RESUMO
Objective: Tapentadol is a drug of choice for neuropathic cancer pain. DN4 questionnaire quickly determines neuropathic pain component. The aim of this study is to determine the correlation between neuropathic malignant pain component by applying tapentadol antidolorose pharmacotherapy in combination with palliative radiotherapy of osseous neuropathic metastatic changes in breast cancer patients before and after palliative radiotherapy. Methods: The first patients group comprised 30 patients with primary breast cancer and proved painful bone secondary deposits with neuropathy for which tapentadol was prescribed, and they underwent palliative radiotherapy. The second group comprised 30 patients with primary breast cancer and proved painful bone metastases with neuropathy treated only with palliative antidolorose radiotherapy. Key findings : After two-months-follow up, tapentadol group patients had lower DN4 score values (Z=2,021; p=0.043). Significantly lower number of tapentadol group patients was without neuropathic pain after a three-month-follow up (χ ²=5,711; p=0.017). Significantly greater number of tapentadol group patients had best ECOG score 0 ( χ² =7,486; p=0.023). There was statistically significant positive correlation between tapentadol dose and DN4 score in patients after a month (ρ=0,471; p=0.009) and three months after the radiotherapy completion (ρ=0,610; p<0.001). Tapentadol is an opioid analgesic efficient for neuropathy relief in these patients and DN4 questionnaire is an efficient pharmacotherapy tool.
Assuntos
Analgésicos Opioides , Neoplasias da Mama , Neuralgia , Fenóis , Tapentadol , Humanos , Tapentadol/administração & dosagem , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/uso terapêutico , Feminino , Pessoa de Meia-Idade , Inquéritos e Questionários , Neuralgia/tratamento farmacológico , Fenóis/administração & dosagem , Idoso , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Dor do Câncer/tratamento farmacológico , Adulto , Neoplasias Ósseas/secundário , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/complicações , Cuidados Paliativos/métodos , Medição da Dor , SeguimentosRESUMO
OBJECTIVE: The aim of this study was to establish the effects of prolonged formulation of tapentadol in combination with palliative radiotherapy on bone metastatic changes in oncology patients with primary breast cancer and proven bone metastases. PATIENTS AND METHODS: The research was conducted as a prospective study at the Clinic for Oncology, University Clinical Center Nis, Nis, Serbia, during a three-month interval of monitoring the patients. The first group comprised 30 patients with mentioned malignancy for which tapentadol was prescribed, and they underwent palliative radiotherapy for bone metastatic changes. The second group comprised 30 patients with the same disease treated only with pain relief radiotherapy to metastatic changes. All the patients were interviewed using the Pain Detect questionnaire. RESULTS: Significantly more patients from the first group had severe pain in comparison to patients from the control group (χ2=16.596; p<0.001) at the second measurement and also at the third measurement (χ2=15.357; p<0.001). At the third measurement, pain with a neuropathic component was significantly more present in patients from the control group (χ2=8.541; p=0.014). There was a significant pain reduction in both groups - Tapentadol group (χ2=59.513; p<0.001) and control group (χ2=60.000; p<0.001) - and also a significant reduction of neuropathic pain component: Tapentadol group (χ2=56.267; p<0.001) and control group (χ2=60,000; p<0.001). There was a statistically significant positive correlation between tapentadol dose and pain intensity according to the numerical pain scale at all three measurements. CONCLUSIONS: Tapentadol prolonged-release formulation is an effective pharmacotherapy solution, along with palliative radiotherapy, for pain relief in patients with skeletal metastatic breast cancer. Palliative radiotherapy in these patients does not provide adequate neuropathic pain component relief.
