RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury.

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia-reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia-reperfusion injury model, the high-dose TNF model, and in A20(-/-) mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia-reperfusion injury model and no benefit in A20(-/-) mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.

Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior.

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Recommended guidelines for the conduct and evaluation of prognostic studies in veterinary oncology.

There is an increasing need for more accurate prognostic and predictive markers in veterinary oncology because of an increasing number of treatment options, the increased financial costs associated with treatment, and the emotional stress experienced by owners in association with the disease and its treatment. Numerous studies have evaluated potential prognostic and predictive markers for veterinary neoplastic diseases, but there are no established guidelines or standards for the conduct and reporting of prognostic studies in veterinary medicine. This lack of standardization has made the evaluation and comparison of studies difficult. Most important, translating these results to clinical applications is problematic. To address this issue, the American College of Veterinary Pathologists' Oncology Committee organized an initiative to establish guidelines for the conduct and reporting of prognostic studies in veterinary oncology. The goal of this initiative is to increase the quality and standardization of veterinary prognostic studies to facilitate independent evaluation, validation, comparison, and implementation of study results. This article represents a consensus statement on the conduct and reporting of prognostic studies in veterinary oncology from veterinary pathologists and oncologists from around the world. These guidelines should be considered a recommendation based on the current state of knowledge in the field, and they will need to be continually reevaluated and revised as the field of veterinary oncology continues to progress. As mentioned, these guidelines were developed through an initiative of the American College of Veterinary Pathologists' Oncology Committee, and they have been reviewed and endorsed by the World Small Animal Veterinary Association.

Expression of the embryonic transcription factor Oct4 in canine neoplasms: a potential marker for stem cell subpopulations in neoplasia.

Neoplastic cells and stem cells share several phenotypic characteristics. Recently, numerous studies have identified adult stem cells that have been hypothesized to be the cellular origin for cancer in several tissues. Oct4 has been consistently associated with pluripotent or stemlike cells, and it is hypothesized that Oct4 is necessary for the maintenance of pluripotency. We hypothesize that Oct4-positive cells are present in all canine neoplasms and that these subpopulations of neoplastic cells might represent "cancer stem" cells. To test this hypothesis, 83 canine neoplasms representing 21 neoplastic diseases were evaluated for Oct4 expression using immunohistochemistry. The results of this study showed that all tumors included in this study contained a subpopulation of Oct4-positive cells, although the proportion of Oct4-positive cells and the intensity of immunoreactivity varied both within and between tumor types. Subpopulations of Oct4-positive cells identified in these tumors are likely to represent "cancer stem" cells and therefore might be responsible for the maintenance and propagation of the tumors. If these cells represent cancer stem cells, and are therefore responsible for the maintenance and growth of the neoplastic cellular population, then these cells should serve as relevant therapeutic targets and offer the greatest potential for curative treatment.

Cellular proliferation in canine cutaneous mast cell tumors: associations with c-KIT and its role in prognostication.

Canine cutaneous mast cell tumor (MCT) is a common neoplastic disease in dogs. Due to the prevalence of canine MCTs and the variable biologic behavior of this disease, accurate prognostication and a thorough understanding of MCT biology are critical for the treatment of this disease. The goals of this study were to evaluate and compare the utility of the proliferation markers Ki67, proliferating cell nuclear antigen (PCNA), and argyrophilic nucleolar organizing region (AgNOR) as independent prognostic markers for canine MCTs and to evaluate the use of these markers in combination, as each marker assesses different aspects of cellular proliferation. An additional goal of this study was to evaluate the associations between cellular proliferation and c-KIT mutations and between cellular proliferation and aberrant KIT protein localization in canine MCTs. Fifty-six MCTs treated with surgical excision alone were included in this study. Each MCT was evaluated for Ki67 expression, PCNA expression, and KIT protein localization using immunohistochemistry; for AgNOR counts using histochemical staining; and for the presence of internal tandem duplication c-KIT mutations using polymerase chain reaction amplification. In this study, increased Ki67 and AgNOR counts were both associated with significantly decreased survival. On the basis of these results, we recommend that the evaluation of cellular proliferation, including evaluations of both Ki67 expression and AgNORs, should be routinely used in the prognostication of canine MCTs. Additionally, the results of this study show that MCTs with aberrant KIT protein localization or internal tandem duplication c-KIT mutations are associated with increased cellular proliferation, further suggesting a role for c-KIT in the progression of canine MCTs.

