Experimental and theoretical analysis of cytokine release for the study of radiation-induced bystander effect

Purpose: To clarify the experimental conditions that might influence the release of cytokines in the culture medium and give some basic input for building a model for cytokine (e.g., Interleukin-6, IL-6) regulation in the case of ‘sham irradiation’ and after ionising radiation exposure.

Materials and methods: The influence of cell type, cell density, medium volume, medium storage temperature and other methodological aspects on IL-6 and Interleukin-8 (IL-8) release were investigated. In addition, the effects over the time of different doses of gamma irradiation on the clonogenic survival of bystander cells and on the secretion of these cytokines were studied.

Results: We observed significant decreases of clonogenic survival in AG01522 and T98G cells after the transfer of medium collected 5 and 20 h after low doses of gamma irradiation. Concerning the Interleukins' measurements, our experiments showed that the aggregate removal modalities tested, and up to 10 freeze-thaw cycles, do not have significant influence on the measurements of IL-6 concentration in the medium. We also observed that the IL-6 accumulated in the medium of human fibroblasts is not degraded when maintained at 37°C. Sets of experiments demonstrated that cell density or medium volume do not influence the release of IL-6. On the contrary, our results showed that IL-8 released by glioblastoma cells strongly depends on the amount of medium. Finally, the exposure of fibroblasts to gamma irradiation has influence on the release kinetics of both IL-6 and IL-8 with peculiar features.

Conclusions: This study solved some of the methodological doubts concerning the study of bystander effects by means of the medium transfer technique; moreover it also highlighted some experimental aspects that need to be considered when approaching this sort of experiments.     

Normal sperm parameters per se do not reliably account for fertility: A case–control study in the real-life setting

A proportion of men are infertile despite having normal medical history/physical examination and normal semen analysis. We aimed to assess whether normal sperm parameters per se account for male factor fertility. 1,957 infertile men were compared with 103 age-comparable fertile controls. Semen analysis was based on 2010 World Health Organization reference criteria. Of all, 12.1% of infertile men and 40.8% of fertile men presented with normal sperm parameters. Among fertile men, 36.9% had isolated sperm abnormalities and 22.3% men showed two or more concomitant sperm abnormalities. Serum total testosterone was higher in infertile men with normal sperm parameters compared to those with ≥2 sperm abnormalities or azoospermia, but similar to those with isolated sperm abnormalities (p ≤ .001). Circulating hormones were similar among sperm parameters groups in fertile men. At multivariable analyses, testicular volume (OR 1.12, p ≤ .001) and FSH (OR 0.8, p ≤ .001) were associated with normal sperm parameters. Overall, the longer the infertility period, the greater the number of sperm parameters abnormalities (p < .01). In conclusion, we found that 12% of infertile men and only 41% of fertile men present with normal sperm parameters. Normal sperm parameters per se do not reliably account for fertility in the real-life setting.     

High cysteine levels in renal transplant recipients: relationship with hyperhomocysteinemia and 5,10-MTHFR polymorphism

BACKGROUND: Long-term survival of renal transplant recipients seems to be influenced by the occurrence of thromboembolic complications and cardiovascular disease. Preliminary data available in the literature found high levels of cysteine (Cy) as a risk factor for deep venous thrombosis independently of high homocysteine (tHcy) levels, but no data are available about Cy levels in renal transplant recipients. METHODS: To investigate Cy, tHcy, and plasminogen activator inhibitor-1 (PAI-1) levels and the prevalence of 5,10-methylenetetrahydrofolate reductase (MTHFR) in renal transplantation, we studied 70 stable renal transplant recipients and 66 age- and sex-matched normal subjects as controls. RESULTS: Cy, tHcy, and PAI-1 levels were significantly higher in renal transplant recipients with respect to controls (Cy: 254 micromol/L [117-466] vs. 198 micromol/L [99-331], P<0.001; tHcy: 17.0 micromol/L [4.0-68] vs. 8.1 micromol/L [2.0-24.0], P<0.00001; PAI-1: 16.8 IU/ml [5.1-45.5] vs. 7.9 IU/ml [4.0-18.0], P<0.00001). High Cy levels were detected in 35.8% of patients. Hyperhomocysteinemia, both in the fasting state and postmethionine loading test, was diagnosed in 90% of cases. The odds ratios for Cy and tHcy levels within the fourth quartile with respect to the other quartiles were markedly increased in renal transplant recipients even after adjustment for prevalent cardiovascular risk factors, glomerular filtration rate, tHcy and, Cy, respectively (Cy: 29.0 micromol/L [95% CI 7.0-111]; tHcy: 29.9 micromol/L [95% CI 7.5-118.1]). Fasting tHcy levels correlated well with PAI-1 (r=0.65; P<0.0001) but not with Cy levels (r=0.10; P=0.4). The prevalence of the MTHFR 677TT genotype in renal transplant recipients was not significantly higher in patients than in controls (mutant allele frequency: 0.48 in patients and 0.47 in controls) and was associated with significantly higher fasting and postmethionine tHcy levels both in controls and patients. After 2 months of vitamin supplementation, tHcy (Pre: 17.0 micromol/L [4.0-68]; Post: 7.5 micromol/L [2.3-21.9]; P<0.0001) and PAI-1 levels (Pre: 16.8 IU/ml [5.1-45.5]; Post: 10 IU/ml [2.0-25]; P<0.001) were significantly decreased, whereas Cy levels showed a small decrease that did not reach statistical significance (Pre: 254 micromol/L [117-466]; Post: 209 micromol/L [168-300]; P=0.3). Patients with the MTHFR 677TT genotype had the major percentage of decrease of tHcy levels with respect to the other genotypes. CONCLUSION: In conclusion, this study demonstrates the presence of elevated Cy plasma levels in renal transplant recipients. Vitamin supplementation reduces tHcy but not Cy levels, and the amount of decrease seems to be influenced by the MTHFR genotype.     

