James A. Thliveris

The relationship of blood concentrations of rapamycin and cyclosporine to suppression of allograft rejection in a rabbit heterotopic heart transplant model.

Heterotopic heart transplants were performed on 50 New Zealand white rabbits. Groups of 5 rabbits were randomly assigned to receive, through an intravenous route, rapamycin (RAPA) or cyclosporine at the following doses: RAPA (0.05, 0.1, 0.5, and 1.0 mg/kg/day); CsA (5.0, 10.0, and 15.0 mg/kg/day). Drug vehicle and saline controls were also included. Trough blood concentrations were monitored in both RAPA- and CsA-treated groups on a weekly basis throughout the study. Biochemical assessment of renal and liver function was performed at the beginning and end of the study. Animals receiving RAPA exhibited excellent allograft survival; only two animals in the lowest dosage group (0.05 mg/kg/day) rejected their grafts. In contrast, no rejection occurred in the CsA-treated groups. Animals that rejected their grafts were maintained on the drug until the endpoint of the study was reached at 60 days posttransplant to monitor drug induced side-effects. In some instances animals were sacrificed prior to this time due to infectious and other complications. No significant changes in renal or liver function were noted in the RAPA-treated group, while in the group of animals receiving the highest dose of CsA (15.0 mg/kg/day) a significant decrease in creatinine clearance was noted. A correlation was shown to exist between dose and the trough concentrations of both drugs. The whole-blood concentrations of RAPA that resulted in maximal efficacy with minimal toxicity was in the range of 10–60 μg/L. Rabbits having trough whole-blood concentrations of <10 μg/L rejected their grafts. A much wider therapeutic range for CsA (50–300 μg/L) was noted. The results suggest that RAPA is as efficacious as CsA in prevention of allograft rejection in the animal model tested. The therapeutic monitoring of trough blood concentrations of RAPA, as with CsA, may be useful in guiding dosage adjustments to maximize the immunosuppressive efficacy while minimizing drug-induced side-effects.

Hyaluronidase 3 (HYAL3) knockout mice do not display evidence of hyaluronan accumulation

Hyaluronidases are endoglycosidases that initiate the breakdown of hyaluronan (HA), an abundant component of the vertebrate extracellular matrix. In humans, six paralogous genes encoding hyaluronidase-like sequences have been identified on human chromosomes 3p21.3 (HYAL2-HYAL1-HYAL3) and 7q31.3 (SPAM1-HYAL4-HYALP1). Mutations in one of these genes, HYAL1, were reported in a patient with mucopolysaccharidosis (MPS) IX. Despite the broad distribution of HA, the HYAL1-deficient patient exhibited a mild phenotype, suggesting other hyaluronidase family members contribute to constitutive HA degradation. Hyal3 knockout (Hyal3-/-) mice were generated to determine if HYAL3 had a role in constitutive HA degradation. Hyal3-/- mice were viable, fertile, and exhibited no gross phenotypic changes. X-ray analysis, histological studies of joints, whole-body weights, organ weights and the serum HA levels of Hyal3-/- mice were normal. No evidence of glycosaminoglycan accumulation, including vacuolization, was identified in the Hyal3-/- tissues analyzed. Remarkably, the only difference identified in Hyal3-/- mice was a subtle change in the alveolar structure and extracellular matrix thickness in lung-tissue sections at 12-14 months-of-age. We conclude that HYAL3 does not play a major role in constitutive HA degradation.

Characterization of Cellular Uptake and Toxicity of Aminosilane-Coated Iron Oxide Nanoparticles with Different Charges in Central Nervous System-Relevant Cell Culture Models

