Mesenchymal Stromal Cells from Human Wharton's Jelly Modulate the Intraocular Immune Response in a Glucocorticoid Hypertension Model: An Exploratory Analysis.

INTRODUCTION: Glaucoma is a neurodegenerative disease characterized by the loss of retinal ganglion cells. Recent research suggests immunological changes such as cytokine imbalance may affect its pathophysiology. This implies that immunomodulation, like that of mesenchymal cells, could be a potential therapeutic avenue for this disease. However, the effects of intravitreal injections of human Wharton's jelly-derived mesenchymal stromal cells (hWJ-MSCs) on intraocular immune response have not been assessed in ocular hypertension (OH) models. METHODS: We explored this by measuring cytokine levels and expression of other markers, such as glial fibrillary acidic protein (GFAP) and T cells, in 15 randomly divided New Zealand rabbits: G1: OH, G2: hWJ-MSCs, and G3: OH+hWJ-MSCs. We analyzed the aqueous humor (IL-6, IL-8, and TNF-α) and vitreous humor (IFN-γ, IL-10, and TGF-ß) using ELISA and flow cytometry (cell populations), as well as TCD3+, TCD3+/TCD4+, and TCD3+/TCD8+ lymphocytes, and GFAP in the retina and optic nerve through immunohistochemistry. RESULTS: We found a decrease in TNF-α, IL-6, IFN-γ, IL-10, and IL-8 in G3 compared to G1 and an increase in TGF-ß in both G2 and G3. TCD3+ retinal infiltration in all groups was primarily TCD8+ rather than TCD4+ cells, and strong GFAP expression was observed in both the retina and optic nerves in all groups. CONCLUSION: Our results suggest that cellular and humoral immune responses may play a role in glaucomatous optic neuropathy and that intravitreal hWJ-MSCs can induce an immunosuppressive environment by inhibiting proinflammatory cytokines and enhancing regulatory cytokines.

Human Platelet Lysate Supports Efficient Expansion and Stability of Wharton's Jelly Mesenchymal Stromal Cells via Active Uptake and Release of Soluble Regenerative Factors.

Manufacturing of mesenchymal stromal cell (MSC)-based therapies for regenerative medicine requires the use of suitable supply of growth factors that enhance proliferation, cell stability and potency during cell expansion. Human blood derivatives such as human platelet lysate (hPL) have emerged as a feasible alternative for cell growth supplement. Nevertheless, composition and functional characterization of hPL in the context of cell manufacturing is still under investigation, particularly regarding the content and function of pro-survival and pro-regenerative factors. We performed comparative analyses of hPL, human serum (hS) and fetal bovine serum (FBS) stability and potency to support Wharton's jelly (WJ) MSC production. We demonstrated that hPL displayed low inter-batch variation and unique secretome profile that was not present in hS and FBS. Importantly, hPL-derived factors including PDGF family, EGF, TGF-alpha, angiogenin and RANTES were actively taken up by WJ-MSC to support efficient expansion. Moreover, hPL but not hS or FBS induced secretion of osteoprotegerin, HGF, IL-6 and GRO-alpha by WJ-MSC during the expansion phase. Thus, hPL is a suitable source of factors supporting viability, stability and potency of WJ-MSC and therefore constitutes an essential raw material that in combination with WJ-MSC introduces a great opportunity for the generation of potent regenerative medicine products.

A new strategy for umbilical cord blood collection developed at the first Colombian public cord blood bank increases total nucleated cell content.

BACKGROUND: The total nucleated cell dosage of umbilical cord blood (UCB) is an important factor in determining successful allogeneic hematopoietic stem cell transplantation after a minimum human leukocyte antigen donor-recipient match. The northern South American population is in need of a new-generation cord blood bank that cryopreserves only units with high total nucleated cell content, thereby increasing the likelihood of use. Colombia set up a public cord blood bank in 2014; and, as a result of its research for improving high total nucleated cell content, a new strategy for UCB collection was developed. STUDY DESIGN AND METHODS: Data from 2933 collected and 759 cryopreserved cord blood units between 2014 and 2015 were analyzed. The correlation of donor and collection variables with cellularity was evaluated. Moreover, blood volume, cell content, CD34+ count, clonogenic capacity, and microbial contamination were assessed comparing the new method, which combines in utero and ex utero techniques, with the conventional strategies. RESULTS: Multivariate analysis confirmed a correlation between neonatal birth weight and cell content. The new collection method increased total nucleated cell content in approximately 26% and did not alter pre-cryopreservation and post-thaw cell recovery, viability, or clonogenic ability. Furthermore, it showed a remarkably low microbial contamination rate (1.2%). CONCLUSION: The strategy for UCB collection developed at the first Colombian public cord blood bank increases total nucleated cell content and does not affect unit quality. The existence of this bank is a remarkable breakthrough for Latin-American patients in need of this kind of transplantation.

Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation.

De novo regeneration of immunity is a major problem after allogeneic hematopoietic stem cell transplantation (HCT). HCT modeling in severely compromised immune-deficient animals transplanted with human stem cells is currently limited because of incomplete maturation of lymphocytes and scarce adaptive responses. Dendritic cells (DC) are pivotal for the organization of lymph nodes and activation of naive T and B cells. Human DC function after HCT could be augmented with adoptively transferred donor-derived DC. In this study, we demonstrate that adoptive transfer of long-lived human DC coexpressing high levels of human IFN-α, human GM-CSF, and a clinically relevant Ag (CMV pp65 protein) promoted human lymphatic remodeling in immune-deficient NOD.Rag1(-/-).IL-2rγ(-/-) mice transplanted with human CD34(+) cells. After immunization, draining lymph nodes became replenished with terminally differentiated human follicular Th cells, plasma B cells, and memory helper and cytotoxic T cells. Human Igs against pp65 were detectable in plasma, demonstrating IgG class-switch recombination. Human T cells recovered from mice showed functional reactivity against pp65. Adoptive immunotherapy with engineered DC provides a novel strategy for de novo immune reconstitution after human HCT and a practical and effective tool for studying human lymphatic regeneration in vivo in immune deficient xenograft hosts.

Toll-like receptor 2/6 agonist macrophage-activating lipopeptide-2 promotes reendothelialization and inhibits neointima formation after vascular injury.

OBJECTIVE: Reendothelialization after vascular injury (ie, balloon angioplasty or stent implantation) is clinically extremely relevant to promote vascular healing. We here investigated the therapeutic potential of the toll-like receptor 2/6 agonist macrophage-activating lipopeptide (MALP)-2 on reendothelialization and neointima formation in a murine model of vascular injury. APPROACH AND RESULTS: The left common carotid artery was electrically injured, and reendothelialization was quantified by Evans blue staining after 3 days. A single injection of MALP-2 (1 or 10 µg, IV) after vascular injury accelerated reendothelialization (P<0.001). Proliferation of endothelial cells at the wound margins determined by 5-ethynyl-2'-deoxyuridine incorporation was significantly higher in MALP-2-treated animals (P<0.05). Furthermore, wire injury-induced neointima formation of the left common carotid artery was completely prevented by a single injection of MALP-2 (10 µg, IV). In vitro, MALP-2 induced proliferation (BrdU incorporation) and closure of an artificial wound of endothelial cells (P<0.05) but not of smooth muscle cells. Protein array and ELISA analysis of isolated primary endothelial cells and ex vivo stimulated carotid segments revealed that MALP-2 stimulated the release of multiple growth factors and cytokines predominantly from endothelial cells. MALP-2 induced a strong activation of the mitogen-activated protein kinase cascade in endothelial cells, which was attenuated in smooth muscle cells. Furthermore, MALP-2 significantly enhanced circulating monocytes and hematopoietic progenitor cells. CONCLUSIONS: The toll-like receptor 2/6 agonist MALP-2 promotes reendothelialization and inhibits neointima formation after experimental vascular injury via enhanced proliferation and migration of endothelial cells. Thus, MALP-2 represents a novel therapeutic option to accelerate reendothelialization after vascular injury.

Early detection of optic nerve head changes using optical coherence tomography after using mesenchymal stromal cells as intravitreal therapy in rabbit models of ocular hypertension.

