[Study on KIR gene polymorphisms in 416 renal transplantation recipients from southern Zhejiang].

OBJECTIVE: To investigate polymorphisms of killer cell immunoglobulin-like receptor gene (KIR) in renal transplant recipients from southern Zhejiang. METHODS: KIR genotypes were analyzed by PCR-SSP in 416 renal transplant recipients, and the genotype frequencies were compared with populations from Eastern China and worldwide. RESULTS: All 16 known KIR genes were detected in the renal transplant recipients, and KIR2DL4, 3DL2-3, 3PD1 were found in all. As a pseudogene, 2DP1 has a high genotype frequency (99%). The frequencies of KIR2DL1, 2DL3, 3DL1, 2DS4 have ranged from 92.1% to 98.8%. Compared with 11 groups in Eastern China and other countries, the KIR2DL2 phenotype frequency was higher (34.6%) than those of Shanghai, Zhejiang and Jiangsu populations (P<0.05). Among 41 genotypes, three have not been reported previously. The most common genotype was AA1, with a frequency of 43.51%, which was significantly lower than those of Jiangsu and Northern Zhejiang. CONCLUSION: Renal transplant recipients from southern Zhejiang share similar features with Eastern China Han population with regard to KIR polymorphisms, but also have unique frequencies for KIR genotypes.

Spatiotemporal characteristics of ground microtremor in advance of rockfalls.

Identifying cliffs that are prone to fall and providing a sufficient lead time for rockfall warning are crucial steps in disaster risk reduction and preventive maintenance work, especially that led by local governments. However, existing rockfall warning systems provide uncertain rockfall location forecasting and short warning times because the deformation and cracking of unstable slopes are not sufficiently detected by sensors before the rock collapses. Here, we introduce ground microtremor signals for early rockfall forecasting and demonstrate that microtremor characteristics can be used to detect unstable rock wedges on slopes, quantitatively describe the stability of slopes and lengthen the lead time for rockfall warning. We show that the change in the energy of ground microtremors can be an early precursor of rockfall and that the signal frequency decreases with slope instability. This finding indicates that ground microtremor signals are remarkably sensitive to slope stability. We conclude that microtremor characteristics can be used as an appropriate slope stability index for early rockfall warning systems and predicting the spatiotemporal characteristics of rockfall hazards. This early warning method has the advantages of providing a long lead time and on-demand monitoring, while increasing slope stability accessibility and prefailure location detectability.

[Bacterial 16S rRNA genes and expression of IL-1ß, TNF-α and IgA in prostate tissues].

OBJECTIVE: To investigate the role of bacteria in the etiology of chronic prostatitis. METHODS: Complete prostate specimens were obtained at autopsy from 192 organ donors (aged 20 - 38 years old) during 2002 to 2008 who died of non-prostatic diseases. One tissue taken from the peripheral prostatic zone according to McNeal was divided into two pieces. One piece of tissue was taken for routine pathological examinations and immunohistochemical studies of interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α) and IgA. Another one was taken for PCR assay to detect the bacterial 16S rRNA genes (16S rDNA). RESULTS: Of 192 prostate specimens, 64 (33.3%) had pathological changes of chronic prostatitis and 38 (19.8%) specimens was positive for bacterial 16S rDNA. Positive rates of 16S rDNA in chronic prostatitis and non-prostatitis specimens were 50.0% (32/64) and 4.6% (6/128) respectively (χ(2) = 55.185, P < 0.001). Expressions of IL-1ß, TNF-α and IgA in specimens of chronic prostatitis were significantly higher than those in non-prostatitis specimens (P < 0.001). A positive correlation could be found among three immunohistochemical indicators (P < 0.01). In 64 specimens with chronic prostatitis, a significant expression of IL-1ß, TNF-α and IgA was more often demonstrated in 16S rDNA positive group than in 16S rDNA negative group (P < 0.001). CONCLUSIONS: The up-regulations of bacterial 16S rDNA, cytokines and immunoglobulin A are involved in inflammatory response of chronic prostatitis. Bacterial infection may be an important cause of chronic prostatitis.

[Expressions of bacterial 16S rRNA, IL-1beta, TNF-alpha and NGF in prostate tissues].

