Direct pulmonary delivery of solubilized curcumin reduces severity of lethal pneumonia.

Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, is associated with reduced lung compliance and hypoxemia. Curcumin exhibits potent anti-inflammatory properties but has poor solubility and rapid plasma clearance. To overcome these physiochemical limitations and uncover the full therapeutic potential of curcumin in lung inflammation, in this study we utilized a novel water-soluble curcumin formulation (CDC) and delivered it directly into the lungs of C57BL/6 mice inoculated with a lethal dose of Klebsiella pneumoniae (KP). Administration of CDC led to a significant reduction in mortality, in bacterial presence within blood and lungs, as well as in lung injury, inflammation, and oxidative stress. The expression of Klebsiella hemolysin gene; TNF-α; IFN-ß; nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3; hypoxia-inducible factor 1/2α; and NF-κB were also decreased following CDC treatment, suggesting modulation of the inflammasome complex and hypoxia signaling pathways as an underlying mechanism by which CDC reduces the severity of pneumonia. On a cellular level, CDC led to diminished cell death, improved viability, and protection of human lung epithelial cells in vitro. Overall, our studies demonstrate that CDC administration improves cell survival and reduces injury, inflammation, and mortality in a murine model of lethal gram-negative pneumonia. CDC, therefore, has promising anti-inflammatory potential in pneumonia and likely other inflammatory lung diseases, demonstrating the importance of optimizing the physicochemical properties of active natural products to optimize their clinical application.-Zhang, B., Swamy, S., Balijepalli, S., Panicker, S., Mooliyil, J., Sherman, M. A., Parkkinen, J., Raghavendran, K., Suresh, M. V. Direct pulmonary delivery of solubilized curcumin reduces severity of lethal pneumonia.

Hepatocellular uptake of cyclodextrin-complexed curcumin during liver preservation: A feasibility study.

The increasing demand for donor organs and the decreasing organ quality is prompting research toward new methods to reduce ischemia reperfusion injury (IRI). Several strategies have been proposed to protect preserved organs from this injury. Before curcumin/dextrin complex (CDC), a potent antioxidant and anti-inflammatory agent, can be used clinically we need to better understand the intracellular uptake under hypothermic conditions on a rat model of liver donation after circulatory death (DCD) and brain death (DBD). To be able to use the fluorescence of CDC for quantification the stability of CDC in different preservation solutions at 4°C or 37°C was investigated. Livers from Wistar rats were procured after being flushed-out through the portal vein using CDC-enriched preservation solutions and stored at 4°C for variable periods. The CDC signal was stable in different preservation solutions over a period of 4 h and allowed the rapid and lasting uptake of curcumin into cells. After 4 h of preservation, CDC was no longer visible microscopically, and HPLC analysis showed very low to non-detectable tissue levels of CDC, proving metabolization during preservation. However, the distribution of CDC was not affected by warm ischemia damage (p = 0.278) nor by flushing the livers before or after 4 h of cold storage and without a warm preflush. Finally, curcumin reduced oxidative stress, lowered histological injury and did not change gene expression after WI/cold storage. Therefore, the use of CDC flush solution for the initial organ flush can offer a promising approach to the enhancement of liver preservation and the maintenance of its quality.

Assessing warm ischemic injury of pig livers at hypothermic machine perfusion.

