Effects of Fatty Acid Amide Hydrolase (FAAH) Inhibitors in Non-Human Primate Models of Nicotine Reward and Relapse

Inhibition of the enzyme fatty acid amide hydrolase (FAAH) counteracts reward-related effects of nicotine in rats, but it has not been tested for this purpose in non-human primates. Therefore, we studied the effects of the first- and second-generation O-arylcarbamate-based FAAH inhibitors, URB597 (cyclohexyl carbamic acid 3’-carbamoyl-3-yl ester) and URB694 (6-hydroxy-[1,1''-biphenyl]-3-yl-cyclohexylcarbamate), in squirrel monkeys. Both FAAH inhibitors: (1) blocked FAAH activity in brain and liver, increasing levels of endogenous ligands for cannabinoid and α-type peroxisome proliferator-activated (PPAR-α) receptors; (2) shifted nicotine self-administration dose–response functions in a manner consistent with reduced nicotine reward; (3) blocked reinstatement of nicotine seeking induced by reexposure to either nicotine priming or nicotine-associated cues; and (4) had no effect on cocaine or food self-administration. The effects of FAAH inhibition on nicotine self-administration and nicotine priming-induced reinstatement were reversed by the PPAR-α antagonist, MK886. Unlike URB597, which was not self-administered by monkeys in an earlier study, URB694 was self-administered at a moderate rate. URB694 self-administration was blocked by pretreatment with an antagonist for either PPAR-α (MK886) or cannabinoid CB₁ receptors (rimonabant). In additional experiments in rats, URB694 was devoid of THC-like or nicotine-like interoceptive effects under drug-discrimination procedures, and neither of the FAAH inhibitors induced dopamine release in the nucleus accumbens shell—consistent with their lack of robust reinforcing effects in monkeys. Overall, both URB597 and URB694 show promise for the initialization and maintenance of smoking cessation because of their ability to block the rewarding effects of nicotine and prevent nicotine priming-induced and cue-induced reinstatement.     

Trimetazidine alone or in combination with gemcitabine and/or abraxane decreased cell viability,migration and ATP levels and induced apoptosis of human pancreatic cells

BackgroundTrimetazidine (TMZ) is an anti-ischemic agent that can inhibit the fatty acid oxidation. It has been stated that inhibition of fatty acid oxidation may be an acceptable approach to cancer treatment.MethodsWe examined the effects of TMZ alone or together with abraxane (ABX) and/or gemcitabine (GEM) on cell viability, apoptosis, adhesion, migration and ATP levels of human pancreatic cancer cell line PANC-1.ResultsTMZ significantly reduced the cell viability at higher concentrations. Lower cell viability values were found in cells co-treated with TMZ + GEM, TMZ + ABX and GEM + ABX. The combined treatment of TMZ with ABX and/or GEM significantly increased the apoptosis rates. The highest percentages of apoptosis were found in TMZ + ABX or TMZ + ABX + GEM treatments. TMZ alone or together with ABX and/or GEM significantly reduced the ATP levels. The lowest migration rates were also found at TMZ + ABX and TMZ + ABX + GEM treatments.ConclusionsOur study is the first study to indicate that TMZ can induce cytotoxicity and apoptosis and reduce migration and ATP levels, especially in cells co-treated with ABX and/or GEM. A combination strategy based on inhibition of fatty acid oxidation and anticancer drugs may be more effective in the treatment of pancreatic cancers.     

