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1.
Antioxid Redox Signal ; 30(2): 213-231, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29463096

RESUMO

Aims: Radiation-induced normal tissue toxicity often precludes the application of curative radiation doses. Here we investigated the therapeutic potential of chemokine C-C motif ligand 2 (Ccl2) signaling inhibition to protect normal lung tissue from radiotherapy (RT)-induced injury. Results: RT-induced vascular dysfunction and associated adverse effects can be efficiently antagonized by inhibition of Ccl2 signaling using either the selective Ccl2 inhibitor bindarit (BIN) or mice deficient for the main Ccl2 receptor CCR2 (KO). BIN-treatment efficiently counteracted the RT-induced expression of Ccl2, normalized endothelial cell (EC) morphology and vascular function, and limited lung inflammation and metastasis early after irradiation (acute effects). A similar protection of the vascular compartment was detected by loss of Ccl2 signaling in lungs of CCR2-KO mice. Long-term Ccl2 signaling inhibition also significantly limited EC loss and accompanied fibrosis progression as adverse late effect. With respect to the human situation, we further confirmed that Ccl2 secreted by RT-induced senescent epithelial cells resulted in the activation of normally quiescent but DNA-damaged EC finally leading to EC loss in ex vivo cultured human normal lung tissue. Innovation: Abrogation of certain aspects of the secretome of irradiated resident lung cells, in particular signaling inhibition of the senescence-associated secretory phenotype-factor Ccl2 secreted predominantly by RT-induced senescent epithelial cells, resulted in protection of the endothelial compartment. Conclusions: Radioprotection of the normal tissue via Ccl2 signaling inhibition without simultaneous protection or preferable radiosensitization of tumor tissue might improve local tumor control and survival, because higher doses of radiation could be used.


Assuntos
Quimiocina CCL2/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Endotélio Vascular/metabolismo , Endotélio Vascular/efeitos da radiação , Pulmão/metabolismo , Transdução de Sinais/efeitos da radiação , Animais , Biomarcadores , Biópsia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Senescência Celular/efeitos da radiação , Quimiocina CCL2/antagonistas & inibidores , Modelos Animais de Doenças , Humanos , Pulmão/patologia , Pulmão/efeitos da radiação , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Knockout , Pneumonia/etiologia , Pneumonia/metabolismo , Pneumonia/patologia , Substâncias Protetoras/farmacologia , Ligação Proteica , Lesões por Radiação/metabolismo , Lesões por Radiação/patologia , Receptores CCR2/genética , Receptores CCR2/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/efeitos da radiação
2.
Int J Cancer ; 141(4): 791-804, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28493326

RESUMO

Epidemiological studies have detected a higher incidence of various tumour entities in diabetic patients. However, the underlying mechanisms remain insufficiently understood. Glucose-derived pericellular and extracellular hyaluronan (HA) promotes tumour progression and development. In our study, we tested the hypothesis that a diabetic metabolic state, characterised by hyperglycaemia and concomitant aberrant insulin signalling, stimulates tumour progression via the induction of HA synthesis. In a streptozotocin-induced diabetic nude mouse tumour xenograft model, hyperglycaemia and lack of insulin caused an increased formation of tumour-associated HA-matrix, which in turn accelerated tumour progression and neoangiogenesis. This process was effectively attenuated by treatment with 4-methylumbelliferone, a pharmacological inhibitor of HA-synthesis. To define the mechanisms behind these in vivo observations, we investigated the impact of hyperglycaemia and insulin on the glucose metabolism in oesophageal squamous cell cancer cells (ESCC). Hyperglycaemia induced HA synthesis while insulin diminished HA production by directing glucose metabolites to glycolysis. Vice versa, inhibition of glycolysis, either by knockdown of the glycolytic key enzyme phosphofructokinase or by an experimental abrogation of insulin signalling (knockdown of the insulin receptor and long-term treatment with insulin) augmented HA synthesis. Consequently, these processes induced invasion, anchorage-independent growth and adhesion of ESCC to endothelial cells in vitro. Thus, the cellular shift in glucose usage from catabolism of glucose to anabolism of HA driven by hyperglycaemia and insulin resistance may represent an important link between diabetes and cancer progression. Hence, therapeutical inhibition of HA synthesis may represent a promising approach for tumour treatment in diabetic patients.