Background and Aim: Stem cell therapy is considered a promising treatment for several neurodegenerative diseases. However, there are very few studies on the use of this therapy in glaucoma models. By detecting the changes produced by glaucoma early, cell therapy could help prevent the events that lead to blindness. In this study, early changes in the optic nerve head (ONH) as detected by optical coherence tomography (OCT) after the application of human Wharton's jelly-derived mesenchymal stromal cells (hWJ-MSCs) in an experimental model of ocular hypertension (OH) were evaluated. Materials and Methods: Fifteen New Zealand rabbits were randomly divided into the following three groups: G1: OH, G2: hWJ-MSCs, and G3: OH + hWJ-MSCs. An OH model was constructed, and the intraocular pressure (IOP) was measured regularly. At week 7, 105/100 µL hWJ-MSCs were intravitreally injected. Retinography and OCT were used to evaluate structural changes in ONH. Results: IOP increased significantly in G1 and G3 from week 3 onward. Retinography revealed more significant optic nerve changes, that is, papillary asymmetry suggestive of optic nerve excavation, vascular alterations, and irregular hypopigmentation peripheral to the optic disk margin, in G1 compared with G3. OH locates the hWJ-MSCs solution in the vitreous in front of the optic nerve. OCT revealed retinal nerve fiber layer (RNFL) reduction in all groups, reduced optic cup volume in G2 and G3 between weeks 1 and 9, and significant ganglion cell layer thickness reduction in G1 and a slight increase in G3. Conclusion: Intravitreal hWJ-MSCs injection produced changes in optic cup volume, which were detected early on by OCT; however, RNFL could not be restored in this OH model.

Interleukin-1 assembles a proangiogenic signaling module consisting of caveolin-1, tumor necrosis factor receptor-associated factor 6, p38-mitogen-activated protein kinase (MAPK), and MAPK-activated protein kinase 2 in endothelial cells.

OBJECTIVE: Interleukin-1ß (IL-1ß) is a major cytokine linking inflammation and angiogenesis in pathological vascular processes, such as atherosclerosis and tumor neoangiogenesis. However, signaling pathways mediating IL-1ß-induced proangiogenic processes in endothelial cells (ECs) have barely been elucidated yet. Therefore, the present study investigated IL-1ß-induced proangiogenic signaling in ECs. METHODS AND RESULTS: IL-1ß potently induced tube formation and migration of ECs. This was associated with and dependent on activation of p38-mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) as determined by pharmacological inhibition and gene silencing. Furthermore, silencing of the adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) (lentiviral short hairpin RNA) inhibited these IL-1ß-induced processes. Moreover, IL-1ß promoted translocation of TRAF6 to insoluble cellular fractions (containing membrane rafts/caveolae) and interaction of TRAF6 with caveolin-1. Accordingly, cellular cholesterol depletion (cyclodextrin) and silencing of caveolin-1 (small interfering RNA) inhibited IL-1ß-induced activation of p38-MAPK and MK2, as well as IL-1ß-induced tube formation and migration. Finally, silencing of TRAF6 and MK2 deficiency inhibited IL-1ß-induced microvessel outgrowth in murine aortic rings ex vivo, and deficiency of MK2 or caveolin-1 significantly reduced IL-1ß-induced angiogenesis in mice in vivo (Matrigel plug assay). CONCLUSIONS: IL-1ß assembles a proangiogenic signaling module consisting of caveolin-1, TRAF6, p38-MAPK, and MK2 in ECs, representing a potential target to intervene into angiogenesis-dependent processes and diseases.

In vitro modelling of local gene therapy with IL-15/IL-15Rα and a PD-L1 antagonist in melanoma reveals an interplay between NK cells and CD4+ T cells.

