In vitro and in vivo tumor growth inhibition by a p16-mimicking peptide in p16INK4A-defective, pRb-positive human melanoma cells

The cell cycle regulatory pathway responsible for the control of the late-G1 checkpoint is found recurrently altered in human malignant melanoma, often due to lack of functional p16 or pRb (pRb-1) proteins. Here we examined the ability of p16-derived peptides to mimic p16 function in two exemplary human melanoma cell lines: the p16-defective, pRb-positive A375M cells and p16-positive, pRb-defective A2058 cells. The synthetic p16-mimicking peptides strongly induced apoptosis in p16-, pRb+ A375M cells in vitro, while they had significantly less activity on p16+, pRb- A2058 cells. The most active p16-mimicking peptide, p16-AP9, also potently inhibited in vivo growth of the A375M melanoma. Treated tumors showed a threefold smaller volume (P < 0.025) and a significant reduction of the mitotic index and of PCNA expression. Growth of A2058 cells in vivo was not affected by treatment with the p16-mimicking peptide. Our results demonstrate that p16-mimicking peptides can induce apoptosis in vitro and that can inhibit tumor growth in vivo in p16-defective, pRb-expressing human melanoma cells, suggesting that p16-mimicking peptides can represent a promising tool for targeted therapy in selected cancer phenotypes.     

Molecular phylogenetics and diagnosis of Anisakis, Pseudoterranova, and Contracaecum from northern Pacific marine mammals

Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic and molecular phylogenetic analyses. Forty-eight new sequences were obtained for this study of 14 Anisakis taxa, 8 Pseudoterranova taxa, 4 Contracaecum taxa, and 4 outgroup species. Partial 28S (LSU) and complete internal transcribed spacer (ITS-1, 5.8S, ITS-2) ribosomal DNA was amplified by the polymerase chain reaction and sequenced. Sequences of ITS indicated that Pseudoterranova specimens from Zalophus californianus (California sea lion), Mirounga angustirostris (northern elephant seal), Phoca vitulina (harbor seal), Enhydra lutris (sea otter), and Eumetopias jubatus (Steller's sea lion) exactly matched P. decipiens s. str., extending the host and geographic range of this species. Anisakis from northern Pacific marine mammals were most closely related to members of the A. simplex species complex. Comparison of Anisakis ITS sequences diagnosed the presence of A. simplex C in 2 M. angustirostris hosts, which is a new host record. Anisakis specimens from Phocoena phocoena (harbor porpoise), Lissodelphis borealis (Pacific rightwhale porpoise), and E. jubatus included 3 ITS sequences that did not match any known species. Contracaecum adults obtained from Z. californianus were most closely related to C. ogmorhini s.l. and C. rudolphii, but ITS sequences of these Contracaecum specimens did not match C. ogmorhini s. str. or C. margolisi. These novel Anisakis and Contracaecum ITS sequences may represent previously uncharacterized species. Phylogenetic analysis of LSU sequences revealed strong support for the monophyly of Anisakinae, Contracaecum plus Phocascaris, Pseudoterranova, and Anisakis. Phylogenetic trees inferred from ITS sequences yielded robustly supported relationships for Pseudoterranova and Anisakis species that are primarily consistent with previously published phenograms based on multilocus electrophoretic data.     

Characterization of tissue specific expression of Notch-1 in human tissues

Signaling through the Notch cell surface receptors is a highly conserved mechanism of cell fate specification. Notch signaling regulates proliferation, differentiation and cell death. In vertebrates, putative gene duplication has originated four Notch genes, Notch-1, -2, -3 and -4. They have been implicated in neurogenesis, hematopoiesis, T-cell development, vasculogenesis and brain cortical growth. We have investigated Notch-1 distribution in normal human tissues by immunohistochemistry and immunoblot. We detected widespread expression of Notch-1 cytoplasmatic staining, with different tissue distributions in the different organs examined. In particular, high expression of Notch-1 was detected in the intermediate suprabasal layers, but not in the dead cells at the extreme periphery of stratified epithelia. Moreover, a low/intermediate level of Notch-1 was observed in lymphocytes in several peripheral lymphoid tissues; in particular the germinal centers of lymph nodes showed the most abundant number of positive cells, which appeared to be centroblasts/immunoblasts based on nuclear morphology. Notch-1 participates in keratinocytes differentiation. We showed by Western blot analysis that Notch-1 level was clearly increased in HaCaT cells after Ca(++) addition and remained substantially elevated until late differentiation stages. These results suggest that Notch-1 may function in numerous cell types in processes beyond cell fate determination, such as neuronal plasticity, muscle hypertrophy, liver regeneration, and germinal center lymphopoiesis during the immune response.     

