Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.

Long-Acting IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Granulocyte Colony-Stimulating Factor or AMD3100.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) has become increasingly important for hematopoietic cell transplantation. Current mobilization approaches are insufficient because they fail to mobilize sufficient numbers of cells in a significant fraction of patients and are biased toward myeloid immune reconstitution. A novel, single drug mobilization agent that allows a more balanced (myeloid and lymphoid) reconstitution would therefore be highly favorable to improve transplantation outcome. In this present study, we tested commercially available IL-33 molecules and engineered novel variants of IL-33. These molecules were tested in cell-based assays in vitro and in mobilization models in vivo. We observed for the first time that IL-33 treatment in mice mobilized HSPCs and common myeloid progenitors more efficiently than clinical mobilizing agents granulocyte colony-stimulating factor (G-CSF) or AMD3100. We engineered several oxidation-resistant IL-33 variants with equal or better in vitro activity. In vivo, these variants mobilized HSPCs and, interestingly, also hematopoietic stem cells, common lymphoid progenitor cells, and endothelial progenitor cells more efficiently than wild-type IL-33 or G-CSF. We then engineered an IL-33-Fc fusion molecule, a single dose of which was sufficient to significantly increase the mobilization of HSPCs after 4 days. In conclusion, our findings suggest that long-acting, oxidation-resistant IL-33 may be a novel approach for HSPC transplantation. IL-33-mobilized HSPCs differ from cells mobilized with G-CSF and AMD3100, and it is possible that these differences may result in better transplantation outcomes.

Blockade of placental growth factor reduces vaso-occlusive complications in murine models of sickle cell disease.

Vaso-occlusive crisis (VOC) is the most common and debilitating complication of sickle cell disease (SCD); recurrent episodes cause organ damage and contribute to early mortality. Plasma placental growth factor (PlGF) levels are elevated in SCD and can further increase under hypoxic conditions in SCD mice. Treatment with a PlGF-neutralizing antibody (anti-PlGF Ab) in SCD mice reduced levels of monocyte chemoattractant protein-3, eotaxin, macrophage colony-stimulating factor, and plasminogen activator inhibitor-1 significantly, and of macrophage-derived chemokine and macrophage inflammatory protein-3ß moderately; this may contribute to inhibition of leukocyte recruitment, activation, and thrombosis. In subsequent experiments, anti-PlGF Ab treatment significantly reduced plasma lactate dehydrogenase levels, indicating possible reduction in cellular destruction and/or hemolysis. Histopathology studies revealed decreased incidence and severity of congestion in the lungs and spleen with repeated anti-PlGF Ab treatment. Furthermore, anti-PlGF Ab significantly reduced vaso-occlusion events under hypoxic conditions in a modified dorsal skinfold chamber model in SCD mice. Therefore, elevated PlGF levels may contribute to recruitment and activation of leukocytes. This can subsequently lead to increased pathology of affected organs in addition to mediating acute hypoxia/reoxygenation-triggered vaso-occlusion under SCD conditions. Thus, targeting PlGF may offer a therapeutic approach to reduce acute VOC and possibly alleviate long-term vascular complications in patients with SCD.

A Near-Atomic Structure of the Dark Apoptosome Provides Insight into Assembly and Activation.

In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade ß-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2' carbon of the deoxyribose ring. Interestingly, ß-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to ß-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway.

A near atomic structure of the active human apoptosome.

In response to cell death signals, an active apoptosome is assembled from Apaf-1 and procaspase-9 (pc-9). Here we report a near atomic structure of the active human apoptosome determined by cryo-electron microscopy. The resulting model gives insights into cytochrome c binding, nucleotide exchange and conformational changes that drive assembly. During activation an acentric disk is formed on the central hub of the apoptosome. This disk contains four Apaf-1/pc-9 CARD pairs arranged in a shallow spiral with the fourth pc-9 CARD at lower occupancy. On average, Apaf-1 CARDs recruit 3 to 5 pc-9 molecules to the apoptosome and one catalytic domain may be parked on the hub, when an odd number of zymogens are bound. This suggests a stoichiometry of one or at most, two pc-9 dimers per active apoptosome. Thus, our structure provides a molecular framework to understand the role of the apoptosome in programmed cell death and disease.

