RESUMEN
BACKGROUND: Standard treatment for diffuse peritonitis due to colorectal perforation may be insufficient to suppress inflammatory reaction in sepsis. Thus, developing new treatments is important. This study aimed to examine whether intraperitoneal irradiation by artificial sunlight suppresses inflammatory reaction in a lipopolysaccharide (LPS)-induced peritonitis model after surgical treatments. MATERIALS AND METHODS: Mice were divided into naive, nontreatment (NT), and phototherapy (PT) groups. In the latter two groups, LPS was intraperitoneally administered to induce peritonitis and removed by intraperitoneal lavage after laparotomy. The PT group was irradiated with artificial sunlight intraperitoneally. We evaluated the local and systemic inflammatory reactions. Murine macrophages were irradiated with artificial sunlight after stimulation by LPS, and cell viability and expression of tumor necrotizing factor-α (TNF-α) were evaluated. RESULTS: As a local inflammatory reaction, the whole cell count, the expression of interleukin-6 and TNF-α in the intra-abdominal fluid, and the peritoneal thickness were significantly lower in the PT group than in the NT group. As a systematic inflammatory reaction, the expression of serum TNF-α, granulocyte macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)-1α, and MIP-1ß were significantly lower in the PT group than in the NT group. Irradiation by artificial sunlight suppressed the expression of TNF-α in murine macrophages without affecting cell viability. CONCLUSIONS: Intraperitoneal irradiation by artificial sunlight could suppress local and systemic inflammatory reactions in the LPS-induced peritonitis murine model. These effects may be associated with macrophage immune responses.
Asunto(s)
Perforación Intestinal/complicaciones , Peritoneo/efectos de la radiación , Peritonitis/terapia , Fototerapia/métodos , Luz Solar , Animales , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Perforación Intestinal/inmunología , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/efectos de la radiación , Masculino , Ratones , Peritoneo/inmunología , Peritonitis/inmunología , Células RAW 264.7RESUMEN
Photodynamic therapy has emerged as an alternative treatment for cutaneous leishmaniasis, and compounds with photocatalytic behavior are promising candidates to develop new therapeutic strategies for the treatment of this parasitic disease. Titanium dioxide TiO2 is a semiconductor ceramic material that shows excellent photocatalytic and antimicrobial activity under Ultraviolet irradiation. Due to the harmful effects of UV radiation, many efforts have been made in order to enhance both photocatalytic and antimicrobial properties of TiO2 in the visible region of the spectrum by doping or through modifications in the route of synthesis. Herein, Fe-, Zn-, or Pt- doped TiO2 nanostructures were synthesized by solution-combustion route. The obtained compounds presented aggregates of 100â¯nm, formed by particles smaller than 20â¯nm. Doping compounds shift the absorption spectrum towards the visible region, allowing production of reactive oxygen species in the presence of oxygen and molecular water when the system is irradiated in the visible spectrum. The Pt (EC50â¯=â¯18.2⯱â¯0.8⯵g/mL) and Zn (EC50â¯=â¯16.4⯱â¯0.3⯵g/mL) -doped TiO2 presented the higher antileishmanial activities under visible irradiation and their application as photosensitizers in photodynamic therapy (PDT) strategies for the treatment of cutaneous leishmaniasis should be considered.
