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1.
Front Pharmacol ; 13: 920336, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36034814

RESUMO

Background: Reference to so-called real-world data is more often made in marketing authorization applications for medicines intended to diagnose, prevent or treat rare diseases compared to more common diseases. We provide granularity on the type and aim of any external data on efficacy aspects from both real-world data sources and external trial data as discussed in regulatory submissions of orphan designated medicinal products in the EU. By quantifying the contribution of external data according to various regulatory characteristics, we aimed at identifying specific opportunities for external data in the field of orphan conditions. Methods: Information on external data in regulatory documents covering 72 orphan designations was extracted. Our sample comprised public assessment reports for approved, refused, or withdrawn applications concluded from 2019-2021 at the European Medicines Agency. Products with an active orphan designation at the time of submission were scrutinized regarding the role of external data on efficacy aspects in the context of marketing authorization applications, or on the criterion of "significant benefit" for the confirmation of the orphan designation at the time of licensing. The reports allowed a broad distinction between clinical development, regulatory decision making, and intended post-approval data collection. We defined three categories of external data, administrative data, structured clinical data, and external trial data (from clinical trials not sponsored by the applicant), and noted whether external data concerned the therapeutic context of the disease or the product under review. Results: While reference to external data with respect to efficacy aspects was included in 63% of the approved medicinal products in the field of rare diseases, 37% of marketing authorization applications were exclusively based on the dedicated clinical development plan for the product under review. Purely administrative data did not play any role in our sample of reports, but clinical data collected in a structured manner (from routine care or clinical research) were often used to inform on the trial design. Two additional recurrent themes for the use of external data were the contextualization of results, especially to confirm the orphan designation at the time of licensing, and reassurance of a large difference in treatment effect size or consistency of effects observed in clinical trials and practice. External data on the product under review were restricted to either active substances already belonging to the standard of care even before authorization or to compassionate use schemes. Furthermore, external data were considered pivotal for marketing authorization only exceptionally and only for active substances already in use within the specific therapeutic indication. Applications for the rarest conditions and those without authorized treatment alternatives were especially prominent with respect to the use of external data from real-world data sources both in the pre- and post-approval setting. Conclusion: Specific opportunities for external data in the setting of marketing authorizations in the field of rare diseases were identified. Ongoing initiatives of fostering systematic data collection are promising steps for a more efficient medicinal product development in the field of rare diseases.

2.
Drug Discov Today ; 25(7): 1223-1231, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32344040

RESUMO

To maintain orphan drug status at the time of market authorization, orphan medicinal products (OMPs) need to be assessed for all criteria, including significant benefit, by the Committee for Orphan Medicinal Products (COMP) of the European Medicines Agency (EMA). Subsequently, health technology assessment (HTA) organizations evaluate the same OMPs in their relative effectiveness assessments (REAs). This review investigates the similarities and differences between the two frameworks for six HTA organizations, including the European Network for HTA. We discuss differences between both assessment frameworks within five domains (clinical evidence used, patient population, intervention, comparators, and outcome measures) for all drugs. Five illustrative cases studies were selected for a qualitative review.


Assuntos
Produção de Droga sem Interesse Comercial/legislação & jurisprudência , Avaliação da Tecnologia Biomédica/legislação & jurisprudência , Europa (Continente) , Política de Saúde/legislação & jurisprudência , Humanos
3.
PLoS One ; 12(4): e0174465, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28380039

RESUMO

BACKGROUND: A relevant proportion of human immunodeficiency virus (HIV) infected patients is co-infected with the hepatitis C virus (HCV). HCV co-infection in HIV-positive patients is associated with faster progression of liver disease in comparison to HCV mono-infection. Natural killer (NK) cells critically modulate the natural course of HCV infection. Both HIV and HCV mono-infection are associated with alterations of the NK cell pool. However, little data is available concerning phenotype and function of NK cells in HIV/HCV co-infection. METHODS: A total of 34 HIV/HCV co-infected, 35 HIV and 39 HCV mono-infected patients and 43 healthy control persons were enrolled into this study. All HIV-positive patients were under effective antiretroviral therapy. NK cell phenotype, IFN-γ production and degranulation were studied by flow cytometry. RESULTS: NK cell frequency in HIV/HCV co-infection was significantly lower than in healthy individuals but did not differ from HIV and HCV mono-infection. HIV/HCV co-infection was associated with significantly decreased expression of the maturation/differentiation markers CD27/62L/127 on NK cells but increased expression of CD57 compared to healthy controls. Of note, expression also differed significantly from HCV mono-infection but was similar to HIV mono-infection, suggesting a pronounced impact of HIV on these alterations. Similar findings were made with regard to the NK cell receptors NKG2A/C and NKp30. More importantly, NK cells in co-infection displayed a highly impaired functional activity with significantly lower IFN-γ production and degranulation than in healthy donors as well as HIV and HCV mono-infection, suggesting a synergistic effect of both viruses. CONCLUSIONS: Our data indicate that HIV/HCV co-infection is associated with significant alterations of the NK cell pool, which might be involved in the rapid progression of liver disease in co-infected patients and which mainly reflect alterations observed in HIV mono-infection.


Assuntos
Coinfecção/imunologia , Infecções por HIV/complicações , Hepatite C/complicações , Células Matadoras Naturais/fisiologia , Adulto , Idoso , Estudos de Casos e Controles , Coinfecção/virologia , Estudos Transversais , Citometria de Fluxo , Infecções por HIV/imunologia , Hepatite C/imunologia , Humanos , Interferon gama/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
4.
PLoS One ; 11(9): e0162068, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27583440

RESUMO

BACKGROUND: Immuno-genetic studies suggest a functional link between NK cells and λ-IFNs. We recently showed that NK cells are negative for the IFN-λ receptor IFN-λR1 and do not respond to IFN-λ, suggesting a rather indirect association between IL-28B genotype and NK cell activity. METHODS: A total of 75 HCV(+) patients and 67 healthy controls were enrolled into this study. IL-28B (rs12979860) and IFNL-4 (rs368234815) genotypes were determined by rtPCR. Total PBMC, monocytes, and NK cells were stimulated with IL-29, the TLR-7/8 agonist R848, or a combination of both. NK cell IFN-γ response was analysed by FACS. IL-12 and IL-18 secretion of monocytes was studied by ELISA. In blocking experiments anti-IL-12/anti-IL-18 were used. RESULTS: Following stimulation of total PBMCs with R848 we found NK cell IFN- γ responses to vary with the IL-28B genotype, with carriers of a T/T genotype displaying the lowest frequency of IFN-γ(+)NK cells. When isolated NK cells were studied no such associations were observed, indicating an indirect association between IL-28B genotype and NK cell activity. Accordingly, we found R848-stimulated monocytes of patients with a T/T genotype to be significantly less effective in triggering NK cell IFN- γ production than monocytes from carriers of a non-T/T genotype. In line with these findings we observed monocytes from T/T patients to secrete significantly lower concentrations of IL-12 than monocytes from non-T/T individuals. CONCLUSIONS: Our data indicate that monocytes from carriers of an IL-28B T/T genotype display a reduced ability to stimulate NK cell activity and, thus, provide a link between IL-28B genotype and NK functions.


Assuntos
Variação Genética , Hepatite C/genética , Hepatite C/imunologia , Interleucinas/genética , Células Matadoras Naturais/imunologia , Ativação Linfocitária/genética , Monócitos/citologia , Feminino , Genótipo , Hepatite C/metabolismo , Humanos , Interferons , Interleucina-12/biossíntese , Células Matadoras Naturais/citologia , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo
5.
AIDS ; 30(3): 355-63, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26558728

RESUMO

OBJECTIVE: Hepatitis C virus (HCV) infection in HIV(+) patients is associated with faster liver disease progression compared with HCV mono-infection. HIV-associated immune defects are considered to play an important role in this context. Here, we analyzed the effects of HIV infection on natural killer (NK)-cell-mediated anti-HCV activity. DESIGN: NK cell phenotype and interferon gamma (IFN-γ) production, NK cell-mediated inhibition of HCV replication and CD4 T-cell/NK cell interactions were studied in treatment naive HIV (n = 22), and HIV patients under combined antiretroviral therapy (n = 29), compared with healthy controls (n = 20). METHODS: NK cell-mediated inhibition of HCV replication was analyzed using the HuH7A2HCVreplicon model. IFN-γ production of NK cells as well as interleukin-2 secretion of CD4 T lymphocytes were studied by flow cytometry. RESULTS: Peripheral blood mononuclear cells from HIV(+) patients displayed a significantly impaired anti-HCV activity, irrespective of combined antiretroviral therapy. This could in part be explained by HIV-associated decline in NK cell numbers. In addition, NK cell IFN-γ production was significantly impaired in HIV infection. Accordingly, we found low frequency of IFN-γ(+) NK cells in HIV(+) patients to be associated with ineffective inhibition of HCV replication. Finally, we show that CD4 T-cell-mediated stimulation of NK cell IFN-γ production was dysregulated in HIV infection with an impaired interleukin-2 response of NK cells. CONCLUSION: HIV infection has a strong suppressive effect on anti-HCV activity of NK cells. This may contribute to low spontaneous clearance rate and accelerated progression of HCV-associated liver disease observed in HIV(+) patients.