Tango7 directs cellular remodeling by the Drosophila apoptosome.

It is now well appreciated that the apoptosome, which governs caspase-dependent cell death, also drives nonapoptotic caspase activation to remodel cells. However, the determinants that specify whether the apoptosome acts to kill or remodel have yet to be identified. Here we report that Tango7 collaborates with the Drosophila apoptosome to drive a caspase-dependent remodeling process needed to resolve individual sperm from a syncytium. In these cells, Tango7 is required for caspase activity and localizes to the active apoptosome compartment via its C terminus. Furthermore, Tango7 directly stimulates the activity of this complex in vitro. We propose that Tango7 specifies the Drosophila apoptosome as an effector of cellular remodeling.

Apoptosome structure, assembly, and procaspase activation.

Apaf-1-like molecules assemble into a ring-like platform known as the apoptosome. This cell death platform then activates procaspases in the intrinsic cell death pathway. In this review, crystal structures of Apaf-1 monomers and CED-4 dimers have been combined with apoptosome structures to provide insights into the assembly of cell death platforms in humans, nematodes, and flies. In humans, the caspase recognition domains (CARDs) of procaspase-9 and Apaf-1 interact with each other to form a CARD-CARD disk, which interacts with the platform to create an asymmetric proteolysis machine. The disk tethers multiple pc-9 catalytic domains to the platform to raise their local concentration, and this leads to zymogen activation. These findings have now set the stage for further studies of this critical activation process on the apoptosome.

Changes in Apaf-1 conformation that drive apoptosome assembly.

Apoptosome assembly is highly regulated in the intrinsic cell death pathway. To better understand this step, we created an improved model of the human apoptosome using a crystal structure of full length Apaf-1 and a single particle, electron density map at ~9.5 Å resolution. The apoptosome model includes N-terminal domains of Apaf-1, cognate ß-propellers, and cytochrome c. A direct comparison of Apaf-1 in the apoptosome and as a monomer reveals conformational changes that occur during the first two steps of assembly. This includes an induced-fit mechanism for cytochrome c binding to regulatory ß-propellers, which is dependent on shape and charge complementarity, and a large rotation of the nucleotide binding module during nucleotide exchange. These linked conformational changes create an extended Apaf-1 monomer and drive apoptosome assembly. Moreover, the N-terminal CARD in the inactive Apaf-1 monomer is not shielded from other proteins by ß-propellers. Hence, the Apaf-1 CARD may be free to interact with a procaspase-9 CARD either before or during apoptosome assembly. Irrespective of the timing, the end product of assembly is a holo-apoptosome with an acentric CARD-CARD disk and tethered pc-9 catalytic domains. Subsequent activation of pc-9 leads to a proteolytic cascade and cell death.

Role of apolipoprotein A-II in the structure and remodeling of human high-density lipoprotein (HDL): protein conformational ensemble on HDL.

High-density lipoproteins (HDL, or "good cholesterol") are heterogeneous nanoparticles that remove excess cell cholesterol and protect against atherosclerosis. The cardioprotective action of HDL and its major protein, apolipoprotein A-I (apoA-I), is well-established, yet the function of the second major protein, apolipoprotein A-II (apoA-II), is less clear. In this review, we postulate an ensemble of apolipoprotein conformations on various HDL. This ensemble is based on the crystal structure of Δ(185-243)apoA-I determined by Mei and Atkinson combined with the "double-hairpin" conformation of apoA-II(dimer) proposed in the cross-linking studies by Silva's team, and is supported by the wide array of low-resolution structural, biophysical, and biochemical data obtained by many teams over decades. The proposed conformational ensemble helps integrate and improve several existing HDL models, including the "buckle-belt" conformation of apoA-I on the midsize disks and the "trefoil/tetrafoil" arrangement on spherical HDL. This ensemble prompts us to hypothesize that endogenous apoA-II (i) helps confer lipid surface curvature during conversion of nascent discoidal HDL(A-I) and HDL(A-II) containing either apoA-I or apoA-II to mature spherical HDL(A-I/A-II) containing both proteins, and (ii) hinders remodeling of HDL(A-I/A-II) by hindering the expansion of the apoA-I conformation. Also, we report that, although endogenous apoA-II circulates mainly on the midsize spherical HDL(A-I/A-II), exogenous apoA-II can bind to HDL of any size, thereby slightly increasing this size and stabilizing the HDL assembly. This suggests distinctly different effects of the endogenous and exogenous apoA-II on HDL. Taken together, the existing results and models prompt us to postulate a new structural and functional role of apoA-II on human HDL.

