Polymorphisms within the ARNT2 and CX3CR1 Genes Are Associated with the Risk of Developing Invasive Aspergillosis.

Invasive aspergillosis (IA) is a life-threatening infection that affects an increasing number of patients undergoing chemotherapy or allo-transplantation, and recent studies have shown that genetic factors contribute to disease susceptibility. In this two-stage, population-based, case-control study, we evaluated whether 7 potentially functional single nucleotide polymorphisms (SNPs) within the ARNT2 and CX3CR1 genes influence the risk of IA in high-risk hematological patients. We genotyped selected SNPs in a cohort of 500 hematological patients (103 of those had been diagnosed with proven or probable IA), and we evaluated their association with the risk of developing IA. The association of the most interesting markers of IA risk was then validated in a replication population, including 474 subjects (94 IA and 380 non-IA patients). Functional experiments were also performed to confirm the biological relevance of the most interesting markers. The meta-analysis of both populations showed that carriers of the ARNT2rs1374213G, CX3CR1rs7631529A, and CX3CR1rs9823718G alleles (where the RefSeq identifier appears as a subscript) had a significantly increased risk of developing IA according to a log-additive model (P value from the meta-analysis [PMeta] = 9.8 · 10-5, PMeta = 1.5 · 10-4, and PMeta =7.9 · 10-5, respectively). Haplotype analysis also confirmed the association of the CX3CR1 haplotype with AG CGG with an increased risk of IA (P = 4.0 · 10-4). Mechanistically, we observed that monocyte-derived macrophages (MDM) from subjects carrying the ARNTR2rs1374213G allele or the GG genotype showed a significantly impaired fungicidal activity but that MDM from carriers of the ARNT2rs1374213G and CX3CR1rs9823718G or CX3CR1rs7631529A alleles had deregulated immune responses to Aspergillus conidia. These results, together with those from expression quantitative trait locus (eQTL) data browsers showing a strong correlation of the CX3CR1rs9823718G allele with lower levels of CX3CR1 mRNA in whole peripheral blood (P = 2.46 · 10-7) and primary monocytes (P = 4.31 · 10-7), highlight the role of the ARNT2 and CX3CR1 loci in modulating and predicting IA risk and provide new insights into the host immune mechanisms involved in IA development.

Polymorphisms in Host Immunity-Modulating Genes and Risk of Invasive Aspergillosis: Results from the AspBIOmics Consortium.

Recent studies suggest that immune-modulating single-nucleotide polymorphisms (SNPs) influence the risk of developing cancer-related infections. Here, we evaluated whether 36 SNPs within 14 immune-related genes are associated with the risk of invasive aspergillosis (IA) and whether genotyping of these variants might improve disease risk prediction. We conducted a case-control association study of 781 immunocompromised patients, 149 of whom were diagnosed with IA. Association analysis showed that the IL4Rrs2107356 and IL8rs2227307 SNPs (using dbSNP numbering) were associated with an increased risk of IA (IL4Rrs2107356 odds ratio [OR], 1.92; 95% confidence interval [CI], 1.20 to 3.09; IL8rs2227307 OR, 1.73; 95% CI, 1.06 to 2.81), whereas the IL12Brs3212227 and IFNγrs2069705 variants were significantly associated with a decreased risk of developing the infection (IL12Brs3212227 OR, 0.60; 95% CI, 0.38 to 0.96; IFNγrs2069705 OR, 0.63; 95% CI, 0.41 to 0.97). An allogeneic hematopoietic stem cell transplantation (allo-HSCT)-stratified analysis revealed that the effect observed for the IL4Rrs2107356 and IFNγrs2069705 SNPs was stronger in allo-HSCT (IL4Rrs2107356 OR, 5.63; 95% CI, 1.20 to 3.09; IFNγrs2069705 OR, 0.24; 95% CI, 0.10 to 0.59) than in non-HSCT patients, suggesting that the presence of these SNPs renders patients more vulnerable to infection, especially under severe and prolonged immunosuppressive conditions. Importantly, in vitro studies revealed that carriers of the IFNγrs2069705C allele showed a significantly increased macrophage-mediated neutralization of fungal conidia (P = 0.0003) and, under stimulation conditions, produced higher levels of gamma interferon (IFNγ) mRNA (P = 0.049) and IFNγ and tumor necrosis factor alpha (TNF-α) cytokines (P value for 96 h of treatment with lipopolysaccharide [PLPS-96 h], 0.057; P value for 96 h of treatment with phytohemagglutinin [PPHA-96 h], 0.036; PLPS+PHA-96 h = 0.030; PPHA-72 h = 0.045; PLPS+PHA-72 h = 0.018; PLPS-96 h = 0.058; PLPS+PHA-96 h = 0.0058). Finally, we also observed that the addition of SNPs significantly associated with IA to a model including clinical variables led to a substantial improvement in the discriminatory ability to predict disease (area under the concentration-time curve [AUC] of 0.659 versus AUC of 0.564; P-2 log likehood ratio test = 5.2 · 10(-4) and P50.000 permutation test = 9.34 · 10(-5)). These findings suggest that the IFNγrs2069705 SNP influences the risk of IA and that predictive models built with IFNγ, IL8, IL12p70, and VEGFA variants can used to predict disease risk and to implement risk-adapted prophylaxis or diagnostic strategies.

