Unraveling the Diagnosis of Kiwifruit Allergy: Usefulness of Current Diagnostic Tests.

OBJECTIVES: To determine the usefulness of the in vitro and in vivo methods used in the diagnosis of kiwifruit allergy and to specifically assess the impact of seed proteins on sensitivity. METHODS: We performed skin prick tests (SPTs) using various commercial extracts, homemade pulp, and seed extracts and prick-prick tests with kiwifruit on 36 allergic patients. The presence of specific IgE (sIgE) was assessed using the ImmunoCAP (kiwifruit extract), ELISA (Act d 1, Act d 2), ISAC, and FABER assays. Immunoblotting of seed extract was carried out, and a single-blind oral food challenge was performed with whole seeds in seed-sensitized individuals. RESULTS: The prick prick test with kiwifruit demonstrated the highest diagnostic capacity (81.8% sensitivity and 94.1% specificity) among the in vivo tests. The sIgE levels measured using ImmunoCAP (kiwifruit extract) showed a similar sensitivity to that of global ISAC and FABER (63.9%, 59.5%, and 58.3%, respectively). Act d 1 was the major allergen. Sensitization to Act d 1 was associated with positive sIgE results to whole kiwifruit extract detected by ImmunoCAP (P<.000). A positive SPT result to kiwifruit seeds was associated with severe symptoms induced by kiwifruit (P=.019) as a marker of advanced disease, but not with clinically relevant sensitization. Challenge testing with kiwifruit seeds performed on 8 seed-sensitized patients yielded negative results. CONCLUSION: Sensitization to Act d 1 is associated with a positive result in conventional diagnostic techniques, whereas kiwifruit seed sensitization does not increase the sensitivity of the diagnostic techniques evaluated.

Unraveling the Diagnosis of Kiwifruit Allergy: Usefulness of Current Diagnostic Tests

Objectives: To determine the usefulness of the in vitro and in vivo methods used in the diagnosis of kiwifruit allergy and to specificallyassess the impact of seed proteins on sensitivity.Methods: We performed skin prick tests (SPTs) using various commercial extracts, homemade pulp, and seed extracts and prick-prick testswith kiwifruit on 36 allergic patients. The presence of specific IgE (sIgE) was assessed using the ImmunoCAP (kiwifruit extract), ELISA(Act d 1, Act d 2), ISAC, and FABER assays. Immunoblotting of seed extract was carried out, and a single-blind oral food challenge wasperformed with whole seeds in seed-sensitized individuals.Results: The prick prick test with kiwifruit demonstrated the highest diagnostic capacity (81.8% sensitivity and 94.1% specificity) amongthe in vivo tests. The sIgE levels measured using ImmunoCAP (kiwifruit extract) showed a similar sensitivity to that of global ISAC andFABER (63.9%, 59.5%, and 58.3%, respectively). Act d 1 was the major allergen. Sensitization to Act d 1 was associated with positivesIgE results to whole kiwifruit extract detected by ImmunoCAP (P<.000). A positive SPT result to kiwifruit seeds was associated withsevere symptoms induced by kiwifruit (P=.019) as a marker of advanced disease, but not with clinically relevant sensitization. Challengetesting with kiwifruit seeds performed on 8 seed-sensitized patients yielded negative results.Conclusions: Sensitization to Act d 1 is associated with a positive result in conventional diagnostic techniques, whereas kiwifruit seedsensitization does not increase the sensitivity of the diagnostic techniques evaluated (AU)

