Development and initial characterization of xenomitochondrial mice.

Xenomitochondrial mice harboring trans-species mitochondria on a Mus musculus domesticus (MD) nuclear background were produced. We created xenomitochondrial ES cell cybrids by fusing Mus spretus (MS), Mus caroli (MC), Mus dunni (Mdu), or Mus pahari (MP) mitochondrial donor cytoplasts and rhodamine 6-G treated CC9.3.1 or PC4 ES cells. The selected donor backgrounds reflected increasing evolutionary divergence from MD mice and the resultant mitochondrial-nuclear mismatch targeted a graded respiratory chain defect. Homoplasmic (MS, MC, Mdu, and MP) and heteroplasmic (MC) cell lines were injected into MD ova, and liveborn chimeric mice were obtained (MS/MD 18 of 87, MC/MD 6 of 46, Mdu/MD 31 of 140, and MP/MD l of 9 founder chimeras, respectively). Seven MS/MD, 1 MC/MD, and 11 Mdu/MD chimeric founder females were mated with wild-type MD males, and 18 of 19 (95%) were fertile. Of fertile females, only one chimeric MS/MD (1% coat color chimerism) and four chimeric Mdu/MD females (80-90% coat color chimerism) produced homoplasmic offspring with low efficiency (7 of 135; 5%). Four male and three female offspring were homoplasmic for the introduced mitochondrial backgrounds. Three male and one female offspring proved viable. Generation of mouse lines using additional female ES cell lineages is underway. We hypothesize that these mice, when crossbred with neurodegenerative-disease mouse models, will show accelerated age-related neuronal loss, because of their suboptimal capacity for oxidative phosphorylation and putatively increased oxidative stress.

The tensor fascia lata free flap in abdominal-wall reconstruction.

The pedicled tensor fascia lata flap (TFL flap) is a method of choice for abdominal-wall reconstruction. Frequently, the size and location of the defect produce this option. Microsurgical transfer may overcome these disadvantages. Therefore, the ability of the TFL free flap to reconstruct complex abdominal wounds was evaluated. Seven patients with full-thickness abdominal-wall defects reconstructed by TFL free flaps were reviewed. Their average age was 44.6 years (range: 27 years to 59 years); follow-up averaged 10.5 months (range: 2 months to 18 months). Fifty-seven percent of the wounds were either infected or contaminated; the defect averaged 15 cm x 26 cm Six 85.7 percent) of the wounds involved the epigastrum. No total flap loss was seen, but three flaps developed distal tip necrosis (42.9%). Microsurgical transfer of the TFL free flap overcomes the limitations of the arc of rotation seen with the pedicled flap. It increases the vascularity of the distal portion of the flap. The TFL free flap is therefore an option in abdominal wounds, particularly those with defects of large size or supraumbilical location.

Planned ventral hernia. Staged management for acute abdominal wall defects.

OBJECTIVE: Analysis of a staged management scheme for initial and definitive management of acute abdominal wall defects is provided. METHODS: A four-staged scheme for managing acute abdominal wall defects consists of the following stages: stage I--prosthetic insertion; stage II--2 to 3 weeks after prosthetic insertion and wound granulation, the prosthesis is removed; stage III--2 to 3 days later, planned ventral hernia (split thickness skin graft [STSG] or full-thickness skin and subcutaneous fat); stage IV--6 to 12 months later, definitive reconstruction. Cases were evaluated retrospectively for benefits and risks of the techniques employed. RESULTS: Eighty-eight cases (39 visceral edema, 27 abdominal sepsis, 22 abdominal wall resection) were managed during 8.5 years. Prostheses included polypropylene mesh in 45 cases, polyglactin 910 mesh in 27, polytetrafluorethylene in 10, and plastic in 6. Twenty-four patients died from their initial disease. The fistula rates associated with prosthetic management was 9%; no wound-related mortality occurred. Most wounds had split thickness skin graft applied after prosthetic removal. Definitive reconstruction was undertaken in 21 patients in the authors' institution (prosthetic mesh in 12 and modified components separation in 9). Recurrent hernias developed in 33% of mesh reconstructions and 11% of the components separation technique. CONCLUSIONS: The authors concluded that 1) this staged approach was associated with low morbidity and no technique-related mortality; 2) prostheses placed for edema were removed with fascial approximation accomplished in half of those cases; 3) absorbable mesh provided the advantages of reasonable durability, ease of removal, and relatively low cost--it has become the prosthesis of choice; and 4) the modified components separation technique of reconstruction provided good results in patients with moderate sized defects.

