Stable neutral radicals of planar N2O2-type dipyrrin platinum complexes: hybrid radicals of the delocalized organic π-orbital and platinum d-orbital.

Neutral radicals of N2O2-dipyrrin platinum complexes were synthesized by the reaction of dipyrrin ligands with PtCl2(cod) and successive one-electron oxidation. The radicals are very stable even under aerobic and ambient conditions. X-ray crystallographic analysis revealed the stacking array of the planar dipyrrin complex moieties. The ESR signals were broadened and significantly downfield shifted. The absorption spectra exhibited NIR bands. These results indicated a delocalized radical character with a contribution by the platinum d-orbital.

Undiminished regulatory T cells in the thymus of patients with myasthenia gravis.

OBJECTIVE: The thymus has been implicated as a possible site of origin that triggers autoimmunity in myasthenia gravis (MG). Although several groups have suggested that the decrease in the number of regulatory T (Treg) cells contributes to the onset of MG, the exact role of Treg cells in MG remains unclear. To address this point, we examined the number and distribution of Treg cells in a large number of patients with MG. METHODS: Immunohistofluorescence analysis of Foxp3 along with CD4 and CD8 was performed in thymic sections of MG (+) (n = 24) and MG (-) patients (n = 27). Circulating CD4(+)CD25(+) cells in the peripheral blood of patients with MG (n = 15) and age-matched healthy subjects (n = 15) were also analyzed. RESULTS: Foxp3(+)CD4(+)CD8(-) cells were predominantly found in the thymic medulla and their number declined with age. There was no significant difference in the number or the distribution of Foxp3(+)CD4(+)CD8(-) cells in the thymus between MG (+) and MG (-) patients. The number of circulating CD4(+)CD25(+) cells in the peripheral blood of patients with MG was not significantly altered compared to that in healthy subjects. CONCLUSION: The cellularity of Treg cells in the thymus and circulation is not diminished in patients with myasthenia gravis.

An acquired inhibitor to the GPVI platelet collagen receptor in a patient with lupus nephritis.

BACKGROUND: GPVI is a major platelet collagen signaling receptor. In rare cases of immune thrombocytopenic purpura (ITP), autoantibodies to GPVI result in receptor shedding. OBJECTIVES: To investigate a possible pathogenic role of plasma anti-GPVI antibody located in a woman with lupus nephritis. METHODS: Measured were (i) platelet aggregation to collagen and convulxin, (ii) platelet GPVI expression (flow cytometry and western blotting), (iii) plasma soluble GPVI (sGPVI, dual antibody ELISA), and (iv) plasma anti-GPVI antibody (ELISA using recombinant sGPVI). RESULTS: In 2006 and early 2007, the patient had a normal platelet count but a virtual absence of platelet aggregation to collagen and convulxin. Her platelets responded normally to other agonists including cross-linking ITAM-dependent FcgammaRIIA by monoclonal antibody, IV.3. Flow cytometry and western blotting showed a platelet deficiency of GPVI. Plasma sGPVI levels were undetectable whereas ELISA confirmed the presence of anti-GPVI antibody. Sequencing revealed a normal GPVI cDNA structure. The patient's plasma and the isolated IgG3 fraction activated and induced GPVI shedding from normal platelets. A deteriorating clinical condition led to increasingly strict immunosuppressive therapy. This was globally associated with a fall in plasma anti-GPVI titres, the restoration of platelet GPVI and the convulxin response, and the loss of her nephrotic syndrome. CONCLUSIONS: Our results show that this patient acquired a potent anti-GPVI IgG3 antibody with loss of GPVI and collagen-related platelet function. Further studies are required to determine whether anti-GPVI antibodies occur in other lupus patients with nephritis.

[Surgically treated pleomorphic carcinoma of the lung].

