Circulating stem cell vary with NYHA stage in heart failure patients

We have investigated the blood levels of sub-classes of stem cells (SCs) [mesenchymal stem cells (MSCs), haematopoietic stem cells (HSCs), endothelial progenitor cells/circulating endothelial cells (EPCs/CECs) and tissue-committed stem cells (TCSCs)] in heart failure (HF) patients at different stage of pathology and correlated it with plasmatic levels of proangiogenic cytokines. Peripheral blood level of SCs were analysed in 97 HF patients (24 in NYHA class I, 41 in class II, 17 in class III and 15 in class IV) and in 23 healthy controls. Plasmatic levels of PDGF-BB, bFGF, HGF, vascular endothelial growth factor (VEGF), SDF-1α, TNF-α and NTproBNP were also measured. Compared with healthy individuals, MSC, and in particular the sub-classes CD45⁻CD34⁻CD90⁺, CD45⁻CD34⁻CD105⁺ and CD45⁻CD34⁻CXCR4⁺ were significantly enhanced in NYHA class IV patients (16.8-, 6.4- and 2.7-fold, respectively). Level of CD45⁻CD34⁻CD90⁺CXCR4⁺cells progressively increased from class II to class IV (fold increases compared with controls: 8.5, 12 and 21.5, respectively). A significant involvement of CXCR4⁺ subpopulation of HSC (CD45⁺CD34⁺CD90⁺CXCR4⁺, 1.4 versus 13.3 cells/μl in controls and NYHA class III patients, respectively) and TCSC (CD45⁻CD34⁺CXCR4⁺, 1.5 cells/ μl in controls versus 12.4 and 28.6 cells/μl in NYHA classes II and IV, respectively) were also observed. All tested cytokines were enhanced in HF patients. In particular, for PDGF-BB and SDF-1α we studied specific ligand/receptors pairs. Interestingly, the first one positively correlated with TCSCs expressing PDGFR (r = 0.52, P = 0.001), whereas the second one correlated with TCSCs (r = 0.34, P = 0.005) and with MSCs CD90⁺ expressing CXCR4 (r = 0.39, P = 0.001). HF is characterized by the increase in the circulating levels of different MSC, HSC, EPC and TCSC subsets. Both the entity and kinetic of this process varied in distinct cell subsets. Specifically, differently from HSCs and EPCs/CECs, MSCs and TCSCs significantly increased with the progression of the disease, suggesting a possible distinct role of these cells in the pathophysiology of HF.     

Vaccines based on the native HIV Tat protein and on the combination of Tat and the structural HIV protein variant DeltaV2 Env

The promising results obtained with the HIV-1 Tat-based vaccines in mice, monkeys and humans, a better understanding of Tat immunomodulatory functions, as well as evidence that vaccination with trimeric V2 loop-deleted HIV-1 Env induces cross-clade neutralizing antibodies led to the rational design of a novel vaccine based on the combination of Tat and V2-deleted Env.     

Complexity and dynamics of HIV-1 quasispecies

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins mediate virus entry into target cells by binding receptors of the cell membrane and fusing viral and cellular structures. In particular, recent crystallographic studies have clarified the complex role of the glycoprotein gp120 in the early phase of the infection. In this context the inter- and intra-host variability of the HIV-1 gp120 poses a major problem for the development of effective methods of immunization against this virus. In the present report, the relevant aspects emerging from the study of HIV-1 variability are addressed and several methodological approaches to evaluate HIV-1 diversity discussed.     

Estrogen metabolism in human colorectal cancer cells

Epidemiological and "in vitro" studies support a direct role of estrogens in the pathogenesis and/or progression of colorectal cancer (CRC). Recent observations suggest a local synthesis of 17beta-estradiol (E(2)). In the present study, the CRC estrogen receptor beta (ERbeta) positive HCT8, HCT116, DLD-1 and LoVo cell lines were evaluated for expression of functional 17beta-hydroxysteroid dehydrogenase (17betaHSD) types 1, 2, 3, and 4. RT-PCR analysis revealed that while 17betaHSD1 and 17betaHSD4 were expressed in all the four cell lines, 17betaHSD2 and 17betaHSD3 were expressed in a cell-specific manner. The interconversion of tritiated estrone (E(1)) or E(2) evaluated by thin layer chromatography of conditioned media revealed that in HCT8, HCT116, and DLD-1 cells both reductive and oxidative activities were present, the latter showing K(m) values (approximately 10 microM) 40-fold higher than the former (approximately 250 nM). On the contrary, in LoVo cells, estrogens were almost (approximately 90%) completely metabolized to hydrophile compounds. Charcoal-dextrane (DC) stripped fetal calf serum (FCS) (10%), E(2) (10nM), Vitamin D(3) (100nM) and the combined E(2) and Vitamin D(3) treatment were evaluated for modulation of 17betaHSD isoenzymes gene expression and activity. Gene expression and activity of 17betaHSD reductive and oxidative isoenzymes were respectively inhibited and enhanced by Vitamin D(3) in HCT8 and LoVo cells. Surprisingly, DC-FCS induced a marked increase of estrogen metabolism toward hydrophile metabolites in all four cell lines. In conclusion, our results clearly show that metabolism of estrogens by 17betaHSD isoenzymes is functional and modulated by external stimuli in continuous neoplastic colonic epithelial cell lines.     