Hypoxic stress induces reversible hypophosphorylation of pRB and reduction in cyclin A abundance independent of cell cycle progression.

The effect of hypoxic stress on the phosphorylation state of the product of retinoblastoma susceptibility gene (pRB) and cyclin A abundance was examined in CV-1P monkey kidney cells. Flow cytometric DNA histogram analysis and [3H]-thymidine incorporation assays demonstrated that hypoxia inhibited cell cycle progression and cell division. Within 6-12 h of hypoxia, pRB became hypophosphorylated and cyclin A abundance fell below detection limits. Hypophosphorylation of pRB and loss of cyclin A detection occurred without progression of cells through S-phase. These effects were found to be reversible by reoxygenation of the hypoxic cultures. Cells were shown to resume DNA synthesis within 12-16 h of reoxygenation concomitant with pRB hyperphosphorylation and an increase in cyclin A detection. These data demonstrate that hypoxic stress blocks the progression of these cells through the phases of the cell cycle and suggests that the effect might arise from the down regulation of key cell cycle controlling elements. The data also raise the possibility that maintaining pRB in a hyperphosphorylated state may be crucial for S-phase progression as well as S-phase entry.

Induction of the proliferative phenotype in differentiated myogenic cells by hypoxia.

The effect of cellular differentiation on the response of cells to hypoxic stress has been evaluated using the myogenic cell line BC3H1. Aerobic myocytes were predominantly in G0/G1 of the cell cycle and could be induced into S and G2/M of the cell cycle only by replating in high serum-containing medium at subconfluent cell density. In contrast, hypoxic myocytes demonstrated marked progression into S and G2/M upon reoxygenation without replating in the presence of serum. This modulation of myocytes by hypoxia was suggested further by the induction of 100-kDa and 9-kDa proteins (PSP 100 and PSP 9) which were otherwise only detectable in myoblasts. Two-dimensional gel analysis of newly synthesized proteins demonstrated that the five major glucose/oxygen-regulated proteins (GRP/ORP 260, 150, 100, 80, and 33) were induced in hypoxic myogenic cells independent of their state of differentiation. In addition to the GRP/ORPs, synthesis of 20 and 23 other major proteins was influenced in myocytes and myoblasts, respectively. The bulk of these alterations in myoblasts (70%) were inhibitions. In contrast, 75% of the alterations in myocyte protein synthesis were either enhancements or inductions. The data show that hypoxia can modulate the myocyte phenotype and invoke proliferative characteristics. Moreover, the data suggest that ischemia will have a different effect on and prognosis for tissues with a high mitotic index compared with differentiated tissues.

31P NMR spectroscopy in vivo of two murine tumor lines with widely different fractions of radiobiologically hypoxic cells.

