RESUMO
Injury to the meniscus is common and frequently leads to the development of post-traumatic osteoarthritis (PTOA). Many times meniscus injuries occur coincident with anterior cruciate ligament (ACL) injuries and lead to a bloody joint effusion. Hemarthrosis, or bleeding into the joint, has been implicated in degeneration of joint tissues. The goal of this review paper is to understand the pathophysiology of blood-induced joint damage, the possible effects of blood on meniscus tissue, and the implications for current meniscus repair techniques that involve the introduction of blood-derived products into the joint. In this review, we illustrate the similarities in the pathophysiology of joint damage due to hemophilic arthropathy (HA) and osteoarthritis (OA). Although numerous studies have revealed the harmful effects of blood on cartilage and synovium, there is currently a gap in knowledge regarding the effects of hemarthrosis on meniscus tissue homeostasis, healing, and the development of PTOA following meniscus injury. Given that many meniscus repair techniques utilize blood-derived and marrow-derived products, it is essential to understand the effects of these factors on meniscus tissue and the whole joint organ to develop improved strategies to promote meniscus tissue repair and prevent PTOA development.
Assuntos
Sangue/metabolismo , Hemartrose/fisiopatologia , Osteoartrite do Joelho/fisiopatologia , Membrana Sinovial/fisiopatologia , Lesões do Menisco Tibial/terapia , Lesões do Ligamento Cruzado Anterior , Reconstrução do Ligamento Cruzado Anterior , Transplante de Medula Óssea/métodos , Hemartrose/etiologia , Hemartrose/metabolismo , Humanos , Osteoartrite do Joelho/etiologia , Osteoartrite do Joelho/metabolismo , Plasma Rico em Plaquetas , Procedimentos de Cirurgia Plástica/métodos , Transplante de Células-Tronco/métodos , Membrana Sinovial/metabolismo , Lesões do Menisco Tibial/complicações , Lesões do Menisco Tibial/fisiopatologia , CicatrizaçãoAssuntos
Rearranjo Gênico/fisiologia , Cadeias Pesadas de Imunoglobulinas/genética , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Idoso , Aberrações Cromossômicas , Estudos de Coortes , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Chronic lymphocytic leukemia (CLL) can be immunosuppressive in humans and mice, and CLL cells share multiple phenotypic markers with regulatory B cells that are competent to produce interleukin (IL)-10 (B10 cells). To identify functional links between CLL cells and regulatory B10 cells, the phenotypes and abilities of leukemia cells from 93 patients with overt CLL to express IL-10 were assessed. CD5(+) CLL cells purified from 90% of the patients were IL-10-competent and secreted IL-10 following appropriate ex vivo stimulation. Serum IL-10 levels were also significantly elevated in CLL patients. IL-10-competent cell frequencies were higher among CLLs with IgV(H) mutations, and correlated positively with TCL1 expression. In the TCL1-transgenic (TCL1-Tg) mouse model of CLL, IL-10-competent B cells with the cell surface phenotype of B10 cells expanded significantly with age, preceding the development of overt, CLL-like leukemia. Malignant CLL cells in TCL1-Tg mice also shared immunoregulatory functions with mouse and human B10 cells. Serum IL-10 levels varied in TCL1-Tg mice, but in vivo low-dose lipopolysaccharide treatment induced IL-10 expression in CLL cells and high levels of serum IL-10. Thus, malignant IL-10-competent CLL cells exhibit regulatory functions comparable to normal B10 cells that may contribute to the immunosuppression observed in patients and TCL1-Tg mice.
Assuntos
Linfócitos B Reguladores/imunologia , Linfócitos B/imunologia , Interleucina-10/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B Reguladores/metabolismo , Linfócitos B Reguladores/patologia , Células Cultivadas , Imunofluorescência , Humanos , Terapia de Imunossupressão , Interleucina-10/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos TransgênicosRESUMO
Rhizobium radiobacter bacteremia is an infrequent cause of human infection. We report a rare manifestation of R. radiobacter infection in which bacteremia occurred in a newborn infant without other risk factors.
