Novel non-stop variant of the NR0B1 gene in two siblings with adrenal hypoplasia congenita.

OBJECTIVES: Mutations in the dosage-sensitive sex reversal-AHC critical region on the X chromosome, gene 1 (DAX-1, officially NR0B1), cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). Salt-losing adrenal insufficiency usually occurs during the neonatal period or early childhood. We report a novel non-stop variant of NR0B1 in two siblings and their unusual clinical course. CASE PRESENTATION: The proband was a boy who presented with an unusual form of AHC with neonatal onset of growth failure and mild salt loss, but without cutaneous pigmentation or plasma ACTH elevation. His 4-year-old elder brother had been growing healthily, but carried an AHC diagnosis. A non-stop variant of NR0B1 (p.*471K) was demonstrated in the patients and their mother. CONCLUSIONS: We identified a novel non-stop variant of NR0B1 in two siblings. Mild salt loss associated with hyperkalemia is a crucial diagnostic clue for AHC, even without apparent symptoms of glucocorticoid deficiency.

Diagnosing Alzheimer's Disease from Circulating Blood Leukocytes Using a Fluorescent Amyloid Probe.

BACKGROUND: Toxic amyloid-ß (Aß) peptides aggregate into higher molecular weight assemblies and accumulate not only in the extracellular space, but also in the walls of blood vessels in the brain, increasing their permeability, and promoting immune cell migration and activation. Given the prominent role of the immune system, phagocytic blood cells may contact pathological brain materials. OBJECTIVE: To develop a novel method for early Alzheimer's disease (AD) detection, we used blood leukocytes, that could act as "sentinels" after trafficking through the brain microvasculature, to detect pathological amyloid by labelling with a conformationally-sensitive fluorescent amyloid probe and imaging with confocal spectral microscopy. METHODS: Formalin-fixed peripheral blood mononuclear cells (PBMCs) from cognitively healthy control (HC) subjects, mild cognitive impairment (MCI) and AD patients were stained with the fluorescent amyloid probe K114, and imaged. Results were validated against cerebrospinal fluid (CSF) biomarkers and clinical diagnosis. RESULTS: K114-labeled leukocytes exhibited distinctive fluorescent spectral signatures in MCI/AD subjects. Comparing subjects with single CSF biomarker-positive AD/MCI to negative controls, our technique yielded modest AUCs, which improved to the 0.90 range when only MCI subjects were included in order to measure performance in an early disease state. Combining CSF Aß42 and t-Tau metrics further improved the AUC to 0.93. CONCLUSION: Our method holds promise for sensitive detection of AD-related protein misfolding in circulating leukocytes, particularly in the early stages of disease.

Preoperative balloon pulmonary angioplasty for chronic thromboembolic pulmonary hypertension led to successful anesthetic management for total hysterectomy under general anesthesia: a case report.

BACKGROUND: Chronic thromboembolic pulmonary hypertension (CTEPH) is a disease of obstructive pulmonary artery remodeling as a consequence of major vessel thromboembolism. Balloon pulmonary angioplasty (BPA) is an alternative treatment for patients with inoperable CTEPH. We report a case of CTEPH which improved following preoperative BPA intervention, allowing total hysterectomy to be performed. CASE PRESENTATION: A 48-year-old woman was transferred to our hospital to undergo total hysterectomy for endometrial cancer. She developed pulmonary embolism 7 months ago at another hospital, and a diagnosis of CTEPH was made based on multiple pulmonary emboli and pulmonary hypertension at our institute. Two BPA sessions for seven branches of the bilateral pulmonary arteries were conducted, resulting in a decrease of mean pulmonary artery pressure from 54 to 33 mmHg. Total hysterectomy was successfully performed under general anesthesia without any complications. CONCLUSIONS: BPA could be effective for reducing PH in patients with CTEPH undergoing noncardiac surgery.

