Development of a generic real-time PCR assay for simultaneous detection of proviral DNA of simian Betaretrovirus serotypes 1, 2, 3, 4 and 5 and secondary uniplex assays for specific serotype identification

Simian betaretroviruses (formerly Type D retroviruses; SRV) are a group of closely related retroviruses for which the natural host species are Asian monkeys of the genus Macaca. Five serotypes have been identified by classical neutralization assays and three additional untyped variants have been reported (SRV_Tsukuba, SRV-6, SRV-7). These viruses may be significant pathogens in macaque colonies, causing a broad spectrum of clinical disease secondary to viral-induced immune suppression. Undetected SRV infections in research macaques also represent a potential confounding variable in research protocols and a concern for human caretakers. Intensive testing efforts have been implemented to identify infected animals in established colonies. A real-time quantitative generic multiplex PCR assay was developed that is capable of simultaneous detection of proviral DNA of SRV serotypes 1, 2, 3, 4 and 5. This assay incorporates amplification of the oncostatin M (OSM) gene for confirmation of amplifiable DNA and allows quantitation of the number of proviral copies per cell analyzed in each multiplex reaction. Detection of multiple serotypes by PCR increases the efficiency and cost-effectiveness of SRV screening programs. A panel of SRV serotype-specific uniplex real-time PCR assays for discrimination among the five recognized serotypes is also described.     

High virus loads in naturally and experimentally SIVagm-infected African green monkeys

A quantitative RT-PCR assay was developed for SIVagm and was used to measure the levels of viral RNA in the plasma of experimentally and naturally infected African green monkeys. The number of productively infected PBMCs and the number of cells carrying integrated provirus were also measured. Plasma virus loads in experimentally infected animals peaked at 2 weeks postinfection, ranging from 2.9 x 10(5) to 4.2 x 10(7) RNA copies/ml plasma. Set points of 2.1 x 10(3) to 2.8 x 10(6) RNA copies/ml plasma were maintained for one year. Similarly, the number of cells carrying integrated SIVagm provirus remained relatively stable in individual animals for one year with set points ranging from 73 to 810 proviral copies per 10(6) PBMC. However, the number of productively infected cells fluctuated considerably during this period. Virus loads in the 26 naturally infected AGMs ranged from 8.3 x 10(3) to 1.1 x 10(7) (mean 1.7 x 10(6)) RNA copies/ml plasma. These levels of viremia are similar to those seen in pathogenic systems (HIV-1, SIVmac), indicating that control of SIVagm replication is not the reason for the natural host's resistance to disease progression.     

The complete genome and genetic characteristics of SRV-4 isolated from cynomolgus monkeys (Macaca fascicularis)

At least 5 serotypes of exogenous simian retrovirus type D (SRV/D) have been found in nonhuman primates, but only SRV-1, 2 and 3 have been completely sequenced. SRV-4 was recovered once from cynomolgus macaques in California in 1984, but its genome sequences are unknown. Here we report the second identification of SRV-4 and its complete genome from infected cynomolgus macaques with Indochinese and Indonesian/Indochinese mixed ancestry. Phylogenetic analysis demonstrated that SRV-4 was distantly related to SRV-1, 2, 3, 5, 6 and 7. SRV/D-T, a new SRV/D recovered in 2005 from cynomolgus monkeys at Tsukuba Primate Center in Japan, clustered with the SRV-4 isolates from California and Texas and was shown to be another occurrence of SRV-4 infection. The repeated occurrence of SRV-4 in cynomolgus monkeys in different areas of the world and across 25 years suggests that this species is the natural host of SRV-4.     

