Implicación del CD5 en la leucemia linfoide crónica de célula B (LLC-B)

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Facultad de Medicina. Departamento de Medicina. Fecha de lectura: 10 de Enero de 200

Evolución de la resistencia antibiótica de Actinobacillus pleuropneumoniae: estudio retrospectivo durante un periodo de tres años

Actinobacillus pleuropneumoniae (App) es el agente causal de la pleuroneumonía porcina, una enfermedad infecciosa del tracto respiratorio que causa considerables pérdidas económicas a la industria porcina a nivel mundial. En el presente trabajo se ha estudiado la evolución de la resistencia antibiótica de App entre 2018 y 2020 a partir de muestras porcinas respiratorias de diferentes regiones de España. Para ello se han realizado pruebas de sensibilidad antibiótica, como la técnica de Kirby-Bauer, Concentración Mínima Inhibitoria por microdilución y E-test, que nos permiten determinar la resistencia o sensibilidad de la bacteria in vitro a los diferentes antibióticos testados. Además, se ha llevado a cabo un ensayo para evaluar la relación entre la producción de biofilm in vitro por parte de App y la resistencia antibiótica, ya que in vivo se ha demostrado que incrementan la resistencia a los antibióticos e interfieren con los mecanismos de defensa inmunológica del hospedador.Los resultados no mostraron diferencias significativas de sensibilidad antibiótica respecto a la situación geográfica y la edad, pero sí a lo largo del tiempo para determinados antibióticos. En general, App muestra alta sensibilidad a anfenicoles, betalactámicos de la clase cefalosporina, macrólidos, pleuromutilinas, quinolonas y sulfonamidas. Por el contrario, presenta sensibilidad reducida a betalactámicos de la clase penicilina, aminoglucósidos y tetraciclinas. Asimismo, no se detectó asociación entre la producción de biofilm y la resistencia antibiótica in vitro.En definitiva, a día de hoy las pruebas de sensibilidad antibiótica son imprescindibles para seleccionar una terapia antimicrobiana efectiva y disminuir el riesgo de desarrollo de resistencias antibióticas. También es necesario evaluar la formación de biofilm para el tratamiento de infecciones en las que esté implicado, ya que puede afectar a la efectividad del mismo.<br /

Importancia de rotavirus tipo A en conejo de cebo: estudio comparativo entre animales con procesos entéricos y animales sanos.

Las enfermedades entéricas causan graves pérdidas económicas en cunicultura debido a la mortalidad, disminución del crecimiento y empeoramiento del índice de conversión. El objetivo de este estudio fue valorar la importancia de rotavirus tipo A dentro del diagnóstico diferencial de diarreas en conejos de cebo y su posible relación con otros agentes. Para ello, se analizaron 90 casos de conejos de cebo (entre 35 y 55 días de vida) con sintomatología digestiva y 40 casos sin aparente sintomatología digestiva. Se realizó el cultivo microbiológico de las muestras; se detectó rotavirus tipo A y gen eae de Escherichia coli mediante PCR a tiempo real (qPCR). Eimeria spp. fue detectada mediante análisis coprológico y se valoró la presencia de Clostridium spiroforme y perfringens mediante tinción de Gram a partir de improntas de digestivo. El 57,8% de los casos de animales enfermos fueron positivos a rotavirus frente al 25% de los animales sanos (p<0,01). La presencia de un sólo agente estudiado no superó el 2,2% de los casos enfermos, evidenciando el carácter multifactorial de los procesos entéricos. La presencia de rotavirus tipo A en animales enfermos conllevó un aumento significativo de la proporción de otros agentes etiológicos (Eimeria spp. y Escherichia coli enteropatógeno). En conclusión, rotavirus tipo A es un agente implicado en los procesos entéricos de los conejos de cebo siendo necesario incluir su análisis en el diagnóstico diferencial

