Juan Bilbao

p53 and cyclin D1 as prognostic factors in squamous cell carcinoma of the larynx

Objectives/Hypothesis Proteins p53 and cyclin D1 play a crucial role in cell cycle control. Protein p53 mutations are one of the most common genetic alterations in human cancer, and cyclin D1 gene amplification has been found to be associated with poor prognosis in different types of tumors. Functional alterations of these proteins may play an important role both in the carcinogenesis of squamous carcinomas of the head and neck and in the clinical evolution of these tumors. The objective of the present study was to evaluate whether the presence of p53 and/or cyclin D1 proteins (detected by immunohistochemical analysis) could serve as relevant variables for the assessment of the prognosis of laryngeal squamous cell carcinoma.

Early cell death in the brain of fetal preterm lambs after hypoxic-ischemic injury.

The objective of the present study was to evaluate using premature fetal lambs the effect of cerebral hypoxia-ischemia induced by partial occlusion of the umbilical cord on the type of cell death which occurs in different brain regions and to ascertain some of the neural pathways which may underlie the associated pathologies. Lambs were sacrificed either immediately after a 1 h hypoxic-ischemic insult or 3 h later. Brains were fixed by perfusion and blocks of the different brain territories were processed for light microscopy (hematoxylin-eosin, Nissl staining), electron transmission microscopy and quantification of apoptosis by the TUNEL method. Other fixed brains were dissociated and labeled by nonyl acridine orange to determine mitochondrial integrity. Non-fixed brains were also used for membrane asymmetry studies, in which cell suspensions were analyzed by flow cytometry to quantify apoptosis. In both hypoxic-ischemic groups, necrotic-like neurons were observed mainly in the mesencephalon, pons, deep cerebellar nuclei and basal nuclei, whereas apoptotic cells were extensively found both in white and gray matter and were not limited to regions where necrotic neurons were present. The 3 h post-partial cord occlusion group, but not the 0 h group, showed a generalized alteration of cell membrane asymmetry and mitochondrial integrity as revealed by Annexin V/PI flow cytometry and nonyl acridine orange studies, respectively. Our results show that the apoptotic/necrotic patterns of cell death occurring early after hypoxic-ischemic injury are brain-region-specific and have distinct dynamics and suggest that therapeutic strategies aimed at rescuing cells from the effects of hypoxia/ischemia should be aimed at blocking the apoptotic components of brain damage.

Selective impairment of spinal mu-opioid receptor mechanism by plasticity of serotonergic facilitation mediated by 5-HT2A and 5-HT2B receptors

Summary Plasticity of serotonergic neurotransmission mediated by 5‐HT2A and 5‐HT2B receptors impairs spinal mu‐opioid of receptor efficacy during neuropathic pain, including upregulation of receptor expression in mu‐opioid receptor containing neurons. ABSTRACT Opioid analgesia is compromised by intracellular mediators such as protein kinase C (PKC). The phosphatidylinositol hydrolysis‐coupled serotonin receptor 5‐HT2 is ideally suited to promote PKC activation. We test the hypothesis that 5‐HT2A and 5‐HT2B receptors, which have been previously shown to become pro‐excitatory after spinal nerve ligation (SNL), can negatively influence the ability of opioids to depress spinal excitation evoked by noxious input. Spinal superfusion with (100 nM) mu‐opioid receptor (MOR)‐agonist DAMGO significantly depressed C fiber‐evoked spinal field potentials. Simultaneous administration of subclinical 5‐HT2AR antagonist 4F 4PP (100 nM) or 5‐HT2BR antagonist SB 204741 (100 nM) significantly reduced the IC50 value for DAMGO in nerve‐ligated rats (97.56 nM ± 1.51 and 1.20 nM ± 1.28 respectively, relative to 104 nM ± 1.08 at the baseline condition), but not in sham‐operated rats. Both antagonists failed to alter depression induced by delta‐opioid receptor (DOR)‐agonist D‐ala2‐deltorphin II after SNL as well as in the sham condition. Western blot analysis of dorsal horn homogenates revealed bilateral upregulation of 5‐HT2AR and 5‐HT2BR protein band densities after SNL. As assessed from double immunofluorescence labeling for confocal laser scanning microscopy, scarce dorsal horn cell processes showed co‐localization color overlay for 5‐HT2AR/MOR, 5‐HT2BR/MOR, 5‐HT2AR/DOR, or 5‐HT2BR/DOR in sham‐operated rats. Intensity correlation‐based analyses showed significant increases in 5‐HT2AR/MOR and 5‐HT2BR/MOR co‐localizations after SNL. These results indicate that plasticity of spinal serotonergic neurotransmission can selectively reduce spinal MOR mechanisms via 5‐HT2A and 5‐HT2B receptors, including upregulation of the latter and increased expression in dorsal horn neurons containing MOR.

