Monitoreo de contaminantes y detección de genes alkb en la Zona Común de Pesca Argentino-Uruguaya

In this study, the presence of diverse pollutants in sediments samples was determined in the Río de la Plata and the Argentine-Uruguayan Common Fishing Zone (AUCFZ 35° S-38° S). In addition, the occurrence of microbial genes alkB, used as functional markers, was analyzed in order to determine the hydrocarbon degradation potential along this marine environment. Heavy metals were detected in sampling sites UY1, RdP4 and AR2. Cr was found in a range from < 5.0 to 20.7 mg kg-1, and Pb from not detectable (ND) to 26.0 mg kg-1. Both Cd and Hg showed values under detection limits (< 0.2 mg kg-1 and < 0.01 mg kg-1, respectively). Cu varied between ND and 24.6 mg kg-1, and it was the only metal that overcame the values recommended by the ISQG (Interim Sediment Quality Guideline), in the guide levels for biota protection in sediments (CCME, Canadian Council of Ministers of the Environment). Hydrocarbons, atrazine, glyphosate + AMPA (aminomethyl phosphonic acid) and pesticides values were under the quantification limits, while PCBs (from < 20.0 to 77.7 µg kg-1) exceeded the action level A for the ‘Recommendations for the Management of Dredging Material in Spanish Ports’. Purified microbial genomic DNA was obtained in eight from the nine analyzed samples, and the catabolic gen alkB was amplified in sampling sites UY2, UY1, RdP4, AR2 and AR1. Further studies are needed to evaluate the microbial biodegradation potential in this area. These researches mean a valuable input in order to investigate the impact of the anthropogenic disturbances on marine ecosystems and to understand the mechanisms of natural attenuation.En este estudio se determinó la presencia de diversos contaminantes en muestras de sedimentos en el Río de la Plata y la Zona Común de Pesca Argentino-Uruguaya (ZCPAU, 35° S-38° S). Además, se analizó la ocurrencia de genes alkB microbianos, utilizados como biomarcadores funcionales para determinar el potencial de degradación de hidrocarburos a lo largo de este ambiente marino. Se detectaron metales pesados en las estaciones UY1, RdP4 y AR2. El Cr se encontró en un rango entre < 5,0 y 20,7 mg kg-1, y el Pb entre no detectable (ND) y 26,0 mg kg-1. Tanto el Cd como el Hg mostraron valores por debajo de los límites de detección (< 0,2 mg kg-1 y < 0,01 mg kg-1, respectivamente). El Cu varió entre ND y 24,6 mg kg-1, y fue el único metal que sobrepasó los valores recomendados por la ISQG (Interim Sediment Quality Guideline), en los niveles guía en sedimentos para la protección de la biota (CCME, Canadian Council of Ministers of the Environment). Los valores de hidrocarburos, atrazina, glifosato + AMPA (ácido aminometil fosfónico) y pesticidas estuvieron por debajo del límite de cuantificación, mientras que los PCBs (desde < 20,0 a 77,7 µg kg-1) excedieron el nivel de acción A de las “Recomendaciones para la Gestión del Material de Dragado en los Puertos Españoles”. Se obtuvo ADN genómico microbiano purificado en ocho de las nueve muestras analizadas y se logró la amplificación del gen catabólico alkB en las estaciones UY2, UY1, RdP4, AR2 y AR1. Es necesario realizar estudios adicionales para evaluar el potencial de biodegradación microbiana en esta área. Estas investigaciones representan un valioso aporte para evaluar el impacto de las alteraciones antropogénicas sobre los ecosistemas marinos y para comprender los mecanismos de la atenuación natural

Zoledronic acid boosts γδ T-cell activity in children receiving αβ+ T and CD19+ cell-depleted grafts from an HLA-haplo-identical donor

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αβ+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αβ T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients

Identification of Specific Plasma Proteins Determining the Agarose Gel Electrophoresis by the Immunosubtraction Technique

Peer Reviewe

The role of the Bcl-2 family of proteins in the pathogenesis of B-cell chronic lymphocytic leukaemia