[Analysis of 61 cases undergoing sphincter-preserving procedure with intersphincteric resection by telescopic anastomosis for ultra-low rectal cancer].

OBJECTIVE: To investigate the efficacy and safety of sphincter-preserving procedure with transabdominal intersphincteric resection for ultra-low rectal cancer. METHODS: Clinical data of 61 cases with ultra-low rectal cancer (distance from anal verge ranged from 4-5 cm) were analyzed retrospectively. The patients underwent sphincter-preserving procedure with intersphincteric resection and telescopic anastomosis. RESULTS: There were 34 males and 27 females. The mean age was 56.7 years. The inferior border of the tumor was 4 cm above the anal verge in 21 cases, and 5 cm in 40 cases. There 55 patients with rectal adenocarcinoma in this cohort. The tumor was well-differentiated in 24 cases, moderately-differentiated in 29 cases, and poorly-differentiated in 2 cases. There were 6 cases with malignant adenoma. The TNM staging was T1N0M0 in 36 cases, T2N0M0 in 23, and T3N1M0 in 2. The ability to control defecation significantly improved in 1-3 months postoperatively, and returned to normal in 6-12 months. Two patients developed anastomotic leak (3.3%), and 3 anastomotic stenosis (4.9%) postoperatively. Fifty-four patients(88.5%) had follow-up. The median follow-up time was 6.2 years. The local recurrence rate was 5.6%, and the 5-year-survival rate was 73.5%. CONCLUSION: Sphincter-preserving procedure with intersphincteric resection and telescopic anastomosis is a safe and effective procedure for ultra-low rectal cancer.

The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3.

Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.

Structure of the Drosophila apoptosome at 6.9 å resolution.

The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ∼6.9 Å resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has "lost" a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution.

High density lipoproteins-based therapies for cardiovascular disease.

Atherosclerosis is the leading cause of death in developed countries. High density lipoproteins (HDL) cholesterol level correlates inversely with the risk of cardiovascular diseases. Thus, HDL has obtained lots of interest for drug development. In this review, we summarized the mechanisms for the antiatherogenic function of HDL, current HDL-based drugs in clinical use and the future direction for HDL-based therapy development.

Structure of an apoptosome-procaspase-9 CARD complex.

Apaf-1 coassembles with cytochrome c to form the apoptosome, which then binds and activates procaspase-9 (pc-9). We removed pc-9 catalytic domains from the holoapoptosome by site-directed thrombinolysis. A structure of the resulting apoptosome-pc-9 CARD complex was then determined at approximately 9.5 A resolution. In our model, the central hub is constructed like other AAA+ protein rings but also contains novel features. At higher radius, the regulatory region of each Apaf-1 is comprised of tandem seven and eight blade beta-propellers with cytochrome c docked between them. Remarkably, Apaf-1 CARDs are disordered in the ground state. During activation, each Apaf-1 CARD interacts with a pc-9 CARD and these heterodimers form a flexibly tethered "disk" that sits above the central hub. When taken together, the data reveal conformational changes during Apaf-1 assembly that allow pc-9 activation. The model also provides a plausible explanation for the effects of NOD mutations that have been mapped onto the central hub.

[Clinical experience of 371 cases of sphincter-preservation with telescopic anastomosis after radical excision for low-middle rectal cancer].

