Correlation between histone H3K4 trimethylation and DNA methylation and evaluation of the metabolomic features in acute rejection after kidney transplantation.

OBJECTIVE: To investigate the difference between trimethylation of monocyte histone H3K4 and DNA methylation in acute rejection (AR) after renal transplantation in rats and reveal the epigenetic mechanism of the AR rats based on metabolomics. METHOD: Peripheral blood mononuclear cells (PBMCs) were isolated, and CD4 +CD25 + Treg cells were sorted by flow cytometry. The Foxp3 mRNA and protein levels of CD4 +CD25 + Treg cells were detected by real-time RT-PCR and Western blotting, respectively. High-throughput screening was applied to evaluate the H3K4 methylation of monocytes using chromatin immunoprecipitation with DNA microarray (ChIP-chip) and verified by ChIP with real-time PCR (ChIP-qPCR). Methylated DNA immunoprecipitation sequencing was combined with real-time PCR (MeDIP-qPCR) to detect the DNA methylation level of positive genes (ABCC4, Mef2d, Tbx1 and Eif6). Real-time quantitative PCR (qRT-PCR) and Western blotting were used to detect the mRNA and protein levels of positive genes. The difference in lipid metabolism in the blood of (non) acute rejection rats was analysed by HPLC/MS. RESULTS: AR rats showed an apparent increase in BUN and Cr levels, as well as IL-2, IL-10 and IFN-γ. Compared with non-AR rats, the expression of CD4 +CD25 + Treg cells and Foxp3 mRNA and protein were significantly lower in AR rats. AR rats also showed an increase in H3K4 trimethylation of CD4 +CD25 + Treg and decrease in DNA methylation. There were significant differences in the DNA methylation level of four positive genes between AR and non-AR rats (P<0.05), in addition to differences in the expression levels of mRNA and protein. Pathological examination of the transplanted kidney indicated that AR rats had more severe pathological injury of the kidney than the non-AR rats. There were significant increase in the contents of several phosphatidylcholines, lysophosphatidylcholine, free fatty acids and carnitine in AR rats which detected by HPLC/MS. CONCLUSION: H3K4 trimethylation increased in PBMCs in AR rats, while DNA methylation decreased, indicating the presence of epigenetic differences between AR and non-AR rats. Metabolomic studies indicated a significant increase in AR rats in the contents of several metabolites, such as phosphatidylcholines, lysophosphatidylcholine, free fatty acids and carnitine, suggesting an increasein phospholipase activity and leading to an energy metabolism imbalance with intensification of ß-oxidation. DNA methylation may be associated with an increase in phosphatidylcholines, lysophosphatidylcholine and free fatty acids in AR rats, which may further affect energy metabolism by enhancing the tricarboxylic acid cycle in AR rats.

Association between Nod-like receptor protein 3 inflammasome and gouty nephropathy.

Crystalized deposits of monosodium urate activate the Nod-like receptor protein 3 (NLRP3) inflammasome, resulting in kidney damage. The present study investigated whether the NLRP3 inflammasome is associated with the progression of hyperuricaemia and gouty nephropathy. Adult male patients were recruited at the Affiliated Baoan Hospital of Shenzhen and divided into three groups of 15 patients each: The control group, the hyperuricaemia group and the gouty nephropathy group. General characteristics and organ function indicators were also measured for each patient. NLRP3, apoptosis-associated speck like protein (ASC) and caspase-1 mRNA and protein expressions in peripheral blood mononuclear cells were detected. The expression of certain downstream inflammatory factors, including interleukin (IL)-1ß and IL-18 were also assessed in plasma. The results demonstrated that the concentration of uric acid and creatinine were increased in the hyperuricaemia and gouty nephropathy groups compared with the control group. NLRP3, ASC and caspase-1 mRNA and protein expression, and IL-1ß and IL-18 expression were increased in the hyperuricaemia and gouty nephropathy groups compared with the control group. In addition, ASC and caspase-1 mRNA and protein expression, and IL-1ß expression were higher in the gouty nephropathy group compared with the hyperuricaemia group. In conclusion, the present results supported the hypothesis that the NLRP3 inflammasome signalling pathway is associated with gouty nephropathy leading to initiation of the inflammatory response and causing renal damage.

Enterovirus 71 Represses Interleukin Enhancer-Binding Factor 2 Production and Nucleus Translocation to Antagonize ILF2 Antiviral Effects.

