Interleukin (IL)‐1β, IL‐1 receptor antagonist,IL‐6, IL‐8, IL‐10, and tumor necrosis factor α gene polymorphisms in patients with febrile seizures

Inflammation and genetics may play a role in the pathogenesis of febrile seizures (FSs). We aimed to test whether interleukin‐1β (IL‐1β), IL‐1 receptor antagonist (IL‐1 Ra), IL‐6 promoter, IL‐8, IL‐10, or tumor necrosis factor (TNF) gene polymorphisms could be used as markers of susceptibility to FSs. An association study was performed among a cohort of 104 patients with FSs and 143 normal control subjects. There was no significant difference between patients and controls in the distribution of allele frequencies of the IL‐1β promoter, IL‐1β exon 5, IL‐6 promoter, IL‐8, IL‐10, or TNF‐α gene polymorphisms. In contrast, the IL‐1 Ra‐I homozygote was more frequent in patients with FSs than in healthy controls (93.2% vs. 83.92%, χ²=4.51, P=0.034). In addition, individuals homozygous for the IL‐1 Ra‐I genotype were more than twice as likely to develop FSs than individuals heterozygous for the IL‐1 Ra‐I/II genotype (OR, 2.63, 95% CI: 1.08–6.39; χ²=4.55, P=0.033). We conclude that the IL‐1 Ra gene might be one of the useful markers for predicting susceptibility to FSs. J. Clin. Lab. Anal. 24:154–159, 2010. © 2010 Wiley‐Liss, Inc.     

Ex vivo expansion of cord blood‐CD34+ cells using IGFBP2 and Angptl‐5 impairs short‐term lymphoid repopulation in vivo

Cord blood‐derived haematopoietic stem cells (CB‐HSCs) are an attractive source for transplantation in haematopoietic disorders. However, the yield of CB‐HSCs per graft is limited and often insufficient, particularly for the treatment of adult patients. Here we compare the capacity of three cytokine cocktails to expand CB‐CD34⁺ cells. Cells were cultured for 5 or 14 days in media supplemented with: (a) SCF, FL, IL‐3 and IL‐6 (SFLIL3/6); (b) SCF, TPO, FGF‐1 and IL‐6 (STFIL6); and (c) SCF, TPO, FGF‐1, IGFBP₂ and Angptl‐5 (STFAI). We observed that STFAI‐culture expansion sustained the most vigorous cell proliferation, maintenance of CD34⁺ phenotype and colony‐forming unit counts. In addition, STFAI‐cultured cells had a potent ex vivo migration activity. STFAI‐expanded cells were able to engraft NSG mice. However, no significant difference in overall engraftment was observed among the expansion cocktails. Assessment of short‐term reconstitution using multilineage markers demonstrated that the STFAI cocktail for HSCs expansion greatly improved total cell expansion but may impair short‐term lymphoid repopulation. Copyright © 2012 John Wiley & Sons, Ltd.     

The CD14++CD16+ monocyte subset and monocyte‐platelet interactions in patients with ST‐elevation myocardial infarction

