Soluble α-synuclein–antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia

Parkinson’s disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn–antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

Parkinson’s disease (PD) is characterized by accumulation of α-synuclein (αSyn; encoded by the SNCA gene) (1). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal cell death in part via microglial activation (2, 3). Moreover, misfolded proteins in general are thought to interact with brain microglia, triggering microglial activation that contributes to neurodegenerative disorders, although microglial phagocytosis may also initially clear aberrant proteins to afford some degree of protection (2, 4). Additionally, in Alzheimer’s disease (AD), amyloid-β peptide (Aβ) is thought to trigger similar processes in microglia (5–7); however, the mechanism for this trigger is still poorly understood.Microglial cells contribute to neuroinflammation, specifically that mediated by the inflammasome. In particular, the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome has been associated with several neurodegenerative disorders, although other types of inflammation may also be important in this regard (8). The NLRP3 inflammasome is a multiprotein complex that responds to cell stress and pathogenic stimuli to promote activation of caspase-1, which in turn mediates maturation and release of proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-18 (9–11). NLRP3 inflammasome activation is a two-step process, involving an initial priming step and a secondary trigger. Priming involves a proinflammatory stimulus, such as endotoxin, a ligand for Toll-like receptor 4 (TLR4), that increases the abundance of NLRP3 and promotes de novo synthesis of pro–IL-1β via nuclear factor κB (11). The secondary trigger promotes inflammasome complex assembly and caspase-1 activation that in turn mediates the cleavage of pro–IL-1β and subsequent release of mature IL-1β. There are various secondary triggers, including adenosine triphosphate (ATP), microparticles, and bacterial toxins, all of which somehow lead to mitochondrial damage and release of oxidized mitochondrial DNA (11). Neuroinflammation has been reported in both human PD and AD brains (12–15), and NLRP3 inflammasome activation in particular has been observed in mouse models of PD and AD (7, 16). Importantly, in these PD models, dopaminergic (DA) neurons in the substantia nigra are resistant to damage in NLRP3-deficient mice compared with wild-type (WT) mice (16). Interestingly, a recent report identified an NLRP3 polymorphism that confers decreased risk in PD (17). Several groups have reported that fibrillar αSyn can activate the NLRP3 inflammasome in mice and in human monocytes (18–22), but it remains unknown if human brain microglia can be activated in this manner. Critically, antibodies targeting misfolded proteins are being tested in human clinical trials for several neurodegenerative diseases, including AD and PD; however, it is still unclear how antibodies to αSyn might affect this inflammatory response. In this study, we characterized the response of human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) to oligomeric/fibrillar αSyn in vitro and in vivo, using engraftment of hiMG in humanized mice. We used these immunocompromised mice because they prevent human cell rejection and express three human genes that support human cell engraftment (23). We show that αSyn and, even more so, αSyn–antibody complexes activate the NLRP3 inflammasome. Moreover, this process is further sensitized by the presence of Aβ and its cognate antibodies. These observations are of heightened interest because recent studies have shown that both misfolded Aβ and αSyn are present in several neurodegenerative disorders such as AD and Lewy body dementia (LBD), a form of dementia that can occur in the setting of PD (24–26).     

Mapping and ablation of ventricular fibrillation—how and for whom?

The involvement of the Purkinje system in a subset of patients with idiopathic ventricular fibrillation or polymorphic VT/VF related to structural heart disease was first demonstrated in the pioneering work of Michel Haissaguerre and co-workers (Circulation 106:962–967, 2002 and Lancet 359:677–678, 2002). It is very important to identify these patients with recurrent episodes of ventricular fibrillation and/or ICD shocks with regard to the presence of triggering premature ventricular contractions (PVC), which may be amenable to mapping and catheter ablation by screening Holter and ICD recordings. The practical problem, which is frequently encountered, is the absence of these PVCs when the patients are brought to the EP lab. However, catheter ablation is an important adjunctive tool to antiarrhythmic drug treatment, beta blocker therapy, and general anesthesia in this setting. Local electrogram criteria related to this phenomenon have been identified guiding mapping and ablation (e.g., low amplitude, high-frequency Purkinje potentials preceding a closely coupled ventricular signal (Fig. 1a)). The favorable long-term follow-up after catheter ablation has been demonstrated in the setting of right and left ventricular Purkinje-related PVCs leading to polymorphic VT/VF (Leenhardt et al., Circulation 89:206–215, 1994) and also following myocardial infarction (Baensch et al., Circulation 108:3011–3016, 2003) and right ventricular outflow tract-associated VF (Noda et al., Journal of the American College of Cardiology 46:1288–1294, 2005). Most recently, epicardial ablation strategies leading to suppression of polymorphic VT/VF episodes related to the Brugada syndrome have been described irrespective to the presence of premature ventricular beats (Nademanee et al., Circulation 123:1270–1279, 2011).     

Isolated Roux loop pancreaticojejunostomy versus pancreaticogastrostomy after pancreaticoduodenectomy: a prospective randomized study

Objectives

The optimal strategy for the reconstruction of the pancreas following pancreaticoduodenectomy (PD) is still debated. The aim of this study was to compare the outcomes of isolated Roux loop pancreaticojejunostomy (IRPJ) with those of pancreaticogastrostomy (PG) after PD.

Methods

Consecutive patients submitted to PD were randomized to either method of reconstruction. The primary outcome measure was the rate of postoperative pancreatic fistula (POPF). Secondary outcomes included operative time, day to resumption of oral feeding, postoperative morbidity and mortality, and exocrine and endocrine pancreatic functions.

