RESUMO
BACKGROUND: Patients with acute ischemic stroke (AIS) are at high risk of adverse cardiovascular events. Until now, the burden of myocardial injury derived from cardiovascular magnetic resonance imaging (CMR) has not been established in this population. METHODS: Patients with AIS underwent CMR at 3 Tesla within 120 h after the index stroke as part of a prospective, single-center study. Patients with persistent atrial fibrillation were excluded. Morphology and function of both cardiac chambers and atria were assessed applying SSFP cine. Myocardial tissue differentiation was based on native and contrast-enhanced imaging including late gadolinium enhancement (LGE) after 0.15 mmol/kg gadobutrol for focal fibrosis and parametric T2- and T1-mapping for diffuse findings. To detect myocardial deformation global longitudinal (GLS), circumferential (GCS) and radial (GRS) strain was measured applying feature tracking. Cardiac troponin was measured using a high-sensitivity assay (99th percentile upper reference limit 14 ng/L). T2 mapping values were compared with 20 healthy volunteers. RESULTS: CMR with contrast media was successfully performed in 92 of 115 patients (mean age 74 years, 40% female, known myocardial infarction 6%). Focal myocardial fibrosis (LGE) was detected in 31 of 92 patients (34%) of whom 23/31 (74%) showed an ischemic pattern. Patients with LGE were more likely to have diabetes, prior myocardial infarction, prior ischemic stroke, and to have elevated troponin levels compared to those without. Presence of LGE was accompanied by diffuse fibrosis (increased T1 native values) even in remote cardiac areas as well as reduced global radial, circumferential and longitudinal strain values. In 14/31 (45%) of all patients with LGE increased T2-mapping values were detectable. CONCLUSIONS: More than one-third of patients with AIS have evidence of focal myocardial fibrosis on CMR. Nearly half of these changes may have acute or subacute onset. These findings are accompanied by diffuse myocardial changes and reduced myocardial deformation. Further studies, ideally with serial CMR measurements during follow-up, are required to establish the impact of these findings on long-term prognosis after AIS.
Assuntos
Cardiomiopatias , AVC Isquêmico , Infarto do Miocárdio , Humanos , Feminino , Idoso , Masculino , Meios de Contraste , AVC Isquêmico/patologia , Estudos Prospectivos , Função Ventricular Esquerda , Imagem Cinética por Ressonância Magnética/métodos , Gadolínio , Cardiomiopatias/patologia , Miocárdio/patologia , Imageamento por Ressonância Magnética , Infarto do Miocárdio/patologia , Fibrose , Valor Preditivo dos TestesRESUMO
PURPOSE: Post-irradiation vasculopathy is a severe form of atherosclerosis and affects the prognosis of head and neck cancer survivors. Sonographic intima-media thickness (IMT) precedes stenosis, plaque formation, and cerebrovascular events. Therefore, IMT may be a valuable screening marker for post-irradiation toxicity. However, the critical irradiation dose and the onset of IMT increase remain unclear. METHODS: The cross-sectional study analysed the carotid artery IMT in 96 irradiated patients and 41 controls regarding irradiation dose, post-irradiation-interval, and cardiovascular risk factors. Distinct irradiation doses to the tumour side and the contralateral hemineck enabled detection of dose depended effects within one patient and control of risk factors. RESULTS: Radiotherapy caused a dose-dependent increase in IMT. The toxicity did not have saturation effects for > 60 Gy. The IMT increase occurred in short-term following radiotherapy and the risk for a pathological value (> 0.9 mm) rose significantly. The correlation between IMT and radiotherapy was comparable to established cardiovascular risk factors. CONCLUSION: Radiotherapists should consider the additional toxicity of high doses for non-metastatic head and neck cancer. If neck metastases require radiotherapy with boost, IMT measurement is suitable for early detection of carotid artery damage.