We experienced 3 resected cases of pleomorphic carcinoma of the lung. Each cases were 74-year-old man (case 1), 74-year-old woman (case 2) and 69-year-old man (case 3). Two patients (case 1 and 2) were histologically diagnosed as pleomorphic carcinoma composed of spindle cell carcinoma with giant cell carcinoma. One patient (case 3) was similarly diagnosed as pleomorphic carcinoma composed of spindle cell carcinoma with adenocarcinoma and squamous cell carcinoma. Although lymph nodes metastasis were not recognized in all patients, invasion to vessels were recognized in 2 patients (case 1 and 3). In one patient (case 1), recurrence was recognized at contralateral side 1 month after surgery and he died of other disease 2 months after surgery. The other 2 patients were alive without recurrence 24 and 5 months after surgery. Recently it is reported that recurrence is recognized at early phase after surgery and prognosis is poor in a case with vessel invasions in spite of pathological NO state. Since one patient (case 3) had nonmetastatic lymph nodes with vessel invasions, careful observation is considered to be necessary.

[Clinical analysis of tracheoplasty and bronchoplasty in primary lung cancer].

In our department, there were 482 thoracic surgeries for primary lung cancer between 1994 and 2007. We clinically reviewed cases that underwent tracheoplasty or bronchoplasty (n = 22, 4.6%). The patients consisted of 21 males and 1 female (66.5 +/- 12.0 years-old). All patients were smokers. The tissue forms were 19 squamous cell carcinomas, 2 adenocarcinomas, 1 large cell carcinoma, 1 adenoid cystic carcinoma and 1 carcinoid, including 2 multiple carcinomas. Sleeve resections involved the trachea in 1, upper lobes in 13, lower lobes in 3, upper-middle lobes in 2 and intermediate bronchus in 1. Wedge resections were performed in the upper lobes in 2. Fourteen reconstructions were performed. We ordinarily sutured the trachea and bronchus in any case, using a single outside knot. There was no leakage at the anastomosis. There were 2 hospital deaths. There were 4 cancer deaths, including 2 local recurrences. There were 4 patients demonstrating stenosis post operatively. There were 3 stenoses among 4 preoperative radiation therapies. We considered that radiation therapy disturbed the repair of the anastomosis. There were 8 pneumonia patients who developed post operatively. There were 2 operative hospital deaths among 3 angio-bronchoplasties without coverage. Recently, we have routinely covered the anastomosis at the reconstruction site and have not experienced any major complications.

Pulmonary atheroembolism via an AV shunt.

Although cholesterol crystal embolism can present with diffuse visceral involvement, lung lesions do not occur unless there is left to right circulatory shunting. Pulmonary atheroembolism was confirmed by histology in an elderly male with recent end-stage renal failure (ESRF) due to atheroembolic renal disease, who presented with massive hemoptysis and intractable respiratory failure. At autopsy, atheromatous degeneration of the aorta was observed and acute cholesterol emboli found in the kidneys, spleen, liver, stomach and lung. Cholesterol clefts were seen in pulmonary arterioles, and ischemic alveolar damage was present. The pulmonary arteries had no atheromatous changes. Intrapulmonary, intracardiac, and aortocaval shunting were not present. Pulmonary atheroembolism arising from a dialysis fistula has not been previously reported.

The balance of VEGF-C and VEGFR-3 mRNA is a predictor of lymph node metastasis in non-small cell lung cancer.

A positive association between vascular endothelial growth factor-C (VEGF-C) expression and lymph node metastasis has been reported in several cancers. However, the relationship of VEGF-C and lymph node metastasis in some cancers, including non-small cell lung cancer (NSCLC), is controversial. We evaluated the VEGF-C and vascular endothelial growth factor receptor-3 (VEGFR-3) expression in NSCLC samples from patients who had undergone surgery between 1998 and 2002 using real-time quantitative RT-PCR and immunohistochemical staining. We failed to find a positive association between VEGF-C and VEGFR-3 mRNA expression and lymph node metastasis in NSCLC. An immunohistological study demonstrated that VEGF-C was expressed not only in cancer cells, but also in macrophages in NSCLC, and that VEGFR-3 was expressed in cancer cells, macrophages, type II pneumocytes and lymph vessels. The VEGF-C/VEGFR-3 ratio of the node-positive group was significantly higher than that of the node-negative group. Immunohistochemical staining showed that VEGFR-3 was mainly expressed in cancer cells. The immunoreactivity of VEGF-C and VEGFR-3 was roughly correlated to the mRNA levels of VEGF-C and VEGFR-3 in real-time PCR. VEGF-C mRNA alone has no positive association with lymph node metastasis in NSCLC. The VEGF-C/VEGFR-3 ratio was positively associated with lymph node metastasis in NSCLC. This suggests that VEGF-C promotes lymph node metastasis while being influenced by the strength of the VEGF-C autocrine loop, and the VEGF-C/VEGFR-3 ratio can be a useful predictor of lymph node metastasis in NSCLC.

