Nutritional management of hyperlipaemia in a jenny: a brief report.

An adult jenny (5-years-old, non-pregnant) was presented to the Veterinary Teaching Hospital (VTH) of the University of Sassari, with a recent history of appetite loss, extreme underweight condition and reluctance to move. On physical inspection, emaciation [body condition score, BCS: 3/9], muscular waste [muscular condition score, MCS: 1/5], loose/running faeces [faecal score, FS: 2/8], and a general state of mild dehydration were found. Blood analyses outlined a general undernourishment condition [circulating albumins, ALB: 17.6 g/L (21.6-31.6 g/L)] with underlying systemic inflammatory profile and moderate increase in circulating enzymes to explore liver function [aspartate amino-transferase, AST: 657 u/L (279-430 u/L); alanine amino-transferase ALT: 60 u/L (5-14 u/L); gamma-glutamyl-transferase, γ-GT: 87 IU/L (14-69 IU/L); total bilirubin close to the upper limit, TB: 0.20 mg/dL(0.07-0.21 mg/dL)]and hyperlipaemia [TG: 8.70 mmol/L (0.60-2.87 mmol/L)], following fat depots mobilisation, with total cholesterol closed to the lower limit of the physiological range. Hyper-phosphataemia was linked to haemolytic anaemia [P:1.81 mmol/L (0.77-1.39 mmol/L) and red blood cells, RBC: 4.14 1012/L (4.40-7.10 1012)] aligned with the TB to the upper limit. On ultrasound abdominal imaging, enlarged and hyper-echogenic liver was observed. Based on the clinical evaluation, a condition of hepatic lipidosis was diagnosed, requiring dedicated nutritional treatment to solve the extreme emaciation along with the metabolic disorder in support of medical therapy. A two-step feeding protocol was planned to support treatments aiming at immediate re-hydration (Ringer lactate solution 2 ml/kg/8 h). The nutritional objectives were meant at first to restart the voluntary feed intake. Gradual increasing energy provision through a palatable hay-based diet was planned to cover one fourth of daily metabolizable energy requirement calculated on the expected metabolic weight, adjusted according to the daily intake of feed and clinical condition. At the conclusion of this first 7-day phase, circulating blood parameters were closer to the reference values and the BCS moved from 3 to 4 out of 9. Bowel motility was restored, and faecal score improved (4/8). In the second phase, allowance to pasture and a combination diet with compound mixed feed were designed. Within four weeks of starting the nutritional plan, blood parameters were re-established to reference values. The gradual feed provision calculated in this two-phase approach proved successful in support of the overall clinical improvement observed after four weeks of treatment, in a severely undernourished jenny with compromised liver functions.

Red Flags for early referral of people with symptoms suggestive of narcolepsy: a report from a national multidisciplinary panel.

OBJECTIVE: Narcolepsy is a lifelong disease, manifesting with excessive daytime sleepiness and cataplexy, arising between childhood and young adulthood. The diagnosis is typically made after a long delay that burdens the disease severity. The aim of the project, promoted by the "Associazione Italiana Narcolettici e Ipersonni" is to develop Red Flags to detect symptoms for early referral, targeting non-sleep medicine specialists, general practitioners, and pediatricians. MATERIALS AND METHODS: A multidisciplinary panel, including patients, public institutions, and representatives of national scientific societies of specialties possibly involved in the diagnostic process of suspected narcolepsy, was convened. The project was accomplished in three phases. Phase 1: Sleep experts shaped clinical pictures of narcolepsy in pediatric and adult patients. On the basis of these pictures, Red Flags were drafted. Phase 2: Representatives of the scientific societies and patients filled in a form to identify barriers to the diagnosis of narcolepsy. Phase 3: The panel produced suggestions for the implementation of Red Flags. RESULTS: Red Flags were produced representing three clinical pictures of narcolepsy in pediatric patients ((1) usual sleep symptoms, (2) unusual sleep symptoms, (3) endocrinological signs) and two in adult patients ((1) usual sleep symptoms, (2) unusual sleep symptoms). Inadequate knowledge of symptoms at onset by medical doctors turned out to be the main reported barrier to diagnosis. CONCLUSIONS: This report will hopefully enhance knowledge and awareness of narcolepsy among non-specialists in sleep medicine in order to reduce the diagnostic delay that burdens patients in Italy. Similar initiatives could be promoted across Europe.

Expression of ATP6V1C1 during oral carcinogenesis.

We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.

Biochemical and molecular characterization of the novel BRAF(V599Ins) mutation detected in a classic papillary thyroid carcinoma.

