Stanley M. Shiigi

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.

Virus-associated deficiencies in the mitogen reactivity in celebes black macaques (Macaca nigra)

Celebes black macaques (Macaca nigra) with a history of diabetes mellitus, recurrent bacterial and protozoal infections, diarrhea, anemia, weight loss, anorexia, and a high mortality were studied to determine their immune status. Two groups of monkeys, healthy and unhealthy, were formed on the basis of a clinical assessment. The proliferative response and the pokeweed-mitogen-induced polyclonal IgG response of peripheral blood mononuclear cells of unhealthy monkeys were significantly less than the responses of healthy monkeys. The percentage of HLA-DR+ cells varied greatly in unhealthy monkeys. The OKT4/OKT8 ratios of unhealthy monkeys were generally greater than the ratios of healthy monkeys. Unhealthy monkeys usually had smaller percentages of OKT8+ cells than did healthy monkeys. The two groups of monkeys were examined for the presence of a syncytial forming retrovirus by a coculture assay involving Raji cells, a human B lymphoblastoid cell line. A type D retrovirus was detected in the unhealthy group but not in the healthy group. Retroperitoneal fibromatosis was detected in several monkeys in the unhealthy group.

Adrenergic Modulation of Apoptosis in Splenocytes of Xenopus laevis in vitro

Impaired and healthy cells undergo suicide using an intrinsic genetic program. Exposure to stress-related α2- or β-adrenergics for 4 or 20 h in vitro had no effect on apoptosis in splenocytes of adult Xenopus laevis, while a 4-hour coincubation of clonidine, an α2-agonist, with a calcium ionophore (A23187) or a phorbol diester (PMA), enhanced apoptosis induced by each apoptogen alone. Clonidine did not affect apoptosis stimulated with dexamethasone (DEX), however. Comparable in vitro exposures to isoproterenol, a β-agonist, reduced apoptotic levels stimulated by all three apoptogens alone. Following 20 h coexposure, clonidine no longer affected A23187-induced apoptosis, but reduced PMA-induced apoptosis, while isoproterenol enhanced apoptosis stimulated with both. Neither agonist modulated apoptosis induced by 20 h of exposure to DEX. Thus, adrenergic agonists modulated apoptosis in cells coexposed to A23187 and PMA, in a time-dependent and adrenoceptor class-dependent fashion. These stress-induced products can affect concurrent apoptosis reversibly over time in vitro, and thus possibly in vivo.

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). Env PCR fragments were readily detected from as few as 10(4) 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.

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.

A monoclonal mouse anti-human IL-2 receptor antibody (anti-Tac) will recognize molecules on the surface of Xenopus laevis immunocytes which specifically bind rIL-2 and are only slightly larger than the human Tac protein

We have made visible the binding of a mouse monoclonal anti-human interleukin 2 (IL-2) receptor (anti-Tac) antibody on the surface of phytohemagglutinin (PHA)-stimulated Xenopus thymocytes using a colloidal gold-conjugated goat anti-mouse antibody and transmission electron microscopy. No binding was found when a different mouse monoclonal antibody (mAb) of the same isotype and subclass was tested, or when the anti-Tac antibody was omitted from the procedure. After metabolic radiolabeling of the IL-2 receptors with [35S]methionine using PHA-stimulated thymocytes of Xenopus laevis, the South African clawed toad, we show that a concentrated preparation of the mouse anti-human Tac antibody will immunoprecipitate a radiolabeled molecule just slightly larger than 55 kDa. Phorbol dibutyrate (PDB), an effective T cell mitogen, and cyclosporin A, an inhibitor of T cell mitogenesis in this species, are both capable of regulating the expression of this IL-2-binding molecule on Xenopus immunocytes. Here, we use the calcium ionophore A23187 to show that the relationship between IL-2 receptor expression and mitogenesis, which was previously established in X. laevis, is associated with a calcium ion flux. Flow cytometry is used for assaying alterations in epitope expression after binding the lectin-stimulated cells under test with a fluorescence (Fl*) conjugate of the anti-Tac antibody or a control mAb, which is either anti-DNP or anti-keyhole limpet hemocyanin (KLH) in specificity, but of the same mouse isotype and subclass as the anti-IL-2 receptor antibody.

Neutralizing antibody in Celebes black macaques recovering from infection with simian acquired immunodeficiency syndrome retrovirus type 2

Neutralizing antibodies that block the ability of simian acquired immunodeficiency syndrome (SAIDS) retrovirus type 2 (SRV-2) to induce syncytium formation in cultures of Raji cells have been found in the serum of nonviremic Celebes black macaques (Macaca nigra). Serum from Celebes macaques that are viremic have little or no neutralizing activity. The neutralizing antibodies were shown to block viral infectivity. The group of monkeys with neutralizing antibodies in their serum exhibited a dramatic improvement in their health from 1982 to 1984. The correlation of neutralizing antibodies with clinical improvement suggests that neutralizing antibodies may play a critical role in limiting the pathogenic effects of SAIDS retrovirus infection and in helping eliminate the infection.