RESUMO
There is an urgent unmet need for human tissue bioassays to predict cytokine storm responses to biologics. Current bioassays that detect cytokine storm responses in vitro rely on endothelial cells, usually from umbilical veins or cell lines, cocultured with freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. These assays therefore comprise cells from 2 separate donors and carry the disadvantage of mismatched tissues and lack the advantage of personalized medicine. Current assays also do not fully delineate mild (such as Campath) and severe (such as TGN1412) cytokine storm-inducing drugs. Here, we report a novel bioassay where endothelial cells grown from stem cells in the peripheral blood (blood outgrowth endothelial cells) and PBMCs from the same donor can be used to create an autologous coculture bioassay that responds by releasing a plethora of cytokines to authentic TGN1412 but only modestly to Campath and not to control antibodies such as Herceptin, Avastin, and Arzerra. This assay performed better than the traditional mixed donor assay in terms of cytokine release to TGN1412 and, thus, we suggest provides significant advancement and a definitive system by which biologics can be tested and paves the way for personalized medicine.
Assuntos
Produtos Biológicos/farmacologia , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Alemtuzumab , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Bevacizumab , Bioensaio/métodos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultura/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Reprodutibilidade dos Testes , Soro/química , Trastuzumab , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Investigation of possible B12 and folate deficiencies requires measurement of these vitamins in serum. There is evidence that holotranscobalamin (holoTC), the active portion of B12 available to cells, is a more specific marker of early B12 deficiency than total B12. The availability of immunoassays for holoTC prompted an international collaborative study to assign a holoTC value to the World Health Organization (WHO) 1st International Standard (IS) for vitamin B12 and serum folate, 03/178. METHODS: The IS, 03/178, and three serum samples with different holoTC levels were assayed by 12 laboratories in eight countries using manual and automated immunoassays for holoTC; one laboratory additionally performed an in-house assay. Fourteen sets of data were analysed. RESULTS: Overall, the IS, 03/178, and the three serum samples demonstrated assay linearity and parallelism. An overall geometric mean (GM) holoTC value of 106.8 pmol/L was obtained for 03/178, with an inter-laboratory geometric coefficient of variation (GCV) of 10.5%. There was a reduction in inter-laboratory variability when the holoTC levels in the serum samples were determined relative to the IS with an assigned holoTC value rather than to the assays' calibration. Accelerated degradation studies showed that 03/178 was sufficiently stable to serve as an IS for holoTC. CONCLUSIONS: The WHO Expert Committee on Biological Standardization endorsed the proposal to assign a holoTC value of 107 pmol/L to 03/178, corresponding to 0.107 pmol per ampoule, for use as the 1st IS for vitamin B12, serum folate, and holoTC.
Assuntos
Ácido Fólico/sangue , Imunoensaio/normas , Laboratórios/normas , Transcobalaminas/análise , Vitamina B 12/sangue , Automação , Calibragem , Humanos , Cooperação InternacionalRESUMO
Intravenous immunoglobulin (IVIG) products are generally safe and efficacious, although treatment-related adverse reactions can occur in recipients. Adverse reactions include hemolysis in non-blood group O recipients linked to the passive transfer of anti-A and/or anti-B present in the fractionated immunoglobulin product. In light of the recent increase in reported cases of severe hemolysis associated with anti-A and/or anti-B, this article traces the development of pharmacopoeial specifications, tests, and reference reagents to control their titers in IVIG products.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Hemólise , Imunoglobulinas Intravenosas/efeitos adversos , Imunoglobulinas Intravenosas/imunologia , Isoanticorpos/efeitos adversos , Isoanticorpos/imunologia , Hemólise/efeitos dos fármacos , Hemólise/imunologia , Humanos , Imunoglobulinas Intravenosas/uso terapêuticoRESUMO
