Long-term impact of atlantoaxial arthrodesis on the pediatric cervical spine

Background  This study was conducted to elucidate radiographically the long-term impact of atlantoaxial arthrodesis on the pediatric cervical spine. Methods  The records of eight children who underwent atlantoaxial arthrodesis for the treatment of upper cervical spinal disorders and were followed up to their adulthood were retrospectively reviewed. Changes in the curvature of the whole cervical spine, and anteroposterior diameters of the spinal canal at C1 and C2 levels were investigated on lateral radiographs before surgery, at 6 months after surgery, and at the final follow-up. Results  The cervical curvature was lordosis or straight before surgery in all children and became sigmoid in six children at 6 months after surgery. At the final follow-up, three of the six children with postoperative sigmoid curvatures regained some degree of lordosis and became straight. The anteroposterior diameters of the spinal canal at C1 and C2 levels did not increase during the follow-up period. Conclusions  Development of postoperative malalignment of the lower cervical spine and impaired growth of the spinal canal at C1 and C2 levels are common after atlantoaxial arthrodesis in children. Postoperative malalignment diminishes during the follow-up period possibly due to remodeling of the pediatric cervical spine, although remodeling of the spinal canal diameter cannot be expected, suggesting the importance of anatomical reduction before or at the time of surgery.     

Comparison of osteoclast precursors in peripheral blood mononuclear cells from rheumatoid arthritis and osteoporosis patients

Osteolytic disorders cause serious problems for quality of life with aging. Osteolysis is performed by osteoclasts of the hematopoietic lineage that share some characteristics with monocytes and macrophages. As osteoclast precursors (pOCs) are present in peripheral blood, their characterization in osteolytic diseases may help us to understand risk factors. Although essential factors for osteoclastogenesis have been reported, the effective induction from pOCs in human peripheral blood mononuclear cells (PBMCs) to mature osteoclasts in culture requires further improvement. The aim of this study was development of an efficient culture system for human osteoclastogenesis and providing a simple system for the enrichment of pOCs from PBMCs. We employed coculturing of human PBMCs with a mouse stromal cell line. Significant numbers of tartrate-resistant acid phosphatase-positive (TRAP⁺) multinucleated osteoclasts (MNCs), which could resorb dentine slices, were efficiently induced in this culture condition. pOCs were enriched in an anti-CD16 antibody column-passed anti-CD14 antibody-bound cell population isolated by magnetic cell sorting. We compared the percentage of the CD14^high CD16^dull cell population, which mainly contained pOCs in PBMCs, from age-matched patients with rheumatoid arthritis (RA) and osteoporosis (OP), but it was comparable. However, the mean number of TRAP⁺ MNCs generated in cultures from PBMCs of RA was higher. In contrast, the frequency of pOCs in PBMCs from OP was relatively higher. These results suggest the characteristics of pOCs from RA and OP may be different, because single pOCs from OP gave rise to lower numbers of osteoclasts than those from RA.     

Heterogeneous transforming growth factor (TGF)-beta unresponsiveness and loss of TGF-beta receptor type II expression caused by histone deacetylation in lung cancer cell lines.

Transforming growth factor (TGF)-beta strongly inhibits epithelial cell proliferation. Alterations of TGF-beta signaling are thought to play a role in tumorigenesis. We show in the present study that most lung cancer cell lines have lost the growth-inhibitory response to TGF-beta signal, and that those with TGF-beta unresponsiveness can be divided into two major groups, TGF-beta type II receptor (TGFbetaRII)(+)/Smad7(+) and TGFbetaRII(-)/Smad7(-), suggesting the heterogeneous mechanisms underlying the TGF-beta responsiveness. The mechanism of the loss of TGFbetaRII expression of the latter group was further studied, identifying aberrant DNA methylation of the promoter region in a limited fraction of cell lines. Interestingly, we found that the alteration of chromatin structure because of histone deacetylation may also be involved, showing a good correlation with loss of TGFbetaRII expression. This notion was supported by the findings of a restriction enzyme accessibility assay, of a chromatin immunoprecipitation assay with anti-acetyl histone antibodies, and of an in vivo induction of TGFbetaRII expression by histone deacetylase inhibitors including trichostatin A (TSA) and sodium butyrate. In vitro induction of TGFbetaRII promoter reporter activity by TSA was also detected and found to require the CCAAT box within the -127/-75 region. A positive regulatory mechanism for TGFbetaRII expression in a TGF-beta-expressing cell line was also investigated, and a TPA-responsive element (TRE)-like motif, TRE2, was detected in addition to the previously reported TRE-like motif Y element in the positive regulatory region. Alterations in two discrete proteins interacting with these two TRE-like motifs were also suspected of being involved in the loss of TGFbetaRII expression. This is the first study to demonstrate that, in addition to the TSA-responsive region and TRE2 motif in the TGFbetaRII promoter, the alteration of histone deacetylation may be involved in the loss of TGFbetaRII expression in lung cancer cell lines.     

