Maria Juliana Majado

Neurotrophic bone marrow cellular nests prevent spinal motoneuron degeneration in amyotrophic lateral sclerosis patients: a pilot safety study.

The objective of this article is to assess the safety of intraspinal infusion of autologous bone marrow mononuclear cells (BMNCs) and, ultimately, to look for histopathological signs of cellular neurotrophism in amyotrophic lateral sclerosis (ALS) patients. We conducted an open single arm phase I trial. After 6 months observation, autologous BMNCs were infused into the posterior spinal cord funiculus. Safety was the primary endpoint and was defined as the absence of serious transplant‐related adverse events. In addition, forced vital capacity (FVC), ALS‐functional rating scale (ALS‐FRS), Medical Research Council scale for assessment of muscle power (MRC), and Norris scales were assessed 6 and 3 months prior to the transplant and quarterly afterward for 1 year. Pathological studies were performed in case of death. Eleven patients were included. We did not observe any severe transplant‐related adverse event, but there were 43 nonsevere events. Twenty‐two (51%) resolved in ≤2 weeks and only four were still present at the end of follow‐up. All were common terminology criteria for adverse events grade ≤2. No acceleration in the rate of decline of FVC, ALS‐FRS, Norris, or MRC scales was observed. Four patients died on days 359, 378, 808, and 1,058 post‐transplant for reasons unrelated to the procedure. Spinal cord pathological analysis showed a greater number of motoneurons in the treated segments compared with the untreated segments (4.2 ± 0.8 motoneurons per section [mns per sect] and 0.9 ± 0.3 mns per sect, respectively). In the treated segments, motoneurons were surrounded by CD90+ cells and did not show degenerative ubiquitin deposits. This clinical trial confirms not only the safety of intraspinal infusion of autologous BMNC in ALS patients but also provides evidence strongly suggesting their neurotrophic activity. STEM CELLS2012;30:1277–1285

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.

Dock10, a novel CZH protein selectively induced by interleukin-4 in human B lymphocytes.

The Dock or CZH proteins are a family of activators for Rho GTPase proteins. The Zizimin subfamily is composed of three members: Dock9, Dock10, and Dock11. We have identified DOCK10 as an interleukin-4 (IL4)-inducible gene in chronic lymphocytic leukemias (CLLs). Subsequently, we have obtained the full-length cDNA sequence, which encodes a 2180 amino acid protein. Dock9 (2069 amino acids) and Dock11 (2073 amino acids) share more identity between them (58%) than with Dock10 (52% and 50%, respectively). Among normal human tissues, DOCK10 and DOCK11 mRNAs were mainly expressed in peripheral blood (PB) leukocytes. Dock10 protein was expressed at similar levels in normal PB-B and PB-T cells. Dock10 protein levels were heterogeneous in CLLs. IL4 consistently increased Dock10 mRNA and protein levels in CLL and normal PB-B cells. In contrast, IL4 did not affect the levels of Dock10 expression in normal PB-T cells. IL4 neither increased DOCK9 or DOCK11 mRNA levels in CLL cells. Dock10 protein distributed in the cytoplasm and nucleus of CLL cells, and IL4 increased its expression in both cellular compartments. The rapid and distinctive induction of Dock10 expression by IL4 in CLL and normal PB-B cells suggests a role for Dock10 in IL4-induced B-cell activation. Dock10 could represent a point of convergence for IL4 signalling and small Rho GTPase function in B cells.

An automatic wash method for dimethyl sulfoxide removal in autologous hematopoietic stem cell transplantation decreases the adverse effects related to infusion

BACKGROUND: Products cryopreserved with dimethyl sulfoxide (DMSO) in stem cell transplant (SCT) often cause many adverse effects during their infusion (major cardiovascular events, dyspnea … even death). These are especially frequent in pediatric patients. We tested if a fully automated and closed wash procedure (Sepax S‐100, Biosafe) allowed us to maintain the absolute CD34+ cell number, cell viability, and engraftment potential, decreasing the untoward reactions.

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.

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.

Human and mouse DOCK10 splicing isoforms with alternative first coding exon usage are differentially expressed in T and B lymphocytes.

