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1.
Medicine (Baltimore) ; 98(33): e16771, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31415377

RESUMO

The cell wall skeleton of Bacillus Calmette-Guérin (BCG-CWS) is a bioactive component that is a strong immune adjuvant for cancer immunotherapy. BCG-CWS activates the innate immune system through various pattern recognition receptors and is expected to elicit antigen-specific cellular immune responses when co-administered with tumor antigens. To determine the recommended dose (RD) of BCG-CWS based on its safety profile, we conducted a phase I dose-escalation study of BCG-CWS in combination with WT1 peptide for patients with advanced cancer.The primary endpoint was the proportion of treatment-related adverse events (AEs) at each BCG-CWS dose. The secondary endpoints were immune responses and clinical effects. A BCG-CWS dose of 50, 100, or 200 µg/body was administered intradermally on days 0, 7, 21, and 42, followed by 2 mg of WT1 peptide on the next day. For the escalation of a dose level, 3 + 3 design was used.Study subjects were 18 patients with advanced WT1-expressing cancers refractory to standard anti-cancer therapies (7 melanoma, 5 colorectal, 4 hepatobiliary, 1 ovarian, and 1 lung). Dose-limiting toxicity occurred in the form of local skin reactions in 2 patients at a dose of 200 µg although no serious treatment-related systemic AEs were observed. Neutrophils and monocytes transiently increased in response to BCG-CWS. Some patients demonstrated the induction of the CD4 T cell subset and its differentiation from the naïve to memory phenotype, resulting in a tumor response.The RD of BCG-CWS was determined to be 100 µg/body. This dose was well tolerated and showed promising clinical effects with the induction of an appropriate immune response.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacina BCG/uso terapêutico , Esqueleto da Parede Celular/uso terapêutico , Mycobacterium bovis , Adjuvantes Imunológicos/administração & dosagem , Adulto , Idoso , Vacina BCG/administração & dosagem , Contagem de Linfócito CD4 , Esqueleto da Parede Celular/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Feminino , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Melanoma/tratamento farmacológico , Pessoa de Meia-Idade , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Resultado do Tratamento
2.
Mol Pharm ; 15(12): 5762-5771, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30380885

RESUMO

The intravesical instillation of live Bacillus Calmette-Guerin (BCG) for treating bladder cancer is a powerful cancer immunotherapy. The BCG cell wall skeleton (BCG-CWS) is the main component of the adjuvant, leading to the induction of antitumor immunity. However, the use of live BCG and BCG-CWS is currently limited to local administration because of the infectiousness of live BCG and the insolubility of BCG-CWS. We previously developed a water-dispersible nanoparticle (NP) formulation of BCG-CWS (CWS-NP), which could be used to apply BCG components for use as a systemically injected adjuvant for the treatment of cancers other than bladder cancer. In the present study, we examined the possible use of CWS-NP for cancer immunotherapy, when intravenously administered. The CWS-NP was a highly uniform dispersion and showed no aggregation in serum. The intravenously injected CWS-NP accumulated in the spleen and was efficiently taken up by dendritic cells, leading to their maturation. The coadministration of CWS-NP and ovalbumin (OVA) loaded NP resulted in the generation of OVA-specific cytotoxic T cells and inhibited the growth of E.G7-OVA tumors. These results represent the first findings related to the use of systemically injected CWS-NP as an adjuvant for cancer immunotherapy.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Esqueleto da Parede Celular/administração & dosagem , Mycobacterium bovis/citologia , Nanopartículas/administração & dosagem , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacocinética , Administração Intravenosa , Animais , Linhagem Celular Tumoral , Esqueleto da Parede Celular/química , Esqueleto da Parede Celular/farmacocinética , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Ensaios de Seleção de Medicamentos Antitumorais , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Neoplasias/imunologia , Neoplasias/terapia , Solubilidade , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Distribuição Tecidual , Água/química
3.
Cytoskeleton (Hoboken) ; 75(12): 498-507, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30160378

