Early Priming Minimizes the Age-Related Immune Compromise of CD8+ T Cell Diversity and Function

The elderly are particularly susceptible to influenza A virus infections, with increased occurrence, disease severity and reduced vaccine efficacy attributed to declining immunity. Experimentally, the age-dependent decline in influenza-specific CD8⁺ T cell responsiveness reflects both functional compromise and the emergence of ‘repertoire holes’ arising from the loss of low frequency clonotypes. In this study, we asked whether early priming limits the time-related attrition of immune competence. Though primary responses in aged mice were compromised, animals vaccinated at 6 weeks then challenged >20 months later had T-cell responses that were normal in magnitude. Both functional quality and the persistence of ‘preferred’ TCR clonotypes that expand in a characteristic immunodominance hierarchy were maintained following early priming. Similar to the early priming, vaccination at 22 months followed by challenge retained a response magnitude equivalent to young mice. However, late priming resulted in reduced TCRβ diversity in comparison with vaccination earlier in life. Thus, early priming was critical to maintaining individual and population-wide TCRβ diversity. In summary, early exposure leads to the long-term maintenance of memory T cells and thus preserves optimal, influenza-specific CD8⁺ T-cell responsiveness and protects against the age-related attrition of naïve T-cell precursors. Our study supports development of vaccines that prime CD8⁺ T-cells early in life to elicit the broadest possible spectrum of CD8⁺ T-cell memory and preserve the magnitude, functionality and TCR usage of responding populations. In addition, our study provides the most comprehensive analysis of the aged (primary, secondary primed-early and secondary primed-late) TCR repertoires published to date.     

Colonic gene expression profile in NHE3-deficient mice: evidence for spontaneous distal colitis

Na+/H+ exchanger 3 (NHE3) provides a major route for intestinal Na+ absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3(-/-) mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2'-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer276-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF-alpha (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3(-/-) mice. NHE3(-/-) mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.     

An assay combining high-performance liquid chromatography and mass spectrometry to measure DNA interstrand cross-linking efficiency in oligonucleotides of varying sequences

The main method of evaluating the DNA interstrand cross-linking ability of cancer chemotherapeutic agents in naked DNA currently involves the electrophoresis of relatively long radiolabeled duplex DNA fragments (typically approximately 2000 bp) on neutral gels after incubation with the agent of interest. Denaturation by heating is carried out prior to loading, and a neutral gel allows reannealing of cross-linked DNA. To avoid the use of radioactivity we have developed a new method based on ion pair reversed phase liquid chromatography (RPLC) and mass spectrometry (MS) that allows characterization and quantitation of drug-DNA interstrand cross-links formed within short oligonucleotide duplexes (i.e., 12 bp). Advantages of this assay include rapid throughput, as compared to electrophoretic methods, and the use of readily available short nonradiolabeled oligonucleotides of any sequence, thereby facilitating investigation of sequence selectivity. A further advantage is that all species separated by the chromatographic process can be positively identified by MS. Using this new method, we have investigated the rate of DNA cross-linking and sequence selectivity of the interstrand cross-linking agent SJG-136, a pyrrolobenzodiazepine (PBD) dimer currently in phase I clinical trials. The assay was found to be sufficiently sensitive and selective to allow separation of the unbound and drug-bound oligonucleotide species by high-performance liquid chromatography (HPLC) and to allow positive identification of these individual species by MS. A further benefit, as compared with electrophoretic methods, is that kinetic information can be obtained and compared for different binding sequences.     

The cel4 Gene of Agaricus bisporus Encodes a β-Mannanase

Mannases have industrial uses in food and pulp industries, and their regulation may influence development of the mushrooms of commercially important basidiomycetes. We expressed an Agaricus bisporus cel4 cDNA, which encodes a mannanase, in Saccharomyces cerevisiae and Pichia pastoris. CEL4 had no detectable activity on cellulose or xylan. This gene is the first isolated from this economically important fungus to encode a mannanase. P. pastoris secreted about three times more CEL4 than S. cerevisiae. The removal of the cellulose-binding domain of CEL4 lowered the secreted specific activity by P. pastoris by approximately 97%. The genomic sequence of cel4 was isolated by screening a cosmid library of A. bisporus C54-carb8. The open reading frame was interrupted by 12 introns. The level of extracellular CEL4 increases dramatically at the postharvest stage in compost extracts of A. bisporus fruiting cultures. In laboratory liquid cultures of A. bisporus, the activity of CEL4 detected in the culture filtrate reached a maximum after 21 days. The levels of CEL4 broadly mirrored the levels of enzyme activity. In the Solka floc-bound mycelium, CEL4 protein showed a maximum after 2 to 3 weeks of culture and then declined. Changes in CEL4 activity during fruiting-body development suggest that hemicellulose utilization plays an important role in sporophore formation. The availability of the cloned gene will further studies of compost decomposition and the extracellular enzymes that fungi deploy in this process.     

Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH₂ groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks.