Topography Influences Adherent Cell Regulation of Osteoclastogenesis.

The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow-derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.

Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs.

Changes in free amino acid concentrations in the blood, brain and muscle of heat-exposed chicks.

An experiment was conducted to analyse the changes in free amino acid concentrations in the blood, brain and muscle of chicks in response to 15 or 30 min exposure to high ambient temperature (HT). Food intake and body weight were not affected, while rectal temperature was significantly increased by short-term HT exposure. Several free amino acid concentrations increased in the blood, brain and muscle even with short-term HT, whereas levels of a few amino acids declined significantly. As well as the nonessential amino acids, essential amino acids also significantly increased with exposure to HT. 3-Methylhistidine, a marker of proteolysis, significantly declined in the muscle of HT chicks, implying a reduction of protein breakdown under HT. These results indicate that alteration of protein metabolism may occur in chicks even with short-term heat exposure.

Cord blood transplantation is associated with rapid B-cell neogenesis compared with BM transplantation.

Hematopoietic cell transplantation (HCT) is used for treatment of hematopoietic diseases. Assessment of T- and B-cell reconstitution after HCT is crucial because poor immune recovery has a major effect on the clinical course. In this study, we retrospectively analyzed T-cell receptor excision circles (TRECs) as well as signal and coding joint kappa-deleting recombination excision circles (sjKRECs and cjKRECs, respectively) as markers of newly produced lymphocytes in 133 patients (56 primary immunodeficient and 77 malignant cases, median (range): 12 (0-62) years old). We analyzed the kinetics of TREC and KREC recovery and determined the factors that contributed to better immune recovery. KRECs became positive earlier than TRECs and increased thereafter. Younger recipient age had a favorable effect on recovery of sjKRECs and cjKRECs. Compared with BM and peripheral blood, our data suggested that cord blood (CB) provided rapid B-cell recovery. CB also provided better B-cell neogenesis in adult HCT recipients. Chronic GVHD was associated with low TRECs, but not increased sjKRECs/cjKRECs. Finally, positive sjKRECs 1 month after HCT were associated with fewer infectious episodes. Monitoring of TRECs and KRECs may serve as a useful tool for assessment of immune reconstitution post HCT.

Intracerebroventricular injection of kynurenic acid attenuates corticotrophin-releasing hormone-augmented stress responses in neonatal chicks.

In the brain of neonatal chicks, tryptophan has a sedative effect, and a part of this effect might be dependent upon its metabolite, serotonin. However, the functional mechanisms have not been fully clarified, since l-tryptophan produces kynurenic acid (KYNA) through the kynurenine pathway. The present study aimed to clarify the effect of KYNA on the stress response upon social isolation. Intracerebroventricular injection of KYNA induced a strong sedative effect under stress compared with the effect of l-tryptophan, with or without intracerebroventricular injection of corticotrophin-releasing hormone (CRH). KYNA dose-dependently induced sedative and hypnotic effects under CRH-augmented social isolation stress. Taken together, these results indicate that KYNA is a likely candidate for the sedative and hypnotic effects of tryptophan under acutely stressful conditions.

Important roles of tachykinins in the development of allergic nasal hyperresponsiveness in guinea-pigs.

BACKGROUND: Although it has been suggested that the use of tachykinin receptor antagonists might prove to be an effective treatment for allergic rhinitis (AR), they are not used clinically. Therefore, we decided to examine the effects of tachykinin receptor antagonists on AR symptoms in an appropriate experimental model. OBJECTIVE: To evaluate newly developed tachykinin receptor antagonists in a Japanese cedar pollen-induced AR model and to determine their effect on allergen-induced sneezing, nasal blockage, and nasal hyperresponsiveness (NHR). METHODS: Sensitized guinea-pigs were challenged by forced inhalation of pollen once every week. Sneezing and nasal blockage were observed after pollen challenges. NHR (nasal blockage) to an intranasal application of leukotriene D(4) was assessed 2 days after an antigen challenge. We also evaluated whether intranasal dosing with a tachykinin causes NHR. NK(1) and NK(2) receptor antagonists were administered before an intranasal treatment with antigen or tachykinin. Amounts of tachykinins present in nasal cavity lavage fluid were measured by an enzyme immunoassay. RESULTS: Although an NK(1) and NK(2) receptor dual antagonist showed no effect on pollen-induced sneezing and biphasic nasal blockage, it did completely suppress the development of NHR. Experiments using specific NK(1) or NK(2) receptor antagonists revealed that NK(2) receptor activation was preferentially involved in the development of hyperresponsiveness. Increases in the levels of substance P (SP) and neurokinin A (NKA) in the nasal tissue were noted 20 min-1 h after the challenge. Intranasal instillation of either SP or NKA-induced NHR, which was almost completely inhibited by NK(2) receptor antagonists and partially inhibited by NK(1) receptor antagonists. CONCLUSIONS: SP and NKA, which are released early after the challenge, mediate the development of NHR by preferentially activating NK(2) receptors. Therefore, NK(2) receptor antagonists might prove to be effective treatment of AR.

