Usefulness of severity scales for cardiogenic shock in-hospital mortality. Proposal for a new prognostic model.

Cardiogenic shock (CS) is a condition comprising multiple etiologies, which associates high mortality rates. Some scoring systems have been shown to be good predictors of hospital mortality in patients admitted to Critical Care Units (CCU). The main objective of this study is to analyze their usefulness and validity in a cohort of CS patients. METHODS: Observational unicentric study of a cohort of CS patients. SOFA, SAPS II and APACHE II scores were calculated in the first 24 h of CCU admission. RESULTS: 130 patients with CS were included. SOFA, SAPS II and APACHE II scores revealed good discrimination for hospital mortality: (AUC) ROC values (AUC: 0.711, 0.752 and 0.742 respectively; P = .6). Calibration, estimated by the Hosmer-Lemeshow test, was adequate in all cases. Acute coronary syndrome, lactate serum values, SAPS II score and vasoactive inotropic score (VIS) were found to be independent predictors for mortality, upon ICU admission. With these variables, a specific prognostic indicator was developed (SAPS-2-LIVE), which improved predictive capability for mortality in our series (AUC) ROC, 0.825 (95% CI 0.752-0.89). CONCLUSION: In this contemporary CS cohort, the aforementioned scores have been shown to have good predictive ability for hospital mortality. These findings could contribute to a more accurate risk stratification in CS.

Late Onset Graft Plasmacytoma-Like PTLD Presenting as Acute Hyperglycemia in a Kidney-Pancreas Transplant Recipient.

Allograft infiltration has been described in up to 20% of all patients with posttransplant lymphoproliferative disorder (PTLD), most representing EBV-positive B-cell lymphomas. Plasma cells are often observed in humoral rejection biopsies, but graft infiltration by plasmacytoma-like PTLD is rare. We report the case of a 54-year-old simultaneous pancreas-kidney transplant recipient (immunosuppression: OKT3, methylprednisolone, cyclosporine, and azathioprine), diagnosed with an IgG-kappa monoclonal gammopathy of undetermined significance eighteen years after transplant. Nine months later, pancreas allograft biopsy performed due to new-onset hyperglycemia (HgA1C 8.6%, C-peptide 6.15ng/mL and anti-GAD 0.9UI/mL) revealed a monotypic plasma cell infiltrate, CD19, CD79a, CD138 positive, with IgG-kappa light chain restriction, and EBV negative. PET-scan FDG uptake was limited to pancreas allograft. Tumor origin could not be established (using DNA microsatellite analysis). Despite treatment with bortezomib and dexamethasone, patient eventually died one month later. This is the first report of a late onset extramedullary plasmacytoma involving a pancreas allograft.

Clinical impact of the subclonal architecture and mutational complexity in chronic lymphocytic leukemia.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

Laser technique for the fabrication of blood vessels-like models for preclinical studies of pathologies under flow conditions.

In this work a method for fabricating functionalized preclinical devices is presented. The manufacturing process combines a laser indirect writing technique to fabricate a soda-lime glass master and soft-lithography methods to obtain the final structure in polydimethylsiloxane (PDMS). The roughness of the device is modified in a controlled manner by applying a post-thermal treatment to the master, and thus devices with different roughness values are created. The PDMS devices are fully covered with human umbilical vein cells in a two-step process. In order to determine the most suitable device to perform bioassays, the cell attachment to the channel is evaluated with regards to the walls roughness when flow experiments are carried out.

A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.

Prospective identification of patients with chronic lymphocytic leukemia (CLL) destined to progress would greatly facilitate their clinical management. Recently, whole-genome DNA methylation analyses identified three clinicobiologic CLL subgroups with an epigenetic signature related to different normal B-cell counterparts. Here, we developed a clinically applicable method to identify these subgroups and to study their clinical relevance. Using a support vector machine approach, we built a prediction model using five epigenetic biomarkers that was able to classify CLL patients accurately into the three subgroups, namely naive B-cell-like, intermediate and memory B-cell-like CLL. DNA methylation was quantified by highly reproducible bisulfite pyrosequencing assays in two independent CLL series. In the initial series (n=211), the three subgroups showed differential levels of IGHV (immunoglobulin heavy-chain locus) mutation (P<0.001) and VH usage (P<0.03), as well as different clinical features and outcome in terms of time to first treatment (TTT) and overall survival (P<0.001). A multivariate Cox model showed that epigenetic classification was the strongest predictor of TTT (P<0.001) along with Binet stage (P<0.001). These findings were corroborated in a validation series (n=97). In this study, we developed a simple and robust method using epigenetic biomarkers to categorize CLLs into three subgroups with different clinicobiologic features and outcome.

