Cost-effectiveness of omalizumab in real world uncontrolled allergic asthma patients.

OBJECTIVE: To estimate the cost-effectiveness of omalizumab compared with standard of care in the treatment and control of severe persistent asthma, using the outcomes from the Portuguese subpopulation of the eXpeRience registry. METHODS: This was a pragmatic cost-effectiveness analysis based on real world data from the eXpeRience registry which recruited 62 patients with uncontrolled persistent allergic asthma from 20 participating centers in Portugal. Response to omalizumab treatment was measured prospectively up to 24 months by the physician's Global Evaluation of Treatment Effectiveness (GETE). Retrospective data on patients' clinical symptoms, asthma control, lung function, exacerbations, and healthcare utilization were available for up to 12 months before omalizumab initiation and served as the standard of care comparator. The number of exacerbations (severe and non-severe), the number of clinical episodes, the number of days absent from work and/or school, and GETE response to therapy were considered as effectiveness outcomes. Following a societal perspective, as cost indicators, both direct and indirect costs were considered. Direct costs relate to the cost of omalizumab, standard of care and clinical episodes (emergency room visits, hospitalizations, and unscheduled doctor visits). Indirect costs relate to the societal cost of work absenteeism. Unit costs for clinical episodes and drugs were taken from official sources within the Portuguese Health Authority. A univariate sensitivity analysis was performed. RESULTS: A rate of 1.5 exacerbations per patient-year was estimated following omalizumab treatment compared with 8.2 exacerbations per patient-year prior to omalizumab initiation, implying an 82.1% reduction in the incidence of exacerbations following omalizumab treatment relative to standard of care alone. A 54.1% reduction in GETE score was also observed in favor of omalizumab treatment. The mean cost per person-year was 3023є in the 12 months of standard of care prior to omalizumab and 16,111є in the period of treatment with omalizumab. The incremental cost-effectiveness ratios were 2244є/exacerbation avoided, and 1750є/unit decrease in GETE classification. CONCLUSION: Our results demonstrate that adding omalizumab to the treatment of patients with uncontrolled severe persistent asthma reduces the number of exacerbations, improving overall treatment effectiveness at an acceptable cost from a societal perspective.

Acute insulin response to arginine in deceased donors predicts the outcome of human islet isolation.

Despite a stringent donor selection, human islet isolation remains frustratingly unpredictable. In this study, we measured acute insulin response to arginine (AIRarg), an in vivo surrogate measure of islet mass, in 29 human deceased donors before organ donation, and correlated values with the outcome of islet isolation. Thirteen isolations (45%) met the threshold for clinical islet transplantation. Among all measured donor characteristics, the only discriminating variable between successful or unsuccessful isolations was donor AIRarg (p < 0.01). Using a threshold of 55 microIU/mL (ROC curve AUC: 72%), isolation was successful in 12/19 donors with high AIRarg and in 1/10 donors with low AIRarg (p < 0.001). The negative and positive predictive values were 90 and 63%, respectively. If used to select donors in the entire cohort, AIRarg would have increased our success rate by 40% and avoided 56% of unsuccessful isolations while missing only 8% of successful preparations. Our results suggest that donor AIRarg is markedly superior to body mass index (BMI) and other criteria currently used to predict isolation outcome. If routinely performed in deceased donors, this simple test could significantly reduce the failure rate of human islet isolation.

1,25-Dihydroxyvitamin D3 protects human pancreatic islets against cytokine-induced apoptosis via down-regulation of the Fas receptor.

Beta cell loss occurs at the onset of type 1 diabetes and after islet graft. It results from the dysfunction and destruction of beta cells mainly achieved by apoptosis. One of the mediators believed to be involved in beta cell apoptosis is Fas, a transmembrane cell surface receptor transducing an apoptotic death signal and contributing to the pathogenesis of several autoimmune diseases. Fas expression is particularly induced in beta cells by inflammatory cytokines secreted by islet-infiltrating mononuclear cells and makes cells susceptible to apoptosis by interaction with Fas-ligand expressing cells. We have previously demonstrated that 1,25(OH)2D3, the active metabolite of vitamin D, known to exhibit immunomodulatory properties and prevent the development of type 1 diabetes in NOD mice, is efficient against apoptosis induced by cytokines in human pancreatic islets in vitro. The effects were mainly mediated by the inactivation of NF-kappa-B. In this study we demonstrated that 1,25(OH)2D3 was also able to counteract cytokine-induced Fas expression in human islets both at the mRNA and protein levels. These results were reinforced by our microarray analysis highlighting the beneficial effects of 1,25(OH)2D3 on death signals induced by Fas activation. Our results provides additional evidence that 1,25(OH)2D3 may be an interesting tool to help prevent the onset of type 1 diabetes and improve islet graft survival.