Asunto(s)
Leishmania/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Fármacos Fotosensibilizantes/farmacología , Titanio/química , Rayos Ultravioleta , Animales , Antracenos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Hierro/química , Leishmania/metabolismo , Leishmania/efectos de la radiación , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/parasitología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/efectos de la radiación , Nanopartículas del Metal/química , Nanopartículas del Metal/uso terapéutico , Ratones , Microscopía Electrónica de Transmisión , Perileno/análogos & derivados , Perileno/química , Perileno/farmacología , Perileno/uso terapéutico , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de Fluorescencia , Difracción de Rayos X , Zinc/químicaRESUMEN
Low-dose irradiation (LDI) has been used in clinics to treat human diseases, including chronic inflammation. This study assessed the effects of LDI on the inflammatory response of activated mouse primary peritoneal macrophages, and the underlying signal pathways. Primary peritoneal macrophages were isolated from mice and then incubated with lipopolysaccharide (LPS)-coated Ti microparticles (Ti-positive control) with or without brief exposure to LDI (X-ray, 0.5 Gy) 1 h later (Ti-LDI group) or left untreated in culture medium (Ti-negative control). The macrophages were then subjected to qRT-PCR, Western blot, cell viability CCK-8 assay, and ELISA. qRT-PCR analysis revealed the Ti-LDI group expressed significantly lower levels of IL-1ß, IL-6, and TNF-α mRNA than those of the Ti-positive control group, while the ELISA data showed that Ti-LDI group had significantly lower secretion of IL-1ß, IL-6, and TNF-α proteins. The most significant reduction associated with LDI was the secretion TNF-α protein, which barely increased from 13 to 25 h after treatment. Western blot data demonstrated that phosphorylation of p65 and ERK was much lower in the Ti-LDI group than in the controls. The data from the current study suggests that LDI of activated mouse macrophages was associated with significantly lower inflammation responses, compared with non-exposed activated macrophages, which was possibly through inhibition of the NF-κB and ERK pathways.
Asunto(s)
Activación de Macrófagos , Macrófagos Peritoneales/efectos de la radiación , Animales , Relación Dosis-Respuesta en la Radiación , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de la radiación , Macrófagos Peritoneales/metabolismo , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Although favorable immune responses to low-dose irradiation (LDI) have been observed in normal mice, i.e., a hormesis effect, little is known about the effects of LDI in infectious diseases. In this study, we examined the effects of LDI on mice with sepsis, a severe and often lethal hyperinflammatory response to bacteria. Female C57BL/6 mice were whole-body irradiated with 10cGy 48h before Escherichia coli infection, and survival, bacterial clearance, cytokines, and antioxidants were quantified. LDI pretreatment significantly increased survival from 46.7% in control mice to 75% in mice with sepsis. The bacterial burden was significantly lower in the blood, spleen, and kidney of LDI-treated mice than in those of control septic mice. The levels of pro-inflammatory cytokines, e.g., IL-1ß and IL-6, as well as anti-inflammatory IL-10 were markedly reduced in pre-LDI septic mice. Nitric oxide production by peritoneal macrophages was also reduced in pre-LDI septic mice. Immune cells in the spleen increased and Nrf2 and HO-1 were induced in pre-LDI septic mice. LDI stimulates the immune response and minimizes lethality in septic mice via enhanced bacterial clearance and reduced initial proinflammatory responses.
Asunto(s)
Infecciones por Escherichia coli/radioterapia , Sepsis/radioterapia , Irradiación Corporal Total , Animales , Recuento de Colonia Microbiana , Citocinas/sangre , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/sangre , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Femenino , Riñón/microbiología , Riñón/efectos de la radiación , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Células RAW 264.7 , Sepsis/sangre , Sepsis/inmunología , Sepsis/microbiología , Bazo/microbiología , Bazo/efectos de la radiaciónRESUMEN
UVR-induced apoptosis in cutaneous antigen presenting cells (APC) causes systemic immune suppression and is dependent on TLR4/MyD88 signalling, but the apoptotic signalling pathways have not been defined. Macrophages pretreated with lipopolysaccharide (LPS) were unresponsive to subsequent LPS treatment, however, but were susceptible to UVR-induced apoptosis. Macrophage survival and apoptotic events after UVR were also unaffected by treatment with TLR4 antagonists, a blocking IgG or a TLR4 analog antagonist, suggesting that UVR cell death is independent of a soluble ligand. After UVR, IRAK4KDKI (catalytically inactive IRAK4) and wild-type (WT) macrophages show equivalent levels of survival, as measured by MTT assay, and apoptosis, as measured by cleaved caspase-3. Furthermore, in macrophages from both mice, UVR activated caspase-8 and PARP, while inactivating Rip3. This finding is supported by a lack of IRAK1 degradation after UVR, compared to treatment with TLR2 or TLR4 agonists. UVR induced association of MyD88 with FADD, an extrinsic apoptotic pathway protein, but not IRAK4. UVR-induced migration of FADD to the cell membrane of WT macrophages, but not MyD88-/- macrophages, was observed (confocal microscopy). Co-immunoprecipitation using an epitope-tagged MyD88 revealed that FADD, but not TRADD, was recruited to MyD88 within 30 minutes of UVR exposure. UVR engages TLR4/MyD88 as a death signalling complex, rather than the classical inflammatory signalling pathway triggered by PAMP recognition of TLR4. These studies provide the rationale for the future development of topical TLR4 modulating therapies to interfere with this UVB-mediated apoptosis and the associated negative consequences of immune suppression.
