bluuebunny/crossref_metadata_2025 huggingface dataset:
STAT4 Polymorphism Is Associated with Early‐Onset Type 1 Diabetes, but Not with Late‐Onset Type 1 Diabetes
Authors: Lee Hye‐Soon , Park Hyewon , Yang Seiwon , Kim Dukhee , Park Yongsoo
Published: August 2009

In an effort to discover non‐HLA genes affecting susceptibility to type 1 diabetes (T1D), we have investigated the association of polymorphisms in STAT4, an important signaling molecule of IL‐12, γIFN, and IL‐23, in a sample of 389 T1D patients and 152 nondiabetic controls in Korea. Four SNPs on chromosome 2q, which were recently found to be associated with rheumatoid arthritis, were examined for association and linkage disequilibrium. We found that neither alleles or genotypes among all four SNPs nor reconstructed haplotypes of the three SNPs within the same LD block (rs7574865, rs8179673, and rs10181656) were associated with susceptibility to T1D. When we stratified T1D patients into early‐onset and late‐onset subgroups on the basis of fewer or more than 7.8 years of age at diagnosis, however, the minor alleles of three SNPs (rs7574865, rs8179673, and rs10181656) showed a significant association with susceptibility to T1D in the early‐onset subgroup (i.e., rs7574865, OR = 1.44 [1.03–2.01], P < 0.05), but not in the late‐onset subgroup, suggesting that STAT4 is related to earlier development of T1D. The analysis of genotypes and haplotypes of the same SNPs (rs7574865, rs8179673, and rs10181656) showed very comparable degrees of risk for T1D. The age at diagnosis is lowest in the patients carrying the homozygotes of a minor allele, middle in the heterozygotes, and highest in the homozygotes of a major allele, suggesting the dosage effects of risk alleles on the age of onset of disease. Recognizing that only the early‐onset cases might represent the true autoimmune T1D in Asian populations, we see that STAT4 alleles and haplotype might influence cytokine signaling and, therefore, development of T1D.
Preparation and Testing of Diabetes Autoantibody Controls
Authors: Brooking H. , Powell M.J. , Amoroso M. , Betterle C. , Pedini B. , Coco G. , Furmaniak J. , Smith B. Rees
Published: August 2009

Stocks of the WHO islet cell antibody, GAD65 antibody, and IA‐2 antibody standard (NIBSC 97/550) are now very limited. We have therefore made and tested a series of control preparations in which human monoclonal autoantibodies to IA‐2 and to GAD65 were diluted in antibody‐negative human serum to different concentrations. Three different diabetes autoantibody controls (DAC 1–3) were made as was a negative control preparation. Aliquots containing 1 mL of autoantibodies in serum were freeze‐dried. After reconstitution (with 1 mL of water) the controls were tested by 125I‐IA‐2 immunoprecipitation assay (IPA), 125I‐GAD65 IPA, GAD65 Ab ELISA and IA‐2 Ab ELISA (kits from RSR Ltd.) and for ICA by immunofluorescence test (IFT). DAC1 is particularly suitable as a control for the 125I IA‐2 IPA; DAC2 is suitable for the 125I‐GAD65 IPA, GAD65 Ab ELISA, and IA‐2 Ab ELISA; and DAC3 is suitable for the ICA IFT. Freeze‐dried preparations showed good stability at 37°C. Reconstituted liquid preparations were stable when stored at 4°C and at 37°C. Availability of an essentially unlimited supply of these reagents should be useful in establishing reproducible and comparable measurements of diabetes autoantibodies in different laboratories using different assays.
TGFβ Plasmid Construction and Delivery for the Prevention of Type 1 Diabetes
Authors: Park Leejin , Lee Eunjig , Lee Sangkyung , Lim Minsu , Hong Hekyung , Shin Geewook , Park Yongsoo
Published: August 2009

Studies of animals with spontaneous autoimmune diabetes have revealed that autoreactive T cells that mediate islet β cell destruction can be manipulated by the administration of Th2 cytokines. Using gene delivery to express the targeted protein, we can overcome the need for frequent administration of cytokines on account of their short half‐lives. In this study, the effect of hTGFβ gene delivery was evaluated both in vitro and in vivo using an adenovirus vector (Ad) constructed with an hTGFβ cDNA. In vitro transfection assays of the construct in HepG2, β cell lines, and islets showed good expression levels of hTGFβ and activation of smad3. Ad‐hTGFβ enhanced differentiation and proliferation in the β cell line or islets without causing apoptosis. Of interest, Ad‐hTGFβ transduction in CD4+CD25− T cells resulted in a significant enhanced expression of CD25 and a regulatory T cell–specific transcription factor, Foxp3. To evaluate in vivo efficacy, Ad‐hTGFβ was intravenously injected into 7‐week‐old NOD mice and compared to the transduction using the vector only. The Ad‐hTGFβ group had persistent gene expression for longer than 5 weeks, and high TGFβ serum level was secreted. There was no difference in the degree of insulitis between the Ad‐hTGFβ group and controls. Although we found favorable in vitro results, such as decrease in islet apoptosis, enhanced proliferation and differentiation, and increase in the level of CD4+CD25+ regulatory T cells, there was no difference in reduction of the development of T1D between controls and Ad‐hTGFβ‐injected mice. Nevertheless, if we find the appropriate mode and timing of TGFβ gene transduction, Ad‐hTGFβ gene therapy might be useful in therapeutic cytokine delivery for the treatment of T1D.
Latent Autoimmune Diabetes in an Adult
Authors: Maruyama Taro , Koyama Ayako , Hampe Christiane S.
Published: August 2009

