A new perspective in sepsis treatment: could RGD-dependent integrins be novel targets?

Sepsis is a life-threatening condition caused by the response of the body to an infection, and has recently been regarded as a global health priority because of the lack of effective treatments available. Vascular endothelial cells have a crucial role in sepsis and are believed to be a major target of pathogens during the early stages of infection. Accumulating evidence suggests that common sepsis pathogens, including bacteria, fungi, and viruses, all contain a critical integrin recognition motif, Arg-Gly-Asp (RGD), in their major cell wall-exposed proteins that might act as ligands to crosslink to vascular endothelial cells, triggering systemic dysregulation resulting in sepsis. In this review, we discuss the potential of anti-integrin therapy in the treatment of sepsis and septic shock.

Identification and validation of a novel anti-virulent that binds to pyoverdine and inhibits its function.

Pseudomonas aeruginosa: causes serious infections in patients with compromised immune systems and exhibits resistance to multiple antibiotics. The rising threat of antimicrobial resistance means that new methods are necessary for treating microbial infections. We conducted a high-throughput screen for compounds that can quench the innate fluorescence of the chromophore region of the P. aeruginosa siderophore pyoverdine, a key virulence factor. Several hits were identified that effectively quench pyoverdine fluorescence, and two compounds considerably improved the survival of Caenorhabditis elegans when worms were challenged with P. aeruginosa. Commercially available analogs of the best hit, PQ3, were tested for their ability to rescue C. elegans from P. aeruginosa and to interact with pyoverdine via fluorescence and solution NMR spectroscopy. 1H-15N and 1H-13C HSQC NMR were used to identify the binding site of PQ3c. The structure model of pyoverdine in complex with PQ3c was obtained using molecular docking and molecular dynamics simulations. PQ3c occupied a shallow groove on pyoverdine formed by the chromophore and N-terminal residues of the peptide chain. Electrostatic interactions and π-orbital stacking drove stabilization of this binding. PQ3c may serve as a scaffold for the development of pyoverdine inhibitors with higher potency and specificity. The discovery of a small-molecule binding site on apo-pyoverdine with functional significance provides a new direction in the search of therapeutically effective reagent to treat P. aeruginosa infections. Abbreviations: NMR: nuclear magnetic resonance; SAR: structure-activity relationship; MD: molecular dynamics; RMSF: root-mean-square fluctuation; HSQC: heteronuclear single quantum correlation; DMSO: dimethyl sulfoxide; Δδavg: average amide chemical shift change; DSS: 2,2-dimethyl-2-silapentane-5-sulfonate; RMSD: root-mean-square deviation; LJ-SR: Lennard-Jones short-range; Coul-SR: Coulombic short-range; FRET: fluorescence resonance energy transfer.

Pharmacologic inhibition of RGD-binding integrins ameliorates fibrosis and improves function following kidney injury.

Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-ß at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease.

cRGD target liposome delivery system promoted immunogenic cell death through enhanced anticancer potency of a thymidine conjugate under UVA activation as a cancer vaccine.

Conventional chemotherapeutic and photodynamic therapy have recently been shown to also elicit immune response against cancer through the immunogenic cell death mechanism, which can be potentially translated into effective cancer vaccines. However, there are few studies on the potential of nanodelivery system to promote the immunogenic cell death as a cancer vaccine. We reported here that cRGD target liposome delivery system was capable to promote the immunogenic cell death through enhanced potency of a thymidine conjugate post UVA activation as a cancer vaccine. Liposomes and cRGD target liposomes were found to significantly increase the cellular accumulation of the thymidine conjugate and subsequently translated into enhanced cytotoxic potency after UVA activation. More importantly, cRGD target liposomes of the thymidine conjugate markedly promoted the early detection of immunogenic cell death markers including ATP, HMGB1 and calreticulin. Subsequent in vivo vaccination-challenge study confirmed effective tumor growth inhibition by the cRGD liposomal thymidine conjugate and UVA treated cancer cells as the cancer vaccine. In addition, liposomes and cRGD target liposomes alone did not shown any induction of the immunogenic cell death markers, revealing the adjuvant nature of the nanodelivery system.