Assuntos
Infecções por HIV/imunologia , Hepacivirus/imunologia , Células Matadoras Naturais/imunologia , Adulto , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade
6.
J Acquir Immune Defic Syndr ; 70(4): 338-46, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26509931

RESUMO

OBJECTIVE: To analyze the role of CD3(+)CD56(+) natural killer (NK)-like T cells in HIV(+) patients with acute hepatitis C. DESIGN: Frequency, phenotype, and anti-hepatitis C virus (HCV) activity of CD3(+)CD56(+) NK-like T cells were studied in 36 HIV(+) patients with acute hepatitis C. As controls, 12 patients with chronic HCV/HIV coinfection, 8 HIV monoinfected patients, and 12 healthy donors were enrolled in this study. METHODS: CD3(+)CD56(+) NK-like T-cell-mediated inhibition of HCV replication was analyzed using the HuH7A2HCVreplicon model. The CD3(+)CD56(+) NK-like T-cell phenotype and interferon (IFN)-γ secretion were studied by flow cytometry. RESULTS: Interleukin 12/interleukin 15 stimulated CD3(+)CD56(+) NK-like T cells from healthy donors effectively block HCV replication in vitro in an IFN-γ dependent manner. Accordingly, we found that blocking of IFN-γ with a specific antibody significantly reduced the antiviral activity of CD3(+)CD56(+) NK-like T cells. However, when CD3(+)CD56(+) NK-like T cells from HIV(+) patients were studied, we found HIV infection to be associated with a significantly impaired IFN-γ production, irrespective of HCV coinfection. Accordingly, CD3(+)CD56(+) NK-like T cells from HIV(+) patients were significantly less effective in blocking HCV replication in vitro than cells from healthy individuals. CONCLUSIONS: Taken together, our data indicate that HIV infection is associated with an impaired anti-HCV activity of CD3(+)CD56(+) NK-like T cells, which might represent a novel mechanism of dysregulated immune response in HIV/HCV-coinfected patients.


Assuntos
Complexo CD3/análise , Antígeno CD56/análise , Infecções por HIV/complicações , Hepacivirus/imunologia , Hepatite C/imunologia , Células T Matadoras Naturais/imunologia , Adulto , Células Cultivadas , Feminino , Citometria de Fluxo , Hepacivirus/fisiologia , Hepatócitos/virologia , Humanos , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Células T Matadoras Naturais/química , Subpopulações de Linfócitos T/imunologia , Replicação Viral , Adulto Jovem
7.
J Hepatol ; 63(6): 1334-44, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26297916

RESUMO

BACKGROUND & AIMS: Natural killer (NK) cells have been shown to exert anti-viral as well as anti-fibrotic functions in hepatitis C virus (HCV) infection. Previous studies, however, analyzed NK cell functions exclusively under atmospheric oxygen conditions despite the fact that the liver microenvironment is hypoxic. Here, we analyzed the effects of low oxygen tension on anti-viral and anti-fibrotic NK cell activity. METHODS: Peripheral (n=34) and intrahepatic (n=15) NK cells from HCV(+) patients as well as circulating NK cells from healthy donors (n=20) were studied with respect to anti-viral and anti-fibrotic activity via co-culture experiments with HuH7 replicon cells and hepatic stellate cells, respectively. RESULTS: Anti-viral activity of resting NK cells from healthy controls was not affected by hypoxia. However, hypoxia significantly reduced the response of healthy NK cells to cytokine stimulation. In contrast to healthy controls, we observed resting and cytokine activated peripheral NK cells from HCV patients to display a significantly decreased anti-viral activity when cultured at 5% or 1% oxygen, suggesting HCV NK cells to be very sensitive to hypoxia. These findings could be confirmed when intrahepatic NK cells were tested. Finally, we show that anti-fibrotic NK cell activity was not affected by low oxygen tension. CONCLUSIONS: Our results show that anti-viral function of NK cells from HCV(+) patients is critically affected by a hypoxic microenvironment and, therefore, indicate that in order to obtain an accurate understanding of intrahepatic NK cell anti-HCV activity, the laboratory modelling should take into account the liver specific levels of oxygen.


