RESUMO
TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/ß-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/ß-catenin signaling correlates with inflammation status. TNF-deficient (Tnf-/-) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-typeâTnfr1/2-/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/ß-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/ß-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced ß-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/ß-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.
Assuntos
Células-Tronco Adultas/fisiologia , Anticorpos Monoclonais/uso terapêutico , Colite/imunologia , Células Epiteliais/fisiologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Sulfato de Dextrana , Humanos , Doenças Inflamatórias Intestinais/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Proteínas Wnt/metabolismo , Cicatrização , beta Catenina/metabolismoRESUMO
An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these low-density granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory proteins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-α synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role.
Assuntos
Adjuvantes Imunológicos/toxicidade , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Infiltração de Neutrófilos/imunologia , Autoantígenos/imunologia , Autoantígenos/toxicidade , Linhagem Celular , Testes Imunológicos de Citotoxicidade , Humanos , Contagem de Leucócitos , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The k-function of Stavroudis describes a solution of the eikonal equation in a region of constant refractive index. Given the k-function describing the optical field in one region of space, and given a prescribed refractive or reflective boundary, we construct the k-function for the refracted or reflected field. This procedure, which Stavroudis calls refracting the k-function, can be repeated any number of times, and therefore extends the usefulness of the k-function formalism to multielement optical systems. As examples, we present an analytic solution for the k-function, wavefronts, and caustics generated by a biconvex thick lens illuminated by a plane wave propagating parallel to the symmetry axis, and numerical results for off-axis plane-wave illumination of a two-mirror telescope.
RESUMO
Autoimmune diseases resulting from MHC class II-restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA323-339 and calcitriol targeted dLN PD-L1hi DCs of immunized mice and reduced their MHC class II expression. OVA323-339/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3+ and IL-10+ peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture's vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.
Assuntos
Doença Antimembrana Basal Glomerular/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Calcitriol/administração & dosagem , Células Dendríticas/imunologia , Epitopos Imunodominantes/administração & dosagem , Transferência Adotiva , Animais , Doença Antimembrana Basal Glomerular/diagnóstico , Doença Antimembrana Basal Glomerular/imunologia , Apresentação de Antígeno/efeitos dos fármacos , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Células CHO , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Cricetulus , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/transplante , Modelos Animais de Doenças , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Tolerância Imunológica/efeitos dos fármacos , Epitopos Imunodominantes/imunologia , Memória Imunológica/efeitos dos fármacos , Injeções Subcutâneas , Lipossomos , Linfonodos/citologia , Camundongos , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Índice de Gravidade de Doença , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
While erosion and tissue necrosis are the end-stage result of inflammatory arthritis, factors that can predict their initiation and severity are unknown. In an effort to identify these prognostic factors we developed contrast-enhanced (CE)-magnetic resonance imaging (MRI) for the mouse knee to assess the pathogenesis of inflammatory arthritis. Using this approach to study synovitis and draining lymph node (LN) function we first demonstrated that the LNs of TNF-Tg mice at 5 months are significantly larger and have greater enhancement in comparison to wild-type (WT) mice. This difference correlated with the abundance of dilated LYVE-1+ sinuses in the draining LNs. Dynamic CE-MRI further demonstrated differences between TNF-Tg and WT mice in the kinetics of LN enhancement. We established an LN capacity (LNcap) measurement that is a function of both volume and CE. We demonstrated that TNF-Tg mice have a 15-fold increase over WT levels at 5 months age (P < 0.001). Amelioration of arthritis with anti-TNF therapy resulted in a significant decrease in LNcap (P < 0.0001) that approached WT levels within 4 weeks. Interestingly, this functional decrease was not associated with a reduction of lymphatic vessels, which persist after therapy in both LNs and synovium. To assess the relationship between draining LN function and synovitis, a regression analysis was performed that demonstrated a significant negative correlation (R(2) = 0.63, P = 0.01) between LNcap and synovial volume. TNF-Tg mice with a lower LNcap display an accelerated progression of arthritis. These results indicate a protective function of enhanced lymphatic drainage in inflammatory arthritis.
Assuntos
Artrite/terapia , Linfonodos/patologia , Linfa/metabolismo , Imageamento por Ressonância Magnética/métodos , Animais , Artrite/patologia , Meios de Contraste/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Drenagem , Feminino , Inflamação/patologia , Linfangiogênese , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Análise de Regressão , Sinovite/metabolismoRESUMO
INTRODUCTION: Contact with the caterpillar of Lonomia achelous causes a hemorrhagic syndrome in humans prompted by two processes, an initial mild DIC that is later masked by overwhelming fibrinolytic activity. Although the venom affects both the hemostatic and inflammatory systems separately, it is not clear whether the hematological and hemostatic disturbances may in part be due to an indirect effect via inflammatory mediators. Here we report results on the crosstalk between these systems, particularly the effect of the pro-inflammatory cytokine TNF-α on hemostatic parameters. MATERIALS AND METHODS: the nitric oxide and TNF-α responses, as well as activation of the coagulation and fibrinolytic systems, were measured in macrophages and endothelial cells treated with Lonomia achelous hemolymph (LAH). The same responses were then determined, in a mouse model of LAH envenomation, after treatment with an anti-TNF-α antibody. RESULTS: Both macrophages and endothelial cells responded strongly to LAH in terms of pro-inflammatory mediator release and fibrinolytic activities as well as pro-coagulant activity (TF activity) in endothelial cells. Treatment with antibody against TNF-α decreased both TNF-α and NO3-/NO2- serum levels in the mice, after LAH injection. Blocking TNF-α also modified significantly the serum levels of plasminogen, fibrinogen and FXIII in mice, as well as decreased TF activity in endothelial cells. CONCLUSIONS: LAH may induce a hemostatic effect through endothelial and macrophage activation. These activated cell release hemostatic enzymes as well as pro-inflammatory mediators, principally TNF-α, that potentiate this release in an autocrine fashion, amplifying the fibrinolytic effect, which may in turn exacerbate the hemorrhagic manifestations. As far as we are aware, this is the first report of the relationship between the hemostatic system and the inflammatory responses in a hemorrhagic syndrome induce by animal secretions.
Assuntos
Hemolinfa/metabolismo , Hemorragia/etiologia , Inflamação/etiologia , Mariposas , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was originally considered to have activity against malignant disease. However, recent studies suggest TNF-alpha may also act as an endogenous tumor promoter. In the present work, mice deficient in TNF-alpha either genetically (TNF-alpha(-/-)) or after blockade with a neutralizing antibody (cV1q) were used to investigate the role of TNF-alpha in skin tumor development. Papillomas were induced in wild-type (wt) mice after treatment of skin with the initiating agent 9,10-dimethyl-1,2-benzanthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 15 weeks. TNF-alpha(-/-) mice were resistant to papilloma development when compared with wt mice on C57Bl/6J, 129/SvEv, and BALB/c genetic backgrounds. Primary murine keratinocytes (newborn keratinocytes) and skin homogenates were used to characterize TPA-stimulated TNF-alpha expression. TPA induced TNF-alpha protein in newborn keratinocytes in vitro and epidermis in vivo. Neutralization of TNF-alpha protein with cV1q in vivo for 0-15 weeks of promotion significantly decreased skin tumor development after 9,10-dimethyl-1,2-benzanthracene/TPA treatment. cV1q treatment during the early stages of tumor promotion (0-6 weeks) was equally effective. These data suggest that early induction of TNF-alpha is critical for skin tumor promotion. cV1q also reduced TPA-stimulated expression of matrix metalloproteinase 9 and granulocyte macrophage colony-stimulating factor, proteins that are differentially regulated in wt and TNF-alpha(-/-) epidermis. Treatment of the 410.4 transplantable breast carcinoma with cV1q reduced tumor growth in vivo, illustrating that inhibition of tumor growth through neutralization of TNF-alpha is not limited to skin carcinogenesis. These results provide further evidence for procancer actions of TNF-alpha and give some rationale for use of TNF-alpha antagonists in cancer prevention and treatment.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Papiloma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Fator de Crescimento Transformador alfa/imunologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Carcinógenos/toxicidade , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Imunidade Inata , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estadiamento de Neoplasias , Papiloma/induzido quimicamente , Papiloma/metabolismo , Ratos , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/metabolismo , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
