Evidence against a role for NLRP3-driven islet inflammation in db/db mice.

OBJECTIVES: Type 2 diabetes (T2D) is associated with chronic, low grade inflammation. Activation of the NLRP3 inflammasome and secretion of its target interleukin-1ß (IL-1ß) have been implicated in pancreatic ß cell failure in T2D. Specific targeting of the NLRP3 inflammasome to prevent pancreatic ß cell death could allow for selective T2D treatment without compromising all IL-1ß-associated immune responses. We hypothesized that treating a mouse model of T2D with MCC950, a compound that specifically inhibits NLRP3, would prevent pancreatic ß cell death, thereby preventing the onset of T2D. METHODS: Diabetic db/db mice were treated with MCC950 via drinking water for 8 weeks from 6 to 14 weeks of age, a period over which they developed pancreatic ß cell failure. We assessed metabolic parameters such as body composition, glucose tolerance, or insulin secretion over the course of the intervention. RESULTS: MCC950 was a potent inhibitor of NLRP3-induced IL-1ß in vitro and was detected at high levels in the plasma of treated db/db mice. Treatment of pre-diabetic db/db mice with MCC950, however, did not prevent pancreatic dysfunction and full onset of the T2D pathology. When examining the NLRP3 pathway in the pancreas of db/db mice, we could not detect an activation of this pathway nor increased levels of its target IL-1ß. CONCLUSIONS: NLRP3 driven-pancreatic IL-1ß inflammation does not play a key role in the pathogenesis of the db/db murine model of T2D.

Inhibiting the NLRP3 inflammasome with MCC950 promotes non-phlogistic clearance of amyloid-ß and cognitive function in APP/PS1 mice.

Activation of the inflammasome is implicated in the pathogenesis of an increasing number of inflammatory diseases, including Alzheimer's disease (AD). Research reporting inflammatory changes in post mortem brain tissue of individuals with AD and GWAS data have convincingly demonstrated that neuroinflammation is likely to be a key driver of the disease. This, together with the evidence that genetic variants in the NLRP3 gene impact on the risk of developing late-onset AD, indicates that targetting inflammation offers a therapeutic opportunity. Here, we examined the effect of the small molecule inhibitor of the NLRP3 inflammasome, MCC950, on microglia in vitro and in vivo. The findings indicate that MCC950 inhibited LPS+Aß-induced caspase 1 activation in microglia and this was accompanied by IL-1ß release, without inducing pyroptosis. We demonstrate that MCC950 also inhibited inflammasome activation and microglial activation in the APP/PS1 mouse model of AD. Furthermore, MCC950 stimulated Aß phagocytosis in vitro, and it reduced Aß accumulation in APP/PS1 mice, which was associated with improved cognitive function. These data suggest that activation of the inflammasome contributes to amyloid accumulation and to the deterioration of neuronal function in APP/PS1 mice and demonstrate that blocking assembly of the inflammasome may prove to be a valuable strategy for attenuating changes that negatively impact on neuronal function.

Inflammasome activity is essential for one kidney/deoxycorticosterone acetate/salt-induced hypertension in mice.

BACKGROUND AND PURPOSE: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1ß and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1ß and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH: Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. KEY RESULTS: 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1ß, as well as protein levels of active caspase-1 and mature IL-1ß. Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. CONCLUSIONS AND IMPLICATIONS: Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1ß pathway as a potential therapeutic target in hypertension.

Strain- and host species-specific inflammasome activation, IL-1ß release, and cell death in macrophages infected with uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1ß (IL-1ß) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1ß secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1ß secretion pathway in human macrophages. This has important implications for understanding UTI in humans.

High-performance liquid chromatographic separation of natural and synthetic desulphoglucosinolates and their chemical validation by UV, NMR and chemical ionisation-MS methods.

Methods are described for the optimised extraction, desulphation and HPLC separation of desulphoglucosinolates. These methods provide rapid separation, identification and quantitative measurements of glucosinolates extracted from Brassica napus L and related crops, of unusual glucosinolates found in crucifer weed species, and also of synthetic alkylglucosinolates. The desulphoglucosinolates used in these studies were either chemically synthesised (at least one example from each major structural class), or purified from various plant sources. Validation of the identities of the desulphoglucosinolates was by comparison of retention times with standards, and by UV, 1H- and 13C-NMR and chemical ionisation MS analysis. A list of useful species, and the specific tissues, from which high concentrations of standards can be extracted is included.

Does labetalol influence the development of proteinuria in pregnancy hypertension? A randomised controlled study.

