Impact of source tissue and ex vivo expansion on the characterization of goat mesenchymal stem cells

Background

There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSCs) have been isolated and characterized from various species, but are poorly characterized in goats.

Results

Goat MSCs isolated from bone marrow (BM-MSCs) and adipose tissue (ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency (CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection. BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture, exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection.

Conclusions

Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.

Electronic supplementary material

The online version of this article (doi:10.1186/2049-1891-6-1) contains supplementary material, which is available to authorized users.     

Role of the “National Reference Centre for Genetically Modified Organisms (GMO) Detection” in the Official Control of Food and Feed

The National Reference Centre for Genetically Modified Organisms (GMO) detection was established in 2002 within the Istituto Zooprofilattico Sperimentale Lazio e Toscana, with the aim of providing scientific and technical support to the National Health System and to the Ministry of Health within the scope of the regulation of GMO use in food and feed.

The recently adopted EU legislation on GMOs (Regulation CE no. 1829/2003 and no. 1830/2003) introduced more rigorous procedures for the authorisation, labelling and analytical control of food and feed consisting, containing or derived from GMOs. The National Reference Centre, besides its institutional tasks as one of the laboratories of the Italian National Health System, collects and analyses data and results of the national official control of GMOs; carries out scientific research aimed at developing, improving, validating and harmonising detection and quantification methods, in cooperation with other scientific institutions, the Community Reference Laboratory and within the European Network of GMOs laboratories (ENGL); collaborates with the Ministry of Health in the definition of control programmes and promotes educational and training initiatives. Objectives defined for 2004–2006, activities in progress and goals already achieved are presented.

     

Comparison of the effects of firocoxib, carprofen and vedaprofen in a sodium urate crystal induced synovitis model of arthritis in dogs

A randomized, placebo-controlled, four-period cross-over laboratory study involving eight dogs was conducted to confirm the effective analgesic dose of firocoxib, a selective COX-2 inhibitor, in a synovitis model of arthritis. Firocoxib was compared to vedaprofen and carprofen, and the effect, defined as a change in weight bearing measured via peak ground reaction, was evaluated at treatment dose levels. A lameness score on a five point scale was also assigned to the affected limb. Peak vertical ground reaction force was considered to be the most relevant measurement in this study. The firocoxib treatment group performed significantly better than placebo at the 3 h post-treatment time point and significantly better than placebo and carprofen at the 7 h post-treatment time point. Improvement in lameness score was also significantly better in the dogs treated with firocoxib than placebo and carprofen at both the 3 and 7 h post-treatment time points.     

Retrobulbar teratoma causing exophthalmos in a cat

A 3-year female-neutered domestic shorthair cat presenting with exophthalmos and an ipsilateral subzygomatic soft tissue mass lesion is described. Magnetic resonance imaging of the mass was performed followed by complete surgical excision. The mass was determined to be a retrobulbar teratoma and complete resection was curative. Teratomas are rare germ-cell tumours that uncommonly form in extragonadal sites. A retrobulbar location has not been previously reported in the cat and should be considered a rare cause of exophthalmos in this species.     

Direct stimulation of the oxidative activity of isolated equine neutrophils by TNF-alpha and IL-1beta

The capacity of the two cytokines TNF-alpha and IL-1beta to directly stimulate the oxidative activity of polymorphonuclear neutrophils remains debated. The purpose of this study was to verify if a direct stimulation of equine neutrophils by TNF-alpha and IL-1beta was possible. Equine neutrophils were isolated from blood by discontinuous density gradient centrifugation. The cell viability after isolation was >98%. The neutrophils were used at 1.25 x 10(6) cells by assay, immediately after isolation. The oxidative activity of neutrophils was measured by luminol- or lucigenin-enhanced chemiluminescence (CL), and the CL was recorded for 60 min. TNF-alpha and IL-1beta were used at concentrations ranging from 0.001 to 100 ng (0.0017-167 ng ml(-1)) for 1.25 x 10(6) neutrophils, and added to the cells just before the CL measurement. Both cytokines highly stimulated the lucigenin-enhanced CL of equine neutrophils in a dose-dependent manner. TNF-alpha was already active at 0.001 ng and IL-1beta at 0.01 ng. The CL response obtained with TNF-alpha was maximal after 5 min and more pronounced with luminol than with lucigenin. With IL-1beta, the luminol-enhanced CL response of neutrophils was short-lived and inversely proportional to the cytokine concentration: the CL response returned to baseline after 12 min, and became even lower than the baseline value for 10 and 100 ng IL-1beta. As luminol (but not lucigenin) enters the cell, we hypothesized that a rapid intracellular consumption of the luminol molecules occurred, explaining the rapid and intense CL response. The choice of the CL enhancer used in previous CL studies of neutrophils stimulation by cytokines could perhaps explain that controversial results were reported. In conclusion, we demonstrated a direct activation of the oxidative activity of equine neutrophils by TNF-alpha and IL-1beta, which was dose-dependent and obtained with very low doses equivalent to the plasma concentrations measured for both cytokines in equine septic shock. TNF-alpha and IL-1beta can thus aggravate neutrophils oxidative activity during septic shock in horses.     

Evaluation of Salmonella-lytic properties of bacteriophages isolated from commercial broiler houses

Because of recent interest in bacteriophage therapy in poultry, information regarding the interaction of bacteriophages and potential host bacteria in the environment should be collected. The present studies were initiated with a rather typical commercial broiler integrator within the south-central United States to examine environmental Salmonella levels in two broiler complexes, attempt to isolate Salmonella-lytic bacteriophages, and elucidate a possible reason for differing apparent Salmonella prevalence. Significantly (P < 0.05) less Salmonella was isolated from houses in complex 1 (15/44 [34%] Salmonella-positive drag swabs) as compared to houses in complex 2 (22/24 [92%]). A total of seven Salmonella-lytic bacteriophages were isolated from Salmonella-positive environments, and two bacteriophages were isolated from a single Salmonella-negative house. During the initial bacteriophage isolation, individual bacteriophages did not replicate in the Salmonella host isolated from the same environment, and lysis of additional Salmonella hosts relied on high numbers of bacteriophage to be present. This suggests that the presence of these bacteriophages in the environment of a commercial broiler house had little to no effect on the presence of Salmonella. This study highlights the need to find additional bacteriophage sources, more effective isolation methods, and more innovative approaches to using bacteriophages to treat enteric disease.     

Comparative analysis of survivin expression in untreated and relapsed canine lymphoma

BACKGROUND: Survivin, a member of the inhibitor of apoptosis protein family, has a dual role in tumor cell proliferative and antiapoptotic pathways. Survivin expression has been shown to be a negative prognostic factor in several cancers of humans, including B-cell non-Hodgkin's lymphoma. HYPOTHESES: High survivin expression will be a negative prognostic factor in dogs with lymphoma (LSA) treated with chemotherapy. In addition, survivin expression will be upregulated in relapsed canine LSA when compared with patient-matched, pretreatment biopsies. ANIMALS: Thirty-one client-owned dogs with stage IIIa or IVa LSA. METHODS: Retrospective evaluation of survivin immunoreactivity was performed on pretreatment lymph node biopsies and patient-matched samples obtained from dogs at relapse after being treated with an abbreviated CHOP-based protocol. RESULTS: In this population of dogs presenting with stage IIIa or IVa B-cell LSA, those dogs that had high survivin immunoreactivity scores had a significantly (P < .01, hazard ratio = 0.30) shorter median disease-free interval than did dogs with low survivin immunoreactivity scores (171 days versus 321 days, respectively). Survivin immunoreactivity was not significantly different in relapsed canine LSA when compared with patient-matched, pretreatment biopsies. CONCLUSIONS AND CLINICAL IMPORTANCE: Survivin expression is a negative prognostic factor that can predict early treatment failure of dogs that present with stage IIIa or IVa, B-cell LSA when treated with a CHOP-based protocol.     

Risk factors for epiploic foramen entrapment colic: an international study

REASON FOR PERFORMING STUDY: Epiploic foramen entrapment (EFE) is one of the most common causes of small intestinal strangulation in the horse. Identification of risk factors would generate hypotheses about causation and may suggest preventive strategies. HYPOTHESIS: Horses exhibiting certain behavioural patterns and those exposed to particular management practices are at increased risk of EFE. METHODS: A matched case-control study was conducted on EFE cases admitted to hospitals in the UK, Ireland and USA. Data on 109 cases and 310 control horses were obtained by telephone questionnaire and conditional logistic regression was used to identify associations between horse- and management-level variables and the risk of EFE. RESULTS: Crib-biting/windsucking behaviour was strongly associated with increased risk of EFE (OR 67.3, 95%CI 15.3-296.5). A history of colic in the previous 12 months (OR 4.4, 95%CI 1.5-12.7) and horses of greater height (OR/cm 1.05, 95%CI 1.01-1.08) were also at increased risk. The person(s) responsible for horses' daily care (nonowner/relative/spouse OR 5.5, 95%CI 2.3-13.3) and a number of behavioural features, including response to a stimulus causing fright (easily frightened OR 0.4, 95%CI 0.1-1.0) or excitement (sweats up easily/occasionally OR 0.3, 95%CI 0.1-0.8), reaction to their surroundings (inquisitive OR 0.4, 95%CI 0.2-0.8) and feeding behaviour when stressed (goes off food in full/part OR 0.3, 95%CI 0.1-1.0) were also associated with altered risk of EFE. CONCLUSIONS: The association between horses of greater height and those with a previous history of colic and increased risk of EFE suggests that some horses may be inherently predisposed to EFE. Furthermore, a behavioural pattern has been characterised that is common to horses at increased risk of EFE. Further research is required to investigate the causal pathway linking behavioural traits with gastrointestinal dysfunction and to determine whether behavioural modification reduces the risk of EFE. POTENTIAL RELEVANCE: The findings of the present study have relevance to horses in the UK, Ireland and USA.     

Effect of dietary supplementation with selenium-enriched yeast or sodium selenite on selenium tissue distribution and meat quality in beef cattle

The objective was to determine the concentration of total Se and the proportion of total Se comprised as selenomethionine (SeMet) and selenocysteine (SeCys) in postmortem tissues of beef cattle offered diets containing graded additions of selenized enriched yeast (SY; Saccharomyces cerevisiae CNCM I-3060) or sodium selenite (SS). Oxidative stability and tissue glutathione peroxidase (GSH-Px) activity of edible muscle tissue were assessed 10 d postmortem. Thirty-two beef cattle were offered, for a period of 112 d, a total mixed ration that had been supplemented with SY (0, 0.15, or 0.35 mg of Se/kg of DM) or SS (0.15 mg of Se/kg of DM). At enrollment (0 d) and at 28, 56, 84, and 112 d following enrollment, blood samples were taken for Se and Se species determination, as well as whole blood GSH-Px activity. At the end of the study beef cattle were killed and samples of heart, liver, kidney, and skeletal muscle (LM and psoas major) were retained for Se and Se species determination. Tissue GSH-Px activity and thiobarbituric acid reactive substances were determined in skeletal muscle tissue (LM only). The incorporation into the diet of ascending concentrations of Se as SY increased whole blood total Se and the proportion of total Se comprised as SeMet, as well as GSH-Px activity. There was also a dose-dependent response to the graded addition of SY on total Se and proportion of total Se as SeMet in all tissues and GSH-Px activity in skeletal muscle tissue. Furthermore, total Se concentration of whole blood and tissues was greater in those animals offered SY when compared with those receiving a comparable dose of SS, indicating an improvement in Se availability and tissue Se retention. Likewise, GSH-Px activity in whole blood and LM was greater in those animals offered SY when compared with those receiving a comparable dose of SS. However, these increases in tissue total Se and GSH-Px activity appeared to have little or no effect in meat oxidative stability.     

Milk yield of primiparous beef cows from three calving systems and varied weaning ages

Primiparous beef cows produced in 3 calving systems were used in a 2-yr study with a completely random design to measure milk yield throughout a 190-d lactation (2002, n = 20; 2003, n = 24 per calving system). Calving occurred in late winter (average calving date = February 4 +/- 2 d), early spring (average calving date = March 30 +/- 2 d), and late spring (average calving date = May 26 +/- 1 d). Additionally, cows used in this study had been weaned at varied ages as calves, creating 6 dam treatments. Dam age at weaning was 140 (late spring), 190 (late winter, early spring, late spring), or 240 (late winter, early spring) d of age. Milk production was measured by using the weigh-suckle-weigh technique at an average of 20, 38, 55, 88, 125, 163, and 190 d in milk. Milk yield for the 190-d lactation period was calculated as area under the curve by trapezoidal summation. Data were analyzed with a model containing treatment, year, and their interaction. Orthogonal contrasts were used to separate effects when treatment was significant (P < 0.10). Total milk yield did not differ (P = 0.42) between cows in the late winter and early spring systems, but cows in the late spring system tended to differ (P = 0.09) from the average of the other 2 systems. Cows in the late spring calving system had increased milk yield in 2002 and lesser milk yield in 2003 compared with the other calving systems (treatment x year interaction, P < 0.001). Cows born in late spring that had been weaned at 140 d of age produced more (P = 0.05) total milk than those weaned at 190 d of age. Peak milk yield was affected (P < 0.001) by treatment and showed a treatment x year interaction (P = 0.006). Day of peak lactation differed among treatments (P = 0.002), with cows in the late winter system peaking later (P = 0.007) than early spring cows, and late spring cows peaking earlier (P = 0.004) than the average of late winter and early spring cows. The average date of peak lactation was May 4 for the late winter system, May 31 for the early spring system, and July 19 for the late spring system. Calf ADG differed (P < 0.001) for the late spring system compared with the average of the late winter and early spring systems, but the relationship interacted with year (P < 0.001). Cow BW and BW change differed among treatments (P < 0.004), with much of the difference associated with the amount of milk produced or the timing of peak lactation. Season of calving affects milk yield of primiparous cows grazing Northern Great Plains rangelands and ADG of their calves.