After a long period of growth, which started in 1994, the Spanish economy entered into a deep structural crisis in 2007. The discussion of the reasons for this situation and the requisite policy response has been intense, with wide variations of opinion. From the conventional point of view, the crisis is mainly a problem of ‘market disequilibrium’. Others believe that there are structural problems related mainly to the weakness of the structure of production. The eruption of the international fi nancial crisis led to the end of a model centred on construction and tourism as its growth drivers, with no other activities looming on the horizon to replace them. The fall in production was refl ected in the labour market in unprecedented unemployment rates and an increase in poverty and inequality that opened a social divide that will be diffi cult to close.

Purpose: Given the association of Toll-like receptor (TLR)-4 to calcific aortic stenosis and the relevant function of the lipid mediator sphingosine-1-phosphate (S1P) in the cardiovascular system, the putative role of TLR4 ligands and S1P in the induction of pro-inflammatory and pro-adhesive responses in endothelial cells from human aortic (AVEC) and pulmonary valves (PVEC) was investigated. Methods: AVEC and PVEC obtained from n =5 heart transplant patients with no valve disease, and AVEC derived from stenotic aortic valves collected from n = 5 patients with degenerative aortic stenosis and indication for valve replacement were studied. Reverse transcriptase PCR assays were used to evaluate the mRNA expression of TLR and S1P receptors. Cytokine secretion and adhesion molecule expression were evaluated by antibody arrays and Western blot, and adhesion of human monocytes to AVEC monolayers was assayed in vitro. Comparison between stenotic and control aortic valve and between aortic and pulmonar valve were performed. Results: Reverse transcriptase PCR assays revealed the expression of TLRs at the mRNA level in AVEC, being TLR4 the most abundant, as well as the expression of S1P receptors, the S1P1 being the most abundant subtype. As regards pro-inflammatory molecules, the exposure of control AVECs to the bacterial lipopolysaccharide (LPS), a TLR4 ligand, induced the secretion of several cytokines such as interleukin (IL)-6, granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte chemotactic protein-1. Moreover, LPS cooperated with S1P to further up-regulate IL-6 and GM-CSF secretion. In addition, AVEC exposure to LPS induced the expression of the adhesion molecule ICAM-1, which was further up-regulated in the presence of S1P. Interestingly, the effect was more prominent in endothelial cells from the aortic valve than from the pulmonary valve, which rarely undergoes stenosis, and more potent in cells from stenotic valves. The cooperative effect on ICAM-1 up-regulation was inhibited by S1P1 receptor antagonists. In adhesion assays, LPS and S1P cooperated to increase the number of monocytes that adhered to the activated AVEC monolayers, and the effect was more marked in stenotic than in control AVECs. Conclusions: TLR4 and S1P1 receptor cooperate to up-regulate pro-inflammatory/pro-adhesive molecules in AVECs, what might be relevant for the pathogenesis of aortic stenosis given the stronger effect on stenotic aortic valves and the lower effect on pulmonary valves, and could open the way for new therapeutic approaches for the disease.

Resumen del poster presentado al 22nd IUBMB & 37th FEBS Congress: From Single Molecules to Systems Biology celebrado en Sevilla (Espana) del 4 al 9 de septiembre de 2012.-- et al.

without inducing autoimmunity. Although these are still early studies, they provide hope that daclizumab may help to restore antitumor immunity , these patients did not develop autoimmunity. regs antigen-specific effector T cell responses. Despite the loss of T and increased levels of vaccine regs experimental vaccine for metastatic breast cancer. These patients had fewer T is consistent with reprogramming to effector T cells. They then treated patients with daclizumab in conjunction with an , which γ treated with daclizumab lost their suppressive function and secreted interferonregs They found in vitro that T and restore the antitumor immune response. regs , could deplete T regs antibody to CD25, which is expressed on T approved − . hypothesized that daclizumab, a Food and Drug Administration et al against the tumor itself. Rech inhibiting immune responses −− are insidious regs causes autoimmunity. However, in the tumor microenvironment, T they prevent immune cells from the attacks on their host that −− perform a critical role in healthy individuals regs T ) that suppress antitumor immune responses. regs delinquent regulatory T cells (T . now extend this concept to the et al measures, is a critical strategy for reforming many types of bad apples. Rech to become sick at even the thought of violence. Indeed, rehabilitation, although carried out with much less extreme , a young criminal undergoes a controversial rehabilitation therapy that reprograms him A Clockwork Orange In regs Rehabilitating T