Objetivos: Determinar la rentabilidad diagnóstica de las técnicas in vitro e in vivo utilizadas en el diagnóstico de alergia al kiwi y estudiarla influencia de las proteínas alergénicas de las semillas en su sensibilidad.Métodos: Se seleccionaron 36 pacientes alérgicos a kiwi. Se les realizó prick test con cuatro extractos comerciales diferentes y prick-prickcon kiwi. Se determinó IgE específica mediante ImmunoCAP (extracto de kiwi), ELISA (Act d 1, Act d 2), las micromatrices ISAC y FABER eImmunoblotting de extracto de semilla de kiwi. Se realizó exposición oral simple ciego frente a semilla de kiwi en pacientes sensibilizadosa la semilla.Resultados: El prick-prick de kiwi fue la prueba in vivo con mayor rendimiento (sensibilidad 81,8%, especificidad 94,1%). El ImmunoCAPde extracto de kiwi mostró una sensibilidad similar a la global del ISAC y del FABER (63,9%, 59,5% y 58,3%, respectivamente). Act d 1fue el alérgeno mayoritario. Se encontró asociación entre los niveles de IgE específica frente a Act d 1 (ISAC) y el extracto de kiwi medianteImmunoCAP (p <0,000). La prueba cutánea positiva con semilla se asoció con mayor gravedad de síntomas frente a kiwi (p = 0,019),como marcador de enfermedad avanzada, pero no como sensibilización clínicamente relevante. La prueba de provocación con semillasfue negativa en los ocho pacientes provocados.Conclusiones: La sensibilización a Act d 1 se asocia con resultados positivos con las técnicas diagnósticas convencionales. La sensibilizaciónfrente a semillas no mejora el rendimiento de las técnicas evaluadas (AU)

In vitro expansion of human cardiac progenitor cells: exploring 'omics tools for characterization of cell-based allogeneic products.

Human cardiac stem/progenitor cells (hCPCs) have been shown to be capable to regenerate contractile myocardium. However, because of their relative low abundance in the heart, in vitro expansion of hCPC is mandatory to achieve necessary quantities for allogeneic or autologous cardiac regeneration therapy applications (10(6)-10(9) cells/patient). Up to now, cell number requirements of ongoing phase I/IIa trials have been fulfilled with production in static monolayer cultures. However, this manufacturing process poses critical limitations when moving to the following clinical phases where hundreds of patients will be enrolled. For this, increased process yield is required, while guaranteeing the quality of the cell-based products. In this work, we developed and validated a robust, scalable, and good manufacturing practice (GMP)-compatible bioprocess for the expansion of high-quality hCPC. We applied platforms extensively used by the biopharmaceutical industry, such as microcarrier technology and stirred systems, and assessed culture conditions' impact on hCPC's quality and potency, as required by regulatory agencies. Complementary analytical assays including gene expression microarrays and mass spectrometry-based approaches were explored to compare transcriptome, proteome, surface markers, and secretion profiles of hCPC cultured in static monolayers and in stirred microcarrier-based systems. Our results show that stirred microcarrier-based culture systems enabled achieving more than 3-fold increase in hCPC expansion, when compared with traditional static monolayers, while retaining cell's phenotype and similar "omics" profiles. These findings demonstrate that this change in the production process does not affect cell's identity and quality, with potential to be translated into a transversal production platform for clinical development of stem-cell therapies.

miR-300 mediates Bmi1 function and regulates differentiation in primitive cardiac progenitors.

B lymphoma Mo-MLV insertion region 1 (Bmi1) is a polycomb-family transcriptional factor critical for self-renewal in many adult stem cells and human neoplasia. We sought to identify microRNAs regulated by Bmi1 that could play a role in multipotent cardiac progenitor cell (CPC) decisions. We found that miR-300, a poorly characterized microRNA mapping in the Dlk1-Dio3 microRNA cluster, was positively regulated by Bmi1 in CPCs. Forced expression of miR-300 in CPCs promoted an improved stemness signature with a significant increase in Oct4 levels, a reduction in senescence progression and an enhanced proliferative status via p19 activation and inhibition of p16 accumulation. Endothelial and cardiogenic differentiation were clearly compromised by sustained miR-300 expression. Additionally, RNA and protein analysis revealed a significant reduction in key cardiac transcription factors, including Nkx2.5 and Tbx5. Collectively, these results suggest that some functions attributed to Bmi1 are due to induction of miR-300, which decreases the cardiogenic differentiation potential of multipotent CPCs in vitro and promotes self-renewal.

Human mesenchymal stem cell-replicative senescence and oxidative stress are closely linked to aneuploidy.

