Immunological response of sheep to injections of plasmids encoding Toxoplasma gondii SAG1 and ROP1 genes.

Infection with the intracellular protozoan parasite Toxoplasma gondii (T. gondii) causes health problems to both humans and livestock and has a large economic impact worldwide. The immune response in sheep following infection with T. gondii was evaluated using six different combinations of plasmid DNA, recombinant antigen and adjuvant. Sheep were generally vaccinated twice by intramuscular injection with plasmid DNA containing gene sequences for either the surface antigen (SAG1) or the rhoptry protein (ROP1) of T. gondii. Two of the groups injected with plasmid DNA SAG1 were boosted with recombinant protein (SAG1). We investigated the efficacy of including oligodeoxynucleotides (ODN) that contain CG motifs (CpG) and the gene coding for ovine granulocyte-macrophage colony stimulating factor (GM-CSF) as potential adjuvants. Administration of the plasmid encoding the ROP1 gene significantly enhanced both IFN-gamma production from peripheral blood cells when cultured in vitro with Toxoplasma antigen, and ROP1-specific IgG1 and IgG2 antibody levels present in serum. However, injection with SAG1 did not stimulate IFN-gamma production. These results indicate the potential of ROP1, given as plasmid DNA, as a potential vaccine candidate to protect sheep against T. gondii infection.

Purification, partial characterization, and clinical evaluation of an adenocarcinoma-associated antigen.

The current investigation describes the purification and partial characterization of a new adenocarcinoma-associated antigen (ACAA). ACAA is a large molecular weight glycoprotein (Mr 790,000 by size chromatography on Sepharose CL-6B) that migrates in the alpha 1 region upon electrophoresis and is eluted from a DEAE-cellulose column at a 0.1 M NaCl concentration. ACAA is immunochemically and biochemically different from carcinoembryonic antigen, alpha-fetoprotein, pancreatic oncofetal antigen, human pancreatic tissue antigen, CA 19-9, ferritin, and acute-phase proteins. Assays for ACAA were carried out using a solid-phase sandwich enzyme immunoassay. The results indicate that ACAA is present in sera of all individuals. Patients with cancer have higher serum levels of ACAA than normal individuals. The greatest frequency of elevated serum values of ACAA was seen in patients with lung and pancreatic cancers followed by colorectal, breast, and prostate cancer. The measurement of ACAA levels may be valuable in the diagnosis and clinical management of patients with certain cancers.

A heterophile system in human renal transplantation. X. HTA sensitivity includes sensitivity to human B lymphocytes.

Antibodies that react with heterophile transplantation antigen (HTA) have been shown previously not to react with HLA-A, B, or C antigens. This paper presents evidence that anti-HTA does react with a subpopulation of human lymphocytes which is comprised primarily of B cells. Anti-HTA reactivity was removed from sera by absorption with each of three different human B lymphocyte cell lines, but it was unaffected by absorption with platelets or thymocytes. Selected high titer anti-HTA sera absorbed with human platelets, human blood group type AB erythrocytes, and sheep erythrocytes caused lysis of a lymphocyte subpopulation principally composed of B lymphocytes. Absorption of these sera with rat erythrocytes removed both lymphocytic activity and anti-HTA activity. Antibody recovered by affinity purification with rat erythrocyte membrane preparations contained both lymphocytic and anti-HTA reactivity. These data, considered with previous studies, seem to establish that B cell sensitization may be acquired by a substantial segment of the population by natural immunization from enteric flora and/or by infections with enteric bacteria.

A heterophile system in human renal transplantation. IX. Comparison of heterophile transplantation antibodies to antivascular endothelial cell antibodies.

Antibodies to heterophile transplant antigen (HTA) were tested for reactivity with antigens on human umbilical cord antigenic specificities on isolated endothelial cells. Furthermore, there are antigens on endothelial cells that are distinct from HLA-A,B, C, and from HTA. It is concluded that the HTA and the VEC antigens are different.

A heterophile system in human renal transplantation. VIII. The morphological distribution of the antigen in rat tissues.

The morphological distribution of heterophile transplant antigen (HTA) was determined in rat tissues using an indirect immunofluorescence technique. Human anti-HTA sera were used to localize HTA in rat kidney, liver, heart, skeletal muscle, spleen, and stomach. HTA was found in basement membrane and supporting stromal elements of all tissues studied. In the kidney, HTA was demonstrated in tubular basement membrane but not glomerular basement membrane. No evidence for cell surface antigen distribution could be ascertained except for erythrocyte membranes. HTA was not found on endothelium of rat blood vessels. We know of no antigens previously implicated in histocompatibility that are stromal in location.