DOCK10 is a member of the dedicator of cytokinesis (DOCK) family of Rho GTPase activators preferentially expressed in lymphocytes. In this paper, we analyzed DOCK10 mRNA diversity produced because of alternative splicing. Alternative first coding exon usage led to 2 main protein-coding transcripts, DOCK10.1 and DOCK10.2. Full-length cDNA clones of both isoforms were obtained from both normal human peripheral blood mononuclear cells and mouse spleen for the first time for human DOCK10.1, mouse DOCK10.1, and mouse DOCK10.2. Human and mouse DOCK10.1 clones corresponded to the protein coding assemblies provided by the National Center for Biotechnology Information as Reference Sequences for DOCK10. Our analysis especially focused on human cDNA clones, of which 63% were alternatively spliced forms involving diverse exons and introns. DOCK10.1 expression was enriched in normal T cells, and DOCK10.2 expression was enriched in normal B cells and chronic lymphocytic leukemia (CLL) B cells. Both isoforms were upregulated in response to interleukin-4 in B cells, both normal and CLL, but not in T cells. Our data suggest that cell-specific mechanisms regulate expression of the alternative first exon variants of DOCK10 in vertebrates.

Splenic rupture in a plasma cell leukemia, mobilized with G‐CSF for autologous stem cell transplant

Splenic rupture (SR) is a rare adverse event observed in patients treated with G‐CSF as a peripheral hematopoietic stem cell (PHSC) mobilizing agent, mostly in myeloma multiple and amiloidosis; to date, to our knowledge, it has not been previously described in plasma‐cell leukemia (PCL). We report a case of a woman with PCL, who presented a SR after PHSC mobilization with Cyclophosphamide+G‐CSF. The spleen removed showed hematopoietic foci and amiloid material. In the course of a second mobilization, 2 months after, the patient died from sepsis. We considered it important to report this case, in order to keep in mind the possibility of SR in patients with malignant gammopathy. J. Clin. Apheresis 25:223–225, 2010.

Tratamiento definitivo del hipoparatiroidismo persistente: alotrasplante de paratiroides

Resumen La hipocalcemia posquirurgica normalmente es una complicacion transitoria de la cirugia del tiroides y paratiroides que se suele resolver en no demasiado tiempo; si el problema se hace persistente la solucion es mas delicada. El tratamiento de eleccion seria el trasplante de paratiroides, pero la necesidad de inmunosupresion y sus efectos secundarios hacen discutible su indicacion. Se presenta un caso de trasplante de tejido paratiroideo de un paciente con hiperparatiroidismo secundario a otro con trasplante renal e hipocalcemia grave resistente a tratamiento medico. El injerto es funcionante despues de 2 anos.

Estudio clínico e inmunólogico del xenorrechazo en el xenotrasplante ortotópico de hígado de cerdo a babuino

Resumen Introduccion La experiencia de xenotrasplante hepatico (Xtoh) de cerdo a primate no humano es muy limitada. Nuestros objetivos han sido: a) comprobar si el higado de un cerdo transgenico h-DAF evita el rechazo hiperagudo; b) estudiar las funciones metabolicas del higado porcino tras el Xtoh; y c) analizar el perfil clinico, bioquimico e inmunologico del rechazo vascular agudo retardado. Animales y metodos Se realizaron 6 Xtoh de cerdo a babuino, 4 de cerdos no modificados y dos de cerdos transgenicos para h-DAF. Se llevaron a cabo determinaciones hematologicas, de coagulacion, de xenoanticuerpos y del complemento. En el babuino que sobrevivio 8 dias, se estudiaron durante los mismos las poblaciones linfocitarias y la actividad litica de los linfocitos. Resultados Los valores de xIgG e IgM descendieron drasticamente a los 3 min de la reperfusion, sobre todo del CH50, C3 y C4. En los higados no modificados geneticamente aparecio una coagulacion intravascular diseminada por rechazo hiperagudo, con una supervivencia inferior a 12 h. Con los higados h-DAF, la coagulacion se normalizo, con una supervivencia de 8 y 4 dias, falleciendo ambos por insuficiencia respiratoria, sin rechazo hiperagudo. El babuino que sobrevivio 8 dias presento a las 36 h un rechazo vascular agudo retardado, detectandose una estimulacion de las HLA clase I sobre los linfocitos CD3+ y CD19+, que respondio al tratamiento. Conclusiones El higado transgenico h-DAAF previene el rechazo hiperagudo y mantiene la coagulacion en rangos normales en el babuino. El rechazo vascular agudo provoca el cese en la produccion de bilis y un patron mixto de citolisis y colostasis. Los valores de expresion de HLA clase I en los linfocitos podrian ser utiles para diagnosticarlo.