Enterovirus 71 (EV71) infection causes hand-foot-mouth disease (HFMD), meningoencephalitis, neonatal sepsis, and even fatal encephalitis in children, thereby presenting a serious risk to public health. It is important to determine the mechanisms underlying the regulation of EV71 infection. In this study, we initially show that the interleukin enhancer-binding factor 2 (ILF2) reduces EV71 50% tissue culture infective dose (TCID50) and attenuates EV71 plaque-formation unit (PFU), thereby repressing EV71 infection. Microarray data analyses show that ILF2 mRNA is reduced upon EV71 infection. Cellular studies indicate that EV71 infection represses ILF2 mRNA expression and protein production in human leukemic monocytes (THP-1) -differentiated macrophages and human rhabdomyosarcoma (RD) cells. In addition, EV71 nonstructural protein 2B interacts with ILF2 in human embryonic kidney (HEK293T) cells. Interestingly, in the presence of EV71 2B, ILF2 is translocated from the nucleus to the cytoplasm, and it colocalizes with 2B in the cytoplasm. Therefore, we present a distinct mechanism by which EV71 antagonizes ILF2-mediated antiviral effects by inhibiting ILF2 expression and promoting ILF2 translocation from the nucleus to the cytoplasm through its 2B protein.

NLRP3 inflammasome and lipid metabolism analysis based on UPLC-Q-TOF-MS in gouty nephropathy.

To determine the differences in plasma metabolism between healthy patients and patients with hyperuricaemia and gouty nephropathy, the present study identified differentially expressed metabolites associated with gouty nephropathy. Furthermore, the NLRP3 inflammasome signalling pathway in gouty nephropathy was explored, and the mechanism of hyperuricaemia­induced renal damage. Adult male patients examined between July 2016 and June 2017 were selected as the patient cohort for the present study from the Affiliated Bao'an Hospital of Shenzhen, Southern Medical University (Shenzhen, China). These patients were divided into three groups of 30 patients each: Control, hyperuricaemia and gouty nephropathy groups. The expression levels of NLRP3, ASC and caspase­1 mRNA and protein were detected in peripheral blood mononuclear cells, and the plasma levels of IL­1ß and IL­18. Ultra­performance liquid chromatography coupled with quadrupole time­of­flight mass spectrometry was used to determine differential levels of metabolites between patients from different groups, in order to identify potential biomarkers. The expression of the NLRP3 inflammasome in peripheral blood mononuclear cells, and the levels of IL­1ß and IL­18 in the plasma were increased in the gouty nephropathy group compared with the control and hyperuricaemia groups. In addition, 46 metabolites were identified as potential plasma metabolic biomarkers that were able to distinguish gouty nephropathy from hyperuricaemia. The majority of these metabolites were involved in lipid metabolism, in particular the activity of phospholipase Α2 and ß­oxidation. These data indicated that lipid metabolism and the NLRP3 inflammasome serve a pivotal role in gouty nephropathy. In addition, the results suggested that lipids may mediate the progression of gouty nephropathy through the activity of phospholipase A2, ß­oxidation and activation of the NLRP3 inflammasome.

Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation.

Activation of the NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 (collectively known as NLRP3) inflammasome plays a key role in host immune response, which is the first line of defense against cellular stresses and pathogen infections. However, excessive inflammasome activation damages host cells, and therefore it must be precisely controlled. Here, we discover that Cullin1 (CUL1), a key component of the Skp1-Cullin1-F-box E3 ligase, plays a critical role in controlling the NLRP3 inflammasome. CUL1 represses inflammasome assembly in cultured cells, suppresses NLRP3 function in human monocytic cell line macrophages, and attenuates inflammatory responses in mouse model. Detailed studies demonstrate that CUL1 interacts with NLRP3 and promotes NLRP3 ubiquitination, but not protein degradation, to repress the NLRP3 inflammasome activation. Moreover, upon inflammatory stimuli, including ATP and nigericin treatments, CUL1 disassociates from NLRP3 to release the repression of the NLRP3 inflammasome. Thus, this study reveals a distinct and unique mechanism underlying the control of systematic activation of the NLRP3 inflammasome.-Wan, P., Zhang, Q., Liu, W., Jia, Y., Ai, S., Wang, T., Wang, W., Pan, P., Yang, G., Xiang, Q., Huang, S., Yang, Q., Zhang, W., Liu, F., Tan, Q., Zhang, W., Wu, K., Liu, Y., Wu, J. Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation.