Soluble HLA class II concentrations in normal individuals and transplant recipients. Comparison with soluble HLA class I concentrations.

We developed an ELISA to quantify soluble HLA class II (S-HLA-II) in 702 sera obtained from normal subjects, patients with end-stage renal disease, and recipients of renal, hepatic, and cardiac transplants. Concentrations of S-HLA-II were detectable in 124 of 126 normal individuals. The distribution of normal values described a monophasic curve with a skewed distribution. In transplant recipients, there were no differences between preoperative and posttransplant values, but values in liver patients were significantly higher than in kidney patients, and values for heart patients were lowest of all groups. There were periodic variations in concentrations in individual patients, but these were unrelated to rejection, infection, or any other apparent clinical event. S-HLA-II was consistently present in the urine. All of these observations contrast with previous observations concerning soluble HLA class I (S-HLA-I) molecules, which were almost the precise reverse. It seems likely that these clear differences in S-HLA-II and S-HLA-I concentrations relate to different physiologic processes in either production, function, or elimination.

HLA in human serum--quantitation of class I by enzyme immunoassay.

A solid-phase, enzyme-linked immunoassay was used to quantitate the soluble fraction of HLA-class I. The sera of 318 individuals were studied, as well as the urine of six individuals with normal renal function. The stability of blood concentrations of the soluble HLA was also evaluated. The data justify the following six conclusions. (1) All normal people have circulating HLA (mean = 357 ng/ml). (2) The population can be divided into one group of low secretors (mean = 162.4 +/- 65.2 ng/ml) and another group of high secretors (mean = 540.7 +/- 185.9 ng/ml) (P less than 0.01). (3) Blood levels in each individual are reasonably consistent over short (days) and long (years) periods of time. (4) The mean concentration of soluble HLA-class I in all renal failure patients was 590 ng/ml, significantly higher than normal (P = less than 0.05); it was highest in patients on peritoneal dialysis (mean = 683 ng/ml) in spite of substantial chronic loss in peritoneal dialysate. (5) Renal allograft recipients with stable allograft function also had mean values greater than normal at 554 ng/ml (P less than 0.05). (6) Soluble HLA-class I was not detected in the urine of individuals with normal renal function.

Serologic allogeneic chimerism.

BACKGROUND: At least some transplanted livers secrete soluble human leukocyte antigens (sHLA) of donor phenotype into the body fluids of recipients. The individuals in whom this phenomenon occurs are by definition serologic allogeneic chimeras. Because an allogeneic transplanted liver may induce tolerance to itself and other organs in animals of the donor strain, and because maintenance of a soluble antigen in the circulation of any animal in sufficient quantity for a sufficient period generally leads to tolerance, this phenomenon may be biologically important. This study was performed to determine how common this phenomenon is and whether it occurs after transplantation of organs other than the liver. METHODS: We studied 445 serum samples obtained from transplant recipients (liver, n=12; kidney, n=18; and heart, n=8) before and at various intervals after transplantation. All patients studied had allografts that had functioned for more than 1 year. We used an enzyme-linked immunosorbent assay to quantitate sHLA-A2 and sHLA-A1/A3/A11 (as a cross-reacting group). Donor and recipient combinations were selected in which measurable allotypes in donors were not present in recipients. In some instances, an additional allotype was present in a recipient but not in a donor. RESULTS: All liver transplant recipients had detectable donor sHLA in their serum samples after transplantation. In 72% of kidney and 50% of heart transplant recipients, donor sHLA was found persistently in serum samples obtained after transplantation. Interestingly, all heart transplant recipients of HLA-A3, but none of HLA-A2, had detectable donor sHLA in their serum samples, a finding that may be due to technical reasons. High and stable serum concentrations of donor sHLA characterize long-term stable allograft function. CONCLUSIONS: Donor sHLA is produced by all transplanted livers, most transplanted kidneys, and at least half of (but probably more) transplanted hearts. The hypothesis that donor sHLA may be tolerogenic to liver transplants can be expanded to include kidney and heart transplants.

Surface distribution of monosialoganglioside GM1 on human blood cells and the effect of exogenous GM1 and neuraminidase on cholera toxin surface labeling. A quantitative immunocytochemical study.