Assuntos
Agrobacterium tumefaciens/isolamento & purificação , Bacteriemia/etiologia , Humanos , Recém-Nascido , MasculinoRESUMO
INTRODUCTION: Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-ß1 (TGF-ß1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-ß1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair. METHODS: A micro-wound assay was used to assess meniscal cell migration and proliferation in response to the following treatments for 0, 24, or 48 hours: 0 to 10 ng/mL IL-1, TNF-α, or TGF-ß1, in the presence or absence of 10% serum. Proliferated and total cells were fluorescently labeled and imaged using confocal laser scanning microscopy and the number of proliferated, migrated, and total cells was determined in the micro-wound and edges of each image. Meniscal cell proliferation was also assessed throughout meniscal repair model explants treated with 0 or 10 ng/mL IL-1, TNF-α, or TGF-ß1 for 14 days. At the end of the culture period, biomechanical testing and histological analyses were also performed. Statistical differences were assessed using an ANOVA and Newman-Keuls post hoc test. RESULTS: IL-1 and TNF-α decreased cell proliferation in both cell and tissue models of meniscal repair. In the presence of serum, TGF-ß1 increased outer zone cell proliferation in the micro-wound and in the cross section of meniscal repair model explants. Both IL-1 and TNF-α decreased the integrative shear strength of repair and extracellular matrix deposition in the meniscal repair model system, while TGF-ß1 had no effect on either measure. CONCLUSIONS: Meniscal cell proliferation in vivo may be diminished following joint injury due to the up-regulation of inflammatory cytokines, thereby limiting native cellular repair of meniscal lesions. Therefore, therapies that can promote meniscal cell proliferation have promise to enhance meniscal repair and improve tissue engineering strategies.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Interleucina-1/farmacologia , Meniscos Tibiais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Fenômenos Biomecânicos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Meniscos Tibiais/patologia , Meniscos Tibiais/fisiopatologia , Microscopia Confocal , Suínos , Técnicas de Cultura de Tecidos , Cicatrização/efeitos dos fármacosRESUMO
Monoclonal B-cell lymphocytosis (MBL) is a hematologic condition wherein small B-cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and 'CLL-like' MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. In all, 91 unique MBL clones were detected: 73 CLL-like MBL (CD5(+)CD20(dim)sIg(dim)), 11 atypical MBL (CD5(+)CD20(+)sIg(+)) and 7 CD5(neg) MBL (CD5(neg)CD20(+)sIg(neg)). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70 and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on seven CLL-like MBL, and showed activation of B-cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.
Assuntos
Linfócitos B/patologia , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Linfocitose/patologia , Biomarcadores Tumorais/metabolismo , Citometria de Fluxo , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
B-cell chronic lymphocytic leukemia (CLL) is characterized by slow accumulation of malignant cells, which are supported in the microenvironment by cell-cell interactions and soluble cytokines such as tumor necrosis factor (TNF). We evaluated the effect of the small molecule TNF inhibitor LMP-420 on primary CLL cells. The mean concentration of LMP-420 required to induce 50% cytotoxicity (ED50) at 72 h was 245 n. LMP-420-induced time- and dose-dependent apoptosis, as shown by annexin V staining, caspase activation and DNA fragmentation. These changes were associated with decreased expression of anti-apoptotic proteins Mcl-1, Bcl-xL and Bcl-2. CLL cells from patients with poor prognostic indicators showed LMP-420 sensitivity equal to that for cells from patients with favorable characteristics. In addition, LMP-420 potentiated the cytotoxic effect of fludarabine and inhibited in vitro proliferation of stimulated CLL cells. Gene expression profiling indicated that the mechanism of action of LMP-420 may involve suppression of nuclear factor-kappaB and immune response pathways in CLL cells. LMP-420 had minimal effects on normal peripheral blood mononuclear cell, B- and T-cell function, and hematopoietic colony formation. Our data suggest that LMP-420 may be a useful treatment for CLL with negligible hematologic toxicities.
