Hypomethylation of NLRP3 gene promoter discriminates glucocorticoid‐resistant from glucocorticoid‐sensitive idiopathic nephrotic syndrome patients

AbstractTo assess whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid (GC)‐resistant from GC‐sensitive idiopathic nephrotic syndrome (INS), patients with minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS), we measured the methylation level of NLRP3 promoter in DNA from peripheral blood cells of 10 adult patients with GC‐resistant FSGS already in hemodialysis and 18 patients with GC‐sensitive INS (13 MCD/5 FSGS) and in 21 pediatric patients with INS with MCD/FSGS before starting any treatment. Association of NLRP3 inflammasome with GC resistance was recapitulated in vitro in monocytic cell lines (THP‐1 and U937). In both adults and pediatric patients, NLRP3 promoter methylation was significantly reduced in GC‐resistant compared with GC‐sensitive patients. Indeed, NLRP3 methylation distinguished GC‐resistant and GC‐sensitive patients (area under the receiver operating characteristic curve [AUROC] 86.7% in adults, p = 0.00019, and 73.5% in children, p = 0.00097). NLRP3 knock‐down augmented sensitivity to GCs in THP‐1 cells, whereas NLRP3 inflammasome activation lowered GC receptor concentration, increasing GC resistance in U937 cells. Our results uncovered a new biological mechanism by which patients with INS may acquire GC resistance, that could be used in future as a novel noninvasive diagnostic tool. Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

☑ Approximately 80% of patients with idiopathic nephrotic syndrome (INS) respond to glucocorticoids, with the remaining 20% being steroid‐resistant.

• WHAT QUESTION DID THIS STUDY ADDRESS?

☑ Whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid‐resistant from glucocorticoid (GC)‐sensitive INS.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

☑ In both adults and children, NLRP3 promoter methylation was significantly reduced in leukocytes of patients with GC‐resistant compared with GC‐sensitive INS. NLRP3 inflammasome activation lowered GC receptor concentration and augmented GC resistance, whereas NLRP3 knockdown increased sensitivity to GCs in cell lines representative of monocytes (U937 and THP1).

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

☑ Our findings uncovered a new biological mechanism whereby patients with INS may develop resistance to GCs that could be used in the future as a novel noninvasive diagnostic tool.     

Expression of neuromedins S and U and their receptors in the hypothalamus and endocrine glands of the rat

Neuromedin S (NMS) and neuromedin U (NMU) are regulatory peptides that share the C-terminal amino-acid sequence and act via common G protein-coupled receptors called NMUR1 and NMUR2. Semiquantitative real time-PCR showed that in the rat hypothalamus and testis NMS gene expression was markedly higher than that of the NMU gene, while the reverse occurred in the anterior pituitary and thyroid gland. Low expression of both genes was detected in the thymus, adrenal gland and ovary, whereas in the pancreatic islets only the expression of NMU mRNA was detected. In the rat hypothalamus the expression of the NMUR2 gene was strikingly higher than that of the NMUR1 gene; in contrast, in the testis and ovary the very low expression of NMUR2 contrasted with the relatively high expression of the NMUR1 gene. In the other glands examined only expression of the NMUR1 gene was found. The marked differences in the level of expression of NMU, NMS and their receptors in the hypothalamus and endocrine glands of the rat suggest that in this species such neuromedins may play different roles in the functional regulation of neuroendocrine axes.     

Cefepime versus carbapenems for the treatment of urinary tract infections caused by extended-spectrum β-lactamase-producing enterobacteriaceae

The aim of this study was to evaluate the effectiveness of cefepime compared with carbapenems for the management of urinary tract infections (UTIs) caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. This was a single-centre, retrospective study among patients with a documented ESBL-producing Enterobacteriaceae UTI between 1 July 2014 and 31 January 2017. Adult patients who received either cefepime or a carbapenem for symptomatic UTI were included in the analysis. The primary endpoint was clinical failure, defined by persistence of initial UTI symptoms that required escalation of therapy. Secondary endpoints included microbiological failure and relapse within 30 days. Of a total of 106 patients included in the study, 17 received cefepime and 89 received a carbapenem. None of the patients in either group experienced clinical or microbiological failure. Relapse occurred in six patients in the carbapenem group and none in the cefepime group. In conclusion, cefepime was comparable with carbapenems in the treatment of UTIs caused by ESBL-producing Enterobacteriaceae. Its use as a carbapenem-sparing agent for this indication should be further explored.     