Brain Natriuretic Peptide and Particular Left Ventricle Segment Asynergy Associated with Cardioembolic Stroke from Old Myocardial Infarction.

BACKGROUND: It is important to determine the usage of anticoagulants by defining the actual risk of cardioembolic stroke in patients with old myocardial infarction. In the present study, we aimed to more precisely evaluate the risks of each segment associated with cardioembolic stroke using a 16-segment model. The usage of the plasma brain natriuretic peptide (BNP) associated with cardioembolic stroke was also evaluated in comparison with a left ventricle ejection fraction less than 40%. METHODS: There were a total of 190 ischemic stroke patients who had premorbid myocardial infarction. The study included a total of 143 ischemic stroke patients with old myocardial infarction who were available for evaluation and excluded patients with atrial fibrillation or acute myocardial infarction. Their left ventricle wall motion abnormality and the level of plasma BNP at their admission were analyzed. RESULTS: Hypertension and a plasma BNP level of 206.9 pg/mL or higher, determined from the receiver operating characteristic curve, were independently associated with cardioembolic stroke (χ(2) = 35.6, R(2) = .30, P < .001). Adjusting for these factors, statistically independent high risk was observed at the basal-inferior, basal-inferolateral, mid-anterior, mid-anteroseptal, apical-anterior, and apical-septal left ventricles. CONCLUSION: High plasma BNP levels and left ventricular wall motion abnormalities in the segments perfused with left anterior descending coronary artery or right coronary artery show a high risk for cardioembolic stroke in patients with old myocardial infarction. Considering these factors, it could be possible to more precisely define the risk of cardioembolic stroke and to perform appropriate antithrombotic treatments in old myocardial infarction patients.

[Associations between ALDH2 and ADH1B Genotypes and Ethanol-Induced Cutaneous Erythema in Young Japanese Women].

OBJECTIVES: The purpose of this study was to identify associations between ALDH2 and ADH1B genotypes and ethanol-induced cutaneous erythema and assess the accuracy of an ethanol patch test in young Japanese women. METHODS: The subjects were 942 female Japanese university students. They were given an ethanol patch test and examined for ethanol-induced cutaneous erythema both immediately after removing the patch and 10 minutes after removing the patch. A saliva sample was used to determine the ALDH2 and ADH1B genotype of each subject by realtime PCR. RESULTS: The sensitivity and specificity of erythema immediately after removing the patch as the marker for the presence of inactive ALDH2 were 69.6% and 87.7%, respectively, and the sensitivity and specificity of erythema 10 minutes after removing the patch were 85.2% and 85.1%, respectively. The sensitivity of erythema after 10 minutes was markedly lower in the ADH1B*1/*1 carriers than in the ADH1B*2 carriers (8.3% vs. 89.7%, p<0.0001), and the specificity was significantly higher in the ADH1B*1/*1 carriers than in the ADH1B*2 carriers (96.9% vs. 84.3%, p<0.05). CONCLUSIONS: Overall, both sensitivity and specificity were satisfactorily high, but having the ADH1B*1/*1 genotype prevented a positive reaction for inactive ALDH2 and caused false-negative results. The data also suggested that having the ADH1B*2/*2 genotype caused a positive reaction in subjects with the ALDH2*1/*1 genotype. Despite these exceptions, the ethanol patch test has enough accuracy and can be used easily to subjects who don't drink alcohol. This is a valuable tool for improving the health literacy of younger generation subjects.

Combination analysis in genetic polymorphisms of drug-metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population.

The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure. We have developed a new straightforward TaqMan PCR genotyping assay to investigate the prevalence of the most common allelic variants of polymorphic CYP enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population. Moreover, we focused on the combination of each genotype for clinical treatment. The genotype analysis identified a total of 139 out of 483 genotype combinations of five genes in the 1,003 Japanese subjects. According to our results, most of subjects seemed to require dose modification during clinical treatment. In the near future, modifications should be considered based on the individual patient genotype of each treatment.