OBJECTIVE: The immune inflammatory disorders rheumatoid arthritis (RA), psoriatic arthritis (PsA) and psoriasis (Ps) share common pathologic features and show responsiveness to anti-tumor necrosis factor (TNF) agents yet they are phenotypically distinct. The aim of this study was to examine if anti-TNF therapy is associated with divergent gene expression profiles in circulating cells and target tissues of patients with these diseases. METHODS: Peripheral blood CD14+ and CD14- cells were isolated from 9 RA, 12 PsA and 10 Ps patients before and after infliximab (IFX) treatment. Paired synovial (n=3, RA, PsA) and skin biopsies (n=5, Ps) were also collected. Gene expression was analyzed by microarrays. RESULTS: 26 out of 31 subjects responded to IFX. The transcriptional response of CD14+ cells to IFX was unique for the three diseases, with little overlap (<25%) in significantly changed gene lists (with PsA having the largest number of changed genes). In Ps, altered gene expression was more pronounced in lesional skin (relative to paired, healthy skin) compared to blood (relative to healthy controls). Marked suppression of up-regulated genes in affected skin was noted 2 weeks after therapy but the expression patterns differed from uninvolved skin. Divergent patterns of expression were noted between the blood cells and skin or synovial tissues in individual patients. Functions that promote cell differentiation, proliferation and apoptosis in all three diseases were enriched. RA was enriched in functions in CD14- cells, PsA in CD14+ cells and Ps in both CD14+ and CD14- cells, however, the specific functions showed little overlap in the 3 disorders. CONCLUSION: Divergent patterns of altered gene expression are observed in RA, PsA and Ps patients in blood cells and target organs in IFX responders. Differential gene expression profiles in the blood do not correlate with those in target organs.
Assuntos
Artrite Psoriásica/genética , Artrite Reumatoide/genética , Infliximab/efeitos adversos , Psoríase/genética , Adulto , Artrite Psoriásica/patologia , Artrite Reumatoide/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Variação Genética , Humanos , Pessoa de Meia-Idade , Biossíntese de Proteínas , Psoríase/induzido quimicamente , Psoríase/tratamento farmacológico , Psoríase/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
OBJECTIVE: Treatment of Crohn's disease (CD) with anti-tumor necrosis factor α (TNFα) decreases intestinal inflammation, but the effect on fibrosis remains unclear. We hypothesized that treatment with rat-specific anti-TNFα will decrease the development of intestinal fibrosis in a rat model of CD. We further hypothesized that magnetization transfer magnetic resonance imaging (MT-MRI) will be sensitive in detecting these differences in collagen content. METHODS: Rats were injected in the distal ileum and cecum with peptidoglycan-polysaccharide (PG-PS) or human serum albumin (control) at laparotomy and then received intraperitoneal injections of rat-specific anti-TNFα or vehicle daily for 21 days after laparotomy. Rats underwent MT-MRI abdominal imaging on day 19 or 20. MT ratio was calculated in the cecal wall. Cecal tissue histologic inflammation was scored. Cecal tissue procollagen, cytokine, and growth factor messenger RNAs were measured by quantitative real-time PCR. RESULTS: PG-PS-injected rats treated with anti-TNFα had less histologic inflammation, and cecal tissue expressed lower levels of proinflammatory cytokine messenger RNAs than vehicle-treated PG-PS-injected rats (IL-1ß: 5.59 ± 1.53 versus 10.41 ± 1.78, P = 0.02; IL-6: 23.23 ± 9.33 versus 45.89 ± 11.79, P = 0.07). PG-PS-injected rats treated with anti-TNFα developed less intestinal fibrosis than vehicle-treated PG-PS-injected rats by tissue procollagen I (2.87 ± 0.66 versus 9.28 ± 1.11; P = 0.00002), procollagen III (2.25 ± 0.35 versus 7.28 ± 0.76; P = 0.0000009), and MT-MRI (MT ratio: 17.79 ± 1.61 versus 27.95 ± 1.75; P = 0.0001). Insulin-like growth factor I (2.52 ± 0.44 versus 5.14 ± 0.60; P = 0.0007) and transforming growth factor ß1 (2.34 ± 0.29 versus 3.45 ± 0.29; P = 0.006) were also decreased in anti-TNFα-treated PG-PS-injected rats. CONCLUSIONS: Anti-TNFα prevents the development of bowel wall inflammation and fibrosis in the PG-PS rat model of CD. MT-MRI measurably demonstrates this decrease in intestinal fibrosis.
