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1.
Arterioscler Thromb Vasc Biol ; 41(6): e338-e353, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33792343
2.
Pharmacol Res ; 161: 105222, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33022407

RESUMO

AIMS: The estrogen-inducible protein Heat Shock Protein 27 (HSP27) as well as anti-HSP27 antibodies are elevated in healthy subjects compared to cardiovascular disease patients. Vaccination of ApoE-/- mice with recombinant HSP25 (rHSP25, the murine ortholog), boosts anti- HSP25 levels and attenuates atherogenesis. As estrogens promote HSP27 synthesis, cellular release and blood levels, we hypothesize that menopause will result in loss of HSP27 atheroprotection. Hence, the rationale for this study is to compare the efficacy of rHSP25 vaccination vs. estradiol (E2) therapy for the prevention of post-menopausal atherogenesis. METHODS AND RESULTS: ApoE-/- mice subjected to ovariectomy (OVX) showed a 65 % increase atherosclerotic burden compared to sham mice after 5 weeks of a high fat diet. Relative to vaccination with rC1, a truncated HSP27 control peptide, atherogenesis was reduced by 5-weekly rHSP25 vaccinations (-43 %), a subcutaneous E2 slow release pellet (-52 %) or a combination thereof (-82 %). Plasma cholesterol levels declined in parallel with the reductions in atherogenesis, but relative to rC1/OVX mice plasma PCSK9 levels were 52 % higher in E2/OVX and 41 % lower in rHSP25/OVX mice (p < 0.0001 for both). Hepatic LDLR mRNA levels did not change with E2 treatment but increased markedly with rHSP25 vaccination. Conversely, hepatic PCSK9 mRNA increased 148 % with E2 treatment vs. rC1/OVX but did not change with rHSP25 vaccination. In human HepG2 hepatocytes E2 increased PCSK9 promoter activity 303 %, while the combination of [rHSP27 + PAb] decreased PCSK9 promoter activity by 64 %. CONCLUSION: The reduction in post-OVX atherogenesis and cholesterol levels with rHSP25 vaccination is associated with increased LDLR but not PCSK9 expression. Surprisingly, E2 therapy attenuates atherogenesis and cholesterol levels post-OVX without altering LDLR but increases PCSK9 expression and promoter activity. This is the first documentation of increased PCSK9 expression with E2 therapy and raises questions about balancing physiological estrogenic / PCSK9 homeostasis and targeting PCSK9 in women - are there effects beyond cholesterol?


Assuntos
Aterosclerose/prevenção & controle , Colesterol/sangue , Estradiol/administração & dosagem , Terapia de Reposição de Estrogênios , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/administração & dosagem , Fígado/efeitos dos fármacos , Chaperonas Moleculares/administração & dosagem , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/metabolismo , Vacinas/administração & dosagem , Animais , Aterosclerose/sangue , Aterosclerose/enzimologia , Aterosclerose/imunologia , Biomarcadores/sangue , Modelos Animais de Doenças , Regulação para Baixo , Implantes de Medicamento , Feminino , Proteínas de Choque Térmico/imunologia , Células Hep G2 , Humanos , Fígado/enzimologia , Menopausa , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Chaperonas Moleculares/imunologia , Ovariectomia , Pró-Proteína Convertase 9/genética , Receptores de LDL/genética , Vacinação
3.
J Gastroenterol Hepatol ; 35(12): 2241-2247, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32386240

RESUMO

BACKGROUND AND AIM: Mitochondrial damage is commonly involved in liver injury. We have previously shown that normal mitochondria can be coated with a carrier protein to form complexes that are specifically taken up by liver cells in culture. The aim of the current study was to determine whether mitochondrial complexes could be specifically delivered to the livers of living rats by intravenous injection. METHODS: Mitochondria were harvested from fresh mouse liver, mixed with an asialoglycoprotein-based carrier, asialoorosomucoid-polylysine (AsOR-PL), and purified to form complexes. To facilitate the release of internalized mitochondria from endosomes, an endosomolytic peptide, listeriolysin O (LLO), was coupled to AsOR to form AsOR-LLO. Mitochondria alone, mitochondrial complexes with AsOR-PL, and mitochondrial complexes plus AsOR-LLO conjugate all containing the same number of mitochondria were injected intravenously. Animals were killed, and organs were removed and analyzed by quantitative polymerase chain reaction of mouse mitochondrial DNA, electron microscopy (EM), and in situ polymerase chain reaction and hybridization followed by immunohistochemical analyses. RESULTS: Calculations revealed that approximately 27% of the total injected mitochondria was detected in the liver, while less than 2% was found in spleen, and < 1% in lungs. Immunohistochemistry showed that mouse mitochondrial DNA staining was minimal with mitochondrial complexes alone, strong periportal with mitochondrial complexes co-injected with AsOR-LLO, and absent with mitochondria alone. CONCLUSIONS: Targetable mitochondrial complexes can be delivered to rat liver, and the efficiency of that process is greatly enhanced by co-injection of a targetable endosomal release agent, AsOR-LLO.


