Computational model of dose response for low-LET-induced complex chromosomal aberrations.

Experiments with full-colour mFISH chromosome painting have revealed high yield of radiation-induced complex chromosomal aberrations (CAs). The ratio of complex to simple aberrations is dependent on cell type and linear energy transfer. Theoretical analysis has demonstrated that the mechanism of CA formation as a result of interaction between lesions at a surface of chromosome territories does not explain high complexes-to-simples ratio in human lymphocytes. The possible origin of high yields of γ-induced complex CAs was investigated in the present work by computer simulation. CAs were studied on the basis of chromosome structure and dynamics modelling and the hypothesis of CA formation on nuclear centres. The spatial organisation of all chromosomes in a human interphase nucleus was predicted by simulation of mitosis-to-interphase chromosome structure transition. Two scenarios of CA formation were analysed, 'static' (existing in a nucleus prior to irradiation) centres and 'dynamic' (formed in response to irradiation) centres. The modelling results reveal that under certain conditions, both scenarios explain quantitatively the dose-response relationships for both simple and complex γ-induced interchromosomal exchanges observed by mFISH chromosome painting in the first post-irradiation mitosis in human lymphocytes.

Complex interchanges as a complex function of chromosome organisation.

A Monte Carlo technique was developed for biophysical modelling of structural organisation of 46 chromosomes within human lymphocyte interphase nucleus. The technique takes into account: different levels of chromatin organisation, non-random localisation of particular chromosomes and chromosome loci dynamics. All chromosomes in a nucleus were modelled as polymer globules. A dynamic pattern of intra/interchromosomal contacts was simulated to predict radiation-induced chromosomal exchange aberrations (CA). Distance dependence of interaction probability was calculated directly with taking DNA break repair into account. Dose-response for simple and complex CA was calculated to analyse mFISH data for human lymphocytes. Calculated simple CA frequencies fitted the experimental data well. Unexpectedly, complex aberrations were underestimated, despite the dense packaging of chromosome territories within a nucleus. To study sensitivity of dose-response to uncertainty of chromosome organisation, CA were recalculated for the alternative concept of nucleus organisation, the SCD model. The simulation showed the underestimation of complex CA yield as well. The movement of damaged loci from different chromosomes to common repair factories was proposed as an additional mechanism of complex CA formation.

Dose-response prediction for radiation-induced chromosomal instability.

Radiation induces chromosome aberrations (CA) that are detected in the first post-irradiation cell cycle and in descendants of irradiated cells. Unstable aberrations in the progeny of exposed cells are referred to as one of the hallmarks of chromosomal instability (CIN). One of the important questions is what is the relationship between the dose response for radiation-induced CA and delayed CA, or CIN. To address this question, a mechanistic model for CIN was developed. Delayed CA are assumed to be formed both by transmission from previous mitotic cycles owing to chromosome breakage-fusion mechanism and by means of generation of DNA/chromosome breakage de novo in each cell cycle of survived cells. Monte Carlo simulation of DNA/chromosome breakage, CA production, cell death due to unstable CA and cell cycle kinetics was performed to predict the dose response for CIN. Different shapes of CIN dose-response curves were predicted for various time points after irradiation and under several assumptions on delayed DNA/chromosome breakage generation. For one of the scenarios studied, the pronounced dose dependence at early time points flattened or even turned into dose independence in a wide dose range after many rounds of replication where a stationary state between CA generation and elimination was achieved. This dose independence was shown to be in concert with the experimental data.

Mechanistic modelling of genetic and epigenetic events in radiation carcinogenesis.

Methodological problems arise on the way of radiation carcinogenesis modelling with the incorporation of radiobiological and cancer biology mechanistic data. The results of biophysical modelling of different endpoints [DNA DSB induction, repair, chromosome aberrations (CA) and cell proliferation] are presented and applied to the analysis of RBE-LET relationships for radiation-induced neoplastic transformation (RINT) of C3H/10T1/2 cells in culture. Predicted values for some endpoints correlate well with the data. It is concluded that slowly repaired DSB clusters, as well as some kind of CA, may be initiating events for RINT. As an alternative interpretation, it is possible that DNA damage can induce RINT indirectly via epigenetic process. A hypothetical epigenetic pathway for RINT is discussed.

