Células madre y sus exosomas como terapia avanzada para tratar la hernia incisional: prueba de concepto en modelo murino / Stem cells and their exosomes as an advanced therapy to treat incisional hernia: proof of concept in a murine model

Una hernia incisional consiste en una protrusión de tejido a través de una cicatriz traumática o quirúrgica en la pared abdominal. El tratamiento de este y de otros tipos de hernias pasa frecuentemente por la implantación de una malla quirúrgica para reforzar el tejido debilitado. Sin embargo, a menudo se produce una respuesta inflamatoria exacerbada que desemboca en diferentes complicaciones, teniendo consecuencias graves para el paciente. Considerando el potencial inmunomodulador de las células madre mesenquimales (MSCs) y de sus exosomas (exo-MSCs), en este estudio planteamos que la administración de ambos productos terapéuticos, conjuntamente con los selladores de fibrina que se utilizan frecuentemente para la fijación de las mallas quirúrgicas, podría ejercer un efecto biológico y terapéutico que ayudara a controlar esa respuesta inflamatoria y mejorara, por tanto, el éxito del tratamiento con mallas quirúrgicas. Los resultados obtenidos en este estudio mostraron, en un modelo murino de hernia incisional, que los exo-MSCs reducen la infiltración de macrófagos inflamatorios M1 y que existe una predominancia de citoquinas relacionadas con la respuesta Th2, y con ello, con la polarización de macrófagos hacia un fenotipo M2 antiinflamatorio, en el tejido circundante a las mallas en las que se vehicularon MSCs o sus exosomas. Este estudio concluye que la fijación de mallas quirúrgicas con selladores de fibrina combinados con MSCs o exo-MSCs tendría un efecto beneficioso en el tratamiento de la hernia incisional, en términos de reducción de la respuesta inflamatoria y modulación de una reacción exacerbada frente a un cuerpo extraño

An incisional hernia constitutes a tissue protrusion through a traumatic or surgical scar in the abdominal wall. Frequently, the treatment of the incisional hernia, as well as other types of hernia, involves the implantation of a surgical mesh to reinforce the weakened tissue. However, an exacerbated inflammatory response is commonly developed after this implantation, having serious consequences for the patient. Considering the immunomodulatory potential of mesenchymal stem cells (MSCs) and their exosomes (exo-MSCs), in this study we proposed that the administration of these two therapeutic products, together with fibrin sealants that are frequently used to fix surgical meshes, could have a beneficial biological and therapeutic effect that could help to modulate the inflammatory response and improve the success of the surgical mesh implantation. The results obtained in this work showed, in a murine model of incisional hernia, that exo-MSCs reduce M1 inflammatory macrophages infiltration and that there is a predominance of Th2- related cytokines in the surrounding tissue of MSCs or exo-MSCs treated meshes, favoring the macrophage polarization towards a M2 anti-inflammatory phenotype. This study concludes that mesh fixation with fibrin sealants co-administered with MSCs or exo-MSCs would have a beneficiary effect on the treatment of incisional hernia in terms of reduction of the inflammatory response and modulation of the foreign body reaction

Chemokine CCL15 Mediates Migration of Human Bone Marrow-Derived Mesenchymal Stem Cells Toward Hepatocellular Carcinoma.

Mesenchymal stem cells (MSCs) possess the ability to migrate toward tumor sites and are regarded as promising gene delivery vehicles for cancer therapeutics. However, the factors that mediate this tropism have yet to be completely elucidated. In this study, through cytokine array analysis, chemokine CCL15 was found to be the most abundant protein differentially expressed in hepatocellular carcinoma (HCC) cell lines compared with a normal liver cell line. Serum CCL15 levels in HCC patients determined by enzyme linked immunosorbent assay were shown to be profoundly elevated compared with healthy controls. Immunohistochemical analysis indicated that CCL15 expression was much stronger in HCC tumor tissues than in adjacent nontumor tissues. Transwell migration assay suggested that CCL15 may be involved in chemotaxis of human MSCs (hMSCs) toward HCC in vitro and that this chemotactic effect of CCL15 is mediated via CCR1 receptors on hMSCs. Orthotopic animal models of HCC were established to investigate the role of CCL15 in hMSCs migration toward HCC in vivo. Both histological and flow cytometric analysis showed that significantly fewer hMSCs localized within 97H-CCL15-shRNA xenografts compared with 97H-green fluorescent protein xenografts after intravenous delivery. Finally, the possible effects of hMSCs on HCC tumor growth were also evaluated. Coculture experiments showed that hMSCs had no apparent effect on the proliferation of HCC cells in vitro In addition, systemic administration of hMSCs did not affect HCC tumor progression in vivo. Our data in this study help to elucidate the mechanism underlying the homing capacity of hMSCs toward HCC.

