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Two monoclonal antibodies (MoAb) 1E4 and ISCR3, which detect class II antigens across species barriers, were studied for their inhibitory effects on human and murine T cell proliferative responses to purified protein derivative of tuberculin (PPD). The 1E4 detected at least a polymorphic determinant on I-A molecules from mice carrying the H-2b haplotype, and the ISCR3 detected the Ia.7 determinant on I-E molecules. Nevertheless, both 1E4 and ISCR3 recognized monomorphic determinants on HLA-DR antigens (human I-E equivalent molecules), but not on HLA-DQ antigens (human I-A equivalent molecules). It was demonstrated that 1E4 significantly inhibited PPD-specific responses of T cells from Ib-bearing mice. In contrast, ISCR3 showed marginal effects on the responses of mice bearing Ia.7. However, in the human system both 1E4 and ISCR3 reduced proliferative responses to PPD. These results suggest that a functional difference exists between humans and mice in the I subregion products involved in the T cell proliferative responses to PPD.

H-2Kk and H-2Dd molecules were specificially purified from a radioiodinated H-2a preparation obtained by papain digestion of spleen cell membranes of A/J strain mice. The molecules were isolated by binding to H-2 alloantisera of the corresponding private specificity followed by precipitation with rabbit anti-mouse IgG antiserum. The specifically precipitated radioiodinated H-2Kk and H-2Dd molecules were dissociated by acid treatment into large and small components of about 37,000 and 11,000 respectively. These were separated by gel filtration at acid pH or by gel isoelectric focusing in the presence of 6 M urea. Each component separated by gel filtration of the acid-dissociated H-2 molecules showed a high degree of size homogeneity as determined by sodium dodecyl sulphate-acrylamide gel electrophoresis. Upon gel isoelectric focusing, however, the small components showed two peaks of radioactivity closely located together at pH 7-8, both of which had a restricted pH range, while the large components gave one peak of a relatively wide pH range of pH 5-6. The H-2Kk and H-2Dd molecules gave essentially the same pattern in terms of the numbers and the positions of the radioactivity bands. Under the iodination conditions used the large components of H-2Kk molecules contained more radioactivity than the small components, while the reverse was true in case of H-2Dd molecules. Such a difference was also found with H-2Kk and H-2Dd molecules isolated by use of alloantisera of the respective public specificity. The assay of binding of the isolated components with H-2 alloantisera of defined specificity revealed that the large components retain most of the allospecificities of the parental H-2 molecules. No H-2 allospecificities were found on the small components. The small components showed extensive binding with rabbit antiserum against mouse beta2-microglobulin. The same antiserum did not show any binding with the large components. On the other hand, both of the components did bind with rabbit antiserum against papain-solubilized H-2 molecules.

The RT1-B/D region-associated antigens which were serologically defined in the previous study (Ohhashi et al., 1981), were partially purified from membranes of a rat B cell leukaemia, KNL-14. Sequential immunoprecipitation test, with the partially purified 125I-B/Dak preparation using four different rat alloantisera, including a monoclonal antibody, disclosed three distinctive populations of beta units of the class II molecules. Highly purified beta units of three discriminable class II molecules were shown to have different structural properties in terms of molecular weights and of electrophoretic profiles on the isoelectric focusing. The beta units shifted to a position of higher molecular weight on SDS-PAGE under reducing condition, thus suggesting to carry intradisulfide bonds. Furthermore, the highly purified beta units cross-reacted with murine anti-Ia sera. The rebinding test revealed that at least two discriminable species of beta units cross-react with anti-I-Ak monoclonal antibody, whereas beta units purified by binding with the 1E4 monoclonal antibody cross-reacted with anti-I-Ab and/or anti-I-Ad antiserum. On the basis of structural and antigenic properties, we have postulated that the rat class II region can be divided into at least three subregions, each containing a locus which encodes a distinctive beta unit of the class II molecule.