Osteopontin-integrin α(v)ß(3) axis is crucial for 5-fluorouracil resistance in oral squamous cell carcinoma.

Clinical applications of a chemotherapeutic agent, 5-fluorouracil (5-FU) in oral squamous cell carcinoma (OSCC) have been limited because of drug resistance. This study aimed to identify novel mechanisms of 5-FU resistance. Here we found increased osteopontin (OPN) gene expression in OSCC tissues with resistance to 5-FU-based chemoradiotherapy. OPN overexpression in OSCC cells led to 5-FU resistance and abrogated the prosurvival effect of the drug in a mouse xenograft model. OPN-induced 5-FU resistance required integrin αvß3. Targeting integrin αvß3 reversed the resistance in a 5-FU-resistant clone highly expressing OPN. Our data suggest that the OPN-integrin αvß3 axis is crucial for 5-FU resistance in OSCC.

Direct detection of single nucleotide polymorphism (SNP) by the TaqMan PCR assay using dried saliva on water-soluble paper and hair-roots, without DNA extraction.

We have developed a new method for directly using unprocessed biological specimens as templates for the TaqMan assay. DNA extraction and purification had been believed to be required for the assay, but our new method could avoid hindering fluorescence detection, even if the templates were used directly. Saliva was needed to be put on water-soluble paper and dried, and hairs were cut to be about 10 mm long. This method could reduce both the time and effort involved, and also the risk of contamination. It should prove to be very valuable for genetic diagnoses in various fields.

Long PCR-based genotyping for a deleted CYP2D6 gene without DNA extraction.

In the post-genome era, a simple and inexpensive method for diagnostic analysis is in high demand. Cytochrome P450 (CYP) 2D6 is one of the most widely investigated CYPs in relation to genetic polymorphism. Detection of CYP2D6*5 is difficult since long PCR is used. Especially for samples without DNA extraction, the detection is not sensitive enough for population analysis. Therefore, we developed a CYP2D6*5 genotyping method that involves nested long PCR, directly using human whole saliva as a template without DNA extraction. This method will be very useful for genetic diagnoses and can be an efficient tool for individualization of drug therapy in clinical studies.

Stromal expression of neutrophil gelatinase-associated lipocalin correlates with poor differentiation and adverse prognosis in oral squamous cell carcinoma.

AIMS: Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily. Although its overexpression in various cancers has been reported, little is known about its expression and clinical significance in oral squamous cell carcinoma (OSCC). This study aimed to elucidate the clinical significance of NGAL in OSCC. METHODS AND RESULTS: We investigated NGAL expression immunohistochemically in tumour cells and stromal cells in 96 OSCC tissues. NGAL expression in tumour cells correlated significantly with histological tumour cell differentiation, as shown by its specific distribution in the horn pearl-forming keratinized tumour cells, but not with other major clinicopathological parameters. We found NGAL(+) cells in the stroma that were predominantly myeloperoxidase-positive neutrophils. The number of such NGAL-expressing stromal cells was associated significantly with poor differentiation and reduced overall survival in OSCC. The prognostic value of stromal NGAL expression was significant in a univariate analysis, while only a trend was found in multivariate analyses. CONCLUSIONS: This study is the first to show the clinical significance of stromal NGAL expression, which may be an indicator of poor prognosis and more aggressive histological grade in OSCC. Our data suggest that NGAL expression in tumour cells and expression in stroma are associated in different ways with OSCC differentiation.

Downregulation of midkine induces cisplatin resistance in human oral squamous cell carcinoma.

