Evaluation of a newly proposed renal risk score for Japanese patients with ANCA-associated glomerulonephritis.

BACKGROUND: We determined the usefulness and prognostic ability of the renal risk score (RRS), proposed in Europe, for Japanese patients with antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (AAGN) and high myeloperoxidase (MPO)-ANCA positivity; these aspects remain to be verified. METHODS: This retrospective study was conducted on 86 Japanese patients with new, biopsy-confirmed AAGN. We calculated the RRS and analyzed the relationship between this classification, and clinicopathological features and prognosis. We also compared the predictive values between RRS for endpoints including renal death and conventional prognostic tools for patients with AAGN. RESULTS: There were 33, 37, and 16 patients in the low-, medium-, and high-risk groups, respectively. All patients were MPO-ANCA positive. The median follow-up period was 33 months; 16 (18.6%) patients progressed to end-stage renal disease (ESRD). In the high-risk group, 9/16 (56.3%) patients progressed to ESRD, and renal prognosis was significantly poorer than that in other groups (low-risk group, P < 0.001; medium-risk group, P = 0.004). In Cox multivariate regression analysis, RRS was an independent, poor renal prognostic factor (hazard ratio 5.22; 95% confidence interval 2.20-12.40; P < 0.001). The receiver-operating characteristic curves of the RRS for each endpoint were comparable with those of the 2010 histological classification and those of the severity classification of Japanese rapidly progressive glomerulonephritis. CONCLUSIONS: This is the first study to report the usefulness of the RRS for predicting renal outcomes among Japanese patients with AAGN. Our predictive value of the RRS was comparable with that of conventional prognostic tools.

Induction of cellular senescence in fibroblasts through ß1-integrin activation by tenascin-C-derived peptide and its protumor effect.

Tenascin-C is upregulated during inflammation and tumorigenesis, and its expression level is correlated with a poor prognosis in several malignancies. Nevertheless, the substantial role of tenascin-C in cancer progression is poorly understood. Previously, we found that a peptide derived from tenascin-C, termed TNIIIA2, acts directly on tumor cells to activate ß1-integrin and induce malignant progression. Here, we show that ß1-integrin activation by TNIIIA2 in human fibroblasts indirectly contributes to cancer progression through the induction of cellular senescence. Prolonged treatment of fibroblasts with TNIIIA2 induced cellular senescence, as characterized by the suppression of cell growth and the induction of senescence-associated-ß-galactosidase and p16INK4a expression. The production of reactive oxygen species and subsequent DNA damage were responsible for the TNIIIA2-induced senescence of fibroblasts. Interestingly, peptide FNIII14, which inactivates ß1-integrin, inhibited fibroblast senescence induced not only by TNIIIA2 but also by H2O2, suggesting that ß1-integrin activation plays a critical role in the induction of senescence in fibroblasts. Moreover, TNIIIA2-induced senescent fibroblasts secreted heparin-binding epidermal growth factor-like growth factor (HB-EGF), which caused preneoplastic epithelial HaCaT cells to acquire malignant properties, including colony-forming and focus-forming abilities. Thus, our study demonstrates that tenascin-C-derived peptide TNIIIA2 induces cellular senescence in fibroblasts through ß1-integrin activation, causing cancer progression via the secretion of humoral factors such as HB-EGF.

Facilitating Community Risk Communication for Wide-Area Evacuation during Large-Scale Floods.

Large-scale floods have been occurring more frequently in Japan as a result of current global weather anomalies, yet evacuation procedures face several issues. These include low evacuation rates of citizens, wide-area evacuation by car, and residents who cannot evacuate on their own. For example, in the Kofu Basin, Yamanashi Prefecture, due to the size of the potential inundation area and a population that exceeds 300,000 people spread across 10 municipalities, a large number of residents would have to evacuate across municipal boundaries by car. The author proposed and applied a risk communication method to the Riverside District, Chuo City (with about 1400 households and a population of about 4000), assisting in developing a community disaster management plan for wide-area evacuation without a single victim in case of floods, which has been in place for three years. The next step was risk communication to key stakeholders, such as national, prefectural, and municipal governments. Finally, a public symposium on large-scale evacuation in the Kofu Basin was held. During the panel discussion with representatives of the Kofu River and National Road Office, prefectural government of Yamanashi, the municipality, community residents, and the author as panelists, the role of each stakeholder in area-wide evacuation was clarified and confirmed.

