Mariarita Dessì

Atherosclerosis, dyslipidemia, and inflammation: the significant role of polyunsaturated Fatty acids.

Phospholipids play an essential role in cell membrane structure and function. The length and number of double bonds of fatty acids in membrane phospholipids are main determinants of fluidity, transport systems, activity of membrane-bound enzymes, and susceptibility to lipid peroxidation. The fatty acid profile of serum lipids, especially the phospholipids, reflects the fatty acid composition of cell membranes. Moreover, long-chain n-3 polyunsatured fatty acids decrease very-low-density lipoprotein assembly and secretion reducing triacylglycerol production. N-6 and n-3 polyunsatured fatty acids are the precursors of signalling molecules, termed “eicosanoids,” which play an important role in the regulation of inflammation. Eicosanoids derived from n-6 polyunsatured fatty acids have proinflammatory actions, while eicosanoids derived from n-3 polyunsatured fatty acids have anti-inflammatory ones. Previous studies showed that inflammation contributes to both the onset and progression of atherosclerosis: actually, atherosclerosis is predominantly a chronic low-grade inflammatory disease of the vessel wall. Several studies suggested the relationship between long-chain n-3 polyunsaturated fatty acids and inflammation, showing that fatty acids may decrease endothelial activation and affect eicosanoid metabolism.

Influence of the human paraoxonase polymorphism (PON1 192) on the carotid-wall thickening in a healthy population.

BACKGROUND Serum paraoxonase (PON1) is a high-density lipoprotein-bound enzyme that can prevent oxidation of low-density lipoprotein and thus exert an anti-atherogenic effect. A polymorphism at codon 192 (Gln/Arg) of the PON1 gene gives rise to two isoforms that differ in substrate-dependent activity. OBJECTIVE To determine any independent contribution of this polymorphism to the variability of intimal-medial thickness (IMT) of the common carotid artery for a sample of asymptomatic adult subjects from southern Italy by ultrasonography. METHODS We studied 196 unrelated asymptomatic subjects (mean age 55.1 years), drawn from participants in a cardiovascular-disease-prevention campaign. Plasma levels of lipids and glucose were measured by routine methods. PON1 polymorphism was determined by polymerase chain reaction. IMT was measured from high-resolution B-mode echo-Doppler ultrasonography images. RESULTS Prevalences of alleles A (Gln) and B (Arg) were 0.68 and 0.32, respectively. We found no significant difference with regard to plasma levels of lipids and glucose and other variables among the PON1 genotypes, although subjects with BB had higher levels of triglycerides and lower levels of high-density lipoprotein cholesterol. Common carotid artery IMT was slightly greater in subjects with BB, although no significant association between PON1 genotypes and common carotid artery IMT was found, even after adjustment for confounding variables. CONCLUSIONS Our findings demonstrate that there is no significant association between PON1 gene polymorphism at codon 192 and common carotid artery IMT for an Italian population. However, the fact that we found slightly greater IMT in subjects with genotype BB would suggest that the study should be performed again with a larger sample.

Determination of kFLC and K Index in cerebrospinal fluid: A valid alternative to assessintrathecal immunoglobulin synthesis

Intrathecal immunoglobulin synthesis is observed in several disorders of the central nervous system, but its detection by current laboratory tests is relatively insensitive and operator depending. We assessed the diagnostic accuracy of a nephelometric assay for k free light chain determination in cerebrospinal fluid and serum. The patients were grouped according to clinical and laboratory criteria. ROC curves for all methods were performed to find the best cut-off value. kFLC Index seems to be more accurate than other parameters. Our data indicate that nephelometric assay for kFLCs in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate multiple sclerosis patients.

Erythrocyte glutathione transferase: a new biomarker for hemodialysis adequacy, overcoming the Kt/V(urea) dogma?