Assuntos
Hipóxia Celular/imunologia , Hepatite C Crônica/imunologia , Células Matadoras Naturais/imunologia , Adulto , Idoso , Antivirais/imunologia , Estudos de Casos e Controles , Linhagem Celular , Técnicas de Cocultura , Citocinas/administração & dosagem , Citocinas/imunologia , Feminino , Fibrose/imunologia , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Células Estreladas do Fígado/imunologia , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Hepatócitos/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon gama/biossíntese , Células Matadoras Naturais/metabolismo , Masculino , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Adulto Jovem
8.
Clin Chem Lab Med ; 53(12): 2031-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26035112

RESUMO

BACKGROUND: Diagnosis of spontaneous bacterial peritonitis (SBP) is based on a differential ascites leukocyte count which does not provide prognostic information. We performed a pilot study to assess calprotectin in ascites as an alternative diagnostic and prognostic marker. METHODS: We collected ascites from patients with liver cirrhosis from March 2012 to July 2013. Routine clinical and laboratory data of the patients were recorded. Ascites calprotectin levels were determined by ELISA. RESULTS: Overall, we collected 120 ascites samples from 100 patients with liver cirrhosis and from eight patients with malignant peritoneal effusion as disease control. Samples without infection had significantly lower calprotectin levels (median 34 ng/mL, range 5-795) than SBP samples (median 928 ng/mL, range 21-110,480; p<0.001) and malignant effusions (median 401, range 47-2596 ng/mL; p<0.001). In non-infected ascites, calprotectin levels were higher in Child-Pugh stage B versus C (median 57 ng/mL vs. 17 ng/mL; p<0.001) and in alcoholic versus viral cirrhosis (median 37 ng/mL vs. 10 ng/mL; p=0.015). The ratio of ascites calprotectin to total protein was a better marker for SBP than calprotectin alone (AUROC=0.93; p<0.001; sensitivity 93%, specificity 79%; positive predictive value 60%; negative predictive value 97%). In addition, high levels of the ratio to total protein were associated with poor 30-day survival (p=0.042). CONCLUSIONS: The ratio of ascites calprotectin to total protein may be a promising new diagnostic and prognostic marker in patients with liver cirrhosis and SBP and should be evaluated further.


Assuntos
Ascite/complicações , Proteína C-Reativa/análise , Complexo Antígeno L1 Leucocitário/análise , Cirrose Hepática/complicações , Peritonite/complicações , Peritonite/diagnóstico , Adulto , Idoso , Ascite/metabolismo , Líquido Ascítico/química , Biomarcadores/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Cirrose Hepática/metabolismo , Masculino , Pessoa de Meia-Idade , Peritonite/metabolismo , Peritonite/microbiologia , Prognóstico
9.
AIDS ; 28(13): 1879-84, 2014 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-24922598

RESUMO

OBJECTIVE: The objective of this study was to analyse the potential role of CD27 in natural killer (NK) cell-mediated control of hepatitis C virus (HCV) infection in HIV-positive patients. DESIGN: Frequency of CD27-expressing CD56 NK cells was analysed in HIV mono-infected individuals and HIV-positive patients with acute or chronic hepatitis C. Anti-HCV activity of CD27(+) and CD27(-) NK cells was compared. METHODS: NK cell mediated inhibition of HCV replication was analysed using the HUH7 HCV Replicon model. NK cell phenotype and interferon (IFN) secretion was studied by flowcytometry. RESULTS: High frequency of CD27(+)CD56 NK cells is associated with spontaneous clearance of acute hepatitis C in HIV-positive patients. Accordingly, we found CD27(+)CD56 NK cells to display strong anti-HCV activity. CONCLUSION: Our results underline the important role of NK cells in modulating outcome of HCV infection.