OBJECTIVE: To evaluate the clinical efficacy, feasibility and safety of sphincter-preservation with telescopic anastomosis of colon and rectal mucosa in low-middle rectal cancer. METHODS: A retrospective analysis was carried out in 371 patients with low-middle rectal cancer in whom telescopic anastomosis was used. There were 224 males and 147 females, with a mean age of 57.9 (21-99) years. The lower margins of the tumors located between 5-8 cm from the anal verge. On histopathology, there were 361 adenocarcinomas, including 138 well-differentiated, 201 moderately differentiated, 11 poorly differentiated, 11 mucinous adenocarcinoma, and 10 adenomas with neoplastic changes. According to the Duke's stage classification, 120 were TNM stage I, 222 stage II, 26 stage III, and 3 stage IV. RESULTS: Three hundred and eighteen (318/371, 85.7%) cases were followed up, and the median follow up time was 5.8 years. Postoperative complications were observed, including 16(4.3%) cases with anastomotic leak, and 8 (2.1%) with anastomotic stenosis. All the patients resumed normal bowel function during 12-24 weeks after operation, with 1-3 times per day. The local recurrence rate was 6.3% (20/318). Hepatic and lung metastasis was 14.5% (46/318) and 2.5% (8/318), respectively. The 5-year survival rate was 69.7%. CONCLUSION: The sphincter-preservation with telescopic anastomosis procedure is safe and effective for low-middle rectal cancer, and the sphincter function can be well-preserved.

Differential stability of high-density lipoprotein subclasses: effects of particle size and protein composition.

High-density lipoproteins (HDLs) are complexes of proteins (mainly apoA-I and apoA-II) and lipids that remove cholesterol and prevent atherosclerosis. Understanding the distinct properties of the heterogeneous HDL population may aid the development of new diagnostic tools and therapies for atherosclerosis. Mature human HDLs form two major subclasses differing in particle diameter and metabolic properties, HDL(2) (large) and HDL(3) (small). These subclasses are comprised of HDL(A-I) containing only apoA-I, and HDL(A-I/A-II) containing apoA-I and apoA-II. ApoA-I is strongly cardioprotective, but the function of the smaller, more hydrophobic apoA-II is unclear. ApoA-II is thought to counteract the cardioprotective action of apoA-I by stabilizing HDL particles and inhibiting their remodeling. To test this notion, we performed the first kinetic stability study of human HDL subclasses. The results revealed that the stability of plasma spherical HDL decreases with increasing particle diameter; which may facilitate preferential cholesterol ester uptake from large lipid-loaded HDL(2). Surprisingly, size-matched plasma HDL(A-I/A-II) showed comparable or slightly lower stability than HDL(A-I); this is consistent with the destabilization of model discoidal HDL observed upon increasing the A-II to A-I ratio. These results clarify the roles of the particle size and protein composition in HDL remodeling, and help reconcile conflicting reports regarding the role of apoA-II in this remodeling.

[Clinical study of 231 cases of radical excision with sphincter preservation by casing anastomosis in low rectal cancer].

OBJECTIVE: To investigate the clinical efficacy, feasibility and safety of sphincter-preserving procedure by casing anastomosis of colon and rectal mucosa in low rectal cancer. METHODS: A retrospective analysis was carried out in 231 cases of low rectal cancer performed casing anastomosis. RESULTS: One hundred and ninety-seven (197/231, 85.3%) cases were followed up, the median time of the follow up was 5.9 years (range, 2 months-14 years). Eight (3.4%) cases of stoma leak and 3 (1.2%) cases of stoma stenosis were found post operation. Defecating function recovered normally (1 - 3 times per day) in 12 - 24 weeks after operation in all patients. Local recurrence was found in 5.1% (10/197) of the cases. Hepatic and lung metastasis was found in 15.2% (30/197) and 2.5% (5/197) of the patients, respectively. The five-year survival rate was 71.6% totally. CONCLUSIONS: The casing anastomosis procedure with sphincter preservation is safe and efficacy for low rectal cancer. With the procedure, the anal function can be preserved well, stoma leak is decreased, and the five-year survival rate is the same as Miles operation.

[Clinical analysis of therapeutic effects of sphincter-preserving operation and Miles operation for rectal cancer].

