Sperm ubiquitination in patients with dysplasia of the fibrous sheath.

BACKGROUND: Human sperm with structural abnormalities display an increased content of the cellular proteolytic marker peptide, ubiquitin. We investigated whether dysplasia of the fibrous sheath (DFS), a severe structural anomaly found in the sperm of some asthenozoospermic patients, is accompanied by (i) increased ubiquitination of the sperm surface and (ii) by increased ubiquitination of the sperm mitochondria. METHODS AND RESULTS: Five DFS patients and eight fertile donors were studied by immunocytochemistry with anti-ubiquitin antibodies. Increased cross-reactivity of the ubiquitinated mitochondrial epitopes was seen in 32-50% of DFS sperm, but only 2-4.1% of sperm from fertile donors. Sperm surface ubiquitination assessed by sperm-ubiquitin tag immunoassay (SUTI) and immunofluorescence demonstrated an increased sperm ubiquitination in all DFS patients. The average median value of ubiquitin-induced fluorescence in DFS patients was 25.8 counts (range 19.8-37.9), as opposed to 13.4 counts range (9.3-16.6) in fertile men. Sperm with 'stump tails', coiled tails, twin and triplet sperm, and clusters of immature spermatogenic cells were common. CONCLUSIONS: DFS sperm have increased cross-reactivity to anti-ubiquitin antibodies, a finding consistent with the ubiquitination of defective sperm shown in animal models. These results justify the use of ubiquitin-based assays for objective semen analysis in infertile men with heritable defects.

Induction of B cell hyperplasia in simian immunodeficiency virus-infected rhesus macaques with the simian homologue of Kaposi's sarcoma-associated herpesvirus.

A simian homologue of Kaposi's sarcoma-associated herpesvirus (KSHV), the eighth human herpesvirus (HHV8), was isolated from a simian immunodeficiency virus (SIV)-infected rhesus macaque (Macaca mulatta) that developed a multicentric lymphoproliferative disorder (LPD). This simian rhadinovirus is genetically similar to a recently described rhesus rhadinovirus (RRV) (Desrosiers, R.C., V.G. Sasseville, S.C. Czajak, X. Zhang, K.G. Mansfield, A. Kaur, R.P. Johnson, A.A. Lackner, and J.U. Jung. 1997. J. Virol. 71:9764-9769) and is designated RRV 17577. RRV 17577 was experimentally inoculated into rhesus macaques with and without SIV(mac239) infection to determine if RRV played a role in development of the LPD observed in the index case. In contrast to control animals inoculated with SIV(mac239) or RRV alone, two animals coinfected with SIV(mac239) and RRV 17577 developed hyperplastic LPD resembling the multicentric plasma cell variant of Castleman's disease, characterized by persistent angiofollicular lymphadenopathy, hepatomegaly, splenomegaly, and hypergammaglobulinemia. Hypergammaglobulinemia was associated with severe immune-mediated hemolytic anemia in one RRV/SIV-infected macaque. Both RRV/SIV-infected macaques exhibited persistent RRV viremia with little or no RRV-specific antibody response. The macaques inoculated with RRV alone displayed transient viremia followed by a vigorous anti-RRV antibody response and lacked evidence of LPD in peripheral blood and lymph nodes. Infectious RRV and RRV DNA were present in hyperplastic lymphoid tissues of the RRV/SIV-infected macaques, suggesting that lymphoid hyperplasia is associated with the high levels of replication. Thus, experimental RRV 17577 infection of SIV-infected rhesus macaques induces some of the hyperplastic B cell LPDs manifested in AIDS patients coinfected with KSHV.

Rhesus rhadinovirus establishes a latent infection in B lymphocytes in vivo.

Recent DNA sequence analysis indicates that rhesus rhadinovirus (RRV) is a member of the lymphotropic gamma-2 herpesvirus family. To determine if RRV is lymphotropic, peripheral blood mononuclear cells from naturally infected monkeys were separated by immunomagnetic bead depletion and analyzed for the presence of RRV by virus isolation and nested PCR. The recovery and consistent detection of RRV in the CD20(+)-enriched fraction clearly demonstrates that B lymphocytes are a major site of virus persistence.