Assuntos
Espessura Intima-Media Carotídea , Neoplasias de Cabeça e Pescoço , Estudos Transversais , Detecção Precoce de Câncer , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Fatores de Risco , UltrassonografiaRESUMO
Early differential diagnosis of parkinsonism is of paramount therapeutic and prognostic importance. In the present review, the diagnostic value of routinely used nuclear medicine procedures is presented and critically discussed. The [(123)I]FP-CIT single-photon emission computed tomography (SPECT) is the method of choice for differentiation between neurodegenerative and non-neurodegenerative parkinsonism. The [(18)F]fluorodeoxyglucose positron emission tomography ([(18)F]FDG-PET) method provides a very high diagnostic accuracy for differentiating between Parkinson's disease (PD) and atypical Parkinsonian syndromes (APS), which is clearly superior to the accuracy of [(123)I]FP-CIT SPECT, [(123)I]IBZM SPECT and [(123)I]MIBG scintigraphy. Furthermore, [(18)F]FDG-PET is the only of the aforementioned techniques that also allows a reliable differentiation between APS subgroups (e.g., multiple system atrophy, progressive supranuclear palsy and corticobasal degeneration). Current studies are investigating the probable value of [(18)F]FDG-PET for risk stratification of dementia in PD.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Transtornos Parkinsonianos/diagnóstico por imagem , Transtornos Parkinsonianos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Receptores Dopaminérgicos/metabolismo , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Diagnóstico Diferencial , Humanos , Imagem Molecular/métodosRESUMO
BACKGROUND AND PURPOSE: Brain imaging with positron emission tomography using [(18) F]fluorodeoxyglucose (FDG-PET) and transcranial B-mode sonography (TCS) improves the differential diagnosis of parkinsonism. The diagnostic merits of these approaches in identifying and differentiating atypical parkinsonian syndromes (APS) are compared. METHODS: Data were included from 36 patients with clinically suspected APS who underwent PET and TCS. FDG-PET scans were analyzed by visual assessment (including voxel-based statistical maps) of a priori defined disease-specific metabolic patterns. Sonographers achieved diagnoses according to pre-defined criteria for echogenicities of the substantia nigra and lenticular nucleus, and third ventricle diameter. Patients with APS were identified and allocated to the subgroups multiple system atrophy (MSA), progressive supranuclear palsy (PSP) or corticobasal degeneration (CBD). RESULTS: After a median follow-up period of 9 months, the final clinical diagnoses (reference standard) were Parkinson's disease, n = 15; MSA, n = 9; PSP, n = 7; and CBD, n = 5 (n = 21 APS in total). Six patients (4 APS) showed an insufficient bone window for TCS. In the remaining 30 patients, sensitivity/specificity for diagnosing APS were 82%/100% and 82%/85% for FDG-PET and TCS, respectively. Diagnostic accuracies did not differ between FDG-PET (90%) and TCS (83%; P = 0.69). Likewise, overall accuracy of subgroup classification (non-APS, MSA, PSP and CBD) did not differ between modalities (FDG-PET 87% and TCS 83%; P = 1.00). CONCLUSIONS: FDG-PET and TCS show comparable accuracies for differential diagnosis of neurodegenerative parkinsonism. This preliminary study supports the use of TCS and warrants further prospective validation.
Assuntos
Encéfalo/diagnóstico por imagem , Atrofia de Múltiplos Sistemas/diagnóstico , Transtornos Parkinsonianos/diagnóstico , Paralisia Supranuclear Progressiva/diagnóstico , Ultrassonografia Doppler Transcraniana , Idoso , Diagnóstico Diferencial , Feminino , Fluordesoxiglucose F18 , Humanos , Masculino , Pessoa de Meia-Idade , Atrofia de Múltiplos Sistemas/diagnóstico por imagem , Transtornos Parkinsonianos/diagnóstico por imagem , Cintilografia , Sensibilidade e Especificidade , Paralisia Supranuclear Progressiva/diagnóstico por imagemAssuntos
Síndrome de Klippel-Trenaunay-Weber/diagnóstico , Angiografia por Ressonância Magnética/métodos , Síndromes Neurocutâneas/diagnóstico , Atrofia , Meios de Contraste , Diagnóstico Diferencial , Eletroencefalografia , Feminino , Gadolínio , Humanos , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios XRESUMO
Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture-produced proteins such as full-size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram-per-liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full-size human anti-HIV antibody 2G12 in N. tabacum cv. BY-2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10-20-fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred-tank bioreactors, where the maximum yield per cultivation volume was 8 mg L(-1) over 7 days. During the stationary phase, antibody levels were 150-fold higher in nitrogen-enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N-glycan analysis.
