Targeting Lipoprotein Biogenesis: Considerations towards Antimicrobials.

Decades have passed without approval of a new antibiotic class. Several companies have recently halted related discovery efforts because of multiple obstacles. One promising route under research is to target the lipoprotein maturation pathway in light of major recent findings and the virulence roles of lipoproteins. To support the future design of selective drugs, considerations and priority-setting are established for the main lipoprotein processing enzymes (Lgt, LspA, and Lnt) based on microbiology, biochemistry, structural biology, chemical design, and pharmacology. Although not all bacterial species will be similarly impacted by drug candidates, several advantages make LspA a top target to pursue in the development of novel antibiotics effective against bacteria that are resistant to existing drugs.

Dietary choline increases brown adipose tissue activation markers and improves cholesterol metabolism in female APOE*3-Leiden.CETP mice.

OBJECTIVES: Studies in mice have recently linked increased dietary choline consumption to increased incidence of obesity-related metabolic diseases, while several clinical trials have reported an anti-obesity effect of high dietary choline intake. Since the underlying mechanisms by which choline affects obesity are incompletely understood, the aim of the present study was to investigate the role of dietary choline supplementation in adiposity. METHODS: Female APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism and cardiometabolic diseases, were fed a Western-type diet supplemented with or without choline (1.2%, w/w) for up to 16 weeks. RESULTS: Dietary choline reduced body fat mass gain, prevented adipocyte enlargement, and attenuated adipose tissue inflammation. Besides, choline ameliorated liver steatosis and damage, associated with an upregulation of hepatic genes involved in fatty acid oxidation. Moreover, choline reduced plasma cholesterol, as explained by a reduction of plasma non-HDL cholesterol. Mechanistically, choline reduced hepatic VLDL-cholesterol secretion and enhanced the selective uptake of fatty acids from triglyceride-rich lipoprotein (TRL)-like particles by brown adipose tissue (BAT), consequently accelerating the clearance of the cholesterol-enriched TRL remnants by the liver. CONCLUSIONS: In APOE*3-Leiden.CETP mice, dietary choline reduces body fat by enhancing TRL-derived fatty acids by BAT, resulting in accelerated TRL turnover to improve hypercholesterolemia. These data provide a mechanistic basis for the observation in human intervention trials that high choline intake is linked with reduced body weight.

Atorvastatin remodels lipid distribution between liver and adipose tissues through blocking lipoprotein efflux in fish.

The regulation of cholesterol metabolism in fish is still unclear. Statins play important roles in promoting cholesterol metabolism development in mammals. However, studies on the role of statins in cholesterol metabolism in fish are currently limited. The present study evaluated the effects of statins on cholesterol metabolism in fish. Nile tilapia (Oreochromis niloticus) were fed on control diets supplemented with three atorvastatin levels (0, 12, and 24 mg/kg diet, ATV0, ATV12, and ATV24, respectively) for 4 wk. Intriguingly, the results showed that both atorvastatin treatments increased hepatic cholesterol and triglyceride contents mainly through inhibiting bile acid synthesis and efflux, and compensatorily enhancing cholesterol synthesis in fish liver (P < 0.05). Moreover, atorvastatin treatment significantly inhibited hepatic very-low-density lipoprotein (VLDL) assembly and thus decreased serum VLDL content (P < 0.05). However, fish treated with atorvastatin significantly reduced cholesterol and triglycerides contents in adipose tissue (P < 0.05). Further molecular analysis showed that atorvastatin treatment promoted cholesterol synthesis and lipogenesis pathways, but inhibited lipid catabolism and low-density lipoprotein (LDL) uptake in the adipose tissue of fish (P < 0.05). In general, atorvastatin induced the remodeling of lipid distribution between liver and adipose tissues through blocking VLDL efflux from the liver to adipose tissue of fish. Our results provide a novel regulatory pattern of cholesterol metabolism response caused by atorvastatin in fish, which is distinct from mammals: cholesterol inhibition by atorvastatin activates hepatic cholesterol synthesis and inhibits its efflux to maintain cholesterol homeostasis, consequently reduces cholesterol storage in fish adipose tissue.

