Rhesus monkeys protected against Plasmodium knowlesi malaria produce antibodies against a 65,000-MrP. knowlesi glycoprotein at the surface of infected erythrocytes.

Sera from 27 rhesus monkeys immunized in various ways against the H strain of Plasmodium knowlesi were analyzed by quantitative crossed immunoelectrophoresis. The reaction of the sera was compared with a reference immune serum only reactive with P. knowlesi-specific 65,000-Mr glycoprotein-immune component 13 (gp65/ic13) in membranes of infected rhesus monkey erythrocytes. Triton X-100-solubilized, 125I-labeled membranes of schizont-infected erythrocytes were used as an antigen. Sera from 9 or 10 monkeys immunized by repeated infections with P. knowlesi reacted with gp65/ic13. In 6 of 10 sera, anti-gp65/ic13 was the only antibody reacting with host cell membrane proteins. In contrast, vaccination of 15 monkeys with predominantly sexual stages or trophozoites of P. knowlesi in Freund complete adjuvant resulted in protection against blood challenges in 7 monkeys, only 2 of which contained precipitating antibody against gp65/ic13. None of the sera from monkeys not protected by infections or vaccinations contained detectable levels of precipitating antibodies against gp65/ic13. Our data indicate that gp65/ic13 acts as a prominent immunogen in vivo during natural p. knowlesi infections of rhesus monkeys. There is a positive correlation suggested between anti-gp65/ic13 antibody and protection in the monkeys analyzed. This correlation does not apply to monkeys protected against P. knowlesi malaria by vaccination, pointing to other effective immune defense mechanisms.     

Identification of antibody epitopes in the CB-11 peptide of bovine type II collagen recognized by sera from arthritis-susceptible and -resistant rhesus monkeys.

Sera from eight rhesus monkeys that had been immunized with native bovine type II collagen were tested for antibodies to cyanogen bromide peptides (CB peptides) of type II collagen by Western blotting. The monkeys produced IgG antibodies to a number of different CB peptides, with five out of eight animals producing antibodies to the CB-11 peptide (four arthritic, one non-arthritic). Antibody epitopes on the CB-11 peptide of bovine type II collagen recognized by these sera were investigated by epitope mapping. Peptides (8-mers overlapping by seven amino acids) representing the CB-11 region were synthesised and the sera screened for binding to these peptides to determine areas of high IgG antibody binding to this region of type II collagen. The profiles obtained were not identical, though there were some epitopes that were commonly recognized. Antibodies to one epitope, also present in human type II collagen, were found only in the sera of two animals with the severest arthritis. The technique of epitope mapping has successfully identified a number of epitopes within the CB-11 peptide of type II collagen recognized by antibodies from bovine type II collagen-immunized monkeys. Studies on the relevance of responses to the identified epitopes can now be undertaken.     

Effect of gonadotropins on ovulation and ovarian histology in the immature mongolian gerbil

The present study is an analysis of the effects of super-ovulatory doses of gonadotropins on the rate and time of ovulation and ovarian histology in immature gerbils. Groups were treated with various combinations of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG). The maximum superovulatory response followed treatment with 10 IU PMSG and 20 IU HCG. High dosages of PMSG inhibited superovulation, as did some combinations involving the highest dose of HCG. Entrapment of ova within corpora lutea was common in groups receiving high doses of either gonadotropin. Luteal regression, appearing by day 3, occurred often in groups receiving high doses of HCG. A dose of 10 IU PMSG and 5 IU HCG resulted in both a near maximal superovulatory response and the least abnormal ovarian alteration.     

Transplacental Alloimmunization to Fetal Erythrocytes in Rhesus Monkeys (Macaca mulatta)

ABSTRACT: Evidence of transplacental immunization in rhesus monkeys was obtained by testing postpartum sera from 252 females for antierythrocyte agglutinins. One-third of the sera contained antibodies reactive with the mates' or the newborns' cells. Fetal erythrocytes were detected in the maternal circulation as early as 8 weeks after conception and as late as 12 days postpartum. Maternal antibodies were detected as early as 2 months after conception and persisted for more than 3 weeks postpartum. It was concluded that the fetal cells stimulated antibody production. Several features of transplacental immunization differ between rhesus monkeys and humans. A parity effect was not observed in rhesus. In fact, 33% of primiparous rhesus females produced antibodies. Also, several of the different allogeneic blood group factors appeared to be immunogenic but differed in immunopotency. Finally, direct antiglobulin tests indicated that erythrocytes of 11% of newborns were coated with maternal antibodies. Nevertheless, in contrast to humans, hemolytic disease was not observed.     