Distribution of type D retrovirus sequences in tissues of macaques with simian acquired immune deficiency and retroperitoneal fibromatosis

Horizontally acquired SAIDS retrovirus type 2 (SRV-2), a type D retrovirus related to the Mason-Pfizer monkey virus, has been associated with the simian acquired immunodeficiency syndrome (SAIDS) including retroperitoneal fibromatosis (RF) in several macaque species at two primate research centers. Virus specific gene sequences are present in lymphoid and RF tissues but not in muscle tissue of diseased macaques or in any tissues of uninfected normal monkeys. Serologic and restriction endonuclease mapping techniques have defined unique SRV-2 strains in the Celebes (SRV-2C) and rhesus (SRV-2R) macaques at the Oregon Regional Primate Center, SRV-2 is related to both MPMV and SAIDS type 1 retroviruses and it has no detectable molecular homology with the human AIDS retroviruses.     

New Simian β Retroviruses from Rhesus Monkeys (Macaca Mulatta) and Langurs (Semnopithecus Entellus) from Rajasthan, India

Natural infection of feral Indian rhesus monkeys (Macaca mulatta) by a new simian β retrovirus, provisionally called simian retrovirus-7 (SRV-7) is described. The virus is capable of in vitro replication in primary human peripheral blood lymphocytes (PBL) and B and T cell lines. We have earlier reported a novel SRV, SRV-6 from Indian langurs (Semnopithecus entellus). Additional sequence analyses from gp20 transmembrane (TM) env genes of SRV-6 and SRV-7 place them in a separate cluster, related to but distinct from known exogenous SRVs and also close to the simian endogenous β retrovirus, (SERV) from African baboon. Phylogenetic analyses of pol gene of SRV-7 place it closer to SERV when the stop codons of the SERV genes are removed. On the other hand, additional sequence data from gp70, surface glycoprotein (SU) region of the env gene of SRV-6 suggest it is more closely related to known exogenous SRVs, (SRV-1 to 3). It is also related to the endogenous langur virus, Po-1-Lu. We hypothesize that SRV-6 and SRV-7 probably originated from a progenitor exogenous SRV which recombined with an endogenous SERV in the TM env and pol genes during evolution, based on the phylogenetic analyses. Genbank Accession numbers of the new sequences: SRV-6 TM (gp20) env: AF401239, SRV-6 SU (gp70) env: AY598468, SRV-7 pol region: AY594212, SRV-7 TM env (gp20): AY594213.     

Transmission of simian acquired immunodeficiency syndrome with a type D retrovirus: immunological aspects

Simian acquired immunodeficiency syndrome (SAIDS) was transmitted to four of four rhesus macaques with blood from rhesus macaques naturally infected with a type D retrovirus, simian retrovirus-2 (SRV-2). Three of the four blood recipients died with SAIDS at 13, 15, and 26 weeks postinoculation. The fourth animal is alive with SAIDS. All four test monkeys became viremic and produced antiviral antibody. None of the inoculated monkeys produced measureable neutralizing antibody to SRV-2. The survivor produced higher levels of antiviral antibody than the monkeys that died. Phytohemagglutinin and concanavalin A reactivity of peripheral blood lymphocytes was depressed from weeks 6 to 12 after inoculation. Clinical findings included development of splenomegaly in all four monkeys, and diarrhea in two monkeys. Blood counts remained within the normal range except for a depression in the number of polymorphonuclear lymphocytes in two monkeys. Hematocrits were decreased in two monkeys just prior to their death. All four test monkeys developed lymph node atrophy and bone marrow hypoplasia. Total proteins and immunoglobulin production were normal. This report provides evidence that SRV-2, as well as other type D retroviruses, causes SAIDS in macaque species.     