Molecular cloning and cell-specific growth characterization of polymorphic variants of type D serogroup 2 simian retroviruses.

Simian retroviruses (SRVs), the etiological agent of a spontaneous Simian acquired immunodeficiency syndrome, endemically infects large percentages of Asian macaques housed in biomedical research colonies and severely compromises the effective use of these species as a viable research animal. We recently described the molecular cloning of a serogroup 2 SRV, D2/RHE/OR, which causes mild immunosuppression in rhesus macaques. A restriction site variant, D2/RHE/OR/V1, has also been recovered from severely ill animals endemically infected with D2/RHE/OR. We now report the complete nucleotide sequences of D2/RHE/OR and D2/RHE/OR/V1. Both infectious molecular clones retain the genetic structure typical of type D SRVs (5' LTR-gag-prt-pol-env-3'LTR) and encode identically sized 8105-bp proviruses. D2/RHE/OR and D2/RHE/OR/V1 are 99.3% similar at the amino acid level, exhibiting only 17 residue differences, of which 10 are located in the envelope glycoproteins. The molecular clones and reciprocal chimeric viruses were used to assess the contribution of different genetic domains to virus infectivity in a T cell infection assay. These experiments indicate that D2/RHE/OR has a reduced ability to infect specific T cell lines, especially Hut-78 and MT-4 cells, and that the envelope gene is not the sole determinant of in vitro tropism.

Simian AIDS type D serogroup 2 retrovirus: isolation of an infectious molecular clone and sequence analyses of its envelope glycoprotein gene and 3' long terminal repeat.

We describe the molecular cloning of a serogroup 2 simian retrovirus (SRV; D2/RHE/OR) and present the sequence of its envelope (env) glycoprotein gene and 3' long terminal repeat region. This report documents the first infectious molecular clone of a serogroup 2 SRV and provides env sequence verification of genetic diversity among serogroup 2 SRV isolates.

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.

Decline in fluorescent low density lipoprotein (LDL) uptake by small and large porcine luteal cells with advancing age of the corpus luteum.

The present study was designed to test the hypothesis that the ability of small luteal cells (SLC) and/or large luteal cells (LLC) to take up low density lipoprotein (LDL) declines with advancing age of the CL. Ovaries from 100-110-kg gilts were classified as early (Days 4-6; n = 5), mid (Days 8-12; n = 6)-, or late (Days 15-18; n = 5) cycle on the basis of gross morphology. Multiple CL from each ovary were pooled and enzymatically dissociated. An aliquot of dispersed luteal cells was reserved for cell culture. Remaining cells were incubated (approximately 4 x 10(5) cells/0.25 ml Dulbecco's Modified Eagle's Medium [DMEM] + 0.1% BSA) for 20 min at 37 degrees C with human LDL (10 micrograms/ml) tagged with the fluorescent probe, Dil (Dil-LDL). Washed and fixed cells were then isolated by flow cytometry into SLC and LLC subpopulations on the basis of forward and 90 degrees light scatter. Cellular fluorescence was analyzed within each subpopulation. The percentage of fluorescent, i.e., Dil-LDL-positive (+), SLC did not differ between early (29.8 +/- 5.9%) and mid (40.5 +/- 6.8%)-cycle, but declined (p < 0.01) in late CL (7.0 +/- 1.6%). Similarly, the percentage of Dil-LDL-(+) LLC was unchanged between early (80.5 +/- 2.0%) and mid (78.6 +/- 4.2%)-cycle, but diminished (p < 0.01) in late (40.2 +/- 1.9%) CL. Moreover, the percentage of total cells isolated in the LLC subpopulation declined dramatically (p < 0.01) between mid (8.0 +/- 0.9%)- and late (1.6 +/- 0.2%) cycle, but the percentage of SLC did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of ovine luteal cell subpopulations by flow cytometry.