RESUMO

In many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. In Caulobacter crescentus and Escherichia coli, MreB is also required to sculpt this morphology de novo. Mycobacteria are rod-shaped but expand their cell wall from discrete polar or subpolar zones. In this genus, the tropomyosin-like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself. Mycobacterium smegmatis rendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms, M. smegmatis also establish and propagate rod shape by cytoskeleton-controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular/fisiologia , Esqueleto da Parede Celular/biossíntese , Mycobacterium smegmatis , Peptidoglicano/metabolismo , Esferoplastos/crescimento & desenvolvimento , Esferoplastos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microscopia , Mycobacterium smegmatis/citologia , Mycobacterium smegmatis/crescimento & desenvolvimento , Esferoplastos/citologia
4.
J Int Med Res ; 46(6): 2398-2409, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29665712

RESUMO

Objective This study was performed to investigate the effect of Nocardia rubra cell wall skeleton (N-CWS) on wound healing of full-thickness skin defects. Methods Two 2- × 2-cm full-thickness wounds, one on each side of the midline, were made on the back of 12 rats. One wound was covered with Vaseline gauze soaked in normal saline, whereas the other was covered with Vaseline gauze and N-CWS. Wound dressings were changed every other day from day 0 (wound creation) to day 11. Four of the 12 rats were killed on day 7, and biopsy samples were obtained for biochemical and histopathological analyses. The expression levels of CD31, CD68, and F4/80 in the tissues were examined immunohistologically. The expression of transforming growth factor (TGF)-ß1 in the wound was determined by western blot. Results N-CWS increased the wound healing rate, reduced the complete wound healing time, and increased the expression levels of CD31, CD68, and F4/80 on day 7. The TGF-ß1 expression level in the wound was significantly higher in the N-CWS group than in the control group on day 7. Conclusions N-CWS can activate macrophages, increase TGF-ß1 expression, and enhance angiogenesis and thus accelerate cutaneous wound healing.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Esqueleto da Parede Celular/uso terapêutico , Ativação de Macrófagos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Nocardia , Cicatrização/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Ativação de Macrófagos/imunologia , Masculino , Neovascularização Fisiológica/imunologia , Ratos , Ratos Sprague-Dawley , Pele/efeitos dos fármacos , Pele/lesões , Pele/fisiopatologia , Fator de Crescimento Transformador beta1/biossíntese , Cicatrização/imunologia
5.
Int J Biol Macromol ; 101: 398-407, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28315436

RESUMO

Several lines of evidences have shown that Nocardia rubra cell wall skeleton (Nr-CWS) has immunoregulatory and anti-tumor activities. However, there is no information about the effect of Nr-CWS on CD4+ T cells. The aim of this study was to explore the effect of Nr-CWS on the phenotype and function of CD4+ T cells. Our results of in vitro experiments showed that Nr-CWS could significantly up-regulate the expression of CD69 and CD25 on CD4+ T cells, promote the proliferation of CD4+ T cells, increase the production of IFN-γ, TNF-α and IL-2 in the supernatants, but has no significant effect on the apoptosis and death of CD4+ T cells. Results of in vivo experiments showed that Nr-CWS could promote the proliferation of CD4+ T cells, and increase the production of IL-2, IFN-γ and TNF-α (Th1 type cytokines). These data suggest that Nr-CWS can enhance the activation of CD4+ T cells, promote the proliferation of CD4+ T cells and the differentiation of CD4+ T cells to Th1 cells.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Esqueleto da Parede Celular/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/sangue , Feminino , Imunomodulação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL
6.
Yakugaku Zasshi ; 136(11): 1477-1484, 2016.
Artigo em Japonês | MEDLINE | ID: mdl-27803478