Detection of SRV/D shedding in body fluids of cynomolgus macaques and comparison of partial gp70 sequences in SRV/D-T isolates

We previously reported the isolation of a novel subtype of SRV/D-Tsukuba (SRV/D-T) from two cynomolgus monkeys (Macaca facicularis) in the breeding colony of Tsukuba Primate Research Center (TPRC). We surveyed for SRV/D infection in the TPRC cynomolgus colony using SRV/D-specific PCR primer sets designed based on the entire gag region sequence. The only SRV/D subtype detected in the colony was SRV/D-T with a positive infection rate of 22.4% (n = 49). It has been reported that the mode of transmission of SRV/D is via contact with virus shed in the body fluids. In this report, to investigate the infection route of SRV/D-T in monkeys at TPRC, we performed virus isolation and PCR for detection of the SRV/D genome from peripheral blood mononuclear cells (PBMCs), plasma, saliva, urine, and feces. Virus isolation and PCR detection were positive in plasma, saliva, urine, and fecal samples from all monkeys on which virus was isolated from PBMCs. This suggests that the spread of SRV/D-T infection in TPRC is via contact with virus shed in saliva, urine, and/or feces. Also, comparison of sequences of gp70 on multiple SRV/D-T isolates revealed that there was little intra- and inter-monkey variation, suggesting that SRV/D-T is fairly stable.     

Simian retrovirus receptor and neutralization mechanism by antibodies to the envelope glycoprotein.

Type D simian retroviruses (SRV) cause an acquired immunodeficiency syndrome (AIDS) in monkeys. Results of infection with SRV range from complete recovery with absence of viremia to a viremic state, which produces AIDS-like symptoms and culminates in death. These varied outcomes render the interaction of the host and SRV an attractive model for the study of immunosuppressive retrovirus resulting in different pathologic consequences. We describe here the isolation and determination of the molecular weight of the receptor for SRV. We demonstrate that a cell receptor with the same molecular weight is bound by the envelope protein of all five serotypes of SRV. We also show that the receptor recognizes a region containing amino acids 142-167 of the envelope protein of SRV serotype 1 (SRV-1). In addition, we show that a different region of SRV serotype 2 (SRV-2) envelope protein containing amino acids 93-106, interacts with a cell receptor of identical molecular weight. Furthermore, polyclonal and monoclonal antibodies that are directed to envelope epitopes 142-167 of SRV-1 or to 93-106 of SRV-2, specifically neutralize only the respective viral serotype. Our results indicate that the neutralization of SRV infectivity by antibodies is achieved through blocking the interaction between the virus and its cell receptor.     

Immunobiological properties of a recombinant simian retrovirus-1 envelope protein and a neutralizing monoclonal antibody directed against it.

We previously reported that an area encompassing amino acids 147-162 of the envelope region of the simian (type D) retrovirus serotype 1 (SRV-1) constitutes an antigenic site for the binding of murine and rhesus neutralizing antibodies. Neutralizing antibodies to SRV-2 are directed to a different area, encompassing residues 96-102 of SRV-2. This paper presents data on the activity of an SRV-1 recombinant envelope protein (rEP) and of monoclonal hybridoma cell line, C11B8, produced from murine spleen cells immunized with SRV-1 rEP. Purified monoclonal antibodies from C11B8 bind to the SRV-1 rEP and to both SRV-1 and SRV-2. However, the monoclonal antibody exhibits strain specificity in the capacity to neutralize SRV-1 infection in vitro. Thus, C11B8 neutralizes SRV-1 infection but fails to neutralize four other known serotypes of the virus. C11B8 also binds to an SRV-1 synthetic peptide representing residues 142-167, which encompasses the previously defined antigenic site of recognition for neutralizing antibodies to SRV-1. This paper also contains evidence that the SRV-1 rEP construct binds the site for SRV-1 attachment to the cell receptor. This is indicated by the ability of SRV-1 rEP to compete with SRV-1 (but not with SRV-2) and inhibit its infectivity in vitro. In addition, SRV-1 rEP inhibits the neutralizing activity of C11B8 against SRV-1 infection in vitro. SRV-1 rEP has no inhibitory effect on rhesus neutralizing antibodies to SRV-2. Taken together, the above findings indicate that immunity conferred at the level of neutralizing antibodies during SRV infection is strain-specific and involves the recognition of envelope sequences unique to each strain.     

