Cidofovir inhibits polyomavirus BK replication in human renal tubular cells downstream of viral early gene expression.

The human polyomavirus BK (BKV) causes nephropathy and hemorrhagic cystitis in kidney and bone marrow transplant patients, respectively. The anti-viral cidofovir (CDV) has been used in small case series but the effects on BKV replication are unclear, since polyomaviruses do not encode viral DNA polymerases. We investigated the effects of CDV on BKV(Dunlop) replication in primary human renal proximal tubule epithelial cells (RPTECs). CDV inhibited the generation of viral progeny in a dose-dependent manner yielding a 90% reduction at 40 microg/mL. Early steps such as receptor binding and entry seemed unaffected. Initial large T-antigen transcription and expression were also unaffected, but subsequent intra-cellular BKV DNA replication was reduced by >90%. Late viral mRNA and corresponding protein levels were also 90% reduced. In uninfected RPTECs, CDV 40 microg/mL reduced cellular DNA replication and metabolic activity by 7% and 11% in BrdU and WST-1 assays, respectively. BKV infection increased DNA replication to 142% and metabolic activity to 116%, respectively, which were reduced by CDV 40 microg/mL to levels of uninfected untreated RPTECs. Our results show that CDV inhibits BKV DNA replication downstream of large T-antigen expression and involves significant host cell toxicity. This should be considered in current treatment and drug development.

Increase in plasma calprotectin during long-distance running.

Running leads to biochemical and hematological changes consistent with an inflammatory reaction to tissue injury. We report changes in the plasma concentration of the leukocyte-derived protein calprotectin after long-distance running. Blood samples were collected from runners before and after a marathon, half-marathon, a 30-km cross-country run, a military ranger-training course and short-term maximal physical exercise until exhaustion, VO2max. Leukocyte counts, plasma calprotectin concentration and calprotectin per neutrophilic granulocyte were assayed using a new method. During the marathon, half-marathon, the 30-km run, the ranger-training course and the VO2max exercise, calprotectin levels increased 96.3-fold, 13.3-fold, 20.1-fold, 7.5-fold and 3.4-fold, respectively. These changes may reflect damage to the tissues or vascular endothelium, causing microthrombi with subsequent activation of neutrophils. These cells are known to phagocytose platelets in microthrombi and may contribute to the prevention of clinical thrombosis. The half-life of calprotectin in plasma was about 5 h. The content of calprotectin per neutrophil remained unchanged during exercise at a level similar to that in healthy blood donors: mean: 25 pg/cell, range 18.8-33.6. A reference interval (mean +/- 2 SD) of 18.6-31.4 pg/cell is suggested.

Isolation and partial characterisation of a new antiproliferative substance from human leucocytes inhibiting growth of Candida albicans.

AIM: To purify and partially characterise a fraction from human leucocytes containing a substance cytotoxic to Candida albicans. METHODS: Leucocytes were isolated from the buffy coats of healthy blood donors. The cytotoxic factor (CF) was isolated from the soluble fraction of the cells. A cell lysate was passed through a filter with a cut off value of 3 kDa, and the filtrate was processed by anionic exchange chromatography and gel filtration. The purified CF was analysed for its chemical and biological properties. The cytotoxicity of CF was tested on C albicans grown on agar plates. RESULTS: Mass spectrometry showed a molecular mass of 2.148 kDa. CF was found in polymorphonuclear neutrophilic cells only. No amino acids were detected, and a low ultraviolet absorbance at 260 nm and resistance to nuclease indicate the absence of nucleic acids. An anthrone test was positive for carbohydrate. The substance was soluble in water. CF showed a dose related cytotoxicity in the range of 0.1-1 mg/ml. The cytotoxic effect was abrogated by zinc ions. Preliminary testing indicated that CF also had cytotoxic effects against some bacteria. CONCLUSIONS: This report describes a factor from isolated human leucocytes that is cytotoxic to C albicans. The substance contains a carbohydrate moiety, whereas no amino acids were detected. The cytotoxicity can be abrogated by zinc ions in vitro. This substance is probably part of the repertoire by which leucocytes prevent infections.

The antimicrobial peptides lactoferricin B and magainin 2 cross over the bacterial cytoplasmic membrane and reside in the cytoplasm.

The localization of immunolabelled antimicrobial peptides was studied using transmission electron microscopy. Staphylococcus aureus and Escherichia coli were exposed to lactoferricin B (17-41), lactoferricin B (17-31) and D-lactoferricin B (17-31). E. coli was also exposed to cecropin P1 and magainin 2. The lactoferricins were found in the cytoplasm of both bacteria. In S. aureus the amount of cytoplasmic lactoferricin B (17-41) was time- and concentration-dependent, reaching a maximum within 30 min. Cecropin P1 was confined to the cell wall, while magainin 2 was found in the cytoplasm of E. coli. The finding of intracellularly localized magainin is not reported previously.