Substance P stimulates release of RANKL via COX-2 expression in human dental pulp cells

Objective: Our previous study found that substance P (SP), a sensory neuropeptide, was expressed in the dental pulp of rats during experimental tooth movement. We examined the effects of SP on the production of prostaglandin (PG) E₂ and the receptor activator of nuclear factor- B ligand (RANKL) by human dental pulp fi broblast-like (HDPF) cells. Materials and methods: SP was added to cultured HDPF cells at concentrations ranging from of 10⁻⁴ to 10⁻¹² mol/L. PGE₂ and soluble RANKL (sRANKL) levels were determined using enzyme-linked immunosorbent assay kits. Gene expression was confi rmed by RT-PCR analysis. Pit formation assays using dentin slices were carried out to examine the effect of SP on osteoclastogenesis. Results: The levels of PGE₂ and sRANKL increased in the presence of SP, though the increases were greater in the experimental groups in both a time- and concentration-dependent manner, and the increase of RANKL was partially mediated by PGE₂ . The gene expression of cyclooxygenase (COX)-2 and RANKL was up-regulated, and conditioned medium samples obtained from HDPF cells treated with SP induced bone resorption. Conclusions: SP stimulated the production of PGE₂ and RANKL, and promoted bone resorption. Therefore, SP may be involved in pulpal inflammation and root resorption during orthodontic tooth movement. Received 11 April 2005; returned for revision 27 July 2005; returned for final revision 20 September 2005; accepted by M. Katori 2 November 2005     

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     

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.     

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.     

The molecular structures of 1,1-diphenylethyl methacrylate and triphenylmethyl methacrylate

The molecular structures of 1,1-diphenylethyl methacrylate (1,1-DPEMA) and triphenylmethyl methacrylate (TrMA) were determined by means of X-ray diffraction. 1,1-DPEMA: monoclinic, space group P2₁/a,a = 9,666(6), b = 19,94(2), c = 8,132(6) Å, β = 104,49(7)°, and Z = 4; TrMA: monoclinic, space group P2₁/n, a = 17,349(3), b = 9,487(2), c = 11,254(2) Å, β = 102,30(2)°, and Z = 4. Both structures were solved by the direct method and refined by the block-diagonal least-squares procedure to R = 0,175 and 0,056 for non-zero reflections, respectively. In both molecules, conformations about the C(1)? C(2) and C(1)? O(1) bonds are all synperiplanar and one of the two or three phenyl groups attached to the C(5) atom is in trans to the O(2).     

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.     

Tissue distribution of the Pk antigen as determined by a monoclonal antibody

Using an anti-Pk monoclonal antibody (mAb) designated CPK-1, the expression of the Pk antigen was assessed on normal human tissue from non-Pk individuals. Although the Pk antigen was detected on fibroblasts and blood vessels as previously reported, it was also found on smooth muscle cells of the digestive tract and the urogenital system. Pk was also found on glandular cells of the stomach, oesophagus and prostate. Additionally, CPK-1 reacted weakly with oesophagus squamous cells, and a small number of glomeruli and tubules in the kidney. The mechanism of expression of the Pk determinant in non-Pk individuals is discussed.     

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.