B7.2-Ig fusion proteins enhance IL-4-dependent differentiation of tumor-sensitized CD8+ cytotoxic T lymphocyte precursors

The B7/CD28 co-stimulatory pathway plays a critical role in T cell activation and differentiation. Our previous study demonstrated that administration of B7.2-Ig fusion proteins to tumor-bearing mice elicits IL-4-dependent, CD8+ T cell-mediated tumor regression. Here, we investigated whether B7.2-Ig stimulation of tumor-sensitized CD8+ CTL precursors during in vitro antigen re-sensitization actually results in their differentiation into mature CTLs and if so, whether such a process depends on IL-4 signals. Splenocytes from tumor-sensitized (tumor-bearing or tumor-immunized) mice exhibited low levels of anti-tumor CTL responses upon culturing alone, but induced strikingly enhanced CTL responses when stimulated in vitro with B7.2-Ig fusion proteins. Because CTLs were not generated from normal splenocytes even by B7.2-Ig stimulation, the expression of the B7.2-Ig effect required the in vivo tumor sensitization of CD8+ CTL precursors. Administration of anti-CD4 or anti-CD40 ligand (CD40L) to mice before tumor sensitization resulted in almost complete inhibition of CTL responses generated in the subsequent culture containing B7.2-Ig. In contrast, anti-IL-4 did not influence in vivo tumor sensitization required for CTL induction. However, B7.2-Ig stimulation of tumor-sensitized splenocytes enhanced IL-4 production and neutralization of this IL-4 with anti-IL-4 potently down-regulated CTL responses. These results indicate that B7.2-Ig enhances IL-4-dependent differentiation of anti-tumor CD8+ CTL precursors that can be sensitized in vivo depending on collaboration with CD4+ T cells involving CD40L function.     

Calcium pump expression in human bone and soft tissue tumours

Ninety-one cases of human bone and soft tissue tumours were studied for calcium pump expression by strepto-avidin-biotin immunohistochemical staining with a monoclonal antibody against sarcoplasmic reticulum calcium-ATPase (mAb6F5). Two out of 5 cases of embryonal rhabdomyosarcoma, 1 out of 5 cases of biphasic synovial sarcoma, 4 of 4 cases of chordoma and all of 3 chondrosarcoma cases were positive for mAb6F5. Although this novel monoclonal antibody can be used as a marker of myogenic tumours, the present positive result for endoplasmic reticulum calcium-ATPase (calcium pump) in other tumours including chordoma, chondrosarcoma and synovial sarcoma indicates a wider immunoreactivity. The findings further suggest that intracellular calcium may play an important role in cell proliferation and/or differentiation.     

Modulation of B-cell abnormalities in lupus-prone (NZB x NZW)F1 mice by normal bone marrow-derived B-lineage cells.

(NZB x NZW)F1(NZB/WF1) mice spontaneously develop an autoimmune disease characterized by abnormality of haemopoietic stem cells. The present study examined a possible regulatory cell interaction between NZB/WF1 and normal bone marrow cells using radiation-induced chimeras. We demonstrated that the ability of NZB/WF1 bone marrow cells to transfer the typical disease with hypergammaglobulinemia including autoantibodies into lethally irradiated normal recipients was prevented by cotransfer of bone marrow from normal CBA/J mice but not from xid CBA/N mice carrying a selective defect in B-cell function. Flow cytometric analysis revealed that the generation of NZB/WF1 cells was reduced in the mixed chimeras given CBA/J but not CBA/N bone marrow cells. Interestingly, radiation chimeras reconstituted with a mixture of NZB/WF1 bone marrow and CBA/J splenic B cells did not show elevation of serum immunoglobulin levels, although most of the spleen cells were dominated by NZB/WF1 cells. On the other hand, NZB/WF1 B cells maturated in vivo in the presence of CBA/J bone marrow or splenic B cells lost the hyper-responsiveness to lipopolysaccharide (LPS) in the autoantibody production in vitro. These results suggest that radiosensitive normal B-lineage cells have the regulatory activity to ameliorate the hypergammaglobulinemia of NZB/WF1 mice by reducing the generation of NZB/WF1 B cells and/or by correcting their hyper-responsiveness, and that NZB/WF1 mice may have a defect(s) in the regulatory cell function. In addition, CBA/J splenic B cells were shown to modulate the B-cell abnormality even when injected into non-irradiated NZB/WF1 mice manifesting autoimmunity.     

