Lineage-Negative Bone Marrow Cells Protect Against Chronic Renal Failure

Progressive renal failure continues to be a challenge. The use of bone marrow cells represents a means of meeting that challenge. We used lineage-negative (Lin(-)) cells to test the hypothesis that Lin(-) cell treatment decreases renal injury. Syngeneic Fischer 344 rats were divided into four groups: sham ( laparotomy only, untreated); Nx (five-sixth nephrectomy and untreated); NxLC1 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy day 15); and NxLC3 (five-sixth nephrectomy and receiving 2 x 10(6) Lin(-) cells on postnephrectomy days 15, 30, and 45). On postoperative day 16, renal mRNA expression of interleukin (IL)-1 beta, tumor necrosis factor-alpha, and IL-6 was lower in NxLC rats than in Nx rats. On postnephrectomy day 60, NxLC rats presented less proteinuria, glomerulosclerosis, anemia, renal infiltration of immune cells, and protein expression of monocyte chemoattractant protein-1, as well as decreased interstitial area. Immunostaining for proliferating cell nuclear antigen showed that, in comparison with sham rats, Nx rats presented greater cell proliferation, whereas NxLC1 rats and NxLC3 rats presented less cell proliferation than did Nx rats. Protein expression of the cyclin-dependent kinase inhibitor p21 and of vascular endothelial growth factor increased after nephrectomy and decreased after Lin(-) cell treatment. On postnephrectomy day 120, renal function (inulin clearance) was significantly better in Lin(-) cell-treated rats than in untreated rats. Lin(-) cell treatment significantly improved survival. These data suggest that Lin(-) cell treatment protects against chronic renal failure. STEM CELLS 2009; 27: 682-692

Multipotent stem cells from umbilical cord: Cord is richer than blood!

The identification of mesenchymal stem cell ( MSC) sources that are easily obtainable is of utmost importance. Several studies have shown that MSCs could be isolated from umbilical cord (UC) units. However, the presence of MSCs in umbilical cord blood (UCB) is controversial. A possible explanation for the low efficiency of MSCs from UCB is the use of different culture conditions by independent studies. Here, we compared the efficiency in obtaining MSCs from unrelated paired UCB and UC samples harvested from the same donors. Samples were processed simultaneously, under the same culture conditions. Although MSCs from blood were obtained from only 1 of the 10 samples, we were able to isolate large amounts of multi-potent MSCs from all UC samples, which were able to originate different cell lineages. Since the routine procedure in UC banks has been to store the blood and discard other tissues, such as the cord and/or placenta, we believe our results are of immediate clinical value. Furthermore, the possibility of originating different cell lines from the UC of neonates born with genetic defects may provide new cellular research models for understanding human malformations and genetic disorders, as well as the possibility of testing the effects of different therapeutic drugs.

Ectonucleotidase activities are altered in serum and platelets of L-NAME-treated rats

It is well known that hypertension is closely associated to the development of vascular diseases and that the inhibition of nitric oxide biosynthesis by administration of N omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME) leads to arterial hypertension. In the vascular system, extracellular purines mediate several effects: thus, ADP is the most important platelet agonist and recruiting agent, while adenosine, all end product Of nucleotide metabolism, is a vasodilator and inhibitor of platelet activation and recruitment. Members of several families of enzymes, known as ectonucleotidases, including E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolase), E-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase) and 5`-nucleotidase are able to hydrolyze extracellular nucleotides until their respective nucleosides. We investigated the ectonuclectidase activities of serum and platelets from rats made hypertensive by oral administration of L-NAME (30 mg/kg/day for 14 days or 30 mg/kg/day for 14 days Plus 7 days of L-NAME washout, in the drinking water) in comparison to normotensive control rats. L-NAME promoted a significant rise in systolic blood pressure from 112 +/- 9.8 to 158 +/- 23 mmHg. The left ventricle weight index (LVWI) was increased in rats treated with L-NAME for 14 days when compared to control animals. In Serum samples, ATP, ADP and AMP hydrolysis were reduced by about 27%, 36% and 27%, respectively. In platelets, the decrease in ATP, ADP and AMP hydrolysis Was approximately 27%, 24% and 32%, respectively. All parameters recovered after 7 days of L-NAME washout. HPLC demonstrated a reduction in ADP, AMP and hypoxanthine levels by about 64%, 69% and 87%, respectively. In this study, we showed that ectonucleotidase activities are decreased in serum and platelets from L-NAME-treated rats, which should represent an additional risk for the development of hypertension. The modulation of ectonucleotidase activities may represent an approach to antihypertensive therapy via inhibition of spontaneous platelet activation and recruitment, as well as thrombus formation. (C) 2008 Elsevier Inc. All rights reserved.

