Stem cell factor supports migration in canine mesenchymal stem cells

Adult Mesenchymal Stem Cells (MSC) are cells that can be defined as multipotent cells able to differentiate into diverse lineages, under appropriate conditions. These cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Initially discovered in bone marrow, MSC can now be isolated from a wide spectrum of adult and foetal tissues. Studies to evaluate the therapeutic potential of these cells are based on their ability to arrive to damaged tissues. In this paper we have done a comparative study analyzing proliferation, surface markers and OCT4, SOX9, RUNX2, PPARG genes expression in MSC cells from Bone marrow (BMMSC) and Adipose tissue (ASC). We also analyzed the role of Stem Cell Factor (SCF) on MSC proliferation and on ASCs metalloproteinases MMP-2, MMP-9 secretion. Healthy dogs were used as BMMSC donors, and ASC were collected from omentum during elective ovariohysterectomy surgery. Both cell types were cultured in IMDM medium with or without SCF, 10% Dog Serum (DS), and incubated at 38 °C with 5% CO2. Growth of BMMSCs and ASCs was exponential until 25–30 days. Flow citometry of MSCs revealed positive results for CD90 and negative for CD34, CD45 and MCH-II. Genes were evaluated by RT-PCR and metalloproteinases by zymografy. Our findings indicate morphological and immunological similarities as well as expression of genes from both origins on analyzed cells. Furthermore, SCF did not affect proliferation of MSCs, however it up-regulated MMP-2 and MMP-9 secretion in ASCs. These results suggest that metalloproteinases are possibly essential molecules pivoting migration.     

Inheritance, mode of inheritance, and candidate genes for primary hyperparathyroidism in Keeshonden

BACKGROUND: Primary hyperparathyroidism (PHPT) is caused by inappropriate secretion of parathyroid hormone (PTH) by autonomously functioning neoplastic or hyperplastic parathyroid "chief" cells. Keeshonden are thought to be over-represented in studies on canine PHPT, but no proof of heritability or mode of inheritance has been published. The canine disease clinically resembles human familial isolated hyperparathyroidism (FIHP). HYPOTHESIS: Primary hyperparathyroidism in Keeshonden is genetically transmitted and is caused by a mutation in 1 of 4 genes implicated in human FIHP: MEN1, CASR, HRPT2, or RET. ANIMALS: Pedigrees consisting of 1647 Keeshonden were created including 219 Keeshonden with known PHPT phenotypes (69 positive). DNA samples were obtained from 176 of the 219 Keeshonden (34 positive). METHODS: Heritability and mode of inheritance were determined by segregation analysis. Canine homologs to the human genes were identified. Exons and surrounding intron regions were sequenced and scanned for sense-altering polymorphisms or polymorphisms that segregated with the disease. Messenger RNA from a parathyroid tumor of an affected Keeshond was analyzed for polymorphisms and splice alterations. RESULTS: PHPT follows an autosomal dominant mode of inheritance in Keeshonden with possible age-dependent penetrance. No polymorphisms identified in the genes analyzed were associated with a change in predicted protein or in hypothesized splice sites. CONCLUSIONS AND CLINICAL IMPORTANCE: PHPT is an autosomal dominant, genetically transmitted disease in Keeshonden. Once the mutation locus is identified, genetic testing should quickly decrease the incidence of PHPT in this breed. It is unlikely that mutations in MEN1, CASR, HRPT2, or RET cause PHPT in Keeshonden.     

Canine bartonellosis: serological and molecular prevalence in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii

The purpose of this study was to determine the serological and molecular prevalence of Bartonella spp. infection in a sick dog population from Brazil. At the S?o Paulo State University Veterinary Teaching Hospital in Botucatu, 198 consecutive dogs with clinicopathological abnormalities consistent with tick-borne infections were sampled. Antibodies to Bartonella henselae and Bartonella vinsonii subsp. berkhoffii were detected in 2.0% (4/197) and 1.5% (3/197) of the dogs, respectively. Using 16S-23S rRNA intergenic transcribed spacer (ITS) primers, Bartonella DNA was amplified from only 1/198 blood samples. Bartonella seroreactive and/or PCR positive blood samples (n=8) were inoculated into a liquid pre-enrichment growth medium (BAPGM) and subsequently sub-inoculated onto BAPGM/blood-agar plates. PCR targeting the ITS region, pap31 and rpoB genes amplified B. henselae from the blood and/or isolates of the PCR positive dog (ITS: DQ346666; pap31 gene: DQ351240; rpoB: EF196806). B. henselae and B. vinsonii subsp. berkhoffii (pap31: DQ906160; rpoB: EF196805) co-infection was found in one of the B. vinsonii subsp. berkhoffii seroreactive dogs. We conclude that dogs in this study population were infrequently exposed to or infected with a Bartonella species. The B. henselae and B. vinsonii subsp. berkhoffii strains identified in this study are genetically similar to strains isolated from septicemic cats, dogs, coyotes and human beings from other parts of the world. To our knowledge, these isolates provide the first Brazilian DNA sequences from these Bartonella species and the first evidence of Bartonella co-infection in dogs.     

Effect of Density at Harvest on the Growth Performance and Profitability of Hatchery‐reared Spotted Rose Snapper,Lutjanus guttatus,Cultured in Floating Net Cages

This study evaluated the effect of the density at harvest on the performance and profitability of hatchery‐reared spotted rose snapper cultured in cages. The fish were stocked at harvest densities of 15, 20, and 22 kg/m³ in cages of 222 and 286 m³. More than 39,000 snapper fingerlings with an initial weight of 14 g were stocked. The fish were fed an extruded diet and cultured over a 360 d period. The thermal growth coefficient ranged from 0.04 to 0.05 and survival was 95% for all treatments, with the highest final weight (436.8 g) observed for fish reared at a density of 20 kg/m³. The allometric value b indicated that hatchery‐raised, cage‐cultured snapper were heavier than their wild counterparts. The major costs were feed (ranging from 44.7–45.9%), labor (22.4–32.6%), and seed costs (20.2–26.1%). The total production cost ranged from US$ 6.5 to US$ 7.5/kg. The baseline scenario was not economically feasible. However, a 10% increase in the sales price resulted in increases in the internal rate of return (183%) and net present value (US$ 97,628.9). These results suggest that L. guttatus has the potential for commercial production in cages.     

Synthesis and toxicological properties of some alkyl and aryl 3,4-methylenedioxyphenyl N,N′-thio-bis-N-methylcarbamates

A series of asymmetrical thio-bis-N-methylcarbamate derivatives were synthesised by combining 3,4-methylenedioxyphenyl N-methylcarbamate with different N-methylcarbamates. Some of these derivatives combined good insecticidal activity against the housefly with reduced acute oral toxicity to mice. The thio-bis-N-methylcarbamates obtained exhibited a lower anti-cholinesterase activity than the more toxic of the two carbamate moieties contained in each combination. The weak synergism of piperonyl butoxide on the insecticidal activity of the derivatives suggests the possibility of a self synergism phenomenon caused by the 3,4-methylenedioxyphenyl group.     

In vitro blastogenic responses of peripheral lymphocytes from marmosets to phytohemagglutinin and to autologous lymphocytes transformed by Epstein-Barr virus

White-lipped marmosets were evaluated for their cell mediated immune (CMI) response to EBV to determine the feasibility of CMI studies in marmoset models for EBV oncogenesis. The mitogen, cell concentrations, the length of incubation period and serum requirements were defined for in vitro lymphocyte stimulation tests. The level of response of each animal was dependent on the concentration of phytohemagglutinin-P (PHA-P) and was independent of cell densities employed. The rate of tritiated-thymidine incorporation by mononuclear cells due to PHA-P increased exponentially between 2–4 days. This test was reproducible for a given batch of PHA-P when the cells were cultured in the presence of 10% heat inactivated fetal bovine serum. The five white-lipped marmosets were seronegative for EBV antigens and did not show lymphocyte stimulation with EBV particles and EBV soluble antigen, but two of these animals exhibited significant stimulation with autologous lymphocytes transformed in vitro by B95-8 virus. Despite the limited amount of blood (3–4 ml) that could be obtained from each animal in a single bleeding, it was possible to perform multiple lymphocyte stimulation assays with the protocol used.     

Fertility and sperm quality of broiler breeder males infected with subgroup J avian leukosis virus

In order to assess the effects of subgroup J avian leukosis virus (ALV-J) on semen quality, broiler breeder males were separated by ALV-J status (ALV-J positive = POS, ALV-J negative = NEG) at 44 wk of age. Of the 249 males originally placed at 1 day of age, 101 (40.6%) died by 43 wk of age. Observations of tumor expression and high mortality suggest that many of the males that died prior to 44 wk of age were infected with ALV-J. From 47 to 56 wk of age, hens were inseminated every third week with 7.5 x 10(7) sperm. Fertility and hatch data were collected by incubating eggs laid during the 2 wk postinsemination (WPI). The number of sperm that penetrated the perivitelline membrane of the ovum was determined from eggs laid on the eighth day postinsemination. Sperm mobility index (SMI) was determined at 58 and 60 wk of age from all males producing semen. Whereas SMI and sperm hole penetration measurements indicated that the sperm quality from treatments POS and NEG were similar, fertility was significantly greater in the POS treatment during the first (89.0% vs. 79.0%) and second WPI (59.3% vs. 45.0%). However, because of numerically higher hatch of fertile from the NEG group, the percentage of hatch of eggs set was similar between groups. These data suggest that ALV-J status of caged males has no influence on sperm quality or hatchability of eggs.     

Dirofilaria repens infection in a dog: diagnosis and treatment with melarsomine and doramectin

Therapy of canine dirofilariois due to Dirofilaria repens is indicated for dogs suffering from clinical signs of this disease, such as dermal swelling, sub-cutaneous nodules and pruritus. It is also important in order to decrease the risk of infection to other dogs and humans in the vicinity of the infected animal when suitable mosquito vectors are present. Combined therapy with the arsenic adulticide melarsomine and the avermectin microfilaricidal doramectin was effective in clearing infection with D. repens in a dog. The number of microfilariae dropped from 17 microl(-1) blood pre-treatment to 7 microl(-1) following the first adulticide injection and reached 0 a day after the microfilaricidal administration. The dog remained negative for D. repens microfilaremia during a follow-up period of 90 days. Euthanasia and necropsy performed 3 months after the initiation of therapy due to a progressive neoplastic disease revealed no evidence of filariae.