Prevalence of dysphagia in a consecutive cohort of subjects with MS using fibre-optic endoscopy.

INTRODUCTION: Multiple sclerosis (MS) refers to chronic inflammation of the central nervous system including the brain and spinal cord. Dysphagia is a symptom that represents challenges in clinical practice. The aim of the present study was to evaluate the prevalence of dysphagia in an Italian cohort of subjects with MS using the Dysphagia Outcome Severity Score (DOSS), based on fibre-optic endoscopy, and determine factors that correlate with the presence of swallowing problems. MATHERIALS AND METHODS: Data were collected in a multicentre study from a consecutive sample of MS patients, irrespective of self-reported dysphagia. The study included 215 subjects. Possible scores for DOSS range from 7 to 1, with 7 indicating normal swallowing. RESULTS: One hundred twenty-four (57.7%) subjects demonstrated abnormal swallowing and 57 (26.5%) of these had swallowing problems that required nutrition/diet modifications when evaluated objectively with fibre-optic endoscopy. Subjects with dysphagia were more severely disabled and more often had a progressive form of MS, compared to MS subjects with normal swallowing. In subjects with EDSS, < 4, 8 (13.3%), had a DOSS < 4. Seventy-five percent of subjects older than 60 years of age had dysphagia. CONCLUSION: In this sample of MS patients, more nearly 60% showed swallowing problems.

Cost of Chronic and Episodic Migraine. A pilot study from a tertiary headache centre in northern Italy.

BACKGROUND: Chronic migraine (CM) has a high impact on functional performance and quality of life (QoL). CM also has a relevant burden on the National Health Service (NHS), however precise figures are lacking. In this pilot study we compared the impact in terms of costs of CM and episodic migraine (EM) on the individual and on the National Health System (NHS). Furthermore, we comparatively evaluated the impact of CM and EM on functional capability and on QoL of sufferers. METHODS: We enrolled 92 consecutive patients attending the Pavia headache centre: 51 subjects with CM and 41 with episodic migraine (EM). Patients were tested with disability scales (MIDAS, HIT-6, SF-36) and with an ad hoc semi-structured questionnaire. RESULTS: The direct mean annual cost (in euro) per patient suffering from CM was €2250.0 ± 1796.1, against €523.6 ± 825.8 per patient with EM. The cost loaded on NHS was €2110.4 ± 1756.9 for CM, €468.3 ± 801.8 for EM. The total economic load and the different sub-items were significantly different between groups (CM vs. EM p = 0.001 for each value). CM subjects had higher scores than EM for MIDAS (98.4 ± 72,3 vs 15.5 ± 17.7, p = 0.001) and for HIT-6 (66.1 ± 8.4 vs 58.7 ± 10.1, p = 0.001). The SF-36 score was 39.9 ± 14,74 for CM and 66.2 ± 18.2 for EM (p = 0.001). CONCLUSIONS: CM is a disabling condition with a huge impact on the QoL of sufferers and a significant economic impact on the NHS. The adequate management of CM, reverting it back to EM, will provide a dual benefit: on the individual and on the society.

Hypoxia: the tumor's gateway to progression along the angiogenic pathway.

Decreased aerobic (hypoxic) conditions in tumors induce the release of cytokines that promote vascularization and thereby enhance tumor growth and metastasis. Recent major advances have provided insight into the role hypoxia plays in cancer biology. The domain structure of the hypoxia-inducible factor 1alpha (HIF-1alpha) has been elucidated, as has the mechanism by which stabilization of HIF-1alpha leads to initiation of the transcription of target genes involved in growth of blood vessels.

Positioning atypical protein kinase C isoforms in the UV-induced apoptotic signaling cascade.