Sepsis-induced selective loss of NMDA receptors modulates hippocampal neuropathology in surviving septic mice.

BACKGROUND: Sepsis-induced neuroinflammation plays an important role in sepsis-related brain dysfunction. However, the molecules that are targeted during neuroinflammation resulting from sepsis-induced brain dysfunction remain unclear. Herein, we tried to investigate the expression and roles of NMDA receptor subunits during sepsis-related brain dysfunction. METHODS: Sepsis was induced by cecal ligation and perforation (CLP) or by a single intraperitoneal injection of lipopolysaccharide (LPS, 8 mg/kg) in C57BL/6J mice. The NMDA receptor co-agonist D-serine was injected intraperitoneally for 3 days (500 mg/kg/day) to compensate for the loss of NMDA receptors. The behaviors of mice were tested in the Barnes maze and in the open field test. The mice were euthanized at the indicated time points. The brains were collected to detect the following: the levels of synaptophysin and NMDA receptor subunits GluN2A, GluN2B and GluN1 (by Western blot and RT-PCR); the number of CA1 neurons (by Nissl staining); neuronal activity (by p-CREB staining); neuroinflammation (by staining of Iba-1 and inflammatory factors IL-1ß, TNF-α, NLRP3); and the levels of oxidative stress [by dihydroethidium (DHE)]. RESULTS: Sepsis selectively decreased the protein and mRNA levels of GluN2A, GluN2B and GluN1 but not the levels of synaptophysin or the neuronal number in the hippocampus of mice in either of the classic CLP-induced or LPS-induced sepsis models during the first 7 days after sepsis. Intraperitoneal injection of D-serine obviously limited the lipopolysaccharide-induced changes, including the impairment of learning and memory, the loss of NMDA receptor subunits, robust neuroinflammation, the levels of ROS stress and the decrease of p-CREB in the hippocampus of mice. CONCLUSION: These data suggest that the sepsis-induced selective loss of NMDA receptors modulates hippocampal neuropathology in the mice that survived sepsis, and the data show that NMDA receptors are potential targets for the improvement of brain dysfunction in sepsis survivors.

Monocytes, endoplasmic reticulum stress and metabolomics in dogs with multiple organ dysfunction syndrome treated by continuous venovenous hemodiafiltration.

OBJECTIVES: We tried to investigate the mechanism of continuous venovenous hemodiafiltration (CVVHDF) treatment in monocytes function, endoplasmic reticulum (ER) stress signaling pathways, metabolomics and histopathological changes of MODS dogs, and aimed to enhance the understanding of pathogenesis and provide novel avenues to potential therapies. METHODS: 12 male Beagle dogs were used to develop the stable models of MODS by using hemorrhagic shock plus resuscitation and endotoxemia, and assigned randomly to CVVHDF group (n=6) and MODS group (n=6). The dogs in CVVHDF group were given the typical CVVHDF treatment for 24h after the completion of endotoxin intravenous infusion, while those in MODS group were offered the i.v heparin instead only. Serum sample were collected at five time points, i.e. before anesthesia, 0h, 6h, 12h and 24h after the endotoxin injection (T1~T5, respectively), and meanwhile, the changes of mRNA, protein and human umbilical vein endothelial cells (HUVECs) apoptosis rates in JNK, CHOP and Caspase-12 were observed before and after interfered by RNA interference technology. RESULTS: The levels of DLA-DR, IL-1ß and IL-4 were higher than those in MODS group after the CVVHDF treatment, and the early and late apoptosis rates showed downward trend compared with MODS group. In vitro and prior to RNA interference (RNAi), the levels of mRNA and protein expression and HUVECs apoptosis rates of JNK, CHOP and Caspase-12 in CVVHDF group were significantly lower compared to T1 and MODS group respectively. However, the levels of mRNA and protein expression and HUVECs apoptosis rates were significantly lower than those before interfered by RNAi in both two groups. The serum levels of LPCs, ornithine, proline, methionine, etc. were down-regulated while carnitines, FFAs, PC, etc. were increased significantly in MODS (T4), and the serum levels of methionine, proline, arginine and lysine were increased while carnitine, LPCs, PCs, SMs and orthophosporic acid were decreased after 12 hours CVVHDF treatment (T4). CONCLUSION: CVVHDF treatment could reduce the apoptosis of the cells by enhancing the antigen presentation, improving the anti-inflammatory and proinflammatory imbalance and even correcting the metabolic disorder of amino acids and phospholipids.

