Maintenance of immune tolerance to a neo-self acetylcholine receptor antigen with aging: implications for late-onset autoimmunity.

Age-related changes in immune regulation are likely to account for the age-associated increase in serum autoantibody levels and in certain autoimmune disorders, such as myasthenia gravis (MG). To demonstrate directly a loss of immune tolerance in older individuals, responses to the acetylcholine receptor, the autoantigen in MG, were assessed in transgenic mice expressing the Torpedo californica acetylcholine receptor (TAChR) alpha-chain as a neo-self Ag. T cells from young transgenic mice had been shown to be tolerant to p146-162, the TAChR alpha-chain peptide that dominated young nontransgenic T cell responses in vitro. The immunodominance of p146-162 was not lost with age; fine specificity was preserved. Moreover, T cell tolerance to p146-162, as well as to other epitopes of the TAChR alpha-chain extracellular domain, was maintained in old transgenic mice. Even multiple TAChR immunizations coupled with the MG-enhancing cytokine, IL-12, did not break tolerance. In addition, T cells exhibiting CD4 upregulation, an early activation marker, were reduced in frequency equivalently in old and young transgenic animals, suggesting that immune regulation in this model was not impacted by aging. Moreover, B cell tolerance was also maintained with age. The persistence of immune tolerance was accompanied by an increase in the proportion of T regulatory cells; it is speculated that this may compensate for deficiencies in central tolerance that occur owing to thymic involution. In summary, our study reveals, for the first time, that some immune tolerance mechanisms do survive aging; this suggests that certain late-onset autoimmune disorders may be induced by a specific insult that disrupts immune homeostasis.

Cortisol release in response to UVB exposure in Xiphophorus fish.

Xiphophorus fishes are comprised of 26 known species. Interspecies hybridization between select species has been utilized to produce experimental models to study melanoma development. Xiphophorus melanoma induction protocols utilize ultraviolet light (UVB) to induce DNA damage and associated downstream tumorigenesis. However, the impact of induced stress caused by the UVB treatment of the experimental animals undergoing tumor induction protocols has not been assessed. Stress is an adaptive physiological response to excessive or unpredictable environmental stimuli. The stress response in fishes may be measured by an assay of cortisol released into the water. Here, we present results from investigations of stress response during an experimental treatment and UVB exposure in Xiphophorus maculatus Jp 163 B, Xiphophorus couchianus, and F1 interspecies hybrids produced from the mating X. maculatus Jp 163 B×X. couchianus. Overall, cortisol release rates for males and females after UVB exposure showed no statistical differences. At lower UVB doses (8 and 16kJ/m(2)), X. couchianus exhibited 2 fold higher levels of DNA damage then either X. maculatus or the F1 hybrid. However, based on the cortisol release rates, none of the fish types tested induced a primary stress response at the UVB lower doses (8 and 16kJ/m(2)). In contrast, at a very high UVB dose (32kJ/m(2)) both X. maculatus and the F1 hybrid showed a 5 fold increase in the cortisol release rate. To determine the effect of pigmentation on UVB induced stress, wild type and albino Xiphophorus hellerii were exposed to UVB (32kJ/m(2)). Albino X. hellerii exhibited 3.7 fold increase in the cortisol release while wild type X. hellerii did not exhibit a significant cortisol response to UVB. Overall, the data suggest the rather low UVB doses often employed in tumor induction protocols do not induce a primary stress response in Xiphophorus fishes.

Alternative strategies for development of a reference transcriptome for quantification of allele specific expression in organisms having sparse genomic resources.

In recent years RNA-Seq technology has been used not only to quantify differences in gene expression but also to understand the underlying mechanisms that lead to these differences. Nucleotide sequence variation arising through evolution may differentially affect the expression profiles of divergent species. RNA-Seq technology, combined with techniques to differentiate parental alleles and quantify their abundance, have recently become popular methods for allele specific gene expression (ASGE) analyses. However, analysis of gene expression within interspecies hybrids may be difficult when one of the two parental genomes represented in the hybrid does not have robust genomic resources or available transcriptome data. Herein, we compare two strategies for analyzing allele specific expression within interspecies hybrids produced from crossing two Xiphophorus fish species. The first strategy relies upon a robust reference transcriptome assembly from one species followed by identification of SNPs and creation of an in silico reference transcriptome for the second species. The second strategy employs de novo assembly of reference transcriptomes for both parental species followed by identification of homologous transcripts prior to mapping hybrid reads to a combined hybrid reference. Our results show that, although both methods are able to achieve balanced allelic distribution upon read mapping of F(1) hybrid fish transcriptomes, the second "de novo" assembly approach is superior for ASGE analyses and leads to results more consistent with those found from quantitative real time PCR assessment of gene expression. In addition, our analysis indicates that indels between the two parental alleles are the major cause of the differences in results observed when employing these two methods.

