Publications by authors named "Paulo Rodrigues-Santos"

26 Publications

  • Page 1 of 1

Innate Lymphoid Cells in Human Pregnancy.

Front Immunol 2020 30;11:551707. Epub 2020 Nov 30.

Faculty of Medicine, Coimbra Institute for Clinical and Biomedical Research (iCBR), University of Coimbra, Coimbra, Portugal.

Innate lymphoid cells (ILCs) are a new set of cells considered to be a part of the innate immune system. ILCs are classified into five subsets (according to their transcription factors and cytokine profile) as natural killer cells (NK cells), group 1 ILCs, group 2 ILCs, group 3 ILCs, and lymphoid tissue inducers (LTi). Functionally, these cells resemble the T helper population but lack the expression of recombinant genes, which is essential for the formation of T cell receptors. In this work, the authors address the distinction between peripheral and decidual NK cells, highlighting their diversity in ILC biology and its relevance to human pregnancy. ILCs are effector cells that are important in promoting immunity, inflammation, and tissue repair. Recent studies have directed their attention to ILC actions in pregnancy. Dysregulation or expansion of pro-inflammatory ILC populations as well as abnormal tolerogenic responses may directly interfere with pregnancy, ultimately resulting in pregnancy loss or adverse outcomes. In this review, we characterize these cells, considering recent findings and addressing knowledge gaps in perinatal medicine in the context of ILC biology. Moreover, we discuss the relevance of these cells not only to the process of immune tolerance, but also in disease.
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http://dx.doi.org/10.3389/fimmu.2020.551707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734178PMC
November 2020

Activated double-negative T cells (CD3CD4CD8HLA-DR) define response to renal denervation for resistant hypertension.

Clin Immunol 2020 Sep 30;218:108521. Epub 2020 Jun 30.

Faculty of Medicine (FMUC), University of Coimbra, Coimbra, Portugal; Department of Cardiology, Coimbra's Hospital and University Centre (CHUC), Coimbra, Portugal; Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Purpose: To explore the cellular immune response of patients with resistant hypertension treated with renal denervation (RDN).

Methods And Results: Twenty-three patients were included and blood samples were obtained in six timings, pre and post procedure. Response was evaluated at six-months and one year and was observed in 69.6% and 82.6% of patients, respectively. Absolute values of HLA-DR+ double negative (DN) T cells were significantly lower in the group of 'responders' at one year, and interaction between the timings were found in three T cell subsets (T CD4, T CD8 and naïve T CD8 cells), with the 'responders' tending to present with lower absolute values and little inter-timing variation.

Conclusions: 'Responders' significantly present with lower absolute values of activated DN T cells and have lower and more stable values of total T CD8+, CD4+, and naïve T CD8+ cells. These cell types may be able to predict response to RDN.
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http://dx.doi.org/10.1016/j.clim.2020.108521DOI Listing
September 2020

Immune Responses after Vascular Photodynamic Therapy with Redaporfin.

J Clin Med 2019 Dec 31;9(1). Epub 2019 Dec 31.

CQC, Chemistry Department, University of Coimbra, 3004-535 Coimbra, Portugal.

Photodynamic therapy (PDT) relies on the administration of a photosensitizer (PS) that is activated, after a certain drug-to-light interval (DLI), by the irradiation of the target tumour with light of a specific wavelength absorbed by the PS. Typically, low light doses are insufficient to eradicate solid tumours and high fluence rates have been described as poorly immunogenic. However, previous work with mice bearing CT26 tumours demonstrated that vascular PDT with redaporfin, using a low light dose delivered at a high fluence rate, not only destroys the primary tumour but also reduces the formation of metastasis, thus suggesting anti-tumour immunity. This work characterizes immune responses triggered by redaporfin-PDT in mice bearing CT26 tumours. Our results demonstrate that vascular-PDT leads to a strong neutrophilia (2-24 h), systemic increase of IL-6 (24 h), increased percentage of CD4 and CD8 T cells producing IFN-γ or CD69 (2-24 h) and increased CD4/CD8 T cell ratio (2-24 h). At the tumour bed, T cell tumour infiltration disappeared after PDT but reappeared with a much higher incidence one day later. In addition, it is shown that the therapeutic effect of redaporfin-PDT is highly dependent on neutrophils and CD8 T cells but not on CD4 T cells.
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http://dx.doi.org/10.3390/jcm9010104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027008PMC
December 2019

NKT-Like (CD3+CD56+) Cells in Chronic Myeloid Leukemia Patients Treated With Tyrosine Kinase Inhibitors.

Front Immunol 2019 22;10:2493. Epub 2019 Oct 22.

Faculty of Medicine (FMUC), Institute of Immunology, University of Coimbra, Coimbra, Portugal.

Therapy with Tyrosine Kinase Inhibitors (TKI) aiming stable deep molecular response is the gold standard to treat Chronic Myeloid Leukemia (CML). NKT-like cells (CD3CD56) combine characteristics of T and NK cells. The physiopathological role of these cells remains unknown although the literature refers their association with inflammation, autoimmune diseases, and cancer. Since the information regarding the role of NKT-like cells in CML is rare, we aimed at the characterization of these cells in CML patients treated with TKIs. Peripheral blood NKT-like cells from 48 CML patients and 40 healthy donors were analyzed by multiparametric flow cytometry. Functional tests consisting of co-culture with leukemic target cells (K562 cell line) were used to measure degranulation and cytokine production. Our results revealed that NKT-like cells are decreased in treated CML patients, although they present increased expression of activation markers (CD69 and HLA-DR), increased degranulation (CD107a) and impaired IFN-γ production. Significantly alterations on the expression of tumor recognition (NCRs and NKp80), and immune regulation receptors (LAG-3, TIM-3, and CD137) by NKT-like cells were observed in CML patients. Second generation TKIs increased cell activation (CD69) and decreased expression of NKp44 and NKp80 by NKT-like cells from CML patients when compared to Imatinib. CML patients that achieved deep molecular response (MR4.5) presented downregulation of NKp44 and LAG-3. Further studies are needed to clarify the role of these cells as biomarkers of therapy response and also to evaluate their value for discrimination of better candidates for sustained treatment-free remission after TKI discontinuation.
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http://dx.doi.org/10.3389/fimmu.2019.02493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817724PMC
September 2020

Effect of Age on NK Cell Compartment in Chronic Myeloid Leukemia Patients Treated With Tyrosine Kinase Inhibitors.