Kt/Vurea ratio is commonly used to assess the delivered dose of dialysis in maintenance hemodialysis (MHD) patients. This parameter only reflects the efficacy of dialytic treatments in removing small toxins, but not middle and protein-bound toxins. Erythrocyte glutathione transferase (e-GST), an enzyme devoted to cell depuration against a lot of large and small toxins, is overexpressed in uremic patients. Aim of the present study is to verify whether e-GST may represent a novel biomarker to assess the adequacy of different dialytic techniques complementary to Kt/Vurea parameter. Furthermore, it will be investigated whether e-GST could reflect the ‘average’ adequacy of multiple dialytic sessions and not of a single one treatment as it occurs for Kt/Vurea. One hundred and three MHD patients and 82 healthy subjects were tested. Fourty four patients were treated with standard bicarbonate hemodialysis (HD) and 59 patients were on online hemodiafiltration (HDF). In all MHD patients e-GST activity was 60% higher than in healthy controls. In HDF, e-GST activity was lower than in HD subgroup (8.2±0.4 versus 10.0±0.4 U/gHb, respectively). Single-pool Kt/Vurea and total weekly Kt/Vurea were higher in HDF than in HD, but no correlation was found between e-GST activity and Kt/Vurea data. e-GST, whose level is stable during the erythrocyte life-span, provides information on the long-term depurative efficacy of dialysis treatments.

Chronic treatment with statins increases the availability of selenium in the antioxidant defence systems of hemodialysis patients.

PROJECT Oxidative stress (OS) is enhanced in hemodialysis (HD) patients. Lipid peroxidation and oxidative damage to glycids, proteins and nucleic acids are the main consequences of OS and are associated with increased cardiovascular risk. Vitamin E and glutathione peroxidase (GSH-Px) represent the main antioxidant systems in human cells. Selenium (Se), bound to the active sites of GSH-Pxs, plays a critical role in this antioxidant defence system. Statins are widely used and extensively investigated in the prevention of cardiovascular disease, notably in high-risk subjects. Several studies show antioxidant effects of statins not related to their lipid-lowering action. Our study aimed to compare serum Se concentration in ESRD patients on maintenance HD and in homogeneous healthy subjects and to investigate whether chronic treatment with statins may interfere with serum Se concentration in HD patients. PROCEDURE A total of 103 HD patients and 69 healthy subjects were enrolled; HD patients were divided into patients who were not treated with statins (group A) and patients who assumed statins since 6 months at least (group B). Serum Se was determined by atomic absorption spectrometry. RESULTS Serum Se was significantly lower in HD patients of group A compared with healthy subjects (81.65+/-19.66 Vs. 96.47+/-15.62 mcg/L, p<0.0040). However, in HD patients who assumed statins serum, Se was significantly higher than in HD patients who did not (111.83+/-18.82 vs. 81.65+/-19.66 mcg/L, p<0.0001). CONCLUSIONS Our results suggest that in HD patients chronic treatment with statins is related to higher-serum Se concentration.

The usefulness of the prognostic inflammatory and nutritional index (PINI) in a haemodialysis population.

BACKGROUND AND AIM Protein-Energy Wasting and inflammation are the principal risk factors of haemodialysis complications. We evaluated the reliability of a simple and non expensive test, the Prognostic Inflammatory and Nutritional Index (PINI), for regular screening of maintenance haemodialysis (MHD) patients in order to detect early onset of inflammation and malnutrition. METHODS AND RESULTS 121 adult patients on maintenance dialysis were followed up for 32 months and screened every 6 months for PINI, calculated as alpha1-Acid Glycoprotein (alpha1-AG)xC-Reactive Protein (CRP)/AlbuminxTransthyretin. PINI score < or =1 was considered normal. Patients were stratified according to their PINI score: 86 patients (71.66%) had a normal score, whereas 35 (28.33%) had PINI > or = 1. The latter also had higher CRP levels, despite no clinical evidence of inflammation at the time of enrolment. Survival in patients with normal PINI was similar to patients with normal CRP, while in patients with abnormal PINI it was significantly lower than in patients with low serum albumin (p<0.05) or elevated CRP (p<0.05). After follow-up, all surviving MHD patients with PINI > or = 1 had at least one cardiovascular event vs 2.5% of patients with PINI > or = 1. CONCLUSION The assessment of PINI can reliably identify MHD patients at higher risk of mortality and morbidity even in the absence of overt Malnutrition-Inflammation Complex Syndrome (MICS). This simple test appears to be more sensitive and specific of the single components, and not expensive, so that it could be routinely used to identify patients with sub-clinical inflammation and/or malnutrition.