Assuntos
Antineoplásicos/farmacologia , Compostos de Boro/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Purinas/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Compostos de Boro/toxicidade , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Prognóstico , Purinas/toxicidade , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
OBJECTIVE: Meniscal tears are a common knee injury and increased levels of interleukin-1 (IL-1) have been measured in injured and degenerated joints. Studies have shown that IL-1 decreases the shear strength, cell accumulation, and tissue formation in meniscal repair interfaces. While mechanical stress and IL-1 modulate meniscal biosynthesis and degradation, the effects of dynamic loading on meniscal repair are unknown. The purpose of this study was to determine the effects of mechanical compression on meniscal repair under normal and inflammatory conditions. EXPERIMENTAL DESIGN: Explants were harvested from porcine medial menisci. To simulate a full-thickness defect, a central core was removed and reinserted. Explants were loaded for 4h/day at 1 Hz and 0%-26% strain for 14 days in the presence of 0 or 100 pg/mL of IL-1. Media were assessed for matrix metalloproteinase (MMP) activity, aggrecanase activity, sulfated glycosaminoglycan (S-GAG) release, and nitric oxide (NO) production. After 14 days, biomechanical testing and histological analyses were performed. RESULTS: IL-1 increased MMP activity, S-GAG release, and NO production, while decreasing the shear strength and tissue repair in the interface. Dynamic loading antagonized IL-1-mediated inhibition of repair at all strain amplitudes. Neither IL-1 treatment nor strain altered aggrecanase activity. Additionally, strain alone did not alter meniscal healing, except at the highest strain magnitude (26%), a level that enhanced the strength of repair. CONCLUSIONS: Dynamic loading blocked the catabolic effects of IL-1 on meniscal repair, suggesting that joint loading through physical therapy may be beneficial in promoting healing of meniscal lesions under inflammatory conditions.
Assuntos
Interleucina-1/farmacologia , Meniscos Tibiais/fisiologia , Estresse Mecânico , Cicatrização/fisiologia , Animais , Endopeptidases/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Traumatismos do Joelho/metabolismo , Metaloproteinases da Matriz/metabolismo , Óxido Nítrico/metabolismo , Suínos , Lesões do Menisco Tibial , Cicatrização/efeitos dos fármacosRESUMO
Monoclonal B-cell lymphocytosis (MBL) is a preclinical hematologic syndrome characterized by small accumulations of CD5(+) B lymphocytes. Most MBL share phenotypic characteristics with chronic lymphocytic leukemia (CLL). Although some MBL progress to CLL, most MBL have apparently limited potential for progression to CLL, particularly those MBL with normal absolute B-cell counts ('low-count' MBL). Most CLL are monoclonal and it is not known whether MBL are monoclonal or oligoclonal; this is important because it is unclear whether MBL represent indolent CLL or represent a distinct premalignant precursor before the development of CLL. We used flow cytometry analysis and sorting to determine immunophenotypic characteristics, clonality and molecular features of MBL from familial CLL kindreds. Single-cell analysis indicated four of six low-count MBL consisted of two or more unrelated clones; the other two MBL were monoclonal. 87% of low-count MBL clones had mutated immunoglobulin genes, and no immunoglobulin heavy-chain rearrangements of V(H) family 1 were observed. Some MBL were diversified, clonally related populations with evidence of antigen drive. We conclude that although low-count MBL share many phenotypic characteristics with CLL, many MBL are oligoclonal. This supports a model for step-wise development of MBL into CLL.
Assuntos
Linfócitos B/patologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfocitose/imunologia , Idoso , Idoso de 80 Anos ou mais , Aberrações Cromossômicas , Feminino , Genes de Imunoglobulinas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/genética , Linfocitose/genética , Masculino , Pessoa de Meia-IdadeRESUMO
Survival of chronic lymphocytic leukemia (CLL) cells requires sustained activation of the antiapoptotic PI-3-K/Akt pathway, and many therapies for CLL cause leukemia cell death by triggering apoptosis. Blood lipoprotein particles are either pro- or antiapoptotic. High-density lipoprotein particles are antiapoptotic through sphingosine-1-phosphate receptor 3-mediated activation of the PI-3-K/Akt pathway. Apolipoprotein E4 (apoE4)-very low density lipoproteins (VLDL) increase apoptosis, but the apoE2-VLDL and apoE3-VLDL isoforms do not. As increased B-cell apoptosis favors longer survival of CLL patients, we hypothesized that APOE4 genotype would beneficially influence the clinical course of CLL. We report here that women (but not men) with an APOE4 genotype had markedly longer survival than non-APOE4 patients. VLDL is metabolized to low-density lipoprotein through lipoprotein lipase. Higher levels of lipoprotein lipase mRNA in these CLL patients correlated with shorter survival. The beneficial effect of APOE4 in CLL survival is likely mediated through APOE4 allele-specific regulation of leukemia cell apoptosis. The APOE allele and genotype distribution in these CLL patients is the same as in unaffected control populations, suggesting that although APOE genotype influences CLL outcome and response to therapy, it does not alter susceptibility to developing this disease.
Assuntos
Apolipoproteína E4/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Apolipoproteína E4/metabolismo , Apoptose/fisiologia , VLDL-Colesterol/sangue , Estudos de Coortes , Feminino , Predisposição Genética para Doença/epidemiologia , Genótipo , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Masculino , Fatores de Risco , Distribuição por Sexo , Análise de SobrevidaRESUMO
OBJECTIVE: Osteoarthritis (OA) is associated with increased levels of reactive nitrogen and oxygen species and pro-inflammatory cytokines, such as interleukin-1 (IL-1). Nitric oxide (NO) can mediate a number of the catabolic effects of IL-1 in articular cartilage. The aims of this study were to determine if OA cartilage shows evidence of DNA damage, and if IL-1 could induce DNA damage in non-OA cartilage by increasing NO or superoxide. METHODS: Articular chondrocytes were isolated from porcine femoral condyles and embedded in 1.2% alginate. The effects of 24h incubation with IL-1, the nitric oxide synthase 2 (NOS2)-selective inhibitor, the free radical scavenger superoxide dismutase (SOD), the NO donor NOC18, or the combined NO and peroxynitrite donor SIN-1 on DNA damage were tested, using the "comet" assay. NO production was measured using the Griess assay. The type of oxidative damage present was assessed using a modified comet assay. RESULTS: OA cartilage had significantly more DNA damage than non-OA cartilage (P<0.001). IL-1 caused an increase in DNA damage (P<0.01), which was associated with increased NO production (P<0.01). Both oxidative DNA strand breaks and base modifications of purines and pyrimidines were observed. IL-1-induced DNA damage was inhibited by an NOS2 inhibitor or by SOD (P<0.01). Furthermore, NOC18 or SIN-1 caused DNA damage (P<0.001). CONCLUSION: Our work shows chondrocytes in osteoarthritic cartilage exhibit DNA damage, and that IL-1 induces DNA damage and reactive oxygen and nitrogen species in non-OA chondrocytes in alginate.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Dano ao DNA , Interleucina-1alfa/farmacologia , Osteoartrite/genética , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Feminino , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , Osteoartrite/metabolismo , Sus scrofaRESUMO
OBJECTIVE: Injury or removal of the knee meniscus leads to progressive joint degeneration, and current surgical therapies for meniscal tears seek to maximally preserve meniscal structure and function. However, the factors that influence intrinsic repair of the meniscus are not well understood. The goal of this study was to investigate the capacity of meniscus tissue to repair a simulated defect in vitro and to examine the effect of pro-inflammatory cytokines on this process. METHODS: Cylindrical explants were harvested from the outer one-third of medial porcine menisci. To simulate a full-thickness defect, a central core was removed and reinserted immediately into the defect. Explants were cultured for 2, 4, or 6 weeks in serum-containing media in the presence or absence of interleukin-1 (IL-1) or tumor necrosis factor alpha (TNF-alpha), and meniscal repair was investigated using mechanical testing and fluorescence confocal microscopy. RESULTS: Meniscal lesions in untreated samples showed a significant capacity for intrinsic repair in vitro, with increasing cell accumulation and repair strength over time in culture. In the presence of IL-1 or TNF-alpha, no repair was observed despite the presence of abundant viable cells. CONCLUSIONS: This study demonstrates that the meniscus exhibits an intrinsic repair response in vitro. However, the presence of pro-inflammatory cytokines completely inhibited repair. These findings suggest that increased levels of pro-inflammatory cytokines post-injury or under arthritic conditions may inhibit meniscal repair. Therefore, inhibition of these cytokines may provide a means of accelerating repair of damaged or injured menisci in vivo.