The use of a second biopsy from the gastric body for the detection of Helicobacter pylori using rapid urease test.

INTRODUCTION: The use of an additional biopsy from the gastric body may help improve the detection of Helicobacter pylori during endoscopy. This study aimed to determine whether such an additional biopsy is necessary in routine rapid urease test (RUT), and whether acid suppression and antibiotic therapy affect RUT results. METHODS: Patients recruited had two gastric mucosal biopsies taken - one from the gastric antrum and the other from the gastric body. Each biopsy was placed into separate RUT kits. Information on previous or current use of proton-pump inhibitors, H2 receptor antagonist, bismuth and antibiotics was obtained. Patients on any of those drugs one week prior to endoscopy were considered to have a positive drug history (PDH). RESULTS: Of the 400 patients recruited, 311 had negative RUTs and 89 had at least one positive RUT. Between the PDH and negative drug history (NDH) groups, there was a significant difference in the distribution of the location of the biopsies that yielded positive RUTs (p = 0.023). The NDH group had a higher proportion of patients who had positive RUTs for both locations, whereas the PDH group had a higher proportion of patients who had positive RUTs for only one location. CONCLUSION: As RUT results are significantly affected by the use of acid suppression and antibiotic therapies, biopsies for RUT should be taken from both the gastric antrum and body to minimise false negative results.

Childhood autoimmune hepatitis in a paediatric unit of a tertiary care hospital.

INTRODUCTION: Although childhood autoimmune hepatitis (AIH) has been extensively investigated in the West, data on AIH in the East is lacking. We aimed to investigate AIH's clinical, biochemical and histological features, as well as its outcomes, in one of Singapore's two major paediatric units. METHODS: This was a retrospective study of children diagnosed with AIH in the paediatric unit of National University Hospital, Singapore, over the last 12 years. Children with de novo AIH after liver transplantation were excluded. The demographic and clinical features of the patients, and their laboratory, treatment and clinical outcomes were reviewed. RESULTS: This study comprised ten patients (six females, four males), with a median age of 5.1 (range 2.1-13.8) years at diagnosis. Five patients had inflammatory bowel disease (IBD). Seven patients had type 1 AIH, and three had autoimmune sclerosing cholangitis (ASC) and IBD; none had type 2 AIH. The median level of aspartate aminotransferase at diagnosis was 183 (range 45-2,649) U/L. Prednisolone 1 mg/kg/day was prescribed at diagnosis for eight patients. Two patients were lost to follow-up and were treated symptomatically when they re-presented with end-stage liver disease. Azathioprine or mycophenolate mofetil was prescribed after 3-7 months of treatment. Normalisation of aminotransferase levels took an average of 5.3 (range 1-39) months. CONCLUSION: AIH is a rare but important cause of liver pathology. Children in this region with elevated aminotransferases or unexplained hepatomegaly should be screened for AIH.

Childhood autoimmune hepatitis in a paediatric unit of a tertiary care hospital

<p><b>INTRODUCTION</b>Although childhood autoimmune hepatitis (AIH) has been extensively investigated in the West, data on AIH in the East is lacking. We aimed to investigate AIH's clinical, biochemical and histological features, as well as its outcomes, in one of Singapore's two major paediatric units.</p><p><b>METHODS</b>This was a retrospective study of children diagnosed with AIH in the paediatric unit of National University Hospital, Singapore, over the last 12 years. Children with de novo AIH after liver transplantation were excluded. The demographic and clinical features of the patients, and their laboratory, treatment and clinical outcomes were reviewed.</p><p><b>RESULTS</b>This study comprised ten patients (six females, four males), with a median age of 5.1 (range 2.1-13.8) years at diagnosis. Five patients had inflammatory bowel disease (IBD). Seven patients had type 1 AIH, and three had autoimmune sclerosing cholangitis (ASC) and IBD; none had type 2 AIH. The median level of aspartate aminotransferase at diagnosis was 183 (range 45-2,649) U/L. Prednisolone 1 mg/kg/day was prescribed at diagnosis for eight patients. Two patients were lost to follow-up and were treated symptomatically when they re-presented with end-stage liver disease. Azathioprine or mycophenolate mofetil was prescribed after 3-7 months of treatment. Normalisation of aminotransferase levels took an average of 5.3 (range 1-39) months.</p><p><b>CONCLUSION</b>AIH is a rare but important cause of liver pathology. Children in this region with elevated aminotransferases or unexplained hepatomegaly should be screened for AIH.</p>

