Algorithm for Treatment of Chronic Spontaneous Urticaria with Omalizumab. / Algoritmo de tratamiento con omalizumab en urticaria crónica espontánea.

BACKGROUND AND OBJECTIVE: Pivotal trials with omalizumab for treatment of chronic spontaneous urticaria (CSU) are generally run over 12 to 24weeks. However, in clinical practice, many patients need longer treatment. In this article, we present an algorithm for treatment with omalizumab. MATERIAL AND METHODS: The consensus document we present is the result of a series of meetings by the CSU working group of "Xarxa d'Urticària Catalana i Balear" (XUrCB) at which data from the recent literature were presented, discussed, compared, and agreed upon. RESULTS: Treatment with omalizumab should be initiated at the authorized dose, and is adjusted at 3-monthly intervals according to the Urticaria Activity Score Over 7days, the Urticaria Control Test, or both. CONCLUSIONS: The algorithm proposed is designed to provide guidance on how to adjust omalizumab doses, how and when to discontinue the drug, and how to reintroduce it in cases of relapse.

[Analysis of clinical relevance applied to 3methods of reducing weight in overweight or obesity followed-up for one year]. / Análisis de relevancia clínica aplicado a 3métodos de reducción de peso en pacientes con sobrepeso u obesidad seguidos durante un año.

OBJECTIVE: To analyse the effect of the use/implementation of 3methods to reduce weight in overweight or obese patients during one year of follow up. MATERIAL AND METHODS: The design corresponds to a double-blind, randomised, controlled clinical trial with 3arms, and 12 months of follow-up. Patients were randomised into 3intervention groups: obesity motivational intervention, with a nurse previously trained in motivational intervention by expert psychologists (G1; n=60); lower intensity consultation, non-motivational group, with digital platform support (G2; N=61), and a third group that received recommendations for weight loss and follow-up in Primary Care Clinic (G3; n=59). Anthropometric variables (weight, height, and abdominal-waist circumference) were measured, and the percentage of patients who managed to reduce their weight ≥5% was considered as the main measurement of treatment effectiveness. RESULTS: All groups significantly decreased body weight at the end of the study, with a reduction in G1 (-5.6kg) followed by G2 (-4.3kg), and G3 (-1.7kg), with an overall mean: -3.9kg. The indicators of clinical relevance were in G1/G3: relative risk (RR): 4.99 (95% CI: from 2.71 to 9.18); relative risk reduction (RRR): 399.1% (171.3 to 818.0); Absolute risk reduction (RAR): 65.3% (from 51.5 to 79.1) and NNT: 2 (from 2 to 2). In the G2/G3 groups: RR: 3.01 (from 1.57 to 5.76); RRR: 200.5% (from 57.0 to 475.5); RAR: 32.8% (from 16.9 to 48.7) and NNT: 4 (from 3 to 6). In the G1/G2 groups: RR: 1.66 (from 1.25 to 2.20); RRR: 66.1% (from 25.3 to 120.1); RAR: 32.5% (from 16.6 to 48.4) and NNT: 4 (from 3 to 7). CONCLUSIONS: All 3groups were able to reduce weight. Although the group with motivational intervention achieved the greatest decrease, as well as the most favourable clinical relevance indicators.

Mitochondrial DNA haplogroup H as a risk factor for idiopathic dilated cardiomyopathy in Spanish population.

Idiopathic dilated cardiomyopathy (IDC) is a structural heart disease with strong genetic background. The different single nucleotide polymorphisms (SNPs) that constitute mitochondrial haplogroups could play an important role in IDC progression. The aim of this study was to test frequencies of mitochondrial haplogroups in healthy controls (n=422) and IDC patients (n=304) of a Caucasian Spanish population. To achieve this, ten major European haplogroups were identified. Frequencies and Odds Ratios for the association between IDC and haplogroups were calculated in both groups. We found that compared to healthy controls, the prevalence of haplogroup H was significantly higher in IDC patients (40.0% vs 50.7%, p-value=0.040).

Analysis of different routes of administration of heterologous 5-azacytidine-treated mesenchymal stem cells in a porcine model of myocardial infarction.