Non-esterified fatty acids are deleterious for human pancreatic islet function at physiological glucose concentration.

AIMS/HYPOTHESIS: Whether excess glucose (glucotoxicity) and excess non-esterified fatty acids (lipotoxicity) act synergistically or separately to alter beta-cell function on Type 2 diabetes remains controversial. We examined the influence of non-esterified fatty acids, with or without concomitant increased glucose concentrations, on human islet function and on the expression of genes involved in lipid metabolism. METHODS: Human islets isolated from non-diabetic and non-obese donors were cultured with 5.5, 16 or 30 mmol/l glucose, and when appropriate with 1 or 2 mmol/l non-esterified fatty acids. After 48 h, glucose-stimulated insulin secretion, insulin content, triglyceride content and expression of different genes were evaluated. RESULTS: Non-esterified fatty acids decreased glucose-stimulated insulin secretion, insulin content and increased triglyceride content of human isolated islets, independently from the deleterious effect of glucose. Increased glucose concentrations also decreased glucose-stimulated insulin secretion and insulin content, but had no influence on triglyceride content. Glucose-stimulated insulin secretion of islets appeared to be significantly correlated with their triglyceride content. Glucose and non-esterified fatty acids modified the gene expression of carnitine palmitoyltransferase-I, acetyl-CoA carboxylase, acyl-CoA oxidase and uncoupling protein 2. CONCLUSION/INTERPRETATION: In our model of isolated human islets, increased glucose and non-esterified fatty acids separately reproduced the two major beta-cell alterations observed in vivo, i.e. loss of glucose-stimulated insulin secretion and reduction in islet insulin content. Our results also suggest that this deleterious effect was, at least in part, mediated by modifications in lipid metabolism gene expression.

Hepatocyte nuclear factor 4 alpha isoforms originated from the P1 promoter are expressed in human pancreatic beta-cells and exhibit stronger transcriptional potentials than P2 promoter-driven isoforms.

The nuclear receptor hepatocyte nuclear factor (HNF) 4 alpha is involved in a transcriptional network and plays an important role in pancreatic beta-cells. Mutations in the HNF4 alpha gene are correlated with maturity-onset diabetes of the young 1. HNF4 alpha isoforms result from both alternative splicing and alternate usage of promoters P1 and P2. It has recently been reported that HNF4 alpha transcription is driven almost exclusively by the P2 promoter in pancreatic islets. We observed that transcripts from both P1 and P2 promoters were expressed in human pancreatic beta-cells and in the pancreatic beta-cell lines RIN m5F and HIT-T15. Expression of HNF4 alpha proteins originating from the P1 promoter was confirmed by immunodetection. Due to the presence of the activation function module AF-1, HNF4 alpha isoforms originating from the P1 promoter exhibit stronger transcriptional activities and recruit coactivators more efficiently than isoforms driven by the P2 promoter. Conversely, activities of isoforms produced by both promoters were similarly repressed by the corepressor small heterodimer partner. These behaviors were observed on the promoter of HNF1 alpha that is required for beta-cell function. Our results highlight that expression of P1 promoter-driven isoforms is important in the control of pancreatic beta-cell function.

Kinetics of diabetes-associated autoantibodies after sequential intraportal islet allograft associated with kidney transplantation in type 1 diabetes.

