Objective evaluation of the performance of surgical trainees on a porcine model of open colectomy.

BACKGROUND: Evaluation of surgical trainee operative performance is rarely objective. A rating system is proposed that assesses trainee performance objectively and quantifies technical improvement. METHODS: General surgery trainees were evaluated while performing porcine segmental colectomy. Initial instruction was provided for the critical operative steps. Evaluations were later repeated without additional instruction. Performance in 17 critical areas was scored. RESULTS: Twenty-three trainees were evaluated. Performance was divided into thirds, with a significant difference detected between tertiles (P < 0.001). Postgraduate year 2 trainees scored lower than those in years 3 and 4 (P < 0.001), but there was no difference between year 3 and 4 trainees (P = 0.557). Mean repeat scores were improved by 35 per cent, with most improvement at postgraduate year 2 level (71 per cent). Mean time taken to complete the operation was reduced by 23 per cent, with the largest reduction in the year 2 group. CONCLUSION: The results support the use of this rating system as a tool for the objective evaluation of trainee operative skill. Instruction in the performance of segmental colectomy using deconstructed, step-by-step direction improved the ability of junior trainees to complete the operation. This evaluation system may be useful in the assessment, instruction and technical development of surgical trainees.

The toxicology of interleukin-12: a review.

Recombinant murine interleukin (IL)-12 (rmIL-12) exhibits antitumor, antiviral, and antimicrobial activities and can modify allergic inflammatory reactions in animal models. Recombinant human IL-12 (rhIL-12) is currently in clinical trials for treatment of cancer, asthma, and viral hepatitis. Principally a phagocyte-derived cytokine, IL-12 targets natural killer cells and T lymphocytes, stimulating their activity and the secretion of interferon (IFN)-gamma. An understanding of the toxicology of IL-12, due in part to effects mediated by IFN-gamma, has emerged from preclinical safety and mechanistic studies and initial clinical trials. Target organs common to several animal species and humans include the lymphohematopoietic system, intestines, liver, and lung.

IRAG working group 2. CAM-based assays. Interagency Regulatory Alternatives Group.

CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of embryonated chicken eggs, were assessed as alternatives to the Draize eye irritation test. Two general types of CAM-based assays are currently in use, the HET-CAM test and the CAMVA assay. Evaluations were made of five data sets produced with three different modifications of the HET-CAM test and two data sets obtained with the same CAMVA protocol. Data sets consisted of 9-133 test chemicals, usually from the sponsor's product line, and also from a validation trial. Each data set and assay protocol were analysed for quality of data, purpose and proposed use of the assay, range of responses covered, range of test materials amenable, current use in safety and risk assessment both in-house and for regulatory purposes. Since the MMAS Draize score was not available for all in vivo data sets, the sigma MMMIS, which correlates well with the MMAS, was used instead. In vitro/in vivo correlations calculated with Pearson's linear coefficient ranged from r = 0.6 to r = 0.9 for six of seven data sets. Corneal opacity and inflammation of the iris showed the best correlation to in vitro data. Prediction rates were significantly improved when partial linear regression was used, and the predictivity of three different HET-CAM protocols was almost the same. HET-CAM assays showed the best prediction with surfactants and surfactant-based formulations, whereas the CAMVA assay provided the best performance with alcohols.

Biologic effects of recombinant human interleukin-12 in squirrel monkeys (Sciureus saimiri).