Asunto(s)
Apoptosis , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Macrófagos Peritoneales/efectos de la radiación , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Femenino , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Lipopolisacáridos , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BLRESUMEN
2-Deoxy-d-glucose (2-DG) has been found to enhance the cytotoxicity of ionizing radiation and chemotherapeutic drugs in several tumor cell lines in vitro. Systemic administration of 2-DG together with localized irradiation of the tumor leads to tumor regression and cure (disease free survival), which correlate with the differential levels of anti-tumor immunity observed in Ehrlich ascites tumor (EAT) bearing mice. Macrophages being a major player of the innate immune system, we investigated the activation status of splenic macrophages during radio-sensitization of EAT in mice as well as in peritoneal macrophages ex vivo and macrophagic cell line (Raw 264.7) in vitro. Results suggest that under in vivo conditions, the combined treatment (2-DG+radiation) restores the M1 phenotype in spleen that correlated with the tumor response. However, 2-DG neither induced significant cytotoxicity nor enhanced radiation-induced cell death in peritoneal macrophages and the macrophage cell line (Raw 264.7). Further, increased arborization and enhanced functional status (expression of MHC class II, CD80, CD86 and phagocytosis) were observed after the combined treatment. Besides this activation, the combined treatment also skewed the macrophages towards M1 phenotype as evidenced by the enhanced secretion of IL-12, IL-2, TNF-α and IFN-γ. These observations suggest that 2-DG not only preserves the survival of normal macrophages during irradiation, but also activates macrophages by polarizing towards M1 phenotype, which is known to be tumoricidal in nature. This study for the first time sheds light on a potential antitumor immune activation by 2-DG involving macrophagic stimulation during in vivo radio-sensitization of tumors, besides the other known antitumor effects of this glucose analogue.
Asunto(s)
Carcinoma de Ehrlich/terapia , Desoxiglucosa/farmacología , Rayos gamma/uso terapéutico , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/efectos de la radiación , Animales , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígeno B7-2/genética , Antígeno B7-2/inmunología , Carcinoma de Ehrlich/genética , Carcinoma de Ehrlich/inmunología , Carcinoma de Ehrlich/patología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de la radiación , Línea Celular , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Masculino , Ratones , Fagocitosis/efectos de los fármacos , Fagocitosis/efectos de la radiación , Cultivo Primario de Células , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Carga Tumoral/efectos de la radiación , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Benign painful and inflammatory diseases have been treated for decades with low/moderate doses of ionizing radiation (LD-X-irradiation). Tissue macrophages regulate initiation and resolution of inflammation by the secretion of cytokines and by acting as professional phagocytes. Having these pivotal functions, we were interested in how activated macrophages are modulated by LD-X-irradiation, also with regard to radiation protection issues and carcinogenesis. We set up an ex-vivo model in which lipopolysaccharide pre-activated peritoneal macrophages (pMΦ) of radiosensitive BALB/c mice, mimicking activated macrophages under inflammatory conditions, were exposed to X-irradiation from 0·01 Gy up to 2 Gy. Afterwards, the viability of the pMΦ, their transmigration and chemotaxis, the phagocytic behaviour, the secretion of inflammatory cytokines and underlying signalling pathways were determined. Exposure of pMΦ up to a single dose of 2 Gy did not influence their viability and phagocytic function, an important fact regarding radiation protection. However, significantly reduced migration, but increased chemotaxis of pMΦ after exposure to 0·1 or 0·5 Gy, was detected. Both might relate to the resolution of inflammation. Cytokine analyses revealed that, in particular, the moderate dose of 0·5 Gy applied in low-dose radiotherapy for inflammatory diseases results in an anti-inflammatory cytokine microenvironment of pMΦ, as the secretion of the proinflammatory cytokine interleukin (IL)-1ß was reduced and that of the anti-inflammatory cytokine transforming growth factor (TGF)-ß increased. Further, the reduced secretion of IL-1ß correlated with reduced nuclear translocation of nuclear factor (NF)-κB p65, starting at exposure of pMΦ to 0·5 Gy of X-irradiation. We conclude that inflammation is modulated by LD-X-irradiation via changing the inflammatory phenotype of macrophages.