Front Immunol 2018 8;9:2587. Epub 2018 Nov 8.

Faculty of Medicine, Institute of Immunology, University of Coimbra, Coimbra, Portugal.

Natural killer (NK) cells are a very important component of the innate immune response involved in the lysis of virus infected and tumor cells. Aging has a profound impact in the frequency, phenotype and function of NK cells. Chronic Myeloid Leukemia (CML) is caused by the BCR-ABL gene formation encoding aberrant oncoprotein tyrosine kinase. Treatment with tyrosine kinase inhibitors (TKIs) induces durable deep molecular response. The response to treatment and life expectancy is lower in older patients with chronic phase of CML than in younger patients. In this work we analyse NK cells from TKI-treated CML patients and healthy controls stratified according to age. We have analyzed the expression of NK receptors, activation markers, NK cell differentiation in CD56 and CD56 NK cell subsets and the expression of CD107a and IFN-γ in NK cells stimulated with K562. Whereas significant differences on the phenotype and function of NK cells were found between middle-aged (35-65 years old) and elderly (older than 65) healthy individuals, NK cells from TKI-treated CML patients do not show significant differences related with age in most parameters studied, indicating that age is not a limitation of the NK cell recovery after treatment with TKI. Our results also revealed differences in the expression of NK receptors, activation markers and functional assays in NK cells from TKI-treated CML patients compared with age-matched healthy controls. These results highlight the relevance of NK cells in TKI-treated patients and the need of an extensive analysis of the effect of aging on NK cell phenotype and function in these patients in order to define new NK-cell based strategies directed to control CML progression and achieve long-term disease remission after TKI cessation.
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http://dx.doi.org/10.3389/fimmu.2018.02587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6246921PMC
October 2019

Repeated Mesenchymal Stromal Cell Treatment Sustainably Alleviates Machado-Joseph Disease.

Mol Ther 2018 09 12;26(9):2131-2151. Epub 2018 Jul 12.

Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Faculdade de Medicina, Rua Larga, Pólo I, 1° andar, 3004-504 Coimbra, Portugal; Faculty of Pharmacy, University of Coimbra, Polo 3, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. Electronic address:

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3, the most common dominant spinocerebellar ataxia (SCA) worldwide, is caused by over-repetition of a CAG repeat in the ATXN3/MJD1 gene, which translates into a polyglutamine tract within the ataxin-3 protein. There is no treatment for this fatal disorder. Despite evidence of the safety and efficacy of mesenchymal stromal cells (MSCs) in delaying SCA disease progression in exploratory clinical trials, unanticipated regression of patients to the status prior to treatment makes the investigation of causes and solutions urgent and imperative. In the present study, we compared the efficacy of a single intracranial injection with repeated systemic MSC administration in alleviating the MJD phenotype of two strongly severe genetic rodent models. We found that a single MSC transplantation only produces transient effects, whereas periodic administration promotes sustained motor behavior and neuropathology alleviation, suggesting that MSC therapies should be re-designed to get sustained beneficial results in clinical practice. Furthermore, MSC promoted neuroprotection, increased the levels of GABA and glutamate, and decreased the levels of Myo-inositol, which correlated with motor improvements, indicating that these metabolites may serve as valid neurospectroscopic biomarkers of disease and treatment. This study makes important contributions to the design of new clinical approaches for MJD and other SCAs/polyglutamine disorders.
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http://dx.doi.org/10.1016/j.ymthe.2018.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127516PMC
September 2018

Revisiting colorectal cancer animal model - An improved metastatic model for distal rectosigmoid colon carcinoma.

Pathophysiology 2018 Jun 13;25(2):89-99. Epub 2018 Mar 13.

Biophysics Unit, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Centre of Investigation on Environment, Genetics and Oncobiology (CIMAGO), Coimbra, Portugal. Electronic address:

Colorectal cancer (CRC) is the second most frequent and fatal cancer in Western countries. Understanding its biology with different incidence along the colon and rectum, genetic profile and how these factors contribute to local/distant progression, has been hampered by the lack of a suitable CRC model. We report a reproducible model, using human CRC cell lines (CL) (WiDr, LS1034, C2BBe1) injected (1 × 10 cells/animal) in RNU rats (n = 55) which underwent cecostomy and descending colostomy with mucosal-cutaneous fistula of the sigmoid colon. CL were characterized by immunohistochemistry: CK20, CDX2, P53, vimentin, Ki67, CD44, CD133, E-cadherin, β-catenin and CEA; cancer stem cells-immune system interaction was studied and tumor progression was assessed with nuclear medicine imaging (Tc-MIBI). Animals developed locally invasive tumors and with WiDr neural invasion was registered. Cancer stem cells were detected in WiDr (CD44 positive). All the cell lines stimulated the immune system, being WiDr the most aggressive. Imaging studies demonstrated tumor uptake. With this CRC model we can study the microenvironment role and tumor-stroma interactions. All CL developed primary disease, but only the WiDR established neural invasion which may represent a metastatic pathway. This model can help unveiling the underlying metastatic mechanisms, and ultimately test better therapeutic approaches for CRC.
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http://dx.doi.org/10.1016/j.pathophys.2018.02.002DOI Listing
June 2018

The effects of physical exercise on nonmotor symptoms and on neuroimmune RAGE network in experimental parkinsonism.

