Platinum-Based Regimens Are Active in Advanced Pediatric-Type Rhabdomyosarcoma in Adults and Depending on HMGB1 Expression.

Rhabdomyosarcoma (RMS) in adults is a rare and aggressive disease, which lacks standard therapies for relapsed or advanced disease. This retrospective study aimed to describe the activity of BOMP-EPI (bleomycin, vincristine, methotrexate and cisplatin alternating with etoposide, cisplatin and ifosfamide), an alternative platinum-based regimen, in adult patients with relapsed/metastatic RMS. In the study, 10 patients with RMS with a median age at diagnosis of 20.8 years and a female/male distribution of 6/4 received a mean of 2.5 cycles of BOMP-EPI. The best RECIST response was a complete response in 1/10 (10%) patients, a partial response in 5/10 (50%), stable disease in 3/10 (30%) and progression in 1/10 (10%). With a median follow-up in the alive patients from the start of therapy of 30.5 months (15.7-258), all patients progressed with a median progression-free survival of 8.47 months (95% CI 8.1-8.8), and 7/10 patients died with a median overall survival of 24.7 months (95% CI 13.7-35.6). BOMP-EPI was an active chemotherapy regimen in adults with pediatric-type metastatic RMS, with outcomes in terms of survival that seem superior to what was expected for this poor-prognosis population. Low HMGB1 expression level was identified as a predictive factor of better response to this treatment.

JNK and NFκB dependence of apoptosis induced by vinblastine in human acute promyelocytic leukaemia cells.

The relationship between the mitogen-activated protein kinase response, nuclear factor-κB (NFκB) expression and the apoptosis in human acute promyelocytic leukaemia NB4 cells treated with vinblastine was investigated in this work. Cell viability, subdiploid DNA and cell cycle were analysed by propidium iodide permeability and flow cytometry analyses. Apoptosis was determined by annexin V-Fluorescein isothiocyanate assays. Western-blot analysis was used for determination of expression levels of apoptotic factors (p53, Bax and Bcl2), intracellular kinases [serine/threonine-specific protein kinase, extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK)], NFκB factor and caspases. Electrophoretic mobility shift assay was usefully applied to study DNA-NFκB interaction. In NB4 cells, vinblastine produces alteration of p53 and DNA fragmentation. Vinblastine treatment had an antiproliferative effect via the induction of apoptosis producing Bax/Bcl-2 imbalance. Vinblastine treatment suppressed NFκB expression and depressed NFκB-DNA binding activity while maintaining JNK activation that subsequently resulted in apoptotic response through caspase-dependent pathway. Our study provides a possible anti-cancer mechanism of vinblastine action on NB4 cells by deregulation of the intracellular signalling cascade affecting to JNK activation and NFκB expression. Moreover, JNK activation and NFκB depression can be very significant factors in apoptosis induction by vinblastine.

Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B. ovis infection.

Brucella ovis causes ram contagious epididymitis, a disease for which a specific vaccine is lacking. Attenuated Brucella melitensis Rev 1, used as vaccine against ovine and caprine brucellosis caused by B. melitensis, is also considered the best vaccine available for the prophylaxis of B. ovis infection, but its use for this purpose has serious drawbacks. In this work, two previously characterized B. ovis attenuated mutants (Δomp25d and Δomp22) were evaluated in mice, in comparison with B. melitensis Rev 1, as vaccines against B. ovis. Similarities, but also significant differences, were found regarding the immune response induced by the three vaccines. Mice vaccinated with the B. ovis mutants developed anti-B. ovis antibodies in serum of the IgG1, IgG2a and IgG2b subclasses and their levels were higher than those observed in Rev 1-vaccinated mice. After an antigen stimulus with B. ovis cells, splenocytes obtained from all vaccinated mice secreted similar levels of TNF-α and IL12(p40) and remarkably high amounts of IFN-γ, a crucial cytokine in protective immunity against other Brucella species. By contrast, IL-1α -an enhancer of T cell responses to antigen- was present at higher levels in mice vaccinated with the B. ovis mutants, while IL-10, an anti-inflammatory cytokine, was significantly more abundant in Rev 1-vaccinated mice. Additionally, the B. ovis mutants showed appropriate persistence, limited splenomegaly and protective efficacy against B. ovis similar to that observed with B. melitensis Rev 1. These characteristics encourage their evaluation in the natural host as homologous vaccines for the specific prophylaxis of B. ovis infection.