In most clinical trials, human mesenchymal stem cells (hMSCs) are expanded in vitro before implantation. The genetic stability of human stem cells is critical for their clinical use. However, the relationship between stem-cell expansion and genetic stability is poorly understood. Here, we demonstrate that within the normal expansion period, hMSC cultures show a high percentage of aneuploid cells that progressively increases until senescence. Despite this accumulation, we show that in a heterogeneous culture the senescence-prone hMSC subpopulation has a lower proliferation potential and a higher incidence of aneuploidy than the non-senescent subpopulation. We further show that senescence is linked to a novel transcriptional signature that includes a set of genes implicated in ploidy control. Overexpression of the telomerase catalytic subunit (human telomerase reverse transcriptase, hTERT) inhibited senescence, markedly reducing the levels of aneuploidy and preventing the dysregulation of ploidy-controlling genes. hMSC-replicative senescence was accompanied by an increase in oxygen consumption rate (OCR) and oxidative stress, but in long-term cultures that overexpress hTERT, these parameters were maintained at basal levels, comparable to unmodified hMSCs at initial passages. We therefore propose that hTERT contributes to genetic stability through its classical telomere maintenance function and also by reducing the levels of oxidative stress, possibly, by controlling mitochondrial physiology. Finally, we propose that aneuploidy is a relevant factor in the induction of senescence and should be assessed in hMSCs before their clinical use.

Comparative phenotypic and molecular characterization of porcine mesenchymal stem cells from different sources for translational studies in a large animal model.

Mesenchymal stem cells have demonstrated their potentiality for therapeutic use in treating diseases or repairing damaged tissues. However, in some cases, the results of clinical trials have been disappointing or have not worked out as well as hoped. These disappointing results can be attributed to an inadequate or insufficient preclinical study. For medical and surgical purposes, the similarities between the anatomy of pig and human make this animal an attractive preclinical model. In this sense, for mesenchymal stem cell-based therapy, it is strongly necessary to have well characterized animal-derived mesenchymal stem cell lines to validate preclinical effectiveness of these cells. In this work, porcine mesenchymal stem cells (pMSCs) were isolated from bone marrow, adipose tissue and peripheral blood and compared in terms of differentiation potential, cell surface markers and gene expression. Our results demonstrated that the isolation and in vitro expansion protocols were feasible and effective. The data presented in this work are relevant because they provide an extensive phenotypic characterization; genetic study and differentiation behavior of the most commonly used stem cell lines for clinical practices. These pMSCs are widely available to scientists and could be a valuable tool to evaluate the safety and efficacy of adoptively transferred cells.

Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis.

Expansion of human stem cells before cell therapy is typically performed at 20% O(2). Growth in these pro-oxidative conditions can lead to oxidative stress and genetic instability. Here, we demonstrate that culture of human mesenchymal stem cells at lower, physiological O(2) concentrations significantly increases lifespan, limiting oxidative stress, DNA damage, telomere shortening and chromosomal aberrations. Our gene expression and bioenergetic data strongly suggest that growth at reduced oxygen tensions favors a natural metabolic state of increased glycolysis and reduced oxidative phosphorylation. We propose that this balance is disturbed at 20% O(2), resulting in abnormally increased levels of oxidative stress. These observations indicate that bioenergetic pathways are intertwined with the control of lifespan and decisively influence the genetic stability of human primary stem cells. We conclude that stem cells for human therapy should be grown under low oxygen conditions to increase biosafety.

miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells.

In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses revealed that miR-335 is significantly downregulated upon hMSC differentiation. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, a positive regulator of MSC self-renewal, and downregulated by interferon-γ (IFN-γ), a pro-inflammatory cytokine that has an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3'UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 downregulation is critical for the acquisition of reparative MSC phenotypes.

'Loss of pigment epithelium-derived factor enables migration, invasion and metastatic spread of human melanoma'.

Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that displays broad anti-tumor activity based on dual targeting of the tumor microenvironment (anti-angiogenic action) and the tumor cells (direct anti-tumor action). Here, we show that PEDF expression is high in melanocytes, but it is lost during malignant progression of human melanoma. Using a high-throughput analysis of the data from microarray studies of molecular profiling of human melanoma, we found that PEDF expression is lost in highly invasive melanomas. In paired cell lines established from the same lesion but representing the high and low extremes of malignant potential, abundant PEDF expression was restricted to the poorly aggressive counterparts. We used RNA interference to directly address the functional consequences of PEDF silencing. PEDF knockdown in poorly aggressive melanoma cell lines augmented migration, invasion and vasculogenic mimicry, which translated into an increased in vivo metastatic potential. PEDF interference also significantly enhanced the migratory and invasive capability of normal melanocytes and moderately increased their proliferative potential. Our results show that loss of PEDF enables melanoma cells to acquire an invasive phenotype and, therefore, modulation of this multifunctional factor could be critical for the malignant progression of human melanoma.

Inhibition of tumor growth in vivo by in situ secretion of bispecific anti-CEA x anti-CD3 diabodies from lentivirally transduced human lymphocytes.

Infiltrating T lymphocytes are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of defective T-cell activation events. Recently, we described a strategy for the tumor-specific polyclonal activation of tumor-resident T lymphocytes based on the in situ production of recombinant bispecific antibodies (bsAbs) by transfected nonhematological cell lines. Here, we have constructed a novel HIV-1-based lentiviral vector for efficient gene transduction into various human hematopoietic cell types. Several myelomonocytic and lymphocytic cell lines secreted the anti-carcinoembryonic antigen (CEA) x anti-CD3 diabody in a functionally active form with CD3(+) T-cell lines being the most efficient secretors. Furthermore, primary human peripheral blood lymphocytes (PBLs) were also efficiently transduced and secreted high levels of functional diabody. Importantly gene-modified PBLs significantly reduced in vivo tumor growth rates in xenograft studies. These results demonstrate, for the first time, the utility of lentiviral vectors for sustained expression of recombinant bsAbs in human T lymphocytes. Such T lymphocytes, transduced ex vivo to secrete the activating diabody in autocrine fashion, may provide a promising route for a gene therapy strategy for solid human tumors.

Transplanted long-term cultured pre-BI cells expressing calpastatin are resistant to B cell receptor-induced apoptosis.

Long-term cultured pre-B cells are able to differentiate into immunoglobulin (Ig)M-positive B cells (IgM(+) cells) when transplanted into severe combined immunodeficient (SCID) mice. Based on previous studies, here we report the development of a reconstitution assay in nonobese diabetic/SCID (NOD/SCID) mice using pre-B cells, which allows us to study the role of calpains (calcium-activated endopeptidases) during B cell development as well as in B cell clonal deletion. Using this model, we show that calpastatin (the natural inhibitor of calpains) inhibits B cell receptor-induced apoptosis in IgM(+) cells derived from transplanted mice. We thus hypothesize an important function for calpain in sculpting the B cell repertoire.

Segregation of leading-edge and uropod components into specific lipid rafts during T cell polarization.

Redistribution of specialized molecules in migrating cells develops asymmetry between two opposite cell poles, the leading edge and the uropod. We show that acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of ganglioside GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively. This segregation to each cell pole parallels the specific redistribution of membrane proteins associated to each raft subfraction. Our data suggest that raft partitioning is a major determinant for protein redistribution in polarized T cells, as ectopic expression of raft-associated proteins results in their asymmetric redistribution, whereas non-raft-partitioned mutants of these proteins are distributed homogeneously in the polarized cell membrane. Both acquisition of a migratory phenotype and SDF-1alpha-induced chemotaxis are cholesterol depletion-sensitive. Finally, GM3 and GM1 raft redistribution requires an intact actin cytoskeleton, but is insensitive to microtubule disruption. We propose that membrane protein segregation not only between raft and nonraft domains but also between distinct raft subdomains may be an organizational principle that mediates redistribution of specialized molecules needed for T cell migration.

A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts.