It is the development of proteinuria in pregnancy-induced hypertension which is associated with an increased perinatal mortality. There is some evidence to suggest that labetalol may diminish the amount of proteinuria in patients who have already developed proteinuric pre-eclampsia. A randomised controlled study design was used to investigate whether labetalol treatment, started when a persistent diastolic blood pressure greater than 90 mmHg was observed, influenced the subsequent development of proteinuria. One hundred and fourteen women with singleton pregnancies and hypertension in the absence of proteinuria were randomised to receive either labetalol or no antihypertensive therapy. At recruitment maternal age, blood pressure and gestation were similar in both the labetalol and control groups. There was no difference in the frequency, quantity or timing of subsequent proteinuria between treatment and control groups. Overall 34% of primigravidae and 10% of parous women developed proteinuria. Labetalol did, however, control the blood pressure in 45 of the 51 treated women (88%) within 24 h. This effect was often shortlived requiring dose escalation after 3 to 5 days in the majority of cases. Labetalol was well tolerated and no significant maternal toxicity was noted.

After the epidemic: follow up study of HIV seroprevalence and changing patterns of drug use.

OBJECTIVE: To follow up known intravenous drug users to determine current health state and drug use, compare characteristics with those of recent drug users, and examine HIV exposure and serostate. DESIGN: Subjects were identified from conventional general practice records and recruited from 1980 to the end of 1985; they were followed up during 1987 and 1988 and compared with drug users identified in the same way but recruited after 1985. SETTING: General practice and community in north west Edinburgh. Follow up conducted throughout the United Kingdom. SUBJECTS: Subjects known to have injected illegal drugs before 1986 (n = 203) and since that time. MAIN OUTCOME MEASURES: Mortality from and prevalence of HIV seropositivity and various parameters indicative of abstinence. RESULTS: Of the 203 subjects in the follow up group, 189 (93%) were traced; 16 (8%) had died and the remaining 173 (85%) were interviewed. In all, 146 (72% of the follow up cohort) had been tested for HIV antibodies, 94 (64%) having positive and 52 (36%) negative results; 57 (28%) had not been tested. Of the 65 subjects in the recently recruited group, 51 (79%) had been tested for HIV, 15 (29%) having positive results. A further 21 (43%) were currently negative for HIV antibody but still at risk. Thirty three (19%) of those followed up were confirmed abstinent, although more (about half) showed evidence of diminished drug injecting. Age correlated strongly with abstinence (p less than 0.001). One third of the group currently used cannabis, buprenorphine, dihydrocodeine, or diazepam. When the two groups were analysed together there was a strong association between the date of starting injecting and HIV seropositivity (chi 3 = 23.81, df = 2, p less than 0.001), with a peak around 1980-3. CONCLUSIONS: Although only a fifth of the followed up group were convincingly abstinent, a much larger group showed evidence of prolonged periods of remission. Overall, much use of oral drugs was confirmed and worrying trends towards taking buprenorphine and benzodiazepines were evident. The peak incidence of starting drug use and the comparatively low seroprevalence of HIV in the newer drug users probably explain the anomalous high seroprevalence in Edinburgh drug users during 1980-5. The epidemic of HIV during the first half of the 1980s in the group suggests that the virus was probably being transmitted because of a pattern of behaviour. Changing patterns of HIV transmission suggest a need to concentrate on heterosexual transmission as the main problem in the future.

Amoxycillin-clavulanic acid (Augmentin) versus metronidazole as prophylaxis in hysterectomy: a prospective, randomized clinical trial.

In order to determine the most effective regimen for the prevention of infection after elective hysterectomy, 300 patients were randomly assigned to receive three perioperative doses of either amoxycillin-clavulanic acid (1.2 g intravenous) or metronidazole (1 g suppository). Of the 280 patients who were assessable 138 were given amoxycillin-clavulanic acid and 142 received metronidazole; 268 underwent abdominal hysterectomy and 12 had vaginal hysterectomy. Patients in the amoxycillin-clavulanic acid group had significantly less infectious morbidity (13.8%) than those in the metronidazole group (33.1%). There were also statistically significant differences in favour of amoxycillin-clavulanic acid with respect to operative site infection, duration of hospital stay, need for postoperative antimicrobials, and surgery for operative site infection. But for one isolate of Bacteroides fragilis, all pathogens isolated from wound infections in the metronidazole group were aerobes. No anaerobes were isolated from patients in the amoxycillin-clavulanic acid group. The results suggest that prophylaxis for hysterectomy should consist of an agent, or combination of agents, with activity against both aerobic and anaerobic bacteria. Amoxycillin-clavulanic acid fulfils this criterion and appears to be effective and safe.