Rabbit autoantibodies to actin induced by immunization with modified homologous actins

Actin is a major antigen involved in the reaction of smooth muscle antibody positive sera from patients with chronic active hepatitis. In the present study, actin extracted from rabbit skeletal muscle was denatured by sodium dodecyl sulfate and was immunized into the rabbit, a homologous animal for actin. The rabbits, thus immunized, produced antibodies reactive with actins of homologous and heterologous animals. In addition, the antibodies showed reactivity with autologous actin. It indicates that the denatured homologous actin is capable of terminating immunological tolerance to actin and induces formation of autoantibody to rabbit actin. This phenomenon may be implicated in the occurrence of anti-actin antibody in sera from patients with chronic liver disease and several other diseases.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Global and temporal regulation of gene expression in Xenopus kidney cells in response to presumed microgravity generated by 3D clinostats.

We documented changes in morphology and gene expression of the renal epithelial cell line A6 derived from Xenopus leavis adult kidney induced by long-term culturing with three dimensional clinostats. An oligo microarray analysis on A6 cells showed that mRNA levels of 52 out of 8091 genes were significantly altered in response to clinorotation. Upregulation or downregulation of gene expression became evident on day 8 and day 10 while there was no significant change on day 5. However, on day 15, expression of 18 out of 52 genes resumed to the levels similar to its original levels while remaining 33 genes maintained altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in mRNA levels of selected genes were found only under clinorotation but not under hypergravity (7 G) and ground control (1 G). Morphological changes including loss of dome-like structures, disassembly of E-cadherin adherence junctions and disassembly of cortical actin were also observed over 10 days of culturing with clinorotation. The results revealed genes which expression was altered specifically in A6 cells cultured under clinorotation.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin     

Two-dimensional flow cytometric analysis of T cell subsets maintained in IL-2 medium after autologous mixed leukocyte reaction activation

Human T cells are stimulated with an autologous mixed leukocyte reaction (AMLR) and can be propagated in interleukin-2. Staining of the cultured cells with the combination of two monoclonal antibodies was evaluated by two-dimensional flow cytometry at weekly intervals. AMLR activation resulted in an initial preservation of the CD4+ (helper/inducer T) subset predominance over the CD8+ (suppressor/cytotoxic T) cells, noted on normal circulating blood lymphocytes. However, during culture in interleukin-2, there was a progressive increase in the percentages of CD8+ Leu 15- cytotoxic T, CD4+ Leu 8- helper T, and CD3+ HLA-DR+ activated T cells, and a concomitant decrease in those of CD4+ Leu 8+ suppressor inducer T and CD8+ Leu 15+ suppressor T cells if the responder sheep red blood cell (SRBC)-rosetting T cells were made up by tris ammonium chloride, but not by hypotonic shock treatment to lyse SRBC. The significant difference between hypotonic shock-T cells and ammonium chloride-T cells in the phenotypic changes of T cell subsets after long-term culture in an interleukin-2 medium may suggest a regulatory role of the ammonium chloride-sensitive T cells in the AMLR.     

Membrane Potentials of Heterotrophically Cultured Tobacco Cells

The electrophysiological properties of the membrane of Nicotianatabacum var. Sarnsun cultured cells were determined using amicroelectrode technique in standard medium containing 1 mMKC1, 1 mM NaCl and 1 mu CaCl₂ at pH 7. Tobacco callus was derivedfrom the pith (E_m=–104.4%16.2 mV). The membrane potentialsof the callus cells did not show a symmetrical Gaussian distributionbut were scattered over a wide range. The percentage of highmembrane potential cells increased as the subculture was continueduntil about 11 months and then decreased. The response of themembrane potential to electric stimulus, ionic composition,metabolic inhibitors, sugars and amino acids was characteristicof high (E_m=–{small tilde}–160 220 mV; H-cells)and low (E_m=–80{small tilde}–90 mV; L-cells) membranepotential cells. The membrane potential of H-cells was largelydepolarized by addition of CN^–, carbonium cyanide m-chlorophenylhydrazone,decyclohexylcarbodiimide, and triphenyltin chloride and transientlydepolarized by addition of glucose, galactose, mannose or sucrose,and D-alanine, L-alanine or Llysine, but the membrane potentialof L-cells was not. (Received December 3, 1982; Accepted March 16, 1983)     

Flavanol glucosides from rhubarb and Rhaphiolepis umbellata

Investigations of rhubarb and the bark of Rhaphiolepis umbellata led to the isolation of new flavan-3-ol glucosides. Their structures were elucidated on the basis of ¹H and ¹³C NMR analysis hydrolytic studies as (+)-catechin 5-O-β-d-glucopyranoside and (?)-catechin 7-O-β-d-glucopyranoside.     

Proliferative kinetics and differentiation of murine blast cell colonies in culture: Evidence for variable G0 periods and constant doubling rates of early pluripotent hemopoietic progenitors

Several investigators have described hemopoietic colonies expressing multilineage differentiation in culture. We recently identified a class of murine hemopoletic progenitors which form blast cell colonies with very high replating efficiencies. In order to clarify further the relationship between progenitors for blast cell colonies and progenitors for the multilineage hemopoietic colonies in culture, we carried out analyses of kinetic and differentiation properties of murine blast cell colonies. Serial observations of the development of blast cell colonies into multilineage (and single lineage) colonies in cultures of spleen cells obtained from 5-fluorouracil (5-FU)-treated mice confirmed the transitional nature of the murine blast cell colonies. The data also suggested that the early pluripotent progenitors are in G₀ for variable periods, and that when triggered into cell cycle, they proliferate at relatively constant doubling rates during the early stages of differentiation. The notion that some of the pluripotent progenitors are in G₀ was also supported by long-term thymidine suicide studies in which spleen cells were exposed to ³H-thymidine with high specific activity for 5 days in culture, washed, and assayed for surviving progenitors. Comparison of replating abilities of day-7 and day-16 blast cell colonies from normal as well as 5-FU-treated mice indicated that some of the day-7 blast cell colonies are derived from maturer populations of progenitors which are sensitive to 5-FU. In contrast, progenitors for the day-16 blast cell colonies are dormant in cell cycle and were not affected by 5-FU treatment. Previously we reported that progenitors for day-16 blast cell colonies have a significant capacity for self-renewal. These observations suggest the hypothesis that the capability for self-renewal is accompanied by long periods of G₀, and that once commitment to differentiation takes place, then active cell division occurs.