Assuntos
Interleucina-1/farmacologia , Meniscos Tibiais/citologia , Meniscos Tibiais/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Sobrevivência Celular , Fluorescência , Técnicas In Vitro , Microscopia Confocal , Estresse Mecânico , Suínos , Lesões do Menisco Tibial , Cicatrização/efeitos dos fármacosRESUMO
Molecular oxygen is required for the production of nitric oxide (NO), a pro-inflammatory mediator that is associated with osteoarthritis and rheumatoid arthritis. To date there has been little consideration of the role of oxygen tension in the regulation of nitric oxide production associated with arthritis. Oxygen tension may be particularly relevant to articular cartilage since it is avascular and therefore exists at a reduced oxygen tension. The superficial zone exists at approximately 6% O2, while the deep zone exists at less than 1% O2. Furthermore, oxygen tension can alter matrix synthesis, and the material properties of articular cartilage in vitro. The increase in nitric oxide associated with arthritis can be caused by pro-inflammatory cytokines and mechanical stress. Oxygen tension significantly alters endogenous NO production in articular cartilage, as well as the stimulation of NO in response to both mechanical loading and pro-inflammatory cytokines. Mechanical loading and pro-inflammatory cytokines also increase the production of prostaglandin E2 (PGE2). There is a complex interaction between NO and PGE2, and oxygen tension can alter this interaction. These findings suggest that the relatively low levels of oxygen within the joint may have significant influences on the metabolic activity, and inflammatory response of cartilage as compared to ambient levels. A better understanding of the role of oxygen in the production of inflammatory mediators in response to mechanical loading, or pro-inflammatory cytokines, may aid in the development of strategies for therapeutic intervention in arthritis.
Assuntos
Artrite Reumatoide/fisiopatologia , Cartilagem Articular/fisiopatologia , Óxido Nítrico/fisiologia , Osteoartrite/fisiopatologia , Oxigênio/fisiologia , Animais , Cartilagem Articular/efeitos dos fármacos , Feminino , Inflamação/etiologia , Mediadores da Inflamação/fisiologia , Oxigênio/farmacologia , Estresse Mecânico , SuínosRESUMO
Alzheimer's disease is characterized by two primary pathological features: amyloid plaques and neurofibrillary tangles. The interconnection between amyloid and tau aggregates is of intense interest, but mouse models have yet to reveal a direct interrelationship. We now show that NO may be a key factor that connects amyloid and tau pathologies. Genetic removal of NO synthase 2 in mice expressing mutated amyloid precursor protein results in pathological hyperphosphorylation of mouse tau, its redistribution to the somatodendritic compartment in cortical and hippocampal neurons, and aggregate formation. Lack of NO synthase 2 in the amyloid precursor protein Swedish mutant mouse increased insoluble beta-amyloid peptide levels, neuronal degeneration, caspase-3 activation, and tau cleavage, suggesting that NO acts at a junction point between beta-amyloid peptides, caspase activation, and tau aggregation.
Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Deleção de Genes , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Caspase 3 , Caspases/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Fosforilação , Proteínas tau/metabolismoRESUMO
The purpose of this study was to examine the lung pathogenesis of murine gammaherpesvirus (MHV-68) infection in mice that lack CC chemokine receptor CCR2, an important receptor for macrophage recruitment to sites of inflammation. BALB/c and CCR2(-/-) mice were inoculated intranasally (i.n.) with MHV-68 and samples were collected during acute infection (6 dpi) and following viral clearance (12 dpi). Immunohistochemistry was used to determine which cells types responded to MHV-68 infection in the lungs. Lung pathology in infected BALB/c mice was characterized by a mixed inflammatory cell infiltrate, necrosis, and increased alveolar macrophages by 12 dpi. Immunohistochemistry showed intense positive staining for macrophages. CCR2(-/-) mice showed greater inflammation in the lungs at 12 dpi than did BALB/c mice, with more necrosis and diffuse neutrophil infiltrates in the alveoli. Immunohistochemistry demonstrated much less macrophage infiltration in the CCR2(-/-) mice than in the BALB/c mice. These studies show that CCR2 is involved in macrophage recruitment in response to MHV-68 infection and illustrates how impairments in macrophage function affect the normal inflammatory response to this viral infection.