Asunto(s)
Quimiotaxis/inmunología , Quimiotaxis/efectos de la radiación , Macrófagos/inmunología , Macrófagos/efectos de la radiación , Fagocitosis/inmunología , Fagocitosis/efectos de la radiación , Radiación Ionizante , Animales , Supervivencia Celular/inmunología , Supervivencia Celular/efectos de la radiación , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/inmunología , Activación de Macrófagos/inmunología , Activación de Macrófagos/efectos de la radiación , Macrófagos/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/efectos de la radiación , Ratones , Transporte de Proteínas , Factor de Transcripción ReIA/metabolismo , Rayos XRESUMEN
AIM: Develop conditions for inactivation of staphylococcus by using photosensibilizator merocyanine 540 (MC540) for the production of antigenic preparation (AP). Study some of immune reactions to AP and the possibility of regulation of DTH reaction to AP under the effect of MC540. MATERIALS AND METHODS: Merocyanine 540 (MC540, Sigma-Aldrich, Switzerland) is used in the study. MC540 and Staphylococcus aureus, strain 78 (Sa78) were irradiated by light of a mercury-quartz lamp DRSH-250 (Zelenograd). C56BL/6 line mice were immunized once by subcutaneous administration of AP. DTH reaction was tested 7 days after the immunization. Functional activity of peritoneal exudate macrophages was determined 1 and 9 days after the immunization. Immune modulating effect of MC540 in DTH was determined after its per os administration to mice 1 hour after AP sensibilization. RESULTS: In order to obtain AP, S. aureus suspension at the concentration of 2.5 x 10(7) CFU/ml in 25 microM MC540 solution and 0.25 M NaCl solution were exposed to irradiation for 5 minutes. During DTH reaction induction its intensity dependence on AP dose was revealed. A persistent increase of a lysosomatic enzyme cathepsin D in macrophages of peritoneal exudate after a single administration of AP was noted. During MC540 irradiation an accumulation of photoproducts that have a pronounced immune suppression effect in DTH reaction had a dose-dependent character. CONCLUSION: Use of saline allows to increase bactericidal potential of a photosensibilizator (PS). However during therapy of localized forms of infection a possible immune modulating effect of PS on macro organism should be considered. By varying PS dose and irradiation time not only maximum bactericidal effect can be achieved but also regulation of inflammatory reactions in the area of PS effect can be ensured.