The PINE study: rationale and design of a randomised comparison of epidural injection of local anaesthetics and steroids versus care-as-usual to prevent postherpetic neuralgia in the elderly [ISRCTN32866390]

BACKGROUND: Postherpetic neuralgia (PHN) is by far the most common complication of herpes zoster (HZ) and one of the most intractable pain disorders. Since PHN is seen most often in the elderly, the number of patients with this disorder is expected to increase in our ageing society. PHN may last for months to years and has a high impact on the quality of life. The results of PHN treatment are rather disappointing. Epidural injection of local anaesthetics and steroids in the acute phase of HZ is a promising therapy for the prevention of PHN. Since randomised trials on the effectiveness of this intervention are lacking, the PINE (Prevention by epidural Injection of postherpetic Neuralgia in the Elderly) study was set up. The PINE study compares the effectiveness and cost-effectiveness of a single epidural injection of local anaesthetics and steroids during the acute phase of HZ with that of care-as-usual (i.e. antivirals and analgesics) in preventing PHN in elderly patients. METHODS / DESIGN: The PINE study is an open, multicenter clinical trial in which 550 elderly (age >/= 50 yr.) patients who consult their general practitioner in the acute phase of HZ (rash < 7 days) are randomised to one of the treatment groups. The primary clinical endpoint is the presence of HZ-related pain one month after the onset of the rash. Secondary endpoints include duration and severity of pain, re-interventions aiming to treat the existing pain, side effects, quality of life, and cost-effectiveness. CONCLUSION: The PINE study is aimed to quantify the (cost-) effectiveness of a single epidural injection during the acute phase of HZ on the prevention of PHN.     

Allozyme and PCR-RFLP markers in anisakid nematodes,aethiological agents of human anisakidosis

In the framework of the researches granted by MURST COFIN97, studies on anisakid nematodes, aethiological agents of human anisakidosis, were carried out. The project was aimed to implement the knowledge on the systematics, genetics, ecology and epizootiology of species of the genera Anisakis and Pseudoterranova by applying genetic markers obtained from multilocus allozyme electrophoresis and from PCR-based techniques. The results obtained by allozyme studies allowed to extend the geographic distribution and to detect new definitive and intermediate/paratenic hosts of two sibling species of the A. simplex complex, i.e. A. simplex s.s. and A. pegreffii and to characterise the species A. simplex C, a new sibling species within the A. simplex complex as well as a new species belonging to the genus Anisakis, A. ziphidarum. Combined allozyme and morphological analyses provided markers for the identification of the four sibling species of the Pseudoterranova decipiens complex and their nomenclatural designations. New markers based on PCR-RFLP analysis were used for the identification of sibling species of the Anisakis simplex complex and of another four species of the genus. Genetic markers based on three diagnostic restriction enzymes allowed the identification as A. pegreffii of a larva obtained by endoscopy in a case of human anisakidosis in Southern Italy.     

The Application of Chironomid Pupal Exuvial Technique (CPET) for Ecological Analysis in a Neotropical Large River System

This field ecological study, based on the chironomid pupal exuvial technique (CPET), is new for the Paraná River and proposes an efficient tool to be used in future ecological approaches and biomonitoring. Drifting of pupal exuviae in a river-floodplain system of the Middle Paraná River floodplain was represented by 34 Chironomidae taxa, being the characteristic association obtained from the CPET: Lopescladius, Onconeura, Paralauterborniella, Polypedilum, and Harnischia complex. Diversity, richness, dominance, total density, and density of dominant taxa were different between the longitudinal and lateral dimensions but not between hydrologic phases, with a greater diversity and richness in the main channel of the river and higher density and dominance in floodplain habitats. The species turnover is the dominant process in structuring studied assemblages in spatial and temporal analysis, increasing in the floodplain habitats and in low-water phase. The results obtained showed that drifting exuviae in the longitudinal axis were coming from different assemblages and environments of a wider area (regional), while exuviae recorded in the connections of the floodplain environments in the lateral dimension could reflect the local assemblages. We demonstrated the ecological value of CPET studies to interpret the attributes of Chironomidae assemblage in river-floodplain systems of large rivers in an integrated way.     

Scleroderma-polymyositis overlap syndrome versus idiopathic polymyositis and systemic sclerosis: a descriptive study on clinical features and myopathology

Introduction

The objective was to characterize the clinical and myopathologic features of patients with scleroderma-polymyositis (SSc-PM) overlap compared with a population of patients with systemic sclerosis (SSc) and polymyositis (PM).

Methods

A three-way comparison of patients with SSc-PM overlap (n = 25) with patients with SSc (n = 397) and PM (n = 40) on clinical and myopathologic features and causes of death. One neuropathologist blinded for the diagnosis evaluated all recent available muscle biopsies. Biopsies were scored for presence of inflammation, necrotic muscle fibers, rimmed vacuoles, fibrosis, and immunohistochemical staining. Clinical or myopathologic characteristics were compared by using the χ² test or one-way analysis of variance (ANOVA).

Results

The prevalence of SSc-PM overlap in the Nijmegen Systemic Sclerosis cohort was 5.9%. The mortality was 32% (eight of 25) in SSc-PM, of which half was related to cardiac diseases. The prevalence of pulmonary fibrosis was significantly increased in SSc-PM (83%) (P = 0.04) compared with SSc (49%) and PM (53%). SSc or myositis-specific antibodies were nearly absent in the SSc-PM group. In almost all biopsies (96%) of SSc-PM patients, necrotic muscle fibers were present, which was significantly increased compared with PM patients (P = 0.02).

Conclusions

Patients with SSc-PM have increased prevalence of pulmonary fibrosis and cardiac disease as the cause of death compared with patients with SSc and PM . In addition, we found that necrotizing muscle fibers with inflammation characterize SSc-PM overlap in muscle biopsies. Further research should focus on underlying mechanisms causing necrosis, inflammation, and fibrosis and their relation to pulmonary involvement and mortality in patients with SSc-PM overlap.