Effective suppression of the modified PHF6 peptide/1N4R Tau amyloid aggregation by intact curcumin, not its degradation products: Another evidence for the pigment as preventive/therapeutic "functional food".

Curcumin is a natural product with multiple biological activities and numerous potential therapeutic applications. In present study, the influence of curcumin and its degradation products (DPs) on the amyloid aggregation of Tau protein and the related PHF6 peptide were investigated. We provided experimental/theoretical evidence for suppressing effects of the compounds on the amyloid formation using far-UV CD as well as AFM, XRD and docking techniques and showed that the parent curcumin displayed stronger inhibition effect against Tau fibril aggregation. The obtained results suggest that the curcumin/DPs binding sites on the Tau molecule are likely to be the same, and provide a good structural basis to explain the efficient aggregation suppressing behavior of the curcumin, compared to the DPs. So, developing more stable curcumin nanoparticle formulations with improved curcumin bioavailability are of great importance. Curcumin's multi-functionality is also highly significant for the therapeutic application of this natural compound against various human diseases.

The Absence of the ACE N-Domain Decreases Renal Inflammation and Facilitates Sodium Excretion during Diabetic Kidney Disease.

BACKGROUND: Recent evidence emphasizes the critical role of inflammation in the development of diabetic nephropathy. Angiotensin-converting enzyme (ACE) plays an active role in regulating the renal inflammatory response associated with diabetes. Studies have also shown that ACE has roles in inflammation and the immune response that are independent of angiotensin II. ACE's two catalytically independent domains, the N- and C-domains, can process a variety of substrates other than angiotensin I. METHODS: To examine the relative contributions of each ACE domain to the sodium retentive state, renal inflammation, and renal injury associated with diabetic kidney disease, we used streptozotocin to induce diabetes in wild-type mice and in genetic mouse models lacking either a functional ACE N-domain (NKO mice) or C-domain (CKO mice). RESULTS: In response to a saline challenge, diabetic NKO mice excreted 32% more urinary sodium compared with diabetic wild-type or CKO mice. Diabetic NKO mice also exhibited 55% less renal epithelial sodium channel cleavage (a marker of channel activity), 55% less renal IL-1ß, 53% less renal TNF-α, and 53% less albuminuria than diabetic wild-type mice. This protective phenotype was not associated with changes in renal angiotensin II levels. Further, we present evidence that the anti-inflammatory tetrapeptide N-acetyl-seryl-asparyl-lysyl-proline (AcSDKP), an ACE N-domain-specific substrate that accumulates in the urine of NKO mice, mediates the beneficial effects observed in the NKO. CONCLUSIONS: These data indicate that increasing AcSDKP by blocking the ACE N-domain facilitates sodium excretion and ameliorates diabetic kidney disease independent of intrarenal angiotensin II regulation.

Effects of MicroRNA-494 on Astrocyte Proliferation and Synaptic Remodeling in the Spinal Cord of a Rat Model of Chronic Compressive Spinal Cord Injury by Regulating the Nogo/Ngr Signaling Pathway.

BACKGROUND/AIMS: Chronic compression of the spinal cord causes the loss of motor neurons in the anterior horn, but the precise and extensive mechanism for the loss is not completely determined. Therefore, this study aims to explore the role of microRNA-494 (miR-494) in the proliferation of astrocytes and in the synaptic remodeling in the spinal cord of a rat model of chronic spinal cord injury (SCI) by regulating the Nogo/NgR signaling pathway. METHODS: A rat model of chronic, compressive SCI was established, and the spinal cord state, blood supply changes, and astrocyte apoptosis were observed. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect expression of miR-494 and the Nogo/NgR signaling pathway-related genes. Fluorescence in situ hybridization (FISH) was used for detecting miR-494 expression and distribution. RESULTS: Higher miR-494 expression was accompanied by the inhibition of astrocyte proliferation and synaptic remodeling. In addition, CDK6 could be regulated by miR-494 and was shown to be one of the target genes of miR-494. Positive expression of miR-494 detected by FISH was consistent with the results from RT-qPCR that miR-494 could downregulate CDK6 gene expression. Moreover, the direct miR-494 target CDK6 plays important inhibitory roles in chronic SCI by suppressing the Nogo/ NgR signaling pathway. CONCLUSIONS: The results demonstrated that miR-494 inhibition can promote astrocyte proliferation and synaptic remodeling by suppressing the Nogo/NgR signaling pathway in a rat model of chronic SCI.