The γ-secretase inhibitor PF-03084014 combined with fludarabine antagonizes migration, invasion and angiogenesis in NOTCH1-mutated CLL cells.

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), especially for the poor prognostic subgroup of NOTCH1-mutated patients. Here, we report that the γ-secretase inhibitor PF-03084014 inhibits the constitutive Notch activation and induces selective apoptosis in CLL cells carrying NOTCH1 mutations. Combination of PF-03084014 with fludarabine has a synergistic antileukemic effect in primary NOTCH1-mutated CLL cells, even in the presence of the protective stroma. At transcriptional level, PF-03084014 plus fludarabine treatment induces the upregulation of the proapoptotic gene HRK and the downmodulation of MMP9, IL32 and RAC2 genes that are related to invasion and chemotaxis. PF-03084014 also overcomes fludarabine-mediated activation of nuclear factor-κB signaling. Moreover, this combination impairs angiogenesis and CXCL12-induced responses in NOTCH1-mutated CLL cells, in particular those related to tumoral migration and invasion. Importantly, all these collaborative effects are specific for NOTCH1 mutation and do not occur in unmutated cases. In conclusion, we provide evidence that Notch is a therapeutic target in CLL cases with NOTCH1-activating mutations, supporting the use of Notch pathway inhibitors in combination with chemotherapy as a promising approach for the treatment of these high-risk CLL patients.

NOTCH1 mutations identify a genetic subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor outcome.

NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.

High resolution melting analysis for the identification of novel mutations in DKC1 and TERT genes in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone-marrow failure syndrome with high clinical heterogeneity. Cells derived from DC patients present short telomeres at early ages, as a result of mutations in genes encoding components of the telomerase complex (DKC1, TERC, TERT, NHP2 and NOP10), or the shelterin complex (TINF2). However, mutations have been identified only in around 50% of the cases, indicating that other genes could be involved in the development of this disease. Indeed, mutations in TCBA1 or chromosome segment C16orf57 have been described recently. We have used HRM technology to perform genetic analysis in the above mentioned genes, in Spanish patients showing both, some clinical features of DC and short telomeres. The mutations have been identified by PCR amplification of DC genes followed by high resolution melting (HRM) and direct DNA sequencing analysis. We have identified seven new families with DC, three with X-linked DC and four with autosomal dominant DC, in which we have found two novel mutations in DKC1 (p.His68Arg and p.Lys390del) and four novel mutations in TERT gene (p.Pro530Leu, p.Arg698Trp, p.Arg971His and p.Arg698Gln). The results show that the use of HRM analysis enables a rapid and inexpensive identification of mutations in dyskeratosis congenita associated genes.

Effective GDNF brain delivery using microspheres--a promising strategy for Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) has shown promise in the treatment of neurodegenerative disorders of basal ganglia origin such us Parkinson's disease (PD). In this study, we investigated the neurorestorative effect of controlled GDNF delivery using biodegradable microspheres in an animal model with partial dopaminergic lesion. Microspheres were loaded with N-glycosylated recombinant GDNF and prepared using the Total Recirculation One-Machine System (TROMS). GDNF-loaded microparticles were unilaterally injected into the rat striatum by stereotaxic surgery two weeks after a unilateral partial 6-OHDA nigrostriatal lesion. Animals were tested for amphetamine-induced rotational asymmetry at different times and were sacrificed two months after microsphere implantation for immunohistochemical analysis. The putative presence of serum IgG antibodies against rat glycosylated GDNF was analyzed for addressing safety issues. The results demonstrated that GDNF-loaded microspheres, improved the rotational behavior induced by amphetamine of the GDNF-treated animals together with an increase in the density of TH positive fibers at the striatal level. The developed GDNF-loaded microparticles proved to be suitable to release biologically active GDNF over up to 5 weeks in vivo. Furthermore, none of the animals developed antibodies against GDNF demonstrating the safety of glycosylated GDNF use.

Sustained release of bioactive glycosylated glial cell-line derived neurotrophic factor from biodegradable polymeric microspheres.

