An Aging Interventions Testing Program: study design and interim report

The National Institute on Aging's Interventions Testing Program (ITP) has developed a plan to evaluate agents that are considered plausible candidates for delaying rates of aging. Key features include: (i) use of genetically heterogeneous mice (a standardized four-way cross), (ii) replication at three test sites (the Jackson Laboratory, TJL; University of Michigan, UM; and University of Texas, UT), (iii) sufficient statistical power to detect 10% changes in lifespan, (iv) tests for age-dependent changes in T cell subsets and physical activity, and (v) an annual solicitation for collaborators who wish to suggest new interventions for evaluation. Mice in the first cohort were exposed to one of four agents: aspirin, nitroflurbiprofen (NFP), 4-OH-alpha-phenyl-N-tert-butyl nitrone (4-OH-PBN), or nordihydroguiaretic acid (NDGA). An interim analysis was conducted using survival data available on the date at which at least 50% of the male control mice had died at each test site. Survival of control males was significantly higher, at the interim time-point, at UM than at UT or TJL; all three sites had similar survival of control females. Males in the NDGA group had significantly improved survival (P = 0.0004), with significant effects noted at TJL (P < 0.01) and UT (P < 0.04). None of the other agents altered survival, although there was a suggestion (P = 0.07) of a beneficial effect of aspirin in males. More data will be needed to determine if any of these compounds can extend maximal lifespan, but the current data show that NDGA reduces early life mortality risks in genetically heterogeneous mice at multiple test sites.     

Effective post-exposure treatment of Ebola infection

Ebola viruses are highly lethal human pathogens that have received considerable attention in recent years due to an increasing re-emergence in Central Africa and a potential for use as a biological weapon. There is no vaccine or treatment licensed for human use. In the past, however, important advances have been made in developing preventive vaccines that are protective in animal models. In this regard, we showed that a single injection of a live-attenuated recombinant vesicular stomatitis virus vector expressing the Ebola virus glycoprotein completely protected rodents and nonhuman primates from lethal Ebola challenge. In contrast, progress in developing therapeutic interventions against Ebola virus infections has been much slower and there is clearly an urgent need to develop effective post-exposure strategies to respond to future outbreaks and acts of bioterrorism, as well as to treat laboratory exposures. Here we tested the efficacy of the vesicular stomatitis virus-based Ebola vaccine vector in post-exposure treatment in three relevant animal models. In the guinea pig and mouse models it was possible to protect 50% and 100% of the animals, respectively, following treatment as late as 24 h after lethal challenge. More important, four out of eight rhesus macaques were protected if treated 20 to 30 min following an otherwise uniformly lethal infection. Currently, this approach provides the most effective post-exposure treatment strategy for Ebola infections and is particularly suited for use in accidentally exposed individuals and in the control of secondary transmission during naturally occurring outbreaks or deliberate release.     

Role of phosphodiesterase type 3A and 3B in regulating platelet and cardiac function using subtype-selective knockout mice

Phosphodiesterase type 3 (PDE3) is an important regulator of cAMP-mediated responses within the cardiovascular system. PDE3 exists as two subtypes: PDE3A and PDE3B, with distinct cellular and subcellular locations. Due to the lack of subtype-specific pharmacological tools, the definitive role of each subtype in regulating cardiovascular function has not been determined. In this study, we investigated platelet and cardiac function, using PDE3A and PDE3B gene knockout (KO) mice. Platelet-rich-plasma was prepared from the blood of KO and age-matched wild-type (WT) mice. PGE1 (1 microg/mL) almost completely inhibited aggregation of platelets from WT, PDE3A KO and PDE3B KO mice. In platelets from WT mice, cilostamide (100 microM), a selective PDE3 inhibitor, blocked collagen- and ADP-induced aggregation. In contrast, cilostamide had no effect on aggregation of platelets from PDE3A KO mice. In PDE3B KO mice, inhibition of collagen- and ADP-induced platelet aggregation was similar to that in WT mice. The resting intra-platelet cAMP concentration in platelets from PDE3A KO mice was twice that in the WT platelets. After PGE1 (0.1 microg/mL) stimulation, intra-cellular cAMP concentration was increased significantly more in platelets from PDE3A KO mice compared to WT mice. In vivo, PDE3A KO mice were protected against collagen/epinephrine-induced pulmonary thrombosis and death, while no such protection was observed in PDE3B KO mice. The heart rate of PDE3A KO mice was significantly higher, compared with age-matched WT mice, while that of PDE3B KO mice was similar to WT. There was no difference in cardiac contractility between PDE3A or PDE3B KO mice. Heart rate and contractility were increased in a similar dose-dependent fashion by isoproterenol in both types of KO mice. Cilostamide increased heart rate and contractility in WT and PDE3B KO but not in PDE3A KO mice. Compared to WT and PDE3B KO mice, cyclic AMP-PDE activity in membrane fractions prepared from the hearts of PDE3A KO mice was lower and not inhibited by cilostamide. The data suggest that PDE3A is the main subtype of PDE3 expressed in platelets and cardiac ventricular myocytes, and is responsible for the functional changes caused by PDE3 inhibition.     

