Flebogamma(®) DIF (intravenous immunoglobulin) purification process effectively eliminates procoagulant activities.

BACKGROUND: Studies have demonstrated that traces of activated factor XI (FXIa) present in specific brands of intravenous immunoglobulin (IVIG) concentrates may pose a thrombogenic risk. AIM: To characterize procoagulant activity during fractionation and the elimination capacity of the Flebogamma(®) DIF (Grifols' IVIG) manufacturing process. METHODS: Flebogamma(®) DIF fractionation steps included cryoprecipitate supernatant (Cryo/S), Fraction (Fr) I supernatant, and Fr II + III suspension. Purification steps included ultrafiltrate I, acid treatment, and pasteurization. Samples were assessed for total protein, IgG, and procoagulant activation markers. RESULTS: Cryo/S showed no procoagulant activity for prekallikrein activator (PKA), kallikrein-like, and non-activated partial thromboplastin time (NaPTT) with normal (-PPP) or FXI-deficient (-FXI) platelet poor plasma. Thrombin generation test (TGT)-PPP and TGT-FXI were <83-148 and <53-197 nM thrombin, respectively. Shortened NaPTTs (100-296 s), high PKA (51-119 IU/mL), kallikrein-like activities (0.043-0.075 ΔAU/min), positive TGTs (98-298 nM), and FXIa (9.5-14.0 ng/mL) were detected in Fr II + III. After pasteurization, no residual evidence of any procoagulant activity marker was observed, including the final IVIG concentrate at 5% or 10% protein. Results were similar in Fr II + III from different IVIG manufacturing facilities. CONCLUSIONS: The Flebogamma(®) DIF production process is capable of eliminating procoagulant activity because of its purification steps.

Locally administered prostaglandin E2 prevents aeroallergen-induced airway sensitization in mice through immunomodulatory mechanisms.

Prostaglandin E2 attenuates airway pathology in asthmatic patients and exerts a protective effect in antigen-sensitized mice when administered systemically. We aimed to establish the consequences of intranasal PGE2 administration on airway reactivity to aeroallergens in mice and reveal the underlying immunoinflammatory mechanisms. PGE2 was administered either daily during a 10-day exposure to house dust mite (HDM) extracts or for limited intervals. Airway hyperreactivity was measured by whole-body and invasive plethysmography. The phenotypes of lung immune cells and cytokine production were analysed by flow cytometry and ELISA, respectively. Airway hyperreactivity was sustainably reduced only when PGE2 administration was restricted to the initial 5 days of exposure to HDM. Lung inflammation, IL-4 production, and airway mast cell activity were also prevented under this early short-term treatment with PGE2. Interestingly, a Th2 response was already committed on day 5 of exposure to HDM. This was paralleled by GM-CSF and osteopontin upregulation and a decreased number of plasmacytoid dendritic and T regulatory cells, as well as a trend towards reduced IL-10 expression. Local PGE2 administration prevented the increase of airway IL-13 and osteopontin and kept lung plasmacytoid dendritic cell counts close to baseline. GM-CSF and Tregs were unaffected by the treatment. These findings suggest that the protection provided by PGE2 is a result of the modulation of early lung immunomodulatory mechanisms, and possibly a shift in the balance of dendritic cells towards a tolerogenic profile.

The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming.

Metabolism is vital to every aspect of cell function, yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis, and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly, the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs, which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs, and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency, and in evaluating iPSC and ESC equivalence.

Rapid and highly efficient generation of induced pluripotent stem cells from human umbilical vein endothelial cells.

