Augmentation of isoproterenol-stimulated tissue cyclic AMP level by cholinergic agonists in rat parotid gland

It was found that methacholine and carbamylcholine, in addition to their known inhibitory effect, augmented the effect of isoproterenol on tissue cyclic AMP accumulation. The effect of methacholine was dose dependent, and significant augmentation was obtained at 0.1 microM with the maximum being attained at about 0.5 microM, whereas more than 10 microM were required to obtain the inhibitory effect. Atropine completely blocked the effect of methacholine. Similar augmentation of isoproterenol effect was obtained by oxotremorine and pilocarpine. Oxotremorine, however, did not inhibit the effect of isoproterenol. Difference in the effect between methacholine or carbamylcholine and oxotremorine was observed in their binding property to cholinergic receptors. A23187 augmented the effect of isoproterenol in a dose-dependent manner. Oxotremorine and A23187 augmented the effect of isoproterenol in the presence of isobutylmethylxanthine, but they did not augment the effect of forskolin and isobutylmethylxanthine on tissue cyclic AMP accumulation. Cholinergic agonist- and A23187-induced augmentation was abolished by omission of calcium in the medium. These results suggest that the augmentation is due to activation of adenylate cyclase, which is mediated by an increase in concentration of intracellular calcium.     

Action of neuropeptide Y on nociceptive transmission in substantia gelatinosa of the adult rat spinal dorsal horn

Effects of neuropeptide Y (NPY) on substantia gelatinosa neurons were investigated in adult rat spinal cord slices using blind whole-cell patch-clamp technique. Bath application of NPY (1 microM) induced a membrane hyperpolarization, resulting in a suppression of the dorsal root stimulation-induced action potentials in 24% of the substantia gelatinosa neurons tested. In voltage clamp mode, NPY produced an outward current dose-dependently in about one third of substantia gelatinosa neurons at the holding potential of -60 mV, which was not affected by tetrodotoxin (1 microM). The NPY-induced current was suppressed by perfusion with a Ba2+-containing external solution and a Cs2SO4 or tetraethylammonium-containing pipette solution. In addition, The NPY-induced outward currents reversed its polarity near the equilibrium potential of K+ ions (-93 mV). The response to NPY recorded with guanosine-5'-O-(2-thiodiphosphate)-beta-S (GDP-beta-S) containing pipette solution was abolished 30 min after patch formation, suggesting that the response was mediated by the G-protein-coupled receptors. Application of an NPY-Y1 selective agonist, [Leu(31), Pro(-34)]-NPY (1 microM), for 30 s also induced an outward current with a similar time course and amplitude to that induced by NPY. On the other hand, the NPY response was blocked by a simultaneous application of NPY-Y1 selective antagonist, BIBP 3226 (1 microM). No significant changes were found in amplitude and frequency of miniature excitatory postsynaptic currents and dorsal root evoked excitatory postsynaptic currents by NPY. In addition, NPY did not affect both of the miniature inhibitory postsynaptic currents and evoked inhibitory postsynaptic currents, mediated by either the GABA or glycine receptor. These findings, taken together, suggest that NPY produces an outward current in substantia gelatinosa neurons through G-protein coupled, and NPY-Y1 receptor-mediated activation of K+ channels without affecting presynaptic components. The inhibition of the synaptic transmission from the primary fibers to the substantia gelatinosa neurons is considered to contribute to the antinociceptive effects of NPY.     

Autologous primary muscle-derived cells transfer into the lower urinary tract

The goal of these experiments was to establish the basic methodology for future clinical applications of muscle-derived cells (MDC) tissue engineering and gene transfer for the treatment of urological dysfunction. Primary MDC isolated via preplating techniques from adult female SD rats were transduced with retrovirus encoding the expression of beta-galactosidase reporter gene. The MDC were injected into the right and left lateral walls of the bladder and proximal urethra of the autologous animals (n = 6) with a 10 microl Hamilton micro syringe. The amount of injected MDC ranged from 1 to 2 x 10(6) cells. The injected tissue was harvested after 7, 14, and 28 days, sectioned and examined histologically for beta-galactosidase and immunohistochemically for fast myosin heavy chain specific to skeletal muscle. The tissues were also stained for anti-CD4 and anti-CD8 antibodies to assess for cellular immune reaction. We have detected a large number of autologous MDC expressing beta-galactosidase and positively stained for fast myosin heavy chain in the bladder and urethral wall. Many injected myoblasts and myotubes were also seen in the bladder and urethral wall at each time point. Staining of lymphocytes with anti-CD4 and anti-CD8 antibodies was negative after MDC injection at each time point. We have demonstrated the long-term survival of autologous MDC and MDC mediated gene transfer into the bladder and urethral wall. Autologous MDC and MDC mediated gene transfer may be a promising treatment to augment bladder and urethral sphincter function.     