Evaluation of the stimulating effect of a low dose of secretin compared to the standard dose on the exocrine pancreas with MRCP: preliminary results in normal subjects (MRCP quantification of secretin stimulation)

Background Secretin administration during MRCP improves depiction of pancreatic ducts and allows assessment of pancreatic exocrine secretions. However, secretin increases the cost of secretin-enhanced MRCP (S-MRCP). The aim of this study was to quantify using MRCP the stimulating effect of 0.3 CU/kg of secretin and to compare it to the standard dose (1 CU/kg). Methods Ten healthy volunteers underwent four S-MRCP (two for each dose). Pancreatic exocrine secretions were quantified by pancreatic flow output (PFO) and total excreted volume (TEV), derived from a linear regression between MR calculated pancreatic exocrine volumes and time. Two readers analysed individually all sets of images. Results A linear increase of pancreatic exocrine volumes was observed after administration of both doses of secretin. Intra-individual and inter-observer differences were not statistically significant (P > 0.05). The mean PFO and TEV, given for reader 1 only, were, respectively, 6.9 ± 1.5 mL/min and 103 ± 26 mL for the standard dose and 6.1 ± 1.2 mL/min and 84 ± 19 mL for the dose of 0.3 CU/kg. Differences between PFO and TEV obtained with the two doses of secretin were statistically significant for both readers (P < 0.05). Conclusions MRCP is a non-invasive, reproducible tool that allows quantification of pancreatic exocrine secretions during secretin stimulation. PFO and TEV calculated with the low dose were still in the range of previously reported reference values. The administration of 0.3 CU/kg of secretin can reduce significantly the cost of S-MRCP.     

Storage-dependent remodeling of the red blood cell membrane is associated with increased immunoglobulin G binding, lipid raft rearrangement, and caspase activation

BACKGROUND: The elucidation of the storage lesion is important for the improvement of red blood cell (RBC) storage. Ex vivo storage is also a model system for studying cell-signaling events in the senescence and programmed cell death of RBCs. The membrane hosts critical steps in these mechanisms and undergoes widespread remodeling over the storage period. STUDY DESIGN AND METHODS: Fresh and CPDA-stored RBCs from 21 blood donors were evaluated as whole cells, membrane ghosts, and cytoskeletons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunofluorescence microscopy, and in situ assays. Band 3 content, immunoglobulin G (IgG) content, specific protein movement to and from the membrane, and caspase system activation were measured. RESULTS: During storage, Band 3 protein was aggregated and its content decreased as did the content of several lipid raft-related proteins. IgG binding to the membrane increased. Sorcin and synexin moved from the cytosol to the membrane, stomatin and flotillins left the membrane, the Fas protein was oligomerized, and caspase was activated. CONCLUSION: The remodeling of the RBC membrane during storage includes loss and oxidative cross-linking of Band 3 as well as IgG binding. This process occurs with lipid raft development and loss and is probably driven by caspase activation. Oxidative injury appears to be an important driver of RBC aging during storage.     

Frequency and location of traumatic ulcerations following placement of complete dentures