BACKGROUND: Interleukin-12 is a novel heterodimeric cytokine that stimulates the proliferation of activated T and NK cells and induces lymphokine-activated killer cell activity in vitro. To investigate the biological effects of recombinant human IL-12 (rHuIL-12) in vivo, two exploratory studies were conducted in squirrel monkeys (Sciureus saimiri), which have been shown to be pharmacologically responsive to rHuIL-12 in vitro. EXPERIMENTAL DESIGN: In the first study, 18 monkeys (3/sex/group) were given daily subcutaneous injections of 0 (vehicle control), 10, or 50 micrograms/kg/day rHuIL-12 for 14 days. In the second study, 18 monkeys were given 0, 0.1, or 1 micrograms/kg/day rHuIL-12 for 14 days The animals were monitored for clinical signs, hematology and clinical chemistry changes, and sacrificed on day 15 to evaluate gross and histopathologic changes. One monkey in the high dose group was sacrificed moribund on day 14. RESULTS: Monkeys given rHuIL-12 had dose-related hematologic changes characterized by mild to moderate anemia and leukocytosis. Serum chemistry changes included hypoproteinemia, hypoalbuminemia, hypophosphatemia, and hypocalcemia. Gross pathologic findings included generalized lymph node enlargement and splenomegaly with pulmonary edema and peritoneal effusions in two high dose monkeys. Dose-related histopathologic findings included thymic cortical atrophy, splenic lymphoid hyperplasia with histiocytic hyperplasia and extramedullary hematopoiesis of red pulp, Kupffer cell hypertrophy and hyperplasia, trilineage bone marrow hyperplasia, and reactive hyperplasia of lymph nodes. Animals in the 10 and 50 micrograms/kg/day dose groups developed high titers of anti-rHuIL-12 antibodies by day 15. CONCLUSIONS: These studies indicate that rHuIL-12 is bioactive over a wide dose range and induces prominent hyperplasia of hematopoietic and lymphohistiocytic tissues in squirrel monkeys. Moreover, positive immunomodulatory activity (enhanced lymphocyte lytic activity) was detected at a dose of rHuIL-12 that is 500-fold less than the dose causing severe toxicity.

Interleukin-12: a cytokine with therapeutic potential in oncology and infectious diseases.

IL-12 is a cytokine that promotes cell-mediated immunity by promoting Th1-type cytokine responses, enhancing the lytic activity of NK/LAK cells, augmenting specific CTL responses, and inducing the production of IFN-gamma. On the other hand, IL-12 suppresses the development of Th2-type cytokine responses and humoral immunity, particularly IgGl and IgE responses. It is likely that IL-12 normally plays an important role in the host defense against intracellular microbial pathogens. In addition, the administration of rIL-12 to mice has been shown to have potent therapeutic effects in several tumour and infectious disease models. IL-12 has been shown to be more efficacious than IL-2 in several murine tumour models, and toxicology studies suggest that it may have a substantially better therapeutic index. In addition, the long serum half-life of IL-12 relative to other cytokines will allow more flexibility in dosing schedules. However, future clinical trials are required to determine whether the efficacy of IL-12 seen in these experimental models is predictive for its use as an immunomodulatory drug in humans.

Species differences in nucleotide pool levels of 2',3'-dideoxycytidine: a possible explanation for species-specific toxicity.

The nucleoside analogue, 2',3'-dideoxycytidine (ddC), a potent inhibitor of human immunodeficiency virus reverse transcriptase (in its anabolized triphosphorylated form), mediates virologic and immunologic improvements in AIDS patients. Clinical studies using ddC have shown various ddC-related toxicities, the most pronounced being a dose-limiting peripheral neuropathy. The dose responsiveness and manifestation of the ddC-related neuropathy vary among species, with greatest sensitivity in human > monkey > rabbit whereas mice and rats are insensitive to ddC-related neuropathy. This study has examined nucleotide pool sizes of ddCTP and its constituents (ddC, ddCMP, ddCDP) in cultured fibroblasts (human, rabbit, mouse) and freshly isolated peripheral lymphocytes (monkey, rabbit, rat, and mouse). Cells were treated with 10 microM [3H]ddC and nucleotide pool sizes analyzed by HPLC. The formation of nucleotide pools increased during the 24-hr assay period. Fibroblast pool formation of phosphorylated metabolites was significantly greater in human > rabbit > mouse. Lymphocytes demonstrated a similar pattern with monkey > rabbit > mouse = rat. Total ddC anabolite pools were also found to be significantly smaller (p < 0.05) in rodent lymphocytes than in those of rabbit or monkey, and rodent fibroblasts were smaller than those of human or rabbit (p < 0.05). These findings indicate that nucleoside phosphorylation and intracellular levels of phosphorylated metabolites may play an important role in determining species sensitivity and manifestation of ddC-related toxicity.