Glial cell-line derived neurotrophic factor (GDNF), a potent neurotrophic factor for dopaminergic neurons, appeared as a promising candidate for treating Parkinson's disease. GDNF microencapsulation could ensure protection against degradation due to the fragile nature of the protein. Poly(lactide-co-glycolide) (PLGA) microparticles loaded with recombinant glycosylated GDNF obtained in a mammalian cell line were prepared by TROMS, a semi-industrial technique capable of encapsulating fragile molecules maintaining their native properties. The effects of several parameters as PLGA copolymer type, PEG 400 quantity co-encapsulated with GDNF or drug loading, on the properties of the particles were investigated. Microparticles showed a mean diameter between 8 and 30 microm, compatible with their stereotaxic implantation. The drug entrapment efficiency ranged from 50.6% to 100% depending on the microsphere composition. GDNF was better encapsulated using hydrophilic polymers with high molecular weight such as RG 503H. In vitro drug release was influenced by the polymer type as well as by the amount of PEG 400 co-encapsulated with GDNF. Microparticles prepared using PLGA RG 503H released 67% of the total protein content within 40 days. Moreover, very low concentrations of poly(vinyl alcohol) were detected after microparticles washing and freeze-drying. Finally, a PC-12 bioassay demonstrated that the in vitro GDNF released was bioactive.

Purification of bioactive glycosylated recombinant glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) neuroprotective effect on dopaminergic neurons has been described in vitro and in vivo, turning up as a promising drug for the treatment of Parkinson's disease. Unglycosylated bacteria-obtained GDNF has already been successfully delivered for a long period of time through an infusion pump directly to the putamen of Parkinsonian patients. Nevertheless, improved distribution and safety issues need to be solved and alternative strategies to long-term delivery seem necessary. The use of glycosylated GDNF could eliminate some safety concerns regarding the presence of antibodies against exogenous unglycosylated GDNF used for the treatment. Therefore, we have chosen a mammalian expression system as a source of glycosylated GDNF. In the present work, we describe the purification of recombinant rat GDNF from the culture media of baby hamster kidney (BHK) cells through several purification steps. Highly pure N-glycosylated recombinant GDNF has been obtained similar to the endogenous protein. Furthermore, the purified protein is biologically active when tested its ability to induce PC12 neurite outgrowth.

[Spanish adaptation of the disease-specific questionnaire MSQOL-54 in multiple sclerosis patients]. / Adaptación al español del cuestionario específico MSQOL-54 para pacientes con esclerosis múltiple.

INTRODUCTION: The Multiple Sclerosis Quality of Life 54 (MSQOL-54) is a health-related quality of life specific questionnaire for multiple sclerosis (MS) patients. The objective of this study was to develop the Spanish version of the MSQOL-54 and to obtain a conceptually equivalent version to the original one for its use in patients with MS in the first phase of the project. METHODS: A transcultural adaptation procedure was designed according to the following phases: a) two independent translations made by bilingual native Spanish speaking translators (forward translation); b) a revision of the items by an expert panel; c) a back translation by a bilingual native English speaking person; d) comparison with the original version (expert panel and advise by the original authors), and e) cognitive debriefing (interviews with subjects with MS) to test the comprehension and feasibility of the instrument. RESULTS: Ten interviews were carried out with 5 men and 5 women with MS, aged 21 to 54 years, with different education levels and EDSS scores ranging from 1,0 to 8,0. Most of the patients found the questionnaire easy to fill out and the understanding favorable. Only one item (item 51) was modified after the cognitive debriefing to improve its comprehension. Finally, a final pretest version was obtained. CONCLUSIONS: The procedure carried out maximizes the conceptual equivalence between the original MSQOL-54 and the translated version and shows that the Spanish pre-test version is comprehensible and its administration feasible in patients with MS. The psychometric properties must be evaluated in the next phase of the project.

Consequences of unilateral nigrostriatal denervation on the thalamostriatal pathway in rats.