Asunto(s)
Antibacterianos/farmacología , Antígenos Bacterianos/inmunología , Hipersensibilidad Tardía/prevención & control , Factores Inmunológicos/farmacología , Fármacos Fotosensibilizantes/farmacología , Pirimidinonas/farmacología , Staphylococcus aureus/inmunología , Administración Oral , Animales , Antígenos Bacterianos/administración & dosificación , Catepsina D/metabolismo , Recuento de Colonia Microbiana , Relación Dosis-Respuesta Inmunológica , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/microbiología , Hipersensibilidad Tardía/patología , Inmunización , Inyecciones Subcutáneas , Luz , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/efectos de la radiaciónRESUMEN
Cutaneous leishmaniasis (CL) is endemic in the tropical and subtropical countries. Antileishmanial drugs that are traditionally used for treatment of CL are mainly toxic, ineffective for some parasite isolates, and mostly expensive. Previous studies showed that some metal and metal oxide nanoparticles have antimicrobial activity. Moreover, the use of nanoparticles together with ultra violet (UV) and infra red (IR) light increases toxic effects of nanoparticles by generation of reactive oxygen species (ROSs) and heat, respectively. There is little information on antileishmanial activity of nanoparticles, alone or together with UV/IR. Thus, the purpose of this research was to study antileishmanial effects of some nanoparticles including silver nanoparticles (Ag NPs), gold nanoparticles (Au NPs), titanium dioxide nanoparticles (TiO2 NPs), zinc oxide nanoparticles (ZnO NPs), and magnesium oxide nanoparticles (MgO NPs) on Leishmania major parasites under UV, IR, and dark conditions. After 24h exposure to nanoparticles, different biological parameters such as cell viability, proliferation, infectivity, and infection index were investigated under UV/IR/dark conditions. In this study, the highest antileishmanial activity was seen for Ag NPs, followed by Au NPs, TiO2 NPs, ZnO NPs, and MgO NPs. Both UV and IR light increased antileishmanial properties of all nanoparticles. In spite of antileishmanial activity of nanoparticles under UV, IR, and dark conditions, these nanoparticles had high cytotoxicity on macrophages, which must be considered in future studies. The authors declare that the use of nanoparticles for treatment of CL may have both positive and negative consequences.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania major/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Nanopartículas , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Oro/farmacología , Rayos Infrarrojos , Leishmania major/patogenicidad , Leishmania major/fisiología , Leishmaniasis Cutánea/tratamiento farmacológico , Macrófagos Peritoneales/microbiología , Macrófagos Peritoneales/efectos de la radiación , Óxido de Magnesio/farmacología , Ratones , Ratones Endogámicos BALB C , Plata/farmacología , Titanio/farmacología , Rayos Ultravioleta , Óxido de Zinc/farmacologíaRESUMEN
In this paper we showed the pH-dependent change in the sensitivity of the membranes of murine peritoneal macrophages to UV-radiation. This relationship is discussed in terms of lipid bilayer membrane stability modification to the action of ROS and lipid peroxidation process (LPP) at different pH. Iron-ascorbate reinforced LPP also led to pH-dependent membranes damage. The increase of the cells incubation medium temperature up to 37 degrees C, which also stimulated LPP, did not change the picture of the pH-dependent damage. Decrease of the incubation medium pH did not reduce H2O2-induced cell damage. Increase of the pH intensified the cells damage.
Asunto(s)
Membrana Celular/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Tolerancia a Radiación/efectos de los fármacos , Animales , Ácido Ascórbico/farmacología , Membrana Celular/efectos de la radiación , Peróxido de Hidrógeno/toxicidad , Concentración de Iones de Hidrógeno , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/efectos de la radiación , Macrófagos Peritoneales/efectos de la radiación , Ratones , Rayos UltravioletaRESUMEN
High mobility group box 1 (HMGB1) protein is a unique non histone nuclear protein that acts extracellularly as a mediator of delayed inflammation. Sub lethal dose of UVB triggers the release of cytokines from macrophages (MΦs). Adding to the panoply of UVB induced cytokines; it is reported that UVB induces HMGB1 release from mouse peritoneal MΦs in time and partially dose dependent manner, independent of TNF-α. UVB also enhanced the transcription of HMGB1 gene and expression of cellular protein, which influences its subsequent release. HMGB1 is secreted by an unconventional secretion pathway of unknown mechanism. Caspase-1 has been shown to function as a general regulator of stress induced unconventional secretion for a number of cytokines. In the present study, we have observed that pharmacological inhibitors specific for caspase-1 (ZVAD and YVAD) abrogated UVB induced HMGB1 release from MΦs. This effect was most likely mediated via physical interaction between HMGB1 and active caspase-1 (p10 and p20) as demonstrated by immunoprecipitation. In addition, it was found that HMGB1 and active caspase-1 p20 release depends on UVB mediated enhancement of intracellular Ca(2+). Thus our data suggests that optimal dose of UVB (50 mJ/cm(2)) induces HMGB1 upregulation and active release from mouse peritoneal MΦs which is mediated by caspase-1 in a Ca(2+) dependent manner.
