We previously demonstrated the therapeutic benefits of pentosan polysulfate (PPS) in a rat model of mucopolysaccharidosis (MPS) type VI. Reduction of inflammation, reduction of glycosaminoglycan (GAG) storage, and improvement in the skeletal phenotype were shown. Herein, we evaluate the long-term safety and therapeutic effects of PPS in a large animal model of a different MPS type, MPS I dogs. We focused on the arterial phenotype since this is one of the most consistent and clinically significant features of the model. MPS I dogs were treated with daily oral or biweekly subcutaneous (subQ) PPS at a human equivalent dose of 1.6 mg/kg for 17 and 12 months, respectively. Safety parameters were assessed at 6 months and at the end of the study. Following treatment, cytokine and GAG levels were determined in fluids and tissues. Assessments of the aorta and carotid arteries also were performed. No drug-related increases in liver enzymes, coagulation factors, or other adverse effects were observed. Significantly reduced IL-8 and TNF-alpha were found in urine and cerebrospinal fluid (CSF). GAG reduction was observed in urine and tissues. Increases in the luminal openings and reduction of the intimal media thickening occurred in the carotids and aortas of PPS-treated animals, along with a reduction of storage vacuoles. These results were correlated with a reduction of GAG storage, reduction of clusterin 1 staining, and improved elastin integrity. No significant changes in the spines of the treated animals were observed. PPS treatment led to reductions of pro-inflammatory cytokines and GAG storage in urine and tissues of MPS I dogs, which were most evident after subQ administration. SubQ administration also led to significant cytokine reductions in the CSF. Both treatment groups exhibited markedly reduced carotid and aortic inflammation, increased vessel integrity, and improved histopathology. We conclude that PPS may be a safe and useful therapy for MPS I, either as an adjunct or as a stand-alone treatment that reduces inflammation and GAG storage.

During embryological limb formation mesenchymal cells condense and differentiate into chondrocytes, in a process known as chondrogenesis. These chondrocytes synthesize glycosaminoglycans (GAGs), thus playing an important role in this process. A simplified system in vitro chondrogenesis, using adult mesenchymal stromal cells (MSCs) has been demonstrated. This differentiation potential is usually assessed by histological staining. Objective. Establishment of a semi-automatic grading system for histochemistry stains and immunohistochemistry assays. Materials and methods. For chondrogenesis cells were cultured for three weeks in aggregates with inducing media. Total GAGs were measured using dimethylmethylene blue (DMB) method. For histological analyses aggregates were stained with Alcian blue for total GAGs detection and immunohistochemistry (IHC) for aggrecan was performed. Semi-automatic grading for all slides was obtained after ImageJ analysis. Results. MSCs cultured as aggregates in chondrogenic differentiation media had similar protein concentrations for all time points, suggesting cellularity remained homogenous during culture. Total GAGs was higher for aggregates cultured in chondrogenic compared to complete media. The same trend was observed for Alcian blue stain grades by blinded observer and analysis using ImageJ software. Aggrecan’s IHC analysis had a decreasing tendency with time for aggregates in chondrogenic media for blinded observer and ImageJ evaluation. Conclusion. We developed a functional system for semi-automatic slide grading. We corroborated these results by biochemical analysis with comparable results. To our knowledge, for in vitro chondrogenesis, this is the first report to evaluate stains usingthis methodology. This procedure might be useful for other applications in the field of Biology and Medical Sciences.Key words: mesenchymal stromal cells, in vitro chondrogenesis, glycosaminoglycans, ImageJ

The use of specialized centers has been the main alternative for an appropriate diagnosis, management and follow up of patients affected by inborn errors of metabolism (IEM). These centers facilitate the training of different professionals, as well as the research at basic, translational and clinical levels. Nevertheless, few reports have described the experience of these centers and their local and/or global impact in the study of IEM. In this paper, we describe the experience of a Colombian reference center for the research, diagnosis, training and education on IEM. During the last 20 years, important advances have been achieved in the clinical knowledge of these disorders, as well as in the local availability of several diagnosis tests. Organic acidurias have been the most frequently detected diseases, followed by aminoacidopathies and peroxisomal disorders. Research efforts have been focused in the production of recombinant proteins in microorganisms towards the development of new enzyme replacement therapies, the design of gene therapy vectors and the use of bioinformatics tools for the understanding of IEM. In addition, this center has participated in the education and training of a large number professionals at different levels, which has contributed to increase the knowledge and divulgation of these disorders along the country. Noteworthy, in close collaboration with patient advocacy groups, we have participated in the discussion and construction of initiatives for the inclusion of diagnosis tests and treatments in the health system.

Introducción. El trasplante es una opción terapéutica en la cardiomiopatía chagásica. Para la detección temprana de una posible reactivación de la infección, se propone el uso de pruebas de reacción en cadena de la polimerasa (PCR) a partir de biopsias endomiocárdicas; el modelo de ratón es una aproximación preliminar para evaluar la aplicación de éstas. Objetivo. Evaluar la aplicación de las pruebas de PCR basadas en los iniciadores TcH2AF-R y S35-S36 para la detección de T. cruzi en tejido cardiaco de ratones infectados con el parásito. Materiales y métodos. Se infectaron por vía intraperitoneal dos grupos de ratones ICR de 15 y 10 individuos con 0,3 ml de PBS que contenían 1x106 tripomastigotes de la cepa MHOM/CO/ 2001/D.A. (T. cruzi I) o 1x104 tripomastigotes de la cepa MHOM/BR/00/Y (T. cruzi II), respectivamente. El seguimiento de la parasitemia se realizó mediante microhematocrito y presencia de parásitos en el corazón a los 30, 60 (modelo agudo), 100 y 150 (modelo crónico) días por medio de histopatología y de las PCR TcH2AF-R y S35-S36. Resultados. La histopatología mostró alteraciones en el miocardio y presencia de amastigotes en los modelos agudo y crónico. En contraste al microhematocrito y al análisis histopatológico, la PCR S35-S36 permitió la detección de ambas cepas del parásito. La PCR TcH2AF-R detectó la cepa T. cruzi I con un desempeño superior al microhematocrito y al análisis histopatológico. Conclusiones. El uso de ambas pruebas de PCR puede ser útil en la confirmación de la reactivación de la infección postrasplante