A mouse model for immunization with ex vivo virus-infected dendritic cells

Dendritic cells (DCs) have been demonstrated to be an important if not essential inducer of cellular immune responses. The ability to grow these cells in vitro may open up new avenues for protective immunizations. In this study we have analyzed the virus-specific memory response generated following immunization with ex vivo-infected bone marrow-derived dendritic cells. We demonstrate that mouse DCs are efficiently infected with influenza virus but do not release infectious progeny virus. Ex vivo-infected DCs secrete interleukin-12 (IL-12) and induce a potent T helper (Th)1-like immune response when injected into mice. This was demonstrated by the generation of cytotoxic T lymphocytes, the production of high levels of gamma-interferon, and undetectable levels of IL-4 upon in vitro restimulation of splenocytes from immunized animals. In addition, the virus-specific antibody response is primarily of the IgG2a isotype, consistent with the expansion of Th1 cells. Animals immunized with DCs infected with X-31 influenza virus and challenged with PR8 influenza virus cleared the infection faster than animals not vaccinated. Thus, infected DCs efficiently activate the cellular immune response and induce heterosubtypic immunity in mice.     

A mouse model of mitochondrial complex III dysfunction induced by myxothiazol

Myxothiazol is a respiratory chain complex III (CIII) inhibitor that binds to the ubiquinol oxidation site Qo of CIII. It blocks electron transfer from ubiquinol to cytochrome b and thus inhibits CIII activity. It has been utilized as a tool in studies of respiratory chain function in in vitro and cell culture models. We developed a mouse model of biochemically induced and reversible CIII inhibition using myxothiazol. We administered myxothiazol intraperitoneally at a dose of 0.56 mg/kg to C57Bl/J6 mice every 24 h and assessed CIII activity, histology, lipid content, supercomplex formation, and gene expression in the livers of the mice. A reversible CIII activity decrease to 50% of control value occurred at 2 h post-injection. At 74 h only minor histological changes in the liver were found, supercomplex formation was preserved and no significant changes in the expression of genes indicating hepatotoxicity or inflammation were found. Thus, myxothiazol-induced CIII inhibition can be induced in mice for four days in a row without overt hepatotoxicity or lethality. This model could be utilized in further studies of respiratory chain function and pharmacological approaches to mitochondrial hepatopathies.     

Iron-mediated inhibition of mitochondrial manganese uptake mediates mitochondrial dysfunction in a mouse model of hemochromatosis

Previous phenotyping of glucose homeostasis and insulin secretion in a mouse model of hereditary hemochromatosis (Hfe(-/-)) and iron overload suggested mitochondrial dysfunction. Mitochondria from Hfe(-/-) mouse liver exhibited decreased respiratory capacity and increased lipid peroxidation. Although the cytosol contained excess iron, Hfe(-/-) mitochondria contained normal iron but decreased copper, manganese, and zinc, associated with reduced activities of copper-dependent cytochrome c oxidase and manganese-dependent superoxide dismutase (MnSOD). The attenuation in MnSOD activity was due to substantial levels of unmetallated apoprotein. The oxidative damage in Hfe(-/-) mitochondria is due to diminished MnSOD activity, as manganese supplementation of Hfe(-/-) mice led to enhancement of MnSOD activity and suppressed lipid peroxidation. Manganese supplementation also resulted in improved insulin secretion and glucose tolerance associated with increased MnSOD activity and decreased lipid peroxidation in islets. These data suggest a novel mechanism of iron-induced cellular dysfunction, namely altered mitochondrial uptake of other metal ions.     

Compromising human skin in vivo and ex vivo to study skin barrier repair

Ex vivo regenerated stratum corneum (SC) after tape-stripping can be used as a model to study the barrier function of compromised skin. Yet, details about how close the regenerated SC model mimics the lipid properties (e.g. lipid composition and lipid ordering) of the in vivo situation are not known. Here, we examined using a comprehensive ceramide analysis whether human ex vivo regenerated SC showed similar lipid properties as human in vivo regenerated SC. Both in vivo and ex vivo regenerated SC had an altered ceramide subclass composition, with increased percentages of sphingosine-based subclass and decreased percentages of phytosphingosine-based subclass ceramides, a reduced mean ceramide chain length, and a higher percentage of unsaturated ceramides. Overall, regenerated SC ex vivo showed more pronounced but similar changes compared to the in vivo response. One of the purposes of these models is to use them to mimic compromised skin of inflammatory skin diseases. The altered lipid properties in regenerated SC were comparable to those observed in several inflammatory skin diseases, which makes them a valuable model for the barrier properties in inflammatory skin diseases.     

