Katherine M. Byrne

Satellite cell regulation following myotrauma caused by resistance exercise.

It is generally accepted that the primary mechanisms governing skeletal muscle hypertrophy are satellite cell activation, proliferation, and differentiation. Specific growth factors and hormones modulate satellite cell activity during normal muscle growth, but as a consequence of resistance exercise additional regulators may stimulate satellite cells to contribute to gains in myofiber size and number. Present knowledge of the regulation of the cellular, biochemical and molecular events accompanying skeletal muscle hypertrophy after resistance exercise is incomplete. We propose that resistance exercise may induce satellite cells to become responsive to cytokines from the immune system and to circulating hormones and growth factors. The purpose of this paper is to review the role of satellite cells and growth factors in skeletal muscle hypertrophy that follows resistance exercise.

Report of the Second Equine Leucocyte Antigen Workshop, Squaw Valley, California, July 1995

The final assignment of antibody clusters for leucocyte antigens and immunoglobulins, as described in detail in Sections 3 and 4, is summarized in Table 4. Together with other mAbs developed outside of ELAW II (Table 9) this pool of reagents represent a powerful array of tools for the study of equine immunity. The Second Equine Leucocyte Antigen Workshop made considerable advances in pursuing the objectives of establishing the specificities of mAbs and achieving consensus on the nomenclature for equine leucocyte and immunoglobulin molecules. Of equal importance, several productive collaborations were fostered among the participating laboratories and observers. Overall, enormous advances have been made in the past decade since mAbs specific for equine leucocyte antigens and immunoglobulins were first reported. There remains enormous scope and need for further studies of equine leucocyte antigens and immunoglobulins, both for the purposes of comparative immunology and for the good of the horse. In the future novel techniques will be required to develop reagents for specific target antigens such as the orthologues of the CD25 or CD45 isoforms. In studies of equine immunoglobulins the functional role of the IgG isotypes must be better established, reagents for IgE must be developed, and cloning of the immunoglobulin heavy chain genes will be essential if the complexities of the IgG sub-isotypes are to be elucidated. The tasks still facing the currently small group of equine immunologists throughout the world remain formidable, and will only be tackled successfully in a spirit of collaboration.

Dietary lutein stimulates immune response in the canine

The possible immuno-modulatory action of dietary lutein in dogs is not known. Female Beagle dogs (17-18-month old; 11.4+/-0.4kg body weight) were supplemented daily with 0, 5, 10 or 20mg lutein for 12 weeks. Delayed-type hypersensitivity (DTH) response to saline, phytohemagglutinin (PHA) and a polyvalent vaccine was assessed on Weeks 0, 6 and 12. Blood was sampled on Weeks 0, 2, 4, 8 and 12 to assess (1) lymphocyte proliferative response to PHA, concanavalin A (Con A), and pokeweed mitogen (PWM), (2) changes in peripheral blood mononuclear cell (PBMC) populations, (3) interleukin-2 (IL-2) production and (4) IgG and IgM production. After the completion of 12-week study, we continued to collect the blood weekly up to 17 weeks to evaluate the changes in immunoglobulin production upon first and second antigenic challenges on Weeks 13 and 15. Plasma lutein+zeaxanthin was undetectable in unsupplemented dogs but concentrations increased (P<0.05) rapidly on Week 2 in lutein-supplemented dogs. Thereafter, concentrations generally continued to increase in dose-dependent manner, albeit at a much slower rate. Dogs fed lutein had heightened DTH response to PHA and vaccine by Week 6. Dietary lutein increased (P<0.05) lymphocyte proliferative response to all three mitogens and increased the percentages of cells expressing CD5, CD4, CD8 and major histocompatibility complex class II (MHC II) molecules. The production of IgG increased (P<0.05) in lutein-fed dogs after the second antigenic challenge. Lutein did not influence the expression of CD21 lymphocyte marker, plasma IgM or IL-2 production. Therefore, dietary lutein stimulated both cell-mediated and humoral immune responses in the domestic canine.