Quorum-sensing and BvrR/BvrS regulation, the type IV secretion system, cyclic glucans, and BacA in the virulence of Brucella ovis: similarities to and differences from smooth brucellae.

Brucella ovis is a rough bacterium--lacking O-polysaccharide chains in the lipopolysaccharide--that is virulent in its natural host and whose virulence mechanisms remain almost unexplored. In a search for additional traits that distinguish B. ovis from smooth Brucella, which require O-polysaccharide chains for virulence, we have analyzed the significance in B. ovis of the main virulence factors described for smooth Brucella. Attempts to obtain strains of virulent B. ovis strain PA that are mutated in the BvrR/BvrS two-component regulatory system were unsuccessful, suggesting the requirement of that system for in vitro survival, while the inactivation of bacA--in contrast to the results seen with smooth Brucella--did not affect splenic colonization in mice or behavior in J774.A1 murine macrophages. Defects in the synthesis of cyclic ß-1,2 glucans reduced the uptake of B. ovis PA in macrophages and, although the intracellular multiplication rate was unaffected, led to attenuation in mice. Growth of strains with mutations in the type IV secretion system (encoded by the virB operon) and the quorum-sensing-related regulator VjbR was severely attenuated in the mouse model, and although the mutant strains internalized like the parental strain in J774.A1 murine macrophages, they were impaired for intracellular replication. As described for B. melitensis, VjbR regulates the transcription of the virB operon positively, and the N-dodecanoyl-dl-homoserine lactone (C(12)-HSL) autoinducer abrogates this effect. In contrast, no apparent VjbR-mediated regulation of the fliF flagellar gene was observed in B. ovis, probably due to the two deletions detected upstream of fliF. These results, together with others reported in the text, point to similarities between rough virulent B. ovis and smooth Brucella species as regards virulence but also reveal distinctive traits that could be related to the particular pathogenicity and host tropism characteristics of B. ovis.

FRAX® tool, the WHO algorithm to predict osteoporotic fractures: the first analysis of its discriminative and predictive ability in the Spanish FRIDEX cohort.

BACKGROUND: The WHO has recently published the FRAX® tool to determine the absolute risk of osteoporotic fracture at 10 years. This tool has not yet been validated in Spain. METHODS/DESIGN: A prospective observational study was undertaken in women in the FRIDEX cohort (Barcelona) not receiving bone active drugs at baseline. Baseline measurements: known risk factors including those of FRAX® and a DXA. Follow up data on self-reported incident major fractures (hip, spine, humerus and wrist) and verified against patient records. The calculation of absolute risk of major fracture and hip fracture was by FRAX® website. This work follows the guidelines of the STROBE initiative for cohort studies. The discriminative capacity of FRAX® was analyzed by the Area Under Curve (AUC), Receiver Operating Characteristics (ROC) and the Hosmer-Lemeshow goodness-of-fit test. The predictive capacity was determined using the ratio of observed fractures/expected fractures by FRAX® (ObsFx/ExpFx). RESULTS: The study subjects were 770 women from 40 to 90 years of age in the FRIDEX cohort. The mean age was 56.8 ± 8 years. The fractures were determined by structured telephone questionnaire and subsequent testing in medical records at 10 years. Sixty-five (8.4%) women presented major fractures (17 hip fractures). Women with fractures were older, had more previous fractures, more cases of rheumatoid arthritis and also more osteoporosis on the baseline DXA. The AUC ROC of FRAX® for major fracture without bone mineral density (BMD) was 0.693 (CI 95%; 0.622-0.763), with T-score of femoral neck (FN) 0.716 (CI 95%; 0.646-0.786), being 0.888 (CI 95%; 0.824-0.952) and 0.849 (CI 95%; 0.737-0.962), respectively for hip fracture. In the model with BMD alone was 0.661 (CI 95%; 0.583-0.739) and 0.779 (CI 95%; 0.631-0.929). In the model with age alone was 0.668 (CI 95%; 0.603-0.733) and 0.882 (CI 95%; 0.832-0.936). In both cases there are not significant differences against FRAX® model. The overall predictive value for major fracture by ObsFx/ExpFx ratio was 2.4 and 2.8 for hip fracture without BMD. With BMD was 2.2 and 2.3 respectively. Sensitivity of the four was always less than 50%. The Hosmer-Lemeshow test showed a good correlation only after calibration with ObsFx/ExpFx ratio. CONCLUSIONS: The current version of FRAX® for Spanish women without BMD analysed by the AUC ROC demonstrate a poor discriminative capacity to predict major fractures but a good discriminative capacity for hip fractures. Its predictive capacity does not adjust well because leading to underdiagnosis for both predictions major and hip fractures. Simple models based only on age or BMD alone similarly predicted that more complex FRAX® models.

