Insights into the evolution of the new variant rabbit haemorrhagic disease virus (GI.2) and the identification of novel recombinant strains.

Rabbit haemorrhagic disease (RHD) is a viral disease that affects the European rabbit. RHD was detected in 1984 in China and rapidly disseminated worldwide causing a severe decline in wild rabbit populations. The aetiological agent, rabbit haemorrhagic disease virus (RHDV), is an RNA virus of the family Caliciviridae, genus Lagovirus. Pathogenic (G1-G6 or variants GI.1a-GI.1d) and non-pathogenic strains (GI.4) have been characterized. In 2010, a new variant of RHDV, RHDV2/RHDVb/GI.2, was detected in France. GI.2 arrived to the Iberian Peninsula in 2011, and several recombination events were reported. Here, we sequenced full genomes of 19 samples collected in Portugal between 2014 and 2016. New GI.2 recombinant strains were detected, including triple recombinants. These recombinants possess a non-structural protein p16 related to a non-pathogenic strain. Evolutionary analyses were conducted on GI.2 VP60 sequences. Estimated time to the most recent common ancestor (tMRCA) suggests an emergence of GI.2 in July 2008, not distant from its first detection in 2010. This is the first study on GI.2 evolution and highlights the need of continued monitoring and characterization of complete genome sequences when studying lagoviruses' evolution.

Epidemiology of RHDV2 (Lagovirus europaeus/GI.2) in free-living wild European rabbits in Portugal.

As the detection of the first outbreak of a novel aetiological agent of rabbit haemorrhagic disease commonly called RHDV2 or RHDVb (Lagovirus europaeus/GI.2, henceforth GI.2) in France in 2010, the virus rapidly spread throughout continental Europe and nearby islands such as Great Britain, Sardinia, Sicily, the Azores and the Canary Islands among others. The outbreaks of this new lagovirus cause important economic losses in rabbitries, and ecological disruptions by affecting the conservation of rabbit-sensitive top predators. We analysed 550 rabbit carcasses collected in the field between May 2013 and March 2016, to investigate the epidemiology of GI.2 in free-living populations and to perform a comparative analysis with the epidemiology of classical rabbit haemorrhagic disease virus forms (RHDV, henceforth GI.1) in Portugal. Rabbits were sexed, aged and liver and blood samples were collected for subsequent RHDV screening and serology. A total of 172 samples were PCR-positive to GI.2, whereas GI.1 strains were not detected in any of the samples. The outbreaks of GI.2 revealed a marked seasonality, with peaks during the breeding season (November-May). We also found that approximately, one-third of free-ranging European rabbits in Portugal have seroconverted to GI.2. We demonstrate that the GI.2 lagovirus is currently widespread in wild populations in Portugal and is affecting a high proportion of adults and juveniles. Therefore, ongoing monitoring and surveillance are required to assess the effects of GI.2 on wild rabbit populations, its evolution, and to guide management actions aimed at mitigating the impacts of rabbit declines in the ecosystem and in rural economies.

Weekly docetaxel versus CMF as adjuvant chemotherapy for older women with early breast cancer: final results of the randomized phase III ELDA trial.

