José María Moraleda

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.

A molecular risk score based on 4 functional pathways for advanced classical Hodgkin lymphoma

Despite improvement in the treatment of advanced classical Hodgkin lymphoma, approximately 30% of patients relapse or die as result of the disease. Current predictive systems, determined by clinical and analytical parameters, fail to identify these high-risk patients accurately. We took a multistep approach to design a quantitative reverse-transcription polymerase chain reaction assay to be applied to routine formalin-fixed paraffin-embedded samples, integrating genes expressed by the tumor cells and their microenvironment. The significance of 30 genes chosen on the basis of previously published data was evaluated in 282 samples (divided into estimation and validation sets) to build a molecular risk score to predict failure. Adequate reverse-transcription polymerase chain reaction profiles were obtained from 262 of 282 cases (92.9%). Best predictor genes were integrated into an 11-gene model, including 4 functional pathways (cell cycle, apoptosis, macrophage activation, and interferon regulatory factor 4) able to identify low- and high-risk patients with different rates of 5-year failure-free survival: 74% versus 44.1% in the estimation set (P < .001) and 67.5% versus 45.0% in the validation set (P = .022). This model can be combined with stage IV into a final predictive model able to identify a group of patients with very bad outcome (5-year failure-free survival probability, 25.2%).

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.

Autologous stem cell transplantation after conditioning with Yttrium-90 ibritumomab tiuxetan plus beam in refractory non-Hodgkin diffuse large B-cell lymphoma: results of a prospective, multicenter, phase II clinical trial

Lymphoma patients with persistent disease undergoing autologous transplantation have a very poor prognosis in the rituximab era. The addition of radioimmunotherapy to the conditioning regimen may improve the outcome for these patients. In a prospective, phase 2 study, we evaluated the safety and efficacy of the addition of 90Y-ibritumomab tiuxetan to the conditioning chemotherapy in patients with refractory diffuse large B-cell lymphoma. Thirty patients with induction failure (primary refractory; n=18) or refractory to salvage immunochemotherapy at relapse (n=12) were included in the study. The median age of the patients was 53 years (range, 25–67). All patients were given 90Y-ibritumomab tiuxetan at a fixed dose of 0.4 mCi/kg (maximum dose 32 mCi) 14 days prior to the preparative chemotherapy regimen. Histological examination showed that 22 patients had de novo diffuse large B-cell lymphoma and eight had transformed diffuse large B-cell lymphoma. All patients had persistent disease at the time of transplantation, with 25 patients considered to be chemorefractory. The median time to neutrophil recovery (>500 white blood cells/μL) was 11 days (range, 9–21), while the median time to platelet recovery (>20,000 platelets/μL) was 13 days (range, 11–35). The overall response rate at day +100 was 70% (95% CI, 53.6–86.4) with 60% (95% CI, 42.5–77.5) of patients obtaining a complete response. After a median follow-up of 31 months for alive patients (range, 16–54), the estimated 3-year overall and progression-free survival rates are 63% (95% CI, 48–82) and 61% (95% CI, 45–80), respectively. We conclude that autologous transplantation with conditioning including 90Y-ibritumomab tiuxetan is safe and results in a very high response rate with promising survival in this group of patients with refractory diffuse large B-cell lymphoma with a very poor prognosis. Study registered at European Union Drug Regulating Authorities Clinical Trials (EudraCT) N. 2007-003198-22.

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.

Bone Marrow Transplantation in Hindlimb Muscles of Motoneuron Degenerative Mice Reduces Neuronal Death and Improves Motor Function

Bone marrow has proved to be an adequate source of stem cells for the treatment of numerous disorders, including neurodegenerative diseases. Bone marrow can be easily and relatively painlessly extracted from a patient or allogenic donor and then transplanted into the degenerative area. Here, the grafted cells will activate a number of mechanisms in order to protect, repair, and/or regenerate the damaged tissue. These properties make the bone marrow a feasible source for cell therapy. In this work, we transplanted bone marrow cells into a mouse model of motoneuron degeneration, with the particularity of placing the cells in the hindlimb muscles rather than in the spinal cord where neuronal degeneration occurs. To this end, we analyze the possibility for the transplanted cells to increase the survival rate of the spinal cord motoneurons by axonal-guided retrograde neurotrophism. As a result, the mice significantly improved their motor functions. This coincided with an increased number of motoneurons innervating the treated muscle compared with the neurons innervating the non-treated contralateral symmetric muscle. In addition, we detected an increase in glial-derived neurotrophic factor in the spinal cord, a neurotrophic factor known to be involved in the rescue of degenerating motoneurons, exerting a neuroprotective effect. Thus, we have proved that bone marrow injected into the muscles is capable of rescuing these motoneurons from death, which may be a possible therapeutic approach for spinal cord motoneuron degenerative diseases, such as amyotrophic lateral sclerosis.

Cell therapy in bisphosphonate-related osteonecrosis of the jaw.

