Heat stress can have a serious impact on the health of both humans and animals. A major question is how heat stress affects normal development and differentiation at both the cellular and the organism levels. Here we use an in vitro experimental system to address how heat shock treatment influences the properties of bovine mesenchymal stem cells (MSCs)-multipotent progenitor cells-which are found in most tissues. Because cattle are sensitive to harsh external temperatures, studying the effects of heat shock on MSCs provides a unique platform to address cellular stress in a physiologically relevant model organism. Following isolation and characterization of MSCs from the cow's umbilical cord, heat shock was induced either as a pulse (1 h) or continuously (3 days), and consequent effects on MSCs were characterized. Heat shock induced extensive phenotypic changes in MSCs and dramatically curtailed their capacity to proliferate and differentiate. These changes were associated with a partial arrest in the G1/S or G2/M checkpoints. Furthermore, MSCs lost their ability to resolve the inflammatory response of RAW macrophages in coculture. A possible explanation for this loss of function is the accumulation of reactive oxygen species and malfunction of the mitochondria in the treated cells. Heat shock treatments resulted in stressinduced premature senescence, affecting the MSCs' ability to proliferate properly for many cell passages to follow. Exposure to elevated external temperatures leads to mitochondrial damage and oxidative stress, which in turn conveys critical changes in the proliferation, differentiation, and immunomodulatory phenotype of heat-stressed MSCs. A better understanding of the effect of heat shock on humans and animals may result in important health and economic benefits.

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Mesenchymal stem cells (MSC) have many roles that are important for the body’s proper functioning. When the MSC pool is damaged, it is often correlated with impaired development or health of the organism. MSC are known for their anti-inflammatory, immunomodulatory and trophic characteristics that play an important role in the physiological homeostasis of many tissues. Heat shock impairs MSC capacity by inducing the generation of reactive oxygen species and mitochondrial dysfunction, which, in turn, send the cells into a state of premature senescence. Here, we pre-exposed MSC to melatonin, resveratrol, or curcumin, which are natural antioxidative compounds, and tested the protective effects of these substances from oxidative stress and aging. Our data showed that pre-exposure of MSC to antioxidants decreased reactive oxygen species while mitochondrial damage remained high. Additionally, although the proliferation of the cells was slow, antioxidants protected the cells from premature senescence, and subsequent cytokine release was prevented. We conclude that while elevated temperatures directly cause mitochondrial damage, senescence is induced by elevated ROS levels. We suggest that heat shock alters cell and tissue homeostasis by several independent mechanisms; however, reducing tissue senescence will reduce damage and provide a pathway to overcome physiological challenges in animals.

The use of mesenchymal stromal cells (MSCs) is emerging as an efficacious and safe treatment for many infectious and non-infectious inflammatory diseases in human and veterinary medicine. Such use could be done to treat mastitis and metritis, which are the most common disease conditions affecting dairy cows leading to considerable economic losses and reduced animal welfare. Currently, both disease conditions are commonly treated using local and systemic administration of antibiotics. However, this strategy has many disadvantages including low cure rates and the public health hazards. Looking for alternative approaches, we investigated the properties of MSCs using in-vitro mammary and endometrial cell systems and in-vivo mastitis and metritis murine model systems. In-vitro, co-culture of mammary and uterus epithelial cells constructed with NF-kB reporter system, the master regulator of inflammation, demonstrated their anti-inflammatory effects in response to.LPS. In vivo, we challenge animals with field strains of mammary and utero pathogenic Escherichia coli and evaluated the effects of local and systemic application of MSC in the animal models. Disease outcome was evaluated using histological analysis, bacterial counts and gene expression of inflammatory markers. We show that MSC treatment reduced bacterial load in metritis and significantly modulated the inflammatory response of the uterus and mammary gland to bacterial infection. Most notably are the immune modulatory effects of remotely engrafted intravenous MSCs, which open new avenues to the development of MSC-based cell-free therapies.

Exposing bovine cumulus-oocyte complexes (COC) to heat shock during in vitro maturation (IVM) reduces oocyte developmental competence. Melatonin has been reported to improve cleavage of heat-shocked oocytes after fertilization. The current study examined whether protective activity of melatonin is associated with nuclear or cytoplasmic maturation events, or both. The COC were aspirated from Holstein ovaries collected at an abattoir and randomly divided into 3 experimental groups. The control COC were matured (22h) in standard oocyte maturation medium (OMM) at 38.5°C and 5% CO2; the treatment COC were matured (22h) in OMM at 41.5°C and 6% CO2 (HS) without or with 10−7 M melatonin (HS+M). Data are presented as least squares means±s.e.m. To examine nuclear maturation, COC were denuded of cumulus cells at the end of maturation, fixed in 2% (vol/vol) paraformaldehyde, and labelled with 10μg mL−1 4′,6-diamidino-2-phenylindole (DAPI) (200 COC, 3 replicates). The meiotic status of denuded oocytes was evaluated under inverted fluorescence microscope using Nis Elements software (Nikon, Tokyo, Japan). The proportion of oocytes that resumed meiosis and reached the metaphase II (MII) stage was lower in the HS group than in the control (13.7v. 55%, respectively; chi-squared, P<0.05); melatonin slightly alleviated this effect, as indicated by a higher proportion of MII-stage oocytes in HS+M than in the HS group (32v. 13.7%, respectively; chi-squared, P<0.05). Cytoplasmic maturation was evaluated according to the distribution pattern of cortical granules (CG). Matured COC were denuded and the oocytes were stained with 100μg mL−1 fluorescein isothiocyanate-conjugated Arachis hypogaea agglutinin (FITC-PNA) and counterstained with 10μg mL−1 DAPI. Oocytes were examined under an inverted fluorescence microscope and classified as 1 of 3 types according to their cortical distribution pattern (3 replicates; 200 COC). The proportion of oocytes classified as type 3, which is associated with cytoplasmic maturation, did not differ between the experimental groups, either when analysed for total oocytes or for nuclear matured oocytes (i.e. MII stage). To evaluate the level of reactive oxygen species (ROS), matured-denuded oocytes were labelled using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and the intracellular fluorescence of DCF was immediately measured by fluorescence microscopy (3 replicates, 425 COC). Heat shock increased ROS level relative to the control (68.8±5.32v. 33.9±4.08 pixels intensity, respectively; P<0.05) and melatonin tended to alleviate the effect of heat stress to some extent (56.0±4.78; P<0.06). In summary, heat shock impairs the process of nuclear maturation, expressed by reduced proportion of MII-stage oocytes at the end of maturation. Melatonin partly overcomes this effect, perhaps through its antioxidant activity.

BackgroundT-cell engagers are bispecific molecules directed against the CD3 complex on one end and a tumor specific antigen on the other end, allowing a physical link of T cell to a tumor cell, resulting in tumor killing and immune activation. Bispecific molecules harnessing and redirecting T-cells towards tumor cells are a promising therapeutic agents. Aptamers are single stranded oligonucleotides with binding and recognition propensities similar to those of antibodies. Aptamers have a number of advantages over bispecific antibodies including shorter generation time and low immunogenicity. Thus, aptamers capable of targeting T cells would have great potential for use as anti-cancer therapeuticsMethodsSystematic evolution of ligands by exponential enrichment (SELEX) methodology was employed in order to identify a novel CD3e binding aptamer. CD3 binding aptamer was subsequently linked into a bispecific T cell engager structure with a tumor-targeting aptameric arm. The tumor-targeting aptamer is developed by Aummune's proprietary tailored therapeutic platform.1 based on identifying functional aptamer sequences capable of specifically killing targeted tumor cells and sparing healthy tissue .Exemplary bispecific aptamers were tested for T cell stimulation by flow cytometry. In vivo antitumor activity was investigated in syngeneic and in xenograft tumor models.ResultsWe have successfully identified a novel CD3e –targeting aptamer with a Kd of 31nM. A bispecific T cell engager comprised of this aptamer and a tumor-targeting aptamer induced a potent stimulation of T cells in vitro, resulting in CD69 upregulation and IFNg secretion.Next, the CD3e targeting aptamer was hybridized to tumoricidal aptamers identified by Aummune's platform (VS12) to target either the human colon carcinoma HCT116 cells or (VS32) the murine triple negative breast cancer 4T1 cells. Both bispecific entities (CS6-VS12 and CS6-VS32) effectively lead to inhibition of tumor growth in vivo and increased survival in the corresponding models.ConclusionsOur data above provide a proof-of-concept for Aummune's Bispecific Aptamer efficacy and provide a framework for the clinical development of this novel tailored immune therapeutic agents. Indeed, we are currently in the process of developing a first-in-human clinical study in subjects with solid tumors.ReferenceMamet N, et al, Commun Biol 2020.