Restricted expression of transgenic HLA-DRA gene in thymic epithelial cells and its role in acquisition of T cell tolerance to self-superantigens and processed DRα-derived peptide

We have established a set of transgenic mouse lines in which the HLA-DRA gene was expressed in different cell types. In one line (DRα-24), DRαEβ^b molecules were expressed on thymic medullary and cortical epithelial cells and all lineages of bone marrow-derived antigen-presenting cells (APC) except for thymic macrophages. By contrast, expression of the molecules in another line (DRα-30) was found on thymic medullary and cortical epithelial cells but not on bone marrow-derived APC in the thymus and periphery. To evaluate the role of thymic epithelial cells in acquisition of T cell tolerance, comparative analysis of DRα-24 and DRα-30 was performed. In DRα-30, T cells expressing TcR Vβ5 and Vβ11 were eliminated to comparable levels to those in DRα-24, suggesting that expression of the DRαEβ^b molecules on thymic epithelial cells are sufficient for clonal deletion of the self-superantigen-reactive T cells. In addition, CD4⁺ T cells from DRa-30 as well as those from DRα-24 were tolerant to DRα-derived peptide/I-A^b complex expressed on spleen cells from DRα-24 even in the presence of exogenous interleukin-2. These observations suggest that expression of the DRα chain in thymic epithelial cells could induce T cell tolerance directed toward naturally processed DRα-derived peptide bound to I-A^b molecules, probably via clonal deletion of the self-reactive T cells.     

Retinoblastoma protein prevents staurosporine-induced cell death in a retinoblastoma-defective human glioma cell line.

OBJECTIVE: To investigate the mechanism of staurosporine-induced glioma cell death and cell cycle arrest using adenovirus-mediated gene transfection, as well as the function of retinoblastoma (Rb) and genetic instability induced by staurosporine. METHODS: Cell cycle regulation, cell death and nuclear abnormalities induced by staurosporine were examined using an adenovirus vector expressing Rb, p16 or p21 genes in human glioma cell lines. RESULTS: The Rb-defective SF-539 cell line was resistant to staurosporine compared with cell lines expressing intact Rb. SF-539 glioma cells exposed to staurosporine became multinucleated and then died. Multinucleation was prevented in SF-539 cells transfected with the Rb gene, thus decreasing the death rate of these cells. CONCLUSIONS: These results imply that enforced Rb expression protects cells from genomic instability induced by staurosporine regardless of its upstream molecular effects.     

The effect of prednisolone on substance P-induced vascular permeability in mice

The effect of prednisolone on the substance P (SP)-induced vascular permeability increase in male ddY, WBB6 F₁–+/+ (control) and WBB6 F₁-W/W^v (no mast cell in skin or internal organs) mice was investigated. 1) SP (1–10 000 pg/site) increased vascular permeability in ddY, WBB6 F₁–+/+ and WBB6 F₁-W/W^v mice ears. 2) SP (100 pg/site)-induced vascular permeability was inhibited by prednisolone (10 mg/kg) administered intraperitoneally 3 to 12 hours prior to the elicitation of the reaction in ddY mice. When dexamethasone at a dose of 1 mg/kg was administered intraperitoneally 2 to 24 hours prior to the elicitation of the reaction, significant inhibition was observed. When prednisolone was administered intraperitoneally 8 hours prior to the elicitation of the reaction, the SP-induced capillary permeability increase in both ddY and WBB6 F₁-W/W^v mice was clearly inhibited by the drug at doses of 5 and 10 mg/kg. 3) Diphenhydramine (1 and 10 mg/kg) inhibited SP-induced vascular reaction in ddY mice but not in WBB6 F₁-W/W^v mice. 4) Atropine (10 mg/kg) inhibited SP-induced vascular reaction in both ddY and WBB6 F₁-W/W^v mice. But acetylcholine did not cause an increase of vascular permeability in ddY and WBB6 F₁-W/W^v mice ears. 5) Prednisolone (5 mg/kg) inhibited histamine- and serotonin-induced vascular permeability in ddY and WBB6 F₁-W/W^v mice ears. 6) Prednisolone (5 and 10 mg/kg) inhibited the SP-induced histamine release from ddY mice peritoneal mast cells. These results suggest that the vascular effect of SP is mediated by both mast cell dependent (release of histamine from mast cells) and mast cell independent mechanisms. Prednisolone inhibits the SP-induced vascular permeability mediated by both mechanisms in mice.     