The presence of drug-resistant cancer cells has been associated with poor clinical outcomes. Cisplatin is one of the most effective chemotherapeutic agents commonly used for several malignancies including oral squamous cell carcinoma (OSCC). Although cisplatin resistance is a major obstacle in cancer treatment, mechanisms by which it develops are not well understood. Midkine (MK), a heparin-binding growth factor, has various cancer-related functions. In this study, we investigated whether MK is involved in cisplatin resistance in OSCC. We demonstrated that the Sa-3R cell line, which is OSCC cisplatin-resistant, exhibited lower MK expression with slow growth compared with its parent, Sa-3 cells. In Sa-3 cells, downregulation of MK expression significantly reduced cisplatin sensitivity, cell growth, and the expression of cyclin D1 and cyclin E1. MK knockdown suppressed cellular cisplatin accumulation via induction of ATP-binding cassette efflux transporters. These data suggest that MK may play important roles in cisplatin resistance in OSCC by modulating both cell growth and intracellular cisplatin accumulation.

A novel functional site of extracellular matrix metalloproteinase inducer (EMMPRIN) that limits the migration of human uterine cervical carcinoma cells.

EMMPRIN (extracellular matrix metalloproteinase inducer)/CD147, a membrane-bound glycoprotein with two extracellular loop domains (termed loops I and II), progresses tumor invasion and metastasis by increasing the production of matrix metalloproteinase (MMP) in peritumoral stoma cells. EMMPRIN has also been associated with the control of migration activity in some tumor cells, but little is known about how EMMPRIN regulates tumor cell migration. In the present study, EMMPRIN siRNA suppressed the gene expression and production of EMMPRIN in human uterine cervical carcinoma SKG-II cells. An in vitro scratch wound assay showed enhancement of migration of EMMPRIN-knockdown SKG-II cells. In addition, the SKG-II cell migration was augmented by adding an E. coli-expressed human EMMPRIN mutant with two extracellular loop domains (eEMP-I/II), which bound to the cell surface of SKG-II cells. However, eEMP-I/II suppressed the native EMMPRIN-mediated augmentation of proMMP-1/procollagenase-1 production in a co-culture of the SKG-II cells and human uterine cervical fibroblasts, indicating that the augmentation of SKG-II cell migration resulted from the interference of native EMMPRIN functions by eEMP-I/II on the cell surface. Furthermore, a systematic peptide screening method using nine synthetic EMMPRIN peptides coding the loop I and II domains (termed EM1-9) revealed that EM9 (170HIENLNMEADPGQYR184) facilitated SKG-II cell migration. Moreover, SKG-II cell migration was enhanced by administration of an antibody against EM9, but not EM1 which is a crucial site for the MMP inducible activity of EMMPRIN. Therefore, these results provide novel evidence that EMMPRIN on the cell surface limits the cell migration of human uterine cervical carcinoma cells through 170HIENLNMEADPGQYR184 in the loop II domain. Finally, these results should provide an increased understanding of the functions of EMMPRIN in malignant cervical carcinoma cells, and could contribute to the development of clinical strategies for cervical cancer therapy.

Therapeutic approaches targeting midkine suppress tumor growth and lung metastasis in osteosarcoma.

Midkine (MK) plays important roles in tumorigenesis, however, the biological function of MK and whether MK can be a therapeutic target in osteosarcoma are unclear. Here, we found that osteosarcoma tissues showed high MK expression. MK knockdown by small interfering RNA significantly induced apoptosis in osteosarcoma cells, whereas recombinant MK increased cell proliferation. Inhibition of MK signaling by anti-MK monoclonal antibody (anti-MK mAb) suppressed growth of osteosarcoma cells both in vitro and in vivo. Moreover, inhibition of MK function significantly suppressed lung metastasis in xenograft transplantation model. Targeting MK by anti-MK mAb may have value in the treatment of osteosarcoma.

Interleukin-6 signalling regulates vascular endothelial growth factor-C synthesis and lymphangiogenesis in human oral squamous cell carcinoma.

