Ciprofloxacin and pegylated G-CSF combined therapy mitigates brain hemorrhage and mortality induced by ionizing irradiation.

Introduction: Brain hemorrhage was found between 13 and 16 days after acute whole-body 9.5 Gy 60Co-γ irradiation (IR). This study tested countermeasures mitigating brain hemorrhage and increasing survival from IR. Previously, we found that pegylated G-CSF therapy (PEG) (i.e., Neulasta®, an FDA-approved drug) improved survival post-IR by 20-40%. This study investigated whether Ciprofloxacin (CIP) could enhance PEG-induced survival and whether IR-induced brain hemorrhage could be mitigated by PEG alone or combined with CIP. Methods: B6D2F1 female mice were exposed to 60Co-γ-radiation. CIP was fed to mice for 21 days. PEG was injected on days 1, 8, and 15. 30-day survival and weight loss were studied in mice treated with vehicles, CIP, PEG, or PEG + CIP. For the early time point study, blood and sternums on days 2, 4, 9, and 15 and brains on day 15 post-IR were collected. Platelet numbers, brain hemorrhage, and histopathology were analyzed. The cerebellum/pons/medulla oblongata were detected with glial fibrillary acidic protein (GFAP), p53, p16, interleukin-18 (IL-18), ICAM1, Claudin 2, ZO-1, and complement protein 3 (C3). Results: CIP + PEG enhanced survival after IR by 85% vs. the 30% improvement by PEG alone. IR depleted platelets, which was mitigated by PEG or CIP + PEG. Brain hemorrhage, both surface and intracranial, was observed, whereas the sham mice displayed no hemorrhage. CIP or CIP + PEG significantly mitigated brain hemorrhage. IR reduced GFAP levels that were recovered by CIP or CIP + PEG, but not by PEG alone. IR increased IL-18 levels on day 4 only, which was inhibited by CIP alone, PEG alone, or PEG + CIP. IR increased C3 on day 4 and day 15 and that coincided with the occurrence of brain hemorrhage on day 15. IR increased phosphorylated p53 and p53 levels, which was mitigated by CIP, PEG or PEG + CIP. P16, Claudin 2, and ZO-1 were not altered; ICAM1 was increased. Discussion: CIP + PEG enhanced survival post-IR more than PEG alone. The Concurrence of brain hemorrhage, C3 increases and p53 activation post-IR suggests their involvement in the IR-induced brain impairment. CIP + PEG effectively mitigated the brain lesions, suggesting effectiveness of CIP + PEG therapy for treating the IR-induced brain hemorrhage by recovering GFAP and platelets and reducing C3 and p53.

Ciprofloxacin Therapy Results in Mitigation of ATP Loss after Irradiation Combined with Wound Trauma: Preservation of Pyruvate Dehydrogenase and Inhibition of Pyruvate Dehydrogenase Kinase 1.

Ionizing radiation exposure combined with wound injury increases animal mortalities than ionizing radiation exposure alone. Ciprofloxacin (CIP) is in the fluroquinolone family of synthetic antibiotic that are available from the strategic national stockpile for emergency use and is known to inhibit bacterial sepsis. The purpose of this study was to evaluate the efficacy of ciprofloxacin as a countermeasure to combined injury mortality and determine the signaling proteins involved in energy machinery. B6D2F1/J female mice were randomly assigned to receive either 9.75 Gy irradiation with Co-60 gamma rays followed by skin wounding (combined injury; CI) or sham procedure (sham). Either ciprofloxacin (90 mg/kg/day) or vehicle (VEH) (water) was administered orally to these mice 2 h after wounding and thereafter daily for 10 days. Determination of tissue adenosine triphosphate (ATP) was conducted, and immunoblotting for signaling proteins involved in ATP machinery was performed. Combined injury resulted in 60% survival after 10 days compared to 100% survival in the sham group. Furthermore, combined injury caused significant reductions of ATP concentrations in ileum, pancreas, brain, spleen, kidney and lung (-25% to -95%) compared to the sham group. Ciprofloxacin administration after combined injury resulted in 100% survival and inhibited reductions in ileum and kidney ATP production. Ileum protein levels of heat-shock protein 70 kDa (HSP-70, a chaperone protein involved in ATP synthesis) and pyruvate dehydrogenase (PDH, an enzyme complex crucial to conversion of pyruvate to acetyl CoA for entrance into TCA cycle) were significantly lower in the CI group (vs. sham group). Using immunoprecipitation and immunoblotting, HSP-70-PDH complex was found to be present in the ileum tissue of CI mice treated with ciprofloxacin. Furthermore, phosphorylation of serine residues of PDH resulting in inactivating PDH enzymatic activity, which occurred after combined injury, was inhibited with ciprofloxacin treatment, thus enabling PDH to increase ATP production. Increased ileum levels of pyruvate dehydrogenase kinase 1 protein (PDK1, an enzyme responsible for PDH phosphorylation) after combined injury were also prevented by ciprofloxacin treatment. Taken together, these data suggest that ciprofloxacin oral administration after combined injury had a role in sustained ileum ATP levels, and may have acted through preservation of PDH by HSP-70 and inhibition of PDK1. These molecular changes in the ileum are simply one of a host of mechanisms working in concert with one another by which ciprofloxacin treatment mitigates body weight loss and drastically enhances subsequent survival after combined injury. To this end, our findings indicate that oral treatment of ciprofloxacin is a valuable therapeutic treatment after irradiation with combined injury and warrants further analyses to elucidate the precise mechanisms involved.