Characterization of T cell epitopes on the envelope glycoprotein of simian retrovirus 1 and 2 (SRV-1 and SRV-2) in several mouse strains.

Various mouse strains were immunized with either SRV-1 or SRV-2 virus adsorbed on alum. Seven to 14 days later spleen cells were removed, and spleen cells were cultured with varying amounts of SRV-1 virus and SRV-2 virus, or varying amounts of selected SRV-1 and SRV-2 synthetic envelope peptides to determine their ability to initiate T cell proliferative responses. Our studies demonstrated that all mouse strains tested gave strong proliferative responses with SRV-2 virus. In contrast, SRV-1 virus induced T cell proliferative responses only in H-2k mouse strains. This apparent major histocompatibility complex (MHC)-restriction of SRV-1 virus-induced T cell proliferation correlates with the increased pathogenicity of SRV-1 virus in rhesus monkeys. The SRV envelope peptide 233-249 which is shared by both SRV-1 and SRV-2 virus initiates strong proliferative responses in both SRV-1 and SRV-2 virus immunized mice. The SRV-2 envelope peptide 96-102 initiates significant proliferative responses in SRV-2 immunized mice, and constitutes both a T and B cell epitope. The SRV-2 envelope peptide 127-152 has a 70% homology with the C-terminal region of SRV-1 peptide 142-167. The ability of SRV-2 peptide 127-152 to initiate T cell proliferation in SRV-1 virus immunized mice and the failure of the SRV-1 peptide 142-162 to initiate proliferation suggests that the region encompassing residues 160-167 must represent a T cell epitope in mice immunized with SRV-1 virus.     

Early immunopathology events in simian retrovirus, type 2 infections prior to the onset of disease

Immunopathology during early simian retrovirus type 2 (SRV-2) infection is poorly characterized. Here, viral dynamics, immune response and disease progression in transiently- or persistently-infected cynomolgus macaques are assessed. Viral nucleic acids were detected in selected lymphoid tissues of both persistently- and transiently-infected macaques, even after viral clearance from the periphery. Immunohistochemical staining of lymphoid tissues revealed alterations in a number of immune cell populations in both transiently- and persistently-infected macaques. The precise pattern depended upon the infection status of the macaque and the marker studied. Gross immunopathological changes in lymphoid tissues were similar between SRV infection and those observed for other simian retroviruses SIV and STLV, suggesting a common immunopathological response to infection with these agents.     

PCR multiplex sur le LightCycler utilisant des sondes d'hydrolyse et d'hybridation : application à la quantification du provirus VIH-1

Quantification of HIV provirus DNA by aid of PCR multiplex using LIGHT Cycler. The quantitation of HIV provirus DNA in peripheral blood mononuclear cells (PBMCs) may be useful to gain insight into the natural history of infection and the continued efficacy of antiretroviral therapy. A quantitative PCR with the Light Cycler strategy has been developed to evaluate HIV DNA in PBMCs together with the amplification of cellular gene (β-globin) used as an internal control. Fluorescent hydrolysis probe and hybridization probes were used to detect HIV DNA and the β-globin sequence, respectively, in the same run. Co-amplification with β-globin did not interfere with HIV-1 quantification and the sensitivity of HIV detection was 5 copies. This assay was compared with an HIV DNA competitive PCR and the correlation between the two methods was good (Δ log = 0.19). The possibility to use simultaneously fluorescent hydrolysis and hybridization probes enlarges the analytic ability of real time PCR LightCycler technology.     

Synthetic peptides of envelope proteins of two different strains of simian AIDS retrovirus (SRV-1 and SRV-2) represent unique antigenic determinants for serum neutralizing antibodies.