Assuntos
Neoplasias da Mama , Dor do Câncer , Dor Crônica , Dor Lombar , Neuralgia , Humanos , Feminino , Tapentadol , Dor do Câncer/tratamento farmacológico , Estudos Prospectivos , Fenóis/uso terapêutico , Dor Lombar/diagnóstico , Neuralgia/tratamento farmacológico , Neoplasias da Mama/complicações , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/induzido quimicamente , Dor Crônica/tratamento farmacológico , Preparações de Ação Retardada , Analgésicos Opioides/uso terapêuticoRESUMO
Following the massive impact of the Covid-19 pandemic on the global economy and on small and medium-sized enterprises (SMEs) in particular, the concept of resilience has experienced a renaissance. As an organizational concept, business model resilience describes the extent to which an organization can maintain or quickly recover its value proposition despite unexpected current or future disruptions (Palzkill-Vorbeck 2018). Although research has been conducted in this area for decades, there is still a lack of a unified framework that brings together the findings from research and links them to organizational practice. The paper addresses this gap by developing a framework for business model resilience and demonstrating its practical relevance for organizational performance during the Covid-19 pandemic in 2020. The framework includes 11 factors that characterize the resilience of an organization's business model. For managers and decision-makers, the framework is an opportunity to assess and improve the resilience of their organizations. For researchers, the framework is an important foundation for transferring the concept of business model resilience into organizational practice.
RESUMO
AIMS: The aim of this study was to investigate whether controlled attenuation parameter (CAP) and liver stiffness measurement (LSM), as assessed by vibration-controlled transient elastography (VCTE), are associated with chronic vascular complications of diabetes mellitus type 2 (T2DM). METHODS: We studied 442 outpatients with established T2DM, and who underwent VCTE and extensive assessment of chronic vascular complications of diabetes. RESULTS: A quarter of analyzed patients had a previous history of myocardial infarction and/or ischemic stroke, and about half of them had at least one microvascular complication (chronic kidney disease (CKD), retinopathy or polyneuropathy). The prevalence of liver steatosis (i.e., CAP ≥ 238 dB/m) and significant liver fibrosis (i.e., LSM ≥ 7.0/6.2 kPa) was 84.2% and 46.6%, respectively. Significant liver fibrosis was associated with an increased likelihood of having myocardial infarction (adjusted-odds ratio 6.61, 95%CI 1.66-37.4), peripheral polyneuropathy (adjusted-OR 4.55, 95%CI 1.25-16.6), CKD (adjusted-OR 4.54, 95%CI 1.24-16.6) or retinopathy (adjusted-OR 1.81, 95%CI 1.62-1.97), independently of cardiometabolic risk factors, diabetes-related variables, and other potential confounders. Liver steatosis was not independently associated with any macro-/microvascular diabetic complications. CONCLUSIONS: Significant liver fibrosis is strongly associated with the presence of macro-/microvascular complications in patients with T2DM. These results offer a new perspective on the follow-up of people with T2DM.
Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Técnicas de Imagem por Elasticidade , Cirrose Hepática , Doenças Cardiovasculares/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/patologia , Humanos , Fígado/diagnóstico por imagem , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/epidemiologia , Cirrose Hepática/etiologia , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
Ever since the introduction of the Market Restructuring Act, the evaluation and price negotiations of drugs have been controversial. While the Federal Joint Committee considers that the process is transparent and in accordance with clear evidence-based criteria, representatives of pharmaceutical companies are particularly critical of the fact that the central association of statutory health insurance is involved in both the determination of added therapeutic benefit of drugs as well as in the subsequent price negotiation. In this study, we investigate these 2 contradictory assessments empirically. For this purpose, we model the benefit assessment and price negotiation processes under AMNOG and analyze their relationship. We show that the number of patients in the target population, and the annual cost of therapy for the appropriate comparator therapy have a negative influence on the determination of the added benefit of the new therapy. The added value itself has a positive (negative) effect on the mark-up for the appropriate comparator therapy (price discount), while the annual treatment costs of the new therapy (which appropriate comparator therapy) have a positive (negative) influence. We find clues, but no significant evidence for the hypothesis that the decisions on the added value of new medicines and the subsequent price negotiations are interdependent.
