ABSTRACT
The use of pyrogen tests to assess the risk of endotoxin in biological products has increased recently due to concerns of some regulatory authorities about products exhibiting low endotoxin recovery (LER). Manufacturers increasingly seek to reduce the use of animals unless essential to assure patient safety. The current study compares the ability of the monocyte activation test (MAT) and the bacterial endotoxin test (BET) to the rabbit pyrogen test (RPT) to detect endotoxin spikes in samples of products shown to exhibit LER. Product samples or water were spiked with endotoxin and held for three days or tested immediately in the BET, the RPT, and two variations of the MAT at the same time. Results show high sensitivity to endotoxin of both the BET and MAT, and much lower sensitivity of the RPT, indicating that much higher levels of reference standard endotoxin are required to induce pyrogenicity in the RPT than the 5 endotoxin units (EU) per kg common threshold. The results of the BET and MAT correlated well for the detection of endotoxin spike in water. We also show that LER (masking of endotoxin) found in the BET is also seen in the MAT and RPT, suggesting that the products themselves elicit a biological inactivation of spiked endotoxin over time, thereby rendering it less or non-pyrogenic. We conclude that the non-animal MAT option is a suitable replacement for the RPT to measure spiked endotoxin in biopharmaceuticals.
Subject(s)
Endotoxins , Pyrogens , Animals , Rabbits , Endotoxins/toxicity , Pyrogens/toxicity , Animal Testing Alternatives , Monocytes , Biological Assay/methodsABSTRACT
Autophagy is a highly conserved and regulated process in eukaryotic cells by which components of the cytoplasm, such as damaged organelles and foreign pathogens, become enveloped into double-membrane autophagosome vesicles that fuse with the lysosome for degradation. Viruses are adept at subverting host cellular pathways for their replication and survival. The human tumor viruses, Epstein-Barr virus (EBV), Kaposi's Sarcoma-Associated Herpesvirus (KSHV), Hepatitis B virus (HBV), and Hepatitis C virus (HCV), have evolved novel ways of modulating autophagy during productive and latent stages of the virus life cycle. This review will discuss how the autophagy pathway becomes activated upon viral infection and the role of viral proteins in regulating the autophagy pathway. Specifically, we will examine how virus-encoded homologs of autophagy proteins evade autophagy-mediated degradation by blocking the induction, elongation, or maturation steps in the autophagy pathway. We will also discuss how certain viruses enhance autophagy induction or usurp autophagic machinery for their own replication. A comprehensive understanding of the autophagic response to tumor viruses may enable the discovery of novel antiviral and/or anticancer drug therapies.
Subject(s)
Autophagy/physiology , Oncogenic Viruses/physiology , Viral Proteins/physiology , Virus Replication/physiology , Animals , Host-Pathogen Interactions , HumansABSTRACT
MicroRNAs (miRNAs) are key regulators of gene expression in higher eukaryotes. Recently, miRNAs have been identified from viruses with double-stranded DNA genomes. To attempt to identify miRNAs encoded by herpes simplex virus 1 (HSV-1), we applied a computational method to screen the complete genome of HSV-1 for sequences that adopt an extended stem-loop structure and display a pattern of nucleotide divergence characteristic of known miRNAs. Using this method, we identified 11 HSV-1 genomic loci predicted to encode 13 miRNA precursors and 24 miRNA candidates. Eight of the HSV-1 miRNA candidates were predicted to be conserved in HSV-2. The precursor and the mature form of one HSV-1 miRNA candidate, which is encoded approximately 450 bp upstream of the transcription start site of the latency-associated transcript (LAT), were detected during infection of Vero cells by Northern blot hybridization. These RNAs, which behave as late gene products, are not predicted to be conserved in HSV-2. Additionally, small RNAs, including some that are roughly the expected size of precursor miRNAs, were detected using probes for miRNA candidates derived from sequences encoding the 8.3-kilobase LAT, from sequences complementary to U(L)15 mRNA, and from the region between ICP4 and U(S)1. However, no species the size of typical mature miRNAs were detected using these probes. Three of these latter miRNA candidates were predicted to be conserved in HSV-2. Thus, HSV-1 encodes at least one miRNA. We hypothesize that HSV-1 miRNAs regulate viral and host gene expression.