There are at least three distinct serotypes of simian type D retrovirus (SRV) which exhibit extensive serological cross-reactivity, but no cross-reactivity exists at the level of serum neutralizing antibodies. Amino acid sequence analysis and hydrophobicity plots of SRV-1 and SRV-2 envelope proteins were compared in order to identify unique potential antigenic determinants to which respective neutralizing antibodies may be directed. Peptides representing residues 147-162 of SRV-1 and 96-102 of SRV-2 were synthesized and assessed for their immunoreactivity. Free peptide inhibition of strain-specific serum (rhesus) neutralizing antibodies to SRV-1 and SRV-2 was demonstrated using the SRV-1 147-162 peptide and the SRV-2 peptide, 96-102, respectively. Inhibition of serum neutralizing activity by these peptides was also strain-specific, showing no cross-inhibition. SRV-1 147-162 conjugated to a protein carrier and cross-linked to Sepharose beads specifically adsorbed neutralizing antibodies from SRV-1 immune rhesus sera. The antibodies eluted from the immunoadsorbent possessed SRV-1 neutralizing activity, but showed no effect on the infectivity of SRV-2. Peptide SRV-1 147-162 also exhibited the capacity to bind specifically with a mouse monoclonal antibody which neutralizes the infectivity of SRV-1. Mice immunized with a recombinant SRV-1 envelope protein or with whole, inactivated SRV-1 produced antibodies which bound the SRV-1 147-162 conjugate, but not the protein carrier itself. Mouse antibodies to the SRV-1 147-162 conjugate exhibited specific binding with both native SRV-1 and with recombinant SRV-1 envelope protein. These findings provide strong evidence that SRV-1 147-162 and SRV-2 96-102 constitute at least two unique antigenic determinants, or parts thereof, which participate in the strain-specific neutralizing antibody response. Moreover, the findings indicate that the SRV-1 neutralizing antibodies produced by monkeys and at least a certain population of neutralizing antibodies produced by mice recognize the same epitope of SRV-1.     

Viremia, antigenemia, and serum antibodies in rhesus macaques infected with simian retrovirus type 1 and their relationship to disease course

The course of simian retrovirus type 1 (SRV-1) infection was studied in 14 experimentally inoculated juvenile rhesus monkeys. Viral transmembrane protein antigenemia and antibodies to whole virus were measured by enzyme linked immunosorbent assay and correlated with the clinical course of disease and virus isolation. Based on these parameters, animals with simian retrovirus type 1-induced disease were divided into three categories: monkeys dying within a few months of fulminating simian acquired immune deficiency syndrome in the face of a high level persistent antigenemia and viremia, and a nondetectable serum antibody response; monkeys that developed a milder form of simian acquired immune deficiency syndrome but remained alive in spite of a chronic low-grade antigenemia and viremia and only a transient initial antibody response; and monkeys that never became ill and that were either transiently or nontransiently viremic and antigenemic. This latter group developed high levels of serum antibodies. The outcome of simian retrovirus type 1-induced disease was similar to that described for feline leukemia virus infection of cats, another retroviral disease of animals. The disease course differed considerably, however, from that reported for retrovirus-induced human acquired immune deficiency syndrome.     

Six mamu-A locus alleles defined by a polymerase chain reaction sequence specific primer method

Rhesus monkeys are relevant models for human diseases and transplantation. In each case, a complete understanding of these models requires knowledge of macaque MHC. Due to high polymorphism and multiple genes per haplotype, it has been difficult to develop a rapid typing method for rhesus monkey MHC class I. We developed a simple and rapid PCR-SSP strategy for rhesus monkey Mamu-A locus typing. Fifty-two rhesus monkeys were included in the study. Six rhesus monkey allel-specific primer pairs were designed based on published Mamu-A locus gene sequences. Allele-specific PCR products ranged in size from 346 to 788 bp; 5' and 3' Mamu-A locus allele specific primers were located in the second and third exons, respectively. Specific PCR product gel purification was followed by direct sequencing, without subcloning, in both directions. Our data showed variability in the number of Mamu-A alleles ranging from 1 to 4 per genotype. The highest frequencies were observed for Mamu-A*02, -A*04, and -A*03 alleles. Thus, we report here the first PCR-SSP typing method for Mamu-A*02, -03, -04, -05, -06, and -07 array of class I alleles. This technique appears to be a highly reproducible and discriminatory method for detecting this subset of class I A locus genes in rhesus monkeys.     

