Prognostic factors for idiopathic pulmonary fibrosis: clinical, physiologic, pathologic, and molecular aspects.

BACKGROUND: Previous studies identified clinical and physiologic factors of idiopathic pulmonary fibrosis (IPF) that are related to an increased risk of mortality. But there are few studies about histologic and molecular approach. OBJECTIVE: We investigated whether the C-reactive protein (CRP), fibroblastic foci, phosphorylated Smad2/3 (p-Smad2/3), tumor growth factor-beta (TGF-beta), TGF-beta receptor II (TbetaRII), and the polymorphism of the TGF-beta1 codon 10 are associated with the progression of IPF patients. DESIGN: Eighty-six IPF patients who underwent surgical lung biopsies were examined. For each patient, clinical and physiologic parameters were investigated, and we performed immunohistochemical staining for p-Smad2/3 and TbetaRII, and genotyping of the TGF-beta1 codon 10 polymorphism. RESULTS: Age at diagnosis, gender, symptom duration, and smoking status did not show a significant association. However, the amount of smoking (p = 0.002), severe reduction in the percentages of predicted forced vital capacity (p = 0.013) and diffusion lung capacity of carbon monoxide (p = 0.023), CRP (p = 0.009) at diagnosis, and fibroblastic foci (p = 0.026) were associated with a poor prognosis. Cellularity, fibrosis, expression level of p-Smad2/3 and TbetaRII, and genotype of the TGF-beta1 codon 10 polymorphism did not have a statistically significant association with the prognosis. CONCLUSION: This study confirmed the amount of smoking, abrupt decrease in follow-up pulmonary function parameters, fibroblastic foci, and increased levels of CRP concentration at diagnosis were significantly associated with poor survival. Larger studies are required to confirm all prognostic factors including CRP.

Anxiety and fear in a cross of C57BL/6J and DBA/2J mice: mapping overlapping and independent QTL for related traits.

Anxiety, like other psychiatric disorders, is a complex neurobehavioral trait, making identification of causal genes difficult. In this study, we examined anxiety-like behavior and fear conditioning (FC) in an F(2) intercross of C57BL/6J and DBA/2J mice. We identified numerous quantitative trait loci (QTL) influencing anxiety-like behavior in both open field (OF) and FC tests. Many of these QTL were mapped back to the same chromosomal regions, regardless of behavior or test. For example, highly significant overlapping QTL on chromosome 1 were found in all FC measures as well as in center time measures in the OF. Other QTL exhibited strong temporal profiles over testing, highlighting dynamic relationship between genotype, test and changes in behavior. Next, we implemented a factor analysis design to account for the correlated nature of the behaviors measured. OF and FC behaviors loaded onto four main factors representing both anxiety and fear behaviors. Using multiple QTL modeling, we calculated the percentage variance in anxiety and fear explained by multiple QTL using both additive and interactive terms. Quantitative trait loci modeling resulted in a broad description of the genetic architecture underlying anxiety and fear accounting for 14-37% of trait variance. Factor analysis and multiple QTL modeling showed both unique and shared QTL for anxiety and fear; suggesting a partially overlapping genetic architecture for these two different models of anxiety.

Curcumin interrupts the interaction between the androgen receptor and Wnt/ß-catenin signaling pathway in LNCaP prostate cancer cells.

Recently, studies have investigated the significance of the Wnt/ß-catenin pathway in prostate cancer. The transcriptional activity of the androgen receptor (AR) is modulated by interaction with coregulators, one of which is ß-catenin. Curcumin, a dietary yellow pigment of Curcuma longa, has emerged as having a chemopreventive role. Although curcumin has been shown to inhibit AR expression, its molecular mechanism has not been fully elucidated. In this study, whether curcumin mediates the Wnt/ß-catenin signaling pathway with regard to AR/ß-catenin interactions was studied. Curcumin was shown to induce significant inhibition of AR expression in a dose-dependent manner. Marked curcumin-induced suppression of ß-catenin was shown in the nuclear and cytoplasmic extracts as well as whole cell lysates. Further analysis revealed that phosphorylation of Akt and glycogen synthase kinase-3ß were attenuated, but phosphorylated ß-catenin was increased after curcumin treatment. Finally, cyclin D1 and c-myc, the target gene of the ß-catenin/T-cell factor transcriptional complex, were also decreased. These findings suggest that curcumin modulates the Wnt/ß-catenin signaling pathway and might have a significant role in mediating inhibitory effects on LNCaP prostate cancer cells.