Assuntos
Anti-Inflamatórios/uso terapêutico , Doença de Crohn/prevenção & controle , Fibrose/prevenção & controle , Enteropatias/prevenção & controle , Imageamento por Ressonância Magnética , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Colite/induzido quimicamente , Colite/patologia , Colite/prevenção & controle , Doença de Crohn/induzido quimicamente , Doença de Crohn/patologia , Citocinas/genética , Modelos Animais de Doenças , Feminino , Fibrose/induzido quimicamente , Fibrose/patologia , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Inflamação/prevenção & controle , Fator de Crescimento Insulin-Like I/genética , Enteropatias/induzido quimicamente , Enteropatias/patologia , Magnetismo , Peptidoglicano/toxicidade , Pró-Colágeno/genética , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase em Tempo Real , Albumina Sérica/toxicidadeRESUMO
Monoclonal antibody (mAb) therapy was first established upon the approval of a mouse antibody for treatment of human acute organ rejection. However, the high incidence of immune response against the mouse mAb restricted therapeutic utility. Development of chimeric, "humanized" and human mAbs broadened therapeutic application to immune-mediated diseases requiring long-term treatment. Indeed, mAb therapeutics targeting soluble cytokines are highly effective in numerous immune-mediated disorders. A recent example is ustekinumab, a first-in-class therapeutic human immunoglobulin G1 kappa mAb that binds to the interleukins (IL)-12 and IL-23, cytokines that modulate lymphocyte function, including T-helper (Th) 1 and Th17 cell subsets. Ustekinumab was generated via recombinant human IL-12 immunization of human immunoglobulin (hu-Ig) transgenic mice. Ustekinumab binds to the p40 subunit common to IL-12 and IL-23 and prevents their interaction with the IL-12 receptor ß1 subunit of the IL-12 and IL-23 receptor complexes. Ustekinumab is approved for treatment of moderate-to-severe plaque psoriasis and has demonstrated efficacy in Crohn disease and psoriatic arthritis. The clinical characterization of ustekinumab continues to clarify our understanding of human immune pathologies and may offer a novel therapeutic option for certain immune-mediated diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Interleucina-12/imunologia , Interleucina-23/imunologia , Psoríase/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Humanos , Camundongos , Psoríase/imunologia , Resultado do Tratamento , UstekinumabRESUMO
BACKGROUND: Niemann-Pick type C (NPC) disease is a lysosomal storage disease characterized by the accumulation of cholesterol and glycosphingolipids. The majority of NPC patients die in their teen years due to progressive neurodegeneration; however, half of NPC patients also suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. We previously showed that a key mediator of NPC liver disease is tumor necrosis factor (TNF) α, which is involved in both proinflammatory and apoptotic signaling cascades. In this study, we tested the hypothesis that blocking TNF action with an anti-TNF monoclonal antibody (CNTO5048) will slow the progression of NPC liver disease. METHODOLOGY/PRINCIPAL FINDINGS: Treatment of wild-type C57BL/6 mice with NPC1-specific antisense oligonucleotides led to knockdown of NPC1 protein expression in the liver. This caused classical symptoms of NPC liver disease, including hepatic cholesterol accumulation, hepatomegaly, elevated serum liver enzymes, and lipid laden macrophage accumulation. In addition, there was a significant increase in the number of apoptotic cells and a proliferation of stellate cells. Concurrent treatment of NPC1 knockdown mice with anti-TNF had no effect on the primary lipid storage or accumulation of lipid-laden macrophages. However, anti-TNF treatment slightly blunted the increase in hepatic apoptosis and stellate cell activation that was seen with NPC1 knockdown. CONCLUSIONS/SIGNIFICANCE: Current therapeutic options for NPC disease are limited. Our results provide proof of principle that pharmacologically blocking the TNF-α inflammatory cascade can slightly reduce certain markers of NPC disease. Small molecule inhibitors of TNF that penetrate tissues and cross the blood-brain barrier may prove even more beneficial.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Modelos Animais de Doenças , Camundongos , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Fator de Necrose Tumoral alfa/imunologia , Animais , Apoptose/efeitos dos fármacos , Colesterol/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Proteínas/genética , Proteínas/metabolismoRESUMO
We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.
