Tenogenic induction of equine mesenchymal stem cells by means of growth factors and low-level laser technology

Tendons regenerate poorly due to a dense extracellular matrix and low cellularity. Cellular therapies aim to improve tendon repair using mesenchymal stem cells and tenocytes; however, a current limitation is the low proliferative potential of tenocytes in cases of severe trauma. The purpose of this study was to develop a method useful in veterinary medicine to improve the differentiation of Peripheral Blood equine mesenchymal stem cells (PB-MSCs) into tenocytes. PB-MSCs were used to study the effects of the addition of some growth factors (GFs) as TGFβ3 (transforming growth factor), EGF2 (Epidermal growth factor), bFGF2 (Fibroblast growth factor) and IGF-1 (insulin-like growth factor) in presence or without Low Level Laser Technology (LLLT) on the mRNA expression levels of genes important in the tenogenic induction as Early Growth Response Protein-1 (EGR1), Tenascin (TNC) and Decorin (DCN). The singular addition of GFs did not show any influence on the mRNA expression of tenogenic genes whereas the specific combinations that arrested cell proliferation in favour of differentiation were the following: bFGF2 + TGFβ3 and bFGF2 + TGFβ3 + LLLT. Indeed, the supplement of bFGF2 and TGFβ3 significantly upregulated the expression of Early Growth Response Protein-1 and Decorin, while the use of LLLT induced a significant increase of Tenascin C levels. In conclusion, the present study might furnish significant suggestions for developing an efficient approach for tenocyte induction since the external administration of bFGF2 and TGFβ3, along with LLLT, influences the differentiation of PB-MSCs towards the tenogenic fate.     

Immunological and pathological investigations in equine experimental uveitis

Background

The pathogenic mechanism of equine recurrent uveitis (ERU) is still poorly defined and many variations between experimental animal models and spontaneous disease exist.

Objectives

The aim of our study was to investigate if Th17 cell-mediated response plays role in the pathogenesis of the used experimental model in horses and to reveal its pathological findings.

Methods

Experimental uveitis was induced in 6 healthy horses. The concentrations of retinal autoantigen CRALBP and IL-17 were measured using ELISA in aqueous humor and vitreous body of the 12 inflamed eyes as well as in 12 control non-inflamed eyes taken from 6 horses in slaughter house. After centrifugation of the two eye media, smears were prepared and cytological investigation was performed. Tissue specimens were taken from all eye globes and were submitted to histopathological investigation.

Results

CRALBP and IL-17 concentrations were significantly elevated in eye media of horses with experimental uveitis in comparison with controls. Cytological and histopathological findings corresponded to the changes characteristic of chronic immune-mediated inflammation with mononuclear cell infiltration of uvea, choroid, retina, and eye media as well as severe retinal destruction.

Conclusions

Our study demonstrated the involvement of the retinal autoantigen CRALBP as well as IL-17 in the pathogenesis of experimental uveitis in horses. These findings suggests that this experimental uveitis in horses may serve as a suitable animal model for investigation of IL-17- mediated immune response during spontaneous autoimmune uveitis in horses as well as in humans.

     

Impaired female fertility in tubulointerstitial antigen-like 1-deficient mice

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a matricellular protein. Previously, we demonstrated that Tinagl1 expression was restricted to extraembryonic regions during the postimplantation period and detected marked expression in mouse Reichert’s membranes. In uteri, Tinagl1 is markedly expressed in the decidual endometrium during the postimplantation period, suggesting that it plays a physical and physiological role in embryo development and/or decidualization of the uterine endometrium during pregnancy. In the present study, in order to determine the role of Tinagl1 during embryonic development and pregnancy, we generated Tinagl1-deficient mice. Although Tinagl1^–/– embryos were not lethal during development to term, homologous matings of Tinagl1^–/– females and Tinagl1^–/– males showed impaired fertility during pregnancy, including failure to carry pregnancy to term and perinatal lethality. To examine ovarian function, ovulation was induced with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); the number of ovulated oocytes did not differ between Tinagl1^–/– and Tinagl1^flox/flox. In vitro fertilization followed by embryo culture also demonstrated the normal developmental potential of Tinagl1-null embryos during the preimplantation period. Our results demonstrate that Tinagl1 deficiency affects female mice and results in subfertility phenotypes, and they suggest that although the potential of Tinagl1^–/– oocytes is normal, Tinagl1 is related to fertility in adult females but is not essential for either fertilization or preimplantation development in vitro.     

