Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit).

PURPOSE: Testicular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model. METHODS: Pieces of immature testicular tissue (1 mm3) were inserted into "E.Vit" devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming. RESULTS: Post-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response. CONCLUSIONS: Vitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue.

MicroRNA Expression Analysis of Centenarians and Rheumatoid Arthritis Patients Reveals a Common Expression Pattern.

Micro-RNA (miRNA) are a family of small non-coding ribonucleic acids that inhibits post-transcriptionally the expression of their target messenger RNA (mRNA). We are interested in studying the involvement of miRNA in longevity and autoimmune diseases. In this study we compared the different expression of seven microRNAs between human plasma healthy controls, plasma samples of centenarians and samples from patients with rheumatoid arthritis. We used the Life Technologies' protocol to quantify seven miRNAs from 62 plasma samples: 20 healthy human controls, 14 centenarians, 28 patients with rheumatoid arthritis. TaqMan MicroRNA assays were used to analyze the expression profiles of miR-125b-5p, miR-425-5p, miR-200b5p, miR-200c-3p, miR-579-3p, miR-212-3p, miR-21-5p and miR-126-3p. The relative expression of mature miRNAs was analyzed using software REST. Our results show that miR-425-5p, miR-21 and miR-212 significantly decreased in centenarians and in patients with rheumatoid arthritis compared with controls. Furthermore in this work we highlight a connection between corticosteroid treatment and miRNAs expression.

Plasma concentrations of transthyretin in older Sardinians including centenarians.

BACKGROUND AND AIMS: Plasma concentrations of transthyretin (TTR), a negative acute-phase protein, can be influenced by many factors including aging. Under physiological circumstances, TTR concentrations are very low in the fetus, increase slowly after birth up to the fifth decade and, then, decrease slowly. Some studies have shown sex-related differences up to about 70 years, when the differences disappear. The aim of this study was to evaluate the change in TTR concentrations in healthy males and females aged more than sixty, including numerous centenarians living in Sardinia, a large Italian island located in the Mediterranean Sea. METHODS: The study sample consisted of 211 healthy subjects grouped by age and sex (male/female ratio: 1:1). Plasma TTR was assessed using a non-competitive enzyme immunoassay (ELISA Assaypro LLC, prealbumin AssayMAX Human ELISA Kit). RESULTS: In subjects aged between 60 and 99 years, plasma TTR concentrations were higher compared to the reference ranges reported by CRM 470. Moreover, unlike other studies, sex-related differences in TTR concentrations were only observed in nonagenarians and centenarians. CONCLUSIONS: We hypothesize that there are TTR-related genetic differences between the Sardinian population and other Caucasian ethnic groups. Further studies and a larger sample are needed to confirm our hypothesis.

Pre-analytical stability of the plasma proteomes based on the storage temperature.

BACKGROUND: This study examined the effect of storage temperature on the protein profile of human plasma. Plasma samples were stored for 13 days at -80°C, -20°C, +4°C and room temperature (20-25°C) prior to proteomic analysis. The proteomic comparisons were based on the differences of mean intensity values of protein spots between fresh plasma samples (named "time zero") and plasma samples stored at different temperatures. To better understand the thermally induced biochemical changes that may affect plasma proteins during storage we identified proteins with different expressions with respect to the time zero sample. RESULTS: Using two-dimensional electrophoresis followed by MALDI-TOF MS and /or LC-MS/MS 20 protein spots representing 10 proteins were identified with significant differences in abundance when stored at different temperatures. Our results, in agreement with various authors, indicate that during storage for a short period (13 days) at four different temperatures plasma proteins were more affected by degradation processes at +4°C compared to the other temperatures analysed. However, we founded that numerous protein spots (vitamin D binding protein, alpha-1-antitrypsin, serotransferrin, apoplipoprotein A-I, apolipoprotein E, haptoglobin and complement factor B) decrease in abundance with increasing temperature up to 4°C, but at room temperature their intensity mean values are similar to those of time zero and -80°C. We hypothesize that these proteins are labile at 4°C, but at the same time they are stable at room temperature (20-25°C). Furthermore we have grouped the proteins based on their different sensitivity to the storage temperature. Spots of serum albumin, fibrinogen gamma chain and haptoglobin are more resistant to the higher temperatures tested, as they have undergone changes in abundance only at room temperature; conversely, other spots of serum albumin, fibrinogen beta chain and serotransferrin are more labile as they have undergone changes in abundance at all temperatures except at -80°C. CONCLUSIONS: Although there are many studies concerning protein stability of clinical samples during storage these findings may help to provide a better understanding of the changes of proteins induced by storage temperature.