OBJECTIVE: To investigate the role of bacteria in the etiology of chronic prostatitis. METHODS: A total of 162 complete prostate specimens were obtained at autopsy from organ donors (aged 20 -38 yr) who died of non-prostatic diseases. Each of the samples from the peripheral zone of the prostate was divided into two parts, one for routine pathological examination and immunohistochemical studies of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and the nerve growth factor (NGF), and the other for PCR assay to detect the bacterial 16S rRNA gene (16S rDNA). RESULTS: Fifty-one (31.5%) of the total specimens presented pathological changes of chronic prostatitis, of which 44 had mild focal stromal, 5 mild focal stromal and periglandular and 2 mild focal periglandular inflammation. The positive rate of 16S rDNA was 19.1% (31/162), 51.0% (26/51) in the chronic prostatitis and 4.5% (5/111) in the non-prostatitis specimens (chi2 = 29.783, P < 0.01). In the specimens with chronic prostatitis, the expressions of IL-1beta, TNF-alpha and NGF were significantly higher in the 16S rDNA positive than in the 16S rDNA negative group (P < 0.01). CONCLUSION: Bacterial inflammation may play an important role in the etiology of chronic prostatitis.

A fast SSP-PCR method for genotyping the ATP-binding cassette subfamily B member 1 gene C3435T and G2677T polymorphisms in Chinese transplant recipients.

AIM: P-glycoprotein, the product of the ATP-binding cassette subfamily B member 1 (ABCB1) gene (or the so-called multidrug resistance 1 gene), is an ATP-driven efflux pump contributing to the pharmacokinetics as well as the pharmacokinetics of drugs that are P-glycoprotein substrates, such as tacrolimus. This paper describes the development of a new method for detection of the 3435C/T and 2677G/T/A single nucleotide polymorphisms of the ABCB1 gene. The method is a simple sequence-specific primer polymerase chain reaction (SSP-PCR). METHODS: 158 Chinese health checkup examinees and 214 transplant recipients were included in the study. Genomic DNA was extracted from peripheral blood and amplified with SSP-PCR to detect the 3435C/T and 2677G/T/A mutations in ABCB1. The SSP-PCR condition was optimized, and the PCR results were compared with those of DNA sequencing. RESULTS: In the optimized condition, the two polymorphisms could be clearly distinguished after one-step PCR and electrophoresis. The ABCB1 3435C/T and 2677G/T/A genotypes of the subjects were scanned, and allele-specific bands were successfully amplified by SSP-PCR, which were in full accordance with the results of sequencing. CONCLUSION: As a fast, simple and inexpensive genotyping tool, the method would be practicable in large clinical studies on interindividual pharmacokinetics.

[Detection of bacterial signal of 16S rRNA gene in prostate tissues obtained by perineal approach from patient with chronic pelvic pain syndrome].

OBJECTIVE: To explore the role of bacteria in etiology of chronic pelvic pain syndrome (CPPS), i.e., chronic prostatitis and the correlation between presence of bacterial signal of 16S ribosomal RNA (16S rRNA) gene and the response to antibiotics. METHODS: Samples of prostatic and subcutaneous tissues were obtained by biopsy via perineal approach from 112 CPPS patients, aged 20 - 48. Polymerase chain reaction was conducted to detect the 16S rRNA gene of bacteria. The patients were treated with gatifloxacin 0.4 g once a day for 4 weeks and then 4 weeks later the effects of treatment were assessed by the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI). RESULTS: PCR was completed in 94 of the 112 patients, and 18 were excluded because their subcutaneous biopsies were positive for 16S rRNA, showing the possible contamination of their prostatic tissues. The total positive rate of bacterial 16S rRNA gene was 63.8% (60/94). The positive rate of bacterial 16S rRNA gene in the patients with IIIa CPPS and IIIb CPPS were 68.3% and 60.3% respectively. The total gatifloxacin effective rate of positive bacterial signal group after the was 55.0%, significantly higher than that of the negative bacterial signal group (14.7%, P < 0.001). The gatifloxacin effective rate of the 16S rRNA positive IIIa CPPS patients was 75%, significantly higher than that of the 6S rRNA negative IIIa CPPS patients (23.1%, P < 0.001), and the gatifloxacin effective rate of the 16S rRNA positive IIIb CPPS patients was 37.5%, significantly higher than that of the 6S rRNA negative IIIb CPPS patients (9.5%, P < 0.05). CONCLUSION: Bacterial infection is related to CPPS in part of the patients. Bacterial signal detection helps predict the effect of antimicrobial therapy.

