In vitro and murine efficacy and toxicity studies of nebulized SCC1, a methylated caffeine-silver(I) complex, for treatment of pulmonary infections.

The expanding clinical challenge of respiratory tract infections due to resistant bacteria necessitates the development of new forms of therapy. The development of a compound composed of silver coupled to a methylated caffeine carrier (silver carbene complex 1 [SCC1]) that demonstrated in vitro efficacy against bacteria, including drug-resistant organisms, isolated from patients with respiratory tract infections was described previously. The findings of current in vitro studies now suggest that bactericidal concentrations of SCC1 are not toxic to airway epithelial cells in primary culture. Thus, it was hypothesized that SCC1 could be administered by the aerosolized route to concentrate delivery to the lung while minimizing systemic toxicity. In vivo, aerosolized SCC1 delivered to mice resulted in mild aversion behavior, but it was otherwise well tolerated and did not cause lung inflammation following administration over a 5-day period. The therapeutic efficacy of SCC1 compared to that of water was shown in a 3-day prophylaxis protocol, in which mice infected with a clinical strain of Pseudomonas aeruginosa had increased survival, decreased amounts of bacteria in the lung, and a lower prevalence of bacteremia. Similarly, by using an airway infection model in which bacteria were impacted in the airways by agarose beads, the administration of SCC1 was significantly superior to water in decreasing the lung bacterial burden and the levels of bacteremia and markers of airway inflammation. These observations indicate that aerosolized SCC1, a novel antimicrobial agent, warrants further study as a potential therapy for bacterial respiratory tract infections.

Synthesis, stability, and antimicrobial studies of electronically tuned silver acetate N-heterocyclic carbenes.

A series of methylated imidazolium salts with varying substituents on the 4 and 5 positions of the imidazole ring were synthesized. These salts were reacted with silver acetate to afford their corresponding silver N-heterocyclic carbene (NHC) complexes. These complexes were then evaluated for their stability in water as well as for their antimicrobial efficacy against a variety of bacterial strains associated with cystic fibrosis and chronic lung infections.

Neutrophil depletion causes a fatal defect in murine pulmonary Staphylococcus aureus clearance.

BACKGROUND: Staphylococcus aureus is the most common cause of healthcare-associated pneumonia. Despite the significant morbidity and mortality associated with the disease, animal models of S. aureus pneumonia are rare. MATERIALS AND METHODS: We examined the pathogenicity of four different strains of S. aureus (both methicillin-sensitive and -resistant as well as Panton-Valentine leukocidin-positive and -negative) in four strains of immunocompetent inbred and outbred mice (FVB/N, C57Bl/6, BALB/c, ND4; n = 148). The immunological basis for the development of murine S. aureus pneumonia was then determined by selectively depleting neutrophils, lymphocytes, or pulmonary macrophages prior to the onset of infection. An additional cohort of animals was rendered immunosuppressed by induction of abdominal sepsis via cecal ligation and puncture 2, 4, or 7 d prior to the onset of pneumonia. RESULTS: Nearly all immunocompetent mice survived, regardless of which strain of S. aureus was used or which strain of mouse was infected. Among animals with immune depletion or prior immunosuppression, survival was decreased only following neutrophil depletion (26% versus 90% alive at 7 d, P < 0.0001). Compared to immunocompetent animals, neutrophil-depleted mice with S. aureus pneumonia had delayed pulmonary bacterial clearance at 16 and 40 h but had no difference in levels of bacteremia. Neutrophil-depleted mice also had elevated levels of pulmonary monocyte chemotactic protein-1 (822 pg/mL versus 150 pg/mL, P < 0.05). In contrast, pulmonary histological appearance was similar in both groups as was dry/wet lung weight. CONCLUSIONS: These results suggest that neutrophils play a critical role in the host response to S. aureus pneumonia, and the survival differences observed in neutrophil-depleted mice are associated with alterations in bacterial clearance and pulmonary cytokine response.

Synthesis from caffeine of a mixed N-heterocyclic carbene-silver acetate complex active against resistant respiratory pathogens.

The bis(N-heterocyclic carbene) (NHC) silver complex, 3, with a methyl carbonate anion was formed from the reaction of the iodide salt of methylated caffeine, 1, with silver (I) oxide in methanol. Attempts to crystallize this complex from a mixture of common alcohols and ethyl acetate led to the formation of an NHC-silver acetate complex, 4. The more direct synthesis of 4 was accomplished by the in-situ deprotonation of 1 by silver acetate in methanol. Complex 4 demonstrated antimicrobial activity against numerous resistant respiratory pathogens from the lungs of cystic fibrosis (CF) patients including members of the Burkholderia cepacia complex that cause a high rate of mortality in patients with cystic fibrosis (CF). Application of this NHC silver complex to primary cultures of murine respiratory epithelial cells followed by microarray analysis showed minimal gene expression changes at the concentrations effective against respiratory pathogens. Furthermore, methylated caffeine without silver showed some antibacterial and antifungal activity.

Entry of Burkholderia organisms into respiratory epithelium: CFTR, microfilament and microtubule dependence.

BACKGROUND: The pathogenesis of infection with Burkholderia cepacia complex (Bcc) organisms may be linked to its capacity to invade respiratory epithelium. METHODS: An antibiotic exclusion assay was used to study B. dolosa AU4459 and B. cenocepacia J2315 invasion into wild-type (WT) and CFTR-deficient respiratory epithelial cells. Inhibitors were used to evaluate Bcc invasion dependency on host microtubule (mt) and microfilament (mf) systems. RESULTS: B. dolosa entered WT-CFTR cells with 5-fold greater efficiency than CFTR deficient cells (25% vs 5%, respectively). Invasion dropped to <0.5% after either mf or mt inhibition. B. cenocepacia entered WT (0.05%) and CFTR-deficient cells (0.07%) with similarly low efficiencies, which significantly decreased with either mf or mt inhibition (0.008% and 0.002%, respectively). CONCLUSION: B. dolosa and B. cenocepacia enter respiratory epithelial cells in a mf and mt dependent fashion. Mutated CFTR leads to less internalization of B. dolosa, but not B. cenocepacia.

Regulation of systemic and local neutrophil responses by G-CSF during pulmonary Pseudomonas aeruginosa infection.

Granulocyte colony-stimulating factor (G-CSF) regulates the production, maturation, and function of neutrophils. Its expression is often induced during infection, resulting in high concentrations of G-CSF in inflammatory exudates and in the blood, suggesting that it may regulate both local and systemic neutrophil responses. Herein, we characterize the neutrophil response in G-CSFR(-/-) mice following intratracheal injection with Pseudomonas aeruginosa-laden agarose beads, modeling the pulmonary infection observed in many patients with cystic fibrosis. G-CSFR(-/-) mice are markedly susceptible to bronchopulmonary P aeruginosa infection, exhibiting decreased survival and bacterial clearance as well as extensive damage to lung tissue. The systemic neutrophil response was mediated primarily by enhanced neutrophil release from the bone marrow rather than increased neutrophil production and was attenuated in G-CSFR(-/-) mice. Despite normal to increased local production of inflammatory chemokines, neutrophil accumulation into the infected lung of G-CSFR(-/-) mice was markedly reduced. Moreover, the percentage of apoptotic neutrophils in the lung was elevated, suggesting that G-CSF signals may play an important role in regulating neutrophil survival at the inflammatory site. Collectively, these data provide new evidence that G-CSF signals play important but specific roles in the regulation of the systemic and local neutrophil response following infection.