BACKGROUND: The measurement of serum IgE aids in the diagnosis and management of atopic allergic disease and hyper-IgE immunodeficiency syndromes. The 2nd World Health Organization (WHO) International Reference Reagent (IRR) for serum IgE (75/502; 5000 IU/ampoule), is widely used to calibrate assays for serum IgE. Exhaustion of stocks of the 2nd IRR necessitated the production of a replacement preparation and its evaluation in an international collaborative study to determine its suitability to serve as the 3rd International Standard (IS) for serum IgE. METHODS: Sera and defibrinated plasma with elevated IgE levels were pooled and lyophilised in ampoules. This preparation, coded 11/234, was assayed by 18 laboratories in 11 countries using commercial assay methodology for IgE, along with the 2nd IRR, 75/502, and two lyophilised serum samples. RESULTS: Overall, there were no consistent differences in the way that the candidate IS (11/234), the IRR (75/502), and the two serum samples behaved in the assays with respect to linearity and parallelism. The mean IgE value of the candidate IS, 11/234, relative to the IRR, 75/502, was 13,411 IU/mL based on parallel line analysis of raw assay data at NIBSC, and 13,551 IU/mL based on the laboratories' own estimates after correcting for the values obtained for 75/502. CONCLUSIONS: The use of 11/234 will ensure that assays for serum IgE continue to be well standardised. The preparation was established by the WHO Expert Committee on Biological Standardization as the 3rd IS for serum IgE with an assigned value of 13,500 IU/mL, corresponding to 6750 IU/ampoule.
Assuntos
Análise Química do Sangue/normas , Imunoensaio/normas , Imunoglobulina E/sangue , Análise de Variância , Análise Química do Sangue/estatística & dados numéricos , Liofilização , Humanos , Imunoensaio/estatística & dados numéricos , Imunoglobulina E/isolamento & purificação , Indicadores e Reagentes/normas , Laboratórios , Proteólise , Padrões de Referência , Organização Mundial da SaúdeRESUMO
The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab')(2) and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants. Superagonistic activity, as measured by cytokine release and proliferation, was assessed by exposing PBMCs to immobilized mAbs/fragments or to aqueous mAbs/fragments in the presence of HUVEC monolayers. Removing the Fc generally curtailed or abolished PBMC activation. However, eliminating detectable FcγR-binding of the IgG4 by mutation (L235E) did not abrogate activity. Stabilizing the "wild-type" IgG4 hinge (S228P) enhanced activity without increasing FcγR binding, which could only partially be explained by inhibition of Fab arm-exchange. Subclass switching the IgG4 mAb to IgG1 decreased activity, whereas switching to IgG2 markedly increased activity. We conclude that the C region strongly influences in vitro CD28-mediated superagonistic signaling. Superagonism requires an intact Fc, as shown by the absence of activity of TGN1412 Fab and F(ab')(2) fragments, but, notably, appears to be relatively independent of FcγR-binding properties. We propose that the Fc, potentially through restricting flexibility, maintains a favorable V region conformation to allow superagonistic activity. These findings have important implications for Ab design strategies.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais/fisiologia , Regiões Constantes de Imunoglobulina/fisiologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados/fisiologia , Sítios de Ligação de Anticorpos/imunologia , Proliferação de Células , Células Cultivadas , Ensaios Clínicos Fase I como Assunto , Técnicas de Cocultura , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Regiões Constantes de Imunoglobulina/química , Regiões Constantes de Imunoglobulina/metabolismo , Fragmentos Fab das Imunoglobulinas/fisiologia , Fragmentos Fc das Imunoglobulinas/fisiologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Macaca fascicularis , Ligação Proteica/imunologia , Receptores de IgG/metabolismo , Receptores de IgG/fisiologia , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Recent work on cognitive-behavioural models of obsessive-compulsive disorder has focused on the roles played by various aspects of self-perception. In particular, moral self-ambivalence has been found to be associated with obsessive-compulsive phenomena. AIMS: In this study we used an experimental task to investigate whether artificially priming moral self-ambivalence would increase participants' deliberation on ethical problems, an index that might be analogous to obsessive-compulsive behaviour. METHOD: Non-clinical participants completed two online tasks designed to prime either moral self-ambivalence, general uncertainty, or neither. All participants then completed a task requiring them to consider solutions to moral dilemmas. We recorded the time participants took to respond to the dilemmas and the length of their responses; we then combined these variables to create a measure of deliberation. RESULTS: Priming moral self-ambivalence led to increases in deliberation, but this was only significant among those participants who scored highly on a baseline measure of moral self-ambivalence. Priming general uncertainty had no significant effect upon deliberation. CONCLUSIONS: The results suggest that moral self-ambivalence may play a role in the maintenance of obsessive-compulsive behaviour. We propose that individuals who are morally self-ambivalent might respond to situations in which this ambivalence is made salient by exhibiting behaviour with obsessive-compulsive characteristics. These findings have implications for the incorporation of ideas about self-concept into theories of obsessive-compulsive disorder.