Critical role of RAGE and HMGB1 in inflammatory heart disease

Autoimmune response to cardiac troponin I (TnI) induces inflammation and fibrosis in the myocardium. High-mobility group box 1 (HMGB1) is a multifunctional protein that exerts proinflammatory activity by mainly binding to receptor for advanced glycation end products (RAGE). The involvement of the HMGB1–RAGE axis in the pathogenesis of inflammatory cardiomyopathy is yet not fully understood. Using the well-established model of TnI-induced experimental autoimmune myocarditis (EAM), we demonstrated that both local and systemic HMGB1 protein expression was elevated in wild-type (wt) mice after TnI immunization. Additionally, pharmacological inhibition of HMGB1 using glycyrrhizin or anti-HMGB1 antibody reduced inflammation in hearts of TnI-immunized wt mice. Furthermore, RAGE knockout (RAGE-ko) mice immunized with TnI showed no structural or physiological signs of cardiac impairment. Moreover, cardiac overexpression of HMGB1 using adeno-associated virus (AAV) vectors induced inflammation in the hearts of both wt and RAGE-ko mice. Finally, patients with myocarditis displayed increased local and systemic HMGB1 and soluble RAGE (sRAGE) expression. Together, our study highlights that HMGB1 and its main receptor, RAGE, appear to be crucial factors in the pathogenesis of TnI-induced EAM, because inhibition of HMGB1 and ablation of RAGE suppressed inflammation in the heart. Moreover, the proinflammatory effect of HMGB1 is not necessarily dependent on RAGE only. Other receptors of HMGB1 such as Toll-like receptors (TLRs) may also be involved in disease pathogenesis. These findings could be confirmed by the clinical relevance of HMGB1 and sRAGE. Therefore, blockage of one of these molecules might represent a novel therapeutic strategy in the treatment of autoimmune myocarditis and inflammatory cardiomyopathy.Inflammatory cardiomyopathy is a relatively common cause of acute heart failure in the young, for which an efficient and specific therapy is lacking. Although most patients recover completely, some present a deteriorating course. Recent work from our laboratory and others has focused on the dysregulation of the immune system as an essential modulator of disease induction and progression in heart failure (1, 2). In this context, release of cardiac troponin I (TnI) from damaged cardiomyocytes into the circulation is believed to trigger an autoimmune response to TnI (3, 4).Our group has established an animal model in which immunization with murine cardiac TnI induces severe myocardial inflammation and fibrosis, followed by severe heart failure (1). However, the exact pathomechanism and immune modulators involved in this inflammatory process are not yet fully elucidated.Extensive work has cast light on the role of high-mobility group box 1 (HMGB1) in the pathogenesis of infectious and noninfectious inflammatory diseases. HMGB1, first described as a DNA binding protein, has subsequently been associated with various pathological conditions such as cardiovascular disease (5, 6), cancer (7), and ischemia/reperfusion (I/R) injury (5). It is a key modulator of innate immune responses and regulates in part adaptive immunity (8). In response to cellular stress, HMGB1 acts as a damage-associated molecular pattern (DAMP) signal after passive release into the extracellular milieu during cell death or active secretion by mononuclear and other cell types (7). It binds to receptors such as receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs) such as TLR-2 and -4, leading to the expression of inflammatory cytokines, chemokines, and corresponding receptors (9).Some studies describe differential effects of HMGB1- or RAGE-dependent signaling with regard to their concentration and release in a particular model or mode of application (10, 11). In a rodent model of myocardial infarction, exogenously administered HMGB1 had a beneficial effect on postinfarct myocardial remodeling (10). Kitahara and colleagues demonstrated reduced necrosis and smaller infarct size after myocardial infarction in transgenic mice overexpressing HMGB1 (12). However, in a murine model of I/R injury, our group recently showed that treatment of wild-type (wt) mice with recombinant HMGB1 increased the infarct size (6). In the same model of I/R injury, RAGE-deficient mice demonstrated significantly reduced myocardial damage compared with wt mice (6).The effect of HMGB1 and RAGE on the pathogenesis of cardiac disorders is not only described in preclinical animal models but is also investigated in human cardiac disorders. Different studies have revealed an elevated HMGB1 level in patients with heart failure correlating with disease severity (13–17). In addition to this, some studies have identified RAGE as a prognostic factor in human heart failure (16, 18, 19).In the present study, we aimed to clarify the role of HMGB1 and RAGE in an experimental model of murine autoimmune myocarditis. This experimental approach enables a reproducible sterile cardiac inflammation, as described previously (1, 20). Furthermore, the clinical relevance of both proteins should be investigated.Hence, we first studied the expression kinetics of HMGB1 in the inflamed myocardium and serum of wt mice. Then, inhibition of HMGB1 by glycyrrhizin (GL) was performed and heart tissue was analyzed in TnI-induced experimental autoimmune myocarditis (EAM) of wt mice. Additionally, RAGE knockout (RAGE-ko) mice were immunized with TnI to further study the role of RAGE signaling in this EAM model. Finally, the role of the HMGB1–RAGE axis in our model was analyzed by an adeno-associated virus (AAV)9-mediated cardiac overexpression of HMGB1. In a last step, we studied the clinical relevance of HMGB1 and RAGE in patients with myocarditis.     