RESUMO

Delivery systems are a powerful technology for enhancing the effect of cancer immunotherapy. We have been in the process of developing lipid-based delivery systems for controlling the physical properties and dynamics of immunofunctional molecules such as antigens and adjuvants. The lipid nanoparticulation of these molecules improves their physical properties, resulting in a good water dispensability, greater stability, and small size. The cell wall skeleton of bacille Calmette-Guerin (BCG-CWS) could be used to replace live BCG as a drug for treating bladder cancer, but problems associated with the physical properties of BCG-CWS have prevented its use. To overcome such problems, we developed a novel packaging method that permits BCG-CWS to be encapsulated into lipid nanoparticles, which induce antitumor responses against bladder cancer. Lipid nanoparticulation also improves the intracellular trafficking and biodistribution of immunofunctional molecules. Cyclic di-GMP (c-di-GMP) is an adjuvant that is recognized by the cytosolic sensor. However, c-di-GMP cannot pass through the cell membrane. We encapsulated c-di-GMP into lipid nanoparticles containing a pH-responsive lipid that was developed in our laboratory and achieved efficient cytosolic delivery and the induction of antitumor immunity. Furthermore, we are attempting to control the functions of immune cells by RNA interference. We have recently succeeded in the efficient delivery of small interfering RNA into mouse dendritic cells (DCs), which led to the enhancement of antitumor activity of DCs. In this review, our recent efforts regarding cancer immunotherapy using lipid-based nanoparticles are reviewed.


Assuntos
Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Imunoterapia , Lipídeos , Nanopartículas , Neoplasias/terapia , Adjuvantes Imunológicos , Animais , Esqueleto da Parede Celular , GMP Cíclico/análogos & derivados , Células Dendríticas , Humanos , Camundongos , Mycobacterium bovis , Interferência de RNA , Neoplasias da Bexiga Urinária/terapia
7.
PLoS One ; 11(11): e0166234, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27829029

RESUMO

The interpretation of high-throughput gene expression data for non-model microorganisms remains obscured because of the high fraction of hypothetical genes and the limited number of methods for the robust inference of gene networks. Therefore, to elucidate gene-gene and gene-condition linkages in the bioremediation-important genus Dehalococcoides, we applied a Bayesian inference strategy called Reverse Engineering/Forward Simulation (REFS™) on transcriptomic data collected from two organohalide-respiring communities containing different Dehalococcoides mccartyi strains: the Cornell University mixed community D2 and the commercially available KB-1® bioaugmentation culture. In total, 49 and 24 microarray datasets were included in the REFS™ analysis to generate an ensemble of 1,000 networks for the Dehalococcoides population in the Cornell D2 and KB-1® culture, respectively. Considering only linkages that appeared in the consensus network for each culture (exceeding the determined frequency cutoff of ≥ 60%), the resulting Cornell D2 and KB-1® consensus networks maintained 1,105 nodes (genes or conditions) with 974 edges and 1,714 nodes with 1,455 edges, respectively. These consensus networks captured multiple strong and biologically informative relationships. One of the main highlighted relationships shared between these two cultures was a direct edge between the transcript encoding for the major reductive dehalogenase (tceA (D2) or vcrA (KB-1®)) and the transcript for the putative S-layer cell wall protein (DET1407 (D2) or KB1_1396 (KB-1®)). Additionally, transcripts for two key oxidoreductases (a [Ni Fe] hydrogenase, Hup, and a protein with similarity to a formate dehydrogenase, "Fdh") were strongly linked, generalizing a strong relationship noted previously for Dehalococcoides mccartyi strain 195 to multiple strains of Dehalococcoides. Notably, the pangenome array utilized when monitoring the KB-1® culture was capable of resolving signals from multiple strains, and the network inference engine was able to reconstruct gene networks in the distinct strain populations.


Assuntos
Esqueleto da Parede Celular/genética , Parede Celular/genética , Chloroflexi/genética , Redes Reguladoras de Genes/genética , Metabolismo/genética , Chloroflexi/metabolismo , Sequência Consenso/genética , Análise de Sequência com Séries de Oligonucleotídeos
8.
J Control Release ; 196: 161-7, 2014 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-25315488

RESUMO

We previously reported on the development of a water soluble formulation of the cell wall skeleton of BCG (BCG-CWS), a major immune active center of BCG, by encapsulating it into a nanoparticle (CWS-NP). The CWS-NP allowed us to clarify the machinery associated with the BCG mediated anti-bladder tumor effect, especially the roles of bladder cancer cells and dendritic cells (DCs) in the initial step, which remains poorly understood. We show herein that the internalization of BCG-CWS by bladder cancer cells, but not DCs, is indispensable for the induction of an antitumor effect against bladder cancer. Tumor growth was significantly inhibited in mice that had been inoculated with mouse bladder cancer (MBT-2) cells containing internalized BCG-CWS. On the other hand, the internalization of BCG-CWS by DCs had only a minor effect on inducing an antitumor effect against MBT-2 tumors. This was clarified for the first time by using the CWS-NP. This finding provides insights into our understanding of the role of bladder cancer cells and DCs in BCG therapy against bladder cancer.