2508 Background: CD4+ regulatory T cells (Tregs) inhibit tumor immunity and represent an obstacle for cancer immunotherapy, but an effective strategy to deplete Tregs in pts has not been identified. In mice, anti-CD25 mAb depletes CD25+ FoxP3+ Tregs and promotes immunity. We therefore evaluated the FDA-approved anti-CD25 mab daclizumab for its effect on Tregs in a phase I clinical study. Methods: Ten HLA-A2+ pts with progressive MBC received an intravenous infusion (1 mg/kg) of daclizumab one week before an experimental vaccine consisting of 4 telomerase and survivin peptides and a cytomegalovirus (CMV) control peptide, emulsified in incomplete Freund's adjuvant (Montanide ISA-51-VG) and administered subcutaneously with GM-CSF every other week (×4) then monthly until disease progression. The pneumococcal-conjugate vaccine (Prevnar), containing CRM-197 antigen to engage T cell help, was also administered. Immune assessment was performed using blood samples obtained at baseline and regularly thereafter. Results: Daclizumab and vaccines were extremely well-tolerated. A median of 4 vaccines per pt (range 2 to 17) were given. Five pts had stable disease as the best response. Both percentage and absolute counts of CD25+ FoxP3+ CD4+ T cells decreased dramatically after daclizumab (p<0.001), based on monitoring with a CD25 antibody not blocked by daclizumab. Total FoxP3+ CD4+ T cells also decreased (p<0.001) but total CD4+ cells did not. The decrease of CD25+ FoxP3+ T cells was rapid (<1 week), marked (mean decrease of 83%), and prolonged (at least 7 weeks). Despite CD25 expression on effector T cells, 80% of pts exhibited CD8 responses to one or more tumor peptides, and 80% had CRM197-specific T cell responses. Two of five CMV seronegative pts exhibited CD8 T cell priming to CMV peptide. Conclusions: A single infusion of anti- CD25 mab daclizumab results in rapid, marked, and prolonged loss of CD25+ FoxP3+ CD4+ Tregs without toxicity in pts with MBC. Robust T cell priming and boosting to all vaccine antigens was observed despite CD25 blockade. These results demonstrate the utility of anti-CD25 mab for achieving extensive depletion of Tregs in patients. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Patent owned by Dana Farber and Whitehead

2505 Background: The telomerase reverse transcriptase hTERT represents a promising target for cancer immunotherapy because of its near universal expression in cancer and its critical role in oncogenesis. CD8+ T lymphocytes recognize peptides derived from hTERT and kill hTERT+ tumors cells of multiple histologies in vitro. Methods: We report the immunologic and biologic effects of vaccinating metastatic BrCa patients with hTERT peptide. Fourteen HLA-A2+ women with metastatic BrCa were vaccinated sq with 10 ug (n=6), 100 ug (n=5), or 1000 ug (n=3) of hTERT I540 peptide in adjuvant with GM-CSF for up to 8 vaccinations. Results: Most patients experienced Grade 1 and 2 injection site reactions and a syndrome of tumor pain or tumor-site itchiness after vaccination. One patient had a minor response following regression chest wall lesions. Three patients had stable disease after completing all 8 vaccinations. Peptide/MHC tetramer analysis of PBMC showed that 7 of 14 patients responded immunologically to the vaccine, including all those with clinical benefit. In freshly isolated PBMC, tetramer+ CD8+ cells were observed after (but not before) vaccination (0.1% to 2.0% of CD8+ T cells, median 0.86%). These cells secreted IFN-gamma in response to hTERT peptide and importantly, secreted IFN-gamma in response to telomerase-positive, HLA-A2+ (but not HLA-A2-negative) wild-type carcinoma cells. For 3 patients undergoing tumor biopsy, hTERT-specific CD8+ tumor infiltrating lymphocytes (TILs) were observed by tetramer analysis after, but not before, vaccination, measuring between 5%–12% tetramer+ cells among CD8+ T cells. By immunohistochemistry, tumor infiltrating lymphocytes observed after vaccination included both CD8+ and CD4+ T cells. TILs were associated in 2 patients with tumor necrosis involving 50%–80% of the biopsy specimens. Conclusions: Vaccination of metastatic BrCa patients against hTERT induces hTERT-specific T cells that are readily identifiable in blood and infiltrate tumors without major clinical toxicity. Tumor regression and tumor necrosis was observed after vaccination. These results suggest that hTERT can serve as a tumor rejection antigen in novel vaccines. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Dana-Farber Cancer Institute Patent Application Pending