A novel Wiskott–Aldrich syndrome protein mutation in an infant with thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) has not yet been reported to be associated with mutations in the Wiskott–Aldrich syndrome (WAS) gene. WAS is an X‐linked recessive disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. A broad spectrum of mutations in the WAS protein (WASP) gene have been identified as causing the disease. In this study, we report on a 2‐month‐old Japanese boy who presented with cytomegalovirus (CMV) infection and TTP. The activity of von Willebrand factor cleaving metalloproteinase, ADAMTS13 was low and the antibody against ADAMTS13 was positive (3.6 Bethesda U/mL). Although TTP was improved by plasma exchange and steroid pulse therapy, thrombocytopenia persisted and regular transfusions of irradiated platelets were needed. Tiny platelets were found on a peripheral blood smear. CMV genome was positive in peripheral blood by polymerase chain reaction and the CMV viremia continued to persist despite intravenous gancyclovir therapy. Through direct sequencing of genomic DNA of the WASP gene in the patient, we identified a novel mutation of WASP gene: the seventh nucleotide in exon 11 (G) had been deleted (1345delG). This mutation causes a frameshift and a stop codon at amino acid 470. Western blotting demonstrated a truncated WAS protein. To our knowledge, this is the first report describing TTP in WAS patients with novel mutation in the WASP gene.     

ESTIMATION OF ABSORPTION OF ENVIRONMENTAL CONTAMINANTS IN LOW-LEVEL EXPOSURE BY PHARMACOKINETIC ANALYSIS

Inhalation pharmacokinetics of the environmental contam inants 1,1,1-trichloroethane, tetrachloroethylene, trichloroethylene, benzene, and p -dichlorobenzene were determined in male Sprague-Dawley rats, and data generated were used to obtain fundamental data for risk assessment of chronic low-level exposures to these substances. Measured amounts of the substances were injected into a closed chamber system in which a rat had been placed, and the concentration changes in the chamber air were examined. The pharmacokinetics of the substances were evaluated using linear or nonlinear compartment models. The metabolic elimination amounts at various exposure concentrations were extrapolated using the estimated pharmacokinetic parameters. At low (1 ppb) concentration exposure to benzene or trichloroethylene the fraction of elimination of these substances was 1.6 and 1.5 times higher, respectively, than that seen at high (10 ppm) concentration exposure. Extrapolation kinetics of low-exposure data showed more trichloroethylene (0.0016 mumol/h/kg), benzene (0.0014 mumol/h/kg), and p -dichlorobenzene (0.00052 mumol/h/kg) was absorbed by the rats than 1,1,1- trichloroethane (0.000019 mumol/h/kg) and tetrachloroethylene (0.000029 mumol/h/kg). Although the exposure concentrations for all chemicals were equal, the differences in absorption quantities need to be considered in evaluation of potential risk assessment.     

Expression of Macrophage Migration Inhibitory Factor in Human Breast Cancer: Association with Nodal Spread

Macrophage migration inhibitory factor (MIF) is known to exert pleiotropic functions including inhibition of macrophage migration, anchoring, and counteraction of the anti-inflammatory and immunosuppressive activity of glucocorticoids. Ninety-three primary breast cancer tissues and 64 sera of primary breast cancer patients were analyzed for the expression of MIF. The clinico-pathological significance of MIF expression was evaluated. It was found that MIF was frequently over-expressed in primary breast cancer tissues. RT-PCR and western blotting analysis confirmed that wild-type MIF is expressed, and immunohistochemical analysis showed that MIF expression was localized at tumor cells as well as stromal cells, including tumor-associated macrophages. Intra-tumoral MIF protein concentrations detected by enzyme-linked immunosorbent assay (ELISA) varied with a median value of 1821 ng/mg protein (range: 8–8126 ng/mg protein), and correlated inversely with nodal involvement ( P =0.039). No significant correlation was observed with other clinico-pathological factors including tumor size, menopausal status and hormone receptors. The circulating level of MIF protein ranged up to 105.7 ng/ml (median: 17.3 ng/ml), and it was also found to correlate inversely with the number of involved nodes ( P =0.02). A comparative study with other soluble inflammatory mediators showed that intratumoral levels of MIF were significantly associated with those of interleukin-1β, suggesting that interactions between tumor cells and tumor-associated macrophages play an important role in the up-regulation of MIF. The multifunctional inflammatory/immune mediator MIF was frequently expressed in primary breast cancer, and its expression level was inversely associated with nodal spread. Thus, MIF seems to play a role in tumor-stroma interactions of primary breast cancers, particularly those with a phenotype of node-negative or minimal nodal spread.     

