Serological response of non-human primates to human melanoma disialoganglioside GD3

Summary The immunogenicity of the disialoganglioside, GD3, a melanoma-tumor-associated antigen, has been evaluated in non-human primates. Sera from four chimpanzees and two monkeys were evaluated for anti-GD3 antibody activity by solid-phase radioimmunoassay using GD3 and control gangliosides as targets. Serum from one monkey, immunized with cells from a melanoma cell line, was strongly reactive with GD3, having a titer of >2500. In contrast, serum from this animal was non-reactive with several other gangliosides including the structurally similar GM3. Anti-GD3 reactivity was also demonstrable, albeit in low titer, in the sera of an additional monkey and a chimpanzee. Each of these animals had likewise been immunized using cells from melanoma cell lines. On the basis of these observations, suggestive of a primate anti-GD3 antibody response, we initiated a series of immunizations of chimpanzee using purified GD3 bound to Salmonella minnesota, R595. IgG reactive with melanoma cells in the cell-binding assay was first detected in sera collected after 4 immunizations and increased in titer against each reactive melanoma cell line during the immunizations. Reactivity of this serum with melanoma cell lines demonstrated a direct correlation with the expression of GD3 by the respective cell line. Anti-GD3 reactivity was evident in solid-phase radioimmunoassay against purified GD3 beginning with serum collected after 11 immunizations. By comparison with its binding to the control ganglioside panel, this serum demonstrated strong specificity for GD3 (titer=640) while having only marginal reactivity with GM3 (titer=40). Immune serum from this animal was also able specifically to block subsequent binding of a murine IgM anti-GD3 antibody (DMab7) to target GD3 in solid-phase radioimmunoassay. Together, these observations suggest that GD3, in the form of a purified molecule bound to a bacterial matrix or as part of the intact melanoma cell membrane, can be immunogenic in non-human primates, and is able to elicit an antibody response of appropriate specificity.Supported in part by grant CA32672 from the National Cancer Institute, Veterans Administration Program 821 and by the Yerkes Regional Primate Center, Atlanta, Georgia. The Yerkes Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care     

A longitudinal investigation of gestural communication in young chimpanzees

A longitudinal study of chimpanzee gestural communication is reported. Subjects were seven 5- to 8-year-old members of a semi-natural group at the Yerkes Field Station. These were the same individuals observed byTomasello et al. (1985) four years previously. Nearly identical operational definitions and observational procedures were used in the two studies. Longitudinal comparisons between the two observation periods revealed that the development of chimpanzee gestural communication is best characterized as a series of ontogenetic adaptations: as particular social functions (e.g., nursing, playing, grooming, etc.) arise, decline, or change, gestural communication follows suit. Most gestures seem to be conventionalized by individuals in direct social interaction with conspecifics. Some gestures may be learned by “second-person imitation”—an individual copying a behavior directed to it by another individual. No evidence was found for “third-person imitation”—an individual copying a gesture used between two other individuals. Implications for the concept of chimpanzee “culture” are discussed. This research was supported in part by NIH Grant RR-00165 from the Division of Research Resources to the Yerkes Regional Primate Research Center. The Yerkes Center is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.     

Population-Level Right Handedness for a Coordinated Bimanual Task in Chimpanzees: Replication and Extension in a Second Colony of Apes

The purpose of this study was to evaluate the reliability of previously published findings on hand preferences in chimpanzees by evaluating hand use in a second colony of captive chimpanzees. We assessed hand preferences for a coordinated bimanual task in 116 chimpanzees (Pan troglodytes) at the University of Texas M. D. Anderson Cancer Center and compared them to previously published findings in captive chimpanzees at the Yerkes National Primate Research Center. The new sample showed significant population-level right handedness, which is consistent with previously published findings in the Yerkes chimpanzees. Combined data on the 2 chimpanzee colonies, revealed a significant effect of rearing history on hand preference, with wild-caught chimpanzees showing less right-handedness than captive-born mother-reared chimpanzees. We discuss the results in terms of the role of early environment on the development of laterality.     