The cholera toxin-colloidal gold-labeled IgG-F(ab')2 anticholera toxin ultrastructural immunocytochemical procedure was used for the localization of GM1 monosialoganglioside on the surface of human blood cells. The number of gold particles per micron of cell surface was counted and the data subjected to statistical analysis. Cholera toxin (CT) binding characteristics assessed in several subjects showed consistent labeling patterns for the various hemic cells, although some quantitative differences were noted in surface labeling densities between subjects. Neutrophils were invariably the most heavily labeled of the hemic cells, while lymphocytes, erythrocytes, and platelets exhibited only limited CT labeling. Exposure of hemic cells to neuraminidase induced a major increase in surface CT labeling that proved to be directly related to cell type and differed in many respects with the CT labeling pattern noted in nonenzyme treated cells. Newly exposed CT binding sites attributed to "masked" GM1 and/or to neuraminidase-transformed GD1a or GT1 gangliosides, showed that the number of new binding sites were nearly twice as abundant on platelet and monocyte sufaces as on the surfaces of neutrophil, lymphocyte, and erythrocyte populations. However, ratios of new CT binding sites to those normally available for CT binding were approximately 10:1 for erythrocytes, approximately 3--7:1 for lymphocytes, monocytes, and platelets, and approximately 1:1 for the neutrophil group. Exogenous GM1 was incorporated into the cell surface of the hemic cells in a differential manner. Platelets showed a dramatic increase in surface CT labeling, viz. approximately 12- to 20-fold, compared to that of other hemic cells; however, neutrophil and erythrocyte GM1 uptake was limited. Our studies have demonstrated that distinct differences exist in the extent of surface CT labeling of the various types of blood cells. They further indicated that the ability of the cell surface to incorporate exogenous GM1 may represent a differential expression of the physiochemical properties of the surface of the individual cell types.

Differential expression of surface monosialoganglioside GM1 in various hemic cell lines of normal human bone marrow. A quantitative immunocytochemical study using the cholera toxin-gold-labeled anti-cholera toxin procedure.

The cholera toxin-colloidal gold-labeled IgG-F(ab')2 anti-cholera toxin ultrastructural immunocytochemical procedure has been used for the localization of GM1 monosialogangliosides on the surface of human bone marrow cells. The number of gold particles per micron of cell surface was counted and the data subjected to statistical analysis. Cholera toxin (CT) binding characteristics assessed in several subjects showed consistent labeling patterns for the various types of marrow cells, although minor quantitative differences were noted in surface labeling densities between subjects. Surface labeling was nonuniformly distributed along the cell membrane of the marrow cells and label clusters or domains were commonly noted. Data analysis indicated that CT labeling was related to cell type, to cell lineage, and to the stage of maturation. Mature neutrophils were the most reactive of the marrow cells and the CT labeling of this cell series increased stepwise from the promyelocyte stage to the segmented neutrophil. A similar pattern occurred during eosinophil maturation and the maturation of the monocyte. A different labeling pattern was found during the differentiation of the erythrocytic cell series with low labeling of proerythroblasts increasing modestly to the early normoblast stage and then decreasing during the final phase of maturation. Exposure to neuraminidase prior to the immunocytochemical sequence induced a major increase in surface CT labeling of the various types of marrow cells, as was particularly evident for the platelet, promyelocyte, myelocyte, monocyte, promonocyte, and erythrocyte cell groups. The data indicated that the number of cryptic GM1 and/or higher gangliosides exposed by neuraminidase in the cell membrane varied during cell differentiation and was directly related to specific cell types. Exogenous GM1 also was demonstrated to be incorporated into the surface of the bone marrow cells in a differential manner and the extent of incorporation was found to be related to specific cell types and to their stage of maturation.

Soluble HLA-I in rheumatic diseases.

