Monocytes-based in vitro assay for a preliminary biocompatibility assessment of blood-contacting devices

The biological evaluation of biomaterials is currently defined by the ISO-10993 norm in which parts four and five are dedicated to emo-compatibility and cell toxicity, respectively. Our study will provide a novel in vitro experimental approach for the biocompatibility assessment of biomaterials or medical devices using human primary monocytes as cellular model. In these new settings, human monocytes are exposed to a medium containing the extractable compounds derived from materials or devices; subsequently, cell toxicity and pro-inflammatory effects are analysed through MTT assay, flow cytometry and enzyme-linked immunosorbent assay (ELISA) methodologies. These experimental procedures offer the advantage to use a human and primary cell context belonging to the immune system, in order to accurately predict the nature of blood/device interaction occurring during a clinical application. To validate the reliability of this method, we also reported a comparative study between two different membranes showing a different level of biocompatibility. On the bases of these data, it is possible to state that this new experimental model represents a good approach to investigate the effects induced by a biomaterial on cell death and inflammation using human, primary monocytes

Radical improvement of signs and symptoms in systemic lupus erythematosus when treated with hemodiafiltration with endogenous reinfusion dialysis

Lupus nephritis is one of the most serious complications of systemic lupus erythematosus (SLE). In the kidney immune complexes (ICs) and autoantibodies activate mesangial cells that secrete the cytokines that further amplify inflammatory processes. We present a case of a 42-year-old woman presented Lupus Nephritis, with periods of exacerbation of SLE, with necrotic-like skin lesions, psoriatic arthritis without skin psoriasis, purpura to lower limb, petechial rash, joint pain, fever, eyelid edema with bilateral conjunctival hyperemia and itching When she was subjected to Hemodiafiltration with Endogenous Reinfusion (HFR) dialysis treatment with super high flux membrane Synclear 02 (SUPRA treatment), fever and joint pain was reduced immediately, subsequently all of her skin damages are reduced and she gradually decreased quantity of prednisone and immunosuppressor per die until completely suspend. As well known that SUPRA treatment remove cytokine from blood ; moreover was used the High-performance liquid chromatography coupled with Quadrupole Time-of-flight Mass Spectrometer (HPLC-QTOF-MS) for identification of proteins captured by resin bed during a dialysis session of the patient. With this technique was identified several biomarker of kidney injuries, uremic toxins, fragments of Immunoglobulins, antigen involved in anti-phospholipid syndrome and a new marker (α-defensin) that correlate significantly with disease activity. The removal of these different proteins can explain the improvement in the patient’s symptoms and the normalization of her LES, confirming that SUPRA are a suitable technique for LN treatment

Identification of the most abundant proteins in equine amniotic fluid by a proteomic approach

Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4\u201312% gels. A pool of the 24 samples, after SDS-PAGE, was cut in 25 slices, trypsin-digested and analysed by mass spectrometry (MS) for protein identification. Mean AF protein concentration was 1.96\ua0\ub1\ua01.12\ua0g/L. Thirty-four proteins were successfully identified by MS and subsequently categorised according to Gene Ontology (GO). Twelve proteins (e.g. fibronectin, lumican, thrombospondin and fibulin) belonged to or interacted with the extracellular matrix (ECM) playing an important role in the development of foetal tissues. Most of the remaining proteins were classified as transport (e.g. albumin, major allergen Equ c1 and alpha-fetoprotein) delivering nutrients, ions and lipids essential for foetal growth and development. Among these proteins, major allergen Equ c1 is widely studied in human medicine because it induces Ig-E mediated type I allergic reaction. The absence of immunoglobulins in equine AF was also confirmed

Validation of potential candidate biomarkers of drug-induced nephrotoxicity and allodynia in medication-overuse headache

Medication-overuse headache (MOH) is a chronic disorder that results from the overuse of analgesics drugs, triptans or other acute headache compounds. Although the exact mechanisms underlying MOH remain still unknown, several studies suggest that it may be associated with development of "central sensitization", which may cause cutaneous allodynia (CA). Furthermore, the epidemiology of drug-induced disorders suggests that medication overuse could lead to nephrotoxicity. The aim of this work was to confirm and validate the results obtained from previous proteomics studies, in which we analyzed the urinary proteome of MOH patients in comparison with healthy non-abusers individuals

Analysis of protein expression in periodontal pocket tissue: a preliminary study

The periodontal disease is caused by a set of inflammatory disorders characterized by periodontal pocket formation that lead to tooth loss if untreated. The proteomic profile and related molecular conditions of pocket tissue in periodontally-affected patients are not reported in literature. To characterize the proteomic profile of periodontally-affected patients, their interproximal periodontal pocket tissue was compared with that of periodontally-healthy patients. Pocket-associated and healthy tissue samples, harvested during surgical therapy, were treated to extract the protein content. Tissues were always collected at sites where no periodontal-pathogenic bacteria were detectable. Proteins were separated using two-dimensional gel electrophoresis and identified by liquid chromatography/mass spectrometry. After identification, four proteins were selected for subsequent Western Blot quantitation both in pathological and healty tissues

Discovery by a proteomic approach of possible early biomarkers of drug-induced nephrotoxicity in medication-overuse headache