Assuntos
Assialoglicoproteínas/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Transplante de Células/métodos , Proteínas de Choque Térmico/administração & dosagem , Proteínas Hemolisinas/administração & dosagem , Fígado , Mitocôndrias Hepáticas/transplante , Orosomucoide/análogos & derivados , Polilisina/administração & dosagem , Animais , Proteínas de Transporte , Endossomos , Feminino , Hepatócitos/citologia , Injeções Intravenosas , Camundongos Endogâmicos , Orosomucoide/administração & dosagem , Ratos Sprague-Dawley
4.
Oncoimmunology ; 9(1): 1749476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32313731

RESUMO

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults with a dismal prognosis. We previously reported that vaccination with heat shock protein peptide complex-96 (HSPPC-96) improves survival in patients with newly diagnosed GBM (NCT02122822). Especially for patients with a strong antitumor immune response after vaccination, a durable survival benefit can be achieved. Here, we conducted T cell receptor (TCR) sequencing to retrospectively examine the TCR repertoires of tumor-infiltrating lymphocytes in long-term survivors (LTS) and short-term survivors (STS). We found that LTS exhibit lower TCR repertoire diversity compared with STS, indicating the prevalence of dominant TCR clones in LTS tumors. Accordingly, the LTS group showed increased inter-patient similarity, especially among high-frequency TCR clones, implying some of these dominant clones are shared among LTS. Indeed, we discovered four TCR clones significantly enriched in the LTS group: the presence of these clones has predictive value for stratifying patients prior to vaccination. Together, these findings uncover a group of preexisting TCR clones shared in LTS that can be utilized as candidate biomarkers to select GBM patients most likely to durably respond to HSPPC-96 treatment.


Assuntos
Neoplasias Encefálicas , Vacinas Anticâncer , Glioblastoma , Proteínas de Choque Térmico , Receptores de Antígenos de Linfócitos T , Adulto , Idoso , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Feminino , Glioblastoma/imunologia , Glioblastoma/terapia , Proteínas de Choque Térmico/administração & dosagem , Proteínas de Choque Térmico/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores de Antígenos de Linfócitos T/imunologia , Estudos Retrospectivos
5.
Infect Immun ; 88(6)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32229617

RESUMO

The major issues in available therapeutic modalities against leishmaniasis are cost, toxicity, and the emergence of drug resistance. The aim of this work was to develop a successful therapeutic adjuvant against drug-resistant Leishmania donovani infection by means of combining Mycobacterium indicus pranii with heat-induced promastigotes (HIP). One-month postinfected BALB/c mice were administered subcutaneously with M. indicus pranii (108 cells) and HIP (100 µg) for 5 days. Spleens were harvested for flow cytometric and reverse transcriptase PCR analysis. The antileishmanial effect of the combination strategy was associated with induction of a disease-resolving Th1 and Th17 response with simultaneous downregulation of CD4+ CD25+ Foxp3+ (nTreg) cells and CD4+ CD25- Foxp3- (Tr1) cells in the spleen. The significant expansion of CD4+ TCM (CD4+ CD44hi CD11ahi CD62Lhi) cells was a further interesting outcome of this therapeutic strategy in the context of long-term protection of hosts against secondary infection. Toll-like receptor 2 (TLR2) was also found instrumental in this antiparasitic therapy. Induced interleukin-6 (IL-6) production from expanded CD11c+ CD8α+ (cDC1) and CD11c+ CD11b+ (cDC2) dendritic cells (DCs) but not from the CD11b+ Ly6c+ inflammatory monocytes (iMOs), was found critical in the protective expansion of Th17 as evidenced by an in vivo IL-6 neutralization assay. It also promoted the hematopoietic conversion toward DC progenitors (pre-DCs) from common dendritic cell progenitors (CDPs), the immediate precursors, in bone marrow. This novel combinational strategy demonstrated that expansion of Th17 by IL-6 released from CD11c+ classical DCs is crucial, together with the conventional Th1 response, to control drug-resistant infection.