Asunto(s)
Caspasa 1/metabolismo , Proteína HMGA1a/metabolismo , Macrófagos Peritoneales/efectos de la radiación , Clorometilcetonas de Aminoácidos/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/efectos de la radiación , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Proteína HMGA1a/genética , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/farmacología , Vías Secretoras/efectos de los fármacos , Vías Secretoras/efectos de la radiación , Activación Transcripcional/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
BACKGROUND: Radiation-induced fibrosis (RIF) is a dose-limiting complication of cancer radiotherapy and causes serious problems, i.e. restricted tissue flexibility, pain, ulceration or necrosis. Recently, we have successfully treated RIF in a mouse model by intraperitoneal administration of chitosan/siRNA nanoparticles directed towards silencing TNF alpha in local macrophage populations, but the mechanism for the therapeutic effect at the lesion site remains unclear. METHODS: Using the same murine RIF model we utilized an optical imaging technique and fluorescence microscopy to investigate the uptake of chitosan/fluorescently labeled siRNA nanoparticles by peritoneal macrophages and their subsequent migration to the inflamed tissue in the RIF model. RESULTS: We observed strong accumulation of the fluorescent signal in the lesion site of the irradiated leg up to 24 hours using the optical imaging system. We further confirm by immunohistochemical staining that Cy3 labeled siRNA resides in macrophages of the irradiated leg. CONCLUSION: We provide a proof-of-concept for host macrophage trafficking towards the inflamed region in a murine RIF model, which thereby suggests that the chitosan/siRNA nanoparticle may constitute a general treatment for inflammatory diseases using the natural homing potential of macrophages to inflammatory sites.
Asunto(s)
Quitosano/química , Inflamación/metabolismo , Macrófagos Peritoneales/metabolismo , Nanopartículas/química , ARN Interferente Pequeño/genética , Neumonitis por Radiación/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Técnicas para Inmunoenzimas , Inflamación/etiología , Inflamación/patología , Inyecciones Intraperitoneales , Macrófagos Peritoneales/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Microscopía Fluorescente , Neumonitis por Radiación/etiología , Neumonitis por Radiación/patología , Enfermedades de la Piel/etiología , Enfermedades de la Piel/metabolismo , Enfermedades de la Piel/patología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Since the beginning of the 20th century, low dose radiotherapy (LD-RT) has been practiced and established as therapy of inflammatory diseases. Several clinical studies already have proven the anti-inflammatory effect of low doses of ionizing irradiation (LDR). However, further research is inevitable to reveal the underlying immune-biological mechanisms. Focus has been set on the modulation of activated macrophages by LDR, since they participate in both, initiation and resolution of inflammation. Here we examined with an ex vivo peritoneal mouse macrophage model how LDR modulates the secretion of the inflammatory cytokines IL-1ß and TNF-α by activated macrophages and whether the basal radiosensitivity of the immune cells has influence on it. Peritoneal macrophages of Balb/c mice responded to exposure of 0.5 or 0.7 Gy of ionizing irradiation (X-ray) with significant decreased release of IL-1ß and slightly, but not significantly, reduced release of TNF-α. Macrophages of the less radiosensitive C57BL/6 mice did not show this anti-inflammatory reaction. This was observed in both wild type and human TNF-α transgenic animals with C57BL/6 background. We conclude that only the inflammatory phenotype of more radiosensitive macrophages is reduced by LDR and that ex vivo and in vivo models with primary cells should be applied to examine how the immune system is modulated by LDR.