Recent studies have documented the involvement of the atypical protein kinase C (aPKC) isoforms in important cellular functions such as cell proliferation and survival. Exposure of cells to a genotoxic stimulus that induces apoptosis, such as UV irradiation, leads to a profound inhibition of the atypical PKC activity in vivo. In this study, we addressed the relationship between this phenomenon and different proteins involved in the apoptotic response. We show that (i) the inhibition of the aPKC activity precedes UV-induced apoptosis; (ii) UV-induced aPKC inhibition and apoptosis are independent of p53; (iii) Bcl-2 proteins are potent modulators of aPKC activity; and (iv) the aPKCs are located upstream of the interleukin-converting enzyme-like protease system, which is required for the induction of apoptosis by both Par-4 (a selective aPKC inhibitor) and UV irradiation. We also demonstrate here that inhibition of aPKC activity leads to a decrease in mitogen-activated protein (MAP) kinase activity and simultaneously an increase in p38 activity. Both effects are critical for the induction of apoptosis in response to Par-4 expression and UV irradiation. Collectively, these results clarify the position of the aPKCs in the UV-induced apoptotic pathway and strongly suggest that MAP kinases play a role in this signaling cascade.

Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.

Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1.

Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.

A dominant negative protein kinase C zeta subspecies blocks NF-kappa B activation.

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a number of genes. NF-kappa B is a heterodimer of 50- and 65-kDa subunits sequestered in the cytoplasm complexed to inhibitory protein I kappa B. Following stimulation of cells, I kappa B dissociates from NF-kappa B, allowing its translocation to the nucleus, where it carries out the transactivation function. The precise mechanism controlling NF-kappa B activation and the involvement of members of the protein kinase C (PKC) family of isotypes have previously been investigated. It was found that phorbol myristate acetate, (PMA) which is a potent stimulant of phorbol ester-sensitive PKC isotypes, activates NF-kappa B. However, the role of PMA-sensitive PKCs in vivo is not as apparent. It has recently been demonstrated in the model system of Xenopus laevis oocytes that the PMA-insensitive PKC isotype, zeta PKC, is a required step in the activation of NF-kappa B in response to ras p21. We demonstrate here that overexpression of zeta PKC is by itself sufficient to stimulate a permanent translocation of functionally active NF-kappa B into the nucleus of NIH 3T3 fibroblasts and that transfection of a kinase-defective dominant negative mutant of zeta PKC dramatically inhibits the kappa B-dependent transactivation of a chloramphenicol acetyltransferase reporter plasmid in NIH 3T3 fibroblasts. All these results support the notion that zeta PKC plays a decisive role in NF-kappa B regulation in mammalian cells.

Evidence of biased immunoglobulin variable gene usage in highly stable B-cell chronic lymphocytic leukemia.

Recognition of biased immunoglobulin variable (IgV) gene usage in B-cell chronic lymphocytic leukemia (B-CLL) may yield insight into leukemogenesis and may help to refine prognostic categories. We explored Ig variable heavy (VH) and light (VL) chain gene usage in highly stable and indolent B-CLL (n=25) who never required treatment over 10 or more years. We observed an unexpectedly high usage of mutated VH3-72 (6/25; 24.0%), a gene that was otherwise rare in B-CLL (7/805; 0.87%; P<0.01), including mutated cases (6/432; 1.39%; P<0.01) and was exceptional among indolent (1/230, 0.435%; P<0.01), and aggressive B-cell lymphomas (0/105; P<0.01). Three of six VH3-72 B-CLL cases utilized the same VL Vkappa4-1 gene. Two V(H)3-72 B-CLL cases had highly homologous VH complementarity determining regions 3 (CDR3s), encoding Cys-XXXX-Cys domains, and utilized Vkappa4-1 genes with homologous IgVL CDR3s. An identical threonine to isoleucine change at codon 84 of V(H)3-72 framework region 3 (FR3) recurred in four cases of highly stable VH3-72 B-CLL. This mutation is expected to cause a conformational change of FR3 proximal to CDR3 that might critically affect high-affinity antigen binding. B-cell receptors encoded by VH3-72 may identify a specific B-CLL group and be implicated in leukemogenesis through an antigen-driven expansion of B cells.

