Osteopontin expression in healing wounds of horses and in human keloids

REASONS FOR PERFORMING STUDY: Convincing evidence shows that persistent or excessive expression of osteopontin (OPN) is linked to fibroproliferation of various organs in laboratory animals and in man, such that its downregulation is a logical therapeutic objective. OBJECTIVES: To investigate OPN expression in an equine model of wound healing and in clinical specimens of equine exuberant granulation tissue and human keloids in an effort to better understand the contribution of this protein to inflammation-associated skin fibrosis. STUDY DESIGN: Description of gene and protein expression in an experimental equine model of wound healing and clinical specimens in horse and man. METHODS: Osteopontin gene expression was evaluated by quantitative PCR, while protein expression was investigated by means of immunohistochemical staining. RESULTS: Quantitative PCR showed that the OPN gene is expressed in normal intact skin of horses and continues to be expressed during the wound-healing process. An increase in gene expression was observed throughout the phases of wound healing, with a final decrease at wound closure. The protein was not detected in normal skin. Keratinocytes in wound-edge samples did not express the protein, whereas dermal immunoreactivity was confined to inflammatory cells. Healed wounds were devoid of staining. Equine exuberant granulation tissue showed immunoreactivity of the surrounding epidermis, infiltrating neutrophils, mononuclear cells, endothelial cells and fibroblasts. Human keloids showed OPN immunoreactivity throughout the epidermis as well as in mononuclear cells and scattered fibroblasts. CONCLUSIONS: Immunohistochemical data show a different pattern of expression between normally healing and fibrotic wounds (exuberant granulation tissue and keloids), thus suggesting a role in fibroproliferation in horses and man

Evaluation of formalin-fixed paraffin-embedded tissues in the proteomic analysis of parathyroid glands

<p>Abstract</p> <p>Background</p> <p>Proteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification.</p> <p>Results</p> <p>The best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin.</p> <p>Conclusions</p> <p>In spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining.</p

Salivary psoriasin (S100A7) correlates with diffusion capacity of carbon monoxide in a large cohort of systemic sclerosis patients

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by progressive fibrosis of the skin and the internal organs. In a previous work we suggested a correlation between levels of salivary psoriasin (S100A7) and pulmonary involvement in SSc patients. The goals of this study are to determine the distribution characteristics of psoriasin in whole saliva (WS) of SSc and healthy donor populations and define its predictive value on diffusion capacity of carbon monoxide (DLCO), along with others clinical parameters. Methods: Salivary level of psoriasin was determined by ELISA kit in 134 SSc patients, 63 Raynaud syndrome patients, 40 patients affected by other connective diseases (non-case) and 74 healthy control subjects. Results: A significant increase of salivary psoriasin was observed in SSc patients when compared with other healthy and pathological controls. Moreover, we confirmed the efficacy of salivary psoriasin to correlate with DLCO in a large cohort of SSc patients. Conclusions: Overall our results suggest a rapid, non invasive and low costing method which can help clinicians in the evaluation of SSc pulmonary involvement

Underuse of Anthracyclines in Women with HER-2+ Advanced Breast Cancer

This article examines how discouraging the use of anthracyclines in combination with trastuzumab in patients with human epidermal growth factor receptor 2 positive metastatic breast cancer because of fears of cardiotoxicity has influenced the use of these agents in this patient setting

Correlation between NK function and response to trastuzumab in metastatic breast cancer patients

<p>Abstract</p> <p>Background</p> <p>Trastuzumab is a monoclonal antibody selectively directed against Her2 and approved for the treatment of Her2 overexpressing breast cancer patients. Its proposed mechanisms of action include mediation of antibody-dependent cellular cytotoxicity (ADCC) by triggering FcγRIII on natural killer (NK) cells. This study addresses the correlation between overall NK function and trastuzumab's clinical activity.</p> <p>Subjects and methods</p> <p>Clinical and immunological responses were assessed in 26 patients receiving trastuzumab monotherapy as maintenance management after chemotherapy (8 mg/kg load and then standard doses of 6 mg/kg every 3 weeks). Cytotoxic activity against the MHC class I-negative standard NK target K562 cell line and HER2-specific ADCC against a trastuzumab-coated Her2-positive SKBR3 cell line were assessed in peripheral blood mononuclear cells (PBMC) harvested after the first standard dose. After six months, seventeen patients were scored as responders and nine as non-responders according to the RECIST criteria, while Progression-Free Survival (PFS) was calculated during a 12 months follow-up.</p> <p>Results</p> <p>The responders had significantly higher levels of both NK and ADCC activities (p < 0.05) that were not different from those of eleven normal controls. The NK activity of the non-responders was significantly (p < 0.05) lower than that of the normal controls. At twelve months, there was a marked correlation between PFS and NK activity only. PFS was significantly longer in patients with high levels of NK activity, whereas its pattern was unrelated to high or low ADCC activity.</p> <p>Conclusion</p> <p>One of the mechanisms of action of trastuzumab is NK cell-mediated ADCC lysis of the Her2-positve target cell. We show here that its potency is correlated with the short-term response to treatment, whereas longer protection against tumor expansion seems to be mediated by pure NK activity.</p

