Expression of the chemokine MIG is a sensitive and predictive marker for antigen-specific, genetically restricted IFN-gamma production and IFN-gamma-secreting cells.

The evaluation of antigen-specific immune responses is critical for understanding the mechanisms of immune protection and for establishing the efficacy of candidate vaccines. Here, we describe a novel assay for IFN-gamma activity which is based on the flow cytometric detection of the chemokine, monokine induced by gamma interferon (MIG) as a sensitive and predictive measure of IFN-gamma-mediated effector function, and a surrogate marker for IFN-gamma-producing cells. Upregulation of MIG expression was demonstrated following in vitro activation of peripheral blood mononuclear cells (PBMCs) with defined CD8+ T-cell epitopes derived from influenza virus, cytomegalovirus (CMV), or Epstein-Barr virus (EBV) and was antigen-specific, genetically restricted and dependent on both CD8+ T cells and IFN-gamma. Furthermore, antigen-specific MIG expression was also demonstrated with Plasmodium falciparum circumsporozoite protein (CSP) peptides, using PBMCs from volunteers immunized with irradiated P. falciparum sporozoites. In multiple parallel experiments, the MIG assay was compared to conventional IFN-gamma ELISPOT, IFN-gamma ELISA, MIG ELISA and intracellular cytokine staining assays. The level of MIG expression was shown to be directly associated with the number of IFN-gamma spot-forming cells (SFCs) detected by ELISPOT (r2=0.94). Moreover, in all instances where cultures were considered positive by ELISPOT, a higher stimulation index was noted with the MIG assay as compared with the ELISPOT assay (on average at least threefold higher) and, in some cases, responses as detected by the MIG assay were significant, but the corresponding response as measured by ELISPOT was not significant. Finally, the flow-based MIG assay offers a number of practical and technical advantages over the ELISPOT assay. Our data validate this novel method for the detection of low as well as high levels of antigen-specific and genetically restricted IFN-gamma activity.

Inverse correlation of telomerase activity/proliferation of CD4+ T lymphocytes and disease progression in simian immunodeficiency virus-infected nonhuman primates.

Both increased lymphocyte renewal with subsequent exhaustion of the immune system and impaired T-cell renewal have been put forth to account for CD4+ T-cell depletion and development of AIDS in HIV-1-infected humans and SIV-infected nonhuman primates. In the present study, telomeric terminal restriction fragment length and telomerase activity were used as measures of proliferative activity of T lymphocytes from three nonhuman primate species before and after being infected with SIV. In peripheral blood T cells, our data show both species and T-cell-subset-specific differences in proliferative activity accompanied by different patterns of disease progression. A significant postinfection increase in telomerase/proliferative activity in CD4+ T cells from seropositive sooty mangabeys and from normal progressor rhesus macaques was associated with asymptomatic infection or delayed disease progression, respectively, whereas a decrease in telomerase/proliferative activity detected in CD4+ T cells postinfection from SIVsmmPBj14-infected pigtailed macaques was associated with rapid CD4+ T-cell depletion and disease progression. The levels of telomerase activity observed in CD4+ T cells from peripheral blood closely parallelled those seen in CD4+ T cells in lymph node samples from selected animals. Our data suggest that an increase in proliferative activity of T lymphocytes in vivo may be associated with a favorable course of SIV infection in nonhuman primates.

A novel role for tumor necrosis factor-alpha in regulating susceptibility of activated CD4+ T cells from human and nonhuman primates for distinct coreceptor using lentiviruses.

Although CD4+ T-cell activation has long been shown to promote infection and replication of simian immunodeficiency virus (SIV) and HIV, recent studies have documented that not all activated CD4+ T cells from human and nonhuman primates are susceptible to infection with HIV/SIV, respectively. Activation of CD4+ T cells with anti-CD3 + anti-CD28 conjugated beads led to induction of a state of anti-viral resistance to infection with strains of viruses that primarily use CCR5 as a coreceptor. The studies reported herein were designed to address the mechanism by which anti-CD3 + anti-CD28-induced stimulation in turn induced antiviral resistance. Results of these studies show that the anti-viral resistance induced by activation of CD4+ T cells with anti-CD3 + anti-CD28 is primarily conferred by the synthesis of tumor necrosis factor-alpha (TNF-alpha), and highlight a unique regulatory role for TNF-alpha in regulating synthesis of MIP-1alpha, MIP-1beta, and regulated-on-activation normal T-expressed and secreted cells, which contributes to this state of antiviral resistance to R5-tropic strains of HIV/SIV. However, while TNF-alpha has a protective role in antiviral resistance of activated CD4+ T cells to R5-tropic viruses, it enhances CXCR4 expression of CD4+ T cells and mediates increased susceptibility to infection with X4-tropic strains of HIV and recombinant SIVs. The results of the studies reported herein also suggest that it is not the Th1 v/s Th2 cytokine profile but the mode of CD4+ T-cell activation that dictates the synthesis of distinct cytokines which regulate the expression of chemokines and chemokine receptors which in turn regulate and confer susceptibility/resistance to R5 v/s X4-tropic HIV and SIV.