Differences in the characteristics of small and large luteal cells, as reported by various laboratories, may be due to species diversity and/or methodological differences in cell preparation. To evaluate whether the method of cell separation affects the properties of luteal cell subpopulations, we sorted and characterized sheep luteal cells by flow cytometry via methods previously used to investigate luteal cell subtypes from the macaque corpus luteum. Corpora lutea were obtained from superovulated ewes on Day 10 after hCG injection and enzymatically dissociated. Dispersed cells were shipped overnight on ice from the University of Arizona to the Oregon Regional Primate Research Center. Viability of cells upon arrival was > or = 80%. When dispersed cells were analyzed by flow cytometry based on forward and 90 degrees light scatter, three distinct subpopulations (P1, P2, P3) were identified. In P1, 35.5 +/- 2.1% of cells, most (97.0 +/- 0.6%; n = 3) of which were 15-22 microns in diameter, stained positive (+) for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. The remainder of P1 cells were 3 beta-HSD negative and < or = 22 microns. The size distribution of P2 was similar to that of P1, but P2 contained more (53.3 +/- 4.2%; n = 4) 3 beta-HSD (+) cells. P3 consisted mostly (88.5 +/- 4.6%; n = 3) of 3 beta-HSD (+) cells > 25 microns in diameter. Cell subpopulations were incubated (n = 6) at 37 degrees C for 3 h with or without hCG (0.1-100 ng/ml), prostaglandin E2 (PGE2; 500 ng/ml), or dibutyryl (db)-cAMP (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin surge increases fluorescent-tagged low-density lipoprotein uptake by macaque granulosa cells from preovulatory follicles.

In the primate ovary, luteal steroidogenesis is largely dependent upon cholesterol derived from receptor-mediated uptake of circulating low-density lipoprotein (LDL). However, granulosa cells (GC) of preovulatory follicles possess few LDL binding sites compared to those present in developing and mature corpora lutea. We recently reported (Endocrinology 1991; 129:3247-3253) that uptake of LDL tagged with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) can be monitored in macaque luteal cells by fluorescence-activated flow cytometry. This study was designed to determine whether an ovulatory stimulus induced uptake of DiI-LDL in GC aspirated from preovulatory follicles of macaques undergoing ovarian stimulation. Development of multiple large follicles was stimulated in adult rhesus macaques with human gonadotropin treatment for 9 days. On Day 10, monkeys received either no ovulatory stimulus or 1000 IU hCG to initiate ovulatory events. GC were aspirated on Day 10 in monkeys receiving no ovulatory stimulus (nonluteinized GC) or 27 h or 34 h after hCG injection (luteinizing GC). GC were resuspended in Ham's F-10 medium + 0.1% BSA and incubated with several concentrations (0-25 micrograms/ml) of DiI-LDL (Biomedical Technologies, Stoughton, MA) for various time intervals (2-60 min). DiI-LDL uptake by GC was time- and concentration-dependent. Coincubation of cells with DiI-LDL and unlabeled LDL dose-dependently suppressed the percentage of fluorescent cells. In contrast, coincubation with up to a 250-fold excess of acetylated LDL or high-density lipoprotein did not alter the percentage of fluorescent GC.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential uptake of fluorescent-tagged low density lipoprotein by cells from the primate corpus luteum: isolation and characterization of subtypes of small and large luteal cells.

Three enriched populations of cells [C alpha, non-steroidogenic cells less than or equal to 15 microns in diameter; R1, small (less than or equal to 15 microns) steroidogenic cells; and R3, large (greater than 20 microns) steroidogenic cells] have been isolated from the macque corpus luteum using flow cytometry based on light scatter properties. To determine whether the cell populations differ in their ability to bind and internalize low-density lipoprotein (LDL), collagenase-dispersed cells were prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. Cells were incubated in Hams F-10 medium containing fluorescent-tagged LDL (DiI-LDL). Optimal labeling occurred at 10 micrograms DiI-LDL/10(6) cells.ml incubated for 20 min at 37 degrees C. Labeled cells were analyzed and sorted by flow cytometry based on light scatter and fluorescence. Only 8.2 +/- 1.0% (n = 10) of C alpha cells exhibited fluorescence intensity greater than the autofluorescence of unlabeled cells. In contrast, 52.3 +/- 3.4% of R1 cells and 83.9 +/- 2.3% of R3 cells were fluorescent. Uptake of DiI-LDL was competitively inhibited when cells were conincubated with either unlabeled monkey or human LDL, but human high-density lipoprotein and very low-density lipoprotein were less effective. Progesterone (P) production by fluorescent [DiI-LDL(+)] R1 luteal cells was increased (P less than 0.001) in the presence of pregnenolone, but not human (h) CG, consistent with earlier results for the R1 population. Surprisingly, basal P production by the nonfluorescent [DiI-LDL(-)] R1 cells was similar to that by fluorescent cells and was stimulated by both hCG (P less than 0.01) and pregnenolone (P less than 0.001). Basal P production by DiI-LDL(+) R3 cells was nearly 10-fold greater than that by DiI-LDL(-) R3 cells. P secretion by both DiI-LDL(+) and (-) R3 cells was stimulated by hCG (P less than 0.01) and pregnenolone (P less than 0.001). DiI-LDL(-) C alpha cells produced barely detectable levels of P, but DiI-LDL(+) C alpha cells secreted P at levels similar to R1 cells. We conclude that: 1) multiparameter (light scatter and fluorescence) cell sorting is a useful method for separating enriched populations of cells from the corpus luteum; and 2) small and large luteal cell populations from the macaque corpus luteum consist of subtypes of steroidogenic cells that differ in lipoprotein uptake and/or gonadotropin sensitivity.

Predominance of T cells that express CD45R in the CD4+ helper/inducer lymphocyte subset of neonates.

Neonates have an increased risk of severe infections. For several in vitro and in vivo immune responses, neonates have been shown to have significant differences when compared to normal adults. To indirectly study immune cellular defects, we compared cell surface markers on cord blood lymphocytes (CBL) from 58 term infants to peripheral blood lymphocytes (PBL) from 17 healthy adults using flow cytometry with standard as well as newly defined monoclonal antibodies (Mab) that distinguish regulatory T cells. CBL had significantly smaller percentages of lymphocytes that express the CD2 and CD8 markers (total T cells, and suppressor/cytotoxic T cells, respectively), although absolute numbers of CD2+ and CD8+ cells were comparable in neonates and adults. CBL and PBL were similar in terms of the percentage of CD4+ cells (helper/inducer T cells), although the absolute numbers of CD4+ cells were higher in CBL than in PBL. The CD4+ population was subdivided into cells bearing the virgin and memory T cell phenotypes using anti-2H4 and anti-4B4 Mab and dual parameter analysis with anti-CD4. Neonates were deficient in the percentage of CD4+, 4B4+ (3.8 +/- 2.8 vs 13.4 +/- 7.5, P less than 0.001), but equivalent to adults in the percentage of CD4+, 2H4+ T cells (21.4 +/- 9.8 vs 18.8 +/- 12.8). In absolute numbers, neonates had fewer CD4+, 4B4+ cells (178 +/- 173 vs 344 +/- 152 cells/microliters, P less than 0.001), but more CD4+,2H4+ cells (978 +/- 572 vs 542 +/- 518 cells/microliters, P less than 0.01) than adults. The predominance of 2H4+ virgin T cells in the CD4 population whose function is associated with that of the induction of suppression rather than the up-regulation of immune responses may contribute to the observed susceptibility of neonates to infection.

Isolation and characterization of cell subpopulations from the monkey corpus luteum of the menstrual cycle.