Cytopenias in HIV infection: mechanisms and alleviation of hematopoietic inhibition.

Hematopoietic abnormalities including anemia, cytopenias, and alterations of the stem cell plasticity in the bone marrow microenvironment commonly occur in HIV infected patients. These observations suggest that HIV-1 infection may affect processes important during early stages of hematopoiesis or stem cell differentiation. Hematopoietic abnormalities may be caused by altered stem cell differentiation possibly due to abnormal lineage specific expression of certain cellular genes such as cytokines relevant to hematopoiesis. These cytokines could affect regulatory signals important in hematopoiesis. However, in HIV infected individuals, it is not only the virus but also the highly active antiretroviral therapy (HAART) that both contribute to persistent hematopoietic suppression and ensuing cytopenias. Even if a lowering of HIV replication by HAART were to occur in infected individuals, prolonged HAART by itself and/or appearance of drug resistant mutants can contribute to hematopoietic suppression and resulting cytopenias. However, confounding factors such as opportunistic infections, immune mediated effects, or the consequences of prolonged physiological stress, which could contribute to decreased hematopoiesis in patients or other individuals, make the causative role of HIV in vivo, uncertain. The severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues (SCID-hu) is a small animal model which mimics HIV infection in humans, and is useful to determine the mechanisms of HIV-1 induced hematopoietic inhibition and development of drug therapies for interventions of stem cell differentiation. Further, SCID mouse serves as a useful small animal recipient of human progenitor cells and also allows us to study the differentiation of these cells in vivo. Results from our studies are expected to provide relief for HIV infected individuals from hematopoietic inhibition and ensuing cytopenias.

Genetic fusions with viral chemokines target delivery of nonimmunogenic antigen to trigger antitumor immunity independent of chemotaxis.

The ideal vaccine carrier should be able to target antigen delivery and possibly recruit antigen-presenting cells (APC) and deliver an activation signal to promote adaptive immune responses. Ligands for chemokine receptors expressed on APC may be attractive candidates, as they can both target and attract APC. To investigate the requirement for APC recruitment, we used a pair of viral chemokines, agonist herpes simplex virus 8-derived macrophage inflammatory protein-I (vMIP-I) and antagonist MC148, which induce and suppress chemotaxis, respectively. Chemokine-antigen fusions efficiently delivered a model nonimmunogenic tumor antigen to APC for processing and presentation to antigen-specific T cells in vitro. Physical linkage of chemokine and antigen and specific binding of chemokine receptor by the fusion protein were required. Mice immunized with vMIP-I or MC148 fusion DNA vaccines elicited protection against tumor challenge. Therefore, vaccine efficacy depends primarily on the ability of the carrier to target antigen delivery to APC for subsequent processing and presentation, and chemotaxis directly induced by the chemokine moiety in the fusion may not be necessary.

A phase II protection study of BB-10010 in patients with high grade non-Hodgkin's lymphoma undergoing intensive chemotherapy.

The aim of this study was to determine whether administration of BB-10010, a synthetic stem cell inhibitor, would allow more intensive chemotherapy to be administered to patients with newly diagnosed high grade NHL. Thirteen patients were randomised to receive BB-10010 concurrently with dose-intensified BEMOP/CA chemotherapy (7 patients) or chemotherapy alone (6 patients). Although the mean neutrophil count of BB-10010 treated patients was higher following cycles 1, 2 and 3 of chemotherapy compared with those receiving chemotherapy alone, there was no difference in the mean number of cycles tolerated, blood component usage and hospital admissions due to infections. No specific toxicity of BB-10010 was identified. Whilst BB-10010 can be administered safely, it does not improve the ability of patients to tolerate intensive chemotherapy for high grade NHL.

Transfection of macrophage inflammatory protein 1 alpha into B16 F10 melanoma cells inhibits growth of pulmonary metastases but not subcutaneous tumors.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha), a CC chemokine, is a chemoattractant for T cells and immature dendritic cells. Plasmacytoma cells expressing MIP-1 alpha generate a cytotoxic T cell response without affecting tumor growth. To understand this discrepancy, we compared a local tumor model with a metastatic one using MIP-1 alpha-transfected B16 F10 melanoma cells. Clonal idiosyncrasies were controlled by selecting three lipotransfected tumor clones and two pcDNA vector transfected control clones with equivalent in vitro proliferative capacities. No significant differences were seen between the MIP-1 alpha-producing and control melanoma cells after s.c. injection in the hind leg. All animals had a leg diameter of 10 cm in 18.5-21.5 days. However, after i.v. injection the number of pulmonary foci was significantly reduced in the MIP-1 alpha-producing clones. Injection of 10(6) control transfected cells resulted in a median of 98.5 tumor foci in 2 wk, whereas the injection of the MIP-1 alpha-producing clones resulted in 89.5, 26.5, and 0 foci. The number of metastatic foci was inversely proportional to the amount of MIP-1 alpha produced by the clone in vitro. Flow cytometry showed a significant increase in CD8(+) cells in lungs of mice with MIP-1 alpha-transfected tumors 3 days after injection. This increase was not maintained 10 days later despite continued production of MIP-1 alpha. The protection offered by transfection with MIP-1 alpha was significantly impaired in beta(2)-microglobulin(-/-) mice. Our findings suggest that MIP-1 alpha is effective in preventing the initiation of metastasis, but not at sustaining an effective antitumor response.