Proteomic analysis of human plasma and peripheral blood mononuclear cells in Systemic Lupus Erythematosus patients.

Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease with a broad spectrum of clinical presentations and incompletely understood pathogenesis. This autoimmune disease is characterized by alterations in both the innate and adaptive immune system that lead to the loss of immunologic tolerance. In autoimmune diseases particularly in SLE, early diagnosis, flare or remission phases can be difficult to identify. Proteomics can help to find new therapeutic targets and it also could help to better understand the cellular mechanisms. The aim of this study was to observe the variations in plasma and Peripheral Blood Mononuclear Cells (PBMCs) proteome in order to increase our knowledge about pathogenesis and to find possible diagnostic markers and/or therapeutic targets for improving diagnosis and treatment. The comparative proteomic analyses showed that several proteins were differentially expressed in the PBMCs from SLE patients. Among these, PRDX2 may be used as candidate biomarker or target protein for further investigations. In plasma, we showed that plasma clusterin levels increased in SLE patients compared to healthy controls, but this increase is not statistically significant. These proteomic results provide suggestions for understanding the molecular mechanisms of SLE, as well as the physiological changes correlated with SLE disease.

Plasma Clusterin and Lipid Profile: A Link with Aging and Cardiovascular Diseases in a Population with a Consistent Number of Centenarians.

The role of Clusterin in attenuation of inflammation and reverse cholesterol transfer makes this molecule a potential candidate as a marker for cancer, cardiovascular disease, diabetes mellitus, and metabolic syndrome. In elderly subjects cardiovascular diseases represent the primary cause of death and different clinical studies have shown a positive correlation of these diseases with changes in the lipid pattern. This work aimed at evaluating the relationship between circulating clusterin and the biochemical parameters that characterize the lipid profile of a Sardinian population divided into five age groups including centenarians; the high frequency in Sardinia of these long-lived individuals gave us the opportunity to extend the range of the age groups to be analyzed to older ages and to better evaluate the changes in the lipid balance during ageing and its relationship with clusterin concentration in plasma. Our results showed that Clusterin concentration values of the youngest group were more similar with the centenarian's group compared to the other age groups, and a positive correlation arises with LDL. Furthermore given the high prevalence of cardiovascular diseases in the population examined and the association of Clusterin with these pathologies we evaluated Clusterin concentration variation in two groups with or without cardiovascular diseases. In presence of cardiovascular disease, Clusterin is significantly related to the most atherogenic components of lipid profile (total cholesterol and LDL), especially in women, suggesting its potential role in modulating cardiovascular metabolic risk factors.

Fetuin-A serum levels are not correlated to kidney function in long-lived subjects.

OBJECTIVES: Serum Fetuin A has been identified as an inhibitor of ectopic calcification. It is reduced in subjects with chronic kidney disease (CKD) and it has been proposed as a potential link between CKD and the higher prevalence of arterial calcification observed in these patients. During aging both the stiffening of arterial wall due to calcification and a decline in kidney function are frequent. The aim of the study is to investigate if Fetuin A serum levels are associated with aging and with AHSG T256S polymorphism. Moreover, we aim at investigate whether serum Fetuin A is correlated to kidney function in this setting of senescence. DESIGN AND METHODS: 256 health long-lived subjects (age 92 [81-100]) were recruited for the study. Serum Fetuin A was evaluated by ELISA, Cystatin C by immune-nephelometry. AHSG T256S was determinated by PCR-RFLP. RESULTS: Serum Fetuin A shows a significant correlation with age (r=0.20; P=0.0048). AHSG TS and SS genotypes are associated to lower levels of serum protein (0.27 [0.19-0.29] g/L vs 0.42 [0.32-0.49] g/L; P<0.027 and 0.34 [0.25-0.41] g/L vs 0.42 [0.32-0.49] g/L; P<0.001, respectively). No significant correlation between Fetuin A and Cystatin C was observed. CONCLUSIONS: Serum Fetuin A increases with age in elder individuals and subjects with the TS or SS AHSG polymorphism have lower levels of the circulating protein. No correlation with kidney function decline was observed. Other mechanisms should be investigated to explain the increase of Fetuin A with age.