Measurement of IgM responses to a subunit influenza A vaccine by sucrose-gradient centrifugation and membrane-filtration enzyme immunoassays

IgM responses to a deoxycholate-split influenza vaccine containing the surface antigens of the H3N2 virus A/Philippines/2/82 were studied in five volunteers, three of whom were seronegative by haemagglutination inhibition (HI) tests. Responses were measured by a sucrose-gradient centrifugation technique, in which IgM-specific HI activity was computed as a proportion of total IgM and IgG-specific HI activity, and by a membrane filtration-enzyme immunoassay (MF-EIA). Responses could be detected in all volunteers when measured by sucrose-gradient centrifugation within 1-2 weeks, and the IgM induced was 5-40% of total HI-specific activity. The response was also observed in the presence of low levels of pre-existing antibody. Levels of IgM antibody, when measured by MF-EIA, could be easily detected in two of the volunteers, while those of two others were very low and there was no response in a fifth. No biphasic virus-specific response to vaccination, involving first IgM and then IgG, could be measured by either technique. From these studies, the sucrose-gradient fractionation technique appears to be the more sensitive procedure.     

Murine Norovirus Infection Has No Significant Effect on Adaptive Immunity to Vaccinia Virus or Influenza A Virus

Murine norovirus (MNV) is endemic in many research mouse colonies. Although MNV infections are typically asymptomatic in immunocompetent mice, the effects of MNV infection on subsequent experimental viral infections are poorly documented. Here, we infected C57BL/6 mice with MNV and then with either vaccinia virus or influenza A virus. MNV infection had no effect on CD8⁺ T-cell or antibody responses to secondary viruses or to secondary virus-induced morbidity or mortality. While our findings suggest that MNV has little influence on host immunity in immunocompetent mice, we would urge caution regarding the potential effects of MNV on immune responses to viruses and other pathogens, which must be determined on a system-by-system basis.Human norovirus (NoV) infections cause greater than 90% of nonbacterial gastroenteritis cases (4, 5) and are an important public health concern. Murine noroviruses (MNV) were recently identified (7) as highly pathogenic agents in immunocompromised mice, and serological studies indicate that over 20% of mice in research colonies are exposed to MNV (6). As with NoV, MNV is spread through the fecal-oral route. While NoV rapidly causes gastrointestinal symptoms and fever in healthy individuals, MNV is typically asymptomatic in immunocompetent mice.MNV isolates are both genetically and biologically diverse (13). In wild-type (wt) mice, some strains of MNV are rapidly cleared, while others persist (13). Controlling MNV infections requires elements of both innate and adaptive immunity. Mice with defects in interferon (IFN) signaling pathways demonstrate increased MNV lethality (7, 9). CD4⁺ and CD8⁺ T cells and B cells are all needed for complete MNV clearance (1, 2). Natural exposure of immunocompromised mice to MNV leads to inflammation of the liver, lungs, and peritoneal and pleural cavities (14).It is well established that infection with natural mouse viruses can greatly impact immune responses to infections with other viruses. The prevalence of MNV in research mouse colonies might therefore lead to irreproducible and variable results that significantly impact research efforts. Indeed, MNV was recently reported to alter disease progression in a mouse model of bacterium-induced inflammatory bowel disease (8). Concern over the potential effects of MNV on viral immunology research prompted a dedicated workshop at the 2008 Keystone Viral Immunity meeting (http://www.keystonesymposia.org). In the present study, we examined the effect of MNV infection on adaptive immune responses in wt mice to influenza A virus (IAV) and vaccinia virus (VV).We infected C57BL/6 mice perorally with a high dose (3 × 10⁷ PFU/mouse) of a plaque-purified MNV stock derived from MNV-CR6p2 (13). The capacity of this plaque-purified virus to persist in wt mice has been confirmed by quantitative PCR analysis and a plaque assay (D. Strong, L. Thackray, and H. Virgin, unpublished observation). We confirmed that the mice were infected by measuring anti-MNV antibodies (Abs) by using an enzyme-linked immunosorbent assay (ELISA) (data not shown). For all experiments, mice were infected with MNV at Washington University and shipped 4 to 5 days later to NIAID for further study. To contain MNV, infected mice were housed in microisolator cages in a quarantine room. In some experiments, control mice were housed in the same room as MNV-infected mice. Sera collected from control mice did not contain anti-MNV Abs as determined by ELISA (data not shown), confirming that transmission of MNV between mice housed in microisolator cages can be prevented by proper cage changing and aseptic handling of samples from infected mice.Upon intraperitoneal (i.p.) infection with either VV or IAV, mice mount robust CD8⁺ T-cell responses that peak, respectively, on day 6 or 7. Anti-VV and anti-IAV CD8⁺ T-cell responses in C57BL/6 mice conform to a well-established immunodominance hierarchy (3, 10). To determine to what extent MNV infection alters the magnitude and/or immunodominance hierarchy of CD8⁺ T-cell responses, we infected C57BL/6 mice i.p. with either VV or IAV 19 days following MNV infection. As controls, naïve mice (MNV negative) were infected with either virus. Lymphocytes were isolated from mice 6 days postinfection with VV and 7 days postinfection with IAV. The fraction of antigen-specific CD8⁺ T cells present in spleen and peritoneal exudate cells (PEC) was determined by intracellular IFN-γ staining after stimulation with synthetic peptides. MNV infection had little effect on the magnitude of splenic or PEC CD8⁺ T cells responding to VV (Fig. 1A and B) or IAV (Fig. 1C and D) infection. Regardless of MNV exposure history, splenic and PEC responses were dominated by B8R- and A8R-specific CD8⁺ T cells following VV infection (Fig. 1A and B) and by PA-specific and NP-specific CD8⁺ T cells following IAV infection (Fig. 1C and D).Open in a separate windowFIG. 1.MNV exposure does not alter CD8⁺ T-cell responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.p. with ∼1 × 10⁶ PFU of VV (A and B) or ∼1 × 10⁷ 50% tissue culture infective dose units of IAV (C and D), and specific CD8⁺ T cells were determined by intracellular IFN-γ staining after restimulating lymphocytes with peptides. Lymphocytes isolated from the spleen (A and C) and peritoneal cavity (B and D) were tested. MNV infections were completed 19 days prior to VV or IAV infections. Means and SEM are shown in panels A and C. A two-way analysis of variance and Bonferroni statistical analysis were completed for these experiments. Cells were pooled for peritoneal lavage samples as shown in panels B and D. Four to five mice/group were used for each experiment; data are representative of two independent experiments.To examine the effect of MNV infection on antiviral Ab responses, MNV-infected and control C57BL/6 mice were infected intranasally (i.n.) with a sublethal dose of either VV or IAV. Three weeks later, levels of anti-VV and anti-IAV Abs were determined by ELISA and hemagglutination inhibition assays, respectively. MNV infection did not significantly modify the magnitude of Ab responses to VV (Fig. ​(Fig.2A)2A) or IAV (Fig. ​(Fig.2B).2B). Next, we determined the effect of MNV infection on heavy chain class switching of anti-VV or anti-IAV Ab responses. Anti-VV and anti-IAV Ab responses exhibited similar heavy chain profiles dominated by immunoglobulin G2b (IgG2b) Abs regardless of MNV status (Fig. 2C and D). Thus, the CD8⁺ T-cell and Ab response to both VV and IAV appears to be essentially unaffected by chronic MNV infection. Since IgG anti-VV or anti-IAV Ab responses are entirely dependent on CD4⁺ T-cell help (11, 12), we can also infer that MNV also does not significantly affect CD4⁺ T-cell responses to VV or IAV.Open in a separate windowFIG. 