Cryopreserved amniotic membrane in the treatment of diabetic foot ulcers: a case series.

OBJECTIVE: The amniotic membrane (AM) is a tissue with low immunogenity and high therapeutic potential due to its anti-inflammatory, anti-fibrotic and antimicrobial effects. This paper describes the use of cryopreserved amniotic membrane allografts to treat diabetic foot ulcers (DFUs) in patients with diabetes. METHOD: In this case series, AM was processed to obtain a final medicinal product: cryopreserved amniotic membrane. cryopreserved AM was applied every 7-10 days until total epithelialisation of the DFUs. RESULTS: A total of 14 patients with DFUs (median size: 12.30cm, (range: 0.52-42.5cm2) were treated and followed up until complete closure (median time: 20 weeks, range: 7-56 weeks). Patients received 4-40 AM applications. All patients in this study achieved complete epithelialisation of the wound. No adverse events were observed. CONCLUSION: AM is a feasible and safe treatment in complex DFUs. Furthermore, the treatment is successful in achieving epithelialisation of long-evolution, unhealed wounds resistant to conventional therapies.

Heterogeneity of mesenchymal cells in human amniotic membrane at term.

There is increasing interest in understanding the tissue biology of human amniotic membrane (hAM) given its applications in medicine. One cellular component is mesenchymal cells, which can be extracted, cultured and differentiated "in vitro" into various cell types. These studies show that there is heterogeneity among mesenchymal cells. The aim of this work is to study the membrane "in situ" to determine whether this cellular heterogeneity exists. The hAMs were obtained from caesarean deliveries at term and analyzed by histological techniques. Types I-III mesenchymal cells and Hofbauer were distinguished by light microscopy. Histochemically, mesenchymal cell types showed successively increasing positivity to: PAS, vimentin, fibronectin, and Concanavalin-A; VGEF, TGF-ß2, PDGF-C, FGF-2. By the semiquantitative point of view, the percentage of Type II cells was 60%, significantly higher than the other types. With transmission electron microscopy, an intermediate cell type between II-III was observed. Strong vesiculation of the rough endoplasmic reticulum (RER) with exocytosis was observed. In addition, an accumulation of a similar material to the extracellular matrix in the RER caused its dilation especially in type III cells. Some of this material acquired a globular structure. These structures were also found free in the extracellular matrix. In conclusion, the mesenchymal cells of the fibroblastic layer of the hAMs studied are heterogeneous, with some undifferentiated and others with a probably senescent fibroblastic phenotype with accumulation in their RER of fibronectin. These results may be of interest to extract mesenchymal cells from hAMs for use in regenerative medicine and to better understand the mechanisms of fetal membrane rupture.

Cryopreservation impact on blood progenitor cells: influence of diagnoses, mobilization treatments, and cell concentration.

BACKGROUND: The aim of this study was to analyze the impact of cryopreservation in series of peripheral blood progenitor cells stratified by diagnosis, mobilization treatments, and cell concentration, as well as the accuracy of the control aliquots. STUDY DESIGN AND METHODS: Viability and colony-forming unit-granulocyte-macrophage (CFU-GM), CD34+ cell, lymphocyte, monocyte, and granulocyte counts and recovery were analyzed in 397 leukapheresis procedures before freezing and after thawing. Data from control cryotubes were compared to those from infusing bags. RESULTS: Cell viability decreased after thawing. Viability recovery was lower in cryotubes than in bags in non-Hodgkin's lymphoma (NHL), in cyclophosphamide plus granulocyte-colony-stimulating factor (Cy+G-CSF) mobilization, and in cell concentration of median or greater. Viability recovery in cryotube was higher in NHL (92.1%) than in Hodgkin's disease (HD; 87.3%) and in G-CSF (95.9%) than Cy+G-CSF mobilization (91.3%). The number of CD34+ cells decreased after thawing in total group, Cy+G-CSF mobilization, and cell concentration less than median subgroups. CD34+ cell recovery was higher in cryotubes (111.3%) than in bags (99.6%) in multiple myeloma (MM; p = 0.015). CFU-GM decreased after thawing in all groups. CFU-GM recovery was lower in cryotubes than in bags in MM (26.0% vs. 59.3%) and in Cy+G-CSF mobilization (49.8% vs. 76.3%). CFU-GM recovery in cryotubes was lower in MM compared with NHL (61.5%), HD (45.1%), and breast cancer (84.0%). Lymphocytes, monocytes, and granulocytes showed differences in the subgroups. CONCLUSION: Cryopreservation negatively impacts in cell viability, CD34+ cell recovery, granulocytes, and CFU-GM, although slight differences between the groups were observed. Cryotubes satisfactorily reflected the quality of the infused cells.

Amniotic membrane induces epithelialization in massive posttraumatic wounds.

