Paula Barros Terraciano

Betacellulin Overexpression in Mesenchymal Stem Cells Induces Insulin Secretion In Vitro and Ameliorates Streptozotocin-Induced Hyperglycemia in Rats

Betacellulin (BTC), a ligand of the epidermal growth factor receptor, has been shown to promote growth and differentiation of pancreatic β-cells and to improve glucose metabolism in experimental diabetic rodent models. Mesenchymal stem cells (MSCs) have been already proved to be multipotent. Recent work has attributed to rat and human MSCs the potential to differentiate into insulin-secreting cells. Our goal was to transfect rat MSCs with a plasmid containing BTC cDNA to guide MSC differentiation into insulin-producing cells. Prior to induction of cell MSC transfection, MSCs were characterized by flow cytometry and the ability to in vitro differentiate into mesoderm cell types was evaluated. After rat MSC characterization, these cells were electroporated with a plasmid containing BTC cDNA. Transfected cells were cultivated in Dulbeccos modified Eagle medium high glucose (H-DMEM) with 10 mM nicotinamide. Then, the capability of MSC-BTC to produce insulin in vitro and in vivo was evaluated. It was possible to demonstrate by radioimmunoassay analysis that 10(4) MSC-BTC cells produced up to 0.4 ng/mL of insulin, whereas MSCs transfected with the empty vector (negative control) produced no detectable insulin levels. Moreover, MSC-BTC were positive for insulin in immunohistochemistry assay. In parallel, the expression of pancreatic marker genes was demonstrated by molecular analysis of MSC-BTC. Further, when MSC-BTC were transplanted to streptozotocin diabetic rats, BTC-transfected cells ameliorated hyperglycemia from over 500 to about 200 mg/dL at 35 days post-cell transplantation. In this way, our results clearly demonstrate that BTC overabundance enhances glucose-induced insulin secretion in MSCs in vitro as well as in vivo.

Cell therapy for chemically induced ovarian failure in mice.

Cell therapy has been linked to an unexplained return of ovarian function and fertility in some cancer survivors. Studies modeling this in mice have shown that cells transplantation generates donor-derived oocytes in chemotherapy-treated recipients. This study was conducted to further clarify the impact of cell transplantation from different sources on female reproductive function after chemotherapy using a preclinical mouse model. Methods. Female mice were administered 7.5 mg/kg cisplatin followed by cell transplantation (one week later) using GFP+ female cell donors. For cell tracking, adipose derived stem cell GFP+ (ADSC), female germline stem cell GFP+/MVH+ (FGSC), or ovary cell suspension GFP+ mice were transplanted into cisplatin-treated wild-type recipients. After 7 or 14 days animals were killed and histological analysis, IHQ for GFP cells, and ELISA for estradiol were performed. Results. Histological examinations showed that ADSC, ovary cell suspension, and FGSC transplant increase the number of follicles with apparent normal structure in the cells recipient group euthanized on day 7. Cell tracking showed GFP+ samples 7 days after transplant. Conclusion. These data suggest that intraovarian injection of ADSCs and FGSC into mice with chemotherapy-induced ovarian failure diminished the damage caused by cisplatin.

Criopreservação de espermatozóides eqüinos comparando duas curvas de congelamento combinadas com diluentes comerciais: uma análise laboratorial

During semen cryopreservation, sperm cells were submitted to several deleterious events leading to membrane damage which result in fertility decrease. This study was designed to compare the effects of two freezing techniques (conventional and automated), and the use of two commercial extenders as cryoprotectants (FR-5® and Botu-Crio®) on total and progressive motility, integrity and functionality of spermatic membranes during the cryopreservation of equine semen. Twenty ejaculates from two stallions were analyzed. The total and progressive motility of fresh and post-thawing semen samples were evaluated by patterns assays. Function of plasmatic membrane was measured by the hipoosmotic swelling test. Integrity of plasmatic membrane was evaluated using carboxifluorescein diacatate and iodidium propide fluorescent probes. There were significant differences between the two freezing techniques and/or between cryoprotectants for all assessed parameters. The combination of Botu-Crio® and automated curves showed better results on total and progressive post-thawing motility. The extender Botu-Crio®, alone, showed to better preserve the membrane integrity and function.

Functional and regenerative effects of local administration of autologous mononuclear bone marrow cells combined with silicone conduit on transected femoral nerve of rabbits

The inoculation of cells into injury sites can accelerate and improve the quality of nerve regeneration. This study aimed to evaluate the functional and regenerative effects of mononuclear autologous bone marrow cells (MABMC) combined with silicon conduit grafting in rabbit femoral nerves. Twenty-eight animals were allocated to one of two groups: treatment group (TG) or control group (CG), divided according to the time of evaluation, at either 50 or 75 days. After neurotmesis of the femoral nerve, surgical repair was performed with nerve autografts in silicon conduits, leaving a 5mm gap in both groups. The TG received MABMC in silicon conduits, and CG received a sham saline inoculum. Histological, clinical and electrophysiological analyses detected no differences between groups, but analysis of leg diameter showed that TG diameters were larger. This cell therapy did not improve regeneration of the femoral nerve, but there was a tendency for better functional recovery.