Assuntos
Anticorpos Monoclonais/farmacologia , Artrite/imunologia , Cartilagem/efeitos dos fármacos , Imunoglobulina G/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Adalimumab , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais Humanizados/farmacologia , Afinidade de Anticorpos , Artrite/induzido quimicamente , Cartilagem/patologia , Modelos Animais de Doenças , Progressão da Doença , Selectina E/genética , Selectina E/metabolismo , Etanercepte , Regulação da Expressão Gênica/efeitos dos fármacos , Hibridomas , Imunoglobulina G/isolamento & purificação , Mediadores da Inflamação/metabolismo , Infliximab , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Conformação Proteica , Receptores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that is synthesized and secreted by cells of the immune system, as well as by certain epithelia and stroma. Based on our previous studies demonstrating TNF-stimulated proliferation of normal and malignant mammary epithelial cells, we hypothesized that TNF might promote the growth of breast cancer in vivo. To test this, we generated bigenic mice that overexpressed activated neu/erbB2 in the mammary epithelium and whose TNF status was wild-type, heterozygous, or null. Mammary tumorigenesis was significantly decreased in TNF-/- mice (n = 30) compared with that in TNF+/+ mice (n = 27), with a palpable tumor incidence of 10.0% and 44.4%, and palpable tumors/mouse of 0.10 +/- 0.06 and 0.67 +/- 0.17, respectively. Tumorigenesis in the heterozygous group fell between that in the TNF+/+ and TNF-/- groups, but was not significantly different from either of the homozygous groups. The decreased tumor development in the TNF-/- mice was associated with a decreased proliferative index in the lobular and ductal mammary epithelium. To further investigate the role of TNF in breast cancer, mammary tumor-bearing mice whose tumors overexpressed wild-type neu/erbB2 were treated with a TNF-neutralizing antibody or a control antibody for 4 weeks (n = 20/group). Mammary tumor growth was significantly inhibited in mice treated with the anti-TNF antibody compared with the control antibody. Together, these data show a stimulatory role for TNF in the growth of breast tumors and suggest that TNF antagonists may be effective in a subset of patients with breast cancer.
Assuntos
Anticorpos Neutralizantes/imunologia , Neoplasias Mamárias Experimentais/patologia , Fator de Necrose Tumoral alfa/deficiência , Animais , Sequência de Bases , Carcinógenos/toxicidade , Primers do DNA , Feminino , Neoplasias Mamárias Experimentais/induzido quimicamente , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Interleukin-6 is a multifunctional cytokine that is critical for T/B-cell differentiation and maturation, immunoglobulin secretion, acute-phase protein production, and macrophage/monocyte functions. Extensive research into the biology of IL-6 has implicated IL-6 in the pathophysiology and pathogenesis of RA. An anti-murine IL-6 mAb that neutralizes mouse IL-6 activities was tested in animal model of collagen-induced arthritis. Prophylactic treatment with anti-IL-6 mAb significantly reduced the incidence and severity of arthritis compared to control mAb treated mice. The mitogenic response of B and T cells isolated from the lymph nodes of anti-IL-6 treated mice was significantly reduced compared to cells isolated from control mAb treated mice. The overall histopathology score for paws from the anti-IL-6 treated mice was significantly reduced when compared to paws from mice treated with control mAb, including both inflammatory (synovitis and pannus) and erosive (erosions and architecture) parameters. Reduced loss of cartilage matrix components was also observed in the anti-IL-6 treated mice. Collectively, these data suggest that IL-6 plays a major role in the pathophysiology of rheumatoid arthritis, and thus support the potential benefit of anti-IL-6 mAb treatment in rheumatoid arthritis patients.