J Appl Physiol (1985) 2017 Jul 6;123(1):161-171. Epub 2017 Apr 6.

Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences, Faculty of Medicine, University of Coimbra, Coimbra, Portugal;

Parkinson's disease (PD) prodromal stages comprise neuropsychiatric perturbations that critically compromise a patient's quality of life. These nonmotor symptoms (NMS) are associated with exacerbated innate immunity, a hallmark of overt PD. Physical exercise (PE) has the potential to improve neuropsychiatric deficits and to modulate immune network including receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs) in distinct pathological settings. Accordingly, the present study aimed to test the hypothesis that PE ) alleviates PD NMS and ) modulates neuroimmune RAGE network in experimental PD. Adult Wistar rats subjected to long-term mild treadmill were administered intranasally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probed for PD NMS before the onset of motor abnormalities. Twelve days after MPTP, neuroimmune RAGE network transcriptomics (real-time quantitative PCR) was analyzed in frontal cortex, hippocampus, and striatum. Untrained MPTP animals displayed habit-learning and motivational deficits without gross motor impairments (cued version of water-maze, splash, and open-field tests, respectively). A suppression of RAGE and neuroimmune-related genes was observed in frontal cortex on chemical and physical stressors (untrained MPTP: RAGE, TLR5 and -7, and p22 NADPH oxidase; saline-trained animals: RAGE, TLR1 and -5 to -11, TNF-α, IL-1β, and p22 NADPH oxidase), suggesting the recruitment of compensatory mechanisms to restrain innate inflammation. Notably, trained MPTP animals displayed normal cognitive/motivational performances. Additionally, these animals showed normal RAGE expression and neuroprotective PD-related gene upregulation in frontal cortex when compared with untrained MPTP animals. These findings corroborate PE efficacy in improving PD NMS and newly identify RAGE network as a neural substrate for exercise intervention. Additional research is warranted to unveil functional consequences of PE-induced modulation of RAGE/DJ-1 transcriptomics in PD premotor stages. This study newly shows that physical exercise (PE) corrects nonmotor symptoms of the intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of experimental parkinsonism. Additionally, we show that suppression of neuroimmune receptor for advanced glycation end products (RAGE) network occurs in frontal cortex on chemical (MPTP) and physical (PE) interventions. Finally, PE normalizes frontal cortical RAGE transcriptomics and upregulates the neuroprotective gene in the intranasal MPTP model of experimental parkinsonism.
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http://dx.doi.org/10.1152/japplphysiol.01120.2016DOI Listing
July 2017

Natural killer cell-based adoptive immunotherapy eradicates and drives differentiation of chemoresistant bladder cancer stem-like cells.

BMC Med 2016 10 21;14(1):163. Epub 2016 Oct 21.

Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Background: High-grade non-muscle invasive bladder cancer (NMIBC) has a high risk of recurrence and progression to muscle-invasive forms, which seems to be largely related to the presence of tumorigenic stem-like cell populations that are refractory to conventional therapies. Here, we evaluated the therapeutic potential of Natural Killer (NK) cell-based adoptive immunotherapy against chemoresistant bladder cancer stem-like cells (CSCs) in a pre-clinical relevant model, using NK cells from healthy donors and NMIBC patients.

Methods: Cytokine-activated NK cells from healthy donors and from high-grade NMIBC patients were phenotypically characterized and assayed in vitro against stem-like and bulk differentiated bladder cancer cells. Stem-like cells were isolated from two bladder cancer cell lines using the sphere-forming assay. The in vivo therapeutic efficacy was evaluated in mice bearing a CSC-induced orthotopic bladder cancer. Animals were treated by intravesical instillation of interleukin-activated NK cells. Tumor response was evaluated longitudinally by non-invasive bioluminescence imaging.

Results: NK cells from healthy donors upon activation with IL-2 and IL-15 kills indiscriminately both stem-like and differentiated tumor cells via stress ligand recognition. In addition to cell killing, NK cells shifted CSCs towards a more differentiated phenotype, rendering them more susceptible to cisplatin, highlighting the benefits of a possible combined therapy. On the contrary, NK cells from NMIBC patients displayed a low density on NK cytotoxicity receptors, adhesion molecules and a more immature phenotype, losing their ability to kill and drive differentiation of CSCs. The local administration, via the transurethral route, of activated NK cells from healthy donors provides an efficient tumor infiltration and a subsequent robust tumoricidal activity against bladder cancer with high selective cytolytic activity against CSCs, leading to a dramatic reduction in tumor burden from 80 % to complete remission.

Conclusion: Although pre-clinical, our results strongly suggest that an immunotherapeutic strategy using allogeneic activated NK cells from healthy donors is effective and should be exploited as a complementary therapeutic strategy in high-risk NMIBC patients to prevent tumor recurrence and progression.
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http://dx.doi.org/10.1186/s12916-016-0715-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5075212PMC
October 2016

Regulation of striatal astrocytic receptor for advanced glycation end-products variants in an early stage of experimental Parkinson's disease.

J Neurochem 2016 08 13;138(4):598-609. Epub 2016 Jun 13.

