Downregulation of Heat Shock Protein 72 Contributes to Fibrostenosis in Crohn's Disease.

Background/Aims: Crohn's disease (CD) with recurrent inflammation can cause intestinal fibrostenosis due to dysregulated deposition of extracellular matrix. However, little is known about the pathogenesis of fibrostenosis. Here, we performed a differential proteomic analysis between normal, inflamed, and fibrostenotic specimens of patients with CD and investigated the roles of the candidate proteins in myofibroblast activation and fibrosis. Methods: We performed two-dimensional difference gel electrophoresis and identified candidate proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and orbitrap liquid chromatography-mass spectrometry. We also verified the levels of candidate proteins in clinical specimens and examined their effects on 18Co myofibroblasts and Caco-2 intestinal epithelial cells. Results: We identified five of 30 proteins (HSP72, HSPA5, KRT8, PEPCK-M, and FABP6) differentially expressed in fibrostenotic CD. Among these proteins, the knockdown of heat shock protein 72 (HSP72) promoted the activation and wound healing of myofibroblasts. Moreover, knockdown of HSP72 induced the epithelial-mesenchymal transition of intestinal epithelial cells by reducing E-cadherin and inducing fibronectin and α-smooth muscle actin, which contribute to fibrosis. Conclusions: HSP72 is an important mediator that regulates myofibroblasts and epithelial-mesenchymal transition in fibrosis of CD, suggesting that HSP72 can serve as a target for antifibrotic therapy.

Association between smoking cessation and obstructive spirometry pattern among Korean adults aged 40-79 years.

Smoking cessation aids in restoring lung function. However, whether long-term cessation can fully restore lung function has not been studied thoroughly, especially in Asian countries. This study aimed to evaluate the association between smoking cessation status and obstructive spirometry pattern among Koreans aged 40-79 years. In total, 6298 men and 8088 women aged 40-79 years from the Korea National Health and Nutrition Examination Survey (2015-2019) were analyzed for smoking cessation status, including the duration after quitting. Current-smokers showed a higher likelihood of having an obstructive spirometry pattern than never-smokers among both men (odds ratio [OR]: 3.15, 95% confidence interval [CI]: 2.32-4.29) and women (OR: 2.60, 95% CI: 1.59-4.23). In men, the effect tended to decrease with longer duration after cessation, but male ex-smokers who had quit smoking ≥ 20 years ago still showed a higher likelihood of having an obstructive spirometry pattern than male never-smokers (OR: 1.40, 95% CI: 1.05-1.89). In female ex-smokers, there was no significant association with the obstructive spirometry pattern, compared to that in female never-smokers. This study emphasizes the benefits of smoking cessation, possibility of long-lasting harm to lung function due to tobacco smoking, and importance of smoking prevention.

Lactobacillus plantarum CBT LP3 ameliorates colitis via modulating T cells in mice.

Lactobacillus plantarum has been identified as a probiotic bacterium owing to its role in immune regulation and maintenance of intestinal permeability. Here, we investigated the anti-colitic effects and mechanism of L. plantarum CBT LP3 (LP3). This in vivo study was performed using dextran sodium sulfate (DSS) to induce colitis in mice. Mice were randomly divided into three groups: a control supplied with normal drinking water, a DSS-treated group followed by oral administration of vehicle, and a DSS-treated group gavaged with LP3 daily for 7 days following DSS administration. An analysis of macrophages and T cell subsets harvesting from peritonium cavity cells and splenocytes was performed using a flow cytometric assay. Gene expression and cytokine profiles were measured using quantitative reverse transcriptase polymerase chain reaction. The administration of LP3 significantly attenuated disease activity and histolopathology compared to control. LP3 had anti-inflammatory effects, with increased induction of regulatory T cells and type 2 helper T cells in splenocytes and restoration of goblet cells accompanied by suppression of proinflammatory cytokine expressions. These findings suggest that L. plantarum CBT LP3 can be used as a potent immunomodulator, which has significant implications for IBD treatment.

Glutathione S-transferase theta 1 protects against colitis through goblet cell differentiation via interleukin-22.

