Enhanced frequency of spontaneous B cell lines from Epstein-Barr virus (EBV) seropositive donors 80 years and older.

Based on the well-recognized decline in immunocompetence which develops with advancing age, we have evaluated the effect of age on the frequency of development of spontaneous Epstein-Barr virus (EBV)-infected B cell lines. Blood mononuclear cells were isolated from 38 clinically healthy seropositive donors. The cells were maintained in vitro according to routine culture conditions for lymphocytes. Eight spontaneously EBV-infected B lymphoblastoid cell lines (LCL) were isolated. The LCL developed in 12.5, 14.3, or 6.3% of the samples derived from donors in the three age groups 20-39, 40-59, 60-79, respectively. In contrast, samples from five of seven (71%) donors 80 years and older yielded LCL. Although the reason(s) for the increased frequency of occurrence of spontaneous LCL from the older adults is yet to be explored, the possible role of the virus-specific T lymphocytes as a contributing factor is discussed.

Comparison of methods for HIV isolation from monocytes and lymphocytes of seropositive asymptomatic hemophiliacs and homosexuals.

1. Since monocyte-macrophages have been recognized as HIV targets in addition to CD4+ T-lymphocytes, we have evaluated HIV infection of purified peripheral blood mononuclear cell fractions obtained from 10 seropositive asymptomatic hemophiliacs and compared it with that of 10 asymptomatic homosexual patients. 2. HIV was isolated more frequently from the lymphocytes than the monocytes of both groups of patients. 3. HIV preferentially replicated in phytohemagglutinin-stimulated lymphocytes compared with growth factor-treated monocytes. Monocytes did not preferentially harbour HIV in either group.

Comparison of methods for HIV isolation from monocytes and lymphocytes of seropositive asymptomatic hemophiliacs and homosexuals

1. Since monocyte-macrophages have been recognized as HIV targets in addition to CD4+ T-lymphocytes, we have evaluated HIV infection of purified peripheral blood mononuclear cell fractions obtained from 10 seropositive asymptomatic hemophiliacs and compared with that of 10 assymptomatic homosexual patients. 2. HIV was isolated more frequently from the lymphocytes than the monocytes of both groups of patients. 3. HIV preferential replicated in phytohemagglutinin-stimulated lymphocytes compared with growth factor-treated monocytes. Monocytes did not preferentially harbour HIV in either group

Synthesis and biological evaluation of prodrugs of zidovudine.

A series of prodrugs of zidovudine (AZT) has been synthesized in an effort to enhance the uptake of the prodrugs by the HIV-1 infected cells and to increase the plasma half-life of AZT. The 5'-OH function of AZT was esterified with various acids in the presence of DCC and 4-(dimethylamino)pyridine (DMAP). The prodrug moieties included (a) morpholine and N-phenylpiperazine-1-acetic acid, (b) 1,4-dihydro-1-methyl-3-nicotinic acid, (c) retinoic acid, and (d) certain amino acids. The anti-HIV-1 activity of the esters was determined in peripheral blood lymphocytes. The IC50 for AZT in this system was 0.12 microM whereas for prodrugs it ranged from 0.05 to 0.2 microM. The prodrugs were generally less cytotoxic than AZT except the retinoic acid ester. In vitro hydrolysis of the various esters in human plasma indicated that these agents were relatively stable toward plasma esterases with t1/2 ranging from 10 to 240 min. Drug uptake studies in H9 cells with radiolabeled analogues demonstrated that the retinoic acid ester achieved approximately 4-fold higher intracellular concentration than [3H]AZT. However, 1,4-dihydro-1-methyl-3-[(pyridylcarbonyl)oxy] ester (5) was the most active agent of this series and had a higher therapeutic index than AZT.

Increased therapeutic efficacy of zidovudine in combination with vitamin E.