Laboratory of Pharmacology and Experimental Therapeutics/IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Convincing evidence indicates that advanced glycation end-products and danger-associated protein S100B play a role in Parkinson's disease (PD). These agents operate through the receptor for advanced glycation end-products (RAGE), which displays distinct isoforms playing protective/deleterious effects. However, the nature of RAGE variants has been overlooked in PD studies. Hence, we attempted to characterize RAGE regulation in early stages of PD striatal pathology. A neurotoxin-based rodent model of PD was used in this study, through administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57BL/6 mice. Animals were killed 6 h post-MPTP to assess S100B/RAGE contents (RT-qPCR, ELISA) and RAGE isoform density (WB) and cellular distribution (immunohistochemistry). Dopaminergic and gliotic status were also mapped (HPLC-ED, WB, immunohistochemistry). At this preliminary stage of MPTP-induced PD in mice, RAGE inhibitory isoforms were increased whereas full-length RAGE was not affected. This putative cytoprotective RAGE phenotype paired an inflammatory and pro-oxidant setting fueling DAergic denervation. Increased RAGE inhibitory variants occur in astrocytes showing higher S100B density but no overt signs of hypertrophy or NF-κB activation, a canonical effector of RAGE. These findings expand our understanding of the toxic effect of MPTP on striatum and offer first in vivo evidence of RAGE being a responder in early stages of astrogliosis dynamics, supporting a protective rather tissue-destructive phenotype of RAGE in the initial phase of PD degeneration. These data lay the groundwork for future studies on the relevance of astrocytic RAGE in DAergic neuroprotection strategies. We report increased antagonistic RAGE variants paralleling S100B up-regulation in early stages of MPTP-induced astrogliosis dynamics . We propose that selective RAGE regulation reflects a self-protective mechanism to maintain low levels of RAGE ligands , preventing long-term inflammation and oxidative stress arising from sustained ligands/flRAGE activation . Understanding loss of RAGE protective response to stress may provide new therapeutic options to halt or slow down dopaminergic axonopathy and, ultimately, neuronal death .
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http://dx.doi.org/10.1111/jnc.13682DOI Listing
August 2016

Monophosphoryl Lipid-A: A Promising Tool for Alzheimer's Disease Toll.

J Alzheimers Dis 2016 04;52(4):1189-202

Laboratório de Farmacologia e Terapêutica Experimental/IBILI, Faculdade de Medicina da Universidade de Coimbra, Portugal.

Neuroinflammation is a two-edged sword in Alzheimer's disease (AD). A certain degree of neuroinflammation is instrumental in the clearance of amyloid-β (Aβ) peptides by activated microglia, although a sustained neuroinflammation might accelerate Aβ deposition, thus fostering the neurodegenerative process and functional decline in AD. There is an increasing body of evidence suggesting that the innate immune system via Toll-like receptor 4 (TLR4) finely orchestrates the highly regulated inflammatory cascade that takes place in AD pathology. Herein we critically review pre-clinical (in vitro and in vivo approaches) and clinical studies showing that monophosphoryl lipid A (MPL), a partial TLR4 agonist, may have beneficial effect on AD physiopathology. The in vivo data elegantly showed that MPL enhanced Aβ plaque phagocytosis thus decreasing the number and the size of Aβ deposits and soluble Aβ in brain from APPswe/PS1 mice. Furthermore, MPL also improved their cognition. The mechanism underlying this MPL effect was proposed to be microglial activation by recruiting TLR4. Additionally, it was demonstrated that MPL increased the Aβ antibody titer and showed a safe profile in mice and primates, when used as a vaccine adjuvant. Clinical studies using MPL as an adjuvant in Aβ immunotherapy are currently ongoing. Overall, we argue that the TLR4 partial agonist MPL is a potentially safe and effective new pharmacological tool in AD.
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http://dx.doi.org/10.3233/JAD-151183DOI Listing
April 2016

The role of immune system exhaustion on cancer cell escape and anti-tumor immune induction after irradiation.

Biochim Biophys Acta 2016 Apr 9;1865(2):168-75. Epub 2016 Feb 9.

Immunology Institute, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Immune surveillance seems to represent an effective tumor suppressor mechanism. However, some cancer cells survive and become variants, being poorly immunogenic and able to enter a steady-state phase. These cells become functionally dormant or remain hidden clinically throughout. Neoplastic cells seem to be able to instruct immune cells to undergo changes promoting malignancy. Radiotherapy may act as a trigger of the immune response. After radiotherapy a sequence of reactions occurs, starting in the damage of oncogenic cells by multiple mechanisms, leading to the immune system positive feedback against the tumor. The link between radiotherapy and the immune system is evident. T cells, macrophages, Natural Killer cells and other immune cells seem to have a key role in controlling the tumor. T cells may be dysfunctional and remain in a state of T cell exhaustion, nonetheless, they often retain a high potential for successful defense against cancer, being able to be mobilized to become highly functional. The lack of clinical trials on a large scale makes data a little robust, in spite of promising information, there are still many variables in the studies relating to radiation and immune system. The clarification of the mechanisms underlying immune response to radiation exposure may contribute to treatment improvement, gain of life quality and span of patients.
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http://dx.doi.org/10.1016/j.bbcan.2016.02.002DOI Listing
April 2016

Gene Association with Leprosy: A Review of Published Data.

Front Immunol 2015 12;6:658. Epub 2016 Jan 12.

Faculty of Medicine, Immunology Institute, University of Coimbra, Coimbra, Portugal; Immunology and Oncology Laboratory, Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal.

