Summary During persistent murine cytomegalovirus (MCMV) infection the T cell response is maintained at extremely high levels for the life of the host. These cells closely resemble human CMV-specific cells which comprise a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation, MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead, we found that most of these cells divide only sporadically in chronically infected hosts and have a short half-life in circulation. The overall population is supported, at least in part, by memory cells primed early in infection as well as recruitment of naïve T cells at late times. These data show that memory inflation is maintained by a continuous replacement of short-lived, functional cells during chronic MCMV infection.

OBJECTIVES Calculation of accurate T1 relaxivity (r1) values for gadolinium-based magnetic resonance contrast agents (GBCAs) is a complex process. As such, often referenced r1 values for the GBCAs at 1.5 T, 3 T, and 7 T are based on measurements obtained in media that are not clinically relevant, derived from only a small number of concentrations, or available for only a limited number of GBCAs. This study derives the r1 values of the 8 commercially available GBCAs in human whole blood at 1.5 T, 3 T, and 7 T. MATERIALS AND METHODS Eight GBCAs were serially diluted in human whole blood, at 7 concentrations from 0.0625 to 4 mM. A custom-built phantom held the dilutions in air-tight cylindrical tubes maintained at 37 ± 0.5°C by a heat-circulating system. Images were acquired using inversion recovery sequences with inversion times from 30 milliseconds to 10 seconds at 1.5 T and 3 T as well as 60 milliseconds to 5 seconds at 7 T. A custom MATLAB program was used to automate signal intensity measurements from the images acquired of the phantom. SigmaPlot was used to calculate T1 relaxation times and, finally, r1. RESULTS Measured r1 values in units of s[BULLET OPERATOR]mM at 1.5 T (3 T/7 T) were 3.9 ± 0.2 (3.4 ± 0.4/2.8 ± 0.4) for Gd-DOTA, 4.6 ± 0.2 (4.5 ± 0.3/4.2 ± 0.3) for Gd-DO3A-butrol, 4.3 ± 0.4 (3.8 ± 0.2/3.1 ± 0.4) for Gd-DTPA, 6.2 ± 0.5 (5.4 ± 0.3/4.7 ± 0.1) for Gd-BOPTA, 4.5 ± 0.1 (3.9 ± 0.2/3.7 ± 0.2) for Gd-DTPA-BMA, 4.4 ± 0.2 (4.2 ± 0.2/4.3 ± 0.2) for Gd-DTPA-BMEA, 7.2 ± 0.2 (5.5 ± 0.3/4.9 ± 0.1) for Gd-EOB-DTPA, and 4.4 ± 0.6 (3.5 ± 0.6/3.4 ± 0.1) for Gd-HP-DO3A. The agents can be stratified by relaxivity, with a significant additional dependency on field strength. CONCLUSIONS This report quantifies, for the first time, T1 relaxivity for all 8 gadolinium chelates in common clinical use worldwide, at current relevant field strengths, in human whole blood at physiological temperature (37°C). The measured r1 values differ to a small degree from previously published values, where such comparisons exist, with the current r1 measurements being that most relevant to clinical practice. The macrocyclic agents, with the exception of Gd-DO3A-butrol, have slightly lower r1 values when compared with the 2 much less stable linear agents, Gd-DTPA-BMA and Gd-DTPA-BMEA. The 2 agents with hepatobiliary excretion, Gd-EOB-DTPA and Gd-BOPTA, have, at 1.5 and 3 T, substantially higher r1 values than all other agents.