OBJECTIVE: Presence or occurrence of pancreas auto-antibodies (aAb) has been shown to be of poor prognosis for islet cell transplantation. The aim of the study was to monitor the kinetics of these aAb after sequential intra-portal islet plus kidney transplantation with pre-Edmonton immunosuppressive regimen in order to determine whether the sequential protocol of transplantation was involved in the occurrence of the immune response. PATIENTS AND METHODS: Three patients with IDDM and a previous (IAK) or simultaneous (SIK) kidney transplantation received 3 or 4 ABO compatible islet preparations. Islets (> 8 000 IEQ/kg post culture) were sequentially transplanted within a 12 day period via a per-cutaneous catheter. Immunosuppressive treatment included cyclosporine, steroïds and mycophenolate. Plasma ICAs, GAD 65, IA2 and C peptide (C-p) levels were monitored. Type II HLA phenotype was determined in donors and recipients. RESULTS: Patient #1 had high anti-GAD levels (26.5 UI/l) before the IAK, while anti-IA2 and ICA levels were low. After the transplantation, C-p levels increased to 4.9 ng/ml at one month before becoming undetectable at 2 months. GAD levels remained high, ICA and IA2 aAb were undetectable. Patients #2 and #3 did not have significant levels of aAb before the islet transplantation. A slight increase in GAD was observed with each islet transplantation, followed by an overt but transient increase in ICA. IA2 levels remained undetectable. Three months after the transplantation and 2 weeks after the increase of ICA, C-p levels, that were >3.4 ng/ml at one month, fell below 0.2 (N: 0.5-2). CONCLUSION: The immunosuppressive regimen used in kidney transplantation is unable to control perfectly anti-pancreas aAb production. Moreover, these results seem to indicate that the benefits of sequential islet transplantation lie more in the increased islet mass they provide than in potential immune benefit.

Human pancreatic ductal cells: large-scale isolation and expansion.

The in vitro differentiation of pancreatic stem cells has recently been shown to represent a new source of beta cells for cell therapy in diabetes. Human ductal cell differentiation, in vitro, has been documented in three-dimensional (3D) culture and recently substantiated. Although encouraging, the optimization of the ductal cell source, expansion and differentiation ex vivo are mandatory for clinical relevance. We compared three sources of human ductal cells (hDC) (method A1-2, B, and C). The classical main duct isolation of hDC by explant (A1), or enzymatic digestion (A2), was compared with two indirect methods: from 3D cultured human islet/duct-enriched fractions (B) and dedifferentiated exocrine fractions (C). Method A: few viable hDC were obtained from the main duct. Method B: embedding islet/duct rich fraction in 3D collagen gels expands the cytokeratin 19 (CK19)-positive ductal component in the form of ductal cysts, as we described previously; monolayers derived from digested cysts were 80% ductal (CK19). Method C: initially adherent amylase-positive exocrine clusters contained 12% (CK19) to 22% (CK7) ductal cells. One-week exocrine cultures were amylase negative and 46% (CK19) to 63% (CK7) ductal. Cell viability varied: <20% (A1), 81+/-12% (B), 91+/-2% (C). Extrapolating total yields we obtained (+/-SEM): 10.5+/-4.6 x 10(3) (A1), 36+/-18 x 10(3) (A2), 292+/-50 x 10(6) (B), 1696+/-526 x 10(6) (C) viable hDC per pancreas. A secondary monolayer expansion of cyst-derived hDC (method B) was achieved with NuSerum (4.2-fold on plastic, 2.6-fold on 804G matrix; p < 0.05 vs. control cells on plastic). First passage exocrine-derived ductal cells also responded to matrix and to growth factors, albeit not significantly. In conclusion, this study demonstrated that an abundant hDC supply can be obtained from islet/duct or exocrine fractions followed by monolayer expansion with NuSerum. If their differentiation capacity is confirmed, in particular exocrine-derived ductal cells may represent a promising abundant source of islets for allogenic and autologous diabetes cell therapy.

Beneficial effect of 1,25 dihydroxyvitamin D3 on cytokine-treated human pancreatic islets.