Assuntos
Proteínas de Choque Térmico/administração & dosagem , Leishmania donovani , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/terapia , Mycobacterium/fisiologia , Proteínas de Protozoários/administração & dosagem , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Terapia Combinada , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Resistência a Medicamentos , Temperatura Alta , Memória Imunológica , Imunofenotipagem , Mediadores da Inflamação , Interleucina-6/biossíntese , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Camundongos , Mycobacterium/imunologia , Baço/imunologia , Baço/metabolismo , Baço/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
6.
Anim Reprod Sci ; 211: 106227, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31785635

RESUMO

Artificial insemination (AI) in pigs is mainly performed with refrigerated boar semen. There is a marked negative seasonal effect on the quality of boar sperm, mainly due to relatively greater ambient temperatures; to counteract this thermal stress, sperm cells possess natural defensive mechanisms such as Heat Shock Proteins (HSPs) that prevent protein denaturation. Thus, the objective of this research was to improve the quality of commercial boar semen collected during the summer when ambient temperatures are greater using recombinant HSPs. For this purpose, different concentrations (0.1, 0.5 and 1 µg/ml) of recombinant heat shock proteins (HSPD1, HSPA8 or HSP86) were added to commercial boar semen and there was cooling for 48 h at 17 °C. After this storage period, sperm quality was assessed by analyzing sperm viability, mitochondrial membrane potential and plasma membrane lipid organization using flow cytometry; additionally, sperm motility was examined using a CASA system. Also, in vitro fertilization (IVF) using HSP-supplemented boar semen was performed and the quality of the embryos produced was evaluated using quantitative real-time polymerase chain reaction (qPCR) analyzing the relative abundance of mRNA transcripts for genes encoding for embryo quality-related proteins (BAX, TFAM, POLG and POG2). Sperm quality variables, blastocyst rates and the abundance of mRNA transcripts for the selected genes were not affected by the presence of recombinant HSPs at any concentration. These results indicate that the supplementation of commercial seminal doses with recombinant HSPs does not improve boar sperm quality or fertility during the summer months when ambient temperatures are greater.


Assuntos
Proteínas de Choque Térmico/farmacologia , Inseminação Artificial/veterinária , Análise do Sêmen/veterinária , Sêmen , Suínos/fisiologia , Animais , Sobrevivência Celular , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico/administração & dosagem , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Recombinantes , Estações do Ano , Motilidade dos Espermatozoides
7.
J Cell Physiol ; 234(11): 21005-21013, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31012118

RESUMO

Heat shock proteins (HSPs) participate in the regulation of different cell activities in response to stimuli. By applying different strategies, the modulation of heat shock proteins is at the center of attention. Conventional delivery approaches are not fully encouraged due to cytotoxicity and immunogenicity issues. Exosomes are touted as bio-shuttles for delivery of distinct biomolecules inside the cells. Here, we aimed to HSP27 small interfering RNA (siRNA)-tagged exosomes for the inhibition of Hsp27 in human neuroblastoma cell line SH-SY5Y and explored differentiation into neuron-like cells. Exosomes were isolated, characterized by scanning electron microscope (SEM) and CD63 then enriched with siRNA against Hsp27. Neuroblastoma cells were incubated with exosomes carrying siRNA for 48 hr. Exosome uptake was monitored by immunofluorescence assay. The cell viability and proliferation were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine/5-bromo-2'-deoxyuridine incorporation assays. The ability of cells to form colonies was evaluated by clonogenic assay. The cell potential to express NeuN, a mature neuron factor, was studied by flow cytometry analysis. SEM showed the nano-sized particles and a high level of CD63 after enrichment. Immunofluorescence imaging revealed an appropriate transfection rate in cell exposed to Hsp27 siRNA tagged exosomes. The cell viability and proliferation were reduced compared to cells received nude exosomes ( p < 0.05). Clonogenic activity of cells was diminished by the inhibition of Hsp27. Flow cytometry analysis revealed that the inhibition of Hsp27 prohibited NeuN content, showing the maturation of SH-SY5Y cells to mature cells compared to control. These data confirmed that exosomes could be used as appropriate bio-shuttles for the inhibition of Hsp27-aborted cell differentiation toward mature neuron.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Choque Térmico/antagonistas & inibidores , Chaperonas Moleculares/antagonistas & inibidores , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Neurônios/citologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Exossomos , Vetores Genéticos , Proteínas de Choque Térmico/administração & dosagem , Humanos , Chaperonas Moleculares/administração & dosagem , Neuroblastoma , Neurônios/metabolismo , RNA Interferente Pequeno/administração & dosagem , Transfecção
8.
Toxicon ; 164: 82-86, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30991063