Association of unexplained infertility with gonadotropin and ovarian antibodies

PROBLEM: To determine the prevalence and characteristics of gonadotropin autoantibodies (GAB) associated with unexplained infertility, and to assess the relationship between ovarian autoantibodies (OVAB) and GAB. METHOD OF STUDY: Ovarian antibodies and GABs in sera of patients with unexplained infertility (n = 53) and a comparison (population) group from a blood bank (n = 40) were detected by immunoassay. Patients with unexplained infertility had either no prior gonadotropin treatment (n = 15) or two or more gonadotropin cycles to induce ovulation (n = 38). RESULTS: The GABs were detected in 67% of women with treatment, 27% of women without treatment and 8% of women in the population. The GABs recognized follicle stimulating hormone (FSH) and luteinizing hormone (LH) and their alpha and beta subunits and to a lesser extent thyroid stimulating hormone (TSH) and prolactin. There was no significant difference in OVAB prevalence between gonadotropin treated or untreated women while GABs were significantly more frequent in gonadotropin treated women (P < or = 0.01). CONCLUSION: Gonadotropin antibodies may represent a separate marker of ovarian autoimmunity in unexplained infertility as they are correlated with OVAB and are present in patients not treated with gonadotropin. However, as a higher frequency of GAB is associated with gonadotropin treatment, patients with ovarian autoimmunity may tend to have an immune response to gonadotropins in addition to an endocrine response.     

Immunity to zona pellucida in women with low response to ovarian stimulation, in unexplained infertility and after multiple IVF attempts

A retrospective study was designed to assess the presence ofantizona pellucida autoantibodies in in-vitro fertilization(IVF) patients in relation to low ovarian response, multipleIVF attempts and unexplained infertility. Antizona pellucidaand antisperm antibodies were determined in serum samples obtainedfrom 37 women undergoing IVF-embryo transfer and 20 fertilewomen. Antizona pellucida antibodies were measured using enzymeimmunoabsorbent assay. Antisperm antibodies were evaluated bythe immunobead binding method. Three of 10 patients with lowresponse to ovarian stimulation had antizona pellucida antibodiesin serum and one patient had antisperm antibodies. None of theother participants in the study and the control groups demonstratedmeasurable levels of serum antigamete antibodies. The resultsin this small group suggest an association between antizonapellucidaantibodies and suboptimal response to gonadotrophins. It isindicated that repeated stimulation and puncture of ovariesin IVF procedures do not elicit auto-immunity to gametes.     

Remote-site stimulation of influenza antibodies in monkeys following intestinal stimulation

Antibodies in nasal secretions and saliva were stimulated in 10 monkeys (Macacus rhesus) which had been immunized orally with a killed influenza vaccine. Prior to immunization, monkeys had no detectable antibody to influenza virus hemagglutinin or neuraminidase in sera or secretions. Oral immunization (6 times within 12 days, total of 0.6 mg hemagglutinin (HA) and 240 neuraminidase units) using intraesophageal intubation induced secretory antibodies to both antigens, but no serum antibody. Six and 8 monkeys reacted with HA-antibodies in nasal secretions and saliva, respectively, whereas neuraminidase antibodies occurred in nasal secretions of all 10 and in the saliva of 9 animals. The results support the concept of a common mucosal immune system in monkeys.     

Borrelia burgdorferi antigens that are targeted by antibody-dependent, complement-mediated killing in the rhesus monkey.

We identified surface antigens of Borrelia burgdorferi that are targeted by antibody-dependent, complement-mediated killing (ADCK) in the rhesus monkey. For this purpose, we had available serum samples from three animals infected with B. burgdorferi JD1 by needle inoculation and from two monkeys that were infected with the same B. burgdorferi strain by Ixodes scapularis tick bite. Sera from monkeys from the first group contained antibodies to OspA and OspB detectable by Western blot (immunoblot) using whole B. burgdorferi antigens, whereas serum samples from animals in the second group did not. The targeting of OspA and OspB by functional antibodies was demonstrated directly by showing that ADCK was partially inhibited when antibodies were preincubated with an excess of soluble recombinant OspA or OspB. Simultaneous addition of OspA and OspB did not result in an additive inhibitory effect on ADCK, a result that suggests that the epitopes on OspA and that on OspB targeted by antibody in this mechanism are the same, or at least cross-reacting. The targeting of non-OspA, non-OspB surface antigens was inferred from the fact that sera from tick-inoculated animals, which did not contain detectable anti-OspA or anti-OspB antibodies, were able to effect ADCK. This killing effect was not inhibitable by the addition of recombinant OspA or OspB or both proteins together. We also showed that both immunoglobulin G and M antibodies participate in the ADCK mechanism in the rhesus monkey. Rhesus complement does not kill B. burgdorferi in vitro in the absence of antibody, and antibody alone is effective in killing only at serum dilutions lower than 1:15. However, such "complement-independent" antibodies were not present in all bleeds. Two main conclusions may be drawn from the analysis of our results. First, both OspA and OspB are targeted by the ADCK mechanism in the rhesus monkey. Second, one or more B. burgdorferi surface antigens that are neither OspA nor OspB also participate in ADCK.     

Modulation of hypothalamic ICSS by concurrent limbic stimulation

Four rhesus monkeys were surgically implanted with arrays of subcortical electrodes and trained to self-administer rewarding electrical stimulation (ICSS) to the hypothalamus. In all four animals, concurrent electrical stimulation of the CA3 region of uncal anterior hippocampus or of the cortico-medial region of amygdala inhibited hypothalamic ICSS. In two of the four animals, concurrent stimulation of the more posterior hippocampus (CA3) facilitated ICSS. Concurrent stimulation of other areas (including basolateral amygdala, hippocampal CA4 region, internal capsule, optic tract, thalamus, and caudate nucleus) had no effect on hypothalamic ICSS. It is suggested that hypothalamic reinforcement processes may vary according to the parameters of limbic involvement.     

Neutralizing Antibodies to Cytomegaloviruses in Normal Simian and Human Sera

Simian and human sera were examined for neutralizing antibodies to simian and human cytomegaloviruses (CMV). Neutralizing antibody to simian CMV was found in sera from 12 of 12 African green monkeys, 8 of 10 rhesus monkeys, and 7 of 7 baboons captured in the wild. The antibody did not cross-react with human CMV strain AD169 but cross-reacted with human strain C87, particularly in the presence of complement. Thirty-six baboons and 10 rhesus monkeys born and hand-reared in captivity remained free of neutralizing antibody both to simian and human CMV for as long as 4 years. Fifteen of 24 human sera (63%) revealed only species-specific neutralizing antibody.     

In Vivo Imaging of Physiological Angiogenesis from Immature to Preovulatory Ovarian Follicles

To develop a model for the study of physiological angiogenesis, we transplanted ovarian follicles onto striated muscle tissue and analyzed the process of microvascularization in vivo using repeated fluorescence microscopy. Follicles were mechanically isolated from unstimulated as well as pregnant mare's serum gonadotropin (PMSG)- or PMSG/luteinizing hormone (LH)-stimulated Syrian golden hamster ovaries and were transplanted as free grafts into dorsal skinfold chambers of untreated or synchronized hamsters. Follicles lacking thecal cell layers did not vascularize regardless whether harvested from unstimulated or PMSG-stimulated animals, but underwent granulosa cell apoptosis, as indicated in vivo by nuclear condensation and fragmentation of bisbenzimide-stained follicular tissue. In contrast, all follicles at 48 hours after PMSG treatment with a multilayered thecal shell exhibited initial signs of angiogenesis within 3 days. Vascularization was completed within 7 to 10 days, comprising a dense glomerulum-like microvascular network. Nature and extent of vascularization of follicles harvested at 72 hours after either PMSG or PMSG/LH treatment did not notably differ from each other when transplanted into the respective synchronized animals. However, follicles with PMSG/LH treatment revealed significantly larger microvessel diameters and higher capillary blood perfusion compared to follicles with sole PMSG treatment, probably reflecting the adaptation to the increased functional demand upon the LH surge. Using the unique experimental approach of ovarian follicle transplantation in the dorsal skinfold chamber of Syrian golden hamsters, we could show in vivo the developmental stage-dependent vascularization of follicular grafts with sustained potential to meet their metabolic demand by increased blood perfusion.     

Influence of gonadal hormones and genital afferents on EEG activity of the hypothalamus in adult male rhesus monkeys

Electrodes were stereotaxically implanted in hypothalamic and cerebral cortical regions of 15 adult male gonadally intact rhesus monkeys. The electrical activity of these regions was recorded electroencephalographically before and after genital stimulation. Observations were also taken after stimulation on skin, abdomen and nipples and repeated daily for 4 to 6 days, before and after intravenous injections of testosterone propionate (0.4 mg/kg). The experiment was repeated on the same animal after gonadectomy. The influence of gential afferents appears to produce focal inhibition of electroencephalographic (EEG) activity in ventromedial nucleus (VMN) in monkeys with intact gonads. Exogenous administration of testosterone propionate produces EEG facilitation in VMN with simultaneous inhibition of the anterior hypothalamic area. With continued increase in amounts of circulating hormone, the EEG activity in the posterior hypothalamic area is also inhibited by the incoming genital afferent information. Gonadectomy brings about EEG slowing with genital stimulation in ventromedial nucleus before hormone injection but this was less marked than that observed in EEG records of intact preparation.     

The causes of false-positives encountered during the screening of old-world primates for antibodies to human and simian retroviruses by ELISA

Sera from 526 old-world primates representing 50 different species were screened by ELISA for antibodies to human T-lymphotropic viruses I and III, and simian retrovirus type 1 (SRV-1). About one-fourth of the sera were positive by ELISA. There was a tendency, however, for the same sera to be positive for all three human and simian retroviruses. Only about one in five of the ELISA antibody-positive sera were confirmed to be positive by Western blotting. False-positive ELISA antibody tests were particularly common among sera from mandrills, crab-eating macaques, lion-tailed macaques, African green monkeys, and DeBrazza's and moustached guenons. Sera that were falsely positive in ELISA antibody tests to the three human and simian retroviruses were found to contain antibodies that reacted at comparable intensity with feline leukemia, infectious peritonitis and panleukopenia viruses. The false anti-viral activity of these sera was found to be due to antibodies that reacted with non-viral proteins that were copurified with all five virus preparations. These proteins were present in normal cat and human cells used to grow the various viruses and in gelatin. The implications of nonspecific cell-protein antibodies in primate sera were discussed in the light of this and previous seroepidemiologic studies of man and old-world monkeys.     

Immunological relationship between delta herpesvirus of patas monkeys and varicells-zoster virus of humans.

The Delta herpesvirus (DHV) which produced outbreaks of exanthematous disease in patas monkeys was shown to have a close immunological relationship to varicella-zoster (V-Z) virus of man. Immunization of rhesus monkeys with DHV or V-Z virus resulted in the development of neutralizing antibodies to both viruses and also in the production of complement-fixing antibodies to V-Z virus. Immunoglobulin M neutralizing antibody to V-Z virus was demonstrated in the serum of a rhesus monkey immunized with DHV, suggesting a primary antibody response rather than recall of antibody to a related virus. Convalescent-phase sera from human zoster cases had comparable levels of neutralizing antibody for both DHV and V-Z virus. Patas monkeys involved in an outbreak of DHV infection showed seroconversion to both DHV and V-Z virus by neutralization tests and to V-Z virus by complement fixation tests. The demonstration of the close antigneic relationship between DHV and V-Z virus suggests that DHV may be useful in an animal model system for studies on the latency and reactivation of V-Z virus.     

