RESUMO
Deficiency of the serine proteinase inhibitor (serpin) alpha 1-antitrypsin (alpha 1AT) is the most common autosomal recessive genetic disorder in Northern Europe. alpha 1AT is the physiological regulator of the proteolytic enzyme neutrophil elastase and severe deficiency states are associated with an increased risk of developing chronic obstructive pulmonary disease (COPD) as a consequence of chronic proteolytic damage to the lungs. Among the known mutations of the alpha 1AT gene causing severe alpha 1AT deficiency and COPD a few alleles are also associated with liver disease. When expressed in cell cultures, all these particular alleles cause intracellular alpha 1AT accumulation which appears to be a prerequisite for the development of hepatic injury. Liver disease is seen in only a small fraction of all patients carrying such alleles, however. The reason for this is not completely clear, but there is evidence that PI ZZ individuals 'susceptible' to liver disease carry an additional defect affecting protein degradation in the endoplasmic reticulum (ER). We characterise a newly identified defective alpha 1AT allele PI Mwürzburg (Pro369 [CCC] to Ser [TCC]) associated with a complete intracellular transport block in cell cultures in vitro. The allele PI Mheerlen, a previously described different amino acid substitution in the same position as PI Mwürzburg (Pro369 [CCC] to Leu [CTC]) is shown to cause complete retention of the mutant alpha 1AT in the ER, too, whereas in the recently described mutant allele PI Q0lisbon (Thr68 [ACC] to Ile [ATC]) a significantly reduced alpha 1AT secretion from the cells was observed. Adenovirus-mediated recombinant expression of mutant Mwürzburg and Mheerlen, and of wild-type alpha 1AT in mouse liver in vivo showed that the mutant human proteins were not secreted into the mouse plasma, in contrast with human wild-type alpha 1AT which circulated at high concentrations over several weeks. In summary, all transportation deficient alpha 1ATs analysed have the potential to cause lung disease in the homozygous state or in heterozygous carriers of another deficiency allele, and they may also cause liver disease in certain patients. The mutant PI Mwürzburg and Mheerlen alpha 1ATs are completely retained within synthesising cells, and the molecular defect of transportation in these two alleles may be similar to that in the common PI Z allele. The molecular defect in the PI Q0lisbon allele (Thr68Ile) shows similarity with the immediately neighbouring Mmineral springs mutation (Gly67Glu).
Assuntos
Alelos , Isoleucina/genética , Leucina/genética , Prolina/genética , Inibidores de Serina Proteinase/genética , Serina/genética , Treonina/genética , alfa 1-Antitripsina/genética , Adulto , Animais , Criança , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , LinhagemRESUMO
The formation of factor VIII antibodies is a major problem for replacement therapy of haemophilia A patients. Antibodies occur in 5-30% of patients with severe haemophilia A. The reason for antibody formation is still unknown. In this study we correlate for the first time different factor VIII gene mutations, stop- and missense mutations, large and small deletions and intrachromosomal intron 22 recombinations to antibody formation. A total of 364 patients with known inhibitor status of our institute, of the database, and of 3 studies representing intron-22-inversion data are included. The results show that the risk for developing factor VIII antibodies is strongly related to stop mutations. large deletions and intrachromosomal recombinations. A probable explanation could be the complete lack of endogenous circulating factor VIII protein in these cases. Other factors that might be important for the pathogenesis of inhibitor formation, e. g. the antenatal period, as well as possible therapeutic effects, are discussed.
Assuntos
Deleção Cromossômica , Fator VIII/imunologia , Hemofilia A/genética , Recombinação Genética , Anticorpos/sangue , Hemofilia A/imunologia , Humanos , Mutação , Fatores de RiscoRESUMO
The objective of this study was to evaluate processing methods for frozen beef subprimals; the effects of freezing and thawing rates on tenderness, sensory properties, and retail display were evaluated. There were 6 treatments: fresh, never frozen 14 d wet aged (14D); fresh, never frozen 21 d wet aged (21D); blast frozen-fast thawed (BF); blast frozen-slow thawed (BS); conventionally frozen-fast thawed (CF); and conventionally frozen-slow thawed (CS). All frozen beef subprimals were aged for 14 d before freezing. Three beef subprimal cuts, rib eye roll (n=90), strip loin (n=90), and top sirloin butt (n=90), were used with 3 replications of 5 samples per treatment per week (total of 9 wk, n=270). Blast freezing occurred by placing spacers between the boxes of meat on pallets at -28°C with high air velocity for 3 to 5 d. Conventional freezing occurred with boxes of meat stacked on pallets and placed in a -28°C freezer with minimal air movement for at least 10 d. Fast thawing of subprimals (to an internal temperature of -1°C to 1°C) occurred by immersion in a circulating water bath (<12°C) for 21 h, and slow thawing of subprimals occurred over a 2-wk period by placing individual subprimals on tables at 0°C. Steaks (2.5 cm thick) were cut from the longissimus thoracis (LT), longissimus lumborum (LL), and gluteus medius (GM) for Warner-Bratzler shear force (WBS), trained sensory evaluation, and retail display. For LL and GM beef steaks, frozen treatments were equal or lower in WBS values to 14D and 21D beef steaks. No differences were detected in WBS among the treatments applied to GM beef steaks (P=0.08). There were no differences in sensory tenderness among the LL, LT, and GM (P>0.05). All LL and LT beef steaks had approximately 4 d to 40% discoloration, and all GM steaks had over 3 d to 40% discoloration. Steaks from the LL and LT began to discolor at about 3 d, and the GM began to discolor after 1 d. For all beef subprimals, purge loss during storage and thawing was significantly greater for the slow-thawed subprimals (P<0.01), and all fast-thawed subprimals were equal or superior to 14D and 21D (P<0.01) in storage and thawing purge. During retail display, the greatest purge loss occurred in fast-thawed treatments (P<0.01). Overall, freezing rate did not affect purge loss, and neither freezing nor thawing rates had significant meaningful effects on WBS, and sensory properties were comparable with fresh, never-frozen subprimals.