Anti-inflammatory actions of interleukin-13: suppression of tumor necrosis factor-alpha and antigen-induced leukocyte accumulation in the guinea pig lung

The Th2 cytokine interleukin (IL)-13 is believed to play an important role in the development of allergy, although it has also been ascribed anti-inflammatory roles in several experimental models. In this study, we have examined the effects of human recombinant IL-13 on eosinophilic lung inflammation in the guinea pig. IL-13 (1 to 100 ng, given by intratracheal instillation) did not elicit airway eosinophil recruitment. A pronounced accumulation of eosinophils, as well as monocyte/macrophages, was elicited by intratracheal instillation of guinea pig tumor necrosis factor alpha (gpTNF-alpha). Intratracheal administration of IL-13 (1 to 100 ng) given immediately prior to exposure to gpTNF-alpha resulted in a dose-related suppression of eosinophil and monocyte/macrophage accumulation in the airways, as assessed by bronchoalveolar lavage (BAL) and eosinophil peroxidase activity in whole-lung homogenates. IL-13 treatment also reduced BAL fluid (BALF) leukocyte accumulation induced by subsequent aerosol antigen challenge of sensitized guinea pigs. Antigen challenge also resulted in elevated levels of immunoreactive eotaxin and eosinophil-stimulating activity in BALF, although only the latter was reduced significantly by IL-13 instillation prior to challenge. In contrast to the suppressive effects of IL-13, instillation of human recombinant IL-4 (100 ng) alone elicited an increase in BALF monocyte/macrophage numbers, and IL-4 was unable to inhibit gpTNF-alpha-induced leukocyte accumulation. Hence, IL-13 (but not human IL-4) exhibits an anti-inflammatory action in the airways of gpTNF-alpha- or antigen-challenged guinea pigs, by mechanisms that may involve the decreased generation of eosinophil-stimulating activity in the airways.     

Interleukin-2 receptor gene expression in kidney transplant recipients treated with cyclosporin A.

We examined the effects of cyclosporin A (CsA) administered in vivo on the capacity of peripheral blood mononuclear cells (PBMC) from kidney transplant recipients to express IL-2 receptor (IL-2R) gene at the level of mRNA after mitogen stimulation in vitro. There were no differences in the percentage of IL-2R+ cells among the groups of normal individuals, azathioprine-prednisolone treated, and CsA-prednisolone-treated recipients, using FITC-labelled monoclonal anti-IL-2R antibody (anti-alpha chain): 40.3 +/- 10.1% and 62.8 +/- 11.1% of normal PBMC (n = 18), 37.0 +/- 9.3% and 61.7 +/- 5.8% of PBMC from azathioprine-prednisolone-treated recipients (n = 20), and 37.7 +/- 9.6% and 60.7 +/- 12.7% of PBMC from CsA-prednisolone-treated recipients (n = 20) expressed IL-2R after 24 h and 48 h of phytohaemagglutinin stimulation, respectively. However, in a study of Northern blotting using cDNA for IL-2R (anti-alpha chain specific), both the 3500 and 1400 bp families of IL-2R mRNA were remarkably decreased in PBMC from CsA-prednisolone-treated recipients compared with azathioprine-prednisolone-treated recipients and normal individuals. These studies demonstrated that CsA could inhibit IL-2R gene expression at the level of mRNA at physiological concentration.     

Idiopathic right ventricular dilation. Special reference to "arrhythmogenic right ventricular dysplasia" and analogous lesions

Two autopsy cases which showed marked depletion of the right ventricular musculature of the heart accompanied with marked infiltration of the adipose tissue were reported. The first cases was an 18-year-old female who died of right sided congestive heart failure after about 4-years clinical course. The autopsy disclosed marked dilation of the right atrium and ventricle. The entire free wall of the right ventricle was markedly thin. Microscopically, most of the myocardial fibers of the right ventricle were replaced by fat and fibrous tissue. The second case, a 15-year-old boy, whose identical twin was previously diagnosed as arrhythmogenic right ventricular dysplasia designated by Fontaine et al., died suddenly during exercise. He showed no cardiac symptoms but electrocardiogram was abnormal. Autopsy revealed majority of the myocardial fibers of the right ventricular free wall were replaced by fatty tissue. In both cases, fatty infiltration was mainly noticed at the epicardial side and some myocardial fibers remained in the fatty tissue showed hypertrophic and/or degenerative changes. Review of the literature on similar cases showing depletion of the right ventricular musculature including so-called adult's Uhl anomaly, ARVD and dilated right ventricular myocardiopathy was conducted and the relationship of the present cases with these lesions was discussed.     

