RESUMO
High concentrations of oxygen are often needed to optimize oxygenation in infants with persistent pulmonary hypertension (PPHN), but this can also increase the risk of hyperoxemia. We determined the occurrence of hyperoxemia in infants treated for PPHN. Medical records of infants ≥ 34 + 0 weeks gestational age (GA) who received inhaled nitric oxide (iNO) were retrospectively reviewed for oxygenation parameters during iNO therapy. Oxygen was manually titrated to target arterial oxygen tension (PaO2) 10-13 kPa and peripheral oxygen saturation (SpO2) 92-98%. The main study outcomes were the incidence and duration of hyperoxemia and hypoxemia and the fraction of inspired oxygen (FiO2). A total of 181 infants were included. The median FiO2 was 0.43 (IQR 0.34-0.56) and the maximum FiO2 was 1.0 in 156/181 (86%) infants, resulting in at least one PaO2 > 13 kPa in 149/181 (82%) infants, of which 46/149 (31%) infants had minimal one PaO2 > 30 kPa. SpO2 was > 98% in 179/181 (99%) infants for 17.7% (8.2-35.6%) of the iNO time. PaO2 < 10 kPa occurred in 160/181 (88%) infants, of which 81/160 (51%) infants had minimal one PaO2 < 6.7 kPa. SpO2 was < 92% in 169/181 (93%) infants for 1.6% (0.5-4.3%) of the iNO time. Conclusion: While treatment of PPHN is focused on preventing and reversing hypoxemia, hyperoxemia occurs inadvertently in most patients. What is Known: ⢠High concentrations of oxygen are often needed to prevent hypoxemia-induced deterioration of PPHN, but this can also increase the risk of hyperoxemia. ⢠Infants with persistent pulmonary hypertension may be particularly vulnerable to the toxic effects of oxygen, and hyperoxemia could further induce pulmonary vasoconstriction, potentially worsening the condition. What is New: ⢠Hyperoxemia occurs in the majority of infants with PPHN during treatment with iNO. ⢠Infants with PPHN spent a considerably longer period with saturations above the target range compared to saturations below the target range.
Assuntos
Hiperóxia , Óxido Nítrico , Síndrome da Persistência do Padrão de Circulação Fetal , Humanos , Recém-Nascido , Hiperóxia/etiologia , Óxido Nítrico/administração & dosagem , Estudos Retrospectivos , Síndrome da Persistência do Padrão de Circulação Fetal/terapia , Masculino , Feminino , Administração por Inalação , Oxigênio/sangue , Oxigênio/administração & dosagem , Saturação de Oxigênio , Oxigenoterapia/métodos , Hipóxia/etiologia , Hipóxia/terapiaRESUMO
IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps.
RESUMO
BACKGROUND: Cyclooxygenase inhibitors are commonly used in infants with patent ductus arteriosus (PDA), but the benefit of these drugs is uncertain. METHODS: In this multicenter, noninferiority trial, we randomly assigned infants with echocardiographically confirmed PDA (diameter, >1.5 mm, with left-to-right shunting) who were extremely preterm (<28 weeks' gestational age) to receive either expectant management or early ibuprofen treatment. The composite primary outcome included necrotizing enterocolitis (Bell's stage IIa or higher), moderate to severe bronchopulmonary dysplasia, or death at 36 weeks' postmenstrual age. The noninferiority of expectant management as compared with early ibuprofen treatment was defined as an absolute risk difference with an upper boundary of the one-sided 95% confidence interval of less than 10 percentage points. RESULTS: A total of 273 infants underwent randomization. The median gestational age was 26 weeks, and the median birth weight was 845 g. A primary-outcome event occurred in 63 of 136 infants (46.3%) in the expectant-management group and in 87 of 137 (63.5%) in the early-ibuprofen group (absolute risk difference, -17.2 percentage points; upper boundary of the one-sided 95% confidence interval [CI], -7.4; P<0.001 for noninferiority). Necrotizing enterocolitis occurred in 24 of 136 infants (17.6%) in the expectant-management group and in 21 of 137 (15.3%) in the early-ibuprofen group (absolute risk difference, 2.3 percentage points; two-sided 95% CI, -6.5 to 11.1); bronchopulmonary dysplasia occurred in 39 of 117 infants (33.3%) and in 57 of 112 (50.9%), respectively (absolute risk difference, -17.6 percentage points; two-sided 95% CI, -30.2 to -5.0). Death occurred in 19 of 136 infants (14.0%) and in 25 of 137 (18.2%), respectively (absolute risk difference, -4.3 percentage points; two-sided 95% CI, -13.0 to 4.4). Rates of other adverse outcomes were similar in the two groups. CONCLUSIONS: Expectant management for PDA in extremely premature infants was noninferior to early ibuprofen treatment with respect to necrotizing enterocolitis, bronchopulmonary dysplasia, or death at 36 weeks' postmenstrual age. (Funded by the Netherlands Organization for Health Research and Development and the Belgian Health Care Knowledge Center; BeNeDuctus ClinicalTrials.gov number, NCT02884219; EudraCT number, 2017-001376-28.).