HIF-1-dependent transcriptional activity is required for oxygen-mediated HIF-1alpha degradation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. In normoxia, HIF-1alpha is a short lived protein, whereas hypoxia rapidly increases HIF-1alpha protein levels by relaxing its ubiquitin-proteasome-dependent degradation. In this study, we show that the p42/p44 MAP kinase cascade, known to phosphorylate HIF-1alpha, does not modulate the degradation/stabilization profile of HIF-1alpha. However, we present evidence that the rate of HIF-1alpha degradation depends on the duration of hypoxic stress. We demonstrate that degradation of HIF-1alpha is suppressed by: (i) inhibiting general transcription with actinomycin D or (ii) specifically blocking HIF-1-dependent transcriptional activity. In keeping with these findings, we postulate that HIF-1alpha is targetted to the proteasome via a HIF-1alpha proteasome targetting factor (HPTF) which expression is directly under the control of HIF-1-mediated transcriptional activity. Although HPTF is not yet molecularly identified, it is clearly distinct from the von Hippel-Lindau protein (pVHL).

Signaling angiogenesis via p42/p44 MAP kinase and hypoxia.

Angiogenesis is associated with a number of pathological situations. In this study, we have focused our attention on the role of p42/p44 MAP (mitogen-activated protein) kinases and hypoxia in the control of angiogenesis. We demonstrate that p42/p44 MAP kinases play a pivotal role in angiogenesis by exerting a determinant action at three levels: i) persistent activation of p42/p44 MAP kinases abrogates apoptosis; ii) p42/p44 MAP kinase activity is critical for controlling proliferation and growth arrest of confluent endothelial cells; and iii) p42/p44 MAP kinases promote VEGF (vascular endothelial growth factor) expression by activating its transcription via recruitment of the AP-2/Sp1 (activator protein-2) complex on the proximal region (-88/-66) of the VEGF promoter and by direct phosphorylation of hypoxia-inducible factor 1 alpha (HIF-1 alpha). HIF-1 alpha plays a crucial role in the control of HIF-1 activity, which mediates hypoxia-induced VEGF expression. We show that oxygen-regulated HIF-1 alpha protein levels are not affected by intracellular localization (nucleus versus cytoplasm). Finally, we propose a model which suggests an autoregulatory feedback mechanism controlling HIF-1 alpha and therefore HIF-1-dependent gene expression.

Signaling angiogenesis via p42/p44 MAP kinase cascade.

Vascular endothelial growth factor (VEGF), a potent agonist secreted by virtually all cells, controls migration and division of vascular endothelial cells. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a key role in vasculogenesis. We analyzed the regulation of VEGF mRNA in normal and transformed CCL39 fibroblasts and then dissected the VEGF promoter to identify the signaling pathway(s) controlling the activation of this promoter in response to growth factors, oncogenes, and hypoxic stress. We demonstrated that the p42/p44 MAP kinase signaling cascade controls VEGF expression at least at two levels. In normoxic conditions, MAPKs activate the VEGF promoter at the proximal (-88/-66) region where Sp-1/AP-2 factors bind. Activation of p42/p44 MAPKs is sufficient to turn on VEGF mRNA. At low O2 tension, hypoxia inducible factor-1 alpha (HIF-1 alpha), a limiting factor rapidly stabilized and phosphorylated, plays a key role in the expression of several genes including VEGF. We demonstrated that p42/p44MAPKs stoichiometrically phosphorylate HIF-1 alpha in vitro and that HIF-1-dependent VEGF gene expression is strongly enhanced by the exclusive activation of p42/p44MAPKs. Finally, we demonstrated that the regulation of p42/p44MAPK activity is critical for controlling proliferation and growth arrest of vascular endothelial cells at confluency. These results point to at least three major targets of angiogenesis where p42/p44 MAP kinases exert a determinant action.

Post-transplant lymphoproliferative disorders. Molecular analysis of histogenesis and pathogenesis.

