The intestinal mucosal barrier (IMB), which consists of mechanical barrier, chemical barrier, biological barrier and immune barrier, plays an important role in the maintenance of intestinal epithelium integrity and defense against invasion of bacteria, endotoxins and foreign antigens. Impaired IMB, characterized by increased intestinal mucosal permeability (IMP) and decreased transmembrane resistance (TR), has been implicated in the pathogenesis of various digestive, urinary, circulatory, neurological and metabolic dysfunctions. Electrophysiological recording of TR in the ex vivo intestinal tissues or cultured epithelial cell monolayers, or biochemical quantification of transepithelial movement of orally-administered molecular probes or specific endogenous protein molecules has frequently been used in the evaluation of IMB. In this paper, the composition and function of IMB will be summarized, with emphasis on the evaluation methods of IMP.
Cells, Cultured ; Inosine Monophosphate/metabolism* ; Intestinal Mucosa ; Permeability

Mycophenolate mofetil (MMF) is a novel new immunosuppressant which suppress proliferation of T and B lymphocytes by inhibiting inosine monophosphate dehydrogenase. Even though the results we can get now are still preliminary but the positive result such as low incidence of acute rejection episode, is very attractive to clinical therapist. It also has certain effect on rescue therapy of steroid resistant acute rejection. There is little proven report of long-term follow up of this drug but the improvement of early graft survival suggest better long-term result. From March 1997, we used MMF as one of the primary immunosuppressant with cyclosporine and steroid in 47 renal allograft recipient (MMF group) and the early result of this protocol is compared with control group using conventional two drug regimen (cyclosporine and steroid)in 96 recipients. The 2 g of MMF were given daily from 2nd post-transplant day. The acute rejection episode within 3 and 6 months are 12.1 and 12.8% in MMF group and these are statistically significant difference with the results of control group (36.5% and 38.5% respectively, P<0.005). The response rate of acute rejection to steroid pulse therapy was 66.7% in MMF group but has no statistical difference with that of conventional group (84.1%). Steroid resistant severe acute rejection that needed to use OKT3 was developed in 1 case (2.1%) in MMF group but 7 (7.3%) in control group. Among the complication, post-transplant infection occurred in 6 cases (12.8%) of MMF group but in 8 cases (8.3%) in control group. Diarrhea that needed medication developed in 8 cases of MMF group (17.0%), but only one of them is necessary to change his immunosuppressive regimen. Leukopenia also developed in 1 case of each group. In summary, the incidence of acute rejection episode and steroid resistant rejection that is necessary to use OKT3 is significantly decreased in MMF group but the response rate to steroid pulse therapy and complications of both groups showed no statistical difference.
Allografts* ; B-Lymphocytes ; Cyclosporine ; Diarrhea ; Graft Survival ; Incidence ; Inosine Monophosphate ; Kidney Transplantation ; Kidney* ; Leukopenia ; Muromonab-CD3 ; Oxidoreductases

PURPOSE: Mesangial cell extracellular matrix (ECM) synthesis plays an important role in various renal diseases. Mycophenolic acid (MPA), which is an inhibitor of inosine monophosphate dehydrogenase (IMPDH), inhibits mesangial cell proliferation and ECM synthesis. However, the exact mechanism of MPA has not been clearly elucidated in mesangial cells. To examine the relative importance of IMPDH on the inhibitory action of MPA, we compared the effects of MPA or IMPDH2 siRNA on platelet-derived growth factor (PDGF)-induced fibronectin secretion and cellular reactive oxygen species (ROS) in mouse mesangial cells (MMC). METHODS: MMC were stimulated with PDGF 10 ng/ml with or without MPA 0.1~10micrometer, IMPDH2 siRNA 10~50 nM, or N-acetylcystein (NAC). IMPDH2 siRNA was transiently transfected by lipofectamine for 24 hours. MPA 0.1~10micrometer, ribavirin 10~100micrometer, and NAC 5 mM were administered 1 hour before the stimulation. Cell viability was measured by methylthiazoletetrazolium (MTT) assay, fibronectin secretion by Western blot analysis, and dichlorofluorescein (DCF)-sensitive cellular ROS by flow cytometry. RESULTS: PDGF 10 ng/ml effectively increased fibronectin secretion and cellular ROS in MMC. MPA and NAC at concentration without affecting basal level of fibronectin and cellular ROS ameliorated PDGF-induced fibronectin secretion and cellular ROS. However, IMPDH2 siRNA only partially reduced PDGF- induced fibronectin secretion and cellular ROS in MMC. CONCLUSION: These results suggest that MPA may inhibit PDGF-induced fibronectin secretion partly through IMPDH2 or cellular ROS in MMC, and there may be other mechanisms on the inhibitory action of MPA in mesenchymal cells.
Animals ; Blotting, Western ; Cell Survival ; Extracellular Matrix ; Fibronectins* ; Flow Cytometry ; Inosine Monophosphate* ; Inosine* ; Mesangial Cells* ; Mice* ; Mycophenolic Acid ; Oxidoreductases* ; Platelet-Derived Growth Factor ; Reactive Oxygen Species* ; Ribavirin ; RNA, Small Interfering

