The cooperation of pharmacologic-dose ascorbate with ceftriaxone against Staphylococcus aureus through bactericidal synergy and enhanced macrophage killing activity.

BACKGROUND: Ascorbate is a low-cost compound with a known bactericidal-synergy to antibitics. However, the synergy depends on concentrations and organisms. Thus, the synergy test by time-kill assay might be appropriate for the screening of the synergy. OBJECTIVE: We aimed to test the adjuvant property of ascorbate with ceftriaxone, a frequently prescribed ß-lactam antibiotic. METHOD: Ascorbate was tested with several bacteria from the American Type Culture Collection (ATCC) including Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli for i) bactericidal property of ascorbate, alone or with ceftriaxone-combination, by time-kill assay, ii) an influence on the killing-activity of bone -marrow-derived macrophage and iii) the attenuation of myositis mouse model. RESULT: The bactericidal synergy (determined with time-kill assay at 24 h) against S. aureus, but not other selected bacteria, was demonstrated in ascorbate (10 and 40 mM) plus ceftriaxone at the minimal inhibitory concentration (1x MIC). Ascorbate alone, without antibiotic, enhanced macrophage killing-activity and directly eliminated bacteria at the concentration 10-40mM and 250mM, respectively (both properties presented against S. aureus and P. aeruginosa, but not other bacteria). Ascorbate with ceftriaxone also reduced bacterial burdens in muscle and serum cytokines of S. aureus -myositis mouse model. Moreover, the synergy against the clinical isolated methicillin resistant S. aureus (MRSA) by time-kill assay and myositis model also presented. CONCLUSION: Ascorbate-ceftriaxone synergy against S. aureus was demonstrated by time-kill assay and myositis model. Time-kill assy might be valuable as a screening test to select the patients that potentially benefit from ascorbate- ceftriaxone adjuvant therapy.

Gold nanoparticles attenuates bacterial sepsis in cecal ligation and puncture mouse model through the induction of M2 macrophage polarization.

BACKGROUND: Gold nanoparticles (AuNP) have several biochemical advantageous properties especially for a candidate of drug carrier. However, the non-conjugated AuNP has a higher rate of cellular uptake than the conjugated ones. Spherical AuNP in a proper size (20-30 nm) is non-toxic to mice and shows anti-inflammatory properties. We tested if the administration of AuNP, as an adjuvant to antibiotics, could attenuate bacterial sepsis in cecal ligation and puncture (CLP) mouse model with antibiotic (imipenem/cilastatin). RESULTS: Indeed, AuNP administration at the time of CLP improved the survival, blood bacterial burdens, kidney function, liver injury and inflammatory cytokines (TNF-α, IL-6, IL-1ß and IL-10). AuNP also decreased M1 macrophages (CD86 + ve in F4/80 + ve cells) and increased M2 macrophages (CD206 + ve in F4/80 + ve cells) in the spleens of sepsis mice. The weak antibiotic effect of AuNP was demonstrated as the reduction of E. coli colony after 4 h incubation. In addition, AuNP altered cytokine production of bone-marrow-derived macrophages including reduced TNF-α, IL-6 and IL-1ß but increased IL-10 at 6 and 24 h. Moreover, AuNP induced macrophage polarization toward anti-inflammatory responses (M2) as presented by increased Arg1 (Arginase 1) and PPARγ with decreased Nos2 (inducible nitric oxide synthase, iNos) and Nur77 at 3 h after incubation in vitro. CONCLUSIONS: The adjuvant therapy of AuNP, with a proper antibiotic, attenuated CLP-induced bacterial sepsis in mice, at least in part, through the antibiotic effect and the induction of macrophage function toward the anti-inflammatory responses.

Fc Gamma Receptor IIB Deficient Mice: A Lupus Model with Increased Endotoxin Tolerance-Related Sepsis Susceptibility.