Antiviral activity and bone marrow toxicity of 3'-azido-3'deoxythymidine (Zidovudine; AZT) was evaluated in the presence of alpha-D-tocopherol acid succinate (ATS) in the MT4 cell line and in murine hematopoietic progenitor cells, respectively. At varying concentrations (.016 to .125 microM) of AZT, addition of ATS (5 to 15 micrograms/ml) showed a dose-dependent increase in anti-HIV activity. The ED90 of AZT in this test system was 0.37 microM, whereas in the presence of ATS (15 micrograms/ml) it was 0.06 microM, thus producing an approximately 6-fold increase in anti-HIV activity. In contrast, in murine bone marrow cells, ATS (4 micrograms/ml) showed significant protection (p less than 0.05) against AZT-induced toxicity as measured by CFU-E and CFU-GM assays. The IC50 values in the presence and absence of ATS for CFU-E were 3.7 and 1.5 microM, whereas for CFU-GM were 6.0 and 2.7 microM, respectively. Overall, these data suggest that AZT in combination with ATS has greater therapeutic efficacy against HIV-1.

A pro-drug of zidovudine with enhanced efficacy against human immunodeficiency virus.

In an attempt to alleviate the drug-related toxicity of zidovudine in patients with AIDS, a pro-drug of zidovudine, 5'-[(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)oxy]-3'-azido-2',3'- dideoxythymidine (DP-AZT), has been evaluated. Cellular uptake by H9 cells and peripheral blood lymphocytes (PBL) with zidovudine and DP-AZT showed at least a 50% greater intracellular concentration of DP-AZT within 2 hr. DP-AZT was significantly less toxic to murine bone marrow cells as measured by CFU-E assay. The ED50 concentration to inhibit the production of HIV specific p24 antigen was 0.05 microM for DP-AZT whereas zidovudine required 0.125 microM. These results demonstrated that DP-AZT has a higher therapeutic ratio than zidovudine as an anti-HIV-1 agent.

Exposure of heterosexuals to human immunodeficiency virus and viremia: evidence for continuing risks in spouses of hemophiliacs.

There is risk of transmission of human immunodeficiency virus (HIV) in sexually active couples, one of whom is seropositive. However, the frequency of such HIV transmission is not known. We have surveyed a population of monogamous hemophiliacs treated with potentially-infected coagulation factor concentrates during 1980-1984. We found high titers of antibodies to HIV in 24 of 30 hemophiliacs and in four of 30 spouses. The duration of HIV exposure from unprotected sexual intercourse ranged from greater than 12 to 78 months. The acquired immunodeficiency syndrome (AIDS) developed in six hemophiliac husbands, and one seropositive wife has lymphadenopathy. We were concerned that viremia with HIV might be the primary determinant of transmission to the men's wives. Circulating HIV was found in all of four hemophiliacs with AIDS, both of two with AIDS-related complex (ARC), four of 14 asymptomatic hemophiliacs, and two of four seropositive spouses. Isolation of HIV was less likely from asymptomatic hemophiliacs (29%) than from asymptomatic seropositive men (71%) in other high-risk groups. Our studies suggest that HIV was transmitted to 17% of the spouses of hemophiliacs. Efforts to educate all such couples about the risk of HIV infection remain imperative.

Immunologic assessment of a cluster of asymptomatic HTLV-I-infected individuals in New Orleans.

PURPOSE: Although clusters of individuals infected with the human T-cell lymphotrophic virus type I (HTLV-I) have been identified in the United States, no systematic evaluation of the immunologic status of these persons has been reported. We therefore studied a group of 11 HTLV-I-infected former intravenous drug abusers who were long-term participants in a methadone maintenance program in New Orleans, Louisiana, to determine the effects of HTLV-I and chronic opiate use on immunity. PATIENTS AND METHODS: Mitogenic responses and results of serologic studies, cell phenotype analysis, and cytotoxicity assays were compared to those in two other HTLV-I seronegative groups: a similar group of 17 methadone users and 15 healthy age-, sex-, and race-matched control subjects. All study participants were seronegative for human immunodeficiency virus type 1. RESULTS: Percentages and numbers of total T lymphocytes (CD2+,CD3+), T-suppressor/cytotoxic lymphocytes (CD8+), cytotoxic lymphocytes (Leu7+, Leu11+, NKH-1+) and B lymphocytes (B4+) were similar among the study groups. Although percentages and numbers of total T-helper lymphocytes (CD4+) were also similar among the groups, HTLV-I-infected subjects had higher percentages and proportions of helper/inducer cells (CD4:4B4+) than did HTLV-I seronegative methadone users. Both methadone using groups had decreased percentages and numbers of suppressor/inducer T lymphocytes (CD4:2H4+). Major histocompatibility complex unrestricted T-cell cytotoxicity (lectin-dependent cellular cytotoxicity), natural killer cell function, and mitogenic responses to the T-cell mitogen phytohemagglutin were similar among the three study groups. Pokeweed mitogen responses were severely depressed in the HTLV-I-infected population. CONCLUSIONS: We conclude that HTLV-I infection is associated with abnormalities in T-cell-dependent B-cell proliferative responses. Furthermore, both long-term methadone use and HTLV-I infection are associated with abnormalities in the distribution of CD4+ cell subpopulations. The increase in the helper/inducer and T-cell cell populations and decrease in the pokeweed mitogenic response noted in HTLV-I-infected subjects appear to be markers for infection with this retrovirus.