Blockade of the immune checkpoint axis consisting of programmed death-1 (PD-1) and its ligand PD-L1 alleviates the functional inhibition of tumor-infiltrating lymphoid cells yet weakly induces their expansion. Exogenous cytokines could further expand lymphoid cells and thus synergize with αPD-L1 therapy. However, systemic delivery of most cytokines causes severe toxicity due to unspecific expansion of immune cells in the periphery. Here, we modelled local delivery of cytokines and αPD-L1 therapeutics to immune cell-containing in vitro melanoma tumors. Three-dimensional tumor models consisting of 624-MEL cells were co-cultured with human peripheral blood lymphoid cells (PBLs) in presence of the cytokines IL-2, IL-7, IL-15, IL-21 and IFN-γ. To model local gene therapy, melanoma tumors were modified with lentiviral vectors encoding IL-15 fused to IL-15Rα (IL-15/IL-15Rα) and K2-Fc, a fusion of a human PD-L1 specific single domain antibody to immunoglobulin (Ig)G1 Fc. To evaluate the interplay between PBL fractions, NK cells, CD4+ T cells or CD8+ T cells were depleted. Tumor cell killing was followed up using real time imaging and immune cell expansion and activation was evaluated with flow cytometry. Among the tested cytokines, IL-15 was the most potent cytokine in stimulating tumor cell killing and expanding both natural killer (NK) cells and CD8+ T cells. Gene-based delivery of IL-15/IL-15Rα to tumor cells, shows expansion of NK cells, activation of NK cells, CD4+ and CD8+ T cells, and killing of tumor spheroids. Both NK cells and CD8+ T cells are necessary for tumor cell killing and CD4+ T-cell activation was reduced without NK cells. Co-delivery of K2-Fc improved tumor cell killing coinciding with increased activation of NK cells, which was independent of bystander T cells. CD4+ or CD8+ T cells were not affected by the co-delivery of K2-Fc even though NK-cell activation impacted CD4+ T-cell activation. This study demonstrates that gene-based delivery of IL-15/IL-15Rα to tumor cells effectively mediates anti-tumor activity and sensitizes the tumor microenvironment for therapy with αPD-L1 therapeutics mainly by impacting NK cells. These findings warrant further investigation of gene-based IL-15 and K2-Fc delivery in vivo.

Toll-like receptor 2/6 stimulation promotes angiogenesis via GM-CSF as a potential strategy for immune defense and tissue regeneration.

Toll-like receptors (TLRs) are known primarily as pathogen recognition receptors of the innate immunity, initiating inflammatory pathways to organize the immune defense. More recently, an involvement of TLRs in various physiologic and pathologic processes has been reported. Because many of these processes implicate angiogenesis, we here elucidated the role of a TLR2/6-dependent pathway on angiogenesis using the TLR2/6 agonist macrophage-activating lipopeptide of 2 kDa (MALP-2), a common bacterial lipopeptide. In vivo and in vitro Matrigel assays demonstrated that MALP-2 promoted angiogenesis in a TLR2/6-dependent manner. Moreover, MALP-2 induced endothelial cell proliferation and migration and a strong secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF release in response to MALP-2 from isolated vascular segments was completely prevented when the endothelium was removed. MALP-2 containing Matrigel implants exhibited vascular structures as well as CD45(+) cells. MALP-2 induced migration of leukocytes and likewise GM-CSF release, particularly from the monocyte population. Inhibition of GM-CSF by siRNA or antibodies suppressed MALP-2-induced angiogenesis in vitro and in vivo. These results clearly identified a TLR2/6-dependent induction of angiogenesis by the bacterial lipopeptide MALP-2, which is mediated by GM-CSF. This might represent a general mechanism to enhance or restore blood flow and recruit immune cells for pathogen defense and tissue regeneration.

Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the FLT3 internal tandem duplication.

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin(-), HLA-DR(+), CD11c(+), CD86(+)) and plasmacytoid DC (pDC: Lin(-), HLA-DR(+), CD123(+), CD86(+)) in diagnostic samples of 47 FLT3-ITD(-) and 40 FLT3-ITD(+) AML patients. The majority of ITD(+) AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD(-) AML samples. Interestingly, mDCs and pDCs sorted out from ITD(+) AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD(+) AML (n = 11) and ITD(-) AML (n = 12) samples analyzed for mixed lineage DCs (Lin(-), HLA-DR(+), CD11c(+), CD123(+)). ITD(+) AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression.

Evolution and Epidemic Spread of SARS-CoV-2 in Colombia: A Year into the Pandemic.

Current efforts to understand the epidemiology, transmission dynamics and emergence of novel SARS-CoV-2 variants worldwide has enabled the scientific community to generate critical information aimed at implementing disease surveillance and control measures, as well as to reduce the social, economic and health impact of the pandemic. Herein, we applied an epidemic model coupled with genomic analysis to assess the SARS-CoV-2 transmission dynamics in Colombia. This epidemic model allowed to identify the geographical distribution, Rt dynamics and predict the course of the pandemic considering current implementation of countermeasures. The analysis of the incidence rate per 100,000 inhabitants carried out across different regions of Colombia allowed visualizing the changes in the geographic distribution of cases. The cumulative incidence during the timeframe March 2020 to March 2021 revealed that Bogotá (8063.0), Quindío (5482.71), Amazonas (5055.68), Antioquia (4922.35) and Tolima (4724.41) were the departments with the highest incidence rate. The highest median Rt during the first period evaluated was 2.13 and 1.09 in the second period; with this model, we identified improving opportunities in health decision making related to controlling the pandemic, diagnostic testing capacity, case registration and reporting, among others. Genomic analysis revealed 52 circulating SARS-CoV-2 lineages in Colombia detected from 774 genomes sequenced throughout the first year of the pandemic. The genomes grouped into four main clusters and exhibited 19 polymorphisms. Our results provide essential information on the spread of the pandemic countrywide despite implementation of early containment measures. In addition, we aim to provide deeper phylogenetic insights to better understand the evolution of SARS-CoV-2 in light of the latent emergence of novel variants and how these may potentially influence transmissibility and infectivity.