Stem cell therapy constitutes an exciting, powerful therapy to repair the heart. Nevertheless, there are numerous doubts about the best route of stem cell administration to achieve implantation into the injured myocardium. Development of a preclinical, large animal model may be useful to obtain a better approach to clinical situations. The aim of this work was to study the effectiveness of various routes of heterologous bone marrow mesenchymal stem cell (MSCs) administration in a porcine model of myocardial infarction. MSC treated with 5-azacytidine were stained with a fluorescent compound (DiO) before their administration to previously infarcted pigs via 3 routes: intracoronary (IC), intramyocardial (IM), or endocardial (EC; n = 5 each group). Healthy, noninfarcted animals were used as a control group. At 30 days after delivery, hearts were divided into 12 parts: infarcted zone (1-6), right-left atria, interatrial and interventricular septa, and right-left ventricles. In each zone we looked for and quantified, injected fluorescence-stained cells. In the animals in which presence of DiO-stained cells was detected, cells were located preferentially in the infarcted zone and not in the atria, ventricles, or septa. Comparing various administration routes, the mean number of engrafted cells within the infarct zone was significantly greater after IC infusion than either IM or EC injection. Fluorescent cells were not observed in healthy zones of the myocardium or in healthy animals.

Gene expression profiles in a porcine model of infarction: differential expression after intracoronary injection of heterologous bone marrow mesenchymal cells.

Myocardial infarction is one of the main causes of mortality in developed countries. Injection of bone marrow mesenchymal stem cells (BMMSC) with the ability to regenerate lost cardiomyocytes is a promising therapy for heart failure. To evaluate this strategy, an in vivo porcine model of infarction was used. Gene expression profiles of 3 groups of pigs (n = 5 each) were analyzed and compared by real-time reverse transcription-polymerase chain reaction (RT-PCR). One of the groups underwent anterior descending coronary occlusion followed by BMMSC injection; a placebo group was injected with culture medium without cells after infarction; and a third group was formed by healthy pigs. Four weeks later, cells or medium was administered by intracoronary injection and, a month later, animals were sacrificed and samples collected. Genes related to cardiomyogenesis (Mef2C, Gata4, Nkx2.5), mobilization and homing of resident or circulating stem cells (Sdf1, Cxcr4, c-Kit), contractibility (Serca2a), and fibrosis (CollA1) were analyzed. Gene expression profiles changed in various heart areas in the 3 groups. Expression of genes related to cardiomyogenesis decreased in infarcted zones compared with homologous regions of healthy hearts. Sdf1 expression increased in the apex of infarcted hearts. Serca2a expression was reduced in the ventricles and atria of infarcted hearts. Also, increases in Cxcr4 and CollA1 expression were observed in infarcted hearts of cell-treated pigs compared with the placebo group. In conclusion, infarction induced changes in genes involved in various biological processes. Intracoronary injection of heterologous BMMSC resulted in localized changes in the expression of Cxcr4 and Col1A1.

Comparison of gene expression profiles in a porcine infarct model after intracoronary, transthoracic, or transendocardiac injection of heterologous bone marrow mesenchymal cells.

An in vivo porcine model of myocardial infarction was developed with the aim of comparing the effectiveness for cardiac repair of intracoronary, transthoracic, or transendocardial delivery strategies for bone marrow mesenchymal stem cells (BMMSC) using an analysis of expression levels of transcripts related to various cellular processes at 8 heart regions using quantitative reverse transcriptase polymerase chain reaction. We observed significant rises in cardiomyogenic markers Mef2C, Gata4 and Nkx2.5, and contractibility marker Serca2A at infarcted regions for cell-treated pigs. We also observed differences in Sdf1 expression related to the organ stress response between delivery strategies. Unexpectedly, increased expression of Col1A1 was detected in 2 cell-treated groups at various heart regions. Our results suggest improvements in both contractility and cardiomyogenic capability of damaged tissue after BMMSC injection, but also warned us about the relevance of the chosen delivery strategy and potential undesired effects like increasing fibrosis after treatment.