Assuntos
Anticorpos Monoclonais/biossíntese , Biotecnologia/métodos , Anticorpos Anti-HIV/biossíntese , Nicotiana , Nitrogênio/metabolismo , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Meios de Cultura/química , Humanos , Nitratos/metabolismo , Proteínas Recombinantes/biossínteseRESUMO
OBJECTIVE: To describe the topography of the N700 component of the scalp-recorded visual event-related potential (ERP) and to provide fundamental knowledge of the conditions under which it occurs. METHODS: We examined the time-course of visual ERP in response to the short (100ms) and prolonged (7s) presentation of simple salient visual stimuli separated by long interstimulus intervals employing high-resolution 64-channel DC-EEG. Current source density (CSD) mapping and spatio-temporal dipole source analysis were performed. RESULTS: CSD analysis revealed highly significant bilateral current sinks over occipito-temporal areas from about 450ms up to 1s after stimulus offset (visual N700). CSD topography and dipole source analysis suggested late prolonged activation of extrastriate visual areas which did not depend merely upon a stimulus offset response, afterimages or blinking, as confirmed by control conditions. CONCLUSIONS: Our findings provide basic knowledge about the time-course of sensory activation. We found that passive watching of rare salient short stimuli automatically evoked sustained activity in the extrastriate visual cortex up to 1s after stimulus offset. SIGNIFICANCE: Visual N700 provides a promising tool for important insights into the cortical mechanisms of stimulus post-processing. Its role in associative learning of temporally non-overlapping stimuli (automatic ultra-short-term sensory memory) should be explored.
Assuntos
Mapeamento Encefálico , Potenciais Evocados Visuais/fisiologia , Couro Cabeludo , Córtex Visual/fisiologia , Adulto , Análise de Variância , Eletroencefalografia , Feminino , Humanos , Masculino , Estimulação Luminosa/métodos , Tempo de Reação/fisiologia , Valores de Referência , Fatores de Tempo , Visão Ocular/fisiologia , Percepção Visual/fisiologiaRESUMO
The influence of a complex microflora residing in the gastrointestinal tract of cattle on the prion protein plays a crucial role with respect to early pathogenesis and the potential infectivity of faeces resulting in contamination of the environment. It is unknown whether infectious prion proteins, considered to be very stable, are inactivated by microbial processes in the gastrointestinal tract of animals during digestion. In our previous study it was shown that the scrapie-associated prion protein was degraded by ruminal and colonic microbiota of cattle, as indicated by a loss of anti-prion antibody 3F4 immunoreactivity in Western blot. Subsequently, in this study hamster bioassays with the pre-treated samples were performed. Although the PrP(Sc) signal was reduced up to immunochemically undetectable levels within 40 h of pre-treatment, significant residual prion infectivity was retained after degradation of infected hamster brain through the gastrointestinal microflora of cattle. The data presented here show that the loss of anti-prion antibody 3F4 immunoreactivity is obviously not correlated with a biological inactivation of PrP(Sc). These results highlight the deficiency of using Western blot in transmissible spongiform encephalopathies inactivation assessment studies and, additionally, point to the possibility of environmental contamination with faeces containing PrP(Sc) following an oral ingestion of prions.