Exponential gain and saturation of a self-amplified spontaneous emission free-electron laser.

Self-amplified spontaneous emission in a free-electron laser has been proposed for the generation of very high brightness coherent x-rays. This process involves passing a high-energy, high-charge, short-pulse, low-energy-spread, and low-emittance electron beam through the periodic magnetic field of a long series of high-quality undulator magnets. The radiation produced grows exponentially in intensity until it reaches a saturation point. We report on the demonstration of self-amplified spontaneous emission gain, exponential growth, and saturation at visible (530 nanometers) and ultraviolet (385 nanometers) wavelengths. Good agreement between theory and simulation indicates that scaling to much shorter wavelengths may be possible. These results confirm the physics behind the self-amplified spontaneous emission process and forward the development of an operational x-ray free-electron laser.

Mammography in the evaluation of masses in breasts reconstructed with TRAM flaps.

Patients with transverse rectus abdominis musculocutaneous (TRAM) flaps may develop physical findings, such as palpable masses, irregularities, and areas of increased tenderness that are suggestive of fat necrosis or recurrent malignancy. The purpose of this study was to evaluate these findings with mammography in an attempt to rule out recurrent malignancy in the autogenously reconstructed breast. Fifteen patients on whom mammography was performed as an aid in the evaluation of suspicious post-TRAM flap findings were reviewed. Common mammographic findings included calcifications believed to demonstrate fat necrosis, benign dermal calcifications, calcified hematoma, and clustered microcalcifications. Areas of increased or decreased density without calcifications were also identified and appeared to be related to surgical changes and fat necrosis. Twenty percent of patients had clustered microcalcifications, and 20% had detectable masses on mammography. One patient had a suspicious mass associated with clustered microcalcifications, leading to a biopsy that revealed fat necrosis. The majority of findings were consistent with normal fat within the TRAM flaps. This study supports mammography as a useful diagnostic tool in patients who have undergone TRAM flap breast reconstruction and who present postoperatively with suspicious physical findings.

Balloon assisted endoscopic harvest of the latissimus dorsi muscle.

In this study, we present our experience with balloon assisted endoscopic harvest of the latissimus dorsi muscle for extremity reconstruction. The balloon performs most of the dissection under the muscle and creates the optical work space used in the endoscopic dissection. Over the course of this series the operative time has been reduced and averaged 2 hours and 44 minutes. The reconstructive goals were met in all cases. The average axillary incision length was 5.6 cm, and there were an average of 1.3 one-centimeter or smaller counter incisions.

Salvage of traumatic below-knee amputation stumps utilizing the filet of foot free flap: critical evaluation of six cases.

Over a 12-year period between 1979 and 1991, 27 patients were operated on at the New York University Medical Center for salvage of below-knee amputation stumps utilizing free flaps. Six different donor sites were used. In 6 patients, the amputated foot was the donor site for a free flap to cover the tibial stump. There were 3 males and 3 females in this group. Five of the patients underwent immediate filet of foot reconstructions, while 1 patient had a reconstruction performed 69 days after injury, electively, when it was determined that below-knee amputation was the best option. All foot flaps survived and ultimately provided the major soft-tissue coverage for the below-knee amputation stump. The length of hospitalization ranged from 24 to 118 days. The time required from foot filet procedure to ambulation was 2, 4, 6, 7, 9, and 12 months in the 6 patients. Five of the 6 patients have resumed work or school after their injury. Foot flaps were based on the posterior tibial artery, anterior tibial artery, or both vessels. Nerve anastomosis of the posterior tibial nerve was performed in 5 patients. In 1 patient it was possible to maintain the continuity of the posterior tibial nerve. Five of the 6 patients were tested over a year after the flap, and all have good cold, pressure, and vibration sensation. Two of the 5 patients have heat sensation, and all 5 patients have at least protective pressure sensation. All the patients ambulate well with a below-knee prosthesis.