A rare case of recurrent cervical cancer presenting multiple metastases to the small intestine is reported. A 69-year-old Japanese woman with a past history of early-stage (Ia) cervical cancer 13 years previously suffered sudden onset of panperitonitis caused by perforation of the small intestine. In the perforated lesion, squamous cell carcinoma, which was histologically similar to that of the primary lesion, was observed, and was diagnosed as a late recurrence of the cervical cancer. Perforation of the small intestine caused by tumor metastasis, especially metastasis from cervical cancer, rerely occurs. Furthermore, recurrence of an early cervical cancer presenting 13 years after hysterectomy is extremely rare. The present case draws attention to the possibility of late recurrence of cervical cancer, even in cases treated at the early-stage.

Seven different alloantisera absorbed with red blood cells (RBC) from appropriate strains of rats detected a series of B cell alloantigenic specificities that could be divided into two groups, presumably coded for by at least two different closely-linked loci in the rat major histocompatibility complex (MHC), RT1. The one locus had two allele codes for a broad specificity and the other locus codes for a unique specificity that was found only in the restricted strains of rats that shared the same mixed lymphocyte reaction (MLR) phenotype. RBC-absorbed alloantisera were monitored against a panel of B cell fractions obtained from sixteen inbred strains. Two alloantisera, ACI anti-W and W anti-TO detected two broad specificities, into either of which all inbred strains tested were classified. Two broad RT1-B region-associated specificities were thus designated provisionally as Ba-1.1 and -1.2. Another five alloantisera detected four respective specificities which have a narrower strain distribution. Sixteen inbred strains were classified into one of five specificities detected by W and F344, F344 anti-SDJ, WKA anti-ACI, W anti-BUF and ACI anti-W absorbed with LEJ lymph node cells. Each specificity was designated provisionally as Ba-2.1, -2.2, -2.4, -2.6, -2.7, respectively. A complete association of Ba-2 specificities with MLR phenotype was observed. Antigenic specificities of Ba-2.2, -2.2 and -2.4 were all classified in a group of Ba-1.1 specificity, whereas Ba-2.6 and -2.7 specificities were associated with Ba-1.2 specificity. This relationship suggested a linkage disequilibrium between the two loci for the Ba antigens.

Autopsy findings of a 34-year-old Japanese male with stomach cancer in a renal allograft are presented. The renal graft was given by his 53-year-old mother, who had the same HLA type as the patient and showed negative response in the Mixed Leukocyte Culture Reaction test. Stomach cancer occurred 4 years and 5 months after the renal graft, and infiltrated widely into the abdominal cavity, causing rectal stricture. In his original left kidney, a small renal adenoma was found. The tumor cells of the adenoma were composed of clear cells. In the adenoma, infiltration of stomach cancer was found. The renal allograft functioned very well until his death in spite of several rejection episodes. This is the seventh reported case in Japan in which a renal allotransplant was accompanied by malignancies.

A population of Fab fragments was identified in the papain-solubilized fraction of membranes of a rat chemically-induced sarcoma, KMT 17. The Fab fragments were partially purified by gel filtration and column electrophoresis. A rabbit antiserum against the partially purified Fab fragments was raised and shown to be specific for Fab fragments by immunoelectrophoresis. Furthermore, four lines of evidence indicated that the KMT 17 Fab fragments carry a cross-reactive idiotype: (1) the antiserum could bind only with a restricted population of normal Fab fragments having a pl of 6.3; (2) an unrelated antibody (WKA anti-SRBC) showed a weak cross-reactivity (less than 6%); (3) syngeneic antisera against the KMT 17 could bind with the 125I-KMT 17 Fab preparation that was purified by an immunoabsorbent column with the rabbit anti-KMT 17 Fab antiserum (anti-idiotype antibody); (4) the rabbit anti-KMT 17 Fab antiserum could bind with neither heavy nor light chains of WKA IgG. The natural antibody carrying the idiotype was found in normal serum and in various organs as well, especially in the lung of the conventional rats. In addition, the fractions containing Fab fragments (Frll) were shown to enhance tumor growth when injected into syngeneic animals, whereas Fab-free fractions that were separated from the Fab fragments by column electrophoresis showed no enhancing activity. These results strongly suggested that a population of Fab fragments carrying a cross-reactive idiotype was responsible for the enhancement of tumor growth. Mechanisms for the biological function of natural anti-tumor antibodies carrying a cross-reactive idiotype are discussed.