Assuntos
Carcinoma de Células Escamosas/patologia , Diabetes Mellitus Experimental/metabolismo , Neoplasias Esofágicas/patologia , Ácido Hialurônico/metabolismo , Hiperglicemia/complicações , Insulina/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Diabetes Mellitus Experimental/complicações , Progressão da Doença , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transdução de Sinais
3.
FASEB J ; 31(7): 2869-2880, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28325757

RESUMO

While radiotherapy is a mainstay for cancer therapy, pneumonitis and fibrosis constitute dose-limiting side effects of thorax and whole body irradiation. So far, the contribution of immune cells to disease progression is largely unknown. Here we studied the role of ecto-5'-nucelotidase (CD73)/adenosine-induced changes in the myeloid compartment in radiation-induced lung fibrosis. C57BL/6 wild-type or CD73-/- mice received a single dose of whole thorax irradiation (WTI, 15 Gy). Myeloid cells were characterized in flow cytometric, histologic, and immunohistochemical analyses as well as RNA analyses. WTI induced a pronounced reduction of alveolar macrophages in both strains that recovered within 6 wk. Fibrosis development in wild-type mice was associated with a time-dependent deposition of hyaluronic acid (HA) and increased expression of markers for alternative activation on alveolar macrophages. These include the antiinflammatory macrophage mannose receptor and arginase-1. Further, macrophages accumulated in organized clusters and expressed profibrotic mediators at ≥25 wk after irradiation (fibrotic phase). Irradiated CD73-/- mice showed an altered regulation of components of the HA system and no clusters of alternatively activated macrophages. We speculate that accumulation of alternatively activated macrophages in organized clusters represents the origins of fibrotic foci after WTI and is promoted by a cross-talk between HA, CD73/adenosine signaling, and other profibrotic mediators.-De Leve, S., Wirsdörfer, F., Cappuccini, F., Schütze, A., Meyer, A. V., Röck, K., Thompson, L. F., Fischer, J. W., Stuschke, M., Jendrossek, V. Loss of CD73 prevents accumulation of alternatively activated macrophages and the formation of prefibrotic macrophage clusters in irradiated lungs.


Assuntos
5'-Nucleotidase/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Pulmão/citologia , Pulmão/efeitos da radiação , Macrófagos Alveolares/efeitos da radiação , Adenosina/metabolismo , Animais , Antígeno CD11b/metabolismo , Adesão Celular , Ácido Hialurônico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/etiologia , Transdução de Sinais
4.
Antioxid Redox Signal ; 26(11): 563-582, 2017 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-27572073

RESUMO

AIMS: Radiation-induced normal tissue toxicity is closely linked to endothelial cell (EC) damage and dysfunction (acute effects). However, the underlying mechanisms of radiation-induced adverse late effects with respect to the vascular compartment remain elusive, and no causative radioprotective treatment is available to date. RESULTS: The importance of injury to EC for radiation-induced late toxicity in lungs after whole thorax irradiation (WTI) was investigated using a mouse model of radiation-induced pneumopathy. We show that WTI induces EC loss as long-term complication, which is accompanied by the development of fibrosis. Adoptive transfer of mesenchymal stem cells (MSCs) either derived from bone marrow or aorta (vascular wall-resident MSCs) in the early phase after irradiation limited the radiation-induced EC loss and fibrosis progression. Furthermore, MSC-derived culture supernatants rescued the radiation-induced reduction in viability and long-term survival of cultured lung EC. We further identified the antioxidant enzyme superoxide dismutase 1 (SOD1) as a MSC-secreted factor. Importantly, MSC treatment restored the radiation-induced reduction of SOD1 levels after WTI. A similar protective effect was achieved by using the SOD-mimetic EUK134, suggesting that MSC-derived SOD1 is involved in the protective action of MSC, presumably through paracrine signaling. INNOVATION: In this study, we explored the therapeutic potential of MSC therapy to prevent radiation-induced EC loss (late effect) and identified the protective mechanisms of MSC action. CONCLUSIONS: Adoptive transfer of MSCs early after irradiation counteracts radiation-induced vascular damage and EC loss as late adverse effects. The high activity of vascular wall-derived MSCs for radioprotection may be due to their tissue-specific action. Antioxid. Redox Signal. 26, 563-582.


Assuntos
Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Pulmão/metabolismo , Pulmão/patologia , Pulmão/efeitos da radiação , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Lesões por Radiação/metabolismo , Superóxido Dismutase-1/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais/patologia , Células Endoteliais/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Fibrose , Expressão Gênica , Células-Tronco Mesenquimais/citologia , Camundongos , Compostos Organometálicos/farmacologia , Fenótipo , Lesões por Radiação/genética , Lesões por Radiação/patologia , Lesões por Radiação/terapia , Lesões Experimentais por Radiação , Salicilatos/farmacologia , Superóxido Dismutase-1/genética
5.
Clin Chem ; 62(11): 1504-1515, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27630154

RESUMO

BACKGROUND: Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions. METHODS: CTCs were enriched based on their EpCAM expression (CellSearch®) or by size and deformability (ParsortixTM), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling. RESULTS: Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8). CONCLUSIONS: Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.


