Differential patterns of large tumor antigen-specific immune responsiveness in patients with BK polyomavirus-positive prostate cancer or benign prostatic hyperplasia

The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as a target of immune response in patients with prostate cancer (PCa) has not been investigated thus far. In this study, we comparatively analyzed humoral and cellular L-Tag-specific responsiveness in age-matched patients bearing PCa or benign prostatic hyperplasia, expressing or not expressing BKV L-Tag-specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) after surgery (prostate specific antigen level of ≥0.2 ng/ml). In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag-positive tumors, stimulation with peptides derived from the BKV L-Tag but not those derived from Epstein-Barr virus, influenza virus, or cytomegalovirus induced a peculiar cytokine gene expression profile, characterized by high expression of interleukin-10 (IL-10) and transforming growth factor β1 and low expression of gamma interferon genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti-BKV L-Tag IgG. Furthermore, in PBMC from these PCa-bearing patients, L-Tag-derived peptides significantly expanded an IL-10-secreting CD4(+) CD25(+(high)) CD127(-) FoxP3(+) T cell population with an effector memory phenotype (CD103(+)) capable of inhibiting proliferation of autologous anti-CD3/CD28-triggered CD4(+) CD25(-) T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag-specific immune response are significantly associated with tumor progression in patients with BKV(+) PCa.     

Do plasma proteins distinguish between liposomes of varying charge density?

Cationic liposomes (CLs) are one of the most employed nonviral nanovector systems in gene therapy. However, their transfection efficiency is strongly affected by interactions with plasma components, that lead to the formation of a “protein corona” onto CL surface. The interactions between nanoparticles entering the body and biomolecules have an essential role for their biodistribution. Because the knowledge of proteins adsorbed onto vector surface could be useful in the screening of new, more efficient and more biocompatible liposomal formulations, the behavior of three CLs with different membrane charge densities was investigated. The proteins of the three coronas were identified by nano-liquid chromatography-tandem mass spectrometry, and quantified with label-free spectral counting strategy. Fibrinogen displayed higher association with CLs with high membrane charge density, while apolipoproteins and C4b-binding protein with CLs with low membrane charge density. These results are discussed in terms of the different lipid compositions of CLs and may have a deep biological impact for in vivo applications. Surface charge of nanoparticles is emerging as a relevant factor determining the corona composition after interaction with plasma proteins. Remarkably, it is also shown that the charge of the protein corona formed around CLs is strongly related to their membrane charge density.     

A comparative proteomic study of human skin suction blister fluid from healthy individuals using immunodepletion and iTRAQ labeling

Aberrations in skin morphology and functionality can cause acute and chronic skin-related diseases that are the focus of dermatological research. Mechanically induced skin suction blister fluid may serve as a potential, alternative human body fluid for quantitative mass spectrometry (MS)-based proteomics in order to assist in the understanding of the mechanisms and causes underlying skin-related diseases. The combination of abundant-protein removal with iTRAQ technology and multidimensional fractionation techniques improved the number of identified protein groups. A relative comparison of a cohort of 8 healthy volunteers was thus recruited in order to assess the net variability encountered in a healthy scenario. The technology enabled the identification, to date, of the highest number of reported protein groups (739) with concomitant relative quantitative data for over 90% of all proteins with high reproducibility and accuracy. The use of iTRAQ 8-plex resulted in a 66% decrease in protein identifications but, despite this, provided valuable insight into interindividual differences of the healthy control samples. The geometric mean ratio was close to 1 with 95% of all ratios ranging between 0.45 and 2.05 and a calculated mean coefficient of variation of 15.8%, indicating a lower biological variance than that reported for plasma or urine. By applying a multistep sample processing, the obtained sensitivity and accuracy of quantitative MS analysis demonstrates the prospective value of the approach in future research into skin diseases.     

Human chorionic villus mesenchymal stromal cells reveal strong endothelial conversion properties

Chorion, amnion and villi are reservoirs of mesenchymal stromal cells (StC) and the hypothesis that StC from fetal tissues retain higher plasticity compared to adult StC has been suggested. Aimed at investigating this aspect, a series of in vitro experiments were performed with StC isolated from first trimester human chorionic villi (CVStC). CVStC were cultured in: (i) standard mesenchymal medium (MM) and (ii) AmniomaxII? (AM), specifically designed to grow amnion-derived cells in prenatal diagnostic procedures. Cells were then exposed to distinct differentiation treatments and distinguished according to morphology, immunophenotype and molecular markers. Human StC obtained from adult bone marrow (BMStC) were used as control. CVStC cultured either in MM or AM presented stromal morphology and immunophenotype, were negative for pluripotency factors (Nanog, Oct-4 and Sox-2), lacked detectable telomerase activity and retained high genomic stability. In AM, however, CVStC exhibited a faster proliferation rate compared to BMStC or CVStC kept in MM. During differentiation, CVStC were less efficient than BMStC in acquiring adipocytes and osteocytes features; the cardiomyogenic conversion occurred at low efficiency in both cell types. Remarkably, in the presence of pro-angiogenic factors, CVStC reprogrammed toward an endothelial-like phenotype at significantly higher efficiency than BMStC. This effect was particularly evident in CVStC expanded in AM. Mechanistically, the reduced CVStC expression of anti-angiogenic microRNA could support this process. The present study demonstrates that, despite of fetal origin, CVStC exhibit restricted plasticity, distinct from that of BMStC and predominantly directed toward the endothelial lineage.     