A new circular RNA–encoded protein BIRC6-236aa inhibits transmissible gastroenteritis virus (TGEV)–induced mitochondrial dysfunction

Transmissible gastroenteritis virus (TGEV), a member of the coronavirus family, is the pathogen responsible for transmissible gastroenteritis, which results in mitochondrial dysfunction in host cells. Previously, we identified 123 differentially expressed circular RNAs (cRNA)from the TGEV-infected porcine intestinal epithelial cell line jejunum 2 (IPEC-J2). Previous bioinformatics analysis suggested that, of these, circBIRC6 had the potential to regulate mitochondrial function. Furthermore, mitochondrial permeability transition, a key step in the process of mitochondrial dysfunction, is known to be caused by abnormal opening of mitochondrial permeability transition pores (mPTPs) regulated by the voltage-dependent anion-selective channel protein 1 (VDAC)–Cyclophilin D (CypD) complex. Therefore, in the present study, we investigated the effects of circBIRC6-2 on mitochondrial dysfunction and opening of mPTPs. We found that TGEV infection reduced circBIRC6-2 levels, which in turn reduced mitochondrial calcium (Ca²⁺) levels, the decrease of mitochondrial membrane potential, and opening of mPTPs. In addition, we also identified ORFs and internal ribosomal entrance sites within the circBIRC6-2 RNA. We demonstrate circBIRC6-2 encodes a novel protein, BIRC6-236aa, which we show inhibits TGEV-induced opening of mPTPs during TGEV infection. Mechanistically, we identified an interaction between BIRC6-236aa and VDAC1, suggesting that BIRC6-236aa destabilizes the VDAC1–CypD complex. Taken together, the results suggest that the novel protein BIRC6-236aa encoded by cRNA circBIRC6-2 inhibits mPTP opening and subsequent mitochondrial dysfunction by interacting with VDAC1.     

Volumetric optoacoustic imaging feedback during endovenous laser therapy – an ex vivo investigation

Endovenous laser therapy (ELT) was introduced in clinical practice for treating incompetent veins about fifteen years ago. Despite the considerable clinical evidence collected so far, no rigorous guidelines are yet available regarding the optimal energy deposition protocols while incidence of recanalization, lack of vessel occlusion and collateral damage remains variable among patients. Online monitoring and feedback‐based control over the lesion progression may improve clinical outcomes. Yet the currently employed monitoring tools, such as Doppler ultrasound, often do not provide sufficient contrast as well as three‐dimensional imaging capacity for accurate lesion assessment during thermal treatments. Here we investigate on the utility of volumetric optoacoustic tomography for real‐time monitoring of the ELT procedures. Experiments performed in subcutaneous veins of an ox foot model revealed the accurate spatio‐temporal maps of the lesion progression and characteristics of the vessel wall. Optoacoustic images further correlated with the temperature elevation measured in the area adjacent to the coagulation spot and made it possible to track the position of the fiber tip during its pull back in real time and in all three dimensions. Overall, we showcase that volumetric optoacoustic tomography is a promising tool for providing online feedback during endovenous laser therapy.

     

Oligomycin-resistant mitochondrial ATPase from mouse fibroblasts.

Fourteen oligomycin-resistant LM(TK-) clones were isolated following the mutagenesis of minicells. In the absence of oligomycin, the mutants grew with population doubling times similar to that of the wild type (1 day). In 3 or 5 microgram oligomycin/ml the doubling times of the mutants were 1.2-2.5 days. Both stable and unstable classes were represented among the oligomycin-resistant mutants. Mitochondrial ATPase activities of the mutants were 1.3-1130 times more resistant to oligomycin than the wild type. The mitochondrial ATPase of OLI 14 was found to be bound firmly to the mitochondrial membrane, showed no alteration in the pH optimum compared to wild-type, and exhibited increased resistance to DCCD and venturicidin. These results are consistent with the conclusion that oligomycin resistance in these mutants results from altered mitochondrial ATPase.     