This study was designed to test the hypothesis that the corpus luteum of primate species consists of cell subpopulations that differ in physical characteristics, function, and regulation by endocrine and paracrine factors. The corpus luteum (n = 25) was removed from rhesus monkeys at the mid-luteal phase of the menstrual cycle (Days 7-8 after the surge of luteinizing hormone, LH) and enzymatically dispersed. Freshly dispersed cells were analyzed and sorted on the basis of their forward and 90 degrees light scatter (FLS and 90LS, respectively) properties using an EPICS C flow cytometer. Freshly dispersed and sorted cells were fixed, stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and measured to determine their diameters. Freshly dispersed (MIX) and sorted cells from corpora lutea during the early (Days 4-5 after the LH surge; n = 4) and mid-luteal phases of the cycle were incubated in vitro and steroid production was assessed. The size distribution of dispersed cells revealed four peaks that corresponded to small (10-15 microns in diameter) 3 beta-HSD-negative, and small, medium (16-20 microns), and large (greater than 20 microns) 3 beta-HSD-positive cells. Analysis of dispersed cells for FLS and 90LS demonstrated two continua (C alpha and C beta). C alpha contained single cells and cell clusters; 99.7 +/- 0.3% (n = 3) of the cells were less than or equal to 15 microns in diameter and 96.7 +/- 0.3% were 3 beta-HSD-negative. C alpha cells produced low levels of progesterone (0.2 +/- 0.1 ng/ml per 5 x 10(4) cells; n = 3) in vitro under basal conditions. C beta consisted of single cells from 10 microns to 40 microns in diameter and contained the lipid-filled and 3 beta-HSD-positive cells. Two regions (R1 and R3) of C beta were defined and their cells separated. In R1, 96 +/- 2% (n = 3) of the cells had diameters of less than or equal to 15 microns, whereas 82 +/- 4% (n = 3) of those in R3 were greater than or equal to 20 microns. Basal progesterone production by R3 cells from early luteal phase of the cycle was 12 times greater than that by R1 cells (n = 3 per group).(ABSTRACT TRUNCATED AT 400 WORDS)

Flow cytometric analysis of I-J expression on murine bone marrow-derived macrophages.

Attempts to analyze bone marrow-derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I-A, I-J, and Mac-1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50-60% Mac 1-positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1-positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I-J until these cells have been in culture 3 to 4 days, and the number of cells expressing I-J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I-A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I-A, and only 20% of peritoneal cells had I-J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I-J positive (up to 70%), and only about 30% of these cells expressed I-A cell surface antigens. The binding of anti-I-J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 micrograms of an IgG2a myeloma protein did not block anti-I-J antibody binding. The addition of 25-200 micrograms of monoclonal anti-Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti-I-Jk antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti-I-J IgM antibody. BMDM provide a pure population of macrophages that express a significant level of cell surface I-J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I-J-positive cells parallels the increase in macrophages in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Simian retrovirus D serogroup 1 has a broad cellular tropism for lymphoid and nonlymphoid cells.

Simian acquired immunodeficiency syndrome is a fatal immunosuppressive disease caused by type D retroviruses such as simian acquired immunodeficiency syndrome retrovirus type 1 (SRV-1). The disease is characterized by generalized lymphadenopathy, opportunistic infections, and lymphoid depletion with defects in both humoral and cell-mediated immunity. To understand how SRV-1 infection relates to the immune defect, we studied in vivo-infected lymphocytes from SRV-1-positive macaques with and without clinical signs of immunosuppressive disease. B and T helper/inducer and T suppressor/cytotoxic lymphocytes were purified by panning or by flow cytometry. Neutrophils were purified by dextran sedimentation, and platelets were purified by low-speed centrifugation. In vitro infection studies were also done with HUT78, H9, K562, rhesus lung fibroblast, rhesus monkey kidney, and bat lung cells. SRV-1 in lymphocytes or culture supernatants was detected by the induction of syncytia in cocultivated Raji cells and was confirmed by immunofluorescence, electron microscopy, or reverse transcriptase assay. We found that B and T helper/inducer lymphocytes were infected in all animals tested. The number of infected T suppressor/cytotoxic cells was generally lower than that of the other cell subsets, and not all animals in this subset had SRV-1 infections. All other cells exposed in vitro to SRV-1, except bat lung cells, were able to be infected. These findings show that SRV-1 has a broad cell tropism for lymphoid and nonlymphoid cell types.