Lymph node metastasis is associated with resistance to conventional therapy and poor survival of patients with oral squamous cell carcinoma (OSCC). Although lymphangiogenesis is well known to be associated with the occurrence of lymph node metastasis in various cancers, the precise mechanisms of lymphangiogenesis in OSCC are largely unknown. IL-6, a potent pro-inflammatory cytokine, has been shown to play active roles in various cancers, including OSCC. This study aimed to investigate the involvement of IL-6 signalling in lymphatic metastasis and to evaluate the efficacy of tocilizumab, a humanized anti-human IL-6 receptor antibody, as an anti-lymphangiogenic agent for OSCC. This investigation confirmed that levels of expression of IL-6 protein and VEGF-C mRNA in OSCC tissues were significantly correlated with lymph node metastasis in patients with OSCC, as assessed by immunohistochemical analysis and real-time quantitative RT-PCR. In vitro studies showed that IL-6 regulated VEGF-C mRNA expression in a human OSCC cell line, SAS cells, through the phosphoinositide 3-kinase-Akt pathway. In addition, treatment with tocilizumab led to markedly reduced VEGF-C mRNA expression and OSCC-related lymphangiogenesis in SAS xenografts. Together, these data suggest that tocilizumab acted as expected: it inhibited lymph node metastasis in OSCC by reducing tumour lymphangiogenesis.

Midkine expression is correlated with an adverse prognosis and is down-regulated by p53 in oral squamous cell carcinoma.

Midkine (MK) expression has been documented to be inversely correlated with the prognosis of patients with various tumors, but the mechanism of this relationship has not been well characterized. Recent studies have also correlated p53 expression with prognosis of patients with oral squamous cell carcinoma (OSCC). We evaluated the relationship between MK expression and clinicopathological features of patients with OSCC to clarify the influence of p53 status on MK expression in OSCC cells. Our results showed that patients with MK over-expression in OSCC cells had a significantly lower 5-year survival rate compared with patients with low MK expression. Immunohistochemical analyses demonstrated overexpression of MK protein in OSCC samples with mutant p53. Cell culture experiments with human lingual squamous cell carcinoma revealed that the MK gene was regulated by the wild-type p53 gene. Thus, MK expression may affect prognosis via the p53 status and mutation of the p53 gene, and MK may be an attractive target for therapeutic intervention in patients with cancer cells with mutant p53.

Midkine expression in malignant salivary gland tumors and its role in tumor angiogenesis.

The aims of this study were to investigate midkine (MK) expression patterns in salivary gland tumors (SGTs) and to evaluate the correlation between MK expression and the degree of malignancy. We performed immunohistochemistry to examine MK expression in specimens of adenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC), and pleomorphic adenoma (PA). In addition, we performed immunohistochemistry for CD31 and measured microvessel density (MVD), which is an indicator of angiogenesis. Immunohistochemistry showed that MK protein expression was significantly higher in specimens of malignant SGTs (ACC [P<0.01] and MEC [P<0.001]) than in benign SGT (PA) samples. Furthermore, MVD values tended to be higher in cases that exhibited high expression of MK, which indicated a significant correlation between the degree of MK expression and MVD (P<0.001). These results suggest that MK may play important roles in malignant transformation and tumor angiogenesis in SGTs.

Humanized anti-interleukin-6 receptor antibody suppresses tumor angiogenesis and in vivo growth of human oral squamous cell carcinoma.