Hematological effects of 2',3'-dideoxycytidine in rabbits.

The antiviral nucleoside analogue 2',3'-dideoxycytidine (ddC) is a DNA chain terminator and/or inhibitor of human immunodeficiency virus (HIV) reverse transcriptase. We evaluated the effects of ddC in 36 New Zealand white rabbits. Three/sex were assigned to a control group and 5 treatment groups (10-250 mg/kg/day) for 13 or 18 weeks. Blood samples were taken 1 week prior to treatment and weekly thereafter to termination with the exception of the 2 highest dose groups, where blood sample collection was terminated at week 13. Selected hematological analytes were measured weekly with the exception of prothrombin time (PT) and activated partial thromboplastin time (APTT). PT and APTT and selected biochemical analytes were measured prior to treatment, at 7 weeks, and after 13 weeks of treatment. All rabbits were necropsied. Giemsa and hematoxylin and eosin sections were prepared from methacrylate-embedded marrow. Hematological effects included decreases in red blood cell count, hemoglobin, hematocrit, and white blood cell count and increases in mean corpuscular volume and red cell distribution width. Platelets, platelet volume, PT, APTT, and mean corpuscular hemoglobin concentration values were variable or unchanged. Effects were dose-related, most were seen at 1 week, and they persisted to term. Bone marrow histopathologic changes included megalocytosis, erythroid hypoplasia, bizarre erythroid nuclear morphology, nuclear-cytoplasmic asynchrony, and increased mitotic figures. Lymphopenia caused by ddC plateaued at 2 weeks and persisted until termination. Heteropenia (neutropenia) was sporadic. Biochemical values for serum analytes were unchanged by treatment. The principal hematological effect of ddC upon the erythron was characterized as a nonregenerative macrocytic anemia with erythroid hypoplasia and megaloblastic erythropoiesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiproliferative effect of interleukin-1 on human ovarian carcinoma cell line (NIH:OVCAR-3).

The human ovarian carcinoma cell line, NIH:OVCAR-3, possesses high affinity receptors for interleukin-1 (IL-1). Binding experiments with 125I-IL-1 alpha indicate a dissociation constant of approximately 55 pM and the presence of approximately 7800 receptors/cell. These receptors bind both IL-1 alpha and IL-1 beta and internalize IL-1. Proliferation is NIH:OVCAR-3 cells is inhibited by IL-1. Half-maximal inhibition is observed with 2-3 units/ml of IL-1 alpha or IL-1 beta. A maximal effect (80% inhibition of cell proliferation) is achieved by treatment of cells with greater than or equal to 10 units/ml of IL-1 for 3 days. The antiproliferative effect of IL-1 is blocked by IL-1 receptor antagonist. Light and electron microscopy studies show that IL-1 treatment causes cytopathological changes and a reduction in the number of mitotic figures in NIH:OVCAR-3. IL-1 stimulates prostaglandin E2 release by NIH:OVCAR-3 cells, but this response is unrelated to the antiproliferative effect of IL-1. Interferon-alpha A (IFN-alpha A) also inhibits growth of NIH:OVCAR-3 cells in a concentration-dependent manner. Combination of IFN-alpha A and IL-1 gives synergistic inhibition of NIH:OVCAR-3 cell proliferation. IL-1 alone or in combination with IFN-alpha A or other agents may be useful for treatment of human ovarian cancer.

Cell-cycle defect of DNA repair in progeria skin fibroblasts.