Inhibition of tau-derived hexapeptide aggregation and toxicity by a self-assembled cyclic d,l-α-peptide conformational inhibitor.

Aggregation and accumulation of amyloid ß and tau proteins to plaques and neurofibrillary tangles are the key pathogenic events in Alzheimer's disease. Here, we studied the capability of the cyclic d,l-α-peptide CP-2 as a conformational inhibitor of different amyloids to cross-interact with tau-derived AcPHF6 peptide and inhibit its aggregation, membrane perturbation and toxicity.

Alamandine attenuates sepsis-associated cardiac dysfunction via inhibiting MAPKs signaling pathways.

Sepsis-induced myocardial dysfunction represents a major cause of death. Alamandine is an important biologically active peptide. The present study evaluated whether alamandine improves cardiac dysfunction, inflammation, and apoptosis, and affects the signaling pathways involved in these events. Experiments were carried out in mice treated with lipopolysaccharide (LPS) or alamandine, and in neonatal rat cardiomyocytes. Alamandine increased the ejection fraction and fractional shortening, both of which were decreased upon LPS infusion in mice. LPS and alamandine reduced blood pressure, and increased the expression of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in the heart in mice. The LPS-induced decrease in α-myosin heavy chain (MHC) and ß-MHC, and increase in S100 calcium binding protein A8 (S100A8) and S100A9, were reversed by alamandine pre-treatment. Alamandine pre-treatment prevented LPS-induced myocardial inflammation, apoptosis and autophagy. LPS increased p-ERK, p-JNK, and p-p38 levels, which were inhibited by alamandine. Dibutyryl cyclic AMP (db-cAMP) increased p-ERK, p-JNK, and p-p38 levels, and reversed the inhibitory effects of alamandine on the LPS-induced increase in p-ERK, p-JNK, and p-p38. Moreover, db-cAMP reduced the expression of α-MHC and ß-MHC in cardiomyocytes, and reversed the almandine-induced attenuation of the LPS-induced decrease in α-MHC and ß-MHC. These results indicate that alamandine attenuates LPS-induced cardiac dysfunction, resulting in increased cardiac contractility, and reduced inflammation, autophagy, and apoptosis. Furthermore, alamandine attenuates sepsis induced by LPS via inhibiting the mitogen-activated protein kinases (MAPKs) signaling pathways.

Honokiol suppresses formyl peptide-induced human neutrophil activation by blocking formyl peptide receptor 1.

Formyl peptide receptor 1 (FPR1) mediates bacterial and mitochondrial N-formyl peptides-induced neutrophil activation. Therefore, FPR1 is an important therapeutic target for drugs to treat septic or sterile inflammatory diseases. Honokiol, a major bioactive compound of Magnoliaceae plants, possesses several anti-inflammatory activities. Here, we show that honokiol exhibits an inhibitory effect on FPR1 binding in human neutrophils. Honokiol inhibited superoxide anion generation, reactive oxygen species formation, and elastase release in bacterial or mitochondrial N-formyl peptides (FPR1 agonists)-activated human neutrophils. Adhesion of FPR1-induced human neutrophils to cerebral endothelial cells was also reduced by honokiol. The receptor-binding results revealed that honokiol repressed FPR1-specific ligand N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein binding to FPR1 in human neutrophils, neutrophil-like THP-1 cells, and hFPR1-transfected HEK293 cells. However, honokiol did not inhibit FPR2-specific ligand binding to FPR2 in human neutrophils. Furthermore, honokiol inhibited FPR1 agonist-induced calcium mobilization as well as phosphorylation of p38 MAPK, ERK, and JNK in human neutrophils. In conclusion, our data demonstrate that honokiol may have therapeutic potential for treating FPR1-mediated inflammatory diseases.

Ligand Strain and Entropic Effects on the Binding of Macrocyclic and Linear Inhibitors: Molecular Modeling of Penicillopepsin Complexes.

Using extensive molecular dynamics simulations, we investigate the structure and dynamics of the complexes formed between penicillopepsin and two peptidomimetic inhibitors: a linear compound, isovaleryl(P4)-valine(P3)-asparagine(P2)-leucine(P1)-phosphonate-phenylalanine(P1'), and its macrocylic analog that includes a methylene bridge between the Asn(P2) and Phe(P1') side chains. The macrocyclic inhibitor, which has a 420-fold stronger affinity than that of the acyclic one, has been considered to lower the entropic penalty for binding. To better understand this binding preference, the solution structure of the inhibitors is studied by molecular dynamics simulations. Subsequently, we assess the influence of the enzyme/inhibitor contacts, the enzyme-induced inhibitor strain, the variation of the ligand configurational entropy and the enzyme reorganization by combining molecular-mechanics Poisson-Boltzmann surface area and normal mode calculations with conformational entropy calculations. We find that there is no relevant entropic stabilization on the binding of the cyclic inhibitor with respect to the acyclic analog because the methylene bridge does not reduce appreciably the conformational flexibility of the free inhibitor. The most important factors explaining the stronger affinity of the macrocyclic inhibitor are the conformational filtering and the lower ligand strain induced by the methylene bridge.

Synergistic Activity of Berberine with Azithromycin against Pseudomonas Aeruginosa Isolated from Patients with Cystic Fibrosis of Lung In Vitro and In Vivo.

BACKGROUND/AIMS: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. METHODS: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. RESULTS: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. CONCLUSION: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.

Furoxan Nitric Oxide Donors Disperse Pseudomonas aeruginosa Biofilms, Accelerate Growth, and Repress Pyoverdine Production.

The use of nitric oxide (NO) as a signal for biofilm dispersal has been shown to increase the susceptibility of many biofilms to antibiotics, promoting their eradication. The delivery of NO to biofilms can be achieved by using NO donors with different kinetics and properties of NO release that can influence their efficacy as biofilm control agents. In this study, the kinetics of three furoxan derivatives were evaluated. The effects of these NO donors, which have an advantageous pharmacological profile of slower onset with an extended duration of action, on Pseudomonas aeruginosa growth, biofilm development, and dispersal were also characterized. Compound LL4254, which showed a fast rate of NO release, induced biofilm dispersal at approximately 200 µM. While LL4212 and LL4216 have a slower rate of NO release, both compounds could induce biofilm dispersal, under the same treatment conditions, when used at higher concentrations. In addition, LL4212 and LL4216 were found to promote P. aeruginosa growth in iron-limited minimal medium, leading to a faster rate of biofilm formation and glucose utilization, and ultimately resulted in early dispersal of biofilm cells through carbon starvation. High concentrations of LL4216 also repressed production of the siderophore pyoverdine by more than 50-fold, via both NOx-dependent and NOx-independent mechanisms. The effects on growth and pyoverdine levels exerted by the furoxans appeared to be mediated by NO-independent mechanisms, suggesting functional activities of furoxans in addition to their release of NO and nitrite. Overall, this study reveals that secondary effects of furoxans are important considerations for their use as NO-releasing dispersal agents and that these compounds could be potentially redesigned as pyoverdine inhibitors.

Antiviral activity of formyl peptide receptor 2 antagonists against influenza viruses.

Influenza viruses are one of the most important respiratory pathogens worldwide, causing both epidemic and pandemic infections. The aim of the study was to evaluate the effect of FPR2 antagonists PBP10 and BOC2 on influenza virus replication. We determined that these molecules exhibit antiviral effects against influenza A (H1N1, H3N2, H6N2) and B viruses. FPR2 antagonists used in combination with oseltamivir showed additive antiviral effects. Mechanistically, the antiviral effect of PBP10 and BOC2 is mediated through early inhibition of virus-induced ERK activation. Finally, our preclinical studies showed that FPR2 antagonists protected mice from lethal infections induced by influenza, both in a prophylactic and therapeutic manner. Thus, FPR2 antagonists might be explored for novel treatments against influenza.

Structure-constrained endomorphin analogs display differential antinociceptive mechanisms in mice after spinal administration.

We previously reported a series of novel endomorphin analogs with unnatural amino acid modifications. These analogs display good binding affinity and functional activity toward the µ opioid receptor (MOP). In the present study, we further investigated the spinal antinociceptive activity of these compounds. The analogs were potent in several nociceptive models. Opioid antagonists and antibodies against several endogenous opioid peptides were used to determine the mechanisms of action of these peptides. Intrathecal pretreatment with naloxone and ß-funaltrexamine (ß-FNA) effectively inhibited analog-induced analgesia, demonstrating that activity of the analogs is regulated primarily through MOP. Antinociception induced by analog 2 through 4 was not reversed by δ opioid receptor (DOP) or κ opioid receptor (KOP) antagonist; antibodies against dynorphin-A (1-17), dynorphin-B (1-13), and Leu5/Met5-enkephalin had no impact on the antinociceptive effects of these analogs. In contrast, antinociceptive effects induced by a spinal injection of the fluorine substituted analog 1 were significantly reversed by KOP antagonism. Furthermore, intrathecal pretreatment with antibodies against dynorphin-B (1-13) attenuated the antinociceptive effect of analog 1. These results indicate that the antinociceptive activity exerted by intrathecally-administered analog 1 is mediated, in part, through KOP with increased release of dynorphin-B (1-13). The chemical modifications used in the present study may serve as a useful tool to gain insight into the mechanisms of endomorphins activity.

Underlying mechanism of the cyclic migrating motor complex in Suncus murinus: a change in gastrointestinal pH is the key regulator.

In the fasted gastrointestinal (GI) tract, a characteristic cyclical rhythmic migrating motor complex (MMC) occurs in an ultradian rhythm, at 90-120 min time intervals, in many species. However, the underlying mechanism directing this ultradian rhythmic MMC pattern is yet to be completely elucidated. Therefore, this study aimed to identify the possible causes or factors that involve in the occurrence of the fasting gastric contractions by using Suncus murinus a small model animal featuring almost the same rhythmic MMC as that found in humans and dogs. We observed that either intraduodenal infusion of saline at pH 8 evoked the strong gastric contraction or continuously lowering duodenal pH to 3-evoked gastric phase II-like and phase III-like contractions, and both strong contractions were essentially abolished by the intravenous administration of MA 2029 (motilin receptor antagonist) and D-Lys3-GHRP6 (ghrelin receptor antagonist) in a vagus-independent manner. Moreover, we observed that the prostaglandin E2-alpha (PGE2-α) and serotonin type 4 (5HT4) receptors play important roles as intermediate molecules in changes in GI pH and motilin release. These results suggest a clear insight mechanism that change in the duodenal pH to alkaline condition is an essential factor for stimulating the endogenous release of motilin and governs the fasting MMC in a vagus-independent manner. Finally, we believe that the changes in duodenal pH triggered by flowing gastric acid and the release of duodenal bicarbonate through the involvement of PGE2-α and 5HT4 receptor are the key events in the occurrence of the MMC.

Triazole RGD antagonist reverts TGFß1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor ß1 (TGFß1) play a central role. When exposed to TGFß1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFß1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvß3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFß1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFß1, and reduces both ALK5/TGFß1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Integration of a 'proton antenna' facilitates transport activity of the monocarboxylate transporter MCT4.

Monocarboxylate transporters (MCTs) mediate the proton-coupled transport of high-energy metabolites like lactate and pyruvate and are expressed in nearly every mammalian tissue. We have shown previously that transport activity of MCT4 is enhanced by carbonic anhydrase II (CAII), which has been suggested to function as a 'proton antenna' for the transporter. In the present study, we tested whether creation of an endogenous proton antenna by introduction of a cluster of histidine residues into the C-terminal tail of MCT4 (MCT4-6xHis) could facilitate MCT4 transport activity when heterologously expressed in Xenopus oocytes. Our results show that integration of six histidines into the C-terminal tail does indeed increase transport activity of MCT4 to the same extent as did coexpression of MCT4-WT with CAII. Transport activity of MCT4-6xHis could be further enhanced by coexpression with extracellular CAIV, but not with intracellular CAII. Injection of an antibody against the histidine cluster into MCT4-expressing oocytes decreased transport activity of MCT4-6xHis, while leaving activity of MCT4-WT unaltered. Taken together, these findings suggest that transport activity of the proton-coupled monocarboxylate transporter MCT4 can be facilitated by integration of an endogenous proton antenna into the transporter's C-terminal tail.