[Clinical analysis of operative and hospital deaths in primary lung cancer].

In our department, there were 313 thoracic surgeries for primary lung cancer from January 1994 to December 2003. We clinically reviewed for the operative and hospital death (n=18, 5.8%). The patients were 16 males and 2 females (70.6 +/- 5.6 years old). The surgical procedures were 4 pneumonectomies, 13 lobectomies (3 bronchoplasties) and 1 partial resection. The mean interval until postoperative death was 122.5 +/- 156.1 days. There were 5 direct operative deaths within 30 days (1.6%). There were 4 cancer deaths, 2 hemoptyses, 2 operative bleeding, 2 thromboses, 2 cerebral hemorrhages, 1 pyothorax, 1 pneumonia, 1 respiratory failure, 1 multiple organ failure after chemotherapy and 2 unexplained deaths. The patients with pneumonectomy or aged significantly had high mortality. For postoperative complications such as hemoptysis or bleeding, perioperative management that takes these issues into consideration is needed. Furthermore, we must carefully review the preoperative evaluation and combined treatment, because there were many cancer deaths among cases showing early recurrence and metastasis.

Estimation of the remaining periodontal ligament from attachment-level measurements.

AIM: Accurate assessment of the remaining area of periodontal attachment assists in determining the prognosis of a tooth. The aim of this study was to determine formulae to estimate the remaining area of periodontal attachment from attachment-level measures. MATERIALS AND METHODS: Roots of 30 extracted teeth of each tooth type were coated with vinyl acetate solution and alpha-cyanoacrylate monomer to produce a membrane. The membrane was removed after guidelines were drawn to simulate various attachment levels. The root surface area on the apical side of each simulated attachment level was measured using image analysis software. The net or percent root surface area and corresponding attachment level data were fitted to a linear, quadratic, cubic, and exponential functions and a growth curve. RESULTS: The linear function provided the most appropriate fit for the data. For net root surface area, a steeper slope was evident for tooth types with a larger mean total root surface area. For percent root surface area, a steeper slope was evident for tooth types with a shorter root length. CONCLUSIONS: A linear function provides the most appropriate formula for estimation of the remaining area of periodontal attachment based on the attachment-level measurement.

Interferon-gamma is produced by CD8 T cells in response to HLA-A24-restricted hepatitis C virus epitopes after sustained virus loss.

Differences in cytotoxic T lymphocyte activity in hepatitis C virus infection may account for the outcome of interferon monotherapy. To investigate this hypothesis, we analysed the response of peripheral CD8(+) T cells that recognized epitopes presented by HLA-A*2402. We synthesized HLA/beta2-microglobulin/peptide complexes using two epitopes. Production of interferon-gamma by CD8(+) T cells in response to plastic-bound monomeric HLA/peptide complex was observed frequently in sustained virus responders (SVR) (n = 13) against all the peptides, NS31296-1304 (the percentage of responding patients, 61.5%) and core 129-137 (53.8%), while no interferon-gamma production was observed in non-responders (NR) (n = 13) for any of the peptides. Tetramer-staining showed the presence of CD8(+) T cells specific for all the peptides except NS31296-1304 in two SVR at the end of interferon monotherapy, although hardly any such cells were found in four NR. Specific killing was observed against peptides NS31296-1304 (3/4) and core 129-137 (1/4) in sustained responders but none in non-responders. These results suggest that the responses of cytotoxic T lymphocytes (CTLs) were induced during interferon therapy in these patients and that interferon-gamma production by CD8(+) T lymphocytes against HCV NS31296-1304 and core 129-137 are well maintained in patients with SVR compared with those with NR. These findings emphasize the importance of the CD8(+) T cell response in controlling HCV infection.

Evidence from mucin core protein expression that some Paget's disease on areola can be of extramammary-like histogenesis and part of multisite disease.