Detection of mitochondrial dysfunction by EPR technique in mouse model of dilated cardiomyopathy

Tgalphaq44 mice with targeted overexpression of activated Galphaq protein in cardiomyocytes mimic many of the phenotypic characteristics of dilated cardiomyopathy in humans. However, it is not known whether the phenotype of Tgalphaq44 mice would also involve dysfunction of cardiac mitochondria. The aim of the present work was to examine changes in EPR signals of semiquinones and iron in Fe-S clusters, as compared to classical biochemical indices of mitochondrial function in hearts from Tgalphaq44 mice in relation to the progression of heart failure. Tgalphaq44 mice at the age of 14 months displayed pulmonary congestion, increased heart/body ratio and impairment of cardiac function as measured in vivo by MRI. However, in hearts from Tgalphaq44 mice already at the age of 10 months EPR signals of semiquinones, as well as cyt c oxidase activity were decreased, suggesting alterations in mitochondrial electron flow. Furthermore, in 14-months old Tgalphaq44 mice loss of iron in Fe-S clusters, impaired citrate synthase activity, and altered mitochondrial ultrastructure were observed, supporting mitochondrial dysfunction in Tgalphaq44 mice. In conclusion, the assessment of semiquinones content and Fe(III) analysis by EPR represents a rational approach to detect dysfunction of cardiac mitochondria. Decreased contents of semiquinones detected by EPR and a parallel decrease in cyt c oxidase activity occurs before hemodynamic decompensation of heart failure in Tgalphaq44 mice suggesting that alterations in function of cardiac mitochondria contribute to the development of the overt heart failure in this model.     

Soluble guanylate cyclase activator BAY 54–6544 improves vasomotor function and survival in an accelerated ageing mouse model

DNA damage is a causative factor in ageing of the vasculature and other organs. One of the most important vascular ageing features is reduced nitric oxide (NO)soluble guanylate cyclase (sGC)—cyclic guanosine monophosphate (cGMP) signaling. We hypothesized that the restoration of NO‐sGC‐cGMP signaling with an sGC activator (BAY 54–6544) may have beneficial effects on vascular ageing and premature death in DNA repair‐defective mice undergoing accelerated ageing. Eight weeks of treatment with a non‐pressor dosage of BAY 54–6544 restored the decreased in vivo microvascular cutaneous perfusion in progeroid Ercc1 ^∆/− mice to the level of wild‐type mice. In addition, BAY 54–6544 increased survival of Ercc1 ^∆/− mice. In isolated Ercc1 ^∆/− aorta, the decreased endothelium‐independent vasodilation was restored after chronic BAY 54–6544 treatment. Senescence markers p16 and p21, and markers of inflammation, including Ccl2, Il6 in aorta and liver, and circulating IL‐6 and TNF‐α were increased in Ercc1 ^∆/− , which was lowered by the treatment. Expression of antioxidant genes, including Cyb5r3 and Nqo1, was favorably changed by chronic BAY 54–6544 treatment. In summary, BAY 54–6544 treatment improved the vascular function and survival rates in mice with accelerated ageing, which may have implication in prolonging health span in progeria and normal ageing.     

Participation of singlet oxygen in ultraviolet-a-induced lipid peroxidation in mouse skin and its inhibition by dietary beta-carotene: an ex vivo study

Dietary beta-carotene acts as a photoprotective agent in the skin, but the exact mechanism of protection is unknown. This ex vivo study is focused on determining the mechanism of action of beta-carotene against UV-A-induced skin damage by characterizing peroxidized phosphatidylcholine (PC) and beta-carotene oxidation products. BALB/c mice were fed with basal or a beta-carotene-supplemented diet, and homogenates from their dorsal skin were prepared after 3 weeks for UV-A irradiation. Analyses revealed that the degree of lipid peroxidation in the beta-carotene group was significantly lower than that in the controls. The isomeric composition of hydroperoxy fatty acids, constituting peroxidized PC, was determined by thin-layer chromatography-blotting followed by gas chromatography/mass spectrometry (MS)/selected ion monitoring analysis. The 9- and 10-isomers of peroxidized PC, resulting from the reaction of singlet molecular oxygen ((1)O(2)) with oleic acid, were elevated in the UV-A-exposed control group compared to the experimental group. Similar results were obtained from methylene-blue-sensitized photooxidation of mouse skin lipids in vitro. Liquid chromatography/MS analysis of the homogenates confirmed the formation of beta-carotene 5,8-endoperoxide, a specific marker for the (1)O(2) reaction. These results indicate that dietary beta-carotene accumulates in the skin and acts as a protective agent against UV-A-induced oxidative damage, by quenching the (1)O(2).     