Analysis of PLA2R1 and HLA-DQA1 sequence variants in Japanese patients with idiopathic and secondary membranous nephropathy.

BACKGROUND: Several recent studies in patients with idiopathic membranous nephropathy (iMN) from Western and Asian counties showed that some single nucleotide polymorphisms (SNPs) within the PLA2R1 and HLA-DQA1 genes are significantly associated with iMN. However, there is only 1 report on analysis of PLA2R1 and HLA regions in Japanese patients with iMN. METHODS: A total of 58 patients with iMN, 26 patients with secondary MN (sMN), and 50 patients with other diseases were enrolled. All patients were Japanese. We selected 6 SNPs within PLA2R1 and 1 SNP within HLA-DQA1, which were significantly associated with iMN in reported white European cohorts, and sequenced these exons using genomic DNA prepared from peripheral mononuclear cells from each patient. We then analyzed differences in PLA2R1 and HLA-DQA1 sequence variants among the 3 groups. RESULTS: Genotypic and allelic frequency distributions for 3 out of 6 SNPs within PLA2R1, rs3749117, rs35771982, and rs2715918 were significantly different between the iMN and control groups. Allelic frequency distributions for SNP rs2187668 within HLA-DQA1 were significantly different between the iMN and control groups. There were no correlations between PLA2R1 and HLA-DQA1 sequence variants and clinical parameters in patients with iMN. There were no significant differences in genotypic or allelic frequency distributions for examined SNPs between the sMN and control groups. CONCLUSIONS: There are some differences in PLA2R1 SNP distributions between previously reported cohorts from other countries and our Japanese cohort of patients with iMN, while there is a significant association between SNP rs35771982 and iMN in most of reported cohorts.

Clinicopathological and long-term prognostic features of membranous nephropathy with crescents: a Japanese single-center experience.

BACKGROUND: Three recent studies from the United States and China reported the clinicopathological features and short-term prognosis in patients with membranous nephropathy (MN) and crescents in the absence of secondary MN, anti-glomerular basement membrane (GBM) antibodies, and anti-neutrophil cytoplasmic antibodies (ANCA). METHODS: We compared clinicopathological and prognostic features in 16 MN patients with crescents (crescent group) and 38 MN patients without crescents (control group), in the absence of secondary MN, anti-GBM antibodies, and ANCA. Median follow-up periods in the crescent and control groups were 79 and 50 months, respectively. RESULTS: Decreased estimated glomerular filtration rates (<50 mL/min/1.73 m2), glomerulosclerosis, and moderate-to-severe interstitial fibrosis were more frequently observed in the crescent group than in the control group (P = 0.043, P = 0.004, and P = 0.035, respectively). Positive staining rates for glomerular IgG2 and IgG4 were significantly different between the 2 groups (P = 0.032, P = 0.006, respectively). Doubling of serum creatinine during follow-up was more frequently observed in the crescent group than in the control group (P = 0.002), although approximately two-thirds of patients in the crescent group were treated with immunosuppressive therapy. Crescent formation and interstitial fibrosis were risks for doubling of serum creatinine [hazard ratio (HR) = 10.506, P = 0.012; HR = 1.140, P = 0.009, respectively]. CONCLUSIONS: This is the first Japanese study demonstrating significant differences in clinicopathological and prognostic features between the 2 groups. Most patients in the crescent group may develop a long-term decline in renal function despite immunosuppressive therapy.

Anti-neutrophil Cytoplasmic Antibody-associated Vasculitis Complicated by Periaortitis and Cranial Hypertrophic Pachymeningitis: A Report of an Autopsy Case.

Anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) is a systemic inflammatory disorder categorized as small-vessel vasculitis. We herein report an elderly Japanese man with AAV (granulomatosis with polyangiitis affecting the eyes, nose, lungs, and kidneys) who also showed periaortitis at the diagnosis and developed cranial hypertrophic pachymeningitis (HP) during steroid maintenance therapy. His consciousness disturbance caused by HP improved after steroid pulse therapy, but he died of aspiration pneumonia. Autopsy findings showed giant cells in the thickened pachymeninges and obsolete inflammatory lesions in the aortic adventitia and renal tubulointerstitium. This is the first case of AAV complicated by periaortitis and cranial HP.

Quantification of Interstitial Fibrosis in Renal Allografts and Clinical Correlates of Long-Term Graft Function.