Summary. Aim: Monocytes contribute to both myocardial damage and repair by virtue of subset heterogeneity. The dynamics and functional characteristics of the three human monocyte subsets, including the unique CD14++CD16+ subset, and their contributions to monocyte platelet aggregates (MPAs) following ST‐elevation myocardial infarction (STEMI) are unknown. We aimed to examine dynamic changes and relation to left ventricular ejection fraction (LVEF) of the three human monocyte subsets and their aggregates with platelets following STEMI. Methods: Three monocyte subsets, CD14++CD16?CCR2+ (‘classical’, Mon1), CD14++CD16+CCR2+ (‘intermediate’, Mon2) and CD14+CD16++CCR2? (‘non‐classical’, Mon3), and their contribution to MPAs were analyzed by flow cytometry in 50 patients with STEMI, 40 patients with stable coronary artery disease (CAD) and 40 healthy volunteers. Study parameters were measured within 24 h of primary percutaneous coronary intervention (PCI) (day1) and on days 3, 7 and 30. Monocyte activation was assessed by measuring the nuclear factor κB (NFκB) pathway. LVEF was assessed 6 weeks after STEMI. Correlations between monocyte subsets/MPAs and plasma cytokines and troponin were assessed. Results: We observed marked differences in subset dynamics, with a prominent increase in Mon2 (P < 0.0001) but no changes in Mon3. Significant increases in Mon2 CD14 (P = 0.002) and CCR2 (P < 0.0001) expression, and reduction in CD16 expression (P = 0.001) were seen. NFκB pathway activity increased most prominently in Mon2 (P = 0.007). Mon2 count correlated with peak troponin (r = 0.31, P = 0.04) and plasma interleukin (IL)‐6 (r = 0.65, P < 0.0001) and IL‐10 (r = 0.34, P = 0.017). Mon1 correlated with IL‐6 (r = 0.55, P < 0.0001). Reduced Mon2 expression of CD16 on day 1 was independently predictive of higher LVEF (β = ?0.37, P = 0.013). The increase in MPA count following STEMI persisted at 1 month. Conclusion: The Mon2 ‘intermediate’ subset has unique dynamic and functional characteristics following STEMI and significant correlations with troponin, plasma cytokines and convalescent left ventricular function. The persistent increase in MPA count 30 days after STEMI may affect monocyte subset functional activity.     

Na+/Ca2+‐exchanger‐mediated Mn2+‐enhanced 1H2O MRI in hypoxic,perfused rat myocardium

Paramagnetic Mn²⁺ has emerged in the search for non‐invasive magnetic resonance imaging (MRI) techniques to monitor Ca²⁺ in diagnostic and prognostic cardiovascular disease tests because it both alters MRI contrast and behaves as a Ca²⁺ ‘surrogate’ in vivo. However, the reliance on macroscopically averaged measurements to infer microscopic processes constitutes a major limitation of MRI. This investigation circumvents this limitation and contributes an MRI‐based myocardial Ca²⁺‐transporter assay, which probes the Na⁺/Ca²⁺‐exchanger involvement in Mn²⁺ (and presumably Ca²⁺) transport by virtue of its response to pharmacological inhibition. In the model employed herein, ex vivo arrested rat hearts underwent normoxia and then hypoxia while a constant (hyperkalemic) perfusion minimized flow (and uncontrolled Ca²⁺‐channel) contributions to Mn²⁺‐enhanced MRI measurements. The results (i) demonstrate that Mn²⁺ (and presumably Ca²⁺) accumulates via Na⁺/Ca²⁺‐exchanger‐mediated transport during hyperkalemic hypoxia and further, (ii) implicate hypo‐perfusion (rather than the diminished participation of an isolated sarcolemmal Ca²⁺‐transporter) as the mechanism that underlies the reported reductions of Mn²⁺ accumulation (relative to healthy myocardium) subsequent to myocardial insults in MRI studies. Although myriad studies have employed Mn²⁺‐enhanced MRI in myocardial investigations, this appears to be the first attempt to assay the Na⁺/Ca²⁺‐exchanger with MRI under highly circumscribed conditions. MRI‐based Ca²⁺‐transporter assays, such as the Na⁺/Ca²⁺‐exchanger assay utilized here, will inevitably impact disciplines in the medical sciences and beyond. Copyright © 2007 John Wiley & Sons, Ltd.     

PTEN inhibits IL-2 receptor-mediated expansion of CD4+ CD25+ Tregs

One of the greatest barriers against harnessing the potential of CD4+ CD25+ Tregs as a cellular immunotherapy is their hypoproliferative phenotype. We have previously shown that the hypoproliferative response of Tregs to IL-2 is associated with defective downstream PI3K signaling. Here, we demonstrate that targeted deletion of the lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) regulates the peripheral homeostasis of Tregs in vivo and allows their expansion ex vivo in response to IL-2 alone. PTEN deficiency does not adversely affect either the thymic development or the function of Tregs, which retain their ability to suppress responder T cells in vitro and prevent colitis in vivo. Conversely, reexpression of PTEN in PTEN-deficient Tregs as well as in activated CD4+ T cells inhibits IL-2-dependent proliferation, confirming PTEN as a negative regulator of IL-2 receptor signaling. These data demonstrate that PTEN regulates the "anergic" response of Tregs to IL-2 in vitro and Treg homeostasis in vivo and indicate that inhibition of PTEN activity may facilitate the expansion of these cells for potential use in cellular immunotherapy.     