Assuntos
Perfilação da Expressão Gênica , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Transcriptoma/genética , Transição Epitelial-Mesenquimal/genética , Humanos , RNA Neoplásico/genética , Células Tumorais Cultivadas
6.
Sci Rep ; 6: 30482, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27460287

RESUMO

Chronic UVB-exposure and declined estradiol production after menopause represent important factors leading to extrinsic and intrinsic aging, respectively. Remodeling of the extracellular matrix (ECM) plays a crucial role in both responses. Whether the dermal ECM is able to recover after cessation of UVB-irradiation in dependence of estradiol is not known, however of relevance when regarding possible treatment options. Therefore, the endogenous sex hormone production was depleted by ovariectomy in female mice. Half of the mice received estradiol substitution. Mice were UVB-irradiated for 20 weeks and afterwards kept for 10 weeks without irradiation. The collagen-, hyaluronan- and proteoglycan- (versican, biglycan, lumican) matrix, collagen cleavage products and functional skin parameters were analyzed. The intrinsic aging process was characterized by increased collagen fragmentation and accumulation of biglycan. Chronic UVB-irradiation additionally augmented the lumican, versican and hyaluronan content of the dermis. In the absence of further UVB-irradiation the degradation of collagen and accumulation of biglycan in the extrinsically aged group was perpetuated in an excessive matter. Whereas estradiol increased the proteoglycan content, it reversed the effects of the perpetuated extrinsic response on collagen degradation. Suspension of the intrinsic pathway might therefore be sufficient to antagonize UVB-evoked long-term damage to the dermal ECM.


Assuntos
Derme/metabolismo , Derme/efeitos da radiação , Estrogênios/farmacologia , Substâncias Protetoras/farmacologia , Raios Ultravioleta , Animais , Biópsia , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Derme/efeitos dos fármacos , Derme/patologia , Feminino , Ácido Hialurônico/metabolismo , Inflamação/patologia , Camundongos Pelados , Ovariectomia , Proteoglicanas/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
PLoS One ; 11(5): e0155126, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27182774

RESUMO

Colorectal cancer (CRC) is one of the major causes of cancer-related death and reliable blood-based prognostic biomarkers are urgently needed. The enumeration and molecular characterization of circulating tumor cells (CTCs) has gained increasing interest in clinical practice. CTC detection by CellSearch® has already been correlated to an unfavorable outcome in metastatic CRC. However, the CTC detection rate in mCRC disease is low compared to other tumor entities. Thus, the use of alternative (or supplementary) assays might help to itemize the prognostic use of CTCs as blood-based biomarkers. In this study, blood samples from 47 mCRC patients were screened for CTCs using the FDA-cleared CellSearch® technology and / or the AdnaTest®. 38 samples could be processed in parallel. We demonstrate that a combined analysis of CellSearch® and the AdnaTest® leads to an improved detection of CTCs in our mCRC patient cohort (positivity rate CellSearch® 33%, AdnaTest® 30%, combined 50%). While CTCs detected with the CellSearch® system were significantly associated with progression-free survival (p = 0.046), a significant correlation regarding overall survival could be only seen when both assays were combined (p = 0.013). These findings could help to establish improved tools to detect CTCs as on-treatment biomarkers for clinical routine in future studies.


Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Testes Genéticos/métodos , Células Neoplásicas Circulantes/metabolismo , Adulto , Idoso , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico
8.
Circ Res ; 119(2): 237-48, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27142162

RESUMO

RATIONALE: In patients after acute myocardial infarction (AMI), the initial extent of necrosis and inflammation determine clinical outcome. One early event in AMI is the increased cardiac expression of atrial natriuretic peptide (NP) and B-type NP, with their plasma levels correlating with severity of ischemia. It was shown that NPs, via their cGMP-forming guanylyl cyclase-A (GC-A) receptor and cGMP-dependent kinase I (cGKI), strengthen systemic endothelial barrier properties in acute inflammation. OBJECTIVE: We studied whether endothelial actions of local NPs modulate myocardial injury and early inflammation after AMI. METHODS AND RESULTS: Necrosis and inflammation after experimental AMI were compared between control mice and littermates with endothelial-restricted inactivation of GC-A (knockout mice with endothelial GC-A deletion) or cGKI (knockout mice with endothelial cGKI deletion). Unexpectedly, myocardial infarct size and neutrophil infiltration/activity 2 days after AMI were attenuated in knockout mice with endothelial GC-A deletion and unaltered in knockout mice with endothelial cGKI deletion. Molecular studies revealed that hypoxia and tumor necrosis factor-α, conditions accompanying AMI, reduce the endothelial expression of cGKI and enhance cGMP-stimulated phosphodiesterase 2A (PDE2A) levels. Real-time cAMP measurements in endothelial microdomains using a novel fluorescence resonance energy transfer biosensor revealed that PDE2 mediates NP/cGMP-driven decreases of submembrane cAMP levels. Finally, intravital microscopy studies of the mouse cremaster microcirculation showed that tumor necrosis factor-α-induced endothelial NP/GC-A/cGMP/PDE2 signaling impairs endothelial barrier functions. CONCLUSIONS: Hypoxia and cytokines, such as tumor necrosis factor-α, modify the endothelial postreceptor signaling pathways of NPs, with downregulation of cGKI, induction of PDE2A, and altered cGMP/cAMP cross talk. Increased expression of PDE2 can mediate hyperpermeability effects of paracrine endothelial NP/GC-A/cGMP signaling and facilitate neutrophil extravasation during the early phase after MI.