RESUMO

Conventional drug delivery systems have many limitations including cytotoxicity and affecting non-specific cells. Cell-targeting peptides (CTPs) as a potential class of targeting moiety have some advantages over previous targeting moieties such as monoclonal antibodies, offer additional benefits to design systems using CTPs. Here we have engineered listeriolysin O (LLO) pore-forming toxin by adding a luteinizing hormone-releasing hormone (LHRH) targeting peptide to its N-terminus. Two versions of the toxin, with and without targeting peptide, were sub-cloned into a bacterial expression plasmid. BL21 DE3 cells were used for induction of expression and recombinant proteins were purified using nickel-immobilized metal affinity chromatography column. In order to treat MDA-MB-231 and SKOV3 cell lines as LHRH receptor positive and negative cells, two mentioned LLO toxins were used to evaluate their cytotoxicity and specificity. Our results reveal that the IC50 of LLO toxin on MDA-MB-231 and SKOV3 cells was 0.32 and 0.41 µg/ml respectively. Furthermore, IC50 of fusion LHRH-LLO toxin on the cells was 0.88 and 19.55 µg/ml. Cytotoxicity of engineered LHRH-LLO toxin on negative cells was significantly 48-fold lower than wild-type LLO toxin. But this difference has been lowered to only 2.7-fold less cytotoxicity in positive cells. To the best of our knowledge, the current work as the first study regarding engineered toxin revealed that CDC family members could be used to target the specific cell-type.


Assuntos
Antineoplásicos/farmacocinética , Toxinas Bacterianas/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Hormônio Liberador de Gonadotropina/farmacocinética , Proteínas de Choque Térmico/farmacocinética , Proteínas Hemolisinas/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/farmacologia , Linhagem Celular Tumoral , Eritrócitos/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Choque Térmico/administração & dosagem , Proteínas de Choque Térmico/farmacologia , Proteínas Hemolisinas/administração & dosagem , Proteínas Hemolisinas/farmacologia , Hemólise , Humanos , Estrutura Molecular , Receptores LHRH/metabolismo , Proteínas Recombinantes
9.
IUBMB Life ; 70(10): 1002-1011, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30171788

RESUMO

Therapeutic human papillomaviruse (HPV) vaccines have the potential to inhibit the tumor growth by targeting HPV E6 and E7 oncoproteins. Among different vaccine strategies, DNA and protein-based approaches are the most effective candidates for stimulation of the immune responses against HPV infections. Our study was designed to assess the efficacy of small heat shock proteins B1 (Hsp27) and B6 (Hsp20) as an adjuvant accompanied by HPV16 E7 and hPP10-E7 antigens in tumor mouse model. A major key for successful DNA and protein transfer into cells is the development of delivery systems with high efficiency and low cytotoxicity. Herein, we used hPP10 and MPG cell penetrating peptides (CPPs) for protein and DNA delivery in vivo, respectively. Our data indicated that the combination of Hsp27 with the recombinant hPP10-E7 protein in homologous protein/protein (hPP10-E7 + Hsp27) and heterologous DNA/protein (pcDNA-E7 + MPG/ hPP10-E7 + Hsp27) significantly enhanced the E7-specific T cell responses. Indeed, these regimens induced high levels of IgG2a, IFN-γ and IL-2 directed toward Th1 responses and also Granzyme B secretion as compared to other immunization strategies, and also displayed complete protection more than 60 days after treatment. These data suggest that the use of Hsp27 as an adjuvant and MPG and hPP10 as a gene and protein carrier would represent promising applications for improvement of HPV therapeutic vaccines. © 2018 IUBMB Life, 70(10):1002-1011, 2018.


Assuntos
Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico/administração & dosagem , Proteínas de Neoplasias/administração & dosagem , Vacinas contra Papillomavirus/administração & dosagem , Neoplasias do Colo do Útero/genética , Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/administração & dosagem , Proteínas de Ligação a DNA/administração & dosagem , Feminino , Granzimas/administração & dosagem , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Proteínas E7 de Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Vacinas contra Papillomavirus/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologia
10.
JCI Insight ; 3(10)2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29769450

RESUMO

BACKGROUND: Heat shock protein peptide complex-96 (HSPPC-96) triggers adaptive and innate antitumor immune responses. The safety and efficacy of HSPPC-96 vaccination was examined in patients with newly diagnosed glioblastoma multiforme (GBM). METHODS: In this open-label, single-arm, phase I study, adult patients were vaccinated with HSPPC-96 in combination with the standard treatment for newly diagnosed GBM after surgical resection. Primary endpoints were frequency of adverse events and progression-free survival (PFS) at 6 months. Secondary endpoints included overall survival (OS), PFS, and tumor-specific immune response (TSIR). RESULTS: A total of 20 patients with newly diagnosed GBM were enrolled from September 2013 to February 2015. No grade 3 or 4 vaccine-related adverse events were noted. After a median follow-up of 42.3 months, PFS was 89.5% (95% CI, 66.9%-98.7%) at 6 months, median PFS was 11.0 months (95% CI, 8.2-13.8), and median OS was 31.4 months (95% CI, 14.9-47.9). TSIR was significantly increased by 2.3-fold (95% CI, 1.7-3.2) after vaccination. Median OS for patients with high TSIR after vaccination was >40.5 months (95% CI, incalculable) as compared with 14.6 months (95% CI, 7.0-22.2) for patients with low TSIR after vaccination (hazard ratio, 0.25; 95% CI, 0.071-0.90; P = 0.034). A multivariate Cox regression model revealed TSIR after vaccination as a primary independent predicator for survival. CONCLUSION: The HSPPC-96 vaccination, combined with the standard therapy, is a safe and effective strategy for treatment of newly diagnosed GBM patients. TSIR after vaccination would be a good indicator predicting the vaccine efficacy. TRIAL REGISTRATION: ClinicalTrials.gov NCT02122822. FUNDING: National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2014BAI04B01, 2014BAI04B02), Beijing Natural Science Foundation (7164253), Beijing Talents Fund (2014000021469G257), and Shenzhen Science and Technology Innovation Committee (JSGG20170413151359491).