Cytomegalovirus (CMV) is a β-herpesvirus that establishes a lifelong latent or persistent infection. A hallmark of chronic CMV infection is the lifelong persistence of large numbers of virus-specific CD8+ effector/effector memory T cells, a phenomenon called “memory inflation”. How the virus continuously stimulates these T cells without being eradicated remains an enigma. The prevailing view is that CMV establishes a low grade “smoldering” infection characterized by tiny bursts of productive infection which are rapidly extinguished, leaving no detectable virus but replenishing the latent pool and leaving the immune system in a highly charged state. However, since abortive reactivation with limited viral gene expression is known to occur commonly, we investigated the necessity for virus reproduction in maintaining the inflationary T cell pool. We inhibited viral replication or spread in vivo using two different mutants of murine CMV (MCMV). First, famcyclovir blocked the replication of MCMV encoding the HSV Thymidine Kinase gene, but had no impact on the CD8+ T cell memory inflation once the infection was established. Second, MCMV that lacks the essential glycoprotein L, and thus is completely unable to spread from cell to cell, also drove memory inflation if the virus was administered systemically. Our data suggest that CMV which cannot spread from the cells it initially infects can repeatedly generate viral antigens to drive memory inflation without suffering eradication of the latent genome pool.

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This paper describes the discovery of synthetic 1,2,4-trioxolane antimalarials and how we established a workable structure-activity relationship in the context of physicochemical, biopharmaceutical, and toxicological profiling. An achiral dispiro-1,2,4-trioxolane (3) in which the trioxolane is flanked by a spiroadamantane and spirocyclohexane was rapidly identified as a lead compound. Nonperoxidic 1,3-dioxolane isosteres of 3 were inactive as were trioxolanes without the spiroadamantane. The trioxolanes were substantially less effective in a standard oral suspension formulation compared to a solubilizing formulation and were more active when administered subcutaneously than orally, both of which suggest substantial biopharmaceutical liabilities. Nonetheless, despite their limited oral bioavailability, the more lipophilic trioxolanes generally had better oral activity than their more polar counterparts. In pharmacokinetic experiments, four trioxolanes had high plasma clearance values, suggesting a potential metabolic instability. The toxicological profiles of two trioxolanes were comparable to that of artesunate.

Several low-grade persistent viral infections induce and sustain very large numbers of virus-specific effector T cells. This was first described as a response to cytomegalovirus (CMV), a herpesvirus that establishes a life-long persistent/latent infection, and sustains the largest known effector T cell populations in healthy people. These T cells remain functional and traffic systemically, which has led to the recent exploration of CMV as a persistent vaccine vector. However, the maintenance of this remarkable response is not understood. Current models propose that reservoirs of viral antigen and/or latently infected cells in lymph nodes stimulate T cell proliferation and effector differentiation, followed by migration of progeny to non-lymphoid tissues where they control CMV reactivation. We tested this model using murine CMV (MCMV), a natural mouse pathogen and homologue of human CMV (HCMV). While T cells within draining lymph nodes divided at a higher rate than cells elsewhere, antigen-dependent proliferation of MCMV-specific effector T cells was observed systemically. Strikingly, inhibition of T cell egress from lymph nodes failed to eliminate systemic T cell division, and did not prevent the maintenance of the inflationary populations. In fact, we found that the vast majority of inflationary cells, including most cells undergoing antigen-driven division, had not migrated into the parenchyma of non-lymphoid tissues but were instead exposed to the blood supply. Indeed, the immunodominance and effector phenotype of inflationary cells, both of which are primary hallmarks of memory inflation, were largely confined to blood-localized T cells. Together these results support a new model of MCMV-driven memory inflation in which most immune surveillance occurs in circulation, and in which most inflationary effector T cells are produced in response to viral antigen presented by cells that are accessible to the blood supply.

Summary Cytomegalovirus (CMV) is a herpesvirus that persists for life and maintains extremely large numbers of T cells with select specificities in circulation. However, it is unknown how viral persistence impacts T cell populations in mucosal sites. We found that many murine (M)CMV-specific CD8s in mucosal tissues became resident memory T cells (TRM). These cells adopted an intraepithelial localization in the salivary gland that correlated with, but did not depend on, expression of the integrin CD103. MCMV-specific TRM cells formed early after infection and spleen-localized cells had reduced capacities to become TRM at late times. Surprisingly however, small numbers of new TRM cells were formed from the circulating pool throughout infection, favoring populations maintained at high levels in the blood and shifting the immunodominance within the TRM populations over time. These data show that mucosal TRM populations can be dynamically maintained by a persistent infection.