Assuntos
Fator Natriurético Atrial/farmacologia , Endotélio Vascular/metabolismo , Mediadores da Inflamação/metabolismo , Infarto do Miocárdio/metabolismo , Animais , Fator Natriurético Atrial/biossíntese , Endotélio Vascular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/farmacologia
9.
Oncotarget ; 7(23): 34930-41, 2016 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-27145459

RESUMO

The prostate specific membrane antigen (PSMA) is the only clinically validated marker for therapeutic decisions in prostate cancer (PC). Characterization of circulating tumor cells (CTCs) obtained from the peripheral blood of PC patients might provide an alternative to tissue biopsies called "liquid biopsy". The aim of this study was to develop a reliable assay for the determination of PSMA on CTCs. PSMA expression was analyzed on tissue samples (cohort one, n = 75) and CTCs from metastatic PC patients (cohort two, n = 29). Specific signals for the expression of PSMA could be seen for different prostate cancer cell line cells (PC3, LaPC4, 22Rv1, and LNCaP) by Western blot, immunohistochemistry (IHC), immunocytochemistry (ICC), and FACS. PSMA expression was found to be significantly increased in patients with higher Gleason grade (p = 0.0011) and metastases in lymph nodes (p = 0.0000085) or bone (p = 0.0020) (cohort one). In cohort two, CTCs were detectable in 20 out of 29 samples (69 %, range from 1 - 1000 cells). Twelve out of 20 CTC-positive patients showed PSMA-positive CTCs (67 %, score 1+ to 3+). We found intra-patient heterogeneity regarding the PSMA status between CTCs and the corresponding primary tumors. The results of our study could help to address the question whether treatment decisions based on CTC PSMA profiling will lead to a measurable benefit in clinical outcome for prostate cancer patients in the near future.


Assuntos
Biomarcadores Tumorais/análise , Células Neoplásicas Circulantes , Antígeno Prostático Específico/análise , Neoplasias da Próstata/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Citometria de Fluxo/métodos , Imunofluorescência/métodos , Humanos , Masculino , Pessoa de Meia-Idade
10.
Oncotarget ; 7(16): 21428-40, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-26870892

RESUMO

Biomarkers for prognosis in radiotherapy-treated breast cancer patients are urgently needed and important to stratify patients for adjuvant therapies. Recently, a role of the receptor of hyaluronan-mediated motility (RHAMM) has been suggested for tumor progression. Our aim was (i) to investigate the prognostic value of RHAMM in breast cancer and (ii) to unravel its potential function in the radiosusceptibility of breast cancer cells. We demonstrate that RHAMM mRNA expression in breast cancer biopsies is inversely correlated with tumor grade and overall survival. Radiosusceptibility in vitro was evaluated by sub-G1 analysis (apoptosis) and determination of the proliferation rate. The potential role of RHAMM was addressed by short interfering RNAs against RHAMM and its splice variants. High expression of RHAMMv1/v2 in p53 wild type cells (MCF-7) induced cellular apoptosis in response to ionizing radiation. In comparison, in p53 mutated cells (MDA-MB-231) RHAMMv1/v2 was expressed sparsely resulting in resistance towards irradiation induced apoptosis. Proliferation capacity was not altered by ionizing radiation in both cell lines. Importantly, pharmacological inhibition of the major ligand of RHAMM, hyaluronan, sensitized both cell lines towards radiation induced cell death. Based on the present data, we conclude that the detection of RHAMM splice variants in correlation with the p53 mutation status could help to predict the susceptibility of breast cancer cells to radiotherapy. Additionally, our studies raise the possibility that the response to radiotherapy in selected cohorts may be improved by pharmaceutical strategies against RHAMM and its ligand hyaluronan.