Fine mapping of QTL for prepulse inhibition in LG/J and SM/J mice using F(2) and advanced intercross lines.

Prepulse inhibition (PPI) of the startle response is a measure of sensorimotor gating, a process that filters out extraneous sensory, motor and cognitive information. Humans with neurological and psychiatric disorders, including schizophrenia, obsessive-compulsive disorder and Huntington's disease, exhibit a reduction in PPI. Habituation of the startle response is also disrupted in schizophrenic patients. In order to elucidate the genes involved in sensorimotor gating, we phenotyped 472 mice from an F(2) cross between LG/J × SM/J for PPI and genotyped these mice genome-wide using 162 single nucleotide polymorphism (SNP) markers. We used prepulse intensity levels that were 3, 6 and 12 dB above background (PPI3, PPI6 and PPI12, respectively). We identified a significant quantitative trait locus (QTL) on chromosome 12 for all three prepulse intensities as well as a significant QTL for both PPI6 and PPI12 on chromosome 11. We identified QTLs on chromosomes 7 and 17 for the startle response when sex was included as an interactive covariate and found a QTL for habituation of the startle response on chromosome 4. We also phenotyped 135 mice from an F(34) advanced intercross line (AIL) between LG/J × SM/J for PPI and genotyped them at more than 3000 SNP markers. Inclusions of data from the AIL mice reduced the size of several of these QTLs to less than 5 cM. These results will be useful for identifying genes that influence sensorimotor gaiting and show the power of AIL for fine mapping of QTLs.

Fine-mapping of muscle weight QTL in LG/J and SM/J intercrosses.

Genetic variation plays a substantial role in variation in strength, but the underlying mechanisms remain poorly understood. The objective of the present study was to examine the mechanisms underlying variation in muscle mass, a predictor of strength, between LG/J and SM/J strains, which are the inbred progeny of mice selected, respectively, for high and low body weight. We measured weight of five hindlimb muscles in LG/J and SM/J males and females, in F(1) and F(2) intercrosses, and in an advanced intercross (AI), F(34), between the two. F(2) mice were genotyped using 162 SNPs throughout the genome; F(34) mice were genotyped at 3,015 SNPs. A twofold difference in muscle mass between the LG/J and SM/J mouse strains was observed. Integrated genome-wide association analysis in the combined population of F(2) and AI identified 22 quantitative trait loci (QTL; genome-wide P < 0.05) affecting muscle weight on Chr 2 (2 QTL), 4, 5, 6 (7 QTL), 7 (4 QTL), 8 (4 QTL), and 11 (3 QTL). The LG/J allele conferred greater muscle weight in all cases. The 1.5-LOD QTL support intervals ranged between 0.3 and 13.4 Mb (median 3.7 Mb) restricting the list of candidates to between 5 and 97 genes. Selection for body weight segregated the alleles affecting skeletal muscle, the most abundant tissue in the body. Combination of analyses in an F(2) and AI was an effective strategy to detect and refine the QTL in a genome-wide manner. The achieved resolution facilitates further elucidation of the underlying genetic mechanisms affecting muscle mass.

Non-synonymous single-nucleotide polymorphisms associated with blood pressure and hypertension.