Assuntos
Bactérias/metabolismo , Colo/microbiologia , Digestão , Encefalopatia Espongiforme Bovina/transmissão , Proteínas PrPSc/metabolismo , Rúmen/microbiologia , Animais , Bovinos , Cricetinae , Encefalopatia Espongiforme Bovina/metabolismo , Proteínas PrPSc/patogenicidadeRESUMO
The high-affinity receptor for IgG, FcgammaRI, shares its capacity to bind IgG2a immune complexes (IgG2a-IC) with the low-affinity receptor FcgammaRIII and complement factors, hampering the definition of its biological role. Moreover, in vivo, FcgammaRI is occupied by monomeric IgG2a, reducing its accessibility to newly formed IgG2a-IC. By using a variety of FcgammaR(-/-) mice, we demonstrate that in the absence of FcgammaRI, the IgG2a-IC-induced cellular processes of phagocytosis, cytokine release, cellular cytotoxicity, and antigen presentation are impaired. FcgammaRI(-/-) mice showed impaired hypersensitivity responses, strongly reduced cartilage destruction in an arthritis model, and impaired protection from a bacterial infection. We conclude that FcgammaRI contributes substantially to a variety of IgG2a-IC-dependent immune functions and immunopathological responses.
Assuntos
Artrite Experimental/imunologia , Bordetella pertussis/imunologia , Hipersensibilidade/imunologia , Receptores de IgG/imunologia , Coqueluche/imunologia , Animais , Artrite Experimental/genética , Artrite Experimental/patologia , Cartilagem/patologia , Feminino , Hipersensibilidade/genética , Imunidade/genética , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/genéticaRESUMO
The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied. IgG-opsonized B. pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN). This process was mediated by the PMN IgG receptors, FcgammaRIIa (CD32) and FcgammaRIIIb (CD16), working synergistically. Furthermore, these FcgammaR triggered efficient PMN respiratory burst activity and mediated transfer of B. pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability. Bacteria opsonized with IgA triggered similar PMN activation via FcalphaR (CD89). Simultaneous engagement of FcalphaRI and FcgammaR by B. pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone. These data provide new insights into host immune mechanisms against B. pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.
Assuntos
Bordetella pertussis/imunologia , Receptores Fc/fisiologia , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/fisiologia , Atividade Bactericida do Sangue , Humanos , Imunidade Celular/imunologia , Imunoglobulina A/sangue , Imunoglobulina A/fisiologia , Imunoglobulina G/sangue , Imunoglobulina G/fisiologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Proteínas Opsonizantes/sangue , Proteínas Opsonizantes/fisiologia , Fagocitose/imunologia , Receptores Fc/sangue , Receptores de IgG/sangue , Receptores de IgG/fisiologia , Explosão Respiratória/imunologiaRESUMO
Infection with Bordetella pertussis, the causative agent of pertussis (whooping cough) in humans, is followed by the production of antibodies of several isotypes, including immunoglobulin A (IgA). Little is known, however, about the role of IgA in immunity against pertussis. Therefore, we studied targeting of B. pertussis to the myeloid receptor for IgA, FcalphaRI (CD89), using either IgA purified from immune sera of pertussis patients or bispecific antibodies directed against B. pertussis and FcalphaRI (CD89 BsAb). Both IgA and CD89 BsAb facilitated FcalphaRI-mediated binding, phagocytosis, and bacterial killing by human polymorphonuclear leukocytes (PMNL) and PMNL originating from human FcalphaRI-transgenic mice. Importantly, FcalphaRI targeting resulted in enhanced bacterial clearance in lungs of transgenic mice. These data support the capacity of IgA to induce anti-B. pertussis effector functions via the myeloid IgA receptor, FcalphaRI. Increasing the amount of IgA antibodies induced by pertussis vaccines may result in higher vaccine efficacy.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos CD/imunologia , Bordetella pertussis/imunologia , Imunoglobulina A/imunologia , Receptores Fc/imunologia , Coqueluche/prevenção & controle , Animais , Antígenos CD/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Fagocitose/imunologia , Receptores Fc/genéticaRESUMO
In the last few years the Pichia pastoris expression system has been gaining more and more interest for the expression of recombinant proteins. Many groups have employed fermentation technology in their investigations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range. A large number of heterologous proteins from different sources has been expressed, but the fermentation process technology has been investigated to a lesser extent. A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fermentations of methylotrophic yeasts. In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products. We have used a commercially available methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of a Mut(+) strain of Pichia pastoris. Specific glycerol feed rates in the range of 38-4.2 mg. g(-1). h(-1) (mg glycerol per gram fresh weight per hour) were investigated. Expression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg. g(-1). h(-1). At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation. These results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research.