Expression of a T cell receptor-like molecule on normal and malignant murine T cells detected by rat monoclonal antibodies to nonclonotypic determinants

A T cell receptor-like molecule with a dimer structure of 45 kilodaltons (Kd) under reducing and 90 Kd under nonreducing conditions was detected on the surface of two murine T lymphoma lines, EL-4 and MBL-2, by two rat monoclonal antibodies. The two antibodies seemed to react with different determinants on the same molecule. The antibodies did not react with the surface of normal T cells as tested by flow cytometric analysis of cell surface staining. Two-dimensional gel electrophoresis (IEF vs SDS-PAGE) and tryptic peptide analysis revealed the molecule to consist of two chains with different isoelectric points and different tryptic peptides. A conventional antiserum was raised against the heterodimer purified from EL-4 cells. The immune serum did not bind to the surface of normal T cells. However, the immune serum as well as the monoclonal antibodies immunoprecipitated the dimer molecules from detergent-solubilized normal thymocytes and spleen cells. The dimer molecule was detected on both immature and mature thymocytes. These results suggest that the antibodies detect non-clonotypic determinants on a T cell receptor-like protein. The determinants are masked on the surface of normal T cells, whereas they are exposed on the surface of at least two T lymphoma cell lines. Three polypeptides of 30 Kd, 25 Kd, and 15 Kd were also coprecipitated with the heterodimer from MBL-2 cells. These proteins may associate with the heterodimer and may be masking the antigenic determinants on normal T cells. The relationship between the heterodimer molecule described here and the T cell antigen receptor or the human T cell antigen 9.3 is still unknown.     

Establishment,maintenance, and behavior of free-ranging chimpanzees on Ossabaw Island,Georgia, U.S.A.

A small number of adult chimpanzees were released on Ossabaw Island, Georgia, in order to evaluate the feasibility of establishing a free-ranging reproductive colony of chimpanzees in a semitropical North American climate. First, three females and one male were released in June 1972. Following the unexpected deaths of two of these females, four more females were added. Except for one newborn that was removed from the colony, the chimpanzee colony remained intact, and was still in existence as of February 1975. Preparation for and maintenance of the chimpanzees is described. Behavioral adaptations to the environment, and some aspects of chimpanzee social behavior are reported. The potential importance of colonies such as this, for reducing over-exploitation of naturally occurring primate populations is discussed. The financial support of Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia, and The Pennsylvania State University, Department of Poultry Science, University Park, Pennsylvania, permitted us to make these observations. Funding for planning and construction of the island facilities, and relocation and maintenance of the chimpanzees was provided by a National Institutes of Health grant toGeoffery Bourne, Director of the Center.     

Association between allo-immunoreactive and xeno-immunoreactive subunits of a glycoprotein tumor-associated antigen

Summary Using allogeneic antibody, we previously described a tumor-associated antigen (TAA) in the urine of 68% of melanoma patients. The TAA was purified from urine of a melanoma patient and used as immunogen to develop a murine monoclonal antibody (AD1-40F4) and xenopolyclonal antibodies in a baboon. Sera from melanoma patients treated with whole melanoma cell vaccine were used as the source of human antibody to the glycoprotein antigen. Treatment with 2-mercaptoethanol and separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis resolved the high-molecular-mass glycoprotein TAA into smaller subunits. Immunoblot analysis indicates that the murine monoclonal antibody (AD1-40F4) recognized a 90–100-kDa subunit of the antigen while human anti-TAA antibodies primarily recognized a 65-kDa subunit in addition to the 90–100-kDa subunit. Baboon polyclonal antibodies recognized the same subunits plus a 120-kDa subunit. Blocking studies indicated that the murine monoclonal and baboon polyclonal antibodies recognized the closely related epitopes on the 90–100-kDa subunit, while human antibodies recognized an epitope entirely distinct from that recognized by the mouse antibody. These results demonstrate the epitope complexity associated with the high-molecular-mass glycoprotein TAA.     

Seroprevalence of West Nile virus in nonhuman primates as related to mosquito abundance at two national primate research centers

West Nile virus (WNV) surfaced as an emerging infectious disease in the northeastern United States in 1999, gradually spread across the continent, and is now endemic throughout North America. Outdoor-housed nonhuman primates at the Tulane National Primate Research Center (TNPRC) in Louisiana were documented with a relatively high prevalence (36%) of antibodies to West Nile virus. We examined the prevalence of antibodies to WNV in a nonhuman primate population housed in outdoor colonies at the Yerkes National Primate Research Center Field Station located near Atlanta, Georgia. We screened rhesus macaques (Macaca mulatta) and sooty mangabeys (Cercocebus atys) that were at least 3 y old by serum neutralization for antibodies to WNV and confirmed these results by hemagglutination-inhibition assay. None of the 45 rhesus monkeys had antibodies to WNV, but 3 of the 45 mangabeys (6.6%) were positive by both serum neutralization and hemagglutination-inhibition tests. The ratio of seroprevalences in the TNPRC and Yerkes primate populations was similar to the ratio of WNV incidences in people in Louisiana and Georgia from 2002 to 2004. The difference in the exposure of nonhuman primates (and possibly humans) to WNV between these 2 regions is consistent with the difference in the abundance of mammal-biting WNV-infectious mosquitoes, which was 23 times lower near Yerkes than around TNPRC in 2003 and 33 times lower in 2004.     

金黄色葡萄球菌LDH多克隆抗体制备及其交叉反应性

目的:在原核载体中表达、纯化金黄色葡萄球菌乳酸脱氢酶,免疫小鼠获得多克隆抗体,使用多克隆抗体分析与其它菌种的交叉反应性。方法:复苏p ET28a-ldh/Bl21重组菌,IPTG诱导重组融合蛋白表达、以抗HIS标签的单克隆抗体进行western-blot鉴定重组蛋白。使用纯化的重组蛋白以及相应的佐剂免疫小鼠,利用ELISA测定血清抗体效价,并使用小鼠抗血清进行免疫印迹法鉴定重组蛋白的反应原性及其与金黄色葡萄球菌、表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌的交叉反应性。结果:SDS-PAGE电泳在39 KDa处可见目的条带,免疫印迹法验证了重组LDH的表达,纯化后获得2.8 mg重组蛋白。纯化蛋白免疫小鼠能诱导产生特异性体液免疫应答,ELISA检测特异性Ig G效价为1:50000,western-blot鉴定显示所制备的多克隆抗血清能分别识别金黄色葡萄球菌重组及天然乳酸脱氢酶,但不识别表皮葡萄球菌、肺炎链球菌、粪肠球菌、大肠埃希菌中天然乳酸脱氢酶。结论:纯化的LDH具有良好的免疫活性,免疫小鼠获得高滴度的多克隆抗体。使用多克隆抗体western-blot显示与其它菌种LDH不存在交叉反应性,为后续使用该重组蛋白进行金黄色葡萄球菌感染的诊断研究奠定基础。     

Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28,400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000-2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine.     

Molecular and antigenic nature of isolated Sm

The Sm antigen was isolated and purified from calf thymus nuclear extract by affinity chromatography. The affinity columns were made with serum antibodies from an SLE patient or an anti-Sm monoclonal antibody derived from a hybridoma cell line. Proteins eluted from these two columns had m.w. of 58,000 and 35,000 by SDS polyacrylamide gel electrophoresis. The natural conformation of this antigen appears to be 95,000 in m.w. with the 58,000 particle containing the Sm antigenic determinant. The affinity column-purified antigen detected by the human anti-Sm antibodies is also recognized by anti-Sm antibodies in murine lupus serum, as shown by solid-phase radioimmunoassay. This study 1) demonstrates the molecular and antigenic nature of the Sm antigen and 2) compares the anti-Sm binding capabilities of antibody populations present in sera from SLE patients and from MRL lpr/lpr mice.     

Extracellular perivascular connective tissue space in the medial terminal nucleus of the accessory optic system in rats

Summary Two types of capillaries were found in the medial terminal nucleus of the rat accessory optic system. Type one capillaries are surrounded by glial processes and lack a perivascular space, whereas the type two capillaries and arterioles show a distinct extracellular perivascular space often filled with collagen fibrils. An internal as well as an external basal lamina lines these spaces. No fenestration of the endothelium was observed.This study was supported by N. I. H. Grant RR-00165 to Yerkes Regional Primate Research Center, and N. I. H. Grants R01-EY 00565-03 and R01-EY 00638-02 to J. Tigges. The excellent technical assistance of Mrs. G. L. Luttrell is gratefully acknowledged. We thank Dr. W. K. O'Steen for providing the rats and Mr. F. H. Kiernan for photographic help. Special thanks are due to Mrs. B. A. Olberding, in charge of the maintenance of the electron microscope.     

Transmission of human TT virus of genotype 1a to chimpanzees with fecal supernatant or serum from patients with acute TTV infection

Fecal supernatant or serum containing TT virus (TTV) of genotype 1a (10(5) copies/ml) from patients with acute TTV infection was inoculated intravenously into two naive chimpanzees. Serum samples were obtained weekly and tested for TTV DNA by genotype 1-specific polymerase chain reaction. TTV DNA was detected in chimpanzee 228 at weeks 5-15 after inoculation with 0.5 ml of serum, and in chimpanzee 234 at weeks 7-19 after inoculation with 1 ml of fecal supernatant. The TTV DNA titer peaked at weeks 12 and 13 in chimpanzee 228 and at weeks 14-16 in chimpanzee 234. Mild biochemical and histological changes in biopsied liver samples were observed in both chimpanzees in association with the reduction in TTV titer. TTV DNA was transient in chimpanzee 228, but in chimpanzee 234 it reappeared at week 21 and persisted through week 30. These results indicate that TTV in feces is infectious and suggest that TTV has hepatitis-inducing capacity.     

The human and murine influenza-specific B cell repertoires share a common idiotope

We have dissected the human influenza-specific B cell repertoire by performing Epstein-Barr virus (EBV) limiting dilution analysis of lymphocytes obtained from donors before and after immunization with a commercially available influenza vaccine. In addition to an analysis of precursor frequency and light chain diversity, we studied sera and culture supernatants containing human anti-influenza antibodies with a panel of murine monoclonal antibodies specific for idiotopes identified on murine anti-PR8 and anti-X-31 antibodies. An idiotypic specificity present on the X-31-specific murine monoclonal PY206 has previously been shown to be shared by murine antibodies specific for PR8, X-31, and other influenza viruses. We observed little correlation among the following parameters: anti-viral titer, serum idiotope content, precursor frequency and immune status. More interestingly, there was a striking predominance of human influenza-specific antibodies that utilized lambda light chains. In addition, 12 of 26 human anti-influenza monoclonals strongly inhibited the binding of one of the murine anti-idiotopes to the labeled murine antibody, PY206. This is the same idiotope that is shared among murine antiinfluenza antibodies and all six individuals studied contained clones reactive with this anti-idiotope. Seven of these 12 idiotope-positive human antibodies gave partial cross-reactivity in a second anti-idiotypic system. These observations imply that a significant level of homology exists between the binding sites of human and murine influenza-specific antibodies and suggest that idiotypic manipulation of the human immune response to influenza virus may have important therapeutic implications.     

人结肠癌(CCA)单克隆抗体及其识别抗原的分析(简报)

正常细胞转化成癌细胞后,其表型发生了一系列不同于正常细胞的变化,成为肿瘤细胞的标志。Gold和Freeman(1965)用人结肠癌组织的抽提物免疫兔,发现有些用人正常结肠组织吸收后的抗血清能够与肿瘤组织和胚胎肠道抽提物起反应,但不与正常组织抽提物起反应,由于这种抗原最初被发现在胚胎组织,故名为癌胚抗原(embryonic carcinoma antigen,简称CEA)。用敏感的放射免疫或免疫酶标方     

An apparatus and procedure for studying the choice component of foraging in a captive group of chimpanzees (Pan troglodytes)

An apparatus is described which was used to investigate the choice component of foraging in a captive group of chimpanzees maintained in a large, outdoor compound at the Yerkes Regional Primate Research Center Field Station. The utilization of more than one apparatus would allow the investigation of other ecological and psychological concepts in nonhuman primates housed under semi-natural conditions.     