A randomized phase-II study of BB-10010 (macrophage inflammatory protein- 1alpha) in patients with advanced breast cancer receiving 5-fluorouracil, adriamycin, and cyclophosphamide chemotherapy.

BB-10010 is a variant of the human form of macrophage inflammatory protein-1alpha (MIP-1alpha), which has been shown in mice to block the entry of hematopoietic stem cells into S-phase and to increase their self-renewal capacity during recovery from cytotoxic damage. Its use may constitute a novel approach for protecting the quality of the stem cell population and its capacity to regenerate after periods of cytotoxic treatment. Thirty patients with locally advanced or metastatic breast cancer were entered into the first randomized, parallel group controlled phase II study. This was designed to evaluate the potential myeloprotective effects of a 7-day regimen of BB-10010 administered to patients receiving six cycles of 5-fluorouracil (5-FU), adriamycin, and cyclophosphamide (FAC) chemotherapy. Patients were randomized, 10 receiving 100 microgram/kg BB-10010, 11 receiving 30 microgram/kg BB-10010, and nine control patients receiving no BB-10010. BB-10010 was well-tolerated in all patients with no severe adverse events related to the drug. Episodes of febrile neutropenia complicated only 4% of the treatment cycles and there was no difference in incidence between the treated and nontreated groups. Studies to assess the generation of progenitor cells in long-term bone marrow cultures were performed immediately preceding chemotherapy and at the end of six dosing cycles in 18 patients. Circulating neutrophils, platelets, CD 34(+) cells, and granulocyte/macrophage colony-forming cell (GM-CFC) levels were determined at serial time points in cycles 1, 3, and 6. The results showed similar hemoglobin and platelet kinetics in all three groups. On completion of the six treatment cycles, the average pretreatment neutrophil levels were reduced from 5.3 to 1.7 x 10(9)/L in the control patients and from 4.3 to 1.9 and 4.5 to 2.5 x 10(9)/L in the 30/100 microgram/kg BB-10010 groups, respectively. Relative to their pretreatment values, 50% of the patients receiving BB-10010 completed the treatment with neutrophil values significantly higher than any of the controls (P = .02). Mobilization of GM-CFC was enhanced by BB-10010 with an additional fivefold increase over that generated by chemotherapy alone, giving a maximal 25-fold increase over pretreatment values. Bone marrow progenitor assays before and after this standard regimen of chemotherapy indicated little long-term cumulative impairment to recovery from chemotherapy. Despite the limited cumulative damage to the bone marrow, which may have minimized the protective value of BB-10010 during this regimen of chemotherapy, better recovery of neutrophils in the later treatment cycles with BB-10010 was indicated in a number of patients.

The growth inhibitory role and potential clinical value of macrophage inflammatory protein 1 alpha in myeloid leukaemias.

The control of primitive haemopoietic progenitor cell proliferation in vitro can be achieved with combinations of growth stimulatory cytokines. Acting in apparent opposition to these growth stimulators are growth inhibitory substances, including prostaglandins, cytokines and chemokines which bind to specific cognate cell surface receptors and promote signal transduction events that interfere with cellular proliferation. Within the bone marrow microenvironment, significant quantities of both growth inhibitors and growth promoters can be detected. The ratio of their concentrations within microenvironmental niches of the marrow may regulate primitive blood cell production. The potential exists, therefore, for the disregulation of haemopoiesis via the disruption of the balance between positive and negative regulators of haemopoietic progenitor proliferation. In one particular disease, chronic myeloid leukaemia (CML), there is a lack of response of leukaemic cells to the chemokine growth inhibitor, Macrophage Inflammatory Protein-1alpha (MIP-1alpha). The role of MIP-1alpha in regulation of haemopoiesis, the response of CML progenitor cells and other myeloid leukaemic cells to this chemokine, and the reasons for lack of response to MIP-1alpha in leukaemic cells are reviewed.

Myeloid progenitor cell proliferation and mobilization effects of BB10010, a genetically engineered variant of human macrophage inflammatory protein-1alpha, in a phase I clinical trial in patients with relapsed/refractory breast cancer.