We report a case of latent autoimmune diabetes in adults (LADA), also known as slowly progressive type 1 diabetes (SPT1D), followed up for changes, including reactivity to the GAD65 antibody epitope for the 9‐year period from impaired glucose tolerance (IGT) to the insulin‐dependent stage (IDDM). This 69‐year‐old male was identified as having IGT by health checkup in 1998. As he was GAD65‐positive (high titer), we initiated close clinical follow‐up. In 2003, a 75‐g oral glucose tolerance test showing a diabetic pattern confirmed that he had progressed to diabetes. During this period, fasting plasma glucose remained within normal range and insulin secretion was unchanged compared to that at the time of IGT diagnosis. His fasting plasma glucose and HbA1c levels began to increase in 2004 and serum C‐peptide began to decrease in 2005. Insulin treatment was started in August 2006. GAD65 antibody titer was high (13900 U/ml) in 1998 and has remained high throughout follow‐up. The patient's GAD65 antibodies were initially directed to the b96.11‐defined epitope only, recognized as an indicator of T1D‐like pathogenesis in our former study. During follow‐up, he developed reactivity to more epitopes (MICA‐3 and MICA‐4, DPC, and DPA). The course of this case suggests that the b96.11‐defined epitope is important for distinguishing LADA patients who progress to IDDM from those who do not and that epitope maturation is restricted to LADA patients who progress to IDDM, an observation similar to that in children at risk for developing typical IDDM.
Hedgehog Signaling during Expansion of Human Pancreatic Islet‐Derived Precursors
Authors: Gallo Rita , Grieco Fabio Arturo , Marselli Lorella , Ferretti Elisabetta , Gulino Alberto , Marchetti Piero , Dotta Francesco
Published: August 2009

A detailed understanding of the molecular process involved in the proliferation of pancreatic precursor cells would provide key elements for developing new therapeutic strategies to cure type 1 diabetes. In the present study we investigated the potential involvement of hedgehog signaling in proliferating human pancreatic islet‐derived mesenchymal (hPIDM) cells, a population of cells that can be successfully expanded and induced to differentiate into an insulin‐secreting phenotype. Here we report that in these precursor cells a hedgehog signaling pathway is activated, as shown by Gli1 expression, and that a dose‐dependent inhibition of such a pathway by cyclopamine results in a significant reduction of cell proliferation.
Tumor Necrosis Factor Alpha Inducing Spatial Interactions between Calcium‐Sensing Receptor and L‐Type Voltage‐Dependent Calcium Channel
Authors: Parkash Jai
Published: August 2009

The temporal and spatial regulation of intracellular calcium concentration ([Ca2+]i) is very crucial for maintaining calcium ion homeostasis within cells, and consequently in the regulation of cellular functions such as β cell replication and differentiation, insulin secretion, and apoptosis. Calcium ion regulatory proteins playing major roles in these processes include L‐type voltage‐dependent calcium channels (L‐type VDCCs) and calcium‐sensing receptors (CaRs). Tumor necrosis factor alpha (TNF‐α), a cytokine, is widely known to activate nuclear factor‐kappa B (NF‐κB) transcription in β cells. Confocal fluorescence imaging data suggest increased co‐localization of CaRs with L‐type VDCCs upon treatment of β cells with TNF‐α, thereby indicating increased membrane‐delimited spatial interactions between these two membrane proteins.
IFN‐α Can Both Protect against and Promote the Development of Type 1 Diabetes
Authors: Wong F. Susan , Wen Li
Published: August 2009

Viral infection has been hypothesized to be one of the environmental triggers for the development of type 1 diabetes. Infection induces a large amount of interferon‐α (IFN‐α) produced by dendritic cells and other cells. To test the role of IFN‐α in the development of diabetes, we have used three different experimental approaches in both diabetes‐prone and ‐resistant animal models for type 1 diabetes. Our results suggested that a viral mimic or IFN‐α can either suppress or promote the development of autoimmune diabetes, depending on the model system. It is likely that IFN‐α plays a complex role in the etiology of type 1 diabetes.
Anti‐CD20 Treatment Prolongs Syngeneic Islet Graft Survival and Delays the Onset of Recurrent Autoimmune Diabetes
Authors: Hu Changyun , Deng Songyan , Wong F. Susan , Wen Li
Published: August 2009

Type 1 diabetes is an autoimmune disease characterized by T cell–mediated destruction of pancreatic islet beta cells. Pancreatic islet transplantation with long‐term immunosuppressive drug treatment is an accepted therapeutic option for patients with type 1 diabetes suffering from disabling hypoglycemia on insulin treatment. Here we investigated the replacement of immunosuppressive drug treatment with immune tolerance establishment induced by temporary B cell–depletion therapy for islet transplantation. The result suggested that the combined therapy of B cell depletion and syngeneic islet transplantation may reverse the disease in hCD20/NOD mice.
Islet Glia, Neurons, and β Cells
Authors: Tsui Hubert , Winer Shawn , Chan Yin , Truong Dorothy , Tang Lan , Yantha Jason , Paltser Geoffrey , Dosch Hans‐Michael
Published: August 2009

Type 1 diabetes (T1D) is caused by autoimmune β cell destruction. The early events triggering T1D and the forces that keep diabetic autoimmunity pancreas specific have been unclear. Our discovery that autoimmune islet destruction is not β‐cell‐exclusive but includes cytotoxic T cell targeting of peri‐islet glia, evoked the possibility that T1D pathogenesis may involve neuronal elements beyond β cell/immune interactions. Recently, we have found that sensory afferent neurons are a critical component in prediabetes initiation, promoting islet inflammation through altered glucose homeostasis and progressive β cell stress. These factors orchestrate a catastrophic cascade culminating in insulin insufficiency mediated by an autoimmune‐prone host. This neuro‐immuno‐endocrinological triad explains diabetic inflammation as a consequence of local neuropeptide deficiency, leading to an innovative concept of disease pathogenesis with novel therapeutic implications.
Co‐transplantation of Stromal Cells Interferes with the Rejection of Allogeneic Islet Grafts
Authors: Jacobson Stella , Kumagai‐Braesch Makiko , Tibell Annika , Svensson Mattias , Flodström‐Tullberg Malin
Published: August 2009