A simple and rapid method was developed based on high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to determine sivelestat and its metabolite XW-IMP-A in human plasma. After a simple protein precipitation, the samples and internal standards were analyzed on a C18 column by a gradient elution program. The mobile phase consisted of 30% acetonitrile in methanol and 5 mmol · L(-1) ammonium acetate at a flow rate of 0.7 mL · min(-1). The mass spectrometric data was collected in multiple reaction monitoring mode (MRM) in the negative electrospray ionization. The standard curves were linear in the range of 10.0-15,000 ng · mL(-1) for sivelestat, and 2.50-1000 ng · mL(-1) for XW-IMP-A. The low limits of quantitation were identified at 10.0 and 2.50 ng · mL for sivelestat and XW-IMP-A, respectively. The intra- and inter-day precision were within 11.3% and 13.1% for sivelestat and XW-IMP-A, and accuracy was 0.3% and 0.6% for sivelestat and XW-IMP-A, within the acceptable limits across all concentrations. The method was successfully validated in the pharmacokinetic study of sivelestat in healthy Chinese volunteers.
Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Glycine ; analogs & derivatives ; blood ; Humans ; Inosine Monophosphate ; blood ; Reproducibility of Results ; Sulfonamides ; blood ; Tandem Mass Spectrometry

BACKGROUND: Triple immunosuppressant therapy including anti-metabolites is the representative immunosuppressive therapy after renal transplantation. This study is to evaluate the factors that influence Mycophenolate sodium (MPS, Myfortic, Novartis, Basel, Switzerland) dosage patterns in renal transplantation patients who take MPS as an inosine monophosphate dehydrogenase (IMPDH) among antimetabolites. METHODS: From May 2007 to April 2008, 16 clinical departments of 14 transplantation centers in Korea retrospectively performed a survey on 650 renal transplantation recipients taking MPS. This survey collected personal information, clinical factors related to transplantation and immunosuppressive therapy. RESULTS: The mean age of the patients was 43.0+/-12.0 (7~75) and the study included 364 males (56.0%) and 286 females (44.0%). The average follow up period after renal transplantation was 49.5+/-53.4 (1~307) months. There were 366 (56.3%) living related cases, 145 (22.3%) living non-related cases and 139 (21.4%) deceased donor cases. Cyclosporine was the most common calcineurin inhibitor (CNI) used in combination therapy with MPS (476 cases, 73.2%) followed by tacrolimus (169 cases, 26.0%). The mean daily dose of MPS was 909.7+/-336.3 (180~1,620)mg and the mean daily dose per kg was 15.3+/-5.9 (2.65~32.73)mg/kg. The daily dose showed significant positive correlation with patient body weight but the daily dose per kg showed negative correlation. The daily dose of MPS was significantly higher in the combination therapy with cyclosporine than that with tacrolimus. The daily dose and the dose per kg decreased with increment of recipient age and post-transplant period. CONCLUSIONS: Our study concluded that MPS dosages correlated with the combined type of CNI, post-transplant period and age.
Body Weight ; Calcineurin ; Cyclosporine ; Female ; Follow-Up Studies ; Humans ; Inosine Monophosphate ; Kidney Transplantation ; Korea ; Male ; Mycophenolic Acid ; Oxidoreductases ; Retrospective Studies ; Sodium ; Tacrolimus ; Tissue Donors ; Transplants

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a key enzyme of de novo GMP biosynthesis. The expression and activity of IMPDH can be affected by diseases and physiological process. It is the drug target for anticancer, antiviral, antimicrobial and immunosuppressive therapeutics. Not only catalytic action but the other biological functions of IMPDH also play an important role in diseases. The basic functions, mechanism of catalysis, classification of inhibitors, biological functions and the latest advances to IMPDH will be illustrated in this review. It is expected to be helpful to the discovery of new inhibitors and biological functions of IMPDH.
Animals ; Binding Sites ; Catalysis ; Drug Design ; Drug Discovery ; Enzyme Inhibitors ; classification ; pharmacology ; Humans ; IMP Dehydrogenase ; antagonists & inhibitors ; genetics ; metabolism ; Inosine Monophosphate ; metabolism ; Molecular Structure ; NAD ; metabolism ; Polymorphism, Genetic