BACKGROUND: Livers originating from donation after circulatory death (DCD) donors are exposed to warm ischemia (WI) before liver transplantation (LTx). Currently, there are no objective tests to evaluate the damage sustained before LTx. This study aims to identify surrogate markers for liver injury that can be assessed during hypothermic machine perfusion (HMP) preservation. In addition, we want to use mathematical equation modeling combining these markers to improve our assessment of DCD livers for transplantation. MATERIALS AND METHODS: Porcine livers were exposed to incremental periods of WI (0-120 min) and subsequently HMP preserved for 4 h. Biochemical and hemodynamic parameters were repeatedly measured in the perfusate during HMP. Subsequently, to mimic LTx, normothermic isolated-liver perfusion was applied for 2 h and the injury assessed using a morphological score. RESULTS: With increasing WI periods, the perfusate became more acidotic, and levels of aspartate aminotransferase (AST), liver fatty acid binding protein, redox-active iron, and arterial vascular resistance increased. A damage index, combining AST and pH (damage index = 2 - 37 × ß(AST) - 257 × ß(pH)) based on multifactorial analysis of the changing pattern of these markers, had increased sensitivity and specificity to reflect WI and reperfusion injury. CONCLUSIONS: This proof of concept study demonstrated the potential role for objective evaluation of DCD porcine livers during HMP and the advantage to use multifactorial analysis on the markers' changing pattern.

Pulmonary administration of a water-soluble curcumin complex reduces severity of acute lung injury.

Local or systemic inflammation can result in acute lung injury (ALI), and is associated with capillary leakage, reduced lung compliance, and hypoxemia. Curcumin, a plant-derived polyphenolic compound, exhibits potent anti-inflammatory properties, but its poor solubility and limited oral bioavailability reduce its therapeutic potential. A novel curcumin formulation (CDC) was developed by complexing the compound with hydroxypropyl-γ-cyclodextrin (CD). This results in greatly enhanced water solubility and stability that facilitate direct pulmonary delivery. In vitro studies demonstrated that CDC increased curcumin's association with and transport across Calu-3 human airway epithelial cell monolayers, compared with uncomplexed curcumin solubilized using DMSO or ethanol. Importantly, Calu-3 cell monolayer integrity was preserved after CDC exposure, whereas it was disrupted by equivalent uncomplexed curcumin solutions. We then tested whether direct delivery of CDC to the lung would reduce severity of ALI in a murine model. Fluorescence microscopic examination revealed an association of curcumin with cells throughout the lung. The administration of CDC after LPS attenuated multiple markers of inflammation and injury, including pulmonary edema and neutrophils in bronchoalveolar lavage fluid and lung tissue. CDC also reduced oxidant stress in the lungs and activation of the proinflammatory transcription factor NF-κB. These results demonstrate the efficacy of CDC in a murine model of lung inflammation and injury, and support the feasibility of developing a lung-targeted, curcumin-based therapy for the treatment of patients with ALI.

Transplantation of allogeneic T cells alters iron homeostasis in NOD/SCID mice.

Iron overload is common in patients undergoing allogeneic hematopoietic cell transplantation (HCT), but the mechanisms leading to overload are unknown. Here, we determined iron levels and the expression of iron regulatory proteins in the liver and gut of nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice that underwent transplantation with syngeneic (histocompatible) or allogeneic (histoincompatible) T lymphocytes. Infusion of histoincompatible T cells resulted in a significant rise in serum iron levels and liver iron content. Iron deposition was accompanied by hepatocyte injury and intestinal villous damage. Feeding of low- or high-iron diet was associated with appropriate ferroportin 1 and hepcidin responses in mice given histocompatible T cells, whereas mice given histoincompatible T cells showed inappropriate up-regulation of duodenal ferroportin 1 and a loss of expression of hepatic hepcidin. These findings suggest that alloreactive T cell-dependent signals induced dysregulation of intestinal iron absorption, which contributed to liver iron overload after HCT.

Multifactorial biological modulation of warm ischemia reperfusion injury in liver transplantation from non-heart-beating donors eliminates primary nonfunction and reduces bile salt toxicity.