Assuntos
Antineoplásicos/farmacologia , Vacina BCG/farmacologia , Esqueleto da Parede Celular/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Emulsões , Feminino , Contagem de Leucócitos , Lipídeos/química , Camundongos , Camundongos Endogâmicos C3H , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Control Release ; 176: 44-53, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24389133

RESUMO

The Mycobacterium bovis Bacille Calmette-Guerin cell wall skeleton (BCG-CWS) could be used to replace live BCG as a bladder cancer drug. However, because BCG-CWS is poorly soluble, has a strong-negative charge, very high molecular weight and heterogeneity in size of tens of µm, it cannot be used in such an application. We report herein on the development of a novel packaging method that permits BCG-CWS to be encapsulated into 166nm-sized lipid particles. The BCG-CWS encapsulated nano particle (CWS-NP) has a high uniformity and can be easily dispersed. Thus, it has the potential for use as a packaging method that would advance the scope of applications of BCG-CWS as a bladder cancer drug. In a functional evaluation, CWS-NP was efficiently taken up by mouse bladder tumor (MBT-2) cells in vitro and inhibited tumor growth in mice bearing MBT-2 tumors. Moreover, intravesically administered CWS-NP showed significant antitumor effects in a rat model with naturally developed bladder cancer. An enhancement in Th1 differentiation by CWS-NP was also confirmed in human T cells. In conclusion, CWS-NP represents a promising delivery system for BCG-CWS for clinical development as a potent bladder cancer drug.


Assuntos
Antineoplásicos/administração & dosagem , Esqueleto da Parede Celular/administração & dosagem , Mycobacterium bovis , Nanopartículas/administração & dosagem , Neoplasias da Bexiga Urinária/tratamento farmacológico , Adulto , Animais , Butilidroxibutilnitrosamina , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Ratos , Ratos Endogâmicos F344 , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Neoplasias da Bexiga Urinária/induzido quimicamente , Adulto Jovem
10.
New Microbiol ; 36(2): 145-51, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23686120

RESUMO

A broad coverage influenza vaccine against multiple viral strains based on the viral nucleoprotein (NP) is a goal pursued by many laboratories. If the goal is to formulate the vaccine with recombinant NP it is essential to count on adjuvants capable of inducing cellular immunity. This work have studied the effect of the monophosphoryl lipid A and trehalose dimycolate, known as the Ribi Adjuvant System (RAS), in the immune response induced in mice immunized with recombinant NP. The NP was formulated with RAS and used to immunize BALB/c mice. Immunizations with NP-RAS increased the humoral and cellular immune responses compared to unadjuvanted NP. The predominant antibody isotype was IgG2a, suggesting the development of a Th1 response. Analysis of the cytokines from mice immunized with NP-RAS showed a significant increase in the production of IFN-g and a decreased production of IL-10 and IL-4 compared to controls without RAS. These results are similar to those usually obtained using Freund's adjuvant, known to induce Th1 and CTL responses when co-administered with purified proteins, and suggest that a similar approach may be possible to enhance the performance of a T-cell vaccine containing NP.