Creatine as an antioxidant.

Creatine monohydrate (Cr), the most diffuse supplement in the sports industry, is receiving greater attention because of its beneficial effects in a wide number of human degenerative diseases and conditions. These effects can be barely explained on the basis of the sole ergogenic role of the Cr/CrP system. Indeed, a wide number of research articles indicate that Cr is capable of exerting multiple, non-energy related, effects on diverse and relevant cellular targets. Among these effects, the antioxidant activity of Cr emerges as an additional mechanism which is likely to play a supportive role in the Cr-cytoprotection paradigm.

Host immune genetic variations influence the risk of developing acute myeloid leukaemia: results from the NuCLEAR consortium.

The purpose of this study was to conduct a two-stage case control association study including 654 acute myeloid leukaemia (AML) patients and 3477 controls ascertained through the NuCLEAR consortium to evaluate the effect of 27 immune-related single nucleotide polymorphisms (SNPs) on AML risk. In a pooled analysis of cohort studies, we found that carriers of the IL13rs1295686A/A genotype had an increased risk of AML (PCorr = 0.0144) whereas carriers of the VEGFArs25648T allele had a decreased risk of developing the disease (PCorr = 0.00086). In addition, we found an association of the IL8rs2227307 SNP with a decreased risk of developing AML that remained marginally significant after multiple testing (PCorr = 0.072). Functional experiments suggested that the effect of the IL13rs1295686 SNP on AML risk might be explained by its role in regulating IL1Ra secretion that modulates AML blast proliferation. Likewise, the protective effect of the IL8rs2227307 SNP might be mediated by TLR2-mediated immune responses that affect AML blast viability, proliferation and chemorresistance. Despite the potential interest of these results, additional functional studies are still warranted to unravel the mechanisms by which these variants modulate the risk of AML. These findings suggested that IL13, VEGFA and IL8 SNPs play a role in modulating AML risk.

Prevalence of human herpesvirus-6 chromosomal integration (CIHHV-6) in Italian solid organ and allogeneic stem cell transplant patients.

The unique phenomenon of human herpesvirus-6 (HHV-6) chromosomal integration (CIHHV-6) may account for clinical drawbacks in transplant setting, being misinterpreted as active infection and leading to unnecessary and potentially harmful treatments. We have investigated the prevalence of CIHHV-6 in 205 consecutive solid organ (SO) and allogeneic stem cell transplant (alloSCT) Italian patients. Fifty-two (38.5%) of 135 solid organ transplant (SOT) and 16 (22.8%) of 70 alloSCT patients resulted positive for plasma HHV-6 DNA by real-time polymerase chain reaction. Seven SOT and three alloSCT patients presented HHV-6-related diseases, requiring antivirals. Two further patients (0.9%) were identified, presenting high HHV-6 loads. The quantification of HHV-6 on hair follicles disclosed the integrated state, allowing the discontinuation of antivirals. Before starting specific treatments, CIHHV-6 should be excluded in transplant patients with HHV-6 viremia by the comparison of HHV-6 loads on different fluids and tissues. Pretransplantation screening of donors and recipients may further prevent the misdiagnosis of CIHHV-6.

Cytarabine-related lung infiltrates on high resolution computerized tomography: a possible complication with benign outcome in leukemic patients.

