Extraction of Immunogenic Murine Mammary Tumor Antigens with 1-Butanol

Solubilized tumor associated antigens (TAA) were prepared by a single phase 1-butanol extraction of a transplantable murine mammary adenocarcinoma (H2712), other syngeneic non-mammary tumors, normal mammary tissue, and embryonic tissues. The crude butanol extract (CBE) of H2712 was found to have tumor-specific activity both in vitro in the leukocyte adherence inhibition (LAI) assay and in vivo in an immunoprophylaxis assay. The failure to detect any of the major mammary tumor virus (MTV) envelope glycoproteins or internal core protein in CBE-H2712 and the failure of tumor-bearing mice to give a LAI response to CBE prepared from embryonic tissues suggests that the CBE antigens which are responsible for LAI activity are not viral or oncofetal antigens. Enzymatic treatment of the CBE-H2712 indicated that a sialated glycoprotein formed the antigenic determinants recognized in CBE by the LAI assay. Gel filtration of LAI-reactive CBE-H2712 on a Sephadex G-75 column isolated both the in vitro and in vivo tumor-specific activity in a fraction pool. Polyacrylamide gel electrophoresis revealed the presence of a 67 kD subunit as the major species in the active fraction.     

Immunology and Immunopathology of the Intestines: Crypt Cell Antigens (Cca): New Carbohydrate Markers for Human Colon Cancer Cells

A panel of monoclonal antibodies produced against surface membrane components of the human colon tumor cell line Caco-2 was found to define oncofetal crypt cell antigens (CCA) expressed by fetal intestinal cells, adult small intestinal crypt cells, and human and rat colonic adenocarcinomas. The epitopes recognized by these antibodies have been identified as O-linked oligosaccharide chains associated with specific glycoproteins in cultured intestinal cells and in colon tumors in vivo. Analysis of a large group of normal and diseased human intestinal specimens has demonstrated a marked heterogeneity in CCA expression which correlated with the degree of organization of the tumor cells in the tissue, suggesting that the CCA represent useful histological and clinical markers for colon cancer.     

抗人肿瘤细胞核单克隆抗体的制备及鉴定

根据Epstein等报告,肿瘤坏死部分的不溶性抗原可以作为放射免疫检测与治疗的靶部位。作者制备了抗人肿瘤细胞核单克隆抗体(McAbs),并对其性质进行了鉴定。放射性同位素标记的抗核McAds的初步生物分布试验表明,抗核McAds确可到达肿瘤坏死部位。     

New Method for DNA Microarrays Development: Applied to Human Platelet Antigens Polymorphisms

DNA microarrays are a powerful experimental tool for the detection of specific genomic sequences and are invaluable to a broad array of applications: clinical diagnosis, personalized medicine, drug research and development, gene therapy, food control technologies, and environmental sciences. Alloimmunization to human platelet antigens (HPAs) is commonly responsible for neonatal alloimmune thrombocytopenia, post-transfusional purpura and platelet transfusion refractoriness. Using DNA microarrays, we developed a diagnosis to type the biallelic HPA-1 platelet group. The region for the human genomic DNA sequence that contains the polymorphism responsible for HPA-1 alleles was amplified by polymerase chain reaction (PCR). The expected DNA fragments were hybridized on DNA microarrays, and the data were analyzed using specially developed software. Our initial results show that the two HPA-1 antigens polymorphisms containing a single base difference were detected using DNA microarrays.     

Tolerance to Self and the Processing and Presentation of Self Antigens

Antigen processing and presentation is critical to the generation and maintenance of self tolerance. The hemoglobin system has provided important data on self antigen processing and presentation in vivo. Hemoglobin/la complexes were detectable in the thymus before the time of positive and negative selection. In addition, thymic epithelial cells were shown to lack the costimulatory factors necessary to trigger T cell clone proliferation. We have extended these findings to the Renal proximal tubule. This class II MHC-expressing epithelial cell was demonstrated to process and present foreign as well as self antigens to T cell hybridomas. Current studies are examining whether this epithelial cell possesses the costimulatory factors Required to fully stimulate T cell clones, or whether the proximal tubule may play an important Role in the maintenance of self tolerance. In addition we describe the exciting model of murine autoimmune myocarditis. We have demonstrated that this is a T cell mediated disease and believe that cardiac antigen presenting cells constitutively process and present the inciting self antigen, myosin. These studies may provide important insights into autoimmunity and self tolerance.     

