An “Escape Clock” for Estimating the Turnover of SIV DNA in Resting CD4+ T Cells

Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an “escape clock” approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT) SIV DNA present in early infection by CTL escape mutant (EM) strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication

Nutritional and toxicological studies of New Zealand Cookia sulcata

Interest in snails as a source of protein and as a delicacy is increasing in many countries. The present study investigated selected nutritional (proximate, amino acid, fatty acid, vitamin E, cholesterol and macro- and trace minerals) and toxic (toxic elements and organochlorine) concentrations of small and large (≤60 and > 60. g whole animal weight, respectively) Captain Cook snails (Cookia sulcata). The major amino acids in C. sulcata muscle were glutamic (13.9. g/100. g protein), arginine (10.2. g/100. g protein), glycine (9.5. g/100. g protein) and taurine (9.5. g/100. g protein). There was no difference in the amino acid profiles related to the snail size. C. sulcata had relatively high amounts of saturated fatty acids (44.4%) and polyunsaturated fatty acids (34.3%), and lesser amounts of mono-unsaturated fatty acids. The major fatty acids detected in C. sulcata were C16:0, C18:0, C20:4 and C22:5, which accounted for more than 60% of the total fatty acids. Snail size had a significant (P < . 0.05) effect on the C16:0 and C18:3 concentrations. The only isoform of vitamin E present in C. sulcata was identified as α-tocopherol at 2.16 and 3.71. mg/100. g fresh weight for the small and large snails, respectively. The average cholesterol concentration in C. sulcata was 1.33. mg/100. g fresh weight. The results indicated that none of the toxic elements, including Al, Ni, As and Pb of C. sulcata, were over the maximum concentration allowed in the Australia New Zealand Food Standard; and the organochlorine pesticides concentrations in C. sulcata were below the detection limit ( < 0.0005. mg/kg). C. sulcata could, therefore, be utilized for special dietary applications requiring higher amounts of Fe, Zn, taurine and tryptophan

Nutritional composition of Mutton bird (Puffinus griseus) meat

Mutton birds (Puffinus griseus) are wild seabird chicks traditionally harvested by Maori but available commercially for seasonal consumption in New Zealand. Little information is available on the nutritional content of the meat from these birds. Proximate analysis and amino and fatty acid composition of Mutton bird breast meat (MBBM) were measured over two harvesting seasons, 2007 and 2008. Protein content was lower, and fat and ash contents were higher (P < 0.05) in meat from birds harvested in 2008 (18.5, 13.0 and 11.7%, respectively) compared with that from 2007 (20.3, 11.8 and 10.3%, respectively). Higher lysine concentrations and lower proline, cysteine and methionine were found in MBBM compared with literature values for beef, lamb and pork. The essential amino acid content in Mutton bird (41.7 and 38.4% for 2008 and 2007, respectively) was slightly lower than those reported for common meats (42–43%). Palmitic, arachidonic, DHA, stearic, EPA, and oleic were the major fatty acids (FA) detected in MBBM and accounted for approximately 60% of the FA. The cholesterol concentration was not affected by season. Seasonal variations MBBM existed which may be of little nutritional consequence but might be a useful indicator for ecological events including changing feed availability

Linking Pig-Tailed Macaque Major Histocompatibility Complex Class I Haplotypes and Cytotoxic T Lymphocyte Escape Mutations in Simian Immunodeficiency Virus Infection

UNLABELLED: The influence of major histocompatibility complex class I (MHC-I) alleles on human immunodeficiency virus (HIV) diversity in humans has been well characterized at the population level. MHC-I alleles likely affect viral diversity in the simian immunodeficiency virus (SIV)-infected pig-tailed macaque (Macaca nemestrina) model, but this is poorly characterized. We studied the evolution of SIV in pig-tailed macaques with a range of MHC-I haplotypes. SIV(mac251) genomes were amplified from the plasma of 44 pig-tailed macaques infected with SIV(mac251) at 4 to 10 months after infection and characterized by Illumina deep sequencing. MHC-I typing was performed on cellular RNA using Roche/454 pyrosequencing. MHC-I haplotypes and viral sequence polymorphisms at both individual mutations and groups of mutations spanning 10-amino-acid segments were linked using in-house bioinformatics pipelines, since cytotoxic T lymphocyte (CTL) escape can occur at different amino acids within the same epitope in different animals. The approach successfully identified 6 known CTL escape mutations within 3 Mane-A1*084-restricted epitopes. The approach also identified over 70 new SIV polymorphisms linked to a variety of MHC-I haplotypes. Using functional CD8 T cell assays, we confirmed that one of these associations, a Mane-B028 haplotype-linked mutation in Nef, corresponded to a CTL epitope. We also identified mutations associated with the Mane-B017 haplotype that were previously described to be CTL epitopes restricted by Mamu-B*017:01 in rhesus macaques. This detailed study of pig-tailed macaque MHC-I genetics and SIV polymorphisms will enable a refined level of analysis for future vaccine design and strategies for treatment of HIV infection. IMPORTANCE: Cytotoxic T lymphocytes select for virus escape mutants of HIV and SIV, and this limits the effectiveness of vaccines and immunotherapies against these viruses. Patterns of immune escape variants are similar in HIV type 1-infected human subjects that share the same MHC-I genes, but this has not been studied for SIV infection of macaques. By studying SIV sequence diversity in 44 MHC-typed SIV-infected pigtail macaques, we defined over 70 sites within SIV where mutations were common in macaques sharing particular MHC-I genes. Further, pigtail macaques sharing nearly identical MHC-I genes with rhesus macaques responded to the same CTL epitope and forced immune escape. This allows many reagents developed to study rhesus macaques to also be used to study pigtail macaques. Overall, our study defines sites of immune escape in SIV in pigtailed macaques, and this enables a more refined level of analysis of future vaccine design and strategies for treatment of HIV infection

Epitope-specific CD8⁺ T cell kinetics rather than viral variability determine the timing of immune escape in simian immunodeficiency virus infection

CD8+ T cells are important for the control of chronic HIV infection. However, the virus rapidly acquires "escape mutations" that reduce CD8+ T cell recognition and viral control. The timing of when immune escape occurs at a given epitope varies widely among patients and also among different epitopes within a patient. The strength of the CD8+ T cell response, as well as mutation rates, patterns of particular amino acids undergoing escape, and growth rates of escape mutants, may affect when escape occurs. In this study, we analyze the epitope-specific CD8+ T cells in 25 SIV-infected pigtail macaques responding to three SIV epitopes. Two epitopes showed a variable escape pattern and one had a highly monomorphic escape pattern. Despite very different patterns, immune escape occurs with a similar delay of on average 18 d after the epitope-specific CD8+ T cells reach 0.5% of total CD8+ T cells. We find that the most delayed escape occurs in one of the highly variable epitopes, and that this is associated with a delay in the epitope-specific CD8+ T cells responding to this epitope. When we analyzed the kinetics of immune escape, we found that multiple escape mutants emerge simultaneously during the escape, implying that a diverse population of potential escape mutants is present during immune selection. Our results suggest that the conservation or variability of an epitope does not appear to affect the timing of immune escape in SIV. Instead, timing of escape is largely determined by the kinetics of epitope-specific CD8+ T cells