Reduced cytokine production by glycogen-elicited peritoneal cells from diabetic rats

IL-1 beta, TNF-alpha, cytokine-induced neutrophil chemoattractant-2 alpha/beta, and IL-10 measurements were performed in elicited peritoneal cells from control, diabetic, and insulin-treated diabetic rats. Production/liberation of these cytokines was decreased in elicited peritoneal cells from diabetic rats. These changes were abolished by insulin treatment of diabetic rats. The alterations observed might be involved in the impaired inflammatory response and high occurrence of apoptosis observed in neutrophils under diabetic states.

Retinoic Acid and VEGF Delay Smooth Muscle Relative to Endothelial Differentiation to Coordinate Inner and Outer Coronary Vessel Wall Morphogenesis

Rationale: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. Objective: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. Methods and Results: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. Conclusion: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels. (Circ Res. 2010;107:204-216.)

TUMOR NECROSIS FACTOR IS NOT ASSOCIATED WITH INTESTINAL ISCHEMIA/REPERFUSION-INDUCED LUNG INFLAMMATION

Intestinal ischemia-reperfusion (I/R) injury may cause acute systemic and lung inflammation. Here, we revisited the role of TNF-alpha in an intestinal I/R model in mice, showing that this cytokine is not required for the local and remote inflammatory response upon intestinal I/R injury using neutralizing TNF-alpha antibodies and TNF ligand-deficient mice. We demonstrate increased neutrophil recruitment in the lung as assessed by myeloperoxidase activity and augmented IL-6, granulocyte colony-stimulating factor, and KC levels, whereas TNF-alpha levels in serum were not increased and only minimally elevated in intestine and lung upon intestinal I/R injury. Importantly, TNF-alpha antibody neutralization neither diminished neutrophil recruitment nor any of the cytokines and chemokines evaluated. In addition, the inflammatory response was not abrogated in TNF and TNF receptors 1 and 2-deficient mice. However, in view of the damage on the intestinal barrier upon intestinal I/R with systemic bacterial translocation, we asked whether Toll-like receptor (TLR) activation is driving the inflammatory response. In fact, the inflammatory lung response is dramatically reduced in TLR2/4-deficient mice, confirming an important role of TLR receptor signaling causing the inflammatory lung response. In conclusion, endogenous TNF-alpha is not or minimally elevated and plays no role as a mediator for the inflammatory response upon ischemic tissue injury. By contrast, TLR2/4 signaling induces an orchestrated cytokine/chemokine response leading to local and remote pulmonary inflammation, and therefore disruption of TLR signaling may represent an alternative therapeutic target.

