Therapeutic Potential in Wound Healing of Allogeneic Use of Equine Umbilical Cord Mesenchymal Stem Cells.

Wound healing after skin injury is a complex process, particularly in equines where leg wounds are prevalent and their repair is complicated due to the anatomical characteristics. Conventional treatments are not effective enough. The umbilical cord offers an unlimited source of adult mesenchymal stem cells (ucMSCs) from Wharton's jelly tissue. The present study aims to demonstrate the safety and therapeutic potential of the allogeneic use of equine ucMSCs (e-ucMSCs) in the healing of severe equine leg wounds. The methods employed were the isolation, culture and expansion of e-ucMSCs. Flow cytometry and a PCR assay were used for cell characterization. This study included an immunomodulation assay, a murine pre-clinical trial and the first phase of an equine clinical trial. Our results showed that e-ucMSCs express a functional HLA-G homolog, EQMHCB2. In the immunomodulation assay, the e-ucMSCs inhibited the proliferation of activated equine peripheral blood mononuclear cells (e-PBMCs). In the murine pre-clinical trial, e-ucMSCs reduced healing time by 50%. In the equine clinical trial, the injection of e-ucMSCs into severe leg lesions improved the closure time and quality of the tissues involved, regenerating them without fibrous tissue scar formation. In conclusion, the results of this study suggest that e-ucMSCs can be used allogeneically for wound healing by creating a tolerogenic environment.

HLA-G gene editing in tumor cell lines as a novel alternative in cancer immunotherapy.

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial.W In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G - cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.

Wound Healing by Allogeneic Transplantation of Specific Subpopulation From Human Umbilical Cord Mesenchymal Stem Cells.

In normal physiological conditions, restoration of a functional epidermal barrier is highly efficient; nevertheless, when it fails, one of the main consequences is a chronic ulcerative skin defect, one of the most frequently recognized complications of diabetes. Most of these chronic venous ulcers do not heal with conventional treatment, leading to the appearance of infections and complications in the patient. Treatments based on the use of autologous mesenchymal stem cells (MSC) have been successful; however, its implementation entails complications. The umbilical cord offers an unlimited source of adult MSC (ucMSC) from the Wharton's jelly tissue with the same relevant features for clinical applicability and avoiding difficulties. It has recently been characterized by one specific subpopulation derived from ucMSC, the differentiated mesenchymal cells (DMCs). This subpopulation expresses the human leukocyte antigen-G (HLA-G) molecule, a strong immunosuppressive checkpoint, and vascular endothelial growth factor (VEGF), the most potent angiogenic factor. Considering the importance of developing a more effective therapy for wound treatment, especially ulcerative skin lesions, we analyzed DMC safety, efficacy, and therapeutic potential. By immunohistochemistry, umbilical cords HLA-G and VEGF positive were selected. Flow cytometry revealed that 90% of the DMC subpopulation are HLA-G+, CD44+, CD73+, CD29+, CD105+, CD90+, and HLA-DR-. Reverse transcription-polymerase chain reaction revealed the expression of HLA-G in all of DMC subpopulations. Upon co-culture with the DMC, peripheral blood mononuclear cell proliferation was inhibited by 50%. In a xenograft transplantation assay, DMC improved wound healing with no signs of rejection of the transplanted cells in immunocompetent mice. This study confirms that HLA-G allows allogeneic cell transplantation, and VEGF is fundamental for the restoration of the failure in blood supply. DMC population has positive effects on wound healing by promoting local angiogenesis in skin lesions. DMC could play a very important role in regenerative medicine and could be a novel allogeneic cell-therapeutic tool for wound healing.

Study of DNA synthesis and mitotic activity of hepatocytes and its relation to angiogenesis in hepatectomised tumour bearing mice.

Partial hepatectomy (PH) alters serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. Furthermore, angiogenesis is mainly stimulated by vascular endothelial growth factor (VEGF) and is a fundamental requirement either in liver regeneration or in tumours growth. This study looks at the expression of VEGF, DNA synthesis (DNAs) and mitotic activity (MA) in hepatectomised (H) and hepatectomised-tumour bearing (HTB) mice throughout a 24 h period. Adult male mice were sacrificed every 4 h from 26 to 50 h post-hepatectomy. H mice show a circadian rhythm in VEGF expression with a maximum value of 2.6 ± 0.1 at 08/46 h of day/hours posthepatectomy (HD/HPH); in DNAs, the maximum value was 3.4 ± 0.3 at 16/30 (HD/HPH) and in MA it was 2.3 ± 0.01 at 12/50 (HD/HPH). In HTB animals the peak of VEGF expression appears at 16/30 (HD/HPH) with a maximum value of 3.7 ± 0.1, the peak of DNAs was at 00/38 (HD/HPH) with a value of 4.6 ± 0.3 and the maximum value of MA of 08/46 (HD/HPH) with a value of 3.01 ± 0.3. We can conclude that the presence of the tumour induces modifications in the intensity and the temporal distribution of the circadian curves of VEGF expression, DNAs and MA of hepatectomised animals.

