix were greater in the vector-transduced cultures than in the controls (nontransduced MSCs stimulated with osteogenic media were used as positive controls, and nontransduced MSCs served as a negative control). The 3-component grafts were used to fill osteotomies created by bone distraction surgery in mongrel dogs. Control groups comprised dogs with bone distraction alone and dogs with nontransduced MSC grafts. The radiography follow-up, performed 10 weeks after distraction, demonstrated a remarkable reduction in the consolidation period compared with controls. Postmortem mandibles submitted for anatomic and histologic analyses showed improved remodeling and bone maturation in the 3C-grafted dogs. Inflammatory infiltrates were not observed in any of the treated areas, and no liver toxicity was detected. CONCLUSIONS: We demonstrated acceleration of osteogenesis in a dog model for bone distraction by using an implant of BMP-2 modified MSCs. These results are helpful for future clinical trials of mandible bone distraction.

Decrease of virulence for BALB/c mice produced by continuous subculturing of Nocardia brasiliensis.

BACKGROUND: Subculturing has been extensively used to attenuate human pathogens. In this work we studied the effect of continuous subculturing of Nocardia brasiliensis HUJEG-1 on virulence in a murine model. METHODS: Nocardia brasiliensis HUJEG-1 was subcultured up to 130 times on brain heart infusion over four years. BALB/c mice were inoculated in the right foot pad with the bacteria subcultured 0, 40, 80, 100 and 130 times (T0, T40, T80 T100 and T130). The induction of resistance was tested by using T130 to inoculate a group of mice followed by challenge with T0 12 weeks later. Biopsies were taken from the newly infected foot-pad and immunostained with antibodies against CD4, CD8 and CD14 in order to analyze the in situ immunological changes. RESULTS: When using T40, T80 T100 and T130 as inoculums we observed lesions in 10, 5, 0 and 0 percent of the animals, respectively, at the end of 12 weeks. In contrast, their controls produced mycetoma in 80, 80, 70 and 60% of the inoculated animals. When studying the protection of T130, we observed a partial resistance to the infection. Immunostaining revealed an intense CD4+ lymphocytic and macrophage infiltrate in healing lesions. CONCLUSIONS: After 130 in vitro passages of N. brasiliensis HUJEG-1 a severe decrease in its virulence was observed. Immunization of BALB/c mice, with these attenuated cells, produced a state of partial resistance to infection with the non-subcultured isolate.

Karwinskia humboldtiana (buckthorn) fruit causes central nervous system damage during chronic intoxication in the rat.

Karwinskia humboldtiana fruit (Kh) causes a neurological disorder 3-4 weeks after ingestion, characterized by flaccid, symmetrical, ascending paralysis, similar to the Guillain-Barre syndrome. In this polyneuropathy the lesion (demyelization) in peripheral nerves has been described in several animal species, both in acute and in chronic intoxication. However, no reports exist about the presence of lesions in the Central Nervous System (CNS), in chronic intoxication. We considered it important to evaluate, with histological techniques, the possible presence of lesions in the brain, by using a model of chronic intoxication that reproduces the same stages present in the human intoxication, to better understanding of this pathological process. In our present work we fed the ground Kh fruit to Wistar rats and samples of brain, cerebellum, and pons were embedded in paraffin. Sections were stained with Hematoxylin & Eosin (HE) and special stains for nerve tissue. Histopathological changes were evaluated in the CNS through the different stages of the polyneuropathy and comparison to a control group. With this methodology, we found lesions in the motor pathway. This is the first report about the presence of neuronal damage caused by Kh in the Central Nervous System in chronic intoxication.