Assuntos
Esqueleto da Parede Celular/administração & dosagem , Fatores Corda/administração & dosagem , Influenza Humana/imunologia , Lipídeo A/análogos & derivados , Proteínas de Ligação a RNA/imunologia , Células Th1/imunologia , Proteínas do Core Viral/imunologia , Animais , Anticorpos Antivirais/imunologia , Esqueleto da Parede Celular/imunologia , Fatores Corda/imunologia , Feminino , Humanos , Imunidade Celular , Imunização , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-4/imunologia , Lipídeo A/administração & dosagem , Lipídeo A/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Ligação a RNA/administração & dosagem , Proteínas de Ligação a RNA/genética , Linfócitos T Citotóxicos/imunologia , Proteínas do Core Viral/administração & dosagem , Proteínas do Core Viral/genética
11.
Drug Discov Ther ; 6(4): 218-25, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23006993

RESUMO

Cell-wall skeleton prepared from Mycobacterium bovis BCG (BCG-CWS) is known as a potent adjuvant and has been shown to possess antitumor activity in many non-clinical and clinical studies. As there are no approved BCG-CWS formulations for cancer therapy, we investigated the potential for cancer immunotherapy of SMP-105, our originally produced BCG-CWS. For optimizing SMP-105 emulsion, we compared the effects of drakeoland squalane-based SMP-105 emulsions on IFN-γ production in rats and evaluated their ability to induce skin reaction in guinea pigs. Both emulsions had the same activity in both experiments. We selected squalane as base material and produced two types of squalane-based formulations (vialed emulsion and pumped emulsion) that can easily be prepared as oil-in-water emulsions. Although the vialed emulsion showed the same pattern of distribution as a usual homogenized emulsion, the pumped emulsion showed more uniform distribution than the other two emulsions. Whereas both emulsions enhanced strong delayed type hypersensitivity (DTH) reaction in a mouse model, the pumped emulsion induced slightly smaller edema. Data on oil droplet size distribution suggest that few micrometer oil droplet size might be appropriate for oil-in-water microemulsion of SMP-105. The antitumor potency of SMP-105 emulsion was stronger than that of some of the launched toll-like receptor (TLR) agonists (Aldara cream, Picibanil, and Immunobladder). Aldara and Picibanil showed limited antitumor effectiveness, while Immunobladder had almost the same effect as SMP-105 at the highest dose, but needed about 10 times the amount of SMP-105. These findings first indicate that SMP-105 has great potential in cancer immunotherapy.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antineoplásicos/farmacologia , Esqueleto da Parede Celular/farmacologia , Mycobacterium bovis/química , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/isolamento & purificação , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Esqueleto da Parede Celular/efeitos adversos , Esqueleto da Parede Celular/isolamento & purificação , Emulsões , Feminino , Cobaias , Hipersensibilidade Tardia/imunologia , Imunoterapia , Interferon gama/biossíntese , Interferon gama/imunologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos Lew , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Reprod Immunol ; 89(1): 46-54, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21397337

RESUMO

Immunological approaches to gender selection have been contemplated since the discovery of the family of male-specific H-Y antigens found only on the surface of male cells. H-Y antigens are able to elicit an immune reaction when cells or tissues from a male donor are grafted to a female recipient. We describe here the development and testing of an inexpensive approach using polyclonal antibodies against four specific H-Y outer membrane proteins male enhanced antigen 1 (MEA 1), male enhanced antigen 2 (MEA 2), sex determining region Y (SRY) and testis determining factor (TDF). Epitopes based on hydrophilic primary sequences of the proteins were synthesized, N-terminal biotin-labeled, linked to streptavidin and mixed with a Ribi adjuvant prior to immunization in rabbits. The antiserum was tested to determine affinity to swine spermatozoa using anti-motility, flow cytometry and motility and sedimentation chambers. Fluorescent microscopy and fluorescent in situ hybridization (FISH) was used to identify the percentage of motile spermatozoa that contained the Y chromosome. We found that the polyclonal antibodies had high affinity to the spermatozoa leading to a cessation of motility. Furthermore, the majority of these non-motile spermatozoa contained the Y chromosome. We conclude that the use of polyclonal antiserum against synthetic H-Y peptide antigens may be an inexpensive and simple means to inhibit the motility of swine spermatozoa bearing the Y chromosome.