Leptin deficiency produces a phenotype of obesity, diabetes, and infertility in the ob/ob mouse. In humans, leptin deficiency occurs in some cases of congenital obesity and in lipodystrophic disorders characterized by reduced adipose tissue and insulin resistance. Cutaneous gene therapy is considered an attractive potential method to correct circulating protein deficiencies, since gene-transferred human keratinocytes can produce and secrete gene products with systemic action. However, no studies showing correction of a systemic defect have been reported. We report the successful correction of leptin deficiency using cutaneous gene therapy in the ob/ob mouse model. As a feasibility approach, skin explants from transgenic mice overexpressing leptin were grafted on immunodeficient ob/ob mice. One month later, recipient mice reached body weight values of lean animals. Other biochemical and clinical parameters were also normalized. In a second human gene therapy approach, a retroviral vector encoding both leptin and EGFP cDNAs was used to transduce HK and, epithelial grafts enriched in high leptin-producing HK were transplanted to immunosuppressed ob/ob mice. HK-derived leptin induced body weight reduction after a drop in blood glucose and food intake. Leptin replacement through genetically engineered HK grafts provides a valuable therapeutic alternative for permanent treatment of human leptin deficiency conditions.

A role for chemokine receptor transactivation in growth factor signaling.

Complex cell responses require the integration of signals delivered through different pathways. We show that insulin-like growth factor (IGF)-I induces specific transactivation of the Gi-coupled chemokine receptor CCR5, triggering its tyrosine phosphorylation and Galpha recruitment. This transactivation occurs via a mechanism involving transcriptional upregulation and secretion of RANTES, the natural CCR5 ligand. CCR5 transactivation is an essential downstream signal in IGF-I-induced cell chemotaxis, as abrogation of CCR5 function with a transdominant-negative KDELccr5A32 mutant abolishes IGF-I-induced migration. The relevance of this transactivation pathway was shown in vivo, as KDELccr5A32 overexpression prevents invasion by highly metastatic tumor cells; conversely, RANTES overexpression confers built-in invasive capacity on a non-invasive tumor cell line. Our results suggest that this extracellular growth factor-chemokine network represents a general mechanism connecting tumorigenesis and inflammation.

New insights into host factor requirements for prokaryotic beta-recombinase-mediated reactions in mammalian cells.

The prokaryotic beta-recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in mammalian cells, both on episomic and chromatin-integrated substrates. Using a specific recombination activated gene expression system, we report the site-specific recombination activity of an enhanced green fluorescent protein (EGFP) fused version of beta-recombinase (beta-EGFP). This allows expression of active beta-recombinase detectable in vivo and in fixed cells by fluorescence microscopy. In addition, cellular viability is compatible with a substantial level of expression of the beta-EGFP protein. Using fluorescence-activated cell sorting, we have been able to enrich cell populations expressing this fusion protein. Application of this strategy has allowed us to study in more depth the host factor requirements for this system. Previous work showed that eukaryotic HMG1 protein was necessary and sufficient to help beta-recombinase activity in vitro. The influence of ectopic expression of HMG1 protein in the recombination process has been analyzed, indicating that HMG1 overexpression does not lead to a significant increase on the efficiency of beta-recombinase-mediated recombination both on episomal substrates and chromatin-associated targets. In addition, beta-recombinase-mediated recombination has been demonstrated in HMG1 deficient cells at the same levels as in wild type cells. These data demonstrate the existence of cellular factors different from HMG-1 that can act as helpers for beta-recombinase activity in the eukaryotic environment.

DNA polymerase mu, a candidate hypermutase?

A novel DNA polymerase (Pol mu) has been recently identified in human cells. The amino-acid sequence of Pol mu is 42% identical to that of terminal deoxynucleotidyl transferase (TdT), a DNA-independent DNA polymerase that contributes to antigen-receptor diversity. In this paper we review the evidence supporting the role of Pol mu in somatic hypermutation of immunoglobulin genes, a T-dependent process that selectively occurs at germinal centres: (i) preferential expression in secondary lymphoid organs; (ii) expression associated to developing germinal centres; and (iii) very low base discrimination during DNA-dependent DNA polymerization by Pol mu, a mutator phenotype enormously accentuated by the presence of activating Mn2+ ions. Moreover, its similarity to TdT, together with extrapolation to the crystal structure of DNA polymerase beta complexed (Pol beta) with DNA, allows us to discuss the structural basis for the unprecedented error proneness of Pol mu, and to predict that Pol mu is structurally well suited to participate also in DNA end-filling steps occurring both during V(D)J recombination and repair of DNA double-strand breaks that are processed by non-homologous end-joining.

DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis.

A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19. Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X. Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis.