Systems Genetics of Metabolism: The Use of the BXD Murine Reference Panel for Multiscalar Integration of Traits.

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.

Effects of pioglitazone on renal calcium excretion.

Context: Glitazones increase fracture risk in long-term users and in postmenopausal women. Studies have demonstrated deleterious effects of glitazones on bone metabolism. Glitazones also have direct renal tubular effects increasing sodium reabsorption. We hypothesized that glitazones may also regulate renal calcium excretion. Design: In this double-blind, randomized, placebo-controlled, four-way, crossover study, we examined the effects of pioglitazone (45 mg/d for 6 wk) or placebo on renal calcium and phosphate excretion and PTH levels during different sodium intakes in 16 individuals (eight with type 2 diabetes and eight with essential hypertension). Results: Pioglitazone had no effect on corrected plasma calcium and phosphate levels but decreased significantly the alkaline phosphatase and PTH levels. Pioglitazone induced on average a 45% increase in urinary calcium excretion. The fractional excretion of calcium rose to the same extent, suggesting a glomerular filtration rate-independent effect. Sodium intake did not influence the calciuric effect of pioglitazone. Changes in diurnal and nocturnal calciuria were similar. There was no effect of pioglitazone on phosphate excretion. Conclusion: Pioglitazone decreases PTH levels and increases urinary calcium excretion, independently from changes in glomerular filtration rate and from the sodium load, suggesting an inhibitory effect of pioglitazone on the tubular reabsorption of calcium. These effects may contribute to the increased fracture risk with glitazone treatment.

Temozolomide combined with bevacizumab in metastatic melanoma. A multicenter phase II trial (SAKK 50/07)

Background: Single agent DTIC is the standard therapy for metastatic melanoma (MM) with response rates of 5−20%. Temozolomide (Tem) as an oral drug has shown equal efficacy in phase III trials. Preclinical models have shown an inhibitory effect for bevacizumab (Bev) on the proliferation of melanoma cells as well as on sprouting endothelial cells. Therefore, a therapeutic approach that combines angiogenesis inhibitors with cytotoxic agents may provide clinical benefit in MM. Methods: Design: Multicenter phase II trial. Primary endpoint: Clinical benefit (CR, PR and SD) at 12 weeks; secondary endpoints: best overall response by RECIST, response duration, progression free survival, adverse events, survival after 6 months and overall survival. Sample size was calculated according to Simon's two stage optimal design (5% significance level and 80% power) with an overall sample size of 62 patients (pts) to test H0: 20% versus H1: 35% rate of clinical benefit. Response assessment was done every 6 weeks (3 cycles). Eligibility: Stage IV MM, ECOG PS 0−2, no prior treatment for metastatic disease. Treatment regimen: One cycle consisted of Tem at 150 mg/m2 days 1−7 po and Bev at 10 mg/kg day 1 over 30 min iv and was repeated every 2 weeks until progression or unacceptable toxicity. Results: Between January 2008 and April 2009, 62 pts (40 male/22 female) at a median age of 61 years (range 30−86) with stage IV (M1a:4, M1b:12, M1c:46) melanoma were enrolled in 9 centers. The first 50 pts, who received 415 cycles are included in this interim report. The overall response rate was 26% (CR: 1 pt, PR: 12 pts; PR not confirmed yet in 3 pts), and 44% (22 pts) had stable disease over 1.5−7.5 months (median: 3). Only 30% (15 pts) had disease progression at the first evaluation at week 6. The hematological grade 3/4 toxicities according to NCI CTAE 3.0 were thrombocytopenia 10% (5 pts), neutropenia 8% (4 pts), lymphopenia and leucocytopenia each 2% (1 pt). Cumulative non-hematological toxicities grade 3/4 were nausea and fatigue each 6% (3 pts), hypertension, vomiting and hemorrhage, each 4% (2 pts), thrombosis/embolism, infection, constipation, anorexia, elevation of alkaline phosphatase, bilirubin, GGT, ALT and AST each 2% (1 pt). Conclusion: In metastatic melanoma the combination of Tem/Bev is a safe regimen with a promising efficacy and few grade 3/4 toxicities. Updated results of all 62 pts will be presented.

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

Developmental changes in the heavy subunit of neurofilaments in the corpus callosum of the cat.

In the corpus callosum of the cat, the heavy subunit of neurofilaments (NFH) can be demonstrated with the monoclonal antibody NE14, as early as P11, not at P3, and only in a few axons. At P18-19 and more markedly at P29, many more callosal axons have become positive to NE14 and this is similar to what is found in the adult. In contrast, callosal axons become positive to the neurofilament antibody SMI-32 only between P29 and P39 and remain positive in the adult. Treatment with alkaline phosphatase prevents axonal staining with NE14, but results in SMI-32 staining of a few callosal axons as early as P11, but not at P3. Between P11 and P19 the number of axons stained with SMI-32 after alkaline phosphatase treatment increases, in parallel with that of axons stained with NE14. Thus NE14 appears to recognize a phosphorylated form of NFH, while SMI-32 appears to recognize an epitope of NFH which is either masked by phosphate or inaccessible until between P29 and P39, unless the tissue is treated with alkaline phosphatase. These two forms of NFH appear towards the end of the period of massive developmental elimination of callosal axons. They are also synchronous with changes in the spacing of neurofilaments quantified in a separate ultrastructural study. These cytoskeletal changes may terminate the juvenile-labile state of callosal axons and allow further axial growth of the axon.

Changes of MAP2 phosphorylation during brain development.

The microtubule-associated protein MAP2 is essential for development of early neuronal morphology and maintenance of adult neuronal morphology. Several splice variants exist, MAP2a-d, with a lack of MAP2a in cat brain. MAP2 is widely used as a neuronal marker. In this study we compared five monoclonal antibodies (MAbs) against MAP2. They show differences in the immunocytochemical distribution of MAP2 isoforms during development of the visual cortex and cerebellum of the cat. Local and temporal differences were seen with MAb AP18, an antibody directed against a phosphorylation-dependent epitope near the N-terminal end. In large pyramidal dendrites in visual cortex, the AP18 epitope remained in parts immunoreactive after treatment with alkaline phosphatase. Three MAbs, AP14, MT-01, and MT-02, recognized the central region of the MAP2b molecule, which is not present in MAP2c and 2d, and reacted with phosphorylation-independent epitopes. During the first postnatal week the immunostaining in cerebellum differed between antibodies in that some cellular elements in external and internal granular layers and Purkinje cells were stained to various degrees, whereas at later stages staining patterns were similar. At early stages, antibody MT-02 stained cell bodies and dendrites in cerebral cortex and cerebellum. With progressing maturation, immunoreactivity became restricted to distal parts of apical dendrites of pyramidal cells and was absent from perikarya and finer proximal dendrites in cortex. MT-02 did not stain MAP2 in cerebellum of adult animals. This study demonstrates that the immunocytochemical detection of MAP2 depends on modifications such as phosphorylation and conformational changes of the molecule, and that MAP2 staining patterns differ between MAbs. Phosphorylation and specific conformations in the molecule may be essential for modulating function and molecular stability of MAP2, and monoclonal antibodies against such sites may provide tools for studying the functional role of modifications.

Phosphorylation-dependent dimerization and subcellular localization of islet-brain 1/c-Jun N-terminal kinase-interacting protein 1.

Islet-brain 1 [IB1; also termed c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1] is involved in the apoptotic signaling cascade of JNK and functions as a scaffold protein. It organizes several MAP kinases and the microtubule-transport motor protein kinesin and relates to other signal-transducing molecules such as the amyloid precursor protein. Here we have identified IB1/JIP-1 using different antibodies that reacted with either a monomeric or a dimeric form of IB1/JIP-1. By immunoelectron microscopy, differences in the subcellular localization were observed. The monomeric form was found in the cytoplasmic compartment and is associated with the cytoskeleton and with membranes, whereas the dimeric form was found in addition in nuclei. After treatment of mouse brain homogenates with alkaline phosphatase, the dimeric form disappeared and the monomeric form decreased its molecular weight, suggesting that an IB1/JIP-1 dimerization is phosphorylation dependent and that IB1 exists in several phospho- forms. N-methyl-D-aspartate receptor activation induced a dephosphorylation of IB1/JIP-1 in primary cultures of cortical neurons and reduced homodimerization. In conclusion, these data suggest that IB1/JIP-1 monomers and dimers may differ in compartmental localization and thus function as a scaffold protein of the JNK signaling cascade in the cytoplasm or as a transcription factor in nuclei.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Influence of distal ileum exclusion on hepatic and renal functions in presence of extrahepatic cholestasis

OBJECTIVE: To verify whether the ileal exclusion interferes with liver and kidney functional changes secondary to extrahepatic cholestasis.METHODS: We studied 24 rats, divided into three groups with eight individuals each: Group 1 (control), Group 2 (ligation of the hepatic duct combined with internal biliary drainage), and Group 3 (bile duct ligation combined with internal biliary drainage and exclusion of the terminal ileum). Animals in Group 1 (control) underwent sham laparotomy. The animals of groups 2 and 3 underwent ligation and section of the hepatic duct and were kept in cholestasis for four weeks. Next, they underwent an internal biliary bypass. In Group 3, besides the biliary-enteric bypass, we associated the exclusion of the last ten centimeters of the terminal ileum and carried out an ileocolic anastomosis. After four weeks of monitoring, blood was collected from all animals of the three groups for liver and kidney biochemical evaluation (albumin, ALT, AST, direct and indirect bilirubin, alkaline phosphatase, cGT, creatinine and urea).RESULTS: there were increased values of ALT, AST, direct bilirubin, cGT, creatinine and urea in rats from Group 3 (p < 0.05).CONCLUSION: ileal exclusion worsened liver and kidney functions in the murine model of extrahepatic cholestasis, being disadvantageous as therapeutic procedure for cholestatic disorders.

Evaluation of an enzyme-linked immunosorbent assay to detect antibodies against Anaplasma marginale

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Anaplasma marginale using a partially soluble antigen prepared from semi-purified initial bodies from erythrocytes with 80.0% of rickettsiaemia. This technique utilized alkaline phosphatase and p-nitrophenyl phosphate as reaction indicators. The high sensitivity (100.0%) was confirmed with sera from 100 calves experimentally-infected with A. marginale. All of these animals showed seroconversion before or at the same time of the first rickettsiaemia or even when it was not detected. Also the elevated specificity (94.0%) was confirmed by the low percentage of cross-reactions with sera from animals experimentally-infected with Babesia bigemina and Babesia bovis (1.4 and 6.6%, respectively). Performances of ELISA and indirect fluorescent antibody test (IFAT) with 324 sera from enzootically stable area did not show statistical difference (P>0.05), since the former showed 96.9% and the latter 97.2% of positive reactions. The advantage of this ELISA is a shorter execution time than others developed until now, allowing more samples to be analyzed.

Serum biochemistry of 4-day-old ostriches (Struthio camelus)

An analysis was made of 30 four-day-old ostriches to evaluate their protein, metabolite, mineral, and serum enzyme profiles, to correlate them with the birds' sex. The values obtained were: Total proteins 3.59±0.72g/dL, albumin 1.04±0.14g/dL, globulins 2.51±0.56g/dL, A:G ratio 0.43± 0.07, total cholesterol 615.10±101.15mg/dL, high density lipoprotein cholesterol (HDL-C) 132.72±20.33mg/dL, low density lipoprotein cholesterol (LDL-C) 454.93±90.81mg/dL, very low density lipoprotein cholesterol (VLDL-C) 27.45±9.96mg/dL, triglycerides 137.23±49.78mg/dL, uric acid 6.24±2.15mg/dL, urea 18.27±12.33mg/dL, creatinine 0.30±0.04mg/dL, total calcium 9.38± 0.76mg/dL, ionized calcium 7.17±0.64mg/dL, phosphorus 6.96±0.91mg/dL, Ca:P ratio 1.37±0.21, iron 24.74±13.02µg/dL, sodium 142.03±6.17mEq/L, chlorides 109.59± 4.99mEq/L, aspartate aminotransferase (AST) 200.67±31.42 U/L, alanine aminotransferase (ALT) 3.90±1.92 U/L, γ-glutamyltransferase (GGT) 1.18±0.73 U/L, alkaline phosphatase (ALP) 597.30± 231.36 U/L, and creatine kinase (CK) 2348.30±755.60 U/L. Males and females showed significant differences in total proteins, globulins, alkaline phosphatase, A:G ratio, and uric acid.