Impact of tumour depth, tumour location and multiple synchronous masses on the prognosis of canine cutaneous mast cell tumours.

The goal of this study was to determine the significance of tumour depth, tumour location and multiple synchronous tumour masses for the prognostic evaluation of canine cutaneous mast cell tumours (MCTs). The study population consisted of 100 formalin-fixed, paraffin-embedded cutaneous MCTs that had been surgically removed from 100 dogs and submitted to the Diagnostic Center of Population and Animal Health at Michigan State University between 1998 and 2001. None of the dogs had received chemotherapy or radiation therapy. For each case the following data were obtained from the referring veterinarians: sex, breed, weight, age at diagnosis, diagnostics performed, adjunct medications given at the time of surgery, tumour location, number of tumour masses, tumour recurrence (development of MCTs at the surgical site), development of additional MCTs at distant sites (outside the surgical margins), tumour duration before removal, survival time and cause of death, if applicable. Tumour depth was determined through microscopic evaluation of 5 microm sections stained with haematoxylin and eosin. Based on univariable and multivariable survival analysis, dogs with multiple synchronous cutaneous MCTs at the time of diagnosis have a worse prognosis compared with dogs with single tumours. Additional treatment beyond surgical excision alone should be considered for these animals. Older dogs and Boxers with cutaneous MCTs were at higher risk to develop additional MCTs at distant sites (outside the surgical margins), and older and male dogs with cutaneous MCTs had significantly shorter survival times. Univariable analysis also determined that dogs with cutaneous MCTs located on the head and neck had an increased risk of additional MCT development at distant sites and that sterilized dogs with cutaneous MCTs had shorter survival times. However, these findings were not confirmed by multivariable analysis. Tumour depth was of no prognostic significance for dogs with cutaneous MCTs.

The use of KIT and tryptase expression patterns as prognostic tools for canine cutaneous mast cell tumors.

Cutaneous mast cell tumors (MCTs) are one of the most common tumors in dogs. Currently, prognostic and therapeutic determinations for MCTs are primarily based on the histologic grade of the tumor, but a vast majority of MCTs are of an intermediate grade, and the prognostic relevance is highly questioned. A more detailed prognostic evaluation, especially of grade 2 canine MCTs, is greatly needed. To evaluate the prognostic significance of KIT and tryptase expression patterns in canine cutaneous MCTs, we studied 100 cutaneous MCTs from 100 dogs that had been treated with surgery only. The total survival and disease-free survival time and the time to local or distant recurrence of MCTs were recorded for all dogs. Using immunohistochemistry, 98 of these MCTs were stained with anti-KIT and antitryptase antibodies. Three KIT- and three tryptase-staining patterns were identified. The KIT-staining patterns were identified as 1) membrane-associated staining, 2) focal to stippled cytoplasmic staining with decreased membrane-associated staining, and 3) diffuse cytoplasmic staining. The tryptase-staining patterns were identified as 1) diffuse cytoplasmic staining, 2) stippled cytoplasmic staining, and 3) little to no cytoplasmic staining. Based on univariate and multivariate survival analysis, increased cytoplasmic KIT staining was significantly associated with an increased rate of local recurrence and a decreased survival rate. The tryptase-staining patterns were not significantly associated with any survival parameter. On the basis of these results, we propose a new prognostic classification of canine cutaneous MCTs, according to their KIT-staining pattern, that can be used for the routine prognostic evaluation of canine cutaneous MCTs.

A nitrogen-free hypocaloric diet and recombinant human growth hormone stimulate postoperative protein synthesis: fasted and fed leucine kinetics in the surgical patient.

