Genomic DNA extraction from whole blood stored from 15- to 30-years at -20 °C by rapid phenol-chloroform protocol: a useful tool for genetic epidemiology studies.

Long-term stored (LTS) whole blood collection can be an important source of DNA without collection costs, but there is a lack of information on methods useful to extract genomic DNA from such type of biological material. Here we report a simple and fast revisited phenol/chloroform extraction method from LTS whole blood. Protocol reliability was assessed by comparison with proteinase K and silica-gel membrane spin column-based DNA extraction methods using LTS -20 °C whole blood from 1980, and by testing it on 82 whole blood samples, collected from 1980 to 1995, with high quality (A(260/280) = 1.79 ± 0.32 O.D., A(260/230) = 1.45 ± 0.52 O.D.) and quantity results. Genotyping efficiency was also checked by performing RFLP-PCR and ASP-PCR of p53 Pro72Arg (rs1042522) SNP and hTERT MNS16A VNTR, respectively, resulting in 100% of samples successfully typed. In addition to the goodness and the efficiency of method proposed here, this protocol achieves working time reduction combining extraction and purification steps, allowing to work at room temperature. Furthermore, phenol is able to inactivate any potential nuclease and potential infective sources from the first step on. Based on these results we also conclude that LTS -20 °C whole blood samples may be considered a reliable and potential resource for future genotyping studies and retrospective analysis in a genetic epidemiological setting.

Administration of DNA Plasmid Coding Protein Aggregating Domain Induces Inflammatory Bone Loss.

BACKGROUND: Plasmids coding protein aggregation polypeptides from different sources have been proposed as genetic adjuvants for DNA vaccines. We reported that a plasmid (pATRex), encompassing the DNA sequence for the von Willebrand A (vWA/A) domain of the Anthrax Toxin Receptor-1 (ANTXR-1, alias TEM8, Tumor Endothelial Marker 8), acts as strong immune adjuvant by inducing formation of insoluble intracellular aggregates and subsequent cell death. OBJECTIVE: In the present study we addressed the question of whether there is any substantial immunotoxicity associated with the use of self-aggregating proteins as genetic adjuvants. METHODS & RESULTS: Here we report, by mean of histology, X-ray and molecular examinations of bone specimens, the unexpected finding that intramuscular injection of pATRex in mice triggers, per se, severe bone loss (osteoporosis) independently from the sex and genotype of the treated animals. CONCLUSION: Even though the study suggests that proteinaceous "sticky " adjuvants are unlikely to find their way into practical vaccination, the information gained is of value as ATRex injections could provide an additional, simplified, mouse model of osteoporosis. Moreover, our results provide experimental support to the hypothesis that proteotoxic aggregates chronically activate the innate immune system in amyloid and aggregosome associated disorders.

The functional polymorphism rs73598374:G>A (p.Asp8Asn) of the ADA gene is associated with telomerase activity and leukocyte telomere length.

Recent evidence demonstrated a relevant role of adenosine deaminase (ADA) in replicative senescence of T cells through its capacity to modulate telomerase activity (TA). Herein, we tested the impact of the functional polymorphism ADA rs73598374:G>A (c.22G>A, p.Asp8Asn) on telomere biology, by measuring TA and leukocyte telomere length (LTL) in healthy subjects selected according to rs73598374 genotype. rs73598374-A carriers showed lower TA (P=0.019) and shorter LTL (P=0.003), respectively, compared to G/G carriers. rs73598374-A carriers showed a stronger cross-sectional age reduction of LTL (r=-0.314, P=0.005) compared to G/G carriers (r=-0.243, P=0.022). The reduced ADA activity associated to rs73598374-A variant predisposes those carriers to display higher levels of adenosine compared to G/G carriers. Consequently, it may lead to an accelerated process of replicative senescence, causing a stronger reduction of TA and in turn shorter LTL. In conclusion, the crucial role played by replicative senescence of the immune system in several human diseases and in the aging process underscores the relevance of the present findings and also spurs interest into the possible involvement of rs73598374 in shaping the susceptibility to several age-related diseases.

Plasmids encoding protein aggregation domains act as molecular adjuvants for DNA vaccines.

BACKGROUND: DNA vaccines provide high tolerability and safety but commonly suffer from suboptimal immunogenicity. We previously reported that a plasmid vector (pATRex), encoding the DNA sequence for the von Willebrand I/A domain of the tumor endothelial marker-8 (TEM8) when given in combination with plasmid-encoded tumor antigens acted as a powerful molecular adjuvant enhancing immunity against breast and melanoma tumors. AIMS: In the present study we addressed two unsolved issues; would the adjuvant action of pATRex extend to a DNA vaccine against infectious disease and, if so, what is the mechanistic basis for pATRex adjuvant action? RESULTS: Here we show in a murine malaria vaccine model that co-administration of pATRex potentiates antibody production elicited by an intramuscular injection of plasmid encoding Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5). pATRex enhanced the B-cell response and induced increased IgG1 production consistent with TH2 polarization of the DNA vaccine response. To explore the mechanism of adjuvant action, cells were transfected in vitro with pATRex and this resulted in formation of insoluble intracellular aggregates and apoptotic cell death. Using a structural modeling approach we identified a short peptide sequence (α3-ß4) within ATRex responsible for protein aggregation and confirmed that transfection of cells with plasmid encoding this self-assembling peptide similarly triggered intracellular aggregates, caspase activation and cell death. CONCLUSION: Plasmids encoding aggregation-promoting domains induce formation of insoluble intracellular aggregates that trigger caspase activation and apoptotic cell death leading to activation of the innate immune system thereby acting as genetic adjuvants.