Assuntos
Gammaherpesvirinae , Infecções por Herpesviridae/patologia , Pulmão/patologia , Macrófagos Alveolares/imunologia , Receptores de Quimiocinas/imunologia , Animais , Infecções por Herpesviridae/imunologia , Pulmão/virologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR2RESUMO
Chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived non-dividing CD5(+) B cells. Nitric oxide (NO) is an important regulator of apoptosis, and the viability of cultured B-CLL cells may be dependent on the autocrine production of nitric oxide by inducible nitric oxide synthase (NOS2). We performed this study to determine whether cytokine factors that prevent spontaneous in vitroapoptosis of B-CLL cells induce B-CLL cell NOS2 enzyme activity. B-CLL cells expressed NOS enzyme activity and NOS2 protein and mRNA. IL-4 and IFN-gamma increased B-CLL cell NOS2 enzyme activity and protein expression during in vitro culture. IFN-gamma, but not IL-4, increased NOS2 mRNA expression in cultured B-CLL cells suggesting that IL-4-mediated changes of NOS2 protein expression occurred at the post-transcriptional level. We were unable to detect increased concentrations of nitrite or nitrate (NO(x)) as surrogate markers of NO production in B-CLL cell cultures treated with IL-4 or IFN-gamma. IL-4 and IFN-gamma diminished NOS inhibitor-induced B-CLL cell death. In summary, we found that B-CLL cells expressed NOS2 and that IL-4 and IFN-gamma increased B-CLL NOS2 expression. Cytokine-mediated expression of NOS2 by B-CLL cells may promote their survival, and therapeutic strategies that target NOS2 or quench NO may be beneficial in patients with B-CLL.
Assuntos
Interleucina-4/farmacologia , Leucemia Linfocítica Crônica de Células B/enzimologia , Óxido Nítrico Sintase/genética , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/imunologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Interleukin-12 (IL-12) is an important regulatory cytokine in infection and immunity. Administration of IL-12 may reduce complications of severe malaria in rodents. Polymorphisms in IL12B, the gene encoding the IL-12 p40 subunit, influence the secretion of IL-12 and susceptibility to Type 1 diabetes. We therefore investigated whether IL12B polymorphisms may affect the outcome of severe malaria. Homozygosity for a polymorphism in the IL12B promoter was associated with increased mortality in Tanzanian children having cerebral malaria but not in Kenyan children with severe malaria. Furthermore, homozygotes for the IL12B promotor polymorphism had decreased production of nitric oxide, which is in part regulated by IL-12 activity. These studies suggest that IL12B polymorphisms, via regulation of IL-12 production, may influence the outcome of malaria infection in at least one African population.
Assuntos
Interleucina-12/genética , Malária Cerebral/mortalidade , Óxido Nítrico/metabolismo , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Animais , Humanos , Subunidade p40 da Interleucina-12 , Quênia/epidemiologia , Malária Cerebral/genética , Dados de Sequência Molecular , Plasmodium/imunologia , Polimorfismo Genético , Tanzânia/epidemiologiaRESUMO
OBJECTIVE: Biomechanical signals play important roles in regulating the homeostasis of articular cartilage, but under abnormal conditions may be a critical factor in the onset and progression of arthritis. Prostaglandin E(2) (PGE(2)) and nitric oxide (NO), derived from the enzymes cyclo-oxygenase 2 (COX2) and NO synthase 2 (NOS2), are inflammatory mediators that modulate numerous physiological and pathophysiological processes and are potentially important pharmacological targets in osteoarthritis. The goal of this study was to determine the effect of mechanical compression on PGE(2) production in the presence of selective NOS2 and COX2 inhibitors. METHODS: Articular cartilage explants harvested from 2-3-year-old pigs were subjected to intermittent compression at 0.5Hz over a range of stress magnitudes. PGE(2) and NO production into the media were determined in the presence and absence of the NOS2 inhibitor 1400W or the COX2 inhibitor NS398. COX2 protein levels were determined by immunoblot analysis. RESULTS: Mechanical compression significantly increased NO and PGE(2) synthesis in a manner that was dependent on the magnitude of stress. The selective COX2 inhibitor blocked compression-induced NO and PGE(2) production. Compression in the presence of 1400W further increased COX2 expression resulting in a 10-fold increase in PGE(2) production compared to uncompressed explants with 1400W and a 40-fold increase in PGE(2) compared to uncompressed explants without 1400W. CONCLUSION: Mechanical compression of articular cartilage increased COX2 and PGE(2) production through a NO-dependent pathway, and therefore pharmacological agents that target the NOS2 pathway in cartilage may have a significant influence on prostanoid production in the joint.
