The first nationwide surveillance of antibacterial susceptibility patterns of pathogens isolated from skin and soft-tissue infections in dermatology departments in Japan.

To investigate the trends of antimicrobial resistance in pathogens isolated from skin and soft-tissue infections (SSTI) at dermatology departments in Japan, a Japanese surveillance committee conducted the first nationwide survey in 2013. Three main organisms were collected from SSTI at 30 dermatology departments in medical centers and 10 dermatology clinics. A total of 860 strains - 579 of Staphylococcus aureus, 240 of coagulase-negative Staphylococci, and 41 of Streptococcus pyogenes - were collected and shipped to a central laboratory for antimicrobial susceptibility testing. The patient profiles were also studied. Among all 579 strains of S. aureus, 141 (24.4%) were methicillin-resistant (MRSA). Among 97 Staphylococcus epidermidis strains, 54 (55.7%) were methicillin-resistant (MRSE). MRSA and MRSE were more frequently isolated from inpatients than from outpatients. Furthermore, these methicillin-resistant strains were also isolated more frequently from patients with histories of taking antibiotics within 4 weeks and hospitalization within 1 year compared to those without. However, there were no significant differences in MIC values and susceptibility patterns of the MRSA strains between patients with a history of hospitalization within 1 year and those without. Therefore, most of the isolated MRSA cases at dermatology departments are not healthcare-acquired, but community-acquired MRSA. S. pyogenes strains were susceptible to most antibiotics except macrolides. The information in this study is not only important in terms of local public health but will also contribute to an understanding of epidemic clones of pathogens from SSTI.

Tumor microenvironment in functional adrenocortical adenomas: immune cell infiltration in cortisol-producing adrenocortical adenoma.

The tumor microenvironment plays pivotal roles in various human neoplasms. However, that of benign tumor, particularly hormone-secreting endocrine tumors, has remained virtually unknown. Therefore, we firstly attempted to analyze the tumor microenvironment of autonomous hormone-secreting adrenocortical adenomas. We first histologically evaluated 21 cortisol-producing adrenocortical adenoma (CPA) and 13 aldosterone-producing adrenocortical adenoma (APA) cases. Quantitative histologic analysis revealed that intratumoral immune cell infiltration (ICI) was more pronounced in CPAs than in APAs. We then evaluated the cytokine and chemokine profiles using polymerase chain reaction arrays in APAs and CPAs. Angiogenic chemokines, C-X-C motif chemokine ligand (CXCL) 1 and CXCL2, were significantly more abundant in CPAs than in APAs using subsequent quantitative polymerase chain reaction and immunohistochemical analyses. We then examined the vascular density between these 2 adenomas, and the density was significantly higher in overt CPAs than in APAs. Of particular interest, CXCL12-positive vessels were detected predominantly in CPAs, and their infiltrating immune cells were C-X-C motif chemokine receptor 4 (CXCR4) positive. These results above indicated that CXCL12-CXCR4 signaling could partly account for ICI detected predominantly in CPAs. We then further explored the etiology of ICI in CPAs by evaluating the senescence of tumor cells possibly caused by excessive cortisol in CPAs. The status of senescence markers, p16 and p21, was significantly more abundant in CPAs than in APAs. In addition, all CPA cases examined were positive for senescence-associated ß-galactosidase. These results all indicated that exposure to local excessive cortisol could result in senescence of tumors cells and play essential roles in constituting the characteristic tissue microenvironment of CPAs.

[Use of a computer-assisted simulation system in undergraduate education of laboratory technologists].

In the undergraduate education of laboratory technologists, practical training courses in a hospital are extremely important to gain knowledge, skills and attitudes. In present laboratories, however, senior technologists have less time for teaching their juniors because of increased demands of hospital tasks. To supplement practical training of students, we therefore decided to employ clinical simulation teaching materials using a computer assisted education system. First, a series of e learning coursewares on laboratory tests for blood transfusion were created using pictures taken during our daily work. The pictures were arranged with questions, answers and commentaries, uploaded to the server in our university, and offered to the students. They were received with high rates of student satisfaction. Since e-learning seemed to be an effective and enjoyable strategy to achieve good learning outcomes, it is desirable to establish an organization to collect peer reviewed high quality e learning materials that can be shared with nationwide educators teaching tomorrow's laboratory technologists.

Expression of endothelin-1 and adrenomedullin was not altered by leptin or resistin in bovine brain microvascular endothelial cells.