Proliferative cell nuclear antigen expression in follicular tumours of the thyroid with special reference to oxyphilic cell lesions

The expression of proliferative cell nuclear antigen (PCNA) in follicular tumours of the thyroid was examined by immunohistochemistry. Both usual nonoxyphilic cell follicular tumours (non-OCT) and oxyphilic cell tumours (OCT) were subdivided into benign, indeterminate, encapsulated carcinoma, and widely invasive carcinoma types. Among non-OCT the percentages of PCNA-positive cells in benign tumours, encapsulated carcinomas, and widely invasive carcinomas was 2.5%–8.6%, 11.8%–39.1%, and 18.6%–20.0%, respectively. There was a statistically significant difference between benign tumours and encapsulated or widely invasive carcinomas, as in previous studies. A value of 10% was appropriate to distinguish benign from malignant lesions. PCNA-positive cells in indeterminate-type non-OCT were not significantly different from those in benign tumours, ranging from 4.3%–19.6%, and occurring at more than 10% in three of six tuours. Among OCT the positivity was less than 10% in benign tumours (4.5%–7.8%) and more than 10% in malignant tumours (14.1%–35.9%) and all the eight indeterminate tumours (12.5%–27.3%), with a statistically significant differences between the benign tumour and each of the latter types. These results indicate that the examination of PCNA is valuable in diagnosis of thyroid follicular tumours and that the use of similar diagnostic criteria may be warranted in both non-OCT and OCT.     

The influence of social environmental condition on the production of stress-induced 22 kHz calls in adult male Wistar rats

Adult rats emit 22 kHz ultrasonic vocalizations (USVs) in response to aversive stimuli, and these sounds are suggested to have communicative information among conspecifics. It is conceivable that social environment during development of rats has relevance to the emission of 22 kHz USVs. To examine the effects of social environment after weaning on production of stress-induced USVs, we compared the amount of emission of USVs among three groups of rats reared under different conditions after weaning. One group of rats was housed individually, and the other two groups were housed in pairs, in which social hierarchy of the pair was determined by social dominance-subordination relationships. The USVs were induced by acute mild somatic stimuli on the back and neck. Individually reared rats emitted much fewer USVs than pair-reared rats. In addition, socially subordinate rats emitted more USVs compared with socially dominant ones. These results suggest that not only social interaction but also the status in social hierarchy may play an important role in the process of the development of USVs induced by somatic stimuli.     

Influence of histochemical and immunohistochemical stains on polymerase chain reaction.