Potentially fatal lung toxicity occurs in 12-20% of leukemic patients treated with cytarabine especially at intermediate to high doses, usually presenting as noncardiogenic pulmonary edema (NCPE). Anecdotally the association between cytarabine and the onset of bronchiolitis obliterans organizing pneumonia (BOOP) has been reported. We describe here three cases of patients affected by acute myeloid leukemia (AML) treated with chemotherapeutic regimens including high dose cytarabine, who developed early onset of fever, mild dyspnea, moderate hypoxemia on arterial blood gas analysis and lung infiltrates documented by high-resolution computerized tomography (HRCT), with a more indolent behaviour and a benign clinical outcome, compared with similar cases previously reported in the literature. Our cases widen the spectrum of clinical features of cytarabine-related toxicity in leukemic patients.

Autosomal dominant polycystic kidney disease: a linkage evaluation of heterogeneity in Italy. Italian Collaborative Group on Polycystic Kidney Disease.

A survey of 29 families with Adult Polycystic Kidney Disease (ADPKD) was performed to evaluate the genetic heterogeneity of the disease in Italy. The approach was through the linkage between the disease and 2 polymorphic DNA fragments as detected by the probes 3'HVR and 24.1. Linkage between the polymorphic markers and the disease was confirmed, with the following lod scores: between 3'HVR and ADPKD1 = 12.974 at theta = 0.02; between 24.1 and ADPKD = 1.716 at theta = 0.07; between 3'HVR and 24.1 = 2.738 at theta = 0.09. No evidence of significant genetic heterogeneity in the examined Italian regions was detected.

Restriction fragment length polymorphism species-specific patterns in the identification of white truffles.

A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction fragment length polymorphism patterns were determined for all five species. The use of PCR in conjunction with restriction enzymes provides a sensitive and efficient tool for use in distinguishing ectomycorrhizal species and monitoring inoculated seedlings or field mycorrhizal populations.

Multiplex PCR for the identification of white Tuber species.

Species-specific primers selected from the internal transcribed spacer region sequence were used to set up a multiplex polymerase chain reaction (PCR) able to simultaneously identify the white truffle species Tuber magnatum, Tuber borchii, Tuber maculatum and Tuber puberulum. Furthermore, a primer specific for the competitive fungus Sphaerosporella brunnea was designed and added to the multiplex PCR set, allowing the detection of the Tuber species and the contaminant fungus in a one-step reaction.

Estimation of fungal biomass and transcript levels in Tilia platyphyllos-Tuber borchii ectomycorrhizae.

Very little information is available to date about the complex truffle life cycle which involves the succession of three developmental phases. In order to gain more knowledge about ectomycorrhizal formation and fruit body development an ectomycorrhizal model system was used to study fungal biomass and plant and fungal transcript levels. They were evaluated in ectomycorrhizal development using the ergosterol assay and the internal transcribed spacer-5.8S ribosomal DNA from Tilia platyphyllos and Tuber borchii as molecular probes respectively. The results obtained from different approaches revealed a decrease in fungal biomass, transcript and protein levels during ectomycorrhizal development.

A new pair of primers designed for amplification of the ITS region in Tuber species.

The alignment of the 28S gene of several species of Pezizales allowed to select two pairs of primers able to amplify the internal transcribed spacer region of ribosomal DNA in mycorrhizal fungi, such as truffles. The higher yield of the amplification product demonstrates a better annealing of the new primers to the rDNA, as compared to the universal primers internal transcribed spacer 1 and internal transcribed spacer 4. Therefore, the new primers can be used as an easier and more sensitive tool for the identification of truffle species in any stage of their life cycle, including the mycorrhizal phase.

Simultaneous high-performance capillary electrophoretic determination of reduced and oxidized glutathione in red blood cells in the femtomole range.

This paper describes a high-performance capillary electrophoretic (HPCE) method which allows a quick, simultaneous and quantitative determination of reduced (GSH) and oxidized (GSSG) glutathione in mammalian red blood cells using a Supelco-bonded hydrophilic phase capillary CElect-P150. The extraction procedure of GSH and GSSG from erythrocytes using Microcon-10 membranes is very simple and allows a correct evaluation of these compounds present in the red blood cells. Furthermore, the HPCE method does not require removal of the excess N-ethylmaleimide used to block the glutathione in its reduced state, making the simultaneous evaluation of GSH and GSSG possible in a very short time (ca. 4 min), with a sensitivity at femtomole level.

Study of the effects on DNA of electromagnetic fields using clamped homogeneous electric field gel electrophoresis.

A clamped homogeneous electric field (CHEF) electrophoresis allowing the separation of DNA molecules in the range of 200 to 3000 kb in size was used to study the biological effects of electric and magnetic fields (EMFs). The results obtained did not show any detectable genomic damage on Saccharomyces cerevisiae.