Assuntos
Reatores Biológicos , Glicerol/metabolismo , Fragmentos de Imunoglobulinas/biossíntese , Metanol/metabolismo , Pichia/genética , Pichia/metabolismo , Biotecnologia/instrumentação , Biotecnologia/métodos , Clonagem Molecular , Fermentação , Expressão Gênica , Fragmentos de Imunoglobulinas/genética , Cinética , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Ressonância de Plasmônio de Superfície , Transformação GenéticaRESUMO
In the absence of opsonizing antibodies, Bordetella pertussis, the causative agent of pertussis, readily binds to phagocytes via complement receptor 3 (CR3). After opsonization with antibodies, binding is mediated by IgG receptors (FcgammaR). The effect of targeting B. pertussis to either FcgammaR or CR3 was studied. The fate of unopsonized B. pertussis, IgG-opsonized B. pertussis, and B. pertussis opsonized with bispecific antibodies (BsAbs) directed to CR3 or FcgammaRII/-III was compared. IgG antibodies mediated binding and phagocytosis of B. pertussis via FcgammaR by polymorphonuclear leukocytes (PMNL) in vitro. Opsonization of B. pertussis with BsAbs directed against either CR3 or FcgammaRII/-III facilitated PMNL phagocytosis; however, in vivo studies with BsAb revealed that FcgammaR-mediated uptake facilitates B. pertussis clearance, in contrast to uptake via CR3. Targeting of B. pertussis to FcgammaRII/-III in mice deficient in FcgammaRII or FcgammaRIII indicated that the protective effect is attributable to FcgammaRIII. Competition between uptake via CR3 or FcgammaR may determine the outcome of natural infection.
Assuntos
Anticorpos Antibacterianos/imunologia , Bordetella pertussis/imunologia , Antígeno de Macrófago 1/imunologia , Fagocitose , Receptores de IgG/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Células Cultivadas , Feminino , Imunoglobulina G/imunologia , Antígeno de Macrófago 1/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Proteínas Opsonizantes/imunologia , Receptores de IgG/genética , Coqueluche/imunologiaRESUMO
Birt-Hogg-Dubé-syndrome, first described 20 years ago, is an autosomal dominant inherited disease, characterized by the occurrence of multiple cutaneous hamartomas with adnexal differentiation. An association with intestinal neoplasms has been mentioned repeatedly. Recently, the cutaneous lesions in this syndrome have been interpreted as different developmental stages of one single hamartoma with sebaceous differentiation, called mantleoma. Numerous dermatohistopathologic studies contribute to the certain diagnosis of Birt-Hogg-Dubé-syndrome. In contrast, little information is available on the therapy of the multiple skin lesions. A father and his daughter with Birt-Hogg-Dubé-syndrome were treated with the CO2 laser, producing satisfactory results.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos , Fibroma/cirurgia , Hamartoma/cirurgia , Terapia a Laser , Neoplasias Primárias Múltiplas/cirurgia , Neoplasias Cutâneas/cirurgia , Adulto , Biópsia , Criança , Aberrações Cromossômicas/genética , Feminino , Fibroma/patologia , Hamartoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/patologia , Pele/patologia , Neoplasias Cutâneas/patologia , Síndrome , Terminologia como AssuntoRESUMO
Clonal T-cell anergy has been proposed as a mechanism to ensure peripheral tolerance in vivo. Anergy has been reported to result from T cell activation with inappropriate antigen-presenting cells (APC) or, in the case of CD4+ T cells, also by altered peptide ligands. This study reveals that altered hapten ligands can also induce anergy in CD8+ T cells. The Kb-restricted, trinitrophenyl (TNP) specific cytotoxic T lymphocyte (CTL) clone E6 was found to lyse target cells presenting the TNP-modified peptides M4L-TNP (derived from mouse serum albumin) or O4TNP (derived from chicken ovalbumin), but not the corresponding dinitrophenol (DNP)-modified peptides. However, whereas M4L-DNP was found totally unreactive, O4DNP antagonistically inhibited M4L-TNP-mediated kill if expressed on the same target cell. Moreover, when presented alone on APC, O4DNP, but not M4L-DNP, induced anergy in clone E6 by preventing its subsequent proliferative response to M4L-TNP. The anergic state did not affect agonist-specific cytolysis or T-cell receptor (TCR) down-modulation by the anergized CTL, and proliferative responses were regained upon addition of interleukin (IL)-2 or IL-12 plus IL-18. These findings substantiate the similarity between hapten-and peptide-recognition by T cells. The induction as well as the reversal of anergy in CD8+ CTL may thus be of relevance not only in autoimmunity or tumour rejection, but also in contact hypersensitivity reactions to haptens.