Assuntos
Neoplasias da Mama/genética , Proliferação de Células/genética , Proteínas da Matriz Extracelular/genética , Receptores de Hialuronatos/genética , Splicing de RNA , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Células MCF-7 , Prognóstico , Interferência de RNA , Tolerância a Radiação/genética , Radiação Ionizante , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida
11.
J Invest Dermatol ; 135(2): 369-377, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25264594

RESUMO

Even though aging and cellular senescence appear to be linked, the biological mechanisms interconnecting these two processes remain to be unravelled. Therefore, microRNA (miRNA/miR) profiles were analyzed ex vivo by means of gene array in fibroblasts isolated from young and old human donors. Expression of several miRNAs was positively correlated with donor age. Among them, miR-23a-3p was shown to target hyaluronan synthase 2 (HAS2). HA is a polysaccharide of the extracellular matrix that critically regulates the phenotype of fibroblasts. Indeed, both aged and senescent fibroblasts showed increased miR-23a-3p expression and secreted significantly lower amounts of HA compared with young and non-senescent fibroblasts. Ectopic overexpression of miR-23a-3p in non-senescent fibroblasts led to decreased HAS2-mediated HA synthesis, upregulation of senescence-associated markers, and decreased proliferation. In addition, siRNA-mediated downregulation of HAS2 and pharmacological inhibition of HA synthesis by 4-methylumbelliferone mimicked the effects of miR-23a-3p. In vivo, miR-23a-3p was upregulated and HAS2 was downregulated in the skin of old mice compared with young mice. Inhibition of HA synthesis by 4-methylumbelliferone in mice reduced dermal hydration and viscoelasticity, thereby mimicking an aged skin phenotype. Taken together, these findings appear to link miR-23a-3p and the HA microenvironment as effector mechanisms in both dermal aging and senescence.


Assuntos
Senescência Celular , Glucuronosiltransferase/genética , MicroRNAs/fisiologia , Envelhecimento da Pele , Regiões 3' não Traduzidas/genética , Adulto , Idoso , Animais , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Feminino , Fibroblastos/metabolismo , Glucuronosiltransferase/antagonistas & inibidores , Humanos , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Himecromona/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade
12.
PLoS One ; 8(11): e80084, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24223213

RESUMO

BACKGROUND: Urothelial bladder cancer is the ninth most common cancer. Despite surgical and chemotherapeutic treatment the prognosis is still poor once bladder cancer progresses to a muscle-invasive state. Discovery of new diagnostic markers and pathophysiologic effectors might help to contribute to novel diagnostic and therapeutic options. The extracellular matrix microenvironment shaped by the extracellular matrix critically affects tumor cell and stroma cell functions. Therefore, aim of the present study was to assess the possible implication of the small leucine-rich proteoglycan biglycan in progression of human urothelial bladder cancer. METHODS AND RESULTS: For this purpose tumor biopsies of 76 bladder cancer patients with different tumor stages (pTa, pT1-T4) were investigated with respect to biglycan expression and correlated with a long-term (10 years) clinical follow-up. Interestingly, higher biglycan mRNA expression was associated with higher tumor stages and muscle invasiveness. In vitro knock-down of endogenous biglycan in human urothelial carcinoma cells (J82 cells) increased proliferation, whereas addition of recombinant biglycan and overexpression of biglycan inhibited tumor cell proliferation. In line with this growth-inhibitory effect of biglycan, transplantation of J82 cells after knock-down of biglycan resulted in significantly increased growth of subcutaneous xenograft tumors in nude mice in vivo. Furthermore, treatment with two anti-proliferative, multi-receptor tyrosine kinase inhibitors-sunitinib and sorafenib-strongly upregulated biglycan expression. Collectively, the experimental data suggest that high biglycan expression is associated with reduced tumor cell proliferation. In accordance, Kaplan-Meier analysis revealed higher 10-year survival in patients with high biglycan mRNA expression in tumor biopsies. CONCLUSION: In conclusion, the present data suggest that biglycan is an endogenous inhibitor of bladder cancer cell proliferation that is upregulated in response to anti-proliferative tyrosine kinase inhibitors. In addition, high biglycan expression is associated with favorable prognosis.


Assuntos
Biglicano/química , Biglicano/metabolismo , Leucina/química , Neoplasias da Bexiga Urinária/metabolismo , Animais , Biglicano/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Indóis/uso terapêutico , Masculino , Camundongos , Camundongos Nus , Niacinamida/análogos & derivados , Niacinamida/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Pirróis/uso terapêutico , RNA Mensageiro/genética , Sorafenibe , Sunitinibe , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética
13.
PLoS One ; 8(9): e75681, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24069434