Pesticide-induced decrease in rat testicular steroidogenesis is differentially prevented by lipoate and tocopherol.

We have previously demonstrated that the sub-chronic administration of low doses of Toc or α-Toc, glyphosate and zineb to rats (i.p. 1/250 LD50, three times a week for 5 weeks) provoked severe oxidative stress (OS) in testicles. These effects were also reflected in plasma. Lipoic acid (LA) and α-tocopherol are considered as antioxidants due to their ability to neutralize reactive oxygenated species (ROS) and reset endogenous antioxidant levels. To investigate the possible protective effect on reproductive function, LA and Toc (i.p. 25, 50 and 100mg/kg) were administered simultaneously with the pesticide mixture (PM) for 5 weeks. Both drugs prevented OS and the damage to proteins and lipids caused by PM in a dose-dependent manner. The PM-induced increase levels of prostaglandins E2 and F2α was completely restored by LA but not by Toc. Similarly, only LA was able to restore the inhibition of testosterone production, the decrease of 3ß- and 17ß-hydroxysteroid dehydrogenases activities, and the elevation of gonatropins (FSH and LH) levels produced by PM. Furthermore, LA was more efficient than Toc in normalizing the histological alterations produced by PM administration, suggesting that pesticides act though other mechanisms that generate oxidative stress. In our experimental model LA displayed a higher protective role against pesticide-induced damage than that observed by Toc administration. Our results suggest that LA administration is a promising therapeutic strategy for coping with disorders suspected to be caused by OS generators - such as pesticides - in male reproductive system.

Changes in VEGF expression and DNA synthesis in hepatocytes from hepatectomized and tumour-bearing mice.

Transplanted tumours could modify the intensity and temporal distribution of the cellular proliferation in normal cell populations, and partial hepatectomy alters the serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. The following experiments were designed in order to study the SI (S-phase index) and VEGF (vascular endothelial growth factor) expression in regenerating liver (after partial hepatectomy) of adult male mice bearing a hepatocellular carcinoma, throughout one complete circadian cycle. We used adult male C3H/S-strain mice. After an appropriate period of synchronization, the C3H/S-histocompatible ES2a hepatocellular carcinoma was grafted into the subcutaneous tissue of each animal's flank. To determine the index of SI and VEGF expression of hepatocytes, we used immunohistochemistry. The animals were divided into two experimental groups: Group I, control, hepatectomized animals; Group II, hepatectomized tumour-bearing animals. The statistical analysis of SI and VEGF expression was performed using Anova and Tukey as a postcomparison test. The results show that in the second group, the curve of SI changes the time points for maximum and minimum activity, and the peak of VEGF expression appears before the first group. In conclusion, in the hepatectomized mice, the increases of hepatic proliferation, measured by the SI index, may produce a rise in VEGF expression with the object of generating a vascular network for hepatic regeneration. Lastly, as we have mentioned, in hepatectomized and tumour-bearing mice, the peak of VEGF expression appears before the one of DNA synthesis.

Comparative study of DNA synthesis and nucleolar organizer regions of sinusoid littoral cells in mouse regenerating liver.

Variations in DNA synthesis (DNAs) and Nucleolar Organizer Regions (NORs) were studied in the littoral cell population from regenerating liver of C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections. Tissue samples were obtained every four hours from the 30th to the 54th hours after a partial hepatectomy. The results showed, in both parameters, a gradual increment of the values during the period studied, with highest values (DNAs 107.1 +/- 16.1 SE; AgNORs 77.3 +/- 3.4 SE) located at 16:00/54 Time of Day / Hours Post-Hepatectomy (TD/HPH), which were significantly different (p <0.001) from the values of the first sample (DNAs 38.1 +/- 9.5 SE; AgNORs 27.3 +/- 1.0 SE) taken at 16:00/30 TD/HPH. The results of our experiment demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU immunohistochemistry and AgNORs numbers in sinusoid littoral cells from mouse regenerating liver.