Assuntos
Custos de Medicamentos , Indústria Farmacêutica , Negociação , Comércio , Custos e Análise de Custo , Alemanha , HumanosRESUMO
In this study, we investigated the relationship between vitamin D, interferon-gamma (IFN-γ), and estradiol (E2) in females of childbearing age with inactive systemic lupus erythematosus (SLE). The study included 22 SLE patients, and 21 age- and gender-matched healthy individuals. Serum concentrations of 25-hydroxyvitamin D3 (25(OH)D3), E2, and IFN-γ were measured by radioimmunoassay using the gamma-counter and ELISA. Patients and control subjects were divided into two groups based on their vitamin D levels (25(OH)D3 ≤ 20 ng/mL; 25(OH)D3 > 20 ng/mL). The median values of IFN-γ and E2 were higher in SLE patients compared to the controls, irrespective of vitamin D level (p = 0.001, p = 0.009, p = 0.003, and p = 0.003, respectively). In SLE patients, there was a negative correlation between IFN-γ and 25(OH)D3 (rs = -0.330; p = 0.03) and a positive correlation between IFN-γ and E2 (rs = 0.404; p = 0.007). This study demonstrates an interesting interplay between vitamin D, INF-γ, and E2 in SLE patients with inactive disease.
Assuntos
Estradiol/sangue , Interferon gama/sangue , Lúpus Eritematoso Sistêmico/sangue , Deficiência de Vitamina D/sangue , Vitamina D/análogos & derivados , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Pessoa de Meia-Idade , Radioimunoensaio , Vitamina D/sangue , Deficiência de Vitamina D/diagnóstico , Adulto JovemAssuntos
Anticorpos Anticardiolipina/imunologia , Síndrome Antifosfolipídica/patologia , Estrogênios/sangue , Lúpus Eritematoso Sistêmico/fisiopatologia , Adulto , Síndrome Antifosfolipídica/imunologia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Fatores de Risco , Índice de Gravidade de Doença , Trombose/etiologia , Trombose/patologia , Adulto JovemRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune connective disease, where vascular lesions are one of the typical symptoms. The differentiation of the type of vascular complications in SLE is very difficult, sometimes impossible, and requires an in-depth immune and histopathological approach, and extensive clinical experience. It may play a key role in the choice of treatment strategy and prediction of patient prognosis. SLE is a prototype of a multisystem autoimmune connective tissue disease, marked by immune complex-mediated lesions of blood vessels in diverse organs. Therefore, awareness of the aetiology, pathophysiology, the clinical and histopathogical setting, and SLE-associated vascular complications is of great clinical significance. In this review, the spectrum of vascular abnormalities and the options currently available to treat the vascular manifestations of SLE are discussed.
Assuntos
Síndrome Antifosfolipídica/complicações , Lúpus Eritematoso Sistêmico/complicações , Vasculite Associada ao Lúpus do Sistema Nervoso Central/terapia , Doenças Vasculares/etiologia , Síndrome Antifosfolipídica/terapia , Humanos , Lúpus Eritematoso Sistêmico/terapia , Doenças Vasculares/terapiaRESUMO
Drug-induced vasculitis is an inflammation of blood vessels caused by the use of various pharmaceutical agents. Vasculitis causes changes in the walls of blood vessels, including thickening, weakening, narrowing and scarring. Inflammation can be short-term (acute) or long-term (chronic) and can be so severe that the tissues and organs supplied by the affected vessels do not get enough blood. The shortage of blood can result in organ and tissue damage, even death. Drug-induced vasculitis is the most common form of vasculitis. The differential diagnosis between drug-induced and idiopathic vasculitic conditions may be difficult in the individual patient. Withdrawal may be helpful to distinguish between these syndromes. Withdrawal of the offending agent alone is often sufficient to induce prompt resolution of clinical manifestations, obviating the need for immunosuppressive and anti-inflammatory drugs. Increasing understanding of the pathophysiological characteristics of all inflammatory vasculitides should lead to better diagnostic and therapeutic approaches to drug-induced vasculitis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Hipersensibilidade a Drogas/complicações , Vasculite/induzido quimicamente , Diagnóstico Diferencial , Progressão da Doença , Glucocorticoides/uso terapêutico , Humanos , Inflamação , Lúpus Eritematoso Sistêmico , Prednisona/uso terapêutico , Medicamentos sob Prescrição/efeitos adversos , Prognóstico , Fatores de Risco , Síndrome de Abstinência a Substâncias , Vasculite/diagnóstico , Vasculite/patologiaRESUMO
Systemic sclerosis is an autoimmune disease characterised by vascular obliteration, excessive extracellular matrix deposition and fibrosis of the connective tissues of the skin, lungs, gastrointestinal tract, heart, and kidneys. The pathogenesis of systemic sclerosis is extremely complex; at present, no single unifying hypothesis explains all aspects. Over the last 20 years increasing evidence has accumulated to implicate infectious agents in the aetiology of systemic sclerosis. Increased antibody titres, a preponderance of specific strains in patients with systemic sclerosis, and evidence of molecular mimicry inducing autoimmune responses suggest mechanisms by which infectious agents may contribute to the development and progression of systemic sclerosis. Here we review the current state of knowledge of infectious risk factors in systemic sclerosis and the possible mechanisms by which infectious exposures might induce pathologic processes.