Side effects associated with current immunosuppressive therapy complicate the use of islet transplantation as a treatment for type 1 diabetes. Multipotent mesenchymal stromal cells (MSCs) have demonstrated immunomodulatory activity. The purpose of this study was to investigate whether a murine stromal cell line affects graft rejection in a fully major histocompatibility complex (MHC)‐mismatched islet transplant model. We show that stromal cells have an inhibitory effect on T cell proliferation in vitro, and that they slow down the rejection of allogeneic islets. These findings indicate a possibility to use MSCs as a treatment to prolong the survival of islet grafts.
Tracking Down Immune Markers from Alternative System Pathway Factors in a Diabetic Population
Authors: Chaudhry Bushra , Islam Najamul , Saboohi Kausar , Ahsen Nazema
Published: August 2009

Hyperglycemia associated with type 1 diabetes (T1D) alters the host immune system, resulting in a predisposition to infectious diseases. The high risk of infection in the diabetic population may lead to life‐threatening situations. The early proteins of the alternative complement system pathway, constituting factors P, B, and D, have been shown to play an important role in preventing infection because they form a membrane attack complex (MAC)‐C5‐9, which debilitates the target microbes and/or molecules via cytotoxic and cytolytic reactions. Patients who are devoid of or contain low levels of these proteins may be susceptible to developing chronic infections. We have observed striking differences in partially fractionated serum proteins in diabetic patients (type 2) relative to controls, through single and two‐dimensional gel electrophoresis. Our data, obtained from 50 diabetic patients in the age group of 25–45 years, who had the disease for fewer than 5 years, indicated patterns in low‐ and high‐molecular‐weight proteins, which could be grouped into five different categories with minor differences in their respective levels of protein expression. Immunoblot assay could barely detect the presence of properdin expression in diabetic patients. Quantization by ELISA in 99 patients indicated low levels of properdin expression in 70% of 50 diabetic patients (6.5 ± 3 μg/mL) when compared to nondiabetic controls (19.5 ± 8.5 μg/mL). This study concluded that patients with low expression of properdin should be advised to take extensive preventive measures and seek early management with appropriate treatments against infection.
Exendin‐4 Therapy in NOD Mice with New‐Onset Diabetes Increases Regulatory T Cell Frequency
Authors: Xue Song , Wasserfall Clive H. , Parker Matthew , Brusko Todd M. , McGrail Sean , McGrail Kieran , Moore Marcus , Campbell‐Thompson Martha , Schatz Desmond A. , Atkinson Mark A. , Haller Michael J.
Published: August 2009

Recent studies, albeit controversial, have suggested that the incretin exendin‐4 (Ex‐4) is capable of inducing β cell proliferation in vivo. Furthermore, this compound has been shown to enhance the ability of other agents (e.g., anti‐CD3, antilymphocyte serum) to reverse type 1 diabetes (T1D) in NOD mice. However, the mechanisms underlying this beneficial action for disease reversal remain largely unclear. Herein, we tested the hypothesis that Ex‐4 therapy may act as a stimulator of regulatory T cells (Tregs). We evaluated the effect of Ex‐4 (Byetta®; 0.2 μg/mouse/day for 30 days) treatment on the frequency and function of Tregs and changes in the cytokine profile of NOD mice with recently diagnosed T1D. In comparison to that of saline‐treated control NOD mice, the frequency of Tregs was increased in Ex‐4‐treated mice. Suppression assays demonstrated a trend towards increased Treg suppression after administration of Ex‐4, but were limited by small sample size. Lastly, Ex‐4 treatment induced production of IL‐10, indicating a possible shift towards a more Th2‐like phenotype. Taken collectively, these data suggest that in addition to its potential effects on β cell proliferation, Ex‐4 may also act as a regulator of the immune response.
Independent Association of Tumor Necrosis Factor Polymorphism with Type 1 Diabetes Susceptibility
Authors: Shin Hyoung Doo , Yang Sei Won , Kim Duk Hee , Park Yongsoo
Published: August 2009

The contribution of SNPs in TNF genes to type 1 diabetes (T1D) is not well established and may be confounded by the linkage disequilibrium within the HLA genes. Seven SNPs in the TNF genes (TNFA and TNFB) were genotyped in a Korean cohort (398 T1D patients and 1422 nondiabetic controls), along with HLA DRB1, DQB1, and MICA (MHC class I chain‐related genes). Among them, three SNPs (TNFB+318, TNFA‐857, and TNFA‐308) and two common TNF haplotypes showed significant association with the risk of T1D (P= 5 × 10−3–10−5). T1D patients were more often heterozygous for the alleles at the TNFB+318 (OR = 1.7, P= 10−3) and TNFA‐308 (OR = 1.7, P < 10−5) than were the controls. Genetic association analyses of the DRB1, DQB1, and MICA alleles with the risk of T1D revealed dramatic associations in several alleles as expected. Independent analyses to discern the genetic effects of TNF polymorphisms on the risk of T1D suggested that these genetic influences might be not totally dependent on the nearby HLA genes. Our results support the hypothesis that two susceptibility loci in the MHC (one in the HLA class II and another in the central MHC region) act epistatically to increase susceptibility to T1D.
Molecular Scanning of the Gene for Thioredoxin, an Antioxidative and Antiapoptotic Protein, and Genetic Susceptibility to Type 1 Diabetes
Authors: Ikegami Hiroshi , Ono Masaya , Fujisawa Tomomi , Hiromine Yoshihisa , Kawabata Yumiko , Yamato Eiji
Published: August 2009