The position of the caudal intralaminar nuclei within basal ganglia circuitry has largely been neglected in most studies dealing with basal ganglia function. During the past few years, there has been a growing body of evidence suggesting that the thalamic parafascicular nucleus in rodents (PF) exerts a multifaceted modulation of basal ganglia nuclei, at different levels. Our aim was to study the activity of the thalamostriatal pathway in rats with unilateral dopaminergic depletion. The experimental approach comprised first unilateral delivery of 6-OHDA in the medial forebrain bundle. Thirty days post-lesioning, animals showing a clear asymmetry were then subjected to bilateral injection of Fluoro-Gold (FG) within the striatum. Subsequently, expression of the mRNA encoding the vesicular glutamate transporter 2 (vGLUT2) was detected within thalamostriatal-projecting neurons (FG-labeled) by in situ hybridization and the results were confirmed by laser-guided capture microdissection microscopy followed by real-time PCR. The data showed that there was a marked neuronal loss restricted to PF neurons projecting to the dopamine-depleted striatum. Moreover, PF neurons innervating the dopamine-depleted striatum were intensely hyperactive. These neurons showed a marked increase on the expression of vGLUT2 mRNA as well as for the mRNA encoding the subunit I of cytochrome oxidase as compared with those neurons projecting to the striatum with normal dopamine content. Thus, the selective neurodegeneration of PF neurons innervating the striatum together with the increased activity of the thalamostriatal pathway coexist after nigrostriatal denervation.

G-CSF increases the number of peripheral blood dendritic cells CD16+ and modifies the expression of the costimulatory molecule CD86+.

Dendritic cells (DC) play a key role in initiating immune reactions after allogeneic stem cell transplantation. The two main peripheral blood DC populations are myeloid (DC1) and lymphoplasmacytoid (DC2). A new subset of myeloid DC, expressing CD16, has been identified. We analyzed the number and CD86 expression of DC subsets in peripheral blood of 18 healthy donors, before and after granulocyte colony-stimulating factor (G-CSF) and in the inoculum of allogeneic peripheral blood transplants (allo-PBT; n=100) and allogeneic bone marrow transplants (allo-BMT; n=22). Granulocyte colony-stimulating factor administration increased the median number of DC1 (P=0.0007), of DC2 (P<0.0001) and of DC CD16+ (P=0.0001). Granulocyte colony-stimulating factor administration was also associated with a significant decrease of CD86 expression on DC1 (P=0.0003) and with a trend for an increase on DC CD16+ (P=0.07). Recipients of allo-PBT received similar quantities of DC1 and higher doses of DC2 and DC CD16+ than recipients of allo-BMT (P=0.5; P=0.0001; P<0.0001, respectively). Granulocyte colony-stimulating factor modifies the number of DC in peripheral blood and the expression of the costimulatory molecule CD86. This resulted in a different composition of DC2 and especially of DC CD16+ in the harvests, which might explain some of the differences observed in allogeneic reactions after allo-PBT with respect to allo-BMT.

[Reliability and validity of the Spanish version of the health and quality-of-life questionnaire, the Vecú et Santé Perçue de l'Adolescent (VSP-A)]. / Fiabilidad y validez del cuestionario de salud y calidad de vida para adolescentes Vecú et Santé Perçue de l'Adolescent (VSP-A).

OBJECTIVE: To assess the psychometric properties of the Spanish version of the Vecú et Santé Perçue de l'Adolescent (VSP-A) in terms of reliability and validity. DESIGN: Cross-sectional study. SETTING: Pilot study parallel to the European Kidscreen project. Two secondary schools, one public and one private, were selected for their suitability in Barcelona and Gerona (Catalonia, Spain). The sample unit was the classroom. PARTICIPANTS: A sample of 354 adolescents aged 12 to 18 years old was selected. MAIN MEASUREMENTS: The Spanish VSP-A questionnaire was administered, and again a week later to check its test-retest stability. The KINDL questionnaire was administered in parallel. RESULTS: The response rate was 82% (n=291). The Spanish version of the VSP-A showed good internal consistency and acceptable test-retest reliability (Cronbach's alpha: 0.69-0.92, intraclass correlation coefficient [ICC]: 0.69-0.74) in most domains. Domains measuring a similar concept in the VSP-A and KINDL had closer correlation coefficients than those measuring different constructs (P<.05), which demonstrates its convergent validity. Girls had worse self-perceived health than boys (lower vitality, physical and emotional well-being and self-esteem, and a lower general score on the VSP-A; p<.01). These differences were more obvious in older teens (16-18 years old). CONCLUSIONS: The Spanish VSP-A showed good psychometric properties and results were consistent with the original French version. The results mean we have an adequate HRQL instrument for teens and for use in primary care and public health.

[Neuroprotective and neurorestorative therapies for the treatment of Parkinson's disease]. / Terapias neuroprotectoras y neurorestauradoras en el tratamiento de la enfermedad de Parkinson.