Hypertension frequently occurs in obese subjects. It has been reported that leptin and resistin induce endothelin-1 expression in vascular endothelial cells. Altered function of brain microvascular endothelial cells may be related to increased occurrences of stroke in hypertensive patients. In the present study, we therefore studied the effects of leptin and resistin on the expression of endothelin-1 and adrenomedullin in bovine brain microvascular endothelial cells. Northern blot analysis showed that leptin (10(-10)-10(-8) mol/l), resistin (10(-10)-10(-8) mol/l) or a combination of leptin and resistin (10(-8) mol/l for each) had no significant effects on the expression of endothelin-1 mRNA or adrenomedullin mRNA in cultured bovine brain microvascular endothelial cells. On the other hand, hypoxia induced, and tumor necrosis factor-alpha (10 ng/ml) decreased, the expression levels of endothelin-1 and adrenomedullin mRNAs, indicating that the bovine brain microvascular endothelial cells were able to respond to hypoxia and tumor necrosis factor-alpha. Consistent with the results of Northern blot analysis, immunoreactive endothelin and immunoreactive adrenomedullin concentrations in the medium were not significantly changed by the treatment with leptin, resistin, or a combination of leptin and resistin. The present study thus showed that neither leptin nor resistin affects the expression of endothelin-1 or adrenomedullin in bovine brain microvascular endothelial cells.

Expression of orexin-A and orexin receptors in the kidney and the presence of orexin-A-like immunoreactivity in human urine.

Orexin-A (hypocretin-1), a neuropeptide with stimulatory actions on arousal and appetite, was originally shown to be specifically expressed in the hypothalamus. We studied expression of orexin-A and orexin receptors in the kidney and the presence of orexin-A-like immunoreactivity in human urine. Immunocytochemistry showed that orexin-A-like immunoreactivity and two types of orexin receptors (types 1 and 2) were localized in the tubules of the human kidney obtained at autopsy. Orexin-A-like immunoreactivity was detected in human kidneys (21.3 +/- 6.2 fmol/g wet weight, mean +/- S.E.M., n = 4) and rat kidneys (16.2 +/- 1.6 fmol/g wet weight, n = 5) by radioimmunoassay, although the levels were much lower than the levels in the brain. Orexin-A-like immunoreactivity was present in the urine obtained from male healthy volunteers (67.8 +/- 4.5 pmol/l, n = 5). Reverse phase high-performance liquid chromatography showed that most of orexin-A-like immunoreactivity of the urine extract was eluted earlier than authentic orexin-A, suggesting that orexin-A-like immunoreactivity in urine was modified to hydrophilic forms. Reverse transcriptase polymerase chain reaction showed expression of orexin receptors 1 and 2 mRNAs in the human kidney. These findings suggest that orexin-A is produced by the renal tubular cells and secreted into urine. Orexin-A may act on the kidney in the autocrine or paracrine fashion, or via the urine (urocrine fashion).

Hypoxia increases endothelin-1 mRNA expression but not immunoreactive endothelin in the medium of T98G glioblastoma cells under cytokine treatment.

Endothelin-1 (ET-1) levels in the culture medium were considered to reflect the transcription of the ET-1 gene and the subsequent secretion of ET-1 from cultured cells. It has not been clarified how different ET-1 mRNA expression levels and immunoreactive (IR)-ET levels in the culture medium are in the cell culture system. We studied ET-1 mRNA expression levels and IR-ET levels in the medium of T98G glioblastoma cells treated with cytokines. T98G glioblastoma cells were cultured with cytokines (interferon-gamma 100 U/ml, tumor necrosis factor-alpha 20 ng/ml and interleukin-1beta 10 ng/ml) under normoxia or hypoxia (1% O(2)). Northern blot analysis showed that ET-1 mRNA expression levels were increased by tumor necrosis factor-alpha alone or a combination of tumor necrosis factor-alpha and interleukin-1beta, or three cytokines, and the increase was further enhanced under hypoxia. Particularly, relative expression levels of ET-1 mRNA were significantly higher under hypoxia than in normoxia in the treatment with a combination of three cytokines. IR-ET levels in the medium were increased by treatment with tumor necrosis factor-alpha, interleukin-1beta or a combination of tumor necrosis factor-alpha and interleukin-1beta, or three cytokines. In contrast to the mRNA expression levels, IR-ET levels in the medium of T98G cells treated with a combination of three cytokines were rather decreased under hypoxia compared with those in normoxia. These findings indicate that hypoxia induces ET-1 mRNA expression in the treatment of three cytokines, but IR-ET levels in the medium do not reflect this induction in T98G glioblastoma cells.

Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney.