BALB/c mice immunized with Nonidet P-40 (NP-40) crude solubilized (CS) extracts of a syngenetic methylcholanthrene-induced BALB/c sarcoma (Meth A) were challenged with viable Meth A cells to determine the ability of the solubilized preparations to induce transplantation rejection. Animals resisting such challenge were then used in agarose microdroplet macrophage migration inhibition (MMI) and tumor cell neutralization (Winn) assays to evaluate the antigenic specificity of these CS extracts. Spleen cells from those animals that rejected Meth A after immunization with the NP-40-solubilized preparations effectively neutralized the tumor-producing capacity of Meth A tumor cells as determined in Winn assays. MMI assays were quite sensitive and detected migration inhibition of peritoneal exudate (PE) cells from immunized mice with extract concentrations as low as picogram quantities. Specificity studies demonstrated that Meth A expressed no antigenic cross-reactivity with similarly prepared extracts of an unrelated SV40-induced sarcoma (mKSA), nor with a mineral oil-induced plasmacytoma (ADJ-PC5) of BALB/c mice. Inhibition of PE cell migration was mediated by culture supernatants (presumably migration inhibition factor [MIF]) generated from a mixture of immune spleen cells and mitomycin C (MMC)-treated Meth A cells as assayed in an indirect MMI test.

Leukocyte subsets that infiltrated into unmodified LEJ(RT1j)2 liver grafts from WKAH(RT1k) hosts were studied. This strain combination is fully allogeneic and yields acute rejections in skin and kidney transplantations. On days 3, 7, and 14, cellular infiltrates gradually increased, and many hepatocytes were degenerative and lost cellular glycogen. Blastoid lymphocytes were frequently seen, and mitotic features of hepatocytes were prominent (on day 14, 3 to 4/10 high-power fields). Later, on days 30, 50, and 300, the cellular infiltrates gradually subsided, and blastoid lymphocytes were rarely seen. Throughout the course, Ia+ cells (RT1.Dk+ and/or RT1.B+ cells) were the major cell populations infiltrating into the grafts. Most of the host RT1.D+ cells were histiocytoid in appearance on immunoelectron microscopy. Histiocytoid cells were the most numerous infiltrating cells, constituting 30-50% of the total infiltrating cells. There were different distributions of T cell subsets between the portal and sinusoidal areas, as we previously observed in acutely rejected rat renal allografts. Both RLyt-1+,2-(Th) and RLyt-1+,2+(Tc/s) cells were found almost equally in the portal area, while RLyt-1+,2+(Tc/s) cells predominated over RLyt-1+,2-(Th) cells in the sinusoidal area. However, a gradual replacement of RLyt-1+,2-(Th) cells by RLyt-1+,2+(Tc/s) cells, as seen in the perivascular area of renal grafts, was not found in the portal areas of the liver grafts. Except for the latter finding, the pattern of cellular infiltrates was similar to that of acute renal rejection, and the significance of these cellular infiltrates is discussed.

Cell-mediated immunity (CMI), as detected by the agarose microdroplet macrophage migration inhibition (MMI) assay, was investigated using peritoneal exudate cells (PEC) of BALB/c mice and several crude membrane (CM) and solubilized preparations of murine plasmacytomas. The MMI assay was quite sensitive and detected inhibition of macrophage migration as low as picogram quantities of CM, NP40 detergent- and papainsolubilized preparations (CS) of ADJ-PC5 and LPC-1 plasmacytomas. The data were highly reproducible from one experiment to the next with the same or different lots of the CM or solubilized extracts. Specificity studies demonstrated that ADJ-PC5 and LPC-1 plasmacytomas expressed cross-reactive tumor-associated antigens (TAA) as detected by MMI and confirmed by tumor challenge and Winn neutralization experiments. No cross-reactivity was observed with similar extracts prepared from an unrelated syngeneic simian virus 40 (SV40)-induced sarcoma. The inhibition of macrophage migration observed was mediated by culture supernatants generated from the mixture of plasmacytoma-immune spleen cells with antigens and then assayed in an indirect MMI assay on normal PEC. The agarose microdroplet MMI assay appeared to be a rapid and sensitive method to measure TAA recognition and to monitor TAA isolation and solubilization with minimum numbers of immune cells.