Assuntos
Antígeno CD56/análise , Infecções por HIV/complicações , Hepacivirus/imunologia , Hepatite C/imunologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Adulto , Idoso , Feminino , Citometria de Fluxo , Humanos , Interferons/metabolismo , Células Matadoras Naturais/química , Subpopulações de Linfócitos/química , Masculino , Pessoa de Meia-Idade
10.
Hepatology ; 59(3): 814-27, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24382664

RESUMO

UNLABELLED: Hepatitis C virus (HCV) coinfection is an increasing health problem in human immunodeficiency virus-positive (HIV(+) ) individuals. However, a considerable proportion of HIV(+) patients manage to overcome acute hepatitis C (AHC) spontaneously. In the present study, we analyzed the role of natural killer (NK) cells in modulating the course of AHC in HIV(+) patients. Twenty-seven HIV(+) patients with AHC (self-limited course: n = 10; chronic course: n = 17), 12 HIV(+) patients with chronic hepatitis C (CHC), 8 HIV monoinfected individuals, and 12 healthy controls were studied. NK cells were phenotypically analyzed by flow cytometry. Interferon-gamma (IFN-γ) secretion, degranulation (CD107a), and anti-HCV (= inhibition of HCV replication) activity of NK subpopulations were analyzed using the HuH7A2 HCVreplicon cell system. NK cell frequency did not differ significantly between HIV(+) patients with chronic and self-limited course of AHC. However, we found NK cells from patients with self-limiting infection to be significantly more effective in inhibiting HCV replication in vitro than NK cells from patients developing CHC. Of note, antiviral NK cell activity showed no significant correlation with NK cell degranulation, but was positively correlated with IFN-γ secretion, and blocking experiments confirmed an important role for IFN-γ in NK cell-mediated inhibition of HCV replication. Accordingly, NK cells from patients that spontaneously cleared the virus displayed a stronger IFN-γ secretion than those developing chronic infection. Finally, we observed high expression of NKG2D and NKp46, respectively, to be associated with self-limiting course of aHCV. Accordingly, we found that blocking of these NK cell receptors significantly impaired antiviral NK cell activity. CONCLUSION: Our data suggest a strong IFN-γ-mediated antiviral NK cell response to be associated with a self-limited course of AHC in HIV(+) patients.


Assuntos
Coinfecção/imunologia , Infecções por HIV/imunologia , Hepacivirus/crescimento & desenvolvimento , Hepatite C/imunologia , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Adulto , Idoso , Linhagem Celular Tumoral , Feminino , Humanos , Imunofenotipagem , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Masculino , Pessoa de Meia-Idade , Replicação Viral/imunologia
11.
PLoS One ; 8(11): e80848, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24260493

RESUMO

BACKGROUND AND AIMS: CXCL1 (CXC chemokine-ligand-1) is a ligand for CXC chemokine receptor 2 expressed on hepatic stellate cells (HSC). Thus, CXCL1 might contribute to HSC activation and fibrogenesis. In the present study, we investigated the influence of the CXCL1 rs4074 polymorphism on the occurrence of alcohol induced liver cirrhosis and hepatocellular carcinoma (HCC). METHODS: The study involved 458 patients with alcoholic cirrhosis (170 with HCC), 115 alcoholics without liver disease and 342 healthy controls. All subjects were genotyped for the CXCL1 rs4074 polymorphism and CXCL1 serum levels of 132 patients were measured. In vitro CXCL1 secretion in TLR-transfected cell lines were studied by ELISA. RESULTS: Distribution of the CXCL1 genotypes (GG/GA/AA) was 159/219/80 in patients with alcoholic cirrhosis, 52/44/19 in alcoholic controls and 158/140/44 in healthy controls. Patients with alcohol-induced cirrhosis were significantly more often carriers of the CXCL1 rs4074 A allele (65.3%) than alcoholics without liver disease (54.8%, OR=1.55; 95%CI=1.025-2.350; p=0.04) and healthy controls (53.8%, OR=1.62; 95%CI=1.212-2.151; p=0.001). Accordingly, the frequency of the CXCL1 rs4074 A allele was significantly higher in the cirrhotic patients than in the subjects without cirrhosis (41.4% vs. 33.9%, OR=1.38, 95% CI:1.14-1.66, p=0.001). Furthermore cirrhotic carriers of the CXCL1 rs4074 A allele had significantly higher CXCL1 serum levels than carriers of the GG genotype. In contrast to sera from healthy controls, sera from patients with alcoholic cirrhosis induced CXCL1 secretion in TLR2- (p=0.016) and TLR4- (p=0.008) transfected HEK293 cells. This finding indicates that sera from patients with alcoholic cirrhosis contain soluble ligands that can induce CXCL1 production via stimulation of TLRs. CONCLUSION: The enhanced CXCL1 serum levels in carriers of the rs4074 A allele together with their increased frequency in patients with alcohol induced cirrhosis suggest the CXCL1 rs4074 A allele as a genetic risk factor for alcoholic cirrhosis.