Asunto(s)
Regulación hacia Abajo/inmunología , Mediadores de Inflamación/antagonistas & inhibidores , Interleucina-1beta/antagonistas & inhibidores , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo/efectos de la radiación , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Activación de Macrófagos/efectos de la radiación , Macrófagos Peritoneales/efectos de la radiación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Rayos XRESUMEN
Our study has shown that the damaging effect of hydroxylated fullerene C60(OH)25 on mouse peritoneal macrophage plasma membranes increased when we enlarged the concentration of fullerene in the incubation media (from 0.005 to 0.5 mg/ml), the incubation temperature (from 22 degrees C to 37 degrees C) and the time of incubation (from 30 to 90 min). In conditions of the H2O2-induced membrane damage, fullerene was observed to intensify the H2O2-induced damaging effect at a concentration of 0.05 mg/ml and reduce it at a concentration of 0.5 mg/ml. In conditions of the UV-induced membrane damage, it was discovered that the damaging effect of UV increased when C60(OH)25 nanoparticles were added to the incubation media before irradiation and decreased when they were added after irradiation. Eventual participation of ROS in damaging effects of C60(OH)25 was discussed.
Asunto(s)
Membrana Celular/efectos de los fármacos , Fulerenos/toxicidad , Macrófagos Peritoneales/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Membrana Celular/metabolismo , Membrana Celular/patología , Membrana Celular/efectos de la radiación , Células Cultivadas , Calor , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/efectos de la radiación , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/efectos de la radiación , Masculino , Ratones , Oxidantes/toxicidad , Estrés Oxidativo/efectos de la radiación , Factores de Tiempo , Rayos Ultravioleta/efectos adversosRESUMEN
Leishmania double transfectants (DTs) expressing the 2nd and 3rd enzymes in the heme biosynthetic pathway were previously reported to show neogenesis of uroporphyrin I (URO) when induced with delta-aminolevulinate (ALA), the product of the 1st enzyme in the pathway. The ensuing accumulation of URO in DT promastigotes rendered them light excitable to produce reactive oxygen species (ROS), resulting in their cytolysis. Evidence is presented showing that the DTs retained wild-type infectivity to their host cells and that the intraphagolysosomal/parasitophorous vacuolar (PV) DTs remained ALA inducible for uroporphyrinogenesis/photolysis. Exposure of DT-infected cells to ALA was noted by fluorescence microscopy to result in host-parasite differential porphyrinogenesis: porphyrin fluorescence emerged first in the host cells and then in the intra-PV amastigotes. DT-infected and control cells differed qualitatively and quantitatively in their porphyrin species, consistent with the expected multi- and monoporphyrinogenic specificities of the host cells and the DTs, respectively. After ALA removal, the neogenic porphyrins were rapidly lost from the host cells but persisted as URO in the intra-PV DTs. These DTs were thus extremely light sensitive and were lysed selectively by illumination under nonstringent conditions in the relatively ROS-resistant phagolysosomes. Photolysis of the intra-PV DTs returned the distribution of major histocompatibility complex (MHC) class II molecules and the global gene expression profiles of host cells to their preinfection patterns and, when transfected with ovalbumin, released this antigen for copresentation with MHC class I molecules. These Leishmania mutants thus have considerable potential as a novel model of a universal vaccine carrier for photodynamic immunotherapy/immunoprophylaxis.
Asunto(s)
Ácido Aminolevulínico/farmacología , Leishmania/genética , Fagocitos/parasitología , Fagosomas/parasitología , Fármacos Fotosensibilizantes/farmacología , Porfirinas/biosíntesis , Vacunación/métodos , Animales , Presentación de Antígeno , Antígenos de Protozoos/inmunología , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Células Dendríticas/efectos de la radiación , Perfilación de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/metabolismo , Leishmania/inmunología , Leishmania/efectos de la radiación , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/parasitología , Macrófagos Peritoneales/efectos de la radiación , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de Oligonucleótidos , Organismos Modificados Genéticamente/inmunología , FotólisisRESUMEN
Effect of Ca2+ ions on UV-induced mice peritoneal macrophage plasma membrane damage has been studied. Drop of the extracellular Ca2+ concentration has been found to result in a reduced expression of this damage. On the contrary, a raised intra- and extra-cellular Ca2+ level is associated with a higher number of cells with damaged plasma membranes. These findings make it possible to suggest that this change in the plasma membrane photosensitivity might be a result of alterations in the membrane lipid matrix electrical stability owing to UV-induced lipid photo-peroxidation. This study has also shown that free radical peroxidation of membrane lipids plays a significant part in UV-induced cell damage.