Transient, 5-HT2B receptor–mediated facilitation in neuropathic pain: Up-regulation of PKCγ and engagement of the NMDA receptor in dorsal horn neurons

Summary 5‐HT2B receptor activation promotes transient translocation of protein kinase C &ggr; (PKC&ggr;) and phosphorylation of NR1, subserving hyersensitivity to thermal and mechanical pain in the rat after spinal nerve ligation. ABSTRACT Spinal nociception can be facilitated by 5‐HT2 receptors in neuropathic pain. We investigated the involvement of glutamate receptors in dorsal neuron hyperexcitation that is promoted by 5‐HT2B receptor (5‐HT2BR) after spinal nerve ligation (SNL) in the rat. Augmentation of C‐fiber–evoked potentials by spinal superfusion with 5‐HT2BR agonist BW 723C86 in nerve‐ligated rats was impeded by co‐administration of NMDA receptor (NMDAR) antagonist D‐AP5, but not by mGluR1/5 antagonist AIDA or mGluR2/3 antagonist LY 341495. Evoked potentials were increased by cis‐ACPD in nerve‐injured rats, irrespective of simultaneous 5‐HT2BR blockade by SB204741. In uninjured rats, NMDAR agonist cis‐ACPD enhanced evoked potentials in the presence of BW 723C86 but not if administered alone or during exposure to protein kinase C &ggr; (PKC&ggr;) inhibitor peptide. Triple immunofluorescence labelings revealed co‐localization of NMDAR and 5‐HT2BR in PKC&ggr;‐expressing perikarya in lamina II neurons. As a result of SNL, PKC&ggr; was transiently and bilaterally up‐regulated in synaptic fraction from dorsal horn homogenates, peaking at day 2 and returning to basal levels by day 9. Chronic blockade of 5‐HT2BR with selective antagonist SB 204741 after SNL bilaterally decreased the following: (i) PKC&ggr; up‐regulation in synaptic fraction, (ii) phosphorylation of NMDAR subunit NR1 (serine 889) in synaptic fraction, and (iii) co‐localization of both PKC&ggr; and phosphorylated NR1 with postsynaptic marker PSD‐95. Chronic delivery of SB 204741 bilaterally attenuated thermal and mechanical allodynia occurring after SNL, particularly at day 2 post injury. These findings suggest that transient activation of the PKC&ggr;/NMDAR pathway is critically involved in 5‐HT2BR‐mediated facilitation in the SNL model of neuropathic pain.

GENESIS AND EVOLUTION OF HIGH-PLOIDY TUMOUR CELLS EVALUATED BY MEANS OF THE PROLIFERATION MARKERS P34CDC2, CYCLIN B1, PCNA AND 3H-THYMIDINE