Characterisation of porcine bone marrow progenitor cells identified by the anti-c-kit (CD117) monoclonal antibody 2B8/BM.

c-kit (CD117) plays an important role in the early stages of haematopoiesis. Previous studies of porcine haematopoietic stem cells have relied for their identification on the use of the c-kit ligand stem cell factor. Here, we describe a new mAb, 2B8/BM, that recognizes a 155-kDa protein expressed on a small subset (2-8%) of bone marrow haematopoietic cells. 2B8/BM(+) cells have a blast appearance, and are mostly negative for lineage-specific markers or express low levels of CD172a or SLA-II. In in vitro colony-forming unit assays these cells were able to give rise to erythroid and myeloid colonies. Altogether these data suggested that the 2B8/BM antigen might be the porcine orthologue of the human c-kit. This specificity was confirmed by the binding of mAb 2B8/BM to CHO cells transfected with a plasmid encoding the porcine c-kit ectodomain. This antibody can facilitate the isolation and enrichment of porcine stem cells to be used in procedures aimed to induce xenograft tolerance or to test their potential to repair damaged tissues and organs.

Phenotypic and functional characterization of porcine granulocyte developmental stages using two new markers.

Here, we describe two new surface antigens, named 6D10 and 2B2, whose expression is restricted to porcine granulocytes. 6D10 is only detected in neutrophils and its expression decreases from promyelocytes to mature cells. By contrast, 2B2 antigen is selectively expressed in mature neutrophils, eosinophils and basophils. The expression of these antigens along granulocyte maturation allows the discrimination of several developmental stages of granulocytes based on phenotypic, morphological and functional characteristics previously established. Moreover, these new markers are useful tools to easily characterize the different granulocytes lineages (neutrophils, eosinophils and basophils). By using multiparameter flow cytometric analysis, we have performed a phenotypic and functional characterization of the granulocyte subsets identified by the combination of 6D10 and 2B2 antigens.

Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation.

Cell transplantation to regenerate injured tissues is a promising new treatment for patients suffering several diseases. Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Our goal was the isolation, characterization, and immortalization of porcine MSCs (pMSCs) to study their potential differentiation "in vitro" into cardiomyocytes. pMSCs were obtained from the aspirated bone marrow of Large-White pigs. After 4 weeks in culture, adherent cells were phenotypically characterized by flow cytometry and immunochemistry by using monoclonal antibodies. Primary pMSCs were transfected with the plasmid pRNS-1 to obtain continuous growing cloned cell lines. Fresh pMSCs and immortalized cells were treated with 5-azacytidine to differentiate them into cardiomyocytes. Flow cytometry analysis of isolated pMSCs demonstrated the following phenotype, CD90(pos), CD29(pos), CD44(pos), SLA-I(pos), CD106(pos), CD46(pos) and CD45(neg), CD14(neg), CD31(neg), and CD11b(neg), similar to that described for human MSC. We derived several stable immortalized MSC cell lines. One of these, called pBMC-2, was chosen for further characterization. After "in vitro" stimulation of both primary or immortalized cells with 5-azacytidine, we obtained different percentages (30%-50%) of cells with cardiomyocyte characteristics, namely, positive for alpha-Actin and T-Troponin. Thus, primary or immortalized pMSCs derived from bone marrow and cultured were able to differentiate "ex vivo" into cardiac-like muscle cells. These elements may be potentials tools to improve cardiac function in a swine myocardial infarct model.

The serum level of xenoantibodies, and hDAF or alphaGAL expression on pig cells, modulate in vitro the protection given by hDAF to primate complement-mediated damage.