Leprosy is a chronic infectious disease caused by an obligate intracellular bacterium known as Mycobacterium leprae. Exposure to the bacillus is necessary, but this alone does not mean an individual will develop clinical symptoms of the disease. In recent years, several genes have been associated with leprosy and the innate immune response pathways converge on the main hypothesis that genes are involved in the susceptibility for the disease in two distinct steps: for leprosy per se and in the development of the different clinical forms. These genes participate in the sensing, main metabolic pathway of immune response activation and, subsequently, on the evolution of the disease into its clinical forms. The aim of this review is to highlight the role of innate immune response in the context of leprosy, stressing their participation in the signaling and targeting processes in response to bacillus infection and on the evolution to the clinical forms of the disease.
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http://dx.doi.org/10.3389/fimmu.2015.00658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709443PMC
January 2016

Can membrane progesterone receptor α on T regulatory cells explain the ensuing human labour?

J Reprod Immunol 2016 Feb 20;113:22-6. Epub 2015 Oct 20.

Faculty of Medicine, University of Coimbra, Portugal.

Progesterone acts as an immunosteroid by contributing to the establishment of a pregnancy-protective milieu. It seems that it is the responsibility of progesterone to evade the inflammatory events that lead to parturition. T regulatory lymphocytes (Treg cells) could further explain the inhibition of the inflammatory mechanisms that lead to labour through the rapid action of progesterone on this cell subset. We investigated Treg cells and the membrane progesterone receptor α (mPRα) in these immune cells with in relationship to human parturition. This pilot cohort study was conducted in a single-centre tertiary obstetrical unit with 20 normal pregnant women. Variation in the absolute and relative frequency of CD4(+) T cells, Treg cells, and of mPR(α+) Treg cells was calculated by flow cytometry on three occasions (second and third trimesters; delivery day). Our results show that during normal pregnancy there is a generalised increase in Treg cells and mPR(α+) Treg cells, from the second to the third trimesters (23.4% vs. 52.3% and 4.3% vs. 8.3%, respectively). On the contrary, on delivery day, compared with the values in the third trimester, there is a sudden decrease in both Treg cells (52.3% vs. 17.4%) and mPR(α+) Treg cells (8.3% vs. 6.1%). Our findings suggest that human labour may develop as a consequence of a decline in mPR(α+) Treg cells, which reduces progesterone anti-inflammatory action through Treg cells.
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http://dx.doi.org/10.1016/j.jri.2015.10.002DOI Listing
February 2016

Functional and molecular characterization of cancer stem-like cells in bladder cancer: a potential signature for muscle-invasive tumors.

Oncotarget 2015 Nov;6(34):36185-201

Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.

Striking evidence associates cancer stem cells (CSCs) to the high recurrence rates and poor survival of patients with muscle-invasive bladder cancer (BC). However, the prognostic implication of those cells in risk stratification is not firmly established, mainly due to the functional and phenotypic heterogeneity of CSCs populations, as well as, to the conflicting data regarding their identification based on a single specific marker. This emphasizes the need to exploit putative CSC-related molecular markers with potential prognostic significance in BC patients.This study aimed to isolate and characterize bladder CSCs making use of different functional and molecular approaches. The data obtained provide strong evidence that muscle-invasive BC is enriched with a heterogeneous stem-like population characterized by enhanced chemoresistance and tumor initiating properties, able to recapitulate the heterogeneity of the original tumor. Additionally, a logistic regression analysis identified a 2-gene stem-like signature (SOX2 and ALDH2) that allows a 93% accurate discrimination between non-muscle-invasive and invasive tumors.Our findings suggest that a stemness-related gene signature, combined with a cluster of markers to more narrowly refine the CSC phenotype, could better identify BC patients that would benefit from a more aggressive therapeutic intervention targeting CSCs population.
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http://dx.doi.org/10.18632/oncotarget.5517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742170PMC
November 2015

Does progesterone administration in preterm labor influence Treg cells?

J Perinat Med 2016 Aug;44(6):605-11

Objectives: The aim of this study was to determine if the actions of progesterone on preterm labor are accomplished through modulation of the percentage of regulatory T-cells (Treg).

Methods: The study was a cohort pilot study made in a single center tertiary obstetrical unit with women in preterm labor arrested with tocolytic treatment. Variation of the number and percentage of Treg cells obtained from peripheral blood samples of women with preterm labor were calculated by flow cytometry, before and after progesterone administration.

Results: In the paired samples for each patient, there was a significant difference in the Treg cell pool after progesterone treatment, with an increase in both their percentage (48.9 vs. 53; P=0.07) and absolute number (14.8 vs. 56.5 cells/μL; P=0.046).

Conclusions: This research demonstrated a considerable increase in the Treg cell pool after progesterone treatment. This indicates a possible mechanism for progesterone treatment benefits in preterm labor, potentially increasing its more rational use.
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http://dx.doi.org/10.1515/jpm-2015-0134DOI Listing
August 2016

Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination.

Biomaterials 2015 Nov 6;69:76-88. Epub 2015 Aug 6.

CNC - Center for Neurosciences and Cell Biology, University of Coimbra, Faculty of Medicine (Polo I), Rua Larga, Coimbra 3004-504, Portugal; FFUC - Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, Coimbra 3000-548, Portugal. Electronic address:

Breast cancer stem cells (CSC) are thought responsible for tumor growth and relapse, metastization and active evasion to standard chemotherapy. The recognition that CSC may originate from non-stem cancer cells (non-SCC) through plastic epithelial-to-mesenchymal transition turned these into relevant cell targets. Of crucial importance for successful therapeutic intervention is the identification of surface receptors overexpressed in both CSC and non-SCC. Cell surface nucleolin has been described as overexpressed in cancer cells as well as a tumor angiogenic marker. Herein we have addressed the questions on whether nucleolin was a common receptor among breast CSC and non-SCC and whether it could be exploited for targeting purposes. Liposomes functionalized with the nucleolin-binding F3 peptide, targeted simultaneously, nucleolin-overexpressing putative breast CSC and non-SCC, which was paralleled by OCT4 and NANOG mRNA levels in cells from triple negative breast cancer (TNBC) origin. In murine embryonic stem cells, both nucleolin mRNA levels and F3 peptide-targeted liposomes cellular association were dependent on the stemness status. An in vivo tumorigenic assay suggested that surface nucleolin overexpression per se, could be associated with the identification of highly tumorigenic TNBC cells. This proposed link between nucleolin expression and the stem-like phenotype in TNBC, enabled 100% cell death mediated by F3 peptide-targeted synergistic drug combination, suggesting the potential to abrogate the plasticity and adaptability associated with CSC and non-SCC. Ultimately, nucleolin-specific therapeutic tools capable of simultaneous debulk multiple cellular compartments of the tumor microenvironment may pave the way towards a specific treatment for TNBC patient care.
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http://dx.doi.org/10.1016/j.biomaterials.2015.08.007DOI Listing
November 2015

Drug transporters play a key role in the complex process of Imatinib resistance in vitro.

Leuk Res 2015 Mar 23;39(3):355-60. Epub 2014 Dec 23.

Applied Molecular Biology and University Clinic of Hematology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Portugal; CIMAGO - Center of Investigation in Environment, Genetics and Oncobiology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Hematology Department, Centro Hospitalar Universitário de Coimbra (CHUC), Portugal. Electronic address:

Imatinib resistance has been associated with BCR-ABL alterations, but other mechanisms might be involved, like drug transporters. Additionally, the impact of poor adherence in resistance has been little explored. Using sensitive and resistance CML cell lines, we investigated the expression of influx/efflux transporters, like P-gP and OCT1. In the therapeutic interruption model, we observed decrease of influx and increase in efflux transporters combined with BCR-ABL over-expression. Comparatively, resistant cells obtained by continuous TKI exposure only demonstrated alterations in drug's transporters. By exploring P-gP expression of resistant cells, we observed the potential of P-gP inhibitor in circumventing Imatinib resistance. Our results revealed the importance of treatment interruptions for expected response levels and show the complexity of Imatinib resistant process. Efflux transports appear as not only relevant for acquisition of resistant phenotype, but also as valid therapeutic tool for managing resistance.
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http://dx.doi.org/10.1016/j.leukres.2014.12.008DOI Listing
March 2015

Conversion to sirolimus ameliorates cyclosporine-induced nephropathy in the rat: focus on serum, urine, gene, and protein renal expression biomarkers.

Biomed Res Int 2014 19;2014:576929. Epub 2014 May 19.

Laboratory of Pharmacology & Experimental Therapeutics, IBILI, Faculty of Medicine, University of Coimbra, Sub-Unit 1 (Pólo III), 3000-548 Coimbra, Portugal.

Protocols of conversion from cyclosporin A (CsA) to sirolimus (SRL) have been widely used in immunotherapy after transplantation to prevent CsA-induced nephropathy, but the molecular mechanisms underlying these protocols remain nuclear. This study aimed to identify the molecular pathways and putative biomarkers of CsA-to-SRL conversion in a rat model. Four animal groups (n = 6) were tested during 9 weeks: control, CsA, SRL, and conversion (CsA for 3 weeks followed by SRL for 6 weeks). Classical and emergent serum, urinary, and kidney tissue (gene and protein expression) markers were assessed. Renal lesions were analyzed in hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome stains. SRL-treated rats presented proteinuria and NGAL (serum and urinary) as the best markers of renal impairment. Short CsA treatment presented slight or even absent kidney lesions and TGF-β, NF- κβ, mTOR, PCNA, TP53, KIM-1, and CTGF as relevant gene and protein changes. Prolonged CsA exposure aggravated renal damage, without clear changes on the traditional markers, but with changes in serums TGF- β and IL-7, TBARs clearance, and kidney TGF-β and mTOR. Conversion to SRL prevented CsA-induced renal damage evolution (absent/mild grade lesions), while NGAL (serum versus urine) seems to be a feasible biomarker of CsA replacement to SRL.
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http://dx.doi.org/10.1155/2014/576929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4055143PMC
September 2015

Transition from cyclosporine-induced renal dysfunction to nephrotoxicity in an in vivo rat model.

Int J Mol Sci 2014 May 20;15(5):8979-97. Epub 2014 May 20.

Laboratory of Pharmacology & Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra 3000-548, Portugal.

Cyclosporin A (CsA), a calcineurin inhibitor, remain the cornerstone of immunosuppressive regimens, regardless of nephrotoxicity, which depends on the duration of drug exposure. The mechanisms and biomarkers underlying the transition from CsA-induced renal dysfunction to nephrotoxicity deserve better elucidation, and would help clinical decisions. This study aimed to clarify these issues, using a rat model of short- and long-term CsA (5 mg/kg bw/day) treatments (3 and 9 weeks, respectively). Renal function was assessed on serum and urine; kidney tissue was used for histopathological characterization and gene and/or protein expression of markers of proliferation, fibrosis and inflammation. In the short-term, creatinine and blood urea nitrogen (BUN) levels increased and clearances decreased, accompanied by glomerular filtration rate (GFR) reduction, but without kidney lesions; at that stage, CsA exposure induced proliferating cell nuclear antigen (PCNA), transforming growth factor beta 1 (TGF-β1), factor nuclear kappa B (NF-κβ) and Tumor Protein P53 (TP53) kidney mRNA up-regulation. In the long-term treatment, renal dysfunction data was accompanied by glomerular and tubulointerstitial lesions, with remarkable kidney mRNA up-regulation of the mammalian target of rapamycin (mTOR) and the antigen identified by monoclonal antibody Ki-67 (Mki67), accompanied by mTOR protein overexpression. Transition from CsA-induced renal dysfunction to nephrotoxicity is accompanied by modification of molecular mechanisms and biomarkers, being mTOR one of the key players for kidney lesion evolution, thus suggesting, by mean of molecular evidences, that early CsA replacement by mTOR inhibitors is indeed the better therapeutic choice to prevent chronic allograft nephropathy.
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http://dx.doi.org/10.3390/ijms15058979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057770PMC
May 2014

Sitagliptin prevents inflammation and apoptotic cell death in the kidney of type 2 diabetic animals.