Large-surface or deep wounds often become senescent in the inflammatory or proliferation stages and cannot progress to reepithelialization. This failure makes intervention necessary to provide the final sealing epithelial layer. The best current treatment is autologous skin graft, although there are other choices such as allogenic or autologous skin substitutes and synthetic dressings. Amniotic membrane (AM) is a tissue of interest as a biological dressing due to its biological properties and immunologic characteristics. It has low immunogenicity and beneficial reepithelialization effects, with antiinflammatory, antifibrotic, antimicrobial, and nontumorigenic properties. These properties are related to its capacity to synthesize and release cytokines and growth factors. We report the use of AM as a wound dressing in two patients with large and deep traumatic wounds. Negative pressure wound therapy followed by AM application was capable of restoring skin integrity avoiding the need for skin graft reconstruction. AM induced the formation of a well-structured epidermis. To understand this effect, we designed some assays on human keratinocyte-derived HaCaT cells. AM treatment of HaCaT induced ERK1/2 and SAP/JNK kinases phosphorylation and c-jun expression, a gene critical for keratinocytes migration; however, it did not affect cell cycle distribution. These data suggest that AM substantially modifies the behavior of keratinocytes in chronic wounds, thereby allowing effective reepithelialization.

Chronic model of tympanic perforation in rats with mitomycin C and dexamethasone.

A rat model of chronic tympanic membrane perforation was developed to be used in the search of new materials for the sealing of these perforations. A longitudinal study was carried out in rats subjected to incisional myringotomy followed by the application of mitomycin C alone or with dexamethasone. Rats were checked at days 3, 7, 10, 14 and weekly thereafter until perforation closure, for up to 6 months. The addition of dexamethasone is a key component in order to obtain a chronic opening. Myringotomies treated with saline had a mean healing time of 8.5 days. At 8 weeks, between 62.5% and 77.7% of tympanic membranes treated with mitomycin C and dexamethasone remained perforated and at 6 months this number fell to 21.4%. This technique is able to maintain most tympanic membrane perforations patent for at least 8 weeks. This rat model is adequate for its use in preclinical or translational research.

Amniotic membrane-derived stem cells: immunomodulatory properties and potential clinical application.

Epithelial and mesenchymal cells isolated from the amniotic membrane (AM) possess stem cell characteristics, differentiation potential toward lineages of different germ layers, and immunomodulatory properties. While their expansion and differentiation potential have been well studied and characterized, knowledge about their immunomodulatory properties and the mechanisms involved is still incomplete. These mechanisms have been evaluated on various target cells of the innate and the adaptive system and in animal models of different inflammatory diseases. Some results have evidenced that the immunomodulatory effect of AM-derived cells is dependent on cell-cell contact, but many of them have demonstrated that these properties are mediated through the secretion of suppressive molecules. In this review, we present an update on the described immunomodulatory properties of the derived amniotic cells and some of the proposed involved mechanisms. Furthermore, we describe some assays in animal models of different inflammatory diseases which reveal the potential use of these cells to treat such diseases.

Autocrine TGF-ß induces epithelial to mesenchymal transition in human amniotic epithelial cells.

Human amniotic epithelial cells (hAECs) have been the object of intense research due to their potential therapeutic use. In this paper, we present molecular evidence of a bona fide epithelial to mesenchymal transition (EMT) undergone by hAECs. Amniotic membrane (AM)-derived hAECs showed the presence of typical epithelial markers such as E-cadherin and cytokeratins. hAECs in culture, however, underwent morphological changes acquiring a mesenchymal shape. Epithelial cell markers were lost and typical mesenchymal markers, such as vimentin and α-SMA, appeared. Several genes associated with EMT, such as SNAI1, MMP9, PAI1, or ACTA2, increased their expression. The expression of the transcription activators KLF4 or MTA3 was consistent with the downregulation of CDH1. We have shown that hAECs undergo EMT due to the autocrine production of TGF-ß. Furthermore, the addition of the TGF-ß receptor I (ALK5) inhibitor SB-431542 or TGF-ß neutralizing antibody to hAECs prevented EMT and preserved the hAECs' epithelial phenotype. Altogether, these results suggest that cultured hAECs undergo EMT through the autocrine production of TGF-ß.

Isolation and characterization of mesenchymal stem cells from the fat layer on the density gradient separated bone marrow.