Assuntos
Doenças Transmissíveis/complicações , Escleroderma Sistêmico/etiologia , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/fisiopatologia , Tecido Conjuntivo/patologia , Citomegalovirus , Progressão da Doença , Matriz Extracelular , Infecções por Helicobacter/complicações , Helicobacter pylori , Humanos , Inflamação/patologia , Parvovirus B19 Humano , Fatores de Risco , Escleroderma Sistêmico/fisiopatologiaRESUMO
Chronic kidney disease (CKD) is a growing public health problem. Individuals in all stages of CKD are at higher risk for development of cognitive impairment and this may be a major determinant in their quality of life (QOL). The prevalence of cognitive deficits is particularly high in subjects with end-stage renal disease (ESRD). While it is sufficiently well documented that ESRD is linked with a change in cognitive function, little is known about the influence of different dialysis modalities on cognitive function. The effect of dialysis modality on risk of cognitive impairment is unclear. Some data suggest that patients with ESRD treated with chronic ambulatory peritoneal dialysis (CAPD) had consistently better cognitive function than patients treated with haemodialysis (HD). We concluded that the previously observed apparent difference between two modalities of dialysis treatments resulted either from very low dialysis delivery or comparison with poorly matched controls. Regarding these data from previous studies we hypothesised that well-dialysed, well-nourished and medically stable HD patients had no cognitive dysfunction in comparison with well-dialysed, well-nourished, medically stable and demographically matched CAPD patients. Also, future studies are needed to differentiate between modality as a risk factor from the factors contributing to selection bias among patients choosing CAPD over HD.
Assuntos
Transtornos Cognitivos/etiologia , Falência Renal Crônica/terapia , Diálise Renal/efeitos adversos , Humanos , Falência Renal Crônica/complicações , Diálise Renal/métodosRESUMO
BACKGROUND: It is found that an antibody directed against DNA topoisomerase I (anti-topo I abs) is detected almost exclusively in systemic sclerosis (SSc). These antibodies are predictors of pulmonary fibrosis and peripheral vascular disease. OBJECTIVE: Metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joints flexion contractures are assessed as markers of active SSc. The aim of this study was to find out is there any relationship between anti-topo I abs and MCP and PIP joints flexion contractures. METHODS: Twenty-eight patients with active disease who fulfilled the American College of Rheumatology criteria for SSc were included in this study. Twenty eight healthy control subjects were also investigated. Clinical and radiological assessments of the hands were carried out. The flexion ranges in the 8 finger joints by goniometric measurement were obtained. Anti-topo I abs with an enzyme linked immunosorbent assay (ELISA) were measured. RESULTS: MCP and PIP joints flexion contractures and the levels of anti-topo I abs were significantly higher in patients with systemic sclerosis than in healthy control. The anti-topo I abs were found in 16 of 28 patients with systemic sclerosis. Sixteen of 28 patients with active disease had MPC and proximal PIP joints flexion contractures. In 16 SSc patients with anti-topo I abs, 13 had metacarpophalangeal and proximal interphalangeal joints flexion contractures. In only 3 patients of 16 with the flexion contractures the levels of anti-topo I abs were negative. The patients with MPC and PIP joints flexion contractures had higher mean value of anti-topo I abs titers (53.718 +/-50.977 vs 8.127 +/- 8.915, P < 0.0001) than did those with no contractures. Furthermore, the titers of anti-topoisomerase I antibody positively correlated with the flexion contractures (r = 0.4252, P = 0.0241). Radiologically, joint space narrowing and flexion contractures of the fingers were seen significantly more frequently in the SSc patients with anti-topo I abs (P < 0.05). CONCLUSION: Serum level of anti-topoisomerase I antibodies is in direct relationship with MPC and PIP joints flexion contractures.