Assuntos
Neoplasias Encefálicas/terapia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Glioblastoma/terapia , Proteínas de Choque Térmico/administração & dosagem , Proteínas de Choque Térmico/efeitos adversos , Adulto , Idoso , Neoplasias Encefálicas/imunologia , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Glioblastoma/imunologia , Humanos , Imunidade Inata , Masculino , Pessoa de Meia-Idade
11.
PLoS One ; 13(5): e0198039, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29795667

RESUMO

Loss of integrity of the blood-brain barrier (BBB) in ischemic stroke victims initiates a devastating cascade of events causing brain damage. Maintaining the BBB is important to preserve brain function in ischemic stroke. Unfortunately, recombinant tissue plasminogen activator (tPA), the only effective fibrinolytic treatment at the acute stage of ischemic stroke, also injures the BBB and increases the risk of brain edema and secondary hemorrhagic transformation. Thus, it is important to identify compounds that maintain BBB integrity in the face of ischemic injury in patients with stroke. We previously demonstrated that intravenously injected phosphorylated recombinant heat shock protein 27 (prHSP27) protects the brains of mice with transient middle cerebral artery occlusion (tMCAO), an animal stroke-model. Here, we determined whether prHSP27, in addition to attenuating brain injury, also decreases BBB damage in hyperglycemic tMCAO mice that had received tPA. After induction of hyperglycemia and tMCAO, we examined 4 treatment groups: 1) bovine serum albumin (BSA), 2) prHSP27, 3) tPA, 4) tPA plus prHSP27. We examined the effects of prHSP27 by comparing the BSA and prHSP27 groups and the tPA and tPA plus prHSP27 groups. Twenty-four hours after injection, prHSP27 reduced infarct volume, brain swelling, neurological deficits, the loss of microvessel proteins and endothelial cell walls, and mortality. It also reduced the rates of hemorrhagic transformation, extravasation of endogenous IgG, and MMP-9 activity, signs of BBB damage. Therefore, prHSP27 injection attenuated brain damage and preserved the BBB in tPA-injected, hyperglycemic tMCAO experimental stroke-model mice, in which the BBB is even more severely damaged than in simple tMCAO mice. The attenuation of brain damage and BBB disruption in the presence of tPA suggests the effectiveness of prHSP27 and tPA as a combination therapy. prHSP27 may be a novel therapeutic agent for ischemic stroke patients whose BBBs are injured following tPA injections.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Proteínas de Choque Térmico/administração & dosagem , Infarto da Artéria Cerebral Média/tratamento farmacológico , Proteínas de Neoplasias/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológico , Ativador de Plasminogênio Tecidual/efeitos adversos , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Isquemia Encefálica/patologia , Proteínas de Choque Térmico/metabolismo , Infarto da Artéria Cerebral Média/patologia , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Fármacos Neuroprotetores/metabolismo , Fosforilação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Acidente Vascular Cerebral/patologia
12.
Clin Exp Med ; 17(2): 209-216, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27160252

RESUMO

Induction of tolerance to autoantigens in vivo is a complex process that involves several mechanisms such as the induction of regulatory T cells and changes in the cytokine and chemokine profiles. This approach represents an attractive alternative for treatment of autoimmune diseases. APL-1 is an altered peptide ligand derived from a novel CD4 + T cell epitope of human heat-shock protein of 60 kDa (HSP60), an autoantigen involved in the pathogenesis of rheumatoid arthritis (RA). We have shown previously that this peptide efficiently inhibited the course of adjuvant-induced arthritis in Lewis rats and induced regulatory T cell (Treg) in ex vivo assay with PBMC isolated from RA patients. This study was undertaken to evaluate the therapeutic effect of APL-1 and its combination with methotrexate (MTX) in collagen-induced arthritis (CIA). CIA was induced in male DBA/1 mice at 8 weeks of age by immunization with chicken collagen. APL, MTX or both were administrated beginning from arthritis onset. Therapeutic effect was evaluated by arthritis and joint pathologic scores. In addition, TNFα and IL-10 in sera were measured by ELISA. Treg induction was assessed by FACS analysis. APL-1 inhibits efficiently the course of arthritis in CIA, similar to MTX. In addition, therapy with APL-1 plus MTX reduced CIA in mice, associated with an increase in Treg. These facts reinforce the therapeutic possibilities of APL-1 as a candidate drug for treatment of RA.