Fludarabine inhibits KV1.3 currents in human B lymphocytes

Fludarabine (F-ara-A) is a purine analog commonly used in the treatment of indolent B cell malignancies that interferes with different aspects of DNA and RNA synthesis. KV1.3 K+ channels are membrane proteins involved in the maintenance of K+ homeostasis and the resting potential of the cell, thus controlling signaling events, proliferation and apoptosis in lymphocytes. Here we show that F-ara-A inhibits KV currents in human B lymphocytes. Our data indicate that KV1.3 is expressed in both BL2 and Dana B cell lines, although total KV1.3 levels were higher in BL2 than in Dana cells. However, KV currents in the plasma membrane were similar in both cell lines and were abrogated by the specific KV1.3 channel inhibitor PAP-1, indicating that KV1.3 accounts for most of the KV currents in these cell lines. F-ara-A, at a concentration (3.5 μM) similar to that achieved in the plasma of fludarabine phosphate-treated patients (3 μM), inhibited KV1.3 currents by 61 ± 6.3% and 52.3 ± 6.3% in BL2 and Dana B cells, respectively. The inhibitory effect of F-ara-A was concentration-dependent and showed an IC50 value of 0.36 ± 0.04 μM and a nH value of 1.07 ± 0.15 in BL2 cells and 0.34 ± 0.13 μM (IC50) and 0.77 ± 0.11 (nH) in Dana cells. F-ara-A inhibition of plasma membrane KV1.3 was observed irrespective of its cytotoxic effect on the cells, BL2 cells being sensitive and Dana cells resistant to F-ara-A cytotoxicity. Interestingly, PAP-1, at concentrations as high as 10 μM, did not affect the viability of BL2 and Dana cells, indicating that blockage of KV1.3 in these cells is not toxic. Finally, F-ara-A had no effect on ectopically expressed KV1.3 channels, suggesting an indirect mechanism of current inhibition. In summary, our results describe the inhibitory effect of F-ara-A on the activity of KV1.3 channel. Although KV1.3 inhibition is not sufficient to induce cell death, further research is needed to determine whether it might still contribute to F-ara-A cytotoxicity in sensitive cells or be accountable for some of the clinical side effects of the drug.This study was supported by MINECO (SAF2013-45800-R, SAF2016-75021-R, RD12/0042/0019, CB/11/00222) and ISCIII (PI12/01135 and PI16/00895). The cost of this publication was paid in part by funds from the European Fund for Economic and Regional Development (FEDER). TG is supported by the Ramón y Cajal Program.Peer reviewedPeer Reviewe

Diversity of Streptococcus equi subsp. zooepidemicus strains isolated from the Spanish sheep and goat population and the identification, function and prevalence of a novel arbutin utilisation system

This work was supported by the Animal Health Trust.The zoonotic bacterium Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a diverse, opportunistic pathogen that can cause mastitis in dairy sheep and goats. We used multilocus sequence typing (MLST) to define the genetic diversity of 60 isolates of S. zooepidemicus, which were recovered from sheep and goats in Spain between 2003 and 2010. We identify a novel clonal complex based on sequence type (ST), ST-236, which accounted for 39 of the 60 isolates. A representative ST-236 strain, S. zooepidemicus strain C7 (SzC7), was sequenced and interrogated for the presence of novel nutritional uptake or utilisation systems, the acquisition of which have previously been shown to be important for environmental adaptation in other streptococcal pathogens. A novel phosphoenolpyruvate sugar phosphotransferase system (PTS), which enabled the utilisation of arbutin, was identified. Functionality of the PTS was confirmed following deletion of the PTS from SzC7. Arbutin is found in multiple animal foodstuffs and we propose that the ability to utilise arbutin may have conferred a selective advantage to strains infecting animals, the diet of which contains this sugar.PostprintPeer reviewe

CD13 as a new tumor target for antibody-drug conjugates: validation with the conjugate MI130110

BACKGROUND: In the search for novel antibody-drug conjugates (ADCs) with therapeutic potential, it is imperative to identify novel targets to direct the antibody moiety. CD13 seems an attractive ADC target as it shows a differential pattern of expression in a variety of tumors and cell lines and it is internalized upon engagement with a suitable monoclonal antibody. PM050489 is a marine cytotoxic compound tightly binding tubulin and impairing microtubule dynamics which is currently undergoing clinical trials for solid tumors. METHODS: Anti-CD13 monoclonal antibody (mAb) TEA1/8 has been used to prepare a novel ADC, MI130110, by conjugation to the marine compound PM050489. In vitro and in vivo experiments have been carried out to demonstrate the activity and specificity of MI130110. RESULTS: CD13 is readily internalized upon TEA1/8 mAb binding, and the conjugation with PM050489 did not have any effect on the binding or the internalization of the antibody. MI130110 showed remarkable activity and selectivity in vitro on CD13-expressing tumor cells causing the same effects than those described for PM050489, including cell cycle arrest at G2, mitosis with disarrayed and often multipolar spindles consistent with an arrest at metaphase, and induction of cell death. In contrast, none of these toxic effects were observed in CD13-null cell lines incubated with MI130110. Furthermore, in vivo studies showed that MI130110 exhibited excellent antitumor activity in a CD13-positive fibrosarcoma xenograft murine model, with total remissions in a significant number of the treated animals. Mitotic catastrophes, typical of the payload mechanism of action, were also observed in the tumor cells isolated from mice treated with MI130110. In contrast, MI130110 failed to show any activity in a xenograft mouse model of myeloma cells not expressing CD13, thereby corroborating the selectivity of the ADC to its target and its stability in circulation. CONCLUSION: Our results show that MI130110 ADC combines the antitumor potential of the PM050489 payload with the selectivity of the TEA1/8 monoclonal anti-CD13 antibody and confirm the correct intracellular processing of the ADC. These results demonstrate the suitability of CD13 as a novel ADC target and the effectiveness of MI130110 as a promising antitumor therapeutic agent.This work was partially supported by grant IPT-2012-0198-090000 (“MARINMAB” project) from Ministerio de Economía y Competitividad (MINECO) and European Regional Development’s funds (ERDF) and by CSIC grant 2019AEP146.S

Case Report: An EGFR-Targeted 4-1BB-agonistic Trimerbody Does Not Induce Hepatotoxicity in Transgenic Mice With Liver Expression of Human EGFR