Oxidative stress has been implicated in the destruction of β cells in type 1 diabetes (T1D). Thioredoxin has been shown to protect cells from oxidative stress and apoptosis. In this study, we screened for sequence variants of the human thioredoxin gene (TXN), and studied the association of the variants in persons with T1D in Japanese. The frequency of the A allele of the G/A SNP in the 3′ flanking region was highest in T1D (8.4%), followed by type 2 diabetes (6.8%), and the lowest in the controls (5.9%), suggesting the contribution of TXN polymorphism to susceptibility to T1D.
Gene Expression Profiles Stratified according to Type 1 Diabetes Mellitus Susceptibility Regions
Authors: Rassi Diane Meyre , Junta Cristina Moraes , Fachin Ana Lúcia , Sandrin‐Garcia Paula , Mello Stephano , Silva Guilherme Liberato , Evangelista Adriane Feijó , Magalhães Danielle Aparecida , Wastowski Isabela Jubé , Crispim Janaína Oliveira , Martelli‐Palomino Gustavo , Fernandes Ana Paula Mores , Deghaide Neife N.H.S. , Foss‐Freitas Maria Cristina , Foss Milton César , Soares Christiane Pienna , Sakamoto‐Hojo Elza Tieme , Passos Geraldo Aleixo Silva , Donadi Eduardo Antônio
Published: August 2009

The MHC region (6p21) aggregates the major genes that contribute to susceptibility to type 1 diabetes (T1D). Three additional relevant susceptibility regions mapped on chromosomes 1p13 (PTPN22), 2q33 (CTLA‐4), and 11p15 (insulin) have also been described by linkage studies. To evaluate the contribution of these susceptibility regions and the chromosomes that house these regions, we performed a large‐scale differential gene expression on lymphomononuclear cells of recently diagnosed T1D patients, pinpointing relevant modulated genes clustered in these regions and their respective chromosomes. A total of 4608 cDNAs from the IMAGE library were spotted onto glass slides using robotic technology. Statistical analysis was carried out using the SAM program, and data regarding gene location and biological function were obtained at the SOURCE, NCBI, and FATIGO programs. Three induced genes were observed spanning around the MHC region (6p21–6p23), and seven modulated genes (5 repressed and 2 repressed) were seen spanning around the 6q21–24 region. Additional modulated genes were observed in and around the 1p13, 2q33, and 11p15 regions. Overall, modulated genes in these regions were primarily associated with cellular metabolism, transcription factors and signaling transduction. The differential gene expression characterization may identify new genes potentially involved with diabetes pathogenesis.
Effect of Hypoxia on Circulating Levels of Retina‐Specific Messenger RNA in Type 2 Diabetes Mellitus
Authors: Wong Amy , Merritt Simon , Butt Asif N. , Williams Adrian , Swaminathan R.
Published: August 2009

Previously it was shown that the circulating rhodopsin mRNA level was higher in diabetic retinopathy (DR). Recent evidence suggests that hypoxia may also be associated with DR. The aim of this study was to investigate the effect of oxygen desaturation on circulating retina‐specific mRNA in type 2 diabetic patients. Thirty‐five type 2 diabetic patients underwent overnight oximetry. Two parameters from oximetry were used to measure oxygen desaturation: the number of times per hour the oxygen saturation decreased by 4% or greater (number of dips/hr) and percentage of sleep time with oxygen saturation (SpO2) <90%. Blood samples were collected into PAXgene Blood RNA tubes. Total RNA was extracted from the samples and reverse‐transcribed into cDNA, and retina‐specific markers were measured by quantitative real time PCR. In patients with ≥5 dips/hr, mRNA values for rhodopsin (P= 0.05) and RPE65 (P= 0.044) were significantly higher than in patients with <5 dips/hr. No change was seen in retinoschisin mRNA expression. In patients with preproliferative or proliferative DR, median levels for rhodopsin mRNA and RPE65 mRNA were 30% and 80% higher and retinoschisin mRNA was lower in patients with ≥5 dips/hr when compared to patients with <5 dips/hr. These results indicate that hypoxia may modulate expression of genes in the retina.
Fetal Nucleic Acids in Maternal Plasma
Authors: Lo Y.M. Dennis
Published: August 2009

The discovery of cell‐free fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Over the last few years, a number of approaches have been demonstrated to allow such circulating fetal nucleic acids to be used for the prenatal detection of chromosomal aneuploidies. One such approach involves the enrichment of fetal DNA, such as by size fractionation or by the controversial formaldehyde treatment technique. A second approach involves the targeting of fetal‐specific nucleic acid molecules, including fetal‐specific epigenetic markers and placenta‐specific mRNA markers. A third approach involves the development of highly discriminatory quantitative methods for chromosome dosage analysis using digital polymerase chain reaction technology. It is likely that these and other methods yet to be developed would allow noninvasive prenatal diagnosis of chromosomal aneuploidies by maternal plasma nucleic acids to be realized in the near future.
Cell‐Free Plasma DNA during Peritoneal Dialysis and Hemodialysis and in Patients with Chronic Kidney Disease
Authors: Korabecna Marie , Opatrna Sylvie , Wirth Jan , Rulcova Kamila , Eiselt Jaromir , Sefrna Frantisek , Horinek Ales
Published: August 2009