Assuntos
Artrite Experimental/tratamento farmacológico , Proteínas de Choque Térmico/administração & dosagem , Fatores Imunológicos/administração & dosagem , Metotrexato/administração & dosagem , Peptídeos/administração & dosagem , Animais , Artrite Experimental/patologia , Modelos Animais de Doenças , Quimioterapia Combinada , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Interleucina-10/sangue , Masculino , Camundongos Endogâmicos DBA , Índice de Gravidade de Doença , Linfócitos T Reguladores/imunologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangue
13.
Artigo em Inglês | MEDLINE | ID: mdl-27881057

RESUMO

BACKGROUND: Heat shock proteins (Hsp) are major chaperone molecules that have recently emerged as cancer therapeutic targets owing to their involvement in tumor cell proliferation, differentiation, invasion and metastasis. High levels of extracellular Hsp90 and Hsp70 have been closely associated with a wide range of human cancers. Accumulating evidence suggests that the pharmacological inhibition of these molecules can play a pivotal role in non-surgical cancer treatment. Efforts have been taken to develop monoclonal antibodies (mAbs) and antibody fragments targeting extracellular Hsp90 and Hsp70, alone or conjugated with standard anticancer agents, to control several types of cancer, such as breast, colon, prostate or melanoma. OBJECTIVE: To provide an overview on the development of monoclonal antibodies and antibody fragments with capacity to bind Hsp90 and Hsp70, aiming at being used for cancer treatment. METHODS: A systematic review was performed using European Patent Office and Google patents databases. RESULTS: Based on the available literature and patents, we report the potential anticancer strategies based on these biological molecules. CONCLUSIONS: Supported by the recent developments in this field, Hsp targeting antibodies therapy may emerge for clinical use in the future for cancer patients, namely as antibody-drug conjugates combining the specificity of these antibodies with the potency of cytotoxic drugs.


Assuntos
Anticorpos Monoclonais/metabolismo , Sistemas de Liberação de Medicamentos/tendências , Proteínas de Choque Térmico/administração & dosagem , Proteínas de Choque Térmico/metabolismo , Patentes como Assunto , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo
14.
Sheng Wu Gong Cheng Xue Bao ; 32(12): 1685-1693, 2016 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-29034636

RESUMO

Type 1 diabetes (T1D), the most prevalent human autoimmune disease, occurs in genetically susceptible individuals. Regulatory T cells (Tregs) are defective in T1D setting. Therefore, efforts to repair or restore Tregs in T1D may prevent or reverse this autoimmune disease. Here, we studied the potential role of rgp96 in preventing T1D, using non-obese diabetic (NOD) mice as an animal model. High-dose rgp96 immunization elicited efficient protection of mice against T1D, as evidenced by stable blood glucose, decreased disease incidence. Significantly increased CD4⁺ CD25⁺ Foxp3⁺ Tregs were observed in immunized mice. In vitro co-culture experiments demonstrated that rgp96 stimulation enhanced Treg proliferation and suppressive function by up-regulation of Foxp3 and IL-10. Our work shows that activation of Tregs by high-dose rgp96 immunization protects against T1D via inducing regulatory T cells and provides preventive and therapeutic potential for the development of an rgp96-based vaccine against T1D.


Assuntos
Antígenos de Neoplasias/imunologia , Diabetes Mellitus Tipo 1/terapia , Proteínas de Choque Térmico/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Neoplasias/administração & dosagem , Técnicas de Cocultura , Diabetes Mellitus Tipo 1/prevenção & controle , Fatores de Transcrição Forkhead , Proteínas de Choque Térmico/administração & dosagem , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos NOD , Regulação para Cima , Vacinação
15.
Chin Med J (Engl) ; 128(16): 2234-41, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26265619

RESUMO

BACKGROUND: To develop a vaccine-based immunotherapy for sarcoma, we evaluated a mixture of heat shock proteins (mHSPs) as a vaccine for sarcoma treatment in a mouse model. Heat shock protein/peptides (HSP/Ps) are autoimmune factors that can induce both adaptive and innate immune responses; HSP/Ps isolated from tumors can induce antitumor immune activity when used as vaccines. METHODS: In this study, we evaluated the effects of mHSP/Ps on prophylactic antitumor immunity. We extracted mHSP/Ps, including HSP60, HSP70, GP96, and HSP110, from the mouse sarcoma cell lines S180 and MCA207 using chromatography. The immunity induced by mHSP/Ps was assessed using flow cytometry, ELISPOT, lactate dehydrogenase release, and enzyme-linked immunosorbent assay. RESULTS: Of S180 sarcoma-bearing mice immunized with mHSP/Ps isolated from S180 cells, 41.2% showed tumor regression and long-term survival, with a tumor growth inhibition rate of 82.3% at 30 days. Of MCA207 sarcoma-bearing mice immunized with mHSP/Ps isolated from MCA207 cells, 50% showed tumor regression and long-term survival with a tumor growth inhibition rate of 79.3%. All control mice died within 40 days. The proportions of natural killer cells, CD8+, and interferon-γ-secreting cells and tumor-specific cytotoxic T-lymphocyte activity were increased in the immunized group. CONCLUSIONS: Vaccination with a polyvalent mHSP/P cancer vaccine can induce an immunological response and a marked antitumor response to autologous tumors. This mHSP/P vaccine exerted greater antitumor effects than did HSP70, HSP60, or tumor lysates alone.