Progression of the Immune Response to Solubilized Tumor Antigens

Local adoptive transfer assays (LATA) were used to analyze and compare the progression of the immune response in C3H/HeJ mice to irradiated tumor cell vaccine and to crude 3M KCl solubilized antigens extracted from a syngeneic methylcholanthrene-induced fibrosarcoma. Sequential LATA performed 2, 6, 9, 12 and 15 days after soluble antigen pretreatment of spleen cell donors revealed a sinusoidal evolution of lymphoid cell activity. An initial brief period of potent tumor facilitation (days 6-9) was followed by a phase of tumor neutralization (days 9-12) which decayed by day 15. On the other hand, spleen cells from donors sensitized with irradiated tumor cells exhibited consistent tumor neutralization which was sustained throughout 15 days. Thus the tumor growth facilitation observed only in CSA-treated donors may represent a qualitative difference in the immune state induced by soluble, as opposed to cellular, forms of tumor antigen.     

Antigens of Human Trophoblast: Trophoblast-Lymphocyte Cross-Reactive Antigens on Platelets*

ABSTRACT: Human and rabbit antibodies to trophoblast-lymphocyte cross-reactive (TLX) antigens were employed in an enzyme-linked immunosorbent assay (ELISA) to identify and characterize the TLX alloantigen system on human platelets. Neither washing nor extraction in chaotrope or acid altered platelet TLX. The antigen was significantly changed by pronase and trypsin digestion, but Folch extraction yielded antigen in the hydrophilic interface, suggesting carbohydrate. Rabbit antibodies prepared to HLA-negative human syncytiotrophoblast TLX antigens were shown by platelet ELISA to have the same specificity and similar allotypy as anti-TLX antibodies from secondary (2°) spontaneously aborting women. Patients with normal pregnancies before becoming 2° aborters had both IgG and IgM antibodies to TLX. Anti-TLX in patients who never had a normal pregnancy were predominantly IgG. ELISA reactions performed with different concentrations of protein in the buffers detected anti-TLX activity in buffers containing high protein concentrations. This has been observed in studies of blocking antibodies in graft-versus-host disease and immune responses to tumor cells. Platelet TLX offers a new genetic and immunological approach to study similarities of the host-parasite relationships in pregnancy, transplantation, and cancer.     

New Automated Chemiluminescence Immunoassay for Simultaneous but Separate Detection of Human Immunodeficiency Virus Antigens and Antibodies

The recently launched Liaison XL Murex HIV Ab/Ag assay (DiaSorin S.p.A) uses chemiluminescence immunoassay technology for the combined qualitative determination of p24 antigen of HIV-1 and specific antibodies to both HIV-1 and HIV-2. We studied 571 serum samples from those submitted to our laboratory for HIV screening. The samples were divided into 3 subsets: subset A, 365 samples collected prospectively during 1 week; subset B, 158 samples from confirmed HIV-positive patients; and subset C, 48 samples with a positive screening result but a negative or indeterminate confirmatory test result. Our standard screening/confirmatory algorithm was used as a reference. In subset A (prospective), 5 samples were positive and 360 negative by the standard procedure. Liaison XL Murex HIV Ab/Ag correctly identified all 5 positive samples (100%) and 357 negative samples (99.2%). In subset B (confirmed positive), all 158 positive samples were in total agreement in both procedures. In subset C (screen positive only), Liaison XL Murex HIV Ab/Ag yielded accurate results in 42 out of 48 samples (87.5%). Global sensitivity and specificity for Liaison XL Murex HIV Ab/Ag (all subsets included) were 98.3% and 98.5%, respectively. Considering only nonselected prospective samples and confirmed positive samples (subsets A and B), the corresponding sensitivity and specificity values were 100% and 99.2%, respectively. The new fully automated HIV screening test showed high sensitivity and specificity compared to our standard algorithm. Its added advantage of being able to detect HIV-1 and HIV-2 antibodies and p24 antigen separately could prove useful in the diagnosis of early infections.     