RESUMO

UNLABELLED: Hyaluronan (HA) is a carbohydrate of the extracellular matrix with tumor promoting effects in a variety of cancers. The present study addressed the role of HA matrix for progression and prognosis of human bladder cancer by studying the expression and function of HA-related genes. METHODS: Tissue samples of 120 patients with different stages of transitional cell bladder cancer, who underwent surgical treatment for bladder cancer at the University Hospital of Essen were analysed. mRNA-expression levels of HA synthases (HAS1-3) and HA-receptors (RHAMM and CD44) were evaluated by real time RT-PCR in comparison to healthy bladder tissue as control. In uni- and multivariate cox proportional hazard survival regression analysis, the impact of the gene expression levels on survival was assessed. In vitro knock-down of RHAMM, CD44 and HAS isoenzymes was achieved by siRNA and lentiviral shRNA in J82 bladder cancer cells. Transfected cells were analysed in vitro with regard to proliferation, cell cycle and apoptosis. J82 cells after knock-down of RHAMM were xenografted into male nu/nu athymic mice to monitor tumor progression in vivo. RESULTS: In invasive tumor stages RHAMM-, HAS1 and HAS2 mRNA-expression levels were elevated whereas HAS3v1 was reduced as compared to non-invasive tumors. Subsequently, Kaplan-Meier analysis revealed reduced bladder cancer specific survival in patients with high RHAMM mRNA and low HAS3v1 expression. Elevated RHAMM in invasive tumors was confirmed by RHAMM immunohistochemistry. Furthermore, multivariate analysis revealed that only RHAMM expression was associated with poor prognosis independent from other survival factors (HR=2.389, 95% CI 1.227-4.651, p=0.01). Lentiviral RHAMM knock-down revealed reduced J82 cell proliferation in vitro and reduced xenograft tumor growth in vivo. CONCLUSION: The data suggest that RHAMM plays a crucial role in mediating progression of muscle-invasive bladder cancer and recommends RHAMM for further evaluation as a prognostic marker or therapeutic target in bladder cancer therapy.


Assuntos
Carcinoma de Células de Transição/genética , Proteínas da Matriz Extracelular/genética , Receptores de Hialuronatos/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/mortalidade , Carcinoma de Células de Transição/patologia , Modelos Animais de Doenças , Progressão da Doença , Proteínas da Matriz Extracelular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Imuno-Histoquímica , Imunofenotipagem , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Fenótipo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Risco , Carga Tumoral/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia
14.
Sci Transl Med ; 5(199): 199ra111, 2013 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-23966300

RESUMO

The epidermal growth factor receptor (EGFR) plays an important role in tissue homeostasis and tumor progression. However, cancer patients treated with EGFR inhibitors (EGFRIs) frequently develop acneiform skin toxicities, which are a strong predictor of a patient's treatment response. We show that the early inflammatory infiltrate of the skin rash induced by EGFRI is dominated by dendritic cells, macrophages, granulocytes, mast cells, and T cells. EGFRIs induce the expression of chemokines (CCL2, CCL5, CCL27, and CXCL14) in epidermal keratinocytes and impair the production of antimicrobial peptides and skin barrier proteins. Correspondingly, EGFRI-treated keratinocytes facilitate lymphocyte recruitment but show a considerably reduced cytotoxic activity against Staphylococcus aureus. Mice lacking epidermal EGFR (EGFR(Δep)) show a similar phenotype, which is accompanied by chemokine-driven skin inflammation, hair follicle degeneration, decreased host defense, and deficient skin barrier function, as well as early lethality. Skin toxicities were not ameliorated in a Rag2-, MyD88-, and CCL2-deficient background or in mice lacking epidermal Langerhans cells. The skin phenotype was also not rescued in a hairless (hr/hr) background, demonstrating that skin inflammation is not induced by hair follicle degeneration. Treatment with mast cell inhibitors reduced the immigration of T cells, suggesting that mast cells play a role in the EGFRI-mediated skin pathology. Our findings demonstrate that EGFR signaling in keratinocytes regulates key factors involved in skin inflammation, barrier function, and innate host defense, providing insights into the mechanisms underlying EGFRI-induced skin pathologies.


Assuntos
Receptores ErbB/imunologia , Pele/imunologia , Animais , Antineoplásicos/efeitos adversos , Quimiocinas/biossíntese , Citocinas/biossíntese , Dermatite/imunologia , Dermatite/patologia , Dermatite/prevenção & controle , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/deficiência , Receptores ErbB/genética , Cloridrato de Erlotinib , Exantema/induzido quimicamente , Exantema/imunologia , Exantema/patologia , Folículo Piloso/imunologia , Folículo Piloso/patologia , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Células de Langerhans/imunologia , Linfócitos/imunologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Quinazolinas/efeitos adversos , Pele/efeitos dos fármacos , Pesquisa Translacional Biomédica
15.
J Biol Chem ; 287(24): 20056-69, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22493503

RESUMO

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.


Assuntos
Senescência Celular/efeitos dos fármacos , Senescência Celular/efeitos da radiação , Fator de Crescimento Epidérmico/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Ácido Hialurônico/metabolismo , Queratinócitos/metabolismo , Raios Ultravioleta/efeitos adversos , Versicanas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Derme/metabolismo , Derme/patologia , Fator de Crescimento Epidérmico/genética , Epiderme/metabolismo , Epiderme/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Técnicas de Silenciamento de Genes , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Hialuronan Sintases , Ácido Hialurônico/genética , Queratinócitos/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Mutantes , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/efeitos da radiação , Versicanas/genética
16.
Basic Res Cardiol ; 106(6): 1099-109, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21901291