Efficient Non-Viral Gene Modification of Mesenchymal Stromal Cells from Umbilical Cord Wharton's Jelly with Polyethylenimine.

Mesenchymal stromal cells (MSC) derived from human umbilical cord Wharton's jelly (WJ) have a wide therapeutic potential in cell therapy and tissue engineering because of their multipotential capacity, which can be reinforced through gene therapy in order to modulate specific responses. However, reported methodologies to transfect WJ-MSC using cationic polymers are scarce. Here, WJ-MSC were transfected using 25 kDa branched- polyethylenimine (PEI) and a DNA plasmid encoding GFP. PEI/plasmid complexes were characterized to establish the best transfection efficiencies with lowest toxicity. Expression of MSC-related cell surface markers was evaluated. Likewise, immunomodulatory activity and multipotential capacity of transfected WJ-MSC were assessed by CD2/CD3/CD28-activated peripheral blood mononuclear cells (PBMC) cocultures and osteogenic and adipogenic differentiation assays, respectively. An association between cell number, PEI and DNA content, and transfection efficiency was observed. The highest transfection efficiency (15.3 ± 8.6%) at the lowest toxicity was achieved using 2 ng/µL DNA and 3.6 ng/µL PEI with 45,000 WJ-MSC in a 24-well plate format (200 µL). Under these conditions, there was no significant difference between the expression of MSC-identity markers, inhibitory effect on CD3+ T lymphocytes proliferation and osteogenic/adipogenic differentiation ability of transfected WJ-MSC, as compared with non-transfected cells. These results suggest that the functional properties of WJ-MSC were not altered after optimized transfection with PEI.

Integrated Analysis of Transcriptome and Secretome From Umbilical Cord Mesenchymal Stromal Cells Reveal New Mechanisms for the Modulation of Inflammation and Immune Activation.

Mesenchymal stromal cells (MSC) have been used in over 800 clinical trials with encouraging results in the field of transplant medicine and chronic inflammatory diseases. Today, Umbilical Cord (UC)-derived MSC are the second leading source used for clinical purposes, mainly due to its easy access and superior immune modulatory effects. Although the underlying molecular mechanisms of immune suppressive activities have not been fully understood, research over the last decade strongly suggests that MSC-mediated benefits are closely related to activation of secretome networks. Nevertheless, recent findings also point to cytokine-independent mechanisms as key players of MSC-mediated immune modulation. Here, we set up a robust in vitro immune assay using phytohemagglutinin- or anti-CD3/CD28-treated human peripheral blood mononuclear cells in cell-to-cell interaction or in cell-contact independent format with UC-MSC and conducted integrated transcriptome and secretome analyses to dissect molecular pathways driving UC-MSC-mediated immune modulation. Under inflammatory stimuli, multiparametric analyses of the secretome led us to identify cytokine/chemokine expression patterns associated with the induction of MSC-reprogrammed macrophages and T cell subsets ultimately leading to immune suppression. UC-MSC transcriptome analysis under inflammatory challenge allowed the identification of 47 differentially expressed genes, including chemokines, anti- and pro-inflammatory cytokines and adhesion molecules found also in UC-MSC-immunosupressive secretomes, including the novel candidate soluble IL-2R. This study enabled us to track functionally activated UC-MSC during immune suppression and opened an opportunity to explore new pathways involved in immunity control by UC-MSC. We propose that identified immunomodulatory molecules and pathways could potentially be translated into clinical settings in order to improve UC-MSC-therapy quality and efficacy.

Strategy for the Generation of Engineered Bone Constructs Based on Umbilical Cord Mesenchymal Stromal Cells Expanded with Human Platelet Lysate.

