Human macrophage and dendritic cell-specific silencing of high-mobility group protein B1 ameliorates sepsis in a humanized mouse model

Hypersecretion of cytokines by innate immune cells is thought to initiate multiple organ failure in murine models of sepsis. Whether human cytokine storm also plays a similar role is not clear. Here, we show that human hematopoietic cells are required to induce sepsis-induced mortality following cecal ligation and puncture (CLP) in the severely immunodeficient nonobese diabetic (NOD)/SCID/IL2Rγ^−/− mice, and siRNA treatment to inhibit HMGB1 release by human macrophages and dendritic cells dramatically reduces sepsis-induced mortality. Following CLP, compared with immunocompetent WT mice, NOD/SCID/IL2Rγ^−/− mice did not show high levels of serum HMGB1 or murine proinflammatory cytokines and were relatively resistant to sepsis-induced mortality. In contrast, NOD/SCID/IL2Rγ^−/− mice transplanted with human hematopoietic stem cells [humanized bone marrow liver thymic mice (BLT) mice] showed high serum levels of HMGB1, as well as multiple human but not murine proinflammatory cytokines, and died uniformly, suggesting human cytokines are sufficient to induce organ failure in this model. Moreover, targeted delivery of HMGB1 siRNA to human macrophages and dendritic cells using a short acetylcholine receptor (AchR)-binding peptide [rabies virus glycoprotein (RVG)-9R] effectively suppressed secretion of HMGB1, reduced the human cytokine storm, human lymphocyte apoptosis, and rescued humanized mice from CLP-induced mortality. siRNA treatment was also effective when started after the appearance of sepsis symptoms. These results show that CLP in humanized mice provides a model to study human sepsis, HMGB1 siRNA might provide a treatment strategy for human sepsis, and RVG-9R provides a tool to deliver siRNA to human macrophages and dendritic cells that could potentially be used to suppress a variety of human inflammatory diseases.Sepsis is an important cause of mortality in intensive-care units, with more than 750,000 individuals developing severe sepsis in North America annually and a mortality rate varying between 35 and 50% (1, 2). The pathogenesis of sepsis includes countless disturbances of the host immune system starting with a harmful, infection-triggered exaggerated inflammatory cascade that results in tissue injury and rapidly leads to massive apoptosis of immune cells (2, 3). This is followed by a secondary immune paralysis phase accompanied by uncontrolled growth of bacteria and tissue damage. Although therapy to suppress the immediate cytokine response, such as treatment with TNF and IL-1β antibodies, have failed in clinical trials (4–6), it has now come to be recognized that, at least in animal models, high-mobility group protein 1 (HMGB1), which is secreted from macrophages and dendritic cells (DCs) but not lymphocytes late in the disease, acts as a master regulator of late and sustained cytokine storm, up-regulating many cytokines including TNF-α, IL-6, IL-1β, and IL-8 (reviewed in refs. 7–11). In fact, injection of mice with HMGB1 is enough to induce the lethal organ damage seen in sepsis (12), whereas treatment with neutralizing HMGB1 antibody can rescue mice and rats from experimental sepsis (13, 14). However, although HMGB1 is also secreted in human sepsis (12), its role in sepsis pathogenesis or the impact of its neutralization on human cells remain unclear.RNA interference can be used to silence virtually any gene, including multiple genes, as long as a way can be found to introduce small interfering (si)RNAs into relevant cell types in vivo without toxicity. Several advances have been made in developing methods to deliver siRNA in vivo to different cell types, most successfully to the liver cells (reviewed in refs. 15–17). A lipid-like nanoparticle called C12-200, which had been developed for liver-specific delivery of siRNA, was recently also shown to deliver siRNA to murine monocytes, and silencing C-C chemokine receptor type 2 (CCR2) in monocytes using this reagent was effective in reducing atherosclerosis, islet transplantation and tumors (18). Whether this reagent also targets human DCs and monocytes/macrophages is unclear. We have reported previously that a short 29-aa peptide derived from the rabies virus glycoprotein (RVG), fused to 9R residues (RVG-9R), can deliver siRNA to murine macrophages and brain cells by specific binding to its ligand acetylcholine receptor (AchR) (19, 20). Because AchR is also expressed on human macrophages and DCs (21) and also because the acetylcholine-binding site on the α7 subunit is highly conserved, we reasoned that RVG-9R might also be used to target human macrophages and DCs. In this study, we validate this hypothesis in vitro, as well as in vivo, using human hematopoietic stem cell–engrafted nonobese diabetic NOD/SCID/IL2Rγ^−/− mice that lack mouse innate and adaptive immune systems (22). More importantly, we also show that silencing human HMGB1 using this delivery reagent in this mouse model substantially reduces human lymphocyte apoptosis and cytokine storm and protects mice from sepsis-induced mortality.     