OBJECTIVE: To design a multifactorial biological modulation approach targeting ischemia reperfusion injury to augment viability of porcine liver grafts from non-heart-beating donors (NHBD). BACKGROUND DATA: Liver Transplantation (LTx) from NHBD is associated with an increased risk of primary nonfunction (PNF) and biliary complications. In porcine NHBD-LTx, we previously reported a 50% risk of PNF and toxic bile formation in grafts exposed to > or =30' warm ischemia (WI). METHODS: Porcine livers exposed to 45' WI were cold stored, transplanted and either modulated (n = 6) or not (controls, n = 9). In the modulation group, donor livers were flushed with warm Ringers (avoiding cold-induced vasoconstriction), streptokinase (eliminating stagnating thrombi), and epoprostenol (vasodilator, platelet aggregation inhibitor) prior to cold storage. In recipients, glycine (Kupffer cell stabilizer), alpha1-acid-glycoprotein (anti-inflammatory protein), MAPKinase-inhibitor (pro-inflammatory cytokine generation inhibitor), alpha-tocopherol and glutathione (anti-oxidants), and apotransferrin (iron chelator) were administrated intravenously. PNF, survival, lactate, transaminase, TNF-alpha, redox-active iron, and biliary bile salt-to-phospholipid ratio were monitored. RESULTS: No PNF was observed in modulated versus 55% in control pigs (P = 0.025). Survival was 83% in modulated versus 22% in control pigs (P = 0.02). At 180' postreperfusion, lactate was lower in modulated (5.4 +/- 1.9 mmol/L) versus control pigs (9.4 +/- 2.2 mmol/L; P = 0.011). At 60' postreperfusion, there was a trend for lower AST in modulated versus control pigs at 60' (939 +/- 578 vs. 1683 +/- 873 IU/L; P = 0.089). Postreperfusion, TNF-alpha remained stable in modulated pigs (49 +/- 27 pg/mL at 15' and 85 +/- 26 pg/mL at 180'; P = 0.399) but increased in control pigs (107 +/- 36 pg/mL at 15' and 499 +/- 216 pg/mL at 180'; P = 0.023). At 180' postreperfusion, redox-active iron was higher in control pigs versus modulated pigs (0.21+/-0.18 vs. 0.042+/-0.062 mum; P = 0.038). Biliary bile salt-to-phospholipid ratio post-LTx was lower in modulated versus control pigs (1128 +/- 447 vs. 4836 +/- 4619; P = 0.05). CONCLUSIONS: A multifactorial biological modulation eliminates PNF, improves liver function and increases survival. Biochemically, TNF-alpha and redox-active iron are suppressed and biliary bile salt toxicity is reduced. Translating this strategy clinically may lead to wider and safer use of NHBD.

Non-transferrin-bound iron in haematological patients during chemotherapy and conditioning for autologous stem cell transplantation.

Free iron induced hydroxyl radical formation is one possible mechanism for tissue injury during cytotoxic therapy. We studied the appearance of free, non-transferrin-bound iron (NTBI) at baseline and during the 20-d period after the onset of cytotoxic chemotherapy in patients with haematological malignancy undergoing intensive chemotherapy or conditioning for autologous stem cell transplantation (aSCT). NTBI was detected on average for 15.6 d in patients treated with chemotherapy only, and for 6.1 d in patients undergoing aSCT. The recovery of the bone marrow function coincided with the disappearance of NTBI. The type of the conditioning regimen was also associated with the appearance of NTBI. The timing of the presence of NTBI accords with the presence of the most important non-infectious complication of intensive chemotherapy and autologous transplantation, mucosal injury, and free iron is likely to contribute to this and probably other complications of the intensive treatments.

Inhibition of ERK1/2 activation by phenolic antioxidants protects kidney tubular cells during cold storage.