Studies of CD40L expression by lymphoid cells from experimentally and naturally SIV-infected nonhuman primate species.

Dysfunction of T lymphocytes is well documented in HIV-1-infected individuals; however, the mechanisms responsible for the noted dysfunction are not well understood. CD40L is an important costimulatory molecule that helps initiate immune responses, and there is controversy regarding whether or not expression of CD40L is compromised in HIV-1-infected individuals. We have utilized the SIV infection of experimentally infected (disease-susceptible) and naturally infected (disease-resistant) nonhuman primates as animal models of human AIDS to address this issue. Little is known concerning the expression of CD40L in nonhuman primates. Studies were conducted to determine the frequency, density, phenotype, and kinetics of CD40L expression by in vitro activated peripheral blood mononuclear cells (PBMCs) from different species of uninfected and SIV-infected monkeys. Data obtained show marked differences in the density and phenotypic lineage that expresses CD40L in lymphoid cells from the three species examined. However, no detectable differences were noted in the frequency and density of CD40L expression by in vitro activated lymphoid cells from uninfected and SIV-infected disease-susceptible rhesus macaques and seropositive as compared to seronegative disease-resistant sooty mangabeys. These data suggest that phenotypic expression of CD40L is not compromised due to SIV infection.

Control mechanisms of virus replication in naturally SIVsmm infected mangabeys and experimentally infected macaques.

As a close cousin to Asian macaques, the African sooty mangabey monkey, a species naturally infected with SIV without ever developing disease, represents an intriguing and thought provoking model for the study of lentiviral infection and disease development. Pursuant to our previous findings that documented the presence of high frequencies of CD8+ T-cells capable of inhibiting lentiviral replication in vitro via a soluble factor, the potential contribution of beta-chemokines and their receptor was evaluated in samples from sooty mangabeys and disease susceptible macaques. Both mangabey and Rhesus macaque PBMC were found to secrete comparable levels of MIP-1alpha, MIP-1beta and RANTES after stimulation in vitro. Constitutive expression of CCR-5 appeared lower in mangabey PBMC but the vast majority of T-cells from mangabeys or macaques were found to express CCR-5 after in vitro activation. Sequence analysis of macaque and mangabey MIP-1alpha and RANTES showed complete homology to the human counterpart. MIP-1beta on the other hand while highly conserved among both monkey species, showed only 93% homology to human MIP-1beta. In addition, CCR-5, CCR-2b and CXCR-4 presented no consistent differences between rhesus and mangabey sequences. Thus, based on current data, differences in disease susceptibility between macaques and mangabeys do not appear to rely on differences in chemokine pathways. However, analyses of the ontogeny of CD8+ T-cell-mediated inhibition of SIV replication showed that macaque PBMC acquire this function shortly after infection, and show a progressive loss thereafter, correlated with progression towards disease. Mangabeys, on the other hand, appear to acquire the CD8+ T-cell inhibitory function long before any evidence of seroconversion to SIV and maintain this function for the lifetime of the animal. In vitro analyses of induction of the CD8+ inhibitory function showed that rhesus CD8+ T-cells have the potential to secrete the inhibitory factor(s) prior to SIV infection.

Development of an animal model for autotransfusion therapy: in vitro characterization and analysis of anti-CD3/CD28 expanded cells.

Previous studies have shown that in vitro culture of human CD4+ T cells with antibodies to CD3 and CD28 immobilized on beads induced an antiviral effect to HIV-1 infection. Herein, we have used CD4+ T cells from nonhuman primates to address issues critical for use of such cells for therapy and immune reconstitution of humans and nonhuman primates infected with HIV and simian immunovirus (SIV). These studies include definition of the kinetics of the antiviral effect, the relative stability of the acquired phenotype, and whether such activated and expanded CD4+ T cells retain their immune function. Results of our studies show that antiviral effect is induced rapidly following activation with anti-CD3/CD28-coated beads. Additionally, the antiviral effect is not stable in these cells and requires continuous culture with anti-CD3/CD28 beads. Removal of CD4+ T cells from anti-CD3/CD28 stimulation renders these cells susceptible to infection, demonstrating that the resistant phenotype is not stable in these cultures. However, anti-CD3/CD28 expanded CD4+ T cells do retain immune function. Thus, although these findings imply a note of caution for therapeutic strategies aimed at providing patients with virus-resistant CD4+ T cells, the present study suggests that transfusion of such cells with retained immune function may have immune restoration capability.

