On the role of antigen in maintaining cytotoxic T-cell memory.

This study evaluated whether T-cell memory reflects increased precursor frequencies of specific long-lived T cells and/or a low-level immune response against some form of persistent antigen. Antivirally protective CD8+ T-cell memory was analyzed mostly in the original vaccinated host to assess the role of antigen in its maintenance. T-cell mediated resistance against reinfection was measured in the spleen and in peripheral solid organs with protocols that excluded protection by antibodies. In vivo protection was compared with detectable cytotoxic T-lymphocyte precursor frequencies determined in vitro. In the spleen, in vitro detectable cytotoxic T-lymphocyte precursor frequencies remained stable independently of antigen, conferring resistance against viral replication in the spleen during reinfection. In contrast, T-cell mediated resistance against reinfection of peripheral solid organs faded away in an antigen-dependent fashion within a few days or weeks. We show that only memory T cells persistently or freshly activated with antigen efficiently extravasate into peripheral organs, where cytotoxic T lymphocytes must be able to exert effector function immediately; both the capacity to extravasate and to rapidly exert effector function critically depend on restimulation by antigen. Our experiments document that the duration of T-cell memory protective against peripheral reinfection depended on the antigen dose used for immunization, was prolonged when additional antigen was provided, and was abrogated after removal of antigen. We conclude that T-cell mediated protective immunity against the usual peripheral routes of reinfection is antigen-dependent.

Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM.

The nonclassical major histocompatibility complex class II molecule HLA-DM (DM) has recently been shown to play a central role in the class II-associated antigen presentation pathway: DM releases invariant chain-derived CLIP peptides (class II-associated invariant chain protein peptide) from HLA-DR (DR) molecules and thereby facilitates loading with antigenic peptides. Some observations have led to the suggestion that DM acts in a catalytic manner, but so far direct proof is missing. Here, we investigated in vitro the kinetics of exchange of endogenously bound CLIP for various peptides on DR1 and DR2a molecules: we found that in the presence of DM the peptide loading process follows Michaelis-Menten kinetics with turnover numbers of 3-12 DR molecules per minute per DM molecule, and with KM values of 500-1000 nM. In addition, surface plasmon resonance measurements showed that DM interacts efficiently with DR-CLIP complexes but only weakly with DR-peptide complexes isolated from DM-positive cells. Taken together, our data provide evidence that DM functions as an enzyme-like catalyst of peptide exchange and favors the generation of long-lived DR-peptide complexes that are no longer substrates for DM.

Gadolinium(III) texaphyrin: a tumor selective radiation sensitizer that is detectable by MRI.

Gadolinium(III) texaphyrin (Gd-tex2+) is representative of a new class of radiation sensitizers detectable by magnetic resonance imaging (MRI). This porphyrin-like complex has a high electron affinity [E1/2 (red.) approximately = -0.08 V versus normal hydrogen electrode] and forms a long-lived pi-radical cation upon exposure to hydrated electrons, reducing ketyl radicals, or superoxide ions. Consistent with these chemical findings, Gd-tex2+ was found to be an efficient radiation sensitizer in studies carried out with HT29 cells in in vitro as well as in in vivo single and multifraction irradiation studies with a murine mammary carcinoma model. Selective localization of Gd-tex2+ in tumors was confirmed by MRI scanning.

An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death.

"Addiction modules" consist of two genes. In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect. Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module. Furthermore, the expression of mazEF is regulated by the cellular level of guanosine [corrected] 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation. These properties suggest that the mazEF system may be responsible for programmed cell death in E. coli and thus may have a role in the physiology of starvation.

Longevity and the genetic determination of collagen glycoxidation kinetics in mammalian senescence.

A fundamental question in the basic biology of aging is whether there is a universal aging process. If indeed such a process exists, one would expect that it develops at a higher rate in short- versus long-lived species. We have quantitated pentosidine, a marker of glycoxidative stress in skin collagen from eight mammalian species as a function of age. A curvilinear increase was modeled for all species, and the rate of increase correlated inversely with maximum life-span. Dietary restriction, a potent intervention associated with increased life-span, markedly inhibited glycoxidation rate in the rodent. On the assumption that collagen turnover rate is primarily influenced by the crosslinking due to glycoxidation, these results suggest that there is a progressive age-related deterioration of the process that controls the collagen glycoxidation rate. Thus, the ability to withstand damage due to glycoxidation and the Maillard reaction may be under genetic control.

Structural features of the invariant chain fragment CLIP controlling rapid release from HLA-DR molecules and inhibition of peptide binding.