Assuntos
Cultura , Desenvolvimento Moral , Transtorno Obsessivo-Compulsivo/psicologia , Priming de Repetição , Autoimagem , Incerteza , Adolescente , Adulto , Feminino , Humanos , Controle Interno-Externo , Masculino , Motivação , Psicometria/estatística & dados numéricos , Inquéritos e Questionários , Adulto JovemRESUMO
AIM: To determine if cytokine release with a solid phase assay is predictive of adverse responses for a range of therapeutic mAbs. METHODS: Cytokine ELISAs and a multi-array system were used to compare responses generated by different therapeutic mAbs using a solid phase assay. Flow cytometry was employed to determine the cellular source of those cytokines. RESULTS: Only TGN1412 and muromonab-CD3 stimulated CD4+ T-cell mediated cytokine release characterized by significant (all P < 0.0001) IFNγ, TNFα, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17 and IL-22 release, comparable with T-cell mitogen. Significantly greater (P < 0.0001) IL-2 release with TGN1412 (2894-6051 pg ml⻹) compared with muromonab-CD3 (62-262 pg ml⻹) differentiated otherwise comparable cytokine responses. Likewise, TGN1412 stimulated significantly more (P = 0.0001) IL-2 producing CD4+ T-cells than muromonab-CD3 and induced Th1, Th2, Th17 and Th22 subsets that co-release this cytokine. Significant TNFα release was observed with bevacizumab (P = 0.0001), trastuzumab (P = 0.0031) and alemtuzumab (P = 0.0177), but no significant IL-2 release. TGN1412 and muromonab-CD3 caused pro-inflammatory cytokine release despite significantly (both P < 0.0001) increasing numbers of T-cells with a regulatory phenotype. CONCLUSIONS: The severity of the adverse response to TGN1412 compared with muromonab-CD3 and other therapeutic mAbs correlates with the level of IL-2 release.
Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais/efeitos adversos , Citocinas/imunologia , Interleucina-2/imunologia , Muromonab-CD3/efeitos adversos , Projetos de Pesquisa/normas , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Ensaios Clínicos como Assunto , Citocinas/efeitos dos fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Muromonab-CD3/imunologia , Medição de RiscoRESUMO
The failure of preclinical testing to predict the severity of the cytokine storm experienced by the recipients of the superagonistic anti-CD28 monoclonal antibody (mAb) TGN1412 during its Phase 1 clinical trial prompted the development of new in vitro experimental approaches for mimicking in vivo cytokine release and lymphoproliferation. Peripheral blood mononuclear cells (PBMC) presented to TGN1412 immobilised on plastic has previously been shown to stimulate a pro-inflammatory cytokine response. The aim of the present study was to investigate a 'co-culture' model for the detection of TGN1412-like immunomodulatory activity in which TGN1412 was presented to PBMC in the presence of monolayers of endothelium-derived cells and other cell types, followed by measurement of cytokine levels in the culture supernatants and proliferation of PBMC. Culturing PBMC with TGN1412 over primary human umbilical vein endothelial cells (HUVEC) and HUVEC-derived cell lines retaining classic endothelial markers, but not cell lines of non-endothelial origin, mediated the specific release of IL-6, IL-8 and TNFα, and proliferation of PBMC. Low levels of IL-2 and IFNγ were also detected in supernatants with most donors of PBMC. An anti-CD28 mAb agonist, i.e., not a superagonist like TGN1412, did not stimulate cytokine release or proliferation of PBMC in co-cultures. In conclusion, co-culture experiments for TGN1412-specific cytokine release required cells of endothelial origin. However, the profile of released cytokines in co-cultures did not mirror that in the clinical trial participants or the responses from PBMC exposed to TGN1412 immobilised on plastic, suggesting that TGN1412 stimulation of PBMC can occur through more than one mechanism.
