Detection and characterization of Pseudomonas syringae pv. actinidifoliorum in kiwifruit in Spain.

AIMS: Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is currently the major threat to its commercial production worldwide. In 2011, the most virulent type (Psa3) was detected for the first time in Northwest-Spain, in the province of Pontevedra. In 2013 surveys, leaves and flower buds with mild symptoms were observed in Actinidia deliciosa 'Hayward' vines in an orchard at the province of A Coruña, suggesting the presence of P. syringae pv. actinidifoliorum (Psaf). METHODS AND RESULTS: Isolates obtained from such orchard were characterized by morphological, biochemical and physiological tests, fatty acids (FA) profile and molecular tests (PCR, BOX-PCR, duplex PCR, multiplex PCR, real-time PCR, PCR-C, phytotoxins, housekeeping and effector genes). Pathogenicity tests were also carried out on plants and fruits of A. deliciosa 'Hayward' and on different cultivated plants and fruits. Results demonstrated the presence of P. syringae pv. actinidifoliorum in Spain. CONCLUSIONS: The work provides new information on the pathovar P. syringae pv. actinidifoliorum, which has only been found previously in New Zealand, Australia and France. SIGNIFICANCE AND IMPACT OF STUDY: The results are relevant for taxonomy of isolates of P. syringae from kiwifruit, especially those of low virulence not belonging to pathovar actinidiae.

Prospective study assessing hypoxia-related proteins as markers for the outcome of treatment with sunitinib in advanced clear-cell renal cell carcinoma.

BACKGROUND: Previous studies suggest that expression of hypoxia markers may be associated with response to antiangiogenic drugs. Thus, we aimed to identify predictors of sunitinib outcome in clear-cell renal cell carcinoma (ccRCC). PATIENTS AND METHODS: The expression of eight key proteins related to hypoxia (CAIX, HIF1A, HIF2A, VEGFA, VEGFR1, VEGFR2, VEGFR3 and PDGFRB) and P-glycoprotein were assessed by immunohistochemistry in 67 primary ccRCC samples from prospectively recruited patients treated with first-line sunitinib. The proteins expression, VHL inactivation and EGLN3 mRNA content were compared with the patients' response to sunitinib. RESULTS: High expression of HIF2A and PDGFRB was associated with better sunitinib RECIST objective response (P = 0.024 and P = 0.026; respectively) and increased VEGFR3 expression was associated with longer progression-free survival (P = 0.012). VEGFR3 overexpression showed a negative correlation with VEGFR3 polymorphism rs307826 (P = 0.002), a sunitinib resistance predictor. With respect to overall survival (OS), high VEGFA was associated with short (P = 0.009) and HIF2A with long (P = 0.048) survival times. High EGLN3 mRNA content was associated with shorter OS (P = 0.023). CONCLUSIONS: We found an association between several proteins involved in hypoxia and sunitinib efficacy. In addition, low VEGFR3 expression was associated with worse outcome and with VEGFR3 rs307826 variant allele, reinforcing VEGFR3 as a marker of sunitinib resistance.

Standard operating procedure for the collection of fresh frozen tissue samples.

Studies using fresh-frozen tissue samples originating from different centres, as is often the case in EORTC related translational research, can show conflicting research results due to heterogeneity in the quality of samples and associated data from each centre. The development of infrastructure for the European Human Frozen Tumour Tissue Bank (TuBaFrost) anticipated this problem and Standard Operating Procedures (SOPs) have been developed to ensure samples collected are of consistent high quality and variation in research results is minimised. The SOPs drew on the best practice standard workflows and operating procedures employed by members of the TuBaFrost Consortium and key tissue bank initiatives worldwide. It was essential to provide workable solutions that reflect the variety in infrastructure and resources at the potential collecting centres and also the fact that it is not necessary to standardise every step of the collection and storage process in order to collect high quality tissue. Hence, the TuBaFrost SOPs detail the compulsory measures that must be implemented in order to become a TuBaFrost collecting centre and also make advisory recommendations regarding the less critical factors. Accordingly, the TuBaFrost SOPs are very flexible and to illustrate this the complete SOP for collecting, freezing and storing tissue at the Erasmus MC Tissue Bank is included. These TuBaFrost SOPs could equally be applicable to centres collecting samples for EORTC related translational research studies in order to standardise sample quality and produce reliable and reproducible research results.