In this study, we determined the association of 1180 non-synonymous single-nucleotide polymorphisms (SNPs) with systolic blood pressure (SBP) and hypertensive status. A total of 8842 subjects were taken from two community-based cohorts--Ansung (n=4183) and Ansan (n=4659), South Korea--which had been established for genome-wide association studies (GWAS). Five SNPs (rs16835244, rs2286672, rs6265, rs17237198 and rs7312017) were significantly associated (P-values: 0.003-0.0001, not corrected for genome-wide significance) with SBP in both cohorts. Of these SNPs, rs16835244 and rs2286672 correlated with risk for hypertension. The rs16835244 SNP replaces Ala288 in arginine decarboxylase (ADC) with serine, and rs2286672 replaces Arg172 in phospholipase D2 (PLD2) with cysteine. A comparison of peptide sequences between vertebrate homologues revealed that the SNPs identified occur at conserved amino-acid residues. In silico analysis of the protein structure showed that the substitution of a polar residue, serine, for a non-polar alanine at amino-acid residue 288 affects a conformational change in ADC, and that Arg172 in PLD2 resides in the PX domain, which is important for membrane trafficking. These results provide insights into the function of these non-synonymous SNPs in the development of hypertension. The study investigating non-synonymous SNPs from GWAS not only by statistical association analysis but also by biological relevance through the protein structure might be a good approach for identifying genetic risk factors for hypertension, in addition to discovering causative variations.

Genetic variations in ATP2B1, CSK, ARSG and CSMD1 loci are related to blood pressure and/or hypertension in two Korean cohorts.

Blood pressure, one of the important vital signs, is affected by multiple genetic and environmental factors. Recently, several genome-wide association (GWA) studies have successfully identified genetic factors that influence blood pressure and hypertension risk. In this study, we report results of the Korean Association REsource (KARE, 8842 subjects) GWA study on blood pressure and hypertension risk. In all, 10 single-nucleotide polymorphisms (SNPs) that showed significant association with hypertension were further analysed for replication associations in the Health2 project (7861 subjects). Among these 10 SNPs, 3 were replicated in the Health2 cohort for an association with systolic or diastolic blood pressure. The most significant SNP (rs17249754 located in ATPase, Ca(++) transporting, plasma membrane 1 (ATP2B1)) has been previously reported, and the other two SNPs are rs1378942 in the c-src tyrosine kinase (CSK) gene and rs12945290 in the arylsulphatase G (ARSG) gene. An additional hypertension case-control study confirmed that rs17249754 (in ATP2B1) increases hypertension risk in both the KARE and Health2 (meta-analysis, P-value=4.25 x 10(-9)) cohorts. One more SNP, rs995322, located in the CUB and Sushi multiple domains 1 (CSMD1), is also associated with increased risk of hypertension (meta-analysis, P-value=1.00 x 10(-4)). Despite the difficulty of obtaining replication results for a complex trait genetic association between blood pressure and hypertension, we were able to identify consistent genetic factors in both the Korean cohorts in ATP2B1, CSK, ARSG and CSMD1 genes.

Comparison of cortical bone thickness and root proximity at maxillary and mandibular interradicular sites for orthodontic mini-implant placement.

OBJECTIVES: To compare maxillary and mandibular cortical bone thickness and rootic proximity for optimal mini-implant placement. SETTING AND SAMPLE POPULATION: CT images from 14 men and 14 women were used to evaluate buccal interradicular cortical bone thickness and root proximity from mesial of the central incisor to the 2nd molar. Cortical bone thickness was measured at 0 degrees , 15 degrees , 30 degrees , and 45 degrees angles relative to the root surface using three-dimensional images. RESULTS: For the cortical bone thickness, there was no statistically significant difference between the maxilla and the mandible in the anterior area; however, there was a significant difference in the posterior area. Cortical bone in the maxilla, mesial and distal to canine interradicular sites, was thickest while thickness in the mandible exhibited a gradual anterior to posterior increase. Cortical bone thickness in the maxilla increased as both level and angle increased, while the cortical bone thickness in the mandible was greatest at 4 mm from the alveolar crest. Root proximity mesial and distal to 2nd premolar interradicular sites was greatest. CONCLUSION: Based on our results, cortical bone thickness depends on the interradicular site rather than sex or individual differences.

Upregulation and function of GADD45gamma in unilateral ureteral obstruction.