Assuntos
Displasia Broncopulmonar , Permeabilidade do Canal Arterial , Enterocolite Necrosante , Ibuprofeno , Conduta Expectante , Humanos , Lactente , Recém-Nascido , Displasia Broncopulmonar/etiologia , Permeabilidade do Canal Arterial/diagnóstico por imagem , Permeabilidade do Canal Arterial/tratamento farmacológico , Permeabilidade do Canal Arterial/mortalidade , Permeabilidade do Canal Arterial/terapia , Ecocardiografia , Enterocolite Necrosante/etiologia , Ibuprofeno/administração & dosagem , Ibuprofeno/efeitos adversos , Ibuprofeno/uso terapêutico , Indometacina/efeitos adversos , Indometacina/uso terapêutico , Lactente Extremamente Prematuro , Recém-Nascido de Baixo Peso , Doenças do Recém-Nascido/tratamento farmacológico , Doenças do Recém-Nascido/terapiaRESUMO
Promising strategies for maximizing IgG effector functions rely on the introduction of natural and non-immunogenic modifications. The Fc domain of IgG antibodies contains an N-linked oligosaccharide at position 297. Human IgG antibodies lacking the core fucose in this glycan have enhanced binding to human (FcγR) IIIa/b, resulting in enhanced antibody dependent cell cytotoxicity and phagocytosis through these receptors. However, it is not yet clear if glycan-enhancing modifications of human IgG translate into more effective treatment in mouse models. We generated humanized hIgG1-TA99 antibodies with and without core-fucose. C57Bl/6 mice that were injected intraperitoneally with B16F10-gp75 mouse melanoma developed significantly less metastasis outgrowth after treatment with afucosylated hIgG1-TA99 compared to mice treated with wildtype hhIgG1-TA99. Afucosylated human IgG1 showed stronger interaction with the murine FcγRIV, the mouse orthologue of human FcγRIIIa, indicating that this glycan change is functionally conserved between the species. In agreement with this, no significant differences were observed in tumor outgrowth in FcγRIV-/- mice treated with human hIgG1-TA99 with or without the core fucose. These results confirm the potential of using afucosylated therapeutic IgG to increase their efficacy. Moreover, we show that afucosylated human IgG1 antibodies act across species, supporting that mouse models can be suitable to test afucosylated antibodies.
RESUMO
Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.
Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Anticorpos Antivirais/sangue , Anticorpos Antivirais/química , COVID-19/fisiopatologia , Células Cultivadas , Estado Terminal , Citomegalovirus/imunologia , Feminino , Fucose/análise , Glicosilação , HIV/imunologia , Vacinas contra Hepatite B/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/química , Inflamação , Interleucina-6/biossíntese , Interleucina-6/imunologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Parvovirus B19 Humano/imunologia , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Adulto JovemRESUMO
Propranolol is an ADRB2 blocker that regulates heart muscle contractions, smooth muscle relaxation, and glycogenolysis. In addition, an increasing number of applications in dermatology have been described, most prominently, the use as a first-line treatment for infantile hemangiomas. We here show that propranolol enhances IL-8-induced neutrophil chemotaxis and reduces the release of ROS after immune complex stimulation. To obtain further molecular insights into the modulatory effects of propranolol in activated neutrophils, we performed RNA sequencing of immune complex-stimulated neutrophils in the absence and presence of the drug. We identified the transcriptomic signature of propranolol and demonstrated an ADR2-independent immunomodulatory effect. To determine if the anti-inflammatory transcriptomic signature of propranolol also translates into clinical effects, we next evaluated the impact of propranolol in a prototypical neutrophil-dependent skin disease, specifically, antibody transfer-induced epidermolysis bullosa acquisita in mice. To validate the identified propranolol gene signature obtained in human neutrophils, we analyzed a selection of genes by RT-PCR in mouse epidermolysis bullosa acquisita skin and confirmed TNF, among others, to be differentially regulated by propranolol treatment. Our data clearly indicate that, based on its molecular impact on immune complex-activated neutrophils, propranolol is a potential treatment option for neutrophil-mediated inflammatory skin diseases.
Assuntos
Antagonistas Adrenérgicos beta/administração & dosagem , Epidermólise Bolhosa Adquirida/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Propranolol/administração & dosagem , Administração Cutânea , Administração Oral , Animais , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Modelos Animais de Doenças , Epidermólise Bolhosa Adquirida/imunologia , Epidermólise Bolhosa Adquirida/patologia , Voluntários Saudáveis , Humanos , Camundongos , Neutrófilos/imunologia , Cultura Primária de Células , RNA-Seq , Receptores Adrenérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/efeitos dos fármacos , Pele/patologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologiaRESUMO
Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.
Assuntos
Adenocarcinoma , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Neoplasias do Colo , Receptores de IgG , Animais , Anticorpos Monoclonais , Modelos Animais de Doenças , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Intravenous immunoglobulins (IVIg) are used in the treatment of different autoimmune and inflammatory diseases, such as immune thrombocytopenia and hemolytic anemia. One of the modes of action of IVIg is preventing phagocytosis of autoantibody-opsonized blood cells by saturation of the Fc-gamma receptors of macrophages in spleen and liver. IgG contains a fixed glycan, which is in most cases biantennary, at the asparagine residue at position 297 in the Fc tail. This glycan consists of a core structure of N-acetyl glucosamine (GlcNAc) and mannose groups, variably extended with core fucose, bisecting GlcNAc as well as terminal galactose and sialic acid. This structural glycan influences the binding affinity of IgG to Fc-gamma receptors. By glyco-engineering, we generated monoclonal IgG antibodies with different Fc-tail glycans and tested both their opsonizing and blocking capacity in a phagocytosis assay of IgG-opsonized erythrocytes with human monocyte-derived macrophages. In contrast to a lack of effect in opsono-phagocytosis, these IgG glycovariants differentially inhibited the uptake of opsonized erythrocytes. The level of bisecting GlcNAc and galactosylation had unexpectedly larger impact than core fucosylation, and suggest that targeted modifications different from the core fucose may well improve the immunomodulating efficacy of IVIg treatment.