NITRIC OXIDE MEDIATES LUNG VASCULAR PERMEABILITY AND LYMPH-BORNE IL-6 AFTER AN INTESTINAL ISCHEMIC INSULT

Acute lung injury following intestinal I/R depends on neutrophil-endothelial cell interactions and on cytokines drained from the gut through the lymph. Among the mediators generated during I/R, increased serum levels of IL-6 and NO are also found and might be involved in acute lung injury. Once intestinal ischemia itself may be a factor of tissue injury, in this study, we investigated the presence of IL-6 in lymph after intestinal ischemia and its effects on human umbilical vein endothelial cells (HUVECs) detachment. The involvement of NO on the increase of lung and intestinal microvascular permeability and the lymph effects on HUVEC detachment were also studied. Upon anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery during 45 min, followed by 2-h intestinal reperfusion. Rats were treated with the nonselective NO synthase (NOS) inhibitor L-NAME (N(omega)-nitro-L-arginine methyl ester) or with the selective inhibitor of iNOS aminoguanidine 1 h before superior mesenteric artery occlusion. Whereas treatment with L-NAME during ischemia increased both IL-6 levels in lymph and lung microvascular permeability, aminoguanidine restored the augmented intestinal plasma extravasation due to ischemia and did not induce IL-6 in lymph. On the other hand, IL-6 and lymph of intestinal I/R detached the HUVECs, whereas lymph of ischemic rats upon L-NAME treatment when incubated with anti-IL-6 prevented HUVEC detachment. It is shown that the intestinal ischemia itself is sufficient to increase intestinal microvascular permeability with involvement of iNOS activation. Intestinal ischemia and absence of constitutive NOS activity leading to additional intestinal stress both cause release of IL-6 and increase of lung microvascular permeability. Because anti-IL-6 prevented the endothelial cell injury caused by lymph at the ischemia period, the lymph-borne IL-6 might be involved with endothelial cell activation. At the reperfusion period, this cytokine does not seem to be modulated by NO.

Up-regulation of NADPH oxidase components and increased production of interferon-gamma by leukocytes from sickle cell disease patients

We have previously demonstrated that mononuclear leukocytes from patients with sickle cell disease (SCD) release higher amounts of superoxide compared with normal controls. The aim of this study was to further study the NADPH oxidase system in these patients by investigating gene expression of NADPH oxidase components, phosphorylation of p47(phox) component, and the release of cytokines related to NADPH oxidase activation in mononuclear leukocytes from patients with SCD. gp91(phox) gene expression was significantly higher in monocytes from SCD patients compared with normal controls (P = 0.036). Monocytes from SCD patients showed higher levels of p47 phox phosphorylation compared with normal controls. INF-gamma release by lymphocytes from SCD patients was significantly higher compared with normal controls, after 48 h culture with phytohemagglutinin (P = 0.02). The release of TNF-alpha by monocytes from SCD patients and normal controls was similar after 24 and 48 h culture with lipopolysaccharide (P > 0.05). We conclude that monocytes from SCD patients show higher levels of gp91(phox) gene expression and p47(phox) phosphorylation, along with increased IFN-gamma release by SCD lymphocytes. These findings help to explain our previous observation showing the increased respiratory burst activity of mononuclear leukocytes from SCD patients and may contribute to inflammation and tissue damage in these patients.

Hematologically important mutations: X-linked chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations. (C) 2010 Elsevier Inc. All rights reserved.

THE ROLE OF INNATE IMMUNITY IN SEPTIC ACUTE KIDNEY INJURIES

Acute kidney injury (AKI) is an important clinical syndrome characterized by abnormalities in the hydroelectrolytic balance. Because of high rates of morbidity and mortality (from 15% to 60%) associated with AKI, the study of its pathophysiology is critical in searching for clinical targets and therapeutic strategies. Severe sepsis is the major cause of AKI. The host response to sepsis involves an inflammatory response, whereby the pathogen is initially sensed by innate immune receptors (pattern recognition receptors [PRRs]). When it persists, this immune response leads to secretion of proinflammatory products that induce organ dysfunction such as renal failure and consequently increased mortality. Moreover, the injured tissue releases molecules resulting from extracellular matrix degradation or dying cells that function as alarmines, which are recognized by PRR in the absence of pathogens in a second wave of injury. Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are the best characterized PRRs. They are expressed in many cell types and throughout the nephron. Their activation leads to translocation of nuclear factors and synthesis of proinflammatory cytokines and chemokines. TLRs` signaling primes the cells for a robust inflammatory response dependent on NLRs; the interaction of TLRs and NLRs gives rise to the multiprotein complex known as the inflammasome, which in turn activates secretion of mature interleukin 1 beta and interleukin 18. Experimental data show that innate immune receptors, the inflammasome components, and proinflammatory cytokines play crucial roles not only in sepsis, but also in organ-induced dysfunction, especially in the kidneys. In this review, we discuss the significance of the innate immune receptors in the development of acute renal injury secondary to sepsis.