Assuntos
Vacinas Anticâncer/uso terapêutico , Proteínas de Choque Térmico/administração & dosagem , Sarcoma Experimental/prevenção & controle , Animais , Feminino , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peptídeos/administração & dosagem , Vacinação
16.
PLoS One ; 10(6): e0130391, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26102080

RESUMO

The human gastric pathogen Helicobacter pylori (H. pylori) is a successful colonizer of the stomach. H. pylori infection strongly correlates with the development and progression of chronic gastritis, peptic ulcer disease, and gastric malignances. Vaccination is a promising strategy for preventing H. pylori infection. In this study, we evaluated the candidate antigens heat shock protein A (HspA) and H. pylori γ-glutamyl transpeptidase (GGT) for their effectiveness in development of subunit vaccines against H. pylori infection. rHspA, rGGT, and rHspA-GGT, a fusion protein based on HspA and GGT, were constructed and separately expressed in Escherichia coli and purified. Mice were then immunized intranasally with these proteins, with or without adjuvant. Immunized mice exhibited reduced bacterial colonization in stomach. The highest reduction in bacterial colonization was seen in mice immunized with the fusion protein rHspA-GGT when paired with the mucosal adjuvant LTB. Protection against H. pylori colonization was mediated by a strong systemic and localized humoral immune response, as well as a balanced Th1/Th2 cytokine response. In addition, immunofluorescence microscopy confirmed that rHspA-GGT specific rabbit antibodies were able to directly bind H. pylori in vitro. These results suggest antibodies are essential to the protective immunity associated with rHspA-GGT immunization. In summary, our results suggest HspA and GGT are promising vaccine candidates for protection against H. pylori infection.


Assuntos
Proteínas de Bactérias/administração & dosagem , Proteínas de Choque Térmico/administração & dosagem , Helicobacter pylori/crescimento & desenvolvimento , gama-Glutamiltransferase/administração & dosagem , Animais , Anticorpos Antibacterianos/biossíntese , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Citocinas/biossíntese , Feminino , Proteínas de Choque Térmico/imunologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , gama-Glutamiltransferase/imunologia
17.
Regul Toxicol Pharmacol ; 71(2): 164-73, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25545317

RESUMO

DroughtGard maize was developed through constitutive expression of cold shock protein B (CSPB) from Bacillus subtilis to improve performance of maize (Zea mays) under water-limited conditions. B. subtilis commonly occurs in fermented foods and CSPB has a history of safe use. Safety studies were performed to further evaluate safety of CSPB introduced into maize. CSPB was compared to proteins found in current allergen and protein toxin databases and there are no sequence similarities between CSPB and known allergens or toxins. In order to validate the use of Escherichia coli-derived CSPB in other safety studies, physicochemical and functional characterization confirmed that the CSPB produced by DroughtGard possesses comparable molecular weight, immunoreactivity, and functional activity to CSPB produced from E. coli and that neither is glycosylated. CSPB was completely digested with sequential exposure to pepsin and pancreatin for 2 min and 30 s, respectively, suggesting that CSPB will be degraded in the mammalian digestive tract and would not be expected to be allergenic. Mice orally dosed with CSPB at 2160 mg/kg, followed by analysis of body weight gains, food consumption and clinical observations, showed no discernible adverse effects. This comprehensive safety assessment indicated that the CSPB protein from DroughtGard is safe for food and feed consumption.