Assuntos
Anticorpos/farmacologia , Epitopos/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Pré-Seleção do Sexo , Espermatozoides/metabolismo , Animais , Afinidade de Anticorpos , Movimento Celular/efeitos dos fármacos , Esqueleto da Parede Celular/administração & dosagem , Células Cultivadas , Fatores Corda/administração & dosagem , Mapeamento de Epitopos , Epitopos/química , Hibridização in Situ Fluorescente , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Masculino , Neoplasia Endócrina Múltipla Tipo 1/imunologia , Neoplasia Endócrina Múltipla Tipo 1/metabolismo , Neoplasia Endócrina Múltipla Tipo 2a/imunologia , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Pré-Seleção do Sexo/métodos , Proteína da Região Y Determinante do Sexo/imunologia , Proteína da Região Y Determinante do Sexo/metabolismo , Espermatozoides/imunologia , Espermatozoides/patologia , Suínos
13.
BJU Int ; 108(9): 1520-6, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21314815

RESUMO

OBJECTIVE: • To conduct a preclinical evaluation of the ability of natural killer cells to cytolyze bladder cancer cells that were modified to show enhanced expression of natural-killer group 2, member D (NKG2D) ligands by R8-liposome-bacillus Calmette-Guéin (BCG)-cell wall skeleton (CWS) treatment. MATERIALS AND METHODS: • The T24 cells and RT-112 cells were co-cultured with R8-liposome-BCG-CWS and BCG for 2, 4, or 6 h, and then the surface expression of NKG2D ligands was analyzed using TaqMan real-time quantitative RT-PCR. • Peripheral blood mononuclear cells were obtained with a conventional preparation kit, and then lymphokine-activated killer (LAK) cells were generated from these purified peripheral blood mononuclear cells via interleukin-2 stimulation. • The anti-tumour effect of LAK cells against untreated and R8-liposome-BCG-CWS co-cultured with cells of the human bladder cancer cell lines T24 and RT-112 was analyzed using the cytotoxic WST-8 assay method at 4 h of culture at various effector/target (E : T) ratios. RESULTS: • Major histocompatibility complex class I-related chain B (MICB) expression was increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. • UL-16-binding protein (ULBP) 1 expression was also increased ≈1.5-fold on T24 cells and RT-112 cells with BCG. R8-liposome-BCG-CWS increased the surface expression of MICB 2.2-fold on T24 cells but did not increase it significantly on RT-112 cells. • ULBP1 expression was increased ≈2.2-fold on RT-112 cells, although no differences were observed between the expression of ULBP2 and 3 with R8-liposome-BCG-CWS. • T24 cells that were co-cultured with R8-liposome-BCG-CWS showed an ≈1.3-fold increase in sensitivity to cytolysis by LAK cells at an E : T ratio of 4 and RT-112 cells showed an ≈1.4-fold increase at an E : T ratio of 2. CONCLUSIONS: • In the present study, the induction of surface NKG2D ligands by R8-liposome-BCG-CWS rendered cancer cells more susceptible to cytolysis by LAK cells. • T24 cells and RT-112 cells, even when cultured singly in the absence of immune cells, can directly respond to R8-liposome-BCG-CWS. • The results obtained in the present study may therefore indicate a novel adoptive immunotherapy against bladder cancers.


Assuntos
Vacina BCG/farmacologia , Esqueleto da Parede Celular/farmacologia , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias da Bexiga Urinária/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Ligantes , Mycobacterium bovis/química , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais Cultivadas/imunologia , Regulação para Cima
14.
J Immunol ; 186(3): 1399-410, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21199899

RESUMO

Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ∼36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.


Assuntos
Adjuvantes Imunológicos/fisiologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Diferenciação Celular/imunologia , Toxina da Cólera/sangue , Toxina da Cólera/fisiologia , Memória Imunológica , Adjuvantes Imunológicos/sangue , Compostos de Alúmen/metabolismo , Compostos de Alúmen/farmacologia , Animais , Subpopulações de Linfócitos B/metabolismo , Esqueleto da Parede Celular/sangue , Esqueleto da Parede Celular/fisiologia , Fatores Corda/sangue , Fatores Corda/fisiologia , Relação Dose-Resposta Imunológica , Feminino , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Imunoglobulina A/biossíntese , Imunoglobulina A/sangue , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Lipídeo A/análogos & derivados , Lipídeo A/sangue , Lipídeo A/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Tempo
15.
Anticancer Res ; 30(10): 4089-96, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21036724

