Xeno-free approach for the expansion of human adipose derived mesenchymal stem cells for ocular therapies.

To restore corneal transparency and vision loss after an injury on the ocular surface, the use of human stem cells from different origins has been recently proposed. Mesenchymal stem cells (MSCs) seem to be an appropriate adult source of autologous stem cells due to their accessibility, high proliferation rate, and multipotent capacity. In this work, we developed a simple culture system to prepare a graft based on a fibrin membrane seeded with human MSCs. A commercial kit, PRGF Endoret®, was used to prepare both, the growth factors used as culture media supplement and the fibrin membrane grafts. Adipose-derived MSCs (Ad-MSCs) were expanded, characterised by flow cytometry and their multilineage differentiation potential confirmed by inducing adipogenesis, osteogenesis and chondrogenesis. Ad-MSCs seeded on the fibrin membranes were grafted onto athymic mice showing good biocompatibility with no adverse reactions observed during the follow up period. These findings support the assumption that a system in which all the biological components (cells, grow factors and carrier) are autologous, could potentially be used for future ex vivo expansion of Ad-MSCs to treat ocular conditions such as an inflammatory milieu, traumatic scars and loss of the regenerative capacity of the corneal epithelium that compromise the quality of vision.

Autologous method for ex vivo expansion of human limbal epithelial progenitor cells based on plasma rich in growth factors technology.

PURPOSE: Develop an autologous culture method for ex vivo expansion of human limbal epithelial progenitor cells (LEPCs) using Plasma Rich in Growth Factors (PRGF) as a growth supplement and as a scaffold for the culture of LEPCs. METHODS: LEPCs were cultivated in different media supplemented with 10% fetal bovine serum (FBS) or 10% PRGF. The outgrowths, total number of cells, colony forming efficiency (CFE), morphology and immunocytochemistry against p63- α and cytokeratins 3 and 12 (CK3-CK12) were analyzed. PRGF was also used to elaborate a fibrin membrane. The effects of the scaffold on the preservation of stemness and the phenotypic characterization of LEPCs were investigated through analysis of CK3-CK12, ABCG-2 and p63. RESULTS: LEPCs cultivated with PRGF showed a significantly higher growth area than FBS cultures. Moreover, the number of cells were also higher in PRGF than FBS, while displaying a better morphology overall. CFE was found to be also higher in PRGF groups compared to FBS, and the p63-α expression also differed between groups. LEPCs cultivated on PRGF membranes appeared as a confluent monolayer of cells and still retained p63 and ABCG-2 expression, being negative for CK3-CK12. CONCLUSIONS: PRGF can be used in corneal tissue engineering, supplementing the culture media, even in a basal media without any other additives, as well as providing a scaffold for the culture.

The chemokine CCL5 induces CCR1-mediated hyperalgesia in mice inoculated with NCTC 2472 tumoral cells.

Although the expression of the chemokine receptor CCR1 has been demonstrated in several structures related to nociception, supporting the nociceptive role of chemokines able to activate it, the involvement of CCR1 in neoplastic pain has not been previously assessed. We have assayed the effects of a CCR1 antagonist, J113863, in two murine models of neoplastic hyperalgesia based on the intratibial injection of either NCTC 2472 fibrosarcoma cells, able to induce osteolytic bone injury, or B16-F10 melanoma cells, associated to mixed osteolytic/osteoblastic bone pathological features. The systemic administration of J113863 inhibited thermal and mechanical hyperalgesia but not mechanical allodynia in mice inoculated with NCTC 2472 cells. Moreover, in these mice, thermal hyperalgesia was counteracted following the peritumoral (10-30µg) but not spinal (3-5µg) administration of J113863. In contrast, hyperalgesia and allodynia measured in mice inoculated with B16-F10 cells remained unaffected after the administration of J113863. The inoculation of tumoral cells did not modify the levels of CCL3 at tumor or spinal cord. In contrast, although the concentration of CCL5 remained unmodified in mice inoculated with B16-F10 cells, increased levels of this chemokine were measured in tumor-bearing limbs, but not the spinal cord, of mice inoculated with NCTC 2472 cells. Increased levels of CCL5 were also found following the incubation of NCTC 2472, but not B16-F10, cells in the corresponding culture medium. The intraplantar injection of CCL5 (0.5ng) to naïve mice evoked thermal hyperalgesia prevented by the coadministration of J113863 or the CCR5 antagonist, d-Ala-peptide T-amide (DAPTA), demonstrating that CCL5 can induce thermal hyperalgesia in mice through the activation of CCR1 or CCR5. However, contrasting with the inhibitory effect evoked by J113863, the systemic administration of DAPTA did not prevent tumoral hyperalgesia. Finally, the peritumoral administration of an anti-CCL5 antibody completely inhibited thermal hyperalgesia evoked by the inoculation of NCTC 2472 cells.