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Current therapies are symptomatic and, although these therapies are efficacious during the early stages of the disease, they present important side effects when they are used for a long time. The ideal therapy would be the one that would slow down or stop the progression of the disease. This can be achieved, for instance, with neuroprotective and neurorestorative therapies. Among them, cell therapy and therapy with trophic factors such as glial cell line derived neurotrophic factor (GDNF) are the most challenging and promising ones for the scientific community. Although the use of GDNF as a treatment for Parkinson s disease was proposed several years ago, it is necessary to develop alternative strategies to deliver GDNF appropriately to concrete areas of the brain. Here, the use of microspheres as the most suitable tool for the administration of this neurotrophic factor is discussed.

[The Spanish version of the German health-related quality of life questionnaire for children and adolescents: the Kindl]. / Versión española del cuestionario alemán de calidad de vida relacionada con la salud en población infantil y de adolescentes: el Kindl.

OBJECTIVES: To obtain a Spanish version of the Kindl semantically and culturally equivalent to the original German version and to test its psychometric properties. MATERIAL AND METHODS: The methodology used in the adaptation process was based on the forward-backward translation method. To assess the psychometric properties of the Spanish Kindl, the pilot test of the project "Screening for and promotion of HRQL in children and adolescents: a European Public Health perspective (Kidscreen)" it was include in. A classroom was selected for each educational level (8-16 years old) from three schools in Gerona and Barcelona. The Spanish Kindl was administered twice, one week apart. Internal consistency was assessed by computing Cronbach alpha and test-retest stability was assessed using intraclass correlation coefficients (ICC). Analysis of variance was performed according to age, sex, type of school, and self-perceived health status. RESULTS: Half of the items (12/24) required minor changes during the adaptation process. The response rate was 91 % (n = 447). Internal consistency was acceptable for most domains (alpha range = 0.40-0.88), as was test-retest stability (ICC range = 0.52-0.80). Girls and older teenagers scored worse in most domains (p < 0.01). No differences were found by type of school. CONCLUSIONS: The Spanish version of the Kindl showed adequate reliability and validity coefficients and represents a new HRQL instrument that can be applied in pediatric clinical practice and public health.

Smoldering multiple myeloma: natural history and recognition of an evolving type.

Patients with smoldering multiple myeloma (SMM) meet the diagnostic criteria of multiple myeloma (MM) but are asymptomatic. Between January 1978 and July 2001, 53 patients (median age 63 years) were diagnosed with SMM. The median serum M-protein and proportion of bone marrow plasma cells were 36 g/l and 27% respectively. Two subsets of SMM were identified: (i) evolving SMM (n = 22), characterized by a progressive increase in serum M-protein, a previously recognized monoclonal gammopathy of undetermined significance (MGUS) and a significant higher proportion of IgA type and (ii) non-evolving SMM (n = 26) with stable M-protein that abruptly increases when symptomatic MM develops. Thirty-four patients developed symptomatic MM. The median time to progression in the overall series was 3.2 years and the only feature associated with a shorter time to progression was the evolving versus non-evolving type (1.3 vs. 3.9 years respectively, P = 0.007). The pattern of progression consisted of anaemia, lytic bone lesions or both, without renal failure, hypercalcaemia or extramedullary plasmacytomas. Fifty-seven per cent of patients that required chemotherapy showed no or minimal response. The median survival from diagnosis and from progression was 8.2 and 3.5 years respectively.

Functionality of enterococci in meat products.

The presence of enterococci in meat fermentation is a constant as reported in the literature. Despite the concern about pathogenicity of enterococci, recent studies point out that food and meat enterococci, especially Enterococcus faecium have a much lower pathogenicity potential than clinical strains. Enterococci possess a competitive advantage over other microbiota in meat fermentations, and many enterococci isolated from sausages have the ability to produce enterocins harbouring antimicrobial activity against pathogens and spoilage microorganisms of meat concern. The application of enterocins producing enterococci or their purified metabolites, as extra hurdles for preservation in sausage fermentation and in sliced-vacuum packed cooked meat products can be beneficial, preventing the outgrowth of Listeria monocytogenes and slime-producing lactic acid bacteria. Enterocins and bacteriocinogenic enterococci hold considerable promise as alternatives to traditional chemical preservatives and they could be exploited for the control of emergent pathogens in meat products. Their inhibitory effect can be increased when used in conjunction with particular physical and chemical processes, but current regulation is hampering the application of purified bacteriocins.