The use of a second biopsy from the gastric body for the detection of Helicobacter pylori using rapid urease test

<p><b>INTRODUCTION</b>The use of an additional biopsy from the gastric body may help improve the detection of Helicobacter pylori during endoscopy. This study aimed to determine whether such an additional biopsy is necessary in routine rapid urease test (RUT), and whether acid suppression and antibiotic therapy affect RUT results.</p><p><b>METHODS</b>Patients recruited had two gastric mucosal biopsies taken - one from the gastric antrum and the other from the gastric body. Each biopsy was placed into separate RUT kits. Information on previous or current use of proton-pump inhibitors, H2 receptor antagonist, bismuth and antibiotics was obtained. Patients on any of those drugs one week prior to endoscopy were considered to have a positive drug history (PDH).</p><p><b>RESULTS</b>Of the 400 patients recruited, 311 had negative RUTs and 89 had at least one positive RUT. Between the PDH and negative drug history (NDH) groups, there was a significant difference in the distribution of the location of the biopsies that yielded positive RUTs (p = 0.023). The NDH group had a higher proportion of patients who had positive RUTs for both locations, whereas the PDH group had a higher proportion of patients who had positive RUTs for only one location.</p><p><b>CONCLUSION</b>As RUT results are significantly affected by the use of acid suppression and antibiotic therapies, biopsies for RUT should be taken from both the gastric antrum and body to minimise false negative results.</p>

Is hybrid surgery of the cervical spine a good balance between fusion and arthroplasty? Pilot results from a single surgeon series.

PURPOSE: Few studies have investigated the role of hybrid surgery (HS) that incorporates anterior cervical discectomy and fusion (ACDF) and artificial disc replacement (ADR) techniques. To our knowledge, this is the first study that provides a direct comparison of all three groups in terms of intra-operative parameters and outcomes with a minimum follow-up of 2 years. METHODS: Seven consecutive patients who underwent HS were matched with another seven patients who underwent ACDF and ADR based on levels of surgery. Prospective data on demographics, pre-operative and post-operative assessments, complications and functional scores (VAS, NDI, EQ-5D health score and index) were analysed using Mann-Whitney U test. Type I error was set at 5 %. RESULTS: Duration of surgery was significantly shorter for ACDF at 135 min (p = 0.025) compared with HS and ADR. ACDF also had greater blood loss when compared with ADR (p < 0.036). ADR has the shortest duration of hospitalization followed by HS and ACDF (p < 0.031). The HS group returned to work fastest (54 days) when compared with both ACDF (107 days) and ADR (73 days) with statistical significance seen between HS and ACDF (p = 0.035). Cervical range of motion (ROM) and functional scores did not show any significant differences. CONCLUSION: HS is comparable to ACDF and ADR in terms of safety and feasibility. Findings of shorter in-hospital stay and earlier return to work in HS group may be further explored in large, randomised controlled trials.

[Oral mucosa for hypospadias repair: a report of 19 cases].

OBJECTIVE: To investigate the indications and clinical effect of using oral mucosa as the graft in hypospadias repair. METHODS: We performed hypospadias repair using oral mucosa for 19 patients, 10 with the urethral opening at the root of the penis, 7 at the scrotum and 2 at the perineum. Of these patients, 8 had received urethroplasty once, and 6 twice, unsuccessfully, and 7 of them were complicated by penile chordee. The lower lip mucosa was used as the graft for 14 cases, and the buccal mucosa for the other 5. Fifteen cases received autologous oral mucosa onlay for repair, with the new urethra covered by the dartos coat of the dorsal skin. For the other 4 cases, whose urethral plates had been damaged in the previous operations, oral mucosa strips were used to substitute the urethral plates to get the penis straightened, followed by urethroplasty 6 months later. RESULTS: Of the 19 patients, 17 (89.5%) achieved satisfactory results, with a desirable shape of the penis and a vertical slit-like meatus at the tip of the glans. Chordee was corrected in all the patients. No urethral stricture was found during the 3 - 18 months follow-up. Urethral fistula occurred in 2 cases because of infection, but cured by surgical repair 6 months later. CONCLUSION: Using oral mucosa as the graft is an effective surgical option for hypospadias repair.