Energy and lipid metabolism as well as tumor pH in two murine tumor lines, the KHT and RIF-1 sarcomas, were studied using 31P NMR spectroscopy. Possible relationships between spectral parameters on the one hand and volume fraction of necrosis and fraction of radiobiologically hypoxic cells on the other were investigated. For both tumor lines the PCr and NTP beta resonances decreased and the Pi resonance increased significantly with increasing tumor volume in the volume range 100-4000 mm3. This decrease in bioenergetic status was accompanied by a decrease in tumor pH from about 7.2 to about 6.8. The NTP beta resonance and the tumor pH tended to be somewhat higher and the Pi resonance somewhat lower for the KHT than for the RIF-1 tumors. Linear relationships were found between tumor pH and Pi or (PCr + NTP beta)/Pi for both tumor lines (P much less than 0.05). The PME resonance increased slightly and the PDE resonance decreased slightly during tumor growth and were not significantly different for the KHT and the RIF-1 tumors. The volume fraction of necrosis was about 5 per cent in both lines at a tumor volume of 100 mm3 and increased to about 30 per cent (KHT) and 50 per cent (RIF-1) at a tumor volume of 4000 mm3. The fraction of radiobiologically hypoxic cells was found to increase from 12 to 23 per cent for the KHT line and from 0.9 to 1.7 per cent for the RIF-1 line when tumor volume was increased from about 200 to about 2000 mm3. The volume-dependence of the 31P NMR spectral parameters indicated increased nutritional deprivation and development of hypoxia and necrosis during tumor growth, and was thus qualitatively in good agreement with the changes observed in necrotic and hypoxic fraction. However, quantitative relationships between any spectral parameter and necrotic or hypoxic fraction across tumor lines were not found, implying that other physiological parameters and/or cellular characteristics may contribute significantly to a 31P NMR tumor spectrum. Consequently, 31P NMR spectra of untreated tumors have to be supplemented with other tumor data, e.g. rate of oxygen consumption, cell survival time under hypoxic stress and/or fraction of metabolically active, non-clonogenic hypoxic cells, to be useful in quantitative determination of tumor hypoxia and hence prediction of tumor radioresistance caused by hypoxia.

Misonidazole: inter-related factors affecting cytotoxicity.

A detailed investigation was undertaken of reported modifiers of the toxicity towards hypoxic cells of misonidazole. The modifiers tested were medium type, cell type, cell density, concentration of misonidazole, addition of serum, addition of sulfhydryl, addition of oxygen and pH. The latter 5 modifiers were found to be most important and were studied in many of the possible combinations. Serum has its greatest protective effect at low concentration (e.g. 0.5 mM) of misonidazole. In the absence of serum, the (log10) survival curve for misonidazole toxicity can be described mathematically as a function of time by a shoulder (DQ) inversely related to misonidazole concentration, and a slope (1/D0) related directly to log10 misonidazole concentration. Sulfhydryl's (cysteamine) protective effect dominates at high concentrations of misonidazole. The protective action of SH can change to potentiative in the absence of serum or at high pH. Addition of oxygen results in overall protection but no relative changes in the effect of the other modifiers. Drugs like ascorbate and disulfide may only potentiate toxicity to the level found in the absence of serum.

Ascorbate anion potentiates cytotoxicity of nitro-aromatic compounds under hypoxic and anoxic conditions.

The nitro-aromatic radiosensitizing drugs are selectively toxic to hypoxic mammalian cells, and this toxicity can be greatly increased by the addition of ascorbate. The ascorbate itself is not toxic to either hypoxic or aerobic cells (as long as catalase is present to prevent the formation of significant concentrations of hydrogen peroxide) and the mixture of ascorbate plus radiosensitizer is not more toxic to aerobic cells. Sulphydryl reducing agents and dithionite have an effect opposite to ascorbate and decrease the toxicity of nitro-aromatic drugs under hypoxic conditions. Sulphydryl reducing agents are also reported to nullify the radiosensitizing properties of nitro-aromatic drugs, in contrast to ascorbate which has no effect on the radiosensitizing properties. The toxicity of nitro-aromatic drugs decreases rapidly with increasing O2 concentration. This decrease is much less rapid when ascorbate is present. The role of ascorbate in this case may be primarily as an O2 scavenger, although it is also possible that the toxic species produced by radiosensitizer-ascorbate mixtures is less easily removed or detoxified by O2.

Gentisuric acid: metabolic formation in animals and identification as a metabolite of aspirin in man.