Limb salvage with microvascular free flap reconstruction using simultaneous polytetrafluoroethylene graft for inflow.

Microvascular free flaps have been successfully used to cover defects of the lower extremity. In patients with peripheral vascular disease and lower extremity defects, revascularization with in situ or reversed saphenous vein bypass graft combined with microvascular tissue transfer can salvage a limb that would otherwise be amputated. However, some of these patients may not have autologous vein available for the bypass procedure. We present a case of a 69-year-old man who underwent revascularization with a long polytetrafluoroethylene (PTFE) graft and a simultaneous microvascular free flap reconstruction using the PTFE graft as the inflow. The patient had undergone coronary artery bypass graft with saphenous vein and experienced a nonhealing wound of the distal saphenous vein harvest site and exposure of 8 cm of tibia. Angiogram revealed a significant stenosis of the common iliac artery, occluded superficial femoral artery, faint filling of the profunda femoris artery, and a faintly reconstituted posterior tibial artery. Because the patient had no available saphenous vein for bypass, he underwent an axillary-profunda and profunda-posterior tibial artery bypass with PTFE. A rectus abdominus microvascular free flap with direct anastomosis of the inferior epigastric artery to the PTFE was used to cover the exposed bone. The patient currently ambulates without difficulty. Limb salvage using bypass with PTFE combined with simultaneous microvascular free flap reconstruction is possible in selected patients.

The in vivo effect of hyaluronan associated protein-collagen complex on wound repair.

Fetal skin wounds heal without scarring, however the underlying mechanisms remain unknown. Immunohistochemical staining and biochemical studies indicate the deposition of a collagen repair matrix that is highly organized. We have previously described a unique hyaluronan associated protein-collagen complex (HA-PC) profile present during the period of scarless healing in the sheep fetus. In this study, we examined the biologic activity of this HA-PC in an in vivo model of adult rat wound repair. A total of 84 incisional and 84 excisional wounds were examined by histology, TGF-beta immunocytochemistry and computer planimetry (excisional wounds only). Planimetry of the excisional wounds demonstrated the mean wound area remaining at day 1 was 88.7% for the control and 63.6% for the treated (p<0.01). At day 2, mean wound area was 81.5% for the control and 63.6% for the treated (p<0.01). At day 4, mean wound area was 56.6% for the control and 41.9% for the treated (p<0.01). At day 7, mean wound area was 26.9% for the control and 16.8% for the treated (p<0.01). At day 14, mean wound area was 7.9% for the control and 3.4% for the treated (p<0.05). Collagen organization was judged to be greater in the treated compared to control wounds, with a mean organization score of 2.3 vs. 1.9 (p=0.0596; Wilcoxon Signed Rank Sum Test). There were significantly more neutrophils at the wound margin of the treated compared to control wounds, 4.0 vs. 2.7 (p=0.038; Paired Two Tailed Student's t-Test). There was no difference in the number of microphages at the wound margin of the treated compared to control wounds, 6.15 vs. 6.0 (p>0.05). TGFbeta1 and beta2 staining was decreased whereas TGFbeta3 staining was increased in the HA-PC treated wounds. These results suggest that compared to control wounds HA-PC treated wounds heal more quickly, with more organized collagen, more neutrophils at the wound margin and increased TGFbeta3 expression. Furthermore, these data suggest that the manipulation of scarring in adult wounds is possible by the addition of proteins present in fetal skin.

Microvascular free-flap salvage of the diabetic foot: a 5-year experience.

This study reviews 21 microvascular free flaps to the diabetic foot in 19 patients over a 65-month period. All flaps were either to the plantar surface of the foot or to cover exposed Achilles tendon. Twenty of the flaps survived. The operations required a long, costly hospitalization with frequent recipient- and donor-site complications. All patients eventually ambulated on their flaps. Five patients came to proximal amputation from 6 to 37 months after surgery. Only one amputation was for flap breakdown.