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Anergia Clonal/imunologia , Haptenos/imunologia , Animais , Técnicas de Cultura de Células , Divisão Celular/imunologia , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Trinitrobenzenos/imunologiaRESUMO
Because of its vascular selectivity, the flashlamp-pumped pulsed dye laser (585 nm) is efficacious in the treatment of vascular lesions and is successfully used for the treatment of port-wine stains and haemangiomas in children. Based on the encouraging results with these cutaneous vascular disorders, the cutaneous lesions of patients with lupus erythematosus (LE) have now also been treated with the pulsed dye laser. Cutaneous lesions in lupus erythematosus are often difficult to treat with readily available local therapeutic methods. We report here on a group of 12 patients whose LE lesions were treated with the pulsed dye laser. In 10 patients, the LE was limited to the skin, while two patients had systemic LE (SLE). Even in the two patients with SLE, a significant improvement of skin lesions was achieved. After a mean number of 51 laser sessions, a median clearance rate of 70% was attained for nine patients. In one case, the laser treatment failed to clear the lesions. Two patients did not show any visible improvement of the lesions, but pain and itching were significantly reduced. There were few side-effects. No prolonged laser-induced scarring occurred and in only two patients was hyperpigmentation seen, which had resolved completely after 4 and 5 months, respectively. During a median follow-up of 7 months (range: 3-32 months), only one patient (after a complete clearance of the skin lesions) had a small relapse. In summary, the pulsed dye laser is an effective therapy for the treatment of superficial skin lesions in LE.
Assuntos
Dermatoses Faciais/radioterapia , Terapia a Laser , Lúpus Eritematoso Cutâneo/radioterapia , Adulto , Dermatoses Faciais/patologia , Feminino , Seguimentos , Humanos , Lúpus Eritematoso Cutâneo/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein found on the surface of normal colon and malignant human adenocarcinomas. Recently, a fusion protein containing two of the seven Ig-like domains present in CEA (N and A3) has been constructed and expressed in Pichia pastoris [You, Hefta, Yazaki, Wu and Shively (1998) Anticancer Res. 18, 3193-3201]. Here, we report the generation and selection of a multi-copy clone expressing this fusion protein, the optimization of the shake-flask expression protocol and the upscaled production of CEA N-A3 using fermentation technology. P. pastoris transformants secreting the CEA N-A3 domain were generated by electrotransformation of the GS115 host strain with the pPIC9K vector containing the CEA N-A3 cDNA [You, Hefta, Yazaki, Wu and Shively (1998) Anticancer Res. 18, 3193-3201] then screened for CEA N-A3 expression and G418 resistance. The recombinant CEA N-A3 domain was detected in the culture supernatant using the monoclonal anti-CEA antibody T84.66. Optimization of methanol-induction conditions resulted in a high-methanol shake-flask expression protocol yielding significantly increased CEA N-A3 levels. Fermentation and culture conditions were optimized for 5-l working-volume fermentations and CEA N-A3 was affinity purified using Ni-IDA (imino di-acetic acid) affinity chromatography from the clarified fermentation supernatant. Peptide N-glycosidase F treatment revealed that the recombinant protein was heavily glycosylated but expressed as a single polypeptide of 28 kDa with no evidence of proteolytic degradation. Our results demonstrate that functional CEA N-A3 domain can be produced in sufficient quantities in P. pastoris for structural analysis or diagnostic applications. To our knowledge, this article represents the first report on the production of a human tumour antigen through fermentation.