ALTERNATIVE SPLICING OF HUMAN PLASMA CHOLESTERYL ESTER TRANSFER PROTEIN MRNA IN CACO-2 CELLS AND ITS MODULATION BY OLEIC ACID

Cholesteryl ester transfer protein (CETP) is a plasma protein involved in the reverse cholesterol transport and expressed in several human tissues and cell lines. We studied CETP expression in Caco-2 cell line, a model of the human enterocyte epithelium. By reverse-transcriptase polymerase chain reaction, we could demonstrate that in basal condition Caco-2 cells have a low rate of expression of active CETP mRNA. Furthermore, we found that even in this cell line CETP mRNA alternative splicing occurs with deletion of exon 9 sequence. Densitometric analysis of the in vitro amplified fragments showed that under basal conditions about 60% of reverse transcribed CETP cDNA corresponds to exon 9-deleted transcripts. After challenge with 50 µM sodium oleate, there is a ∼2 fold increase in the transcription rate of the full-length CETP cDNA, as measured by competitive PCR, which is accompanied to an increased activity measured in the cell-conditioned medium. On the contrary, no significant change is seen in the amount of exon 9-deleted cDNA. Consequently, an inversion in the ratio of full-length and exon 9-deleted CETP cDNA is evident, suggesting that sodium oleate selectively enhances the expression of full-length CETP mRNA.

KFLC Index utility in multiple sclerosis diagnosis: Further confirmation

The Multiple Sclerosis (MS) diagnosis is based on dissemination of focal lesions in time and space. The free light chains (FLCs) determination might be a sensitive alternative to oligoclonal bands assay. The study aim was to redefine sensitivity, specificity of the kFLC Index cut-off. We analyzed serum and cerebrospinal fluid of 176 patients, with different neurological disorders. We obtained a cut off of 12,3 for kFLC Index with a sensitivity and specificity of 93% and 100% respectively. Our data confirm that the kFLC Index is a valid tool in the diagnosis of MS.

Minimal tumour burden in haematological diseases: a step forward with quantitative assessment of Bence‐Jones in nephelometry?