Effects of YM358, an angiotensin II type 1 (AT1) receptor antagonist, and enalapril on blood pressure and vasoconstriction in two renal hypertension models

The effects of the angiotensin II type 1 receptor antagonist YM358 on blood pressure were compared to those of the angiotensin converting enzyme inhibitor enalapril in one-kidney, one-clip renal hypertensive rats (1K1C RHR), two-kidney, one-clip renal hypertensive rats (2K1C RHR) and normotensive rats (NTR). Additionally, the local drug actions in peripheral tissues were investigated using isolated mesenteric arteries from these rats. In 2K1C RHR, YM358 and enalapril produced a long-lasting hypotensive effect in a dose-dependent manner. In 1K1C RHR, YM358 (30 mg/kg) also produced an antihypertensive effect, whereas enalapril (30 mg/kg) had no effect. Administration of YM358, but not enalapril, to 1K1C RHR, 2K1C RHR and NTR did not affect heart rate. In isolated mesenteric arteries from 1K1C RHR and 2K1C RHR, angiotensin II (Ang II), angiotensin I (Ang I) and tetradecapeptide (TDP), a physiologically active renin substrate, produced concentration-dependent vasoconstriction. YM358 (10(-7) M) inhibited the vasoconstricting responses to Ang II, Ang I and TDP in isolated mesenteric arteries. In contrast, enalaprilat (10(-7) M), an active metabolite of enalapril, did not completely inhibit the response to Ang I and TDP. These results indicate that YM358 has higher efficacy than enalapril for the treatment of hypertension.     

Decreased expression of CD44, alpha-catenin, and deleted colon carcinoma and altered expression of beta-catenin in ulcerative colitis-associated dysplasia and carcinoma, as compared with sporadic colon neoplasms

BACKGROUND: To clarify the cell adhesion status in ulcerative colitis (UC)-associated colon neoplasm, expression of cell adhesion molecules were investigated and compared with that of sporadic colon neoplasm. METHODS: A total of 14 low grade dysplasias, 16 high grade dysplasias, and 8 adenocarcinomas associated with UC and 17 sporadic adenomas with mild to moderate dysplasia, 22 adenomas with severe dysplasia, and 15 invasive adenocarcinomas were immunohistochemically examined using monoclonal antibodies against CD44, E-cadherin, alpha- and beta-catenin, and deleted colon carcinoma (DCC). RESULTS: CD44, especially its standard form, and DCC expression was stronger in the sporadic colon neoplasms than in the UC-associated lesions. Although E-cadherin did not show significant differences between the two cases, alpha-catenin was more expressed in sporadic colon adenomas with severe dysplasia and carcinomas than in their UC-associated counterparts. Membranous beta-catenin staining was stronger in UC-associated neoplasms, whereas sporadic lesions had greater cytoplasmic and nuclear expression. CONCLUSIONS: The differences in cell adhesion molecule expression suggests that UC-associated and sporadic colon neoplasms arise from different pathways of tumorigenesis.     

Methylation of the hMLH1 promoter in familial gastric cancer with microsatellite instability

Microsatellite instability (MSI), which is recognized as an important mechanism in tumorigenesis, has been reported in familial gastric cancers (FGC). However, genetic defects responsible for this phenotype, that is, mutations in mismatch-repair genes such as hMLH1 and hMSH2, have not been detected in most FGC cases. Earlier studies have shown that the promoter region of the hMLH1 gene was methylated in some sporadic colorectal and endometrial cancers. To determine how FGC acquire MSI, we examined the MSI status, hMLH1-protein expression and methylation status of the hMLH1-promoter region in FGC cases. Out of 9 cancers, 6 from 8 FGC kindreds showed MSI at one or more loci; no germline mutations in the hMLH1 or hMSH2 genes were detected; 4 cancers exhibiting MSI displayed aberrant hMLH1 expression: complete loss in one, decreased level in another, and partially staining pattern in the remaining 2. Methylation in the hMLH1-promoter region was found in these 4 cases. In contrast, the cancers displaying hMLH1-protein expression were not methylated in the hMLH1-promoter region. Our data show a significant association between the absence of hMLH1 expression and methylation of its promoter in FGC cases with MSI. This suggests that the mechanism of inactivation of hMLH1 is epigenetic and that there are other genes responsible for FGC.     

In vivo pharmacologic profile of YM158, a new dual antagonist for leukotriene D4 and thromboxane A2 receptors

The antagonistic activity of oral YM158 (3-[(4-tert-butylthiazol-2-yl)methoxy]-5'-[3-(4-chlorobenzenesu lfonyl)propyl]-2'-(1H-tetrazol-5-ylmethoxy)benzanilide monosodium salt monohydrate), a new dual antagonist for leukotriene (LT) D4 and thromboxane (TX) A2 receptors, was investigated. Oral YM158 caused dose-dependent inhibition of LTD4-induced increases in plasma leakage and LTD4- or U46619-induced increases in airway resistance, with ED50 values of 6.6, 8.6 and 14 mg/kg, respectively. The dose-range of YM158's inhibitions was almost the same for both LTD4 and TXA2 receptors, and repeated oral doses did not affect its efficacy. Furthermore, oral YM158 inhibited antigen-induced bronchoconstriction. Although the potency of pranlukast for LTD4 receptor antagonism (ED50 = 0.34 mg/kg) is greater than that of YM158 (ED50 = 8.6 mg/kg), the doses of both pranlukast and YM158 for significant inhibition of the antigen-evoked airway response were the same, indicating that the TXA2 receptor antagonism of YM158 plays an important role in its anti-asthmatic effects. In conclusion, YM158 promises to be a novel agent for treating bronchial asthma.