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Despite major advances in the β-lactamase inhibitor field, certain enzymes remain refractory to inhibition by agents recently introduced. Most important among these are the class B (metallo) enzyme NDM-1 of Enterobacteriaceae and the class D (OXA) enzymes of Acinetobacter baumannii. Continuing the boronic acid program that led to vaborbactam, efforts were directed toward expanding the spectrum to allow treatment of a wider range of organisms. Through key structural modifications of a bicyclic lead, stepwise gains in spectrum of inhibition were achieved, ultimately resulting in QPX7728 (35). This compound displays a remarkably broad spectrum of inhibition, including class B and class D enzymes, and is little affected by porin modifications and efflux. Compound 35 is a promising agent for use in combination with a β-lactam antibiotic for the treatment of a wide range of multidrug resistant Gram-negative bacterial infections, by both intravenous and oral administration.

In recognition of the need for effective oral therapies to treat Gram-negative bacterial infections, efforts were directed toward identifying an oral prodrug of β-lactamase inhibitor clinical candidate QPX7728. Seventeen prodrugs were synthesized; key properties investigated were rates of cleavage to the active form in vitro, pharmacokinetics across species, and crystallinity. Compound 5-Na (QPX7831 Sodium) emerged with optimal properties across all key attributes.

Resistance to beta-lactams has created a major clinical issue. QPX7728 is a novel ultrabroad-spectrum cyclic boronic acid beta-lactamase inhibitor with activity against both serine and metallo-beta-lactamases developed to address this resistance for use in combination with beta-lactam antibiotics. The objective of these studies was to evaluate the activity of QPX7728 in combination with multiple beta-lactams against carbapenem-resistant Klebsiella pneumoniae isolates in a neutropenic mouse thigh infection model. Neutropenic mice were infected with strains with potentiated beta-lactam MICs of ≤2 mg/liter in the presence of 8 mg/liter QPX7728. Two strains of carbapenem-resistant K. pneumoniae were tested with aztreonam, biapenem, cefepime, ceftazidime, ceftolozane, and meropenem alone or in combination with 12.5, 25, or 50 mg/kg of body weight of QPX7728 every 2 hours for 24 hours. Treatment with all beta-lactams alone either was bacteriostatic or allowed for bacterial growth. The combination of QPX7728 plus each of these beta-lactams produced bacterial killing at all QPX7728 doses tested. Overall, these data suggest that QPX7728 administered in combination with different partner beta-lactam antibiotics may have utility in the treatment of bacterial infections due to carbapenem-resistant K. pneumoniae.

Acinetobacter baumannii infections are difficult to treat and have limited treatment options. Carbapenems, including meropenem, are currently considered the first-line agents for the treatment of infections caused by Acinetobacter spp. The percentage of a 24-hour period that the concentration of free drug in plasma is above the MIC (%24-h fT>MIC) to achieve stasis, 1 log CFU, or 2 log CFU of bacterial killing against A. baumannii has not been studied previously for meropenem. The objective of this study was to determine these parameters for meropenem against A. baumannii in a neutropenic mouse thigh infection model. Six A. baumannii clinical isolates with MICs ranging from 0.25 to 16 mg/liter were tested. Meropenem produced a bacteriostatic effect with a %24-h fT>MIC of 7 to 24% and produced 1 log CFU of bacterial killing with a %24-h fT>MIC of 15 to 37%.

Burkholderia cepacia complex is an opportunistic pathogen capable of causing chronic pulmonary infections. These studies were conducted to demonstrate the activity of aerosolized levofloxacin in a chronic mouse lung infection model caused by B. cepacia isolates from patients with cystic fibrosis. Treatment with aerosolized levofloxacin for 4 days produced at least 1 log CFU of bacterial killing against all strains tested, suggesting possible utility in the treatment of lung infections caused by B. cepacia isolates.

Minocycline is currently approved in the United States for the treatment of infections caused by susceptible isolates of Acinetobacter spp. The objective of these studies was to determine the minocycline exposures associated with an antibacterial effect against Acinetobacter baumannii in a rat pneumonia model. Rats received minocycline doses as 30-min intravenous infusions. In the rat pneumonia model, six clinical isolates of A. baumannii with MICs ranging from 0.03 to 4 mg/liter were studied. In this model, minocycline produced a bacteriostatic effect with a free 24-h area under the concentration-time curve (AUC)/MIC ratio of 10 to 16 and produced 1 log of bacterial killing with a free 24-h AUC/MIC of 13 to 24. These exposures can be achieved with the current FDA-approved dosage regimens of intravenous minocycline.