Phospho-regulation of human protein kinase Aurora-A: analysis using anti-phospho-Thr288 monoclonal antibodies.

Mammalian Aurora-A is related to a serine/threonine protein kinase that was originally identified by its close homology with Saccharomyces cerevisiae Ipl1p and Drosophila melanogaster aurora that are key regulators in the orchestration of mitotic events. The protein level of Aurora-A, its peak kinase activity during mitosis, and its activation have been attributed to phosphorylation. Here we show that this enzyme is an arginine-directed kinase and define its substrate specificity. We also found that Thr288 within the activation loop is a critical residue for activating phosphorylation events in vitro and that it is spatiotemporally restricted to a brief window at mitosis on duplicated centrosomes and on spindle microtubules proximal to the poles in vivo. Immunodepletion assays indicated that an upstream kinase(s) of Aurora-A might exist in mammalian cells in addition to autophosphorylation. Furthermore, human activated Aurora-A forms complexes with the negative regulator protein serine/threonine phosphatase type 1 (PP1) that was negatively phosphorylated on Thr320. Interestingly, phospho-specific Aurora-A monoclonal antibodies restrain Aurora-A kinase activity in vitro, providing further therapeutic avenues to explore.

Hematopoietic stem cell transplantation for 30 patients with primary immunodeficiency diseases: 20 years experience of a single team.

We retrospectively analyzed our results of 30 patients with three distinctive primary immunodeficiency diseases (PIDs)--severe combined immunodeficiency (SCID, n = 11), Wiskott-Aldrich syndrome (WAS, n = 11) and X-linked hyper-immunoglobulin M (IgM) syndrome (XHIM, n = 8)--who underwent hematopoietic SCT (HSCT) during the past 20 years. Until 1995, all donors were HLA-haploidentical relatives with T-cell depletion (TCD) (n = 8). Since 1996, the donors have been HLA-matched related donors (MRD) (n = 8), unrelated BM (UR-BM) (n = 7) and unrelated cord blood (UR-CB) (n = 7). Twenty-seven of 30 patients had various pre-existing infections with or without organ damages before HSCT. Conditioning regimen and GVHD prophylaxis were determined according to disease, donor and pretransplant status. Although one of eight patients transplanted with TCD is alive with full engraftment, the other seven died. On the other hand, 18 of 22 patients transplanted without TCD are alive and well, including six of eight transplanted from MRD, seven of seven from UR-BM and five of seven from UR-CB. All 19 survivors did not require Ig supplementation after HSCT. These results indicate that UR-CBT as well as UR-BMT provides good results for PID comparable to MRD-SCT, and that early diagnosis, HSCT at early stage, careful supportive therapy and monitoring for various pathogens are important for the successful HSCT.

A case of gastrointestinal stromal tumour of the ampulla of Vater.

Gastrointestinal stromal tumour rarely develops in the duodenal ampulla region. We report here a case of gastrointestinal stromal tumour of the ampulla of Vater found in a 44-year-old Japanese man presenting with biliary obstruction. He died of hepatic failure with diffuse liver metastasis. The postmortem examination showed a large Borrman type III-like tumour in the duodenal ampullary region with direct invasion of the pancreas and extrahepatic bile duct as well as metastases to the liver and regional lymph nodes. The duct orifice was located at the centre of the tumour. Microscopically, the tumour consisted of anaplastic spindle cells with high mitotic activity (90 mitoses per 50 high-power fields). Immunohistochemically, the spindle cells were positive for KIT and CD34. The final diagnosis was high-grade malignant gastrointestinal stromal tumour of the ampulla of Vater. Considering the recent advances in the diagnosis and treatment of gastrointestinal stromal tumour, this neoplasm should be included in the differential diagnosis of the tumours appearing in the duodenal ampulla region.

The transradial approach for selective carotid and vertebral angiography.