Assuntos
Anemia Hemolítica/terapia , Imunoglobulina G/metabolismo , Imunoglobulinas Intravenosas/uso terapêutico , Macrófagos/fisiologia , Púrpura Trombocitopênica Idiopática/terapia , Acetilglucosamina/química , Afinidade de Anticorpos , Autoanticorpos/metabolismo , Células Cultivadas , Humanos , Imunoglobulina G/química , Fagocitose , Ligação Proteica , Engenharia de Proteínas , Receptores de IgG/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Much controversy exists about the optimal management of a patent ductus arteriosus (PDA) in preterm infants, especially in those born at a gestational age (GA) less than 28 weeks. No causal relationship has been proven between a (haemodynamically significant) PDA and neonatal complications related to pulmonary hyperperfusion and/or systemic hypoperfusion. Although studies show conflicting results, a common understanding is that medical or surgical treatment of a PDA does not seem to reduce the risk of major neonatal morbidities and mortality. As the PDA might have closed spontaneously, treated children are potentially exposed to iatrogenic adverse effects. A conservative approach is gaining interest worldwide, although convincing evidence to support its use is lacking. METHODS: This multicentre, randomised, non-inferiority trial is conducted in neonatal intensive care units. The study population consists of preterm infants (GA < 28 weeks) with an echocardiographic-confirmed PDA with a transductal diameter > 1.5 mm. Early treatment (between 24 and 72 h postnatal age) with the cyclooxygenase inhibitor (COXi) ibuprofen (IBU) is compared with an expectative management (no intervention intended to close a PDA). The primary outcome is the composite of mortality, and/or necrotising enterocolitis (NEC) Bell stage ≥ IIa, and/or bronchopulmonary dysplasia (BPD) defined as the need for supplemental oxygen, all at a postmenstrual age (PMA) of 36 weeks. Secondary outcome parameters are short term sequelae of cardiovascular failure, comorbidity and adverse events assessed during hospitalization and long-term neurodevelopmental outcome assessed at a corrected age of 2 years. Consequences regarding health economics are evaluated by cost effectiveness analysis and budget impact analysis. DISCUSSION: As a conservative approach is gaining interest, we investigate whether in preterm infants, born at a GA less than 28 weeks, with a PDA an expectative management is non-inferior to early treatment with IBU regarding to the composite outcome of mortality and/or NEC and/or BPD at a PMA of 36 weeks. TRIAL REGISTRATION: This trial is registered with the Dutch Trial Register NTR5479 (registered on 19 October 2015), the registry sponsored by the United States National Library of Medicine Clinicaltrials.gov NCT02884219 (registered May 2016) and the European Clinical Trials Database EudraCT 2017-001376-28 .
Assuntos
Inibidores de Ciclo-Oxigenase/uso terapêutico , Permeabilidade do Canal Arterial/tratamento farmacológico , Ibuprofeno/uso terapêutico , Lactente Extremamente Prematuro , Doenças do Prematuro/tratamento farmacológico , Conduta Expectante , Análise Custo-Benefício , Permeabilidade do Canal Arterial/complicações , Permeabilidade do Canal Arterial/mortalidade , Permeabilidade do Canal Arterial/cirurgia , Enterocolite Necrosante/etiologia , Humanos , Recém-Nascido , Doenças do Prematuro/mortalidade , Ligadura , Projetos de Pesquisa , Tempo para o Tratamento , Conduta Expectante/economiaRESUMO
Central venous catheters (CVCs) in neonates are associated with an increased risk of thrombosis. Most reports focus on umbilical venous catheters (UVCs) and peripherally inserted central catheters (PICCs), whereas data available on femoral venous catheters (FVCs) are limited. We performed a retrospective cohort study in all neonates (gestational age ≥34 wk) with CVCs. The primary outcome was the occurrence of thrombosis in CVCs. The secondary outcomes were possible risk factors for thrombosis, the thrombotic incidence in FVCs, UVCs, and PICCs, and clinical aspects of thrombosis in these groups. A total of 552 neonates received a total of 656 catheters, including 407 (62%) UVCs, 185 (28%) PICCs, and 64 (10%) FVCs. Thrombosis was detected in 14 cases, yielding an overall incidence of 2.1% or 3.6 events per 1000 catheter days. FVC was significantly associated with the occurrence of thrombosis when compared with UVC (P=0.02; odds ratio, 3.8; 95% confidence interval, 1.2-12.0) and PICC (P=0.01; odds ratio, 8.2; 95% confidence interval, 1.6-41.7). The incidence of thrombosis was higher in FVCs than in UVCs and PICCS, that is, 7.8% (5/64), 1.7% (7/407), and 1.1% (2/185), respectively (P<0.01). The number of thrombotic events per 1000 catheter days was 12.3 in FVCs, 3.2 in UVCs, and 1.5 in PICCs (P<0.05). We concluded that thrombosis occurs more frequently in FVCs than in other CVCs.