The polymerase chain reaction (PCR) analysis of DNA extracted from tissue sections can be applied to a variety of research and diagnostic protocols. To analyze selectively the specific areas of tissue, a direct microdissection of histochemically or immunohistochemically stained sections, if satisfactory for PCR, is helpful. However, the influence of various staining methods on PCR has been poorly investigated. In this study, paraffin sections of formalin-fixed lymph node samples were histochemically stained with Mayer's hematoxylin, eosin Y, methyl green, or May-Grunwald solution and immunostained for CD45 using 3,3'-diaminobenzidine (DAB), DAB with cobalt ion (DAB-Co), or new fuchsin as the chromogen. In addition, unstained sections were treated with trypsin, microwave, or pressure cooker, the techniques frequently used in immunostains for antigen unmasking. DNA was extracted from each section, and the PCR efficiency in amplifying a 110 bp portion of the beta-globin gene was evaluated by two parameters: the cycle count in which the first visible band was obtained (CYCLE(min)) and the maximum amount of PCR products (CONC(max)). The hematoxylin stain showed a significantly prolonged CYCLE(min) (P < .01) and lower CONC(max) (P < .05) in comparison with unstained and untreated control sections. The May-Grunwald stain showed a prolonged CYCLE(min) (P < .01), although the CONC(max) was not significantly different from that of the control (P = .051). The eosin and methyl green stains showed no effects against PCR. In immunostains, the DAB-Co method showed a lower CONC(max) (P < .05), whereas the CYCLE(min) was not prolonged. The DAB and new fuchsin methods had no untoward effects. Antigen-unmasking treatments showed deteriorating effects on PCR. The trypsin treatment significantly prolonged the CYCLE(min) (P < .01), and the PCR amplification did not reach the "plateau" level with a maximum of 60 cycles. The PCR efficiency was worse in microwave or pressure cooker treatment, with neither CYCLE(min) nor CONC(max) being obtained. When target areas from sections for subsequent PCR amplification are microdissected, methyl green is most suitable as a dye for nuclear staining. The immunohistochemical visualization with DAB or new fuchsin yields no unfavorable effects. A successful PCR amplification may not be expected in sections that are pretreated in a microwave oven or pressure cooker.     

Inhibition of passive cutaneous anaphylaxis-associated scratching behavior by mu-opioid receptor antagonists in ICR mice

BACKGROUND: Itching in humans is attenuated by mu-opioid receptor antagonists. ICR mice display increases in scratching behavior upon induction of IgE-mediated passive cutaneous anaphylaxis (PCA), or intradermal injection of compound 48/80 or histamine. METHODS: Cutaneous reactions were induced in ICR mice by IgE-mediated PCA, compound 48/80 and histamine, and the scratching behavior associated with the cutaneous reactions was evaluated. RESULTS: Naloxone and nalmefene reduced the incidence of scratching behavior associated with PCA. Naloxone also inhibited the induction of scratching behavior caused by compound 48/80 and histamine. Naloxone did not affect the increase in vascular permeability caused by PCA and injection of compound 48/80. CONCLUSION: Scratching behavior in mice may be induced by a sensation or a mechanism similar to itching in humans and should become a useful model for examining itching in humans.     

Closed-loop identification of carotid sinus baroreflex transfer characteristics using electrical stimulation

Although random aortic pressure (AOP) perturbation according to a binary white noise sequence enables us to estimate open-loop dynamic characteristics of the carotid sinus baroreflex under closed-loop conditions, the necessity of arterial catheter implantation limits the applicability of this method in freely moving animal experiments. Thus, we explored a closed-loop system identification method using electrical stimulation. In 6 anesthetized and vagotomized rabbits, we stimulated the aortic depressor nerve with a binary white noise sequence (0-10 Hz) under baroreflex closed-loop conditions while measuring cardiac sympathetic nerve activity (SNA) and AOP. We used a closed-loop identification method to estimate the peripheral arc transfer function from SNA to AOP. The peripheral arc transfer function approximated a second-order low-pass filter and its fitted parameters did not differ from those obtained by an open-loop identification method (dynamic gain: 1.16+/-0.32 vs. 1.02+/-0.11; natural frequency: 0.08+/-0.03 vs. 0.09+/-0.03 Hz; damping ratio: 1.53+/-0.15 vs. 1.57+/-0.21). In 6 different rabbits, we applied intermittent rapid pacing (396 beats/min) under baroreflex closed-loop conditions to estimate the neural arc transfer function from AOP to SNA. The neural arc transfer function approximated a first-order high-pass filter and its fitted parameters did not differ from those obtained by an open-loop identification method (dynamic gain: -1.15+/-0.45 vs. -1.06+/-0.05; corner frequency: 0.12+/-0.05 vs. 0.13+/-0.03 Hz). In conclusion, the closed-loop identification method using electrical stimulation is effective to estimate the neural and peripheral arc transfer functions.