New serum-derived albumin scaffold seeded with adipose-derived stem cells and olfactory ensheathing cells used to treat spinal cord injured rats.

Recent advances in spinal cord injury (SCI) research and cell culture techniques and biomaterials predict promising new treatments for patients with SCI or other nerve injuries. Biomaterial scaffolds form a substrate within which cells are instructed to form a tissue in a controlled manner. This study was designed to assess axon regeneration and locomotor recovery in rats with spinal cord injury treated with a novel serum-derived albumin scaffold seeded with adipose derived stem cells (ADSCs) and olfactory ensheathing cells (OECs). OECs are considered promising candidates for the treatment of SCI, and ADSCs have the ability to differentiate into neural lineages. In vitro experiments revealed that ADSCs and OECs adhered to the scaffold, remained viable and expressed specific markers of their cell types when cultured in the scaffold. Rats treated with scaffold plus cells showed locomotor skills at several time points from 45 days post-injury that were improved over those recorded in control injured, untreated animals. Astrocytic scars and tissue regeneration, identified using histological and immunohistochemical techniques, revealed that although the scaffold itself appeared to play a significant role in reducing glial scar formation and filling of the lesion cavity with cells, the presence of ADSCs and OECs in the scaffold led to the appearance of cells expressing markers of neurons and axons at the injury site. Our findings point to the clinical feasibility of an albumin scaffold seeded with ADSCs and OECs as a treatment candidate for use in spinal cord injury repair studies.

In vivo behavior of complete human oral mucosa equivalents: characterization in athymic mice.

BACKGROUND AND OBJECTIVE: The interest in tissue engineering as a way to achieve repair of damaged body tissues has led to the carrying out of many studies whose results point to the potential effectiveness of these methods. In a previous study, we reported the obtaining of complete autologous oral mucosa equivalents (CAOMEs), characterized by oral immature keratinocytes and stem cells on an autologous plasma and fibroblast scaffold. The purpose of this study is to show their behavior in vivo, by using them as free grafts in experimental animals, and to demonstrate their potential capacity to regenerate oral mucosa. MATERIAL AND METHODS: We engineered CAOMEs, as previously described. All CAOMEs thus obtained were used as free grafts in nu/nu mice. To assess their evolution in vivo, we studied their histological and immunohistochemical features by using AE1/AE3 pancytokeratin, the 5/6 cytokeratin pair, cytokeratin 13, laminin 5, collagen IV, vimentin, p-63 and Ki-67, at 7, 14 and 21 d. RESULTS: The structure became progressively closer to that of oral mucosa samples. Cytokeratin 5/6 staining became increasingly intense in the basal and suprabasal layers, and cytokeratin 13 was exclusively positive in the superficial layers. The basal membrane was completed in 21 d. Vimentin showed a correct formation of the chorion. The increasingly positive staining of p-63 and Ki-67 indicated that the regeneration process was taking place. CONCLUSION: The present study shows the potential regenerative capacity of the CAOMEs by their ability to reach maturity similar to that seen in oral mucosa.

In vitro engineering of complete autologous oral mucosa equivalents: characterization of a novel scaffold.

BACKGROUND AND OBJECTIVE: Restoration of oral mucosa defects by means of in vitro-cultured equivalents has become a valid alternative in the field of oral and periodontics surgery. Although different techniques have been described, none has been able to provide an equivalent with an autologous scaffold for the epithelium. The purpose of this study was to obtain complete autologous oral mucosa equivalents (CAOME) using the patient's own fibroblasts and plasma and to characterize these equivalents both morphologically and immunohistochemically. MATERIAL AND METHODS: We acquired cell types (keratinocytes and fibroblasts) from the same mucosal samples, which were taken from healthy patients who underwent oral surgery. To construct the CAOME, a small sample of blood was obtained from the patient and subsequently processed to obtain a fibrin glue scaffold. All CAOME thus obtained were stained using the standard hematoxylin and eosin method to study their morphological characteristics. To establish the type of cells in the epithelial layer, CAOME were stained with pancytokeratin AE1/AE3, cytokeratins 5/6 and 13, p-63 and Ki-67. Finally, laminin 5 and collagen IV were used to reveal the presence of a basal membrane. RESULTS: The CAOME featured a monolayer of cube-shaped epithelial cells similar to that found on the basal layer of the oral mucosa. Close to the epithelial layer lay the fibrin and fibroblasts-embedded scaffold. The CAOME was positive to pancytokeratin AE1/AE3, cytokeratin 5/6 and p-63. No reaction was found to cytokeratin 13 and Ki-67. There was staining to laminin 5 but not to collagen IV. CONCLUSIONS: It is possible to engineer a CAOME with an epithelium of basal-like and immature keratinocytes, which could potentially reconstruct in vivo loss of tissue.

Visceral leishmaniasis and other severe infections in an adult patient with p47-phox-deficient chronic granulomatous disease.

We report a rare case of a male patient without known immunodeficiency consecutively diagnosed with visceral leishmaniasis, brain abscess and cavitating pneumonia in the 3rd decade of life. Chronic granulomatous disease (CGD) was diagnosed by a nitroblue tetrazolium test. A p47-phox mutation of the NADPH oxidase of the leukocytes was suspected by immunoblotting and confirmed by DNA analysis. The patient was homozygous for this mutation while his mother and sister were heterozygous asymptomatic carriers. After the CGD diagnosis the patient started a chronic prophylactic regimen with subcutaneous interferon-gamma (0.05 mg/m2 of body surface/three times a week), and oral trimethoprim-sulfamethoxazole and itraconazole (both at 5 mg/kg/day) with no subsequent infections after 12 months of follow-up.

Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter.

Cultured epithelial grafts have proven to be life-saving in the treatment of large skin losses. It has become apparent that one of the main difficulties of this technology is the overall poor take of the grafts as a consequence of severely damaged dermal beds. Skin substitutes providing both cultured keratinocytes, as an epidermal layer, and a dermal analogous offer a more suitable material for skin repair. Ex vivo transfer of stroma regeneration-promoting genes to keratinocytes appears to be an attractive strategy for improving the therapeutic action of these grafts. The use of epidermal-specific promoters as expression drivers of exogenous genes results in both high expression levels and stratum specificity, as shown in transgenic mice studies. Most current gene transfer protocols to primary keratinocytes involve transduction of epidermal cells with retroviral vectors. However, transfer of gene constructs harboring these long DNA fragment promoters cannot be achieved through viral transduction. In this paper, we describe a protocol consisting of lipid-mediated transfection, G418 selection and an enhanced green fluorescence protein (EGFP)-based enrichment step for obtaining high levels of transgene-expressing primary keratinocytes. Using this protocol, the cDNA for vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen driven by the 5.2 kb bovine keratin K5 promoter, was stably transfected into pig primary keratinocytes. Genetically modified keratinocytes, expanded on live fibroblast-containing fibrin gels and transplanted to nude mice as a composite material, elicited a strong angiogenic response in the host stroma as determined by fresh tissue examination and CD31 immunostaining. Since the formation of a well-vascularized wound bed is a crucial step for permanent wound closure, the use of an 'angiogenic' composite material may improve wound bed preparation and coverage with cultured keratinocyte grafts.

Natural infection by gastrointestinal and bronchopulmonary nematodes in mouflons (Ovis musimon) and their response to netobimin treatment.

Gastrointestinal and bronchopulmonary nematode infections and the efficacy of netobimin (Hapasil) were analyzed by way of fecal examination in 10 female mouflons (Ovis musimon), in central Spain, February 1993. Before treatment all 10 mouflons had Trichostrongylus axei, Teladorsagia circumcincta and Marshallagia spp.; sic had Nematodirus spp., two had Trichuris sp., one had Capillaria sp., seven had bronchopulmonary Dictyocaulus filaria and 10 mouflons had protostrongylid lungworms (Muellerius capillaris, Protostrongylus rufescens, Cystocaulus ocreatus or Neostrongylus linearis). Netobimin (7.5 mg/kg) was 100% effective against T. axei, T. circumcincta, Marshallagia spp., and D. filaria infections whereas one animal continued eliminating Nematodirus spp. eggs. The drug also was effective against Capillaria spp. but not against Trichuris spp. or protostrongylid infections.

[Pathology of the phagocytic mononuclear system. Apropos of 4 cases]. / Patología del sistema mononuclear fagocítico. A propósito de cuatro observaciones.

Four patients, with disorders belonging to mononuclear phagocyte system diseases are described: a case of malignant histiocytosis; one of Weber-Christians disease and two siblings affected by familiar erythrophagocytic lymphohistiocytosis. An attempt is made to update classification of this group of diseases previously known as reticulosis, reticulohistiocytosis, reticuloendotheliosis, etcetera.