BACKGROUND: Evidence on adjuvant chemotherapy in older women with breast cancer is poor. We tested whether weekly docetaxel is more effective than standard chemotherapy. PATIENTS AND METHODS: We carried out a multicenter, randomized phase III study. Women aged 65-79, operated for breast cancer, with average to high risk of recurrence, were allocated 1 : 1 to CMF (cyclophosphamide 600 mg/m², methotrexate 40 mg/m², fluorouracil 600 mg/m², days 1, 8) or docetaxel (35 mg/m(2) days 1, 8, 15) every 4 weeks, for four or six cycles according to hormone receptor status. Primary end point was disease-free survival (DFS). A geriatric assessment was carried out. Quality of life (QoL) was assessed with EORTC C-30 and BR-23 questionnaires. RESULTS: From July 2003 to April 2011, 302 patients were randomized and 299 (152 allocated CMF and 147 docetaxel) were eligible. After 70-month median follow-up, 109 DFS events were observed. Unadjusted hazard ratio (HR) of DFS for docetaxel versus CMF was 1.21 [95% confidence interval (CI) 0.83-1.76, P = 0.32]; DFS estimate at 5 years was 0.69 with CMF and 0.65 with docetaxel. HR of death was 1.34 (95% CI 0.80-2.22, P = 0.26). There was no interaction between treatment arms and geriatric scales measuring patients' ability or comorbidities. Hematological toxicity, mucositis and nausea were worse with CMF; allergy, fatigue, hair loss, onychopathy, dysgeusia, diarrhea, abdominal pain, neuropathy, cardiac and skin toxicity were worse with docetaxel. One death was attributed to CMF and two to docetaxel. Increasing age, impairment in instrumental daily living activities, number of comorbidities and docetaxel treatment were independently associated with severe nonhematological toxicity. QoL was worse with docetaxel for nausea-vomiting, appetite loss, diarrhea, body image, future perspective, treatment side-effects and hair loss items. CONCLUSIONS: Weekly docetaxel is not more effective than standard CMF as adjuvant treatment of older women with breast cancer and worsens QoL and toxicity. CLINICALTRIALSGOV: NCT00331097.

Bone effect of adjuvant tamoxifen, letrozole or letrozole plus zoledronic acid in early-stage breast cancer: the randomized phase 3 HOBOE study.

BACKGROUND: To measure bone mineral density (BMD) reduction produced by letrozole as compared with tamoxifen and the benefit of the addition of zoledronic acid. PATIENTS AND METHODS: A phase 3 trial comparing tamoxifen, letrozole or letrozole+zoledronic acid in patients with hormone receptor-positive early breast cancer was conducted; triptorelin was given to premenopausal patients. Two comparisons were planned: letrozole versus tamoxifen and letrozole+zoledronic acid versus letrozole. Primary end point was the difference in 1-year change of T-score at lumbar spine (LTS) measured by dual energy X-ray absorptiometry scan. RESULTS: Out of 483 patients enrolled, 459 were available for primary analyses. Median age was 50 (range 28-80). The estimated mean difference (95% confidence interval [CI]) in 1-year change of LTS was equal to -0.30 (95% CI -0.44 to -0.17) in the letrozole versus tamoxifen comparison (P<0.0001) and to +0.60 (95% CI +0.46 to +0.77) in the letrozole+zoledronic acid versus letrozole comparison (P<0.0001). Bone damage by letrozole decreased with increasing baseline body mass index in premenopausal, but not postmenopausal, patients (interaction test P=0.004 and 0.47, respectively). CONCLUSIONS: In the HOBOE (HOrmonal BOne Effects) trial, the positive effect of zoledronic acid on BMD largely counteracts damage produced by letrozole as compared with tamoxifen. Letrozole effect is lower among overweight/obese premenopausal patients.

No evidence that wild red deer (Cervus elaphus) on the Iberian Peninsula are a reservoir of Mycobacterium avium subspecies paratuberculosis infection.

The potential role of red deer (Cervus elaphus) as a reservoir of Mycobacterium avium subspecies paratuberculosis (MAP) infection is largely unknown. A total of 332 wild red deer were investigated using post-mortem examination, bacteriology and serology. Only three animals (1.12%) were found to have lesions on histopathological examination and no MAP bacteria were recovered on culture. The results suggest it is unlikely that wild red deer make a significant contribution to the maintenance of MAP infection in the region. The cross-reactivity of the ELISAs used indicates this diagnostic modality is ineffective in the detection of MAP infection in this species. The implications of these results for the control of this important pathogen in both livestock and wildlife are discussed.

Processing/structure/property relationship of multi-scaled PCL and PCL-HA composite scaffolds prepared via gas foaming and NaCl reverse templating.

In this study, we investigated the processing/structure/property relationship of multi-scaled porous biodegradable scaffolds prepared by combining the gas foaming and NaCl reverse templating techniques. Poly(ε-caprolactone) (PCL), hydroxyapatite (HA) nano-particles and NaCl micro-particles were melt-mixed by selecting different compositions and subsequently gas foamed by a pressure-quench method. The NaCl micro-particles were finally removed from the foamed systems in order to allow for the achievement of the multi-scaled scaffold pore structure. The control of the micro-structural properties of the scaffolds was obtained by the optimal combination of the NaCl templating concentration and the composition of the CO2-N2 mixture as the blowing agent. In particular, these parameters were accurately selected to allow for the fabrication of PCL and PCL-HA composite scaffolds with multi-scaled open pore structures. Finally, the biocompatibility of the scaffolds has been assessed by cultivating pre-osteoblast MG63 cells in vitro, thus demonstrating their potential applications for bone regeneration.