Genes Involved in Immune Reinduction May Constitute Biomarkers of Response for Metastatic Melanoma Patients Treated with Targeted Therapy.

Targeted therapy in metastatic melanoma often achieves a major tumour regression response and significant long-term survival via the release of antigens that reinduce immunocompetence. The biomarkers thus activated may guide the prediction of response, but this association and its mechanism have yet to be established. Blood samples were collected from nineteen consecutive patients with metastatic melanoma before, during, and after treatment with targeted therapy. Differential gene expression analysis was performed, which identified the genes involved in the treatment, both in the first evaluation of response and during progression. Although clinical characteristics of the patients were poorer than those obtained in pivotal studies, radiological responses were similar to those reported previously (objective response rate: 73.7%). In the first tumour assessment, the expression of some genes increased (CXCL-10, SERPING1, PDL1, and PDL2), while that of others decreased (ARG1, IL18R1, IL18RAP, IL1R1, ILR2, FLT3, SLC11A1, CD163, and S100A12). The analysis of gene expression in blood shows that some are activated and others inhibited by targeted therapy. This response pattern may provide biomarkers of the immune reinduction response, which could be used to study potential combination treatments. Nevertheless, further studies are needed to validate these results.

Dequalinium induces apoptosis in peripheral blood mononuclear cells isolated from human chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is an abnormal neoplasic proliferation of B cells, which accumulate mainly in the bone marrow and blood preventing both B cells development in the lymph nodes and the ability to fight against infection. The antitumor agents used in chemotherapy are aimed at inducing malignant cell death, thus limiting the growth and spreading of these cells. However, the lack of specificity for tumor cells exhibited by these agents causes undesirable side effects that have led to the investigation of new therapeutic strategies designed to specifically target malignant cells and thus trigger selective cell destruction. Dequalinium (DQA) is an antitumoral agent that selectively accumulates in the mitochondria and has been shown to display anticancer activity in cells from different malignancies. In the present study, the DQA-induced cytotoxicity in B-CLL cells was analyzed by measuring cell viability and cell death, either by necrosis or apoptosis. Our results support the importance of DQA as a selective and potential antileukemic drug with a higher cytotoxic effect on peripheral blood mononuclear cells from B-CLL patients than in those from healthy donors and encourage the performance of further studies in combination with other agents.

Measuring health-related quality of life in men with osteoporosis or osteoporotic fracture.