Respiratory chain analysis of skin fibroblasts in mitochondrial disease

NADH:ubiquinone dehydrogenase (complex I) deficiency can be diagnosed from cultured skin fibroblasts using a number of methods, the most commonly used is a linked assay of rotenone-sensitive complex I + III activity (NADH:cytochrome c reductase). Because of interference from diaphorases, this method requires either the isolation of mitochondria (or at least partial purification). For a suitable mitochondrial preparation from skin fibroblasts, this requires the culturing of 4-20 individual 100mm tissue culture plates, depending on the purity of preparation required. These assays are therefore time-consuming, and do not assist in a rapid diagnosis. There is also no clear demarkation between the normal range of activity and the deficient range since mild mutations can produce only partial decreases in complex I activity. Equally, assaying patient cells that do not have a specific deficiency may prove to be time-wasting in the process of providing a quick, definitive clinical diagnosis. The lactate/pyruvate ratio of fibroblasts has been used to indicate the extent of respiratory chain involvement, as cells with a metabolic defect usually produce more lactate with an increased ratio from 25:1 to much higher values [Methods Enzymol. 264 (1996) 454]. This measurement may not always be conclusive, as the values can fluctuate as a result of culture medium, cell passage number, cell number and viability. In this report, we evaluate the use of pyruvate oxidation measurements from whole cells prepared from a single plate of cultured fibroblasts as an alternative to lactate/pyruvate ratios, or other methods both direct and indirect as indicators of the extent of respiratory chain involvement and the possibility of a defect within complex I. Whole cell 2-14C pyruvate oxidation appears to indicate the presence of a complex I defect in patients compared to normal controls more reliably than L/P ratios, but this has some puzzling exceptions.     

Genotoxic effects of butadiene in mouse lung cells detected by an ex vivo micronucleus test

Lung fibroblasts from BD-exposed mice have been analysed for the occurrence of micronuclei. Primary cultures set up 24h after the end of exposure were treated with cytochalasin B and micronuclei scored in binucleate cells. A three-fold statistically significant increase of micronucleated cells was detected after exposure to 500ppm, the lowest tested concentration. A linear dose effect relationship was observed between 500 and 1300ppm. Immunofluorescent staining of kinetochore proteins was applied to distinguish between acentric micronuclei produced by chromosome breaks and micronuclei containing a centromeric region, most likely induced by chromosome loss. A statistically significant increase of both types of MN in 1300ppm-exposed females and a significant increase in centromeric MN in 500ppm-exposed males were detected. These data demonstrate that an intermediate of BD metabolism with a potential for clastogenic and aneugenic effects is active in lung cells after inhalation exposure. These effects can play a role in the initiation and promotion of BD-induced lung tumours.     

A comparative study of ex vivo skin optical clearing using two‐photon microscopy

Multiphoton tomography (MPT) is a prospective tool for imaging the skin structure. Aiming to increase the probing depth, a comparative ex vivo study of optical clearing of porcine ear skin was performed by using two optical clearing agents (OCAs), i.e., glycerol and iohexol (Omnipaque^TM) at different concentrations, which exhibit different osmotic properties. The results show that a topical application of glycerol or Omnipaque^TM solutions onto the skin for 60 min significantly improved the depth and contrast of the MPT signals. By utilizing 40%, 60% and 100% glycerol, and 60% and 100% Omnipaque^TM it was demonstrated that both agents improve autofluorescence and SHG (second harmonic generation) signals from the skin. At the applied concentrations and agent time exposure, glycerol is more effective than Omnipaque^TM. However, tissue shrinkage and cell morphology changes were found for highly concentrated glycerol solutions. Omnipaque^TM, on the contrary, increases the safety and has no or minimal tissue shrinkage during the optical clearing process. Moreover Omnipaque^TM allows for robust multimodal optical/X‐ray imaging with automatically matched optically cleared and X‐ray contrasted tissue volumes. These findings make Omnipaque^TM more prospective than glycerol for some particular application.