Consequences of targeted treatments for second-line therapy.

The paradigm for first-line treatment of relapsed or metastatic non-small cell lung cancer (NSCLC) is changing. Large phase III trials demonstrated that, in 2010, we cannot select a therapy without an accurate definition of tumor histology and epidermal growth factor receptor (EGFR) status. Patients harboring an EGFR-activating mutation have a better prognosis and certainly are extremely sensitive to EGFR-tyrosine kinase inhibitors, while other agents, such as bevacizumab or pemetrexed, are more effective and less toxic in patients with non-squamous histology. Moreover, data from large phase III trials demonstrated that maintenance therapy with pemetrexed, docetaxel or erlotinib is an effective strategy against metastatic NSCLC. Overall, the changing paradigm in first-line treatment of NSCLC inevitably is changing the second-line strategy. In addition, the emerging role of maintenance therapy is leading to early use of all agents potentially active in a second- or third-line setting, with the consequence that very few options are available at disease progression. The aim of this article is to discuss the consequences of targeted treatments for second-line therapy in metastatic NSCLC.

Design of porous polymeric scaffolds by gas foaming of heterogeneous blends.

One of the challenges in tissue engineering scaffold design is the realization of structures with a pre-defined multi-scaled porous network. Along this line, this study aimed at the design of porous scaffolds with controlled porosity and pore size distribution from blends of poly(epsilon-caprolactone) (PCL) and thermoplastic gelatin (TG), a thermoplastic natural material obtained by de novo thermoplasticization of gelatin. PCL/TG blends with composition in the range from 40/60 to 60/40 (w/w) were prepared by melt mixing process. The multi-phase microstructures of these blends were analyzed by scanning electron microscopy and dynamic mechanical analysis. Furthermore, in order to prepare open porous scaffolds for cell culture and tissue replacement, the TG and PCL were selectively extracted from the blends by the appropriate combination of solvent and extraction parameters. Finally, with the proposed combination of gas foaming and selective polymer extraction technologies, PCL and TG porous materials with multi-scaled and highly interconnected porosities were designed as novel scaffolds for new-tissue regeneration.

Engineered mu-bimodal poly(epsilon-caprolactone) porous scaffold for enhanced hMSC colonization and proliferation.

The use of scaffold-based strategies in the regeneration of biological tissues requires that the design of the microarchitecture of the scaffold satisfy key microstructural and biological requirements. Here, we examined the ability of a porous poly(epsilon-caprolactone) (PCL) scaffold with novel bimodal-micron scale (mu-bimodal) porous architecture to promote and guide the in vitro adhesion, proliferation and three-dimensional (3-D) colonization of human mesenchymal stem cells (hMSCs). The mu-bimodal PCL scaffold was prepared by a combination of gas foaming (GF) and selective polymer extraction (PE) from co-continuous blends. The microarchitectural properties of the scaffold, in particular its morphology, porosity distribution and mechanical compression properties, were analyzed and correlated with the results of the in vitro cell-scaffold interaction study, carried out for 21days under static conditions. Alamar Blue assay, scanning electron microscopy, confocal laser scanning microscopy and histological analyses were performed to assess hMSC adhesion, proliferation and 3-D colonization. The results showed that the combined GF-PE technique allowed the preparation of PCL scaffold with a unique multiscaled and highly interconnected microarchitecture that was characterized by mechanical properties suitable for load-bearing applications. Study of the cell-scaffold interaction also demonstrated the ability of the scaffold to support hMSC adhesion and proliferation, as well as the possibility to promote and guide 3-D cell colonization by appropriately designing the microarchitectural features of the scaffold.

p53 and Ki-67 expression in renal cell carcinomas of pregnant women and their correlation with prognosis: a pilot study.