Assuntos
Cateterismo Venoso Central/efeitos adversos , Veia Femoral , Trombose Venosa Profunda de Membros Superiores/epidemiologia , Cateterismo Periférico/efeitos adversos , Cateteres Venosos Centrais/efeitos adversos , Estudos de Coortes , Feminino , Humanos , Incidência , Recém-Nascido , Masculino , Estudos Retrospectivos , Fatores de Risco , Veias Umbilicais , Trombose Venosa Profunda de Membros Superiores/etiologiaRESUMO
Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.
RESUMO
BACKGROUND & AIMS: Although tumor necrosis factor (TNF) antagonists reduce many clinical features of inflammatory bowel disease, complete mucosal healing occurs in fewer than 50% of patients. The Fc-region of monoclonal antibodies against TNF has immunosuppressive properties via effects on macrophage polarization. We examined the interaction between the anti-TNF Fc-region and Fcγ receptors (FcγR), and whether the absence of the Fc core fucose (which increases binding to FcγRIIIa) increases the efficacy of anti-TNF in mice with colitis. METHODS: We generated Rag1-/- mice that lack all activating FcγRs (FcγRI, FcγRIII, and FcγRIV; called FcγR-/-Rag1-/- mice). We produced hypo-fucosylated antibodies against mouse and human TNF (adalimumab). Colitis was induced in mice by transfer of CD4+CD45RBhi to FcγR-/-Rag1-/- or Rag1-/- littermates; mice were given different antibodies against TNF or isotype (control) antibodies and disease activity index scores were determined. Colon tissues were collected and analyzed by histology. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors. T-cell proliferation and proportions of CD206+ (immune regulatory) macrophages were measured in mixed lymphocyte reactions. Human PBMCs were genotyped for FCGR3A158 (the FcγRIIIa-158F allotype displays a lower Fc binding affinity) using the TaqMan single nucleotide polymorphism genotype assay. RESULTS: Rag1-/- mice with colitis given anti-TNF had near complete mucosal healing and Rag1-/- mice given an isotype control antibody developed severe colitis. In contrast, FcγR-/-Rag1-/- mice were refractory to the effects of anti-TNF: their histological colitis scores were as severe as those from FcγR-/-Rag1-/- mice given a control antibody. Colons from Rag1-/- mice that received anti-TNF had an increased number of CD206+ macrophages compared with Rag1-/- mice given control antibody; in FcγR-/-Rag1-/- mice given anti-TNF these numbers were as low as FcγR-/-Rag1-/- given the control antibody. In human PBMCs, anti-TNF increased the number of CD206+ macrophages: this required expression of FcγRIIIa; numbers of these cells were reduced in PBMCs with the low-affinity FcγRIIIa-158F genotype. A hypo-fucosylated form of adalimumab bound human FcγRIIIa with a higher affinity than control adalimumab. When hypo-fucosylated adalimumab was added to PBMCs, a larger number of CD206+ macrophages formed and T-cell proliferation was reduced, compared with addition of a control adalimumab. Hypo-fucosylated adalimumab increased the number of CD206+ macrophages in PMBCs that expressed the low-affinity FcγRIIIa. In mice with colitis, hypo-fucosylated anti-TNF significantly increased the number of CD206+ macrophages in the colon compared with control anti-TNF and was more effective in reducing colitis severity as measured by histology. CONCLUSIONS: In a study of the in vitro and in vivo mechanisms of anti-TNF, we found FcγR engagement by anti-TNF to be required for reduction of colitis in mice and development of CD206+ macrophages. A hypo-fucosylated form of anti-TNF binds FcγRIIIa with higher affinity and induces development of CD206+ macrophages in human PBMCs, especially PBMCs that express low-affinity FcγRIIIa. Hypo-fucosylated anti-TNF might be more effective in patients with inflammatory bowel disease.