Optimization of ex vivo activation and expansion of macaque primary CD4-enriched peripheral blood mononuclear cells for use in anti-HIV immunotherapy and gene therapy strategies

The rhesus macaque model is a useful experimental system to evaluate effects of T-cell autotransfusion and gene therapies for HIV-1 infection and AIDS prior to a clinical trial. To obtain sufficient numbers of primary macaque CD4 T lymphocytes for this purpose, we examined the culture conditions that were needed to optimize ex vivo activation and expansion of macaque primary CD4-enriched peripheral blood mononuclear cells (PBMCs). In this report, we compared the effects of various stimulants on cell expansion, surface expression of CCR5 and CXCR4, and levels of transduction with a Moloney leukemia virus (MoLV) vector encoding the phenotypic selection marker truncated human nerve growth factor receptor (deltaNGFR) alone or with the human anti-HIV-1 tat intrabody sFvhutat2. The use of feeder cells strikingly increased the proliferation rate of macaque CD4-enriched PBMCs in vitro. In the presence of an irradiated rhesus macaque B-lymphoblastoid cell line (BLCL), the highest cell expansion over 21 days was achieved with cells activated by Con A (9648-fold), in turn, from high to low, phytohemagglutinin (PHA) (4855-fold), and anti-CD3/CD28-coated beads (2367-fold). Further studies showed that BLCL feeder cells were more effective than human PBMCs (hPBMCs) in promoting proliferation of macaque CD4-enriched PBMCs activated with Con A and anti-CD3/CD28, respectively. The combined use of both BLCL and hPBMC feeder cells did not further increase cell expansion when compared with the use of BLCL cells alone. In addition, the addition of BLCL-conditioned medium (CM) and hPBMC-CM induced cell growth at a rate higher than did the culture medium alone but not as high as with feeder cells. Con A-activated macaque CD4-enriched PBMCs retained 88% of CXCR4 and 39% of CCR5 expression over 17 days compared with PHA-activated cells (50% for CXCR4, 16% for CCR5) and anti-CD3/CD28-activated cells (34% for CXCR4, 37% for CCR5). Finally, PHA, Con A, and CD3/CD28-coated beads supported comparable levels of MoLV transduction. The results should improve the utility of the rhesus macaque model for the testing of T-cell autotransfusion and gene therapies for HIV-1 infection/AIDS.     

Natural distribution of squirrel monkey retrovirus proviral sequences in primate DNAs.

3H-labelled 70S RNA of squirrel monkey retrovirus (SMRV) hybridized to a high degree (greater than 52%) to the DNA of various tissues of two squirrel monkeys. Hybridization of the same probe to DNAs of other primates including New World monkeys (Woolly monkey, capuchin, owl monkey), Old World monkeys (rhesus, African green), apes (gibbon, chimpanzee), and human (A204 cells infected with MPMV) revealed no significant hybridization. Analysis of the kinetics of hybridization indicated that SMRV provirus was present in multiple copies in various squirrel monkey tissues (C0t 1/2 = 120 to 400) and in SMRV-infected A204 cells at a low number of copies (C0t 1/2 = 1500). These results demonstrate that SMRV is an endogenous virus of squirrel monkeys and the first isolated from a New World monkey.     

Protective ribosomal preparation from Shigella sonnei as a parenteral candidate vaccine.

