Performance of MycAssay Aspergillus DNA real-time PCR assay compared with the galactomannan detection assay for the diagnosis of invasive aspergillosis from serum samples.

Invasive aspergillosis (IA) is a major problem in the immunocompromised population, and its diagnosis is difficult due to the low sensitivity of available tests. Detection of Aspergillus nucleic acid by polymerase chain reaction (PCR) in serum samples is a promising diagnostic tool; however, use of multiple "in-house" methods precludes standardization. The first commercial PCR assay, MycAssay Aspergillus (Myconostica, Ltd), became available recently, and its performance in the diagnosis of IA was evaluated and compared with the galactomannan (GM) assay. Serum samples obtained from patients with hematological cancer were tested retrospectively with MycAssay Aspergillus PCR. Per-episode and per-test analyses were undertaken with 146 sera from 35 hematological patients. Sixteen patients had proven or probable IA and 19 had possible or no IA. In per-episode analysis, MycAssay Aspergillus had a sensitivity of 43.8% (95% confidence interval [CI], 19.8%-70.1%) and a specificity of 63.2% (95% CI, 38.4%-83.7%) for IA diagnosis. In per-test analyses, MycAssay Aspergillus had a lower specificity than the GM assay (83.3% vs. 93.1%, P = 0.04). The addition of PCR to routine clinical practice would have permitted the diagnosis of one additional probable IA in our cohort. Use of PCR instead of GM assay would have delayed the diagnosis in two cases. Aspergillus DNA detection by PCR with serum specimens using MycAssay showed a lower specificity than the GM assay and was associated with a low sensitivity for IA diagnosis. More studies are needed to determine the exact role of MycAssay in IA diagnosis in patients with hematological malignancy.

Folate deficiency and ionizing radiation cause DNA breaks in primary human lymphocytes: a comparison.

DNA double-strand breaks, the most serious DNA lesion caused by ionizing radiation, are also caused by several vitamin or mineral deficiencies, such as for folate. Primary human lymphocytes were either irradiated or cultured at different levels of folate deficiency to assess cell proliferation, apoptosis, cell cycle, DNA breaks, and changes in gene expression. Both radiation and folate deficiency decreased cell proliferation and induced DNA breaks, apoptosis, and cell cycle arrest. Levels of folate deficiency commonly found resulted in effects similar to those caused by 1 Gy of radiation, a relatively high dose. Though both radiation and folate deficiency caused DNA breaks, they affected the expression of different genes. Radiation activated excision and DNA double-strand break repair genes and repressed mitochondrially encoded genes. Folate deficiency activated base and nucleotide excision repair genes and repressed folate-related genes. No DNA double-strand break repair gene was activated by folate deficiency. These findings suggest that a diet poor in folate may pose a risk of DNA damage comparable to that of a relatively high dose of radiation. Our results also suggest that research on biological effects of low-dose radiation should take into account the nutritional status of the subjects, because folate deficiency could confound the effects of low-dose radiation.

Screening for polyomavirus associated nephropathy in renal transplantation with blood viral load measurement.

BACKGROUND: Polyomavirus associated nephropathy (PVAN) is an important cause of graft failure in the renal transplant population. It has been shown that viremia precedes PVAN, suggesting that measurement of blood viral load could be used for PVAN screening. OBJECTIVES: To verify the utility of BK virus (BKV) blood viral load measurement for PVAN screening in the renal transplant population, establish a threshold value, and determine the sensitivity and specificity of the test. STUDY DESIGN: We developed a real-time PCR assay for BKV blood viral load measurement and included this assay in the PVAN screening protocol of the renal transplant recipients of our institution. We report results for 60 patients who had a blood viral load measurement concomitantly with an allograft biopsy with immunohistochemistry for polyomavirus. RESULTS: 14 patients were found to have a PVAN on allograft biopsy together with a viral load above 3.0x10(3)copies/ml. None of the patients with a viral load under 3.0x10(3)copies/ml had a PVAN on allograft biopsy. The area under the receiver operating characteristic (ROC) curve was 0.95 (95% CI: 0.91-1.00) and using a threshold value of 3.0x10(3)copies/ml yielded a sensitivity of 100% (95% CI: 76.8-100%) and a specificity of 89.6% (95% CI: 77.3-96.5%) for PVAN screening. CONCLUSIONS: BKV blood viral load measurement is sensitive and specific for PVAN screening when a threshold value is precisely determined.

Zinc deficiency induces oxidative DNA damage and increases p53 expression in human lung fibroblasts.