Purpose: Delineation of the long-term follow-up data on a series of patients with malignant mesothelioma, who received a single intrapleural dose of a nonreplicative adenoviral (Ad) vector encoding the herpes simplex virus thymidine kinase “suicide gene” (Ad.HSVtk) in combination with systemic ganciclovir. Experimental Design: This report focuses on the 21 patients receiving “high-dose” therapy, defined by an intrapleural dose of vector (≥1.6 × 1013 viral particles), where transgene-encoded tk protein was reliably identified on immunohistochemical staining. In 13 patients, the vector was deleted in the E1 and E3 regions of the Ad; in the other eight patients, the vector had deletions in the Ad genes E1 and E4. Safety, immunologic responses, transgene expression, and clinical responses were evaluated. Results: Both the E1/E3-deleted vector and the E1/E4-deleted vector were well tolerated and safe, although production of the E1/E4 vector was more difficult. Posttreatment antibody responses against the tumors were consistently seen. Interestingly, we observed a number of clinical responses in our patients, including two long-term (>6.5 year) survivors, both of whom were treated with the E1/E4-deleted vector. Conclusions: Intrapleural Ad.HSVtk/ganciclovir is safe and well tolerated in mesothelioma patients and resulted in long-term durable responses in two patients. Given the limited amount of gene transfer observed, we postulate that Ad.HSVtk may have been effective due to induction of antitumor immune responses. We hypothesize that approaches aiming to augment the immune effects of Ad gene transfer (i.e., with the use of cytokines) may lead to increased numbers of therapeutic responses in otherwise untreatable pleural malignancies.

7572 Background: Imatinib mesylate (Gleevec) is a selective tyrosine kinase inhibitor with multiple targets including the KIT and PDGR receptors. Preclinical studies suggest that the combination of Gleevec (G) may have activity against melanoma, based in part on the effects of G on c-kit, as well as its antivascular effects via alterations in the PDGF receptor. Novel strategies are needed to improve upon the results of single agent chemotherapy in patients with melanoma. Methods: This phase I study was initiated to define the safety profile and the maximum tolerated dose of Gleevec combined with Temozolomide (T) in patients with locally advanced melanoma or metastatic disease. No prior treatment with T was allowed. Results: Sixty-three percent of patients had received prior therapy. Gleevec was given daily starting on Day 1 and T was given daily × 5 on Days 1–5, administered every 28 days. To date 8 patients have been entered onto the study. Overall therapy has been well tolerated. Predictable side effects of T include mild nausea, myelosuppression, and fatigue. Toxicities related to Gleevec are primarily fluid retention. No unexpected toxicities have been seen to date. MTD has not been reached. There have been no grade 4 toxicities. There was only one grade 3 toxicity related to treatment, which was edema. Conclusions: Preliminary analysis of the data shows a favorable toxicity profile. As the MTD is established, a phase II trial will begin to further define the overall activity of this combination therapy. Accrual to the clinical trial continues and updated results will be presented. No significant financial relationships to disclose.

Previous studies of auditory-nerve fiber (ANF) representation of vowels in cats and rodents (chinchillas and guinea pigs) have shown that, at amplitudes typical for conversational speech (60-70 dB), neuronal firing rate as a function of characteristic frequency alone provides a poor representation of spectral prominences (e.g., formants) of speech sounds. However, ANF rate representations may not be as inadequate as they appear. Here, it is investigated whether some of this apparent inadequacy owes to the mismatch between animal and human cochlear characteristics. For all animal models tested in earlier studies, the basilar membrane is shorter and encompasses a broader range of frequencies than that of humans. In this study, a customized speech synthesizer was used to create a rendition of the vowel [E] with formant spacing and bandwidths that fit the cat cochlea in proportion to the human cochlea. In these vowels, the spectral envelope is matched to cochlear distance rather than to frequency. Recordings of responses to this cochlear normalized [E] in auditory-nerve fibers of cats demonstrate that rate-based encoding of vowel sounds is capable of distinguishing spectral prominences even at 70-80-dB SPL. When cochlear dimensions are taken into account, rate encoding in ANF appears more informative than was previously believed.

This article explores two related topics: the recent effects of women in Spain entering the labour market and the current transformation of time management at home and at work. There are important movements in both fields with correlating consequences. These changes will greatly affect the role of women in our society determining the quality and evolution of not only women's lives, but also of our society in general. This article discusses some basic questions about time management and its impact on people's lives, the important changes in the culture and work of women in the last two decades and the transformations in the management of work time with specific attention given to its effects on women and domestic care.