Although cell polyploidization is not an infrequent event in mammalian cells and is common in tumours, the mechanisms involved are not well understood. Using the murine B16 cell line as a model, we evaluated the role of some key proteins involved in cell cycle progression: p34cdc2, cyclin B1 and PCNA. By means of flow cytometry, we showed that both in modal‐ and in high‐ploidy subpopulations, almost all cells were p34cdc2‐positive. In the modal‐ploidy subpopulation only 17.1% cells were cyclin B1‐positive and 85.6% PCNA‐positive; in contrast, in the high‐ploidy subpopulation up to 91.8% cells were cyclin B1‐positive and 97.3% cells were PCNA‐positive (P < 0.001). Immunofluorescence microscopy showed that PCNA was located in the nucleus; p34cdc2, both in the nucleus and cytoplasm; and cyclin B1 yielded a cytoplasmic spotted pattern with a perinuclear reinforcement. After a 24‐h incubation with 3[H]‐thymidine followed by withdrawal of the isotope, high‐ploidy cells remained labelled 8 days after thymidine withdrawal, in contrast to modalploidy cells. Taken together, our results suggest that polyploid cells are not quiescent, their cell cycle is longer than that of the modal‐ploidy population, and they maintain cyclin B1 throughout the cycle, which may contribute to their genesis by impeding the exit from mitosis.

Valoración de factores sociales y clínicos en el síndrome de fibromialgia

Resumen Introduccion La depresion y la ansiedad son estados afectivos negativos que con frecuencia se asocian con el dolor cronico. Por el contrario, factores psicosociales como el apoyo social influyen en la percepcion de dolor y contribuyen al bienestar del paciente con dolor cronico. Objetivos Valorar el tipo de terapia que recibe y el apoyo social que percibe de su entorno el paciente con sindrome de fibromialgia (SFM) y determinar su relacion con sus indices de depresion y de ansiedad. Material y metodos Se han recabado datos de 229 pacientes de SFM mediante un cuestionario autoaplicado que ha incluido los aspectos mas preocupantes de la enfermedad, el efecto de las terapias empleadas, la satisfaccion con la atencion sanitaria recibida, la percepcion de apoyo social, asi como indices de ansiedad y de depresion. Resultados y conclusiones Las personas que han valorado mas positivamente el apoyo de su entorno social y familiar demuestran mayor proteccion frente al estres psicologico en terminos de ansiedad y depresion. La intervencion sobre el apoyo del entorno inmediato del paciente con SFM podria contribuir a la reduccion del dolor reduciendo los niveles de ansiedad y de depresion.

Morphine‐induced depression of spinal excitation is not altered following acute disruption of GABAA or GABAB receptor activity

Loss of spinal inhibitory mechanisms is thought to contribute to the pathophysiology of abnormal pain states, including neuropathic pain. By using an evoked spinal field potential technique, the hypothesis was tested here that decreased spinal GABAergic control underlies poor response to morphine (MOR) that often accompanies neuropathic pain. Therefore, field potentials evoked by electrical peripheral nerve stimulation during spinal superfusion with MOR were recorded in rats rendered neuropathic by a spinal nerve ligation (SNL) procedure, and compared to responses recorded in naïve rats. MOR effects on evoked field potentials were then assessed in rats in which spinal GABAergic inhibition had been acutely reduced by treatment with GABAA and GABAB receptor‐antagonists.

Depression of C fibre-evoked spinal field potentials by the spinal δ opioid receptor is enhanced in the spinal nerve ligation model of neuropathic pain: Involvement of the μ-subtype

The depression rate of C fibre-evoked spinal field potentials by spinally applied morphine is increased in two states of spinal hyperexcitation, namely the spinal ligation model (SNL) of neuropathic pain and long-term potentiation (LTP) of C fibre-evoked spinal field potentials. This present work sought to determine opioid receptor subtypes involved in such increase in the SNL model. We recorded spinal field potentials during spinal superfusion with increasing, cumulative concentrations of selective subtype-specific agonists in rats subjected to SNL, as well as in non-ligated animals. The mu opioid receptor (MOR) agonist DAMGO significantly depressed field potentials evoked by C (100 nM) or Adelta fibres (1 microM) both in neuropathic and non-ligated rats, whereas the kappa receptor opioid (KOR) agonist +/-U-50488 was ineffective. The delta opioid receptor (DOR) (D-Ala2)-Deltorphin II was more effective in reducing C fibre-evoked spinal field potentials in rats subjected to SNL (100 nM) than in non-ligated rats (100 microM). Subclinical MOR activation (10 nM DAMGO) produced a leftward shift in (D-Ala2)-Deltorphin II dose-response curve in non-ligated rats (IC50 16.59 +/- 0.99 microM vs 120.3 +/- 1.0 microM in the absence of DAMGO), and isobolar analysis revealed synergistic interaction (interaction index 0.25). MOR blockade (100 microM CTOP) disinhibited C fibre-evoked potentials in neuropathic, but not in basal animals, and partially impeded DOR depression in both groups. DOR blockade (1 mM naltrindole) was ineffective in either group. We show that DOR-mediated depression of spinal responses to peripheral unmyelinated fibre-input is increased in the SNL model, an increase that is contributed to by positive interaction with the spinal MOR.

