Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 ؋ 10 2 CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 ؋ 10 3 CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.Bovine mastitis (BM) is an inflammation of the mammary gland, usually due to a microbial infection (28), which causes North American dairy producers to lose billions of dollars every year. These losses are primarily due to lower milk yields, reduced milk quality, and higher production costs. BM often becomes chronic, and it is important to identify quickly the new clinical cases in order to control infection in the herd. The bacteria responsible for BM can be classified as environmental (Escherichia coli, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis) or contagious (Staphylococcus aureus and Streptococcus agalactiae) depending of their primary reservoir (environment versus infected mammary gland quarter) (11,25).The suitability of a detection method for routine diagnosis depends on several factors, such as specificity, sensitivity, expense, amount of time, and applicability to large numbers of milk samples. The most common but unspecific method (2) to identify potential chronic infections is a somatic cell count: the California Mastitis Test in field conditions and the automated method in the diagnosis laboratory. Currently, the method of identification of the mammary gland pathogens is by in vitro culture, which provides the "gold standard"; however, this technique is labor-intensive and time-consuming. Two other problems can be encountered when these methods of identification are used: first, 2 to 3 days are required to grow, isolate, and identify the pathoge...
It was previously demonstrated that fluid liposomal-encapsulated tobramycin, named Fluidosomes, was successful in eradicating mucoid Pseudomonas aeruginosa in an animal model of chronic pulmonary infection, whereas free antibiotic did not reduce colony-forming unit (CFU) counts (C. Beaulac et al., Antimicrob. Agents Chemother. 40 (1996) 665-669; C. Beaulac et al., J. Antimicrob. Chemother. 41 (1998) 35-41). These liposomes were also shown to be bactericidal in in vitro tests against strong resistant P. aeruginosa 64 microg/ml). The time needed to reach the maximal fusion rate was about 5 h for the resistant strain comparatively to much shorter time for the sensitive strain. The specific characteristics of Fluidosomes could help overcome bacterial resistance related to permeability barrier and even enzymatic hydrolysis considering the importance of synergy in the whole process of antibiotic resistance.
The in vitro activities of 10 families of antimicrobial agents alone and in combination with a synthetic polycationic polymer, polyethylenimine (PEI), against a resistant clinical isolate of Pseudomonas aeruginosa were investigated by MIC assays, checkerboard testing, and killing curve studies. At a concentration of 250 nM, PEI (10 kDa) was not directly bactericidal or bacteriostatic; but when it was used in combination with novobiocin, ceftazidime, ampicillin, ticarcillin, carbenicillin, piperacillin, cefotaxime, chloramphenicol, rifampin, or norfloxacin, it significantly reduced the MICs of these antibiotics by 1.5-to 56-fold. However, the MICs of aminoglycosides, polymyxins, and vancomycins were increased by 1.2-to 5-fold when these drugs were combined with PEI; and the MICs of tetracycline, erythromycin, ciprofloxacin, and ofloxacin were not affected when these drugs were combined with PEI. In the killing curve studies, combinations of PEI with novobiocin, ceftazidime, chloramphenicol, or rifampin resulted in 5-to 8-log 10 CFU/ml reductions in bacterial counts when 25% of the MIC of each antibiotic was used. These results indicate that infections due to resistant Pseudomonas strains could be treated by the use of a synergistic combination of PEI and antimicrobial drugs.Over the past two decades, Pseudomonas aeruginosa has attracted attention as an opportunistic pathogen in hospitalized, immunocompromised, and cystic fibrosis patients (6,15,20). Despite aggressive antibiotic therapy, P. aeruginosa is rarely eradicated owing to its high level of intrinsic resistance to many drugs (14,19,27). This resistance is due to the effective permeability of the outer membrane (OM) of gram-negative bacteria to both hydrophobic antibiotics and high-molecular-weight hydrophilic drugs (for reviews, see references 4, 25, 28, and 30). Unlike most cell types, gram-negative bacteria surround themselves with a double membrane. The outermost of these two membranes is asymmetric, with the inner leaflet composed of glycerophospholipids and the outer leaflet predominantly composed of lipopolysaccharide (LPS). Tight interactions between the highly negatively charged LPS molecules are believed to form an effective barrier against hydrophobic compounds. For these polyanionic molecules to form a stable "tiled roof" on the surface of the OM, adjacent LPS molecules are linked electrostatically by divalent cations (Ca 2ϩ , Mg 2ϩ ) present in the OM. Polyethylenimine (PEI) is a weakly basic, aliphatic, nontoxic synthetic polymer which is polycationic owing to the presence of primary, secondary, and tertiary amino groups. It is well known that certain polycationic agents such as polymyxin and its derivatives polylysines and protamine can increase the permeability of the gram-negative bacterial OM to solutes that are normally unable to penetrate (25). Helander et al. found that, when it was applied alone, PEI has a strong permeabilizing effect but no bactericidal effect on gram-negative bacteria (8). Escherichia coli, P. aeruginosa, and Salmon...
Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV ؉ and EBV ؊ B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the ␣ and  chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cellcell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl--cyclodextrin (MCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DRinduced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not. Although major histocompatibility complex (MHC)1 class II molecules do not possess any known signaling motifs in their cytoplasmic and transmembrane domains, they act as signal transducers in addition to playing a critical role in antigen presentation and autoimmune disease susceptibility and severity (1, 2). Corley et al. (3) reported that the recognition of MHC class II-peptide complexes by specific T cell receptors (TCR) leads to the activation of both T lymphocytes and antigenpresenting cells (APCs), suggesting that MHC class II molecules can act as signal transducers. To confirm this possibility, anti-MHC class II antibodies (Abs) and superantigens (SAgs), which act as natural MHC class II ligands, were used to mimic MHC class II-peptide complex recognition by specific TCRs. This ligation led to various cellular events such as homotypic and heterotypic cell-cell adhesion (4), B cell proliferation and differentiation (5), cytokine production, and expression of costimulatory molecules (6) and, under certain conditions, cell death (7). Like other ligand-receptor interactions, some of these events are dependent on MHC class II ligand dimerization (8, 9), generate cAMP and intracellular calcium flux (1), and are mediated by signaling pathways and secondary messengers including protein kinase C (PKC), protein tyrosine kinase (PTK), cyclooxygenase 2,...
The human-specific p35 isoform of the invariant chain (Ii) includes an R-X-R endoplasmic reticulum (ER) retention motif that is inactivated upon HLA-DR binding. Although the masking is assumed to involve the cytoplasmic tails of class II molecules, the mechanism underlying this function remains to be investigated. Moreover, in light of the polymorphic nature of the class II cytosolic tails, little is known about the capacity of various isotypes or alleles to overcome the retention signal of Iip35. To gain further insights into these issues, we first addressed the proposed role of the HLA-DR cytoplasmic tails. As shown by flow cytometry, the presence of Iip35 in transfected HeLa cells prevented surface expression of HLA-DR molecules lacking their cytoplasmic tails (DRalphaTM/betaTM). These truncated class II molecules and Iip35 accumulated in the ER, and co-localized with calnexin, as determined by confocal microscopy. Sensitivity of DRalphaTM/betaTM to endoglycosidase H treatment confirmed that these molecules do not reach the trans-Golgi network when associated with Iip35. Further characterization revealed that the beta chain cytosolic tail is critical for efficient ER egress of class II/Iip35 complexes. Interestingly, our results clearly demonstrate for the first time that DP and DQ isotypes can also overcome the retention motif of Iip35 through a mechanism involving their very distinctive polymorphic beta chain cytoplasmic tails. Altogether, these results further dissect the masking of di-basic retention signals, and emphasize the interplay between class II molecules and Ii for the transport of the complex to the endocytic pathway.
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