Triple extramammary Paget's disease, which consists ordinarily of bilateral axillary and genital lesions, is uncommon. Triple extramammary Paget's disease involving other sites has never been reported, although solitary extramammary Paget's disease can occur at various sites around the body. Erythematous plaques on the areola, axilla and genitalia of a 91-year-old man were surgically removed under the clinical diagnosis of multiple extramammary Paget's disease. Histology revealed that all three lesions consisted of intraepidermal nests of Paget cells and other isolated Paget cells scattered in the epidermis. Although adnexal invasion was observed in the genital lesion, neither intraductal invasion nor underlying breast carcinoma was detected in the areolar lesion. Immunohistochemically, the Paget cells in all lesions expressed simple epithelial cytokeratins (CK8, 18 and 19), mucin (MUC)1 and MUC5AC, but neither CK20 nor MUC2. From the histological findings, the present case was interpreted as triple extramammary Paget's disease rather than synchronous mammary and extramammary Paget's disease. Furthermore, the mucin core protein expression pattern, which was identical to that observed in extramammary Paget's disease, supported the above interpretation.

Prostaglandin D2 inhibits fibroblast migration.

Fibroblasts play an important role in the repair and remodelling processes following injury. Prostaglandin D2 (PGD2) is a potent mediator in inflammatory processes. In this study, the effect of the PGD2 on human foetal lung fibroblasts (HFL-1) chemotaxis induced by human plasma fibronectin (HFn) was investigated using the blindwell chamber technique. PGD2 inhibited HFL-1 chemotaxis to HFn (20 microg x mL(-1)) by 20.8 +/- 3.8% (p<0.05). Checkerboard analysis of HFn-directed migration confirmed that PGD2 inhibited both chemotaxis and chemokinesis. The effect of PGD2 was concentration-dependent and the inhibitory effect diminished with time. The PGD2 receptor (DP) agonist BW245C (500 nM) had a similar effect, inhibiting chemotaxis to 39.4 +/- 6.3%. The inhibitory effects of both PGD2 and BW245C on HFL-1 chemotaxis were blocked by the DP receptor antagonist AH6809 (2 microM). The inhibitory effect of PGD2 on fibroblast chemotaxis was also blocked by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitor, KT5720, suggesting a DP receptor-initiated, cAMP-dependent effect mediated by PKA. Prostaglandin D2 appears to inhibit fibroblast chemotaxis, perhaps by modulating the rate of fibroblast migration. Such an effect may contribute to regulation of the wound healing response following injury in asthma patients.

Potentiation of human lung fibroblast chemotaxis by the thromboxane A(2) analog U-46619.

Fibroblast production of extracellular matrix is crucial not only for normal tissue development and maintenance of tissue structure but also for the repair and remodeling processes after injury. Thromboxane A(2) (TXA(2)) is a potent mediator in inflammatory processes. The aim of this study was to investigate the effect of TXA(2) on chemotaxis of human fetal lung (HFL-1) fibroblasts induced by human plasma fibronectin or platelet-derived growth factor BB (PDGF-BB). By using the blindwell chamber technique, the TXA(2) agonist U-46619 alone had no chemotactic activity. However, U-46619 (200 nmol/L) stimulated HFL-1 fibroblast chemotaxis to human plasma fibronectin (20 microg/mL; 161.8% +/- 13.4%; P <.005) and to PDGF-BB (10 ng/mL; 188.5% +/- 7.0%; P <.005). Checkerboard analysis of human plasma fibronectin-directed migration confirmed that the TXA(2) agonist increased both chemotaxis and chemokinesis. The stimulatory effect of the TXA(2) agonist was concentration dependent and increased with time. Another agent known for stimulating the protein kinase C pathway, phorbol 12-myristate 13-acetate (10(-8) mol/L), had a similar effect, stimulating chemotaxis to fibronectin (146.2% +/- 8.6%). The stimulatory effect of the TXA(2) agonist on HFL-1 fibroblast chemotaxis was inhibited by the synthetic thromboxane receptor antagonist SQ29,548 (10(-5) mol/L) and the protein kinase C inhibitor calphostin (10(-7) mol/L). In summary, TXA(2) appears to stimulate fibroblast chemotaxis to fibronectin and PDGF, perhaps by modulating the rate of fibroblast migration. Such an effect may contribute to regulation of wound healing and the development of fibrotic disorders.