A parenteral Shigella ribosomal vaccine (SRV) was investigated in animals for safety, antibody-inducing capacity, and protective activity. Ribosomal preparations from a Shigella sonnei phase I avirulent strain were obtained and shown to possess chemical, sedimentation, and other properties typical of bacterial ribosomes. No endotoxin contamination was revealed by a ketodeoxyoctonate assay, although the presence of some kind of O antigen was evidenced by serological findings and the high activity of SRV in inducing the O-antibody response and immunological memory in animals. SRV was nontoxic in mice, guinea pigs, and monkeys and induced no local reactions when injected subcutaneously in reasonable doses. Significant protection against a local Shigella infection (Sereny test) was seen in guinea pigs injected with SRV (efficiency index, about 60%) and the specificity of the protection was evident from cross-challenge experiments. The protective efficiency of SRV was especially high in rhesus monkeys challenged orally with virulent Shigella cells (89%, as calculated from the summarized data of several experiments in 71 animals). Protection in monkeys was long lasting and could be demonstrated several months after injection of SRV. An inexpensive technique can be used for the production of SRV on a large scale. The high immunogenicity of SRV is discussed in terms of the amplifying effect of the ribosome, which serves as a delivery system for polysaccharide O antigen. Further study of SRV as a candidate vaccine for humans seems justified by the data obtained.     

Polymerase chain reaction detection of type D simian retrovirus proviral DNA from infected macaques

A simple polymerase chain reaction (PCR) approach was developed for detection of Type D simian retrovirus (SRV) serogroup 2 proviral DNA using peripheral blood lymphocytes (PBLs) obtained from infected macaques. PCR primer pairs were developed against serogroup 2 envelope (env) gene sequence, and fidelity of PCR fragment amplification was determined using molecularly cloned SRV serogroup 2 (D2/RHE/OR) DNA, and genomic DNA from Raji cells independently infected with different SRV serogroups. One primer pair exhibiting high fidelity was then utilized for PCR detection of serogroup 2 proviral DNA from PBLs, and from cells sorted into immune cell subpopulations by fluorescent-activated cell sorting (FACS). Enυ PCR fragments were readily detected from as few as 10⁴ PBLs or immune cell subpopulations. In addition, highly specific PCR primers against serogroups 1 and 3 were utilized to detect proviral DNA from Raji cells infected with SRV serogroups. In all cases, primers designed to amplify serogroups 1,2, and 3 proviral DNA were specific for their intended serogroup. This primer information and development of a PCR approach for detection of specific SRV proviral DNA will be of potential utility as a rapid surveillance tool in monitoring type D simian retrovirus infection within Asian macaque colonies.     

Cytokine responses in young and old rhesus monkeys: effect of caloric restriction.

Caloric restriction (CR) is the only known intervention demonstrated to retard a great variety of aging processes, extend median and maximum life-span, and decrease the incidence of age-associated diseases in mammals. Paralleling findings from rodent studies, studies in rhesus monkeys (Macaca mulatta) suggest that CR may retard many age-sensitive parameters in primates. A recent study in rhesus monkeys showed age-related dysregulation of cytokine levels. Specifically, age-related increases in interleukin-10 (IL-10) and IL-6 proteins were observed in supernatants from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs), and interferon-gamma (IFN-gamma) protein exhibited an age-related decrease in phytohemagglutinin (PHA)-stimulated PBMCs. To investigate effects of CR on age-related changes in cytokine production, we obtained PBMCs from control and CR rhesus monkeys aged 6-7 and 22-25 years. We evaluated IL-10 and IL-6 protein and gene expression after exposure to LPS and IFN-gamma protein and gene expression after PHA stimulation. The results revealed significantly higher levels of IFN-gamma protein and gene expression in aged monkeys on CR for 2 years compared with controls. No significant CR effects were observed on IL-10 and IL-6 protein levels. IFN-gamma plays an important role in the initial defense mechanism against viral and microbial disease and cancer. Altered regulation of IFN-gamma in old CR rhesus monkeys may be a key factor in reducing cancer incidence and other age-associated diseases.