An evaluation of the phosphorus storage capacity of an anaerobic/aerobic sequential batch biofilm reactor.

The aim of this work was to assess the phosphorus storage capability of the polyphosphate (poly-P) accumulating organisms (PAO) in the biofilm using a sequential batch biofilm reactor (SBBR). In the anaerobic phase, the specific COD uptake rates increases from 0.05 to 0.22 (mg-COD/mg-biomass/h) as the initial COD increases and the main COD uptake activity occurs in the initial 30 min. The polyhydroxyalkanoates (PHAs) accumulation from 18 to 38 (mg-PHA/g-biomass) and phosphorus release from 20 to 60 (mg-P/L) share a similar trend. The adsorbed COD cannot be immediately transformed to PHAs. Since the PHAs' demand per released phosphorus is independent of the initial COD, the enhancement of the PHA accumulation would be of benefit to phosphorus release. The only requirement is to have an initial amount of substrate that will result in sufficient PHA accumulation (approximately 20 mg-PHA/g-biomass) for phosphorus release. During the aerobic phase, the aeration should not only provide sufficient dissolved oxygen, but should also enhance the mass transfer and the diffusion. In other words, the limitation to the phosphorus storage capability always occurs during the anaerobic phase, not the aerobic phase.

Representing the retinal line spread shape with mathematical functions.

OBJECTIVE: To report a mathematical function that characterizes the double-pass line spread function (LSF) of the human eye. Determining analytical functions that represent the double-pass LSF is important because it allows modeling the optical performance of the eye. METHODS: Optical section retinal images, generated in normal human eyes using a modified slit-lamp biomicroscope, were analyzed to derive the double-pass LSF by plotting the intensity distribution of laser light reflected/ scattered from the vitreoretinal interface. Three mathematical functions (Lorentzian, Gaussian, exponential) were fitted to the double-pass LSF and the root mean square error (RMSE) was calculated to provide a measure of the goodness of fit. RESULTS: The Lorentzian function provided the best representation of the double-pass LSF of normal human eyes. The full width at half maximum (FWHM) of the Lorentzian fitted curve was positively correlated with age, indicating that the double-pass LSF broadens with age. Furthermore, the goodness of fit of the Lorentzian function was significantly better in younger subjects as compared with older subjects, suggesting that the fitted function to the double-pass LSF may vary according to age. CONCLUSION: The results demonstrate an age-related change in the double-pass LSF width and the goodness of fit of the Lorentzian function.

[Detection of 16S ribosomal RNA gene of bacteria in prostate tissues of adults].

OBJECTIVE: To investigate the role of bacteria in chronic prostatitis. METHODS: Complete specimens of prostate were obtained from 140 organ donors, aged 20 - 35, at autopsy. A piece of tissue was collected from the peripheral zone of prostate from each specimen and was divided into 2 parts to undergo pathological examination and PCR so as to detect the 16S ribosomal RNA (16S rRNA) gene of bacteria. RESULTS: Focal mild inflammation was shown in 46 of the 104 specimens (32.9%), including interstitial inflammation in 42 specimens, inflammation in both interstitial and body of gland in 3 specimens, and perigladulitis in 1 specimen. Twenty-seven of the 140 specimens (19.3%) were positive in 16S rRNA gene. The positive rate of 16S rRNA gene of the specimens with prostatitis was 48.9%, significantly higher than that of the specimens without prostatitis (5.3%, P < 0.001). CONCLUSION: Bacteria may play an important role in the pathogenesis of chronic prostatitis.

[Application of HLAMatchmaker analysis eplets mismatch of renal transplant matching].

OBJECTIVE: Eplets mismatch based on HLAMatchmaker software evaluates the clinical application of kidney transplantation. METHODS: In 239 cases of renal transplant,merits of methods of the traditional HLA six antigen matcheing criteria, cross reaction groups standard and Eplets mismatch based on HLAMatchmaker standard were compared respectively. RESULTS: The number of mismatchs with three methods in 239 cases, were grouped according to low-high mismatchs. The results revealed that HLAMatchmaker algorithm could significantly increase the number of low mismatchs group 54 (22.6%), compared with the HIA group 19(7.9%) and CREGs group 32 (13.4%). The comparison was discovered statistical significance among the three groups (P<0.001), so the comparison between each group was. CONCLUSION: HLAMachmaker of donor-recipients matching, is a more efficient, time-saving and high sensitivity matching solution to allograft renal transplantation.