Dephosphorylation of cardiac proteins in vitro - a matter of phosphatase specificity

Protein phosphorylation is reversibly regulated by the interplay between kinases and phosphatases. Recent developments within the field of proteomics have revealed the extent of this modification in nature. To date there is still a lack of information about phosphatase specificity for different proteomes and their conditions to achieve maximum enzyme activity. This information is important per se, and in addition often requested in functional and biochemical in vitro studies, where a dephosphorylated sample is needed as a negative control to define baseline conditions. In this study, we have addressed the effectiveness of two phosphatases endogenously present in the heart (protein phosphatases 1 and 2A) and two generic phosphatases (alkaline phosphatase and lambda protein phosphatase) on three cardiac subproteomes known to be regulated by phosphorylation. We optimized the dephoshorylating conditions on a cardiac tissue fraction comprising cytosolic and myofilament proteins using 2DE and MS. The two most efficient conditions were further investigated on a mitochondrial-enriched fraction. Dephosphorylation of specific proteins depends on the phosphatase, its concentration, as well as sample preparation including buffer composition. Finally, we analyzed the efficiency of alkaline phosphatase, the phosphatase with the broadest substrate specificity, using TiO(2) peptide enrichment and 2DLC-MS/MS. Under these conditions, 95% of the detected cardiac cytoplasmic-enriched phospho-proteome was dephosphorylated. In summary, targeting dephosphorylation of the cardiac muscle subproteomes or a specific protein will drive the selection of the specific phosphatase, and each requires different conditions for optimal performance.     

Metabolic syndrome and the role of dietary lifestyles in Alzheimer's disease

Since Alzheimer's disease (AD) has no cure or preventive treatment, an urgent need exists to find a means of preventing, delaying the onset, or reversing the course of the disease. Clinical and epidemiological evidence suggests that lifestyle factors, especially nutrition, may be crucial in controlling AD. Unhealthy lifestyle choices lead to an increasing incidence of obesity, dyslipidemia and hypertension – components of the metabolic syndrome. These disorders can also be linked to AD. Recent research supports the hypothesis that calorie intake, among other non-genetic factors, can influence the risk of clinical dementia. In animal studies, high calorie intake in the form of saturated fat promoted AD-type amyloidosis, while calorie restriction via reduced carbohydrate intake prevented it. Pending further study, it is prudent to recommend to those at risk for AD – e.g. with a family history or features of metabolic syndrome, such as obesity, insulin insensitivity, etc. – to avoid foods and beverages with added sugars; to eat whole, unrefined foods with natural fats, especially fish, nuts and seeds, olives and olive oil; and to minimize foods that disrupt insulin and blood sugar balance.     

CpG drives human transitional B cells to terminal differentiation and production of natural antibodies

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.     

Interaction of DDSDEEN peptide with N-CAM protein. Possible mechanism enhancing neuronal differentiation

DDSDEEN chromatin peptide, after dansylation, was studied for its ability to bind N-CAM protein. The binding causes a quenching of the Dns-peptide fluorescence emission. Dose- and time-dependent binding of Dns-peptide with N-CAM has been shown. Fluorescence quenching is completely lost if the Dns-peptide is subjected to carboxypeptidase digestion. Moreover the undansylated peptide pEDDSDEEN competes with the DnsDDSDEEN peptide for the binding with the N-CAM protein. The Dns-peptide-N-CAM bond has been related to the peptide biological activity probably involved in the promotion of neuronal differentiation. An attempt to recognize a possible N-CAM binding site for Dns-peptide was performed by alignment of N-CAM from various sources with some sequences that have been previously reported as binding sites for the pEDDSDEEN and DDSDEEN peptides. Interestingly, the alignment of N-CAM from various sources with the peptides WHPREGWAL and WFPRWAGQA recognizes on rat and human N-CAM a unique sequence that could be the specific binding site for chromatin peptide: WHSKWYDAK. This sequence is present in fibronectin type-III domain of N-CAM. In addition molecular modeling studies indicate the N-CAM sequence WHSKWYDAK as, probably, the main active site for DnsDDSDEEN (or pEDDSDEEN) peptide ligand. Accordingly the binding experiments show a high affinity between WHSKWYDAK and DnsDDSDEEN peptides.