Activating mutations of the BRAF gene are the most common genetic alterations in papillary thyroid carcinomas (PTCs) and the T1799A transversion, resulting in BRAFV600E, appeared virtually unique in this cancer type. Here, we report on the identification in a classic PTC of a novel BRAF mutation, namely a 1795GTT insertion, resulting in BRAFV599Ins, and describe its biochemical and molecular characterization. Kinase assays carried out on BRAFV599Ins and BRAFV600E revealed a three- to five-fold increase in the enzymatic activity of both mutants with respect to BRAFWT. Similarly, evaluation of BRAF-induced phosphorylation of MEK, MAPK and RSK revealed a significant MAPK cascade activation in cells expressing BRAFV599Ins or BRAFV600E, but not in cells expressing BRAFWT. Molecular dynamic simulations showed a destabilization of the inactive conformation of the enzyme in both BRAFV599Ins and BRAFV600E mutants, but not in BRAFWT. The analysis of the interaction energies inside the catalytic site allowed to demonstrate the presence of repulsive electrostatic forces acting on the activation loop and moving from inward to outward of the mutant enzymes. Finally, focus assays in NIH-3T3 cells confirmed a high transformation rate in the cells transfected either with BRAFV599Ins or BRAFV600E. In conclusion, this study demonstrated that BRAFV599Ins, as BRAFV600E, is a 'gain of function' mutation, characterized by a constitutive catalytic activation, which accounts for its causative role in the studied PTC.

Accentuated response to phenylhydrazine and erythropoietin in mice genetically impaired for their GATA-1 expression (GATA-1(low) mice).

The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.

Erythropoietin-dependent suppression of the expression of the beta subunits of the interleukin-3 receptor during erythroid differentiation.

To clarify how erythroid cells lose their response to interleukin-3 (IL-3), we analyzed the expression of the alpha (alpha(IL-3)) and beta (beta(IL-3)/beta(com)) subunits of its receptor in a panel of murine cell lines immortalized at different stages of hemopoietic differentiation. The panel was composed by the mast cell line 32D and by its granulo-monocytic (32D GM), granulocytic (32D G), and erythroid (32D Epo1.1 and Epo) subclones. The 32D Epo cells grow only in erythropoietin (EPO) while the Epo1.1 subclone grows in either EPO or IL-3. The phenotype of these cells is that of early (expression of globins and erythroid-specific carbonic anhydrase II) and late (also expression of the erythroid-specific band 4.1 mRNA) erythroblasts when they grow in IL-3 or EPO, respectively. All the cell lines expressed comparable levels of alpha(IL-3). In contrast, the expression of beta(IL-3)/beta(com) was restricted to cells growing in IL-3 and was barely detectable in 32D Epo and 32D Epo1.1 cells growing in EPO. When switched from EPO to IL-3, 32D Epo1.1 cells expressed 10 times more beta(IL-3)/beta(com) by rapidly activating (within 1 h) their transcription rate. When reexposed to EPO, 32D Epo1.1 cells first expressed (1-6 h) more beta(IL-3)/beta(com) (2 times) but suppressed such an expression at later time points (by 48 h). The beta(IL-3)/beta(com) mRNA half-life was also different when 32D Epo1.1 cells grew in EPO or IL-3 (2-3 h vs >5 h, respectively). These results indicate that EPO specifically induces transcriptional and posttranscriptional downmodulation of beta(IL-3)/beta(com) expression in late erythroid cells.

In vivo expansion of purified hematopoietic stem cells transplanted in nonablated W/Wv mice.

We have evaluated the in vivo amplification potential of purified murine hematopoietic stem cells, identified as Wheat Germ Agglutinin+ (WGA+), 15-1.1(-) , Rhodamine 123 Dull (Rho-dull) cells, by serial transplantation into stem cell defective nonmyeloablated W/Wv mice. C57BL Rho-dull cells (250/ 500 cells/mouse) permanently engrafted nonablated W/Wv mice as defined by the presence of > 95% red and > 20% white donor-derived circulating cells for at least 1.5 years following transplantation. At this time, approximately 61% of Rho-dull cells and all the Rho-bright progenitor and colony forming cells of the engrafted mice were found to be donor-derived by c-Kit genotyping and by their response to stem cell factor (SCF). Retransplantation of 250-1000 Rho-dull cells from primary into secondary W/Wv recipients generated C57BL hematopoiesis in 40%-64% of animals revealing the presence of donor derived hematopoietic stem cells (HSC) in the bone marrow of the primary recipients. One and half years after transplantation, the bone marrow of the secondary engrafted animals contained C57BL Rho-dull cells approximately = 51% by genotype), which were capable of reconstituting tertiary W/Wv recipients. In this respect, 25% of tertiary mice expressed C57BL hematopoiesis when transplanted with 250-1000 Rhodull cells purified from secondary W/Wv recipients. On the basis of the number of Rho-dull cells purified from a single mouse, we calculate that approximately 7.3x10(4) Rho-dull cells, which are genotypically and functionally defined as C57BL long-term repopulating stem cells, were generated in the marrow of reconstituted primary W/Wv recipients transplanted 1.5 years earlier with 250-500 C57BL Rho-dull cells. We conclude that murine HSC have extensive amplification capacity in nonmyeloablated animals.

The use of intermediate and high purity factor VIII products in the treatment of von Willebrand disease.