Asunto(s)
Calcio/metabolismo , Membrana Celular/efectos de la radiación , Macrófagos Peritoneales/efectos de la radiación , Rayos Ultravioleta , Animales , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Iones/metabolismo , RatonesRESUMEN
The plasma membrane dose-dependent damage of UV(B)-irradiated mouse peritoneal macrophages was investigated using laser interference microscopy (LIM). LIM is a method which allows one to estimate morphological and functional parameters of a cell without dyeing or introduction of other substances which can affect the cell condition. This makes it possible to reduce and accelerate the procedure of counting the damaged cells as compared with the methods using different dyes. The value of optical path difference (OPD)--a variable proportional to the object thickness and the difference in the refractive indices of the object and the surrounding medium was used for estimation of the cell damage. Also compared was usability of LIM and microfluorimetry assay in investigations of the UV(B)-irradiated macrophage plasma membrane.
Asunto(s)
Membrana Celular/efectos de la radiación , Macrófagos Peritoneales/efectos de la radiación , Microscopía Confocal/métodos , Microscopía de Interferencia/métodos , Rayos Ultravioleta , Animales , Membrana Celular/ultraestructura , Relación Dosis-Respuesta en la Radiación , Macrófagos Peritoneales/citología , RatonesRESUMEN
The influence of weak magnetic fields of different types on the rate of the formation of reactive oxygen species in mouse peritoneal neutrophils has been studied. It was found that the exposure of neutrophils activated by phorbol 12-myristate 13-acetate to the magnetic field tuned to the parametric resonance for Ca2+ ions leads to a decrease in the rate of the reactive oxygen species (ROS) generation by 23%. Conversely, the generation of ROS in neutrophils exposed to the same field but stimulated by the bacterial peptide FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) increased by about 21%. Pulsed magnetic fields also changed the rate of ROS generation in phorbol-stimulated neutrophils by about 20%, but the sign of the effects observed in this case was opposite to those induced by the magnetic field tuned to the parametric resonance for Ca2+ ions.
Asunto(s)
Campos Electromagnéticos , Macrófagos Peritoneales/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Animales , Técnicas In Vitro , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Magnetoterapia , Ratones , N-Formilmetionina Leucil-Fenilalanina/farmacologíaRESUMEN
It was demonstrated on macrophages treated with UV irradiation dose 9 J/sm2 (lamda max =306 nm) that intra- or extracellular pH reducing lead to decrease the number of cells with damaged membranes in macrophage population. An intra- or extracellular pH elevation leads to increase of UV-irradiation membrane-acting effect. It was also shown that pH-dependence of UV-irradiation damage effect has been lost after preliminary osmotic swelling of cells. The cells survived after UV-irradiation in doses 8 and 10 J/sm2 (lamda max =297 nm) have an intracellular pH lower than non-irradiated cells.
Asunto(s)
Membrana Celular/efectos de la radiación , Macrófagos Peritoneales/efectos de la radiación , Tolerancia a Radiación , Rayos Ultravioleta , Animales , Membrana Celular/fisiología , Concentración de Iones de Hidrógeno , Macrófagos Peritoneales/fisiología , RatonesRESUMEN
The effect of 30-min vector potential variation on the phagocytosis of macrophages from laboratory mice was investigated. It was found that the effect changes the characteristics of mice immunity: the quantity and percentage of neutrophils and lymphocytes in the blood, the activity of capture, degranulation, the splitting of bacterial agents, and the dynamics of formation of reactive oxygen species.