PURPOSE: To determine the location of mucosal injuries that appear following placement of complete dentures, as well as the number of adjustments necessary to achieve patient comfort. The frequency of mucosal injuries in female and male patients and their connection with clinical anatomic features were also investigated. MATERIALS AND METHODS: Sixty-one completely edentulous healthy patients who wore dentures (47 women and 14 men) took part in the study; 122 newly fabricated complete maxillary and mandibular dentures were investigated. All patients were seen for a 1-week adjustment appointment. Areas where signs of denture-induced mucosal injuries appeared were marked on an anatomic illustration. The follow-up period was in 1-week increments as deemed necessary by the patient. Associations between variables were analyzed with analysis of variance. Results were recorded as mean + SD. Statistical significance was set at P < or = .05. RESULTS: Eighty-seven percent of the dentures required adjustment at week 1, 50% at week 2, and only 7% at week 3. No patients required a further visit. Most frequently injured maxillary areas were the vestibular sulcus (41%), maxillary tuberosity (21%), and hamular notch (12%). In the mandible, the most frequently injured areas were the retromylohyoid area (17%), lingual sulcus (14%), and vestibular sulcus (13%). Denture-induced irritations were detected in a higher ratio in the mandible (P < .001), especially in male denture wearers at the first adjustment (P < .05). Men had a higher ratio of lesions at the region of the maxillary vestibular sulcus between the labial and buccal frenum and at the mandibular vestibular sulcus of the buccal shelf region (P < .001). CONCLUSIONS: Denture-induced irritations appeared most often in the vestibular sulcus of the maxilla and mandible, indicating that it is necessary to evaluate the area of the facial seal of the prosthesis by applying a medium- or a heavy-pressure indicator paste to the borders, and to make adjustments at the delivery stage and subsequent adjustment appointments. Denture placement must not be the final patient-clinician encounter when treating with complete dentures. Denture adjustments are very important clinical phases of denture fabrication and essential in patient care.     

Protection from angiotensin II-mediated vasculotoxic and hypertensive response in mice lacking PI3Kgamma

Hypertension affects nearly 20% of the population in Western countries and strongly increases the risk for cardiovascular diseases. In the pathogenesis of hypertension, the vasoactive peptide of the renin-angiotensin system, angiotensin II and its G protein-coupled receptors (GPCRs), play a crucial role by eliciting reactive oxygen species (ROS) and mediating vessel contractility. Here we show that mice lacking the GPCR-activated phosphoinositide 3-kinase (PI3K)gamma are protected from hypertension that is induced by administration of angiotensin II in vivo. PI3Kgamma was found to play a role in angiotensin II-evoked smooth muscle contraction in two crucial, distinct signaling pathways. In response to angiotensin II, PI3Kgamma was required for the activation of Rac and the subsequent triggering of ROS production. Conversely, PI3Kgamma was necessary to activate protein kinase B/Akt, which, in turn, enhanced L-type Ca(2+) channel-mediated extracellular Ca(2+) entry. These data indicate that PI3Kgamma is a key transducer of the intracellular signals that are evoked by angiotensin II and suggest that blocking PI3Kgamma function might be exploited to improve therapeutic intervention on hypertension.     

Studies in Emergency Department Data Collection: Shared versus Split Responsibility for Patient Enrollment

OBJECTIVES: To compare patient enrollment in six clinical studies using shared coverage (24 emergency department [ED] rooms-two students share enrollment responsibility) with enrollment using split coverage (12 rooms each per student). The academic associate (AA) program uses undergraduate students to collect data for clinical studies in the ED by providing double coverage 16 hours/day, seven days/week. Prior studies have shown that this system captures >85% of eligible patients. Methods to obtain closer to 100% enrollment are desired. METHODS: During consecutive 15-day periods with the same 24 AAs, the daily ED census, hours of AA coverage, and enrollment in each of six studies were evaluated prospectively in the ED. Data are presented as means with 95% confidence intervals (CIs). RESULTS: There was no difference between the shared and split enrollment periods with respect to hours of AA coverage (30.3 vs. 30.7 hours/day; p = 0.7) or average daily ED census (133.7 vs. 141.8; p = 0.15). Overall, the percentages of ED patients recruited for study participation were not different depending on whether the split versus shared recruitment strategy was used (907 patients recruited out of 2005 ED patients (45.2%; 95% CI = 43.0 to 47.4) vs. 937 of 2127 (44.0%; 95% CI = 41.9 to 46.1). The 95% CI for the 1.2% difference was -1.8% to 4.2%. Patient enrollments in six individual studies were similar regardless of recruitment strategy. Following the 30-day trial, AAs were surveyed: 17 of 24 (71%) found the split strategy to be "more helpful in enrolling subjects," and 20 of 24 (83%) found split strategy helped them "keep better track" of patients. CONCLUSIONS: Study subject enrollment was not affected by the use of either the shared or split responsibility strategy for recruitment. Students generally preferred the split strategy because it was more helpful and easier to monitor. Therefore, this may be the best option for similar student-oriented data collection programs.