The enzyme glutathione S-transferase theta 1 (GSTT1) is involved in detoxifying chemicals, including reactive oxygen species (ROS). Here, we provide a significant insight into the role of GSTT1 in inflammatory bowel disease (IBD). We identified decreased expression of GSTT1 in inflamed colons from IBD patients compared to controls. We intrarectally or intraperitoneally delivered Gstt1 gene to mice with dextran sodium sulfate (DSS)-induced colitis and noted attenuation of colitis through gene transfer of Gstt1 via an IL-22 dependent pathway. Downregulation of GSTT1 by pathogen-associated molecular patterns (PAMPs) of microbes reduced innate defense responses and goblet cell differentiation. The GSTT1 mutation in intestinal epithelial cells (IECs) and IBD patients decreased its dimerization, which was connected to insufficient phosphorylation of signal transducer and activator of transcription-3 and p38/mitogen-activated protein kinase by their common activator, IL-22. GSTT1 ameliorated colitis and contributed as a modulator of goblet cells through sensing pathogens and host immune responses. Its mutations are linked to chronic intestinal inflammation due to its insufficient dimerization. Our results provide new insights into GSTT1 mutations that are linked to chronic intestinal inflammation due to its insufficient dimerization and their functional consequences in IBDs.

Deep Resequencing of Ulcerative Colitis-Associated Genes Identifies Novel Variants in Candidate Genes in the Korean Population.

Background: Genome-wide association studies and meta-analyses have revealed the genetic background of ulcerative colitis (UC) by identifying common variants. However, these variants do not fully explain the disease variance in UC. To identify novel variants, we performed deep resequencing of UC-associated genes in Korean UC patients and subsequently investigated the functional roles of identified susceptibility genes. Methods: We performed targeted deep resequencing of 108 genes in 24 Korean UC patients and then performed association analysis with data from 126 healthy controls. We validated these variants using 2-stage replication studies including 793 UC patients and 783 controls. We performed in silico and pathway analyses and functional analyses. Results: The combined analysis including 2 replication studies identified 6 novel susceptibility loci and reconfirmed 10 previously reported loci. Among the novel single nucleotide variants (SNVs), rs10035653 in C5orf55 (P = 2.08 × 10-3; OR = 1.50), rs41417449 in BTNL2 (P = 1.27 × 10-2; OR = 1.32), rs3117099 in HCG23 (P = 9.98 × 10-6; OR = 1.40), rs7192 in HLA-DRA (P = 6.95 × 10-9; OR = 1.57), and rs3744246 in ORMDL3 (P = 2.21 × 10-2; OR = 1.21) were identified as causal variants, whereas rs713669 in IL17REL (P = 2.69 × 10-2; OR = 0.84) as a protective variant for UC. When correcting multiple testing, 3 novel SNVs (rs41417449 in BTNL2, rs3744246 in ORMDL3, and rs713669 in IL17REL) and 4 previously reported SNVs did not reach a statistical significance. Functional study suggested that SNVs of BTNL2 and C5orf55 exacerbated the inflammatory response both in vitro and in vivo. Conclusions: This study identified 3 novel susceptibility loci and validated 6 previously reported SNVs for UC through deep resequencing in Koreans and revealed the functional roles of BTNL2 and C5orf55.

Identification of genetic susceptibility loci for intestinal Behçet's disease.

Several recent genome-wide association studies (GWAS) identified susceptibility loci/genes for Behçet's disease (BD). However, no study has specifically investigated the genetic susceptibility loci associated with intestinal involvement in BD. We aimed to identify distinctive genetic susceptibility loci/genes associated with intestinal involvement in BD and determine their roles in intestinal inflammation as well as their interactions with genes involved in inflammatory bowel disease (IBD). GWAS and validation studies showed intestinal BD-specific associations with an NAALADL2 gene locus (rs3914501, P = 3.8 × 10-4) and a YIPF7 gene locus (rs6838327, P = 3.5 × 10-4). Validation, haplotype, and pathway analyses showed distinct genetic architectures between intestinal BD and BD without intestinal involvement. Furthermore, network analysis revealed shared pathogenic pathways between intestinal BD and IBD. Gene functional analyses indicated that down-regulation of NAALADL2 and YIPF7 expression was associated with exacerbating intestinal inflammatory responses both in vitro and in vivo. Our results provide new insights into intestinal BD-specific genetic variations, which represents a distinct pathway from BD without intestinal involvement. Functional consequences of the intestinal BD-specific NAALADL2 and YIPF7 expression patterns proved a suggestive association with intestinal inflammation risk, which warrants further validation.

Neurometabolic profiles of the substantia nigra and striatum of MPTP-intoxicated common marmosets: An in vivo proton MRS study at 9.4 T.