The detergent Nonidet P40 was used to solubilize tumor-specific transplantation antigens (TSTA) from crude membranes obtained from dissociated cells of simian virus 40-induced sarcoma of BALB/c mice. A good recovery of specific tumor rejection activity was observed. One fraction, fraction V, was obtained following polyacrylamide-agarose filtration of the solubilized material, and this fraction contained most of the activity. An increased specific activity followed gel filtration. Preliminary data from lectin column chromatography of the active fraction V indicated a separation of TSTA activity from H-2 activity.

The immune response to bovine insulin (BI) in the rat is controlled by the major histocompatibility complex (Mhc)-linked immune response gene (Ir-BI) and immune suppression gene (Is-BI). In the present study, we investigated the low responsiveness to BI in the WKAH rat (RT1k) and attempted to explore the functional link between Is-BI and Mhc class II molecules. Lymph node cells (LNC) from the low responder (WKAH) rats responded well to BI when a large amount of antigen was added to the culture in vitro or after OX8-bearing (OX8+) T cells were eliminated. These LNC, after the elimination of OX8+ cells, could show the RT1.Dk-restricted proliferative response upon in vitro challenge with BI, BI-B chain, or pork insulin. In addition, OX8+ T cells, which were activated with BI and antigen-presenting cells (APC) in vitro, suppressed the anti-BI response of W3/25-bearing proliferating T cells from BI-immunized rats. The results have demonstrated that proliferating T-cell repertoires do exist to BI, which recognize BI-B chain in the context of RT1.Dk molecules in the WKAH rat, and that the state of low responsiveness is mediated to a great extent by antigen-specific OX8+ suppressor T (Ts) cells. Furthermore, the elimination of APC or the addition to RT1.Bk-specific monoclonal antibody in the in vitro secondary activation culture of Ts cells diminished the suppressive activity of OX8+ Ts cells. In the induction phase of Ts cells it therefore seems to be necessary for these cells to recognize BI together with RT1.Bk molecules on APC.

A 56-year-old Japanese woman who had an ectopic liver attached to the inner surface of the gallbladder is reported. The ectopic nodule measuring 1.2 X 0.5 X 0.8 cm was found incidentally when her gallbladder was resected because of cholelithiasis. Ectopic liver tissue in reported cases war located on the serosal surface, or in the wall of the gallbladder. This report described, to our knowledge, the first case of an ectopic liver nodule attached to the mucosal surface of the gallbladder with a thin pedicle.

A mouse plasma substance carrying beta-2-microglobulin activity and lacking in H-2 alloantigenic activity was separated from the blood plasma of A/J strain female mice. The plasma substance had a molecular size of about 300,000-400,000 daltons and an electrophoretic mobility of alpha-globulin. The plasma substance was split by papain digestion to a fragment of about 50,000-60,000 daltons that still carried beta2-microglobulin activity. The papain-split plasma substance was devoid of H-2 alloantigenic activities and Thy-1 and TL alloantigenic activities as well, but yet had a two-component structure that was similar to papain-solubilized H-2 molecules. It contained a 37,000-dalton component linked non-covalently to an 11,00-dalton component, i.e. mouse beta2-microglobulin. This plasma substance appears to be different from Ss protein or Slp protein, both found in mouse serum.

Rat serum was fractionated on a column of Sephacryl-300 and tested with a rabbit anti-rat beta-2-microglobulin (B2m) antiserum. This antiserum was directed against B2m purified from rat liver, and its specificity was confirmed by immunoprecipitation procedures. The antiserum recognized three peaks in the fractionated rat serum: a 200- to 300-kd (kilodalton) fraction, a 40- to 70-kd component, and the free 12-kd B2m. Indirect immunoprecipitation from the 200- to 300-kd fraction led to the identification of a 43-kd polypeptide associated with B2m. A xenoantiserum against RT1 class I antigen also precipitated a similar polypeptide from the same fraction, but this molecule differed in size and antigenic specificity from the one precipitated by anti-rat B2m.