Assuntos
Alcoolismo/genética , Carcinoma Hepatocelular/genética , Quimiocina CXCL1/genética , Predisposição Genética para Doença , Cirrose Hepática Alcoólica/genética , Neoplasias Hepáticas/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Idoso de 80 Anos ou mais , Alcoolismo/sangue , Alcoolismo/etnologia , Alelos , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/etnologia , Estudos de Casos e Controles , Quimiocina CXCL1/sangue , Etanol/efeitos adversos , Feminino , Heterozigoto , Humanos , Cirrose Hepática Alcoólica/sangue , Cirrose Hepática Alcoólica/etnologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/etnologia , Masculino , Pessoa de Meia-Idade , Fatores de Risco , População Branca
12.
Int J Oral Maxillofac Implants ; 28(5): e230-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24066334

RESUMO

PURPOSE: Classic tissue engineering consists of three components: scaffold, cells, and growth or differentiation factors. Currently, expensive bone morphogenetic proteins are the most common substance used for hard tissue regeneration. An alternative could be gamma-aminobutyric acid/lactam (GABA-lactam) analogs. MATERIALS AND METHODS: The effects of gabapentin-lactam, cis- and trans-8-tertbutyl-GABA-pentinlactam (trans-TB-GBP-L), and phenyl-GABA-lactam were tested in this study on ovine mesenchymal stem cell (MSC) proliferation. MSCs were selected from bone marrow aspirate concentrate by plastic adherence and amplified. Aliquots of the cells were incubated in medium, with four different concentrations of the GABA-lactam analogs dissolved in dimethyl sulfoxide. Cells in medium with and without dimethyl sulfoxide served as controls. Cell proliferation was tested with a nonradioactive assay. Before and after GABA-lactam analog influence, the MSC character was evaluated by the ability of the cells to differentiate into osteoblasts, chondrocytes, and adipocytes. RESULTS: Proliferation was significantly increased under the influence of the analogs, depending on their concentration. MSCs cultured in 1 nmol/L trans-TB-GBP-L showed the highest proliferation rate. The MSC character was not altered. CONCLUSIONS: GABA-lactam analogs could be suited to stimulate MSC proliferation for tissue engineering applications. Further in vivo studies are necessary to evaluate the possible clinical potential of GABA-lactam analogs for hard tissue regeneration.


Assuntos
Compostos Aza/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Compostos de Espiro/farmacologia , Ácido gama-Aminobutírico/análogos & derivados , Animais , Células da Medula Óssea/citologia , Condrócitos/citologia , Condrócitos/metabolismo , Meios de Cultura/química , Dimetil Sulfóxido/farmacologia , Células-Tronco Mesenquimais/citologia , Fenótipo , Ovinos , Engenharia Tecidual/métodos , Ácido gama-Aminobutírico/farmacologia
13.
J Hepatol ; 59(3): 427-33, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23665286

RESUMO

BACKGROUND & AIMS: HIV/HCV co-infection is characterized by a faster progression to liver fibrosis compared to HCV mono-infection. Epidemiologic studies found an association between low CD4(+) T cell counts and advanced stages of liver fibrosis. However, the mechanisms underlying this association remain unclear. CD4(+) T cells critically modulate NK cell activity. Of note, NK cells have been shown to display anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Thus, we speculated that CD4(+) T cells might modulate fibrosis progression by interacting with NK cells. METHODS: NK cells from HCV(+) (n=35), HIV(+)/HCV(+) (n=28), HIV(+) (n=8) patients, and healthy controls (n=30) were used in this study. NK cells were cultured in the presence or absence of supernatants from CD3/CD28-stimulated CD4(+) cells. Then, NK cells were co-incubated with activated HSC and studied for degranulation, IFN-γ secretion, and induction of HSC apoptosis. RESULTS: Following incubation with CD4(+) T cell supernatants, NK cells displayed a significantly increased activity against primary HSC as compared to unstimulated NK cells. This effect was, at least in part, mediated via an IL-2 dependent upregulation of NKG2D expression. HCV/HIV co-infection was associated with an impaired IL-2 secretion of CD4(+) T cells resulting in an ineffective stimulation of anti-fibrotic NK cell function. CONCLUSIONS: Here, we show that CD4(+) T cells are able to stimulate anti-fibrotic NK cell activity via IL-2 mediated upregulation of NKG2D. HIV-induced loss of CD4(+) T cells together with an impaired activity of CD4(+) T cells may contribute to accelerate progression of liver fibrosis observed in co-infection.