The ability of human complement regulatory molecules to prevent xenograft rejection following pig-to-primate xenotransplantation is limited. We assayed the efficacy of transgenic human decay accelerating factor (hDAF) expressed on porcine cells to inhibit the in vitro complement activity of primate sera. We measured the cytotoxic activity of baboon or human sera against peripheral blood lymphocytes (PBLs) from hDAF or nontransgenic pigs using a flow cytometry complement-mediated cytotoxicity assay (FCCA). We also analyzed the anti-Galalpha1-3Gal (alphaGal) antibody titer of the baboon sera by ELISA and the expression of hDAF and alphaGal on the PBL surface by immunofluorescence. Transgenic hDAF expression was capable of protecting pig cells against injury produced by both baboon and human serum. However, the hDAF molecule was more efficient against human than baboon sera. The humoral cytotoxicity capacity correlated with the level of both IgG and IgM anti-alphaGal antibodies. In addition, inhibition of complement-mediated cytotoxicity of hDAF pig cells correlated with the expression of hDAF and alphaGal molecules on target cells. These results confirm in vitro the protective role of hDAF in pig cells to heterologus complement mediated damage, but they also suggest that protection decreases in the presence of high levels of anti-porcine antibodies in serum, low expression of hDAF, or high expression of alphaGal on pig cells.

Flow cytometry complement-mediated cytotoxicity assay detects baboon xenoantibodies directed to porcine epitopes undetected by hemolytic assay.

The pig-to-primate model is increasingly being utilized as the final preclinical means of assessing therapeutic strategies aimed at allowing discordant xenotransplantation. To obtain information about the nature of cytotoxic response in pig-to-baboon xenotransplants, we sought to determine if serum cytotoxicity in this model was assay dependent. Sera from nine kidney or heart xenotransplanted baboons were obtained before transplantation and at the time of acute humoral xenograft rejection (AHXR). Cytotoxicity was measured by an anti-pig haemolytic assay (APHA) and by a flow cytometry complement-dependent assay (FCCA), using pig blood lymphocytes (PBLs). Serum samples showing inter-assay differences were absorbed with pig erythrocytes and assayed by APHA and FCCA, as well as by measuring anti-alphaGal and total anti-pig xenoantibodies. The results showed that in four AHXR samples, FCCA cytotoxicity was higher than APHA cytotoxicity. Absorption with pig erythrocytes diminished FCCA and removed APHA cytotoxicity. Residual FCCA activity was due to total anti-pig and IgM anti-alphaGal and non-Gal antibodies. Our results indicate that some cytotoxic antibodies present in the sera of xenotransplanted baboons at time of AHXR are IgM antibodies directed against pig PBL antigens not detected by APHA.

The CaMV 35S promoter is highly active on floral organs and pollen of transgenic strawberry plants.

We have evaluated the expression of the reporter beta-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter in flowers and pollen from 14 independent transgenic strawberry lines. Of the 14 lines evaluated, 13 (92.8%) showed GUS activity--as estimated by the histochemical GUS assay--in some floral organs, with expression being most common in the flower stem, sepals, petals, ovary and stigma. Ten of these thirteen transgenic lines (77%) showed GUS activity in pollen, although the percentages of positive pollen per flower varied greatly among the different lines. A study of the GUS expression during pollen maturation showed that the (CaMV 35S) promoter showed low expression in pollen from flower buds before anthesis but was activated in mature pollen following anther dehiscence. The percentages of pollen grains that showed GUS activity ranged from 2.1% to 46.3%. These percentages were similar or even higher when mature pollen was stored dry at room temperature for 2 weeks. After 5 weeks of storage, the percentages of GUS-positive pollen decreased in two of the six lines analysed but remained at similar values in the other four lines. GUS activity was also measured in protein extracts of mature pollen by means of the fluorometric GUS assay, with the values obtained ranging from 3.8 micromol MU mg protein(-1) h(-1) to 0.26 micromol MU mg protein(-1) h(-1). Contrary to the generally held view that the CaMV 35S promoter is virtually silent in pollen, we conclude that it is highly expressed in transgenic strawberry pollen.

A new epitope on swine CD5 molecule detected by monoclonal antibody 5F12/9.

This paper describes the production and characterization of a monoclonal antibody (MAb), 5F12/9, that recognizes a new epitope on porcine CD5. Conformation of its CD5 specificity was obtained by means of sequential immunoprecipitation and Western blot experiments in combination with anti-CD5 MAb 1H6/8, whereas cross-blocking experiments with both MAbs showed that they reacted with different epitopes.