The mechanisms of clearance of circulating plasma DNA are not fully understood, and so we aimed to examine it in patients with impaired renal function compared with healthy individuals. We also assessed the effect of peritoneal dialysis and hemodialysis on circulating plasma cell‐free DNA (cfDNA) in our treated patients. Overall, 20 healthy volunteers, 20 patients with chronic kidney disease (CKD), 18 patients undergoing peritoneal dialysis (PD), and 17 patients on hemodialysis (HD; high‐flux polysulfone membrane) were examined. Cell‐free DNA levels were determined using real‐time GADPH gene sequence amplification. The levels of cfDNA in all groups of our patients did not differ significantly from those of healthy volunteers. In HD patients, cfDNA levels were significantly increased compared with those of CKD patients (P < 0.05) and PD‐treated patients (P < 0.01). In PD‐treated patients, cfDNA was detectable in overnight effluent, with its levels correlating inversely with the duration of PD treatment (r=−0.619, Spearman's coefficient, P= 0.008). Factors contributing to these differences may include changes in the quality and quantity of the cell population of the peritoneum, highlighting the need for additional studies clarifying the dynamics of cfDNA during PD. The plasma levels of cfDNA do not seem to be dramatically altered even in CKD patients or those on PD or HD (as long as they are measured prior to the procedure in the latter two). Our data suggest renal elimination is not the main mechanism of circulating cfDNA clearance.
Transrenal Nucleic Acids: From Proof of Principle to Clinical Tests
Authors: Melkonyan Hovsep S. , Feaver W. John , Meyer Erik , Scheinker Vladimir , Shekhtman Eugene M. , Xin Zhenghan , Umansky Samuil R.
Published: August 2009

In spite of numerous publications on potential diagnostic application of circulating DNA and transrenal nucleic acid (Tr‐NA) analysis, few, if any, tests based on this technology are available in clinical labs. This delay in test development and implementation is caused, at least in part, by the deficit in robust methods for isolation of short nucleic acid fragments from bodily fluids, as well as in techniques for analyzing these fragments. We have developed a new anion exchanger‐based method for the isolation of cell‐free nucleic acid fragments from large volumes of bodily fluids, and analyzed these fragments by PCR techniques specially designed to amplify “ultrashort” templates. The combination of these two techniques not only revealed the presence in urine of 10–150 bases or bp DNA and RNA fragments in addition to previously observed 150–200‐bp DNA fragments and high molecular weight DNA, but also significantly increased the sensitivity of Tr‐DNA detection. Additionally, we detected in urine a variety of miRNAs, including those excreted transrenally, thereby opening new diagnostic possibilities for Tr‐NA analysis.
Removal of High‐Molecular‐Weight DNA by Carboxylated Magnetic Beads Enhances the Detection of Mutated K‐ras DNA in Urine
Authors: Su Ying‐Hsiu , Song Janet , Wang Zhili , Wang Xiao‐He , Wang Mengjun , Brenner Dean E. , Block Timothy M.
Published: August 2009

We have previously demonstrated that mutated DNA derived from the circulation can be detected in urine and predominantly exists as DNA fragments <1 kb. To preferentially isolate the trans‐renal DNA from urine, we developed a method using carboxylated beads to separate high‐MW (1 kb or larger) from low‐MW DNA in urine. A primer set for 18s rRNA (generating a PCR product of 872 bp) was designed and optimized for real‐time PCR quantification of high‐MW DNA templates. To evaluate the method, urine samples from 5 volunteers with no known diseases and 36 patients with various colorectal diseases were collected and tested. It was found that the average removal efficiency of high‐MW DNA from total urine DNA using carboxylated beads is 92.72%± 1.42%. Furthermore, compared with using total urine DNA, our method provides a greater ability to detect mutated K‐ras in the urine of colorectal cancer patients. The concurrence of K‐ras mutations detected in disease tissue and the corresponding urine specimen is significantly higher (P= 0.0015) when the samples were enriched in low‐MW DNA.
Circulating DNA
Authors: Van Der Vaart Maniesh , Pretorius Piet J.
Published: August 2009

Circulating DNA is present in the blood of all individuals, but it has been found that cancer patients and patients with a variety of other conditions have increased amounts of these circulating DNA fragments in their blood. Even though more than 30 years of research have been done on this subject, the origin of these nucleic acid molecules is still not clear. We present evidence that does not support the general notion that apoptosis or necrosis is the major source of circulating free DNA. Active release of free circulating DNA by living cells may be a plausible mechanism. Disturbance of the equilibrium between the release of DNA by living cells and the mechanisms used for clearing this DNA may play the main role in the appearance of increased amounts of circulating DNA in the blood of individuals with different ailments. Elucidating the origin and the mechanism that cells use to release free circulating DNA into the blood may enhance the diagnostic and prognostic value of these nucleic acid molecules.
The Effect of Protein Transport Inhibitors on the Production of Extracellular DNA
Authors: Morozkin Evgeniy S. , Babochkina Tatyana I. , Vlassov Valentin V. , Laktionov Pavel P.
Published: August 2009

