Studies on the recovery from tolerance to tumor antigens

C3H/He mice were injected i.v. with heavily X-irradiated syngeneic X5563 tumor cells three times at 4-day intervals. This regimen resulted in the abrogation of the potential to generate X5563 tumor-specific T cell-mediated immunity as induced by i.d. inoculation of viable X5563 tumor cells followed by surgical resection of the tumor, representing the tolerance induction. Although such a tumor-specific tolerant state was long-lasting, the recovery of anti-X5563 effector T cell responses was observed when the above ordinary immunization procedure was performed 6 months after the tolerance induction. The present study investigated whether the recovery from the tolerance can be accelerated by applying a helper-effector T-T cell interaction model in which enhanced anti-X5563 immunity is obtained by priming mice with BCG and by immunizing X5563 tumor cells modified with BCG cross-reactive MDP hapten (designated as L4-MDP) in the presence of anti-L4-MDP helper T cells preinduced with BCG. The results demonstrated that BCG-primed mice which received the tolerance regimen failed to generate anti-X5563 immunity when the ordinary immunization was performed 2 or 3 months after the tolerance induction. In contrast, the immunization of BCG-primed and X5563-tolerant mice with L4-MDP-coupled X5563 tumor cells at comparable timing to that of the ordinary immunization were capable of generating potent X5563-specific in vivo protective T cell-mediated immunity. As control groups, BCG-primed or unprimed tolerant mice did not develop anti-X5563 immunity when immunized with L4-MDP-uncoupled or L4-MDP-coupled tumor cells, respectively. These results indicate that immunization of BCG-primed, tumor-tolerant mice with L4-MDP-modified tumor cells results in accelerated recovery from the tumor tolerance.     

The role of tumor-specific Lyt-1+2− T cells in eradicating tumor cells in vivo

Summary The present study investigates some of mechanisms for tumor-specific Lyt-1⁺2^– T cell-mediated tumor cell eradication in vivo through analyses of tumor specificity in the afferent tumor recognition and efferent rejection phases. When C3H/He mice which had acquired immunity against syngeneic MH134 hepatoma were challenged with other syngeneic X5563 plasmacytoma cells, these mice failed to exhibit any inhibitory effect on the growth of X5563 tumor cells. However, the inoculation of X5563 tumor cells into the MH134-immune C3H/He mice together with the MH134 tumor cells resulted in appreciable growth inhibition of antigenically distinct (bystander) X5563 tumor cells. Although the growth of X5563 cells was inhibited in an antigen-nonspecific way in mice immunized to antigenically unrelated tumor cells (bystander effect), the activation of Lyt-1⁺2^– T cells leading to this effect was strictly antigen-specific. Such a bystander growth inhibition also required the admixed inoculation of the bystander (X5563) and specific target (MH134) tumor cells into a single site in mice immunized against the relevant MH134 tumor cells. Furthermore, the results demonstrated that Lyt-1⁺2^– T cells specific to MH134 tumor cells were responsible for mediating the growth inhibition of antigenically irrelevant (bystander) and relevant tumor cells. These results are discussed in the context of cellular and molecular mechanisms involved in the Lyt-1⁺2^– T cell-initiated bystander phenomenon.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture     

Type II collagen-induced murine arthritis. I. Induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity

Immunization of DBA/1 mice with type II collagen resulted in typical and progressive arthritis, which is associated with the production of high titer of anti-collagen antibody and the induction of cell-mediated immunity as exemplified by delayed type hypersensitivity response as well as lymphokine production. In contrast, administration of heat-denatured collagen into DBA/1 mice failed to induce the arthritis. These mice produced only marginal antibody, whereas they developed comparable cell-mediated immunity to that induced by immunization with native collagen, and therefore the inoculation of heat-denatured collagen provided the regimen capable of inducing preferentially cell-mediated immunity without the generation of high level of antibody. Inasmuch as administration of antibody induced only marginal and transient joint swelling not associated with typical histologic lesion, the synergistic effect of humoral and cell-mediated immunities was investigated using antibody preparation and the regimen to induce selectively cell-mediated immunity. The results demonstrate that administration of antibody into DBA/1 mice pre-sensitized with heat-denatured collagen resulted in potent and progressive arthritis. Such synergy was further confirmed by the induction of arthritis in T cell-depleted DBA/1 mice that had been adoptively transferred with antibody and lymphoid cells from heat-denatured collagen-sensitized mice. Moreover, it was revealed that the nature of cells capable of transferring cell-mediated immunity was of Thy-1+ and L3T4+ Lyt-2-. These results indicate that anti-collagen antibody and L3T4+ T cell-mediated cellular immunity are crucially required for the perpetuated development of type II collagen-induced arthritis.     