ELISA for detection of group-common and virus-specific antibodies in human and simian sera induced by herpes simplex and related simian viruses

A rapid (3.5 h) enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to herpes simplex virus type 1 (HSV-1) and to two antigenically related monkey viruses, simian agent 8 (SA8) and Herpesvirus simiae (B virus). Crude preparations of detergent solubilized infected cells and similarly treated control mock-infected cells served as antigens for coating wells in microplates. Biotinylated protein A and avidin-conjugated alkaline phosphatase were used to detect antibodies in sera from different species (humans, monkeys and rabbits). Three prototype assays are described with three degrees of specificity. Common or specific determinants on the viral antigens could be assayed in simple competition tests using similar antigen preparations to those coating the wells. The specific assays permitted rapid differential serodiagnosis of antibodies to human and simian herpesviruses.     

Delivery rates following IVF treatment, using two recombinant FSH preparations for ovarian stimulation

BACKGROUND: A prospective, semi-randomized, open, clinical study was conducted to compare ovarian response, pregnancy outcome and delivery rates using two recombinant FSH preparations (Gonal-F and Puregon) for IVF. METHODS: We analysed stimulation parameters and outcome data in 812 initiated treatment cycles where 292 women used Gonal-F and 200 used Puregon. Embryo transfer was carried out in 676 cycles. In the two preparation groups we also compared 170 women previously treated with FSH for IVF with 266 previously untreated. RESULTS: The pregnancy rate with Gonal-F was 26% and with Puregon 28%. Delivery rates were identical, 22%. Clinical pregnancy and delivery rates per cycle with embryo transfer in earlier untreated women were 29.0 and 24.0%, whereas in previously treated women they were 23.5 and 18.8% respectively. After repeated cycles, delivery frequencies consecutively decreased, independent of the FSH preparation used. CONCLUSION: Gonal-F and Puregon seem to be equally potent in achieving follicular development and equally effective, in terms of delivery rates, for use in an IVF programme. Repeated cycles resulted in equally large consecutive decreases in delivery rates, regardless of preparation choice, but were considered worthwhile for up to three stimulation cycles in selected patients.     

Recombinant luteinizing hormone supplementation to recombinant follicle-stimulating hormone induced ovarian hyperstimulation in the GnRH-antagonist multiple-dose protocol

BACKGROUND: Suppression of endogenous LH production by mid-follicular phase GnRH-antagonist administration in controlled ovarian hyperstimulation protocol using recombinant (rec) FSH preparations void of LH activity may potentially affect ovarian response and the outcome of IVF treatment. The present study prospectively assessed the effect of using a combination of recFSH and recLH on ovarian stimulation parameters and treatment outcome in a fixed GnRH-antagonist multiple dose protocol. METHODS: 127 infertile patients with an indication for IVF or ICSI were recruited and randomized (using sealed envelopes) to receive a starting dose of either 150 IU recFSH (follitropin alpha) or 150 IU recFSH plus 75 IU recLH (lutropin alpha) for ovarian hyperstimulation. GnRH-antagonist (Cetrorelix) 0.25 mg was administered daily from stimulation day 6 onwards up to and including the day of the administration of recombinant HCG (chorion gonadotropin alpha). Gonadotropin dose adjustments were allowed from stimulation day 6 onwards, HCG was administered as soon as three follicles > or =18 mm were present. The primary outcome parameter was treatment duration until administration of HCG. RESULTS: Exogenous LH did not shorten the time necessary to reach ovulation induction criteria. Serum estradiol (E(2)) and LH levels were significantly higher on the day of HCG administration in the recLH-supplemented group (1924.7 +/- 1256.4 vs 1488.3 +/- 824.0 pg/ml, P < 0.03), and 2.1 +/- 1.4 vs 1.4 +/- 1.5 IU/l, P < 0.01, respectively). CONCLUSIONS: Except for higher E(2) and LH levels on the day of HCG administration, no positive trend in favour of additional LH was found as defined by treatment outcome parameters.     

Action of PMSG and asialo-PMSG on rat Leydig and granulosa cells.