Mesenchymal Stem Cells Attenuate Renal Fibrosis Through Immune Modulation and Remodeling Properties in a Rat Remnant Kidney Model

Mesenchymal stem cells (MSCs) have regenerative properties in acute kidney injury, but their role in chronic kidney diseases is still unknown. More specifically, it is not known whether MSCs halt fibrosis. The purpose of this work was to investigate the role of MSCs in fibrogenesis using a model of chronic renal failure. MSCs were obtained from the tibias and femurs of male Wistar-EPM rats. Female Wistar rats were subjected to the remnant model, and 2 vertical bar x vertical bar 10(5) MSCs were intravenously administrated to each rat every other week for 8 weeks or only once and followed for 12 weeks. SRY gene expression was observed in female rats treated with male MSCs, and immune localization of CD73(+)CD90(+) cells at 8 weeks was also assessed. Serum and urine analyses showed an amelioration of functional parameters in MSC-treated animals at 8 weeks, but not at 12 weeks. Masson`s trichrome and Sirius red staining demonstrated reduced levels of fibrosis in MSC-treated animals. These results were corroborated by reduced vimentin, type I collagen, transforming growth factor beta, fibroblast specific protein 1 (FSP-1), monocyte chemoattractant protein 1, and Smad3 mRNA expression and alpha smooth muscle actin and FSP-1 protein expression. Renal interleukin (IL)-6 and tumor necrosis factor alpha mRNA expression levels were significantly decreased after MSC treatment, whereas IL-4 and IL-10 expression levels were increased. All serum cytokine expression levels were decreased in MSC-treated animals. Taken together, these results suggested that MSC therapy can indeed modulate the inflammatory response that follows the initial phase of a chronic renal injury. The immunosuppressive and remodeling properties of MSCs may be involved in the decreased fibrosis in the kidney. STEM CELLS 2009;27:3063-3073

Enhanced Neural Progenitor/Stem Cells Self-Renewal via the Interaction of Stress-Inducible Protein 1 with the Prion Protein

Prion protein (PrPC), when associated with the secreted form of the stress-inducible protein 1 (STI1), plays an important role in neural survival, neuritogenesis, and memory formation. However, the role of the PrP(C)-STI1 complex in the physiology of neural progenitor/stem cells is unknown. In this article, we observed that neurospheres cultured from fetal forebrain of wild-type (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were maintained for several passages without the loss of self-renewal or multipotentiality, as assessed by their continued capacity to generate neurons, astrocytes, and oligodendrocytes. The homogeneous expression and colocalization of STI1 and PrP(C) suggest that they may associate and function as a complex in neurosphere-derived stem cells. The formation of neurospheres from Prnp(0/0) mice was reduced significantly when compared with their wild-type counterparts. In addition, blockade of secreted STI1, and its cell surface ligand, PrP(C), with specific antibodies, impaired Prnp(+/+) neurosphere formation without further impairing the formation of Prnp(0/0) neurospheres. Alternatively, neurosphere formation was enhanced by recombinant STI1 application in cells expressing PrP(C) but not in cells from Prnp(0/0) mice. The STI1-PrP(C) interaction was able to stimulate cell proliferation in the neurosphere-forming assay, while no effect on cell survival or the expression of neural markers was observed. These data suggest that the STI1-PrP(C) complex may play a critical role in neural progenitor/stem cells self-renewal via the modulation of cell proliferation, leading to the control of the stemness capacity of these cells during nervous system development. STEM CELLS 2011;29:1126-1136