Studies on the induction of tolerance to alloantigens. III. Induction of antibodies directed against alloantigen-specific delayed-type hypersensitivity T cells by a single injection of allogeneic lymphocytes via portal venous route

BALB/c mice were inoculated with normal C3H/He spleen cells via the portal venous (p.v.) route. Intravenous injection of serum from these BALB/c mice into naive syngeneic mice resulted in almost complete abrogation of their ability to generate anti-C3H/He delayed-type hypersensitivity (DTH) responses as induced by s.c. immunization with C3H/He cells. Since a portion of the same serum did not inhibit the development of anti-C57BL/6 DTH responses, the suppressive effect of the transferred serum was alloantigen-specific. Such serum factor(s) was produced in normal but not in nude mice and the suppressive activity was transferred in H-2- or immunoglobulin allotype-incompatible combinations. Immunochemical analyses of this serum suppressive factor have revealed that its m.w. was approximately 150,000, corresponding to the size of immunoglobulin (Ig)G, and that the activity was trapped by protein A or by an anti-immunoglobulin column. Although the absorption of the serum from anti-C3H/He-tolerant BALB/c mice with C3H/He target spleen cells did not abrogate the suppressive activity, the additional absorption with spleen cells from anti-C3H/He hyperimmune BALB/c mice almost completely eliminated the suppressive potential. Moreover, pretreatment of BALB/c anti-C3H/He DTH effector spleen cells with the above serum from tolerant mice induced the inhibition of anti-C3H/He DTH responses. Taken together, these results indicate that a single injection of allogeneic cells via the p.v. route results in the production of antibody capable of inhibiting the capacity of DTH effector cells specific for alloantigens used for the p.v. presensitization.     

Enhanced production of IL-6 in tumor-bearing mice and determination of cells responsible for its augmented production

IL-6 is a cytokine secreted in normal individuals by monocytes, fibroblasts, and endothelial cells. We have found increased levels of IL-6 in the sera from MH134 hepatoma- and CSA1M fibrosarcoma-bearing mice. Concerning the capacity of these tumor cells themselves to produce IL-6 in vitro, they exhibited the distinct contrast, i.e., the MH134 tumor cells produced high levels of IL-6 whereas the CSA1M generated a marginal level of IL-6. It was, however, demonstrated that appreciably enhanced IL-6 production was observed in spleen cell culture supernatants from both types of tumor-bearing mice when compared to those obtained from normal mice. More importantly, in contrast to the production of IL-6 by non-T cell compartment of normal spleen cells, enhanced IL-6 production of spleen cells from tumor-bearing mice was ascribed to T cell compartment. Analysis of T cell phenotype has revealed that enhanced IL-6 production was mediated predominantly by Lyt-2+ but not by L3T4+ T cell subset. Thus, these results indicate that increased circulating IL-6 is elicited in the tumor-bearing state and that irrespective of the potential of tumor cells themselves to produce IL-6, T cells, especially Lyt-2+ T cells from tumor-bearing mice are responsible for such a high level of IL-6 production.     

Upwelling-like bottom intrusion enhances the pelagic–benthic coupling by a fish predator in a coastal food web

Upwelling regions where nutrients are transported from deep to surface waters are among the most productive in the oceans. Although it is well known that the upwelling affects fishery production through bottom-up trophic cascading, it remains unexplored how temporal variation in its intensity alters overall trophic energy flows within a focal food web. In the present study, we demonstrate that inter-annual variation in the intensity of upwelling-like bottom intrusion alters food web properties in coastal waters of the Uwa Sea by focusing on the levels of δ¹³C and δ¹⁵N for a demersal fish predator, Acropoma japonicum. This approach integrates information on prey–predator interactions. In the season following a stratification period when pelagic productivity is limited by nutrient availability, A. japonicum showed lower levels of δ¹³C in years with high bottom intrusion intensity than in those with low intensity. One possible cause for this isotopic depletion is that the bottom intrusion-induced nutrient supply enhances pelagic productivity and consequently facilitates a foraging shift by A. japonicum from ordinary benthic prey to supplementary pelagic prey with a lower δ¹³C. In conclusion, the increased intensity of bottom intrusion results in coupling of two major trophic energy flows, pelagic and benthic food chains, through the demersal predator’s foraging shift.     

Local increase in trapezius muscle oxygenation during and after acupuncture

Purpose

This study aimed to compare the trapezius muscle blood volume and oxygenation in the stimulation region and in a distant region in the same muscle during acupuncture stimulation (AS). We hypothesized that AS provokes a localized increase in muscle blood volume and oxygenation in the stimulation region.

