Maria José Carvalho

Amniotic membrane: from structure and functions to clinical applications

Amniotic membrane (AM) or amnion is a thin membrane on the inner side of the fetal placenta; it completely surrounds the embryo and delimits the amniotic cavity, which is filled by amniotic liquid. In recent years, the structure and function of the amnion have been investigated, particularly the pluripotent properties of AM cells, which are an attractive source for tissue transplantation. AM has anti-inflammatory, anti-bacterial, anti-viral and immunological characteristics, as well as anti-angiogenic and pro-apoptotic features. AM is a promoter of epithelialization and is a non-tumorigenic tissue and its use has no ethical problems. Because of its attractive properties, AM has been applied in several surgical procedures related to ocular surface reconstruction and the genito-urinary tract, skin, head and neck, among others. So far, the best known and most auspicious applications of AM are ocular surface reconstruction, skin applications and tissue engineering. However, AM can also be applied in oncology. In this area, AM can prevent the delivery of nutrients and oxygen to cancer cells and consequently interfere with tumour angiogenesis, growth and metastasis.

Selective cytotoxicity and cell death induced by human amniotic membrane in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a worldwide high incidence and mortality. For this reason, it is essential to invest in new therapies for this type of cancer. Our team already proved that human amniotic membrane (hAM) is able to inhibit the metabolic activity of several human cancer cell lines, including HCC cell lines. Taking into account the previously performed work, this experimental study aimed to investigate the pathways by which hAM protein extracts (hAMPEs) act on HCC. Our results showed that hAMPE reduce the metabolic activity, protein content and DNA content in a dose- and time-dependent manner in all HCC cell lines. This therapy presents selective cytotoxicity, since it was not able to inhibit a non-tumorigenic human cell line. In addition, hAMPE induced cell morphology alterations in all HCC cell lines, but death type is cell line dependent, as proved by in vitro and in vivo studies. In conclusion, hAMPE have a promising role in HCC therapy, since it is capable of inducing HCC cytotoxicity and cell death.

Oxidative Stress, DNA, Cell Cycle/Cell Cycle Associated Proteins and Multidrug Resistance Proteins: Targets of Human Amniotic Membrane in Hepatocellular Carcinoma

The anticancer effects of human amniotic membrane (hAM) have been studied over the last decade. However, the action mechanisms responsible for these effects are not fully understood until now. Previously results reported by our team proved that hAM is able to induce cytotoxicity and cell death in hepatocellular carcinoma (HCC), a worldwide high incident and mortal cancer. Therefore, this experimental study aimed to investigate the cellular targets of hAM protein extracts (hAMPE) in HCC through in vitro studies. Our results showed that hAMPE is able to modify oxidative stress environment in all HCC cell lines, as well as its cell cycle. hAMPE differently targets deoxyribonucleic acid (DNA), P21, P53, β-catenin and multidrug resistance (MDR) proteins in HCC cell lines. In conclusion, hAMPE has several targets in HCC, being clear that the success of this treatment depends of a personalized therapy based on the biological and genetic characteristics of the tumor.

Amniotic membrane extract differentially regulates human peripheral blood T cell subsets, monocyte subpopulations and myeloid dendritic cells

The discovery of the immunoregulatory potential of human amniotic membrane (hAM) propelled several studies focusing on its application for the treatment of immunological disorders. However, there is little information regarding the effects of hAM on distinct activation and differentiation stages of immune cells. Here, we aim to investigate the effect of human amniotic membrane extract (hAME) on the pattern of cytokine production by T cells, monocytes and myeloid dendritic cells (mDCs). For this purpose, peripheral blood mononuclear cells (PBMCs) from eight healthy individuals were stimulated in vitro in the presence or absence of hAME. Mitogen-induced proliferation of PBMCs and cytokine production among the distinct T cell functional compartments, monocyte subpopulations and mDCs were evaluated. hAME displayed an anti-proliferative effect and decreased the frequency of T cells producing tumor necrosis factor (TNF)α, interferon (IFN)γ and interleukin (IL)-2, for all T cell functional compartments. The frequency of IL-17 and IL-9-producing T cells was also reduced. The inhibition of mRNA expression of granzyme B, perforin and NKG2D by CD8+ T cells and γδ T cells and the augment of FoxP3 and IL-10 in CD4+ T cells and IL-10 in regulatory T cells were also observed. Furthermore, hAME inhibited IFNγ-induced protein (IP)-10 expression by classical and non-classical monocytes, without hampering the production of TNFα and IL-6 by monocytes and mDCs. These results suggest that hAME exerts an anti-inflammatory effect on T cells, still at a different extent for distinct T cell functional compartments.

Cognition, Grammaticalization and Syntactic Change. The emergence of Compound Tenses in Portuguese

This article analyzes the historical process of the grammaticalization of aver/ter ‘to have’ in compound tenses, since Portuguese is somewhat isolated among Romance languages in this respect. Our research has shown that the change in auer/ter, from indicating possession to acting as auxiliaries, involved a process of recategorization similar to that seen with the verbs “go”, “come” and “have” in some languages, sharing with them a fundamental characteristic: the fact that in one phase of the transition constructions developed which were capable of being interpreted in both ways (expressing both result and time). Thus, as in those languages, the process of recategorizing involves an intermediate step of structural ambiguity whereby a given construction can be interpreted in two different ways. We will try to clarify the gradual process of reanalysis of ter in the medieval period, based on a corpus of original juridical sources (13th – 16th centuries) which we have transcribed, originating from a particularly important centre in medieval Portuguese culture, the monastery of Alcobaca (between Coimbra and Lisbon). The corpus consists of 153 original documents not only from the monastery but also from the outlying areas under its jurisdiction, dating from between 1289 and 1565.

Phenotypic analysis of breast cell lines – triple negative cells vs expressing hormonal receptors

Breast cancer patients are stratified in 3 groups: expressing hormonal receptors (HR), which respond to therapies targeting estrogen receptors; HER2+, candidates to trastuzumab; and triple negative (TN), for which, despite its more aggressive clinical behavior, chemotherapy is therapy available. In vitro studies proved that cells subjected to specific growth factors form spherical colonies of stem-cells in suspension designated mammospheres (MS). The aim of this work is to assess the capacity to form MS in TN (HCC1806) comparing with HR+ (MCF7) breast cancer cell lines. Cell lines were propagated according to ATCC. MS forming protocol consisted on cell culturing in DMEMF12 supplemented with bFGF and EGF. Media was renewed every 2 days. Flow cytometry analysis, with antibodies anti-CD44, anti-CD24 and anti CD133 was made. Microscopic observation showed a differential phenotype. HR+ cells formed spherical colonies in suspension while TN maintained adherent appearing only a few groups of MS. This may be related with late recurrence. Controls of both cell lines analyzed showed a low CD44 markup. For both cell lines CD133 did not show significant changes. HR+ cells had higher expression of CD44 and CD133 when compared with TN. In TN cells was identified 2 populations of cells in suspension. The major one was CD44+ and in 1% this expression was even more relevant and may be representative of a progenitor cell population. The HR+ showed stronger staining for CD24 than TN. The HR+ also differentiated 2 populations, a major one CD44+ and 9% with minor expression of CD44. The results suggest a significant fraction of HR+ harboring CD44+, representing a phenotype of progenitor cells.