The presence of various genomic sequences in the pool of extracellular DNA was studied by cloning and sequencing the DNA eluted from the surface of HeLa cells. Sequences of 19 genes, 10 pseudogenes, and 41 repeated elements were found in 103 clones. Sequences of LINE repeats were found in 17% of the clones; consequently real‐time PCR assay for quantification of LINE repeats was chosen to study the influence of protein transport inhibitors on the concentration of free and cell‐surface‐bound DNA in the cultures of HUVEC and HeLa cells. Treatment of both cell lines with the inhibitors did not interfere with the concentration of extracellular DNA in the growth medium, except for chloroquine, which doubled the concentration of extracellular DNA in HUVEC culture. The treatment of HUVECs with monensin, glyburide, or methylamine decreases the cell‐surface‐bound DNA concentration by 30, 35, and 19%, respectively. The incubation of HeLa cells with monensin reduces the concentration of cell‐surface‐bound DNA by 15%; however, the treatment with glyburide increases cell‐surface‐bound DNA concentration by 50%. The data obtained demonstrate the involvement of vesicular transport in generation of extracellular DNA.
Hepatitis B Virus X‐DNA
Authors: Zhang Wei , Li Yan‐Hong , Zhu Shao‐Jun , Zhang Yan , Gong Li , Wang Shu‐Mei , Hacker Hans Jörg , Schröder Claus Hobe , Cheng Dong‐Sheng , Feng Ying‐Ming
Published: August 2009

HBV genome replication intermediates blocked at early stages of minus strand synthesis have been identified in a study on circulating DNA and RNA during short‐term lamivudine therapy. This suggested that the inhibition of HBV replication processes in the liver are mirrored in the blood. Levels of circulating HBV mRNA remained largely unaffected. Here we followed therapy with two patients (patients 1 and 2) up to stages without apparent replication. As in the earlier study, DNA segments produced successively during replication were used as targets for quantitative PCR: X (early minus strand), C (completed minus strand), and preC (nascent plus strand). Corresponding RNA was quantified by RT/PCR. Polyadenylated viral RNA were assayed as full‐length (f) and as truncated (tr) RNAs. Blocked X‐region intermediates persisted for about one year. After a period of undetectable HBV DNA viral replication resumed in patient 1 because of the emergence of drug‐resistant mutants and in patient 2 because of the discontinuation of therapy. In the former case, X‐region intermediates reappeared first, then C‐ and, finally, preC‐region intermediates. Stopping therapy, in contrast, led to a simultaneous reappearance of all three types of intermediates. At low replication levels or its absence, trRNA represented the only polyadenylated viral RNA. Apparently, HBV serum nucleic acid markers allow a study of replication and transcription separately. Specifically, it is concluded (1) that PCR assays for monitoring lamivudine therapy must target the X‐gene region and (2) that in the absence of HBV replication, trRNA may constitute a serum marker for HBV expression.
The Housekeeping Gene (GA3PDH) and the Long Interspersed Nuclear Element (LINE) in the Blood and Organs of Rats Treated with Cocaine
Authors: Voskresenskiy Anatoliy M. , Sun Lena S.
Published: August 2009

Housekeeping genes are necessary for maintenance of the vital activity of the cells of any phylum of organisms. Transposons or mobile genetic elements are eurysynusic in nature. Thus, the role of these and other genes in the pathogenesis of many diseases and of drug addiction in particular is being investigated. The goal of the work is to determine the influence of cocaine on the activity of GA3PDH and on a representative of the LINE family (L1Rn) in plasma, and in a pellet of blood cells, and in the organs of rats. Gene expression was evaluated by RT‐PCR. The GA3PDH (452‐bp fragment) was predictably found in plasma, in a pellet of blood cells, and in organs. Its quantity in plasma was greater in the experimental groups than in the control. In a pellet of blood cells and in organs, the GA3PDH activity between the different groups of animals essentially did not differ. The L1Rn fragment (319 bp) in plasma was not found. The expression of L1Rn was much higher in a pellet of blood cells and in organs of experimental animals. These experiments have shown the presence of GA3PDH in the plasma of the controls and an increase in quantity in the plasma of experimental animals. The activation of LINE in a pellet of blood cells of rats and in organs under the influence of cocaine has been demonstrated. Apparently, a recruitment phenomenon of housekeeping genes and transposons is possible in the pathogenesis of drug addiction.
Circulating Nucleic Acids in Plasma and Serum
Authors: Gahan Peter B. , Swaminathan R.
Published: August 2009

DNA and RNA fractions have been isolated from the whole blood, serum, plasma, the surface of blood cells, and urine of both healthy individuals and patients. The ability to isolate, quantify, and analyze these molecules has led to the identification of specific nucleic acid fragments related to particular disorders such as diabetes, cancer, myocardial infarction, and stroke, threby permitting their early diagnosis. Currently, a number of methods for isolating the nucleic acids are employed and although a start has been made to compare the efficiencies of these methods, there is still a way to go before there are precise protocols for nucleic acid extraction. The older chemical methods of extraction still outperform some of the available kits. Some progress is being made to determine the origin of the circulating nucleic acids, although there are still many questions to be answered, including whether the source is through the spontaneous release of newly synthesized nucleic acid or whether it just derived from necrotic and apoptotic cells. In addition, it can be demonstrated that the nucleic acids can enter cells and exhibit a biological activity in the recipient cells. Hence, the question remains: Are the circulating nucleic acids freely entering tissues and cells from the blood and inducing changes in those tissues and cells? Further work is needed to elucidate these areas, and the various protocols must be standardized if the new methodology is to be widely and accurately applied in the diagnosis of disease and the monitoring of therapy. This chapter summarizes the work reported in this volume.
Asthma and Infectious Respiratory Disease in Relation to Residence near Hazardous Waste Sites
Authors: Carpenter David O. , Ma Jing , Lessner Lawrence
Published: August 2009