Background Aminosilane-coated iron oxide nanoparticles (AmS-IONPs) have been widely used in constructing complex and multifunctional drug delivery systems. However, the biocompatibility and uptake characteristics of AmS-IONPs in central nervous system (CNS)-relevant cells are unknown. The purpose of this study was to determine the effect of surface charge and magnetic field on toxicity and uptake of AmS-IONPs in CNS-relevant cell types. Methods The toxicity and uptake profile of positively charged AmS-IONPs and negatively charged COOH-AmS-IONPs of similar size were examined using a mouse brain microvessel endothelial cell line (bEnd.3) and primary cultured mouse astrocytes and neurons. Cell accumulation of IONPs was examined using the ferrozine assay, and cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results No toxicity was observed in bEnd.3 cells at concentrations up to 200 μg/mL for either AmS-IONPs or COOH-AmS-IONPs. AmS-IONPs at concentrations above 200 μg/mL reduced neuron viability by 50% in the presence or absence of a magnetic field, while only 20% reductions in viability were observed with COOH-AmS-IONPs. Similar concentrations of AmS-IONPs in astrocyte cultures reduced viability to 75% but only in the presence of a magnetic field, while exposure to COOH-AmS-IONPs reduced viability to 65% and 35% in the absence and presence of a magnetic field, respectively. Cellular accumulation of AmS-IONPs was greater in all cell types examined compared to COOH-AmS-IONPs. Rank order of cellular uptake for AmS-IONPs was astrocytes > bEnd.3 > neurons. Accumulation of COOH-AmS-IONPs was minimal and similar in magnitude in different cell types. Magnetic field exposure enhanced cellular accumulation of both AmS- and COOH-AmS-IONPs. Conclusion Both IONP compositions were nontoxic at concentrations below 100 μg/mL in all cell types examined. At doses above 100 μg/mL, neurons were more sensitive to AmS-IONPs, whereas astrocytes were more vulnerable toward COOH-AmS-IONPs. Toxicity appears to be dependent on the surface coating as opposed to the amount of iron-oxide present in the cell.

The Cardioprotective Role of Probucol Against Anthracycline and Trastuzumab-Mediated Cardiotoxicity

OBJECTIVE Although the combination of doxorubicin (Dox) and trastuzumab (Trz) reduces breast cancer progression and recurrence, it is limited by significant cardiotoxic side effects. Little is known about the utility of antioxidants in the prevention of this drug-induced cardiomyopathy. The aim of the study was to determine whether the antioxidant probucol (Prob) would be useful in attenuating Dox and Trz-mediated cardiotoxicity. METHODS A total of 114 mice were randomized to treatment with Trz, Dox, or Dox+Trz. Within each arm, mice received prophylactic treatment with placebo or Prob. Serial murine echocardiography with tissue Doppler imaging was performed daily for 10 days. At 10 days posttreatment, the hearts were removed for histopathologic and Western blot analyses. RESULTS Left ventricular cavity dimensions and systolic parameters were preserved in mice prophylactically treated with Prob after the administration of Dox+Trz. Although the combination of Dox+Trz demonstrated >80% mortality at day 5, prophylactic treatment with Prob reduced mortality to 40% at day 10. There was decreased histologic evidence of cardiac damage and reduced apoptosis due to Dox+Trz in mice pretreated with Prob. CONCLUSION The cardiotoxic effects of Dox+Trz are partially attenuated by the prophylactic administration of the antioxidant Prob.

TOXICITY OF CYCLOSPORINE METABOLITES

Eight cyclosporine (CsA) metabolites were isolated from the urine of renal transplant recipients. The structure and purity of the metabolites were characterized by fast atom bombardment/mass spectroscopy as well as by proton and 13C nuclear magnetic resonance. The in vitro toxicity of the metabolites were tested using a porcine renal epithelial cell line (LLC-PK1). None of the metabolites was as effective as CsA in inhibiting cell growth and DNA, RNA, or protein synthesis, with the majority of them exhibiting activity less than 10% of that of CsA when the IC50 (the concentration required for 50% inhibition of that particular metabolic function) values were compared. The exception to this was the demethylated metabolite M-21, which exhibited a potency of 17–50% of CsA for the various metabolic parameters examined. The results suggest that the immunosuppressive activity of metabolites may be dissociated from their toxicity. Morphologically, CsA and the metabolite M-21 resulted in changes consistent with the vacuolization seen in tubular cells exposed to CsA in vivo. In contrast, M-17 up to the maximum concentration tested (25,000 μg/L) was found not to cause such changes.