We examined whether 1,25 dihydroxyvitamin D(3) (1,25 D(3)), the active form of vitamin D involved in the regulation of the immune system, may also protect human pancreatic islet cells from destruction induced by cytokines. In this study, we specifically investigated the effect of 1,25 D(3) on oxidative stress and major histocompatibility complex (MHC) induction, both implicated in cytokine-induced islet cell dysfunction and destruction. We also investigated the effects of 1,25 D(3) on interleukin (IL)-6, a pleiotropic cytokine implicated in the pathogenesis of immunoinflammatory disorders. Human pancreatic islets, isolated from heart-beating donors, were treated with a combination of three cytokines, IL-1beta+tumor necrosis factor alpha+interferon gamma, in the presence or absence of vitamin D, and compared with with untreated control cells. Metabolic activity was assessed by cell viability and insulin content. Oxidative stress was estimated by heat shock protein 70 (hsp70) expression, cell manganese superoxide dismutase (MnSOD) activity and nitrite release, a reflexion of nitric oxide (NO) synthesis. Variation of immunogenicity of islet preparations was determined by analysis of the MHC class I and class II transcripts. Inflammatory status was evaluated by IL-6 production. After 48 h of contact with cytokines, insulin content was significantly decreased by 40% but cell viability was not altered. MHC expression significantly increased six- to sevenfold as well as NO and IL-6 release (two- to threefold enhancement). MnSOD activity was not significantly induced and hsp70 expression was not affected by the combination of cytokines. The addition of 1,25 D(3) significantly reduced nitrite release, IL-6 production and MHC class I expression which then became not significantly different from controls. These results suggest that the effect of 1,25 D(3) in human pancreatic islets cells may be a reduction of the vulnerability of cells to cytotoxic T lymphocytes and a reduction of cytotoxic challenge. Hence, 1,25 D(3) might play a role in the prevention of type 1 diabetes and islet allograft rejection.

Identification and purification of functional human beta-cells by a new specific zinc-fluorescent probe.

Pancreatic beta-cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-sensitive fluorescent probe able to selectively label labile zinc in viable beta-cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number (r = 0.98) and between zinc fluorescence and insulin content (r = 0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resulted in a clear separation of beta-cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the visible spectrum, appears very promising for beta-cell experimentation, both clinically and for basic research.

Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in normal human pancreatic islet cells.

AIMS/HYPOTHESIS: Thiazolidinediones are reported to improve pancreatic islet morphology and beta-cell function in rodents, supporting the hypothesis of a direct action of thiazolidinediones on endocrine islet cells. In this study we examined the expression of the peroxisome proliferator-activated receptor gamma, a nuclear receptor that is activated by naturally occurring fatty acids and synthetic thiazolidinediones, in normal human endocrine pancreatic cells. METHODS: Human islets were isolated from pancreata harvested in ten brain-dead lean non-diabetic adult donors. We analysed the gene and protein expression of the human peroxisome proliferator-activated receptor gamma and evaluated the effects of peroxisome proliferator-activated receptor gamma agonist on insulin secretion in human islet preparations. RESULTS: The RT-PCR carried out on total RNA from four distinct human islet preparations demonstrated the presence of peroxisome proliferator-activated receptor gamma mRNA. Western blot analysis showed the consistent expression of peroxisome proliferator-activated receptor gamma protein. Peroxisome proliferator-activated receptor gamma was shown to be present in all three endocrine cell types studied (alpha, beta and delta cells) by immunohistochemistry. CONCLUSION/INTERPRETATION: We found that peroxisome proliferator-activated receptor gamma is highly expressed in human islet endocrine cells, both at the mRNA and protein levels. These results support the hypothesis of a direct influence of peroxisome proliferator-activated receptor gamma agonist on human pancreatic endocrine cells.

Adult human cytokeratin 19-positive cells reexpress insulin promoter factor 1 in vitro: further evidence for pluripotent pancreatic stem cells in humans.