Vaborbactam is a novel beta-lactamase inhibitor with activity against important beta-lactamases, in particular, serine carbapenemases, and is currently approved in combination with meropenem as Vabomere for the treatment of complicated urinary tract infections, including pyelonephritis. This combination is highly active against Gram-negative pathogens, especially Klebsiella pneumoniae carbapenemase (KPC)-producing carbapenem-resistant Enterobacteriaceae. The objective of these studies was to evaluate vaborbactam pharmacokinetics (PK) and pharmacodynamics (PD) relationships for efficacy in a neutropenic mouse thigh infection model, as well as in an in vitro hollow-fiber infection model, in combination with a fixed exposure of meropenem using KPC-containing strains of Enterobacteriaceae. For both models, the meropenem dosage regimen was designed to simulate a 2-g dose administered every eight hours (q8h) by 3-h infusion. Vaborbactam dosage regimens were designed to produce a wide range of 24-h areas under the concentration-time curves (AUCs) in the thigh infection model. However, for the hollow-fiber model, the AUCs were limited to values of 192, 320, or 550 mg · h/liter. In both the animal and in vitro models, the PK-PD parameter that best described the antibacterial activity of vaborbactam, when administered in combination with meropenem at exposures equivalent to 2 g dosed q8h by 3-h infusion in humans, was the 24-h free vaborbactam AUC/meropenem-vaborbactam (with vaborbactam at 8 mg/liter) MIC ratio. The magnitude of this ratio for bacteriostasis was 9 to 12 and the magnitude to observe a 1-log kill was 18 to 38. In addition, a magnitude greater than 24 suppressed the development of resistance in the in vitro hollow-fiber model.

We have evaluated the activity of meropenem-vaborbactam against clinical isolates of Pseudomonas aeruginosa and Acinetobacter baumannii in a neutropenic mouse thigh infection model. Data show that meropenem-vaborbactam regimens equivalent to 3-h infusions every 8 h with 2 g meropenem and 2 g vaborbactam produced bacterial killing against strains with MICs of 2 to 16 mg/liter and suggests that this combination may have utility in the treatment of infections caused by P. aeruginosa and A. baumannii .

Minocycline is currently approved in the United States for the treatment of infections caused by susceptible isolates of Acinetobacter spp. The objective of these studies was to determine the minocycline exposures associated with an antibacterial effect against Acinetobacter baumannii in a rat pneumonia model. Rats received minocycline doses as 30-min intravenous infusions. In the rat pneumonia model, six clinical isolates of A. baumannii with MICs ranging from 0.03 to 4 mg/liter were studied. In this model, minocycline produced a bacteriostatic effect with a free 24-h area under the concentration-time curve (AUC)/MIC ratio of 10 to 16 and produced 1 log of bacterial killing with a free 24-h AUC/MIC of 13 to 24. These exposures can be achieved with the current FDA-approved dosage regimens of intravenous minocycline.

Background Currently available polymyxins are limited by toxicity and poor efficacy at tolerated doses. We have developed a new series of polymyxin derivatives with improved safety profiles and in vitro potency against major MDR bacteria. The following describes studies on the in vivo antimicrobial activity and toxicity of QPX9003 in mice and rats. Methods Mouse studies. The minimum lethal dose (MLD by IV bolus) and nephrotoxicity (6 IP doses administered 2 hours apart) of QPX9003 and polymyxin B (PMB) were determined in Swiss mice. For the neutropenic mouse thigh infection using A. baumannii, Swiss mice were infected with ~106 CFU/thigh. Doses were administered IP at various intervals starting 2-hour post-infection and continued over 24 hours. Rat studies. For the rat lung infection model, Sprague-Dawley rats were infected with ~107 CFU/lung. QPX9003 and PMB were administered IV every 4 hours starting 2 hours post-infection and continued over 24 hours. Bacteria. For both infection models, animals were infected with A. baumannii AB1016 (QPX9003 MIC of 0.5 mg/L and PMB MIC of 1.0 mg/L). Untreated control groups were sacrificed at the start of treatment and both untreated and treated groups were sacrificed 24 hours after the start of treatment, infected tissues harvested, homogenized, and plated to determine colony counts. Results QPX9003 had reduced acute toxicity and nephrotoxicity compared with PMB in mice. QPX9003 showed better bacterial killing of A. baumannii than PMB at similar plasma exposures in both the mouse thigh model (−0.41 vs. +0.83 log CFU/thigh) and rat lung infection model (−1.10 vs. +1.44 log CFU/lung). Conclusion QPX9003 was less acutely toxic, less nephrotoxic, and was more efficacious in mouse and rat infection models compared with PMB. QPX9003 is a promising new polymyxin. (This work was supported in part by federal funds from the National Institutes of Allergy and Infectious Diseases [R01AI098771], and the Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response; Biomedical Advanced Research and Development Authority (BARDA), under OTA number HHSO100201600026C). Disclosures All authors: No reported disclosures.

The objective of these studies was to evaluate the exposures of meropenem and vaborbactam that would produce antibacterial activity and prevent resistance development in carbapenem-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae strains when tested at an inoculum of 10 8 CFU/ml. Thirteen K. pneumoniae isolates, three Enterobacter cloacae isolates, and one Escherichia coli isolate were examined in an in vitro hollow-fiber model over 32 h. Simulated dosage regimens of 1 to 2 g of meropenem with 1 to 2 g of vaborbactam, with meropenem administered every 8 h by a 3-h infusion based on phase 1 or phase 3 patient pharmacokinetic data, were studied in the model. A dosage of 2 g of meropenem in combination with 2 g of vaborbactam was bactericidal against K. pneumoniae , E. cloacae , and E. coli strains, with meropenem-vaborbactam MICs of up to 8 mg/liter. When the vaborbactam exposure was adjusted to the levels observed in patients enrolled in phase 3 trials (24-h free AUC, ∼550 mg · h/liter, versus 320 mg · h/liter in the phase 1 studies), 2 g of meropenem with 2 g of vaborbactam was also bactericidal against strains with meropenem-vaborbactam MICs of 16 mg/liter. In addition, this level of vaborbactam also suppressed the development of resistance observed using phase 1 exposures. In this pharmacodynamic model, exposures similar to 2 g of meropenem in combination with 2 g of vaborbactam administered every 8 h by a 3-h infusion in phase 3 trials produced antibacterial activity and suppressed the development of resistance against carbapenem-resistant KPC-producing strains of Enterobacteriaceae .

Meropenem-vaborbactam (Vabomere) is highly active against Gram-negative pathogens, especially Klebsiella pneumoniae carbapenemase (KPC)-producing, carbapenem-resistant Enterobacteriaceae . The objective of these studies was to evaluate the efficacy of meropenem alone and in combination with vaborbactam in mouse thigh and lung infection models. Thighs or lungs of neutropenic mice were infected with KPC-producing carbapenem-resistant Enterobacteriaceae , with meropenem MICs ranging from ≤0.06 to 8 mg/liter in the presence of 8 mg/liter vaborbactam. Mice were treated with meropenem alone or meropenem in combination with vaborbactam every 2 h for 24 h to provide exposures comparable to 2-g doses of each component in humans. Meropenem administered in combination with vaborbactam produced bacterial killing in all strains tested, while treatment with meropenem alone either produced less than 0.5 log CFU/tissue of bacterial killing or none at all. In the thigh model, 11 strains were treated with the combination of meropenem plus vaborbactam (300 plus 50 mg/kg of body weight). This combination produced from 0.8 to 2.89 logs of bacterial killing compared to untreated controls at the start of treatment. In the lung infection model, two strains were treated with the same dosage regimen of meropenem and vaborbactam. The combination produced more than 1.83 logs of bacterial killing against both strains tested compared to untreated controls at the start of treatment. Overall, these data suggest that meropenem-vaborbactam may have utility in the treatment of infections due to KPC-producing carbapenem-resistant Enterobacteriaceae .

The recently approved combination of meropenem and vaborbactam (Vabomere) is highly active against Gram-negative pathogens, especially Klebsiella pneumoniae carbapenemase (KPC)-producing, carbapenem-resistant Enterobacteriaceae . We evaluated the efficacy of meropenem-vaborbactam against three clinically relevant isolates in a murine pyelonephritis model. The data indicate that the combination of meropenem and vaborbactam significantly increased bacterial killing compared to that with the untreated controls. These data suggest that this combination may have utility in the treatment of complicated urinary tract infections due to KPC-producing, carbapenem-resistant Enterobacteriaceae .

Bitkilere yonelik gerceklestirilen floristik calismalar, her bir ulkenin dogal kaynaklarini korumada en onemli rollerden faktorlerden biridir. Arastirma bolgesinde onemli yetisme alanlarini temsil eden konumlardan, bitki turleri toplanmistir. Bu kapsamdaki bitki toplama calismalari, 2013 ve 2014 yillarinda bitkilerin aktif buyume donemlerinde gerceklestirilmistir. Toplamda 140 adet tibbi bitki turu belgelenmistir. Bu turler, 39 aileye ve 116 cinse ayrilmistir. 26 ture sahip Lamiaceae (Ballibabagiller), 21 tur ile Asteraceae (Papatyagiller) ve 13 tur ile Rosaceae (Gulgiller) aileleri, arastirma bolgesinde en fazla rastlanan tibbi bitki aileleri olmustur. Hemikriptofitler %40, terofitler %18,4, geofitler %14,25, fanerofitler %13,75 ve kamefitler %6,42 oranindadir. Tibbi bitkilerin fitocografya ozelligi, bu turlerin IranTuran, Avrupa-Sibirya ve Akdeniz bolgelerine ait oldugunu gostermistir. Mevcut arastirmanin sonuclari, bu arastirmada belgelenen Kereste Disi Orman Urunleri olarak tibbi bitkilerin ve yabani meyvelerin, kirsal toplumun refahinda ve surdurulebilir orman yonetiminde onemli rol oynadigini gostermistir.

The increasing dissemination of carbapenemases in Gram-negative bacteria has threatened the clinical usefulness of the β-lactam class of antimicrobials. A program was initiated to discover a new series of serine β-lactamase inhibitors containing a boronic acid pharmacophore, with the goal of finding a potent inhibitor of serine carbapenemase enzymes that are currently compromising the utility of the carbapenem class of antibacterials. Potential lead structures were screened in silico by modeling into the active sites of key serine β-lactamases. Promising candidate molecules were synthesized and evaluated in biochemical and whole-cell assays. Inhibitors were identified with potent inhibition of serine carbapenemases, particularly the Klebsiella pneumoniae carbapenemase (KPC), with no inhibition of mammalian serine proteases. Studies in vitro and in vivo show that RPX7009 (9f) is a broad-spectrum inhibitor, notably restoring the activity of carbapenems against KPC-producing strains. Combined with a carbapenem, 9f is a promising product for the treatment of multidrug resistant Gram-negative bacteria.

Cholera toxin (CT) elicits a mucosal immune response in mice when used as a vaccine adjuvant. The mechanisms by which CT exerts its adjuvant effects are incompletely understood. We show that protection against inhalation anthrax by an irradiated spore vaccine depends on CT-mediated induction of IL-17-producing CD4 Th17 cells. Furthermore, IL-17 is involved in the induction of serum and mucosal antibody responses by CT. Th17 cells induced by CT have a unique cytokine profile compared with those induced by IL-6 and TGF-β, and their induction by CT requires cAMP-dependent secretion of IL-1β and β-calcitonin gene-related peptide by dendritic cells. These findings demonstrate that Th17 cells mediate mucosal adjuvant effects of CT and identify previously unexplored pathways involved in Th17 induction that could be targeted for development of unique mucosal adjuvants.

Bacillus anthracis is a National Institute of Allergy and Infectious Diseases Category A priority pathogen and the causative agent of the deadly disease anthrax. We applied a transposon mutagenesis system to screen for novel chromosomally encoded B. anthracis virulence factors. This approach identified ClpX, the regulatory ATPase subunit of the ClpXP protease, as essential for both the hemolytic and proteolytic phenotypes surrounding colonies of B. anthracis grown on blood or casein agar media, respectively. Deletion of clpX attenuated lethality of B. anthracis Sterne in murine subcutaneous and inhalation infection models, and markedly reduced in vivo survival of the fully virulent B. anthracis Ames upon intraperitoneal challenge in guinea pigs. The extracellular proteolytic activity dependent upon ClpX function was linked to degradation of cathelicidin antimicrobial peptides, a front-line effector of innate host defense. B. anthracis lacking ClpX were rapidly killed by cathelicidin and α-defensin antimicrobial peptides and lysozyme in vitro. In turn, mice lacking cathelicidin proved hyper-susceptible to lethal infection with wild-type B. anthracis Sterne, confirming cathelicidin to be a critical element of innate defense against the pathogen. We conclude that ClpX is an important factor allowing B. anthracis to subvert host immune clearance mechanisms, and thus represents a novel therapeutic target for prevention or therapy of anthrax, a foremost biodefense concern.

Immunostimulatory oligonucleotide (ISS-ODN) used as adjuvants are commonly modified with phosphorothioate (PS). The PS backbone prevents nuclease degradation, but confers undesired side effects, including systemic cytokine release. Previously, R10-60, a phosphodiester (PO) ISS-ODN, was structurally optimized as an intracellular Toll-like receptor-9 agonist. Here intravenous, intradermal and intranasal administration of PO R10-60 elicit local or adaptive immune responses with minimal systemic effects compared to a prototypic PS ISS-ODN in mice. Furthermore, prophylactic intranasal administration of PO R10-60 significantly delayed death in mice exposed to respiratory anthrax comparable to the PS ISS-ODN. The pattern of cytokine release suggested that early IL-1beta production might contribute to this protective effect, which was replicated with recombinant IL-1beta injections during infection. Hence, the transient effects from a PO TLR-9 agonist may be beneficial for protection in a bacterial bioterrorism attack, by delaying the onset of systemic infection without the induction of a cytokine syndrome.

A newly emerged periodontopathic pathogen Tannerella forsythia (formerly Bacteroides forsythus), a Gram-negative, filament-shaped, strict anaerobic, non-pigmented oral bacterium, possesses a surface (S-) layer. In our previous studies, the S-layer has been isolated, and shown to mediate hemagglutination, adhesion/invasion of epithelial cell, and murine subcutaneous abscess formation. In the present study, biochemical and molecular genetic characterization of the S-layer are reported. Amino acid sequencing and mass spectrometry indicated that the S-layer is composed of two different proteins, termed 200 and 210 kDa proteins. It was also shown that these proteins are glycosylated. The genes encoding the core proteins of these glycoproteins, designated as tfsA and tfsB, have been identified in silico, cloned, and their sequences have been determined. The tfsA (3.5 kb) and tfsB (4.1 kb) genes are located in tandem, and encode for 135 and 152 kDa proteins, respectively. An apparent discrepancy in molecular weights, 135 vs. 200 kDa and 152 vs. 210 kDa, is accounted for carbohydrate residues attached to the core proteins. Amino acid sequence comparison exhibited a 24% similarity between the 200 and 210 kDa proteins. Further sequence analyses showed that TfsA and TfsB possess putative signal peptide sequences with cleavage sites at alanine residues, and transmembrane domains on the C-terminal region. Northern blot and RT-PCR analyses confirmed an operon structure of tfsAB, suggesting co-regulation of these genes in producing the S-layer. Putative promoter sequences and transcription termination sequences for this operon have also been identified. Comparison with database indicates that the S-layer of T. forsythia has a unique structure exhibiting no homology to other known S-layers of prokaryotic organisms. The present study shows that the T. forsythia S-layer is very unique, since it appears to be composed of two large glycoproteins, and it does not reveal any homology to other known S-layer proteins or glycoproteins of prokaryotic organisms.

Bacteroides forsythus has emerged as a crucial periodontal pathogen with possible implications for systemic disease. The aim of this study was to isolate the S-layer from B. forsythus and examine its virulence potential as a part of efforts to characterize virulence factors of B. forsythus. The role of the S-layer in the haemagglutinating and adherent/invasive activities was evaluated. It was observed that the S-layer alone was able to mediate haemagglutination. In adherent and invasive studies, transmission electron microscopy clearly revealed that B. forsythus cells were able to attach to and invade KB cells, showing the formation of a microvillus-like extension around adherent and intracellular bacteria. The quantitative analysis showed that five different B. forsythus strains exhibited attachment (1·9–2·3 %) and invasion (0·4–0·7 %) capabilities. It was also observed through antibody inhibition assays that adherent/invasive activities of B. forsythus are mediated by the S-layer. Furthermore, an in vivo immunization study adopting a murine abscess model was used to prove that the S-layer is involved in the infectious process of abscess formation. While mice immunized with purified S-layer and B. forsythus whole cells did not develop any abscesses when challenged with viable B. forsythus cells, unimmunized mice developed abscesses. Collectively, the data obtained from these studies indicate that the S-layer of B. forsythus is a virulence factor.

Introduction: Human immunodeficiency virus infection / acquired immune deficiency syndrome (HIV/AIDS) and tuberculosis are the most important communicable diseases in vulnerable populations. Objectives: The aim of this study was to determine the prevalence of HIV/AIDS and tuberculosis among homeless individuals in Shiraz, Iran. Patients and Methods: This is a cross-sectional study which was carried out on 589 homeless men and women in Shiraz city. The data collection tool was a structurally standard questionnaire. After filling questionnaire, ELISA test was taken each person. If the results of the ELISA test were positive, then, a complementary Western blot test was conducted for HIV. Accordingly a PPD was conducted for individuals suspected to tuberculosis. Data were analyzed by ANOVA and student t test. Results: HIV prevalence in this population was 7.6%. Around 8.88% of HIV positive cases were men. Around 2.37% of the cases had an experience of drug injection. The difference between cases with positive test of HIV in drug users and those who did not use drugs was statically significant (P < 0.05). Around 34.7% of men had experienced homosexual relations. Conclusion: In this study, prevalence of HIV in participants was 7.6%, therefore much more attention needs to be paid to the health of homeless people.

Dogal ormanin ve orman sakinlerinin refahinin surdurulmesi, biyocesitliligin korunmasi acisindan son derece onemlidir. Bu amac dogrultusunda, orman disindaki tarimsal faaliyetlerde bir yenilik olarak safran yetistiriciligine baslanmasi onemli bir faktor olabilir. Bu hedefin gerceklestirilebilmesi icin, safran yetistirmenin tarimsal uygunlugunu ve uygun mekansal modelin belirlenmesi gerekmektedir. Iran’in Kuzey Horasan Eyaleti’nde safran yetistirmeye uygun alanlarin belirlenmesi icin Cografi Bilgi Sistemi (GIS) kullanilmistir. Iklim faktorleri (sicaklik ve yagis miktari), topografya (Sayisal Yukseklik Modeli ve egim) ve arazi kullanimi gibi cevre bilesenleri dikkate alinmistir. Bu calismanin sonuclari, ozellikle safranin yayginlasmasi icin potansiyel yetistirme bolgelerine ve safran yetistiriciligi icin uygun bir haritanin gelistirilmesine yoneliktir. Iran’in Kuzey Horasan Eyaleti’nde tarimsal arazinin %69’unun (1.887 ha) halihazirda safrana uygun oldugunu gorduk. Gecim kaynaklarinin iyilestirilmesi ve ormanlarin korunmasinin saglanmasi icin safranin yetistirilmesine uygun olan arazinin haritasi uzerinde bir calisma yapilabilir.

Iran with a wide diversity of wild medicinal plants (8,000 Medicinal plants) is increasingly becoming a valuable source of household income for many rural people. We showed that economic potential of the wild medicinal plant and their contribution to the local people’s livelihoods. This study identified the economic importance of medicinal plants and evaluated with Geographical Information System (GIS) tool to develop spatial maps covering medicinal plants prevalent in Darkesh area, Northern Khorasan, Iran. Then provide the coordinates of the identified area as your results. 10 medicinal plants belonging to 4 families were collected and its geographical distribution is illustrated in the GIS map. More than 50 species were collected and 10 species being the most frequently collected in this area. Most plants were collected in the form of whole plant. It was estimated that at least 18.000 USD are being annually traded in study area, the 10 species most frequently collected contributing most of the retail value. Results from this study indicate that the collection of certain medicinal plants could be increased livelihood for edge community of natural resources. Also, the geographical location of medicinal plants facilitates easy access of medicinal plant's natural habitat and would help to find out potential of medicinal plants in the study area. Keywords: Medicinal plants, Geographical distribution, GIS Mapping, Darkesh, Iran

Pulmonary administration of Toll-like receptor (TLR) ligands protects hosts from inhaled pathogens. However, systemic side effects induced by TLR stimulation limit clinical development. Here, a small-molecule TLR7 ligand conjugated with phospholipid, 1V270 (also designated TMX201), was tested for innate immune activation and its ability to prevent pulmonary infection in mice. We hypothesized that phospholipid conjugation would increase internalization by immune cells and localize the compound in the lungs, thus avoiding side effects due to systemic cytokine release. Pulmonary 1V270 administration increased innate cytokines and chemokines in bronchial alveolar lavage fluids, but neither caused systemic induction of cytokines nor B cell proliferation in distant lymphoid organs. 1V270 activated pulmonary CD11c+ dendritic cells, which migrated to local lymph nodes. However, there was minimal cell infiltration into the pulmonary parenchyma. Prophylactic administration of 1V270 significantly protected mice from lethal infection with Bacillus anthracis, Venezuelan equine encephalitis virus and H1N1 influenza virus. The maximum tolerated dose of 1V270 by pulmonary administration was 75 times the effective therapeutic dose. Therefore, pulmonary 1V270 treatment can protect the host from different infectious agents by stimulating local innate immune responses while exhibiting an excellent safety profile.

Inflammation resulting from chronic bacterial infection in the lung contributes to long-term pulmonary complications in chronic pulmonary infections such as cystic fibrosis. Aerosol administration of levofloxacin as in the form of the investigational formulation MP-376 results in higher concentrations in lung tissues that are higher than those that can be attained with oral or intravenous dosing of levofloxacin. The objective of this study was to evaluate the effect of high concentrations of levofloxacin achieved with aerosol administration of MP-376 on proinflammatory cytokine secretion by immortalized human bronchial epithelia cells in vitro. Additionally, we investigated the potential mechanisms of the immunomodulatory effect of levofloxacin. In vitro studies in human lung epithelial cell lines showed that levofloxacin led to a dose-related reduction in IL-6 and IL-8 concentrations, with 300 μg mL(-1) resulting in the reduction of levels of IL-6 by fourfold and IL-8 by twofold (P

Progressive respiratory failure due toPseudomonas aeruginosais the leading cause of morbidity and mortality in patients with cystic fibrosis. The pulmonary delivery of antimicrobial agents provides high concentrations of drug directly to the site of infection and attains pharmacokinetic-pharmacodynamic indices exceeding those which can be achieved with systemic dosing. MP-376 is a new formulation of levofloxacin that enables the safe aerosol delivery of high concentrations of drug to pulmonary tissues. In vivo studies were conducted to demonstrate the efficacy of MP-376 in models of mouse pulmonary infection. The superiority of aerosol dosing over systemic dosing was demonstrated in models of both acute and chronic lung infection. In a model of acute lung infection, aerosol treatment with MP-376 once or twice daily reduced the lung bacterial load to a greater extent than aerosol tobramycin or aztreonam did when they were administered at similar or higher doses. The bacterial killing by aerosol MP-376 observed in the lung in the model of acute pulmonary infection translated to improved survival (P< 0.05). In a model of chronic pulmonary infection, aerosol MP-376 had antimicrobial effects superior to those of aztreonam (P< 0.05) and effects similar to those of tobramycin (P> 0.05). In summary, these data show that aerosol MP-376 has in vivo activity when it is used to treat acute and chronic lung infections caused byP. aeruginosa.

We report on the identification of a novel small molecule inhibitor of anthrax lethal factor using a high-throughput screening approach. Guided by molecular docking studies, we carried out structure-activity relationship (SAR) studies and evaluated activity and selectivity of most promising compounds in in vitro enzyme inhibition assays and cellular assays. Selected compounds were further analyzed for their in vitro ADME properties, which allowed us to select two compounds for further preliminary in vivo efficacy studies. The data provided represents the basis for further pharmacology and medicinal chemistry optimizations that could result in novel anti-anthrax therapies.

Background: Anthrax meningitis is the main neurological complication of systemic infection with Bacillus anthracis approaching 100% mortality. The presence of bacilli in brain autopsies indicates that vegetative bacteria are able to breach the blood-brain barrier (BBB). The BBB represents not only a physical barrier but has been shown to play an active role in initiating a specific innate immune response that recruits neutrophils to the site of infection. Currently, the basic pathogenic mechanisms by which B. anthracis penetrates the BBB and causes anthrax meningitis are poorly understood. Methodology/Principal Findings: Using an in vitro BBB model, we show for the first time that B. anthracis efficiently invades human brain microvascular endothelial cells (hBMEC), the single cell layer that comprises the BBB. Furthermore, transcriptional profiling of hBMEC during infection with B. anthracis revealed downregulation of 270 (87%) genes, specifically key neutrophil chemoattractants IL-8, CXCL1 (Groα) and CXCL2 (Groβ), thereby strongly contrasting hBMEC responses observed with other meningeal pathogens. Further studies using specific anthrax toxin-mutants, quantitative RT-PCR, ELISA and in vivo assays indicated that anthrax toxins actively suppress chemokine production and neutrophil recruitment during infection, allowing unrestricted proliferation and dissemination of the bacteria. Finally, mice challenged with B. anthracis Sterne, but not the toxin-deficient strain, developed meningitis. Conclusions/Significance: These results suggest a significant role for anthrax toxins in thwarting the BBB innate defense response promoting penetration of bacteria into the central nervous system. Furthermore, establishment of a mouse model for anthrax meningitis will aid in our understanding of disease pathogenesis and development of more effective treatment strategies. © 2008 van Sorge et al.

In this study, we analyzed a series of rhodanine derivatives, as potential inhibitors of bacterial toxins, namely the proteases anthrax lethal factor and the botulinum neurotoxin type A. Conducting an extensive structure-activity relationship study on rhodanine derivatives, we profiled their selectivity against the two bacterial toxins and two related human metalloproteases using in vitro assays. In addition, we examined initial in vitro ADME-Tox properties of selected compounds and their ability to protect lethal factor-induced cell death of macrophages. These data allowed the selection of one additional drug candidate for which preliminary in vivo efficacy studies against anthrax spores were conducted. Integration of these results with our structure-activity relationship studies provides a framework for the development of potential drug candidates against anthrax and botulinum.

Molecular pathogenesis of Tannerella forsythia, a putative periodontal pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here, identification of in vivo expressed antigens of T. forsythia is reported using in vivo-induced antigen technology (IVIAT). Among 13 000 recombinant clones screened, 16 positive clones were identified that reacted reproducibly with sera obtained from patients with periodontal disease. DNA sequences from 12 of these in vivo-induced genes were determined. IVIAT-identified protein antigens of T. forsythia include: BspA, a well-defined virulence factor of T. forsythia; enzymes involved in housekeeping functions (tRNA synthetases, glycine hydroxymethyltransferase, and glucoside glucohydrolase); enzymes implicated in tissue destruction (dipeptidyl peptidase IV); a DNA mismatch repair protein; and putative outer membrane proteins of unknown function. The in vivo gene expression of these IVIAT-identified antigens was confirmed by a quantitative real-time PCR analysis. This is, to the best of the authors' knowledge, the first report using IVIAT in T. forsythia. It is anticipated that detailed analysis of the in vivo-induced genes identified by IVIAT in this study will lead to a better understanding of the molecular mechanisms mediating periodontal infection by T. forsythia.