Assuntos
Anticorpos Monoclonais/farmacologia , Células Endoteliais/citologia , Leucócitos Mononucleares/citologia , Anticorpos Monoclonais Humanizados , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Fatores de Tempo , Veias Umbilicais/citologiaRESUMO
The beneficial effect of antibody therapy in human disease has become well established mainly for the treatment of cancer and immunological disorders. The inherent monospecificity of mAbs present limitations to mAb therapy which have become apparent notably in addressing complex entities like infectious agents or heterogenic endogenous targets. For such indications mixtures of antibodies comprising a combination of specificities would convey more potent biological effect which could translate into therapeutic efficacy. Recombinant polyclonal antibodies (rpAb) consisting of a defined number of well-characterized mAbs constitute a new class of target specific antibody therapy. We have developed a cost-efficient cell banking and single-batch manufacturing concept for the production of such products and demonstrate that a complex pAb composition, rozrolimupab, comprising 25 individual antibodies can be manufactured in a highly consistent manner in a scaled-up manufacturing process. We present a strategy for the release and characterization of antibody mixtures which constitute a complete series of chemistry, manufacturing, and control (CMC) analytical methods to address identity, purity, quantity, potency, and general characteristics. Finally we document selected quality attributes of rozrolimupab based on a battery of assays at the genetic-, protein-, and functional level and demonstrate that the manufactured rozrolimupab batches are highly pure and very uniform in their composition.
Assuntos
Biotecnologia/normas , Imunoglobulina G/biossíntese , Proteínas Recombinantes/biossíntese , Biotecnologia/métodos , Linhagem Celular , Humanos , Imunoglobulina G/química , Imunoglobulina G/uso terapêutico , Controle de Qualidade , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Recent interest in the role of vulnerability factors in obsessional washing has suggested that disgust sensitivity, danger expectancy and health anxiety may be of interest. AIMS: This study explores the differential impact of these factors on both behavioural and cognitive measures of washing behaviour and is based on a replication of the Jones and Menzies (1997) experiment, during which participants immersed their hands in a noxious compound while rating themselves on a range of measures: the time they subsequently took to wash their hands was measured and danger expectancies were found to be the best predictor of this. METHOD: The present study added measures of disgust sensitivity and health anxiety to this experimental methodology while removing factors they found to be of little import to compulsive washing. Thirty non-clinical participants took part. RESULTS: Results confirmed that disgust sensitivity was related to the behavioural measure of washing time, but that this relationship was almost entirely mediated by the danger expectancy concerning judgements of severity of consequent disease. However, a different pattern emerged when the outcome measure was questionnaire based: danger expectancy was not at all related to this. Disgust sensitivity mediated the relationship between health anxiety and scores on a questionnaire measure of washing compulsions. Interestingly, these scores were not related to the behavioural measure of washing time. CONCLUSIONS: The implications of these relationships to the further development of subtypes of Obsessive Compulsive Disorder (OCD) are discussed.