Molecular cloning and expression of rhesus macaque and sooty mangabey interleukin 16: biologic activity and effect on simian immunodeficiency virus infection and/or replication.

Interleukin 16 (IL-16) has been shown to diminish HIV and SIV replication through inhibition of HIV and SIV mRNA transcription. To evaluate its role further, we compared IL-16 cloned from disease-susceptible rhesus macaques and disease-resistant sooty mangabeys. Recombinant rhesus macaque (rr) IL-16 was compared with recombinant sooty mangabey (rm), human, and other nonhuman primate IL-16 sequences and evaluated for its ability to induce chemotaxis and inhibit the mixed lymphocyte response (MLR). Also, rrIL-16 and rmIL-16 were evaluated for suppression of SIVmac251, which replicates efficiently in T cells and monocyte/macrophages (dual tropic), and cloned SIVmac239, which replicates efficiently in T cells (T tropic). Sequence comparison of rrIL-16 and rmIL-16 with human IL-16 showed >97% amino acid identity. Biocharacterization of rrIL-16 revealed potent induction of chemotaxis (p < 0.05) and marked inhibition of MLR (73 +/- 0.6%,p < 0.05) in rhesus and human cell systems. Using rrIL-16 and rmIL-16, p27 antigen production from PBMCs infected with SIVmac251 was decreased up to 70% (p < 0.05 and p < 0.01, respectively). In similar cultures infected with SIVmac239, rrIL-16 and rmIL-16 reduced p27 levels by 96 and 100%, respectively. These data demonstrate the biologic and antiviral functionality of rrIL-16 and rmIL-16.

Expression and in vitro evaluation of rhesus macaque wild type (wt) and modified CC chemokines.

Several human CC chemokines have been shown to inhibit HIV/ SIV infection in vitro, providing the rationale for their potential use in vivo. However, because of their inherent physiological effect, such chemokines are reasoned to be of limited therapeutic value due to potential side effects. The knowledge that amino terminus modified or deleted human RANTES retains its receptor binding properties but loses its signaling properties has provided a means to use such modified chemokines in vivo for possible therapeutic benefits. In efforts to test the efficacy of such modified chemokines, our laboratory has cloned, sequenced, and prepared recombinant forms of wild-type (wt) and amino-terminus modified rhesus macaque chemokines MIP-1alpha, MIP-1beta, and RANTES. These sets of chemokines were tested for their potential to inhibit SIV infection and induce signaling. The data showed that whereas wt chemokines retained both virus inhibitory and signaling functions, corresponding amino-terminus modified chemokines only showed virus inhibitory effects without detectable signaling effects. Such reagents will be valuable for evaluation of their therapeutic potential in vivo, either alone or as adjuncts to other chemotherapeutic drugs.

Immune and hematopoietic parameters in HIV-1-infected chimpanzees during clinical progression toward AIDS.

Until recently, chimpanzees were considered susceptible to human immunodeficiency virus type 1 (HIV-1) infection, but refractory to disease induction based on the asymptomatic status of all experimentally infected chimpanzees after over 10 years postinfection (PI). However, a decline in peripheral CD4+ T cells was noted in one chimpanzee (C499) of the Yerkes cohort of HIV-1 infected apes, after 11 years PI concurrent with increasing plasma viral load. These clinical signs were followed by the occurrence of opportunistic infections, thrombocytopenia, and progressive anemia leading to euthanasia. A second chimpanzee (C455) was transfused with blood from C499 collected during the symptomatic stage. Shortly thereafter, this second animal showed a rapid decline in peripheral CD4+ T-cell levels and sustained high viral load. Hematological analyses showed a 50% decrease in CFU-GM for both apes during the symptomatic phase and a reduction of 40% and 73% of the total CFU despite normal levels of CD34+ cells in the bone marrow. Cryopreserved sequential PBMC samples from these two chimpanzees were analyzed for constitutive and PHA-P induced levels of cytokines and chemokines. Data show that whereas there were no detectable constitutive levels of mRNA coding for IL-2, 4, and 10, there appears to be a transient increase in IFN-gamma message level coincident with increased viremia and this IFN-gamma synthesis decreased with disease progression. PHA-induced cytokine mRNA analysis showed low or undetectable levels of IL-4 and IL-10 mRNA in all samples and a marked decrease in the levels of IL-2 shortly after HIV infection. In addition, there was also a gradual decrease in IFN-gamma mRNA with progression of disease. Of interest were the findings of high to normal levels of PHA-induced synthesis of the chemokines MIP-1alpha, MIP-1beta, and RANTES in samples during the asymptomatic and early symptomatic period, which also dramatically decreased at late stages of the disease. These data suggest important roles for IL-2, IFN-gamma, and the chemokines in the regulation of immune responses in HIV-1-infected chimpanzees.