AIM: Post-transplant lymphoproliferative disorders (PTLD) represent a serious complication of solid organ transplantation. Despite several advances in the biological categorization of PTLD, current classifications are not fully predictive of the clinical behavior of the disease. This study assessed a comprehensive molecular analysis of the clinico-pathologic spectrum of PTLD in order to better clarify the physiopathology of these disorders. METHODS: Fifty-two monoclonal PTLD were investigated for: 1). somatic hypermutation of IgV genes by direct sequencing of IgV rearrangements; 2). expression of BCL6, MUM1 and CD138 proteins by immunohistochemistry; 3). aberrant hypermethylation of DAP-kinase gene by methylation-specific polymerase chain reaction (PCR); 4). genotypic characterization of Epstein Barr virus (EBV) in EBV infected PTLD by PCR analysis of the prevalence of deletions in the carboxyterminal portion of the LMP1 gene and for the definition of type-1/type-2 EBV infection. RESULTS: We report that virtually all monoclonal PTLD originate from B cells that have experienced the germinal center (GC) reaction reflecting different stages of mature B cell differentiation and that tumor development seems frequently associated with EBV and/or other molecular lesions preventing apoptosis of cells that have failed the physiological process of germinal center reaction. CONCLUSION: To date, classification of PTLD is mainly based on morphology and conventional immunophenotyping. Because current classification schemes are not fully predictive of prognosis, knowledge of PTLD histogenesis and pathogenesis may potentially contribute to refine the distinction of PTLD into more homogeneous categories with prognostic relevance.

Molecular characterization of common variable immunodeficiency-related lymphomas.

Common variable immunodeficiency (CVI) patients are at high relative risk of developing non-Hodgkin lymphomas (NHL), mainly represented by B-lineage diffuse large cell lymphomas. The molecular pathogenesis and histogenesis of CVI-related NHL are poorly understood. We have thus attempted to provide a detailed molecular characterization of their histogenesis and pathogenesis. A panel of 5 CVI-related NHL was subjected to detailed analysis of histogenetic markers (mutations of immunoglobulin variable heavy chain-IgVH and of BCL-6 genes) acquired by B-cells at the time of germinal center transit. Somatic hypermutation of IgVH and BCL-6 genes occurred in 5/5 cases; in all cases, mutations were stable with no evidence of ongoing mutation processes. In 3/5 cases, the pattern of IgVH mutations was consistent with selection and stimulation of the tumor clone by antigen. To further clarify the pathogenesis, samples were tested for inactivation by promoter hypermethylation of the genes 0(6)-methylguanine-DNA-methyltransferase (MGMT) and glutathione S-transferase (GST) p1, which code for detoxifying enzymes, as well as of death-associated protein (DAP)-kinase, coding for a proapoptotic molecule. Promoter hypermethylation of MGMT, GSTp1 and DAP-kinase was detected in 2/5, 3/5 and 3/5 CVI-related NHL, respectively. Overall, these data indicate that: i) similarly to other immunodeficiency-related NHL, CVI-related NHL derive from germinal center-related B-cells, namely centrocytes or post-germinal center B-cells; ii) antigen stimulation and selection are involved in the development of at least a fraction of these cases; iii) hypermethylation of the MGMT, DAP-kinase and GSTp1 genes occurs at sustained frequencies in CVI-related NHL and may provide novel prognostic markers and therapeutic targets for the clinical management of these lymphomas.

Interaction between PARP-1 and HIF-2α in the hypoxic response.

Hypoxia-inducible factors (HIFs) mediate the transcriptional adaptation of hypoxic cells. The extensive transcriptional programm regulated by HIFs involves the induction of genes controlling angiogenesis, cellular metabolism, cell growth, metastasis, apoptosis, extracellular matrix remodeling and others. HIF is a heterodimer of HIF-α and HIF-ß subunits. In addition to HIF-1α, HIF-2α has evolved as an isoform that contributes differently to the hypoxic adaptation by performing non-redundant functions. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein involved in the control of DNA repair and gene transcription by modulating chromatin structure and acting as part of gene-specific enhancer/promoter-binding complexes. Previous results have shown that PARP-1 regulates HIF-1 activity. In this study, we focused on the cross-talk between HIF-2α and PARP-1. By using different approaches to suppress PARP-1, we show that HIF-2α mRNA expression, protein levels and HIF-2-dependent gene expression, such as ANGPTL4 and erythropoietin (EPO), are regulated by PARP-1. This regulation occurs at both the transcriptional and post-trancriptional level. We also show a complex formation between HIF-2α with PARP-1. This complex is sensitive to PARP inhibition and seems to protect against the von Hippel-Lindau-dependent HIF-2α degradation. Finally, we show that parp-1(-/-) mice display a significant reduction in the circulating hypoxia-induced EPO levels, number of red cells and hemoglobin concentration. Altogether, these results reveal a complex functional interaction between PARP-1 and the HIF system and suggest that PARP-1 is involved in the fine tuning of the HIF-mediated hypoxic response in vivo.