Isolation and chemiluminescent properties of ferret (Mustela putorius furo) polymorphonuclear cells

Ferret polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation. Using a 50% Percoll solution (density=1.066), PMNs and PBMCs were successfully isolated after centrifugation; the purities of the PMNs and PBMCs were 94.2% and 95.6%, respectively. To evaluate the function of isolated ferret PMNs, we measured the superoxide generation with a MCLA-dependent chemiluminescence assay. The isolated ferret PMNs responded to phorbol 12-myristate 13-acetate (PMA) with kinetics similar to that of human PMNs. The ferret PMNs did not respond to N-formyl-Met-Leu-Phe (fMLF), unlike human PMNs, which rapidly responded. Thus, authors established a method for the rapid separation of highly purified populations of functional PMNs from the whole blood of ferrets.     

Comparison of dendritic cell-mediated immune responses among canine malignant cells

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.     

Using midday stem water potential for scheduling deficit irrigation in mid–late maturing peach trees under Mediterranean conditions

Irrigation techniques that reduce water applications are increasingly applied in areas with scarce water resources. In this study, the effect of two regulated deficit irrigation (RDI) strategies on peach [Prunus persica (L.) Batsch cv. “Catherine”] performance was studied over three growing seasons. The experimental site was located in Murcia (SE Spain), a Mediterranean region. Two RDI strategies (restricting water applications at stage II of fruit development and postharvest) based on stem water potential (Ψ_s) thresholds (?1.5 and ?1.8 MPa during fruit growth and ?1.5 and ?2.0 MPa during postharvest) were compared to a fully irrigated control. Soil water content (θ_v), Ψ_s, gas exchange parameters, vegetative growth, crop load, yield and fruit quality were determined. RDI treatments showed significantly lower values of θ_v and Ψ_s than control trees when irrigation water was restricted, causing reductions in stomatal conductance and photosynthesis rates. Vegetative growth was reduced by RDI, as lower shoot lengths and pruning weights were observed under those treatments when compared to control. However, fruit size and yield were unaffected, and fruit quality was slightly improved by RDI. Water savings from 43 to 65 % were achieved depending on the year and the RDI strategy, and no negative carryover effect was detected during the study period. In conclusion, RDI strategies using Ψ_s thresholds for scheduling irrigation in mid–late maturing peach trees under Mediterranean conditions are viable options to save water without compromising yield and even improving fruit quality.     

Measured and estimated water use and crop coefficients of grapevines trained to overhead trellis systems in California’s San Joaquin Valley

The use of overhead trellis systems for the production of dry-on-vine (DOV) raisins and table grapes in California is expanding. Studies were conducted from 2006 to 2009 using Thompson Seedless grapevines grown in a weighing lysimeter trained to an overhead arbor trellis and farmed as DOV raisins for the first two years and for use as table grapes thereafter. Maximum canopy coverage for the two lysimeter vines across years was in excess of 80 %. Seasonal (15 March–31 October) evapotranspiration for the lysimeter vines (ET_Lys) was 952 mm in 2007 (farmed as DOV raisins) and 943 and 952 mm (when farmed as table grapes). The maximum crop coefficient (K _cLys) across all 4 years ranged from 1.3 to 1.4. These maximum values were similar to those estimated using the relationship where K _c is a function of the amount of shaded area measured beneath the canopy at solar noon (K _c = 0.017 × percent shaded area). Covering the lysimeter’s soil surface with plastic (and then removing it) numerous times during the 2009 growing season (1 June–14 September) reduced ET_Lys from an average of 6.4 to 5.6 mm day^?1 and the K _c from 1.07 to 0.93. A seasonal basal K _c (K _cb) was calculated for grapevines using an overhead trellis system with a 13 % reduction in the K _cLys across the growing season.     

Crop response of drought-tolerant and conventional maize hybrids in a semiarid environment

In the High Plains, corn (Zea mays L.) is an important commodity for livestock feed. However, limited water resources and drought conditions continue to hinder corn production. Drought-tolerant (DT) corn hybrids could help maintain high yields under water-limited conditions, though consistent response of such hybrids is unverified. In this two-year study, the effects of three irrigation treatments were investigated for a DT and conventional maize hybrid, Pioneer AQUAMax P0876HR and Pioneer 33Y75, respectively. In 2013, the drier of the 2 years, irrigation amounts and crop water use (ET_c) were greater for the conventional hybrid, but grain water use efficiency (WUE) and harvest index were significantly greater for the DT hybrid. In 2014, grain yields and WUE were not significantly different between hybrids. However, irrigation amounts, ET_c and biomass yields were greater for the conventional hybrid. Results from both years indicate that the DT hybrid required less water to maximize grain yield as compared to the conventional hybrid. Producing relatively high yields with reduced amounts of water may provide a means for producers to continue corn production in a semiarid environment with declining water supplies.