Twelve otherwise healthy patients undergoing elective surgery for resection of rectosigmoid adenocarcinoma were randomly allocated to two groups: one group receiving intravenous dextrose 5% 600 to 800 kcal.d-1 (DX, n = 6) and the other group receiving the same amount of dextrose intravenously plus recombinant human growth hormone (DX + rGH, n = 6). Supplementation with rGH started on the day of surgery and continued postoperatively for 5 days. No nitrogen was provided in the diet. This regimen was started 3 days before surgery and continued for 5 days after surgery. Protein kinetics were studied over a period of 8 hours in all patients. Following an overnight fast, a primed constant infusion of L-[1-13C]leucine was maintained for 4 hours (fasted state) and continued for a further 4 hours (fed state) during which 5% beet dextrose (low 13C content) with or without rGH was administered. The isotope studies were performed on the day before surgery and 6 days after surgery. Other measurements included urinary nitrogen excretion, gaseous exchange, and plasma concentrations of insulin, GH, and insulin-like growth factor-I (IGF-I). Addition of rGH to the dextrose diet had a significant positive effect on protein synthesis (P = .02). Surgery was responsible for a significant increase in postoperative whole-body protein breakdown and synthesis and leucine oxidation (P < .01), although lesser changes were observed in the DX group. An interaction between rGH and surgery was associated with a significant increase in protein synthesis (P = .009), but not with changes in either protein breakdown or leucine oxidation. Carbohydrate provision in the form of beet dextrose during the fed state of the isotopic study did not attenuate the significant decrease in protein synthesis (P = .01) or breakdown (P = .003) either before or after surgery, probably reflecting the absence of nitrogen in the diet. No significant interaction was found between rGH and feeding. These results of leucine kinetics indicate that addition of rGH to a low-dextrose intake in the absence of dietary nitrogen can actually promote protein synthesis. The low levels of leucine oxidation could be explained by the fact that amino acids resulting from protein degradation were directed preferentially toward resynthesis of new proteins rather than to oxidative pathways. There was a significant increase in plasma insulin and GH in the group receiving rGH (P < .05). The postoperative plasma concentration of IGF-I did not change in the latter group compared with the DX group, in which IGF-I concentration decreased significantly (P < .05) as part of the response to combined surgery and dietary restriction. Although both IGF-I and insulin are independently capable of stimulating protein synthesis, elevated levels of either hormone or GH itself may primarily modulate protein synthesis, even with a low intake of carbohydrates.

Growth hormone modulates amino acid oxidation in the surgical patient: leucine kinetics during the fasted and fed state using moderate nitrogenous and caloric diet and recombinant human growth hormone.

Twelve patients (aged 70 +/- 9 years) who were scheduled for resection of rectosigmoid colon adenocarcinoma but were otherwise healthy were randomly allocated after surgery to receive either peripheral parenteral nutrition alone ([PPN] n = 6) or in combination with recombinant human growth hormone (rGH) at a daily dose of 0.15 U x kg(-1) x d(-1)(PPN + rGH, n = 6). The daily nutritional regimen was 0.1 g nitrogen x kg(-1) x d(-1) and 20 kcal x kg(-1) x d(-1) (nonprotein energy was supplied as 60% lipid and 40% carbohydrate), and it was maintained for 6 days before and 6 days after surgery. Protein kinetics were studied in all 12 patients during the fasted and fed states before and 6 days after surgery using an 8-hour 13C-leucine tracer infusion. Daily urinary nitrogen, gaseous exchange, and plasma insulin, growth hormone, and insulin-like growth factor-I (IGF-I) were determined before and after surgery. Surgery was responsible for significant increases in postabsorptive whole-body protein flux and synthesis and leucine oxidation (P < .01). Supplementation of PPN with rGH contributed to a significant attenuation of the postoperative increase in leucine oxidation (P = .02), with a significant increase in whole-body protein synthesis (P = .02) and no effect on protein breakdown (P = .40). During the fed state, leucine oxidation increased significantly (P = .005), with the greatest change occurring in the PPN group. Feeding was associated with a significant decrease in whole-body protein breakdown before and after surgery in both groups (P = .001). Postoperative urinary nitrogen excretion was lower but was not statistically significant in the PPN + rGH group compared with the PPN group. There was a significant increase in oxygen consumption (VO2) and carbon dioxide production (VCO2) as a result of feeding and surgery (P < .01). Supplementation with rGH caused a decrease in the respiratory quotient (RQ) (P = .04), particularly after surgery, indicating a direct effect of rGH on fatty acid oxidation. Circulating plasma insulin increased significantly in both groups with feeding and rGH supplementation (P < .05). This was enhanced after surgery, particularly in the rGH group (P < .05). Plasma growth hormone decreased after surgery in the PPN group (P < .05), but did not change as a result of feeding. The circulating levels increased in the PPN + rGH group following subcutaneous administration before or after surgery. Plasma IGF-I decreased after surgery in the PPN group (P < .05), and no changes occurred in the PPN + rGH group with feeding. The present findings suggest a distinct positive effect of rGH on protein synthesis in catabolic patients receiving a moderate intake of nitrogen and calories. This is achieved by modulation of amino acid oxidation. The acute effect of intravenous (IV) nutrients on protein metabolism during the catabolic phase of surgical stress caused a direct decrease in protein breakdown with no effect on protein synthesis.