Comparative study of DNA synthesis and nucleolar organizer regions of sinusoid littoral cells in mouse regenerating liver

Variations in DNA synthesis (DNAs) and Nucleolar Organizer Regions (NORs) were studied in the littoral cell population from regenerating liver of C3HS inbred mice standardized for periodicity analysis.Immunohistochemieal detection of Bromodeoxyuridine (BrdU) with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections. Tissue samples were obtained every four hours from the 30th to the 54th hours after a partial hepatectomy. The results showed, in both parameters, a gradual increment of the values during the period studied, with highest values (DNAs 107.1 ± 16.1 SE; AgNORs 77. 3± 3.4 SE) located at 16:00/54 Time of Day / Hours Post Hepatectomy (TD/HPH), which were significantly different (p <0.001) from the values of the first sample (DNAs 38.1 ± 9.5 SE; AgNORs 27.3 ± 1.0 SE) taken at 16:00/30 TD/HPH. The results of our experiment demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU immunohistoehemistry andAgNORs numbers in sinusoid littoral cells from mouse regenerating liver

Comparative study of DNA synthesis and nucleolar organizer regions of sinusoid littoral cells in mouse regenerating liver

Variations in DNA synthesis (DNAs) and Nucleolar Organizer Regions (NORs) were studied in the littoral cell population from regenerating liver of C3HS inbred mice standardized for periodicity analysis.Immunohistochemieal detection of Bromodeoxyuridine (BrdU) with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections. Tissue samples were obtained every four hours from the 30th to the 54th hours after a partial hepatectomy. The results showed, in both parameters, a gradual increment of the values during the period studied, with highest values (DNAs 107.1 ± 16.1 SE; AgNORs 77. 3± 3.4 SE) located at 16:00/54 Time of Day / Hours Post Hepatectomy (TD/HPH), which were significantly different (p <0.001) from the values of the first sample (DNAs 38.1 ± 9.5 SE; AgNORs 27.3 ± 1.0 SE) taken at 16:00/30 TD/HPH. The results of our experiment demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU immunohistoehemistry andAgNORs numbers in sinusoid littoral cells from mouse regenerating liver

Comparative study of DNA synthesis and nucleolar organizer regions of sinusoid littoral cells in mouse regenerating liver.

Variations in DNA synthesis (DNAs) and Nucleolar Organizer Regions (NORs) were studied in the littoral cell population from regenerating liver of C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections. Tissue samples were obtained every four hours from the 30th to the 54th hours after a partial hepatectomy. The results showed, in both parameters, a gradual increment of the values during the period studied, with highest values (DNAs 107.1 +/- 16.1 SE; AgNORs 77.3 +/- 3.4 SE) located at 16:00/54 Time of Day / Hours Post-Hepatectomy (TD/HPH), which were significantly different (p <0.001) from the values of the first sample (DNAs 38.1 +/- 9.5 SE; AgNORs 27.3 +/- 1.0 SE) taken at 16:00/30 TD/HPH. The results of our experiment demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU immunohistochemistry and AgNORs numbers in sinusoid littoral cells from mouse regenerating liver.

DNA synthesis and nucleolar organizer regions circadian rhythm in mouse regenerating liver hepatocytes

DNA synthesis and Nucleolar Organizer Regions (NORs) were studied in C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) incorporated into DNA with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections of regenerating liver. Tissue samples were obtained at different times after hepatectomy along a circadian span. The results showed a strong correlation of values between DNA synthesis (BrdU labelling index) and AgNOR numbers, with higher counts during the activity period of animals at 00:00/38 and 04:00/42 hours Time of Day/Hours Post-Hepatectomy (TD/HPH), being the differences with other time points highly significant. Our observations demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU incorporation and AgNOR numbers with a defined circadian rhythm in mouse regenerating hepatocytes.

DNA synthesis and nucleolar organizer regions circadian rhythm in mouse regenerating liver hepatocytes

DNA synthesis and Nucleolar Organizer Regions (NORs) were studied in C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) incorporated into DNA with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections of regenerating liver. Tissue samples were obtained at different times after hepatectomy along a circadian span. The results showed a strong correlation of values between DNA synthesis (BrdU labelling index) and AgNOR numbers, with higher counts during the activity period of animals at 00:00/38 and 04:00/42 hours Time of Day/Hours Post-Hepatectomy (TD/HPH), being the differences with other time points highly significant. Our observations demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU incorporation and AgNOR numbers with a defined circadian rhythm in mouse regenerating hepatocytes.(AU)