Modulation of humoral and cell-mediated immune responses by dietary lutein in cats.

The immuno-modulatory role of dietary lutein in domestic cats is unknown. Female Tabby cats (10-month old; n=56) were supplemented daily for 12 weeks with 0, 1, 5 or 10mg lutein. Blood was collected on Weeks 0, 2, 4, 8 and 12 to assess the following: (1) mitogen-induced peripheral blood mononuclear cells (PBMCs) proliferation, (2) changes in PBMC subpopulations, (3) interleukin-2 (IL-2) production and (4) plasma immunoglobulin (Ig)G production. In addition, delayed-type hypersensitivity (DTH) response to concanavalin A (Con A) or a polyvalent vaccine was performed on Weeks 0, 6 and 12. Dietary lutein increased plasma lutein concentrations in a dose-dependent manner (p<0.001) and concentrations had not reached steady state after 12 weeks of feeding in cats given 5 or 10mg lutein. Concentrations of plasma retinol and alpha-tocopherol were not influenced by diet. The DTH response to vaccine but not to Con A increased (p<0.05) in a dose-dependent manner on Week 6. Compared to control, cats fed lutein also showed enhanced Con A- and pokeweed mitogen-stimulated PBMCs proliferation. Dietary lutein also increased the percentages of CD4+ and CD21+ lymphocytes on Week 12 but had no significant effect on pan T, CD8 and MHC class II markers. Plasma IgG was higher (p<0.05) in cats fed 10mg lutein on Weeks 8 and 12. These results support the immuno-modulatory action of lutein in domestic cats.

A standardized gating technique for the generation of flow cytometry data for normal canine and normal feline blood lymphocytes.

Flow cytometry is becoming a commonly used technique to characterize a variety of cells. It provides a powerful application to rapidly determine the relative percentages of T-lymphocyte subsets and B-lymphocytes. The effectiveness of its application, however, is dependent on standardization, especially in a clinical setting. Application of flow cytometry to veterinary diagnostics has been limited by the unavailability of reagents and by the unstandardized characterization of normal values using antibodies not commercially available, but typically provided through the generosity of other researchers. This paper presents a standardized gating protocol, and average values and ranges observed for normal canine and feline blood lymphocytes using commercially available antibodies to cell surface markers for CD5, CD3, CD4, CD8, MHC II, and B lymphocytes. The averages for these markers on gated lymphocytes were as follows: Canine CD5 83.3%, Canine CD4 45.0%, Canine CD8 28.8%, Canine MHC II 98.0%, Canine B Cell 12.9%, Canine CD4/CD8 ratio 1.87, Feline T lymphocytes 77.3%, Feline CD4 44.5%, Feline CD8 25.7%, Feline B Cell 24.1%, Feline CD4/CD8 Ratio 1.75. Normal values were also established for a mixed breed group of dogs, and old versus young dogs. This information will provide researchers and clinicians with a standardized protocol for gating, which establishes a basis for comparison between techniques, and a measure of phenotypic percentages for flow cytometry in normal dogs and cats based on this standardization and commercially available antibodies.

Equine infectious anaemia virus proteins with epitopes most frequently recognized by cytotoxic T lymphocytes from infected horses

Efficacious lentiviral vaccines designed to induce cytotoxic T lymphocytes (CTL) in outbred populations with a diverse repertoire of MHC class I molecules should contain or express multiple viral proteins. To determine the equine infectious anaemia virus (EIAV) proteins with epitopes most frequently recognized by CTL from seven horses infected for 0.5 to 7 years, retroviral vector-transduced target cells expressing viral proteins were used in CTL assays. Gag p15 was recognized by CTL from 100% of these infected horses. p26 was recognized by CTL from 86%, SU and the middle third of Pol protein were each recognized by 43%, TM by 29%, and S2 by 14%. Based on these results, it is likely that a construct expressing the 359 amino acids constituting p15 and p26 would contain epitopes capable of stimulating CTL in most horses.