The effects of topical hypothermia and steroids on ATP levels in an in vivo liver ischemia model.

Complex hepatic surgery often requires occlusion of the portal triad in order to decrease parenchymal bleeding. This study was undertaken to evaluate the effects of topical hypothermia and intravenous steroids on liver ischemia by measuring adenosine triphosphate (ATP) levels within the hepatic parenchyma. Forty New Zealand white rabbits were divided into four experimental and four control groups. All experimental animals underwent laparotomy and ligation of the porta hepatis. Serial liver biopsy specimens were obtained at predetermined time intervals. Group I received no further intervention. Group II were topically cooled until intrahepatic temperature reached 30 degrees C. Group III received preligation intravenous methylprednisolone (30 mg/kg). Group IV received both steroids and topical hypothermia. The corresponding control groups underwent laparotomy and isolation of the porta without ligation. Adenosine triphosphate was extracted from the liver parenchyma and quantified by high-performance liquid chromatography (HPLC). The data were analyzed using a three-factor mixed analysis of variance (ANOVA). There was a statistically significant protective effect on ATP levels provided by topical hypothermia at 15 and 30 minutes of ischemia (p < 0.01), but not at 60 minutes (p > 0.05). Steroids were not found to have any protective effect on ATP levels at any time point. The combination of steroids and topical hypothermia provided significant preservation of hepatic parenchymal ATP levels, although less than that of hypothermia alone, at 15 and 30 minutes of ischemia (p < 0.01).

Serial quantitation of hyaluronan and sulfated glycosaminoglycans in fetal sheep skin.

Fetal wounds are abundant in hyaluronic acid (HA), but little is known as to the total HA content of fetal tissues as a function of gestational age. Previous studies demonstrated scarless healing prior to approximately 130 days gestation, after which disorganized collagen deposition became prevalent. Glycosaminoglycans (GAGs) have been shown to play an important role in wound healing, cytodifferentiation, and morphogenesis. In this study, we examined the HA and GAG content of fetal sheep skin of increasing gestational age. We found that the total amount of HA and GAGs declined from a high at 80 days gestation (528 +/- 9 micrograms/gm) to a low at 130 days (174 +/- 11). Analysis of the various GAG species revealed that HA comprised the largest fraction (75-96%). The sulfated GAGs, Heparan Sulfate (HS) and Dermatan Sulfate (DS), were not present in the extracellular matrix until 120 days gestation. Both the trough of HA content and the appearance of sulfated GAGs in the extracellular fraction correspond to the appearance of scarring in fetal sheep wound repair.

Isolation and partial characterization of hyaluronan-protein-collagen complex (HA-PC) from fetal sheep skin of different gestational ages.

Hyaluronic acid (HA) has a positive effect on cell migration, differentiation and wound healing. Earlier work from our laboratory has shown the presence of biologically active proteins associated with HA. The protein associated with HA of fetal sheep skin varies in molecular weight depending on its gestational age. Specifically, the protein profile changes at 125 days of gestation, from a 60 KDa protein to a smaller protein of about 21 KDa. This time period coincides with the time that scarring becomes apparent in fetal sheep skin wounds. In this study, we have quantified changes in the proteins associated with HA with increasing gestational age, obtained amino acid profiles of these proteins with increasing gestational age, and proposed the existence of an HA-associated protein-collagen complex (HA-PC) which may serve as a scaffold for wound healing. Our results indicate that HA-PC content decreases from 42% of the dry weight at 75 days of gestation to 22% at 125 days of gestation. Protein content, in contrast, increases to 40% of the dry weight at 140 days of gestation. At the same time, collagen content increases from < 1% of the dry weight at 75 days to > 10% at 140 days. The increase in collagen content may account for the increase in total protein seen at 140 days. The expression of varying HA-PC's at different gestational ages may influence the kinetics of collagen fibrillogenesis and thus account for the previously noted late gestational change from "scarless" wound healing to "adult-like" wound healing in fetal sheep.