RESUMO
Inflammation is associated with blood vessel and lymphatic vessel proliferation and remodeling. The microvasculature of the mouse trachea provides an ideal opportunity to study this process, as Mycoplasma pulmonis infection of mouse airways induces widespread and sustained vessel remodeling, including enlargement of capillaries into venules and lymphangiogenesis. Although the mediators responsible for these vascular changes in mice have not been identified, VEGF-A is known not to be involved. Here, we sought to determine whether TNF-alpha drives the changes in blood vessels and lymphatics in M. pulmonis-infected mice. The endothelial cells, but not pericytes, of blood vessels, but not lymphatics, were immunoreactive for TNF receptor 1 (TNF-R1) and lymphotoxin B receptors. Most TNF-R2 immunoreactivity was on leukocytes. Infection resulted in a large and sustained increase in TNF-alpha expression, as measured by real-time quantitative RT-PCR, and smaller increases in lymphotoxins and TNF receptors that preceded vessel remodeling. Substantially less vessel remodeling and lymphangiogenesis occurred when TNF-alpha signaling was inhibited by a blocking antibody or was silenced in Tnfr1-/- mice. When administered after infection was established, the TNF-alpha-specific antibody slowed but did not reverse blood vessel remodeling and lymphangiogenesis. The action of TNF-alpha on blood vessels is probably mediated through direct effects on endothelial cells, but its effects on lymphangiogenesis may require inflammatory mediators from recruited leukocytes. We conclude that TNF-alpha is a strong candidate for a mediator that drives blood vessel remodeling and lymphangiogenesis in inflammation.
Assuntos
Vasos Sanguíneos , Inflamação/imunologia , Linfangiogênese/imunologia , Vasos Linfáticos , Sistema Respiratório , Fator de Necrose Tumoral alfa/imunologia , Animais , Vasos Sanguíneos/anatomia & histologia , Vasos Sanguíneos/fisiologia , Perfilação da Expressão Gênica , Glicoproteínas/metabolismo , Vasos Linfáticos/anatomia & histologia , Vasos Linfáticos/fisiologia , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/patologia , Mycoplasma pulmonis , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Sistema Respiratório/anatomia & histologia , Sistema Respiratório/imunologia , Transdução de Sinais/fisiologiaRESUMO
A simple expression is given for the k-function associated with the general solution of Stavroudis to the eikonal equation for refraction or reflection of a plane wave from an arbitrary surface. Using this result, we specialize the solution to derive analytic expressions for the wavefront and caustic surfaces after refraction of a plane wave from any rotationally symmetric surface. The method is applied to evaluating and comparing the wavefront and caustic surfaces formed both by a planospherical lens and a planoaspheric lens used for laser beam shaping, which provides understanding of how the irradiance is redistributed over a beam as the wavefront folds back on itself within the focal region.
RESUMO
OBJECTIVE: While bone marrow edema (BME) detected by magnetic resonance imaging (MRI) is a biomarker of arthritis, its nature remains poorly understood due to the limitations of clinical studies. In this study, MRI of murine arthritis was used to elucidate its cellular composition and vascular involvement. METHODS: BME was quantified using normalized bone marrow intensity (NBMI) from precontrast MRI and normalized marrow contrast enhancement (NMCE) following intravenous administration of gadopentate dimeglumine. Wild-type (WT) and tumor necrosis factor (TNF)-transgenic mice were scanned from 2 to 5 months of age, followed by histologic or fluorescence-activated cell sorting (FACS) analysis of marrow. In efficacy studies, TNF-transgenic mice were treated with anti-TNF or placebo for 8 weeks, and then were studied using bimonthly MRI and histologic analysis. RESULTS: NBMI values were similar in WT and TNF-transgenic mice at 2 months. The values in WT mice steadily decreased thereafter, with mean values becoming significantly different from those of TNF-transgenic mice at 3.5 months (mean +/- SD 0.29 +/- 0.08 versus 0.46 +/- 0.13; P < 0.05). Red to yellow marrow transformation occurred in WT but not TNF-transgenic mice, as observed histologically at 5 months. The marrow of TNF-transgenic mice that received anti-TNF therapy converted to yellow marrow, with lower NBMI values versus placebo at 6 weeks (mean +/- SD 0.26 +/- 0.07 versus 0.61 +/- 0.22; P < 0.05). FACS analysis of bone marrow revealed a significant correlation between NBMI values and CD11b+ monocytes (R2 = 0.91, P = 0.0028). Thresholds for "normal" red marrow versus pathologic BME were established, and it was also found that inflammatory marrow is highly permeable to contrast agent. CONCLUSION: BME signals in TNF-transgenic mice are caused by yellow to red marrow conversion, with increased myelopoiesis and increased marrow permeability. The factors that mediate these changes warrant further investigation.