We examined the temporal regulation of DNA repair during synchronous cell proliferation in normal and progeroid human fibroblasts. Ultraviolet light-induced (254 nm, 20 J/m2) unscheduled DNA synthesis was measured at 4-h intervals after serum stimulation, for up to 32 h. Normal cells regulated DNA repair in a defined temporal sequence, showing a peak in the induction of DNA repair just before DNA synthesis. Progeroid skin fibroblasts failed to show an increase in nucleotide excision repair before scheduled DNA synthesis, but the background level of DNA repair was not significantly different from that in controls. Regulation of repair in progeroid human fibroblasts appeared similar, but not identical to that previously reported by Gupta and Sirover (1984b) for xeroderma pigmentosum complementation group C. Our results suggest that patients with Hutchinson-Gilford progeria may have a defect in DNA repair; the results offer nominal evidence that the average level of UV-induced DNA is decreased, and that individuals with this disease lack both the normal enhancement of DNA repair before scheduled DNA synthesis and the temporal control of DNA repair.

The influence of dietary restriction on DNA repair in rodents: a preliminary study.

A range study was undertaken to determine if dietary restriction (DR) affects DNA repair in rodents. Unscheduled DNA synthesis (UDS) was examined in two strains of rat (Brown Norway, BN and Brown Norway X Fischer 344 F1 hybrid, BNF) at 18 months of age. O(6)-Methylguanine-acceptor protein activity (MGAP) was measured across species using rat (Brown Norway X Fischer F-344 F1 hybrid, 18 months) and mouse (B6CB F1 hybrid, 30 months). The rodents were maintained on either an ad libitum (AL) or a restricted diet (60% of the caloric intake of AL rodents). UDS increased approximately 48-65% in freshly isolated skin cells from DR animals opposed to their AL controls after challenge with ultraviolet light (254 nm, 20 J/m2 UV). After treatment with methylmethane sulfonate (0.5 mM MMS), a significant increase in UDS was observed (P less than 0.01, approx, 55% for BN and 52% for BNF rats). Results of measurements for MGAP activity found levels to increase 73% in DR rats and approximately 28% in DR mice when compared to their AL counterparts. In addition MGAP levels in phase shifted mice were examined at three time points during a 24-h period where significant changes were found to occur in the metabolism of DR rodents. The activity of MGAP changed in a circadian fashion with significant increases in MGAP activity in DR mice occurring during the period of highest metabolic activity.

Fluorophotometric quantitation of DNA in articular cartilage utilizing Hoechst 33258.

A sensitive fluorophotometric assay was developed for the measurement of DNA in articular cartilage. The tissue was digested with Proteinase K and dodecyl sodium sulfate, followed by analysis with Hoechst 33258 dye. DNA content was determined on both fresh and lyophilized material containing as little as 50 ng DNA. The results are comparable to values for other fluorophotometric and spectrophotometric methods reported in the literature. In addition, this method can be incorporated into existing methodology, allowing quantitation of specific glycosaminoglycans in the same cartilage sample in terms of DNA.

Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 2. Characterization of proteoglycans.

The effect of ascorbate on the proteoglycans synthesized by rabbit articular chondrocytes was studied in first- and third-passage cultures for 12 and 26 days total duration, respectively. L-Ascorbate (0.2 mM) was added daily to half of the flasks after attachment of the cells. The cultures were labeled with Na2[35S]O4 or [14C]-glucosamine and [3H]-proline. Proteoglycans were isolated from the media and pericellular matrices by dissociative extraction and associative density gradient centrifugation. There was a large decline in the amount of proteoglycan synthesized between early and late cultures. Ascorbate increased the DNA content, amount of radiosulfate incorporated into glycosaminoglycans per microgram of DNA, and the proportion of labeled proteoglycan in the pericellular fraction of both short- and long-term cultures. The proteoglycans of the media and matrices of all cultures, with and without ascorbate, eluted as aggregates under associative column chromatographic conditions. The proteoglycans of 26-day cultures exhibited a higher degree of polydispersity in size than those of the short-term culture and contained small amounts of keratan (2-5%) and dermatan sulfate (4-8%) as assessed by keratanase and chondroitinase digestions, respectively. The effect of ascorbate, therefore, was to increase the amount of proteoglycan formed and to direct it into matrix deposition rather than to alter its quality.