Tumour banking: the Spanish design.

In the last decade the technical advances in high throughput techniques to analyze DNA, RNA and proteins have had a potential major impact on prevention, diagnosis, prognosis and treatment of many human diseases. Key pieces in this process, mainly thinking about the future, are tumour banks and tumour bank networks. To face these challenges, diverse suitable models and designs can be developed. The current article presents the development of a nationwide design of tumour banks in Spain based on a network of networks, specially focusing on its harmonization efforts mainly regarding technical procedures, ethical requirements, unified quality control policy and unique sample identification. We also describe our most important goals for the next years. This model does not correspond to a central tumour bank, but to a cooperative and coordinated network of national and regional networks. Independently from the network in which it is included, sample collections reside in their original institution, where it can be used for further clinical diagnosis, teaching and research activities of each independent hospital. The herein described 'network of networks' functional model could be useful for other countries and/or international tumour bank activities.

Sistema personalizado de dosificación (SPD), más allá del blíster: presentación de un caso clínicoen un paciente con enfermedad de Alzheimer / Personalized dosification systems (PDS), beyond the blister: presentation of a clinical case in a patient with Alzheimer’s disease

Los sistemas personalizados de dosificación (SPD) constituyen un recurso primordial para mejorar la adherencia farmacoterapéutica desde la farmacia comunitaria.El farmacéutico comunitario (FC), al ofrecer el SPD como servicio profesional farmacéutico asistencial, debe aprovechar todo su potencial para alcanzar objetivos terapéuticos, además del acondicionamiento de la medicación en el blíster desechable, e incorporar Revisiones del Uso de los Medicamentos (RUM), seguimiento farmacoterapéutico y un contacto frecuente con familia y demás equipo de salud que atienden al paciente. En este caso, conscientes de la importancia de un seguimiento cercano, especialmente durante el confinamiento por la pandemia de COVID-19 y en una paciente con enfermedad de Alzheimer, se consigue mejorar la adherencia terapéutica gracias a un servicio farmacéutico que, más allá del blíster, incluye entrevistas con cuidadora y familia, revisiones del uso de medicación semanales, indicación farmacéutica y contacto directo con el médico de atención primaria (MAP) en situaciones complejas como, por ejemplo: aparición de nuevos problemas de salud y reacciones adversas a los medicamentos (RAM), provocados por medicamentos o dificultad para tragar sólidos. En este caso, debido al aislamiento provocado por el confinamiento, se incluyen además servicios de Atención Farmacéutica Domiciliaria (AFD) y dispensación a domicilio. Protocolizar y realizar una revisión de mediación semanal en el servicio de SPD mejora la adherencia y la consecución de objetivos terapéuticos. La colaboración entre médico, farmacéutico y familia facilita el seguimiento de los pacientes adheridos al servicio para conseguir resultados en salud. (AU)

TuBaFrost 3: regulatory and ethical issues on the exchange of residual tissue for research across Europe.

The regulatory regimes for research with residual tissue and accompanying data differ widely between countries in the European Union (EU): from specific consent to opt-out or even no consent at all. This could greatly hamper research where the exchange of tissue and accompanying data has become the gold standard, like in TubaFrost. Instead of adhering to international guidelines, which have a democratic deficit, or an attempt for a new set of possible harmonising rules, TubaFrost chose to create a coordinating rule: if tissue may legitimately be used for a certain kind of research in the country where it was taken and under whose jurisdiction the patient falls, it may also be used for such research in the country where it is sent to in the context of a scientific program even if in that other country other regulations would apply for research with residual tissue taken from patients under their jurisdiction. This coordinating rule has a sound basis in EU law in general and will solve the problems related to diverging national regulatory regimes in the case of cross national research with residual tissue.