RESUMO

Epidemiological and clinical data suggest that estrogen retards the progression of atherosclerosis. This study aims to elucidate whether the phenotypic regulation of human vascular smooth muscle cells (VSMC) by estrogen may involve effects on the hyaluronan matrix. VSMC were synchronized by serum withdrawal and subsequently stimulated with 0.001, 0.01, 0.1 and 1 µM estradiol (E(2)) in the presence or absence of platelet-derived growth factor BB (PDGF-BB) for 24 h. E(2) reduced mRNA-expression of hyaluronic acid synthase (HAS) 1 in the presence and absence of PDGF-BB. In contrast, HAS2- and HAS3-mRNA-expression were not affected. This E(2)-mediated effect on HAS1 mRNA-expression was accompanied by reduced hyaluronan secretion and a shift of HA toward lower molecular weight as evidenced by molecular sieve chromatography. The downregulation of HAS1 was abrogated by the estrogen receptor (ER) α and ß antagonist ICI182780 and could be mimicked by the ERα-agonist propyl-pyrazole triol (PPT). On the contrary, the ERß-agonist diarylpropionitrile (DPN) had no effect on HAS1 mRNA-expression. To investigate whether the downregulation of HAS1 was causally involved in the phenotypic regulation of human VSMC by E(2), lentiviral overexpression of HAS1 was conducted. Overexpression of HAS1 abrogated the inhibition of sustained ERK1/2 phosphorylation and in turn inhibition of DNA-synthesis by E(2). For the first time this study provides strong evidence that HAS1-driven HA-synthesis is a target of E(2) in human VSMC and that E(2) mediates part of its anti-proliferative effects through an ERα-dependent inhibition of HA-synthesis.


Assuntos
Estradiol/metabolismo , Glucuronosiltransferase/biossíntese , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Becaplermina , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Estradiol/farmacologia , Estrogênios/metabolismo , Estrogênios/farmacologia , Humanos , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
J Biol Chem ; 286(20): 18268-76, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21454612

RESUMO

UVB irradiation causes characteristic features of skin aging including remodeling of the dermal extracellular matrix. A key feature during this process is the up-regulation of matrix metalloproteinases and cleavage of collagen. Hyaluronic acid (HA), a major component of the dermal matrix, decreases after chronic UVB exposure. However, the factors that govern the decline of HA synthesis during the course of actinic aging are largely unknown. The aim of the present study was to explore whether collagen degradation causes inhibition of HA synthesis in human skin fibroblasts. After treatment of fibroblasts with collagen fragments (CF) in vitro, resolution of the actin cytoskeleton and inhibition of HA secretion occurred because of specific down-regulation of hyaluronan synthase 2 (HAS2) expression. The α(v)ß(3)-agonist, RGDS, latrunculin A, and an inhibitor of Rho-activated kinase inhibited HAS2 expression. Conversely, blocking antibodies to α(v)ß(3) abolished the down-regulation of HAS2 and the cytoskeletal effects. Furthermore, inhibition of cofilin phosphorylation in response to CF was prevented by α(v)ß(3)-blocking antibodies. The key role of ERK signaling was shown by reduced nuclear accumulation of phosphoERK and of ELK-1 phosphorylation in response to CF. In addition, the ERK inhibitor PD98059 reduced HAS2 expression. Also, UVB irradiation of fibroblasts caused down-regulation of HAS2, which was sensitive to matrix metalloproteinase inhibitors and to α(v)ß(3)-blocking antibodies. In conclusion, these data suggest that CF activate α(v)ß(3)-integrins and in turn inhibit Rho kinase (ROCK) signaling and nuclear translocation of phosphoERK, resulting in reduced HAS2 expression. Therefore, a novel mechanism is presented how proteolytic collagen cleavage may inhibit HA synthesis in dermal fibroblasts during extrinsic skin aging.


Assuntos
Derme/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Ácido Hialurônico/biossíntese , Raios Ultravioleta/efeitos adversos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos da radiação , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Envelhecimento/patologia , Envelhecimento/efeitos da radiação , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Células Cultivadas , Colágeno , Derme/patologia , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/sangue , Glucuronosiltransferase/genética , Humanos , Hialuronan Sintases , Integrina alfaVbeta3/biossíntese , Integrina alfaVbeta3/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Masculino , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
18.
Circulation ; 122(22): 2313-22, 2010 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-21098434