DNA repair by articular chondrocytes. IV. Measurement of Micrococcus luteus endonuclease-sensitive sites by alkaline elution in rabbit articular chondrocytes.

The ability of resting and dividing rabbit articular chondrocytes to repair low doses of ultraviolet (UV) damage was measured through removal of UV endonuclease-sensitive sites (ESS, pyrimidines dimers) as measured by alkaline elution. The repair of damage was significantly (P less than 0.001) greater in dividing than non-dividing cells. An age-related decrease in repair capability was found in resting chondrocytes, but not in their dividing counterpart. These results support earlier findings of unscheduled DNA synthesis by the same cells. (Mech. Ageing Dev., 32: 39-55, 1985).

DNA repair by articular chondrocytes. V. O6 methylguanine-acceptor protein activity in resting and cultured rabbit and human chondrocytes.

The level of O6-methylguanine acceptor protein activity was examined in rabbit and human articular chondrocytes of different ages. The activity per microgram of DNA in rabbit chondrocytes was 5-fold lower than in humans. There was no age-dependent decrease in the activity of resting or cultured chondrocytes of either species. The values for resting cells were comparable to those of cultured cells. The lack of age-related differences in methyltransferase activity, in contrast to nucleotide excision repair [Mech. Ageing Dev. 32 (1985) 39-55], indicates that separate repair systems behave differently with respect to chronological aging. The methyl transferase activity may be more essential for survival of articular chondrocytes and therefore more highly conserved with age.

DNA repair by articular chondrocytes. III. Unscheduled DNA synthesis following ultraviolet light irradiation of resting cartilage.

Unscheduled DNA synthesis (UDS) was demonstrated both in fresh slices of rabbit articular cartilage and in resting chondrocytes dissociated from it. Uptake of [3H]thymidine by chondrocytes treated with 10 mM hydroxyurea and irradiated with 254 nm ultraviolet light (20 J/m2) was measured by autoradiography. The UDS in the resting chondrocytes was minuscule compared with that of chondrocytes growing in monolayer culture. It was greater in resting cells from 3- than in 18-month old rabbits but persisted in animals up to 52 months of age. In both resting and cultured chondrocytes, UDS was greatly inhibited by aphidicolin (5 micrograms/ml). The finding of age-related decrease in UDS of resting chondrocytes, contrasted with previously demonstrated lack of it in dividing ones, raises questions about in vitro artefacts in studies of cellular senescence.

Xenografts of articular chondrocytes in the nude mouse.

Subcutaneous transplantation of articular chondrocytes isolated enzymatically from immature rabbits and dogs into athymic (nu/nu) mice resulted in the formation of hyaline cartilaginous nodules. Graft rejection was seen when the cells were injected into heterozygous (nu/+) mice. Radiosulfate-labeled proteoglycan extracted from the xenografts had a high buoyant density and was digested by chondroitinase ABC. A monomeric preparation of proteoglycan (A1-D1) contained a small quantity of aggregate as assessed by gel chromatography and gel electrophoresis. Almost no incorporation of 3H-thymidine was found by autoradiography. The matrix did not become calcified over the course of 42 days. The nude mouse system lends itself to testing a variety of problems in the biology of cartilage. These include the redifferentiation of chondrocytes following dedifferentiation in vitro. Species differences were found in this regard. The nodules formed by rabbit articular chondrocytes, grown in monolayer culture for up to 14 days, had a hyaline chondroid character. Dog chondrocytes that had "dedifferentiated" during 14 days of culture prior to transplantation, formed a graft that had a sparse fibrous rather than hyaline matrix.

DNA repair by articular chondrocytes. I. Unscheduled DNA synthesis following ultraviolet irradiation in monolayer culture.