OBJECTIVE: To study serum levels of Class I soluble HLA (sHLA-I) in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis or dermatomyositis (PM/DM) or scleroderma and to assess the possible influence of ethnic factors on concentration in each disease group. METHODS: Solid-phase enzyme linked immunoassay was used to measure sHLA-I in the serum of 385 patients with varied ethnic backgrounds (American-Caucasians, African-Americans, Georgian-Caucasians) with rheumatic diseases. Studies on patients were compared to similar measurements of 189 healthy individuals. RESULTS: Mean sHLA-I levels were significantly higher in patients with SLE than those observed in healthy individuals or other rheumatic diseases. Highest concentrations were present in Georgian-Caucasian patients with SLE. American-Caucasian patients with RA or scleroderma had higher sHLA-I levels than normal Caucasian individuals. The majority of patients with PM/DM in all ethnic subgroups were low secretors of sHLA-I. CONCLUSION: Mechanisms underlying the secretion of sHLA-I appear to differ among the rheumatic diseases studied and various ethnic groups. These genetic differences in sHLA-I secretion could be associated with ethnic and pathophysiologic differences among these rheumatic diseases.

Malignant lymphoma occurring in a family.

Three patients, a 57-year-old man, his 60-year-old sister, and 81-year-old mother, developed non-Hodgkin's lymphoma within an interval of 12 months. There were histologic similarities of the biopsy material from these patients, but the clinical response was variable. Two patients achieved a sustained remission, and one expired after similar chemotherapy. Studies revealed slightly decreased numbers of circulating T lymphocytes in both surviving patients, and decreased cellular reactivity to mitogens in the man. HLA typing was not conclusive; HLA haplotypes were not the same as those reported in other families with non-Hodgkin's lymphoma. Electron microscopy of biopsy material revealed no viral inclusions. Except for the familial relationship, no common etiologic factors were identified.

Development of a transplantable model of pancreatic duct adenocarcinoma.

We report here the first development of a continuous cell line in tissue culture of an animal pancreatic duct adenocarcinoma that is histologically similar to human pancreatic duct adenocarcinoma. A primary pancreatic duct adenocarcinoma, induced in a male Syrian golden hamster after 23 weeks of weekly subcutaneous injection of N-nitrobis (2-hydroxypropyl)amine, was minced and injected subcutaneously into three hamsters. After 8 weeks, a single tumor was apparent. Subsequent passages of fragments into the cheek pouches were performed at 3- to 4-week intervals. After five passages, minced fragments of a tumor were placed in tissue culture. Colonies appeared by 7 days; an epitheloid cell line, without fibroblasts, was established by 60 days. Single-cell suspensions, injected into hamster cheek pouches or subcutaneously, produced tumors in a dose-dependent fashion. Spent culture medium of tissue culture cells and saline extracts of freshly excised tumors contained pancreatic oncofetal antigen-like activity.

Correlation between sex hormone binding and peritumoral edema in intracranial meningiomas.

Estrogen and progesterone receptor binding activity was measured in 22 intracranial meningioma surgical specimens. None of the tumors was estrogen receptor-positive, whereas 19 were progesterone receptor-positive. Of these 19 patients, all demonstrated significant computed tomographic (CT) evidence of peritumoral edema. None of the 3 patients who lacked progesterone receptor binding had CT evidence of peritumoral edema (P less than 0.005). Peritumoral edema associated with intracranial meningiomas seems to be related, at least in part, to progesterone binding activity. This implicates the potential use of progesterone antagonists for the treatment of incompletely resected or recurrent meningiomas.

Response to exogenous cholecystokinin of six human gastrointestinal cancers xenografted in nude mice.

Gastrointestinal hormones regulate growth of cancers as well as normal tissues. We investigated whether long-term cholecystokinin (CCK) administration might affect growth or metabolism of human tumors xenografted in nude mice. In each experiment, approximately 20 nude mice bearing subcutaneous xenografts of the particular cancer line being studied were used. Half received CCK and half received saline solution intraperitoneally twice daily for 14 days. Tumor volume and body weight were measured every 3 days. If the tumors produced marker substances, these were measured in nude mouse serum and also in the xenografts. Tumor growth was significantly retarded by CCK in two of the six cancers studied. In each case, DNA, RNA, and protein reflected tumor volumes. In one of these tumors (SLU 077), serum carcinoembryonic antigen (CEA) levels paralleled the tumor volumes. In another tumor (SLU 132), serum CEA levels and tumor immunolabeling for CEA and pancreatic oncofetal antigen increased in response to CCK administration, whereas tumor volumes did not. These findings suggest that exogenous highdose CCK altered the growth and metabolism in two of six human cancers studied.

Serum tumor markers for pancreatic carcinoma.