We performed differential display analysis to determine transcriptional activity in the rat kidney, following unilateral ureteral obstruction and found a 12-fold increase in the expression of Growth Arrest and DNA Damage-45gamma (GADD45gamma), a stress-responsive molecule that interacts with cell-cycle proteins. GADD45gamma was strongly expressed in as little as 6 h following ureteric obstruction in the renal tubules, and was also found in kidney tissue of patients with chronic glomerulonephritis. Adenovirus-mediated expression of GADD45gamma in cultured renal tubular cells activated p38 along with a significant upregulation of C-C and C-X3-C chemokine ligands and fibrosis-related factors such as several matrix metalloproteinases, transforming growth factor-beta1, decorin, and bone morphogenetic protein 2. Silencing of GADD45gamma expression significantly blunted the upregulation of these inflammatory and fibrogenic mediators and monocyte infiltration in the ureteral obstructed rat kidney. Our study shows that GADD45gamma is quickly upregulated in the kidney with an obstructed ureter, enhancing the production of factors regulating the pathogenesis of kidney disease.

Tryptophan hydroxylase 2 (TPH2) haplotypes predict levels of TPH2 mRNA expression in human pons.

Tryptophan hydroxylase isoform 2 (TPH2) is expressed in serotonergic neurons in the raphe nuclei, where it catalyzes the rate-limiting step in the synthesis of the neurotransmitter serotonin. In search for functional polymorphisms within the TPH2 gene locus, we measured allele-specific expression of TPH2 mRNA in sections of human pons containing the dorsal and median raphe nuclei. Differences in allelic mRNA expression--referred to as allelic expression imbalance (AEI)--are a measure of cis-acting regulation of gene expression and mRNA processing. Two marker SNPs, located in exons 7 and 9 of TPH2 (rs7305115 and rs4290270, respectively), served for quantitative allelic mRNA measurements in pons RNA samples from 27 individuals heterozygous for one or both SNPs. Significant AEI (ranging from 1.2- to 2.5-fold) was detected in 19 out of the 27 samples, implying the presence of cis-acting polymorphisms that differentially affect TPH2 mRNA levels in pons. For individuals heterozygous for both marker SNPs, the results correlated well (r=0.93), validating the AEI analysis. AEI is tightly associated with the exon 7 marker SNP, in 17 of 18 subjects. Remarkably, expression from the minor allele exceeded that of the major allele in each case, possibly representing a gain-of-function. Genotyping of 20 additional TPH2 SNPs identified a haplotype block of five tightly linked SNPs for which heterozygosity is highly correlated with AEI and overall expression of TPH2 mRNA. These results reveal the presence of a functional cis-acting polymorphism, with high frequency in normal human subjects, resulting in increased TPH2 expression levels. The SNPs that correlate with AEI are closely linked to TPH2 SNPs previously shown to associate with major depression and suicide.

A recombinant human enzyme for enhanced interstitial transport of therapeutics.

Subcutaneously injected therapeutics must pass through the interstitial matrix of the skin in order to reach their intended targets. This complex, three-dimensional structure limits the type and quantity of drugs that can be administered by local injection. Here we found that depolymerization of the viscoelastic component of the interstitial matrix in animal models with a highly purified recombinant human hyaluronidase enzyme (rHuPH20) increased the dispersion of locally injected drugs, across a broad range of molecular weights without tissue distortion. rHuPH20 increased infusion rates and the pattern and extent of appearance of locally injected drugs in systemic blood. In particular, rHuPH20 changed the pharmacokinetic profiles and significantly augmented the absolute bioavailability of locally injected large protein therapeutics. Importantly, within 24 h of injection, the interstitial viscoelastic barriers were restored without histologic alterations or signs of inflammation. rHuPH20 may function as an interstitial delivery enhancing agent capable of increasing the dispersion and bioavailability of coinjected drugs that may enable subcutaneous administration of therapeutics and replace intravenous delivery.

CD14 receptor gene promoter polymorphism: awakening maternal immunity during pregnancy.

