Marta Alonso-Guervós

Genomic profiling of sinonasal squamous cell carcinoma.

Sinonasal squamous cell carcinomas (SCCs) are rare tumors with no etiologic link to tobacco and alcohol, as opposed to other SCCs of the head and neck (HNSCC). Little is known about the genetic changes in sinonasal SCC.

Screening of Bifidobacteria and Lactobacilli Able to Antagonize the Cytotoxic Effect of Clostridium difficile upon Intestinal Epithelial HT29 Monolayer

Clostridium difficile is an opportunistic pathogen inhabiting the human gut, often being the aetiological agent of infections after a microbiota dysbiosis following, for example, an antibiotic treatment. C. difficile infections (CDI) constitute a growing health problem with increasing rates of morbidity and mortality at groups of risk, such as elderly and hospitalized patients, but also in populations traditionally considered low-risk. This could be related to the occurrence of virulent strains which, among other factors, have high-level of resistance to fluoroquinolones, more efficient sporulation and markedly high toxin production. Several novel intervention strategies against CDI are currently under study, such as the use of probiotics to counteract the growth and/or toxigenic activity of C. difficile. In this work, we have analyzed the capability of twenty Bifidobacterium and Lactobacillus strains, from human intestinal origin, to counteract the toxic effect of C. difficile LMG21717 upon the human intestinal epithelial cell line HT29. For this purpose, we incubated the bacteria together with toxigenic supernatants obtained from C. difficile. After this co-incubation new supernatants were collected in order to quantify the remnant A and B toxins, as well as to determine their residual toxic effect upon HT29 monolayers. To this end, the real time cell analyser (RTCA) model, recently developed in our group to monitor C. difficile toxic effect, was used. Results obtained showed that strains of Bifidobacterium longum and B. breve were able to reduce the toxic effect of the pathogen upon HT29, the RTCA normalized cell-index values being inversely correlated with the amount of remnant toxin in the supernatant. The strain B. longum IPLA20022 showed the highest ability to counteract the cytotoxic effect of C. difficile acting directly against the toxin, also having the highest capability for removing the toxins from the clostridial toxigenic supernatant. Image analysis showed that this strain prevents HT29 cell rounding; this was achieved by preserving the F-actin microstructure and tight-junctions between adjacent cells, thus keeping the typical epithelium-like morphology. Besides, preliminary evidence showed that the viability of B. longum IPLA20022 is needed to exert the protective effect and that secreted factors seems to have anti-toxin activity.

Genetic model of transformation and neoplastic progression in laryngeal epithelium.

The aim of this study was to analyze genetic alterations in the transformation‐progression model of laryngeal tumors.

Análisis genético molecular con MLPA en los adenocarcinomas nasosinusales

Introduccion y objetivo Los adenocarcinomas nasosinusales (ACNS) son tumores epiteliales raros, primarios de las fosas nasales y los senos paranasales, que se caracterizan por presentar estructuras glandulares. Los objetivos de este trabajo son: determinar las alteraciones genicas en los ACNS mediante MLPA (multiplex ligation-dependent probe amplification) y establecer la relacion entre los cambios genicos del tumor y el comportamiento clinico-evolutivo de los pacientes. Material y metodo Realizamos un estudio longitudinal prospectivo a 20 pacientes con ACNS controlados en nuestro servicio entre 1998 y 2004. A las muestras tumorales se les extrajo el ADN para realizar la MLPA. Resultados La categoria T de los pacientes fue para T2: 4 (20 %), T3: 6 (30%), T4a: 3 (15 %) y T4b: 7 (35 %). Todos eran inicialmente N0 y M0. El 85 % habia estado expuesto, en el medio laboral, al polvo de madera. El tratamiento indicado fue quirurgico (100 %) con radioterapia complementaria (70 %). La supervivencia global a los 5 y 10 anos fue del 42 y el 22 %, respectivamente. Las ganancias genicas mas frecuentes fueron: PTP4A3 y PDCD8 (65 %), TNRFSF7 (50 %), RECQL4 y LMO2 (45 %); las perdidas: BCL2 (70 %), IL13 (55 %), ABCB1 y RB1 (50 %), PIK3CA y CDH1 (45 %). Conclusiones La aparicion de metastasis se correlaciono de forma significativa con perdidas en F3, MIF y BRCA1. La peor supervivencia con perdidas en F3, BCRA1 y MIF y ganancias en PIK3CA, UTY y RELA. En el analisis multivariable alcanzaron significacion estadistica las perdidas en BRCA1 y F3.

Protein in culture and endogenous lipid interact with embryonic stages in vitro to alter calf birthweight after embryo vitrification and warming.

Short-term protein removal in vitro improves long-term blastocyst competence to survive vitrification. We investigated the mechanisms and effects underlying protein removal. Day-6 morulae and early blastocysts were cultured individually with and without protein for 24h. Development and lipid content were analysed in expanded blastocysts derived from morulae (M-XB) and from early blastocysts (EB-XB). Expression of genes involved in lipid metabolism, stress responses and apoptosis was analysed in fresh and vitrified-warmed M-XB produced with and without protein. Pregnancy rates, birth rates and birthweight (BW) were recorded after transfer of embryos. Day-7 EB-XB production rates (with, 66.9±6.2 and without, 68.8±6.0 protein) were higher than M-XB rates (with, 21.4±4.6 and without, 9.4±4.6 protein; P<0.005). EB-XB showed fewer lipids than M-XB (P=0.03). In fresh M-XB, expression of sterol regulatory element binding protein (SREBP1) was lower with (4.1±2.2) than without (13.6±2.2) protein, contrary to results obtained for Patatin-like phospholipase domain containing 2, Hormone-sensitive lipase and Bcl-2-associated X protein (P<0.05). Protein did not affect pregnancy rates and birth phenotypes (P>0.05). However, BW was higher (P<0.01) in calves born from vitrified M-XB (48.6±3.4kg) than from EB-XB (39.8±2.9kg). Such effects were more pronounced in females (P<0.001). Calves from fresh embryos did not show BW differences. These results indicate that embryonic kinetics and vitrification impact birth phenotypes, at least in females. Alterations might involve exogenous protein and mobilisation of lipid stocks.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