Various biochemical substances are being evaluated for use as serum tumor markers of adenocarcinoma of the pancreas. The currently established markers, CA 19-9 and POA, have an important but limited role in the diagnosis of pancreatic carcinoma. The role could be expanded if the specificity of these tests for pancreatic cancer could be increased, and this may be possible for each of these tests if several recent findings can be confirmed. Most of the new tumor markers are blood-group-related substances, in that the cancer-associated substances share epitopes that are similar to those of the Lewis blood group system. It seems likely that a "panel" of these markers could improve the specificity of these tests for pancreatic carcinoma. However, improvement of the clinical specificity appears unlikely since each of these markers has a high false-positive rate in patients with other cancers, liver diseases, and nonmalignant diseases of the pancreas. Additional study will be required to determine the optimal group of these tests to be used as a pancreatic cancer test panel. Also, more emphasis should be directed to the identification of tumor markers that can be used to detect pancreatic cancer at a stage when it can be treated effectively. The use of tumor markers for monitoring patients does not result in longer patient survival times or a higher survival rate because salvage therapies for this disease are ineffective. If effective salvage therapies can be developed, monitoring with serum tumor markers will become more significant. Thus, continued emphasis should be given to the development of serum tumor markers that have diagnostic utility.

Fibronectin gelatin-binding activity: effects of platelet thrombospondin, serum, and plasma.

Platelet thrombospondin and an unidentified but biologically similar plasma protein were shown to inhibit the gelatin-binding activity of fibronectin. Inhibition of fibronectin gelatin-binding activity was identified and quantitated by using latex-fibronectin particles in combination with latex-gelatin particles in a new competitive aggregation assay. Inhibition was expressed as the reciprocal of the dilution of test sample required to produce a 50% return of baseline control aggregation rate (inhibitor units). Serum and plasma from healthy donors (n = 60) showed similar reductions in fibronectin gelatin-binding activity (47.9 +/- 12.9 and 49.4 +/- 12.7 inhibitor units per milliliter, respectively). However, serum fibronectin gelatin-binding activity per milligram of fibronectin was significantly less than that of plasma. The addition of calcium chloride to platelet-rich plasma resulted in a similar reduction in fibronectin gelatin-binding activity per milligram of fibronectin. No change was observed after recalcification of platelet-poor plasma. Washed platelets (1 X 10(9)/ml) in Tris HCl buffer released 18 +/- 8 fibronectin inhibitor units per milliliter after calcium ionophore A23187 addition. When inhibitor-rich preparations from platelets and plasma were chromatographed on Sepharose CL-6B, the inhibitors eluted at the same location. Inhibitor-rich eluates from both sources bound to heparin-Sepharose and eluted with 0.45 mol/L NaCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inhibitor preparations demonstrated a major protein band with an approximate molecular weight of 185 kd. Western blot analyses using antiplatelet thrombospondin identified the platelet-derived inhibitor as thrombospondin but failed to react with the plasma-derived inhibitor. These data demonstrated that platelet-released thrombospondin was responsible for the reduction in fibronectin gelatin-binding activity seen in serum. An unidentified plasma factor also inhibits fibronectin gelatin binding.

A human myeloma cell line that does not express immunoglobulin but yields a high frequency of antibody-secreting hybridomas.

We selected an 8-azaguanine-resistant variant of a human myeloma cell line (RPMI 8226) by cloning the parental cells on a feeder layer of mouse spleen cells in the presence of increasing concentrations of 8-azaguanine. Culture media and cellfree extracts of both the parental and variant (8226 AR/NIP4-1) cell lines were assayed for production of immunoglobulin heavy and light chains by double immunodiffusion and for lambda-chain by radioimmunoassay. Secretion of free lambda-chain by the parental cell line was confirmed. In contrast, no immunoglobulin heavy or light chains were detected in culture medium of the variant cell line by either immunodiffusion or radioimmunoassay. No intracellular lambda-chain could be detected in the variant cells by radioimmunoassay of cellfree extracts or by immunofluorescence of fixed cells. Hybridomas were produced by fusion of 8226AR/NIP4-1 cells with lymphocytes from a mesenteric lymph node recovered at surgery from a hypertransfused renal transplant recipient. Twenty hybrid culture supernatants were assayed for immunoglobulin by double immunodiffusion, and 15 contained either IgG (lambda) or IgG (kappa). None produced IgM or IgA. An IgG (kappa)-producing hybridoma was shown by immunofluorescence not to express lambda-chain. A second fusion between the variant cell line and spleen cells from a renal transplant patient produced a stable hybridoma secreting IgM (lambda) antibody specific for the I antigen.