2.MNV exposure does not alter Ab responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with ∼1 × 10³ PFU of VV (A and C) or ∼50 50% tissue culture infective dose units of IAV (B and D), and virus-specific Abs were determined by ELISA (A, C, and D) or hemagglutination inhibition (B). The ELISA results shown in panel A measured the total IgG, while the ELISA results shown in panels C and D measured the individual isotype indicated. MNV infections were completed 19 days prior to VV or IAV infections. Means and standard errors of the means are shown in panels A, C, and D. Means are shown as lines in panel B. A two-way analysis of variance and Bonferroni statistical analysis were completed for experiments shown in panels A, C, and D, and t tests were completed for the experiment shown in panel B. Four to five mice/group were used for each experiment. O.D., optical density; HAI, hemagglutination inhibition.T-cell and Ab responses, together with innate immune mechanisms, collaborate to control viral replication and limit pathogenesis. To examine the effect of chronic MNV infection on VV-induced or IAV-induced pathogenesis, we infected C57BL/6 mice i.n. with a lethal or sublethal dose of VV or IAV and monitored body weight over a 16-day period. MNV-CR6p2 infection had no significant effect on morbidity or mortality from either virus (Fig. ​(Fig.33 and ​and4).4). Since MNV isolates are highly diverse, we decided to examine the effects of a second strain of MNV (MNV-CW3) which is fully cleared in immunocompetent mice. Mice that cleared MNV-CW3 (19 days post-MNV infection) were infected i.n. with VV or IAV. Once again, this strain of MNV had no effect on VV-induced or IAV-induced morbidity or mortality (Fig. ​(Fig.33 and ​and4).4). Future studies should address the extent to which other MNV strains affect the generation of adaptive immune responses to secondary viral infections.Open in a separate windowFIG. 3.MNV does not increase morbidity following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with a sublethal dose of VV (∼1 × 10³ PFU) (A) or IAV (∼50 50% tissue culture infective dose units) (B), and weight loss was recorded for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. A two-way analysis of variance and Bonferroni statistical analysis were completed. Four to five mice/group were used for each experiment.Open in a separate windowFIG. 4.MNV does not increase mortality following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with VV (∼1 × 10⁴ PFU) (A) or IAV (∼500 50% tissue culture infective dose units) (B), and survival was monitored for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. Eight to 10 mice/group were used for each experiment.Taken together, these data demonstrate that MNV infection has no significant effects on the measured immune response to VV or IAV. Our results cannot, however, be simply extrapolated to other viruses or microorganisms. Rather, the effect of MNV infection on host immunity in mouse model disease systems needs to be established on a system-by-system basis. Without this knowledge, the possible confounding effects of MNV infection will continue to undermine the confidence in results obtained using mice in colonies in which MNV infections are endemic.     

