Grading score system: A method for evaluation of the degree of senescence in Senescence Accelerated Mouse (SAM)

For evaluation of the degree of senescence in SAM-P, accelerated senescence prone mouse, formerly called SAM or prone series or P-series, consisting of SAM-P/1, SAM-P/2, SAM-P/3 and SAM-P/4 corresponding to P-1, P-2, P-3 and P-4 series, respectively, in the previous reports, and in SAM-R, accelerated senescence resistant mouse, formerly called resistant series or R-series, consisting of SAM-R/1, SAM-R/2 and SAM-R/3 corresponding to R-1, R-2 and R-3 series, respectively, in the previous reports, the grading score system was adopted. The items to be examined in this system include 11 categories selected from the clinical signs and gross lesions considered to be associated with the aging process. The degree of the senescence in each category was graded from 0 to 4 according to the detailed criteria devised in our laboratory. After 8 months of age each mouse was examined every 4 months, and some of the mice were examined after 2 months of age.In almost all categories, the grading score and incidence began to increase from 4 or 6 months of age and continued to increase with advancing age in both SAM-P and SAM-R. The increase, however, was more marked in SAM-P than in SAM-R. The slow but steady increase in the SAM-R levelled out at 24 months of age and was comparable to that of 12 months of age in SAM-P. In both SAM-P/1 at 8 months of age and SAM-R/2 at 12 months of age, there was a significant reverse correlation between total score of this grading score system and length of residual life after examination.Systematic and extensive studies using the grading score system showed that if the validity of the system is, based on “irreversibility” and “universality” of the changes in     

Neuropeptides stimulate production of interleukin-1β, interleukin-6, and tumor necrosis factor-α in human dental pulp cells

Objective:Orthodontic tooth movement causes inflammatory reactions in the periodontal membrane and dental pulp. It has been reported that substance P (SP) and calcitonin gene-related peptide (CGRP), both sensory neuropeptides, are manifested in the dental pulp of rats during experimental tooth movement, suggesting that they might be involved in the dental pulp inflammation during orthodontic tooth movement. However, the relationships between neuropeptides and pro-inflammatory cytokines have not been fully elucidated.Materials and methods:Human dental pulp (HDP) fibroblasts were prepared from 6 healthy young volunteers (3 males, 3 females; 15–25 years old) during the course of orthodontic treatment. HDP cells were incubated for 24 h in fresh medium containing 2% FCS in the presence of various concentrations of CGRP (10^–12 to 10^–4 M) and SP (10^–12 to 10^–4 M), and the levels of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)- present in the media were determined using commercially available high-sensitivity enzyme-linked immunosorbent assay kit.Results:We examined the effects of stimulation by these neuropeptides on the production of inflammatory cytokines in HDP fibroblasts, and found that the levels of IL-1, IL-6, and TNF- increased in a time- and concentration-dependent manner. However, the neuropeptides did not act synergistically to increase cytokine secretion in HDP cells or significantly modify LPS-induced cytokine production by HDP cells.Conclusions:Our results suggest that human pulp fibroblasts may be involved in the progress of inflammation in pulp tissue during orthodontic tooth movement, as they produced large amounts of IL-1, IL-6, and TNF- following stimulation with neuropeptides.Received 2 March 2003; returned for revision 14 July 2003; accepted by M.J. Parnham 17 December 2003     

Topical application of FK506 (tacrolimus) ointment inhibits mite antigen-induced dermatitis by local action in NC/Nga mice

BACKGROUND: FK506 ointment (tacrolimus ointment, protopic) is a new drug therapeutically effective for patients with atopic dermatitis (AD). However, the mechanism of action of FK506 ointment on AD is not fully understood. METHODS: We examined the effect of FK506 ointment on mite antigen-induced dermatitis in NC/Nga mice. Clinical symptoms and ear thickness were recorded, and histopathological studies and in vitro analyses were performed. RESULTS: Topical application of FK506 ointment (0.03-0.3%) suppressed the development of dermatitis. In the lesional skin, both interleukin (IL)-4 and interferon (IFN)-gamma were detected, even though the IL-4+/IFN-gamma- T helper 2 (Th2) population was predominant in the regional lymph nodes (LNs). Topical application of FK506 treatment reduced the elevated level of both IL-4 and IFN-gamma in the skin, but did not decrease the expansion of the Th2 population in the LNs. CONCLUSIONS: Topical application of FK506 ointment suppresses dermatitis by inhibiting the activation of inflammatory cells locally, without systemic immune suppression, in this AD model.     

Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of ∼7 s even though exocytosis was induced with large increases in cytosolic Ca²⁺ concentration by uncaging of a caged-Ca²⁺ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.     