TuBaFrost 6: virtual microscopy in virtual tumour banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated Virtual Microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting bio-repositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

TuBaFrost 1: Uniting local frozen tumour banks into a European network: an overview.

TuBaFrost is the consortium responsible for the creation of a virtual European human frozen tumour tissue bank: a collection of high quality frozen residual, accurately classified tumour tissue samples, which are stored in European cancer centres and universities. This virtual tissue bank, searchable on the internet, has rules for access and use, and a code of conduct to comply with the various legal and ethical regulations in European countries. The easy accessibility and the European scale of the bank will result in the availability of a large number of samples even of rarer tumour types. Standardisation of collection, storage and quality control throughout the network is achieved minimising inter-institutional variability. A website providing access to upload, search and request samples is a key tool of the tissue bank. The search engine makes use of virtual microscopy. An overview of the development of the European virtual frozen tissue bank infrastructure is described in this paper. The various key aspects are described in more detail in a series of articles to appear in this Journal.

TuBaFrost 2: Standardising tissue collection and quality control procedures for a European virtual frozen tissue bank network.

Tumour Bank Networking presents a great challenge for oncological research as in order to carry out large-scale, multi-centre studies with minimal intrinsic bias, each tumour bank in the network must have some fundamental similarities and be using the same standardised and validated procedures. The European Human Frozen Tumour Tissue Bank (TuBaFrost) has responded to this need by the promotion of an integrated platform of tumour banks in Europe. The operational framework for TuBaFrost has drawn upon the best practice of standard workflows and operating procedures employed by members of the TuBaFrost project and key initiatives worldwide.

TuBaFrost 4: access rules and incentives for a European tumour bank.

When designing infrastructure for a networked virtual tumour bank (samples remain at the collector institutes and sample data are collected in a searchable central database), it is apparent that this can only function properly after developing an adequate set of rules for use and access. These rules must include sufficient incentives for the tissue sample collectors to remain active within the network and maintain sufficient sample levels in the local bank. These requirements resulted in a key TuBaFrost rule, stating that the custodianship of the samples remains under the authority of the local collector. As a consequence, the samples and the decision to issue the samples to a requestor are not transferred to a large organisation but instead remain with the collector, thus allowing autonomous negotiation between collector and requestor, potential co-authorship in publications or compensation for collection and processing costs. Furthermore, it realises a streamlined cost effective network, ensuring tissue visibility and accessibility thereby improving the availability of large amounts of samples of highly specific or rare tumour types as well as providing contact opportunities for collaboration between scientists with cutting edge technology and tissue collectors. With this general purpose in mind, the rules and responsibilities for collectors, requestors and central office were generated.

TuBaFrost 5: multifunctional central database application for a European tumor bank.

Developing a tissue bank database has become more than just logically arranging data in tables combined with a search engine. Current demand for high quality samples and data, and the ever-changing legal and ethical regulations mean that the application must reflect TuBaFrost rules and protocols for the collection, exchange and use of tissue. To ensure continuation and extension of the TuBaFrost European tissue bank, the custodianship of the samples, and hence the decision over whether to issue samples to requestors, remains with the local collecting centre. The database application described in this article has been developed to facilitate this open structure virtual tissue bank model serving a large group. It encompasses many key tasks, without the requirement for personnel, hence minimising operational costs. The Internet-accessible database application enables search, selection and request submission for requestors, whereas collectors can upload and edit their collection. Communication between requestor and involved collectors is started with automatically generated e-mails.

TuBaFrost: European virtual tumor tissue banking.

