Establishment of a tumor-specific immunotherapy model utilizing TNP-reactive helper T cell activity and its application to the autochthonous tumor system

Preinduction of potent hapten-reactive helper T cell activity and subsequent immunization with hapten-coupled syngeneic tumor cells result in enhanced induction of tumor-specific immunity through T-T cell collaboration between anti-hapten helper T cells and tumor-specific effector T cells. On the basis of this augmenting mechanism, a tumor-specific immunotherapy protocol was established in which a growing tumor regresses by utilizing a potent trinitrophenyl (TNP)-helper T cell activity. C3H/He mice were allowed to generate the amplified (more potent) TNP-helper T cell activity by skin painting with trinitrochlorobenzene (TNCB) after pretreatment with cyclophosphamide. Five weeks later, the mice were inoculated intradermally with syngeneic transplantable X5563 tumor cells. When TNCB was injected into X5563 tumor mass, an appreciable number of growing tumors, in the only group of C3H/He mice in which the amplified TNP-helper T cell activity had been generated were observed to regress (regressor mice). These regressor mice were shown to have acquired tumor-specific T cell-mediated immunity. Such immunity was more potent than that acquired in mice whose tumor was simply removed by surgical resection. These results indicate that in situ TNP haptenation of the tumor cells in TNP-primed mice can induce the enhanced tumor-specific immunity leading to the regression of a growing tumor. Most importantly, the present study further investigates the applicability of this TNP immunotherapy protocol to an autochthonous tumor system. The results demonstrate that an appreciable percent of growing methylcholanthrene-induced autochthonous tumors regressed by the above TNP immunotherapy protocol. Thus, the present model provides an effective maneuver for tumor-specific immunotherapy in syngeneic transplantable as well as autochthonous tumor systems.     

Retention of Metabolic and Differentiation Potentials of Green Lavandula vera Callus after Freeze-Preservation

Green Lavandula vera callus that produced biotin was preservedsuccessfully in liquid nitrogen for up to 3 weeks. We foundthat the callus recovered after the freeze-preservation, retainingnot only the biosynthetic capability for biotin but also differentiationpotentials such as chloroplast development and plantlet formation.The significance of retention of the metabolic and differentiationpotentials of the callus is discussed in terms of the freeze-preservationof plant genetic resources. ^* This study is dedicated to the late Professor J. Ashida. (Received September 1, 1982; Accepted November 5, 1982)     

Serotonin Stimulates Both Cytosolic and Membrane-Bound Guanylate Cyclase in NG108–15 Cells

The cyclic GMP (cGMP) content was rapidly (greater than 30 s) increased by serotonin [5-hydroxytryptamine (5-HT)] (EC50 = 10 microM), and the increase lasted for greater than 10 min in NG108-15 cells. The 5-HT-induced elevation of cGMP level (EC50 = 10 microM) at 20 s ("fast" elevation) was inhibited by ICS 205-930 or MDL 72,222 and by Ca2+ deficiency in the reaction medium but not by organic Ca2+ antagonists. The 5-HT effect at 10 min ("slow" elevation) was not inhibited by several antagonists for 5-HT receptors of the 1A, 1B, 1C, 1D, 2, and 3 subtypes and was independent from external Ca2+ concentration. The fast and slow effects of 5-HT were similar to the effects of bradykinin and atrial natriuretic peptide (ANP), respectively, in aspects of both Ca2+ dependency and time course of the effects. Bradykinin transiently stimulated formation of inositol phosphates as well as accumulation of cGMP, a finding suggesting that intracellular Ca2+ is involved in bradykinin-induced cGMP accumulation as shown in the fast response to 5-HT. ANP, an activator of membrane-associated guanylate cyclase (mGC), slowly (approximately 60 s) increased the cGMP content (EC50 = 10 nM), a result lasting for greater than 10 min, and the effects were independent from external Ca2+, as shown in the slow response to 5-HT. 5-HT and ANP did not induce formation of inositol phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatitis B core antigen-specific IFN-gamma production of peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection

To evaluate the specificity of cellular immune response to hepatitis B virus (HBV) Ag in patients with chronic HBV infection, we have measured IFN-gamma production and proliferation of PBMC of 16 patients with chronic active hepatitis (CAH), 17 asymptomatic carriers of HBV (ASC), 6 anti-hepatitis B surface (HBs)-positive subjects, and 6 control individuals with ELISA procedure and [3H]thymidine incorporation. There was no significant increase in the mean proliferative response to recombinant HB surface and core Ag (rHBsAg and rHBcAg), nor was IFN-gamma production elicited with rHBsAg in any group. In contrast, PBMC of HBeAg-positive and anti-HBe-positive CAH patients, and anti-hepatitis B "e" Ag (HBe)-positive ASC showed significantly enhanced IFN-gamma production in response to HBcAg, whereas those of HBeAg-positive ASC and anti-HBs-positive subjects did not respond to HBcAg. The maximal response was observed in a 5-day culture with 500 ng/ml of rHBcAg when assessed by stimulation index value. Monocytes did not demonstrate an increased suppressor or helper activity for IFN-gamma production in these patients. T cell subset fractionation revealed that CD4+ cells were main population of IFN-gamma production specific for HBcAg and CD8+ cells did not suppress IFN-gamma production of CD4+ cells. Furthermore, CD4+ cells of HBeAg-positive ASC generated lesser amounts of IFN-gamma than HBeAg-positive CAH patients did. These results show that the measurement of IFN-gamma production is useful to determine cellular immune response to HBV Ag and suggest that IFN-gamma production depends on the helper activity of CD4+ T cells sensitized to HBcAg.     

Ontogenesis of α2-Adrenoceptor Coupling with GTP-Binding Proteins in the Rat Telencephalon

The ontogenesis of alpha 2-adrenoceptors and GTP-binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese-induced elevation of [3H]clonidine binding was increased in an age-dependent manner but the guanosine 5'-O-(3-thio)triphosphate-induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age-dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP-ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39-kDa proteins ADP-ribosylated by pertussis toxin (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.     

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.     

Cetacean relaxin. Isolation and sequence of relaxins from Balaenoptera acutorostrata and Balaenoptera edeni

The tendency toward extremely high variability among relaxins derived from purportedly closely related species has come to an abrupt end with the discovery of quasi-porcine relaxin in the minke whale (Balaenoptera acutorostrata) and the Bryde's whale (Balaenoptera edeni). An aqueous abstract of the corpora lutea of the two baleen whales contained significant amounts of relaxin-like activity as determined by a mouse bioassay and by cross-reactivity with anti-pig relaxin antibodies. The activity could be isolated and purified to homogeneity. Sequence analysis revealed that both whale relaxins differed from each other by about 3 residues, whereas the relaxin of B. edeni differed at only one position from that of pig relaxin. The similarity appears to include even the chain length heterogeneity observed at the C-terminal end of the B chain in porcine relaxin which is produced by a peculiar mode of connecting peptide removal from the pro-hormone. This finding may well represent one of the better documented challenges to the current paradigm of molecular evolution.