BACKGROUND: The present study investigated interstitial fibrosis (IF) in 144 kidney recipients 0 h and 1 year post transplantation and assessed relationships with Banff code scores, clinical parameters, and long-term graft function. METHODS: A quantitative analysis of IF was performed using the computer-assisted imaging of Sirius red-stained biopsy samples. Percent IF (%IF) in the cortical region was assessed at 0 h and 1 year, and an increase in the ratio of %IF from 0 h to 1 year was calculated. The relationship between %IF and Banff code scores was analyzed. Demographics and trough concentrations of tacrolimus were tested as risk factors in the top 20 patients with increases in %IF. The influence of increases in the ratio of %IF at 1 year on long-term graft function and survival was also assessed in these 20 patients. RESULTS: Median %IF at 0 h and 1 year were 1.55 and 2.80%, respectively. No correlation was found between %IF and Banff code scores. The mean increase in the ratio of %IF from 0 h to 1 year was 4.31-fold. The increase in %IF in the top 20 patients correlated with diabetes mellitus. Graft function, but not graft survival, was lower in the top 20 patients for 10 years post transplantation. CONCLUSIONS: A correlation was not found between %IF and Banff code scores. Greater increases in %IF within 1 year post transplantation may influence long-term graft survival. Computer-analyzed increases in %IF at 1 year may be a surrogate marker for long-term graft function.

Different Expression Patterns of Toll-Like Receptor mRNAs in Blood Mononuclear Cells of IgA Nephropathy and IgA Vasculitis with Nephritis.

Mucosal immunity may play a key role in IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN). IgAVN is characterized by the presence of non-thrombocytopenic palpable purpura, associated with glomerulonephritis with IgA-dominant immune deposits. Recent studies have shown the up-regulation of Toll-like receptors (TLRs) in patients with IgAN or IgAVN. Among TLRs that mediate innate immune reactions, TLR2, TLR4, and TRL5 recognize bacterial components, while TLR3, TLR7, and TLR9 recognize viral components. Here we compared the expression levels of TLR mRNAs in peripheral blood mononuclear cells (PBMCs) from 49 IgAN patients, 20 IgAVN patients, and 20 patients with thin basement membrane nephropathy (TBMN), unrelated to immune-mediated pathogenesis, as a control. The real-time RT-PCR analysis revealed the significantly higher expression levels of TLR2, TLR3, TLR5, TLR7, and TLR9 mRNAs in PBMCs of IgAN and IgAVN patients, compared to TBMN patients. Importantly, TLR4 mRNA levels were significantly higher in IgAN patients than in IgAVN patients, while its expression levels were comparable in IgAVN patients and TBMN patients. In contrast, TLR5 and TLR9 mRNA levels were significantly higher in IgAVN patients than in IgAN patients. In IgAN patients, expression levels of TLR2, TLR3, TLR5, or TLR9 mRNA were correlated with proteinuria levels, and TLR4 mRNA levels were correlated with serum IgA levels. In IgAVN patients, however, there was no such correlation. The up-regulated expression of TLR mRNAs in PBMCs may be related to the development of IgAN and IgAVN. The distinct expression patterns between these two diseases may reflect their different pathogenetic mechanisms.

Effect of CYP3A5 and ABCB1 polymorphisms on the interaction between tacrolimus and itraconazole in patients with connective tissue disease.

PURPOSE: The aim of this study was to investigate the effect of itraconazole (ITCZ), a potent inhibitor of CYP3A4 and P-glycoprotein, on the blood concentration 12 h after tacrolimus administration (C 12h) in relation to CYP3A5 6986A>G and ABCB1 3435C>T genotype status in patients with connective tissue disease (CTD). METHODS: Eighty-one CTD patients taking tacrolimus (Prograf®) once daily at night (2100 hours) were enrolled in this study. Whole blood samples were collected 12 h after tacrolimus administration at steady state. RESULTS: The dose-adjusted tacrolimus C 12h with or without ITCZ co-administration was significantly higher in patients with CYP3A5*3/*3 than in those with the CYP3A5*1 allele [CYP3A5 *1/*1 vs. *1/*3 vs. *3/*3 = 1.67 vs. 2.70 vs. 4.83 ng/mL/mg (P = 0.003) and 0.68 vs. 0.97 vs. 2.20 ng/mL/mg (P < 0.001), respectively], but differences were not observed for ABCB1 genotypes. However, there was no difference in the increase rate of the dose-adjusted C 12h of tacrolimus between CYP3A5 or ABCB1 genotypes (P = 0.378 and 0.259). On the other hand, reduction of the estimated glomerular filtration rate exhibited a correlation with the C 12h of tacrolimus after ITCZ co-administration (r = -0.482, P = 0.009). CONCLUSIONS: In CYP3A5*3/*3 patients, because the metabolic pathway for tacrolimus occurs only through CYP3A4, the combination with ITCZ seems to lead to a higher risk of acute renal dysfunction. Therefore, we suggest that the target blood tacrolimus concentration be set as low as possible through dose-adjustment for patients with the CYP3A5*3/*3 allele.