Hyper-elevated immune response of FcGRIIb-/- mice, a lupus model with an inhibitory-signaling defect, can become exhausted (less subsequent immune-response than the first response) with sequential lipopolysaccharide (LPS) stimulation. Endotoxin tolerance-related modifications of inflammatory response were investigated in FcGRIIb-/- mice in both an in vivo sepsis model and in vitro using cultured macrophages. Serum cytokine concentrations, after the second LPS injection (at 5-fold higher levels than the first dose), did not exceed the first dose levels in either FcGRIIb-/- or wild-type mice. These data indicated an endotoxin-tolerance response in both genetic backgrounds. However, the difference of cytokine levels between the first and second LPS injection was more prominent in FcGRIIb-/- mice. More importantly, CLP-induced sepsis after LPS-preconditioning (two separated doses of LPS administration) was more severe in FcGRIIb-/- mice (as measured by mortality rate, bacteria count in blood, serum cytokines, creatinine, and alanine transaminase). An attenuated response was demonstrated after two sequential LPS stimulations of bone-marrow-derived macrophages. Cytokine production was reduced and lower bacterial killing activity occurred with macrophages from FcGRIIb-/- mice relative to wild-type macrophages. Thus, there is a more prominent effect of endotoxin-tolerance in FcGRIIb-/- macrophages relative to wild-type. In conclusion, repeated-LPS administrations induced quantitatively greater endotoxin-tolerance responses in FcGRIIb-/- mice both in vivo and in vitro. Endotoxin-tolerance in vivo was associated with more severe sepsis, at least in part, due to macrophage-dysfunction.

The role of macrophages in the susceptibility of Fc gamma receptor IIb deficient mice to Cryptococcus neoformans.

Dysfunctional polymorphisms of FcγRIIb, an inhibitory receptor, are associated with Systemic Lupus Erythaematosus (SLE). Cryptococcosis is an invasive fungal infection in SLE, perhaps due to the de novo immune defect. We investigated cryptococcosis in the FcγRIIb-/- mouse-lupus-model. Mortality, after intravenous C. neoformans-induced cryptococcosis, in young (8-week-old) and older (24-week-old) FcγRIIb-/- mice, was higher than in age-matched wild-types. Severe cryptococcosis in the FcγRIIb-/- mice was demonstrated by high fungal burdens in the internal organs with histological cryptococcoma-like lesions and high levels of TNF-α and IL-6, but not IL-10. Interestingly, FcγRIIb-/- macrophages demonstrated more prominent phagocytosis but did not differ in killing activity in vitro and the striking TNF-α, IL-6 and IL-10 levels, compared to wild-type cells. Indeed, in vivo macrophage depletion with liposomal clodronate attenuated the fungal burdens in FcγRIIb-/- mice, but not wild-type mice. When administered to wild-type mice, FcγRIIb-/- macrophages with phagocytosed Cryptococcus resulted in higher fungal burdens than FcγRIIb+/+ macrophages with phagocytosed Cryptococcus. These results support, at least in part, a model whereby, in FcγRIIb-/- mice, enhanced C. neoformans transmigration occurs through infected macrophages. In summary, prominent phagocytosis, with limited effective killing activity, and high pro-inflammatory cytokine production by FcγRIIb-/- macrophages were correlated with more severe cryptococcosis in FcγRIIb-/- mice.

Cilostazol attenuates intimal hyperplasia in a mouse model of chronic kidney disease.

Intimal hyperplasia (IH) is a common cause of vasculopathy due to direct endothelial damage (such as post-coronary revascularization) or indirect injury (such as chronic kidney disease, or CKD). Although the attenuation of coronary revascularization-induced IH (direct-vascular-injury-induced IH) by cilostazol, a phosphodiesterase III inhibitor, has been demonstrated, our understanding of the effect on CKD-induced IH (indirect-vascular-injury-induced IH) is limited. Herein, we tested if cilostazol attenuated CKD-induced IH in a mouse model of ischemic-reperfusion injury with unilateral nephrectomy (Chr I/R), a normotensive non-proteinuria CKD model. Cilostazol (50 mg/kg/day) or placebo was orally administered once daily from 1-week post-nephrectomy. At 20 weeks, cilostazol significantly attenuated aortic IH as demonstrated by a 34% reduction in the total intima area with 50% and 47% decreases in the ratios of tunica intima area/tunica media area and tunica intima area/(tunica intima + tunica media area), respectively. The diameters of aorta and renal function were unchanged by cilostazol. Interestingly, cilostazol decreased miR-221, but enhanced miR-143 and miR-145 in either in vitro or aortic tissue, as well as attenuated several pro-inflammatory mediators, including asymmetrical dimethylarginine, high-sensitivity C-reactive protein, vascular endothelial growth factor in aorta and serum pro-inflammatory cytokines (IL-6 and TNF-α). We demonstrated a proof of concept of the effectiveness of cilostazol in attenuating IH in a Chr I/R mouse model, a CKD model with predominantly indirect-vascular-injury-induced IH. These considerations warrant further investigation to develop a new primary prevention strategy for CKD-related IH.