In vitro effects of cysteine protease inhibitors on Trichomonas foetus-induced cytopathic changes in porcine intestinal epithelial cells.

OBJECTIVE To investigate the effects of specific cysteine protease (CP) inhibitors on cytopathic changes to porcine intestinal epithelial cells induced by Tritrichomonas foetus isolated from naturally infected cats. SAMPLE T foetus isolates from 4 naturally infected cats and nontransformed porcine intestinal epithelial cells. PROCEDURES T foetus isolates were treated with or without 0.1 to 1.0mM of the CP inhibitors antipain, cystatin, leupeptin, and chymostatin and the vinyl sulfone inhibitors WRR-483 and K11777. In-gel gelatin zymography was performed to evaluate the effects of these inhibitors on CP activity of T foetus isolates. Each treated or untreated isolate was also cocultured with monolayers of porcine intestinal epithelial cells for 24 hours, and cytopathic effects of T foetus were evaluated by light microscopy and crystal violet spectrophotometry. RESULTS Results of in-gel gelatin zymography suggested an ability of WRR-483, K11777, and cystatin to target specific zones of CP activity of the T foetus isolates. These inhibitors had no effect on T foetus growth, and the cytopathic changes to the intestinal epithelium induced by all 4 T foetus isolates were significantly inhibited. CONCLUSIONS AND CLINICAL RELEVANCE This study revealed that certain protease inhibitors were capable of inhibiting regions of CP activity (which has been suggested to cause intestinal cell damage in cats) in T foetus organisms and of ameliorating T foetus-induced cytopathic changes to porcine intestinal epithelium in vitro. Although additional research is needed, these inhibitors might be useful in the treatment of cats with trichomonosis.

Effects of neuropeptide FF and related peptides on the antinociceptive activities of VD-hemopressin(α) in naive and cannabinoid-tolerant mice.

Neuropeptide FF (NPFF) system has recently been reported to modulate cannabinoid-induced antinociception. The aim of the present study was to further investigate the roles of NPFF system in the antinociceptive effects induced by intracerebroventricular (i.c.v.) administration of mouse VD-hemopressin(α), a novel endogenous agonist of cannabinoid CB1 receptor, in naive and VD-hemopressin(α)-tolerant mice. The effects of NPFF system on the antinociception induced by VD-hemopressin(α) were investigated in the radiant heat tail-flick test in naive mice and VD-hemopressin(α)-tolerant mice. The cannabinoid-tolerant mice were produced by given daily injections of VD-hemopressin(α) (20 nmol, i.c.v.) for 5 days and the antinociception was measured on day 6. In naive mice, intracerebroventricular injection of NPFF dose-dependently attenuated central analgesia of VD-hemopressin(α). In contrast, neuropeptide VF (NPVF) and D.NP(N-Me)AFLFQPQRF-NH2 (dNPA), two highly selective agonists for Neuropeptide FF1 and Neuropeptide FF2 receptors, enhanced VD-hemopressin(α)-induced antinociception in a dose-dependent manner. In addition, the VD-hemopressin(α)-modulating activities of NPFF and related peptides were antagonized by the Neuropeptide FF receptors selective antagonist 1-adamantanecarbonyl-RF-NH2 (RF9). In VD-hemopressin(α)-tolerant mice, NPFF failed to modify VD-hemopressin(α)-induced antinociception. However, both neuropeptide VF and dNPA dose-dependently potentiated the antinociception of VD-hemopressin(α) and these cannabinoid-potentiating effects were reduced by RF9. The present works support the cannabinoid-modulating character of NPFF system in naive and cannabinoid-tolerant mice. In addition, the data suggest that a chronic cannabinoid treatment modifies the pharmacological profiles of NPFF, but not the cannabinoid-potentiating effects of neuropeptide VF and dNPA.