BACKGROUND: Osteoporosis is a serious health problem that worsens the quality of life and the survival rate of individuals with this disease on account the osteoporotic fractures. Studies have long focused on women, and its presence in men has been underestimated. While many studies conducted in different countries mainly assess health-related quality of life and identify fracture risks factors in women, few data are available on a Spanish male population. METHODS/DESIGN: Observational study. STUDY POPULATION: Men ≥ 40 years of age with/without diagnosed osteoporosis and with/without osteoporotic fracture included by their family doctor. MEASUREMENTS: The relationship between customary clinical risk factors for osteoporotic fracture and health-related quality of life in a Spanish male population. A telephone questionnaire on health-related quality of life is made. STATISTICAL ANALYSIS: The association between qualitative variables will be assessed by the Chi-square test. The distribution of quantitative variables by Student's t-test. If the conditions for using this test are not met, the non-parametric Mann-Whitney's U test will be used.The validation of the results obtained by the FRAX™ tool will be performed by way of the Hosmer-Lemeshow test and by calculating the area under the Receiver Operating Characteristic (ROC) curve (AUC). All tests will be performed with a confidence intervals set at 95%. DISCUSSION: The applicability and usefulness of Health-related quality of life (HRQOL) studies are well documented in many countries. These studies allow implementing cost-effective measures in cases of a given disease and reducing the costly consequences derived therefrom. This study attempts to provide objective data on how quality of life is affected by the clinical aspects involved in osteoporosis in a Spanish male population and can be useful as well in cost utility analyses conducted by health authorities.The sample selected is not based on a high fracture risk group. Rather, it is composed of men in the general population, and accordingly comparisons should not lead to erroneous interpretations.A possible bias correction will be ensured by checking reported fractures against healthcare reports and X-rays, or by consulting health care centers as applicable.

Prediction of absolute risk of fragility fracture at 10 years in a Spanish population: validation of the WHO FRAX ™ tool in Spain.

BACKGROUND: Age-related bone loss is asymptomatic, and the morbidity of osteoporosis is secondary to the fractures that occur. Common sites of fracture include the spine, hip, forearm and proximal humerus. Fractures at the hip incur the greatest morbidity and mortality and give rise to the highest direct costs for health services. Their incidence increases exponentially with age.Independently changes in population demography, the age - and sex- specific incidence of osteoporotic fractures appears to be increasing in developing and developed countries. This could mean more than double the expected burden of osteoporotic fractures in the next 50 years. METHODS/DESIGN: To assess the predictive power of the WHO FRAX™ tool to identify the subjects with the highest absolute risk of fragility fracture at 10 years in a Spanish population, a predictive validation study of the tool will be carried out. For this purpose, the participants recruited by 1999 will be assessed. These were referred to scan-DXA Department from primary healthcare centres, non hospital and hospital consultations. STUDY POPULATION: Patients attended in the national health services integrated into a FRIDEX cohort with at least one Dual-energy X-ray absorptiometry (DXA) measurement and one extensive questionnaire related to fracture risk factors. MEASUREMENTS: At baseline bone mineral density measurement using DXA, clinical fracture risk factors questionnaire, dietary calcium intake assessment, history of previous fractures, and related drugs. Follow up by telephone interview to know fragility fractures in the 10 years with verification in electronic medical records and also to know the number of falls in the last year. The absolute risk of fracture will be estimated using the FRAX™ tool from the official web site. DISCUSSION: Since more than 10 years ago numerous publications have recognised the importance of other risk factors for new osteoporotic fractures in addition to low BMD. The extension of a method for calculating the risk (probability) of fractures using the FRAX™ tool is foreseeable in Spain and this would justify a study such as this to allow the necessary adjustments in calibration of the parameters included in the logarithmic formula constituted by FRAX™.

Durable Response to Vemurafenib and Cobimetinib for the Treatment of BRAF-Mutated Metastatic Melanoma in Routine Clinical Practice.