PURPOSE: The transradial approach is not so popular in cerebral angiography. The purpose of this study was therefore to present our experience of success rate and safety of this method. MATERIAL AND METHODS: From December 1998 to June 2001, 526 carotid and vertebral angiographies with DSA were performed via the radial artery. A 1.4-mm catheter was used through a 1.4-mm introducer sheath. We evaluated the procedure as successful if sufficient images for diagnosis were obtained of the bilateral carotid arteries and unilateral vertebral artery. Each patient was reassessed for any complications, occurring until the next morning. The length of time needed for an examination was measured in the last 10 cases. RESULTS: In all but 5 cases, the procedures were evaluated as successful (99.0%). Unsuccessful cases manifested severe pain at the radial puncture, angiospasm at the radial artery, loop formation at the radial artery, occlusion at the subclavian artery, and an aberrant right subclavian artery. No severe complications including neurological ones were encountered. Minor complications were noted in 17 cases (3.2%): 4 cases of thrombus at the ulnar artery, 1 angiospasm at the radial artery, and 12 cases of small hematoma at the puncture site. The radial approach took 14 min less in the common carotid study and 3 min 30 s less in the internal carotid study than by the femoral approach. CONCLUSION: The transradial approach enabled selective studies for carotid and vertebral angiography with a high success rate and safety with few complications.

Invasive subglottal aspergillosis in a patient with severe aplastic anemia: a case report.

A 19-year-old female with aplastic anemia who developed subglottal aspergillosis is reported. She presented with fever, cough and stridor. Inspiratory dyspnea progressed rapidly and emergent tracheostomy was performed, which confirmed the diagnosis. In spite of intensive anti-fungal treatment combined with adoptive immunotherapy, Aspergillus infection expanded and she died of pulmonary aspergillosis. Autopsy revealed the fungal mass obstructing the trachea and disseminated pulmonary aspergillosis. Difficulties in diagnosis and management of subglottal Aspergillus infection are discussed.

[A case of Mycobacterium intracellulare infection complicated by immotile cilia syndrome].

A 25-year-old woman with a history of immotile cilia syndrome (ICS) was admitted to our hospital with dyspnea. Chest roentgenography revealed dense infiltrates in both lower lung fields in addition to bronchiectasis and small nodular opacities, which had been observed previously. Transbronchial lung biopsy demonstrated evidence of non-caseating epithelioid cell granuloma. Sputum specimens were examined, and isolates were identified as Mycobacterium intracellulare. The patient was given antituberculous therapy and clarithromycin, which induced clinical improvement. It is well known that bronchial mucociliary transport is severely impaired in patients with ICS. However, to our knowledge, cases of M. intracellulare infection complicated by ICS have not been reported in Japan. We must pay close attention to the concurrence of these diseases.

Nationwide survey shows that methicillin-resistant Staphylococcus aureus strains heterogeneously and intermediately resistant to vancomycin are not disseminated throughout Japanese hospitals.

A total of 6,625 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates obtained from 278 hospitals throughout Japan were obtained between November and December 1997 and were examined for their sensitivities to vancomycin using Mueller Hinton (MH), brain heart infusion (BHI), agar plates, or the broth microdilution method. A concentrated inoculum of an MRSA strain or the use of highly enriched medium, such as BHI medium, allows an individual cell to grow on agar plates containing a vancomycin concentration greater than the MIC for the parent strain. However, cells of the colonies which grew on BHI agar plates containing the higher vancomycin concentrations did not acquire a level of vancomycin resistance greater than that of the parent strain and were not subpopulations of heterogeneously vancomycin-resistant MRSA. There was no significance in the fact that these colonies grew on the higher concentration of vancomycin: none showed stable resistance to vancomycin at a concentration above the MIC for the parent strain, and no cell from these colonies showed a relationship between the MIC and the ability of these colonies to grow on higher concentrations of vancomycin. The vancomycin MIC was not above 2 microg/ml for any of the cells originating from these colonies. No Mu3-type heterogeneously resistant MRSA strains, which constitutively produce subpopulations from MRSA clinical isolates with intermediate vancomycin resistance at a high frequency, were detected. There was a unipolar distribution of the MICs ranging from 0.25 to 2 microg of vancomycin/ml among the 6,625 MRSA clinical isolates, indicating that there was no Mu50-type intermediately vancomycin-resistant MRSA (MIC, 8 microg/ml by National Committee for Clinical Laboratory Standards criteria) among the clinical isolates, and there was no evidence of dissemination of Mu3-type MRSA heteroresistant to vancomycin.

Accumulation of high levels of the p53 and p130 growth-suppressing proteins in cell lines stably over-expressing cyclin-dependent kinase 6 (cdk6).