Establishment and genetic characterization of six unique tumor cell lines as preclinical models for sinonasal squamous cell carcinoma

Sinonasal squamous cell carcinomas (SCC) are rare tumors, etiologically related to occupational exposure to wood and leather dust. In spite of surgical and radiotherapeutic advances, the 5 year survival is still 30–50%. Therefore, alternative treatment options are needed. We report the establishment and characterization of six unique human sinonasal SCC cell lines, named SCCNC1, 2, 4, 5, 6 and 7. In vitro growth and invasion characteristics were evaluated and genetic profiles were compared to those of the original primary tumors. The population doubling times ranged from 21 to 34 hours. Cell lines SCCNC2 and 7 were highly invasive in matrigel. Five cell lines carried a high number of copy number alterations, including amplifications and homozygous deletions, while one showed only three abnormalities. Sequence analysis revealed three cell lines with TP53 mutation and none with KRAS or BRAF. Overexpression of p53 was observed in five, and of EGFR in four cell lines. None of the cell lines showed strong immunopositivity of p16 or presence of human papilloma virus. In conclusion, we have created six new cell lines that are clinically and genetically representative of sinonasal SCC and that will be a useful tool for the preclinical testing of new therapeutic agents.

From laryngeal epithelial precursor lesions to squamous carcinoma of the larynx: the role of cell cycle proteins and β-catenin

Novel markers to accurately predict the risk of malignant transformation in laryngeal epithelial precursor lesions (EPL) are needed. We tried to identify some molecular alterations occurring in laryngeal tumorigenesis. In this study, 60 paraffin-embedded EPL and 17 metachronous invasive carcinomas were immunostained for markers associated with proliferation (Ki67), cell cycle control (p53, p21, p16, p27, cyclin D1), and cell adhesion and invasion (laminin and β-catenin). Aberrant expression of p16 and p53 and positivity at cytoplasm for β-catenin and cyclin D1 were detected significantly in EPL with progression to invasive laryngeal carcinoma. All cases with basal and suprabasal reactivity of p53 showed β-catenin overexpression. We found that β-catenin protein expression increased significantly with the grade of dysplasia. This is one of the studies with the largest number of laryngeal EPL and invasive carcinoma studied sequentially. Our data confirm the role of some cell cycle regulatory proteins in the development of laryngeal carcinoma. Cytoplasmic retention of β-catenin in EPL seems to be related with more aggressive biological behavior. Combined increased p53 and cytoplasmic β-catenin protein expression could be biologically important in laryngeal tumorigenesis. Further research is required to clarify the involvement of β-catenin in the mechanism associated with malignant transformation in laryngeal tissues.

Evidence of Nestin-Positive Cells in the Human Cutaneus Meissner and Pacinian Corpuscles

Nestin is an intermediate filament protein expressed in neuroepithelial stem cells during development and it is later replaced by cell specific neuronal or glial filaments. Nevertheless, nestin⁺ cells remain within adult tissues and they can be regarded as potential neural stem cell (NSC). Nestin⁺ cells have been detected in Schwann cells related with sensory corpuscles of rodent and they have been demonstrated to be NSC. We have investigated the existence of nestin⁺ in human cutaneous cells Meissner and Pacinian corpuscles through the use of immunohistochemistry techniques and in situ hybridization. S100 protein (also regarded as a marker for NSC) and vimentin (the intermediate filament of mature Schwann cells in sensory corpuscles) were also investigated. The results show that the adult human cutaneous sensory Meissner and Pacinian corpuscles contains a small population of Schwann-related cells (vimentin⁺) which on the basis of their basic immunohistochemical characteristics (S100 protein⁺, nestin⁺) can be potential NSCs. Cells sharing identical immunohistochemical profile were also found in the close vicinity of Meissner corpuscles. Because their localization they are easily accessible and may represent a peripheral niche of NSC to be used for therapeutic goals.

Genetic and protein markers related to laryngeal epithelial precursor lesions and their neoplastic progression

Abstract Conclusion: Various biomarkers might ultimately prove to have prognostic value and could be clinically relevant. It is mandatory confirm the prognostic power of these markers in large, well-designed, and prospective studies. Objective: The aim of this study was to investigate the possible role of specific genes and proteins in laryngeal tumorigenesis. Methods: Genetic analysis by multiple ligation-dependent probe amplification and analysis of protein expression by immunohistochemistry were carried out in a series of 50 tissue samples. Results: In the smoker normal mucosa group TP53 loss was predominant, whereas in the epithelial precursor lesions (EPLs) CDKN2A loss and BCL2L1 gain were most frequent. EPL with progression presented CTNNB1 loss. Positivity at cytoplasm for β-catenin, cyclin D1 and p53 was detected in all EPL cases with progression to invasive carcinoma. Multivariate analysis showed that expression of β-catenin and loss of CTTNB1 were associated with laryngeal cancer risk.