Initiation of ethanol self-administration by the sucrose-substitution method with HAS and LAS rats.

This study was performed to examine ethanol self-administration in rats bred for different sensitivities to the sedative effects of alcohol [the Colorado High Alcohol Sensitive (HAS) and Low Alcohol Sensitive (LAS) rats]. Four rats from each replicate line of the HAS and LAS rats (n = 16) were obtained from the University of Colorado, and initiation to self-administer ethanol by the sucrose-substitution procedure was attempted. Before the initiation procedure was conducted, home-cage ethanol intake and preference ratio did not differ between LAS and HAS rats. During the initiation procedure, the LAS rats came to self-administer 10% ethanol (v/v) at similar levels as outbred Wistar rats initiated with the same procedure (approximately 0.4 g/kg/session). The HAS rats, however, failed to initiate (approximately 0.08 g/kg/ session after completing the sucrose-substitution procedure) and lever pressing was reduced even more in the HAS rats when the ethanol concentration presented was > 10% (v/v). Three of the eight HAS rats stopped lever pressing completely when the ethanol concentration was raised to 15%. After initiation, home-cage preference ratio differed significantly between the LAS and HAS rats (LAS > HAS, p < 0.03). That the LAS rats did not consume greater amounts of ethanol compared with outbred Long-Evans or Wistar rats is contrary to our hypothesis, based on recent human data suggesting that a lower sensitivity to ethanol could result in increased alcohol intake. The finding that the HAS rats could not be initiated, while selectively bred ethanol nonpreferring rats can, is also contrary to our hypothesis. Further studies related to ethanol self-administration with the HAS line could provide important information related to the genetics of alcohol nonacceptance.

Detection of intracellular signal transduction molecules in PBMC from rhesus macaques and sooty mangabeys.

One of the manifestations of human HIV-1 and nonhuman primate SIV infection that lead to disease is reasoned to be secondary to generalized T-cell dysfunction. The molecular mechanisms associated with the T-cell dysfunction remain to be elucidated. To address this issue, we sought to utilize the nonhuman primate model to study intracellular signaling events in cells from disease-susceptible rhesus macaques and disease-resistant sooty mangabeys. Because relatively little is known about these events in nonhuman primates, our laboratory defined optimal conditions, reagents, and assays for the study of signal transduction events in cells from nonhuman primates. The protein phosphorylation patterns in the two monkeys exhibited quantitative, qualitative, and kinetic differences. Antibodies to Stat6 detected a unique band in macaque cell lysates. This band is markedly decreased human cell lysates and never seen in mangabey cell lysates. Detection of various other intracellular signaling proteins is also described.

Sucrose, ethanol, and sucrose/ethanol reinforced responding under variable-interval schedules of reinforcement.

Adding sweeteners to ethanol solutions is a common method of inducing rats to consume ethanol. However, it has usually been assumed that it is the sweet taste and/or the calories contained in the sweet solution that controls consumption. The present experiment examined the role of ethanol in controlling responding reinforced by ethanol or an ethanol/sucrose mixture compared with sucrose solutions of various concentrations. After initiation to self-administer 10% (v/v) ethanol using the sucrose-substitution method, rats were trained to respond under a concurrent VI 5" VI 5" schedule. During one condition, responding on one lever was reinforced by the presentation of 10% ethanol, and responding on a second lever was reinforced by water or one of the following sucrose solutions: 1% (w/v), 1.5%, 2%, 2.5%, 3%, and 5%. During a subsequent condition, responding reinforced by a 10% ethanol/2% sucrose mixture was compared under the concurrent schedule with responding reinforced by water, 2%, 2.5%, 3%, 5%, or 10% sucrose (w/v). The results indicated that the ethanol or ethanol/sucrose mixture maintained more responding than did sucrose solutions that were sweeter. Data support the conclusion that, after initiation, the taste and/or pharmacological effects of ethanol had become an important component of the reinforcing stimulus independent of the sweetener.