2‐(Trimethylammonium)ethyl (R)‐3‐methoxy‐3‐oxo‐2‐stearamidopropyl phosphate promotes megakaryocytic differentiation of myeloid leukaemia cells and primary human CD34+ haematopoietic stem cells

In this study we showed that 2‐(t rimethylammonium)e thyl (R)‐3‐m ethoxy‐3‐o xo‐2‐s tearamidopropyl pho sphate [(R)‐TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient differentiation of megakaryocytes. Specifically, (R)‐TEMOSPho induces cell cycle arrest, cell size increase and polyploidization from K562 and HEL cells, which are used extensively to model megakaryocytic differentiation. In addition, megakaryocyte‐specific cell surface markers showed a dramatic increase in expression in response to (R)‐TEMOSPho treatment. Importantly, we demonstrated that such megakaryocytic differentiation can also be induced from primary human CD34⁺ haematopoietic stem cells. Activation of the PI3K–AKT pathway and, to a lesser extent, the MEK–ERK pathway appears to be required for this process, as blocking with specific inhibitors interferes with the differentiation of K562 cells. A subset of (R)‐TEMOSPho‐treated K562 cells undergoes spontaneous apoptosis and produces platelets that are apparently functional, as they bind to fibrinogen, express P‐selectin and aggregate in response to SFLLRN and AYPGFK, the activating peptides for the PAR1 and PAR4 receptors, respectively. Taken together, these results indicate that (R)‐TEMOSPho will be useful for dissecting the molecular mechanisms of megakaryocytic differentiation, and that this class of compounds represents potential therapeutic reagents for thrombocytopenia. Copyright © 2012 John Wiley & Sons, Ltd.     

Electromagnetic fields counteract IL‐1β activity during chondrogenesis of bovine mesenchymal stem cells

Osteoarthritis (OA) is a common joint disease associated with articular cartilage degeneration. To improve the therapeutic options of OA, tissue engineering based on the use of mesenchymal stem cells (MSCs) has emerged. However, the presence of inflammatory cytokines, such as interleukin‐1β (IL‐1β), during chondrogenesis reduces the efficacy of cartilage engineering repair procedures by preventing chondrogenic differentiation. Previous studies have shown that electromagnetic fields (EMFs) stimulate anabolic processes in OA cartilage and limit IL‐1β catabolic effects. We investigated the role of EMFs during chondrogenic differentiation of MSCs, isolated from bovine synovial fluid, in the absence and presence of IL‐1β. Pellets of MSCs were differentiated for 3 and 5 weeks with transforming growth factor‐β3 (TGFβ3), in the absence and presence of IL‐1β and exposed or unexposed to EMFs. Biochemical, quantitative real‐time RT–PCR and histological results showed that EMFs alone or in the presence of TGFβ3 play a limited role in promoting chondrogenic differentiation. Notably, in the presence of IL‐1β and TGFβ3 a recovery of proteoglycan (PG) synthesis, PG content and aggrecan and type II collagen mRNA expression in the EMF‐exposed compared to unexposed pellets was observed. Also, histological and immunohistochemical results showed an increase in staining for alcian blue, type II collagen and aggrecan in EMF‐exposed pellets. In conclusion, this study shows a significant role of EMFs in counteracting the IL‐1β‐induced inhibition of chondrogenesis, suggesting EMFs as a therapeutic strategy for improving the clinical outcome of cartilage engineering repair procedures, based on the use of MSCs. Copyright © 2012 John Wiley & Sons, Ltd.     

Resveratrol counteracts IL‐1β‐mediated impairment of extracellular matrix deposition in 3D articular chondrocyte constructs

When aiming at cell‐based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL‐1β need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti‐inflammatory properties. However, long‐term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long‐term model cultures for cell‐based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL‐1β (1–10 ng/ml) or RSV (50 μM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL‐1β (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL‐1β‐treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL‐1β treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL‐1β‐mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long‐term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques.