Mannheimia haemolytica and lipopolysaccharide induce airway epithelial inflammatory responses in an extensively developed ex vivo calf model.

Pulmonary infection is associated with inflammation and damage to the bronchial epithelium characterized by an increase in the release of inflammatory factors and a decrease in airway barrier function. Our objective is to optimize a method for the isolation and culture of primary bronchial epithelial cells (PBECs) and to provide an ex vivo model to study mechanisms of epithelial airway inflammation. PBECs were isolated and cultured from the airways of calves in a submerged cell culture and liquid-liquid interface system. A higher yield and cell viability were obtained after stripping the epithelium from the bronchial section compared to cutting the bronchial section in smaller pieces prior to digestion. Mannheimia haemolytica and lipopolysaccharide (LPS) as stimulants increased inflammatory responses (IL-8, IL-6 and TNF-α release), possibly, by the activation of "TLR-mediated MAPKs and NF-κB" signaling. Furthermore, M. haemolytica and LPS disrupted the bronchial epithelial layer as observed by a decreased transepithelial electrical resistance and zonula occludens-1 and E-cadherin expression. An optimized isolation and culture method for calf PBECs was developed, which cooperated with animal use Replacement, Reduction and Refinement (3R's) principle, and can also contribute to the increased knowledge and development of effective therapies for other animal and humans (childhood) respiratory diseases.

Effect of particulate matter less than 10µm (PM10) on mortality in Bogota, Colombia: a time-series analysis, 1998-2006 / Efecto del material particulado menor a 10µm (PM10) sobre la mortalidad en Bogotá, Colombia: un análisis de series de tiempo 1998-2006

Objective. To analyze the association between daily mortality from different causes and acute exposure to particulate matter less than 10 microns in aerodynamic diameter (PM10), in Bogota, Colombia. Materials and methods. A time-series ecological study was conducted from 1998 to 2006. The association between mortality (due to different causes) and exposure was analyzed using single and distributed lag models and adjusting for potential confounders. Results. For all ages, the cumulative effect of acute mortality from all causes and respiratory causes increased 0.71% (95%CI 0.46-0.96) and 1.43% (95%CI 0.85-2.00), respectively, per 10µg/m³ increment in daily average PM10 with a lag of three days before death. Cumulative effect of mortality from cardiovascular causes was -0.03% (95%CI -0.49-0.44%) with the same lag. Conclusions. The results suggest an association between an increase in PM10 concentrations and acute mortality from all causes and respiratory causes.

Objetivo. Analizar la asociación entre la mortalidad diaria debida a distintas causas y la exposición aguda a partículas menores de 10 micras de diámetro aerodinámico (PM10), en Bogotá, Colombia. Material y métodos. Se realizó un estudio ecológico de series de tiempo (1998-2006). La asociación entre mortalidad y exposición se analizó ajustando modelos de retraso simple y retraso distribuido para diferentes causas de mortalidad. Resultados. En todas las edades, el riesgo acumulado en la mortalidad aguda por todas las causas y causa respiratoria aumentó 0.71% (IC95% 0.46-0.96) y 1.43% (IC95% 0.85-2.00), respectivamente, por incremento de 10µg/m³ en el promedio diario de PM10, tomando un retraso de tres días anteriores al deceso, mientras el riesgo acumulado en la mortalidad por causa cardiovascular fue de -0.03% (IC95% -0.49-0.44), para el mismo retraso. Conclusiones. Los resultados sugieren asociación entre el incremento de las concentraciones de PM10 y la mortalidad aguda por todas las causas y causa respiratoria.

Bacterial Ghosts as antigen and drug delivery system for ocular surface diseases: Effective internalization of Bacterial Ghosts by human conjunctival epithelial cells.

The purpose of the presented investigation was to examine the efficiency of the novel carrier system Bacterial Ghosts (BGs), which are empty bacterial cell envelopes of Gram-negative bacteria to target human conjunctival epithelial cells, as well as to test the endocytic capacity of conjunctival cells after co-incubation with BGs generated from different bacterial species, and to foreclose potential cytotoxic effects caused by BGs. The efficiency of conjunctival cells to internalize BGs was investigated using the Chang conjunctival epithelial cell line and primary human conjunctiva-derived epithelial cells (HCDECs) as in vitro model. A high capacity of HCDECs to functionally internalize BGs was detected with the level of internalization depending on the type of species used for BGs generation. Detailed analysis showed no cytotoxic effect of BGs on HCDECs independently of the used bacterial species. Moreover, co-incubation with BGs did not enhance expression of both MHC class I and class II molecules by HCDECs, but increased expression of ICAM-1. The high rates of BG's internalization by HCDECs with no BG-mediated cytotoxic impact designate this carrier system to be a promising candidate for an ocular surface drug delivery system. BGs could be useful for future therapeutic ocular surface applications and eye-specific disease vaccine development including DNA transfer.

[Fatty acid composition of cells and lipopolysaccliarides of Mannheimia haemolytica, Mannheimia glucosida and Bibersteinia trehalosi strains].

The Mannheimia haemolytica, Mannheimia glucosida and Bibersteinia trehalosi strains and the similar fatty acid composition of cells with domination of C(16:1) and C(16:0), which were in almost equal quantities, C(14:0 and C(18:1) + C(18:2). The fatty acid composition of lipopolysaccharides (LPS) of the studied bacteria had no essential differences too. It was mainly represented by C(14:0) and 3-OH-C(14:0) which consisted of more than 80% of all LPS fatty acids. C(12:0), C(16:1) and C(16:0) were presented in LPS in small quantities. The M. haemolytica, M. glucosida and B. trehalosi strains did not differ essentially by fatty acid compositions of cells and LPS from earlier studied strains of genera Pasteurella (P. multocida), Haemophilus (H. influenzae and other species), Actinobacillus (A. pleuropneumoniae). This shows the close phylogenetic relationship of the mentioned bacteria and significance of investigated signs as chemotaxonomic markers for differentiation of taxons of the above genus level. The paper is presented in Russian.

Structural basis for iron binding and release by a novel class of periplasmic iron-binding proteins found in gram-negative pathogens.

We have determined the 1.35- and 1.45-A structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A. M. haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever. The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host. The ferric (Fe(3+)) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion. The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion. Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm.

Pharmacological inhibition of Mannheimia haemolytica lipopolysaccharide and leukotoxin-induced cytokine expression in bovine alveolar macrophages.

The inflammatory cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) are believed to contribute to the pathogenesis of lung injury in bovine pneumonic mannheimiosis (BPM) caused by Mannheimia (Pasteurella) haemolytica. Inflammatory cytokines may, therefore, represent therapeutic targets to be modulated for the purpose of treating or preventing this important disease of cattle. The purpose of this study was to evaluate the ability of six pharmacological agents to suppress the expression of TNFalpha, IL-1beta, and IL-8 genes and proteins in bovine alveolar macrophages (AM) exposed to M. haemolytica lipopolysaccharide (LPS) and leukotoxin (LktA) in vitro. The compounds tested included dexamethasone (DEX), tetrahydropapaveroline (THP), pentoxifylline (PTX), rolipram (ROL), SB203580 (SB), and thalidomide (THL). Cytokine expression was induced by the addition of purified M. haemolytica LPS and LktA to AM cell cultures following pretreatment with inhibitor compounds. Secretion of TNFalpha, IL-1beta, and IL-8 proteins into the cell culture supernatant was measured using enzyme-linked immunosorbent assays, and steady-state accumulation of cytokine-specific mRNA was measured by northern blot analysis. Dose-dependent inhibition of cytokine secretion occurred in response to pretreatment of AM with DEX (TNFalpha, IL-1beta, IL-8), THP (TNFalpha, IL-1beta, IL-8), PTX (TNFalpha, IL-1beta, IL-8), ROL (TNFalpha, IL-1beta), and SB (TNFalpha, IL-8). Significant dose-dependent inhibition of cytokine mRNA expression occurred in response to pretreatment with DEX (TNFalpha, IL-1beta, IL-8), THP (TNFalpha, IL-1beta, IL-8), and PTX (TNFalpha). DEX was the most effective inhibitor by far; pretreatment with this compound yielded greater than 95% inhibition of cytokine gene and protein expression over a broad range of concentrations. These findings demonstrate that DEX, THP, PTX, ROL, and SB are capable of suppressing inflammatory cytokine secretion by bovine AM in vitro. If pulmonary cytokine secretion may be similarly inhibited in vivo, anti-cytokine therapy may represent a novel strategy for the management of BPM.

p53 and c-myc activation by Pasteurella haemolytica leukotoxin is correlated with bovine mononuclear cells apoptosis.

To analyse the role of Pasteurella haemolytica Leukotoxin (LKT) in the mechanism of apoptotic cell death of bovine lymphocytes, we evaluated DNA fragmentation and p53 and c-myc expression. P. haemolytica strain ATCC 14003 was cultivated for LKT production. DNA fragmentation was analysed by electrophoresis on Agarose gel. DNA strand breaks in individual apoptotic cells were also detected by an in situ Terminal deoxy nucleotidyl Transferase (TdT). The Polymerase Chain Reaction (PCR) procedure was used for verified p53 and c-myc activation by P. haemolytica LKT. LKT was able to induce DNA fragmentation in a dose and time-dependent fashion. The greatest apoptotic effect was obtained using LKT at a concentration of 0.25 U. The results show that p53 and c-myc activation by LKT is correlated with apoptosis of bovine lymphocytes and monocytes. Our data suggest that LKT may have an important role in the bacterial virulence of Pasteurella haemolytica.

Pulmonary expression of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-8 in the acute phase of bovine pneumonic pasteurellosis.

Inflammatory cytokines are suspected to contribute to the pathogenesis of bovine pneumonic pasteurellosis (BPP) through neutrophil recruitment, leukocyte activation, and the induction of a broad array of soluble inflammatory mediators. An in vivo experimental model of BPP was used to characterize the pulmonary expression kinetics of tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) genes and proteins during the acute phase of disease development. Cytokine expression in bronchoalveolar lavage (BAL) fluid, BAL cells, and pneumonic lung parenchyma was quantitated by northern blot analysis, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization at 2, 4, 8, 16, and 24 hours after endobronchial inoculation of Pasteurella (Mannheimia) haemolytica. Expression of TNFalpha, IL-1beta, and IL-8 was significantly increased in the airways and lung lesions of infected calves as compared with mock-infected controls. Although kinetic patterns varied, peak levels of cytokine mRNA occured within 8 hours postinfection (PI), and peak cytokine concentrations occurred within 16 hours PI. In all samples, IL-8 was expressed to the greatest extent and TNFalpha was least expressed. Expression of TNFalpha was restricted to alveolar macrophages. Alveolar and interstitial macrophages produced IL-1beta and IL-8 in the first 4 hours; bronchial and bronchiolar epithelial cells were also significant sources of IL-8 during this period. By 8 hours PI, neutrophils were the dominant source of both IL-1beta and IL-8. These findings demonstrate a spatial and temporal association between pulmonary expression of inflammatory cytokines and acute lung pathology, supporting the hypothesis that cytokines contribute to inflammatory lung injury in BPP.

Purification and characterization of the Pasteurella haemolytica 35 kilodalton periplasmic iron-regulated protein.

Pasteurella haemolytica serovar A1 is the causative agent of acute fibrinohemorrhagic pneumonia also known as shipping fever. Many pathogens, including P. haemolytica, survive in their respective hosts through the up-regulation of an iron acquisition system. In this study we identified, purified and characterized a 35-kDa periplasmic iron-regulated protein. The N-terminal sequence of the iron-regulated protein ANEVNVYSYRQP YLIEPMLK was identical to the deduced amino acid sequence of the ferric binding protein, FbpA, of P. haemolytica. Growth of P. haemolytica in a synthetic medium (RPMI-1640), without iron and supplemented with 50 gM 2,2' dipyridyl, facilitated the expression, isolation and purification of the native P. haemolytica FbpA. The protein was purified to homogeneity by using ammonium sulfate precipitation followed by CM-Sepharose ion exchange chromatography. SDS-PAGE showed a single band with a molecular weight of 35,369. Isoelectric focusing showed multiple bands with pIs of 5.5, 5.6, 5.8, and one major band with pI of 6.4. The molecular weight obtained by electrospray mass spectrometry was 35,822. Equilibrium velocity ultracentrifugation established that the protein existed as a monomer under native conditions with an apparent molecular weight of 33,795. Analysis of secondary structure of FbpA by circular dichroism showed 42.1% alpha helical structure. This protein is the second periplasmic iron-regulated protein described for P. haemolytica.

Conservation of expression and N-terminal sequences of the Pasteurella haemolytica 31-kilodalton and Pasteurella trehalosi 29-kilodalton periplasmic iron-regulated proteins.

This study examined the conservation of expression of a 31-kDa iron-regulated protein by serotypes of Pasteurella haemolytica and Pasteurella trehalosi associated with pasteurellosis of cattle and sheep. A polyclonal antibody prepared against the purified 31-kDa periplasmic iron-regulated protein from P. haemolytica serotype A1 showed that all P. haemolytica serotypes expressed similar 31-kDa proteins with identical N-terminal sequences, whereas P. trehalosi serotypes expressed immunologically different 29-kDa proteins with a different N-terminal sequence. Antibody to the 31-kDa iron-regulated protein was a useful tool to distinguish similarities and differences of the iron-regulated proteins of P. haemolytica and P. trehalosi.

Discrimination between apo and iron-loaded forms of transferrin by transferrin binding protein B and its N-terminal subfragment.

Many pathogens of the Pasteurellaceae and Neisseriaceae possess a surface receptor that binds transferrin (Tf) as an initial step in an iron acquisition process. This receptor is comprised of two proteins, transferrin binding protein A (TbpA) and transferrin binding protein B (TbpB). Since the ability to recognize the iron-loaded form of Tf preferentially would be a useful attribute of these receptors, we examined this property in a number of bacterial species. In solid-phase binding assays with isolated membranes, only the receptor from Moraxella catarrhalis was capable of preferentially binding iron-loaded Tf. In a competitive affinity isolation assay which enabled us to resolve TbpA and TbpB, TbpA from all tested species was shown to bind both apo and iron-loaded Tf. Under these assay conditions TbpB from M. catarrhalis, Haemophilus somnus and Pasteurella haemolytica discriminated between apo and holo Tf, whereas TbpB from Neisseria meningitidis showed no discrimination. The ability of TbpB from N. meningitidis to bind iron-saturated hTf preferentially became evident in a TbpA- background or by using recombinant TbpB. In binding assays with recombinant fusion proteins, both intact TbpB and the N-terminal half of TbpB from all the tested species preferentially bound Fe-loaded Tf, indicating that this may be a conserved mechanism by which these organisms optimize their ability to acquire iron.

Molecular and biochemical mechanisms of Pasteurella haemolytica leukotoxin-induced cell death.

Pasteurella haemolytica leukotoxin (LKT) is a member of the RTX family of pore-forming toxins that kill bovine immune cells. Several studies have suggested that RTX toxins kill target cells by the induction of apoptosis. In the present study, BL3 bovine leukaemia cells were exposed to LKT and assessed by molecular and flow cytometric techniques that measure different aspects of apoptotic cell death. The intoxicated cells demonstrated morphological, light scatter and Hoechst 33258 staining characteristics consistent with cells undergoing apoptosis. The cells also exhibited internucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, both indicators of apoptosis. LKT-treated cells bound annexin-V-FITC indicating that phosphatidylserine groups were translocated from the inner to the outer leaflet of the cell membrane. The effect of LKT on cells was dose dependent and inhibitable by incubation with anti-LKT monoclonal antibody. Finally, an early step for induction of apoptosis appears to be the binding of LKT to a beta2 integrin since pre-incubating cells with anti-beta2 integrin antibodies inhibited LKT-induced apoptosis. This study provides new insights into understanding the pathogenesis of bovine pasteurellosis and could lead to the development of both preventative and therapeutic strategies for disease management.

Purification and characterization of a 31-kilodalton iron-regulated periplasmic protein from Pasteurella haemolytica A1.

A prominent iron-regulated periplasmic protein was purified from Pasteurella haemolytica grown in an iron-deficient chemically defined medium. The protein was purified by anion exchange chromatography and appeared as a single band by SDS-PAGE with a molecular weight of 32,000. A yield of five mg was obtained from 91 mg of protein extract. The iron-regulated protein existed as a monomer in the native state with an average molecular weight of 29,877 as determined by analytical ultracentrifugation. The protein had a molecular weight of 30,880 as determined by matrix-assisted laser desorption mass spectrometry, hence the protein is referred to as the 31 kDa protein. Isoelectric focusing showed four bands with pIs of 7.15, 6.8, 6.6, and 5.9. The secondary structure of the protein was determined by circular dichroism and contained 16% alpha-helical structure. The N-terminal sequence, EPFKVVTTFTVIQDIAQNVAGDKAT, showed a 95% identity with the 31 kDa iron-binding protein from Haemophilus influenzae. Isolation and characterization of iron-regulated proteins are of particular interest because of their potential roles in iron assimilation and microbial virulence.