Ramiro E. Toribio

Blood Arginine Vasopressin, Adrenocorticotropin Hormone, and Cortisol Concentrations at Admission in Septic and Critically Ill Foals and their Association with Survival

BACKGROUND Sepsis is an important cause for neonatal foal mortality. The hypothalamic-pituitary-adrenal axis (HPAA) responses to sepsis are well documented in critically ill humans, but limited data exist in foals. The purpose of this study was to evaluate the HPAA response to sepsis in foals, and to associate these endocrine changes with survival. HYPOTHESIS Blood concentrations of arginine vasopressin (AVP), adrenocorticotropin hormone (ACTH), and cortisol will be higher in septic foals as compared with sick nonseptic and healthy foals. The magnitude of increase in hormone concentration will be negatively associated with survival. ANIMALS Fifty-one septic, 29 sick nonseptic, and 31 healthy foals of < or =7 days of age were included. METHODS Blood was collected at admission for analysis. Foals with positive blood culture or sepsis score > or =14 were considered septic. Foals admitted with disease other than sepsis and healthy foals were used as controls. AVP, ACTH, and cortisol concentrations were measured using validated immunoassays. RESULTS AVP, ACTH, and cortisol concentrations were increased in septic foals. Septic nonsurvivor foals (n = 26/51) had higher plasma ACTH and AVP concentrations than did survivors (n = 25/51). Some septic foals had normal or low cortisol concentrations despite increased ACTH, suggesting relative adrenal insufficiency. AVP, ACTH, and cortisol concentrations were higher in sick nonseptic foals compared with healthy foals. CONCLUSIONS AND CLINICAL IMPORTANCE Increased plasma AVP and ACTH concentrations in septic foals were associated with mortality. Several septic foals had increased AVP : ACTH and ACTH : cortisol ratios, which indicates relative adenohypophyseal and adrenal insufficiency.

The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice.

The functions of parathyroid hormone‐related protein (PTHrP) on morphogenesis, cell proliferation, apoptosis, and calcium homeostasis have been attributed to its N terminus. Evidence suggests that many of these effects are not mediated by the N terminus but by the midregion, a nuclear localization sequence (NLS), and C terminus of the protein. A knock‐in mouse lacking the midregion, NLS, and C terminus of PTHrP (Pthrp△/△) was developed. Pthrp△/△ mice had craniofacial dysplasia, chondrodysplasia, and kyphosis, with most mice dying by d 5 of age. In bone, there were fewer chondrocytes and osteoblasts per area, bone mass was decreased, and the marrow was less cellular, with erythroid hypoplasia. Cellular proliferation was impaired, and apoptosis was increased. Runx2, Ocn, Sox9, Crtl1, ß‐catenin, Runx1, ephrin B2, cyclin D1, and Gata1 were underexpressed while P16/ Ink4a, P21, GSK‐3ß, Il‐6, Ffg3, and Ihh were overexpressed. Mammary gland development was aberrant, and energy metabolism was deregulated. These results establish that the midregion, NLS, and C terminus of PTHrP are crucial for the commitment of osteogenic and hematopoietic precursors to their lineages, and for survival, and many of the effects of PTHrP on development are not mediated by its N terminus. The down‐regulation of Runx1, Runx2, and Sox9 indicates that PTHrP is a modulator of transcriptional activation during stem cell commitment. Toribio, R E., Brown, H. A., Novince, C. M., Marlow, B. Hernon, K., Lanigan, L. G., Hildreth III, B. E., Werbeck, J. L., Shu, S. T., Lorch, G., Carlton, M., Foley, J., Boyaka, P., McCauley, L. K., Rosol, T. J. The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice. FASEB J. 24, 1947–1957 (2010). www.fasebj.org

Animal Models of Bone Metastasis

Bone is one of the most common sites of cancer metastasis in humans and is a significant source of morbidity and mortality. Bone metastases are considered incurable and result in pain, pathologic fracture, and decreased quality of life. Animal models of skeletal metastases are essential to improve the understanding of the molecular pathways of cancer metastasis and growth in bone and to develop new therapies to inhibit and prevent bone metastases. The ideal animal model should be clinically relevant, reproducible, and representative of human disease. Currently, an ideal model does not exist; however, understanding the strengths and weaknesses of the available models will lead to proper study design and successful cancer research. This review provides an overview of the current in vivo animal models used in the study of skeletal metastases or local tumor invasion into bone and focuses on mammary and prostate cancer, lymphoma, multiple myeloma, head and neck squamous cell carcinoma, and miscellaneous tumors that metastasize to bone.

Disorders of calcium and phosphate metabolism in horses.