Diminution of experimental autoimmune uveoretinitis (EAU) in mice depleted of NK cells

To evaluate the potential role of NK1.1 (CD161c) cells in autoimmune uveoretinitis, we treated experimental autoimmune uveoretinitis (EAU)-susceptible mice with anti-CD161c antibodies (PK136) to deplete natural killer (NK) cells. Injection of anti-CD161c antibodies deleted NK cells from the peripheral blood of EAU-susceptible mice. The T cell proliferative response against the ocular autoantigen K2 was not suppressed in mice treated with anti-CD161c antibody when compared with T cells from control mice. Although mice treated with anti-CD161c developed EAU, the clinical severity on days 17 and 19 after induction of EAU was significantly mild in anti-CD161c-treated mice compared with control mice. In addition, the histopathological severity of EAU was significantly milder in mice treated with anti-CD161c antibodies than controls 21 days after induction of EAU. Our results indicate that the severity of EAU is augmented by NK1.1(+) NK cells.     

Immunoregulatory defects of V alpha 24V+ beta 11+ NKT cells in development of Wegener's granulomatosis and relapsing polychondritis

The frequency of either CD4(-)8(-) (double negative; DN) or CD4(+) V alpha 24(+)V beta 11(+) NKT cells, the expression of CD1d and the binding of CD1d-tetramer loaded with alpha-galactosylceramide (alpha-GalCer) to NKT cells were analysed in peripheral blood mononuclear cells (PBMCs) of patients with Wegener's granulomatosis (WG), relapsing polychondritis (RP) and healthy subjects (HS). DN and CD4(+) V alpha 24(+)V beta 11(+) NKT cells as well as CD1d-alpha-GalCer tetramer-positive NKT cells, were significantly decreased in number in both WG and RP patients compared to those from HS. When cytokine profiles were analysed in these PBMCs upon stimulation with phorbol ester and calcium ionophore, CD4(+) T cells from patients with WG and RP exhibited a Th1 bias, whereas CD4(+) NKT cells from WG patients in remission showed a Th2 bias. These findings suggest that NKT cells (especially CD4(+) NKT cells) play a regulatory role in Th1 autoimmunity in patients with WG and RP. The reduction in NKT cell counts appears to be associated with the low responsiveness to alpha-GalCer. The dysfunction of NKT cells to recognize ligands such as alpha-GalCer may also contribute to the defects observed in NKT cells from WG and RP patients.     

The effects of temperature on the mechanical performance in fatigued single muscle fibers of the frog induced by twitch and tetanus

Muscle fatigue induced by consecutive twitches or tetani was studied in single skeletal muscle fibers of the frog, Rana japonica. The fatigue by twitch appeared sooner after the start of stimulation at lower temperatures (2-5 degrees C) than at higher ones (15-20 degrees C), while the fatigue by tetanus appeared sooner at higher temperatures. When a twitch-fatigued fiber was bathed in a solution with caffeine (15 mM), the contracture force was much higher than the fatigued force, while in tetanus fatigue, the force by caffeine was not different from the fatigued force. The length-force relation in fatigued fibers was compared with that in pre-fatigue at low and high temperatures. It was noticed that the ascending limb of the length-force curve in fatigued fibers by twitch was lower than that in pre-fatigue at the low temperatures; namely, the fatigue by twitch was more marked in shorter muscle length, while no marked change in the length-force relation was detected in the tetanus fatigue at the low and high temperatures. The maximum shortening velocity, measured by the slack test, decreased in both types of fatigue. These results suggest that the fatigue by twitch may be mainly due to the failure of activation of the contractile system, while in the fatigue by tetanus, the rate of the interaction between actin and myosin may be impaired due to the change in intracellular chemical environment.     

Relationship between muscle fatigue and oxygen uptake during cycle ergometer exercise with different ramp slope increments

