Assessment of CD200R Activation in Combination with Doxycycline in a Model of Melioidosis.

Antimicrobial resistance continues to be a global issue. Pathogens, such as Burkholderia pseudomallei, have evolved mechanisms to efflux certain antibiotics and manipulate the host response. New treatment strategies are therefore required, such as a layered defense approach. Here, we demonstrate, using biosafety level 2 (BSL-2) and BSL-3 in vivo murine models, that combining the antibiotic doxycycline with an immunomodulatory drug that targets the CD200 axis is superior to antibiotic treatment in combination with an isotype control. CD200-Fc treatment alone significantly reduces bacterial burden in lung tissue in both the BSL-2 and BSL-3 models. When CD200-Fc treatment is combined with doxycycline to treat the acute BSL-3 model of melioidosis, there is a 50% increase in survival compared with relevant controls. This benefit is not due to increasing the area under the concentration-time curve (AUC) of the antibiotic, suggesting the immunomodulatory nature of CD200-Fc treatment is playing an important role by potentially controlling the overactive immune response seen with many lethal bacterial infections. IMPORTANCE Traditional treatments for infectious disease have focused on the use of antimicrobial compounds (e.g. antibiotics) that target the infecting organism. However, timely diagnosis and administration of antibiotics remain crucial to ensure efficacy of these treatments especially for the highly virulent biothreat organisms. The need for early antibiotic treatment, combined with the increasing emergence of antibiotic resistant bacteria, means that new therapeutic strategies are required for organisms that cause rapid, acute infections. Here, we show that a layered defense approach, where an immunomodulatory compound is combined with an antibiotic, is better than an antibiotic combined with a relevant isotype control following infection with the biothreat agent Burkholderia pseudomallei. This approach has the potential to be truly broad spectrum and since the strategy includes manipulation of the host response it's application could be used in the treatment of a wide range of diseases.

The expression of ferritin, lactoferrin, transferrin receptor and solute carrier family 11A1 in the host response to BCG-vaccination and Mycobacterium tuberculosis challenge.

Iron is an essential cofactor for both mycobacterial growth during infection and for a successful protective immune response by the host. The immune response partly depends on the regulation of iron by the host, including the tight control of expression of the iron-storage protein, ferritin. BCG vaccination can protect against disease following Mycobacterium tuberculosis infection, but the mechanisms of protection remain unclear. To further explore these mechanisms, splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR (P≤0.05 and ≤0.01, respectively). The mechanisms of this down-regulation were shown to involve TNFα and nitric oxide. A more in depth analysis of the mRNA expression profiles, including genes involved in iron metabolism, was performed using a guinea pig specific immunological microarray following ex vivo infection with M. tuberculosis of splenocytes from BCG-vaccinated and naïve guinea pigs. M. tuberculosis infection induced a pro-inflammatory response in splenocytes from both groups, resulting in down-regulation of ferritin (P≤0.05). In addition, lactoferrin (P≤0.002), transferrin receptor (P≤0.05) and solute carrier family 11A1 (P≤0.05), were only significantly down-regulated after infection of the splenocytes from BCG-vaccinated animals. The results show that expression of iron-metabolism genes is tightly regulated as part of the host response to M. tuberculosis infection and that BCG-vaccination enhances the ability of the host to mount an iron-restriction response which may in turn help to combat invasion by mycobacteria.

An activating combination of CD2 antibodies stimulates tyrosine phosphorylation in a T lymphocyte cell line.

The activating combination of CD2 antibodies Leu-5b plus 9.1 stimulates tyrosine phosphorylation in the human T cell line Jurkat. The tyrosine phosphorylation has the same molecular weight pattern as that seen when cells are stimulated on the CD3 receptor with OKT3 antibodies. These data provide evidence that signal transduction by the CD2 receptor is coupled to an increase in tyrosine phosphorylation that is similar to that coupled to the CD3 receptor.

Pertussis toxin activates protein kinase C and a tyrosine protein kinase in the human T cell line Jurkat.

Pertussis toxin activates T lymphocytes by a mechanism that is independent of its ADP-ribosylation activity. The toxin stimulates increases in diacylglycerol and intracellular calcium apparently by interacting with a cell surface receptor. Consistent with the production of these second messengers we have found that pertussis toxin activates protein kinase C in the Jurkat cell line. The toxin was also found to activate a tyrosine protein kinase in these cells in a manner similar to that observed with phytohemagglutinin. These results provide evidence that the mechanism of activation of T cells by pertussis toxin involves stimulating the activity of protein kinase C and a tyrosine protein kinase.

Demonstration that LSTRA cells have an elevated level of proteins phosphorylated on tyrosine residues.

The LSTRA cell line has been shown to have an exceptionally high level of a tyrosine protein kinase (pp56lck). We now report that LSTRA cells also have a much higher level of proteins phosphorylated on tyrosine residues in comparison to several other cell lines with normal levels of pp56lck. The level of phosphotyrosine-containing proteins in LSTRA cells was comparable to that seen in K562 cells, a cell line known to have a constitutively active tyrosine protein kinase. These results provide evidence that LSTRA cells have an elevated level of in vivo tyrosine protein kinase activity, probably due to the overexpression and activation of pp56lck.

Role for an episulfonium ion in S-(2-chloroethyl)-DL-cysteine-induced cytotoxicity and its reaction with glutathione.

The cysteine S conjugate of 1,2-dichloroethane, S-(2-chloroethyl)-DL-cysteine (CEC), is hepatotoxic, nephrotoxic, and mutagenic. To determine the cellular and chemical mechanisms involved in CEC-induced toxicity and to assess the role of an episulfonium ion, the effect of CEC on the viability of isolated rat hepatocytes was studied. CEC addition resulted in both a time- and concentration-dependent loss of cell viability. Depletion of intracellular glutathione concentrations (greater than 70%) and inhibition of microsomal Ca2+ transport and Ca2+-ATPase activity preceded the loss of cell viability, and initiation of lipid peroxidation paralleled the loss of viability. The depletion of glutathione concentrations was partially attributable to a reaction between glutathione and CEC to form S-[2-(DL-cysteinyl)ethyl]glutathione, which was identified by NMR and mass spectrometry. N-Acetyl-L-cysteine, vitamin E, and N,N'-diphenyl-p-phenylenediamine protected against the loss of cell viability. N,N'-Diphenyl-p-phenylenediamine inhibited CEC-initiated lipid peroxidation but did not protect against cell death at 4 h, indicating that lipid peroxidation was not the cause of cell death. The analogues S-ethyl-L-cysteine, S-(3-chloropropyl)-DL-cysteine, and S-(2-hydroxyethyl)-L-cysteine, which cannot form an episulfonium ion, were not cytotoxic, thus demonstrating a role for an episulfonium ion in the cytotoxicity associated with exposure to CEC and, possibly, 1,2-dichloroethane. These results show that an alteration in Ca2+ homeostasis and the generation of an electrophilic intermediate may be involved in the mechanism of cell death.