Lymphocyte subpopulations and T-cell subsets in human oral cancer tissues: immunohistologic analysis by monoclonal antibodies

A series of T-cell-specific monoclonal antibodies (Leu-1, Leu-2a, and Leu-3a) and B-cell-specific monoclonal antibody (HLB-1) were used to detect the localization and intensity of infiltration of lymphocyte subpopulations and T-cell subsets in frozen sections of 17 patients with the oral cancer. The vast majority of the lymphocyte infiltrates in the oral cancer tissues were reactive with Leu-1. In contrast, B cells were detectable with HLB-1 in only 2 of 17 cases. Leu-2a-positive cells were dominant in four cases, whereas Leu-3a positive cells were dominant in only three cases. In seven cases, both cells infiltrated to the same degree. Leu-2a positive cells tended to be dominant in the cases with earlier clinical stages.     

Ebselen prevents noise-induced excitotoxicity and temporary threshold shift

This investigation tested the hypothesis that a noise-induced temporary threshold shift (TTS) can be attenuated by a peroxynitrite scavenger, ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one). Guinea pigs received an oral dose of the vehicle or 10 mg/kg ebselen 1 h before exposure to 115 dB SPL 4-kHz octave band noise for 3 h. In controls, auditory brainstem response (ABR) thresholds increased by 25–45 dB immediately after noise and returned to pre-exposure baseline thresholds 7 days later. Ebselen eliminated this ABR threshold shift following noise exposure. In controls, swelling of the afferent dendrites beneath the inner hair cells was evident immediately after noise, whereas ebselen significantly reduced this pathology. These findings suggest that scavenging peroxynitrite can attenuate noise-induced excitotoxicity and, thereby, TTS.     

Development of the subclavian artery in the loggerhead turtle (Caretta caretta) studied by the injection method]

In the turtle, the left aorta and the pulmonary trunk originate from the right ventricle, while the right aorta takes its origin from the left ventricle as a functional systematic arch. The subclavian artery arises from the brachiocephalic artery on each side, and passes ventral to the vagus nerve and the jugular vein. These features are basically the same as in birds, and the subclavian artery of the adult turtle corresponds to a secondary artery from the viewpoint of comparative anatomy. Many investigators, including one of the present authors (Suzuki, 1987), have studied the development of the aortic arch and the subclavian artery in the chick embryo, but not in the turtle. The present authors examined it in Loggerhead turtle (Caretta caretta) embryos, from 14 days of incubation to completion of the aortic arch (27 days incubation). All blood vessels were injected with Berlin blue solution using a fine glass needle inserted into the aortic trunk through the ventricle of the heart. The following results were obtained. 1. In the turtle embryo the primary subclavian artery develops first, but is replaced by the secondary subclavian artery as in the chick. 2. The primary subclavian artery arises from the 12th dorsal intersegmental artery and passes dorsal to the posterior cardinal vein. In the 16-day embryo, it gives rise to capillary nets both cranially and caudally at the base of the forelimb bud along the inner surface of the thoracic wall. 3. At 19 days of incubation, a small blood vessel arises from the aortic sac at the origin of the third aortic arch and passes laterally, ventral to the anterior cardinal vein. The vessel then extends caudally, and finally, at 21 days of incubation, connects to the cranial part of the capillary net of the primary subclavian artery at about the middle of the lateral thoracic wall. After the completion of the connection, the vessel from the aortic sac is called by the name "the secondary subclavian artery." 4. The secondary subclavian artery gradually increases in size, while the proximal part of the primary one begins to atrophy and finally disappears at 27 days of incubation. After this, the forelimb bud receives its blood supply only from the newly-formed secondary subclavian artery. 5. In conclusion, in the turtle, the secondary subclavian artery is formed by connection of the primary artery with the caudally extending artery arising from the aortic sac, while in the chick it is derived from an outgrowth of the primary artery.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppressor activity in the supernatant of T cell clones derived from chimeric mouse spleen cells

The supernatant from cultures of T cell clones derived from (BALB/c----C3H/He) chimeras suppresses BALB/c anti-C3H/He or BALB/c anti-C57BL/6 MLRs. When we studied the alloantigen specificity of the suppressor activity in culture supernatant, we observed three types of the suppressor activity (i.e., the suppressor activity against BALB/c anti-C3H/He MLR, against BALB/c anti-C57BL/6 MLR, and against both MLRs) on day 3 after stimulation of the T cell clones with 20% crude IL2 and feeder cells. Since the alloantigen specificity fluctuated somewhat with time, we considered that a time-course study was needed to determine it correctly. We thought it unlikely that any IFN-gamma or PGE2 in the culture supernatant of the T cell clones would have mediated the suppression. Our results suggest that alloantigen specific and non-specific suppressor T cells exist in bone marrow chimeras. The former appears to play an important role in inducing and maintaining transplantation tolerance, while the latter seems to have a rather harmful effect upon chimeras.