Methods

Two sets of near-infrared spectrometer (NIRS) probes, with 40-mm light-source detector spacing, were placed on the right trapezius muscle, with a 50-mm distance between the probes. Changes in muscle oxygenation (oxy-Hb) and blood volume (t-Hb) in stimulation and distant regions (50 mm away from the stimulation point) were measured using NIRS. Nine healthy acupuncture-experienced subjects were chosen as the experimental (AS) group, and 10 healthy acupuncture-experienced subjects were chosen for the control (no AS) group. Measurements began with a 3-min rest period, followed by "Jakutaku" (AS) for 2 min, and recovery after stimulation.

Results

There was a significant increase in oxy-Hb (60.7 μM at maximum) and t-Hb (48.1 μM at maximum) in the stimulation region compared to the distant region. In the stimulation region, a significant increase in oxy-Hb and t-Hb compared with the pre-stimulation level was first noted at 58.5 s and 13.5 s, respectively, after the onset of stimulation.

Conclusion

In conclusion, oxygenation and blood volume increased, indicating elevated blood flow to the small vessels, not in the distant region used in this study, but in the stimulation region of the trapezius muscle during and after a 2-min AS.

     

A study of passive weight-bearing lower limb exercise effects on local muscles and whole body oxidative metabolism: a comparison with simulated horse riding,bicycle, and walking exercise

Background

We have developed an exercise machine prototype for increasing exercise intensity by means of passively exercising lower limb muscles. The purpose of the present study was to compare the passive exercise intensity of our newly-developed machine with the intensities of different types of exercises. We also attempted to measure muscle activity to study how these forms of exercise affected individual parts of the body.

Methods

Subjects were 14 healthy men with the following demographics: age 30 years, height 171.5 cm, weight 68.3 kg. They performed 4 types of exercise: Passive weight-bearing lower limb exercise (PWLLE), Simulated horse riding exercise (SHRE), Bicycle exercise, and Walking exercise, as described below at an interval of one week or longer. Oxygen uptake, blood pressure, heart rate, and electromyogram (EMG) were measured or recorded during exercise. At rest prior to exercise and immediately after the end of each exercise intensity, the oxygenated hemoglobin levels of the lower limb muscles were measured by near-infrared spectroscopy to calculate the rate of decline. This rate of decline was obtained immediately after exercise as well as at rest to calculate oxygen consumption of the lower limb muscles as expressed as a ratio of a post-exercise rate of decline to a resting one.

Results

The heart rate and oxygen uptake observed in PWLLE during maximal intensity were comparable to that of a 20-watt bicycle exercise or 2 km/hr walking exercise. Maximal intensity PWLLE was found to provoke muscle activity comparable to an 80-watt bicycle or 6 km/hr walking exercise. As was the case with the EMG results, during maximal intensity PWLLE, the rectus femoris muscle consumed oxygen in amounts identical to that of an 80-watt bicycle or a 6 km/hr walking exercise.

Conclusion

Passive weight-bearing lower limb exercise using our trial machine could provide approximately 3 MET of exercise and the thigh exhibited muscle activity equivalent to that of 80-watt bicycle or 6 km/hr walking exercise. Namely, given the same oxygen uptake, PWLLE exceeded bicycle or walking exercise in muscle activity, thus PWLLE is believed to strengthen muscle power while reducing the load imposed on the cardiopulmonary system.

     

Two translation products of Yersinia yscQ assemble to form a complex essential to type III secretion

The bacterial flagellar C-ring is composed of two essential proteins, FliM and FliN. The smaller protein, FliN, is similar to the C-terminus of the larger protein, FliM, both being composed of SpoA domains. While bacterial type III secretion (T3S) systems encode many proteins in common with the flagellum, they mostly have a single protein in place of FliM and FliN. This protein resembles FliM at its N-terminus and is as large as FliM but is more like FliN at its C-terminal SpoA domain. We have discovered that a FliN-sized cognate indeed exists in the Yersinia T3S system to accompany the FliM-sized cognate. The FliN-sized cognate, YscQ-C, is the product of an internal translation initiation site within the locus encoding the FliM-sized cognate YscQ. Both intact YscQ and YscQ-C were found to be required for T3S, indicating that the internal translation initiation site, which is conserved in some but not all YscQ orthologs, is crucial for function. The crystal structure of YscQ-C revealed a SpoA domain that forms a highly intertwined, domain-swapped homodimer, similar to those observed in FliN and the YscQ ortholog HrcQ(B). A single YscQ-C homodimer associated reversibly with a single molecule of intact YscQ, indicating conformational differences between the SpoA domains of intact YscQ and YscQ-C. A "snap-back" mechanism suggested by the structure can account for this. The 1:2 YscQ-YscQ-C complex is a close mimic of the 1:4 FliM-FliN complex and the likely building block of the putative Yersinia T3S system C-ring.