Madalena Costa

Macrophage polarization following chitosan implantation

Macrophages are a key cell in the host response to implants and can be polarized into different phenotypes capable of inducing both detrimental and beneficial outcomes in tissue repair and remodeling, being important in tissue engineering and regenerative medicine. The objective of this study was to evaluate the macrophage response to 3D porous chitosan (Ch) scaffolds with different degrees of acetylation (DA, 5% and 15%). The M1/M2 phenotypic polarization profile of macrophages was investigated in vivo using a rodent air-pouch model. Our results show that the DA affects the macrophage response. Ch scaffolds with DA 5% induced the adhesion of lower numbers of inflammatory cells, being the M2 the predominant phenotypic profile among the adherent macrophages. In the inflammatory exudates F4/80(+)/CD206(+) cells (M2 macrophages) appeared in higher numbers then F4/80(+)/CCR7(+) cells (M1 macrophages), in addition, lower levels of pro-inflammatory cytokines together with higher levels of anti-inflammatory cytokines were found. Ch scaffolds with DA 15% showed opposite results, since M1 were the predominant macrophages both adherent to the scaffold and in the exudates, together with high levels of pro-inflammatory cytokines. In conclusion, Ch scaffolds with DA 5% induced a benign M2 anti-inflammatory macrophage response, whereas Ch scaffolds with DA 15% caused a macrophage M1 pro-inflammatory response.

Prostate cancer cell proliferation and angiogenesis in different obese mice models

Obesity has been associated with increased incidence and aggressiveness of prostate cancer. Although controversial, several studies suggest that leptin could influence tumour cell growth and proliferation. The main goal of this study was to assess cellular growth of prostate adenocarcinoma cells in obese mice with different endogenous hormonal environments in what relates to leptin circulating levels and sensitivity. Four groups of mice (n = 6/group) were used, namely obese mice with congenital non‐functioning leptin receptor OBR (db/db), obese mice with congenital leptin deficiency (ob/ob), mice with diet induced obesity (DIO) and normal weight C57BL/6J mice (control). All groups of mice were injected subcutaneously with 3.0 × 105 RM1 cells/500 μl PBS (murine prostate carcinoma androgen insensitive cells) and tumour growth and angiogenesis were evaluated 14 days after inoculation. The tumours induced in ob/ob and DIO mice were significantly larger (P < 0.001) while those induced in db/db mice were significantly smaller (P = 0.047), when compared with controls. Morphometric analysis revealed that mitotic index and Ki‐67 positive nuclear density, both cell proliferation markers, were also significantly lower in the tumours of db/db mice (P < 0.001) when compared to controls. An inverse correlation was observed between leptin plasma levels and tumour weight (r = −0.642, P < 0.001), mitotic index (r = −0.646, P < 0.01) and Ki‐67 positive nuclear density (r = −0.795, P < 0.001). These results suggest that high leptin concentrations are not favourable to RM1 cell growth and proliferation. On the contrary, high plasma leptin levels were associated with less cellular proliferation and angiogenesis in vivo.

Adipocyte secreted factors enhance aggressiveness of prostate carcinoma cells.

Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.

Detailed characterization of incretin cell distribution along the human small intestine

BACKGROUND Incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), are physiologic stimulants of insulin release that have been implicated in diabetes remission after bariatric surgery. The detailed distribution of incretin cells along the human small gut, so far unknown, is of utmost importance for the understanding of the metabolic changes observed after bariatric surgery because diabetes remission rate varies according to the type of anatomic rearrangement. OBJECTIVE To characterize the distribution of incretin producing cells along the human jejunum-ileum. SETTING Academic public institution. METHODS Small intestines (n = 30) from autopsies were sampled every 20 cm along their entire length and tissue microarrays were constructed. The percentage of immunohistochemistry-stained cell areas for GLP-1, GIP, and chromogranin A at each segment length was quantified using a computer-aided analysis tool. RESULTS The percentage of stained area for GLP-1 immunoreactive cells was found to be significantly higher from 200 cm from Treitz ligament onward compared with the first 80 cm of the small intestine, whereas GIP immunoreactive cells were predominant expressed in the first 80 cm. In contrast, chromogranin A expression was constant along the entire jejunum-ileum. CONCLUSION The uneven distribution of GLP-1-expressing cells, with a higher density from 200 cm of the jejunum-ileum, could contribute to explain the improvement of glycemic profile of diabetic patients observed after anatomic rearrangement of the intestinal tract, in particular when subjected to gastric bypass with longer biliopancreatic limbs.