A comparison of biologically variable ventilation to recruitment manoeuvres in a porcine model of acute lung injury

BackgroundBiologically variable ventilation (return of physiological variability in rate and tidal volume using a computer-controller) was compared to control mode ventilation with and without a recruitment manoeuvre – 40 cm H2O for 40 sec performed hourly; in a porcine oleic acid acute lung injury model.MethodsWe compared gas exchange, respiratory mechanics, and measured bronchoalveolar fluid for inflammatory cytokines, cell counts and surfactant function. Lung injury was scored by light microscopy. Pigs received mechanical ventilation (FIO2 = 0.3; PEEP 5 cm H2O) in control mode until PaO2 decreased to 60 mm Hg with oleic acid infusion (PaO2/FIO2 <200 mm Hg). Additional PEEP to 10 cm H2O was added after injury. Animals were randomized to one of the 3 modes of ventilation and followed for 5 hr after injury.ResultsPaO2 and respiratory system compliance was significantly greater with biologically variable ventilation compared to the other 2 groups. Mean and mean peak airway pressures were also lower. There were no differences in cell counts in bronchoalveolar fluid by flow cytometry, or interleukin-8 and -10 levels between groups. Lung injury scoring revealed no difference between groups in the regions examined. No differences in surfactant function were seen between groups by capillary surfactometry.ConclusionsIn this porcine model of acute lung injury, various indices to measure injury or inflammation did not differ between the 3 approaches to ventilation. However, when using a low tidal volume strategy with moderate levels of PEEP, sustained improvements in arterial oxygen tension and respiratory system compliance were only seen with BVV when compared to CMV or CMV with a recruitment manoeuvre.

Comparison of the effect of rapamycin and FK506 on release of prostacyclin and endothelin in vitro

Alterations in mesangial and endothelial cell production of vasoactive substances may be a contributing factor to the decreased renal blood flow and glomerular thrombosis associated with FK506 nephrotoxicity. In preliminary studies Rapamycin (RAPA) appears to induce fewer renal side-effects than FK506, although further documentation is required. In this study, the effects of FK506 and RAPA on release of prostacyclin, a vasodilator, and endothelin, a vasoconstrictor, were investigated in cultured rabbit mesangial and endothelial cells. In general, the effects of both RAPA and FK506 on the basal or stimulated release of prostacyclin (as measured by release of its stable metabolite, 6-keto-PGF1 alpha) or endothelin from mesangial cells and endothelial cells were similar with the following exceptions: RAPA resulted in a significant (p < 0.05) increase in the release of prostacyclin (PGI2) from endothelial cells, while in contrast, FK506 resulted in a significant decrease in the release of this analyte from these cells. The similar effects both drugs have on release of vasodilatory and vasoconstrictor substances in vitro does not explain the differences in renal side-effects of the drugs in vivo.

Bnip3 mediates doxorubicin-induced cardiac myocyte necrosis and mortality through changes in mitochondrial signaling