Given the strong coupling between the substantia nigra (SN) and striatum (STR) in the early stage of Parkinson's disease (PD), yet only a few studies reported to date that have simultaneously investigated the neurochemistry of these two brain regions in vivo, we performed longitudinal metabolic profiling in the SN and STR of 1-methyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated common marmoset monkey models of PD (n = 10) by using proton MRS (1 H-MRS) at 9.4 T. T2 relaxometry was also performed in the SN by using MRI. Data were classified into control, MPTP_2weeks, and MPTP_6-10 weeks groups according to the treatment duration. In the SN, T2 of the MPTP_6-10 weeks group was lower than that of the control group (44.33 ± 1.75 versus 47.21 ± 2.47 ms, p < 0.05). The N-acetylaspartate to total creatine ratio (NAA/tCr) and γ-aminobutyric acid to tCr ratio (GABA/tCr) of the MPTP_6-10 weeks group were lower than those of the control group (0.41 ± 0.04 versus 0.54 ± 0.08 (p < 0.01) and 0.19 ± 0.03 versus 0.30 ± 0.09 (p < 0.05), respectively). The glutathione to tCr ratio (GSH/tCr) was correlated with T2 for the MPTP_6-10 weeks group (r = 0.83, p = 0.04). In the STR, however, GABA/tCr of the MPTP_6-10 weeks group was higher than that of the control group (0.25 ± 0.10 versus 0.16 ± 0.05, p < 0.05). These findings may be an in vivo depiction of the altered basal ganglion circuit in PD brain resulting from the degeneration of nigral dopaminergic neurons and disruption of nigrostriatal dopaminergic projections. Given the important role of non-human primates in translational studies, our findings provide better understanding of the complicated evolution of PD.

Behavior, PET and histology in novel regimen of MPTP marmoset model of Parkinson's disease for long-term stem cell therapy.

Stem cell technologies are particularly attractive in Parkinson's disease (PD) research although they occasionally need long-term treatment for anti-parkinsonian activity. Unfortunately, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) widely used as a model for PD has several limitations, including the risk of dose-dependent mortality and the difficulty of maintenance of PD symptoms during the whole experiment period. Therefore, we tested if our novel MPTP regimen protocol (2 mg/kg for 2 consecutive days and 1 mg/kg for next 3 consecutive days) can be maintained stable parkinsonism without mortality for long-term stem cell therapy. For this, we used small-bodied common marmoset monkeys (Callithrix jacchus) among several nonhuman primates showing high anatomical, functional, and behavioral similarities to humans. Along with no mortality, the behavioral changes involved in PD symptoms were maintained for 32 weeks. Also, the loss of jumping ability of the MPTP-treated marmosets in the Tower test was not recovered by 32 weeks. Positron emission tomography (PET) analysis revealed that remarkable decreases of bindings of 18F-FP-CIT were observed at the striatum of the brains of the marmosets received MPTP during the full period of the experiment for 32 weeks. In the substantia nigra of the marmosets, the loss of tyrosine hydroxylase (TH) immunoreactivity was also observed at 32 weeks following the MPTP treatment. In conclusion, our low-dose MPTP regimen protocol was found to be stable parkinsonism without mortality as evidenced by behavior, PET, and TH immunohistochemistry. This result will be useful for evaluation of possible long-term stem cell therapy for anti-parkinsonian activity.

Modeling Parkinson's disease in the common marmoset (Callithrix jacchus): overview of models, methods, and animal care.

The common marmoset (Callithrix jacchus) is a small-bodied, popular New World monkey and is used widely in reproductive biology, neuroscience, and drug development, due to its comparative ease of handling, high reproductive efficiency, and its unique behavioral characters. In this review, we discuss the marmoset models in Parkinson's disease (PD), which is a neurological movement disorder primarily resulting from a degeneration of dopaminergic neurons with clinical features of tremor, rigidity, postural instability, and akinesia. The most common PD models involve the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 6-hydroxydopamine to study the pathogenesis and to evaluate novel therapies. Following the systemic or local administration of these neurotoxins, the marmosets with very severe Parkinson's symptoms are recommended to be placed in an intensive care unit with artificial feeding to increase survival rate. All procedures with MPTP should be conducted in a special room with enclosed cages under negative-pressure by trained researchers with personal protection. Behavioral tests are conducted to provide an external measure of the brain pathology. Along with several biomarkers, including α-synuclein and DJ-1, non-invasive neuroimaging techniques such as positron emission tomography and magnetic resonance imaging are used to evaluate the functional changes associated with PD. With the recent growing interest in potential and novel therapies such as stem cell and gene therapy for PD in Korea, the marmoset can be considered as a suitable non-human primate model in PD research to bridge the gap between rodent studies and clinical applications.