ObjectiveBisphosphonate-related osteonecrosis of the jaw (BRONJ) is a clinical condition found in patients who have received intravenous or oral bisphosphonate therapy for various diseases related to bone. This report describes a novel treatment of BRONJ using autologous bone marrow stem cells, platelet-rich plasma, beta tricalcium phosphate, and demineralized bone matrix. Study DesignWe report a 71-year-old woman with history of multiple myeloma treated with intravenous zoledronic acid during 4 years. After a tooth extraction, the patient presented with a painful BRONJ lesion with no healing wound and cortical bone exposure. The patient was surgically managed with a standardized protocol of autologous stem cell therapy combining bone marrow harvest, cell concentration procedures, and intraoral surgery. ResultsCT scan performed 6 months later showed improvement of bone and concentric ossification. Cellular therapy might be considered a new strategy to heal BRONJ lesions.

Amniotic Membrane Modifies the Genetic Program Induced by TGFß, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds

Background Post-traumatic large-surface or deep wounds often cannot progress to reepithelialisation because they become irresponsive in the inflammatory stage, so intervention is necessary to provide the final sealing epidermis. Previously we have shown that Amniotic Membrane (AM) induced a robust epithelialisation in deep traumatic wounds. Methods and Findings To better understand this phenomenon, we used keratinocytes to investigate the effect of AM on chronic wounds. Using keratinocytes, we saw that AM treatment is able to exert an attenuating effect upon Smad2 and Smad3 TGFß-induced phosphorylation while triggering the activation of several MAPK signalling pathways, including ERK and JNK1, 2. This also has a consequence for TGFß-induced regulation on cell cycle control key players CDK1A (p21) and CDK2B (p15). The study of a wider set of TGFß regulated genes showed that the effect of AM was not wide but very concrete for some genes. TGFß exerted a powerful cell cycle arrest; the presence of AM however prevented TGFß-induced cell cycle arrest. Moreover, AM induced a powerful cell migration response that correlates well with the expression of c-Jun protein at the border of the healing assay. Consistently, the treatment with AM of human chronic wounds induced a robust expression of c-Jun at the wound border. Conclusions The effect of AM on the modulation of TGFß responses in keratinocytes that favours proliferation together with AM-induced keratinocyte migration is the perfect match that allows chronic wounds to move on from their non-healing state and progress into epithelialization. Our results may explain why the application of AM on chronic wounds is able to promote epithelialisation.

Stage IV and age over 45 years are the only prognostic factors of the International Prognostic Score for the outcome of advanced Hodgkin lymphoma in the Spanish Hodgkin Lymphoma Study Group series

Abstract The International Prognostic Score (IPS) is the most widely used system to date for identifying risk groups for the outcome of patients with advanced Hodgkin lymphoma, although important limitations have been recognized. We analyzed the value of the IPS in a series of 311 patients with advanced classical Hodgkin lymphoma (cHL) (Ann Arbor stage III, IV or stage II with B symptoms and/or bulky masses) treated with first-line chemotherapy including adriamycin (adriamycin, bleomycin, vinblastine, dacarbazine [ABVD] or equivalent variants). In univariate and multivariate analyses, stage IV disease and age ≥ 45 years were the only factors with independent predictive significance for overall survival (OS) (p = 0.002 and p < 0.001, respectively). Stage IV was still significant for freedom from progression (FFP) (p = 0.001) and age ≥ 45 years was borderline significant (p = 0.058). IPS separates prognostic groups, as in the original publication, but this is mainly due to the high statistical significance of stage IV and age ≥ 45 years. Moreover, the combination of these two factors enables a simpler system to be constructed that separates groups with different FFP and OS. In conclusion, in our series, stage IV and age ≥ 45 years are the key prognostic factors for the outcome of advanced cHL.

Amniotic membrane application for the healing of chronic wounds and ulcers

Wound healing usually follows a predictable sequence and prognosis of events. Its evolutionary process is the result of a complicated interaction between patient-related factors, the wound, the treatment used and the skills and knowledge of the professionals who treat them. Only through a meticulous initial assessment of the wound is it possible to identify the factors that contribute to its complexity. The challenge for professionals will be to implement efficient therapies at the right time and in the most cost-efficient way in order to reduce associated problems, treat the symptoms and expectations of the patients and achieve adequate wound healing whenever possible. This is particularly evident in big chronic wounds with considerable tissue loss, which become senescent in the process of inflammation or proliferation losing the ability to epithelialize. Generally, chronic wounds do not respond to current treatments, therefore they need special interventions. AM is a tissue of particular interest as a biological dressing and it has well-documented reepithelialization effects which are in part related to its capacity to synthesize and release biological active factors. Our studies have demonstrated that amniotic membrane (AM) is able to induce epithelialization in chronic wounds that were unable to epithelialize. AM induces several signaling pathways that are involved in cell migration and/or proliferation. Additionally, AM is able to selectively antagonize the anti-proliferative effect of transforming growth factor-ß (TGF-β) by modifying the genetic program that TGF-β induces on keratinocytes. The combined effect of AM on keratinocytes, promoting cell proliferation/migration and antagonizing the effect of TGF-β is the perfect combination, allowing chronic wounds to move out of their non-healing state and progress into epithelialization.