Characterization of the human ABO gene promoter in erythroid cell lineage.

BACKGROUND: The human ABO blood group system is important in transfusion and organ transplantation. Although the molecular basis of the ABO gene has been established, recent studies have begun to characterize the mechanism of the ABO gene expression. MATERIALS AND METHODS: Transient transfection assays were carried out in human erythroleukaemia HEL cells and human gastric cancer KATOIII cells. Electrophoretic mobility shift assays were performed using nuclear extracts derived from both cells. RESULTS: Our characterization of the 5'-upstream sequence of the ABO genes indicated that the region between -117 and +31 is essential to direct expression of a reporter gene in erythroid cells. We show that a sequence located between positions -22 and -14 of the ABO promoter binds a ubiquitous transcription factor Sp1 or Sp1-like protein(s). Mutation of this site abrogates binding of those factors and reduces the ability of the ABO promoter to function in erythroleukaemia cells and gastric cancer cells. CONCLUSION: The expression of the ABO promoter appears to be influenced by the binding of Sp1 or Sp1-like protein(s) in both erythroid and epithelial cell lineages.

MHC-dependent shaping of the inhibitory Ly49 receptor repertoire on NK cells: evidence for a regulated sequential model.

Engagement of MHC class I-specific inhibitory receptors regulates natural killer (NK) cell development and function. Using both new and previously characterized anti-Ly49 monoclonal antibodies, we comprehensively determined expression and co-expression frequencies of four Ly49 receptors by NK cells from MHC-congenic, MHC class I-deficient, and Ly49A-transgenic mice to study mechanisms that shape the inhibitory Ly49 repertoire. All Ly49 receptors were expressed on partially overlapping subsets. Significantly, in the absence of class I MHC, several receptor pairs were co-expressed more frequently than predicted from a purely random expression model, indicating that biases independent of MHC class I underlie receptor co-expression in some cases. MHC interactions were found to inhibit Ly49 co-expression variably depending on the MHC allele and the receptor pair examined. These data extend previous evidence that interactions with MHC shape the repertoire. It was previously proposed that developing NK cells express Ly49 receptors sequentially and cumulatively, until self-MHC specific receptors are expressed and inhibit new receptor expression. Fulfilling a major prediction of this model, we found that class I recognition by a Ly49A transgene expressed by all developing NK cells equivalently inhibited expression of endogenous self-specific and nonself-specific Ly49 receptors.

A family with liddle's syndrome caused by a mutation in the beta subunit of the epithelial sodium channel.

Liddle's syndrome is a rare form of autosomal-dominant salt-sensitive hypertension. Constitutive activation of the amiloride-sensitive distal renal epithelial sodium channel (ENaC) is essential for salt-sensitive hypertension. Recently, several DNA analysis studies have indicated that there is a mutation of C-terminus of either the beta or y subunit. We sequenced the C-termini of the beta and -gamma subunits of the ENaC in a Japanese family with hypertension and hypopotassemia without excess minerarocorticoids, clinically diagnosed as Liddle's syndrome. The mutation of the ENaC of this family was beta R564X. Since such case seem to be rare in the literature, detailed data are shown in this report.

Gender difference in the relationships among hyperleptinemia, hyperinsulinemia, and hypertension.

Studies reveals that plasma leptin levels (LEP) in females are higher than those in males, and that LEP in hypertensive subjects are higher than those in BMI-matched normotensive subjects. To investigate the relationships among LEP, blood pressure (BP) and insulin sensitivity, we studied these relationships in 133 Japanese males and 263 females. LEP were positively correlated with BP, body mass index, body fat mass (FM) and homeostasis model assessment (HOMA). Regression analysis in which age and FM were adjusted showed LEP were associated with BP and HOMA. Even with adjustment by age, FM and HOMA, LEP were still positively correlated BP in males. LEP in insulin-resistant hypertensives was significantly higher than those in insulin-sensitive hypertensives, in insulin-sensitive normotensives and in insulin-resistant normotensives in males. However, in females, a significantly higher LEP was observed in insulin-resistant subjects than in insulin-sensitive subjects regardless of hypertension. These data suggest that it would be sexual difference in the relationships among hyperleptinemia, hyperinsulinemia and hypertension.