RESUMO

BACKGROUND: Hyaluronan is thought to mediate neointimal hyperplasia but also vasoprotection as an integral component of the endothelial glycocalyx. The present study addressed for the first time the effects of long-term pharmacological inhibition of hyaluronan synthesis on vascular function and atherosclerosis. METHODS AND RESULTS: Four-week-old apolipoprotein E-deficient mice on a Western diet received orally an inhibitor of hyaluronan synthesis, 4-methylumbelliferone (4-MU; 10 mg/g body wt), resulting in 600 nmol/L 4-MU in plasma. As a result, aortic plaque burden was markedly increased at 25 weeks. Furthermore, acetylcholine-dependent relaxation of aortic rings was decreased and mean arterial blood pressure was increased in response to 4-MU. However, hydralazine blunted the hypertensive effect of 4-MU without inhibiting the proatherosclerotic effect. A photothrombosis model revealed a prothrombotic state that was not due to increased platelet activation or increased thrombin activation as monitored by CD62P expression and the endogenous thrombin potential. Importantly, increased recruitment of macrophages to vascular lesions was detected after 2 and 21 weeks of 4-MU treatment by immunohistochemistry, by intravital microscopy, and in a peritonitis model. As a potential underlying mechanism, severe damage of the endothelial glycocalyx after 2 and 21 weeks of treatment with 4-MU was detected by electron microscopy of the innominate artery and myocardial capillaries. Furthermore, 600 nmol/L 4-MU inhibited hyaluronan synthesis in cultured endothelial cells. CONCLUSIONS: The data suggest that systemic inhibition of hyaluronan synthesis by 4-MU interferes with the protective function of the endothelial glycocalyx, thereby facilitating leukocyte adhesion, subsequent inflammation, and progression of atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Progressão da Doença , Ácido Hialurônico/antagonistas & inibidores , Ácido Hialurônico/metabolismo , Acetilcolina/farmacologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Feminino , Glicocálix/efeitos dos fármacos , Glicocálix/metabolismo , Himecromona/análogos & derivados , Himecromona/farmacologia , Camundongos , Camundongos Knockout , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologia
19.
Dermatology ; 221(3): 219-28, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20820101

RESUMO

BACKGROUND: Over the past decade injectable hyaluronan (HA) formulations have been widely used to decrease the visibility of skin aging. However, little basic research has been performed to address their effect on dermal skin fibroblasts. OBJECTIVE: The aim of the present study was to investigate whether human skin fibroblasts are affected by exogenous non-cross-linked HA with respect to proliferation, migration and extracellular matrix composition. METHODS: The effect of a non-cross-linked HA on proliferation, migration and gene expression of human dermal fibroblasts was determined. Furthermore, affinity histochemistry of pericellular HA was performed. RESULTS: Proliferation was significantly stimulated by HA whereas migration was not affected. Importantly, exogenous HA was incorporated into fine HA filaments of the pericellular fibroblast matrix. CONCLUSION: This is the first evidence that a HA formulation used in humans for cosmetic reasons stimulates fibroblast proliferat


Assuntos
Fibroblastos/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Pele/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Ácido Hialurônico/metabolismo , Pele/citologia
20.
Antimicrob Agents Chemother ; 52(11): 3994-4000, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18779351

RESUMO

During antibiotic drug development, media are frequently spiked with either serum/plasma or protein supplements to evaluate the effect of protein binding. Usually, previously reported serum or plasma protein binding values are applied in the analysis. The aim of this study was to evaluate this approach by experimentally measuring free, unbound concentrations for antibiotics with reportedly high protein binding and their corresponding antimicrobial activities in media containing commonly used protein supplements. Free, unbound ceftriaxone and ertapenem concentrations were determined in bacterial growth medium with and without bovine/human serum albumin, as well as adult bovine serum and human plasma using in vitro microdialysis. The corresponding antimicrobial activity was determined in MIC and time-kill curve experiments using Escherichia coli ATCC 25922 and Streptococcus pneumoniae ATCC 6303 as test strains. A semimechanistic maximum effect model was simultaneously fitted to the data and respective EC(50) (concentration at half-maximum effect) values compared. Protein binding differed significantly for ceftriaxone (P < 0.05) between human plasma (76.8 +/- 11.0%) and commercially available bovine (20.2 +/- 8.3%) or human serum albumin (56.9 +/- 16.6%). Similar results were obtained for ertapenem (human plasma, 73.8 +/- 11.6%; bovine serum albumin, 12.4 +/- 4.8%; human serum albumin, 17.8 +/- 11.5%). The MICs and EC(50)s of both strains were significantly increased (P < 0.05) for ceftriaxone when comparing human and bovine serum albumin, whereas the EC(50)s were not significantly different for ertapenem. Free, unbound antibiotic concentrations differed substantially between plasma and protein supplements and correlated well with antimicrobial efficacy. Therefore, free, active concentrations should be measured in the test system instead of correcting for literature protein binding values.


Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas Sanguíneas/metabolismo , Animais , Bovinos , Ceftriaxona/metabolismo , Ceftriaxona/farmacologia , Descoberta de Drogas , Ertapenem , Escherichia coli/efeitos dos fármacos , Humanos , Técnicas In Vitro , Modelos Biológicos , Ligação Proteica , Albumina Sérica/metabolismo , Soroalbumina Bovina/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , beta-Lactamas/metabolismo , beta-Lactamas/farmacologia
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