Assuntos
Artrite/diagnóstico , Doenças da Medula Óssea/diagnóstico , Edema/diagnóstico , Imageamento por Ressonância Magnética/métodos , Fatores de Necrose Tumoral/farmacologia , Animais , Artrite/tratamento farmacológico , Medula Óssea/efeitos dos fármacos , Meios de Contraste , Gadolínio DTPA , Camundongos , Camundongos Transgênicos , Mielopoese/efeitos dos fármacos , Inibidores do Fator de Necrose TumoralRESUMO
OBJECTIVE: To develop longitudinal 3-dimensional (3-D) measures of outcomes of inflammation and bone erosion in murine arthritis using contrast-enhanced magnetic resonance imaging (CE-MRI) and in vivo microfocal computed tomography (micro-CT) and, in a pilot study, to determine the value of entry criteria based on age versus synovial volume in therapeutic intervention studies. METHODS: CE-MRI and in vivo micro-CT were performed on tumor necrosis factor-transgenic (TNF-Tg) mice and their wild-type littermates to quantify the synovial and popliteal lymph node volumes and the patella and talus bone volumes, respectively, which were validated histologically. These longitudinal outcome measures were used to assess the natural history of erosive inflammatory arthritis. We also performed anti-TNF versus placebo efficacy studies in TNF-Tg mice in which treatment was initiated according to either age (4-5 months) or synovial volume (3 mm(3) as detected by CE-MRI). Linear regression was performed to analyze the correlation between synovitis and focal erosion. RESULTS: CE-MRI demonstrated the highly variable nature of TNF-induced joint inflammation. Initiation of treatment by synovial volume produced significantly larger treatment effects on the synovial volume (P = 0.04) and the lymph node volume (P < 0.01) than did initiation by age. By correlating the MRI and micro-CT data, we were able to demonstrate a significant relationship between changes in synovial and patellar volumes (R(2) = 0.75, P < 0.01). CONCLUSION: In vivo CE-MRI and micro-CT 3-D outcome measures are powerful tools that accurately demonstrate the progression of erosive inflammatory arthritis in mice. These methods can be used to identify mice with arthritis of similar severity before intervention studies are initiated, thus minimizing heterogeneity in outcome studies of chronic arthritis seen between genetically identical littermates.
Assuntos
Artrite/patologia , Osso e Ossos/patologia , Linfonodos/patologia , Membrana Sinovial/patologia , Animais , Artrite/diagnóstico por imagem , Biomarcadores , Osso e Ossos/diagnóstico por imagem , Modelos Animais de Doenças , Edema/diagnóstico por imagem , Edema/patologia , Inflamação/diagnóstico por imagem , Inflamação/patologia , Linfonodos/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Transgênicos , Osteíte/diagnóstico por imagem , Osteíte/patologia , Projetos Piloto , Membrana Sinovial/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genéticaRESUMO
We consider four families of functions--the super-Gaussian, flattened Gaussian, Fermi-Dirac, and super-Lorentzian--that have been used to describe flattened irradiance profiles. We determine the shape and width parameters of the different distributions, when each flattened profile has the same radius and slope of the irradiance at its half-height point, and then we evaluate the implicit functional relationship between the shape and width parameters for matched profiles, which provides a quantitative way to compare profiles described by different families of functions. We conclude from an analysis of each profile with matched parameters using Kirchhoff-Fresnel diffraction theory and M2 analysis that the diffraction patterns as they propagate differ by small amounts, which may not be distinguished experimentally. Thus, beam shaping optics is designed to produce either of these four flattened output irradiance distributions with matched parameters will yield similar irradiance distributions as the beam propagates.
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Although cytokines are critical in maintaining normal physiology, excessive production of these proteins can lead to pathological consequences. Inhibitors of cytokine production or action are therefore widely investigated as potential therapeutic agents in a variety of immune and inflammatory diseases. Indeed, the successful application of inhibitors of tumor necrosis factor-alpha in rheumatoid arthritis and Crohn's disease heralds the great therapeutic potential of biopharmaceutical agents to counteract cytokine activities. Emerging opportunities for new therapeutics, as well as the challenges we face in their use and development, are described in this review.