The hypothesis that simply living near a hazardous waste site increases risk of exposure to chemicals was tested. Using data from the New York Statewide Planning and Research Cooperative System, which provides information on hospitalized patients, plus information on the location and contents of every known hazardous waste site in New York, the rates of hospitalization for asthma (ICD‐9 493), infectious respiratory disease (ICD‐9 460–466, 480–487, and 490–491), and Chronic obstructive pulmonary disease (COPD) (ICD‐9 490–492 and 494–496) were determined among individuals who lived in (a) zip codes containing or abutting a hazardous waste site with persistent organic pollutants (POPs), (b) zip codes containing or abutting a hazardous waste site, but not one with POPs, and (c) zip codes that do not contain or abut an identified hazardous waste site. After adjustment for MHI, race, gender and urban or rural residence, there was a significantly elevated risk of asthma (rate ratio (RR) = 1.09), infectious respiratory disease (RR = 1.15), and COPD (RR = 1.19) in individuals living in a zip code with a POP waste site, and a significantly elevated risk of asthma (RR = 1.09), infectious respiratory disease (RR = 1.12), and COPD (RR = 1.13) associated with residence in a zip code containing a waste site, but not one with POPs, both relative to residence in a zip code without a waste site. These observations are consistent with the hypothesis that simply living near a hazardous waste site increases risk of exposure to substances that contribute to respiratory disease.
Electrochemical Heterogeneous Catalytic Degradation of Wastewater Containing Phenol
Authors: Jia Baojun , Zhang Dong , Zhang Ai L. , Zhou Ji T.
Published: August 2009

An electrochemical–heterogeneous catalytic process was applied to remove phenol from wastewater. Three key factors—applied potential, supporting electrolyte concentration, and pH—were tested for their effects on the phenol removal. The optimal condition was determined as an applied potential of 25 V, Na2SO4 concentration of 500 mg L−1, and a pH of 2.5. Under the optimal condition, the phenol and chemical oxygen demand (COD) removals were 83.7% and 42.9%, respectively. The apparent kinetics of phenol removal could be expressed as a pseudo first‐order reaction. In addition, a possible mechanism of phenol degradation was proposed based on the intermediate products.
Photochemical Oxidation of Thiophene by O2 in an Oil/Acetonitrile Two‐Phase Extraction System
Authors: Li Fa‐tang , Zhao Di‐Shun , Li Hong‐Xia , Liu Rui‐Hong
Published: August 2009

Photochemical oxidation of thiophene in an n‐octane/acetonitrile extraction system using O2 as oxidant was studied. Results obtained here can be used as a reference for desulfurization of gasoline, because thiophene is one of the main components containing sulfur in fluid catalytic cracking gasoline. A 500‐W high‐pressure mercury lamp was used as a light source for irradiation, and air was introduced by a gas pump to supply O2. Thiophene dissolved in nopolar n‐octane solvent was photodecomposed and removed into the polar acetonitrile phase. The desulfurization rate of thiophene in n‐octane was 65.2% under photoirradiation for 5 h under the conditions of air flow at 150 mL min−1, and V(n‐octane):V(acetonitrile) = 1:1. This can be improved to 96.5% by adding 0.15 g Na‐ZSM‐5 zeolite into the 100‐mL reaction system, which is the absorbent for O2 and thiophene. Under such conditions, the photooxidation kinetics of thiophene with O2 and Na‐ZSM‐5 zeolite is first‐order with an apparent rate constant of 0.6297 h−1 and half‐time of 1.10 h. The sulfur content can be reduced from 800 μL L−1 to 28 μL L−1.
The Role of Calcium Ions in the Photocatalytic Oxidation of Humic Acid at Neutral pH
Authors: Mariquit Eden G. , Salim Chris , Hinode Hirofumi
Published: August 2009

Humic acids (HAs) are natural organic matter derived from the decomposition of plant, algal, and microbial materials. They belong to the group of the most predominant type of natural organic matter present in ground and surface waters. HAs affect the mobility and bioavailability of aquatic contaminants. However, if they are left unremoved from the water before water treatment processes, they can form carcinogenic disinfection by‐products, such as trihalomethanes, haloacetic acids, and other halogenated disinfection by‐products, that can pose a threat to human beings. An advanced oxidation process using UV light and a commercially available titanium dioxide was used to oxidize HA at a pH that is similar to that of natural water. The effect of adding calcium ions to the adsorption and the photocatalytic oxidation of HAs was studied. The effect of varying the TiO2 load was also investigated. The experiment was done using a photochemical batch reactor equipped with a mercury lamp emitting light with wavelengths of 310–580 nm. The absorbances by the samples were determined at wavelengths of 254 nm and 436 nm, which represent the aromatic‐compound content of and the color of the solution, respectively. Results indicated calcium ions have an effect on both the adsorption and the photocatalytic oxidation of HA at a pH within 8.0 ± 0.5. Calcium ions facilitated adsorption of HA onto the surface of TiO2 and resulted to faster photocatalytic oxidation. The data were plotted with respect to the normalized absorbances and irradiation time.
POPs Hot Spot
Authors: Khwaja Mahmood A.
Published: August 2009