BACKGROUND: The combination of BRAF and MEK inhibitors delays the onset of resistance and provides more sustained and dramatic responses in comparison with a BRAF inhibitor in monotherapy. The objective of the study was to evaluate the effectiveness of the combination therapy with vemurafenib/cobimetinib in terms of durability, and to describe differential characteristics in patients associated to durable responses in real-world settings. PATIENTS AND METHODS: Retrospective, observational, cross-sectional, multicenter study involving 41 patients with advanced melanoma harboring a BRAF V600 mutation who initiated a combination therapy with vemurafenib/cobimetinib between May 2018 and March 2019. Participants were differentiated regarding the durability of the response: durable (complete response, CR, or a partial response, PR, for at least 12 months) and non-durable (stable disease, SD, progressive disease, PD, or CR/PR <12 months). Secondary endpoints included treatment adherence, labor productivity, anxiety/depression, and safety profile. RESULTS: During the combination therapy, 12 patients (29.3%) had a CR, 19 a PR (46.3%), 5 showed SD (12.2%), and 5 had PD. A total of 12 patients (29.3%) were considered as achieving a durable response and 29 (70.7%) as a non-durable one. Practically all sociodemographic and clinical characteristics were similar between patients. Body mass index was the only differential factor (with higher body mass index achieving a non-durable response). The treatment adherence was 100% in patients with durable response and 66.7% in those with non-durable. CONCLUSION: The combination treatment with vemurafenib/cobimetinib results in an important impact on long-term survival, leading to a steady CR in one-third of the patients.

Erratum: Hindi, N., et al. Trabectedin Plus Radiotherapy for Advanced Soft-Tissue Sarcoma: Experience in Forty Patients Treated at a Sarcoma Reference Center. Cancers 2020, 12, 3740.

The authors noticed that content of "Conflicts of Interest" in the original version [...].

Trabectedin Plus Radiotherapy for Advanced Soft-Tissue Sarcoma: Experience in Forty Patients Treated at a Sarcoma Reference Center.

Symptomatic control and tumoral shrinkage is an unmet need in advanced soft-tissue sarcoma (STS) patients beyond first-line. The combination of trabectedin and radiotherapy showed activity in a recently reported clinical trial in this setting. This retrospective series aims to analyze our experience with the same regimen in the real-life setting. We retrospectively reviewed advanced sarcoma patients treated with trabectedin concomitantly with radiotherapy with palliative intent. Growth-modulation index (GMI) was calculated as a surrogate of efficacy. Forty metastatic patients were analyzed. According to RECIST, there was one (2.5%) complete response, 12 (30%) partial responses, 18 (45%) disease stabilizations, and nine (22.5%) progressions. After a median follow-up of 15 months (range 2-38), median progression-free survival (PFS) and overall survival (OS) were 7.5 months (95% CI 2.8-12.2) and 23.5 months (95% CI 1.1-45.8), respectively. Median GMI was 1.42 (range 0.19-23.76), and in 16 (53%) patients, it was >1.33. In patients with GMI >1.33, median OS was significantly longer than in those with GMI 0-1.33 (median OS 52.1 months (95% CI not reached) vs. 8.9 months (95% CI 6.3-11.6), p = 0.028). The combination of trabectedin plus radiotherapy is an active therapeutic option in patients with advanced STS, especially when tumor shrinkage for symptomatic relief is needed.

Analysis of the occurrence and distribution of the Omp25/Omp31 family of surface proteins in the six classical Brucella species.

Members of the Omp25/Omp31 family of surface proteins were previously shown to participate in the virulence of some Brucella species and a different distribution of the seven proteins of this family among species could be related to the difference in pathogenicity and host preference they exhibit. Accordingly, in this work we have analyzed the expression of the genes coding for the Omp25/Omp31 family in the six classical Brucella species and a set of B. ovis mutant strains with each omp gene inactivated. Immunoblot of whole-cell lysates with antibodies raised against the purified recombinant outer membrane proteins (OMPs) did not show the simultaneous presence of the seven OMPs in any of the Brucella strains studied, but different Omp25/Omp31 profiles were detected, in our experimental conditions, between the Brucella strains representative of the six classical species. Transcripts for omp31, omp25 and omp25c were, in general, the most abundant of the family and some hits were found in B. ovis for a posttranscriptional regulation mechanism and for a compensatory mechanism increasing the synthesis of a protein to compensate for the absence of another one. Finally, the potential interest of Omp25c and Omp31b as subcellular vaccines, considering their occurrence in the Brucella strains studied and their antigenic relatedness with other proteins of the family, is discussed.