In the present study, we reported two cases of renal cell carcinoma (RCC) diagnosed in pregnant women (Pt) that were submitted to radical nephrectomy, in both cases within the fourth month. The patients, after 13 and 3 years, respectively, did not show evidence of recurrent disease. We performed an immunohistochemical study on RCC specimens in comparison to seven age-matched controls (Cl). The panel of antibodies included Ki-67, p53, bcl-2, ER, PgR, PCNA, and IGF-1. We describe a difference in the expression of p53 and Ki-67. Specifically, p53 was highly expressed in RCC of both Pt but scarcely present or absent in Cl; by contrast, Ki-67 was hardly expressed or negative in RCC of both Pt, being commonly positive in Cl. These results may correlate with a good outcome of the disease in Pt. Although the limited number of cases did not permit any statistical evaluation, we postulate that these differences have not to be underestimated since they may disclose a correlation between pregnancy and biological behavior of tumoral disease. Further study may (dis)prove this hypothesis.

Methodology of clinical trials with new molecular-targeted agents: where do we stand?

In recent years, we have witnessed growing interest in the methodology of clinical trials with molecular-targeted agents. In phase I studies, alternative end points to toxicity have been proposed to define the optimal biological dose: the identification of a 'target effect', the measurement of 'surrogates' for biological activity and the assessment of drug plasma levels. However, these end points are not routinely incorporated into the study design and have rarely formed the primary basis for dose selection. In phase II studies, response rate remains the preferred end point in the early evaluation of new drugs. However, this approach might lead to rejection of potentially useful drugs when significant tumor shrinkage cannot be demonstrated. Therefore, a number of alternative end points have been proposed for agents that are not expected to cause a major tumor regression: time to progression, progression-free survival, overall survival, early progression rate and growth modulation index. In phase III trials, where efficacy in terms of survival remains the most important goal of the research, the major issues are the adequate selection of patients and the optimal clinical setting of evaluation of drugs. In conclusion, many important questions regarding the methodology of clinical research with target-based agents remain open and need to be defined by research in the near future.

Is epirubicin effective in first-line chemotherapy of metastatic breast cancer (MBC) after an epirubicin-containing adjuvant treatment? A single centre phase III trial.

The aim of the study was to demonstrate the superiority of docetaxel and epirubicin vs docetaxel alone as first-line therapy in metastatic breast cancer patients pretreated with adjuvant or neoadjuvant epirubicin. We compared single agent docetaxel 100 mg m-2 (D) with the combination of docetaxel 80 mg m-2 and epirubicin 75 mg m-2 (ED). The response rate (72 vs 79%), the progression-free survival (median 9 vs 11 months) and the overall survival (median 18 vs 21 months) were not significantly different between the ED (n=26) and D arms (n=25), respectively. Leucopaenia, nausea and stomatitis were significantly worse with ED. In conclusion, epirubicin should not be administered in combination with taxanes in metastatic breast cancer patients relapsed after an anthracycline-based adjuvant or neoadjuvant therapy.

Supportive care in patients with advanced non-small-cell lung cancer.

The present study describes supportive care (SC) in patients with advanced non-small-cell lung cancer (NSCLC), evaluating whether it is affected by concomitant chemotherapy, patient's performance status (PS) and age. Data of patients enrolled in three randomised trials of first-line chemotherapy, conducted between 1996 and 2001, were pooled. The analysis was limited to the first three cycles of treatment. Supportive care data were available for 1185 out of 1312 (90%) enrolled patients. Gastrointestinal drugs (45.7%), corticosteroids (33.4%) and analgesics (23.8%) were the most frequently observed categories. The mean number of drugs per patient was 2.43; 538 patients (45.4%) assumed three or more supportive drugs. Vinorelbine does not produce substantial variations in the SC pattern, while cisplatin-based treatment requires an overall higher number of supportive drugs, with higher use of antiemetics (41 vs 27%) and antianaemics (10 vs 4%). Patients with worse PS are more exposed to corticosteroids (42 vs 30%). Elderly patients require drugs against concomitant diseases significantly more than adults (20 vs 7%) and are less frequently exposed to antiemetics (12 vs 27%). In conclusion, polypharmacotherapy is a relevant issue in patients with advanced NSCLC. Chemotherapy does not remarkably affect the pattern of SC, except for some drugs against side effects. Elderly patients assume more drugs for concomitant diseases and receive less antiemetics than adults.