Adipose-derived stem cells improve full-thickness skin grafts in a rat model

To investigate the effects of heterologous adipose-derived stem cells (ADSCs) on autologous full-thickness skin grafts, we designed a first-intention healing model using Wistar rats. We harvested and sutured two full-thickness skin grafts in the dorsal recipient beds of 15 rats, randomized into three groups. In the treatment group, 1 × 106 ADSCs resuspended in saline solution (200 μL) were administered subcutaneously to the skin graft. The control group received only saline solution subcutaneously, whereas the negative control group did not receive any treatment. Compressive dressings were maintained until postoperative day 5. The grafts were assessed by two observers, who checked for the presence of epidermolysis on day 14. Planimetry showed the relative areas of normal skin, redness, ulceration, and contraction. Graft samples were obtained on day 14 and stained with hematoxylin and eosin and Massons trichrome. Epidermal analysis evaluated thickening, keratosis, acanthosis, hydropic degeneration, and inflammatory infiltrate. Dermal evaluation investigated the absence of hair follicles, granulation tissue formation, presence of inflammatory infiltrate, and collagen deposition. Immunohistochemistry was performed for dermal anti-VEGF and epidermal anti-Ki-67 staining. The ADSC group presented better macroscopic aspects, lower incidence of epidermolysis, and less loss of hair follicles. In addition, the ADSC group presented the lowest frequency of histopathological changes in the dermis and epidermis, as well as the largest subcutaneous and granulation tissue VEGF averages and the weakest Ki-67 staining of the epidermal basal layer. Subcutaneous administration of ADSCs may improve the integration of skin grafts, reducing the deleterious effects of ischemia and reperfusion injury.

Comparison of the characteristics of canine adipose tissue-derived mesenchymal stem cells extracted from different sites and at different passage numbers

Mesenchymal stem cells (MSCs) have desirable characteristics for use in therapy in animal models and veterinary medicine, due to their capacity of inducing tissue regeneration and immunomodulation. The objective of this study was to evaluate the differences between canine adipose tissue-derived MSCs (AD-MSCs) extracted from subcutaneous (Sc) and visceral (Vs) sites. Surface antigenic markers, in vitro differentiation, and mineralized matrix quantification of AD-MSCs at different passages (P4, P6, and P8) were studied. Immunophenotypic analysis showed that AD-MSCs from both sites were CD44+, CD90+, and CD45−. Moreover, they were able, in vitro, to differentiate into fat, cartilage, and bone. Sc-AD-MSCs preserve in vitro multipotentiality up to P8, but Vs-AD-MSCs only tri-differentiated up to P4. In addition, compared to Vs-AD-MSCs, Sc-AD-MSCs had greater capacity for in vitro mineralized matrix synthesis. In conclusion, Sc-AD-MSCs have advantages over Vs-AD-MSCs, as Sc AD-MSCs preserve multipotentiality during a greater number of passages, have more osteogenic potential, and require less invasive extraction.