Assuntos
Adalimumab/farmacologia , Anticorpos Monoclonais/farmacologia , Colite/tratamento farmacológico , Colo/efeitos dos fármacos , Imunossupressores/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Transferência Adotiva , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/metabolismo , Colo/imunologia , Colo/metabolismo , Colo/patologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Receptores de IgG/deficiência , Receptores de IgG/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Fatores de Tempo , Fator de Necrose Tumoral alfa/imunologia , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: Current anti-cancer therapeutic antibodies that are used in the clinic are predominantly humanized or fully human immunoglobulin G1 (IgG1). These antibodies bind with high affinity to the target antigen and are efficient in activating the immune system via IgG Fc receptors and/or complement. In addition to IgG1, three more isotypes are present in humans, of which IgG3 has been found to be superior compared to human IgG1 in inducing antibody dependent cell cytotoxicity (ADCC), phagocytosis or activation of complement in some models. Nonetheless, no therapeutic human IgG3 mAbs have been developed due to the short in vivo half-life of most known IgG3 allotypes. In this manuscript, we compared the efficacy of V-gene matched IgG1 and IgG3 anti-tumour mAb (TA99) in mice, using natural variants of human IgG3 with short- or long half-life, differing only at position 435 with an arginine or histidine, respectively. RESULTS: In vitro human IgG1 and IgG3 did not show any differences in opsonisation ability of B16F10-gp75 mouse melanoma cells. IgG1, however, was superior in inducing phagocytosis of tumour cells by mouse macrophages. Similarly, in a mouse peritoneal metastasis model we did not detect an improved effect of IgG3 in preventing tumour outgrowth. Moreover, replacing the arginine at position 435 for a histidine in IgG3 to enhance half-life did not result in better suppression of tumour outgrowth compared to wild type IgG3 when injected prior to tumour cell injection. CONCLUSION: In conclusion, human IgG3 does not have improved therapeutic efficacy compared to human IgG1 in a mouse tumour model.
Assuntos
Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Melanoma Experimental/imunologia , Monócitos/imunologia , Neoplasias Peritoneais/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Meia-Vida , Humanos , Masculino , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/patologia , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/terapia , Fagocitose , RadioimunoterapiaRESUMO
Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents â¼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be â¼10% in six serum-derived IgG3 samples and â¼13% in two monoclonal IgG3 allotypes.
Assuntos
Imunoglobulina G/análise , Peptídeos/análise , Treonina/química , Adulto , Sequência de Aminoácidos , Sequência de Carboidratos , Feminino , Expressão Gênica , Glicosilação , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Proteólise , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Treonina/metabolismo , Tripsina/químicaRESUMO
Sotos syndrome (SoS) is characterized by tall stature, characteristic craniofacial features and mental retardation. It is caused by haploinsufficiency of the NSD1 gene. In this study, our objective was to identify downstream effectors of NSD1 and to map these effectors in signaling pathways associated with growth. Genome-wide expression studies were performed on dermal fibroblasts from SoS patients with a confirmed NSD1 abnormality. To substantiate those results, phosphorylation, siRNA and transfection experiments were performed. A significant association was demonstrated with the Mitogen-Activated Protein Kinase (MAPK) pathway. Members of the fibroblast growth factor family such as FGF4 and FGF13 contributed strongly to the differential expression in this pathway. In addition, a diminished activity state of the MAPK/ERK pathway was demonstrated in SoS. The Ras Interacting Protein 1 (RASIP1) was identified to exhibit upregulated expression in SoS. It was shown that RASIP1 dose-dependently potentiated bFGF induced expression of the MAPK responsive SBE reporter providing further support for a link between NSD1 and the MAPK/ERK signaling pathway. Additionally, we demonstrated NSD1 expression in the terminally differentiated hypertrophic chondrocytes of normal human epiphyseal growth plates. In short stature syndromes such as hypochondroplasia and Noonan syndrome, the activation level of the FGF-MAPK/ERK-pathway in epiphyseal growth plates is a determining factor for statural growth. In analogy, we propose that deregulation of the MAPK/ERK pathway in SoS results in altered hypertrophic differentiation of NSD1 expressing chondrocytes and may be a determining factor in statural overgrowth and accelerated skeletal maturation in SoS.