The emerging role of the molecular marker p27 in the differential diagnosis of adrenocortical tumors

Malignant adrenocortical tumors (ACTs) are rare and highly aggressive; conversely, benign tumors are common and frequently found incidentally (the so-called incidentalomas). Currently, the use of molecular markers in the diagnosis of ACTs is still controversial. The aim of this study was to analyze the molecular profile of different ACTs with the purpose of identifying markers useful for differentiating between these tumors. The ACTs that were studied (n=31) included nonfunctioning adenomas (ACAn)/incidentalomas (n=13), functioning adenomas with Cushings syndrome (ACAc) (n=7), and carcinomas (n=11); normal adrenal glands (n=12) were used as controls. For each sample, the percentage area stained for the markers StAR, IGF2, IGF1R, p53, MDM2, p21, p27, cyclin D1, Ki-67, β-catenin, and E-cadherin was quantified using a morphometric computerized tool. IGF2, p27, cyclin D1, and Ki-67 were the markers for which the percentage of stained area was significantly higher in carcinoma samples than in adenoma samples. Ki-67 and p27 were the markers that exhibited the highest discriminative power for differential diagnosis between carcinomas and all type of adenomas, while IGF2 and StAR were only found to be useful for differentiating between carcinomas and ACAn and between carcinomas and ACAc respectively. The usefulness of Ki-67 has been recognized before in the differential diagnosis of malignant tumors. The additional use of p27 as an elective marker to distinguish benign ACTs from malignant ACTs should be considered.

Mesenchymal Stem/Stromal Cells seeded on cartilaginous endplates promote Intervertebral Disc Regeneration through Extracellular Matrix Remodeling.

Intervertebral disc (IVD) degeneration is characterized by significant biochemical and histomorphological alterations, such as loss of extracellular matrix (ECM) integrity, by abnormal synthesis of ECM main components, resultant from altered anabolic/catabolic cell activities and cell death. Mesenchymal Stem/Stromal Cell (MSC) migration towards degenerated IVD may represent a viable strategy to promote tissue repair/regeneration. Here, human MSCs (hMSCs) were seeded on top of cartilaginous endplates (CEP) of nucleotomized IVDs of bovine origin and cultured ex vivo up to 3 weeks. hMSCs migrated from CEP towards the lesion area and significantly increased expression of collagen type II and aggrecan in IVD, namely in the nucleus pulposus. Concomitantly, hMSCs stimulated the production of growth factors, promoters of ECM synthesis, such as fibroblast growth factor 6 (FGF-6) and 7 (FGF-7), platelet-derived growth factor receptor (PDGF-R), granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin-like growth factor 1 receptor (IGF-1sR). Overall, our results demonstrate that CEP can be an alternative route to MSC-based therapies for IVD regeneration through ECM remodeling, thus opening new perspectives on endogenous repair capacity through MSC recruitment.

Lymph Node Metastases in Papillary and Medullary Thyroid Carcinoma Are Independent of Intratumoral Lymphatic Vessel Density

Background: Blood and lymph vessel invasion are well-recognized markers of tumor aggressiveness, as these are the routes that lead to metastases. Thyroid tumors, depending on the histological variant, tend to have distinctive biological behaviors and use different vascular routes to metastasize, yet the mechanisms underlying the metastatic process are still poorly understood. Objectives: The aim of this study was to assess how the lymph vessel density (LVD) in different histological types of thyroid tumors, and in their surrounding tissue, correlate with the presence of lymph node metastases (LNM) and tumor pathological features. Methods: Lymph vessels of papillary thyroid carcinomas (PTC), of the classical (CVPTC, n = 50) and follicular variants (FVPTC, n = 18), and medullary thyroid carcinomas (MTC, n = 34) were immunohistochemically stained against antigen D2-40. The stained area was quantified using a computerized morphometric analysis tool and correlated with the tumor pathological characteristics. Results: LVD within all analyzed thyroid tumor subtypes was significantly lower than in the surrounding thyroid tissues (p < 0.001). Despite intratumoral LVD being significantly higher in CVPTC than in FVPTC, and peritumoral LVD being significantly higher in MTC than in PTC (p < 0.05), no correlations were found between LVD (either intratumoral or peritumoral) and the presence of lymph node metastasis. Conclusions: As no LVD differences were found amongst thyroid tumors with or without LNM, dissemination is more likely to depend on the tumor ability to invade the abundant lymph vessel network of the surrounding thyroid tissue than on the ability of the tumor to promote de novo lymphangiogenesis.