A study on the experimentally induced yolk sac tumor in the rat was made in special regard to the characteristics and origin of tumor cells. Pregnant rats which fetuses were removed on the 12th day of gestation, developed tumors derived from the fetal membranes left outside the uterus, which were composed of differentiated teratomas and yolk sac tumors. Serial observation of the oncogenesis revealed that an early lesion of the yolk sac tumor appeared in a nodule found as early as 3 weeks after the fetectomy and production of alpha-phetoprotein (AFP) was observed histochemically in the tumor cells 5 weeks after the fetectomy. A cultured cell line established after cloning from the transplantable yolk sac tumor which had been induced similarly and converted into ascitic form was also investigated. Light and electron microscopic studies on both induced tumors and the cultured cells indicated a similarity of AFP producing tumor cells with parietal yolk sac cells and of PAS-positive hyaline-like substance with the Reichert membrane. It is reasonable to conclude that the yolk sac tumor observed is regarded as a parietal yolk sac carcinoma described by Pierce. Histogenesis of the tumor is also discussed.

Distributions and ultrastructures of T cell subsets within grafts were studied in unmodified, first-set rat renal transplantation systems (F344----WKA, TO----WKA). Rejections took place within 9.0 +/- 0.5 and 12.2 +/- 1.6 days, respectively. Mouse monoclonal antibodies, R1-3B3 and R1-10B5, were used here for the detection of RLyt-1 and RLyt-2 antigenic systems of rat T cells, respectively. RLyt-1 and RLyt-2 antigens are chemical homologues of mouse Lyt-1 and Lyt-2,3 antigens. In the perivascular areas, RLyt-1+,2- cells were the predominant T cells in earlier stages of rejection. However, toward the end of rejection, RLyt-1+,2- cells decreased in number, with the increase in RLyt-1+,2+ cells. In the interstitial peritubular areas, RLyt-1+,2+ cells were the predominant T cells throughout the course of rejection. Thus, the major T cells in perivascular areas were helper T cells in earlier stages and cytotoxic/suppressor T cells in the later stage, whereas in interstitial peritubular areas, cytotoxic/suppressor T cells were the major T cell population during the entire course of rejection. Ultrastructural studies revealed that many RLyt-2+ blast cells had clustered dense bodies, and that RLyt-2+ cells were found among renal tubular epithelial cells. Renal tubular cells adjacent to RLyt-2+ cells were degenerative in most cases. In addition, membranous structures seen in grafts were RLyt-1+,2+,Ia-, suggesting that they might derive from the destruction of cytotoxic/suppressor T cells after several target cell lyses in vivo.

Pancreatic tumor antigen (PTA) was purified to apparent homogeneity from hamster pancreatic adenocarcinomas by a rapid and simple procedure using column chromatography on Mono P and Superose 6. Amino acid analysis indicated essential similarities between hamster PTA and human pancreatic cancer-associated antigen (PCAA, PCAAc), although differences in the contents of several amino acid residues between the two proteins did suggest species variation. Investigation of the binding pattern of antibody raised against PTA by peroxidase-labeled antibody immunocytochemistry revealed that, whereas PTA is absent or only faintly expressed in normal epithelium, it is very strongly positive in transplantable hamster pancreatic adenocarcinomas induced by nitrosamines. PTA was found to be distributed over the entire surface of neoplastic cells and it was concluded that the presence of PTA on the basolateral surface of malignant pancreatic epithelial cells, where it might have access to the blood circulation, could be one explanation for the observed elevated concentrations of PTA in the serum. PTA is thus a potential marker for hamster neoplastic pancreatobiliary duct-type cells.

Tumor-specific transplantation antigen (TSTA) was solubilized from cell membranes of sarcoma Meth-A with non-ionic detergent Nonidet P40. Soluble TSTA was partially characterized by chromatographic separation and electrophoresis. The antigen responsible for tumor rejection activity had a molecular weight of approximately 70,000 daltons in the presence of detergent and an electrophoretic mobility of alpha-globulin. TSTA was well separated from mouse histocompatibility antigen H-2 by a sequence of procedures, including gel filtration, lectin affinity chromatography, column electrophoresis, and rechromatography on agarose, showed only three major bands on polyacrylamide gel electrophoresis. TSTA was specific for sarcoma Meth-A.