TuBaFrost is a consortium responsible for the task to create a virtual European human frozen tumor tissue bank, composed of high quality frozen tumor tissue collections with corresponding accurate diagnosis stored in European cancer centers and universities, searchable on the Internet, providing rules for access and use and a code of conduct to comply with the various legal and ethical regulations in European countries. Such infrastructure would enlarge tissue availability and accessibility in large amounts of specified or even rare tumor samples. Design of an infrastructure for European residual tissue banking with the described characteristics, clear focus points emerge that can be broken down in dedicated subjects: (1) standardization and quality assurance (QA) to avoid inter-institute quality variation; (2) law and ethics enabling exchange of tissue samples possible between institutes in the different European countries, where law and ethics are characterized by a strong variability; (3) rules for access, with sufficient incentives for collectors; (4) central database application containing innovations on search and selection procedures; (5) support when needed with histology images; and (6) Internet access to search and upload, with in addition a solid website giving proper information on the procedures, intentions and activities not only to the scientific community, but also to the general public. One consortium decision, part of the incentives for collectors, had major impact on the infrastructure; custodianship over the tissues as well as the tissues stay with the collector institute. Resulting in specimens that are not given to an organization, taking decisions on participation of requests, but instead the local collected tissues stay very easy to access by the collector and allows autonomous negotiation between collector and requestor on cooperation, coauthorship in publication or compensation in costs. Thereby, improving availability of large amounts of high quality samples of a highly specified or rare tumor types and contact opportunities for cooperation with other institutes.

Virtual microscopy in virtual tumor banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated virtual microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting biorepositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

Monitoring of West Nile virus, Usutu virus and Meaban virus in waterfowl used as decoys and wild raptors in southern Spain.

In the last decade, the number of emerging flaviviruses described worldwide has increased considerably, with wild birds acting as the main reservoir hosts of these viruses. We carried out an epidemiological survey to determine the seroprevalence of antigenically related flaviviruses, particularly West Nile virus (WNV), Usutu virus (USUV) and Meaban virus (MBV), in waterfowl used as decoys and wild raptors in Andalusia (southern Spain), the region considered to have the highest risk of flaviviruses circulation in Spain. The overall flaviviruses seroprevalence according to bELISA was 13.0% in both in decoys (n=1052) and wild raptors (n=123). Specific antibodies against WNV, USUV and MBV were confirmed by micro virus neutralization tests in 12, 38 and 4 of the seropositive decoys, respectively. This is the first study on WNV and USUV infections in decoys and the first report of MBV infections in waterfowl and raptors. Moreover we report the first description of WNV infections in short-toed snake eagle (Circaetus gallicus) and Montagu's harrier (Circus pygargus). The seropositivity obtained indicates widespread but not homogeneous distribution of WNV and USUV in Andalusia. The results also confirm endemic circulation of WNV, USUV and MBV in both decoys and wild raptors in southern Spain. Our results highlight the need to implement surveillance and control programs not only for WNV but also for other related flaviviruses. Further research is needed to determine the eco-epidemiological role that waterfowl and wild raptors play in the transmission of emerging flaviviruses, especially in decoys, given their close interactions with humans.

Loss of JUNB/AP-1 promotes invasive prostate cancer.

Prostate cancer is a frequent cause of male death in the Western world. Relatively few genetic alterations have been identified, likely owing to disease heterogeneity. Here, we show that the transcription factor JUNB/AP-1 limits prostate cancer progression. JUNB expression is increased in low-grade prostate cancer compared with normal human prostate, but downregulated in high-grade samples and further decreased in all metastatic samples. To model the hypothesis that this downregulation is functionally significant, we genetically inactivated Junb in the prostate epithelium of mice. When combined with Pten (phosphatase and tensin homologue) loss, double-mutant mice were prone to invasive cancer development. Importantly, invasive tumours also developed when Junb and Pten were inactivated in a small cell population of the adult anterior prostate by topical Cre recombinase delivery. The resulting tumours displayed strong histological similarity with human prostate cancer. Loss of JunB expression led to increased proliferation and decreased senescence, likely owing to decreased p16(Ink4a) and p21(CIP1) in epithelial cells. Furthermore, the tumour stroma was altered with increased osteopontin and S100 calcium-binding protein A8/9 expression, which correlated with poor prognoses in patients. These data demonstrate that JUNB/AP-1 cooperates with PTEN signalling as barriers to invasive prostate cancer, whose concomitant genetic or epigenetic suppression induce malignant progression.