HIV-1 Tat-based vaccines: from basic science to clinical trials

Vaccination against human immunodeficiency virus (HIV)-1 infection requires candidate antigen(s) (Ag) capable of inducing an effective, broad, and long-lasting immune response against HIV-1 despite mutation events leading to differences in virus clades. The HIV-1 Tat protein is more conserved than envelope proteins, is essential in the virus life cycle and is expressed very early upon virus entry. In addition, both humoral and cellular responses to Tat have been reported to correlate with a delayed progression to disease in both humans and monkeys. This suggested that Tat is an optimal target for vaccine development aimed at controlling virus replication and blocking disease onset. Here are reviewed the results of our studies including the effects of the Tat protein on monocyte-derived dendritic cells (MDDCs) that are key antigen-presenting cells (APCs), and the results from vaccination trials with both the Tat protein or tat DNA in monkeys. We provide evidence that the HIV-1 Tat protein is very efficiently taken up by MDDCs and promotes T helper (Th)-1 type immune responses against itself as well as other Ag. In addition, a Tat-based vaccine elicits an immune response capable of controlling primary infection of monkeys with the pathogenic SHIV89.6P at its early stages allowing the containment of virus spread. Based on these results and on data of Tat conservation and immune cross-recognition in field isolates from different clades, phase I clinical trials are being initiated in Italy for both preventive and therapeutic vaccination.     

Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.

During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant. In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells). Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present. Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression. This is due to the different concentrations of exogenous Tat required for the two effects. The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration. In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein. These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.     

G6PD Ferrara I has the same two mutations as G6PD A(-) but a distinct biochemical phenotype

The cloning and sequencing of the normal glucose-6-phosphate dehydrogenase (G6PD) gene has led to the study of the molecular defects that determine enzymatic variants. In this paper, we describe the mutations responsible for the Ferrara I variant in an Italian man with a family history of favism, from the Po delta. Nucleotide sequencing of this variant showed a GA mutation at nucleotide 202 in exon IV causing a ValMet amino acid exchange, and a second AG mutation at nucleotide 376 in exon V causing an AsnAsp amino acid substitution. Although on the basis of its biochemical properties this variant was classified as G6PD Ferrara I, it has the same two mutations as G6PD A(-), which is common in American and African blacks, and as the sporadic Italian G6PD Matera. The mutation at nucleotide 202 was confirmed by NlaIII digestion of a polymerase chain reaction amplified DNA fragment spanning 109 bp of exon IV. The 109-bp mutated amplified sequence is not distinguishable from the normal sequence in single strand conformation polymorphism analysis.     

Activation of the complement system during immunotherapy of cancer with interleukin-2: a possible explanation of the capillary leak syndrome

The capillary leak syndrome, responsible for fluid loss into the interstitial space, represents one of the major cardiovascular toxicities of IL-2 during the immunotherapy of cancer. The mechanisms involved in the increased vascular permeability have still to be better understood. The present study was carried out to investigate the role of the complement system in mediating the IL-2 vascular toxicity. The study was performed in metastatic renal cancer patients, treated with IL-2 through a 24-hour i.v. infusion at a daily dose of 3 x 10(6) U/m2 for 5 consecutive days, corresponding to one IL-2 course. Six IL-2 courses were evaluated. C3 and C4 were measured daily during IL-2 infusion, and 2 and 5 days after its interruption. IL-2 administration induced a significant decrease in both C3 and C4 mean levels, which became within the normal range 5 days after the end of IL-2 infusion. These results show that IL-2 administration may directly activate the complement system through the classical pathway, which might play a role in determining the increased vascular permeability.     

Molecular characterisation of the glucose-6-phosphate dehydrogenase (G6PD) Ferrara II variant

During the last ten years, molecular biological techniques such as cloning and sequencing and, more recently, polymerase chain reaction (PCR) amplification have led to the identification of the molecular defects responsible for more than fifty glucose-6-phosphate dehydrogenase (G6PD) variants. In this paper, we report the identification of the molecular abnormality underlying the G6PD Ferrara II variant, present in the Po delta area of Northern Italy. Biochemical characterisation shows an enzymatic activity of about 15% of normal (WHO class III), slow electrophoretic mobility, low Km for G6P, high percentage substrate analogue utilisation and a biphasic pH optimum curve. After PCR amplification, non-radioiso-topic single-strand conformation polymorphism analysis carried out for the entire coding region has revealed a mobility shift in exon 8. Nucleotide sequencing has demonstrated a missense 844 G>C mutation, causing an Asp>His amino-acid replacement, known as being responsible for G6PD Seattle, G6PD Modena and G6PD Lodi.     

A new Upper Cretaceous titanosaur nesting site from La Rioja (NW Argentina), with implications for titanosaur nesting strategies

Cretaceous titanosaur nesting sites are currently known only from Europe, Asia and South America. In the latter, only the Auca Mahuevo and Sanagasta nesting sites have been confidently assigned to this clade of sauropod dinosaurs. Here we report the discovery of the first eggs and egg clutches found at Tama, a new Upper Cretaceous fossiliferous locality in the Los Llanos Formation, Sierra de Los Llanos (La Rioja, NW Argentina). At least five egg clutches, several partially preserved, isolated eggs and many eggshell fragments were discovered in a single outcrop of a sandstone horizon which represents a cumulative palaeosol profile. Although the mechanical and digital preparation of eggs did not reveal any embryonic remains in ovo, the morphology of the eggs and eggshells closely matches that of titanosaur eggs and eggshells found worldwide. The morphology and spatial grouping of the titanosaur eggs from Tama, along with geological observations support a burrow‐nesting strategy for these dinosaurs. Although the Sanagasta and Tama eggs were found in the same stratigraphical unit and share several morphological characters, they clearly differ in shell thickness and egg size. This, coupled with the interpretation of different sedimentary contexts for these nesting sites, strongly suggests that at least two different titanosaur species nested in La Rioja during the Late Cretaceous, using different nesting strategies. The occurrence of this new titanosaur nesting site in a semiarid palaeoenvironment represents an interesting case study for the reproductive biology of the titanosaur dinosaurs, particularly their labile nesting behaviour.