Epstein-Barr virus-related herpesvirus from a rhesus monkey (Macaca mulatta) with malignant lymphoma.

A herpesvirus (RhEBV) was isolated from a lymphoblastoid cell line (LCL) that became established from a malignant lymphoma in a rhesus monkey. The predominant cell marker in the LCL was that of B lymphocytes. RhEBV-induced viral capsid (VCA) and nuclear antigens (NA) in the LCL were serologically related to similar antigens known to be induced by human Epstein-Barr virus (EBV). RhEBV was of nonhuman primate origin and was clearly differentiated from EBV in the anti-complement immunofluorescence reaction using human and non-human primate sera with antibodies to the NA induced by the respective viruses. While human sera reacted with NA induced by both EBV and RhEBV, monkey sera failed to recognize the NA induced by EBV. RhEBV-induced NA was present in nearly all the cells of a suspension prepared from the tumor tissue mass, but not in the monolayer fibroblasts derived from the tumor tissue or in the blood and lymph-node lymphocytes of clinically healthy animals. RhEBV induced in vitro transformation and establishment of LCLs from peripheral blood lymphocytes of normal rhesus and cynomolgus monkeys but not from those of 6 other non-human primate species tested. The LCLs, with predominant B-lymphocyte markers, established after treatment with RhEBV, all had evidence of the virus infection since nearly all cells in the culture expressed the virus-induced NA.

Transmissible lymphoma and simian acquired immunodeficiency syndrome in rhesus monkeys.

Four rhesus monkeys (Macaca mulatta) were inoculated with a homogenate of a cutaneous lepromatous leprosy lesion from a mangabey monkey (Cercocebus atys). One died of B-cell lymphoma, and another died of an immunodeficiency syndrome. Cell suspensions prepared from the tumor and spleen of the monkey with lymphoma induced lymphoma or an immunodeficiency syndrome when inoculated into additional young rhesus monkeys. The immunodeficiency syndrome was similar to simian acquired immunodeficiency syndrome and consisted of opportunistic infections, lymphoid hyperplasia or atrophy, wasting, and syncytial cell formation. Mitogen responses and percentages of T4- and T8-positive lymphocytes were normal until the animals were moribund. Lymphoblastoid cell lines became established in vitro from tumor cell suspensions. These cells were infected with a herpesvirus related to Epstein-Barr virus. In addition, a retrovirus morphologically similar to human T-cell lymphotrophic virus type III (HTLV-III) and simian T-lymphotrophic virus type III (STLV-III) was isolated from one of the lymphoblastoid cell lines (LCL). Type D retroviruses could not be demonstrated in the monkeys in the transmission study; however, a retrovirus similar to that in the LCL was isolated from 4 animals by coculture of peripheral blood lymphocytes with the human cell line H9. These results suggest that this retrovirus, STLV-III/Delta, may be associated with the immunodeficiency syndrome in these macaques and may be of mangabey origin.

Isolation of an HTLV-III-related retrovirus from macaques with simian AIDS and its possible origin in asymptomatic mangabeys.