The development and utility of a defined muscle and fat co-culture system

The utility of co-culture systems in defining the interactions between two different cell types has been well documented in the literature but is a relatively new tool for use in the study of cells derived from normal muscle tissue from meat animals. The majority of myonuclei in postnatal skeletal muscle cells (myofibers) result from satellite cell proliferation, differentiation and fusion with existing myofibers. As such, satellite cell culture systems that mimic postnatal myofibers have great potential for delineating the process of growth and repair of muscle mass. Other ways to simulate the environment of postnatal myofibers might include the development of co-culture systems using satellite cells, or satellite cell-derived myotubes, and other mesodermal cells commonly found associated with muscle tissue in vivo. This brief review describes our initial efforts to develop a defined satellite cell and preadipocyte co-culture system. We provide useful information about defined media requirements and requirements for proper cell orientation and growth on two different growth planes. We present summary data to suggest that differences were found between members of the insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) families of polypeptides, when conditioned media samples were analyzed from co-cultures composed of 3T3-L1 preadipocytes and satellite cells (with different propensities to undergo morphological fusion to form multinucleated myotubes). We also provide information about potential problems to avoid when initiating and conducting co-culture experiments. Such a co-culture system has application in the study of human obesity and also in the regulation of fat deposition in meat animals.

Traditional and emerging methods for analyzing cell activity in cell culture.

The selection of appropriate techniques to assay for markers of cell activity is important for obtaining optimal results in cell culture-based research. This paper is intended as a guide to many of the assays currently available and new techniques that have been recently introduced in the literature. This paper addresses both manual assay techniques, including the use of hemocytometers, phase contrast microscopy, cell staining, and the immunofluorescent antibody assay (IFA), and automated assays for cell activity, including stained optical density, proliferating cell nuclear antigen, creatine kinase assay, DNA quantification, electronic cell counting, flow cytometry, magnetic cell sorting, image analysis, chemiluminescence, radioisotope labeling, precursor incorporation, in-situ hybridization/ligand binding, and enzyme-linked immuno-culture assay (ELICA). Advantages/disadvantages and applicability of these assays to different areas of cell culture research are discussed, and guidelines for selecting an appropriate assay are suggested.

Ten commandments for preventing contamination of primary cell cultures.

Procedures for preventing contamination in primary cell cultures must be carefully defined and strictly followed in order to obtain healthy cells. Protocols have been developed and refined in our laboratory for establishing primary cultures of muscle and fat stem cells without contamination from a variety of animals. Contamination of cell cultures is not only frustrating, but is also very expensive both in time and loss of materials. Through the consistent use of proper aseptic techniques, most instances of contamination may be avoided. We suggest that the basic principles detailed here will find wide applicability in the culturing of primary cells without contamination from many different types of animals and tissues.

In vitro model of equine muscle regeneration

Equine satellite cells are responsible for muscle healing and regeneration in the mature horse. We describe the in vitro cell culture conditions required for clonal populations of equine satellite cells to undergo both proliferation and differentiation. Our hypothesis is that these in vitro conditions model regeneration of muscle and can be used to evaluate potential therapeutics. In this study, 2 areas of satellite cell response were tested: proliferation of clones induced by growth factors, and fusion induced by culture conditions. Equine satellite cell clones showed differences in their response to growth factors as well as accumulation of cellular protein concentrations. Equine satellite cells proliferate in response to both human and bovine FGF. IGF-1, a powerful mitogen of other satellite cell culture systems, was not as effective for inducing equine satellite cell proliferation. Protein concentrations were also measured in satellite cell cultures. Clones differed in cellular protein produced depending on growth conditions. Conditions inducing differentiation into myotubes was also determined for a 96 well assay and can be used to study the final stage of functioning muscle production. This in vitro model is the first step in identifying potential therapeutics to speed wound healing and promote muscle regeneration in horses.