Mediators Inflamm 2014 8;2014:538737. Epub 2014 Apr 8.

Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal ; Center of Ophthalmology and Vision Sciences, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal.

This study aimed to evaluate the efficacy of sitagliptin, a dipeptidyl peptidase IV (DPP-IV) inhibitor, in preventing the deleterious effects of diabetes on the kidney in an animal model of type 2 diabetes mellitus; the Zucker diabetic fatty (ZDF) rat: 20-week-old rats were treated with sitagliptin (10 mg/kg bw/day) during 6 weeks. Glycaemia and blood HbA1c levels were monitored, as well as kidney function and lesions. Kidney mRNA and/or protein content/distribution of DPP-IV, GLP-1, GLP-1R, TNF-α, IL-1β, BAX, Bcl-2, and Bid were evaluated by RT-PCR and/or western blotting/immunohistochemistry. Sitagliptin treatment improved glycaemic control, as reflected by the significantly reduced levels of glycaemia and HbA1c (by about 22.5% and 1.2%, resp.) and ameliorated tubulointerstitial and glomerular lesions. Sitagliptin prevented the diabetes-induced increase in DPP-IV levels and the decrease in GLP-1 levels in kidney. Sitagliptin increased colocalization of GLP-1 and GLP-1R in the diabetic kidney. Sitagliptin also decreased IL-1β and TNF-α levels, as well as, prevented the increase of BAX/Bcl-2 ratio, Bid protein levels, and TUNEL-positive cells which indicates protective effects against inflammation and proapoptotic state in the kidney of diabetic rats, respectively. In conclusion, sitagliptin might have a major role in preventing diabetic nephropathy evolution due to anti-inflammatory and antiapoptotic properties.
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http://dx.doi.org/10.1155/2014/538737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000968PMC
December 2014

Sitagliptin prevents aggravation of endocrine and exocrine pancreatic damage in the Zucker Diabetic Fatty rat - focus on amelioration of metabolic profile and tissue cytoprotective properties.

Diabetol Metab Syndr 2014 Mar 20;6(1):42. Epub 2014 Mar 20.

Laboratory of Pharmacology & Experimental Therapeutics, IBILI, Faculty of Medicine, Sub-Unit 1 (Polo III), University of Coimbra, 3000-548 Coimbra, Portugal.

Background: The purpose of this study was to investigate some of the possible mechanisms underlying the protective effects of a dipeptidyl peptidase IV (DPP-IV) inhibitor, sitagliptin, on pancreatic tissue in an animal model of type 2 diabetes mellitus (T2DM), the Zucker Diabetic Fatty (ZDF) rat, focusing on glycaemic, insulinic and lipidic profiles, as well as, on apoptosis, inflammation, angiogenesis and proliferation mediators.

Methods: Male obese diabetic ZDF (fa/fa) rats, aged 20 weeks, were treated with sitagliptin (10 mg/kg bw/day) during 6 weeks and compared to untreated diabetic and lean control littermates. Metabolic data was evaluated at the beginning and at the end of the treatment, including glycaemia, HbA1c, insulinaemia, HOMA-beta and TGs. Endocrine and exocrine pancreas lesions were assessed semiquantitatively by histopathological methods. Pancreas gene (mRNA) and protein expression of mediators of apoptotic machinery, inflammation and angiogenesis/proliferation (Bax, Bcl2, IL-1β, VEGF, PCNA and TRIB3) were analyzed by RT-qPCR and/or by immunohistochemistry.

Results: Sitagliptin treatment for 6 weeks (between 20 and 26 week-old) was able to significantly (p < 0.001) ameliorate all the metabolic parameters, by preventing the increase in blood glucose and in serum TGs contents (16.54% and 37.63%, respectively, vs untreated), as well as, by preventing the decrease in serum insulin levels and in the functional beta cells capacity accessed via HOMA-beta index (156.28% and 191.74%, respectively, vs untreated). Sitagliptin-treated diabetic rats presented a reduced pancreas Bax/Bcl2 ratio, suggestive of an antiapoptotic effect; in addition, sitagliptin was able to completely reduce (p < 0.001) the pancreas overexpression of IL-1β and TRIB3 found in the untreated diabetic animals; and promoted a significant (p < 0.001) overexpression of VEGF and PCNA.

Conclusion: In this animal model of obese T2DM (the ZDF rat), sitagliptin prevented β-cell dysfunction and evolution of pancreatic damage. The protective effects afforded by this DPP-IV inhibitor may derive from improvement of the metabolic profile (viewed by the amelioration of glucose and TGs levels and of insulin resistance) and from cytoprotective properties, such as antiapoptotic, anti-inflammatory, pro-angiogenic and pro-proliferative.
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http://dx.doi.org/10.1186/1758-5996-6-42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998187PMC
March 2014

The need for transparency and good practices in the qPCR literature.