Results

Ninety patients treated by PD were included in the study. The median total operative time was significantly longer in the IRPJ group (320 min versus 300 min; P = 0.047). Postoperative pancreatic fistula developed in nine of 45 patients in the IRPJ group and 10 of 45 patients in the PG group (P = 0.796). Seven IRPJ patients and four PG patients had POPF of type B or C (P = 0.710). Time to resumption of oral feeding was shorter in the IRPJ group (P = 0.03). Steatorrhea at 1 year was reported in nine of 42 IRPJ patients and 18 of 41 PG patients (P = 0.029). Albumin levels at 1 year were 3.6 g/dl in the IRPJ group and 3.3 g/dl in the PG group (P = 0.001).

Conclusions

Isolated Roux loop PJ was not associated with a lower rate of POPF, but was associated with a decrease in the incidence of postoperative steatorrhea. The technique allowed for early oral feeding and the maintenance of oral feeding even if POPF developed.     

The effect of low concentrations versus high concentrations of local anesthetics for labour analgesia on obstetric and anesthetic outcomes: a meta-analysis

Introduction

The influence that different concentrations of labour epidural local anesthetic have on assisted vaginal delivery (AVD) and many obstetric outcomes and side effects is uncertain. The purpose of this meta-analysis was to determine whether local anesthetics utilized at low concentrations (LCs) during labour are associated with a decreased incidence of AVD when compared with high concentrations (HCs).

Methods

We searched PubMed, Ovid EMBASE, Ovid MEDLINE, CINAHL, Scopus, clinicaltrials.gov, and Cochrane databases for randomized controlled trials of labouring patients that compared LCs (defined as ≤ 0.1% epidural bupivacaine or ≤ 0.17% ropivacaine) of epidural local anesthetic with HCs for maintenance of analgesia. The primary outcome was AVD and secondary outcomes included Cesarean delivery, duration of labour, analgesia, side effects (nausea and vomiting, motor block, hypotension, pruritus, and urinary retention), and neonatal outcomes. The odds ratios (OR) or weighted mean differences (WMD) and 95% confidence intervals (CI) were calculated using random effects modelling. An OR < 1 or a WMD < 0 favoured LCs.

Results

Eleven studies met our criteria (eight bupivacaine and three ropivacaine studies), providing 1,145 patients in the LCs group and 852 patients in the HCs group for analysis of the primary outcome. Low concentrations were associated with a reduction in the incidence of AVD (OR = 0.70; 95% CI 0.56 to 0.86; P < 0.001). There was no difference in the incidence of Cesarean delivery (OR 1.05; 95% CI 0.82 to 1.33; P = 0.7). The LCs group had less motor block (OR 3.9; 95% CI 1.59 to 9.55; P = 0.003), greater ambulation (OR 2.8; 95% CI 1.1 to 7.14; P = 0.03), less urinary retention (OR 0.42; 95% CI 0.23 to 0.73; P = 0.002), and a shorter second stage of labour (WMD ?14.03; 95% CI ?27.52 to ?0.55; P = 0.04) compared with the HCs group. There were no differences between groups in pain scores, maternal nausea and vomiting, hypotension, fetal heart rate abnormalities, five-minute Apgar scores, and need for neonatal resuscitation. One-minute Apgar scores < 7 favoured the HCs group (OR 1.53; 95% CI 1.07 to 2.21; P = 0.02), and there was more pruritus in the LCs group (OR 3.36; 95% CI 1.00 to 11.31; P = 0.05).

Conclusion

When compared with HCs of local anesthetics, the use of LCs for labour epidural analgesia reduces the incidence of AVD. This may be due to a reduction in the amount of local anesthetic used and the subsequent decrease in motor blockade. We therefore recommend the use of LCs of local anesthetics for epidural analgesia to optimize obstetric outcome.     

A randomized study of open-flap surgery of 32 intrabony defects with and without adjunct bovine bone mineral treatment

Background: Bovine bone mineral (BBM) is extensively used as a filler material in periodontal reconstructive surgery of intrabony defects. Data are mostly available on the combined use of BBM with other biomaterials. The aim of this study is to evaluate healing after open‐flap debridement (OF) of intrabony periodontal defects alone or with adjunct treatment with BBM. Methods: After initial treatment, 32 patients with 32 intrabony periodontal defects participated in the study. Full‐thickness flaps were raised and root surfaces and defects were debrided. Patients were then randomly assigned to treatment groups, either OF alone or combined with defect fill with BBM, and followed in a strict postoperative maintenance care program for 12 months. Results: At 12 months, a mean ± SE gingival recession of 1.1 ± 0.3 mm in OF and 0.9 ± 0.4 mm in BBM occurred. Probing depth reduction was 4.0 ± 0.5 mm in OF and 3.2 ± 0.7 mm in BBM. Gain in clinical attachment level was 2.8 ± 0.6 mm in OF and 2.3 ± 0.8 mm in BBM. Probing bone level was reduced by 2.7 ± 0.7 mm in OF and 1.8 ± 1.1 mm in BBM. None of the above parameters showed significant intergroup differences. In contrast, radiographic defect depth change was significantly greater in BBM (3.4 ± 2.3 mm) than in OF (1.9 ± 1.7 mm). Conclusions: Both treatments resulted in improved periodontal conditions. The adjunctive use of BBM in this study did not enhance the clinical result compared to OF alone.