Identifying developmental toxicity pathways for a subset of ToxCast chemicals using human embryonic stem cells and metabolomics.

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information, including Stemina Biomarker Discovery's predictive DevTox® model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity.

The influence of nitric oxide synthase 2 on cutaneous wound angiogenesis.

BACKGROUND: Inducible nitric oxide synthase (nitric oxide synthase 2, NOS 2) inhibition significantly suppresses chronically ischaemic skin flap survival, possibly because of reduced angiogenesis. OBJECTIVES: To investigate the effect of genetic NOS 2 inhibition on cutaneous wound angiogenesis in two in vivo murine models. The impact of NOS 2 manipulation on vascular endothelial growth factor (VEGF)-A stimulated and fibroblast growth factor (FGF)-2 stimulated angiogenesis was also investigated in the Matrigel(®) plug assay. METHODS: (i) Matrigel plugs/incisional wounds: two groups of NOS 2-/- mice and two groups of wild-type (WT) mice had bilateral Matrigel plugs containing 500 ng mL(-1) VEGF-A or 1000 ng mL(-1) FGF-2 injected subcutaneously in the abdomen. A 2·5 cm long dorsal incisional skin wound was created and sutured closed in the same animals. Wounds and plugs were explored at 7 or 12 days. (ii) Excisional wounds: dorsal 0·5 × 1·0 cm excisional skin wounds were created in four groups (two NOS 2-/- and two WT) and explored at 7 or 14 days. Wounds and Matrigel plugs were examined histologically and morphometrically for determination of percentage vascular volume (PVV). RESULTS: The PVV in NOS 2-/- incisional wounds and excisional wounds was significantly less than in WT wounds (P = 0·05 and P < 0·001, respectively). The PVV was significantly less in VEGF-A stimulated Matrigel plugs compared with FGF-2 stimulated plugs in NOS 2-/- mice (P < 0·01), but not in WT mice. CONCLUSIONS: NOS 2 is significantly involved in angiogenic signalling in healing skin wounds, particularly within the first 7 days. However, Matrigel plug vascularization suggests that the role of NOS 2 in angiogenesis is related to VEGF-A but not FGF-2 stimulated angiogenesis.

Hypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor-A expression and downregulation of miRNA-1, miRNA-206 and angiopoietin-1.

Vascularization is a major hurdle for growing three-dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR-1 and miR-206 (p < 0.05) and angiopoietin-1 (p < 0.05) with upregulation of vascular endothelial growth factor-A (VEGF-A; p < 0.05). The miR-1 and angiopoietin-1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR-206 in L6 myoblasts caused a significant increase in VEGF-A expression (p < 0.05), further establishing that changes in VEGF-A expression are influenced by miR-206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel™ into isolated bilateral tissue engineering chambers incorporating a flow-through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin-1, miR-1 and miR-206. The relatively simple strategy of hypoxic preconditioning of implanted cells - including non-stem cell types - has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.

Radiation injuries to the bowel associated with the treatment of carcinoma of the cervix.

Advances in radiation techniques and increased dosage have improved the cure rate of patients with cancer of the cervix to 65 percent. Associated with this increased dosage (betatron, 5,250 r and intracavitary 137-cesium, 4,000 r at point A) has been a serious complication incidence of 10 percent. Major intestinal complications usually become manifest within an 8 to 24 month period following radiation. Few are associated with tumor and the majority are amenable to surgical correction. Rectosigmoid stenosis is a common and frequently unrecognized complication. The 8 to 12 cm. segment of rectosigmoid, with its rigid wall and narrowed lumen, can be recognized on barium examination. The symptoms are those on incomplete obstruction and deterioration, frequently confused with tumor progression. Thirty-one patients have been treated by resection and low anterior anastomosis with relief of symptoms. Rectosigmoid stenosis progressing to necrosis, perforation, or fistula (an additional 29 patients) is treated best by the Hartmann operation as a first stage. This procedure has been less complicated than either colostomy alone or resection and anastomosis. Fifteen patients with low level rectovaginal fistula or stenosis were treated by defunctioning sigmoid colostomy. A loop transverse colostomy was unsatisfactory. Ileorectovaginal fistulas occurred in an additional six patients. Preoperative investigation should establish the presence or absence of an ileal component in all fistulas. Radiation ileitis is rare as an isolated finding but frequently is associated with severe rectosigmoid damage. Surgical treatment is seldom necessary but, if indicated (ten patients), resection appears to be preferable to bypass.

Head and neck reconstruction following resection of carcinoma, using microvascular free flaps.