Evaluation of human trophoblasts and ovine testis cell lines for the study of the intracellular pathogen Brucella ovis.

Since pathogenic Brucella survive and replicate inside phagocytes, cellular models of infection constitute important tools in brucellosis research. We describe the behavior of B. ovis PA (which causes a type of ovine brucellosis mainly affecting the male reproductive tract) and representative attenuated mutants in two commercially available cell lines of non-professional phagocytes related to Brucella tissue preference: OA3.Ts ovine testis cells and JEG-3 human trophoblasts. In comparison with J774.A1 macrophages and HeLa cells, intracellular bacteria were enumerated at several post-infection time points and visualized by confocal microscopy. Replication of B. ovis in OA3.Ts and JEG-3 cells was equivalent to that observed in J774.A1 macrophages-despite the more efficient internalization in the latter-and better than in HeLa cells. Multiplication and/or survival in all phagocytes was dependent on virB2 and vjbR but independent of cgs, despite the attenuation in mice of the Δcgs mutant. However, Omp25c was required for B. ovis internalization only in HeLa cells, and removal of Omp31 increased bacterial internalization in human HeLa and JEG-3 cells. The results presented here demonstrate variability in the interaction of B. ovis with different host cells and provide advantageous models of non-professional phagocytes to study the intracellular behavior of B. ovis.

Characterization of Cell Envelope Multiple Mutants of Brucella ovis and Assessment in Mice of Their Vaccine Potential.

Brucella ovis is a non-zoonotic Brucella species lacking specific vaccine. It presents a narrow host range, a unique biology relative to other Brucella species, and important distinct surface properties. To increase our knowledge on its peculiar surface and virulence features, and seeking to develop a specific vaccine, multiple mutants for nine relevant cell-envelope-related genes were investigated. Mutants lacking Omp10 plus Omp19 could not be obtained, suggesting that at least one of these lipoproteins is required for viability. A similar result was obtained for the double deletion of omp31 and omp25 that encode two major surface proteins. Conversely, the absence of major Omp25c (proved essential for internalization in HeLa cells) together with Omp25 or Omp31 was tolerated by the bacterium. Although showing important in vitro and in vivo defects, the Δomp10Δomp31Δomp25c mutant was obtained, demonstrating that B. ovis PA survives to the simultaneous absence of Omp10 and four out seven proteins of the Omp25/Omp31 family (i.e., Omp31, Omp25c, Omp25b, and Omp31b, the two latter naturally absent in B. ovis). Three multiple mutants were selected for a detailed analysis of virulence in the mouse model. The Δomp31Δcgs and Δomp10Δomp31Δomp25c mutants were highly attenuated when inoculated at 106 colony forming units/mouse but they established a persistent infection when the infection dose was increased 100-fold. The Δomp10ΔugpBΔomp31 mutant showed a similar behavior until week 3 post-infection but was then totally cleared from spleen. Accordingly, it was retained as vaccine candidate for mice protection assays. When compared to classical B. melitensis Rev1 heterologous vaccine, the triple mutant induced limited splenomegaly, a significantly higher antibody response against whole B. ovis PA cells, an equivalent memory cellular response and, according to spleen colonization measurements, better protection against a challenge with virulent B. ovis PA. Therefore, it would be a good candidate to be evaluated in the natural host as a specific vaccine against B. ovis that would avoid the drawbacks of B. melitensis Rev1. In addition, the lack in this attenuated strain of Omp31, recognized as a highly immunogenic protein during B. ovis infection, would favor the differentiation between infected and vaccinated animals using Omp31 as diagnostic target.

Dequalinium induces cell death in human leukemia cells by early mitochondrial alterations which enhance ROS production.