Assuntos
Atitude Frente a Saúde , Transtornos Cognitivos/psicologia , Cultura , Comportamento Perigoso , Emoções , Desinfecção das Mãos , Transtorno Obsessivo-Compulsivo/psicologia , Adulto , Ansiedade/diagnóstico , Ansiedade/psicologia , Nível de Alerta , Transtornos Cognitivos/diagnóstico , Feminino , Humanos , Julgamento , Masculino , Transtorno Obsessivo-Compulsivo/diagnóstico , Inventário de Personalidade/estatística & dados numéricos , Psicometria , Reprodutibilidade dos Testes , Adulto JovemRESUMO
BACKGROUND: The usefulness of serum transferrin receptor (sTfR) as a marker of iron deficiency is limited by lack of standardization of commercial immunoassays for sTfR. An international collaborative study was performed to evaluate a lyophilized preparation of recombinant soluble transferrin receptor (rsTfR) for its suitability to serve as a World Health Organization (WHO) Reference Reagent to standardize immunoassays for sTfR. METHODS: The concentration of pure rsTfR was determined from the A(280 nm) using the adjusted theoretical extinction coefficient and molecular weight calculated from its sequence, before dilution and lyophilization in a sTfR-depleted serum matrix. Six manufacturers and a health protection laboratory assayed the candidate Reference Reagent, coded 07/202, along with three lyophilized serum samples, using commercial assays for sTfR. RESULTS: Dose-response plots demonstrated acceptable overall parallelism between 07/202, manufacturers' in-house standards, and serum samples. However, there was poor agreement on the estimated (r)sTfR content of 07/202 and serum samples. Expressing the sTfR content of the serum samples relative to 07/202 markedly improved agreement between methods. CONCLUSIONS: Use of 07/202 would reduce inter-method variability. The preparation was established as the 1st WHO Reference Reagent for sTfR with assigned free rsTfR monomer values of 21.7 mg/L and 303 nmol/L (0.5 mL reconstitution).
Assuntos
Imunoensaio/normas , Receptores da Transferrina/sangue , Proteínas Recombinantes/normas , Imunoensaio/métodos , Indicadores e Reagentes , Estabilidade Proteica , Receptores da Transferrina/genética , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Padrões de Referência , Reprodutibilidade dos Testes , Organização Mundial da SaúdeRESUMO
Anaemia is a major global health problem. Although the main cause is iron deficiency, anaemia also results from other nutritional deficiencies (folate and vitamin B12), haemolytic disorders including haemoglobinopathies, and bone marrow disorders. Accurate diagnosis of anaemia is dependent on reliable diagnostic tests and reference ranges, which in turn are dependent on effective standardisation. Standardisation is achieved through the availability of reference materials and reference measurement procedures. International biological reference materials have therefore been developed to standardise and control diagnostic tests for anaemia for a diverse range of analytes including total haemoglobin and haemoglobin types, ferritin, the serum transferrin receptor, serum vitamin B12 and folate, whole blood folate, and alloantibodies which mediate immune haemolytic anaemia.
Assuntos
Anemia/sangue , Anemia/diagnóstico , Ferritinas/sangue , Ferritinas/normas , Ácido Fólico/sangue , Ácido Fólico/normas , Hemoglobinas/normas , Humanos , Cooperação Internacional , Receptores da Transferrina/sangue , Padrões de Referência , Sensibilidade e Especificidade , Vitamina B 12/sangue , Vitamina B 12/normasRESUMO
Memory failures reported by obsessive-compulsive (OC) checkers often seem to be errors of "reality-monitoring", or misremembering whether one performed or imagined performing an action. To examine these memory processes in the context in which such errors are said to occur, an in-home reality-monitoring experiment involving bothersome and non-bothersome actions was conducted with 21 OC checkers and 24 non-clinical controls. OC checkers reported poorer confidence in memory, but both groups performed similarly on tests of immediate and delayed free and prompted recall. Among OC checkers (but not controls), accuracy in recall and confidence in memory were correlated. Theoretical implications are discussed.