Highly purified pregnant mares serum gonadotropin (PMSG) was nearly as potent as ovine luteinizing hormone (LH) and human follicle stimulating hormone (hFSH) when bioassayed in vitro in systems known to respond primarily to LH or FSH. An analogue of human chorionic gonadotropin treated with neuraminidase, galactosidase, beta-N-acetylglucosaminidase, and mannosidase (hCG) inhibited the stimulatory effects of hCG, LH, and PMGS on cAMP accumulation in rat Leydig cells but did not inhibit the stimulatory effects of FSH or PMSG on cAMP accumulation in ovarian granulosa cells obtained from immature rats fed diethylstillbestrol. Thus PMSG appeared to form functional complexes with both LH and FSH receptors and may be unique among mammalian gonadotropins. Treatment of PMGS with neuraminidase increased its potency nearly tenfold in vitro apparently by increasing its affinity for both LH and FSH receptors. Although the kinetics of PMSG binding were not investigated with radiolabeled materials, indirect functional binding studies are described that suggest that hCG more rapidly forms stable hormone-receptor complexes than PMSG, asialo-PMSG, FSH, and LH when all hormones are incubated under the same conditions.     

恒河猴实验感染庚型肝炎病毒的实验研究

目的研究庚型肝炎病毒（ＨＧＶ）在恒河猴中的实验感染状态。方法用一名ＨＧＶＲＮＡ阳性、ＨＢＶ、ＨＣＶ均阴性的健康献血员血浆实验感染２只恒河猴，并取第一代猴感染后６周的血再感染１只第二代恒河猴，然后用以第二代猴感染６周后血继续感染２只第三代恒河猴。分别用逆转录聚合酶链反应（ＲＴ－ｎＰＣＲ）检测受感染猴血清中的ＨＧＶＲＮＡ，并每周抽血测定血清中丙氨酸转氨酶（ＡＬＴ）。结果感染１周后猴血清ＨＧＶＲＮＡ阳转，最长持续阳性２８周以上。不同感染个体血清ＡＬＴ水平有明显差异，其中１号猴有短期轻度升高，５号猴血清ＡＬＴ较长时间在１００Ｕ／Ｌ以上。肝活检发现，感染后１６周猴肝组织出现明显的病毒性肝炎样病理改变。进一步对该献血员血浆和感染后猴血清中的ＨＧＶ５’端部分非编码区基因ＰＣＲ产物进行测序，结果显示感染用献血员血浆和猴血清中ＨＧＶ序列与国外株ＨＧＵ４４４０２的同源性分别为９８３３％和９５８３％；与ＨＧＵ３６３８０株的同源性分别为９２５０％和８９１７％；感染猴血清中ＨＧＶ序列与献血员ＨＧＶ序列同源性为９５８３％。结论恒河猴对ＨＧＶ敏感，可以做为实验模型动物     

Features associated with reproductive ageing in female rhesus monkeys

BACKGROUND: The specific aims were to determine the effects of maternal age on the meiotic and developmental competence of oocytes and incidence of chromosomal anomalies in oocytes from a population of fertile rhesus monkeys. METHODS: Monkeys were divided into two age groups (4-15 and 16-26 years of age) and underwent ovarian stimulation for collection of oocytes. RESULTS: In the older, compared with younger, monkeys, serum basal concentrations of FSH were elevated (P < 0.05), peak concentrations of estradiol during a stimulation cycle were diminished (P < 0.05), and mean numbers of oocytes retrieved following ovarian stimulation were markedly (P < 0.05) reduced. There were no significant maternal age-related impairments in oocyte maturation, fertilization or blastocyst development. Both abnormal numbers of whole chromosomes, as well as free chromatids, were detected in a limited number of rhesus oocytes. CONCLUSIONS: Similarities between female rhesus monkeys and women in several features associated with reproductive ageing, in conjunction with our ability to perform IVF and other assisted reproductive techniques in monkeys, demonstrate the suitability of these animals for studies on human reproductive ageing and maternal age-related infertility. Although maternal age-related impairments in oocytes were not evident prior to implantation, further studies may reveal more subtle impairments, manifested during post-implantation development.