Adrenomedullin 2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) peptide family. AM2/IMD has a vasodilator action, and antidiuretic and antinatriuretic effects in mice. The aim of the present study is to clarify immunolocalization of AM2/IMD in human hypothalamus, heart and kidney obtained at autopsy. Immunocytochemistry showed AM2/IMD-immunoreactive cell bodies in the paraventricular and supraoptic nuclei of human hypothalamus. Both parvocellular and magnocellular cells in the paravetricular nucleus are immunostained with AM2/IMD. Immunostaining of serial sections showed co-localization of AM2/IMD-like immunoreactivity and vasopressin in the paraventricular nucleus. Myocardial cells of the heart and renal tubular cells were positively immunostained with AM2/IMD, whereas neither renal glomeruli nor vasculature in the heart and kidney were immunostained. Reverse-transcriptase polymerase chain reaction confirmed expression of AM2/IMD mRNA in the brain, pituitary, heart and kidney. The present study has shown the wide expression of AM2/IMD in human hypothalamus, heart and kidney, raising the possibility that this novel peptide may be related to the central and peripheral regulation of the circulation and water-electrolyte metabolism.

Effects of adipokines on expression of adrenomedullin and endothelin-1 in cultured vascular endothelial cells.

Obesity is a major risk factor for the development of hypertension. Adipokines may cause hypertension by acting both centrally and directly on the vascular vessels. We wished to clarify whether three adipokines, leptin, resistin and tumor necrosis factor-alpha, affect expression of adrenomedullin and endothelin-1 in vascular endothelial cells. Human umbilical vein endothelial cells were cultured for 24 h with leptin (1-10 nmol/l), resistin (1-10 nmol/l) or tumor necrosis factor-alpha (1-10 ng/ml). Expression of adrenomedullin and endothelin-1 was examined by radioimmunoassay and northern blot analysis. Immunoreactive-adrenomedullin in the medium and adrenomedullin mRNA expression levels were decreased by treatment of tumor necrosis factor-alpha time- and dose-dependently, whereas endothelin-1 secretion was not significantly changed by it. Leptin or resistin had no significant effects on expression of adrenomedullin or endothelin-1 in human umbilical vein endothelial cells. Under hypoxic conditions (1% O2), expression of both adrenomedullin and endothelin-1 was induced in these cells. Immunoreactive-adrenomedullin levels in the medium were decreased by treatment of tumor necrosis factor-alpha under hypoxia. Leptin or resistin had no significant effects on adrenomedullin or endothelin-1 expression also in hypoxia. These findings have raised the possibility that decreased expression of adrenomedullin by tumor necrosis factor-alpha may be related to the increased risk of hypertension and other cardiovascular diseases in obese subjects.

Suppression of cytokine-induced expression of endothelin-1 by dexamethasone in human retinal pigment epithelial cells.

We have recently shown that retinal pigment epithelial cells secrete endothelin-1 (ET-1). In the present study, we studied effects of cytokines and dexamethasone on expression of ET-1 in two types of cultured human retinal pigment epithelial cells, ARPE- 19 cells and D407 cells. Treatment for 24 hours with a combination of three cytokines, interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta, induced expression of ET-1 in both cells. When treated with dexamethasone (10(-7) mol/L) alone, the immunoreactive endothelin level in the medium increased about three-fold in D407 cells, but not in ARPE-19 cells. Dexamethasone (10(-7) mol/L), however, suppressed the cytokine-mediated increases in ET-1 mRNA expression levels and immunoreactive endothelin levels in the medium. These findings raise the possibility that antiinflammatory effects of dexamethasone in eyes may be partly mediated or modulated by its effects on expression of ET-1.

Voltage-gated calcium channels in the human adrenal and primary aldosteronism.

Calcium channel blockers can efficiently be used in the treatment of primary aldosteronism (PA) related hypertension, but details on the localization of calcium channel (CC) in the human adrenal and its disorders, including PA, have remained unclear. Therefore, in this study we analyzed the known α subunits of L-, N- and T-type CCs in 74 adrenocortical aldosterone-producing adenomas (APA) and 16 cortisol-producing adenomas (CPA) using quantitative RT-PCR (qPCR). We also examined the status of L-(CaV1.2, CaV1.3), N-(CaV2.2) and T-(CaV3.2) CC subunits in five non-pathological adrenals (NA), five idiopathic hyperaldosteronism (IHA) cases, and 50 APA using immunohistochemistry. After qPCR evaluation, only CaV1.2, CaV1.3, CaV2.2, and CaV3.2 mRNA levels could be detected in APA and CPA. Among those, only CaV3.2 mRNA levels were significantly correlated with plasma aldosterone levels (P=0.0031), CYP11B2 expression levels (P<0.0001) and the presence of KCNJ5 mutations (P=0.0019) in APA. The immunolocalization of CCs in NA and IHA was detected in the zona glomerulosa (ZG), with a predominance of CaV3.2 in APA. These findings suggest that different types of CC can be involved in calcium-related aldosterone biosynthesis.

PCP4: a regulator of aldosterone synthesis in human adrenocortical tissues.

Purkinje cell protein 4 (PCP4) is a calmodulin (CaM)-binding protein that accelerates calcium association and dissociation with CaM. It has been previously detected in aldosterone-producing adenomas (APA), but details on its expression and function in adrenocortical tissues have remained unknown. Therefore, we performed the immunohistochemical analysis of PCP4 in the following tissues: normal adrenal (NA; n=15), APA (n=15), cortisol-producing adenomas (n=15), and idiopathic hyperaldosteronism cases (IHA; n=5). APA samples (n=45) were also submitted to quantitative RT-PCR of PCP4, CYP11B1, and CYP11B2, as well as DNA sequencing for KCNJ5 mutations. Transient transfection analysis using PCP4 siRNA was also performed in H295R adrenocortical carcinoma cells, following ELISA analysis, and CYP11B2 luciferase assays were also performed after PCP4 vector transfection in order to study the regulation of PCP4 protein expression. In our findings, PCP4 immunoreactivity was predominantly detected in APA and in the zona glomerulosa of NA and IHA. In APA, the mRNA levels of PCP4 were significantly correlated with those of CYP11B2 (P<0.0001) and were significantly higher in cases with KCNJ5 mutation than WT (P=0.005). Following PCP4 vector transfection, CYP11B2 luciferase reporter activity was significantly higher than controls in the presence of angiotensin-II. Knockdown of PCP4 resulted in a significant decrease in CYP11B2 mRNA levels (P=0.012) and aldosterone production (P=0.011). Our results indicate that PCP4 is a regulator of aldosterone production in normal, hyperplastic, and neoplastic human adrenocortical cells.

Glutamate receptors and the regulation of steroidogenesis in the human adrenal gland: the metabotropic pathway.

BACKGROUND: l-glutamate is a major excitatory neurotransmitter in the mammalian brain. Glutamate receptors have been reported in the rat adrenal cortex and in human aldosterone-producing adenomas (APA). However, details regarding the expression levels and functions of these receptors in human adrenocortical tissues remain unknown. METHODS: The mRNA levels of glutamate receptors were evaluated by qPCR in: 12 normal adrenal cortex (NAC), 11 APA, and 12 cortisol-producing adenoma (CPA) tissues. Protein localization was evaluated by immunohistochemistry for 15 NAC, 5 idiopathic hyperaldosteronism cases, 15 APA and 15 CPA. H295R cells were treated with angiotensin-II or forskolin alone or combined with the GRM2/3 agonist LY354740. RESULTS: The level of GRM3 mRNA was higher in APA than in CPA (P=0.0086) or NAC (P=0.0022). GRM1, IGLUR2, and IGLUR3 were also detected in adrenocortical tissues. When added to angiotensin-II/forskolin treatments, LY354740 decreased aldosterone and cortisol production in H295R cells. CONCLUSIONS: GRM3 is considered to regulate steroidogenesis in adrenocortical tissues.

Adrenal CYP11B1/2 expression in primary aldosteronism: immunohistochemical analysis using novel monoclonal antibodies.

CYP11B1 and CYP11B2 play pivotal roles in adrenocorticosteroids synthesis. We performed semi-quantitative immunohistochemical analysis of these proteins in adrenals from patients with primary aldosteronism using novel monoclonal antibodies. Clusters of cortical cells positive for CYP11B2 were detected in the zona glomerulosa (ZG) of normal adrenal gland (NA), idiopathic hyperaldosteronism (IHA) and the adjacent adrenal of aldosterone-producing adenoma (APA). In APA, heterogenous immunolocalization of CYP11B2 and diffuse immunoreactivity of CYP11B1 were detected in tumor cells, respectively. The relative immunoreactivity of CYP11B2 in the ZG of adjacent adrenal of APA was significantly lower than that of NA, IHA and APA tumor cells, suggestive of suppressed aldosterone biosynthesis in these cells. These findings did indicate the regulatory mechanisms of aldosterone biosynthesis were different between normal/hyperplastic and neoplastic aldosterone-producing cells in human adrenals. CYP11B2 immunoreactivity in the ZG could also serve as a potential immunohistochemical marker differentiating morphologically hyperplastic ZG of IHA and APA adjacent adrenal.