Serum and urine free light chains (FLCs) monoclonal immunoglobulin (k and k) are important markers in the diagnosis and monitoring of B cell proliferative disorders, such as Multiple Myeloma (MM), Monoclonal Gammopathy (MG), Monoclonal Gammopathy of Undetermined Significance (MGUS), Amyloidosis (AL) and related disorders (Bird et al, 2009; Kyle et al, 2010). MM is a progressive neoplastic disease characterized by bone marrow plasmacytosis (plasma cell tumour) and overproduction of an intact monoclonal immunoglobulin (IgG, IgA, IgM, IgD or IgE) and/or Bence Jones protein. The presence of one of the three following criteria allows the diagnosis of myeloma in affected patients: layers or clusters of plasma cells in the bone, osteolytic lesions (without the presence of metastatic cancer or granulomatous disease) or Bence Jones proteinuria (Durie et al, 2006). Determination of the Bence Jones protein (BJP) is essential when clinical suspicion of MG persists and serum tests are negative; moreover it is useful to establish a diagnosis of MG (Bird et al, 2009; Dimopoulos et al, 2011) and to monitor response to therapy in MM. It has been suggested that the FLCs assay can replace the traditional 24-h urine protein electrophoresis and immunofixation as part of a diagnostic screen (International Myeloma Working Group 2003; Eslick & Talaulikar, 2013). The excess production of FLCs (over 10–30 g/day) by a neoplastic clone of plasma cells saturates their absorption in the proximal renal tubules, causing damage and proteinuria, with large amounts of FLCs detectable in the urine. Urine FLCs (BJP) analysis can probably still be useful for initiating therapy and to highlight minimal tumour burden in patients with MM (Eslick & Talaulikar, 2013; Paiva et al, 2015a) and related disorders. There is growing interest in Minimal Residual Disease (MRD) monitoring, which can be used as a prognostic factor and to predict patients’ outcomes in MM (Paiva et al, 2015b). Guidelines recommend that immunofixation (IFE) of urine should be used to detect BJP (Durie et al, 2006; Bird et al, 2009). Currently, BJP quantification must be carried out on spot urine sample by densitometry of the corresponding immunoelectrophoretic band. This method is complicated by the different affinity for used dyes, the coincidental migration of other proteins and is operator dependent; therefore, quantitative analysis could be performed by nephelometric/turbidimetric assay. Although IFE is the ‘gold standard’ for the determination of BJP, a new nephelometric method, based on monoclonal antibodies, has been developed (Pretorius et al, 2012). The monoclonal antibodies provide a more accurate measurement reducing a possible overestimation when polyclonal antibodies are used. The aim of the present study was to compare the performance of nephelometric assay to IFE agarose gel in urine samples and to assess the correlation between the results of these two tests and the clinical diagnosis (negative or positive for B cell proliferative disorders). Serum and urine samples were collected from 378 patients (186 female and 192 male; one sample for each patient: no follow-up sample needed for this type of analysis) who presented to “Tor Vergata University Hospital”. Patients were evaluated with serum electrophoresis (SPE), urine immunofixation (IFE) and nephelometric assays for urine FLCs and additional routine analyses. Exclusion criteria were history and/or signs of chronic disease as primary renal failure or patients undergoing dialysis. FLCs measurement was performed using the N Latex FLC kit based on a mixture of monoclonal antibodies for use on the BN ProSpec System analyser (Siemens GmbH, Marburg, Germany) (Pretorius et al, 2012). BJP was determined by immunofixation on a semi-automated agarose electrophoresis system (Hydragel 4 BJ and Hydrasys; Sebia, Florence, Italy). The gels were evaluated for the presence of FLCs by three independent operators and reported as positive or negative. The evaluation was performed without knowledge of the outcome from the nephelometric assays. Data obtained show a significant correlation between nephelometric and IFE assays (Spearman correlation r = 0 82, P < 0 001). We analysed the Receiver-Operator Characteristic (ROC) curves for kappa and lambda nephelometric FLCs assay with respect to diagnosis (Fig 1A and B). The areas under ROC curves were 1 for both kappa and lambda BJP. We found 99% and 100% sensitivity and 100% and 99% specificity, for kappa and lambda FLCs, respectively, and the optimal cutoff for kappa FLCs was 0 25 mg/l and 0 11 g/l for lambda FLCs. We made a new correlation of the nephelometric data with respect to diagnosis that was re-assessed and shown to be more significant (r = 0 98, P < 0 001; Spearman correlacorrespondence

K Index in cerebrospinal fluid: a valid tool in multiple sclerosis diagnosis

SummaryBackground.Detection of oligoclonal IgG bands in cerebrospinal fluid by isoelectrocfocusing and immunodetection is the current gold standard to detect an inflammatory process in the central nervous system. It has been proposed that the presence of free light chains (FLCs) in CSF was associated with recent demyelination activity in MS and might be used as a prognosis marker. Our study’s objective is assessing the diagnostic accuracy of a new highly sensitive latex-enhanced nephelometric assay for k free light chain (kFLC) determination in CSF/serum as an alternative to traditional tests and its clinical application.Methods.kFLCs were measured in CSF/serum pairs from 80 patients by the use of a new highly sensitive latex-enhanced nephelometric automated immunoassay for detection of immunoglobulin FLC. The eighty patients were split into three groups according to the neurological diagnosis. In this study we confirm even more the use of the k Index as a diagnostic aid in multiple sclerosis.Results.kFLC Index seems to be more accurate parameter respect the determination of oligoclonal immunoglobulin bands (OCBs). We recalculate the K Index sensitivity and specificity respect the precedent published result. Two patients previously diagnosed with leukoencephalopathy have gone to group 3 as confirmed the diagnosis of MS.Conclusions.These new data reinforce even more the use of the k Index to diagnose MS in comparison to classical methods and to the reference method, the OCBs.