The bone marrow in murine AIDS

In order to increase the understanding of blood cytopenias in HIV infection we have investigated the bone marrow in murine AIDS. C57BL/6 mice infected with the LP-BM5 retrovirus show a decrease in cellularity, numerous haemophagocytic histiocytes, a reduction of all erythroid precursor cells, an increase in eosinophil number and an increase in lymphocytes. Immunostaining with an anti-Pr60gag antibody shows that the majority of bone marrow cells express the viral protein. Thus, the bone marrow in MAIDS has many similarities with the bone marrow from patients with advanced AIDS and may prove useful as a model for therapy aimed at treating blood cytopenias.     

In vivo characterization of renal auto-antigens involved in human auto-immune diseases: the case of membranous glomerulonephritis

Renal auto-immune diseases represent a major source of morbidity in humans. For many years the knowledge on mechanisms of auto-immunity involving the kidney has been uniquely based on animal models. However, these findings often could not be readily translated to humans owing to notably difference in antigen expression by human podocytes. One example is Heymann nephritis (HN), the experimental model of human membranous glomerulonephritis (MGN), which is obtained in rats by injecting antibodies against megalin, a protein that is not present in human glomeruli. Human studies could not be done in the past since sequencing required too much material exceeding what obtainable from tissue biopsies in vivo. Research is now on the way to identify auto-antigens and isolate specific auto-antibodies in humans. New technology developments based on tissue microdissection and proteomical analysis have facilitated the recent discoveries, allowing direct analysis of human tissue in vivo. Major advances on the pathogenesis of MGN, the prototype for the formation and glomerular deposition of auto-antibodies, are now in progress. Two independent groups have, in fact, demonstrated the existence of specific IgG(4) against phospholipase A2 receptor, aldose reductase and Mn-superoxide dismutase in glomerular eluates and in plasma of a prominent part of patients with MGN, suggesting a major role of these proteins as auto-antigens in human MGN. This review will focalize these aspects outlining the contribution of proteomics in most recent developments.     

Oris: a tool for modeling, verification and evaluation of real-time systems

Oris is a tool for qualitative verification and quantitative evaluation of reactive timed systems, which supports modeling and analysis of various classes of timed extensions of Petri Nets. As most characterizing features, Oris implements symbolic state space analysis of preemptive Time Petri Nets, which enable schedulability analysis of real-time systems running under priority preemptive scheduling; and stochastic Time Petri Nets, which enable an integrated approach to qualitative verification and quantitative evaluation. In this paper, we present the current version of the tool and we illustrate its application to two different case studies in the areas of qualitative verification and quantitative evaluation, respectively.     

Microscopic overview of crinoid regeneration.

Crinoids are well known for their striking regenerative potential and can rapidly and completely regenerate arms lost following self-induced or traumatic amputation. Thus they provide a valuable experimental model for investigation of the regenerative process from the macroscopic to the molecular level. In these last years we have studied in detail the overall process of arm regeneration in the comatulid Antedon mediterranea. This phenomenon can be described on the whole as a typical blastemal regeneration in which new structures develop from migratory pluripotential, actively proliferating cells in the presence of presumptive regulatory factors. The overall process can be subdivided into three main phases: a repair phase, an early regenerative phase, and an advanced regenerative phase, whose crucial aspects are related to common fundamental mechanisms such as cell migration and proliferation, intervention of stem cells and/or dedifferentiated cells, contribution of putative growth factors, particularly in terms of specific neurally derived factors, and mechanisms of pattern formation. This article focuses on the main aspects of the phenomenon and gives a brief account of the most recent and relevant results. Our approach employs classical methods of light (LM) and electron (TEM and SEM) microscopy, immunocytochemistry, and histofluorescence on experimentally induced arm regenerations of standard or abnormal type obtained in significantly different experimental conditions, including extreme mutilations (explants) or exposure to pseudo-estrogenic environmental contamination.     

Leucine transport in Xenopus laevis oocytes: functional and morphological analysis of different defolliculation procedures

L-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence of 100 mM NaCl, L-leucine uptake was reduced by 67.5% +/- 5.7 when defolliculation was performed enzymatically by collagenase treatment, whereas the reduction was 30.5% +/- 6.4 after mechanical defolliculation. The Na(+)-dependent uptake of 0.1 mM L-leucine was 18.6 +/- 4.6 pmol oocyte-1 40 min-1 in folliculated oocytes and 5.6 +/- 1.9 in collagenase defolliculated oocytes (means +/- SE). L-leucine uptake was not affected by the removal of the follicular layer if defolliculation occurred after the transport period; radiolabeled L-leucine is therefore not taken up into a compartment that is removed by the defolliculation process. The different L-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific L-leucine binding to membranes. L-leucine kinetics showed that the L-leucine Vmax and Km values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The Vmax and Km values of Na(+)-dependent L-leucine transport, calculated from data obtained the day after defolliculation by collagenase treatment, were: 16 +/- 1.5 pmol oocyte-1 40 min-1 and 57 +/- 21 mumol (mean +/- SD). The Na(+)-activation curve of 0.1 mM L-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experiments showed that after defolliculation, the fibers forming the vitelline membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase treatment. Mechanical defolliculation did not appreciably affect the oocyte microvilli, however this procedure did not completely remove all follicle cells. The damage to collagenase treated oocytes was reversible, and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to involve protein synthesis in view of the increased value of L-leucine Vmax, and microscopic observation showing recovery of the microvillar apparatus.     

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5′UTR and 3′UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband’s group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3′UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.     

Serenoa repens and Urtica dioica Fixed Combination: In-Vitro Validation of a Therapy for Benign Prostatic Hyperplasia (BPH)

Benign prostatic hyperplasia (BPH) is an age-related chronic disorder, characterized by the hyperproliferation of prostatic epithelial and stromal cells, which drives prostate enlargement. Since BPH aetiology and progression have been associated with the persistence of an inflammatory stimulus, induced both by Nuclear Factor-kappa B (NF-κB) activation and reactive oxygen species (ROS) production, the inhibition of these pathways could result in a good tool for its clinical treatment. This study aimed to evaluate the antioxidant and anti-inflammatory activity of a combined formulation of Serenoa repens and Urtica dioica (SR/UD) in an in vitro human model of BPH. The results confirmed both the antioxidant and the anti-inflammatory effects of SR/UD. In fact, SR/UD simultaneously reduced ROS production, NF-κB translocation inside the nucleus, and, consequently, interleukin 6 (IL-6) and interleukin 8 (IL-8) production. Furthermore, the effect of SR/UD was also tested in a human androgen-independent prostate cell model, PC3. SR/UD did not show any significant antioxidant and anti-inflammatory effect, but was able to reduce NF-κB translocation. Taken together, these results suggested a promising role of SR/UD in BPH and BPH-linked disorder prevention.     

D-Amino acids in reconstituted infant formula: A comparison between conventional and microwave heating

It was recently claimed that microwave heating of reconstituted formulas for infants could induce racemisation of amino acids, in particular, of proline. In the present work we investigated the presence of D-amino acids in different samples of a reconstituted infant formula heated by a conventional heat-transfer way (oil bath), in a commercially available domestic microwave oven and in a wave-guide device. The analyses were carried out both by gas chromatography and by high performance liquid chromatography. The presence of D-enantiomers was determined both in proteins after hydrolysis (6 M HCl at 100°C for 6 h) and in the free amino acid pool. Regarding protein-bound amino acids, there is no evidence of racemisation in all samples examined under the different experimental conditions (conventionally and microwave-treated samples). In particular, D-proline was not detected at least above 1 mg litre^?1 in proteins and above 20 μg litre^?1 in free amino acids. Although small percentages (D/D + L %) of D-glu and D-asp and higher percentages of D-ala were detected among free amino acids, in all samples, no significant differences could be found between untreated and treated samples.