Tumor-induced osteomalacia: experience from a South American academic center.

The majority of tumor-induced osteomalacia cases have been reported in the Northern Hemisphere and Asia. In this first series of South American patients, we show that the clinical presentation and sensitivity of plasmatic fibroblast growth factor 23 and somatostatin analog-based imaging are similar to those described in other populations. INTRODUCTION: Describe the experience of clinical presentation, diagnostic study, and treatment of patients with tumor-induced osteomalacia (TIO) in a South American academic center in comparison to literature. METHODS: Analysis of the records of patients diagnosed with TIO. The clinical presentation, diagnostic studies, and treatment were analyzed. Fibroblast growth factor 23 (FGF23) was measured by ELISA. RESULTS: Six patients were diagnosed with TIO during the studied period. The patients' median age was 53 years (range 22-64). All patients presented with weakness and pain in the extremities. Four experienced fractures during their evolution. The median time to diagnosis was 4.5 years (1-20). Biochemical studies showed hypophosphatemia, median of 1.4 mg/dL (1.2-1.6), with low maximum rates of tubular reabsorption of phosphate adjusted for glomerular filtration rate. FGF23 was elevated in 4/6 patients and inappropriately normal in the other two. In three patients, the location of the tumor was clinically evident and confirmed with anatomical imaging. In the remaining patients, two tumors were located with 68Ga DOTATATE-PET/CT and one with OctreoScan. The causal tumors were located in the lower extremities in five patients and invading the frontal sinus in one patient. In all patients, tumors were successfully removed. Within 14 days, there was normalization of phosphate and FGF23 levels and resolution of clinical symptoms in all patients. In all cases, the histopathology was compatible with a phosphaturic mesenchymal tumor. CONCLUSIONS: The clinical presentation, delay time to diagnosis, FGF23 diagnostic sensitivity and histopathology in this first series of South American patients is similar to those described in other populations. The success of localization by somatostatin analog-based imaging, suggests this may the optimal imaging modality.

Repeatability and reproducibility of the Obel grading system for equine laminitis.

The aim of this study was to evaluate both the repeatability (intraobserver agreement) and reproducibility (interobserver agreement) of Obel grading, the most widely accepted method for describing the severity of equine laminitis. The study comprised two parts. In study A, to test intraobserver agreement, 25 ponies were graded twice by a single blinded experienced equine veterinary surgeon, on the first occasion via clinical examination and on the second occasion via a video recording of the initial clinical examination. In study B, to test interobserver agreement, video recordings of 13 ponies were graded independently by 58 blinded experienced equine veterinary surgeons. A weighted kappa (kappa(w)) statistic was used to determine the measure of agreement between gradings. Intraobserver agreement of Obel grading was shown to be moderate (kappa(w)=0.54), and interobserver agreement was found to be substantial (kappa(w)=0.65). In addition, intraobserver agreement improved from moderate to substantial (kappa(w) increased from 0.54 to 0.70) when the Obel grades were grouped and horses were categorised as either sound, or as having mild (grade 1 or 2) or severe (grade 3 or 4) lameness.

Dietary fructan carbohydrate increases amine production in the equine large intestine: implications for pasture-associated laminitis.

Pasture-induced laminitis in the horse is associated with the overconsumption of fermentable carbohydrate, in the form of simple sugars, fructans, or starch. The fermentation of carbohydrate in the cecum and large intestine results in the production of lactic acid and other toxins or "laminitis trigger factors." Vasoactive amines have been suggested as possible initiating factors. The aim of this study was to feed a commercially available form of fructan carbohydrate (inulin, 3 g/kg of BW per day) to normal ponies and to ponies predisposed to laminitis, to mimic a change from a basal hay diet to lush spring-summer pasture. Five normal and 6 laminitis-prone, native-breed ponies were acclimated to a basal hay diet before the inclusion of inulin and chopped dried grass. Blood samples, fecal samples, and foot temperature measurements were taken throughout the study. Amines were measured in the feces and plasma by HPLC and liquid chromatography-mass spectrometry, respectively. The pH of the fecal samples decreased from 6.89 +/- 0.11 on the hay diet to a minimum of 6.18 +/- 0.11 with the addition of inulin (P <0.05). An increase was observed in the fecal concentrations of a number of amines, including tryptamine (2.5-fold increase, P <0.05) and tyramine (2-fold increase, P <0.05). No changes were noted in plasma amine concentrations or plasma D- or L-lactate, indicating that there may be a threshold of hindgut pH change before mucosal damage can result in the release of these factors into the circulation. No differences in pH or any of the measured compounds were observed between the group of normal ponies and those predisposed to laminitis. This indicates that differences in the intestinal microflora do not account for this predisposition. However, the results from this study indicate that moderate increases in dietary fructan carbohydrate can produce increases in bacterial fermentation products and other compounds in the large intestine, which may be relevant to the pathogenesis of acute laminitis in ponies on pasture.

The role of protein kinase C in regulating equine eosinophil adherence and superoxide production.