Umbilical cord mesenchymal stromal cells (UC-MSC) are promising candidates for cell therapy due to their potent multilineage differentiation, enhanced self-renewal capacity, and immediate availability for clinical use. Clinical experience has demonstrated satisfactory biosafety profiles and feasibility of UC-MSC application in the allogeneic setting. However, the use of UC-MSC for bone regeneration has not been fully established. A major challenge in the generation of successful therapeutic strategies for bone engineering lies on the combination of highly functional proosteogenic MSC populations and bioactive matrix scaffolds. To address that, in this study we proposed a new approach for the generation of bone-like constructs based on UC-MSC expanded in human platelet lysate (hPL) and evaluated its potential to induce bone structures in vivo. In order to obtain UC-MSC for potential clinical use, we first assessed parameters such as the isolation method, growth supplementation, microbiological monitoring, and cryopreservation and performed full characterization of the cell product including phenotype, growth performance, tree-lineage differentiation, and gene expression. Finally, we evaluated bone-like constructs based on the combination of stimulated UC-MSC and collagen microbeads for in vivo bone formation. UC-MSC were successfully cultured from 100% of processed UC donors, and efficient cell derivation was observed at day 14 ± 3 by the explant method. UC-MSC maintained mesenchymal cell morphology, phenotype, high cell growth performance, and probed multipotent differentiation capacity. No striking variations between donors were recorded. As expected, UC-MSC showed tree-lineage differentiation and gene expression profiles similar to bone marrow- and adipose-derived MSC. Importantly, upon osteogenic and endothelial induction, UC-MSC displayed strong proangiogenic and bone formation features. The combination of hPL-expanded MSC and collagen microbeads led to bone/vessel formation following implantation into an immune competent mouse model. Collectively, we developed a high-performance UC-MSC-based cell manufacturing bioprocess that fulfills the requirements for human application and triggers the potency and effectivity of cell-engineered scaffolds for bone regeneration.

Ex vivo expanded hematopoietic progenitor cells improve cardiac function after myocardial infarction: role of beta-catenin transduction and cell dose.

Cell-based therapy after myocardial infarction (MI) is a promising therapeutic option but the relevant cell subsets and dosage requirements are poorly defined. We hypothesized that cell therapy for myocardial infarction is improved by ex vivo expansion and high-dose transplantation of defined hematopoietic progenitor cells (HPCs). Since beta-catenin promotes self-renewal of stem cells we evaluated the therapeutic efficacy of beta-catenin-mediated ex vivo expansion of mouse HPCs in a mouse model of myocardial ischemia/reperfusion followed by intraarterial cell delivery. The impact of cell dose was determined by comparing a low-dose (LD, 5 x 10(5) cells) vs. a high-dose (HD, 1 x 10(7) cells) cell transplantation regimen of beta-catenin-HPCs. The impact of beta-catenin modification of HPCs was determined by comparing control-transduced HPCs (GFP-HPCs) vs. transgenic beta-catenin-HPCs. HD beta-catenin-HPCs significantly improved LV function and end-systolic and end-diastolic dimensions as compared to saline and LD beta-catenin-HPCs. Furthermore, while treatment with HD GFP-HPC resulted in a modest cardiac improvement the application of beta-catenin-HPCs was superior, resulting in a significant improvement in EF, FS and LVESD over saline and control GFP-HPC treatment. Although myocardial engraftment of HPCs was only transient, as determined by cell quantification after dye labeling, beta-catenin-HPC treatment significantly decreased infarct size, reduced cardiomyocyte apoptosis and increased capillary angiogenesis in vitro and in vivo. Ex vivo expanded HPCs improve cardiac function and remodeling post MI in a cell number- and beta-catenin-dependent manner.

Renovascular hypertension by two-kidney one-clip enhances endothelial progenitor cell mobilization in a p47phox-dependent manner.