Increased number of multi-oocyte follicles (MOFs) in juvenile p27Kip1 mutant mice: potential role of granulosa cells

STUDY QUESTION Why are female mice that lack a functional p27 protein infertile? SUMMARY ANSWER The absence of a functional p27 leads to a dramatic increase in the number of multi-oocyte follicles (MOFs) in juvenile female mice; p27 would promote the individualization of follicles favoring the development of fertile eggs. WHAT IS KNOWN ALREADY p27-/- female mice are infertile. p27 suppresses excessive follicular endowment and activation and promotes follicular atresia in mice. MATERIALS AND METHODS Ovaries from wild type (WT) and p27Kip1 mutant mice aged 2, 4 and 12 weeks were subjected to immunohistochemistry/immunofluorescence. The slides with whole organs serially sectioned were scanned and examined by image analysis. MAIN RESULTS AND THE ROLE OF CHANCE Compared with WT, p27Kip1 mutant pre-pubertal mice had a greater number of oocytes, a greater number of growing follicles and a greater number of MOFs. These differences were statistically significant (P < 0.05), particularly in the case of MOFs (P > 0.001). The unusually large number of MOFs in juvenile p27-deficient mice is a novel observation. In WT mice p27 protein remains present in the oocyte nucleus but gradually decreases in the ooplasm during follicular growth, while granulosa cells show dynamic, follicle stage-related changes. LIMITATIONS, REASONS FOR CAUTION These results have been obtained in mice and they cannot be directly extrapolated to humans. WIDER IMPLICATIONS OF THE FINDINGS The dramatic increase in the numbers of MOFs in juvenile p27 mutants has not been previously reported. The number of MOFs declines sharply as the mice become sexually mature, pointing to their negative selection. These findings open a new approach to the study of sterility. STUDY FUNDING/COMPETING INTERESTS This study has been funded by the Basque Government, Dept. of Health grant 2007111063 and Dept. of Industry (Saiotek) grant S-PC11UN008. Jairo Perez-Sanz was the recipient of a grant from Fundación Jesús de Gangoiti Barrera. The authors have no conflicts of interest to declare.

Is psychological distress intrinsic to fibromyalgia syndrome? Cross-sectional analysis in two clinical presentations

Clinical presentation of fibromyalgia syndrome (FMS) is heterogeneous and often involves psychological comorbidities. Clinical subgrouping of FMS patients has been proposed as a strategy to improve patients’ long-term outcomes by helping identify specific treatment needs. Using the 90 Symptom Checklist Revised (SCL-90-R), we have assessed emotional distress in two FMS patient subpopulations discriminated on the basis of their differences in scores on specific items of the Fibromyalgia Impact Questionnaire (FIQ). Subjects classed as type II exhibited high emotional distress on all ten dimensions studied, which included somatization, obsessive–compulsive, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, psychoticism, and additional items subscales, as well as on the global severity index (GSI), positive symptom total (PST), and positive symptom distress index (PDSI). T-scores in these patients were above diagnostic cutoff level of 60 on somatization, obsessive–compulsive, and depression subscales. In contrast, the profile exhibited by type I subjects fell entirely within normal values for nonpsychiatric population. Emotional status was significantly inversely correlated with present clinical pain in type I-, but not in type II-fibromyalgia patients. Regression analysis revealed a model based on phobic anxiety, paranoid ideation, and depression subscales as best contributing to classification. The present data suggest that associated psychological distress and maladaptive emotional responses that are commonly attributed to the general FMS population may be largely a distinguishing feature of one subset of patients.