Effectiveness of a new inflatable balloon device for gluing dissected layers in an experimental model of aortic dissection

OBJECTIVES:

In the surgical treatment of acute aortic dissection, tissue glues are widely used to reinforce the adhesion between the dissected aortic layers. A new inflatable balloon device was developed to compress the dissected aortic wall during gluing to increase adhesion between the dissected layers. The present study used an ex vivo experimental animal model to test the hypothesis that this device is effective when gluing the true and false channels of dissected aortas.

METHODS:

In the ex vivo experimental model, aortic dissection was simulated surgically on 12 fresh bovine aorta samples. In six samples (group I), the inflatable balloon device was inserted into the aorta to reinforce and fuse the dissected layers during gluing. The other six fresh bovine aortic samples (group II) were compressed between the surgeon’s fingers during gluing. Aortic samples were evaluated and compared macroscopically and histologically.

RESULTS:

In group I, adhesion between the dissected layers was easily achieved during gluing. All false cavities were perfectly closed, with no deleterious effects related to the device. In group II, the adhesion between the dissected layers was not complete and some false cavities remained patent.

CONCLUSIONS:

The inflatable balloon device can increase the adhesive effect of tissue glues via homogenous compression of the dissected aortic layers. In addition, the balloon can prevent distal embolization of the glue.     

The Effect of Mitomycin-C in Reducing Pericardial Adhesion After Cardiac Surgery in Rabbits

Background

Mitomycin-C has been in clinical use primarily as a chemotherapeutic agent and is well known for antifibrotic properties. It has been widely used to prevent postoperative fibroblast proliferation and reduce scar adhesion in ophthalmologic and otolaryngologic operations. We investigated the efficacy of mitomycin-C in reducing postoperative pericardial adhesions in a rabbit model.

Methods

New Zealand white rabbits were used and categorized into 2 groups, study (use of mitomycin-C) and control. Group 1 (n = 8) was treated with a sponge impregnated with mitomycin-C solution that was applied over the abraded epicardium. In group 2 (control group), the sponge was impregnated with 0.9% isotonic NaCl solution and was applied with the same protocol as the mitomycin-C–impregnated sponge in group 1 (n = 8). Rabbits were humanely killed at a mean of 2 weeks. The scores of adhesion were graded by macroscopic examination, and the pericardial tissues were analyzed microscopically in terms of inflammation and fibrosis.

Results

In group 1, the adhesion scores were significantly lower than the control group's. No significant difference was found between the groups in terms of the severity of inflammation. There was a difference between groups in terms of the degree of fibrosis.

Conclusions

The use of mitomycin-C to prevent postoperative pericardial adhesions reduced adhesion and fibrosis scores in an experimental rabbit model. However, efficacy in reducing inflammation was not demonstrated.     