Post-surgery epidural blockade with local anaesthetics attenuates the catecholamine and thermogenic response to perioperative hypothermia.

Core (aural canal) and mean skin (15 sites) temperatures, plasma adrenaline, noradrenaline and metabolites, and gaseous exchange were measured before, during and for 4 h after surgery in sixteen patients scheduled for elective colorectal surgery. All patients received general anaesthesia and no measures were taken to prevent the perioperative loss of body heat. At time of abdominal wall closure, when the core temperature was below 35.0 degrees C, the patients were randomly allocated to receive either 20-30 mg of papaveretum i.v. (papaveretum group, n = 8) or 15 ml of bupivacaine 0.75% via thoracic (T9) epidural route to obtain a T4-S5 sensory blockade (epidural group, n = 8). Continuous infusion of either i.v. papaveretum or epidural 0.25% bupivacaine was continued after surgery. During the recovery period of four hours the rate of increase in core and mean skin temperatures was significantly slower in the epidural group compared with the papaveretum group (P < 0.01). Plasma catecholamine concentrations remained elevated after surgery in the papaveretum group, whilst they decreased significantly once epidural blockade was established (P < 0.001). There was a lower trend, however not significant, in the rise of postoperative oxygen consumption and plasma glucose concentration in the epidural group compared with the papaveretum group.

Metabolic effects and secretory properties of a radiation-induced transplantable rat insulinoma.

The metabolic effects and secretory properties of a radiation-induced transplantable insulinoma were examined in 16-17 week old NEDH rats. Subcutaneous subscapular implantation of tumour fragments resulted in hyperphagia, increased body weight gain, marked hyperinsulinaemia and severe hypoglycaemia, with the resulting death of the recipient by 27 days. Ultimate tumour size was 2.1 +/- 0.4 g (mean +/- SEM). At 3 days after transplantation, plasma glucose and insulin responses to intraperitoneal glucose, insulin, arginine and adrenaline were similar to control rats. At 20 days, plasma glucose concentrations of insulinoma-bearing rats remained low throughout glucose tolerance tests, and insulin responsiveness to glucose stimulation was absent. 2-Deoxy-D-glucose produced only a small rise of glucose concentrations in tumour-bearing rats. Insulin sensitivity was not appreciably impaired at 20 days despite severe hyperinsulinaemia and hypoglycaemia. The ability of adrenaline and propranolol to suppress plasma insulin and raise plasma glucose concentrations was also retained. At 20 days, glucagon evoked a marked plasma insulin response with no change in plasma glucose concentrations. In contrast, arginine and glibenclamide failed to stimulate insulin above high basal concentrations.

Brachial plexus neuropathy from metastatic testicular seminoma. Prolonged survival after surgery and radiation therapy.

Prolonged survival and cure of brachial neuropathy were accomplished following subtotal removal and radiation therapy to metastatic seminoma involving the soft tissues of the right upper arm. Metastasis developed six years after orchiectomy for seminoma, and the patient is free of disease six years after treatment of metastatic disease.