Effects of CYP3A5 polymorphism and the tacrolimus 12 h concentration on tacrolimus-induced acute renal dysfunction in patients with lupus nephritis.

1. The purpose of the present study was to investigate the effect of tacrolimus concentration in blood 12 h after administration (C12h) on acute renal dysfunction in patients with lupus nephritis (LN) taking tacrolimus once daily. 2. Five of the 35 LN patients experienced tacrolimus-induced acute renal dysfunction. 3. The average annual C12h of tacrolimus was higher for patients with events of elevated serum creatinine level than for patients not experiencing these events [6.4 (5.6-8.8) versus 2.8 (2.2-4.6) ng/mL, respectively, p=0.001]. 4. The average annual tacrolimus C12h was higher for patients with CYP3A5*3/*3 (PM) than for patients with the CYP3A5*1 allele (EM) [4.6 (3.2-6.6) versus 2.5 (2.0-3.1) ng/mL, respectively, p=0.002]. 5. The area under the receiver operating characteristic was 0.887, which gave the best sensitivity (80.0%) and specificity (86.7%) at a tacrolimus C12h (average annual C12h or C12h at events) threshold of 5.2 ng/mL. 6. C12h measurements, CYP3A5 genotyping and dose adjustments of tacrolimus should be performed to prevent acute renal dysfunction in LN patients taking tacrolimus once daily.

Validation of the Japanese histologic classification 2013 of immunoglobulin A nephropathy for prediction of long-term prognosis in a Japanese single-center cohort.

BACKGROUND: A new Japanese histologic classification (JHC) of immunoglobulin A nephropathy (IgAN) for prediction of long-term prognosis was proposed in 2013. The goal of this study was to validate the JHC system in a Japanese single-center cohort. METHODS: A retrospective study was conducted in 198 Japanese adult patients with IgAN. Clinical findings including blood pressure, urinary protein, estimated glomerular filtration rate (eGFR), and outcomes were evaluated in these patients. The glomerular lesion percentage score (GLPS) [number of glomeruli with cellular crescents, fibrocellular crescents, global sclerosis, segmental sclerosis, or fibrous crescents/number of total obtained glomeruli × 100 (%)] was assessed in each patient and categorized into histologic grades (HGs) of HG1 (<25 %), HG2 (25-49 %), and HG3/4 (≥50 %). Associations of GLPS (HG) with disease progression (50 % eGFR decline or end-stage renal disease requiring dialysis) within 10 years after biopsy and the rate of annual eGFR decline were examined. RESULTS: During a median follow-up period of 12.0 years after biopsy, disease progression occurred in 12.8 % (12/94) of HG1 patients, 32.3 % (21/65) of HG2 patients, and 46.2 % (18/39) of HG3/4 patients. The risk of disease progression was significantly higher in the HG2 and HG3/4 groups than in the HG1 group (odds ratios: 3.3 and 5.9 vs. 1). A higher GLPS was significantly associated with a higher risk of disease progression and a greater annual eGFR decline. CONCLUSION: The newly proposed JHC system 2013 based on GLPS (HG) was well correlated with long-term prognosis in our cohort of Japanese adult patients with IgAN.

Validation of the 2010 histopathological classification of ANCA-associated glomerulonephritis in a Japanese single-center cohort.

OBJECTIVES: To validate the 2010 histopathological classification system of anti-neutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis (GN) in a Japanese single-center cohort. METHODS: We retrospectively studied 54 patients (28 renally limited pauci-immune GN, 25 microscopic polyangiitis, and one Churg-Strauss syndrome). RESULTS: There were 17 patients with focal GN, eight patients with crescentic GN, 19 patients with mixed GN, and 10 patients with sclerotic GN. Detailed information regarding treatment was available in 39 patients. All these patients were treated with steroids with or without immunosuppressive agents. Hemodialysis was introduced in two patients with crescentic GN and three patients with sclerotic GN. During the follow-up period, 27 of 54 patients died. The major cause of death was pneumonia. Significant differences were observed in estimated glomerular filtration rate among patients with focal, crescentic, mixed, and sclerotic GN at entry and 1- and 5-year follow-up. Patients with focal GN had preserved renal function and favorable outcome. CONCLUSIONS: Our validation study suggests that the 2010 histopathological classification of ANCA-associated GN might aid in prognostication of patients at the time of diagnosis and in therapy selection.

Serum interleukin 6 levels as a useful prognostic predictor of clinically amyopathic dermatomyositis with rapidly progressive interstitial lung disease.

OBJECTIVES: Rapidly progressive interstitial lung disease (RP-ILD) is life-threatening in patients with clinically amyopathic dermatomyositis (CADM). Useful prognostic markers are necessary for treatment selection. This study aimed to investigate differences in clinical and laboratory characteristics between surviving and non-surviving patients. METHODS: Twelve CADM patients with RP-ILD were enrolled. Six patients lived (Group A) and six patients died (Group B) after immunosuppressive treatment for RP-ILD. Clinical manifestations and laboratory data before treatment were compared between the two groups. RESULTS: Among the clinical manifestations and laboratory data examined, serum interleukin 6 (IL-6) levels in Group B were significantly higher than those in Group A (mean ± SD 28.5 ± 21.0 vs. 7.2 ± 1.6 pg/mL; p = 0.009). Simple regression analysis showed that serum IL-6 was the only significant prognostic factor (p = 0.032). Kaplan-Meier estimates showed that the cumulative survival rate was significantly lower in patients with serum IL-6 levels of ≥ 9 pg/mL than in patients with those of < 9 pg/mL (p = 0.04). CONCLUSIONS: Serum IL-6 levels may predict the prognosis of CADM patients with RP-ILD. The intensity of immunosuppressive treatment can be decided according to serum IL-6 levels at an early phase of the disease.

Validation of the 2010 histopathological classification of ANCA-associated glomerulonephritis in a Japanese single-center cohort.

OBJECTIVES: To validate the 2010 histopathological classification system of anti-neutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis (GN) in a Japanese single-center cohort. METHODS: We retrospectively studied 54 patients (28 renally limited pauci-immune GN, 25 microscopic polyangiitis, and one Churg-Strauss syndrome). RESULTS: There were 17 patients with focal GN, eight patients with crescentic GN, 19 patients with mixed GN, and 10 patients with sclerotic GN. Detailed information regarding treatment was available in 39 patients. All these patients were treated with steroids with or without immunosuppressive agents. Hemodialysis was introduced in two patients with crescentic GN and three patients with sclerotic GN. During the follow-up period, 27 of 54 patients died. The major cause of death was pneumonia. Significant differences were observed in estimated glomerular filtration rate among patients with focal, crescentic, mixed, and sclerotic GN at entry and 1- and 5-year follow-up. Patients with focal GN had preserved renal function and favorable outcome. CONCLUSIONS: Our validation study suggests that the 2010 histopathological classification of ANCA-associated GN might aid in prognostication of patients at the time of diagnosis and in therapy selection.

Renal biopsy in patients aged 80 years and older: a single-center experience in Japan.

BACKGROUND: There is a paucity of data on renal biopsy in a large number of the very elderly (age ≥ 80 years) worldwide. METHODS: Clinicopathological features in 73 patients aged ≥ 80 years were evaluated and compared with control groups of 172 patients aged 60 - 61 years and 128 patients aged 70 - 71 years. RESULTS: The common indications for biopsy in the very elderly were nephrotic syndrome (NS), followed by proteinuria without NS and/or hematuria, and acute kidney injury (AKI). Histological diagnoses were considered to potentially modify treatment in 57 cases (78.1%): the most frequent diagnosis was membranous nephropathy, followed by minimal change disease, and various other diseases. There were no biopsy procedure-related serious complications. Clinical assessment of treatments was evaluated in 38 of 54 patients with AKI and/or NS. Improvement in renal dysfunction or NS was observed in 24 of 30 (80%) patients who received immunosuppressive therapy. There were statistically significant differences in the disease spectrum between the very elderly and control groups. CONCLUSIONS: This is the first report of renal biopsy findings in a relatively large number of Japanese very elderly patients. Histological observations are useful aids in estimating the prognosis and therapy selection for renal disorders, even in the very elderly.

Distribution of glomerular IgG subclass deposits in patients with membranous nephropathy and anti-U1 ribonucleoprotein antibody.

BACKGROUND: It is known that a predominant glomerular deposition of IgG4 is characteristic of idiopathic membranous nephropathy (MN) and that significant deposition of other IgG subclasses is also observed in lupus MN. However, there is no report focusing on the distribution of glomerular IgG subclass deposits in MN patients with anti-U1 ribonucleoprotein (RNP) antibody. METHODS: We evaluated clinicopathological features and the distribution patterns of glomerular IgG subclass deposits in seven MN patients with positive anti-RNP antibody and negative antibodies to double-stranded DNA (dsDNA) and Smith antigen (Sm) (RNP-MN group) and in seven age- and sex-matched lupus MN patients with positive anti-dsDNA antibody and negative antibodies to RNP and Sm (L-MN group). RESULTS: Mixed connective tissue disease was diagnosed in four patients in the RNP-MN group. Two patients in the RNP-MN group and three patients in the L-MN group developed nephrotic syndrome. Renal insufficiency was not present in all patients in both groups. Hypocomplementemia was found in two patients in the RNP-MN group and six patients in the L-MN group. In the RNP-MN group, positive stainings for glomerular IgG1, IgG2, IgG3 and IgG4 were observed in one, seven, zero and five patients, respectively. On the contrary, in the L-MN group, positive stainings for glomerular IgG1, IgG2, IgG3 and IgG4 were observed in seven, seven, seven, and six patients, respectively. CONCLUSIONS: This is the first study showing striking differences in the distribution of glomerular IgG subclass deposits between RNP-MN and L-MN groups. RNP-MN and L-MN may result from different immunological mechanisms.

Serum procalcitonin levels in patients with myeloperoxidase-antineutrophil cytoplasmic antibodies-associated glomerulonephritis.

INTRODUCTION: High serum procalcitonin (PCT) levels (≥0.5 ng/mL) commonly occur with systemic bacterial and fungal infections. Although several studies suggested that measuring serum PCT levels may serve as a useful marker to distinguish between active antineutrophil cytoplasmic antibodies (ANCA)-associated diseases and invasive infections, there is no information on PCT in myeloperoxidase (MPO)-ANCA-associated glomerulonephritis. METHODS: The authors measured serum PCT concentrations before initiation of immunosuppressive therapy in 67 patients with biopsy-proven MPO-ANCA-associated glomerulonephritis. The authors compared complications and clinicopathological parameters between patients with serum PCT levels of <0.5 ng/mL (group A: 58 patients) and ≥0.5 ng/mL (group B: 9 patients). RESULTS: All 58 patients in group A did not show any clinical sign of systemic infection. On the other hand, 3 of 9 patients in group B had bacterial or fungal infections of the respiratory or urinary tact. One patient had a history of chronic urinary tract infection. In the remaining 5 patients in group B, there were 3 patients with concurrent malignancies and 1 postoperative patient with malignancy. Another in group B had a long history of interstitial pneumonia of unknown origin and severe renal insufficiency. Serum levels of C-reactive protein and creatinine were significantly higher in group B than in group A. CONCLUSIONS: In patients with MPO-ANCA-associated glomerulonephritis, serum PCT levels of ≥0.5 ng/mL are recommended as cutoff for consideration of bacterial and fungal infections. Elevated serum PCT levels could also be observed in some patients with severe injury of the kidneys and/or lungs in the absence of infection.

Acute stress increases the synthesis of 7α-hydroxypregnenolone, a new key neurosteroid stimulating locomotor activity, through corticosterone action in newts.

7α-Hydroxypregnenolone (7α-OH PREG) is a newly identified bioactive neurosteroid stimulating locomotor activity in the brain of newt, a wild animal, which serves as an excellent model to investigate the biosynthesis and biological action of neurosteroids. Here, we show that acute stress increases 7α-OH PREG synthesis in the dorsomedial hypothalamus (DMH) through corticosterone (CORT) action in newts. A 30-min restraint stress increased 7α-OH PREG synthesis in the brain tissue concomitant with the increase in plasma CORT concentrations. A 30-min restraint stress also increased the expression of cytochrome P450(7α) (CYP7B), the steroidogenic enzyme of 7α-OH PREG formation, in the DMH. Decreasing plasma CORT concentrations by hypophysectomy or trilostane administration decreased 7α-OH PREG synthesis in the diencephalon, whereas administration of CORT to these animals increased 7α-OH PREG synthesis. Glucocorticoid receptor was present in DMH neurons expressing CYP7B. Thus, CORT appears to act directly on DMH neurons to increase 7α-OH PREG synthesis. We further investigated the biological action of 7α-OH PREG in the brain under stress. A 30-min restraint stress or central administration of 7α-OH PREG increased serotonin concentrations in the diencephalon. Double immunolabeling further showed colocalization of CYP7B and serotonin in the DMH. These results indicate that acute stress increases the synthesis of 7α-OH PREG via CORT action in the DMH, and 7α-OH PREG activates serotonergic neurons in the DMH that may coordinate behavioral responses to stress. This is the first demonstration of neurosteroid biosynthesis regulated by peripheral steroid hormone and of neurosteroid action in the brain under stress in any vertebrate class.

Biosynthesis, mode of action, and functional significance of neurosteroids in the purkinje cell.

The brain has traditionally been considered to be a target site of peripheral steroid hormones. In addition to this classical concept, we now know that the brain has the capacity to synthesize steroids de novo from cholesterol, the so-called "neurosteroids." In the middle 1990s, the Purkinje cell, an important cerebellar neuron, was identified as a major site for neurosteroid formation in the brain of mammals and other vertebrates. This discovery has provided the opportunity to understand neuronal neurosteroidogenesis in the brain. In addition, biological actions of neurosteroids are becoming clear by the studies using the Purkinje cell, an excellent cellular model, which is known to play an important role in memory and learning processes. Based on the studies on mammals over the past decade, it is considered that the Purkinje cell actively synthesizes progesterone and estradiol from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. Both progesterone and estradiol promote dendritic growth, spinogenesis, and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Such neurosteroid actions mediated by neurotrophic factors may contribute to the formation of cerebellar neuronal circuit during neonatal life. 3α,5α-Tetrahydroprogesterone (allopregnanolone), a progesterone metabolite, is also synthesized in the cerebellum and considered to act as a survival factor of Purkinje cells in the neonate. This review summarizes the current knowledge regarding the biosynthesis, mode of action, and functional significance of neurosteroids in the Purkinje cell during development in terms of synaptic formation of cerebellar neuronal networks.

Simultaneous determination of warfarin and 7-hydroxywarfarin enantiomers by high-performance liquid chromatography with ultraviolet detection.

Warfarin is administered clinically as a racemic mixture of two enantiomers, (R)-warfarin and (S)-warfarin. (S)-Warfarin has more potent anticoagulant activity than (R)-warfarin and is metabolized mainly to (S)-7-hydroxywarfarin by CYP2C9. A simple, rapid, and sensitive high-performance liquid chromatography method with ultraviolet detection was developed for the simultaneous quantitative determination of the (R)- and (S)-enantiomers of warfarin and 7-hydroxywarfarin in human plasma. Analytes and the internal standard (p-chlorowarfarin) were separated using a mobile phase of 0.5% KH2PO4 (pH 3.5)-methanol (41:59, v/v) on a Chiral CD-Ph column at a flow rate of 0.5 mL/min and were detected at a ultraviolet absorbance of 305 nm. Analysis required 200 µL of plasma and involved a simple and rapid solid-phase extraction with an Oasis HLB cartridge, which gave high recovery (greater than 91.8%) and good selectivity for all analytes. The lower limit of quantification for the (R)- and (S)-enantiomers of warfarin and 7-hydroxywarfarin was 2.5 ng/mL for each analyte. Inter- and intraday coefficients of variation for all analytes were less than 14.2% and accuracies were within 6.6% over the linear range. Our results indicate that this method is applicable to the simultaneous monitoring of the enantiomers of warfarin and 7-hydroxywarfarin in human plasma. The S/R-enantiomer ratio of warfarin and the (S)-warfarin/(S)-7-hydroxywarfarin ratio 3 hours after administration in 67 CYP2C9*1/*1 patients ranged from 0.24 to 0.75 and from 1.83 to 19.02, respectively, whereas these ratios in a CYP2C9*3/*3 patient were 1.12 and 17.02, respectively.