The invariant chain (Ii) prevents binding of ligands to major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum and during intracellular transport. Stepwise removal of the Ii in a trans-Golgi compartment renders MHC class II molecules accessible for peptide loading, with CLIP (class II-associated Ii peptides) as the final fragment to be released. Here we show that CLIP can be subdivided into distinct functional regions. The C-terminal segment (residues 92-105) of the CLIP-(81-105) fragment mediates inhibition of self- and antigenic peptide binding to HLA-DR2 molecules. In contrast, the N-terminal segment CLIP-(81-98) binds to the Staphylococcus aureus enterotoxin B contact site outside the peptide-binding groove on the alpha 1 domain and does not interfere with peptide binding. Its functional significance appears to lie in the contribution to CLIP removal: the dissociation of CLIP-(81-105) is characterized by a fast off-rate, which is accelerated at endosomal pH, whereas in the absence of the N-terminal CLIP-(81-91), the off-rate of C-terminal CLIP-(92-105) is slow and remains unaltered at low pH. Mechanistically, the N-terminal segment of CLIP seems to prevent tight interactions of CLIP side chains with specificity pockets in the peptide-binding groove that normally occurs during maturation of long-lived class II-peptide complexes.

Thermotolerance and extended life-span conferred by single-gene mutations and induced by thermal stress.

We have discovered that three longevity mutants of the nematode Caenorhabditis elegans also exhibit increased intrinsic thermotolerance (Itt) as young adults. Mutation of the age-1 gene causes not only 65% longer life expectancy but also Itt. The Itt phenotype cosegregates with age-1. Long-lived spe-26 and daf-2 mutants also exhibit Itt. We investigated the relationship between increased thermotolerance and increased life-span by developing conditions for environmental induction of thermotolerance. Such pretreatments at sublethal temperatures induce significant increases in thermotolerance and small but statistically highly significant increases in life expectancy, consistent with a causal connection between these two traits. Thus, when an animal's resistance to stress is increased, by either genetic or environmental manipulation, we also observe an increase in life expectancy. These results suggest that ability to respond to stress limits the life expectancy of C. elegans and might do so in other metazoa as well.

Codominance and toxins: a path to drugs of nearly unlimited selectivity.

The effectiveness of drugs is often limited by their insufficient selectivity. I propose designs of therapeutic agents that address this problem. The key feature of these reagents, termed comtoxins (codominance-mediated toxins), is their ability to utilize codominance, a property characteristic of many signals in proteins, including degradation signals (degrons) and nuclear localization signals. A comtoxin designed to kill cells that express intracellular proteins P1 and P2 but to spare cells that lack P1 and/or P2 is a multidomain fusion containing a cytotoxic domain and two degrons placed within or near two domains P1* and P2* that bind, respectively, to P1 and P2. In a cell containing both P1 and P2, these proteins would bind to the P1* and P2* domains of the comtoxin and sterically mask the nearby (appropriately positioned) degrons, resulting in a long-lived and therefore toxic drug. By contrast, in a cell lacking P1 and/or P2, at least one of the comtoxin's degrons would be active (unobstructed), yielding a short-lived and therefore nontoxic drug. A comtoxin containing both a degron and a nuclear localization signal can be designed to kill exclusively cells that contain P1 but lack P2. Analogous strategies yield comtoxins sensitive to the presence (or absence) of more than two proteins in a cell. Also considered is a class of comtoxins in which a toxic domain is split by a flexible insert containing binding sites for the target proteins. The potentially unlimited, combinatorial selectivity of comtoxins may help solve the problem of side effects that bedevils present-day therapies, for even nonselective delivery of a comtoxin would not affect cells whose protein "signatures" differ from the targeted one.

Very-long-baseline radio interferometry surveys of the compact structure in active galactic nuclei.

Very-long-baseline radio interferometry (VLBI) imaging surveys have been undertaken since the late 1970s. The sample sizes were initially limited to a few tens of objects but the snapshot technique has now allowed samples containing almost 200 sources to be studied. The overwhelming majority of powerful compact sources are asymmetric corejects of one form or another, most of which exhibit apparent superluminal motion. However 5-10% of powerful flat-spectrum sources are 100-parsec (pc)-scale compact symmetric objects; these appear to form a continuum with the 1-kpc-scale double-lobed compact steep-spectrum sources, which make up 15-20% of lower frequency samples. It is likely that these sub-galactic-size symmetric sources are the precursors to the large-scale classical double sources. There is a surprising peak around 90 degrees in the histogram of misalignments between the dominant source axes on parsec and kiloparsec scales; this seems to be associated with sources exhibiting a high degree of relativistic beaming. VLBI snapshot surveys have great cosmological potential via measurements of both proper motion and angular size vs. redshift as well as searches for gravitational "millilensing."