Assuntos
Autoanticorpos/sangue , Contratura/imunologia , DNA Topoisomerases Tipo I/imunologia , Articulações dos Dedos/fisiopatologia , Articulação Metacarpofalângica/fisiopatologia , Escleroderma Sistêmico/imunologia , Adulto , Idoso , Contratura/etiologia , Contratura/fisiopatologia , Feminino , Mãos/diagnóstico por imagem , Humanos , Pessoa de Meia-Idade , Radiografia , Amplitude de Movimento Articular , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/fisiopatologiaAssuntos
Eletrocardiografia , Cardiopatias/complicações , Cardiopatias/fisiopatologia , Pancreatite Necrosante Aguda/complicações , Pancreatite Necrosante Aguda/fisiopatologia , Arritmias Cardíacas/complicações , Arritmias Cardíacas/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Frequência Cardíaca , Humanos , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by beta 2 glycoprotein I (beta 2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-beta 2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.
Assuntos
Anticorpos Antinucleares/química , Anticorpos Antifosfolipídeos/química , Apoptose/imunologia , Glicoproteínas/imunologia , Fragmentos de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Fosfatidilserinas/imunologia , Sequência de Aminoácidos , Anticorpos Antinucleares/genética , Anticorpos Antinucleares/imunologia , Anticorpos Antifosfolipídeos/genética , Anticorpos Antifosfolipídeos/imunologia , DNA/imunologia , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Células Jurkat , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , beta 2-Glicoproteína IRESUMO
Receptor editing is a means by which immature bone marrow B cells can become self-tolerant. Rearrangements of heavy (H) and/or light (L) chain genes are induced by encounter with autoantigens to change the specificity from self to nonself. We have developed site-directed transgenic mice (sd-tg) whose transgenes code for the H chain of antibodies that bind DNA. B cells that express the transgenic H chain associate mainly with four of the 93 functional Vkappa genes of the mouse. Numerous aspartate residues that might inhibit DNA binding by the V(H) domain distinguish these L chain Vkappa sequences, but engaging these Vkappa editors often requires multiple rearrangements. Among the edited B cells is a subset of multispecific cells that express multiple receptors. One consequence of multispecificity is partial autoreactivity; these multispecific B cells may contribute to autoimmunity.