Gentisuric acid was synthesized from gentisic acid and glycine ethyl ester. NMR, mass spectrometric and elemental analysis confirmed the product as GU, and physicochemical characteristics were determined. A TLC-densitometric technique was developed to estimate GU and other metabolites of aspirin. Conjugation of gentisic acid with glycine to form GU was catalyzed by a mitochondrial fraction of rat and beef liver. GU was also formed by the rat liver microsomal hydroxylation of salicyluric acid, and phenobarbital pretreatment increased this formation. A random survey showed GU in 76% of SA-positive urines from aspirin-treated patients. Identity of GU in urine from two aspirin-treated patients was confirmed by TLC and mass spectrometric analysis, and hydrolysis of the compound from one patient yielded glycine and gentisic acid. Urine from controls or post-aspirin treatment patients did not show GU by TLC analysis. These results demonstrate for the first time the metabolic formation of GU in animals and its occurrence as a metabolite of aspirin in man.

The multicellular spheroid as a model tumor allograft. II. Characterization of spheroid-infiltrating cytotoxic cells.

Alloimmune lymphoid cells infiltrating multicellular spheroids of EMT6 mammary sarcoma cells (a solid tumor allograft model) have been characterized according to their morphological and functional properties. Both lymphocytes and macrophages were found within spheroids at the time of peak tumor cell damage. Cytotoxic cells specific for allograft antigens were also present. Using a short-term 51-Cr release assay, the cells responsible for cytotoxicity were characterized as a nonadherent, nonphagocytic T cell population. Velocity sedimentation cell separation further demonstrated that these cytotoxic cells had the physical properties of small lymphocytes. Some evidence for selective spheroid infiltration by specifically alloimmune cells was also obtained. The possible relationship of this cellular infiltrate to graft damage is discussed.

The multicellular spheroid as a model tumor allograft. I. Quantitative assessment of spheroid destruction in alloimmune mice.

A quantitative model for the assessment of in situ immunity to solid tumor allografts has been developed. Multicellular spheroids of murine EMT6 mammary sarcoma cells were implanted in the peritoneal cavity of normal or specifically alloimmune mice. Damage to spheroids was quantitatively assessed at various times by trypsinizing the recovered spheroids and assaying for surviving EMT6 cells by a cloning technique. In alloimmune mice, significant destruction of spheroids was observed within 24 hr of implantation, and a 99% reduction in the number of clonogenic MT6 cells in spheroids was consistently found after 48 hr. In contrast, little or no cytotoxic effect was observed when spheroids were implanted for 48 hr in nonimmune mice or in mice immunized against unrelated alloantigens. Implantation of spheroids in alloimmune athymic (nu/nu) mice did not result in appreciable spheroid damage as compared with littermate controls. Histological analysis of spheroids taken from alloimmune mice at the time of maximum tumor cell destruction indicated that large numbers of mononuclear cells had infiltrated the spheroid. These results suggests that multicellular spheroids will be a useful model for quantitative studies of the cellular mechanisms responsible for tissue-damaging reactions in vivo.

Effect of weak magnetic fields on growth of cells in tissue culture.

Normal Chinese hamster V79 cells were grown in vitro for more than one year in a magnetic field of 10(-7) tesla to test recently hypothesized effects of weak magnetic fields on biological systems. No significant difference was observed between growth rate in such fields and that in the ambient geomagnetic field.

Multicellular spheroids: a new model target for in vitro studies of immunity to solid tumor allografts.

Multicellular spheroids of EMT6 mammary sarcoma cells of BALB/c origin were incubated with normal spleen cells or alloimmune spleen cells generated in vitro in mixed leukocyte cultures (MLC). After 24 hours, spheroids were trypsinized and assayed for surviving tumor cells by use of a cloning technique. Under these conditions a 60-80% reduction in clone-forming tumor cells was observed after incubation of spheroids with immune lymphocytes as compared to normal lymphocyte controls. This cytotoxic effect occurred in situ, and alloimmune cells sensitized against unrelated antigens were much less cytotoxic than were specifically sensitized cells. In parallel autoradiographic studies, some immune lymphoid cells that had been labeled with tritiated thymidine during the proliferative phase of the MLC could be demonstrated within spheroids after 24 hours. These results suggested that multicellular spheroids will be a useful in vitro model for more detailed analysis of the factors controlling infiltration in in situ destruction of solid tumor grafts.