The ability to induce somatic cells to pluripotency by ectopic expression of defined transcription factors (e.g. KLF-4, OCT4, SOX2, c-MYC, or KOSM) has transformed the future of regenerative medicine. Here we report somatic cell reprogramming of human umbilical vein endothelial cells (HUVECs), yielding induced pluripotent stem (iPS) cells with the fastest kinetics, and one of the highest reprogramming efficiencies for a human somatic cell to date. HUVEC-derived iPS (Huv-iPS) cell colonies appeared as early as 6 days after a single KOSM infection, and were generated with a 2.5-3% reprogramming efficiency. Furthermore, when HUVEC reprogramming was performed under hypoxic conditions in the presence of a TGF-beta family signaling inhibitor, colony formation increased an additional ∼2.5-fold over standard conditions. Huv-iPS cells were indistinguishable from human embryonic stem (ES) cells with regards to morphology, pluripotent marker expression, and their ability to generate all embryonic germ layers in vitro and in vivo. The high efficiency and rapid kinetics of Huv-iPS cell formation, coupled with the ease by which HUVECs can be collected, expanded and stored, make these cells an attractive somatic source for therapeutic application, and for studying the reprogramming process.

A high proliferation rate is required for cell reprogramming and maintenance of human embryonic stem cell identity.

Human embryonic stem (hES) cells show an atypical cell-cycle regulation characterized by a high proliferation rate and a short G1 phase. In fact, a shortened G1 phase might protect ES cells from external signals inducing differentiation, as shown for certain stem cells. It has been suggested that self-renewal and pluripotency are intimately linked to cell-cycle regulation in ES cells, although little is known about the overall importance of the cell-cycle machinery in maintaining ES cell identity. An appealing model to address whether the acquisition of stem cell properties is linked to cell-cycle regulation emerged with the ability to generate induced pluripotent stem (iPS) cells by expression of defined transcription factors. Here, we show that the characteristic cell-cycle signature of hES cells is acquired as an early event in cell reprogramming. We demonstrate that induction of cell proliferation increases reprogramming efficiency, whereas cell-cycle arrest inhibits successful reprogramming. Furthermore, we show that cell-cycle arrest is sufficient to drive hES cells toward irreversible differentiation. Our results establish a link that intertwines the mechanisms of cell-cycle control with the mechanisms underlying the acquisition and maintenance of ES cell identity.

High-efficient generation of induced pluripotent stem cells from human astrocytes.

The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells, including fibroblasts, keratinocytes and peripheral blood cells, with variable reprogramming efficiencies and kinetics. Here, we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells, with similar efficiencies to keratinocytes, which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure.

Activity of the cyclooxygenase 2-prostaglandin-E prostanoid receptor pathway in mice exposed to house dust mite aeroallergens, and impact of exogenous prostaglandin E2.

BACKGROUND: Prostaglandin E2 (PGE2), experimentally administered to asthma patients or assayed in murine models, improves allergen-driven airway inflammation. The mechanisms are unknown, but fluctuations of the endogenous cyclooxygenase (COX)-2/prostaglandin/E prostanoid (EP) receptor pathway activity likely contribute to the clinical outcome. We analyzed the activity of the pathway in mice sensitized to aeroallergens, and then studied its modulation under exogenous PGE2. METHODS: Mice were exposed to house dust mite (HDM) aeroallergens, a model that enable us to mimic the development of allergic asthma in humans, and were then treated with either subcutaneous PGE2 or the selective EP1/3 receptor agonist sulprostone. Simultaneously with airway responsiveness and inflammation, lung COX-2 and EP receptor mRNA expression were assessed. Levels of PGE2, PGI2, PGD2 were also determined in bronchoalveolar lavage fluid. RESULTS: HDM-induced airway hyperreactivity and inflammation were accompanied by increased COX-2 mRNA production. In parallel, airway PGE2 and PGI2, but not PGD2, were upregulated, and the EP2 receptor showed overexpression. Subcutaneous PGE2 attenuated aeroallergen-driven airway eosinophilic inflammation and reduced endogenous PGE2 and PGI2 production. Sulprostone had neither an effect on airway responsiveness or inflammation nor diminished allergen-induced COX-2 and PGE2 overexpression. Finally, lung EP2 receptor levels remained high in mice treated with PGE2, but not in those treated with sulprostone. CONCLUSION: The lung COX-2/PGE2/EP2 receptor pathway is upregulated in HDM-exposed mice, possibly as an effort to attenuate allergen-induced airway inflammation. Exogenous PGE2 downregulates its endogenous counterpart but maintains EP2 overexpression, a phenomenon that might be required for administered PGE2 to exert its protective effect.