RESUMO

AIM: Bacillus Calmette-Guerin (BCG) is one of therapeutic options for urothelial carcinoma (UC). The objectives of this study were to determine the direct effect of viable or heat-killed BCG and BCG cell wall skeleton (BCG-CWS) on UC cells in vitro. MATERIALS AND METHODS: UC cell lines were co-cultured with viable or heat-killed BCG Immunobladder® (Tokyo 172 strain) and BCG-CWS. Viability of the cells, apoptosis and BrdU incorporation were estimated. RESULTS: BCG induced cell growth retardation in highly malignant UC bearing integrin α5ß1 (VLA5). VLA5-blocking antibody partially abrogated this effect. BCG treatment induced a modest increase in the sub-G(1) fraction of cells and a decrease of BrdU incorporation. Cell growth retardation effect of viable BCG was reproduced by both heat-killed BCG and BCG-CWS. CONCLUSION: The results indicate that VLA5 may be a biomarker of UC with sensitivity to BCG. Moreover, BCG-CWS is a promising substance which might replace BCG, preventing life-threatening complications of viable BCG treatment.


Assuntos
Vacina BCG/farmacologia , Mycobacterium bovis/imunologia , Neoplasias da Bexiga Urinária/terapia , Processos de Crescimento Celular/imunologia , Linhagem Celular Tumoral , Esqueleto da Parede Celular/imunologia , Esqueleto da Parede Celular/farmacologia , Quinase 1 de Adesão Focal/biossíntese , Quinase 1 de Adesão Focal/imunologia , Fase G1/imunologia , Humanos , Integrina alfa5beta1/biossíntese , Integrina alfa5beta1/imunologia , Transdução de Sinais , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/imunologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologia
16.
Vaccine ; 28(50): 7873-80, 2010 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-20937311

RESUMO

Mycobacterial cell-wall skeleton (CWS) is an immunoactive and biodegradable particulate adjuvant and has been used for immunotherapy in patients with cancer. The CWS of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS) was studied as a universal vaccine vehicle for antigen conjugation, to develop potentially effective and safe vaccines. Here, we describe experiments in which protein antigens, such as keyhole limpet haemocyanin (KLH), ovalbumin (OVA) and bovine serum albumin (BSA) were highly efficiently coupled to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS)-activated carboxyl groups of BCG-CWS, and tested the immunogenicity of OVA-conjugated BCG-CWS vaccine. We found that a strong immune response was induced in mice immunised with OVA-conjugated BCG-CWS, which was similar to the enhancement of the immune responses in mice immunised with OVA and complete Freund's adjuvant. Covalent conjugation of OVA to BCG-CWS was essential for Th1-skewed immune responses, with prominent expression of IFN-γ. Furthermore, antigen-conjugated BCG-CWS vaccine is simple to manufacture, safe, and easy to use. Our results suggest that mycobacterial CWS as a universal vaccine vehicle for conjugation of a wide variety of antigens constitutes a breakthrough for development of the most promising vaccines for infections, allergic diseases, and cancer.


Assuntos
Adjuvantes Imunológicos/farmacologia , Vacina BCG/imunologia , Esqueleto da Parede Celular/imunologia , Animais , Formação de Anticorpos , Proliferação de Células , Esqueleto da Parede Celular/farmacologia , Ensaio de Imunoadsorção Enzimática , Etildimetilaminopropil Carbodi-Imida/farmacologia , Adjuvante de Freund/imunologia , Imunoglobulina G/sangue , Interferon gama/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/imunologia , Ovalbumina/imunologia , Baço/citologia , Baço/imunologia , Succinimidas/farmacologia , Células Th1/imunologia
17.
Trends Microbiol ; 18(8): 348-56, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20598544