Regulated structural transitions unleash the chaperone activity of αB-crystallin

The small heat shock protein αB-crystallin is an oligomeric molecular chaperone that binds aggregation-prone proteins. As a component of the proteostasis system, it is associated with cataract, neurodegenerative diseases, and myopathies. The structural determinants for the regulation of its chaperone function are still largely elusive. Combining different experimental approaches, we show that phosphorylation-induced destabilization of intersubunit interactions mediated by the N-terminal domain (NTD) results in the remodeling of the oligomer ensemble with an increase in smaller, activated species, predominantly 12-mers and 6-mers. Their 3D structures determined by cryo-electron microscopy and biochemical analyses reveal that the NTD in these species gains flexibility and solvent accessibility. These modulated properties are accompanied by an increase in chaperone activity in vivo and in vitro and a more efficient cooperation with the heat shock protein 70 system in client folding. Thus, the modulation of the structural flexibility of the NTD, as described here for phosphorylation, appears to regulate the chaperone activity of αB-crystallin rendering the NTD a conformational sensor for nonnative proteins.Molecular chaperones share the ability to bind nonnative, aggregation-prone polypeptides and assist their folding and assembly (1–3). Among these, the small heat shock protein (sHsp) αB-crystallin (also HspB5) is one of the major constituents of the vertebrate eye lens where it functions both as a chaperone and structural protein (4, 5). In nonlenticular tissues, αB-crystallin (αB) participates in sustaining cellular proteostasis. The involvement in neurodegenerative diseases (6, 7), multiple sclerosis (8), myopathies (9), as well as in cell cycle control, apoptosis, and cancer (10, 11) underlines its importance for cellular proteostasis.αB exhibits a tripartite organization (Fig. 1A) with a central α-crystallin domain (ACD) flanked by an N-terminal domain (NTD) and a short C-terminal extension (CTE) (12, 13). The ACD forms stable dimers (14–16) that further assemble into higher-order oligomers via interactions mediated by the NTD and CTE (17–19). αB forms dynamic populations of multimers with a variable number of subunits (20, 21). Structural studies indicate that the variety of oligomeric states including a symmetric 24-mer (22) is created by addition of subunits to (or subtraction from) existing oligomers (17, 18). As for many other sHsps (23), the polydispersity of αB is coupled to spontaneous subunit exchange of yet undetermined units. αB quaternary dynamics was attributed to fluctuations of the intersubunit contacts mediated by the C-terminal IXI motif (24). However, given its involvement in oligomer formation, the NTD must also play a decisive role.Open in a separate windowFig. 1.Oligomer size, hydrophobicity, and quaternary dynamics of αB phosphomimics. (A) Domain organization of human αB: NTD (orange) with the three major in vivo phosphorylation sites Ser19, Ser45, and Ser59, ACD (blue), and CTE (green). (Lower Left) Surface representation of the cryo-EM density map of the αB 24-mer with the docked pseudoatomic model according to Braun et al. (17) superimposed (ribbon representation, domains colored as above). The molecule is viewed along a molecular threefold symmetry axis perpendicular to the area harboring the NTDs (closed threefold area, 3_c). (Lower Right) Close-up view of the pseudoatomic model at the area 3_c. The colored spheres depict residues S19 (green), S45 (blue), and S59 (cyan). (B) Analysis of αB-WT and its triple Glu mutant (αB-3E) by sedimentation velocity AUC. The s-value distributions of αB-WT (black) and αB-3E (red) obtained by c(s) analysis. (C) Transmission electron micrograph of αB-3E (0.02 mg/mL, pH 7.4) negatively stained with ammonium molybdate [1.5% (wt:vol), pH 5.5]. (Scale bar, 100 nm.) (Inset) TEM image of αB-WT (0.05 mg/mL) prepared as described for αB-3E. (D) Subunit exchange kinetics of αB-WT and αB-3E. Shown are the temporal changes in the donor fluorescence intensities at 415 nm due to reversal of FRET on adding excess amounts of unlabeled αB-WT (black) and αB-3E (red) to FRET-equilibrated heterooligomers of AIAS- and LYI-labeled αB-WT-S153C. αB-3E shows a higher subunit exchange rate (0.369 ± 0.028 min⁻¹) than αB-WT (0.053 ± 0.013 min⁻¹). The respective fluorescence spectra are depicted as insets in the same color code.In general, sHsps including αB recognize aggregation-prone, partially unfolded substrates (4, 25, 26) and keep them in a refolding-competent state (27, 28). The substrate binding sites of sHsps have not been defined yet. Recent studies suggest the involvement of multiple sites from all three sequence regions (29–32). Due to the lack of structural information for the NTD, however, it is still not possible to propose a molecular mechanism for the mode of action of αB or sHsps in general. The emerging view is that their structural plasticity may be an important element in substrate recognition and binding (25, 29, 33–35). In this context, a key issue is to define how changes in the structural ensemble of αB correlate with chaperone function.Because sHsps do not possess ATPase activity, their chaperone function is regulated by different means. For many mammalian sHsps, phosphorylation plays a major role in this context (36). For αB, the three major phosphorylation sites, Ser19, Ser45, and Ser59, all located within the NTD (Fig. 1A), are phosphorylated in response to various kinds of stresses (37, 38). Phosphorylation of αB has been shown to correlate with a reduction in average oligomer size and enhanced substrate binding (39–42). However, its influence on the molecular architecture and the underlying activation mechanism remained elusive.To probe the role of the NTD of αB in oligomer assembly, equilibrium, and dynamics, as well as its impact on chaperone function, we used phosphomimicking mutants and phosphorylated αB. By destabilization of NTD interactions in larger oligomers, we were able to populate and identify the chaperone-active species in the remodeled ensemble and determine the structures of the active oligomers in which the structural flexibility and accessibility of the NTD emerged to be the key element for efficient binding as well as subsequent folding of clients.     