Calcium and phosphate have structural and nonstructural functions, and their concentrations in the extracellular compartment are affected by the physiologic status of the animal as well as diseases. Important progress in understanding calcium and phosphorus metabolism in healthy and diseased horses and foals has been made in recent years. For example, several studies have confirmed that hypocalcemia is frequent in horses with gastrointestinal disease and that calcium endocrine dysregulation is associated with survival in foals. One critical point in the homeostasis of these minerals is their interaction and interdependence with other ions, including potassium and magnesium. In this review, the author provides a clinical overview on disorders of calcium and phosphate in the horse.

Canine Prostate Carcinomas Express Markers of Urothelial and Prostatic Differentiation

Prostate carcinoma and transitional cell carcinoma (TCC) occur in the prostate gland of older dogs and have morphologic similarities when evaluated by light microscopy. The dog is a commonly used animal model for studying human prostate carcinoma; therefore, it is important to accurately differentiate canine prostate carcinomas from TCCs. We investigated whether keratin 7 (K7) and arginine esterase (AE) would aid differentiation of canine prostate carcinoma from TCC. K7 expression was evaluated in normal and neoplastic canine prostate and bladder tissues using immunohistochemistry. The expression of AE messenger ribonucleic acid (mRNA) in normal and neoplastic canine prostate and bladder was detected using northern blots and reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, AE enzyme activity was measured in normal and neoplastic canine prostate and bladder tissues. We found marked similarities in K7 expression in prostate carcinomas and TCCs. AE mRNA was present in high levels in normal prostatic tissue but was reduced in prostate carcinoma by northern blot assay. Nested RT-PCR detected AE mRNA both in TCCs (13 of 15) and in prostate carcinomas (13 of 13). Enzymatic activity of AE was high in normal prostate gland and in some prostate carcinomas, whereas normal bladder and TCCs produced lower levels of AE. In conclusion, K7 and AE cannot be used to differentiate TCC from prostate carcinoma in dogs.

Molecular cloning and expression of equine calcitonin, calcitonin gene-related peptide-I, and calcitonin gene-related peptide-II

In this study, we describe the cloning and tissue expression of equine calcitonin (CT), calcitonin-gene related peptide (CGRP)-I, and CGRP-II cDNA. We also describe a novel divergent form of CGRP (CGRP-I). Equine CT has greatest homology (>85%) to human, rat and mouse subgroups of calcitonins. Equine CGRP-I has low homology (<59%) to CGRPs of other species. The signal and N-terminal peptides for equine CT and CGRP-I were identical, indicating that these peptides are encoded by a gene equivalent to the human CALC-I gene. Equine CGRP-II has >80% homology to chicken, human, rat, ovine, swine, and bovine CGRPs. The homology between equine CGRP-I and CGRP-II is low (56%). The high homology of equine CGRP-II and the low homology of equine CGRP-I to CGRP in other species were unexpected findings. Northern blot analysis revealed that CT mRNA expression was restricted to the thyroid gland; however, RT-PCR revealed that CT mRNA expression was also present in the pituitary gland and in the liver. CGRP-I and CGRP-II mRNA expression was present in several regions of the nervous system and other tissues of neuroectodermal origin. An unexpected finding was CGRP-I expression in the kidney by both Northern analysis and by RT-PCR. Based on these results, CT gene expression in the horse was not restricted to the thyroid gland, and CT may be important in regulating pituitary cell function. CGRPs are widely expressed in tissues of the central and peripheral nervous system. Information from this study will be valuable to study the role of CT, CGRP-I, and CGRP-II in equine health and disease.

Insulin, Glucagon, and Leptin in Critically Ill Foals

BACKGROUND Endocrine dysregulation of hormones of energy metabolism is well documented in critically ill humans, but limited information exists in septic foals. The purpose of this study was to provide information on the hormonal response to energy metabolism in critically ill foals, focusing on insulin, glucagon, and leptin. HYPOTHESIS Concentrations of insulin, glucagon, leptin, and triglycerides will be higher, whereas glucose concentration will be lower in septic foals than in healthy and sick nonseptic foals. The magnitude of these differences will be associated with severity of disease and nonsurvival. ANIMALS Forty-four septic, 62 sick nonseptic, and 19 healthy foals <7 days of age. METHODS In this prospective multicenter cross-sectional study, blood samples were collected at admission. Foals with positive blood culture or sepsis score ≥12 were considered septic. RESULTS Septic foals had lower glucose and insulin and higher triglyceride and glucagon concentrations than did healthy foals. Glucagon concentrations were not different between septic foals that died (n = 14) or survived (n = 30). Higher insulin and lower leptin concentrations were associated with mortality. Quantitative insulin-sensitivity check index was higher in septic foals. CONCLUSIONS AND CLINICAL IMPORTANCE Energy metabolism and the endocrine response of related hormones in septic foals are characterized by hypoglycemia, hypertriglyceridemia, low insulin concentration, and high glucagon concentration. Leptin and insulin may have prognostic value for nonsurvival in septic foals. The hormonal response related to energy metabolism in critical illness differs between foals and humans.