Terminalia catappa attenuates phenylhydrazine-induced anaemia and hepato-renal toxicity in male Wistar rat by boosting blood cells, modulation of lipoproteins and up-regulation of in vivo antioxidant armouries.

AIM: This study investigated the haematinic, antihyperlipidaemic, hepato-renal protective effects of Terminalia catappa aqueous leaf extract on male Wistar rats exposed to phenylhydrazine toxicity. METHODS: Animals were exposed to phenylhydrazine (PHZ) 50 mg/kg intraperitoneal for two consecutive days thereafter, treated with T. catappa extract (100 mg/kg and 200 mg/kg) orally for 21 days. After the experimentation, animals were sedated with ketamine (70 mg/kg) and euthanized by cervical dislodgement. Blood and organs were collected for haematology and biochemical studies following standard laboratory methods. RESULTS: Our study showed that T. catappa significantly increased erythrocytes, haemoglobin, haematocrit and high density lipoprotein as well as down-regulating leukocytes, thrombocytes, ALP AST, ALT creatinine, urea, total cholesterol as well as low density lipoprotein. The liver, kidney and spleen antioxidant defence were also up-regulated against the adverse effect caused by phenylhydrazine exposure. CONCLUSION: Terminalia catappa attenuated Phenylhydrazine-induced anaemia and hepato-renal toxicity in male Wistar rat by boosting blood cells, modulation of lipoproteins and up-regulation of in vivo antioxidant armouries.

Beneficial Effect of Cuban Policosanol on Blood Pressure and Serum Lipoproteins Accompanied with Lowered Glycated Hemoglobin and Enhanced High-Density Lipoprotein Functionalities in a Randomized, Placebo-Controlled, and Double-Blinded Trial with Healthy Japanese.

This study evaluated the efficacy and safety of 20 mg of Cuban policosanol in blood pressure (BP) and lipid/lipoprotein parameters of healthy Japanese subjects via a placebo-controlled, randomized, and double-blinded human trial. After 12 weeks of consumption, the policosanol group showed significantly lower BP, glycated hemoglobin (HbA1c), and blood urea nitrogen (BUN) levels. The policosanol group also showed lower aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transferase (γ-GTP) levels at week 12 than those at week 0: A decrease of up to 9% (p < 0.05), 17% (p < 0.05), and 15% (p < 0.05) was observed, respectively. The policosanol group showed significantly higher HDL-C level and HDL-C/TC (%), approximately 9.5% (p < 0.001) and 7.2% (p = 0.003), respectively, than the placebo group and a difference in the point of time and group interaction (p < 0.001). In lipoprotein analysis, the policosanol group showed a decrease in oxidation and glycation extent in VLDL and LDL with an improvement of particle shape and morphology after 12 weeks. HDL from the policosanol group showed in vitro stronger antioxidant and in vivo anti-inflammatory abilities. In conclusion, 12 weeks of Cuban policosanolconsumption in Japanese subjects showed significant improvement in blood pressure, lipid profiles, hepatic functions, and HbA1c with enhancement of HDL functionalities.

Effects of Cilostazol and Isosorbide Mononitrate on Cerebral Hemodynamics in the LACI-1 Randomized Controlled Trial.

BACKGROUND AND PURPOSE: Cerebral small vessel disease-a major cause of stroke and dementia-is associated with cerebrovascular dysfunction. We investigated whether short-term isosorbide mononitrate (ISMN) and cilostazol, alone or in combination, improved magnetic resonance imaging-measured cerebrovascular function in patients with lacunar ischemic stroke. METHODS: Participants were randomized to ISMN alone, cilostazol alone, both ISMN and cilostazol, or no medication. Participants underwent structural, cerebrovascular reactivity (to 6% carbon dioxide) and phase-contrast pulsatility magnetic resonance imaging at baseline and after 8 weeks of medication. RESULTS: Of 27 participants (mean age, 68±7.7; 44% female), 22 completed cerebrovascular reactivity and pulsatility imaging with complete datasets. White matter cerebrovascular reactivity increased in the ISMN (ß=0.021%/mm Hg [95% CI, 0.003-0.040]) and cilostazol (ß=0.035%/mm Hg [95% CI, 0.014-0.056]) monotherapy groups and in those taking any versus no medication (ß=0.021%/mm Hg [95% CI, 0.005-0.037]). CONCLUSIONS: While limited by small sample size, we demonstrate that measuring cerebrovascular function with magnetic resonance imaging is feasible in clinical trials and that ISMN and cilostazol may improve cerebrovascular function. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02481323. URL: www.isrctn.com; Unique identifier: ISRCTN12580546. URL: www.clinicaltrialsregister.eu; Unique identifier: EudraCT 2015-001953-33.