The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells.

Cultures of normal spleen cells with anti-idiotypic antibody (anti-Id) or antigen B (AgB)-specific T suppressor factor (Tsf1) in mini-Marbrook chambers for 4 days at 37 degrees C lead to the in vitro induction of AgB-specific T suppressor (TS) cells. These TS cells significantly suppress a secondary AgB-specific IgE response, but they do not affect a secondary AgB-specific IgG response. Depletion of both B cells and macrophages from normal spleen cells by panning on anti-Ig-coated petri dishes provides an enriched T cell population. These enriched T cells when cultured with anti-Id or Tsf1 in mini-Marbrook chambers do not produce AgB-specific TS cells, and mice treated with cells harvested from the mini-Marbrook chambers have normal secondary AgB-specific IgG and IgE responses. The addition of as few as 1000 bone marrow-derived macrophages (BMDM) to cultures of the enriched T cells with anti-Id, or Tsf1 restores the ability of these cultures to produce significant levels of AgB-specific TS cells. Further studies reveal that the macrophage population must be histocompatible and express a cell surface I-J antigen. Attempts to pulse BMDM with anti-Id or Tsf1 at 4 degrees C and to culture in mini-Marbrook chambers 10(3) pulsed BMDM with enriched T cells were unsuccessful in producing AgB-specific TS cells. However, pulsing BMDM with anti-Id or Tsf1 at 37 degrees C, and adding 10(3) of these pulsed BMDM to enriched T cells in culture led to the formation of significant levels of AgB-specific TS cells.

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.

Neutralizing antibody prevents type D retrovirus viremia in Celebes black macaques.

The Celebes black macaque (Macaca nigra) colony at the Oregon Regional Primate Research Center has a high incidence of simian acquired immunodeficiency syndrome (SAIDS-RF) that may be caused by type D retrovirus type 2 (SRV-2). During the spring and autumn screening of the colony, seven monkeys previously aviremic were found to be viremic on the basis of the Raji co-culture assay. These monkeys and control groups were selected for further study, which included titration of neutralizing antibody activity and immunofluorescent antibody (IFA) activity before and at the time that the animals became viremic. Results indicated that neutralizing antibody was not present before or at the time that monkeys became viremic and that control monkeys who were IFA+ and did not become viremic had high levels of neutralizing antibody. The IFA titre did not change significantly or predictably at the time the animals became viremic.

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.

Relationship of mitogen reactivity to type D retrovirus infection in Celebes black macaques (Macaca nigra).

The Celebes black macaque (Macaca nigra) colony at the Oregon Regional Primate Research Center has a high incidence of an immunodeficiency syndrome characterized by recurrent diarrhea and the development of retroperitoneal fibromatosis (RF). We have examined the relationship of type D viral infection to the immunodeficiency syndrome by surveying the colony for viral infection and for mitogen reactivity. Type D virus-positive monkeys (28% of the colony) have a higher prevalence of diarrhea, splenomegaly, lymphadenopathy and weight loss than do virus-negative monkeys, and RF has been found to occur only in virus-positive animals. Comparison of the concanavalin A (con-A) and phytohemagglutinin reactivities of the virus-positive and -negative populations has revealed no significant difference. However, within the virus-positive population, those with RF have reduced con-A reactivity and there are both high and low mitogen responders in the groups lacking RF. Thirty-two percent of the virus-positive monkeys are free of clinical symptoms, 40% have clinical symptoms but no RF, and 27% have clinical symptoms and RF. Five of the six monkeys with RF are older than the RF-free monkeys but monkeys are susceptible to type D retrovirus infection regardless of age or sex. The progressive nature of this immunodeficiency syndrome, its broad age range, and the probability that the etiological agent is also a type D retrovirus and the similarity of RF to Kaposi's sarcoma make this a potentially useful model for human AIDS.