OBJECTIVE: To determine if protein kinase C (PKC) regulates equine eosinophil function. MATERIAL OR SUBJECTS: Blood eosinophils were obtained from healthy ponies. METHODS: IL-5- and histamine-induced adherence to serum-coated plastic was measured as the eosinophil peroxidase content of adherent cells and serum treated zymosan (STZ)-and IL-5-induced superoxide production by the reduction of cytochrome C. Eosinophil PKC activity was quantitated as the rate of transfer of (32)P from ATP to substrate. The effects of Ro31-8220 (isotype non-selective PKC inhibitor), Go6976 (conventional PKC inhibitor), and rottlerin (PKCdelta inhibitor) were determined by ANOVA and Bonferroni's or Dunnett's test. RESULTS: Ro31-8220 and Go6976 reduced superoxide production whereas only Go6976 inhibited adherence. Rottlerin inhibited histamine-induced adherence and increased STZ-induced superoxide production. Ro31-8220 and Go6976, but not rottlerin, inhibited PKC activity. CONCLUSIONS: PKC is involved in regulating equine eosinophil adherence and superoxide production. The role of PKCdelta appears to depend upon the stimulus used and response measured.

Demonstration of regional differences in equine ventricular myocardial velocity in normal 2-year-old Thoroughbreds with Doppler tissue imaging.

REASONS FOR PERFORMING STUDY: Doppler tissue imaging (DTI) is a novel noninvasive method by which myocardial velocity can be assessed directly and it allows regional, rather than global, cardiac function to be evaluated. HYPOTHESIS: That regional differences in myocardial velocities exist within the equine ventricle. OBJECTIVES: To develop a repeatable examination technique for DTI in horses, describe DTI findings in various regions of the normal equine ventricle, compare colour (CDTI) and spectral (SDTI) techniques of DTI, and document regional differences in myocardial velocity. METHODS: Five regions of the ventricles (right ventricular wall, interventricular septum and left, right and caudal regions of the left ventricle) were evaluated using SDTI and CDTI in 20 clinically normal Thoroughbreds age 2 years. Individual repeatability of the method was determined by examination of one 6-year-old Thoroughbred on 6 occasions. RESULTS: Three major movements were observed in the ventricular walls in systole, early diastole and late diastole. The interventricular septum had a complex pattern of movement. The left region of the left ventricle and interventricular septum had the most rapid movement. The individual repeatability of CDTI was poor, while in systole and early diastole, but not late diastole, SDTI produced repeatable estimates of maximal myocardial velocity. The different velocity estimates obtained with SDTI and CDTI are not interchangeable. Regional differences in the peak mean and maximal myocardial velocities were found in systole and early diastole (P<0.05), but were not identified in late diastole. CONCLUSIONS: The SDTI modality appears to produce the most repeatable data. There are regional differences in myocardial velocity within the equine ventricles for systole and early diastole. POTENTIAL RELEVANCE: DTI shows potential as a tool for studying regional myocardial movement both in clinical cases suspected of having myocardial dysfunction and in a research setting. In particular, SDTI offers potential as a direct and noninvasive means to study early diastolic function of the equine ventricles.

Protein kinase C (PKC) isotype profile in eosinophils from ponies with sweet itch and role in histamine-induced eosinophil activation.

Eosinophils have been implicated in the pathogenesis of the seasonal equine allergic skin disease, sweet itch. Protein kinase C (PKC) is involved in regulating eosinophil function and antigen challenge has been reported to alter PKC isotype expression in blood eosinophils from allergic human subjects. Here we have compared the pattern of PKC isotype expression in eosinophils from sweet itch ponies with that in cells from normal ponies both during the active and inactive phases of the disease. A role for PKC in histamine-induced eosinophil activation was also investigated. Conventional PKCs alpha and beta, novel PKCs delta and epsilon and atypical PKCs iota and zeta were identified in eosinophils pooled from four allergic ponies during the inactive phase, when no clinical signs were evident. The PKC isotypes, like those in eosinophils from normal ponies, were located primarily in the particulate fraction of the cell. Isotype expression in cells from normal and allergic animals did not appear to be different. In contrast, during the active phase of the disease, when the sweet itch ponies had clinical signs, the expression of PKCs beta, epsilon and iota in eosinophils from these animals appeared to be increased relative to that in cells from normal ponies. When PKC expression in eosinophils from five individual normal and sweet itch ponies was compared, small, but statistically significant, increases in PKC epsilon and PKCdelta expression were evident in eosinophils from the sweet itch ponies during the active and inactive phases, respectively. The non-selective PKC inhibitors, staurosporine and Ro31-8220, significantly reduced histamine-induced superoxide production. Use of Gö6976, an inhibitor of conventional PKCs, suggested that PKCalpha and/or beta were involved and that there was significantly greater inhibition of the response in eosinophils obtained from sweet itch ponies during the active phase. There was no significant difference in histamine-induced superoxide production by eosinophils from allergic and normal ponies and the functional significance of the increased PKC isotype expression in eosinophils from sweet itch ponies relative to that in cells from healthy animals remains to be established.

Cloning, expression and biological activity of equine interleukin (IL)-5.

The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.