Significance We provide new, exciting evidence for a previously unidentified signaling pathway that mechanistically links mitochondrial respiratory chain defects to necrosis and heart failure induced by the chemotherapy agent doxorubicin (DOX). We specifically show that DOX disrupts protein complexes between the key respiratory chain proteins, including uncoupling protein 3 and cytochrome c oxidase, resulting in abnormal mitochondrial respiration and necrosis through a mechanism contingent on Bcl-2-like 19kDa-interacting protein 3 (Bnip3). Perhaps most compelling is our finding that inhibiting Bnip3 completely abrogated the cardiotoxic effects of DOX. These exciting findings have important clinical implications not only for preventing heart failure by targeting Bnip3 in cancer patients undergoing chemotherapy, but also for understanding the pathogenesis of other diseases in which mitochondrial function is compromised. Doxorubicin (DOX) is widely used for treating human cancers, but can induce heart failure through an undefined mechanism. Herein we describe a previously unidentified signaling pathway that couples DOX-induced mitochondrial respiratory chain defects and necrotic cell death to the BH3-only protein Bcl-2-like 19kDa-interacting protein 3 (Bnip3). Cellular defects, including vacuolization and disrupted mitochondria, were observed in DOX-treated mice hearts. This coincided with mitochondrial localization of Bnip3, increased reactive oxygen species production, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, and necrosis. Interestingly, a 3.1-fold decrease in maximal mitochondrial respiration was observed in cardiac mitochondria of mice treated with DOX. In vehicle-treated control cells undergoing normal respiration, the respiratory chain complex IV subunit 1 (COX1) was tightly bound to uncoupling protein 3 (UCP3), but this complex was disrupted in cells treated with DOX. Mitochondrial dysfunction induced by DOX was accompanied by contractile failure and necrotic cell death. Conversely, shRNA directed against Bnip3 or a mutant of Bnip3 defective for mitochondrial targeting abrogated DOX-induced loss of COX1-UCP3 complexes and respiratory chain defects. Finally, Bnip3−/− mice treated with DOX displayed relatively normal mitochondrial morphology, respiration, and mortality rates comparable to those of saline-treated WT mice, supporting the idea that Bnip3 underlies the cardiotoxic effects of DOX. These findings reveal a new signaling pathway in which DOX-induced mitochondrial respiratory chain defects and necrotic cell death are mutually dependent on and obligatorily linked to Bnip3 gene activation. Interventions that antagonize Bnip3 may prove beneficial in preventing mitochondrial injury and heart failure in cancer patients undergoing chemotherapy.

The FGF-2-triggered protection of cardiac subsarcolemmal mitochondria from calcium overload is mitochondrial connexin 43-dependent

AIMS Fibroblast growth factor 2 (FGF-2) protects the heart from ischaemia- and reperfusion-induced cell death by a mechanism linked to protein kinase C (PKC)ε-mediated connexin 43 (Cx43) phosphorylation. Cx43 localizes predominantly to gap junctions, but has also been detected at subsarcolemmal (SSM), but not interfibrillar (IFM), mitochondria, where it is considered important for cardioprotection. We have now examined the effect of FGF-2 administration to the heart on resistance to calcium-induced permeability transition (mPTP) of isolated SSM vs. IFM suspensions, in relation to mitochondrial PKCε/Cx43 levels, phosphorylation, and the presence of peptide Gap27, a Cx43 channel blocker. METHODS AND RESULTS FGF-2 perfusion increased resistance to calcium-induced mPTP in SSM and IFM suspensions by 2.9- and 1.7-fold, respectively, compared with their counterparts from vehicle-perfused hearts, assessed spectrophotometrically as cyclosporine A-inhibitable swelling. The salutary effect of FGF-2 was lost in SSM, but not in IFM, in the presence of Gap27. FGF-2 perfusion increased relative levels of PKCε, phospho(p) PKCε, and Tom-20 translocase in SSM and IFM, and of Cx43 in SSM. Phospho-serine (pS) 262- and pS368-Cx43 showed a 30- and 8-fold increase, respectively, in SSM from FGF-2-treated, compared with untreated, hearts. Stimulation of control SSM with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased both calcium tolerance and mitochondrial Cx43 phosphorylation at S262 and S368. The PMA-induced phosphorylation of mitochondrial Cx43 was prevented by εV1-2, a PKCε-inhibiting peptide. CONCLUSIONS SSM are more responsive than IFM to FGF-2-triggered protection from calcium-induced mPTP, by a mitochondrial Cx43 channel-mediated pathway, associated with mitochondrial Cx43 phosphorylation at PKCε target sites.

Dynamic MR lymphangiography and carbon dye for sentinel lymph node detection: A solution for sentinel lymph node biopsy in mucosal head and neck cancer†

The practical application of sentinel lymph node biopsy in squamous cell carcinoma of the head and neck is restricted by the time sensitivity of blue dye and lack of spatial resolution and nonspecific node enhancement with radiocolloid. This study evaluates the use of magnetic resonance (MR) lymphangiography and carbon dye labeling to circumvent these limitations.