We investigated whether monocyte CD14 receptor gene promoter polymorphisms were associated with the development and severity of pre-eclampsia. We genotyped the CD14 -260 C/T polymorphism in 36 pre-eclamptic patients and 52 healthy pregnant controls. A total of 30.6% and 69.4% of pre-eclamptic patients had the C and T alleles, respectively, and 48.0% and 52.0% of the controls, respectively. More pre-eclamptic patients were TT homozygotes compared with controls (50.0% versus 13.5%). In pre-eclamptic patients, the TT homozygotes exhibited a significantly higher mean systolic blood pressure compared with the non-TT homozygotes (173 +/- 28 mmHg versus 153 +/- 22 mmHg). We also noted a tendency towards increased proteinuria and placental abruption in the TT homozygotes compared with the non-TT homozygotes. We conclude that CD14 gene promoter polymorphisms appear to be a risk factor for pre-eclampsia. With further research, these findings might form the basis of a prognostic tool for pre-eclampsia.

Allelic expression of serotonin transporter (SERT) mRNA in human pons: lack of correlation with the polymorphism SERTLPR.

An insertion/deletion polymorphism in the SERT linked promoter region (SERTLPR), previously reported to regulate mRNA expression in vitro, has been associated with mental disorders and response to psychotropic drugs. Contradictory evidence, however, has raised questions about the role of SERTLPR in regulating mRNA expression in vivo. We have used analysis of allelic expression imbalance (AEI) of SERT mRNA to assess quantitatively the contribution of SERTLPR to mRNA expression in human post-mortem pons tissue sections containing serotonergic neurons of the dorsal and median raphe nuclei. Any difference in the expression of one allele over the other indicates the presence of cis-acting elements that differentially affect transcription and/or mRNA processing and turnover. Using a marker SNP in the 3' untranslated region of SERT mRNA, statistically significant differences in allelic mRNA levels were detected in nine out of 29 samples heterozygous for the marker SNP. While the allelic expression differences were relatively small (15-25%), they could nevertheless be physiologically relevant. Although previous results had suggested that the long form of SERTLPR yields higher mRNA levels than the short form, we did not observe a correlation between SERTLPR and allelic expression ratios. Also in contrast to previous results, we found no correlation between SERTLPR and allelic expression ratios or SERT mRNA levels in B-lymphocytes. This study demonstrates that regulation of SERT mRNA is independent of SERTLPR, but could be associated with polymorphisms in partial linkage disequilibrium with SERTLPR.

Evaluation of pentoxifylline in the prevention of haze after photorefractive keratectomy in the rabbit.

BACKGROUND: Pentoxifylline (PTX) is a methylxanthine derivative that, besides its hemorrheologic properties, possesses multiple physiologic effects at the cellular level. It has been used in keloid prevention due to its ability to inhibit the secretion of collagen and glycosaminoglycans from activated fibroblasts. METHODS: Ten New Zealand White (NZW) rabbits underwent a -7.00 diopters, 6.0 mm diameter photorefractive keratectomy after laser ablation of the epithelium with a VISX 20/20 excimer laser. The bare stroma was stained with fresh 0.5% dichlorotriazinyl aminofluorescein (DTAF). The procedure was performed on both eyes, 4 days apart. One eye received 1% Pentoxifylline qid and the other balanced salt solution qid as a control for 4 weeks, starting the same day of surgery. Two masked observers graded the amount of haze at 2, 4, 6, and 8 weeks after surgery using slit-lamp biomicroscopy. Three rabbits were sacrificed at 2 and 4 weeks followed by two rabbits at 6 and 8 weeks. The area between the DTAF-stained collagen to the base of the epithelium was measured using a digital image analyzer. RESULTS: There was no statistically significant difference in the amount of haze either by slit-lamp microscopy or by histological analysis between the pentoxifylline-treated eyes and the controls at any time interval (Student's t-test: 0.16 to 0.92) CONCLUSION: Pentoxifylline did not seem to affect haze formation in a PRK rabbit model. As no signs of toxicity were observed, further studies might examine higher concentrations or dose frequencies.