RESUMO

FtsZ, the best-known prokaryotic division protein, assembles at midcell with other proteins forming a ring during septation. Widely conserved in bacteria, FtsZ represents the ancestor of tubulin. In the presence of GTP it forms polymers able to associate into multi-stranded flexible structures. FtsZ research is aimed at determining the role of the Z-ring in division, describing the polymerization and potential force-generating mechanisms and evaluating the roles of nucleotide exchange and hydrolysis. Systems to reconstruct the FtsZ ring in vitro have been described and some of its mechanical properties have been reproduced using in silico modeling. We discuss current research in FtsZ, some of the controversies, and finally propose further research needed to complete a model of FtsZ action that reconciles its in vitro properties with its role in division.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Divisão Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Bactérias/genética , Esqueleto da Parede Celular/química , Esqueleto da Parede Celular/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Modelos Biológicos , Conformação Proteica , Estrutura Terciária de Proteína
18.
Vaccine ; 28 Suppl 5: F17-22, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20362624

RESUMO

In a series of studies in SPF and conventional guinea pigs, various adjuvants (larifan, polyoxidonium-PO, natrium thiosulphate-NT, TNF-ß and Ribi adjuvant system-RAS) were evaluated for their ability to enhance immune responses to the live brucellosis vaccine, Brucella abortus strain 82-PS (penicillin-sensitive). Combining adjuvants with S82-PS increased synthesis of antibodies against rough (R) and smooth (S) Brucella antigens. Dynamics and levels of antibodies differed dependent upon the adjuvant. Adjuvants enhanced cell-mediated responses to S82-PS, and phagocytosis by macrophages. Humoral and cellular immune responses stimulated by the adjuvants correlated with increased vaccine protection against experimental challenge. The highest protection was demonstrated by combining TNF-ß or PO with S82-PS. Our data demonstrates the potential of adjuvants to improve immunogenic properties of live brucellosis vaccines.


Assuntos
Adjuvantes Imunológicos/farmacologia , Vacina contra Brucelose/imunologia , Brucella abortus/imunologia , Brucelose/prevenção & controle , Animais , Anticorpos Antibacterianos/imunologia , Brucelose/imunologia , Proliferação de Células , Esqueleto da Parede Celular/imunologia , Fatores Corda/imunologia , Cobaias , Imunidade Celular , Imunidade Humoral , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Linfotoxina-alfa/imunologia , Macrófagos Peritoneais/imunologia , Masculino , Compostos Orgânicos/imunologia , Fagocitose , Piperazinas/imunologia , Polímeros
19.
J Microbiol Methods ; 80(3): 302-5, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20079769

RESUMO

A unique hydrolysis method using a two-layer solution, consisting of diluted hydrochloric acid and toluene was developed to isolate whole arabinose mycolates from the cell wall skeleton of Mycobacterium bovis BCG Tokyo 172 (SMP-105) in order to reveal its pivotal role in enhancing immune responses against tumors.


Assuntos
Arabinose/análogos & derivados , Esqueleto da Parede Celular/química , Mycobacterium bovis/química , Ácidos Micólicos , Adjuvantes Imunológicos/química , Arabinose/química , Ácido Clorídrico/química , Hidrólise , Estrutura Molecular , Ácidos Micólicos/química , Ácidos Micólicos/isolamento & purificação , Tóquio , Tolueno/química
20.
Virol J ; 7: 20, 2010 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-20102602

RESUMO

BACKGROUND: Infectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (Pneumoviridae), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins. RESULTS: Three compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats S.hispidus and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease. CONCLUSIONS: This work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease.


Assuntos
Antivirais/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Dedos de Zinco , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/metabolismo , 2,2'-Dipiridil/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Antivirais/metabolismo , Esqueleto da Parede Celular/administração & dosagem , Fatores Corda/administração & dosagem , Dissulfetos/metabolismo , Dissulfetos/farmacologia , Dissulfiram/metabolismo , Dissulfiram/farmacologia , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Ligação Proteica , Ratos , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Sigmodontinae/virologia , Tiram/metabolismo , Tiram/farmacologia , Vacinas Atenuadas/imunologia , Proteínas Virais/metabolismo
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