Influence of the Flushing Response in the Relationship between Alcohol Consumption and Cardiovascular Disease Risk.

BACKGROUND: The purpose of this study was to examine the relationship between cardiovascular disease risk and alcohol consumption according to facial flushing after drinking among Korean men. METHODS: The subjects were 1,817 Korean men (non-drinker group, 283 men; drinking-related facial flushing group, 662 men; non-flushing group, 872 men) >30 years who had undergone comprehensive health examinations at the health promotion center of a Chungnam National University Hospital between 2007 and 2009. Alcohol consumption and alcohol-related facial flushing were assessed through a questionnaire. Cardiovascular disease risk was investigated based on the 2008 Framingham Heart Study. With the non-drinker group as reference, logistic regression was used to analyze the relationship between weekly alcohol intake and cardiovascular disease risk within 10 years for the flushing and non-flushing groups, with adjustment for confounding factors such as body mass index, diastolic blood pressure, low density lipoprotein cholesterol, triglycerides, and exercise patterns. RESULTS: Individuals in the non-flushing group with alcohol consumption of ≤4 standard drinks (1 standard drink = 14 g of alcohol) per week had significantly lower moderate or high cardiovascular disease risk than individuals in the nondrinker group (adjusted odds ratio, 0.51; 95% confidence interval, 0.37 to 0.71). However, no significant relationship between the drinking amount and cardiovascular disease risk was observed in the flushing group. CONCLUSION: Cardiovascular disease risk is likely lowered by alcohol consumption among non-flushers, and the relationship between the drinking amount and cardiovascular disease risk may differ according to facial flushing after drinking, representing an individual's vulnerability.

Effect of Lead(IV) Acetate on Procoagulant Activity in Human Red Blood Cells.

Lead (Pb) is a ubiquitously occurring environmental heavy metal which is widely used in industry and human life. Possibly due to a global industrial expansion, recent studies have revealed the prevalent human exposure to Pb and increased risk of Pb toxicity. Once ingested by human, 95% of absorbed Pb is accumulated into erythrocytes and erythrocytes are known to be a prime target for Pb toxicity. Most of the studies were however, focused on Pb2+ whereas the effects of Pb4+, another major form of Pb on erythrocytes are poorly understood yet. In this study, we investigated and compared the effects of Pb4+, Pb2+ and other heavy metals on procoagulant activation of erythrocytes, an important factor for the participation of erythrocytes in thrombotic events in an effort to address the cardiovascular toxicity of Pb4+. Freshly isolated erythrocytes from human were incubated with Pb4+, Pb2+, Cd2+ and Ag+ and the exposure of phosphatidylserine (PS), key marker for procoagulant activation was measured using flow cytometry. As a result, while Cd2+ and Ag+ did not affect PS exposure, Pb4+ and Pb2+ induced significantly PS exposure in a dose-dependent manner. Of a particular note, Pb4+ induced PS exposure with a similar potency with Pb2+. PS bearing microvesicle (MV), another important contributor to procoagulant activation was also generated by Pb4+. These PS exposure and MV generation by Pb4+ were well in line with the shape change of erythrocyte from normal discocytes to MV shedding echinocytes following Pb4+ treatment. Meanwhile, nonspecific hemolysis was not observed suggesting the specificity of Pb4+-induced PS exposure and MV generation. These results indicated that Pb4+ could induce procoagulant activation of erythrocytes through PS exposure and MV generation, suggesting that Pb4+ exposure might ultimately lead to increased thrombotic events.

Acute liver toxicity by carbon tetrachloride in HSP70 knock out mice.

The effects of carbon tetrachloride (CCl(4)) treatment on acute liver damage in knock out (heat shock proteins -- HSP70-/-) mice and wild-type (C57BL/6) mice were examined. Acute liver injury was induced by a single intraperitoneal injection of 0.3 ML/kg CCl(4) in olive oil. Mice were sacrificed at 12, 24, 48 and 72 h after treatment. To assess hepatotoxicity, alanine transaminase, neutrophil infiltration and degree of necrosis were measured. Western blot analysis was employed for heat shock proteins. The result revealed that HSP70-/- mice showed higher alanine transaminase levels and a more severe degree of neutrophilic infiltration and necrosis than those of wild-type mice. Furthermore, HSP70-/- mice recovered more slowly from CCl(4) treatment. In HSP70-/- mice, HSP47 was overexpressed. Therefore, HSP70-/- mice could be an adequate model of acute liver toxicity study.