The effect of the mammalian neuropeptide, gastrin-releasing peptide (GRP), on gastrointestinal and pancreatic hormone secretion in man.

Gastrin-releasing peptide, a newly isolated mammalian peptide similar in its structure and actions to the amphibian peptide, bombesin, has recently been localized to nerves in the brain, gut and pancreas. The present study investigates its effects on gut and pancreatic peptides in man. Intravenous infusion of 0.7 and 2.9 pmol min-1 kg-1 produced significant elevation of plasma gastrin, cholecystokinin-like immunoreactivity and neurotensin. It was found also to potentiate glucose-dependent insulin secretion. Its specific location in nerve fibres in the proximal gut and pancreas and its selective effect on gastroenteropancreatic peptides may favour its role as a physiological regulatory neuropeptide.

Abnormalities of growth hormone release in response to human pancreatic growth hormone releasing factor (GRF (1-44) ) in acromegaly and hypopituitarism.

Human pancreatic growth hormone releasing factor (GRF (1-44)) is the parent molecule of several peptides recently extracted from pancreatic tumours associated with acromegaly. A study was conducted to examine its effects on the release of growth hormone in normal volunteers and in patients with hypopituitarism and acromegaly. GRF (1-44) dose dependently stimulated the release of growth hormone in normal people and produced no appreciable side effect. This response was grossly impaired in patients with hypopituitarism and, although similar to the growth hormone response to hypoglycaemia, was of quicker onset and a more sensitive test of residual growth hormone function. Patients with acromegaly appeared to fall into (a) those with a normal response to GRF, whose growth hormone suppressed significantly with oral glucose, and (b) those who had an exaggerated response to GRF (1-44), whose growth hormone had not suppressed previously after oral glucose. Present methods for testing growth hormone deficiency entail using the insulin stress test, which is time consuming, unpleasant, and sometimes dangerous. A single intravenous injection of GRF now offers the possibility of an easier, safer, and more reliable routine test for growth hormone deficiency. It has the further advantage of being free of side effects and readily performed in outpatients. Hence it seems likely to become the standard test and take the place of the insulin stress test.

Human pancreatic growth-hormone-releasing factor selectively stimulates growth-hormone secretion in man.

A growth-hormone-releasing factor has been characterised and sequenced from a pancreatic tumour removed from a patient with acromegaly. It is a 40-residue linear peptide. Synthetic human pancreatic growth-hormone-releasing factor (hpGRF-40), 1 microgram/kg bodyweight, was administered as an intravenous bolus to six healthy men. hpGRF-40 selectively stimulated growth-hormone secretion. Serum growth-hormone concentrations were increased within 5 min, reaching a peak between 30 and 60 min (20 . 4 +/- 6 . 5 ng/ml compared with 2 . 1 +/- 0 . 1 ng/ml after placebo). Serum levels of prolactin, thyrotropin, luteinising hormone, and corticotropin (measured indirectly through plasma cortisol) were not increased after administration of hpGRF-40. Similarly, the concentrations of blood glucose, plasma insulin, glucagon, pancreatic polypeptide, cholecystokinin, gastrin, gastric inhibitory peptide, motilin, and somatostatin were unaffected by hpGRF-40. There were no changes in blood pressure, pulse rate, or body temperature, and no side-effects were noted. The characteristics of this peptide fulfil many of the criteria required of the hypophysiotropic growth-hormone-releasing hormone. hpGRF holds promise for a new approach to the diagnosis and treatment of various disorders of growth-hormone secretion.

Acute nickel intoxication by dialysis.

Nickel intoxication was observed in a group of 23 dialyzed patients when leaching of nickel-plated stainless steel water heater tank contaminated the dialysate. Symptoms occurred during and after dialysis at plasma nickel concentrations of approximately 3 mg/L. Symptoms included nausea (37 of 37), vomiting (31 of 37), weakness (29 of 37), headache (22 of 37), and palpitation (two of 37). Remission of symptoms occurred spontaneously, generally 3 to 13 hours after cessation of dialysis. The evidence indicated that the nickel became bound in the plasma after crossing the membrane, resulting in a higher concentration in the plasma than in the dialysate and preventing its removal by dialysis.