Procoagulant activities of skeletal and cardiac muscle myosin depend on contaminating phospholipid.

Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine-binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by ∼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.

Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance.IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

Heparanase Level and Procoagulant Activity Are Increased in Thalassemia and Attenuated by Janus Kinase 2 Inhibition.

Patients with thalassemia exhibit an increased risk of thrombotic events that is augmented after splenectomy. Heparanase protein enhances cancer progression, angiogenesis, and inflammation; it also activates the coagulation system through direct interaction with tissue factor (TF). Additionally, erythropoietin, which is elevated in anemic patients, up-regulates heparanase expression via the Janus kinase 2 (JAK-2) pathway. This study aimed was to explore the heparanase profile in thalassemia. Coagulation factors were analyzed via immunostaining, enzyme-linked immunosorbent assay, and heparanase procoagulant activity assay. In spleen specimens of thalassemia major patients, a higher level of heparanase staining was observed compared with control spleens resected after trauma (P < 0.001). Higher heparanase levels, heparanase and TF procoagulant activity, and erythropoietin levels were found in the plasma of 67 thalassemia major patients compared with 29 control subjects. No difference was found in pediatric patients (23 of 67) compared with adults or splenectomized versus nonsplenectomized patients. Higher levels of heparanase, TF, TF pathway inhibitor, and TF pathway inhibitor-2 were observed in liver, spleen, heart, and kidney tissues of thalassemia intermedia mice (Hbbth3/+). These protein levels significantly reduced when mice were treated with the JAK-2 inhibitor ruxolitinib (P < 0.0001). In summary, heparanase levels are elevated in thalassemia, which may contribute to thrombotic phenomena in these patients. Inhibition of heparanase or the JAK-2 pathway may reduce thrombotic risk in thalassemia.

Biological and Physico-Chemical Characteristics of Arginine-Rich Peptide Gemini Surfactants with Lysine and Cystine Spacers.

Ultrashort cationic lipopeptides (USCLs) and gemini cationic surfactants are classes of potent antimicrobials. Our recent study has shown that the branching and shortening of the fatty acids chains with the simultaneous addition of a hydrophobic N-terminal amino acid in USCLs result in compounds with enhanced selectivity. Here, this approach was introduced into arginine-rich gemini cationic surfactants. l-cystine diamide and l-lysine amide linkers were used as spacers. Antimicrobial activity against planktonic and biofilm cultures of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) strains and Candida sp. as well as hemolytic and cytotoxic activities were examined. Moreover, antimicrobial activity in the presence of human serum and the ability to form micelles were evaluated. Membrane permeabilization study, serum stability assay, and molecular dynamics were performed. Generally, critical aggregation concentration was linearly correlated with hydrophobicity. Gemini surfactants were more active than the parent USCLs, and they turned out to be selective antimicrobial agents with relatively low hemolytic and cytotoxic activities. Geminis with the l-cystine diamide spacer seem to be less cytotoxic than their l-lysine amide counterparts, but they exhibited lower antibiofilm and antimicrobial activities in serum. In some cases, geminis with branched fatty acid chains and N-terminal hydrophobic amino acid resides exhibited enhanced selectivity to pathogens over human cells.

A method for gene knockdown in the retina using a lipid-based carrier.

Purpose: The use of small non-coding nucleic acids, such as siRNA and miRNA, has allowed for a deeper understanding of gene functions, as well as for development of gene therapies for complex neurodegenerative diseases, including retinal degeneration. For effective delivery into the eye and transfection of the retina, suitable transfection methods are required. We investigated the use of a lipid-based transfection agent, Invivofectamine® 3.0 (Thermo Fisher Scientific), as a potential method for delivery of nucleic acids to the retina. Methods: Rodents were injected intravitreally with formulations of Invivofectamine 3.0 containing scrambled, Gapdh, Il-1ß, and C3 siRNAs, or sterile PBS (control) using a modified protocol for encapsulation of nucleic acids. TdT-mediated dUTP nick-end labeling (TUNEL) and IBA1 immunohistochemistry was used to determine histological cell death and inflammation. qPCR were used to determine the stress and inflammatory profile of the retina. Electroretinography (ERG) and optical coherence tomography (OCT) were employed as clinical indicators of retinal health. Results: We showed that macrophage recruitment, retinal stress, and photoreceptor cell death in animals receiving Invivofectamine 3.0 were comparable to those in negative controls. Following delivery of Invivofectamine 3.0 alone, no statistically significant changes in expression were found in a suite of inflammatory and stress genes, and ERG and OCT analyses revealed no changes in retinal function or morphology. Injections with siRNAs for proinflammatory genes (C3 and Il-1ß) and Gapdh, in combination with Invivofectamine 3.0, resulted in statistically significant targeted gene knockdown in the retina for up to 4 days following injection. Using a fluorescent Block-It siRNA, transfection was visualized throughout the neural retina with evidence of transfection observed in cells of the ganglion cell layer, inner nuclear layer, and outer nuclear layer. Conclusions: This work supports the use of Invivofectamine 3.0 as a transfection agent for effective delivery of nucleic acids to the retina for gene function studies and as potential therapeutics.

Total Synthesis of A54145 Factor D.

An efficient total synthesis of A54145 factor D (A5D), a member of the A54145 family of cyclic lipodepsipeptide antibiotics, is reported. The peptide was constructed by attaching the peptide to the 2'-chlorotrityl polystyrene resin via Sar5 and developing conditions that avoided diketopiperazine formation upon subsequent elaboration using 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. This route allowed for facile formation of the crucial depsi bond. A branched acyclic precursor was cyclized off-resin and then globally deprotected to obtain A5D. Consistent with recent studies by others, we found that the MeOAsp residue has the 2S,3R configuration. We also established that the configuration of the stereocenter in the anteiso-undecanoyl lipid tail does not affect biological activity.

Trained Immunity Characteristics Are Associated With Progressive Cerebral Small Vessel Disease.

Background and Purpose- Cerebral small vessel disease (cSVD) is the major vascular cause of cognitive decline and dementia. The pathogenesis of cSVD remains largely unknown, although several studies suggest a role for systemic inflammation. In certain pathophysiological situations, monocytes can reprogram toward a long-term proinflammatory phenotype, which has been termed trained immunity. We hypothesize that trained immunity contributes to the progression of cSVD. Methods- Individuals with mild-to-severe cSVD participated in the study. Severity of cSVD was determined by the white matter hyperintensities (WMH) volume (mL) on magnetic resonance imaging in 2006, 2015, and the progression between 2006 and 2015 (ΔWMH). Cytokine production was assessed after ex vivo stimulation of peripheral blood mononuclear cells and monocytes. Additionally, monocyte subsets were identified by flow cytometry. Results- Fifty-one subjects (70±6 years, 60% men, 5.1±6.4 mL ΔWMH) were included. Circulating hsIL (high-sensitivity interleukin)-6 correlated with cSVD ( P=0.005, rs=0.40). Cytokine production capacity by monocytes was associated with cSVD progression. Basal IL-8 and IL-17 production ( P=0.08, rs=0.25; P=0.03, rs=0.30) and IL-6 production after Pam3Cys stimulation in monocytes was associated with cSVD (n=35: P=0.008, rs=0.44). Conversely, interferon (IFN)-γ production in Candida albicans stimulated peripheral blood mononuclear cells was negatively correlated with cSVD ( P=0.009, rs=-0.36). Flow cytometry revealed a correlation of the intermediate monocyte subset with cSVD ( P=0.01, rs=0.36). Conclusions- Severity and progression of cSVD are not only correlated with systemic inflammation (hsIL-6) but also with trained immunity characteristics of circulating monocytes, in terms of an altered cytokine production capacity and a shift toward the proinflammatory intermediate monocyte subset.

Regulation of systemic tissue injury by coagulation inhibitors in B6.MRL/lpr autoimmune mice.

Impaired fibrinolysis and complement activation in Systemic Lupus Erythematosus contributes to disease amplification including increased risk of thrombosis and tissue Ischemia/Reperfusion (IR) injury. Previous work has demonstrated complement is a key regulator of tissue injury. In these studies inhibitors had varying efficacies in attenuating injury at primary versus systemic sites, such as lung. In this study the role of coagulation factors in tissue injury and complement function was evaluated. Tissue Factor Pathway Inhibitor (TFPI), an extrinsic pathway inhibitor, and Anti-Thrombin III, the downstream common pathway inhibitor, were utilized in this study. TFPI was more effective in attenuated primary intestinal tissue injury. However both attenuated systemic lung injury. However, ATIII treatment resulting in enhanced degradation of C3 split products in lung tissue compared to TFPI. This work delineates the influence of specific early and late coagulation pathway components during initial tissue injury versus later distal systemic tissue injury mechanism.

Reconstituted Discoidal High-Density Lipoproteins: Bioinspired Nanodiscs with Many Unexpected Applications.

PURPOSE OF REVIEW: Summarize the initial discovery of discoidal high-density lipoprotein (HDL) in human plasma and review more recent innovations that span the use of reconstituted nanodisc HDL for membrane protein characterization to its use as a drug carrier and a novel therapeutic agent for cardiovascular disease. RECENT FINDINGS: Using a wide variety of biophysical techniques, the structure and composition of endogenous discoidal HDL have now largely been solved. This has led to the development of new methods for the in vitro reconstitution of nanodisc HDL, which have proven to have a wide variety of biomedical applications. Nanodisc HDL has been used as a platform for mimicking the plasma membrane for the reconstitution and investigation of the structures of several plasma membrane proteins, such as cytochrome P450s and ABC transporters. Nanodisc HDL has also been designed as drug carriers to transport amphipathic, as well as hydrophobic small molecules, and has potential therapeutic applications for several diseases. Finally, nanodisc HDL itself like native discoidal HDL can mediate cholesterol efflux from cells and are currently being tested in late-stage clinical trials for cardiovascular disease. The discovery of the characterization of native discoidal HDL has inspired a new field of synthetic nanodisc HDL, which has offered a growing number of unanticipated biomedical applications.

Monophosphoryl Lipid A and Pam3Cys Prevent the Increase in Seizure Susceptibility and Epileptogenesis in Rats Undergoing Traumatic Brain Injury.

Five percent of all epilepsy cases are attributed to traumatic brain injury (TBI), which are known as post-traumatic epilepsy (PTE). Finding preventive strategies for PTE is valuable. Remarkable feature of TBI is activation of microglia and subsequent neuroinflammation, which provokes epileptogenesis. The toll-like receptor agonists monophosphoryl lipid A (MPL) and tri-palmitoyl-S-glyceryl-cysteine (Pam3Cys) are safe, well-tolerated and effective adjuvants existing in prophylactic human vaccines. We examined the impact of early injection of MPL and Pam3Cys to rats, on the rate of kindled seizures acquisition following TBI. Rats received a single dose (1 µg/rat) of MPL or Pam3Cys through intracerebroventricular injection. 5 days later, trauma was exerted to temporo-parietal cortex of rats by controlled cortical impact device. After 24 h, traumatic rats underwent amygdala kindling. Brain level of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) was also measured in traumatic rats by immunoblotting. Compared to non-traumatic (sham-operated) rats, traumatic rats showed three times lower seizure threshold (133 ± 5 µA vs. 416.3 ± 16 µA, p < 0.001); about three times less number of stimuli to become kindled (5 ± 1 vs. 14 ± 2, p < 0.01); longer duration of kindled seizure parameters including entire seizure behavior, generalized seizures, and afterdischarges (p < 0.001); and a two times increase in the TNF-α level. MPL and Pam3Cys did not change kindling rate and the seizure parameters in sham-operated rats. The MPL- and Pam3Cys-pretreated traumatic rats displayed seizure threshold, speed of kindling, and duration of kindled seizure parameters, similar to the non-traumatic rats. Pretreatment by MPL and Pam3Cys prevented the increase in TNF-α level by trauma. Given that MPL and Pam3Cys currently have clinical use as well-tolerated vaccines with reliable safety, they have the potential to be used in prevention of PTE.