Like other chemicals of the persistent organic pollutant (POP) group, dichlorodiphenyltrichloroethane (DDT), a pesticide, is also persistent in nature and does not readily degrade in the environment by biological, physical, or chemical processes. In view of the known toxicity, accumulative, persistent characteristics, and adverse environmental and health impacts of DDT, an extensive survey of the soil was undertaken in and around a demolished DDT factory at Nowshera, NWFP, to examine DDT levels of the contaminated soil. For the present study, 81 soil samples were collected within a half‐kilometer distance from the old gate of the factory and at different depths in eight different directions. Analytical data indicated that 90.91% of the soil samples studied were contaminated with DDT, with 66.6% of the samples indicating residual DDT levels higher than DDT minimum risk level (MRL) in soil (0.05 μg/g). Soil in the southeast direction appeared to be the most contaminated (average 6.70 ± 1.25 μg/g), showing 5.19 μg/g residual DDT in a soil sample collected as far away as 520 m from the factory. Soil in the south direction also appeared highly contaminated, with an average DDT residual level of 7.16 ± 1.70 μg/g between 65 and 390 m from the factory. For soil between the surface and a depth of 0.60 m, the highest residual DDT level (5.78 ± 3.94 μg/g) was observed in samples from the northwest direction, followed by samples from the west direction (4.88 ± 3.80 μg/g).
Analysis and Removal of Organic Pollutants in Biologically Treated Landfill Leachate by an Inorganic Flocculent Composite of Al(III)‐Mg(II)
Authors: Sang Yimin , Gu Qingbao , Sun Tichang , Li Fasheng , Pan Yiting
Published: August 2009

A novel inorganic flocculent composite of Al(III)‐Mg(II) poly‐magnesium‐aluminum‐sulfate (PMAS) is used to remove organic matter from biologically treated leachate in some landfills in Beijing, China. Jar‐test experiments are employed to determine the optimum conditions for the removal of organic matter, which is represented as UV254. Under optimum conditions, the removal of COD, BOD5, and color is also determined. Moreover, gas chromatography coupled with mass spectrometry (GC–MS) is used to analyze the organic matter in the biologically treated leachate before and after treatment by the coagulant. The experimental results indicate that the removal of COD, BOD5, UV254, and color by coagulation with PMAS can reach above 65%, 60%, 85%, and 85%, respectively, under optimal conditions. This greatly weakens its pollution extent and improves its visual appeal. Forty‐one kinds of organic pollutants in the biologically treated leachate were determined. Some of them belong to the Black List of water environmental preferred controlled pollutants, as judged by the United States and China. The species of alkyl, alkene, acyclic alcohol, and acyclic acyl amines are about 85% removed, some of them are removed completely, while the species of acids, esters, and ketones are removed at about 65%. Those contaminants with benzene rings, such as aromatic hydrocarbons, hydroxybenzene, aromatic alcohol, and aromatic acyl amine, are about 50% partially removed.
Solid‐Waste Management Practices of Households in Manila, Philippines
Authors: Bernardo Eileen C.
Published: August 2009

The experiences and practices of household waste management of people in a barangay (village) in Manila, Philippines are documented. The data were gathered through an interview with household members using open‐ended questions. Interviews were also conducted with garbage collectors as well as scavengers. Results showed that the households generated an average of 3.2 kg of solid waste per day, or 0.50 kg/capita/day. The types of wastes commonly generated are food/kitchen wastes, papers, PET bottles, metals, and cans, boxes/cartons, glass bottles, cellophane/plastics, and yard/garden wastes. The respondents segregate their wastes into PET bottles, glass bottles, and other waste (mixed wastes). No respondents perform composting. It is worth noting, however, that burning of waste is not done by the respondents. The households rely on garbage collection by the government. Collection is done twice daily, except Sundays, and household members bring their garbage when the garbage truck arrives. However, there are those who dump their garbage in nondesignated pick‐up points, usually in a corner of the street. The dumped garbage becomes a breeding ground for disease‐causing organisms. Some household respondents said that it is possible that the dumping in certain areas caused the dengue fever suffered by some of their family members. Mothers and household helpers are responsible for household waste management. Scavengers generally look for recyclable items in the dumped garbage. All of them said that it is their only source of income, which is generally not enough for their meals. They are also aware that their work affects their health. Most of the respondents said that garbage collection and disposal is the responsibility of the government. The results of the study showed that RA 9003, also known as the Ecological Solid Waste Management Act of 2000, is not fully implemented in Metro Manila.
Susceptibility of Children to Environmental Pollutants
Authors: Sly Peter D. , Flack Felicity
Published: August 2009

Children are more vulnerable to adverse environmental exposures. The unique ways in which they interact with their environment and their dynamic developmental physiology mean that they generally receive a higher dose of toxicant for a given level of environmental exposure. In addition, children are frequently more likely to suffer adverse health outcomes from exposures. The developmental stage of the child during which the exposure occurs has a major influence on the consequences of the exposure. For example, exposures during organogenesis may result in permanent structural changes, whereas exposures once organogenesis is complete are more likely to result in functional consequences. The immune, respiratory, and central nervous systems are immature at birth and have a prolonged period of postnatal maturation. Thus, these organ systems are vulnerable to postnatal exposures.
Leaves of Orange Jasmine (Murraya paniculata) as Indicators of Airborne Heavy Metal in Bangkok, Thailand
Authors: Titseesang Teerawet , Wood Timothy , Panich Noppaporn
Published: August 2009

Orange jasmine (Murraya paniculata) leaves were collected from three different sites in the area of Bangkok and in a remote area as a control site. The leaf samples were digested and the concentrations of Cu, Fe, Pb, Mn, Ni, Cr, and Zn were then quantified by using inductively coupled plasma–atomic emission spectrometry (ICP–AES). All three Bangkok sites were polluted with heavy metals compared with the background site. Fe was found as the highest mean concentration of the metals studied, while Pb was the lowest. There was a high correlation coefficient between metals Cu‐Mn, Cu‐Zn, Cu‐Pb, and Mn‐Zn. However, Fe was not correlated with other metals. There was no significant difference in Pb between sites. The significant difference in other metals found in the study could be attributed to different anthropogenic activities between sites. The principal‐component analysis (PCA) identified two factors according to the sources of metals making up the anthropogenic (traffic) and natural (soil) sources. Traffic emission was found to be the main source of metal pollution in the atmosphere of Bangkok.