BACKGROUND: Cold storage of tissues induces reactive oxygen species (ROS), which contribute to cell injury. We have compared different antioxidants in protection of renal tubular cells against hypothermia injury and studied their effect on cold-induced mitogen-activated protein (MAP) kinase activation. METHODS: Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin solution supplemented with compounds tested for 16 hr at 4 degrees C. Release of lactate dehydrogenase and cellular adenosine triphosphate were measured. Activation of MAP kinases was determined by Western blotting. Intracellular ROS were monitored with a fluorescent probe. RESULTS: Cold storage resulted in a substantial loss of cell viability. The simple phenol butylated hydroxyanisol (BHA) most effectively prevented hypothermia-induced cell injury, whereas about 100-fold higher concentration of the polyphenol epigallocatechin gallate (EGCG) was needed, although EGCG most effectively scavenged intracellular ROS elicited by serum withdrawal. The MEK inhibitor U0126 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyleneiodonium effectively protected the cells against hypothermia injury. ERK1/2 was rapidly activated during chilling of the cells and this was inhibited by BHA but not by EGCG. CONCLUSION: The results suggest that chilling of renal epithelial cells induces ROS generation by NADPH oxidase, which leads to rapid activation of the MEK-ERK1/2 cascade and initiation of cell injury. This can be prevented by antioxidants.

Exogenous alpha-1-acid glycoprotein protects against renal ischemia-reperfusion injury by inhibition of inflammation and apoptosis.

BACKGROUND: Although ischemia-reperfusion (I/R) injury represents a major problem in posttransplant organ failure, effective treatment is not available. The acute phase protein alpha-1-acid glycoprotein (AGP) has been shown to be protective against experimental I/R injury. The effects of AGP are thought to be mediated by fucose groups expressed on the AGP protein inhibiting neutrophil infiltration. However, the precise mechanism of protection remains to be established. We therefore studied the effects of exogenous human AGP (hAGP) in a mouse model of ischemic acute renal failure. METHODS: Mice were subjected to renal I/R and treated with hAGP, fucose-depleted hAGP, or control treated. Also, transgenic mice over-expressing rat AGP or wild-type controls were subjected to renal I/R. RESULTS: Treatment was with hAGP as well as fucose-depleted hAGP protected mice against I/R-induced acute renal failure. Surprisingly, AGP-over-expressing mice were not protected against I/R injury. Both natural and fucose-depleted hAGP inhibited the activation of the complement system, as determined by renal C3 deposition and influx of neutrophils measured by immunohistochemistry and myeloperoxidase-enzyme-linked immunoadsorbent assay. Tubular epithelial cell structure (actin cytoskeleton) and cell-cell interaction (tight-junction architecture) were completely preserved in AGP-treated mice. Also, epithelial caspase activation and apoptotic DNA cleavage were prevented by AGP treatment. CONCLUSIONS: Both natural and fucose-depleted hAGP protect against renal I/R injury by preservation of tubular epithelial structure and inhibition of apoptosis and subsequent inflammation. Therefore, hAGP can be regarded as a potential new therapeutic intervention in the treatment of acute renal failure, as seen after transplantation of ischemically injured kidneys.

Reduction of circulating redox-active iron by apotransferrin protects against renal ischemia-reperfusion injury.

BACKGROUND: Warm ischemia-reperfusion (I/R) injury plays an important role in posttransplant organ failure. In particular, organs from marginal donors suffer I/R injury. Although iron has been implicated in the pathophysiology of renal I/R injury, the mechanism of iron-mediated injury remains to be established. The authors therefore investigated the role of circulating redox-active iron in an experimental model for renal I/R injury. METHODS: Male Swiss mice were subjected to unilateral renal ischemia for 45 min, followed by contralateral nephrectomy and reperfusion. To investigate the role of circulating iron, mice were treated with apotransferrin, an endogenous iron-binding protein, or iron-saturated apotransferrin (holotransferrin). RESULTS: Renal ischemia induced a significant increase in circulating redox-active iron levels during reperfusion. Apotransferrin, in contrast to holotransferrin, reduced the amount of circulating redox-active iron and abrogated renal superoxide formation. Apotransferrin treatment did not affect I/R-induced renal apoptosis, whereas holotransferrin aggravated apoptotic cell death. Apotransferrin, in contrast to holotransferrin, inhibited the influx of neutrophils. Both apo- and holotransferrin reduced I/R-induced complement deposition, indicating that the effects of transferrin are differentially mediated by its iron and protein moiety. Finally, apotransferrin, in contrast to holotransferrin, dose-dependently inhibited the loss of renal function induced by ischemia. CONCLUSIONS: Redox-active iron is released into the circulation in the course of renal I/R. Reducing the amount of circulating redox-active iron by treatment with apotransferrin protects against renal I/R injury, inhibiting oxidative stress, inflammation, and loss of function. Apotransferrin could be used in the treatment of acute renal failure, as seen after transplantation of ischemically damaged organs.