The hypothesis that aging of articular chondrocytes at a cellular level results from loss of DNA repair capability was studied by measuring unscheduled DNA synthesis (UDS). Cultured rabbit and human articular chondrocytes were irradiated with 254 nm ultraviolet light (20 J/m2) following treatment with 10 mM hydroxyurea. Neither the "in vitro senescence" nor spontaneous transformation that developed during serial passage of rabbit chondrocytes was accompanied by diminution of UDS. Synthesis of sulfated glycosaminoglycans declined more rapidly than the ability of the cells to divide. Levels of UDS by chondrocytes from old donors, rabbit or human, were comparable to those of younger individuals. UDS was greater in human than rabbit chondrocytes. Similar data have been reported previously for dermal fibroblasts but do not necessarily indicate that there is a direct or causative relationship between UDS capability and the longevity of mammalian species. X-Irradiation of rabbit chondrocytes or cartilage explants, in doses up to 40 000 rads, yielded no measurable UDS.

DNA repair by articular chondrocytes. II. Direct measurements of repair of ultraviolet and X-ray damage in monolayer cultures.

The abilities of human and rabbit articular chondrocytes to repair ultraviolet (UV) and X-ray damage were measured in terms of the removal of UV--endonuclease-sensitive sites (pyrimidine dimers) and single-strand breaks, respectively. The initial 3-h rate of dimer repair in human cells, incubated in medium containing 10% human serum, was about 2.5 times as large as in rabbit cells incubated in medium containing 10% or 20% fetal bovine serum. Similar results have been previously reported for unscheduled DNA synthesis (UDS), indicating that UDS is a valid quantitative measure of repair in this cell system. The repair of single-strand breaks was rapid (approx. 50% completed in less than 10 min). An estimate, from the measured numbers of lesions and patch sizes, indicated that the amount of UDS following 20 krad would be 100 to 300 times less than that in 3 h following 10 J/m2 of 254 nm and hence would not be detectable radioautographically.

Effect of purified growth factors on rabbit articular chondrocytes in monolayer culture. II. Sulfated proteoglycan synthesis.

The effect of 3 purified peptide growth factors--platelet-derived growth factor (PDGF), epidermal growth factor (EGF), pituitary fibroblast growth factor (FGF)--heat-inactivated fetal bovine serum (FBS), insulin, and 0.2 mM ascorbate on synthesis of sulfated proteoglycan by rabbit articular chondrocytes was studied in monolayer culture. Growth of the cells increased linearly as the concentration of heat-inactivated FBS rose from 0 to 30%. Glycosaminoglycan (GAG) synthesis (35SO4/micrograms DNA) was enhanced as the concentration of heat-inactivated FBS went from 0 to 5%. At higher levels of serum, radiosulfate incorporation declined progressively. Two modes of study of the test factors were used: 1) the dose of the factor was increased while the serum concentration was fixed at a low basal level (1% heat-inactivated FBS); 2) the dose of the test factor was kept constant but the level of heat-inactivated FBS varied from 0 to 10%. There was an inverse relationship between GAG and DNA synthesis when proliferation of cells was increased by EGF and platelet lysate. PDGF (1 U/ml) stimulated radiosulfate incorporation as well as DNA formation in the serum-free medium; the values for GAG synthesis did not increase as the serum concentration increased, but the cell mass did. The action of FGF was intermediate between that of EGF and PDGF: with 50 ng FGF/ml, increasing concentrations of serum caused a large progressive reduction of radiosulfate incorporation as growth was stimulated. In basal medium, however, FGF caused mild enhancement of GAG synthesis. Insulin increased aggregatable proteoglycan production far out of proportion to its growth-promoting activity in the presence of 1% heat-inactivated FBS. The response was effaced when higher concentrations of serum were employed. Ascorbate had a unique anabolic effect, increasing both cell growth and proteoglycan synthesis that is not suppressed by higher concentrations of serum. The content of serum and its several peptide and hormonal components thus have divergent effects on growth and proteoglycan synthesis in cell culture. This phenomenon must be taken into account in studying biochemical processes and pharmacologic reactions of articular chondrocytes in vitro.