BACKGROUND: Enhanced mechanical forces, e.g. in arterial hypertension, stimulate the formation of reactive oxygen species (ROS) by the NAD(P)H oxidase. Since bone marrow derived endothelial progenitor cells (EPCs) contribute to vascular remodeling and repair, we investigated whether renovascular hypertension stimulates EPC mobilization in a NAD(P)H oxidase-dependent manner. METHODS: Renovascular hypertension was induced by two-kidney one-clip (2K1C) in C57BL/6 (WT) and in mice lacking the p47phox subunit of the NAD(P)H oxidase (p47phox-/-). RESULTS: In WT, 2K1C increased blood pressure levels by 32.4 +/- 4 mmHg, which was associated with a four-fold increase in circulating EPCs (Sca-1+;Flk-1+). In p47phox-/- mice, the increase in blood pressure was significantly reduced (15.1 +/- 1.8 mmHg, P < 0.05) and not associated with increased EPCs. Inhibitors of the renin-angiotensin system (RAS) and nonspecific vasodilators normalized blood pressure and inhibited EPC mobilization in WT mice after 2K1C. In addition, p47phox deficiency and pharmacological ROS blockage abrogated 2K1C-induced blood pressure elevation and EPC mobilization. Stromal cell derived factor (SDF)-1 and matrix metalloproteinase (MMP)-9 activity in the bone marrow, required for EPC mobilization, were modulated in WT mice after 2K1C. In contrast, no alterations in SDF-1 and MMP-9 were observed in p47phox-/- mice. Moreover, enhanced migration of Lin- bone marrow mononuclear cells was observed when stimulated with plasma from 2K1C WT mice but not when stimulated with plasma from 2K1C p47phox-/- mice. CONCLUSION: Enhanced mechanical stretch in renovascular hypertension induces EPC mobilization in a p47phox-dependent manner, involving bone marrow SDF-1 and MMP-9 which may contribute to compensatory vascular adaptation in renovascular hypertension.

Critical role for p47phox in renin-angiotensin system activation and blood pressure regulation.

OBJECTIVE: Renin-angiotensin system (RAS) activation leads to increased production of NAD(P)H oxidase-derived reactive oxygen species (ROS), and both have been implicated in the initiation and progression of arterial hypertension, atherosclerosis, and cardiac hypertrophy. The cytosolic subunit p47phox is critically involved in agonist-induced NAD(P)H oxidase activation. Here, we investigated the role of p47phox in blood pressure control, endothelium-dependent relaxation, cardiac hypertrophy, RAS activation, and renal oxidative stress under resting conditions. METHODS AND RESULTS: Mice deficient in p47phox (on C57BL/6 background) developed significantly higher systolic blood pressure levels compared to C57BL/6 wild-type animals (136.0+/-3.0 mmHg vs. 112.2+/-2.6, P<0.01, n=16) as measured by the tail cuff method from week 6 up to week 12 post partum. The increase in blood pressure in p47phox-/- mice was associated with an impaired endothelium-dependent relaxation (P<0.005 vs. wild-type, n=11). At the age of 12 weeks p47phox-/- mice showed increased plasma renin activity as analyzed by radioimmunoassay (14.5+/-1.8 ng/mL/h vs. 9.6+/-1.7 ng/mL/h, P<0.05, n=10) and enhanced angiotensin converting enzyme (ACE) activity in the kidney and aorta as measured by Hip-His-Leu cleavage (7.6+/-0.8 vs. 4.8+/-0.9 nmol/L His-Leu/mg protein, P<0.05, n=5) compared to wild-type mice. No differences in oxygen radical formation was determined in kidney samples by lucigenin- and luminol-enhanced chemiluminescence or by electron spin resonance spectroscopy. Consistently, treatment with the radical scavenger tempol did not lower blood pressure in p47phox-/- mice, whereas ACE and angiotensin II type I receptor inhibition normalized blood pressure. CONCLUSION: Deficiency of the NAD(P)H oxidase subunit p47phox leads to RAS activation, which subsequently contributes to blood pressure increase in a ROS-independent manner.

MLH1 and MSH2 mutations in Colombian families with hereditary nonpolyposis colorectal cancer (Lynch syndrome)--description of four novel mutations.

This study searched for mutations in the MLH1 and MSH2 genes in 23 unrelated Colombian families with suspected hereditary nonpolyposis colorectal cancer (HNPCC). The families were grouped according to the fulfillment of the Amsterdam II criteria or the Bethesda guidelines. We screened all probands by single-strand conformational polymorphism (SSCP) and direct DNA sequencing. Eleven families fulfilled the Amsterdam criteria II and 12 families the Bethesda guidelines. Germline mutations were detected in 11 families, which corresponds to a mutation detection rate of 48%. When only families fulfilling the Amsterdam II criteria were analyzed, the mutation detection rate rose to 82%. Only 8% of the mutation detection rate was found in families following the Bethesda guidelines. Three mutations were shared by two different families, which corresponds to a total of eight different mutations, seven of them found in the MLH1 gene and one in the MSH2 gene. We have identified four mutations that have not been previously reported to the International Collaborative Group of HNPCC. Three of these are pathogenic, a single base substitution (C > T) at codon 640, exon 17, a G deletion at codon 619, exon 16 and in the MLH1 gene and a two-nucleotide deletion (TG) at codon 184, exon 3 in the MSH2. Also, an unclassified variant, a substitution (C > G) at the codon 141, exon 5 of the MLH1, was detected.