PURPOSE: Mycophenolic acid (MPA) is the active agent of mycophenolate mofetil (MMF), which is an immunosuppressive drug. MPA is a selective inhibitor of inosine monophosphate dehydrogenase. The aim of this study was for demonstrate that mycophenolic acid induces apoptosis in human Jurkat cells via the generation of reactive oxygen species (ROS). METHODS: The cells were cultured in the presence or absence of MPA. Flow cytometric analysis was performed after propidium iodide staining. Western blotting for caspase 3, Bcl-2 and Bax proteins was also performed. RESULTS: MPA decreased the viability of Jurkat cells in a dose- and time-dependent manner. The MPA induced apoptotic cell death displayed nuclear fragmentation and sub G0/G1 phase arrest in the Jurkat cells. The expression of caspase-3 proteases in the MPA treated-Jurkat cells increased in a time-dependent manner. Treatment with MPA resulted in increased ROS generation in the Jurkat cells. There was a decreased expression of Bcl-2 and an increased expression of Bax protein in the MPA treated Jurkat cells. CONCLUSION: This result suggests that MPA-induced cytotoxicity is associated with a direct increase of both ROS generation and the expression of Bax protein.
Apoptosis ; bcl-2-Associated X Protein ; Blotting, Western ; Caspase 3 ; Cell Death ; Humans ; Inosine Monophosphate ; Jurkat Cells ; Mycophenolic Acid ; Oxidoreductases ; Peptide Hydrolases ; Propidium ; Reactive Oxygen Species

PURPOSE: Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase (IMPDH), is the active metabolite of the immunosuppressive drug, mycophenolate mofetil (MMF). MMF is used to prevent an immune- mediate rejection response following organ transplantation via the inhibition of the IMPDH and GTP biosynthesis pathway. This study was designed to elucidate the mechanism by which MPA exerts its cytotoxic effect on human T lymphocytic and monocytic cell lines. METHODS: MOLT-4 and U937 cell lines were treated with MPA. Cell viability, expression of Bcl2 family proteins and Fas/Fas-L, effects of antioxidants and intracellular Ca2+ regulating agents and apoptosis were measured using a variety of microscopic and biochemical techniques. RESULTS: MPA induced the death of U937 and MOLT-4 cells in dose and time dependent manners, which was revealed an apoptosis with a characteristic ladder pattern of DNA fragmentation. In addition, BAPTA/AM, an intracellular Ca2+ chelator protected MOLT-4 cells from MPA treated apoptosis, although it did not have an additive with thapsigargin, and increases cytosolic Ca2+ stores. However, antioxidants including reduced glutathione (GSH) and N-acetyl-L-cysteine (NAC) did not inhibit the apoptosis of cells by MPA. Furthermore, guanosine suppressed MPA induced apoptosis of MOLT-4 lymphocytes, although adenosine did not. MPA also increased the catalytic activity of caspase family cysteine proteases including caspase-8, 9 and 3 proteases in MOLT-4 cells. Sequential activation indicated that the cleavage of caspase-8 and 9 precedes those of caspase-3. CONCLUSION: The results suggest that MPA induces the apoptotic death of MOLT-4 lymphocytes via the activations of caspase family proteases and the depletion of GTP.
Acetylcysteine ; Adenosine ; Antioxidants ; Apoptosis ; Caspase 3 ; Caspase 8 ; Cell Line ; Cell Survival ; Cysteine Proteases ; Cytosol ; DNA Fragmentation ; Glutathione ; Guanosine ; Guanosine Triphosphate ; Humans ; Inosine Monophosphate ; Lymphocytes ; Mycophenolic Acid* ; Organ Transplantation ; Oxidoreductases ; Peptide Hydrolases ; Signal Transduction* ; T-Lymphocytes* ; Thapsigargin ; Transplants ; U937 Cells