The representation of Schroeder-phase harmonic complex sounds in the ventral cochlear nucleus (VCN) of the anesthetized chinchilla was studied. Stimuli consisted of a series of harmonically related sinusoids, multiples of a fundamental frequency (f0), summed in either negative (-SCHR) or positive (+SCHR) Schroeder phase. Psychoacoustic experiments performed in humans by other investigators have revealed that masking effects of -SCHR stimuli are larger than those found using +SCHR stimuli as maskers. In our laboratory, basilar membrane measurements at the base of the chinchilla cochlea show that responses to -SCHR stimuli are less "peaked," or modulated, than responses to +SCHR stimuli. We also found that suppression of a characteristic-frequency (CF) tone by -SCHR stimuli is larger than that evoked by +SCHR stimuli. Rate-intensity functions display higher firing rates in responses to -SCHR stimuli than in those produced by +SCHR stimuli. Firing rates evoked by either -SCHR or +SCHR stimuli saturate at lower values than those obtained in responses to CF tones. Rate and synchrony suppressions by -SCHR stimuli were larger than those evoked by +SCHR stimuli. Auditory nerve fiber responses to Schroeder complex stimuli share most of the properties of VCN responses, indicating little additional processing by the VCN.

Multicomponent stimuli consisting of two to seven tones were used to study suppression of basilar-membrane vibration at the 3-4-mm region of the chinchilla cochlea with a characteristic frequency between 6.5 and 8.5 kHz. Three-component stimuli were amplitude-modulated sinusoids (AM) with modulation depth varied between 0.25 and 2 and modulation frequency varied between 100 and 2000 Hz. For five-component stimuli of equal amplitude, frequency separation between adjacent components was the same as that used for AM stimuli. An additional manipulation was to position either the first, third, or fifth component at the characteristic frequency (CF). This allowed the study of the basilar-membrane response to off-CF stimuli. CF suppression was as high as 35 dB for two-tone combinations, while for equal-amplitude stimulus components CF suppression never exceeded 20 dB. This latter case occurred for both two-tone stimuli where the suppressor was below CF and for multitone stimuli with the third component=CF. Suppression was least for the AM stimuli, including when the three AM components were equal. Maximum suppression was both level- and frequency dependent, and occurred for component frequency separations of 500 to 600 Hz. Suppression decreased for multicomponent stimuli with component frequency spacing greater than 600 Hz. Mutual suppression occurred whenever stimulus components were within the compressive region of the basilar membrane.

The response of chinchilla basilar membrane in the basal region of the cochlea to multicomponent (1, 3, 5, 6, or 7) stimuli was studied using a laser interferometer. Three-component stimuli were amplitude-modulated signals with modulation depths that varied from 25% to 200% and the modulation frequency varied from 100 to 2000 Hz while the carrier frequency was set to the characteristic frequency of the region under study (approximately 6.3 to 9 kHz). Results indicate that, for certain modulation frequencies and depths, there is enhancement of the response. Responses to five equal-amplitude sine wave stimuli indicated the occurrence of nonlinear phenomena such as spectral edge enhancement, present when the frequency spacing was less than 200 Hz, and mutual suppression. For five-component stimuli, the first, third, or fifth component was placed at the characteristic frequency and the component frequency separation was varied over a 2-kHz range. Responses to seven component stimuli were similar to those of five-component stimuli. Six-component stimuli were generated by leaving out the center component of the seven-component stimuli. In the latter case, the center component was restored in the basilar-membrane response as a result of distortion-product generation in the nonlinear cochlea.

Measurements from the 1-4-mm basal region of the chinchilla cochlea indicate the basilar membrane in the hook region (12-18 kHz) vibrates essentially as it does more apically, in the 5-9-kHz region. That is, a compressive nonlinearity in the region of the characteristic frequency, amplitude-dependent phase changes, and a gain relative to stapes motion that can attain nearly 10,000 at low levels. The displacement at threshold for auditory-nerve fibers in this region (20 dB SPL) was approximately 2 nm. Measurements were made at several locations in individual animals in the longitudinal and radial directions. The results indicate that there is little variability in the phase of motion radially and no indication of higher-order modes of vibration. The data from the longitudinal studies indicate that there is a shift in the location of the maximum with increasing stimulus levels toward the base. The cochlear amplifier extends over a 2-3-mm region around the location of the characteristic frequency.

Responses of neurons in the ventral cochlear nucleus (VCN) of anesthetized chinchillas to six synthetic vowel sounds (/a/, /e/, /ϵ/, /i/, /o/ and /u/) were recorded at several intensity levels. Stimuli were synthesized with a fundamental frequency of 100 Hz or 181.6 Hz and had formant values at integer multiples of 100 Hz. Responses came from most neuron types in the VCN (with the exception of onset cells with an I-shaped pattern). Population studies, performed only on primary-like (PL) and chopper neurons, showed that PL neurons provide a better temporal representation than do chopper neurons. At the lowest level of stimulation, all neuron types provide an accurate rate–place representation of vowel spectra. With an increase in stimulus level, the rate–place representation of PL neurons becomes inferior to that of chopper neurons, either sustained choppers or transient choppers.