Congenital hypofibrinogenemia: characterization of two missense mutations affecting fibrinogen assembly and secretion

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by abnormally low levels of fibrinogen in plasma, generally due to heterozygous mutations in one of the three fibrinogen genes (FGA, FGB, and FGG, coding for Aα, Bβ, and γ chain, respectively). Hypofibrinogenemic patients are usually asymptomatic, whereas individuals bearing similar mutations in the homozygous or compound heterozygous state develop a severe bleeding disorder: afibrinogenemia. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations affecting fibrinogen assembly or secretion, distributed throughout the 50-kb fibrinogen gene cluster. In this study, we report the mutational screening of two unrelated hypofibrinogenemic patients leading to the identification of two missense mutations, one hitherto unknown (αCys45Phe), and one previously described (γAsn345Ser). The involvement of αCys45Phe and γAsn345Ser in the pathogenesis of hypofibrinogenemia was investigated by in-vitro expression experiments. Both mutations were demonstrated to cause a severe impairment of intracellular fibrinogen processing, either by affecting half-molecule dimerization (αCys45Phe) or by hampering hexamer secretion (γAsn345Ser).     

Low density lipoprotein retention by aortic tissue. Contribution of extracellular matrix

We compared in vitro heparin binding activity and in vivo intravascular clearance and aortic uptake in rabbits of native, reductively methylated and heparin-complexed low density lipoprotein (LDL) in order to explore the extracellular matrix binding vs cellular metabolism of LDL. Reductively methylated LDL formed soluble and insoluble complexes with heparin which was comparable to native LDL. Reductive methylation of LDL produced only 30% reduction in aortic uptake vs 60% reduction in plasma clearance, reflecting the relatively smaller contribution of receptor-mediated pathway in aortic tissue vs whole animal. The intravascular clearance of native and heparin-complexed LDL remained essentially the same, indicating similarities in cellular metabolism of LDL in both cases. But the aortic uptake of the heparin bound LDL was 30% less than the native LDL, suggesting an inhibition in binding of heparin-complexed LDL to tissue proteoglycans. Saline extraction accounted for only part (53-66%) of the LDL preparations that were retained by the tissue while subsequent collagenase and elastase treatments extracted 3-5% and 17-22% of the materials respectively. These results favor the contribution of arterial extracellular matrix components to the retention of LDL.     

The 'Open-Artery Hypothesis': new clinical and pathophysiologic insights

The open-artery hypothesis states that myocardial reperfusion, even if late for myocardial salvage, provides benefits and prevents adverse cardiac remodeling. While observational data in humans regarding the deleterious impact of a permanent infarct-related artery occlusion and the benefits of spontaneous reperfusion are quite consistent, the reports regarding late revascularization are inconclusive in order to prove such a hypothesis. The observational studies tend to have selection biases, while randomized trials to date are too small to be conclusive. Moreover, the pathophysiological mechanisms underlying presumed benefits of reperfusion are still unclear. However, although the open-artery hypothesis remains unproven, the current evidence suggesting benefits calls for additional studies. Limitations of ischemic left ventricular dilatation and myopathy could markedly reduce cardiovascular morbidity and mortality after acute myocardial infarction.     

Evaluating the landscape of gene cooperativity with receptor tyrosine kinases in liver tumorigenesis using transposon-mediated mutagenesis

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