Effect of Preexisting Immunity on an Adenovirus Vaccine Vector: In Vitro Neutralization Assays Fail To Predict Inhibition by Antiviral Antibody In Vivo

A major obstacle to the use of adenovirus vectors derived from common human serotypes, such as human adenovirus 5 (AdHu5), is the high prevalence of virus-neutralizing antibodies in the human population. We previously constructed a variant of chimpanzee adenovirus 68 (AdC68) that maintained the fundamental properties of the carrier but was serologically distinct from AdC68 and resisted neutralization by AdC68 antibodies. In the present study, we tested whether this modified vector, termed AdCDQ, could induce transgene product-specific CD8⁺ T cells in mice with preexisting neutralizing antibody to wild-type AdC68. Contrary to our expectation, the data show conclusively that antibodies that fail to neutralize the AdCDQ mutant vector in vitro nevertheless impair the vector''s capacity to transduce cells and to stimulate a transgene product-specific CD8⁺ T-cell response in vivo. The results thus suggest that in vitro neutralization assays may not reliably predict the effects of virus-specific antibodies on adenovirus vectors in vivo.Adenovirus (Ad) vectors are effective at inducing potent CD8⁺ T-cell responses to immunogens. In animal models, Ad vectors encoding antigens of simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV), used in combination with plasmid-based DNA vectors, generate CD8⁺ T-cell responses that attenuate infection by SIV (9) and by HIV-SIV chimeras (16). In humans, Ad vectors derived from human serotype 5 (AdHu5) are immunogenic and are well tolerated at immunogenic doses; however, in a recent clinical trial, an AdHu5-based HIV-1 vaccine failed to prevent (and may have facilitated) infection (1a). It is not clear whether CD8⁺ T-cell responses will be sufficient to prevent or control HIV infection and disease. However, it seems likely that the induction of effective immune responses against HIV will require multiple doses of antigen, with a priming dose followed by one or more booster immunizations. Prime-boost regimens based on the sequential use of DNA and AdHu5 vectors are being tested clinically, and regimens involving the sequential administration of serologically distinct Ad vectors are being explored in preclinical animal models (1, 5, 8, 9).One major obstacle to the use of vectors derived from AdHu5 and other common human serotypes is the high prevalence of virus-neutralizing antibodies (VNAs) in humans. Preexisting VNAs to the vaccine carrier prevent the vector from transducing target cells, which reduces the amount of vaccine antigen that can be produced and dampens the resultant adaptive immune responses (2, 3, 12). Approximately 40 to 45% of the U.S. population has VNAs to AdHu5, and seroprevalence rates are even higher in Asia and Africa (6, 24).We developed vectors derived from chimpanzee Ads to which humans lack preexisting immunity. When tested in a rodent model, one such vector, AdC68, induces potent transgene product-specific CD8⁺ T-cell responses that can be increased by booster immunizations with serologically distinct Ad vectors (3, 19, 23). However, because the use of multiple serotypes in a prime-boost regimen may prove cumbersome in clinical applications, we have attempted to modify the major neutralizing binding sites within the AdC68 capsid. It has been suggested that the binding sites for Ad-neutralizing antibodies preside primarily within the major capsid protein hexon (4, 10, 14, 15, 17). We defined a single hexon surface loop as the major neutralization site on AdC68 and showed that a mutant vector, AdCDQ, which incorporates a 3-amino-acid mutation within this loop, resists in vitro neutralization by polyclonal antisera obtained from animals immunized against AdC68 (10). Because it is serologically distinct from its parent vector, we expected that AdCDQ could be used in combination with AdC68 in an effective prime-boost regimen.In the present study, we tested whether the AdCDQ vector induces a transgene product-specific CD8⁺ T-cell response in mice with preexisting neutralizing antibody to wild-type AdC68. Contrary to our expectation, the data show conclusively that antibodies that fail to neutralize the AdCDQ vector in vitro nevertheless impair the vector''s capacity to transduce cells and to stimulate a transgene product-specific CD8⁺ T-cell response in vivo. The results thus suggest that in vitro neutralization assays may not reliably predict the effects of virus-specific antibodies on Ad vectors in vivo.     

Adenosine 5'-diphosphate-dependent subunit dissociation of bovine dopamine beta-hydroxylase

In a previous study, it was shown that purified soluble bovine dopamine beta-hydroxylase exhibits pH-dependent reversible tetramer-dimer dissociation (Saxena, A., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1985) J. Biol. Chem. 260, 3386-3392). Here we report evidence for the dissociation of this enzyme by magnesium-adenosine diphosphate independent of pH in the pH range 5-7. Quantitative binding of ADP to dopamine beta-hydroxylase revealed that there are two binding sites/dimeric species of hydroxylase and that ADP is tightly bound with a KD less than 10(-8) M. Kinetic data obtained at pH 5.5, the pH inside the chromaffin granule, shows that the apparent Km values for both the substrates tyramine and ascorbate are lowered by the presence of ADP without affecting the Vmax of the enzyme. The ADP-dependent lowering of apparent Km values results from a dissociation of the enzyme to the dimeric species which has inherently lower apparent Km values for substrates.     