Zoledronic acid reduces bone loss and tumor growth in an orthotopic xenograft model of osteolytic oral squamous cell carcinoma.

Squamous cell carcinoma (SCC) is the most common form of oral cancer. Destruction and invasion of mandibular and maxillary bone frequently occurs and contributes to morbidity and mortality. We hypothesized that the bisphosphonate drug zoledronic acid (ZOL) would inhibit tumor-induced osteolysis and reduce tumor growth and invasion in a murine xenograft model of bone-invasive oral SCC (OSCC) derived from an osteolytic feline OSCC. Luciferase-expressing OSCC cells (SCCF2Luc) were injected into the perimaxillary subgingiva of nude mice, which were then treated with 100 μg/kg ZOL or vehicle. ZOL treatment reduced tumor growth and prevented loss of bone volume and surface area but had no effect on tumor invasion. Effects on bone were associated with reduced osteolysis and increased periosteal new bone formation. ZOL-mediated inhibition of tumor-induced osteolysis was characterized by reduced numbers of tartrate-resistant acid phosphatase-positive osteoclasts at the tumor-bone interface, where it was associated with osteoclast vacuolar degeneration. The ratio of eroded to total bone surface was not affected by treatment, arguing that ZOL-mediated inhibition of osteolysis was independent of effects on osteoclast activation or initiation of bone resorption. In summary, our results establish that ZOL can reduce OSCC-induced osteolysis and may be valuable as an adjuvant therapy in OSCC to preserve mandibular and maxillary bone volume and function.

Calcium Regulating Hormones and Serum Calcium and Magnesium Concentrations in Septic and Critically Ill Foals and their Association with Survival

BACKGROUND Disorders of calcium regulation are frequently found in humans with critical illness, yet limited information exists in foals with similar conditions including septicemia. The purpose of this study was to determine whether disorders of calcium exist in septic foals, and to determine any association with survival. HYPOTHESIS Blood concentrations of ionized calcium (Ca(2+)) and magnesium (Mg(2+)) will be lower in septic foals with concomitant increases in parathyroid hormone (PTH), calcitonin (CT), and parathyroid-related peptide (PTHrP) compared with healthy foals. The magnitude of these differences will be negatively associated with survival. ANIMALS Eighty-two septic, 40 sick nonseptic, and 24 healthy foals of <or=7 days were included. METHODS Prospective, observational study. Blood was collected at initial examination for analysis. Foals with positive blood culture or sepsis score >or=14 were considered septic. Foals with disease other than sepsis and healthy foals were used as controls. Hormone concentrations were measured with validated immunoassays. RESULTS Septic foals had decreased Ca(2+) (5.6 versus 6.1 mg/dL, P < .01) and increased serum PTH (16.2 versus 3.2 pmol/L, P < .05), and phosphorus concentrations (7.1 versus 6.3 mg/dL, P < .01). No differences in serum Mg(2+), PTHrP, and CT concentrations were found. Nonsurviving septic foals (n = 42/82) had higher PTH concentrations (41.1 versus 10.7 pmol/L, P < .01) than survivors (n = 40/82). CONCLUSIONS AND CLINICAL IMPORTANCE Septic foals were more likely to have disorders of calcium regulation compared with healthy foals, where hyperparathyroidemia was associated with nonsurvival.

Hysteresis and calcium set-point for the calcium parathyroid hormone relationship in healthy horses.

Abnormalities in calcium (Ca(2+)) homeostasis are reported in horses with several pathological conditions; however, there is little information on Ca(2+) regulation in horses. The objectives of the present study were to determine the Ca(2+) set-point in healthy horses, to determine whether the Ca(2+)/parathyroid hormone (PTH) response curves were characterized by hysteresis, and to determine if the order of experimentally induced hypocalcemia or hypercalcemia had an effect on PTH secretion. The Ca(2+) set-point and hysteresis were determined in 12 healthy horses by infusing Na(2)EDTA and calcium gluconate. The Ca(2+) set-point was 1.37 +/- 0.05 mmol/L, which is higher than values reported for humans and dogs (1.0-1.2 mmol/L). Hysteresis was present during hypocalcemia and hypercalcemia. Horses in which hypocalcemia was followed by hypercalcemia secreted more PTH (7440 +/- 740 pmol min/L) than horses in which hypercalcemia was followed by hypocalcemia (5990 +/- 570 pmol min/L). This study has demonstrated that the Ca(2+) set-point in the horse is higher than in other domestic animals and man. We have shown that the Ca(2+)/PTH relationship in horses is sigmoidal and displays hysteresis during both hypocalcemia and hypercalcemia, and that extracellular Ca(2+) concentrations may affect the response of the parathyroid gland to hypocalcemia.