Assuntos
Antígeno Carcinoembrionário/biossíntese , Pichia/genética , Sequência de Bases , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/isolamento & purificação , Cromatografia de Afinidade , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fermentação , Glicosilação , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Transformação GenéticaRESUMO
Bordetella pertussis can attach, invade and survive intracellularly in human macrophages in vitro. To study the significance of this bacterial feature in vivo, we analyzed the presence of viable bacteria in broncho-alveolar lavage (BAL) cells of mice infected with B. pertussis. We found B. pertussis to be present in a viable state in BAL fluid cells until at least 19 days after infection, suggesting B. pertussis to be able to survive in those cells. This intracellular niche may play an important role in the pathogenesis of pertussis. Pertussis toxin and the RGD sequence of the virulence factor filamentous hemagglutinin (FHA) both play a role in the attachment of B. pertussis to human and mouse macrophages in vitro and we hypothesized these virulence factors to be required for invasion and subsequent intracellular survival of B. pertussis in macrophages in vivo. A B. pertussis double mutant, in which the FHA RGD motif was changed to RAD and the ptx genes were deleted, was also found in a viable state in BAL fluid cells, albeit at lower levels than the wild-type strain. In our model, uptake of B. pertussis by alveolar phagocytes in vivo is thus, at least in part, determined by the bacterial virulence factors FHA and pertussis toxin.
Assuntos
Bordetella pertussis/crescimento & desenvolvimento , Líquido da Lavagem Broncoalveolar/microbiologia , Coqueluche/microbiologia , Adesinas Bacterianas/fisiologia , Animais , Bordetella pertussis/isolamento & purificação , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Colônia Microbiana , Hemaglutininas/fisiologia , Pulmão/microbiologia , Macrófagos Peritoneais/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Toxina Pertussis , Fatores de Virulência de BordetellaRESUMO
Plant-suspension cells are an in vitro system that can be used for recombinant protein production under carefully controlled certified conditions. Plant-suspension cells can be grown in shake flasks or fermenters to produce secondary metabolites, like vincristine and vinblastine, and to produce recombinant proteins after transformation. This review article focuses on discussing the generation of transformed suspension-cell lines expressing recombinant proteins, like antibodies, and recombinant-protein downstream processing and purification.
Assuntos
Células Vegetais , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Reatores Biológicos , Células Cultivadas , Fermentação , Plantas/genética , Plantas/metabolismo , Plantas Tóxicas , Proteínas Recombinantes/metabolismo , Nicotiana/citologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
This review article focuses on the use of the methylotrophic yeast Pichia pastoris as a recombinant protein-expression system. P. pastoris is a useful system for the expression of milligram-to-gram quantities of a protein, which can be scaled up to fermentation to meet greater demands. Compared with mammalian cells, Pichia do not require a complex growth medium or culture conditions, they are as easy to manipulate genetically as Escherichia coli and have a eukaryotic protein-synthesis pathway. They seem suited to laboratory-scale production of recombinant proteins for in-house use or, in some cases, molecular farming of recombinant products. This review article focuses on the use of P. pastoris, describes a fermentation production run of a single-chain antibody fragment and includes a discussion of fermentation as a production strategy.