Macrophage Inflammatory Protein (MIP)-1alpha is myelosuppressive in vitro and in vivo for hematopoietic stem and immature subsets of myeloid progenitor cells, demonstrates some myeloprotective effects in mice treated with Ara-C and hydroxyurea, and has stem/progenitor cell mobilizing activity in mice. Based on these observations, BB10010, a genetic variant of MIP-1alpha, was assessed for effects on marrow and blood myeloid progenitor cells in patients with relapsed/refractory breast cancer. MIP-1alpha readily polymerizes, whereas BB10010 has a reduced tendency to form large polymers at physiological pH and ionic strength and retains biological activity. Patients were injected with 5, 10, 30 or 100 microg/kg BB10010 s.c. daily for 3 days. BB10010 significantly reduced the cycling status of marrow myeloid progenitors from pretreatment levels of 39-58% to 0 - 11% one day after the third and last injection of BB10010. This was associated with significant decreases in frequency of marrow progenitors (number of colonies formed per number of cells plated) and percent biopsied marrow CD34+ cells. The suppressive effects were reversible in patients and the rapidity of this reversal demonstrated in mouse studies. BB10010 had no effect on nucleated cellularity or on the proliferation of nucleated cells as assessed in marrow biopsies from the patients. These latter effects may in part reflect the noted decreased apoptosis of nucleated cells by BB10010. BB10010 also demonstrated significant but modest myeloid progenitor cell mobilizing capacity. Blood progenitors were in a slow or non-cycling state prior to treatment and this did not change after administration of BB10010. The above effects of BB10010 were similar at the four different dosage levels assessed. These results demonstrate in humans the suppressive and mobilizing effects of MIP-1alpha and BB10010 previously noted in vivo in mice.

C-C chemokine RANTES and HIV long terminal repeat-driven gene expression.

The C-C chemokines RANTES, MIP-1alpha, and MIP-1beta have been characterized as constituents of an HIV- and SIV-suppressive factor released by CD8+ cells. Furthermore, it has been demonstrated that chemokine receptors cooperate in HIV entry. However, these proteins are also known to have an effect on multiple intracellular signaling cascades that may affect the process of transcription. In the present study we demonstrate that treatment of CD4+ T cells with these chemokines or with cell supernatants from HTLV-I-immortalized CD8+ T cells results in significant reduction in the abundance of HIV-1-specific RNA as analyzed by Northern blot hybridization. To examine the possibility that such suppressive factors may inhibit HIV RNA transcription, we studied the effect of RANTES, the most effective HIV-suppressive chemokine, on basal and Tat-induced HIV-directed LTR expression of a reporter gene. Neither recombinant RANTES nor conditioned medium from CD8+ cells significantly altered HIV-1 LTR-directed chloramphenicol acetyltransferase expression in either transiently or stably transfected CD4+ T cell lines, either in the presence or in the absence of Tat. These results suggest that C-C chemokines do not inhibit viral RNA transcription.

A randomized phase II study of BB-10010: a variant of human macrophage inflammatory protein-1alpha for patients receiving high-dose etoposide and cyclophosphamide for malignant lymphoma and breast cancer.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that can inhibit the cell cycle progression of both primitive haemopoietic and epidermal progenitor cells. This property could potentially be exploited to attenuate both the myelosuppressive effects of chemotherapy as well as mucositis. We evaluated both the biological and clinical effects of BB-10010, a genetically engineered variant of MIP-1alpha, in patients with malignant lymphoma or breast cancer receiving high-dose etoposide (VP 3.6 g/m2) and cyclophosphamide (Cy 200 mg/kg). 52 patients were randomized to one of three cohorts. Cohort A received no BB-10010; cohorts B and C received 10 microg/kg and 100 microg/kg of BB-10010, respectively. All patients received post-chemotherapy G-CSE BB-10010 was well tolerated. There were no significant differences between groups in recovery to an ANC > 0.5 x 10(9)/l, 1 x 10(9)/l or 1.5 x 10(9)/l, the number of days with an ANC < 0.5 x 10(9)/l, days to a platelet count > 50 x 10(9)/l or 100 x 10(9)/l, or the incidence and severity of mucositis. There was no evidence of any effect of BB-10010 on colony-forming cell (CFC) or long-term culture-initiating cell (LTC-IC) mobilization, cycling activity in the marrow or on chemotherapy-induced changes in CFC or LTC-IC number both of which were in the normal range by 22 d after completion of the chemotherapy. To our knowledge this is the first report of a myelointensive regimen having no apparent long-term effect on the LTC-IC compartment. In summary, BB-10010 is safe when used in patients receiving high-dose therapy but has no effect on reducing the toxicity of such therapy.