BACKGROUND: Medication-overuse headache (MOH) is a chronic headache condition that results from the overuse of analgesics drugs, triptans, or other antimigraine compounds. The epidemiology of drug-induced disorders suggests that medication overuse could lead to nephrotoxicity, particularly in chronic patients. The aim of this work was to confirm and extend the results obtained from a previous study, in which we analyzed the urinary proteome of 3 MOH patients groups: non-steroidal anti-inflammatory drugs (NSAIDs), triptans and mixtures abusers, in comparison with non-abusers individuals (controls). METHODS: In the present work we employed specialized proteomic techniques, namely two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS), and the innovative Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry (SELDI-TOF-MS), to discover characteristic proteomic profiles associated with MOH condition. RESULTS: By 2-DE and MS analysis we identified 21 over-excreted proteins in MOH patients, particularly in NSAIDs abusers, and the majority of these proteins were involved in a variety of renal impairments, as resulted from a literature search. Urine protein profiles generated by SELDI-TOF-MS analysis showed different spectra among groups. Moreover, significantly higher number of total protein spots and protein peaks were detected in NSAIDs and mixtures abusers. CONCLUSIONS: These findings confirm the presence of alterations in proteins excretion in MOH patients. Analysis of urinary proteins by powerful proteomic technologies could lead to the discovery of early candidate biomarkers, that might allow to identify MOH patients prone to develop potential drug overuse-induced nephrotoxicity

Proteomic research of proteins involved in pain expression in an animal model of chronic pain

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Serum protein changes in a rat model of chronic pain show a correlation between animal and humans

In previous works we showed the overexpression of some proteins in biological fluids from patients suffering chronic pain. In this proteomic study we analysed serum from a rat model of neuropathic pain obtained by the chronic constriction injury (CCI) of sciatic nerve, at two time intervals, 2 and 5 weeks after the insult, to find proteins involved in the expression or mediation of pain. Sham-operated and CCI rats were treated with saline or indomethacin. Two weeks after ligation, we identified three serum proteins overexpressed in CCI rats, two of which, alpha-1-macroglobulin and vitamin D-binding protein (VDBP), remained increased 5 weeks post-surgery; at this time interval, we found increased levels of further proteins, namely apolipoprotein A-I (APOA1), apolipoprotein E (APOE), prostaglandin-H2 D-isomerase (PTGDS) and transthyretin (TTR), that overlap the overexpressed proteins found in humans. Indomethacin treatment reversed the effects of ligation. The qPCR analysis showed that transcript levels of APOA1, APOE, PTGDS and VDBP were overexpressed in the lumbar spinal cord (origin of sciatic nerve), but not in the striatum (an unrelated brain region), of CCI rats treated with saline 5 weeks after surgery, demonstrating that the lumbar spinal cord is a possible source of these proteins

dynamic thiol disulphide homeostasis before and after radical prostatectomy in patients with prostate cancer

AbstractThiol groups are important anti-oxidants and essential molecules protecting organism against the harmful effects of reactive oxygen species (ROS). The aim of our study is to evaluate thiol–disulphide homeostasis with a novel recent automated method in patients with localized prostate cancer (PC) before and six months after radical prostatectomy (RP). 18 patients with PC and 17 healthy control subjects were enrolled into the study. Blood samples were collected from the controls subjects and patients before and six months after RP. Thiol–disulphide homeostasis was determined using a recently developed novel method. Prostate-specific antigen (PSA), albumin, total protein, total thiol, native thiol, disulphide and total antioxidant status (TAS) were measured and compared between the groups. Native thiol, total thiol and TAS levels were significantly higher in the control group than the patients before RP (p < .001). There was a non-significant increase in the native thiol, total thiol and TAS levels i..

Enriched sera protein profiling for detection of non-small cell lung cancer biomarkers

<p>Abstract</p> <p>Background</p> <p>Non Small Cell Lung Cancer (NSCLC) is the major cause of cancer related-death. Many patients receive diagnosis at advanced stage leading to a poor prognosis. At present, no satisfactory screening tests are available in clinical practice and the discovery and validation of new biomarkers is mandatory. Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-ToF-MS) is a recent high-throughput technique used to detect new tumour markers. In this study we performed SELDI-ToF-MS analysis on serum samples treated with the ProteoMiner™ kit, a combinatorial library of hexapeptide ligands coupled to beads, to reduce the wide dynamic range of protein concentration in the sample. Serum from 44 NSCLC patients and 19 healthy controls were analyzed with IMAC30-Cu and H50 ProteinChip Arrays.</p> <p>Results</p> <p>Comparing SELDI-ToF-MS protein profiles of NSCLC patients and healthy controls, 28 protein peaks were found significantly different (p < 0.05), and were used as predictors to build decision classification trees. This statistical analysis selected 10 protein peaks in the low-mass range (2-24 kDa) and 6 in the high-mass range (40-80 kDa). The classification models for the low-mass range had a sensitivity and specificity of 70.45% (31/44) and 68.42% (13/19) for IMAC30-Cu, and 72.73% (32/44) and 73.68% (14/19) for H50 ProteinChip Arrays.</p> <p>Conclusions</p> <p>These preliminary results suggest that SELDI-ToF-MS protein profiling of serum samples pretreated with ProteoMiner™ can improve the discovery of protein peaks differentially expressed between NSCLC patients and healthy subjects, useful to build classification algorithms with high sensitivity and specificity. However, identification of the significantly different protein peaks needs further study in order to provide a better understanding of the biological nature of these potential biomarkers and their role in the underlying disease process.</p