Nat Methods 2013 Nov;10(11):1063-7

Postgraduate Medical Institute, Anglia Ruskin University, Chelmsford, UK.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
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http://dx.doi.org/10.1038/nmeth.2697DOI Listing
November 2013

Alterations in the peripheral blood B cell subpopulations of multidrug-resistant tuberculosis patients.

Clin Exp Med 2014 Nov 26;14(4):423-9. Epub 2013 Sep 26.

Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marques de Pombal, 3004-517, Coimbra, Portugal.

The function of B cells in the immune response against Mycobacterium tuberculosis (Mtb) is still regarded as secondary, although major findings in mouse models of tuberculosis (TB) support their participation as regulators and antibody producers. However, studies in cohorts of TB or multidrug-resistant TB (MDR-TB) patients have failed to clearly identify changes in the circulating B cell pool. Therefore, in the present study we aimed at identifying alterations in the different B cell subpopulations in peripheral blood samples of HIV-negative pulmonary MDR-TB patients when compared to healthy donors. The data show, for the first time, that MDR-TB patients, similarly to what has been observed in other chronic inflammatory diseases, have a much lower frequency of peripheral blood unswitched IgD(+)CD27(+) memory B cells. Equally novel are the findings that in MDR-TB patients there is a reduction in the circulating plasma cell pool and that in MDR-TB there is an increased frequency of circulating type 1 transitional IgD(+)CD38(++), CD69(+) and TLR9(+) B cells. These results document disease-related shifts in peripheral blood B cell subsets in MDR-TB and suggest that such changes should be taken into account when designing new strategies to boost the cellular and humoral immune response against Mtb.
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http://dx.doi.org/10.1007/s10238-013-0258-1DOI Listing
November 2014

Monoclonal anti-CD8 therapy induces disease amelioration in the K/BxN mouse model of spontaneous chronic polyarthritis.

Arthritis Rheum 2010 Oct;62(10):2953-62

Instituto Gulbenkian de Ciência, Oeiras, Portugal.

Objective: CD8+ T cells are part of the T cell pool infiltrating the synovium in rheumatoid arthritis (RA). However, their role in the pathogenesis of RA has not been fully delineated. Using the K/BxN mouse model of spontaneous chronic arthritis, which shares many similarities with RA, we studied the potential of CD8+ T cell depletion with monoclonal antibodies (mAb) to stop and reverse the progression of experimental arthritis.

Methods: CD8+ T cells from the blood and articular infiltrate of K/BxN mice were characterized for cell surface phenotypic markers and for cytokine production. Additionally, mice were treated with specific anti-CD8 mAb (YTS105 and YTS169.4), with and without thymectomy.

Results: CD8+ T cells from the peripheral blood and joints of K/BxN mice were mainly CD69+ and CD62L-CD27+ T cells expressing proinflammatory cytokines (interferon-γ [IFNγ], tumor necrosis factor α [TNFα], interleukin-17a [IL-17A], and IL-4), and granzyme B. In mice receiving anti-CD8 mAb, the arthritis score improved 5 days after treatment. Recovery of the CD8+ T cells was associated with a new increase in the arthritis score after 20 days. In thymectomized and anti-CD8 mAb-treated mice, the arthritis score improved permanently. Histologic analysis showed an absence of inflammatory infiltrate in the anti-CD8 mAb-treated mice. In anti-CD8 mAb-treated mice, the serologic levels of TNFα, IFNγ, IL-6, and IL-5 normalized. The levels of the disease-related anti-glucose-6-phosphate isomerase antibodies did not change.

Conclusion: These results indicate that synovial activated effector CD8+ T cells locally synthesize proinflammatory cytokines (IFNγ, TNFα, IL-17, IL-6) and granzyme B in the arthritic joint, thus playing a pivotal role in maintaining chronic synovitis in the K/BxN mouse model of arthritis.
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http://dx.doi.org/10.1002/art.27729DOI Listing
October 2010

Preventive but not curative efficacy of celecoxib on bladder carcinogenesis in a rat model.

Mediators Inflamm 2010 13;2010:380937. Epub 2011 Feb 13.

Institute of Pharmacology & Experimental Therapeutics, IBILI, Medicine Faculty, Sub-Unit 1 (Polo III), Coimbra University, 3000-354 Coimbra, Portugal.

To evaluate the effect of a cyclooxygenase 2 inhibitor, celecoxib (CEL), on bladder cancer inhibition in a rat model, when used as preventive versus as curative treatment. The study comprised 52 male Wistar rats, divided in 5 groups, during a 20-week protocol: control: vehicle, carcinogen: 0.05% of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), CEL: 10  mg/kg/day of the selective COX-2 inhibitor Celebrex, preventive CEL (CEL+BBN-P), and curative CEL (BBN+CEL-C) groups. Although tumor growth was markedly inhibited by the preventive application of CEL, it was even aggravated by the curative treatment. The incidence of gross bladder carcinoma was: control 0/8(0%), BBN 13/20(65%), CEL 0/8(0%), CEL+BBN-P 1/8(12.5%), and BBN+CEL-C 6/8(75%). The number and volume of carcinomas were significantly lower in the CEL+BBN-P versus BBN, accompanied by an ample reduction in hyperplasia, dysplasia, and papillary tumors as well as COX-2 immunostaining. In spite of the reduction of tumor volumes in the curative BBN+CEL-C group, tumor malignancy was augmented. An anti-inflammatory and antioxidant profile was encountered only in the group under preventive treatment. In conclusion, preventive, but not curative, celecoxib treatment promoted a striking inhibitory effect on bladder cancer development, reinforcing the potential role of chemopreventive strategies based on cyclooxygenase 2 inhibition.
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http://dx.doi.org/10.1155/2010/380937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042634PMC
July 2011