Assuntos
Coinfecção/imunologia , Infecções por HIV/complicações , Infecções por HIV/imunologia , Hepatite C Crônica/complicações , Hepatite C Crônica/imunologia , Células Matadoras Naturais/imunologia , Cirrose Hepática/etiologia , Cirrose Hepática/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Técnicas de Cocultura , Coinfecção/patologia , Meios de Cultivo Condicionados , Progressão da Doença , Feminino , Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/patologia , Hepatite C Crônica/patologia , Humanos , Interleucina-2/metabolismo , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Adulto Jovem
14.
BMC Cancer ; 12: 85, 2012 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-22401174

RESUMO

BACKGROUND: Tumour surveillance via induction of TRAIL-mediated apoptosis is a key mechanism, how the immune system prevents malignancy. To determine if gene variants in the TRAIL receptor I (DR4) gene affect the risk of hepatitis C virus (HCV)-induced liver cancer (HCC), we analysed DR4 mutations C626G (rs20575) and A683C (rs20576) in HCV-infected patients with and without HCC. METHODS: Frequencies of DR4 gene polymorphisms were determined by LightSNiP assays in 159 and 234 HCV-infected patients with HCC and without HCC, respectively. 359 healthy controls served as reference population. RESULTS: Distribution of C626G and A683C genotypes were not significantly different between healthy controls and HCV-positive patients without HCC. DR4 variants 626C and 683A occurred at increased frequencies in patients with HCC. The risk of HCC was linked to carriage of the 626C allele and the homozygous 683AA genotype, and the simultaneous presence of the two risk variants was confirmed as independent HCC risk factor by Cox regression analysis (Odds ratio 1.975, 95% CI 1.205-3.236; p = 0.007). Furthermore HCV viral loads were significantly increased in patients who simultaneously carried both genetic risk factors (2.69 ± 0.36 × 10(6) IU/ml vs. 1.81 ± 0.23 × 10(6) IU/ml, p = 0.049). CONCLUSIONS: The increased prevalence of patients with a 626C allele and the homozygous 683AA genotype in HCV-infected patients with HCC suggests that these genetic variants are a risk factor for HCC in chronic hepatitis C.


Assuntos
Carcinoma Hepatocelular/genética , Predisposição Genética para Doença , Hepatite C/complicações , Neoplasias Hepáticas/genética , Polimorfismo Genético , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Carcinoma Hepatocelular/virologia , Feminino , Frequência do Gene , Genótipo , Humanos , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Modelos de Riscos Proporcionais
15.
Artigo em Inglês | MEDLINE | ID: mdl-19813017

RESUMO

Many flowers display colour patterns comprising a large peripheral colour area that serves to attract flower visitors from some distance, and a small central, contrastingly coloured area made up by stamens or floral guides. In this study, we scaled down the size of floral guides to detect the minimal size bumblebees (Bombus terrestris) and honeybees (Apis mellifera) require for guidance. We analyzed the approach and the precise contact of the antennal tips with the floral guide of artificial flowers which precedes landing and inspection. Both bumblebees and honeybees were able to make antennal contact with circular floral guides which were 2 mm in diameter; bumblebees performed better than honeybees and antennated also at floral guides smaller than 2 mm. In discrimination experiments with bumblebees, a minimum floral guide size of 2 mm was required for discrimination between artificial flowers with and without floral guides. With increasing experience bumblebees targeted close to the site of reward instead of making antennal contact with the floral guide, whereas honeybees did not alter their initial behaviour with growing experience. Bumblebees and honeybees spontaneously target diminutive floral guides to achieve physical contact with flowers by means of their antennae which helps them to inspect flowers.


Assuntos
Abelhas/fisiologia , Visão de Cores/fisiologia , Comportamento Exploratório/fisiologia , Flores/fisiologia , Aprendizagem/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Animais , Células Quimiorreceptoras/fisiologia , Comportamento Alimentar/fisiologia , Feminino , Voo Animal/fisiologia , Flores/anatomia & histologia , Orientação/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Percepção Espacial/fisiologia , Especificidade da Espécie , Tato/fisiologia
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