Assuntos
Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Síndrome de Sotos/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Pele/metabolismo , Síndrome de Sotos/metabolismo , Regulação para CimaRESUMO
Dendritic cells (DCs) are promising antigen presenting cells for cancer treatment. Previously, we showed that the combination of monophosphoryl lipid A (MPLA) with IFNgamma generates mature DCs that produce IL-12 and polarize CD4(+) T cells towards a Th1 phenotype. Here, we extended these observations by showing that the DCs generated with the clinical grade maturation cocktail of MPLA/IFNgamma induce superior tumour antigen-specific CD8(+) CTL responses compared to the cytokine cocktail matured DCs that are currently used in the clinic. MPLA/IFNgamma DCs can induce CTL responses in healthy individuals as well as in melanoma patients. The CTL induction was mainly dependent on the IL-12 produced by the MPLA/IFNgamma DCs. The high amounts of induced CTLs are functional as they produce IFNgamma and lyse target cells and this cytolytic activity is antigen specific and HLA restricted. Furthermore, the CTLs proved to kill tumour cells expressing endogenous tumour antigen in vitro. Therefore, MPLA/IFNgamma DCs are very promising for the use in future cancer immunotherapy.
Assuntos
Adjuvantes Imunológicos/farmacologia , Células Dendríticas/metabolismo , Lipídeo A/análogos & derivados , Melanoma/imunologia , Linfócitos T Citotóxicos/metabolismo , Antígenos de Neoplasias/imunologia , Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/patologia , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Humanos , Interferon gama/farmacologia , Interleucina-12/metabolismo , Lipídeo A/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Melanoma/patologia , Melanoma/terapia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Células Th1/imunologia , Receptores Toll-Like/agonistasAssuntos
Proteínas de Transporte/genética , Anormalidades Craniofaciais/genética , Transtornos do Crescimento/genética , Mutação , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Genes da Neurofibromatose 1 , Histona Metiltransferases , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Deficiências da Aprendizagem/genética , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Fenótipo , Síndrome , Ubiquitina-Proteína Ligases , Adulto JovemRESUMO
Sotos syndrome (SoS, OMIM#117550) is an overgrowth disorder characterized by excessive growth-especially in the first years of childhood-distinctive craniofacial features, and various degrees of mental retardation. Haploinsufficiency of the nuclear receptor binding SET domain containing protein 1 (NSD1) gene, due to either intragenic mutations or whole-gene microdeletions, is found in the majority of patients with SoS. However, in approximately 10-40% of patients with a typical SoS phenotype, no abnormalities are detected. In this study, hemizygous hypermethylation or genomic sequence abnormalities of the promoter region of NSD1 were hypothesized to be the underlying cause in patients with a SoS phenotype, but without confirmed NSD1 alterations. In 18 patients, including one patient with a reported hepatocellular carcinoma, the promoter region of NSD1 was analyzed. However, no hypermethylation or sequence abnormalities in the promoter region could be detected. It therefore seems unlikely that such abnormalities of NSD1 are a major culprit in patients with phenotypical SoS. Additional methods are necessary for detection of other genetic or epigenetic causes of SoS.
Assuntos
Transtornos do Crescimento/genética , Regiões Promotoras Genéticas , Sequência de Bases , Metilação de DNA , Primers do DNA , Humanos , Mutação , Fenótipo , Reação em Cadeia da Polimerase , SíndromeRESUMO
PURPOSE OF REVIEW: Sotos syndrome (SoS) (OMIM #117550) is a childhood overgrowth syndrome characterized by excessive growth, distinctive craniofacial features, developmental delay, and advanced bone age. Recently, haploinsufficiency of the NSD1 gene has been identified as the major cause of SoS, with intragenic mutations or submicroscopic microdeletions being found in about 60 to 75% of clinically diagnosed patients with SoS. RECENT FINDINGS: Recent reports provided much information about the genetic background of SoS, the NSD gene family, and genotype-phenotype correlation. They also added new perspectives in the discussion about a possible association between SoS and neoplasia. SUMMARY: This review focuses on recent genetic developments in SoS. Clinical features and associated anomalies are reviewed in relation to possible functional roles of NSD1. Genotype-phenotype correlation between patients with SoS harboring either intragenic mutations or microdeletions is discussed as well as their implication for possible revision of the diagnostic criteria of SoS. Furthermore, future prospects in genetic research of SoS are presented.