Pathological, immunological, and molecular features of Hodgkin's disease associated with HIV infection. Comparison with ordinary hodgkin's disease.

The aim of the present study was to analyze in a series of 24 HIV-positive Hodgkin's disease (HD) patients the morphological and immunological features, the presence of rearrangements in the immunoglobulin heavy chain (IgH) gene, expression of the Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1), and the existence of deletions in the intracytoplasmic domain of the LMP-1 gene. The results obtained were compared with those from a parallel series of 56 patients with ordinary HD. Briefly, comparison of the two series showed a predominance of unfavorable histological subtypes in HIV-positive HD patients. The mixed cellularity subtype was more frequent in HIV-positive than in HIV-negative HD patients: the difference in percentage was statistically significant (p = 0.04). Neoplastic cell-rich cases were significantly more frequent (p = 0.40) in HIV patients (59%) than in ordinary HD patients (34%). In 25% of HIV-infected and in 14% of ordinary HD patients, the neoplastic cells were CD20+, a difference that was not statistically representative. Clonal IgH rearrangements were detected in 33% of HIV-infected patients and in 23% of ordinary HD patients, a nonsignificant difference. LMP-1 expression was detected in 100% of HIV-positive patients and in 57% of ordinary HD patients (p = 0.004). A 30-base-pair deletion in the carboxy-terminal domain of the LMP-1 gene was found in 16 of 18 HIV-infected patients (89%), whereas it was identified in only 8 of 25 ordinary HD patients (32%) (p = 0.008). In conclusion, HD in HIV-infected patients as compared with HD in HIV-negative individuals is associated with morphological features of aggressivity, with a higher frequency of neoplastic cells, and with constant LMP-1 expression. The fact that LMP-1 is expressed in all HIV-infected patients suggests that EBV plays an etiological role in the pathogenesis of HIV-associated HD. Furthermore, the presence of EBV strains carrying deletions near the 3' end of the LMP-1 gene in the majority of cases may be related with the morphological and clinical aggressivity of HD in immunocompromised patients.

Persistent and generalized lymphadenopathy: a lesion of follicular dendritic cells? An immunohistologic and ultrastructural study.

An immunohistochemical and ultrastructural study of 14 cases of persistent and generalized lymphadenopathy (PGL), acquired immune deficiency syndrome (AIDS) related, revealed florid follicular hyperplasia, follicular dendritic cell (FDC) lysis, lymphoid follicle invaginations, increased presence of T8 cells in germinal center, immature sinus histiocytosis (monocytoid B-cells), and inversion of T4/T8 ratio in the paracortical area. Electron microscopic examination showed viral particles of morphologic characteristics consistent with human immunodeficiency virus (HIV) virions attached to the processes of FDC in three of the nine cases studied. Lesions of the germinal center dendritic cell network are the cardinal feature of PGL. This finding lends support to the idea of a viral aggression directed against FDC as the cause of disregulation of the B-cells.

Human tonsil intraepithelial B cells: a marginal zone-related subpopulation.