PURPOSE: The biological effect of interleukin-6 (IL-6) signaling in oral squamous cell carcinoma (OSCC) and whether IL-6 receptor (IL-6R)-mediated signaling can be a therapeutic target for OSCC are unclear. The aim of this study was to investigate the effects of inhibition of IL-6R-mediated signaling on OSCC progression and to evaluate the availability of tocilizumab, a humanized antihuman IL-6R antibody, as a therapeutic agent for OSCC. EXPERIMENTAL DESIGN: We evaluated expression levels of IL-6 and IL-6R in 58 OSCC tissues and 4 OSCC cell lines by real-time quantitative reverse transcription-PCR and/or immunohistochemstry. We investigated the effects of tocilizumab on OSCC growth in vitro and in xenografts. Xenografts were analyzed by immunohistochemistry for phosphorylated signal transducer and activator of transcription 3 (pSTAT3), Ki-67, and CD31, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was done. RESULTS: Expression levels of IL-6 at both mRNA and protein levels in OSCC tissues were significantly higher than those in normal mucosal tissues. In addition, OSCC cell lines expressed higher levels of both IL-6 and IL-6R mRNA than did HaCaT keratinocytes. Tocilizumab significantly reduced in vivo growth of SAS cells with a drastic reduction of STAT3 phosphorylation in tumor cells in mice. Inhibition of IL-6 signaling significantly decreased vascular endothelial growth factor mRNA expression in SAS, and microvessel density and vessel diameter in SAS tumors in tocilizumab-treated mice. CONCLUSIONS: Therapeutic approaches targeting IL-6R by tocilizumab may be effective for OSCC treatment by at least inhibiting angiogenesis.

Identification of an active site of EMMPRIN for the augmentation of matrix metalloproteinase-1 and -3 expression in a co-culture of human uterine cervical carcinoma cells and fibroblasts.

OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN) is highly expressed on malignant tumor cell surface and accelerates tumor invasion. We previously reported that human uterine cervical carcinoma SKG-II cells exhibit the progression of in-vitro invasiveness by utilizing the enhanced production of matrix metalloproteinase (MMP) in human uterine cervical fibroblasts (HUCF) under an in-vitro co-culture model (Sato T et al., Gynecol Oncol 2004; 92:47-56). The aim of this study was to clarify the active site of EMMPRIN in the augmentation of MMP production in the co-culture of SKG-II cells and HUCF. METHODS: Western and Northern blot analyses were used to examine EMMPRIN and MMP expression in a co-culture of SKG-II cells or EMMPRIN-transfected COS-7 cells and HUCF. A systematic peptide screening method using nine synthetic EMMPRIN peptides was used to identify active site(s) of EMMPRIN for MMP induction. RESULTS: SKG-II cells constitutively expressed 53-kDa EMMPRIN on the cell surface and EMMPRIN production was enhanced under the co-culture. The concomitant augmentation of proMMP-3 production was diminished by adding an EMMPRIN antibody. EMMPRIN-transfected COS-7 cells stimulated HUCF to predominantly augment proMMP-1 and -3 expressions. A systematic peptide screening method revealed that (42)SLNDSATEVTGHRWLK(57) in the first loop domain of EMMPRIN participated in the augmentation of proMMP-1 production. CONCLUSIONS: These results provide a novel mechanism of malignancy of uterine cervical carcinoma, in that the augmentation of EMMPRIN expression by tumor-stromal cell interaction progresses tumor invasion along with the increase of MMP expression via an active site of EMMPRIN, (42)SLNDSATEVTGHRWLK(57).

Aberrant aquaporin 5 expression in the sweat gland in aquagenic wrinkling of the palms.

Aquagenic wrinkling of the palms (AWP) is an uncommon disease characterized by the rapid and transient formation of edematous whitish plaques on the palms on exposure to water. Although this disease is occasionally accompanied by hyperhidrosis, the pathophysiology of AWP remains unknown. Herein we describe a patient with AWP. The location of wrinkling was limited to the areas positive for iodine-starch test after water exposure, which suggests that AWP is etiologically related to hyperhidrosis. Histologic examination revealed hyperplastic and papillated eccrine glandular epithelium with the enlarged diameter of eccrine coils. Immunohistochemically, while aquaporin 5 (AQP5), one of the water channel AQP families, was present exclusively in the dark cells of sweat glands of healthy donors, an aberrant AQP5 staining, extending to the clear cells, was found in the patient with AWP. The hyperplastic glandular epithelium and aberrant AQP5 staining in the patient's sweat glands suggest that AWP stems from dysregulation of sweating.