OBJECTIVE: To investigate and compare therapeutic effects of sphincter-preserving operation and Miles operation for rectal cancer. METHODS: A retrospective analysis was carried out in 572 cases of rectal cancer operations performed from January 1980 to December 2006. RESULTS: Sphincter-preserving operation was carried out in 403 cases and Miles procedure in 169 cases. The follow-up rate was 76.2% (436/572) with a period of 0.5 - 25.0 years (median, 9.5 years). Local recurrence occurred in 6.3% (20/317) of sphincter-preserving operation and 7.6% (9/119) of Miles operation, the differences was not significant (chi2 = 1.3942, P > 0.05). Distal metastasis was found in 50 cases (15.7%) of sphincter-preserving operation and 19 cases (16.2%) of the Miles operation with no significant difference (chi2 = 0.6672, P > 0.05). There was no significant difference in five-year survival rate between the two groups, with 67.8% in sphincter-preserving operation and 67.2% in Miles operation. CONCLUSIONS: Sphincter-preserving operations can improve the quality of life in rectal cancer although with the same five-year survival rate and recurrence rate as Miles operation. The operation for rectal cancer should be performed individually according to the location, the bionomics and the clinical stage.

[Extended parietal cell vagotomy in the treatment of acute perforation of duodenal ulcer in 176 cases].

OBJECTIVE: To evaluate the long-term therapeutic efficacy of extended parietal cell vagotomy (EPCV) in the treatment of duodenal ulcer complicated with acute perforation. METHODS: Therapeutic efficacy of EPCV in 176 cases subjected to duodenal ulcer with acute perforation since 1979 was evaluated, including postoperative complication, ulcer recurrence rate, gastric empting function, endoscopic and radiographical examination, nutritional status and Visick classification. RESULTS: Among 176 patients, 153 (86.9%) cases were successfully followed-up for 5 years after operation. No operative death was found. Postprandial superior belly fullness occurred in 13 cases (8.5%) and heartburn in 12 cases (7.8%), which could be relieved by Domperidone. Adhesive ileus was noted in 4 cases (2.6%) which was cured by adhesiolysis. The total ulcer recurrence rate was 2.6% (4 cases) within 2 to 3 years after operation. Superficial gastritis occurred in 21 cases (13.7%) and duodenal bulb in 31 cases (20.3%). Sinus ventriculi vermicular motion was good and gastric emptying was normal. No anemia was found. Body weight gained in 116 cases (75.8%). One hundred and forty-six cases(95.4%) were reforming Visick grade I and II , 3 cases(2.0%) grade III , and 4 cases (2.6%) IV . CONCLUSIONS: EPCV is convenient for performance with low postoperative complication rate. Its long-term efficacies are quite good, which including normal nutritional status, high quality of life and low ulcer recurrence rate. EPCV is one of effective and safe treatments for duodenal ulcer complicated with acute perforation.

[Analysis of 618 cases of radical resection of rectal carcinoma].

OBJECTIVE: To investigate and analyse curative effects of Miles operation and anal sphincter preserving operation for rectal carcinoma in 20 years. METHODS: From 1984 to 2004, 618 cases of rectal carcinoma that underwent radical resection including Miles operation and anal sphincter preserving procedures were analysed retrospectively each 10 years, earlier 10 years from 1984 to 1994, and later 10 years from 1994 to 2004. RESULTS: Among the 618 cases, 492 (79.6%) were followed up. The median of the follow-up time was 5.4 years. In the earlier 10 years, local recurrence rate of post operation was 6.9% (14/201), for Miles operation and anal sphincter preserving procedures the local recurrence rate was 6.7% and 7.1% respectively. In the later 10 years, the local recurrence rate was 5.1% (15/291), 4.8% for Miles operation, 5.2% for anal sphincter preserving procedures. With the procedure of canular anastomosis of colon and rectal mucosa, the local recurrence rate was 4.9%. Overall five-year survival rate was 64.7% (130/201) in the earlier 10 years, 66.3% (59/89) for Miles operation, 63.4% (71/112) for anal sphincter preserving procedures. In the later 10 years, the five-year survival rate was 68.0% (198/291) in all, for Miles operation 66.3% (55/83), for anal sphincter preserving procedures 68.7% (143/208). With the procedure of canular anastomosis of colon and rectal mucosa, the five-year survival rate was 71.3% (62/87). CONCLUSIONS: The operation for rectal cancer should be chosen individually according to locus, biological character, and clinical stages. Anal sphincter preserving procedures are performed increasingly, and they provide the same five-year survival rate as Miles operation does, and the patient's quality of life can be improved obviously.