Our experience with 15 microvascular free-flap transfers is documented and discussed. We feel that the procedure is an effective means of reconstruction for complex and extensive defects. Rapid advances have been made in all areas of cancer surgery. It is important that these patients be afforded a speedy and, preferably, immediate reconstruction and rehabilitation. Conventional techniques do not satisfy all the required goals.

Efficacy of needle biopsy in postradiation thyroid disease.

Retrospective review was carried out of 124 patients with nodular disease of the thyroid gland and a history of radiation exposure who had undergone needle aspiration biopsy. Latency period from time of radiation varied from 2 to 50 years; but in 92 patients it exceeded 2 decades. Our patient group included those with occupational exposure and a past history of radiation for cancer. Incidence of cancer in the entire group was 49% but, for solitary lesions, this was increased to 56%, while only a 30% incidence of cancer was found in cases of multinodular goiters. Accuracy of needle aspiration biopsy overall was 74%: for the group with cancer--90%, for the group with adenomas--65%, and for the group with "benign" tumors--83%. Further assessment of needle technique indicated a sensitivity of 70%, specificity of 90%, positive predictive value of 90%, and negative predictive value of 83% to 65%. The accuracy could be increased to 84% if all adenomas were considered as possible malignancies. Eighteen percent of our patients had second tumors in the head and neck or breast area. Near-total thyroidectomy was considered to be the preferred procedure without accidental nerve injury and was done in one case of hypoparathyroidism after excision of an extensive tracheal invasive cancer. No evidence of death, recurrence, or metastasis as a result of thyroid cancer has been noted. While needle biopsy is indispensable to intelligent management, the history of radiation to the head and neck area must be preeminent in the selection of patients for surgical treatment. Conservative management appears to be reasonable in those patients with "benign" cytology, a less than 1 cm nodule, multinodularity, a functioning thyroid scan result, but persistence in the face of a lack of response to conservative management does not appear to be warranted.

The surgical aspects of hyperparathyroidism.

We followed up 250 patients surgically treated for hyperparathyroidism. Selective venous catheterization with radioimmunoassay was an effective method of preoperative localization, but its greatest contribution was in patients needing reexploration of the neck. Because of a 15 percent incidence of multiple gland involvement, we tried to identify all glands, if possible. If more than one gland was abnormal, it also was removed. If three or four glands were abnormal, a subtotal parathyroidectomy was done. Five patients showed persistent hyperparathyroidism because of failure to find or remove all hyperfunction tissue: two patients were successfully treated at a third operation; one has not undergone reexploration, and two have parathyromatosis. Only one patient developed late recurrent hyperparathyroidism. The removal of a single adenoma is adequate providing the remaining glands are grossly and histologically normal. Hypoparathyroidism is a potential, although uncommon, risk following subtotal parathyroidectomy.

Soft tissue hematoma as a cause of fever in the adult.

Hematoma as an isolated cause of temperature elevation in adult patients is rarely reported. We describe a patient with a large hematoma involving his right leg that caused significant pyrexia. The computerized axial tomography findings are discussed, as well as the possible mechanisms responsible for the temperature elevation.

Fibroosseous tumors of the facial skeleton in association with primary hyperparathyroidism: an endocrine syndrome or coincidence?

In a retrospective study of 350 cases of primary hyperparathyroidism at the University of Toronto, 5 patients had associated benign fibroosseous lesions of the facial skeleton which did not regress after successful parathyroidectomy and restoration of normocalcemia. Two of these patients were siblings and an offspring of one of these patients was recently shown to have hyperparathyroidism, suggesting that this group of patients does represent a syndrome with familial and genetic implications. At present bone disease in patients with hyperparathyroidism should be viewed with some consideration of unrelated process, and appropriate biopsy and treatment should be undertaken after its recognition.

Reoperative surgery for hyperparathyroidism.

The difficulties created by an unsuccessful initial operation can usually be avoided by choosing experienced surgeons, identifying four glands, and selectively excising grossly abnormal parathyroid tissue. Patients with multiple endocrine adenomatosis or familial hyperparathyroidism should have a subtotal parathyroidectomy. Reoperative surgery is difficult and although it is successful in approximately 70 to 80 percent of patients, it is associated with a significant morbidity which includes persistent hypercalcemia, hypoparathyroidism, and nerve injury. Reoperative parathyroid surgery should be done in specialized centers where the sophisticated methods of preoperative localization and surgeons experienced in this field are available.

Vascular problems associated with carotid body tumors.