Human immune response to multiple injections of murine monoclonal IgG

Murine monoclonal antibody infusions in humans should induce a human anti-mouse immunoglobulin (mIgG) immune response, especially if multiple infusions over an extended period of time are necessary for therapeutic efficacy. We have administered multiple infusions of the murine monoclonal antibody T101 to patients with cutaneous T cell lymphoma (CTCL) or chronic lymphocytic leukemia (CLL). Five of 10 CTCL patients, compared with zero of six CLL patients, developed antibodies to mIgG. In those CTCL patients who did not demonstrate anti-mIgG antibodies, we were unable to correlate the lack of response to any of a large number of clinical parameters. Anti-mIgG antibodies were of both the mu and gamma isotypes and were detectable 14 days after the first infusion. Multiple infusions were associated with elevated titers. The anti-idiotypic portion of the anti-mIgG titer steadily increased with each infusion until eventually, in one patient receiving eight weekly infusions, well over one-half the serum anti-mIgG recognized only T101 and not four other murine IgG2AK antibodies tested. To increase our confidence in these findings, four separate assay systems were used to make these determinations. The identification of anti-idiotype antibodies as the dominant species of the immune response to multiple infusions of murine monoclonal antibody has major implications for future work with monoclonal antibodies. Although it has been suggested that human monoclonal antibodies would obviate an immune response, our work implies that such antibodies might still induce anti-idiotype antibodies if multiple infusions are administered.     

Critical factors for liposome-incorporated tumour-associated antigens to induce protective tumour immunity to SL2 lymphoma cells in mice

Physical and immunogenic properties of reconstituted membranes designed for the presentation of tumour-associated antigens (TAA) to the immune system are described. Proteins and lipids of crude membranes of SL2 murine lymphosarcoma cells were partially solubilized with octylglucoside. Reconstituted membranes, consisting mainly of unilamellar vesicles with a diameter of 0.03–0.15 μm, were formed by detergent removal and were purified by floatation in a discontinuous sucrose gradient to remove non-lipid-bound protein. Subcutaneous immunization of syngeneic mice with reconstituted membranes or with purified reconstituted membranes induced protection against an intraperitoneal challenge with 10³ viable SL2 cells. Reconstituted membranes were more immunogenic than crude membranes in immunoprotection experiments when compared on the basis of protein dose. Detergent removal was required to obtain an immunogenic presentation form of SL2 membrane antigens and to avoid toxicity associated with the detergent. Reconstitution of SL2 membranes in the presence of exogenous phospholipid slightly increased the fraction of protein that associated with the reconstituted membranes. However, the immunogenicity of the solubilized membrane TAA was not significantly affected by the presence of exogenous phospholipid. The reconstitution procedure described may be useful in identifying membrane factors required for the induction of immune responses against TAA. The versatility of the system may be employed to develop safe alternatives for whole-cell vaccines.     

Monoclonal antibodies to human tumor necrosis factor alpha: in vitro and in vivo application.

Three stable murine hybridoma cell lines, which secrete monoclonal antibodies (mAb) to human tumor necrosis factor alpha (TNF alpha), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon gamma (IFN gamma). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNF alpha. This antibody was further used during in vivo studies to neutralize human TNF alpha in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNF alpha than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNF alpha of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNF alpha, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7 alpha has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7 alpha in serum samples, with a limit of detection of 5 to 10 pg/mL.     

Isolation of tumor-associated antigen from sera of bovine leukemia virus-infected cattle

Tumor-associated antigens (TAA) expressed on the surface of enzootic bovine leukemia (EBL) cells were detected and separated from sera of bovine leukemia virus (BLV)-positive cattle using monoclonal antibody-conjugated immunoaffinity matrix. Eluted fraction from these sera showed 3 polypeptides with molecular weights of 70K, 52K, and 30K daltons, and these polypeptides reacted with a monoclonal antibody against TAA. However, only 70K peptide was isolated from culture supernatant of EBL B-cell line. We also tried to examine a reversed passive hemagglutination test to develop a rapid screening system of serum TAA level, but its sensitivity was below the level of detection when EBL sera was applied directly. This is the first report on the existence of tumor antigens in sera from leukemic cattle.     

Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein

A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.