Simultaneous monitoring of CMV and BKV by quantitative PCR in renal transplant recipients.

Polyomavirus (BKV) and cytomegalovirus (CMV) are associated with renal graft failure. The aim was to establish a quantitative PCR method (Q-PCR) to detect BKV and CMV simultaneously. The conserved sequences of BKV and CMV were amplified and cloned into the plasmids as standards. The sensitivity, specificity and the precision of the assay were evaluated. Q-PCR was used to detect BKV and CMV DNA simultaneously in 480 blood samples of renal transplantation recipients. The sensitivity of the Q-PCR assay to detect BKV or CMV DNA reached 5×10(3)copies/mL. The use of control DNA verified that the assay could specifically detect the target DNA. The precision of the assay to quantify target DNA copies was acceptable (ICV 3.44% for BKV and 2.23% for CMV; differences between batches ICV 4.98% for BKV and 3.76% for CMV). In 480 samples, 130 samples (27.08%) were CMV DNA positive, which was significantly higher than the 64 BKV DNA positive samples (13.33%, p<0.05). BKV or CMV DNA positivity was significantly associated with high concentrations of Tacrolimus (TAC) (p value<0.05). The Q-PCR assay to detect both CMV and BKV DNA simultaneously was developed successfully with high sensitivity, precision, and time-effectiveness for clinical measurement.

The progression of the tubulointerstitial fibrosis driven by stress-induced "proliferation-death" vicious circle.

Several hypotheses have been developed to interpret the progression of tubulointerstitial fibrosis (TF), including senescence, epithelial-mesenchymal transition, inflammation, chronic hypoxia, and reactive oxygen species. All of these hypotheses are based on persistent cell injury and localized cell death. Proliferation of neighboring renal tubular epithelial cells (RTECs) is beneficial for organ function recovery from acute injury. However, compensatory proliferation is not always advantageous, as the proliferating cells are vulnerable to ongoing detrimental stimuli, such as inflammation, endocrine stress, high blood pressure, hypoxia/ischemia, and the like. Cell injury and death promotes secretion of growth factors, which evokes proliferation of RTECs; entering the cell cycle makes the RTECs more vulnerable to injury and death. Under persistent stress, death and proliferation are mutually promoted and form the vicious circle that triggers, maintains, and augments the inflammation and progression of TF. We hypothesize that the "proliferation-death" circle is another important pathophysiologic mechanism of TF onset. Through this hypothesis, this paper interprets the development and progression of TF. Moreover, the vicious circle may be universal, underlying the development of inflammation and fibrosis in various organs and tissues. The hypothesis also suggests a potential therapy strategy for the inhibition of fibrosis.

Timing, conditions, and complications of post-operative conception and pregnancy in female renal transplant recipients.

The aim of this study was to explore the timing, conditions, and complications of post-operative conception and pregnancy among female renal transplant recipients in China. A cohort of 25 female renal transplant recipients who subsequently had successful pregnancies was randomly selected from eight organ transplantation centers in China. In this cohort, there were 38 post-transplant conceptions and 25 live births. The effects of conception and pregnancy on renal function as well as any effects of transplantation on delivery, prematurity, and maternal and infant health were investigated. Out of 38 conceptions after transplantation, seven ended in spontaneous abortion, six in artificial abortion, and 25 in single births, seven of which were premature (28%). The growth and development of all of the infants were normal. All the 25 received artificial (formula) feeding. Six patients had to return to hemodialysis therapy at 1-41 months after conception due to reduced function of the transplanted kidney. It appears best for female renal transplant recipients to wait at least for 2 years post-transplant before pregnancy. We found no significant effect on fetal growth and development. The incidence of premature births among female renal transplant recipients was high which might have an effect on transplant renal function and maternal health. Breast feeding is not considered suitable for these patients and was therefore not studied.

[Correlation of HSP90 mRNA expression with migration ability of human multiple myeloma cells].