Erratum to "Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron". [FEMS Immunol. Med Microbiol. 37 (2003) 45-51].

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.

Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron.

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.

Preanalytical factors and reference intervals for serum hepcidin LC-MS/MS method.

BACKGROUND: Hepcidin is a potential biomarker for anemia of chronic diseases and disorders of iron metabolism. Thus, data of preanalytical factors, reference values and method characteristics are critical for clinical use of hepcidin determination. METHODS: We studied the effects of sample storage, sampling device and a meal on hepcidin levels using a liquid-chromatography tandem mass spectrometric (LC-MS/MS) method for serum hepcidin. Age- and gender-dependent reference values were determined using serum samples from healthy volunteers (n=231). The results were also compared with those obtained by a commercial competitive ELISA for hepcidin. RESULTS: In serum samples, hepcidin is stable for one day at room temperature, six days at +4°C and at least 42 days at -20°C. Breakfast or type of sampling device does not affect hepcidin concentration. Significantly lower hepcidin concentrations were observed in women ≤50 than >50 years of age or in men (p<0.0001 for both). Reference values for females aged 18-50 years were 0.4-9.2 nmol/L, for those >50 years 0.7-16.8 nmol/L and for males ≥18 years 1.1-15.6 nmol/L. Comparison with a competitive ELISA showed poor correlation. CONCLUSIONS: Fasting before sampling and type of blood collection were not critical. Samples can be transported to laboratory at room temperature if they arrive within a day. Significantly lower concentrations of serum hepcidin were observed in menstruating than in post-menopausal women and in men.

Addition of a water-soluble propofol formulation to preservation solution in experimental kidney transplantation.

BACKGROUND: Novel interventions that protect against ischemia and reperfusion injury are needed to improve early graft function after kidney transplantation. Propofol, a widely used anesthetic, has proven an efficient membrane-targeted antioxidant and cytoprotective agent. METHODS: The cytoprotective effects of propofol and its reaction intermediate dipropofol on hypothermic proximal tubular epithelial cells were compared with other phenolic antioxidants. For delivery of propofol into kidney grafts, a water-soluble cyclodextrin complex of propofol was prepared. The therapeutic effects of this propofol formulation were studied in a porcine autotransplantation model using 45 min of warm ischemia and 22 hr of hypothermic preservation. RESULTS: Propofol and dipropofol effectively protected tubular cells from hypothermic injury in vitro. Delivery of propofol to porcine kidneys was achieved by adding the cyclodextrin complex of propofol to the preservation solution during machine perfusion. This preservation strategy significantly prevented lipid peroxidation and tended to attenuate the increase in renovascular resistance during the early reperfusion period after autologous kidney transplantation. The antioxidant effects of propofol were followed by a modest improvement in renal function in the first 10 days after transplantation. Treatment with propofol during organ preservation did not reduce neutrophil infiltration into the graft. CONCLUSION: We consider propofol to be a promising renoprotective agent that may attenuate hypothermic and ischemic acute kidney injury in renal transplantation. The novel application of cyclodextrin carrier systems enabled delivery of the water-insoluble propofol to the graft during hypothermic preservation.

Cyclodextrin-complexed curcumin exhibits anti-inflammatory and antiproliferative activities superior to those of curcumin through higher cellular uptake.