Subcutaneous prostaglandin E(2) restrains airway mast cell activity in vivo and reduces lung eosinophilia and Th(2) cytokine overproduction in house dust mite-sensitive mice.

BACKGROUND: Prostaglandin (PG) E(2) is thought to exert protective effects in the lungs. Accordingly, aerosolized PGE(2) prevents the experimentally induced airway response to allergen challenge in asthmatics. In vitro evidence indicating that functional PGE(2) receptors (EP) are expressed on human mast cells and that PGE(2) can alter cytokine production suggests that these phenomena may be involved in its beneficial effect in asthma. However, in vivo evidence is scarce. METHODS: We assessed the effects of exogenous PGE(2) and of the EP1/EP3 agonist sulprostone on the murine airway response to house dust mite (HDM) allergens, a model that accurately reproduces the spontaneous exposure of allergic asthma patients to aeroallergens. We also analyzed the in vivo impact of PGE(2) on production in the murine airway of mast cell protease (mMCP)-1, a specific marker of lung mast cell activity, and on local production of cytokines. RESULTS: Exogenous PGE(2), but not sulprostone, reduced eosinophilic infiltration in HDM-sensitized mice by half and led to a strong reduction in airway Th(2) cytokine expression. These anti- inflammatory effects were accompanied in vivo by a substantial reduction in HDM-induced upregulation of airway mMCP-1. Neither PGE(2) nor sulprostone had any effect on airway hyperresponsiveness to methacholine. CONCLUSIONS: Our results indicate that the anti-inflammatory effect of PGE(2) can be reproduced in vivo in HDM-sensitized mice and suggest that this protective effect is dependent in vivo on inhibition of the allergen-triggered proinflammatory activity of bronchial mast cells. Finally, the effect of PGE(2) is linked to reduced upregulation of airway Th(2) cytokines.

An intranasal selective antisense oligonucleotide impairs lung cyclooxygenase-2 production and improves inflammation, but worsens airway function, in house dust mite sensitive mice.

BACKGROUND: Despite its reported pro-inflammatory activity, cyclooxygenase (COX)-2 has been proposed to play a protective role in asthma. Accordingly, COX-2 might be down-regulated in the airway cells of asthmatics. This, together with results of experiments to assess the impact of COX-2 blockade in ovalbumin (OVA)-sensitized mice in vivo, led us to propose a novel experimental approach using house dust mite (HDM)-sensitized mice in which we mimicked altered regulation of COX-2. METHODS: Allergic inflammation was induced in BALBc mice by intranasal exposure to HDM for 10 consecutive days. This model reproduces spontaneous exposure to aeroallergens by asthmatic patients. In order to impair, but not fully block, COX-2 production in the airways, some of the animals received an intranasal antisense oligonucleotide. Lung COX-2 expression and activity were measured along with bronchovascular inflammation, airway reactivity, and prostaglandin production. RESULTS: We observed impaired COX-2 mRNA and protein expression in the lung tissue of selective oligonucleotide-treated sensitized mice. This was accompanied by diminished production of mPGE synthase and PGE2 in the airways. In sensitized mice, the oligonucleotide induced increased airway hyperreactivity (AHR) to methacholine, but a substantially reduced bronchovascular inflammation. Finally, mRNA levels of hPGD synthase remained unchanged. CONCLUSION: Intranasal antisense therapy against COX-2 in vivo mimicked the reported impairment of COX-2 regulation in the airway cells of asthmatic patients. This strategy revealed an unexpected novel dual effect: inflammation was improved but AHR worsened. This approach will provide insights into the differential regulation of inflammation and lung function in asthma, and will help identify pharmacological targets within the COX-2/PG system.