The functional VNTR MNS16A of the TERT gene is associated with human longevity in a population of Central Italy.

BACKGROUND: Telomerase, encoded by TERT, is the ribonucleoprotein polymerase that maintains telomere ends and it plays a crucial role in cellular senescence. TERT single nucleotide polymorphisms (SNPs) have been associated both with various malignancies and telomere length (TL). The association of TERT SNPs with longevity remains uncertain and varies with ethnicity. The aim of this study was to investigate whether the functional variable number of tandem repeat (VNTR) MNS16A of TERT is associated with longevity. METHODS: MNS16A genotypes have been determined for 1072 unrelated healthy individuals from Central Italy (18-106 years old) divided into three gender-specific age classes defined according to demographic information and accounting for the different survivals between sexes: for men (women), the first class consists of individuals <66 years old (<73 years old), the second class of individuals 66-88 years old (73-91 years old), and the third class of individuals >88 years old (>91 years old). TL was assessed using genomic DNA from whole blood of 72 selected individuals by a multiplex real-time PCR assay. RESULTS: MNS16A appears associated to longevity, showing significant associations in Comparison 2 (Age Class 3 vs. Age Class 2) under both additive (odds ratio [O.R.] 0.749; p=0.019) and dominant (O.R. 0.579; p=0.011) models. The MNS16A*L allele is significantly underrepresented in Age Class 3 (O.R. 0.759; p=0.020) compared to Age Class 2. A significant telomere attrition is reported along the three age classes (p=0.0001), that remains significant only in L*/L* genotype carriers (p=0.002) when the analysis was conducted according to MNS16A genotype. CONCLUSIONS: The TERT MNS16A*L allele appears negatively associated with longevity. The concomitant significant telomere cross sectional attrition rate observed for L*/L* genotype suggests that this polymorphism could influence human longevity by affecting TL.

Haptoglobin (HP) polymorphisms and human longevity: a cross-sectional association study in a Central Italy population.

BACKGROUND: Haptoglobin (HP), which scavenges free, cell-toxic hemoglobin and has anti-inflammatory and immune-modulatory function in extravascular tissues, may represent an excellent candidate gene to investigate the life-span expectancy. METHODS: HP 1/2 polymorphism has been determined for 1072 (569 females, 503 males) unrelated healthy individuals from Central Italy, 18-106 years old, divided into three gender-specific age classes defined according to demographic information and accounting for the different survivals between sexes. HP*1F/S subtyping was also performed to check the possible existence for a preferential advantage of HP*1F or HP*1S allele. RESULTS: HP*1/*1 genotype results associated to increased probability of young subjects of attaining longevity (Comparison 1: O.R. 1.709, p=0.0114) with a concomitant advantage of HP*1 allele (Comparison 1: O.R. 1.273, p=0.0194). On the other side, carriers of HP*2 allele displayed an overall significant disadvantage in reaching Age Class 2 (O.R. 0.585, p=0.0092). No significant differences were noticed between age groups either considering total HP*1F and HP*1S allele frequencies or according to HP 1/2 genotypes. CONCLUSION: The crucial role played by HP in aging process is warranted by its many established functions and its related phenotypes so that it may be considered an important gene involved in the determination of human survival.

Insights into the role of Fc gamma receptors (FcgammaRs) genetic variations in monoclonal antibody-based anti-cancer therapy.

Recently, the field of oncology has witnessed the introduction of several effective chemotherapeutic agents. Still, not all cancers respond to the use of conventional chemotherapy and thus combination therapy is an emerging weapon in the battle against cancer. There is emerging evidence in support of the use of Monoclonal antibodies (MoAbs) in cancer therapy. The mechanisms behind their efficacy are multi-faceted; they can kill tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis as well as target ligands or growth factor receptors favoring tumor growth. The interaction of the Fc domains of antibodies with the Fcgamma (gamma) receptors is an essential checkpoint in ADCC. This interaction is strongly regulated and is largely dependent upon receptor conformation and number. It is accepted that germ-line single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) have the potential to predict the outcome of therapy. The possibility of predicting patients response to monoclonal antibody therapy is of particular importance, as response rates are moderate, with the risk of serious side effects all at a high financial cost. This patent review provides an insight into the role of Fcgamma receptors (FcgammaRs) genetic variation in Monoclonal Antibody-based anti-cancer therapy.