Safety of dental extractions in coronary drug-eluting stenting patients without stopping multiple antiplatelet agents

Background:

The risk of excessive bleeding prompts physicians to stop multiple antiplatelet agents before minor surgery, which puts coronary stenting patients at risk for adverse thrombotic events.

Hypothesis:

We hypothesized that most dental extractions can be carried out safely without stopping multiple antiplatelet agents.

Methods:

All dental extraction patients who had undergone coronary stenting and who were also on oral multiple antiplatelet agents therapy were enrolled. One hundred patients underwent dental procedures without stopping antiplatelet agents. All wounds were sutured and followed up at 24 hours, 1 week, and 1 month after the procedure. There were 2233 patients who had not taken oral antiplatelet agents from a health promotion center and had teeth extracted by the same method. After performing propensity‐score matching for the entire population, a total of 100 matched pairs of patients were created. The primary outcome was a composite of excessive intraextraction blood loss, transfusion, and rehospitalization for bleeding, and the secondary outcome was a composite of death, nonfatal myocardial infarction, target lesion revascularization, and stent thrombosis within 1 month after the procedure.

Results:

There were 2 excessive intraextraction bleeding cases that continued at the extraction site for 4 and 5 hours, respectively, in the coronary stenting patients, and 1 excessive intraextraction bleeding case that continued for 3 hours in the control patients. There were no cases of transfusion, rehospitalization for bleeding, or major cardiovascular events for the 2 propensity‐matched groups.

Conclusions:

We found that most dental extractions in coronary stenting patients can be carried out safely without stopping multiple antiplatelet agents. The authors have no conflicts of interest to disclose. This study was supported in part by a grant from Yuhan Corporation, Ltd., Seoul, The Republic of Korea.     

Change of coronary flow velocity during the cold pressor test is related to endothelial markers in subjects with chest pain and a normal coronary angiogram

Background:

Several studies demonstrated that endothelial or atherosclerotic biomarkers, including plasma free insulin‐like growth factor‐I(IGF‐I), soluble CD40 ligand (sCD40L), adiponectin, and leptin have an influence on coronary endothelial function.

Hypothesis:

The aim of the present study was to investigate whether change of coronary flow velocity of the distal left anterior descending artery (LAD) during the cold pressor test (CPT) with transthoracic Doppler echocardiography (TTE) was associated with these biomarkers in subjects with chest pain and a normal coronary angiogram.

Methods:

In 190 subjects (mean age, 54±11 years; male:female, 113:77) with chest pain and a normal coronary angiogram, peak diastolic velocity (PDV) of the distal LAD during the CPT with TTE was assessed. Acetylcholine provocation test was performed in 58 subjects (mean age, 51±10 years) who were clinically suspected of vasospasm. CPT%PDV was defined as the percent change in PDV during the CPT. Associations between CPT%PDV and clinical parameters were analyzed.

Results:

According to multiple regression analysis, CPT%PDV was associated with plasma free IGF‐I in the entire study population (β=0.295, P<0.001 in all subjects; β=0.341, P=0.001 in males; β=0.243, P=0.037 in females; β=0.303, P=0.002 in nonsmokers; and β=0.256, P=0.047 in smokers), and sCD40L in males (β=?0.269, P=0.008)and smokers (β=?0.261, P=0.046). Subjects with vasospasm to intracoronary acetylcholine had lower plasma free IGF‐I(6.9±3.3 vs 8.9±3.4, P=0.026) and CPT%PDV (8.8±24.9 vs 52.7±26.0, P<0.001) than the others. Plasma adiponectin and leptin were not associated with CPT%PDV.

Conclusions:

Change of coronary flow velocity assessed using the CPT with TTE may be related to endothelial markers, especially plasma free IGF‐I. © 2011 Wiley Periodicals, Inc. Additional Supporting Information may be found in the online version of this article. The authors have no funding, financial relationships, or conflicts of interest to disclose.

     

Endoscopic Submucosal Dissection of Gastric Neoplasia Involving the Pyloric Channel by Retroflexion in the Duodenum

Background  

Tumors involving the pyloric channel have been considered as difficult lesions for successful endoscopic resection. We studied the feasibility of endoscopic submucosal dissection (ESD) using retroflexion in the duodenum to resect the gastric neoplasia involving the pyloric channel.