Acquired immune deficiency syndrome (AIDS) has become a worldwide epidemic, so the development of vaccines and antiviral agents effective against the causative agent, human T-lymphotropic virus type III (HTLV-III), is vital. This work would be greatly simplified if a suitable animal model could be developed. Here we report the isolation of an HTLV-III-related retrovirus, STLV-III/Delta, from rhesus macaques (Macaca mulatta) with transmissible simian AIDS (SAIDS) and from asymptomatic sooty mangabeys (Cercocebus atys). SAIDS was initially diagnosed in several macaques previously inoculated with tissue homogenates of mangabey origin. Western blot analysis of both the mangabey and macaque sera demonstrated the presence of antibody cross-reactive primarily with the HTLV-III proteins p24 and p61. In a related experiment, analysis of these same sera revealed simian antibody to STLV-III/Delta proteins similar, but not identical, to those of HTLV-III with estimated relative molecular masses (Mrs) of 16,000 (16K), 26K, 35K, 45K, 60K and 110K. Infection of the mangabey, an African primate, with an HTLV-III-related virus may provide a clue to the origin of HTLV-III in humans. The apparent difference in susceptibility to SAIDS-like disease between infected macaques and mangabeys suggests that these species may respond differently to STLV-III infection.

Immunology of pyelonephritis in the primate model. II. Effect on immunosuppression.

Nonobstructive pyelonephritis was produced in the rhesus monkey (Macaca mulatta) by means of retrograde inoculation of Escherichia coli to the point of pyelotubular backflow. The effects of treatment with cyclophosphamide or azathioprine were determined. Commensal renal viruses were not activated by the immunosuppression, and thus did not complicate our results. Both cyclophosphamide and azathioprine prolonged the bacteriuria and produced more severe pathology. Cyclophosphamide decreased the leukocytic response and partically suppressed the antibody response. The increased amount of acute pyelonecytic response and partially suppressed the antibody response. The increased amount of acute pyelonephritic lesions after treatment with this drug suggest that the antibody and inflammatory responses may be important protective mechanisms, particularly regarding prevention of abscesses. In contrast, azathioprine did not decrease the leukocytosis nor the antibody responses, but resulted in decreased in vitro responsiveness of lymphocytes to mitogens. The increased severity of chronic pyelonephritic lesions after azathioprine treatment suggests that the cellular immune response also may be an important protective mechanism during late stages of the disease. The results thus indicate that the immune response is protective and is not directly responsible for the chronic scarring of pyelonephritis.

Virus associated papillomas in colobus monkeys (Colobus guereza).

Numerous papillomas ranging in size from 1--10 mm were seen in four colobus monkeys (Colobus guereza). The lesions were confined to the fingers and toes and to the dorsal-lateral aspects of the hands and feet distal to the carpus and tarsus. Electron microscopy of two of the lesions revealed compactly arranged crystalline arrays of intranuclear virus particles in the epidermis. The particles were seen only in cells of the stratum granulosum and stratum corneum. By the negative stain technique, the non-enveloped spherical particles measured 52--58 mm in diameter. The arrangement of the capsomeres was consistent with an icosahedral symmetry. The observations suggested a viral etiology for the cutaneous papillomas similar to that of other mammalian species.

Social and reproductive behaviors in surrogate-reared squirrel monkeys (Saimiri sciureus).

A colony of 16 surrogate-reared squirrel monkeys was observed for the adequacy of social and reproductive behaviors at maturity. Aside from some self-directed abnormalities centering around nonnutritive orality, this group was behaviorally normal in spite of having no contacts with mother-reared, older conspecifics. Half (N=5) of the females gave birth, with four infants viable. One infant has remained with the colony and is thriving. The other three remained with the group for as long as 42 days before death or removal. Maternal care appeared adequate. Mean estimated conception age for females was 35 months, and the mean impregnation age for males was estimated to be 42 months.

Herpesvirus saimiri-induced lymphoproliferative disease in howler monkeys.

Four of 5 howler monkeys (Alouatta caraya) experimentally infected with Herpesvirus saimiri (HVS) developed a rapidly fatal malignant lymphoma accompanied by peripheral T-cell lymphocytosis. HVS was isolated from fresh and tissue cultured blood and tissue lymphocytes and from cell cultures derived from nonlymphoid organs. Humoral antibodies against HVS-induced antigens were detected in the sera of the animals. The in vitro response of the peripheral blood lymphocytes to mitogenic stimulants was depressed following HVS infection.

Congenital transmission of a papovavirus of the stump-tailed macaque.

Stump-tailed macaque virus, a newly recognized papovavirus of the SV40 polyoma subgroup, was demonstrated in kidney cultures from each of five stump-tailed macaque fetuses in the second half of gestation and from six adult stump-tailed macaques. Such regular presence of virus in the fetus is an unusual feature for a papovavirus.