Cyclin-dependent kinase 6(cdk6) is present in randomly proliferating cultures of 3T3 cells but has little detectable enzymatic activity. Significant activity is detected only during a short period in early G1 phase. To examine the possible functions of cdk6 in 3T3 cells, lines stably over-expressing cdk6 were constructed and compared to normal 3T3 cells or cell lines with reduced cdk6 levels due to expression of a dominant-negative form of the protein. Over-expression of cdk6 in cells, which led to high levels of activity even in proliferating cultures, had dramatic effects. Cell lines stably over-expressing wild-type cdk6 had a markedly reduced growth rate compared to parental 3T3 cells or lines expressing a dominant-negative form of cdk6. They also over-produced the p53 and p130 proteins and had increased sensitivity to UV-irradiation. Irradiation resulted in accumulation of the Bax protein and rapid cell death. Levels of p53 and p130 proteins were down-regulated and the growth rate of the cells was increased by introduction of the dominant-negative form of cdk6 into cells over-expressing cdk6, indicating that cdk6 is involved in the overproduction of p53 and p130. The results suggest that cdk6, through regulation of growth-suppressing molecules, may play a role in halting cellular growth when proliferation is inappropriate.

Effect of a combined administration of 5-fluorouracil and medroxyprogesterone acetate on pyrimidine nucleoside phosphorylases and thymidylate synthetase in 7,12-dimethylbenz.

The effect of a combined therapy of medroxyprogesterone acetate (MPA) and 5-fluorouracil (5-FU) on tumor size, pyrimidine nucleoside phosphorylase (PyNPase) activity, and thymidylate synthetase (TS) activity was examined in Sprague-Dawley (SD) rats with 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors. MPA augmented the antitumor activity of 5-FU and protected against body weight-loss due to 5-FU administration. PyNPase activity of both the MPA group and the MPA+5-FU group tended to increase compared with that of the 5-FU alone group. TS inhibition levels in the MPA+5-FU group tended to increase compared with those in the 5-FU alone group. These results indicate that MPA tended to augment antitumor activity of 5-FU and to reduce the side effects caused by 5-FU.

Thymoma. Analysis of prognostic factors.

OBJECTIVE: We evaluated the prognostic factors for thymoma that remain controversial. METHODS: We studied 72 consecutive patients treated for thymoma during the period between 1966 and 1997. Recurrence-free interval rates and overall survival rates calculated by the Kaplan-Meier method were compared using log-rank test by the Masaoka stage, extent of surgical resection, histology, or associated disease(s). Multivariate analysis was performed using Cox's proportional hazards model. RESULTS: Thirty-two thymomas were at Masaoka stage I, 9 at stage II, 15 at stage III, and 16 were at stage IV. There were 56 complete resections, 7 incomplete resections (2 at stage III and 5 at stage IV), and 9 biopsies (1 at stage III and 8 at stage IV). Forty-one thymomas were cortical, 16 medullary, and 15 were mixed form. Association of myasthenia gravis was found in 20 patients, and pure red cell aplasia in 7. After an average follow-up period of 103 months, the recurrence-free 5-, 10-, 15-year interval rate was 89%, 80%, 80%, respectively, and overall 5-, 10-, 15-year survival rate was 86%, 71%, 59%, respectively. Factors influencing the recurrence-free interval and overall survival included the Masaoka stage, extent of surgical resection, and association with pure red cell aplasia. Multivariate analysis revealed stage IV tumor and association with pure red cell aplasia as risk factors for recurrence. Pure red cell aplasia indicated poor prognosis for overall survival. CONCLUSIONS: Masaoka stage, extent of surgical resection, and association with pure red cell aplasia were prognostic factors for thymoma. Multidisciplinary treatment for stage IV tumors and better control of pure red cell aplasia, if associated, should be investigated.

Identification of a novel chloride channel expressed in the endoplasmic reticulum, golgi apparatus, and nucleus.

MID-1 is a Saccharomyces cerevisiae gene encoding a stretch-activated channel. Using MID-1 as a molecular probe, we isolated rat cDNA encoding a protein with four putative transmembrane domains. This gene encoded a protein of 541 amino acids. We also cloned the human homologue, which encoded 551 amino acids. Messenger RNA for this gene was expressed abundantly in the testis and moderately in the spleen, liver, kidney, heart, brain, and lung. In the testis, immunoreactivity of the gene product was detected both in the cytoplasm and the nucleus. When expressed in Chinese hamster ovary cells, the gene product was located in intracellular compartments including endoplasmic reticulum and the Golgi apparatus. When microsome fraction obtained from the transfected cells, but not from mock-transfected cells, was incorporated into the lipid bilayer, an anion channel activity was detected. Unitary conductance was 70 picosiemens in symmetric 150 mm KCl solution. We designated this gene Mid-1-related chloride channel (MCLC). MCLC encodes a new class of chloride channel expressed in intracellular compartments.