Assuntos
Imaginação , Transtornos da Memória/diagnóstico , Transtorno Obsessivo-Compulsivo/diagnóstico , Teste de Realidade , Atitude , Grupos Controle , Manual Diagnóstico e Estatístico de Transtornos Mentais , Feminino , Seguimentos , Humanos , Entrevistas como Assunto , Masculino , Transtornos da Memória/psicologia , Memória de Curto Prazo , Rememoração Mental , Modelos Psicológicos , Testes Neuropsicológicos , Transtorno Obsessivo-Compulsivo/psicologia , Inventário de Personalidade , Escalas de Graduação Psiquiátrica , Reconhecimento Psicológico , Retenção Psicológica , Autoimagem , Análise e Desempenho de TarefasRESUMO
It has been suggested that VH4-34 gene segment expression is counter-selected in multiple myeloma (MM) due to a self-tolerance mechanism. We cloned and sequenced a VH4-34 gene segment from bone marrow mononuclear cells of a stage III MM patient. We show that VH4-34 was expressed by the serum IgA myeloma (M)-protein, as demonstrated by reactivity with the VH4-34 specific 9G4 mAb and mass spectrometry (MS). The M-protein had weak reactivity with nuclei. These results demonstrate that VH4-34 may be expressed in secreted IgA M-protein with weak autoreactivity. Thus, counter-selection of VH4-34 is pronounced but not absolute in MM. Mechanisms of how VH4-34 can occasionally be expressed in MM and clinical implications are discussed.
Assuntos
Imunoglobulina A/imunologia , Mieloma Múltiplo/imunologia , Proteínas do Mieloma/imunologia , Idoso , Sequência de Aminoácidos , Anticorpos Antinucleares/genética , Anticorpos Antinucleares/imunologia , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Autoimunidade , Linfócitos B/imunologia , Linfócitos B/patologia , Sequência de Bases , Células Clonais/imunologia , Células Clonais/patologia , Análise Mutacional de DNA , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Rearranjo Gênico de Cadeia Leve de Linfócito B , Genes de Imunoglobulinas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Espectrometria de Massas , Dados de Sequência Molecular , Mieloma Múltiplo/patologia , Proteínas do Mieloma/genética , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/imunologia , Tolerância a Antígenos Próprios , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
We have shown that two of the matrix metalloproteinases (MMPs), matrilysin and stromelysin-1, are capable of cleaving all of the human IgG subclasses. The cleavage occurs at a conserved site in the CH(2) domain of the heavy chain of IgG, releasing a single chain Fc-like fragment. We have not been able to demonstrate cleavage of IgA, IgD, IgM or IgE classes, which lack the cleavage site, nor could we show cleavage of IgG by collagenase, gelatinase, macrophage metalloelastase or membrane-type (MT)-MMP. This cleavage of IgG, by separating the antigen-binding (Fabprime prime or minute)(2) from the Fc portion, will remove much of the immunoglobulins' functionality, e.g. complement fixation, Fc receptor binding. In the context of a tumour producing matrilysin or stromelysin, this may represent a way in which the tumour protects itself from ADCC. In inflamed or damaged tissues where plasma protein leakage occurs, degradation by MMPs may be a mechanism for clearance of IgG.