Mycoplasma lipoproteins are major determinants of neutrophil extracellular trap formation.

Neutrophil granulocytes are paramount to innate responses as major effectors of acute inflammation. Among the various strategies enacted by neutrophils to eliminate microbes NETosis is a novel distinct antimicrobial activity in which an interlacement of chromatin fibres rich in granule-derived antimicrobial peptides and enzymes is extruded (NETs, neutrophils extracellular traps ). NETs contribute to the pathogenesis of acute and chronic inflammatory disorders. The interactions of mycoplasmas and innate immune cells, in particular neutrophil granulocytes, are poorly defined. Here, we describe NET formation in vivo in the mammary gland and milk of sheep naturally infected by Mycoplasma agalactiae. Also, we assess the contribution of liposoluble proteins, the most abundant component of the Mycoplasma membrane, in inducing NETosis. We demonstrate that Mycoplasma liposoluble proteins induce NET release at levels comparable to what observed with other stimuli, such as lipopolysaccharides and phorbol 12-myristate 13-acetate. Stimulation of neutrophils with synthetic diacylated lipopeptides based on the M. agalactiae P48, P80, and MAG_1000 proteins, combined in a mix or used individually, suggests that NETosis might not be dependent on a specific lipopeptide sequence. Also, NETosis is partially abolished when TLR2 is blocked with specific antibodies. The results presented in this work provide evidences for the mechanisms underlying NET activation in mycoplasma infections, and on their contribution to pathogenesis of mycoplasmosis.

A multicomponent macrocyclization strategy to natural product-like cyclic lipopeptides: synthesis and anticancer evaluation of surfactin and mycosubtilin analogues.

A multicomponent macrocyclization strategy towards cyclic lipopeptides is described. The approach relies on the utilization of the Ugi and Passerini multicomponent reactions for the cyclization of peptides and oxo-peptides, and here it is employed for the construction of a small library of analogues of the natural products mycosubtilin and surfactin A. A key feature of this method is the simultaneous incorporation of either one or two exocyclic lipid tails along with the macrocyclic ring closure, which is only possible due to the multicomponent nature of the macrocyclization step. The evaluation of the anticancer activity of the lipopeptide library showed that the installation of a second lipid moiety in the surfactin scaffold leads to a more potent cytotoxicity in cancer cells. This is a new example of the multicomponent reaction potential in rapidly producing natural product analogues for biological screening.

TLR2 stimulation regulates the balance between regulatory T cell and Th17 function: a novel mechanism of reduced regulatory T cell function in multiple sclerosis.

CD4(+)CD25(hi) FOXP3(+) regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the pathogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2 heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity and progression.

Membrane Binding and Oligomerization of the Lipopeptide A54145 Studied by Pyrene Fluorescence.

A54145 is a lipopeptide antibiotic related to daptomycin that permeabilizes bacterial cell membranes. Its action requires both calcium and phosphatidylglycerol in the target membrane, and it is accompanied by the formation of membrane-associated oligomers. We here probed the interaction of A54145 with model membranes composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, using the steady-state and time-resolved fluorescence of a pyrene-labeled derivative (Py-A54145). In solution, the labeled peptide was found to exist as a monomer. Its membrane interaction occurred in two stages that could be clearly distinguished by varying the calcium concentration. In the first stage, which was observed between 0.15 and 1 mM calcium, Py-A54145 bound to the membrane, as indicated by a strong increase in pyrene monomer emission. At the same calcium concentration, excimer emission increased also, suggesting that Py-A54145 had oligomerized. A global analysis of the time-resolved pyrene monomer and excimer fluorescence confirmed that Py-A54145 forms oligomers quantitatively and concomitantly with membrane binding. When calcium was raised beyond 1 mM, a distinct second transition was observed that may correspond to a doubling of the number of oligomer subunits. The collective findings confirm and extend our understanding of the action mode of A54145 and daptomycin.