B. anthracis is the causative agent of anthrax. Pathogenesis is primarily mediated through the exotoxins lethal factor and edema factor, which bind protective antigen (PA) to gain entry into the host cell. The current anthrax vaccine (AVA, Biothrax™) consists of aluminum-adsorbed cell-free filtrates of unencapsulated B. anthracis, wherein PA is thought to be the principle target of neutralization. In this study, we evaluated the efficacy of the natural adjuvant, C3d, versus alum in eliciting an anti-PA humoral response and found that C3d conjugation to PA and emulsion in incomplete Freund's adjuvant (IFA) imparted superior protection from anthrax challenge relative to PA in IFA or PA adsorbed to alum. Relative to alum-PA, immunization of mice with C3d-PA/IFA augmented both the onset and sustained production of PA-specific antibodies, including neutralizing antibodies to the receptor-binding portion (domain 4) of PA. C3d-PA/IFA was efficacious when administered either i.p. or s.c., and in adolescent mice lacking a fully mature B cell compartment. Induction of PA-specific antibodies by C3d-PA/IFA correlated with increased efficiency of germinal center formation and plasma cell generation. Importantly, C3d-PA immunization effectively protected mice from intranasal challenge with B. anthracis spores, and was approximately 10-fold more effective than alum-PA immunization or PA/IFA based on dose challenge. These data suggest that incorporation of C3d as an adjuvant may overcome shortcomings of the currently licensed aluminum-based vaccine, and may confer protection in the early days following acute anthrax exposure.

The immunotherapeutic activity of Toll-like receptor (TLR) activators has been difficult to exploit because of side effects related to the release and systemic dispersion of proinflammatory cytokines. To overcome this barrier, we have synthesized a versatile TLR7 agonist, 4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde (UC-1V150), bearing a free aldehyde that could be coupled to many different auxiliary chemical entities through a linker molecule with a hydrazine or amino group without any loss of activity. UC-1V150 was covalently coupled to mouse serum albumin (MSA) at a 5:1 molar ratio to yield a stable molecule with a characteristically altered UV spectrum. Compared with the unconjugated TLR7 agonist, the UC-1V150/MSA was a 10- to 100-fold more potent inducer of cytokine production in vitro by mouse bone marrow-derived macrophage and human peripheral blood mononuclear cells. When administrated to the lung, the conjugate induced a prolonged local release of cytokines at levels 10-fold or more higher than those found in serum. Under the same conditions, the untethered TLR7 ligand induced quick systemic cytokine release with resultant toxicity. In addition, two pulmonary infectious disease models were investigated wherein mice were pretreated with the conjugate and then challenged with either Bacillus anthracis spores or H1N1 influenza A virus. Significant delay in mortality was observed in both disease models with UC-1V150/MSA-pretreated mice, indicating the potential usefulness of the conjugate as a localized and targeted immunotherapeutic agent.

Helicobacter pyloriis a gram-negative microaerophilic bacterium that colonizes the gastric mucosa, leading to disease conditions ranging from gastritis to cancer. Toll-like receptors (TLRs) play a central role in innate immunity by their recognition of conserved molecular patterns on bacteria, fungi, and viruses. Upon recognition of microbial components, these TLRs associate with several adaptor molecules, including myeloid differentiation factor 88 (MyD88). To investigate the contribution of the innate immune system toH. pyloriinfection, bone marrow-derived macrophages from mice deficient in TLR2, TLR4, TLR9, and MyD88 were infected withH. pyloriSS1 and SD4 for 24 or 48 h. We demonstrate that MyD88 was essential forH. pyloriinduction of all cytokines investigated except alpha interferon (IFN-α). The secretion of IFN-α was substantially increased from cells deficient in MyD88.H. pyloriinduced interleukin-12 (IL-12) and IL-10 through TLR4/MyD88 signaling. In addition,H. pyloriinduced less IL-6 and IL-1β in TLR2-deleted macrophages, suggesting that the MyD88 pathway activated by TLR2 stimulation is responsible forH. pyloriinduction of the host proinflammatory response (IL-6 and IL-1β). These observations are important in light of a recent report on IL-6 and IL-1β playing a role in the development ofH. pylori-related gastric cancer. In conclusion, our study demonstrates thatH. pyloriactivates TLR2 and TLR4, leading to the secretion of distinct cytokines by macrophages.