Agonistic monoclonal antibodies (mAbs) targeting the co-stimulatory receptor 4-1BB are among the most effective immunotherapeutic agents across pre-clinical cancer models. However, clinical development of full-length 4-1BB agonistic mAbs, has been hampered by dose-limiting liver toxicity. We have previously developed an EGFR-targeted 4-1BB-agonistic trimerbody (1D8N/CEGa1) that induces potent anti-tumor immunity without systemic toxicity, in immunocompetent mice bearing murine colorectal carcinoma cells expressing human EGFR. Here, we study the impact of human EGFR expression on mouse liver in the toxicity profile of 1D8N/CEGa1. Systemic administration of IgG-based anti-4-1BB agonist resulted in nonspecific immune stimulation and hepatotoxicity in a liver-specific human EGFR-transgenic immunocompetent mouse, whereas in 1D8N/CEGa1-treated mice no such immune-related adverse effects were observed. Collectively, these data support the role of FcγR interactions in the major off-tumor toxicities associated with IgG-based 4-1BB agonists and further validate the safety profile of EGFR-targeted Fc-less 4-1BB-agonistic trimerbodies in systemic cancer immunotherapy protocols.This study was supported by grants from the European Union [IACT Project (602262)], the Spanish Ministry of Science and Innovation; the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, PID2019-110405RB-100, RTC-2016-5118-1, RTC-2017-5944-1), partially supported by the European Regional Development Fund; the Carlos III Health Institute (PI16/00357), co-founded by the Plan Nacional de Investigación and the European Union; the CRIS Cancer Foundation (FCRIS-IFI-2018), and the Spanish Association Against Cancer (AECC, 19084).Peer reviewe

Polymorphism in the CD5 Gene Promoter in B-Cell Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Despite the low incidence of microsatellite instability (MSI) in lymphoid malignant neoplasms, it has been reported that the CD5 promoter MSI was relatively frequent among B-cell chronic lymphoproliferative disorders. We studied the presence of MSI in the CD5 promoter in 134 cases of B-cell chronic lymphocytic leukemia (B-CLL) and 47 of mantle cell lymphoma (MCL) by comparing the pattern of microsatellite repeats on autologous germline and tumor DNA samples. Microsatellite alterations were not observed in any case. However, the allele distribution of this polymorphism showed a higher frequency of the 18 CA allele (0.585) in MCL cases (P = .026; odds ratio [OR], 1.75; 95% confidence interval [CI], 1.07–2.87) and of the 19 CA allele (0.179) in B-CLL cases (P = .005; OR, 2.26; 95% CI, 1.27–4.01) compared with control cases (0.442 and 0.087, respectively). This suggests that although MSI seems not to be involved in the pathogenesis of these 2 lymphoid malignant neoplasms, the polymorphic CD5 promoter is associated with increased susceptibility to these disorders.Supported by the LAIR Foundation, Madrid, Spain, and by grant SAF2002-04329 from the Ministerio de Ciencia y Tecnología, Spain

Association of the microsatellite in the 3' untranslated region of the CD154 gene with rheumatoid arthritis in females from a Spanish cohort: a case-control study

CD40–CD154 interaction is an important mediator of inflammation and has been implicated in T helper type 1-mediated autoimmune diseases including rheumatoid arthritis (RA). Linkage studies have shown association of markers in the proximity of the CD154 gene. In the present work we investigated whether specific allele variants of the microsatellite in the 3' UTR of the CD154 gene might modulate the risk of RA. The study, in a case-control setting, included 189 patients and 150 healthy controls from the Canary Islands, Spain. The 24CAs allele was less represented in female patients than in controls (0.444 in controls versus 0.307 in patients, P = 0.006, odds ratio (OR) 0.556, 95% confidence interval (CI) 0.372 to 0.831) but not in males (0.414 versus 0.408), and only when homozygous (P = 0.012; OR 0.35, 95% CI 0.16 to 0.77). We also verified that CD154 association with RA was independent of human leukocyte antigen (HLA) phenotype. A further functional study showed that after stimulation anti-CD3, CD154 mRNA was more stable in CD4+ T lymphocytes from patients with RA bearing the 24CAs allele (mRNA half-life 208 minutes) than in patients without the 24CAs allele (109 minutes, P = 0.009). However, a lower percentage of CD154+CD4+ T lymphocytes was seen in freshly isolated peripheral blood mononuclear cells from patients carrying 24CAs alleles (mean 4.28 versus 8.12; P = 0.033), and also in CD4+ T lymphocytes stimulated with anti-CD3 (median 29.40 versus 47.60; P = 0.025). These results were concordant with the smaller amounts of CD154 mRNA isolated from stimulated T lymphocytes with 24CAs alleles. The CD154 microsatellite therefore seems to affect the expression of the gene in a complex manner that implies not only mRNA stability. These data suggest that the CD154 microsatellite contributes to the regulation of mRNA and protein expression, although further studies will be necessary to elucidate its role in disease predisposition.This study was supported by Fundación LAIR (P1310). T.M. was supported by a grant of Comunidad de Madrid.Peer reviewe