Liquid and Gel Platelet Rich Plasma as Skin Healing Adjuvant

Background: In recent decades, many researches have been conducted on processes involved in tissue repairing, mainly in the development of resources and technology designed to improve the wound healing progress. Platelet rich plasma (PRP) derived from autologous blood is defined as a plasma volume with platelet concentration higher than physiological level. It is an autogenous and low cost source of growth factors, which are essential for tissue regeneration due to their angiogenic, mitogenic, and chemotactic properties. The aim of this study was evaluate two forms of PRP- liquid and gel - regarding their capacity to influence quality and repair time of standardized skin injuries. Materials, Methods & Results: New Zealand healthy rabbits were distributed in three groups (n = 6): control group (CG), liquid platelet rich plasma group (LIQPRP), and gel platelet rich plasma group (GELPRP). Acute skin lesions were inducted in two areas approximately 2 cm close to scapular edge and depth including epidermis, dermis, and hypodermis to external muscular fascia. Animals received treatment according to each group. Injuries were measured with digital pachymeter in two directions: longer length (l) and longer width (w), every two days. Areas and healing rates were calculated. Microscopic analysis samples were collected on days seven and 14 and evaluated through hematoxylin and eosin staining (HE) for global tissue examination, and through Masson’s trichrome (MT) to collagen fibers present within the interstice. These analyzes considered: angiogenesis, inflammation infiltrated and collagen fibers quantity. Immunohistochemistry with anti-Ki-67 antibody was utilized for proliferative profile assessment. Kruskal-Wallis’ non-parametric tests of independent samples was performed for comparison of values obtained through platelet count, referring to evaluation of platelet increase on treatments. Scar contraction rate (CR) was evaluated through Shapiro-Wilk’s normality test, and then submitted to mixed models test. Results obtained by histopathological and immunohistochemistry were also evaluated by Shapiro-Wilk’s normality test (for all tests a 5% level of significance was considered). Platelet concentration achieved with liquid PRP was 8.64 and gel PRP reached 5.62 times higher than physiological values. Platelet increase mean for both groups was 7.95. No statistical significance was observed between groups. No side-effects or adverse reactions related to PRP usage were observed while study was conducted. Discussion: In the present study, there was a need to raise platelet poor plasma volume in order to obtain autogenous thrombin required for gel PRP. After this modification, a stable and reasonable platelet concentration gel was produced. However, this form of PRP application requires more time for sample preparation, increasing the production cost. Furthermore, injection of liquid PRP directly in the wound site activates platelets by generated substances due to needle perforation, and mainly due to tissue trauma generated at the lesion site. Relating to the therapies administered, gel PRP was considered more manageable, since 3D structure could easily adapt to wound site after simply deposition of it. Liquid PRP was administered with needle and syringe, which required the surgeon to be more careful and perform a slow injection in order to avoid any spill and loss of material. Furthermore, histopathological analysis did not point any clot traces formed by gel PRP dehydration, although it is not possible to ensure that the clot was eliminated, reabsorbed, or even removed by the animal. By this protocol, a stable and reasonable platelet concentration gel was produced. Further studies are encouraged as well as employment of alternative diagnostic tools, in order to better understand found results.

Cell therapy in the treatment of bipolar mania in an animal model: a proof of concept study

INTRODUCTION The rationale of mesenchymal stem cells (MSCs) as a novel therapeutic approach in certain neurodegenerative diseases is based on their ability to promote neurogenesis. Hippocampal atrophy has been related to bipolar disorder (BD) in preclinical, imaging and postmortem studies. Therefore, the development of new strategies to stimulate the neurogenesis process in BD is crucial. OBJECTIVES To investigate the behavioral and neurochemical changes induced by transplantation of MSCs in a model of mania-like behavior induced by lisdexamfetamine dimesylate (LDX). METHODS Wistar rats (n=65) received one oral daily dose of LDX (10 mg/kg) or saline for 14 days. On the 8th day of treatment, the animals additionally received intrahippocampal saline or MSC (1 µL containing 25,000 cells) or lithium (47.5 mg/kg) as an internal experimental control. Two hours after the last administration, behavioral and neurochemical analyses were performed. RESULTS LDX-treated rats had increased locomotor activity compared to saline-saline rats (p=0.004), and lithium reversed LDX-related hyperactive behavior (p<0.001). In contrast, the administration of MSCs did not change hyperlocomotion, indicating no effects of this treatment on LDX-treated rats (p=0.979). We did not find differences between groups in BDNF levels (p>0.05) in the hippocampus of rats. CONCLUSION Even though these results suggest that a single intrahippocampal injection of MSCs was not helpful to treat hyperactivity induced by LDX and neither influenced BDNF secretion, we cannot rule out the possible therapeutic effects of MSCs. Further research is required to determine direct effects of LDX on brain structures as well as in other pathophysiological targets related to BD.

Chlamydia trachomatis infection in infertile and pregnant women in southern Brazil

Introduction: Chlamydia trachomatis (CT) is the most prevalent sexually transmitted bacterial infection, affecting mainly young, sexually active women. Untreated infection may lead to reproductive complications due to tubal damage. Infections during pregnancy may cause preterm labor, low birth weight, perinatal death, and neonatal conjunctivitis and pneumonia. There are few data on CT infection in Brazil. The aim of this study was to determine CT prevalence in infertile and pregnant women. Methods: A cross-sectional study included 77 infertile and 60 asymptomatic pregnant women. First-void urine was tested for CT using PCR (Polymerase Chain Reaction). Blood samples were collected for CT IgG antibodies testing using indirect immunofluorescence. A questionnaire about medical, gynecological, and sexual history was completed by all participants. Results: We found statistically similar prevalence of PCR and IgG antibodies between the groups. There was a 61% prevalence of CT IgG antibodies in infertile women and 56.7% in pregnant women. PCR was positive in only one (1.3%) infertile woman and in none pregnant women. Conclusion: There is a high prevalence of CT IgG antibody in Brazilian pregnant and infertile women, but we found a low prevalence of positive PCR in the urine samples. CT antibodies were associated with sexual behavior and smoking. Keywords: Chlamydia trachomatis , Chlamydia infections, prevalence, nucleic acid amplification techniques; infertility, female; fluorescent antibody technique