Conformation and spin state in methemoglobin.

The properties of human methemoglobin have been investigated under a wide variety of conditions to determine its conformation and to test for evidence of the T state conformation which has been proposed by Perutz to exist in the presence of high spin ligands and inositol hexaphosphate (IHP). Subunit dissociation was measured as a criterion for the T state since marked differences in the tetramer-dimer equilibrium exist for oxyhemoglobin (R state) and deoxyhemoglobin (T state). In the absence of IHP, complexes of methemoglobin with both high spin ligands (water, fluoride) or low spin ligands (azide, cyanide) show extensive dissociation in 2,2-bis(hydroxymethyl)-2,2',2"-nitriloethanol buffers, pH 6, 0.1 M NaCl, with values of the tetramer-dimer dissociation constant (K4,2) near 10-5 M. The addition of IHP lowers K4,2 to a value near 10-5 M for all forms of methemoglobin. Combination of IHP with methemoglobin promotes a conformational change, but the change is apparently independence of spin state. The conformation acquired in the presence of IHP is not identical with the T state (K4,2 similar to 10-12 M) and can also occur with hemoglobin in the ferrous form, as revealed by a substantial reduction in K4,2 for CO-hemoglobin upon addition of IHP. Subunit dissociation has also been measured using the haptoglobin reaction, since haptoglobin binds only to hemoglobin dimers. The haptoglobin experiments give results that are qualitatively in agreement with the conclusions reached by ultracentrifuge measurements. Similar results are also obtained by estimating the degree of dissociation on the basis of the material which aggregates following mixing with dithionite. The effect of IHP on azide-binding kinetics with methemoglobin has also been examined. Changes in reactivity is observed upon addition of IHP, but the principal effect is observed upon addition of IHP, but the principal effect is an enhancement of the rate of reaction of the beta chains. Changes in the reactivity of the beta93 sulfhydryl group of methemoglobin also accompany addition of IHP, but in a manner which is largely independent of the spin state of the iron. Similar changes are again found with CO-hemoglobin upon addition of IHP. The rate of binding of bromthymol blue also shows some changes upon addition of IHP, but the changes are more pronounced for deoxyhemoglobin than for methemoglobin. Since the results obtained did not appear to indicate a significant role for spin state in the changes observed, additional studies were undertaken using EPR spectroscopy.     

On the detection of homotropic effects in enzymes of low co-operativity. Application to modified aspartate transcarbamoylase

In contrast with the ease of observing heterotropic effects in allosteric enzymes of low co-operativity, the detection of homotropic effects is often difficult. As a consequence, erroneous conclusions about the uncoupling of homotropic and heterotropic effects can result unless sensitive techniques are used for analyzing the kinetic data. Simulations of experiments as well as actual measurements on the allosteric enzyme, aspartate transcarbamoylase, of Escherichia coli and some of its modified forms, were performed in attempts to develop stringent diagnostic procedures for the detection of homotropic effects in enzymes of low co-operativity. The analyses show that direct saturation plots (velocity versus substrate concentration), double reciprocal plots, and Hill plots yield misleading results in that the co-operativity known to be present is not observed. In contrast, Eadie plots (velocity/substrate concentration versus velocity) are much more sensitive in revealing homotropic effects. Since the observed co-operativity depends on both the allosteric equilibrium constant, L, and the number of active sites, n, simulations were performed on the effect of those parameters. The maxima in the Eadie plots increased as L was lowered and conversely the maxima decreased as n was reduced. These changes were confirmed with a mutant aspartate transcarbamoylase which had the same specific activity as the wild-type enzyme and a lower value of L, and also with a hybrid enzyme containing fewer active sites and the same L value. Analogous experiments on nitrated aspartate transcarbamoylase derivatives of decreasing activity showed that Eadie plots were of value in distinguishing between the changes in L and n values resulting from the inactivation. Data from the literature were analyzed in the form of Eadie plots and in all cases homotropic effects were readily detectable for aspartate transcarbamoylase derivatives previously claimed to be devoid of co-operativity.