The magic of the hypoxia-signaling cascade.

All organisms respond to changes in their environment by activating complex signaling cascades. The "hypoxia-signaling cascade" is activated in response to low oxygen availability and this activation is central to maintaining oxygen homeostasis and hence to survival. By regulating the transcriptional complex hypoxia-inducible factor, hypoxia is associated with several physiopathological processes. Several strategies, based on the targeting of the hypoxia-signaling cascade, have been developed to treat these pathologies. Our review summarize different aspects of the hypoxic pathway.

Enzyme-linked immunosorbent assay for pharmacological studies targeting hypoxia-inducible factor 1alpha.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of a wide range of genes related to oxygen delivery and metabolic adaptation under hypoxic (low-oxygen) conditions. HIF-1 is, in fact, a heterodimer of two subunits, HIF-1alpha and HIF-1beta. The only analytical methods available for measuring HIF-1alpha levels in tumors are immunohistochemistry and Western blotting. Immunohistochemistry has the advantage of allowing the identification and direct examination of HIF-1alpha-expressing cells, but has the intrinsic limitation, as for Western blotting, of being nonquantitative. We developed and validated an enzyme-linked immunosorbent assay (ELISA) approach to measure HIF-1alpha levels in cultured tumor cell lines in vitro. HIF-1alpha was expressed in thirteen tumor cell lines grown under hypoxic conditions; however, the levels differed strongly between cell lines. These data point to intrinsic differences between cell lines for the induction of HIF-1alpha under hypoxic conditions. The ELISA developed in the present study is thus an interesting alternative to other analytical methods used to measure HIF-1alpha protein levels and should be useful in preclinical pharmacological studies targeting HIF-1alpha.

MAP kinases and hypoxia in the control of VEGF expression.

Vascular endothelial growth factor (VEGF), a potent cytokine secreted by virtually all cells plays a key role in tumor angiogenesis. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a very tight regulation of this gene. This commentary reviews the mechanisms whereby VEGF mRNA is controlled within the tumor environment by hypoxia and the MAP kinase signaling cascades. Using hamster fibroblasts as a cellular model, we demonstrated that the Ras-mediated activation of p42/p44 MAP kinases exerts a prominent action at the transcriptional level. In normoxic conditions, p42/p44 MAPKs activate the VEGF promoter at the proximal (-88/-66) region where Sp 1/AP-2 transcriptional factor complexes are recruited. At low O2 tension, the stabilized and nuclear hypoxia inducible factor- 1alpha (HIF-1alpha) is directly phosphorylated by p42/p44 MAPKs, an action which enhances HIF-1-dependent transcriptional activition of VEGF. In addition, MAPKs activated under various cellular stresses (p38MAPK and JNK), contribute to the increased expression of this angiogenic growth and survival factor by stabilizing the VEGF mRNA.

Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells.

Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of genes such as vascular endothelial growth factor (VEGF) and Erythropoietin in low oxygen conditions (hypoxia). VEGF is strongly induced at both the mRNA and protein expression level by a number of hormones and growth factors in vascular smooth muscle cells (VSMC) independently of the oxygen environment. However, the role of HIF-1alpha in this induction has not been studied. We report here that HIF-1alpha protein levels are strongly increased by fetal calf serum in quiescent VSMC. More interestingly, Angiotensin II (Ang II), thrombin, platelet-derived growth factor, and other hormones can also increase HIF-1alpha in VSMC to levels that are substantially more elevated than the hypoxic treatment. HIF-1alpha induced by Ang II is located in the nucleus, binds to the hypoxic response element, and is transcriptionally active. The induction of HIF-1alpha by hormones is mediated through the production of reactive oxygen species (ROS), since it can be blocked by the ROS inhibitors, diphenyleneiodonium and catalase. Finally, strong induction of VEGF mRNA by Ang II can also be inhibited by these ROS inhibitors. These results implicate HIF-1alpha and HIF-1-dependent transcriptional activity in the induction of VEGF expression after agonist stimulation and define novel hypoxia-independent mechanisms that should play a major role in vascular remodeling.