Curcumin, a yellow pigment present in the spice turmeric (Curcuma longa), has been linked with multiple beneficial activities, but its optimum potential is limited by poor bioavailability, in part due to the lack of solubility in aqueous solvents. To overcome the solubility problem, we have recently developed a novel cyclodextrin complex of curcumin (CDC) and examined here this compound for anti-inflammatory and antiproliferative effects. Using the electrophoretic mobility shift assay, we found that CDC was more active than free curcumin in inhibiting TNF-induced activation of the inflammatory transcription factor NF-kappaB and in suppressing gene products regulated by NF-kappaB, including those involved in cell proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF). CDC was also more active than free curcumin in inducing the death receptors DR4 and DR5. Annexin V staining, cleavage of caspase-3 and PARP, and DNA fragmentation showed that CDC was more potent than free curcumin in inducing apoptosis of leukemic cells. Antiproliferative assays also demonstrated that CDC was more active than free curcumin in suppressing proliferation of various cancer cell lines. The cyclodextrin vehicle had no effect in these assays. Compared with free curcumin, CDC had a greater cellular uptake and longer half-life in the cells. Overall we demonstrated that CDC had superior attributes compared with free curcumin for cellular uptake and for antiproliferative and anti-inflammatory activities.

Inhibition of erythroid and granulocyte-macrophage colony formation by non-transferrin-bound iron in vitro: protective effect of apotransferrin.

OBJECTIVES: The aim of the study was to investigate in vitro the effect of free iron on erythroid and granulocyte-macrophage colony formation and the effect of binding free iron with apotransferrin. METHODS: Normal haematopoietic progenitors were cultured in vitro with different concentrations of free iron in the form of ferric nitrilotriacetic acid (FeNTA). Parallel cultures were performed after the preincubation of FeNTA with apotransferrin. RESULTS: Free iron inhibited colony formation by erythroid and granulocyte-macrophage progenitors and reduced the size of the colonies in a dose-dependent manner. Preincubation of FeNTA with apotransferrin diminished the inhibitory effect of FeNTA on colony formation increasing both the number and the size of colonies. CONCLUSIONS: Free iron was toxic to haematopoietic progenitors in in vitro cultures; the toxic effect could be reduced with apotransferrin.

Primary graft nonfunction and Kupffer cell activation after liver transplantation from non-heart-beating donors in pigs.

More extensive use of non-heart-beating donors (NHBD) could reduce mortality on liver transplantation waiting lists, but this is associated with more primary nonfunction (PNF). We assessed which parameters are involved in the development of PNF in livers from NHBD in a previously validated pig liver transplantation model, in which livers were transplanted after exposure to incremental periods of warm ischemia. The risk of PNF was unacceptably high (>50%) when livers were exposed to >30 minutes' warm ischemia before a short cold ischemic period. This study examined how PNF is affected by Kupffer cell activation (beta-galactosidase), the generation of cytokines tumor necrosis factor alpha and interleukin 6, antioxidant mechanisms (ascorbic acid, alpha-tocopherol, reduced glutathione), circulating redox-active iron, and sinusoidal endothelial cell function (hyaluronic acid clearance). Kupffer cells were more activated in PNF recipients, as suggested by higher beta-galactosidase levels (15 minutes after reperfusion), and secondarily, by higher production of tumor necrosis factor alpha and interleukin 6 (180 minutes after reperfusion). In addition, alpha-tocopherol and reduced glutathione were lower, and ascorbic acid and redox-active iron higher in PNF recipients. Finally, PNF grafts displayed progressively decreasing hyaluronic acid clearance (suggesting sinusoidal endothelial cell dysfunction) and parenchymal edema. Consequently, a reduced-flow phenomenon was documented. In grafts from NHBD that are destined to fail, beta-galactosidase activity (a surrogate of Kupffer cell activation) is higher, proinflammatory cytokines are overproduced, some antioxidant mechanisms fail, and circulating redox-active iron is more rapidly released. A no-flow phenomenon is eventually observed in these failing grafts.