Transcriptomic analysis of cultured whale skin cells exposed to hexavalent chromium [Cr(VI)].

Hexavalent chromium Cr(VI) is known to produce cytotoxic effects in humans and is a highly toxic environmental contaminant. Interestingly, it has been shown that free ranging sperm whales (Phyester macrocephalus) may have exceedingly high levels of Cr in their skin. Also, it has been demonstrated that skin cells from whales appear more resistant to both cytotoxicity and clastogenicity upon Cr exposure compared to human cells. However, the molecular genetic mechanisms employed in whale skin cells that might lead to Cr tolerance are unknown. In an effort to understand the underlying mechanisms of Cr(VI) tolerance and to illuminate global gene expression patterns modulated by Cr, we exposed whale skin cells in culture to varying levels of Cr(VI) (i.e., 0.0, 0.5, 1.0 and 5.0 µg/cm²) followed by short read (100 bp) next generation RNA sequencing (RNA-seq). RNA-seq reads from all exposures (≈280 million reads) were pooled to generate a de novo reference transcriptome assembly. The resulting whale reference assembly had 11K contigs and an N50 of 2954 bp. Using the reads from each dose (0.0, 0.5, 1.0 and 5.0 µg/cm²) we performed RNA-seq based gene expression analysis that identified 35 up-regulated genes and 19 down-regulated genes. The experimental results suggest that low dose exposure to Cr (1.0 µg/cm²) serves to induce up-regulation of oxidative stress response genes, DNA repair genes and cell cycle regulator genes. However, at higher doses (5.0 µg/cm²) the DNA repair genes appeared down-regulated while other genes that were induced suggest the initiation of cytotoxicity. The set of genes identified that show regulatory modulation at different Cr doses provide specific candidates for further studies aimed at determination of how whales exhibit resistance to Cr toxicity and what role(s) reactive oxygen species (ROS) may play in this process.

Characterization of telomeres and telomerase expression in Xiphophorus.

Research investigating telomere lengths and telomerase expression in vertebrates has progressively become important due to the association of these two biological endpoints with cellular aging and cancer in humans. Studies that rely upon the traditional use of laboratory mice have been faced with limitations largely due to inbred mice possessing large telomeres and ubiquitous expression of telomerase. Recently, a number of small fish species have been shown to provide potentially informative models for examining the role of telomeres and telomerase within intact vertebrate animals. Xiphophorus fishes represent a new world live-bearing genus that has not previously been assessed for telomere length or telomerase expression. To add to the knowledge base of telomere and telomerase biology in vertebrates we assessed telomere length and telomerase expression among several species of Xiphophorus. The telomere lengths in several organs (gill, brain, eyes, testis, ovary and liver) in three species (Xiphophorus hellerii, Xiphophorus maculatus, Xiphophorus couchianus) and also in F(1) interspecies hybrids were approximately 2-6 kb. This size was consistent within the same organs of the same species, as well as between species and F(1) hybrids. Despite possessing relatively short telomere lengths compared to humans, the consistency of size among Xiphophorus species and organs may allow experimental detection of telomere shortening. The relative expression of telomerase reverse transcriptase (TERT) was determined by quantitative real-time PCR. Expression levels of TERT was measured in seven organs (ovary, testis, liver, gill, brain, heart, skin) from X. maculatus, X. hellerii and in control and ultraviolet light (UVB) exposed skin samples from X. maculatus, X. hellerii, and F(1) interspecies hybrids. TERT gene expression was significantly higher in ovary and testis, while all other organs showed low relative TERT expression. Detectable increases in TERT expression were found in skin samples upon UVB exposure. Our findings suggest that Xiphophorus may serve as a suitable model for future studies investigating the association of telomere length and telomerase expression in regard to aging and disease.

An age-old paradigm challenged: old baboons generate vigorous humoral immune responses to LcrV, a plague antigen.

Immune senescence in the elderly results in decreased immunity with a concomitant increase in susceptibility to infection and diminished efficacy of vaccination. Nonhuman primate models have proven critical for testing of vaccines and therapeutics in the general population, but a model using old animals has not been established. Toward that end, immunity to LcrV, a protective Ag from Yersinia pestis, was tested in young and old baboons. Surprisingly, there was no age-associated loss in immune competence; LcrV elicited high-titer, protective Ab responses in the older individuals. The primary responses in the younger baboons were lower, but they did show boosting upon secondary immunization to the levels achieved in the old animals. The LcrV Ag was also tested in mice and, as expected, age-associated loss of immunity was seen; older animals responded with lower-titer Abs and, as a result, were more susceptible to Yersinia challenge. Thus, although age-related loss in immune function has been observed in humans, rodents, and some nonhuman primates, baboons appear to be unusual; they age without losing immune competence.