Identification of H-2Kb Binding and Immunogenic Peptides from Human Papilloma Virus Tumour Antigens E6 and E7

Peptides can be used to induce MHC class I restricted cytotoxic T cells (CTL) tbrough in vivo immunization. This approach may enable the development of peptide vaccination schemes for immunization against viral infection in humans. Human papillomavirus (HPV) is one of a few viruses associated with human cancer and the development of an anti-cancer vaccine seems possible. As a model approach, we searched the E6 and E7 proteins of the human papillomavirus type 16 for possible murine MHC class I restricted peptide epitopes. We utilized the mouse H2-K^b peptide binding motif which consists of phenylalanine or tyrosine at position five and leucine at the carboxy-terminus with the modification that leucine could be replaced by other aliphatic but non-aromatic amino acids. Four peptide sequences from E6 and two from E7 were selected. These peptides were tested for their ability to bind and stabilize K^b and for their immunogenicity in vivo . It was shown tbat one peptide from E6, E6.1 (50–57), bound K^b, but was not able to prime mice in vivo . In contrast, the two selected E7 peptides E7.1 (21–28) and E7.2 (48–55) bound K^b and were immunogenic in vivo . The peptide induced CTL lysed syngeneic EL-4 cells transfected with the open reading frame of E7 but not vector only transfectants. This implies tbat both peptides were naturally processed and presented by K^b on the surface of target cells. MHC class I peptide binding motifs therefore appear to be an effective and useful tool to predict peptide epitopes of proteins associated with cancer.     

Participation of CD1 Molecules in the Presentation of Bacterial Protein Antigens in Humans

Human CD1 molecules, expressed on the surface of professional antigen-presenting cells (including dendritic cells, Langerhans' cells, B cells and activated monocytes) are structurally homologous to major histocompatibility complex (MHC) class I and class II molecules. CD1b and CD1c have been shown to present nonpeptide bacterial antigens to T cells. We hypothesized that CD1 molecules may also be involved in the presentation of bacterial protein antigens. Human peripheral blood mononuclear cells (PBMC) were exposed to two medically important proteins, tetanus toxoid (TT) and purified protein derivative (PPD), with and without murine monoclonal antibodies (MoAbs) specific for CD1a, CD1b and CD1c. All the MoAbs substantially inhibited the proliferative responses of PBMC to TT and PPD. Simultaneous interaction of CD1 and MHC class II molecules was even more inhibitory to these antigen-specific proliferative responses. In contrast, neither mixed lymphocyte reaction nor superantigen and mitogenic responses were affected by CD1-specific antibodies, indicating a certain restriction pattern in antigen presentation. Our findings suggest that, besides MHC class I and II molecules, there is a family of nonpolymorphic cell surface molecules that is able to present certain bacterial protein antigens to T cells.     

Antigens of the Human Syncytiotrophoblast Microvillus Plasma Membrane*

ABSTRACT: Further data are presented on the isolation and biochemical analysis of syncytiotrophoblast microvillus plasma membrane preparations from normal full-term human placentas. No evidence has been obtained that IgG associated with these membranes is maternal antibody to trophoblast antigen. An oncotrophoblast plasma membrane antigen was previously identified by immunofluorescence, and an indirect radioisotopic antibody assay has now been used to confirm the detection of a cell surface antigen common to breast carcinoma cells and normal trophoblast. The relation of trophoblast plasma membrane antigens to possible maternal immunization in pregnancy is discussed.