Assuntos
Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/isolamento & purificação , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Choque Térmico/administração & dosagem , Proteínas de Choque Térmico/isolamento & purificação , Zea mays , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Proteínas de Transporte/efeitos adversos , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Proteínas de Escherichia coli/efeitos adversos , Feminino , Proteínas de Choque Térmico/efeitos adversos , Masculino , Camundongos , Proteínas de Ligação a RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Zea mays/efeitos adversos
18.
J Biotechnol ; 192 Pt A: 130-5, 2014 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-25270023

RESUMO

Archaeosomes are a type of liposomes prepared from the polar lipids of various Archaeobacteria. These have unique structural features that increase the lipid bilayer's stability even under high temperatures, low or high pH, presence of phospholipases and bile salts. This makes them ideal as basis for the development of new drug, gene and vaccine delivery systems. In this study we prepared large unilamellar archaeosomes (400nm size) from Aeropyrum pernix K1 and demonstrated their potential as base for the development of an efficient and universal system for drug or therapy delivery to epithelial cells. Our archaeosomes may be used to deliver small fluorescent molecules (calcein), smaller proteins (60kDa listeriolysin), large protein aggregates (e.g. keratin 14) and plasmid DNA, into epithelial cells grown in culture. The delivery efficiency for small molecules is already quite high at this initial stage of development, around 40%. Our unilamellar archaeosomes are also not toxic to keratinocytes even at high doses (500µg/ml).


Assuntos
Aeropyrum , Sistemas de Liberação de Medicamentos , Toxinas Bacterianas/administração & dosagem , Linhagem Celular , Linhagem Celular Tumoral , DNA/administração & dosagem , Células Epiteliais/metabolismo , Fluoresceínas/administração & dosagem , Proteínas de Choque Térmico/administração & dosagem , Proteínas Hemolisinas/administração & dosagem , Humanos , Queratina-14/genética , Queratinócitos/metabolismo , Lipídeos/química , Lipossomos , Plasmídeos , Proteínas Recombinantes/administração & dosagem
19.
J Immunol ; 191(8): 4456-65, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24048898

RESUMO

Select members of the heat shock proteins (HSPs) family, such as gp96, elicit immune responses specific to their chaperoned peptides. Although immunologic effects of HSPs on APCs described to date have largely been demonstrated with cell lines or primary cells in culture, their collective responses in vitro have been consistent with priming immune responses. In this study, we examine the physiologically relevant APCs in mice that are targeted after vaccination with native, murine HSPs, and we characterize those cells. Gp96 accesses the subcapsular region of the draining lymph node, and it is internalized predominantly by CD11b(+) cells in this locale. Cells acquiring gp96 can transfer protective antitumor immunity to naive mice by actively cross-presenting gp96-chaperoned peptides and providing costimulation. Our studies illustrate how HSPs act to alert the immune system of cellular damage and will be of paramount importance in immunotherapy of patients with cancer and infectious disease.


Assuntos
Proteínas de Choque Térmico/imunologia , Linfonodos/imunologia , Neoplasias/imunologia , Transferência Adotiva , Animais , Antígenos de Neoplasias/imunologia , Antígeno CD11b/metabolismo , Linhagem Celular , Proteínas de Choque Térmico/administração & dosagem , Imunização , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/imunologia
20.
Vaccine ; 31(35): 3564-71, 2013 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-23727004

RESUMO

Heat shock proteins (HSPs) play important roles in the pathogenesis of pneumococcal infection, and they are considered as potential protein vaccine antigens. In this study, we investigated the efficacy of immunization with pneumococcal HSPs, including ClpP (hsp100/Clp peptidase subunit), DnaJ (hsp40) and GroEL (hsp60), to protect against pneumococcal carriage, lung colonization and sepsis in mouse models using different serotypes of Streptococcus pneumoniae. In a nasopharyngeal colonization model by serotype 6B or 14 and in a lung colonization model by serotype 19F, immunization with pneumococcal HSPs could elicit effective protection. Likewise, vaccination with ClpP, DnaJ or GroEL allowed significantly longer mouse survival times after lethal intranasal challenge with serotype pneumococcal 2, 3 or 4. Interestingly, combinations of these HSPs could consistently enhance the protection against nasopharynx carriage, lung colonization as well as invasive infection caused by different pneumococcal serotypes. In an in vitro killing assay, anti-sera against ClpP, DnaJ or GroEL could kill S. pneumoniae by polymorphonuclear leukocytes in a complement-dependent way, and combinations of multiple anti-sera against these HSPs could increase the killing ability compared with single anti-sera. Finally, passive immunization studies with anti-sera against pneumococcal HSPs also demonstrated that an additive effect could be achieved by using multiple anti-sera when compared with single anti-sera. Thus, inclusion of multiple pneumococcal HSPs is important for the development of protein-based pneumococcal vaccines.


Assuntos
Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Proteínas de Choque Térmico HSP40/imunologia , Vacinas Pneumocócicas/imunologia , Pneumonia Pneumocócica/imunologia , Serina Endopeptidases/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Carga Bacteriana/imunologia , Endopeptidase Clp , Proteínas de Choque Térmico/administração & dosagem , Proteínas de Choque Térmico/imunologia , Imunização Passiva , Pulmão/imunologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Doenças Nasofaríngeas/imunologia , Doenças Nasofaríngeas/prevenção & controle , Pneumonia Pneumocócica/prevenção & controle , Streptococcus pneumoniae/imunologia , Vacinação
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