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

BACKGROUND: PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. RESULTS: Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. CONCLUSIONS: The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria

A multidisciplinary approach to study a couple of monozygotic twins discordant for the chronic fatigue syndrome: a focus on potential salivary biomarkers

BACKGROUND: Chronic Fatigue Syndrome (CFS) is a severe, systemic illness characterized by persistent, debilitating and medically unexplained fatigue. The etiology and pathophysiology of CFS remains obscure, and diagnosis is formulated through the patient’s history and exclusion of other medical causes. Thereby, the availability of biomarkers for CFS could be useful for clinical research. In the present study, we used a proteomic approach to evaluate the global changes in the salivary profile in a couple of monozygotic twins who were discordant for CFS. The aim was to evaluate differences of salivary protein expression in the CFS patient in respect to his healthy twin. METHODS: Saliva samples were submitted to two-dimensional electrophoresis (2DE). The gels were stained with Sypro, and a comparison between CFS subject and the healthy one was performed by the software Progenesis Same Spot including the Analysis of variance (ANOVA test). The proteins spot found with a ≥2-fold spot quantity change and p<0.05 were identified by Nano-liquid chromatography electrospray ionization tandem mass spectrometry. To validate the expression changes found with 2DE of 5 proteins (14-3-3 protein zeta/delta, cyclophilin A, Cystatin-C, Protein S100-A7, and zinc-alpha-2-glycoprotein), we used the western blot analysis. Moreover, proteins differentially expressed were functionally analyzed using the Ingenuity Pathways Analysis software with the aim to determine the predominant canonical pathways and the interaction network involved. RESULTS: The analysis of the protein profiles allowed us to find 13 proteins with a different expression in CFS in respect to control. Nine spots were up-regulated in CFS and 4 down-regulated. These proteins belong to different functional classes, such as inflammatory response, immune system and metabolism. In particular, as shown by the pathway analysis, the network built with our proteins highlights the involvement of inflammatory response in CFS pathogenesis. CONCLUSIONS: This study shows the presence of differentially expressed proteins in the saliva of the couple of monozygotic twins discordant for CFS, probably related to the disease. Consequently, we believe the proteomic approach could be useful both to define a panel of potential diagnostic biomarkers and to shed new light on the comprehension of the pathogenetic pathways of CFS

P2‐239: POTENTIAL DIAGNOSTIC VALUE OF RED BLOOD CELLS α‐SYNUCLEIN HETEROAGGREGATES IN ALZHEIMER'S DISEASE

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Vinorelbine-based salvage therapy in HER2-positive metastatic breast cancer patients progressing during trastuzumab-containing regimens: a retrospective study

<p>Abstract</p> <p>Background</p> <p>The vinka-alkaloyd vinorelbine is a potentially valuable treatment in patients with HER2-positive, trastuzumab-resistant advanced breast cancer. We sought to document the clinical activity of vinorelbine-based salvage treatments in this clinical setting.</p> <p>Methods</p> <p>We analyzed a cohort of 424 consecutive women receiving trastuzumab-based therapy for HER2-positive advanced breast cancer. Of these, 299 were identified as progressing during the initial trastuzumab-based treatment, and 77 received vinorelbine-based therapy as first salvage treatment. Central review of pathological specimens revealed that 70 patients had HER2-amplification detected by FISH. For these patients we determined overall response rate (ORR = complete-CR + partial-PR) and clinical benefit (CB = CR+PR+ Stable disease lasting at least 6 months), time to progression (TTP) and overall survival (OS) from the initiation of vinorelbine-based salvage therapy.</p> <p>Results</p> <p>In 60 patients who were evaluable for tumor response, ORR and CB rates were 28% (95% C.I. 18%-41%) and 50% (95% C.I. 38%-62%), respectively. Median follow-up from the initiation of salvage therapy was 15 months (range 1–63 months). Median TTP and OS were 7.1 months (95% C.I. 6.6–7.7 months) and 21 months (95% C.I. 14.3–27.7 months), respectively. No differences in clinical outcomes were observed according to whether vinorelbine was administered as a single agent or in combination with other cytostatics, or whether trastuzumab was stopped or continued beyond disease progression.</p> <p>Conclusion</p> <p>our findings suggests that vinorelbine-based combinations are active and should be further evaluated in studies conducted in trastuzumab-resistant patients, including those evaluating newer HER2-targeting agents.</p