Vascular and apoptotic changes in the placode of myelomeningocele mice during the final stages of in utero development

OBJECT Myelomeningocele (MMC) is a primary neurulation defect that is associated with devastating neurological disabilities in affected newborns. To better characterize the in utero neurodegenerative process of MMC, the authors investigated the changes in vascular organization, apoptosis, and the presence of inflammatory cells during gestation by using a mutant mouse model of MMC. METHODS The curly tail/loop tail (ct/lp) mutant mouse model of MMC was chosen to obtain fetuses at different stages of gestation. Mouse fetuses harboring MMC were harvested by caesarean section at embryonic Days 14.5, 16.5, and 18.5 (complete mouse gestation at 19 days, 6 mice/group); littermate fetuses with the same gestational age but without an MMC were used as controls. Samples of the MMC placode or normal spinal cord were stained for immunocytochemical labeling with caveolin antibody (endothelium marker) and activated caspase-3 antibody (apoptosis marker). Samples were morphometrically analyzed with a computer-assisted image analyzer. RESULTS The MMC mice presented with an increase in vascular density from embryonic Days 16.5-18.5 and an enhanced number of apoptotic cells at embryonic Day 18.5, compared with controls. There were scarce signals of an inflammatory reaction in the MMC placode, as a few infiltrating neutrophils were seen only at embryonic Day 18.5. CONCLUSIONS Fetal placodes in MMC mice showed evidence of increased vascular density since embryonic Day 16.5 and increased apoptosis at embryonic Day 18.5. These new data support the view that in utero changes of the MMC placode, occurring during the last stages of gestation, contribute to the neuropathological manifestations in full-term newborns with MMC.

Inflammatory granulocytes decrease subcutaneous growth of melanoma in mice.

Growing melanomas invade the subcutaneous tissues. We have compared the size of tumors implanted in the subcutaneous cavities of C57BL/6 mice where inflammatory reactions were induced before the injection of 5×105 melanoma cells (B16F10 cell line). Granulocytic inflammation of the subcutaneous cavities resulted in a significant decrease in the growth of the implanted melanomas, whereas monocytic inflammation had no effect on tumor growth. We conclude that granulocytes, but not monocytes/macrophages, have anti-tumor action on melanoma that invade the subcutaneous tissues.

Immunocytochemical Characterization of Astrocytosis along the Spinal Cord of Loop-Tail/Curly-Tail Mice with Myelomeningocele

Background: The neurological deficits of myelomeningocele (MMC) have been attributed both to a primary neurulation defect and to a secondary injury of the placode in the intrauterine environment. Since astrocytes are involved in glial scar formation after spinal cord injury, the characterization of astrocyte density along the spinal cord upstream of the MMC can be used as a surrogate marker of the extension of the injury beyond the MMC. Methods: The curly-tail/loop-tail murine model was applied to obtain newborn mice with MMC. The astrocyte density and topography both at the MMC placode level and at the upper segments of the spinal cord was characterized by immunolabeling using the anti-glial fibrillary acidic protein antibody. This was followed by a qualitative evaluation of immunolabeled cells and morphometric analysis of the samples. Results: The topography of astrocytes in the spinal cord of MMC newborn mice was compared with that of newborn control mice (without spina bifida aperta) (n = 8/group). The anti-glial fibrillary acidic protein immunoreactivity was significantly increased in the MMC samples in comparison with the normal spinal cord, indicating the presence of an astrocytic response. Increased astrocytosis was also observed in the transitional area located above the MMC. The astrocytosis decreased progressively along the MMC spinal cord until matching the pattern of the control spinal cords. This transitional area involved a segment of the spinal cord with a length of 240 µm in the newborn mouse. Conclusions: MMC newborn mice show spinal cord injury that is located upstream of the exposed placode and is characterized by proliferation of astrocytes. This finding offers further support for the hypothesis of a tethering mechanism as part of the spinal cord injury observed in MMC newborns.