Human pancreatic cells with a typical ductal phenotype and potential to proliferate can be obtained in vitro, but the differentiation capacity of these putative human pancreatic stem cells remains to be documented. We investigated the protein and mRNA expression of insulin promoter factor 1 (IPF-1) (or pancreas/duodenal homeobox 1), a transcription factor critical for pancreatic development and endocrine cell neogenesis, in human pancreatic ductal cells derived from cultured exocrine tissue. In vitro, exocrine cells rapidly adhered (within 12 h) and were de-/transdifferentiated to ductal cells after 3 days with a dramatic loss of amylase protein (n = 4, 92 +/- 3.3%, P < 0.05 vs. day 1) and a simultaneous increase of ductal cytokeratin 19 protein (n = 4, 3.4-fold on day 3 and 7-fold on day 9, P < 0.05 vs. day 1). IPF-1 protein and mRNA levels were low to undetectable in exocrine preparations before culture. After 2 days of culture, a 3.2-fold increase in IPF-1 protein was observed, corresponding to the characteristic 46-kDa protein in Western blots. Reverse transcriptase-polymerase chain reaction confirmed a 10.5-fold increase in IPF-1 mRNA levels after 3 days of culture (n = 5, P < 0.001 vs. day 1). Double immunocytochemistry showed direct evidence that IPF-1 appeared during culture in these exocrine-derived ductal cells (cytokeratin 7-positive) and was not merely in contaminating endocrine cells (chromogranin A-positive). In conclusion, we describe herein the first converging evidence on both the molecular and protein level that human cells with a typical ductal phenotype derived ex vivo from pancreatic exocrine tissue (obtained from healthy donors) can reexpress IPF-1 in culture, suggesting their pancreatic precursor/stem cell potential.

[Human pancreatic stem cell and diabetes cell therapy]. / Cellules souches du pancréas humain et thérapie cellulaire du diabète.

Cell therapy offers today important perspectives for the treatment of type 1 diabetes. The current utilization of primary human islets of Langerhans nevertheless forbids all hope of developing this treatment on a large scale. The recent description of the persistence of stem cells capable of proliferating and differentiating in the adult pancreas offers an attractive alternative for the production in vitro of homologous insulin-secreting cells. We first reproduced in vitro from human islet preparations the proliferation of ductal epithelial structures and their progressive organization. Thereafter, we focused on the description of a reproducible source of human ductal cells by the transdifferentiation of exocrine preparations. More recently we described in these exocrine derived ductal cells the the expression the of insulin promoter factor-1 (IPF-1/otherwise known as PDX-1), a transcription factor essential for the differentiation of ductal cells into endocrine cells during both development and pancreatic regeneration. If the proliferation and differentiation of these cells is confirmed, this approach could lead to the description of an abundant source of human pancreatic stem cells for the production ex vivo of human insulin secreting cells and may even allow autologous cell therapy, in the absence of immunosuppression.

Human pancreatic islet quality control: easy assessment of metabolic functions.

We describe simplified and rapid methods to assess islet function with the aim to develop better protocols for islet isolation and to determine islet characteristics before transplantation. These methods are also useful in the assessment of the potentially beneficial or deleterious effects of compounds added to the culture media in stimulation experiments. To this end, we took advantage of the multiscreen assay system produced by Millipore SA. This 96-well unit allowed the free-floating culture of islets on filter membranes, the rapid vacuuming and collection of conditioned media or reaction buffer and thus successive testing of the same number of islets, possibly at different culture times. We estimated islet viability by determination of the metabolic activity of cells, normal function of islets by their ability to metabolize glucose and to synthesize and secrete insulin and of nitrite release, a reflection of nitric oxide (NO) status of cells, which may be involved in a signaling pathway during glucose-stimulated insulin secretion or in cytokine inducible pathway. Assays may be performed either on selected islets or on aliquots of semi-purified preparations designated for grafting, allowing thus the rapid estimation of graft function of the entire preparation. This herein described system may be also extended to many other functional tests.

Effects of vitamin D3 derivatives on growth, differentiation and apoptosis in tumoral colonic HT 29 cells: possible implication of intracellular calcium.

In addition to the effects on tumor cell differentiation and growth inhibition, vitamin D3 derivatives may exert other cellular actions such as the inhibition of angiogenesis or the induction of apoptosis. In this study, we demonstrated that vitamin D3 derivatives, 1,25-dihydroxyvitamin D3, the natural derivative and Ro 23-7553, a synthetic derivative, displayed complex effects in tumoral colonic HT 29 cells. Indeed, as a function of the stage of culture, they induced either apoptosis or differentiation along with a constant cell cycle blockade in G1. Intracellular calcium analysis indicated that treatment resulted in disturbance in the distribution of calcium suggesting a possible role for intracellular calcium in the observed effects. The association of 9-cis-retinoic acid, the ligand of RXR, with vitamin D3 derivatives modified the demonstrated effects, indicating in our model, a preferential effect of vitamin D3 derivatives via the heterodimeric form of the receptor.