Early-onset breast cancer patients in the South and Southeast of Brazil should be tested for the TP53 p.R337H mutation

Abstract Germline TP53 mutations are associated with Li-Fraumeni syndrome (LFS), a disease that predisposes carriers to a wide variety of early onset tumors. In southern and southeastern Brazil, a high frequency of a germline TP53 mutation, p.R337H, was diagnosed in 0,3% of the population due to a founder effect. Carriers are at risk for developing cancer but the penetrance is lower than in typical DNA binding domain mutations. To date, only a few families were detected and diagnosis of carriers remains a challenge. Therefore, the inclusion of additional criteria to detect p.R337H carriers is necessary for the Brazilian population. We assessed the A.C. Camargo Cancer Center Oncogenetics Department database in search of common characteristics associated with p.R337H families that did not fulfill LFS/LFL clinical criteria. Among 42 p.R337H families, three did not meet any LFS/LFL criteria. All cases were young female patients with breast cancer diagnosed before age 45 and with no family history of LFS linked-cancers. Our results suggest that screening for the germline TP53 p.R337H mutation should be indicated, along with BRCA1 and BRCA2 genetic testing, for this group of patients, especially in the South and Southeast of Brazil

Characterization of Equine Adipose Tissue-Derived Progenitor Cells Before and After Cryopreservation

In horses, stem cell therapies are a promising tool to the treatment of many injuries, which are common consequences of athletic endeavor, resulting in high morbidity and often compromising the performance. In spite of many advantages, the isolation of stem cells similar to human, from equine adipose tissue, occurred only recently. The aim of this study was to isolate equine adipose tissue-derived progenitor cells (eAT-PC), to characterize their proliferative potential, and to study their differentiation capacity before and after cryopreservation. The cells, isolated from horse adipose tissue, presented similar fibroblast-like cell morphology in vitro. Their proliferation rate was evaluated during 63 days (23 passages) before and after cryopreservation. After the induction of osteogenic differentiation, von Kossa staining and positive immunostaining studies revealed the formation of calcified extracellular matrix confirming the osteogenic potential of these cells. Adipogenic differentiation was induced using two protocols: routine and other one developed by us, while our protocol requires a shorter time (Oil Red O staining revealed significant accumulation of lipid droplets after 7 days). Chondrogenic differentiation was observed after 21 days of induced pellet culture, as evidenced by histological (toluidine blue) and immunohistochemistry studies. Our data demonstrate that eAT-PC can be easily isolated and successfully expanded in vitro while presenting significant proliferating rate. These cells can be maintained undifferentiated in vitro and can efficiently undergo differentiation at least into mesodermal derivates. These eAT-PC properties were preserved even after cryopreservation. Our findings classify eAT-PC as a promising type of progenitor cells that can be applied in different cell therapies in equines.FAPESPApplied Genetics, Veterinary Activities Ltda[PIPE 1

Changes in expression pattern of selected endometrial proteins following mesenchymal stem cells infusion in mares with endometrosis.

Mesenchymal stem cells (MSCs) due to their self-renewal potential and differentiation capacity are useful for tissue regeneration. Immunomodulatory and trophic properties of MSCs were demonstrated suggesting their use as medicinal signaling cells able to positively change local environment in injured tissue. Equine endometrosis is a progressive degenerative disease responsible for glandular alterations and endometrial fibrosis which causes infertility in mares. More precisely, this disease is characterized by phenotypic changes in the expression pattern of selected endometrial proteins. Currently, no effective treatment is available for endometrosis. Herein, we aimed at the evaluation of expression pattern of these proteins after allogeneic equine adipose tissue-derived multipotent mesenchymal stem cells (eAT-MSCs) infusion as well as at testing the capacity of these cells to promote endometrial tissue remodeling in mares with endometrosis. eAT-MSC (2 × 10(7)/animal) were transplanted into mares' uterus and control animals received only placebo. Uterine biopsies were collected before (day 0) and after (days 7, 21 and 60) cells transplantation. Conventional histopathology as well as expression analysis of such proteins as laminin, vimentin, Ki-67-antigen, α-smooth muscle actin (α-SMA) and cytokeratin 18 (CK18) have been performed before and after eAT-MSCs transplantation. We demonstrated that eAT-MSCs induced early (at day 7) remodeling of endometrial tissue microenvironment through changes observed in intra cellular and intra glandular localization of aforementioned proteins. We demonstrated that eAT-MSCs were able to positively modulate the expression pattern of studied secretory proteins as well as, to promote the induction of glandular epithelial cells proliferation suggesting local benefits to committed endometrial tissue environment after eAT-MSCs transplantation

Smooth-muscle-α-actin (SMA) expression before (at day 0) and after (at day 7) eAT-MSCs intrauterine transplantation.

<p>A, B) At day 0, SMA expression was observed in uterine glands (white arrowhead) and in periglandular fibroblasts (black arrow). A1) At day 7, SMA showed no signs of expression. B) Pattern of SMA expression is similar to A and B. LM. Scale bars: A = 25 µm; A1, B, B1 = 50 µm.</p

Proliferation rate analyzed by Ki-67 antigen expression.

<p>Proliferation rate analyzed by Ki-67 antigen expression.</p

Histological analysis of alterations in mares’ endometrium following eAT-MSCs intrauterine transplantation.

<p>A–D3) Mares which received the cells. DE–F3) Control mares. A–F) Morphology of endometrial surface prior eAT-MSCs intrauterine cells transplantation. A1–D1) Day 7 after eAT-MSCs intrauterine transplantation. A2–D2) Same as in (A1–D1) at day 21. A3–D3) Same as in (A1–D1) at day 60. E1–F3) Respective controls. LM. Scale bars: A–F3 = 50 µm.</p

Antibodies used in immunohistochemistry.

1<p>IgG polyclonal goat anti-mouse + goat anti-rabbit HRP (secondary antibody).</p>2<p>Polyclonal rabbit anti-goat HRP (secondary antibody).</p

Cytokeratin 18 (CK18) and Ki-67 expression before (at day 0) and after (at days 7, 21 and 60) eAT-MSCs intrauterine transplantation: A–A3 and D–D3 - experimental group; B–B3 and D–D3 - control.

<p>A, B) At day 0, CK18 (black arrows) localized in damaged epithelia of glands (G). A1–A3) At days 7, 21 and 60, the absence of CK18 expression was observed. B1, B2) At days 7 and 21, CK18 expression was still observed (black arrows) in control. B3) At day 60, control mares showed no signs of CK18 expression. C, D) At day 0, none or a few Ki-67 positive cells were observed. C1, D1) At day 7, amount of Ki-67 positive cells (black arrow) was increased. C2, D2) At day 21, both groups showed positive Ki-67 staining. C3) At day 60, the expression of Ki-67 was still observed. D3) In control, the absence of Ki-67 expression. LM. Scale bars: A–D1 = 50 µm; C2, C3, D2, D3 = 25 µm.</p