Assuntos
Anticorpos Antinucleares/genética , Autoantígenos/imunologia , Autoimunidade/genética , Subpopulações de Linfócitos B/imunologia , DNA/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Rearranjo Gênico de Cadeia Leve de Linfócito B , Genes de Imunoglobulinas , Cadeias kappa de Imunoglobulina/fisiologia , Tolerância a Antígenos Próprios/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Antinucleares/química , Anticorpos Antinucleares/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Subpopulações de Linfócitos B/metabolismo , Códon/genética , Hibridomas/imunologia , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/genética , Ponto Isoelétrico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransgenesRESUMO
This study analyzes the gene repertoire coding for antibodies to an evolutionary novel immunogenic carbohydrate antigen in mice. The alpha-gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R) is an autoantigen, abundantly expressed in wild type mice, but absent in alpha 1,3galactosyltransferase knock-out (KO) mice, where it can induce the production of the anti-Gal antibody. Hybridoma clones secreting anti-Gal were isolated from different mice and their immunoglobulin genes were analyzed. All anti-Gal clones were found to be encoded by the heavy chain gene VH22.1 and light chain gene VK5.1. Moreover, one 'forbidden' anti-Gal clone, produced in a wild type mouse, was also encoded by VH 22.1 and VK 5.1. The genes coding for the different anti-Gal clones were found to contain somatic mutations and different CDR3 domains. These data imply that a highly restricted gene usage combined with junctional diversity and somatic mutations can generate new antibodies that have not been produced in the course of the evolution of a species.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Evolução Molecular , Galactose/imunologia , Galactosiltransferases/deficiência , Genes de Imunoglobulinas/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Diversidade de Anticorpos/genética , Diversidade de Anticorpos/imunologia , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Sequência de Carboidratos , Células Clonais/imunologia , Células Clonais/metabolismo , Proteínas do Sistema Complemento/imunologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Epitopos/imunologia , Eritrócitos/imunologia , Galactosiltransferases/genética , Genes de Imunoglobulinas/imunologia , Mutação em Linhagem Germinativa/genética , Humanos , Hibridomas/imunologia , Hibridomas/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Imunoglobulina M/química , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Alinhamento de Sequência , Baço/imunologiaRESUMO
Autoantibodies to DNA and chromatin employ junctional diversity and somatic mutations to generate or enhance antigen recognition. To define the role of diversity generating mechanisms in the etiology of autoantibodies to nuclear antigens, the heavy (H) chain of a murine autoantibody, 3H9, was used in its somatically mutated or germ-line form in conjunction with its own or with heterologous CDR3 (H3) domains. The resulting H chains were expressed together with the 3H9 light (L) chain as single-chain Fv (scFv) in Escherichia coli and assayed for binding to DNA, nucleosomes, or cardiolipin by enzyme-linked immunosorbent assay. All recombinant scFv exhibited nearly identical binding to cardiolipin. In contrast, the binding to nuclear antigens was drastically reduced by the reversion of mutations in 3H9 or the exchange of H3, such that only 3H9 itself bound strongly to single-stranded DNA, double-stranded DNA and nucleosomes. The results illustrate diverse interactions between a single combining site and different autoantigens. The analysis of these interactions suggests that the 3H9 VH domain, as encoded by the germ line, directs binding to cardiolipin, whereas structural determinants of H3, in concert with the remainder of the combining site, guide the maturation of antibody binding toward nuclear autoantigens.
Assuntos
Cardiolipinas/metabolismo , Regiões Determinantes de Complementaridade/metabolismo , DNA/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Nucleossomos/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Cadeias Pesadas de Imunoglobulinas/química , Dados de Sequência MolecularRESUMO
OBJECTIVE: To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). METHODS: A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. RESULTS: DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. CONCLUSION: Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.
Assuntos
DNA/imunologia , Adulto , Anticorpos Antinucleares/genética , Arginina/química , Sequência de Bases , Células da Medula Óssea/imunologia , DNA/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Isotipos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Lúpus Eritematoso Sistêmico/imunologia , Biblioteca de Peptídeos , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Antibodies to DNA define an important autospecificity that arises in systemic lupus erythematosus (SLE). To elucidate the molecular features that may explain the pathogenesis of SLE, a heterologous system for expression of cloned V genes is often desirable. Here, a single-chain Fv coding domain was constructed by using the heavy- and light-chain V genes of a high-affinity site-directed mutant of the murine anti-dsDNA autoantibody, 3H9. This scFv was joined in frame to the c-jun leucine zipper for dimerization, and to two affinity tags, domain B of the staphylococcal protein A and a pentahistidine peptide, for purification. Dimerization of the scFv was determined by size-exclusion chromatography. The yields of the scFv following affinity purification on IgG agarose or Ni-NTA agarose were compared, and the activities of the resulting protein fractions were determined. A two-step purification of periplasmic extracts on Ni-NTA agarose and IgG agarose, followed by elution with 3.5 M MgCl(2), yielded scFv with the highest specific activity. The final purified material bound DNA by ELISA, electrophoretic mobility shift assay, and immunofluorescence of fixed Hep-2 cells. Antibodies purified in this fashion should have applications in structure/function studies in which it is essential to generate highly purified antigen-combining sites.