Laparoscopic versus open splenectomy in dogs

In the last few years, the use of laparoscopy in veterinary medicine has expanded and consequently so was the need for studies that establish the advantages, disadvantages and possible complications of each procedure. The purpose of the current study was to describe a laparoscopic splenectomy technique and the alterations due to this access, and compare it to the open procedure in dogs. A total of 15 healthy female mongrel dogs were used, with mean weight of 17.4±2.5kg. The animals were distributed into three groups: Group IA of open splenectomy (laparotomy) using double ligation of the vessels of the splenic hilum with poliglicolic acid, Group IB of open splenectomy (laparotomy) with bipolar electrocoagulation of the splenic hilum, and Group II of laparoscopic access with bipolar electrocoagulation of the splenic hilum. Operative time, blood loss, size of incisions, complications during and after surgery were evaluated. Other parameters included pain scores, white blood cell (WBC) counts and postoperative serum concentrations of alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), C-reactive protein (CRP), glucose and cortisol. No differences were found in the evaluation of parameters between both open splenectomy techniques employed. Laparoscopic access presented significant differences (p<0,05) when compared with open surgery: Longer operative time, smaller abdominal access, decrease in blood loss, lower concentrations of CRP, higher levels of CK and ALP, and lower scores in the pain scale. Laparoscopic surgery showed fewer complications of the surgical wound. No significant differences were observed between groups in the postoperative temperature, WBC, ALT, cortisol and glucose concentrations. In conclusion, the laparoscopic technique is useful for splenectomy in dogs, being advantageous in terms of blood loss, surgical stress and surgical wounds. However, it expends more operative time and causes transitory increase in hepatic and muscular enzymes.

Nutritional fibrous osteodystrophy in goats

Seven out of 25 goats from a southern Brazilian flock developed nutritional fibrous osteodystrophy. Affected animals were younger than 1 year of age and were confined in stalls and fed a concentrate ration containing 1:6 calcium:phosphorus ratio. The remaining flock (35 goats) was managed at pasture and showed no disease. Clinical signs were characterized by mandibular and maxillary enlargements, varying degrees of mouth opening and protruding tongue, dyspnea, apart of abnormalities of prehension and mastication. Affected animals had increased seric levels of phosphorus and parathormone, as well as higher alkaline phosphatase activity. Postmortem examination on three succumbed goats revealed bilateral enlargement of the maxilla and mandibula, and loose teeth, apart of multiple incomplete rib fractures in one of them. Severe diffuse proliferation of loose connective tissue surrounded the osteoid trabeculae, many of which were partially or completely non-mineralized. Mineralized osteoid trabeculae showed osteoclasts in the Howship's lacunae.

Passive immunity transfer and serum constituents of crossbred calves

Passive immunity transfer (PIT) evaluation is an essential tool for the maintenance of healthy calves during the first months of life. Since lactation number and breed have been proven to influence immunoglobulin levels in colostrum, the aim of this study was to evaluate PIT from primiparous and multiparous Canchim cows to their calves. Blood samples were collected from the calves before colostrum intake and 1, 2, 7, 15 and 30 days thereafter, while colostrum samples from the cows were taken immediately after parturition. Activities of gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), and concentrations of total protein, albumin, globulins, immunoglobulin A (IgA), immunoglobulin G (IgG), total and ionized calcium, inorganic phosphorus, magnesium, sodium and potassium were evaluated in calves' serum and activities of GGT and ALP and concentrations of total protein, IgA and IgG were assessed in cow's colostrum whey. Immunoglobulins concentrations were evaluated by electrophoresis in polyacrylamide gels. Serum biochemistry evaluations revealed an increase in gamma-glutamyl transferase and alkaline phosphatase activities and in total protein, globulins, immunoglobulin A and immunoglobulin G levels in calves' serum after colostrum intake. Only total protein and light chain immunoglobulin G levels in colostrum whey were affected by the cows' lactation number. Phosphorus and magnesium levels in blood serum increased after colostrum intake, while sodium and potassium levels oscillated in the experimental period. PIT was influenced by the cows' lactation number but was efficient in both groups.