Incidence of latent infection of Epstein–Barr virus in lung cancers—an analysis of EBER1 expression in lung cancers by in situ hybridization

To evaluate the presence of Epstein–Barr virus (EBV) in lung cancers of Japanese patients, 81 lung cancers were examined using a highly sensitive in situ hybridization (ISH) method, employing an antisense oligonucleotide probe for EBV-encoded small nuclear RNA-1 (EBER). EBER1 expression was demonstrated in one poorly differentiated squamous cell carcinoma associated with marked lymphoid stroma (PDSCC-LS), two well differentiated adenocarcinomas, and two moderately differentiated squamous cell carcinomas, but was not detectable in other lung cancers, including small cell carcinomas. Unlike lymphoepithelioma-like undifferentiated carcinoma (LELC) of the lung, the PDSCC-LS consisted of poorly differentiated cells with distinct cell borders and nuclei with a coarse chromatin pattern and some prominent nucleoli. Most of the cancer cells expressed intense EBER1 signals. Although small to moderate numbers of cells positive for EBER1 were present in two adenocarcinomas and two squamous cell carcinomas, EBER1 signals varied in intensity and number in these four cases. Although polymerase chain reaction (PCR) and Southern blot hybridization with a ³²P-labelled probe internal to the primers were conducted to detect the EBV genome in 24 lung cancers, including five EBER1-positive cases, the genome was found to be positive in the five cases with EBER1-positive staining, including the PDSCC-LS, two adenocarcinomas and two squamous cell carcinomas, but not in the other cases. This study indicates that the morphological features of EBV-associated lung cancers are not restricted to the typical LELC type.     

Regulation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor expression in human neutrophils

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, which is capable of inducing apoptosis in many cell types, including tumour and virus-infected cells, but rarely in normal cells. Expression of TRAIL mRNA and TRAIL receptors has previously been detected in neutrophils; however, the expression of TRAIL protein and the regulation of TRAIL and TRAIL receptor expression in these cells remain unknown. Here we report, for the first time, that neutrophils constitutively express TRAIL protein on their cell surface and that the TRAIL protein is shed during culture. TNF-alpha is a down-regulator of TRAIL expression, whereas IFN-gamma up-regulates the expression of TRAIL. Neutrophils did not express a detectable level of TRAIL-R1 or -R4, but constitutively expressed a low, but substantial, level of TRAIL-R2 and a high level of TRAIL-R3. Although the level of TRAIL-R2 was not significantly altered during culture under different experimental conditions, approximately 30% of TNF-alpha-treated cells rapidly lost their high-level TRAIL-R3 expression, whereas the majority of IFN-gamma-treated cells retained a high level of TRAIL-R3 expression. Anti-TRAIL neutralizing antibody significantly inhibited neutrophil apoptosis during cultures in medium alone, or in the presence of TNF-alpha or IFN-gamma. Thus, our study identified human neutrophils as a cellular source of TRAIL and suggests that neutrophil-derived TRAIL may play a role in immune surveillance. Our results also suggest a role for the TRAIL/TRAIL receptor system in neutrophil apoptosis.     

Effect of Mycobacterium bovis BCG vaccination on Mycobacterium-specific cellular proliferation and tumor necrosis factor alpha production from distinct guinea pig leukocyte populations

In this study, we focused on three leukocyte-rich guinea pig cell populations, bronchoalveolar lavage (BAL) cells, resident peritoneal cells (PC), and splenocytes (SPC). BAL cells, SPC, and PC were stimulated either with live attenuated Mycobacterium tuberculosis H37Ra or with live or heat-killed virulent M. tuberculosis H37Rv (multiplicity of infection of 1:100). Each cell population was determined to proliferate in response to heat-killed virulent H37Rv, whereas no measurable proliferative response could be detected upon stimulation with live mycobacteria. Additionally, this proliferative capacity (in SPC and PC populations) was significantly enhanced upon prior vaccination with Mycobacterium bovis BCG. Accordingly, in a parallel set of experiments we found a strong positive correlation between production of antigen-specific bioactive tumor necrosis factor alpha (TNF-alpha) and prior vaccination with BCG. A nonspecific stimulus, lipopolysaccharide, failed to induce this effect on BAL cells, SPC, and PC. These results showed that production of bioactive TNF-alpha from mycobacterium-stimulated guinea pig cell cultures positively correlates with the vaccination status of the host and with the virulence of the mycobacterial strain.