Assuntos
Imunoglobulina G/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Clonagem Molecular , Cricetinae , Humanos , Dados de Sequência Molecular , Papaína/metabolismo , Pepsina A/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Botulinum type A toxin (BoNT/A) is defined by its specific endopeptidase cleavage of SNAP25 between Gln(197) and Arg(198) under reducing conditions. The neurotoxin is widely used for therapeutic or cosmetic purposes, but should not contain other toxin serotypes or unwanted protease activities. Using a neoepitope endopeptidase immunoassay, additional cleavage between Arg(198) and Ala(199) was detected with a range of therapeutic BoNT/A products confirming an earlier report of an unidentified proteolytic component. By developing the assay and making it insensitive to BoNT/C1, any activity due to the type C1 toxin was excluded. Therapeutic preparations consist of ng quantities of toxin protein which are typically stabilised by 0.125-30 mg of HSA. An excellent correlation (R(2)=0.993) between HSA content per vial and measured activity was obtained within the therapeutic BoNT/A products tested. No activity was detected in any of the non-albumin formulated preparations, thereby identifying HSA as the source of the unknown protease for the first time. To investigate the cause of this activity, either as an intrinsic molecular activity of albumin or due to an albumin-associated purification contaminant, further studies on a variety of commercial plasma-derived HSA products or recombinant HSA materials free from potential plasma contaminants were carried out. The measured proteolytic levels were highly consistent amongst preparations, and could all be partially inhibited by the presence of zinc and blocked by PKSI-527 and aprotinin. By contrast, the data did not support the role of plasmin, kallikrein, trypsin, α(2)-antiplasmin-plasmin complexes or HSA purification contaminants, PKA (prekallikrein activator) or kallikrein-like activity. Taken together, these findings indicate a new intrinsic proteolytic activity of the albumin molecule revealed under reducing conditions as the source of the unexpected Arg-Ala cleaving activity.
Assuntos
Endopeptidases/química , Imunoensaio/métodos , Albumina Sérica/análise , Albuminas/química , Toxinas Botulínicas/química , Cisteína/química , Relação Dose-Resposta a Droga , Epitopos/química , Humanos , Neurotoxinas/química , Peptídeos/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Proteína 25 Associada a Sinaptossoma/química , Calicreínas Teciduais/química , Ácido Tranexâmico/análogos & derivados , Ácido Tranexâmico/química , Zinco/químicaRESUMO
Two methods for predicting the risk of pro-inflammatory clinical infusion reactions during monoclonal antibody therapy were evaluated. In the first, the antibody of interest is immobilised by air-drying onto 96-well plates prior to the addition of human peripheral blood mononuclear cells (PBMCs). In the second, the antibody is added in aqueous phase to a co-culture of human PBMCs and human endothelium-derived cells. In both methods the cells are incubated with the antibody to allow the accumulation of pro-inflammatory cytokines, quantified by enzyme-linked immunosorbent assay (ELISA). The antibodies associated with clinical infusion reactions, Herceptin, Campath-1H and TGN1412, gave the largest responses taking into account the data for all readouts (tumour necrosis factor-α, TNF, interleukin-6, IL-6, IL-8, IL-2 and cell proliferation) for both methods. Overall, the antibodies tested could be ranked as follows: TysabriAssuntos
Anticorpos Monoclonais/efeitos adversos
, Imunoterapia/efeitos adversos
, Inflamação/etiologia
, Anticorpos Imobilizados
, Anticorpos Monoclonais/administração & dosagem
, Anticorpos Monoclonais/uso terapêutico
, Proliferação de Células
, Técnicas de Cocultura
, Citocinas/análise
, Citocinas/biossíntese
, Células Endoteliais/imunologia
, Ensaio de Imunoadsorção Enzimática/métodos
, Humanos
, Imunoensaio/métodos
, Inflamação/imunologia
, Mediadores da Inflamação/análise
, Mediadores da Inflamação/metabolismo
, Infusões Intravenosas
, Leucócitos Mononucleares/imunologia
, Leucócitos Mononucleares/patologia
, Fatores de Risco
RESUMO
A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999-2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991-1994 and NHANES 1999-2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007-2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.
Assuntos
Ácido Fólico/sangue , Inquéritos Nutricionais , Biomarcadores/sangue , Cromatografia Líquida , Eritrócitos/química , Humanos , Técnicas Microbiológicas , Ensaio Radioligante , Padrões de Referência , Espectrometria de Massas em TandemRESUMO
A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12-related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES--serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)--and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12-related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.