In the last 25 years at the University of Toronto, 27 patients with carotid body tumors were encountered. All patient problems appeared to be related to delay in treatment, size of the tumor, and previous operations. To minimize hazard in this procedure, one should avoid operating on elderly and unfit patients, and otherwise operate early to avoid the problem of a bulky tumor, using a simple subadventitial approach while reserving complete carotid excision and vascular replacement for complicated adherent lesions. Preoperative angiography is mandatory for definitive diagnosis, which permits proper planning of available operating room time, blood replacement, and availability of workers with expertise in vascular surgery. Adequate surgery is effective in tumor clearance, preventing malignant transformation and the lethal effects of local growth.

Induction of hyperparathyroidism by radioactive iodine.

External irradiation to the head and neck has been associated with the emergence of hyperparathyroidism, due both to adenoma and hyperplasia since a single case report was published in 1975. A recent report indicates that there may be some association between hyperparathyroidism and radioiodine administered for thyroid disease. Eight patients are presented herein, all female and in middle age, who presented with hyperparathyroidism due to adenoma (six patients) or hyperplasia (one patient who had undergone radioiodine treatment). In three patients, the dose of radioiodine exceeded 30 mCi. The duration of latency ranged from 4 to 20 years. Associated thyroid disease included cancer, colloid nodules, and thyroiditis. All patients became eucalcemic and have remained so. Experimental results that have supported the influence of radioiodine on parathyroid hyperactivity do not appear completely applicable to the human model. It is believed that the low mitotic rate equivalent to low radiosensitivity may have protected the parathyroid gland somewhat from neoplastic change, although the association of thyroid carcinoma and parathyroid adenoma appears to be a significant expression of radiation exposure. It may be the lack of a pituitary-stimulating hormone and the absence of an abscopal mechanism, as illustrated by irradiation to the thyroid, that explains the relative immunity of parathyroid behavior in the face of radioiodine exposure. Regardless of the frequency and validity of the association, it is important to recognize the possibility of the association illustrated by our case reports and scrutinize appropriate patients by intermittent serum calcium sampling, both for confirmation and possible detection of occult hyperparathyroidism.

Management of medullary carcinoma of the thyroid.

Total thyroidectomy is universally advised for the familial variety of MCT. Although total thyroidectomy is also recommended for sporadic cases, partial thyroidectomy may be adequate. Cervical and upper mediastinal nodes should be sampled for microscopic study, even when they are small and appear to be normal. Appropriate neck or mediastinal nodes should be sampled for microscopic study, even when they are small and appear to be normal. Appropriate neck or mediastinal dissection is done if metastasis is present. External radiation is a valuable adjuvant to surgical excision following the apparent complete resection of the tumor, and is beneficial in the management of unresectable disease. Despite local control, patients continue to die from disseminated disease; therefore, there must be a continued search for an effective chemotherapeutic program. Much remains to be learned from calcitonin monitoring of MCT patients.

Effectiveness of subgingival scaling and root planing: single versus multiple episodes of instrumentation.

This study evaluates the effectiveness of subgingival scaling and root planing comparing the effect of a single instrumentation to the effect of three instrumentations. A total of 35 teeth in 15 patients were selected; 15 were scaled once (Group A), 15 were scaled three times (Group B), and 5 were used as controls (Group C), representing teeth that were not instrumented. The Group A and B teeth were chosen in the same patient based on random selection. All the teeth were scored by the calculus index of the periodontal disease index. Six surface locations were probed to determine probing depth. The level of the gingival margin was marked on the teeth to locate supra- and subgingival calculus after extraction. The Group A and B teeth received the initial episode of scaling and root planing for not more than 10 minutes, then only the Group B teeth received two additional instrumentations of not more than 5 minutes each. The additional instrumentations were performed 24 hours after the initial scaling. The scaled and control teeth were extracted immediately after the third instrumentation period. The teeth were washed with water and stained with methylene blue. They were viewed under a stereomicroscope which had a tenth grid on its eyepiece. Assessments were made involving the total counts and percents of the surfaces covered with calculus on the scaled and unscaled teeth. The results demonstrated no significant difference in the effectiveness of calculus removal between single and multiple episodes of scaling and root planing. Similar results were found for the total amount of calculus removed, the calculus removed from individual surfaces, and the calculus removed from various probing depth levels.

Factors influencing inhibitory stimulus control: discrimination training and prior non-differential reinforcement.

In Exp. I, shallow U-shaped gradients of inhibition in the line-orientation dimension were obtained from birds that had a vertical (0 degrees ) line on a green surround correlated with extinction and a blank green surround correlated with reinforcement. Birds that had massed extinction in the presence of the 0 degrees line showed flat gradients. Thus, discrimination training, but not massed extinction, appears to generate inhibitory control. In Exp. II, as in studies of control by a stimulus correlated with punishment, non-differential training across the line-orientation dimension preceded further sessions. Steep inverted gradients about the 0 degrees line were obtained after discrimination training with the 0 degrees line correlated with extinction. Gradients obtained after massed extinction tended to be flat. Again, discrimination training was critical in obtaining negative gradients of stimulus control.