[Detection mutations in the DNA mismatch repair genes of hMLH1 and hMSH2 genes in Colombian families with suspicion of hereditary non-polyposis colorectal carcinoma (Lynch syndrome)]. / Detección de mutaciones de los genes hMLH1 y hMSH2 del sistema de reparación de malos apareamientos del ADN en familias colombianas sospechosas de cancer colorrectal no polipósico hereditario (síndrome de Lynch).

INTRODUCTION: Colorectal cancer (CRC) is the second highest cause of cancer mortality in developed countries. In Colombia, CRC ranks fifth as a cause of cancer death. Approximately 75% of CRC appear to be spontaneous and 25% are familial, with 5% of the latter clearly hereditary. Of these, hereditary non-polyposis colorectal carcinoma (HNPCC)-or Lynch syndrome is the most important. OBJECTIVE: Herein, the two most important genes involved in Lynch syndrome, the hMLH1 and hMSH2 were analyzed for presence of mutations. MATERIALS AND METHODS: Seventeen Colombian families that fulfilled the Amsterdam II criteria or Bethesda guidelines for Lynch syndrome were selected. The of 35 exons of hMLH1 and hMSH2 genes were screened by SSCP and those with electrophoretic variants were sequenced. RESULTS: Eight germinal mutations were detected, corresponding to a 47% detection mutation rate. Six of the eight mutations have previously been reported. These consisted of the following mutations: a single base substitution at the donor splicing site of exon 9, a single base substitution (A>G) at codon 755 of the exon 17, and another single base substitution (G>A) at codon 681 of exon 18. The two novel mutations consisted of a single base substitution (C>T) at codon 640 of exon 17 of the hMLH1 gene and a two-nucleotide deletion (TG) at codon 184 of exon 3 of hMSH2 gene. In addition, two families were observed with a polymorphism in the intron 13 (G>A) nt 1558+14, of hMLH1 gene. CONCLUSIONS: This study represented the first survey for detecting mutations associated with Lynch syndrome in Colombia, and is intended to lead to the establishment of a management and prevention program.

Allogeneic CD4+CD25high T cells regulate obliterative bronchiolitis of heterotopic bronchus allografts in both porcinized and humanized mouse models.

BACKGROUND: Bronchiolitis obliterans syndrome is caused by a fibroproliferative process in lung allografts resulting in irreversible damage. In this study, we induced obliterative bronchiolitis and studied the contribution of regulatory T cells to its development in immune-deficient mice receiving heterotopic porcine bronchus transplants, and major histocompatibility complex-mismatched porcine peripheral blood mononuclear cell. Furthermore, we aimed to corroborate our findings in a humanized mouse model. METHODS: Heterotopic bronchus transplantation was performed in 33 NOD.rag(−/−)γc(−/−) mice, using miniature pigs as tissue donors.The recipient mice then either received saline (negative control), unsorted MHC-mismatched PBMC (positive control), PBMC enriched with CD4(+)CD25(high) cells or PBMC depleted of CD4(+)CD25(high) cells for reconstitution. The results were validated in 28 NOD.rag(−/−)γc(−/−) mice undergoing heterotopic human bronchus transplantation and reconstitution with allogeneic human PBMC. RESULTS: Histological lesions similar to those typical for obliterative bronchiolitis developed in vivo after reconstitution with allogeneic PBMC and were more severe in animals engrafted with PBMC depleted of CD4(+)CD25(high) cells. In contrast, the group reconstituted with PBMC enriched with CD4(+)CD25(high) cells showed well-preserved histology. The results of the humanized model confirmed those obtained in the porcinized model. CONCLUSIONS: In conclusion, both porcinized and humanized mouse models of heterotopic subcutaneous bronchus transplantation imitate the in vivo development of bronchiolitis obliterans syndrome-like lesions and reveal its sensitivity to T-cell regulation.