BACKGROUND: Mizoribine (MZR) is an imidazole nucleoside isolated from Eupenicillium brefeldianum. MZR is currently in clinical use for patients who have undergone renal transplantation. Therapeutic efficacy of MZR has also been demonstrated in rheumatoid arthritis and lupus nephritis. MZR has been shown to inhibit the proliferation of lymphocytes by interfering with inosine monophosphate dehydrogenase. Since the exact mechanism by which MZR benefits rheumatoid arthritis (RA) is not clear, we investigated the ability of MZR to direct its immunosuppressive influences on other antigen presenting cells, such as macrophages. METHODS: Mouse macrophage RAW264.7 cells were stimulated with lipopolysaccharide in the presence of MZR. To elucidate the mechanism of the therapeutic efficacy in chronic inflammatory diseases, we examined the effects of MZR on the production of pro-inflammatory cytokines, nitric oxide (NO) and prostaglandin E2 (PGE2) in macrophages. RESULTS: MZR dose-dependently decreased the production of nitric oxide and pro- inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukins 1beta (IL-beta) and IL-6 PGE2. Examination of gene expression levels showed that the anti-inflammatory effect correlated with the down-regulation of inducible nitiric oxide synthase expression, cycloxygenase-2 expression and TNF-alpha gene expression. CONCLUSION: In this work, we resulted whether MZR (1.25~10 microgram/ml) inhibited macrophage activation by inhibiting secretion of pro-inflammatory cytokines, NO and PGE2. These findings provide an explanation for the therapeutic efficacy of MZR in chronic inflammation- associated diseases.
Animals ; Antigen-Presenting Cells ; Arthritis, Rheumatoid ; Cytokines* ; Dinoprostone* ; Down-Regulation ; Eupenicillium ; Gene Expression ; Humans ; Inosine Monophosphate ; Interleukin-6 ; Interleukins ; Kidney Transplantation ; Lupus Nephritis ; Lymphocytes ; Macrophage Activation ; Macrophages* ; Mice ; Nitric Oxide ; Oxidoreductases ; Tumor Necrosis Factor-alpha

OBJECTIVES: To study the clinical features of children with carbapenem-resistant Enterobacterales (CRE) infection and the molecular characteristics of isolated strains. METHODS: A retrospective analysis was performed on the clinical data and infection status of the children who were hospitalized in Guangdong Provincial People's Hospital from January 2018 to June 2021. A total of 1 098 non-repetitive strains of Enterobacterales were obtained. Drug sensitivity test, PCR amplification, and resistance-related gene sequencing were performed for 66 isolated CRE strains to observe molecular epidemiology. RESULTS: Among the 1 098 strains of Enterobacterales, the detection rate of CRE was 6.01% (66/1 098). The 66 CRE strains were isolated from 66 children, among whom there were 37 boys (56%) and 29 girls (44%), with an age of 2 days to 14 years. Among these 66 children, 16 (24%) had an age of <1 month, 28 (42%) had an age of 1-12 months, 11 (17%) had an age of 12-36 months, and 11 (17%) had an age of >36 months. The children with CRE were mainly distributed in the department of neonatology (38 children, 58%) and the pediatric intensive care unit (17 children, 26%). The top three types of specimens with CRE detection were respiratory specimens (48%), midstream urine specimens (21%), and blood specimens (17%). The CRE strains were mainly Klebsiella pneumoniae (45 strains, 68%), Escherichia coli (12 strains, 18%), and Enterobacter cloacae (6 strains, 9%), with high resistance to carbapenems (such as imipenem and ertapenem), penicillin, and cephalosporins, slightly high resistance to commonly used antibiotics, and relatively low resistance to amikacin (14%), levofloxacin (23%), and tobramycin (33%). The carbapenemase genotypes of Klebsiella pneumoniae strains were mainly bla_NDM (20 strains, 44%), bla_IMP (10 strains, 22%), and bla_KPC (5 strains, 11%), and the carbapenemase genotypes of Escherichia coli strains were mainly bla_NDM (10 strains, 83%). After sequencing, there were 24 bla_NDM-1 strains, 6 bla_NDM-5 strains, 5 bla_IMP-4 strains, and 3 bla_KPC-2 strains, and some genotypes were not identified. CONCLUSIONS There is a high incidence rate of CRE infection among children, mainly those aged 1-12 months. CRE generally has high resistance to antibacterial drugs, and metalloenzymes are the main type of carbapenemases for CRE strains in children.
Adolescent ; Anti-Bacterial Agents ; Bacterial Proteins ; Carbapenems ; Child ; Child, Preschool ; Escherichia coli ; Female ; Humans ; Infant ; Infant, Newborn ; Inosine Monophosphate ; Klebsiella pneumoniae ; Male ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Retrospective Studies ; beta-Lactamases