Effects of type of exercise on depression in recovering substance abusers.

This experiment investigated the effects of three types of structured exercise (aerobics, bodybuilding, and circuit training) on depressive symptoms of 45 clients undergoing a 4-wk., inpatient rehabilitation program for substance abuse. Pre- and posttest measures included the Center of Epidemiological Studies--Depression, resting pulse rate, blood pressure, maximum strength on incline bench press, and estimates of aerobic fitness and body fat. The bodybuilding program produced a significant decrease in depressive symptoms. Physiological and psychological explanations are discussed.

Childhood abuse as a factor in attrition from drug rehabilitation.

The following factors were examined as possible influences of clients' attrition from inpatient and outpatient drug-rehabilitation programs: depression (Center of Epidemiological Studies-Depression test), attributional style (Attributional Style Questionnaire), primary drug of choice, family incidence of substance abuse, and history of childhood physical abuse. A step-wise regression analysis indicated that a history of childhood abuse was a statistically reliable predictor of program noncompletion for 92 substance abusers who entered a drug-rehabilitation program.

The role of endocannabinoids in pain modulation and the therapeutic potential of inhibiting their enzymatic degradation.

The need for new pain therapies that provide greater relief without unwanted side-effects drives the search for new drug targets. The identification of endogenous lipid ligands for the two known cannabinoid receptors (CB(1) and CB(2)) has led to numerous studies investigating the role of these endocannabinoids in pain processes. The two most widely studied endocannabinoids are anandamide (AEA; arachidonoyl ethanolamide) and 2-arachidonoylglycerol (2-AG), but there are also a number of structurally related endogenous lipid signaling molecules that are agonists at cannabinoid and noncannabinoid receptors. These lipid signaling molecules are not stored in synaptic vesicles, but are synthesized and released on-demand and act locally, as they are rapidly inactivated. This suggests that there may be therapeutic potential in modulating levels of these ligands to only have effects in active neural pathways, thereby reducing the potential for side-effects that result from widespread systemic cannabinoid receptor activation. One approach to modulate the levels and duration of action of these lipid signaling molecules is to target the enzymes responsible for their hydrolysis. The two main enzymes responsible for hydrolysis of these lipid signaling molecules are fatty acid amide hydrolase (FAAH) and monoacylglyceride lipase (MGL). This article will discuss the role of the endocannabinoid system in the modulation of pain and will review the current understanding of the properties of the hydrolytic enzymes and the recent advances in developing inhibitors for these targets, with particular relevance to the treatment of pain.

Fatty acid amide hydrolase inhibition enhances the anti-allodynic actions of endocannabinoids in a model of acute pain adapted for the mouse.

Cannabinoid ligands have been shown to be anti-nociceptive in animal models of acute and chronic pain by acting at the two known cannabinoid receptors, cannabinoid-1 receptor (CB-1) and cannabinoid-2 receptor (CB-2). A major concern with the use of cannabinoids for pain relief is that they activate receptors at sites other than those involved in the transmission of nociceptive stimuli. An alternative approach is to target the naturally occurring endocannabinoids, such as anandamide (AEA), 2-arachidonylglycerol (2-AG) and N-arachidonylglycine (N-AG). However in vivo results obtained with these compounds appear to be weak, most probably due to their rapid degradation and subsequent short half-life. The predominant enzyme responsible for the hydrolysis of anandamide (and some other endocannabinoids) in the brain is fatty acid amide hydrolase (FAAH). Recently, the alpha-ketoheterocycle OL135 has been synthesized and shown to be a highly potent and selective inhibitor of FAAH with efficacy in pain models in vivo. In the present study, we have adapted the mild thermal injury (MTI) model of acute pain for the mouse and pharmacologically characterized this model by showing significant reversal of the tactile allodynia by morphine (3, 5 and 10 mg kg(-1) s.c.), gabapentin (100 and 300 mg kg(-1) i.p.), ibuprofen (100 mg kg(-1) i.p.) and OL135 (10, 30 and 100 mg kg(-1) i.p.). Furthermore we have demonstrated, using this model, that a subtherapeutic dose of OL135 can enable the endocannabinoids AEA and 2-AG, but not N-AG to be active at doses where they are otherwise nonanalgesic (20 mg kg(-1) i.p.). The implications of this model in the study of pain in mice, and the therapeutic potential of FAAH inhibition to provide analgesia without the undesirable side effects of direct agonism of cannabinoid receptors are discussed.