     

Absence of metabolic rate allometry in an ex vivo model of mammalian skeletal muscle

Within mammalian species, standard metabolic rate (SMR) increases disproportionately with body mass (Mb), such that the mass-specific SMR correlates negatively with Mb. This phenomenon can be explained in part by reduced cellular metabolic rates in larger species. To better understand the cause(s) of this cellular metabolic rate allometry we have used an ex vivo approach to isolate and identify potential contributors. Skeletal myoblasts from mammalian species ranging inMb from 30 g to over 300,000 g were isolated and differentiated into myotubes in vitro. Oxygen consumption rates, citrate synthase (CS) activity, and lactate dehydrogenase (LDH) activity were measured in myotubes under standardized conditions. No correlation of any of these parameters was observedwith speciesMb, suggesting that there is no genetic contribution to between-species differences in cellular metabolic rates. Myotubes were incubated in serum from species ranging from 30 g to 400,000 g to determine whether between-species differences in the levels of metabolically important hormones might produce allometric trends in the cultured cells. However, there was no observed effect of serum donor Mb on any of the metabolic characteristicsmeasured. Thus, there is no evidence for a relationship between skeletal muscle oxidative metabolism and Mb in an ex vivo model.     

Endogenous mitochondrial oxidative stress in MnSOD-deficient mouse embryonic fibroblasts promotes mitochondrial DNA glycation

The accumulation of somatic mutations in mitochondrial DNA (mtDNA) induced by reactive oxygen species (ROS) is regarded as a major contributor to aging and age-related degenerative diseases. ROS have also been shown to facilitate the formation of certain advanced glycation end-products (AGEs) in proteins and DNA and N(2)-carboxyethyl-2'-deoxyguanosine (CEdG) has been identified as a major DNA-bound AGE. Therefore, the influence of mitochondrial ROS on the glycation of mtDNA was investigated in primary embryonic fibroblasts derived from mutant mice (Sod2(-/+)) deficient in the mitochondrial antioxidant enzyme manganese superoxide dismutase. In Sod2(-/+) fibroblasts vs wild-type fibroblasts, the CEdG content of mtDNA was increased from 1.90 ± 1.39 to 17.14 ± 6.60 pg/μg DNA (p<0.001). On the other hand, the CEdG content of nuclear DNA did not differ between Sod2(+/+) and Sod2(-/+) cells. Similarly, cytosolic proteins did not show any difference in advanced glycation end-products or protein carbonyl contents between Sod2(+/+) and Sod2(-/+). Taken together, the data suggest that mitochondrial oxidative stress specifically promotes glycation of mtDNA and does not affect nuclear DNA or cytosolic proteins. Because DNA glycation can change DNA integrity and gene functions, glycation of mtDNA may play an important role in the decline of mitochondrial functions.     

3‐photon fluorescence imaging of sulforhodamine B‐labeled elastic fibers in the mouse skin in vivo

Elastic fibers are key constituents of the skin. The commonly adopted optical technique for visualizing elastic fibers in the animal skin in vivo is 2‐photon microscopy (2 PM) of autofluorescence, which typically suffers from low signal level. Here we demonstrate a new optical methodology to image elastic fibers in animal models in vivo: 3‐photon microscopy (3 PM) excited at the 1700‐nm window combining with preferential labeling of elastic fibers using sulforhodamine B (SRB). First, we demonstrate that intravenous injection of SRB can circumvent the skin barrier (encountered in topical application) and preferentially label elastic fibers, as verified by simultaneous 2 PM of both autofluorescence and SRB fluorescence from skin structures. Then through 3‐photon excitation property characterization, we show that 3‐photon fluorescence can be excited from SRB at the 1700‐nm window, and 1600‐nm excitation is most efficient according to our 3‐photon action cross section measurement. Based on these results and using our developed 1600‐nm femtosecond laser source, we finally demonstrate 3 PM of SRB‐labeled elastic fibers through the whole dermis in the mouse skin in vivo, with only 3.7‐mW optical power deposited on the skin surface. We expect our methodology will provide novel optical solution to elastic fiber research.