Poor zinc nutrition may be an important risk factor in oxidant release and the development of DNA damage and cancer. Approximately 10% of the United States population ingests <50% of the recommended daily allowance for zinc, a cofactor in proteins involved in antioxidant defenses, electron transport, DNA repair and p53 protein expression. This study examined the effects of zinc deficiency on oxidative stress, DNA damage and the expression of DNA repair enzymes in primary human lung fibroblasts by the use of DNA microarrays and functional assays. Cellular zinc was depleted by 1) growing cells in a zinc-deficient medium and 2) exposuring cells to an intracellular zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine. Array data revealed upregulation of genes involved in oxidative stress and DNA damage/repair and downregulation of other DNA repair genes. Zinc deficiency in cells caused an increase in oxidant production (dichlorofluoroscein fluorescence) and a significant induction of single-strand breaks (Comet assay) and p53 protein expression (Western blot analysis). Thus, zinc deficiency not only caused oxidative stress and DNA damage, but also compromised the cells' ability to repair this damage. Zinc adequacy appears to be necessary for maintaining DNA integrity and may be important in the prevention of DNA damage and cancer.

Folate deficiency inhibits the proliferation of primary human CD8+ T lymphocytes in vitro.

Folate is required for one-carbon transfer reactions and the formation of purines and pyrimidines for DNA and RNA synthesis. Deficiency of folate can lead to many clinical abnormalities, including macrocytic anemia, cardiovascular diseases, birth defects, and carcinogenesis. The nucleotide imbalance due to folate deficiency causes cell cycle arrest in the S phase and uracil misincorporation into DNA, which may result in DNA double-strand breaks during repair. The role of folate in the immune system has not been fully characterized. We cultured PHA-activated human T lymphocytes in varying concentrations of folate, and measured proliferation, cell cycle, apoptosis, uracil misincorporation, and proportions of Th cells (CD4(+)) and cytotoxic T (CD8(+)) cells. Folate deficiency reduced proliferation of T lymphocytes, induced cell cycle arrest in the S phase, induced apoptosis, and increased the level of uracil in DNA. Folate deficiency also increased the CD4(+) to CD8(+) ratio due to a marked reduction of CD8(+) cell proliferation. Folate or nucleoside repletion of folate-deficient cells rapidly restored T lymphocyte proliferation and normal cell cycle, reduced the DNA uracil content, and lowered the CD4(+) to CD8(+) ratio. These data suggest that folate status may affect the immune system by reducing the capacity of CD8(+) cells to proliferate in response to activation.

The fetoprotein transcription factor (FTF) gene is essential to embryogenesis and cholesterol homeostasis and is regulated by a DR4 element.

The fetoprotein transcription factor (FTF) gene was inactivated in the mouse, with a lacZ gene inserted inframe into exon 4. LacZ staining of FTF+/- embryos shows that the mFTF gene is activated at initial stages of zygotic transcription. FTF gene activity is ubiquitous at the morula and blastocyst stages and then follows expression patterns indicative of multiple FTF functions in fetal development. FTF-/- embryos die at E6.5-7.5, with features typical of visceral endoderm dysfunction. Adult FTF+/- mice are hypocholesterolemic, and express liver FTF at about 40% of the normal level. Overexpression of liver FTF in transgenic mice indicates in vivo that FTF is an activator of CYP7A1. However, CYP7A1 expression is increased in FTF+/- liver. Gene expression profiles indicate that higher CYP7A1 expression is caused by attenuated liver cell stress signaling. Diet experiments support a model where FTF is quenched both by activated c-Jun, and by SHP as a stronger feedback mechanism to repress CYP7A1. A DR4 element is conserved in the FTF gene promoter and activated by LXR-RXR and TR-RXR, qualifying the FTF gene as a direct metabolic sensor. Liver FTF increases in rats treated with thyroid hormone or a high cholesterol diet. The FTF DR4 element tightens functional links between FTF and LXRalpha in cholesterol homeostasis and can explain transient surges of FTF gene activities during development and FTF levels lower than predicted in FTF+/- liver. The FTF-lacZ mouse establishes a central role for FTF in developmental, nutritive, and metabolic functions from early embryogenesis through adulthood.

Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes.

Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. We improved a previously described gas chromatography-mass spectrometry (GC-MS) assay for uracil in DNA and validated the assay by analyzing the DNA-uracil content of normal, primary human lymphocytes that were cultured in 0-3000 nM folic acid. In addition, the effects of nucleoside mixtures T or TdCA (T, thymidine; A, adenosine; dC, deoxycytidine) were investigated. Over 4 consecutive days, the inter- and intraassay coefficients of variation (CVs) were 2.3-3.9 and 0.6-2.2%. Mean recovery was 99.4%. Oligonucleotides containing 100 pg of uracil yielded a mean uracil measurement of 110.1 pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12 nM, and minima at 0 and 3000 nM folate. Extremely folate-deficient cells may incorporate less uracil because DNA synthesis is reduced. A wide response to folate deficiency was seen in cells from different donors, suggesting that genetic background plays a critical role in individual susceptibility to DNA damage and cancer risk. Unexpectedly, TdCA supplementation caused increased DNA-uracil (vs 3000 nM folate for 10 days, P > 0.05), probably due to the conversion of deoxycytidine to deoxyuridine by cytidine deaminase, leading to elevated dUMP/dTMP ratios. This improved uracil assay could serve as a useful tool in the study of the mechanism of uracil misincorporation into DNA. The assay requires 3 microg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans.