Novel markers of graft outcome in a cohort of kidney transplanted patients: a cohort observational study.

Renal biopsy (RBx) informs about kidney transplantation (KTx) prognosis. In our observational study the prevalence of histological anomalies and the prognostic role of CD45, vimentin (VIM) and periostin (POSTN) in KTx-RBx have been evaluated. One hundred forty-six KTx-RBx (2009-2012) were analysed for general histology and in immunohistochemistry for CD45, VIM and POSTN. Clinical data of the 146-KTx patients were collected at the RBx time (T0), 6 and 12 months before and after RBx. Follow-up time was 21 ± 14 months. Glomerulosclerosis was 20% glomeruli/biopsy. Tubular atrophy (TA), Interstitial infiltrate (I-Inf) and interstitial fibrosis (IF) were slight in 21-18% and 25%, moderate in 22-30% and 26% and severe in 30-18% and 28% of patients. Fifty-eight percent of patients had lesions compatible with IF-TA. CD45, VIM and POSTN correlated to each-other and to TA, I-Inf and IF. VIM and POSTN correlated to GS. CD45 and VIM correlated directly to renal function (RF) and 25(OH)VitD, while POSTN inversely to 25(OH)VitD. Thirty patients restarted dialysis (HD+). HD+ had lower T0-eGFR, and higher CD45, VIM and POSTN than HD-. POSTN resulted the strongest in discriminate for HD+ . CD45, VIM and POSTN correlate to each-other and predict graft outcome. POSTN was the strongest in discriminate for HD+. 25(OH)VitD might influence inflammation and fibrosis in KTx.

FGF23-regulated production of Fetuin-A (AHSG) in osteocytes.

INTRODUCTION: AHSG, a serum glycoprotein with recognized anti-calcification activity, has also been suggested to modulate both bone formation and resorption. Though the bulk of AHSG is mostly synthesized in the liver, it has been claimed that also bone cells might produce it. However, the extent of the bone AHSG production and the potential controlling factors remain to be definitively proven. A relevant number of studies support the notion that FGF23, a bone-derived hormone, not only regulates the most important mineral metabolism (MM) related factors (phosphate, parathyroid hormone, vitamin D, etc.), but might be also involved in cardiovascular (CV) outcome, both in chronic kidney disease (CKD) patients and in the general population. Furthermore, in addition to some direct autocrine and paracrine effects in bone, FGF23 has been suggested to interact with AHSG. In this study we investigated if AHSG is really produced by bone cells, and if its bone production is related and/or controlled by FGF23, using cultured bone cells, according to a new method recently published by our group. RESULTS: Our data show that AHSG is consistently produced in osteocytes and to a far lesser extent in osteoblasts. Both FGF23 addition to the culture medium and its over-expression in osteocytes were associated with a consistent increase of both AHSG mRNA and protein, while FGF23 silencing was followed by opposite effects. Though most of these results were largely affected by the blockage of FGF23 receptors, the role of these receptors in the different experimental sets is still not completely clarified. In addition, we found that FGF23 and AHSG proteins co-localized both in cytoplasm and nucleus, which suggests a possible reciprocal interactivity. CONCLUSIONS: Our data not only confirm that AHSG is produced in bone, mainly in osteocytes, but show for the first time that its production is modulated by FGF23. Since both proteins play important roles in the bone and cardiovascular pathology, these results add new pieces to the puzzling relationship between bone and vascular pathology, in particular in CKD patients, prompting future investigations in this field.

An aeration control strategy for oxidation ditch processes based on online oxygen requirement estimation.

A feedforward-feedback aeration control strategy based on online oxygen requirements (OR) estimation is proposed for oxidation ditch (OD) processes, and it is further developed for intermittent aeration OD processes, which are the most popular type in Japan. For calculating OR, concentrations of influent biochemical oxygen demand (BOD) and total Kjeldahl nitrogen (TKN) are estimated online by the measurement of suspended solids (SS) and sometimes TKN is estimated by NH4-N. Mixed liquor suspended solids (MLSS) and temperature are used to estimate the required oxygen for endogenous respiration. A straightforward parameter named aeration coefficient, Ka, is introduced as the only parameter that can be tuned automatically by feedback control or manually by the operators. Simulation with an activated sludge model was performed in comparison to fixed-interval aeration and satisfying result of OR control strategy was obtained. The OR control strategy has been implemented at seven full-scale OD plants and improvements in nitrogen removal are obtained in all these plants. Among them, the results obtained in Yumoto wastewater treatment plant were presented, in which continuous aeration was applied previously. After implementing intermittent OR control, the total nitrogen concentration was reduced from more than 5 mg/L to under 2 mg/L, and the electricity consumption was reduced by 61.2% for aeration or 21.5% for the whole plant.

A novel model of in vitro osteocytogenesis induced by retinoic acid treatment.

Despite recent research which more and more stresses the importance of osteocytes in regulating bone and systemic mineral metabolism, current molecular and functional knowledge of osteocyte properties are still incomplete, mostly due to limited availability of in vitro models. Osteocytes are terminally differentiated dendritic cells, and therefore are not easy to obtain and maintain in primary cultures. As an alternative, osteocyte differentiation can be induced by progressive osteoblast embedding in mineralised extracellular matrix. In this model, which is suitable for reproduction of bone development, the presence of calcified matrix prevents several cell biological methods from being used. Therefore, the osteocyte-like MLO-Y4 cell line continues to be the most widely used cellular system. Here we show that treatment of primary osteoblasts or MC3T3-E1 cells with retinoic acid generates a homogeneous population of ramified cells with osteocyte features, as confirmed by morphological and molecular analyses. The first morphological changes are detectable in primary cells after 2 days of treatment, and in the cell line after 4 days of treatment. Differentiation is complete in 5 and 10 days, respectively, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers, and up-regulation of osteocyte-specific molecules, most notably among them sclerostin. Compared to other published protocols, our method has a number of advantages. It is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in the complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Inhibition of UV-induced G1 arrest by exposure to 50 Hz magnetic fields in repair-proficient and -deficient yeast strains.

PURPOSE: To assess the possibility that extremely low frequency (ELF) magnetic fields obstruct the damage repair process, the gene conversion frequency and cell cycle kinetics in a DNA repair-proficient and nucleotide excision repair (NER)-deficient strain of diploid yeast Saccharomyces cerevisiae. MATERIALS AND METHODS: DNA repair- or NER-deficient cells were irradiated with sublethal doses of ultraviolet light (UV) radiation followed by exposure to 50 Hz magnetic fields up to 30 mT for 48 h. After exposure, colony-forming ability was scored as revertants in which gene conversion had restored the functional allele of the ARG4 gene conversion hotspot. Cell cycle analysis was performed using flow cytometry. RESULTS: Gene conversion rate was increased by the combined exposure in DNA repair-proficient cells, whereas it remained unchanged between UV alone and the combined exposure in NER-deficient cells. The UV-induced G1 arrest was inhibited by exposure to 30 mT ELF magnetic fields in both repair-proficient and -deficient cells. CONCLUSIONS: The results suggest that exposure to high-density (30 mT) ELF magnetic fields decreases the efficiency of NER by suppressing G1 arrest, which in turn led to enhancement of the UV-induced gene conversion.

Induction of micronuclei in mice exposed to static magnetic fields.

The aim of this experiment was to investigate whether static magnetic fields (SMFs) have cytogenetic effects in mouse bone marrow cells. The frequency of micronuclei was significantly increased by exposure of mice to 3.0 T for 48 and 72 h and 4.7 T for 24, 48 and 72 h. The increase in micronucleus frequency was dose dependent at all times. Micronucleus frequency at 4.7 T was higher than at 3.0 T. We consider that the increased numbers of micronuclei may be attributable to a stress reaction caused by SMFs or a direct clastogenic/spindle disturbance effect of SMFs.

Involvement of eddy currents in the mutagenicity of ELF magnetic fields.

Possible carcinogenic and/or mutagenic activity of extremely low frequency magnetic fields was examined using somatic mutation and recombination test system of Drosophila melanogaster. An X-linked semi-dominant DNA repair defective mutation mei-41(D5) was introduced into the conventional mwh/flr test system to enhance mutant spot frequency. Virgin females of w mei-41(D5)/FM6; flr/TM6 were crossed with w mei-41(D5)/Y; mwh jv; spa(pol) males. The F(1) third instar larvae were exposed to a 50Hz, 20mT sinusoidal AC magnetic field for 24h. After moulting from pupal cases, their wings were examined under a bright field microscope to detect hair spots with mwh or flr mutant morphology. The exposure caused a statistically significant enhancement in somatic recombination spot frequency. Mutant spots arising due to chromosomal non-disjunction or terminal deletion also increased but the frequency of spots resulting from point mutation was not altered. The enhancement in the recombination spot frequency was suppressed to the control level when a culture medium without electrolytes was used during exposure. When larvae were exposed to a magnetic field in an annular dish, flies from the outer ring showed more mutant spots compared to those from the inner ring. These results suggest that the detected mutagenic activity was that of the induced eddy current, rather than that of the magnetic field itself.

A 50 Hz, 14 mT magnetic field is not mutagenic or co-mutagenic in bacterial mutation assays.

We used bacterial mutation assays to assess the mutagenic and co-mutagenic effects of power frequency magnetic fields (MF). For the former, we exposed four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and two strains of Escherichia coli (WP2 uvrA, WP2 uvrA/pKM101) to 50Hz, 14mT circularly polarized MF for 48h. All results were negative. For the latter, we treated S. typhimurium (TA98, TA100) and E. coli (WP2 uvrA, WP2 uvrA/pKM101) cells with eight model mutagens (N-ethyl-N'-nitro-N-nitrosoguanidine, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, 4-nitroquinoline-N-oxide, 2-aminoanthracene, N(4)-aminocytidine, t-butyl hydroperoxide, cumen hydroperoxide, and acridine orange) with and without the MF. The MF induced no significant, reproducible enhancement of mutagenicity. We also investigated the effect of MF on mutagenicity and co-mutagenicity of fluorescent light (ca. 900lx for 30min) with and without acridine orange on the most sensitive tester strain, E. coli WP2 uvrA/pKM101. Again, we observed no significant difference between the mutation rates induced with and without MF. Thus, a 50Hz, 14mT circularly polarized MF had no detectable mutagenic or co-mutagenic potential in bacterial tester strains under our experimental conditions. Nevertheless, some evidence supporting a mutagenic effect for power frequency MFs does exist; we discuss the potential mechanisms of such an effect in light of the present study and studies done by others.

Analysis of Chlamydia trachomatis serovars in endocervical specimens derived from pregnant Japanese women.

The polymerase chain reaction (PCR) method has been employed to amplify a chlamydial genome encoding four variable segments of the major outer membrane protein and genotyping of different Chlamydia trachomatis serovars was successfully achieved by means of restriction fragment length polymorphism (RFLP) analysis and sequencing of amplified DNA. These methods were applied to identify the serotypes of C. trachomatis in endocervical specimens obtained from asymptomatic pregnant Japanese women at 28-30 weeks of gestation. Among the 218 specimens, 207 were serotyped 43 (19.3%) as serovar D, 53 (24.3%) as E, 24 (11.0%) as F, 39 (17.9%) as G, 15 (6. 9%) as H, 15 (6.9%) as I, five (2.3%) as J, nine (4.1%) as K and four (1.8%) as mixed. Among the 11 unclassified strains by RFLP, six (2.8%) were identified as serovar B variants and five (2.3%) were identified as D/IC-Cal-8. It was suggested that variants of endemic trachoma serovars also have affinity for the urogenital tract of Japanese pregnant women.

Subtyping of cytomegalovirus strains obtained from immunocompetent children.

We analyzed 33 clinical isolates of cytomegalovirus (CMV) obtained from immunocompetent Japanese children. DNA was extracted from infected MRC-5 cells and we amplified CMV glycoprotein-B (gB), major IE (MIE), DNA polymerase and glycoprotein-H (gH) genes using PCR. Among the 33 clinical isolates, 24 were identified as gB group 1, one as group 2, 6 as group 3, and 2 as simultaneous infections with group 1 and group 3 strains. Clinical isolates of CMV have also been classified into 18 MIE, 8 DNA polymerase and 15 gH genotype patterns by restriction fragment length polymorphism (RFLP). RFLP analysis is useful in molecular epidemiological studies of CMV infection in immunocompetent individuals.

Mutagenicity and co-mutagenicity of static magnetic fields detected by bacterial mutation assay.

Possible mutagenic and co-mutagenic effects of strong static magnetic fields were estimated using bacterial mutagenicity test. Mutagenic potential of static magnetic fields up to 5T (T:1T=10,000 G) was not detected by the bacterial mutagenicity test using four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA either in the pre-incubation method or in the plate incorporation method. In the co-mutagenicity test, E. coli WP2 uvrA cells were treated with various chemical mutagens and were simultaneously exposed to a 2T or a 5T static magnetic field. Mutation rate in the exposed group was significantly higher than that in the non-exposed group when cells were treated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), 4-nitroquinoline-N-oxide (4-NQO), 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ) or 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2). The mutagenicity of 2-aminoanthracene (2-AA), 9-aminoacridine (9-AA), N4-aminocytidine and 2-acetoamidofluorene (2-AAF) was not affected by the magnetic field exposure. Possible mechanisms of the co-mutagenicity of magnetic fields are discussed.

CMV retinitis after cessation of ganciclovir therapy for CMV antigenemia in an unrelated BMT recipient.

An 11-year-old boy with severe aplastic anemia underwent unrelated BMT following TBI, antithymocyte globulin and CY. On day +23, CMV antigenemia was detected which resolved with ganciclovir. Eight days after discontinuing ganciclovir, he complained of impaired visual acuity. Ophthalmologic findings and a positive PCR study using anterior chamber fluid from the right eye confirmed the presumptive diagnosis of CMV retinitis, although CMV antigenemia and PCR studies using PBMC were then negative. He was successfully re-treated with ganciclovir. CMV retinitis should be considered even when CMV antigenemia is not present or PCR using PBMC is negative.

Detection of cytokines and cytomegalovirus DNA in serum as test for congenital infection.

Maternal serum samples at 10 and 22 weeks of gestational age and cord blood samples were available from six cases of asymptomatic congenital human cytomegalovirus (HCMV) infection. Meaningful rises of serum IgG-antibody titers by ELISA occurred in three cases. Serum interferon (IFN)-gamma activity was detected in all six cases. Serum cell free soluble interleukin-2 receptor (sIL-2R) activity rose above the normal range (145-519 U/ml) in one IgG and IgM antibody-positive and three IgG antibody-positive woman. Serum levels of sIL-2R and IFN-gamma were not elevated in anti-HCMV antibody-negative healthy pregnant women. No HCMV IE DNA was detected by PCR in the serum of any of the pregnant women. HCMV DNA was detected in the serum of one of six infants with asymptomatic congenital HCMV infection. Assessment of the changes of serum cytokines such as sIL-2R and IFN-gamma in HCMV antibody-positive pregnant women may be useful for the prenatal diagnosis of active HCMV infection during pregnancy.