Geldanamycin inhibits hemorrhage-induced increases in caspase-3 activity: role of inducible nitric oxide synthase.

Hemorrhage has been shown to increase inducible nitric oxide synthase (iNOS) and deplete ATP levels in tissues and geldanamycin limits both processes. Moreover, it is evident that inhibition of iNOS reduces caspase-3 and increases survival. Thus we sought to identify the molecular events responsible for the beneficial effect of geldanamycin. Hemorrhage in mice significantly increased caspase-3 activity and protein while treatment with geldanamycin significantly limited these increases. Similarly, geldanamycin inhibited increases in proteins forming the apoptosome (a complex of caspase-9, cytochrome c, and Apaf-1). Modulation of the expression of iNOS by iNOS gene transfection or siRNA treatment demonstrated that the level of iNOS correlates with caspase-3 activity. Our data indicate that geldanamycin limits caspase-3 expression and protects from organ injury by suppressing iNOS expression and apoptosome formation. Geldanamycin, therefore, may prove useful as an adjuvant in fluids used to treat patients suffering blood loss.

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel.

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca2+], was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca2+]i in Jurkat T cells was 70 +/- 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca2+]i to 52 +/- 2 nM (n = 23), indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca2+]i by 30 +/- 5% (P < 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca2+]i responsiveness to EBE. Removal of external Ca2+ or Na+, but not Mg2+, inhibited the EBE-induced increase in [Ca2+]i. Dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, also inhibited the EBE-induced increase in [Ca2+]i in a concentration-dependent manner with an IC50 of 0.11 +/- 0.02 nM. When external [K+] was increased from 4.5 mM to 25 mM, EBE decreased [Ca2+]i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca2+ channel blocker. These results suggest that the EBE-induced [Ca2+]i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca2+]i is mediated by activation of Na+/Ca2+ exchangers and opening of L-type voltage-gated Ca2+ channels.

Heat stress protection against mesenteric I/R-induced alterations in intestinal mucosa in rats.

Prior induction of heat shock protein 70 (HSP70) protects against ischemia-reperfusion (I/R) mucosal injury, but the ability of HSP70 to affect I/R-induced alterations in epithelial cell function is unknown. Rats subjected to whole body hyperthermia (41.5-42 degrees C for 6 min) increased HSP70 and heat shock factor 1 mRNA expression, reaching a maximum 2 h after heat stress and declining thereafter. HSP70 production was maximally elevated at 4 h after heat stress and remained elevated until after 12 h. Heat stress alone had no effect on mucosal function except to enhance secretion in response to ACh. Heat stress provided complete morphological protection against I/R-induced mucosal injury but did not confer a similar protection against I/R-induced decreases in mucosal resistance, sodium-linked glucose absorption, or tachykinin-mediated chloride secretion. Heat stress, however, attenuated the I/R-induced suppression of ACh response, and this effect was dependent on enteric nerves. Thus induction of heat shock protein 70 is associated with the preservation of mucosal architecture and attenuation of some specific functional alterations induced by I/R.