AIMS: To determine if intraepithelial B cells in reactive human palatine tonsils were similar to the marginal zone cells of the spleen and Peyer's patches. METHODS: Reactive human palatine tonsils were studied using conventional methods of light microscopy, electron microscopy, and a panel of monoclonal antibodies for leucocyte common antigens. RESULTS: Clinically important numbers of marginal zone-related B cells around the mantle zone were absent in lymphoid follicles, but in the cryptal epithelium there were abundant lymphoid cells with centrocyte-like nuclei and clear cytoplasm, intermingled with macrophages and plasma cells. The immunophenotype of these intraepithelial B cells was distinctive and similar to that found in the splenic marginal zone cells (IgM+, IgD-, CD23-, CD10-, CD35+, CD21+, bc12+, KB61+). CONCLUSIONS: Intraepithelial B cells in human tonsil could represent the counterpart of the marginal zone described in Peyer's patches. Their presence within the epithelium could reflect the destination for the malignant B cells in the lymphoepithelial lesion of mucosa associated lymphoid tissue (MALT) lymphomas. Human palatine tonsil lymphoid tissue has morphological, immunophenotypic, and pathological features similar to those of MALT.

Epstein-Barr virus-latent membrane protein 1 expression has a favorable influence in the outcome of patients with Hodgkin's Disease treated with chemotherapy.

The effect of molecular factors in the outcome of Hodgkin's Disease (HD) is being currently studied. In a previous series of HD, including patients treated only with radiotherapy and patients treated with chemotherapy (with or without radiotherapy), we found that a high proliferation index had an adverse influence in overall survival (OS) and in the achievement of a complete remission (CR). Loss of Rb expression also had an adverse prognostic influence in achievement of CR. On the other hand LMP1-EBV expression had a favorable influence for OS. The expression of other molecular factors, p53, bcl2 and CD15 did not show prognostic influence. In the present paper we have studied the effect of these molecular variables in 110 patients, of the previous series who had been treated with chemotherapy. A retrospective study was performed in these 110 patients with HD treated with chemotherapy (ABVD or variants, 62%, or regimes not containing adriamycin, 38%) with or without adjutant radiotherapy, collected at the 11 centers belonging to the Spanish Collaborative Group for the Study of Hodgkin's Disease. The prognostic value of clinical variables and the expression of p53, bcl2, CD15, Rb, LMP 1-EBV and proliferative fraction demonstrated with sensitive immunohistochemical methods were studied. Cox's multivariate analysis was performed to assess their influence in failure-free survival (FFS) and OS. A multivariate logistic regression analysis was performed for studying the effect of the variables in the achievement of a CR. Of the clinical variables, only advanced stage (III/IV) had a significant independent adverse influence in FFS, in OS and in the achievement of CR and advanced age in OS. Of the molecular variables, LMP1-EBV had an independent and strong favorable influence in FFS, in OS and in the achievement of CR. Rb expression had a modest favorable influence in CR. The rest of the molecular variables had no independent influence on the outcome of the disease. In conclusion these results confirm the favorable prognostic value of LMP1-EBV expression in the subset of patients with HD treated with chemotherapy.

[Low-grade B cell gastric lymphoma originated in mucosa-associated lymphoid tissue. Relationship of tumor cells with the marginal zone and monocytoid B lymphocytes. An immunohistochemical and ultrastructural study]. / Linfoma B gástrico de bajo grado originado en tejido linfoide asociado a mucosas. Relación de las células tumorales con la zona marginal y los linfocitos B monocitoides. Estudio inmunohistoquímico y ultraestructural.

Seven cases of gastric B-cell low-grade lymphomas were characterized by morphology, immunohistology and electron microscopy. All them were immunophenotyped with a panel of monoclonal antibodies against immunoglobulins and other B-cell determinants. Histologic study of gastric B-cell low-grade lymphomas showed germinal centers of lobated shape and polyclonal nature, mainly polyclonal subepithelial plasma cell (except in one case) and neoplastic interfollicular B-cells of monoclonal character. Light-chain restriction supports the neoplastic nature of gastric lymphoma of low-grade malignancy, a distinctive tumour of extranodal B-cell origin. Interfollicular B-cells share with marginal zone cells a perifollicular localization, morphology and phenotype, suggesting a possible relation between these two cellular subtypes. In two cases, the tumour appears constituted by monocytoid B-lymphocytes (MBL), which suggests a relation of tumoral interfollicular B-cells with this subpopulation.