Assuntos
Dinoprostona/biossíntese , Isoenzimas/farmacologia , Óxido Nítrico Sintase/farmacologia , Prostaglandina-Endoperóxido Sintases/farmacologia , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2 , Feminino , Immunoblotting , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II , Estresse Mecânico , SuínosRESUMO
Fludarabine is active but not curative in the treatment of chronic lymphocytic leukemia (B-CLL). Nitric oxide (NO) supplied from exogenous, NO-donating pro-drugs can also induce apoptosis and death of acute leukemia cells. This study investigated combinations of fludarabine with NO-donating pro-drugs for their cytotoxicity against freshly isolated B-CLL lymphocytes following a 72 h exposure in vitro. The median IC(50)for fludarabine was 2.2 microM (n = 85). The nitric oxide donors DETA-NO, PAPA-NO, and MAHMA-NO were also cytotoxic, and their effects were inversely related to rates of NO release. Neither DETA-NO depleted of NO nor DETA itself was effective, indicating that NO was required for cytotoxicity. Drug interactions were evaluated by a modified combination index method. Synergy was observed in combinations of fludarabine or nelarabine (506U78) with DETA-NO in 52% and 88% of samples, respectively. Interestingly, the combination of fludarabine and DETA-NO was more cytotoxic in B-CLL cells less sensitive to fludarabine. DETA-NO did not enhance the activity of other DNA anti-metabolites, topoisomerase I and II inhibitors, or alkylating agents. Finally, the anti-leukemic activity of fludarabine alone or in combination with DETA-NO was found to correlate with inhibition of cellular RNA synthesis. These results indicate that NO donors could enhance fludarabine therapy for B-CLL.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Óxido Nítrico/farmacologia , Vidarabina/análogos & derivados , Vidarabina/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Triazenos/farmacologiaRESUMO
Depending on sequence, bacterial and synthetic DNAs can activate the host immune system and influence the host response to infection. The purpose of this study was to determine the abilities of various phosphorothioate oligonucleotides with cytosine-guanosine-containing motifs (CpG DNA) to activate macrophages to produce nitric oxide (NO) and prostaglandin E(2) (PGE(2)) and to induce expression of NO synthase 2 (NOS2) and cyclooxygenase 2 (COX2). As little as 0.3 microg of CpG DNA/ml increased NO and PGE(2) production in a dose- and time-dependent fashion in cells of the mouse macrophage cell line J774. NO and PGE(2) production was noted by 4 to 8 h after initiation of cultures with the CpG DNA, with the kinetics of NO production induced by CpG DNA being comparable to that induced by a combination of lipopolysaccharide and gamma interferon. CpG DNA-treated J774 cells showed enhanced expression of NOS2 and COX2 proteins as determined by immunoblotting, with the relative potencies of the CpG DNAs generally corresponding to those noted for the induction of NO and PGE(2) production as well as to those noted for the induction of interleukin-6 (IL-6), IL-12, and tumor necrosis factor. Extracts from CpG DNA-treated cells converted L-arginine to L-citrulline, but the NOS inhibitor N(G)-monomethyl-L-arginine (NMMA) inhibited this reaction. The COX2-specific inhibitor NS398 inhibited CpG DNA-induced PGE(2) production and inhibited NO production to various degrees. The NOS inhibitors NMMA, 1400W, and N-iminoethyl-L-lysine effectively blocked NO production and increased the production of PGE(2) in a dose-dependent fashion. Thus, analogues of microbial DNA (i.e., CpG DNA) activate mouse macrophage lineage cells for the expression of NOS2 and COX2, with the production of NO and that of PGE(2) occurring in an interdependent manner.