B-cell chronic lymphocytic leukaemia (B-CLL) is an acquired neoplastic disease characterised by a clonal accumulation of long-lived, functionally immature and CD5+ B-lymphocytes, which particularly accumulate in the lymphatic system, peripheral blood, bone marrow, spleen and liver. Symptoms include lymphocytosis, immune system dysfunction and autoimmune disease, but transformation to more aggressive forms of neoplastic disease occur and development of a second malignancy is not uncommon. The disease is one of later years being unusual before 50 years of age, the rates of incidence vary on a racial basis, and it has a highly variable prognosis. Some patients die within months of diagnosis despite intensive treatment, whilst others survive for 30-plus years without any form of medical intervention and die of unrelated causes. The principal causes of death in patients whose deaths are directly related to the disease are opportunistic infection due to the impairment of immune system function and bleeding disorders. No treatment has been shown to cure the disease or consistently extend life expectancy. It has been recognised for more than 30 years that the accumulation of malignant cells in B-CLL is at least as important in the pathogenesis of the disease as their neoplastic proliferation. With the discoveries that Bcl-2 extended the life of follicular lymphoma cells by conferring resistance to apoptosis and was commonly expressed in B-CLL cells, it was extrapolated that Bcl-2 might play a similar role in the development of the disease by extending the life span of B-CLL cells. Bcl-2 has frequently been shown to be over expressed in B-CLL cells and genetic translocations and/or malfunction of Bcl-2 family regulating molecular entities may play a part in this. However, since its discovery, Bcl-2 has been shown to be part of a large family of genes which is highly and evolutionarily conserved. Members of the bcl-2 family are defined by sequence homology in four Bcl-2 homology (BH) regions and a hydrophobic membrane-spanning domain, with the possession of specific BH domains determining whether individual proteins have pro- or anti-apoptotic activity. Family members such as Bcl-2 and Bcl-XL extend the life span of cells, whilst others such as Bax and Bak shorten it. Oltvai, Milliman and Korsmeyer have proposed a general model of apoptosis, in which the cell's apoptotic fate is determined by the cellular balance between pro- and anti-apoptotic bcl-2 family members. The effect of unregulated expression of Bcl-2 family members in B-CLL cells conforms to this paradigm and resistance to apoptosis appears to be conferred through a cellular imbalance of power between pro- and anti-apoptotic bcl-2 family members, particularly Bcl-2 and Bax, which is tilted in favour of cell survival. However, the apoptotic fate of B-CLL cells, and hence the neoplasm, may be influenced by other family members, with Mcl-1, Bcl-XL, Bak, with the non-family but Bcl-2-associated protein, Bag-1, also found expressed in B-CLL cells. Similarities between the structure of the more conserved family members and other pore-forming proteins, along with the ability of Bcl-2, Bcl-XL, and Bax to form pores in synthetic membranes, suggest that they may exert their influence through pore-forming activities in intracellular membranes, particularly mitochondrial membranes. Bcl-2 family members may regulate apoptosis by changing the permeability of membranes to ions and apoptosis-inducing molecules, and physical interactions between Bcl-2 family proteins mediated by the BH domains may be important in both pore-forming and pore-inhibiting activities. Research findings suggest that the levels of Bcl-2 family members in B-CLL cells may be modulated by a wide range of largely extracellular influences, including the cytokines interleukin-4 (IL-4), IL-8, IL-10, interferon-a (IFN-?), IFN-?, and basic fibroblast growth factor (bPGF). Levels of Bcl-2 family members may also be modulated by contact between B-CLL cells and bone marrow (BM) stromal cells, activation of lgM, CD95, CD40 or CD6, the p53 gene product, and co-cultivation with CDw32-transfected murine fibroblasts. Such modulation may offer some insight into the pathogenesis of the disease, an explanation for the higher level expression of Bcl-2 family members in B-CLL, and an explanation for the highly variable prognosis. Additionally, if Bcl-2 family members can be shown unequivocally to be controlled by any of these molecular entities, the existence of these influences may offer the opportunity to reduce the neoplastic cells' apoptotic threshold by manipulating the relative levels of pro- and anti-apoptotic Bcl-2 family members as a treatment regime, or prior to more conventional treatment regimes

C. elegans expressing D76N β 2 -microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

Funder: Ministero dell'Istruzione, dell'Università e della Ricerca Dipartimenti di Eccellenza 2018-2022 grant to the Molecular Medicine Department (University of Pavia)Abstract: The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β2-m expressing worms. We also demonstrated the specificity of the β2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates

Normal cervical changes in parous women during the second half of pregnancy - a prospective, longitudinal ultrasound study

OBJECTIVE: To determine what constitutes normal cervical changes during the second half of pregnancy in parous women delivering at term. DESIGN: The study comprises 21 healthy, pregnant parous women who all gave birth at term. They were examined with transvaginal ultrasound every two weeks from 24 gestational weeks until delivery. Cervical length and width were measured. The inner cervical os was assessed as being closed or open, the length and width of any opening were measured, and dynamic cervical changes (i.e. opening and closing of the inner cervical os during examination) were noted. RESULTS: Median cervical length was 41 mm (range 26-55) at the first examination and 29 mm (range 8-56) at the last examination. The corresponding figures for cervical width were 38 mm (range 29-47) and 46 mm (range 38-64). Cervical length decreased in 18 women but remained unchanged in three. Three patterns of change in cervical length were observed: in 12 women there was a steady, continuous decrease in cervical length (median decrease rate 1.1 mm/week, range 0.6-2.4); in four women the decrease rate accelerated towards the end of pregnancy, the median decrease rate after the change being 3.0 mm/week (range 1.5-4.8); and in two women there was a sudden drop in cervical length at term. Cervical width increased in 16 women but remained unchanged in five. Two patterns of change in cervical width were seen: 14 women manifested a steady continuous increase in cervical width (median 0.8 mm/week, range 0.4-1.8); in two women the increase rate accelerated from around 34 gestational weeks, the increase rate after the change being 4.1 and 5.9 mm/week, respectively. Opening of the internal cervical os was observed at least once in 11 (52%) women and was seen as early as at 24 and 25 gestational weeks in two women. The opening was always V-shaped (median length 6 mm, range 4-17; median width 7 mm, range 3-20). Dynamic changes of the internal cervical os were seen in three women (14%) at 25, 30 and 41 gestational weeks, respectively. CONCLUSION: The cervix of parous women decreases in length and increases in width from midpregnancy to term, but the pattern of change varies between individuals. Knowledge of the different patterns of normal change forms the basis of transvaginal ultrasound studies of pathological cervical changes during pregnancy