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-kappa B ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-kappa B (NF-kappa B) signaling pathway in Escherichia coli LIPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, iv., 10 days) and controls were instilled intratracheally with saline containing LPS (750 mu g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LIPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1 beta, TNF-alpha, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1 beta, TNF-alpha, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1 beta (40%), TNF-alpha (similar to 30%), and IL-10 (similar to 40%) mRNA levels and reduced concentrations of IL-1 beta (52%), TNF-alpha (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1 beta, TNF-alpha) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-kappa B signaling pathway.

Essential role of nuclear factor-kappa B for NADPH oxidase activity in normal and anhildrotic ectodermal dysplasia leukocytes

This work investigated the functional role of nuclear factor-kappa B (NF-kappa B) in respiratory burst activity and in expression of the human phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase genes CYBB, CYBA, NCF1, and NCF2. U937 cells with a stably transfected repressor of NF-kappa B (IKB alpha-S32A/S36A) demonstrated significantly lower superoxide release and lower CYBB and NCF1 gene expression compared with control U937 cells. We further tested Epstein-Barr virus (EBV)-transformed B cells from patients with anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), an inherited disorderof NF-kappa B function. Superoxide release and CYBB gene expression by EDA-ID cells were significantly decreased compared with healthy cells and similar to cells from patients with X-linked chronic granulomatous disease (X91 degrees CGD). NCF1 gene expression in EDA-ID S321 cells was decreased compared with healthy control cells and similar to that in autosomal recessive (A47 degrees) CGD cells. Gel shift assays demonstrated loss of recombinant human p50 binding to a NF-kappa B site 5` to the CYBB gene in U937 cells treated with NF-kappa B inhibitors, repressor-transfected U937 cells, and EDA-ID patients cells. Zymosan phagocytosis was not affected by transfection of U937 cells with the NF-kappa B repressor. These studies show that NF-kappa B is necessary for CYBB and NCF1 gene expression and activation of the phagocyte NADPH oxidase in this model system.

Alveolar macrophages from susceptible mice are more competent than those of resistant mice to control initial Paracoccidioides brasiliensis infection

Alveolar macrophages ( AM) are the first host cells to interact with Paracoccidioides brasiliensis (Pb), a primary human pathogen that causes severe pulmonary infections in Latin America. To better understand innate immunity in pulmonary paracoccidioidomycosis, we decided to study the fungicidal and secretory abilities of AM from resistant (A/J) and susceptible (B10.A) mice to infection. Untreated, IFN-gamma and IL-12 primed AM from B10. A and A/J mice were challenged with P. brasiliensis yeasts and cocultured for 72 h. B10. A macrophages presented an efficient fungicidal ability, were easily activated by both cytokines, produced high levels of nitric oxide ( NO), IL-12, and MCP-1 associated with low amounts of IL-10 and GM-CSF. In contrast, A/J AM showed impaired cytokine activation and fungal killing, secreted high levels of IL- 10 and GM-CSF but low concentrations of NO, IL- 12, and MCP-1. The fungicidal ability of B10. A but not of A/J macrophages was diminished by aminoguanidine treatment, although only the neutralization of TGF-beta restored the fungicidal activity of A/J cells. This pattern of macrophage activation resulted in high expression of MHC class II antigens by A/J cells, while B10. A macrophages expressed elevated levels of CD40. Unexpectedly, our results demonstrated that susceptibility to a fungal pathogen can be associated with an efficient innate immunity, while a deficient innate response can ultimately favor the development of a resistant pattern to infection. Moreover, our data suggest that different pathogen recognition receptors are used by resistant and susceptible hosts to interact with P. brasiliensis yeasts, resulting in divergent antigen presentation, acquired immunity, and disease outcomes.