Tamoxifen in the treatment of hepatocellular carcinoma: 5-year results of the CLIP-1 multicentre randomised controlled trial.

BACKGROUND: In 1998, when data of a meta-analysis on tamoxifen in the treatment of hepatocellular carcinoma (HCC) had suggested a little advantage for this treatment, we published the results of a multicenter randomised controlled trial, that showed no survival benefit for tamoxifen vs. control. Here we report an updated analysis of the study results 4.5 years after the closure of enrollment. METHODS: The study had a planned sample size of 480 patients. Patients with any stage HCC were eligible, irrespective of locoregional treatment. Tamoxifen was given orally, 40 mg/die, from randomisation until death. RESULTS: 496 patients were randomised by 30 Institutions from January 1995 to January 1997. Information was available for 477 patients. As of July 2001, 374 deaths (78%) were recorded, and median survival times were 16 and 15 months (p=0.54), in the control and tamoxifen arm. Data were further analysed separately for advanced patients and for those eligible to potentially curative locoregional treatments: relative hazard of death for patients receiving tamoxifen was equal to 0.98 (95% CI 0.76-1.25) for the former group and 1.38 (95% CI 0.95-2.01) for the latter. The prognostic score recently devised by our group (CLIP score) was, as expected, strictly correlated (p<0.0001) to the locoregional treatment received and strongly correlated with prognosis. CONCLUSIONS: the update of the present study confirms that tamoxifen is not effective in prolonging survivals, both in advanced patients and in those potentially curable and that the CLIP score is able to predict prognosis.

Recurrent oral condylomata acuminata and hairy leukoplakia: an early sign of myelodysplastic syndrome in an HIV-seronegative patient.

Oral hairy leukoplakia (OHL) has been observed in all risk groups seropositive for HIV infection. Recently, this lesion has also been described in HIV-seronegative patients with immunosuppression of iatrogenic origin. We report on a HIV-1 and HIV-2 seronegative, heterosexual man affected by refractory anemia with ringed sideroblasts (myelodysplastic syndrome), who developed recurrent oral condylomata acuminata and OHL as an early clinical manifestation. The diagnosis of OHL was confirmed by identifying Epstein-Barr viral particles by electron microscopy and by in situ DNA hybridization. HIV infection was ruled out using polymerase chain reaction and testing for HIV-1 and HIV-2 antibodies.

Intrathecal synthesis of anti-HIV antibodies in AIDS patients.

We studied the production within the CNS of anti-HIV antibodies, of non-specific IgG, and the presence of HIV antigens in the serum and CSF of 28 HIV infected patients belonging to group IV in the Center for Disease Control classification. CSF and serum were diluted under optimal conditions to equalize their IgG content, to enable us to better interpret serum and CSF reactivity by means of Western blot and ELISA. Under these conditions, no patient displayed a limited immunological response profile in CSF as compared to serum. On the contrary, there was intrathecal synthesis (ITS) of anti HIV-antibodies in Western blot test in 21 patients for gp160 and ITS was demonstrable for env, gag, and pol products. ITS of anti-HIV antibodies occurred in 17 patients when measured by ELISA. ITS of non specific IgG and HIV-antigens in CSF were less frequent. A marked anti-HIV response is evident in the CSF-CNS compartment in the later phases of the HIV infection.

Modification of muscle proteins IEF patterns in a fish Lebistes reticulatus, related to diet, immunization and genetic selection.

The effects of some environmental and genetic factors on the isoelectric focusing (IEF) patterns of muscle proteins from a fish Lebistes reticulatus (Cyprinodonts) were investigated. Of two diets applied the restricted one resulted in a loss of bands in the IEF patterns of the treated fishes muscle homogenate. Instead immunization gave an increase in both number and intensity of IEF bands in (4.0-6.0; 7.0-7.9) pH ranges which are typical of immunoglobulins. Lastly IEF patterns from random and assortative mating animals were compared, resulting in loss of IEF-bands in the inbred lines. Results showed that IEF of muscle proteins could be an excellent method to evaluate the response by fish phenotypes to environmental or genetic factors.