Summary The surface electromyogram (EMG) from active muscle and oxygen uptake ( ) were studied simultaneously to examine changes of motor unit (MU) activity during exercise tests with different ramp increments. Six male subjects performed four exhausting cycle exercises with different ramp slopes of 10, 20, 30 and 40 W · min^–1 on different days. The EMG signals taken from the vastus lateralis muscle were stored on a digital data recorder and converted to obtain the integrated EMG (iEMG). The was measured, with 20-s intervals, by the mixing chamber method. A non-linear increase in iEMG against work load was observed for each exercise in all subjects. The break point of the linear relationship of iEMG was determined by the crossing point of the two regression lines (iEMG_bp). Significant differences were obtained in the exercise intensities corresponding to maximal oxygen uptake ( ) and the iEMG_bp between 10 and 30, and 10 and 40 W · min –1 ramp exercises (P < 0.05). However, no significant differences were obtained in and corresponding to the iEMG_bp during the four ramp exercises. With respect to the relationship between and exercise intensity during the ramp increments, the -exercise intensity slope showed significant differences only for the upper half (i.e. above iEMG_bp). These results demonstrated that the and at which a nonlinear increase in iEMG was observed were not varied by the change of ramp slopes but by the exercise intensity corresponding to and the iEMG_bp was varied by the change of ramp slopes. In addition, the significant differences in the exercise intensity slopes for the upper half of the tests would suggest that the recruitment patterns of MU and/or muscle metabolic state might be considerably altered depending upon the ramp slope increments.     

Expression of cytoplasmic TFF2 is a marker of tumor metastasis and negative prognostic factor in gastric cancer

Trefoil factor family 2 (TFF2) is a small peptide constitutively expressed in the gastric mucosa, where it plays a protective role in restitution of gastric mucosa. TFF2 has also been shown to be expressed in some gastric cancers, but its role in tumor metastasis and patient prognosis has not been examined. In this study, we examined TFF2 expression at both the mRNA and protein levels and correlated these results with the clinicopathologic characteristics and prognosis of gastric cancer patients. Among the 144 curatively resected samples, 43 (30%) were positive for TFF2. TFF2 expression was preferentially observed in the infiltrating tumor cells sparing the superficial cells. Significantly increased expression of TFF2 was noted in large tumors of the diffuse type. An increased prevalence of TFF2 expression was also found in tumors with advanced T and N stage and in patients with lymphatic and venous invasion. Accordingly, patients with TFF2-expressing tumors had a significantly worse disease-free survival, and in multivariate analysis, this finding remained significant as an independent prognostic factor. Taken together, our results suggest that TFF2 expression may play a role in gastric cancer invasion and as such could be a useful target for therapeutic intervention.     

Background conductance attributable to spontaneous opening of muscarinic K+ channels in rabbit sino-atrial node cells.

Single myocytes were dissociated from the rabbit sino-atrial node, and the membrane background conductance produced by spontaneous opening of the muscarinic K+ channels was investigated by recording whole-cell and single channel currents in both normal K+ (5.4 mM) and high-K+ (145 mM) external solutions. Increasing external K+ concentration ([K+]o) from 5.4 to 145 mM induced a large inward shift of the whole-cell current accompanied by considerable current fluctuations at -50 mV. The high-K(+)-induced current was both K+ selective and voltage dependent, which was examined by varying [K+]o. This current was almost completely suppressed by 1-5 mM Ba2+ or 2-10 mM Cs+ and it was partly blocked by 10 microM atropine. In high-K+ (145 mM) solution, 20 nM acetylcholine (ACh) further increased the K+ conductance as well as the current noise. The power density spectrum of the noise was fitted with a sum of two Lorentzian functions. The corner frequencies of both the slow (approximately 5 Hz) and fast (approximately 120 Hz) components were comparable between the noise before and during the ACh application. Internal dialysis with a non-hydrolysable derivative of ATP, 5'-adenylylimido-diphosphate (AMP-PNP) or Mg(2+)-free solution markedly decreased both the amplitude and fluctuations of the high-K(+)-induced current. The relation between the variance of the current fluctuations and the mean current amplitude was linear in every experiment using dialysis of AMP-PNP or Mg(2+)-free internal solution, or using superfusion of ACh. The slopes of these relations gave comparable single channel current amplitudes of -0.7 pA at -50 mV. These results indicate that the spontaneous opening of the muscarinic K+ channels is largely responsible for the high-K(+)-induced current. In the high-K+ solution, the variance-mean relation at -50 mV showed that the muscarinic K+ channel provides an inward current of 3.12 +/- 2.13 pA pF-1 (n = 23), which was about 60% of the total inward background current. In the normal K+ solution, the variance-mean relation at -50 mV indicated that an outward current of 6.0 +/- 2.0 pA (0.33 +/- 0.28 pA pF-1, n = 8) was provided by the K+ channel. The single channel current amplitude was estimated to be 0.06 +/- 0.02 pA (n = 9). Cell-attached recordings in the absence of ACh demonstrated sporadic and brief openings of channels identical to the ACh-induced channels. The power density spectra of the single channel currents exhibited kinetic properties comparable with those of the whole-cell currents.(ABSTRACT TRUNCATED AT 400 WORDS)