Osteochondritis dissecans

Osteochondritis dissecans (OCD) is a disease of unknown etiology, characterized by separation of necrotic bone from its bony bed. While the juvenile form seen in patients with open physes has a 60-90% rate of spontaneous resolution, the adult form has virtually no chance of spontaneous healing. Plain X-rays are sufficient for the diagnosis, and magnetic resonance imaging (MRI) is essential for evaluation of disease progression and/or healing. The clinical correlation of MRI criteria defined in recent years to determine stability of the lesion is high. Juvenile OCD can be treated conservatively if there are no signs of instability on magnetic resonance images. Adult patients or unstable lesions in children should be treated surgically. For stable lesions, arthroscopic antegrade perforation is indicated to increase vascularity and stimulate healing. Unstable or displaced lesions should be treated with debridement, internal fixation, and cancellous bone grafting. Although cannulated metal screws are the most widely used implants for internal fixation, biodegradable implants have also been utilized in recent years. Loose fragments that are too deformed to be internally fixed should be removed and cartilage reconstruction techniques should be employed for the remaining crater. Long-term results of loose body removal alone are unsatisfactory. Modern cartilage restoration techniques are technically demanding due to the large, deep, and unconfined nature of the defect in the femoral condyle.     

Laparoscopic repair of peptic ulcer perforation without omental patch versus conventional open repair

BACKGROUND: Laparoscopic surgery, a minimally invasive technique, has recently begun to be used on perforated peptic ulcers effectively and frequently. Nevertheless, most studies have shown that the disadvantages of the laparoscopic treatment of peptic ulcers are a long operation time, a high reoperation rate, and a need for an experienced surgeon. Thus, the objective of the current study was to compare the safety and efficacy of optimized laparoscopic surgery without an omental patch for a perforated peptic ulcer within a shorter operational time with conventional open surgery in a 4-year period. PATIENTS AND METHODS: From May 2002 to June 2006, 35 consecutive patients with a clinical diagnosis of a perforated peptic ulcer were prepared prospectively to undergo either an open or optimized laparoscopic surgery. RESULTS: Seventeen patients with a perforated peptic ulcer underwent simple laparoscopic repair without an omental patch. Three patients (17.6%) who were begun by the laparoscopic approach had to be converted to open surgery. Eighteen patients underwent conventional open surgery. The mean operative time for laparoscopic repair was 42.10 minutes (range, 35-60), which was significantly shorter than the 55.83 minutes for open repair (range, 35-72; P = 0.001). Postoperative parenteral analgesic requirements were lower after laparoscopic repair (75.0 mg) than that after an open repair procedure (101.39 mg; P = 0.02). There was no statistically significant difference between the procedures in terms of hospital stay (5 vs. 5.33 days; P = 0.37) and the timing of access to normal daily activity (6.8 vs. 7.1 days) (P = 0.54). CONCLUSIONS: Laparoscopic surgery, when optimized by a simple repair without an omental patch and 10 mm of a large-channel aspirator-irrigator, may be safely and effectively applied to the patients with small duodenal perforated peptic ulcers (<10 mm) and because of its having low risk factors. The procedure may be an alternative treatment to other procedures when in experienced hands.