CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipidspecific T cells has been hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d-glycolipid tetramers from fully denatured human CD1d molecules by using the technique of oxidative refolding chromatography. We demonstrate that chaperone-and foldase-assisted refolding of denatured CD1d molecules and 2-microglobulin in the presence of synthetic lipids is a rapid method for the generation of functional and specific CD1d tetramers, which unlike previously published protocols ensures isolation of CD1d tetramers loaded with a single lipid species. The use of human CD1d-␣-galactosylceramide tetramers for ex vivo staining of peripheral blood lymphocytes and intrahepatic T cells from patients with viral liver cirrhosis allowed for the first time simultaneous analysis of frequency and specificity of natural killer T cells in human clinical samples. Application of this protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in disease.
Using patient data from a unique single source outbreak of hepatitis B virus (HBV) infection, we have characterized the kinetics of acute HBV infection by monitoring viral turnover in the serum during the late incubation and clinical phases of the disease in humans. HBV replicates rapidly with minimally estimated doubling times ranging between 2.2 and 5.8 d (mean 3.7 ± 1.5 d). After a peak viral load in serum of nearly 1010 HBV DNA copies/ml is attained, clearance of HBV DNA follows a two or three phase decay pattern with an initial rapid decline characterized by mean half-life (t
1/2) of 3.7 ± 1.2 d, similar to the t
1/2 observed in the noncytolytic clearance of covalently closed circular DNA for other hepadnaviruses. The final phase of virion clearance occurs at a variable rate (t
1/2 of 4.8 to 284 d) and may relate to the rate of loss of infected hepatocytes. Free virus has a mean t
1/2 of at most 1.2 ± 0.6 d. We estimate a peak HBV production rate of at least 1013 virions/day and a maximum production rate of an infected hepatocyte of 200–1,000 virions/day, on average. At this peak rate of virion production we estimate that every possible single and most double mutations would be created each day.
We present the design and development of the automata processor, a massively parallel non-von Neumann semiconductor architecture that is purpose-built for automata processing. This architecture can directly implement non-deterministic finite automata in hardware and can be used to implement complex regular expressions, as well as other types of automata which cannot be expressed as regular expressions. We demonstrate that this architecture exceeds the capabilities of high-performance FPGA-based implementations of regular expression processors. We report on the development of an XML-based language for describing automata for easy compilation targeted to the hardware. The automata processor can be effectively utilized in a diverse array of applications driven by pattern matching, such as cyber security and computational biology.
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