The density gradient centrifugation method was originally designed for the isolation of mononuclear peripheral blood cells and rapidly adapted to fractionate bone marrow (BM) cells. This method involves the use of gradient density solutions with low viscosity and low osmotic pressure that allows erythrocytes and more mature cells gravitate to the bottom at a density fraction superior to 1.080 g/dL; mononuclear cells (MNCs) held in the plasma-solution to interphase at a density between 1.053 and 1.073 g/dL; plasma, dilution medium and anticoagulant to occupy a density less than 1.050 g/dL and the fat cells to float due to their very low density. BM-mesenchymal stem cells (MSCs) are usually obtained after the separation and cultures of BM-MNCs from the plasma-solution interphase, which is traditionally considered the only source of progenitor cells (hematopoietic and nonhematopoietic). In this study evidences that MSCs could be isolated from the very low-density cells of the fat layer are presented. In addition, we demonstrated that the MSCs obtained from these cells have similar immunophenotypic characteristics, and similar proliferative and differentiation potential to those obtained from the MNCs at plasma-solution interphase. The method represents a simple and cost effective way to increase the MSCs yield from each BM donor, without the need to look for other sources, additional manipulation of cells, and risks of contamination or disturbances of the potential of differentiation. These cells might serve as a complementary source of MSCs to facilitate preclinical and clinical application in tissue engineering and cell therapy.

The amniotic membrane as a source of stem cells.

Cellular therapy has emerged as a new potential tool for curing a wide range of degenerative diseases and tissue necrosis. Embryonic stem cells possess potential for differentiation into a wide range of cell lineages, but the ethical issues associated with establishment of this human cell line have to be resolved prior to any use. The bone marrow (BM) is the usual source of adult stem cells for hematopoietic stem cell transplants and cellular therapy, but the BM harvest is a surgical procedure that requires general anesthesia or sedation, and there seems to be a reduction of the proliferative potential and differentiation capacity of the marrow mesenchymal stem cells in older donors. For these reasons there is an increasing interest in other sources of stem cells from adult and fetal tissues. The amniotic membrane (AM) or amnion is a tissue of particular interest because its cells possess characteristics of stem cells with multipotent differentiation ability, and because of low immunogenicity and easy procurement from the placenta, which is a discarded tissue after parturition, thus avoiding the current controversies associated with the use of human embryonic stem cells. Therefore, amniotic membrane has been proposed as a good candidate to be used in cellular therapy and regenerative medicine.

Evaluación del Protocolo 86 para el tratamiento de las Leucemias Mieloblásticas Agudas (P 86 L.M.A.) / Evaluation of the Protocolo 86 for the treatment of Acute Mielogenous Leukemia (P 86 A.M.L)

Se presentan los resultados obtenidos en 56 pacientes con Leucemia Mieloblastica Aguda, menores de 60 años, Tratados con el Protocolo 86 (P 86 L.M.A.) en la Unidad de Hematología y Banco de Sangre del Hospital Universitario de Caracas, durante el período de 1986-1990. Veintitres pacientes (41%) obtuvieron remisión completa (RC) en un tiempo promedio de 50 días; veinte pacientes (36%) murieron por hemorragias e infecciones durante la pancitopenia post inducción y 13 (23%) no respondieron. La sobrevida libre de enfermedad para el 50%de los pacientes de 12 meses con sólo el 11%de los pacientes libres de enfermedad a los 33 meses. La sobrevida total de 21 meses para el 50%de los pacientes que alcanzaron remisión. Se analizan las posibles causas de estos resultados

Leucemia linfoblástica aguda en niños evaluación del protocolo nacional 80 (P.N.80) / Leukemia lymphoblastic acute in child evaluation of the national protocol (P.N.80)

Se presentan los resultados obtenidos en 58 pacientes con Leucemia Lnfoblástica Aguda, menos de 18 años, tratados con el Protocolo nacional 80 (P.N. 80) en la Unidad de Hematología y Banco de sangre del Hospital Universitario de Caracas, durante el período 1980-1985. Veintinueve pacientes de Riesgo Estandard (R.E) y veintinueve de Alto Riesgo (A.R). El porcentaje de Remisión Completa en los R.E fue de 86%, con una sobrevida libre de enfermedad de 11 meses en el 50% de los pacientes, mientras que en los de A.R la Remisión Completa fue de 66% y la sobrevida libre de enfermedad de 6 meses. La sobrevida Total a los 3 años fue de un 50% para los pacientes de R.E y se mantuvo igual durante los 5 años de observación, y de sólo 20% para los pacientes de A.R. Se analizan las posibles causas de estos resultados

Aglutininas antialbúminas / Antialbumin agglutinins

Se presentan dos casos de donantes cuyos sueros presentaron Aglutininas Antialbúminas con diferentes patrones de reactividad serológica. Ambos casos representan los primeros observados en el Banco de Sangre del Hospital Universitario de Caracas desde su fundación en 1956. Se hace una revisión de la evolución y progresión que en el conocimiento de estos anticuerpos se ha adquirido en el transcurso de los años y se señala la necesidad de conocerlos, a fin de evitar la confusión con anticuerpos específicos para los antígenos eritrocitarios de gran importancia en la terapia transfusional