Since 1985, viral-attenuated blood products have been available for the treatment of patients with hemophilia. Unfortunately, similar viral-attenuated blood products, enriched for von Willebrand factor (vWF), have not been readily available for the treatment of patients with von Willebrand disease (vWD). In the current study, we examined the clinical efficacy and in vivo properties of two viral-attenuated factor VIII products, Koate-HS and Koate-HP, in the treatment of patients with vWD. Twenty-one (21) infusions were evaluated in 17 different vWD patients (4 with type IA; 8 with Type IIA; 1 with Type IID; 4 with type III). Seven (7) patients received Koate-HS and 12 patients received Koate-HP (2 patients received both products; 1 patient was studied three times). Von Willebrand factor antigen, ristocetin cofactor, bleeding time, and the multimeric composition of vWF were determined pre- and post-infusion. Complete or partial correction of prolonged bleeding times was observed in 2 of the 6 patients tested following treatment with Koate-HS and in 7 out of 11 patients tested following treatment with Koate-HP. Surgery was performed on five of these patients, two of whom were treated with Koate-HS and three of whom were treated with Koate-HP. In the surgical patients, clinical hemostasis was achieved regardless of whether the bleeding time was corrected. We conclude that both Koate-HS and Koate-HP can be utilized successfully in the treatment of patients with vWD in spite of the lack of high molecular weight multimers of vWF in these products.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal diagnosis of haemophilia A by factor VIII gene analysis.

Cloned factor VIII deoxyribose nucleic acid (DNA) sequences were used as probes in the prenatal diagnosis of haemophilia A. Fetal DNA from cultured amniotic fluid cells was examined for a DNA polymorphism within the factor VIII gene which marked the haemophilia A gene in the pregnant obligate carrier. The fetus was predicted to be an affected male, and the diagnosis of haemophilia A was confirmed both in utero and after termination of the pregnancy.

Prenatal diagnosis of classic hemophilia (hemophilia A) by immunoradiometric assays.

During the period from 1978 to 1983, 92 pregnancies have been evaluated by fetoscopy for the prenatal diagnosis of hemophilia A. Satisfactory fetal plasma samples were obtained in 80 instances and the diagnosis--or exclusion--of hemophilia was made by immunoradiometric assay of the factor VIII coagulant protein (VIII:CAg). The accuracy of the diagnosis established by fetoscopy has been verified after delivery or termination, and there have been no misdiagnoses resulting from laboratory error. Additional evidence for the accuracy of the analysis was the observation that the frequency of hemophilia in pregnancies of obligate carriers of the hemophilia gene, and of women whose plasma assays were indicative of the carrier state, was 29 of 59 fetuses at risk. In one case of cross-reacting material-positive hemophilia, samples obtained at fetoscopy and from the newborn infant had normal VIII:CAg levels but the infant had decreased factor VIII procoagulant activity. There were five fetal deaths resulting from fetoscopy in 55 pregnancies not intentionally terminated. Although only a small percentage of pregnant hemophilia carriers in the United States have elected to undergo fetoscopy for prenatal diagnosis, this procedure has allowed a number of pregnancies to go to term with delivery of normal males in families that were not willing to accept the risk of a hemophilic child. In eight instances, fetoscopic evaluation was sought for two successive pregnancies.

Von Willebrand factor multimer patterns in von Willebrand's disease.

The von Willebrand factor antigen (factor VIII-related antigen, VIIIR:Ag) multimer pattern has been analysed by SDS-agarose electrophoresis of plasmas from 116 patients (47 families) with von Willebrand's disease. In addition to previously recognized patterns, a subclassification was established between plasmas that had a type Ia pattern (VIIIR:Ag multimer pattern like that of normal plasma) and those that had a type Ib pattern in which there was a relative reduction in the concentration of the larger VIIIR:Ag multimers even though all multimeric forms were present. The different patterns were consistent within families and were inherited by autosomal dominant transmission. Von Willebrand's disease heterogeneity was apparent in the distribution of these plasmas: type Ia, 43 patients in 18 families; type Ib, 39 patients in 15 families; type II, 22 patients in 10 families, one of which was further classified as type IIB, one of which was type IIC, and three were IIA. Seven patients with severe von Willebrand's disease were also studied. In general, the interpretation of SDS-agarose multimer patterns corresponded to those previously obtained by crossed immunoelectrophoresis, but the former technique was more sensitive and could identify differences that were not apparent by crossed immunoelectrophoresis.

Detection of hemophilia carriers during pregnancy.

The accuracy of hemophilia A carrier detection during pregnancy has been determined using combined measurement of VIII:CAg and VIIIR:Ag. These immunoassays detect determinants that are sufficiently stable in plasma that the assays could be done on frozen samples that had been obtained when women were seen for antenatal diagnosis studies (carrier women) or for routine prenatal care (controls). A linear discriminant was calculated that best separated the data for 32 normal women and 25 obligate carriers of the hemophilia gene. Twenty-three of 25 carriers (92%) and all 32 control women were correctly identified in this analysis. The overall classification accuracy (55/57, 96%) is comparable to that obtained by VIII:C and VIIIR:Ag measurements using freshly drawn blood samples in nonpregnant individuals. This study demonstrates that hemophilia A carriers can be detected during pregnancy with sufficient accuracy that the information may be used for genetic counseling.