Dequalinium (DQA) has been proposed as a selective antitumoral agent due to its preferential accumulation in mitochondria of cancer cells. Our aim was a better understanding of DQA cytotoxicity. DQA-induced NB4 and K562 cell alterations are initiated within the first 30 min of treatment at a high DQA concentration with a mitochondrial membrane depolarization. Cytochrome c release to cytoplasm, superoxide anion overproduction and ATP depletion in NB4 cells induce, 16 h later, apoptosis by a typical caspase-9/caspase-3-dependent intrinsic pathway. K562 cells were more resistant to the DQA effect than NB4 cells, remaining viable for longer time periods.

Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae.

Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cell lines. Mitochondrial implication in cell death.

Dequalinium (DQA) is a delocalized lipophylic cation that selectively targets the mitochondria of carcinoma cells. However, the underlying mechanisms of DQA action are not yet well understood. We have studied the effects of DQA on two different leukemia cell lines: NB4, derived from acute promyelocytic leukemia, and K562, derived from chronic myeloid leukemia. We found that DQA displays differential cytotoxic activity in these cell lines. In NB4 cells, a low DQA concentration (2microM) induces a mixture of apoptosis and necrosis, whereas a high DQA concentration (20microM) induces mainly necrosis. However, K562 cell death was always by necrosis as the cells showed a resistance to apoptosis at all time-periods and DQA concentrations assayed. In both cell lines, the cell death seems to be mediated by alterations of mitochondrial function as evidenced by loss of mitochondrial transmembrane potential, O2*- accumulation and ATP depletion. The current study improves the knowledge on DQA as a novel anticancer agent with a potential application in human acute promyelocytic leukemia chemotherapy.

In vivo biodistribution of erythrocytes and polyethyleneglycol-phosphatidylethanolamine micelles carrying the antitumour agent dequalinium.

Dequalinium (DQA), a lipophilic drug with anti-cancer activity has been incorporated into mouse red blood cells (DQA-RBCs) and polyethylene glycol phosphatidylethanolamine micelles (DQA-PEG-PE-micelles) in order to overcome the drug's solubility problems and to make it suitable for in vivo applications. The incorporation of DQA into erythrocytes, the release of DQA from RBCs in the presence of autologous plasma and the biodistribution of 51Cr-DQA-RBCs and 111In-DQA-PEG-PE micelles in mice has been studied. Under optimal conditions, up to 84.9% of 0.2 mM dequalinium can be incorporated into erythrocytes. The incubation of DQA-RBC with serum leads to the release of DQA over a period of 24 h. Since 51Cr-DQA-RBCs were found to have a long circulation half-life (5-6 days), the use of RBCs as a sustained release system for DQA can be suggested. In contrast to DQA containing erythrocytes, however, DQA loaded 111In-PEG-PE micelles displayed a shorter half-life (4 h) due to their quick uptake by the liver. The further exploration of PEG-PE micelles as a fast acting release system for DQA appears warranted.

Microbiological characterization of food residues for animal feeding.

A description is offered of microbiological characterization of the biodegradable fractions present in food wastes so that those fractions can be transformed in such a way that they will fulfil the specifications involved in their use as raw materials in other production areas. In this way the wastes can be converted into sub-products, hence minimizing the amount of them eventually sent to rubbish dumps. Of all the types of residues analyzed, only those obtained by separate collection from fishmongers' and greengrocers' sections of large supermarkets and small shops were valid for the objectives of the project and were subjected to a heat treatment to test whether or not this treatment was capable of reducing their microbiological content to the point of converting them into acceptable raw materials for animal feed. Residues from butchers' sections of supermarkets and small shops, and residues from restaurants were not included in the final study because of the prohibition by the European legislation in force of using any kind of meat containing wastes for feeding farm animals. In the present work we made a one-year analysis of representative samples of such wastes. We observed that after thermal treatment at a temperature of at least 65 degrees C for 20 min the nutritional and microbiological parameters remained suitable for their possible use as animal feed and that their harmlessness was ensured, with no loss of nutritional characteristics. Regarding the microbiological study of the meals which have been obtained from residues for the production of the feed and the feed itself, and in accordance with the data for nutritional composition, we consider valid and sanitarily adequate their use as animal feed with the concomitant consequent minimization of waste, which has become a priority in view of the recent legislation enacted by the European Union.