This study was aimed to investigate the correlation of heat shock protein 90 (HSP90) expression with migration ability of human multiple myeloma cells. The HSP90 mRNA expression and migration change of human multiple myeloma cell line (U266) were detected by RT-PCR and Transwell chamber respectively after treatment of U266 cells with final concentration 50, 100, 150, 200 nmol/L of bortezomib (proteosome inhibitor) for 4 hours. The results indicated that along with the increasing of bortezomib concentration, the expression level of HSP90alpha mRNA in U266 cells was enhanced, while no obvious increase of HSP90beta mRNA expression was observed in spite of statistical difference as a whole (p<0.05), but with the increasing of drug concentration in cells, their migration ability gradually decreased (p<0.05). It is concluded that the correlation of HSP90 expression with migration ability of human multiple myeloma cells exists.

[Effects of SDF-1/CXCR4 on the chemotaxis of cord blood AC133(+) cells].

The aim of this study was to explore the effects of the stromal cell-derived factor (SDF-1) and chemokine receptors (CXCR4) on chemotaxis of cord blood AC133(+) cells. The optimal SDF-1 concentration was determined in Transwell System. The cell migration was calculated from the number of cells passing through polycarbonate membrane with 8 microm pore. The expressions of CXCR4 in fresh and cultured cord blood AC133(+) cells were analyzed by flow cytometry with two-color direct immunofluorescence. The results showed that the chemotactic rate of fresh cord blood AC133(+) cells increased along with increasing concentrations of SDF-1, however, it tended to be stable when the concentration of SDF-1 reached 150 ng/ml. There was no difference in the chemotactic rate of cord blood AC133(+) cells between the group with SDF-1 adding CXCR4-blocking antibody and the group without SDF-1. When AC133(+) cells were cultured in vitro with hemopoietic growth factors, the expression of CXCR4 increased at the early stage, but decreased gradually along with time extending. In conclusion, there was correlation between the chemotactic rate of AC133(+) cells and the expression of chemokine receptor CXCR4.

[Mechanism of C-reactive protein on proliferation of multiple myeloma U266 cells].

This study was purposed to investigate the mechanism of C-reactive protein (CRP) on proliferation of U266 cells. The human multiple myeloma cell line U266 was incubated with human CRP (0, 5, 10, 20 mg/L) for 24 hours, then the proliferation level of U266 cells was detected by using blood analyser. The mRNA expressions of survivin and HSP90alpha were examined by RT-PCR. The results showed that the proliferation ratio was increased, as compared with the control group (p<0.05); furthermore, the mRNA levels of survivin and HSP90alpha were up-regulated in proportion to the increased CRP concentrations. There was significant correlation between expression of survivin and HSP90alpha (r=0.737, p<0.0001) in incubated cells. It is concluded that CRP can stimulate the proliferation of MM cells directly by up-regulating the expression of survivin and HSP90alpha in MM cells. CRP can be regarded as a potential target for MM treatment.

[Establishment and preliminary application of polymerase chain reaction with confronting two-pair primers for the single nucleotide polymorphisms of metabolic enzymes].

OBJECTIVE: To develop a simple, accurate, rapid, economic, large-scale detection method for the detection of single nucleotide polymorphisms (SNPs) metabolic enzymes, using polymerase chain reaction with confronting two-pair primers (PCR-CTPP). METHODS: The primers of CYP1A1 (A4889G), EPHX1 (A416G) and NQO1 (C609T) were designed for PCR-CTPP, and the PCR conditions were optimized. The results of genotyping were verified by DNA sequencing. The above SNPs were detected by the PCR-CTPP detection method in a randomly selected 183 healthy individuals of Han ethnicity. The genotype frequencies were analyzed and compared with people from other ethnicities. RESULTS: The allele-specific bands of CYP1A1 (A4889G), EPHX1 (A416G) and NQO1 (C609T) were successfully amplified by PCR-CTPP under the optimal conditions and the results of genotyping were consistent with DNA sequencing. Among 183 healthy Han individuals, the genotypic distributions of CYP1A1 (A4889G) , EPHX1 (A416G) and NQO1 (C609T) showed that the wild-type, homozygous variants, and heterozygotes were 103 (56.3%), 8 (4.4%), 72 (39.3%) and 142 (77.6%), 4 (2.2%), 37(20.2%), 60(32.8%), 32 (17.5%), 91 (49.7%) respectively. The distributions of genotypes were all in accordance with the Hardy-Weinberg equilibrium (P > 0.05), with statistical differences and with other ethnic populations (P < 0.05). CONCLUSION: The SNPs of metabolic enzymes can be detected by PCR-CTPP method which is simple, accurate, rapid, economic and with large scale. PCR-CTPP can be used for large scale clinical and epidemiological screening.