Assuntos
Armazenamento de Alimentos/métodos , Congelamento , Carne/normas , Animais , Bovinos , Análise de Alimentos , Embalagem de Alimentos , Humanos , Carne/análise , Músculo Esquelético/química , Sensação , Paladar , Fatores de Tempo , ÁguaRESUMO
The multidisciplinary care, at different referral centers of cystic fibrosis, is aimed at monitoring and treating cystic fibrosis patients. Mortality attributed to this hereditary disease is high, since it affects the exocrine glands, involving multiple organs, and evolves in a chronic, progressive way. However, systemized care and the improved, shared understanding of gastroenterologists, nutritionists and pulmonologists, contribute to prolonged survival and abated morbimortality. The aim of this study is to describe the main aspects of clinical and nutritional intervention in cystic fibrosis patients so that monitoring by a multidisciplinary team is optimized and performed as early as possible. The review was carried out on articles indexed in the Medline, Lilacs, SciELO, Current Contents and Cochrane databases, finding 189 articles in Portuguese, English and Spanish, with emphasis on articles published between 2000 and 2011. Due to the scientific relevant contribution, some publications before 2000 were included totalized 77 related to the multidisciplinary care. The reviewed studies suggest that multidisciplinary care is essential for knowledge integration in order to impose permanent update of scientific information, thereby contributing to the development of intervention strategies that enhance survival and motivate the development of skills to cope with the complex treatment regimen that is necessary for cystic fibrosis treatment and prevention of related complications.
Assuntos
Fibrose Cística/terapia , Apoio Nutricional , Fibrose Cística/diagnóstico , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Humanos , Fenômenos Fisiológicos da Nutrição , Equipe de Assistência ao Paciente , Modalidades de FisioterapiaRESUMO
Environmental enrichment has been shown to be neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). Because PD patients are not typically diagnosed until later neuropathological stages, the current study investigated the capacity of an enriched environment (EE) to stimulate restoration of neurons in the substantia nigra pars compacta (SNpc) and locomotor recovery after lesioning, as opposed to before. A low-dose chronic MPTP regimen was used to achieve a partial, less severe lesion of the nigrostriatal pathway not seen in acute MPTP models. Both young adult (10 weeks) and aged (12 months) C57BL/6J male mice were used to assess the effects of aging on recovery with EE intervention. After the first week of either MPTP (7 mg/kg/d in young; 5 mg/kg/d in aged) or saline injection, animals from both groups were housed in a standard environment (SE) or an EE for 3 weeks, with continued daily administration of MPTP. We are the first to report that following 3 weeks exposure to an EE, young and aged MPTP-lesioned mice showed a significant 53% and 52% restoration of tyrosine hydroxylase (TH)-labeled neurons in the SNpc, respectively. This increase in TH-labeled cells in the MPTP+EE group was correlated with recovery of free-standing rear (FSR) behavior in both age groups; however, improved locomotor control as measured by foot faults (FF) per total activity was only seen in the aged MPTP+EE group. Our data demonstrate that an EE promotes neurorestoration in TH protein expression in SNpc neurons as well as some locomotor recovery in both young and aged animals in this mouse model of PD.
Assuntos
Comportamento Animal/fisiologia , Ambiente Controlado , Neurônios/enzimologia , Transtornos Parkinsonianos/terapia , Recuperação de Função Fisiológica/fisiologia , Substância Negra/enzimologia , Animais , Modelos Animais de Doenças , Meio Ambiente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/patologia , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/fisiopatologia , Estimulação Física/métodos , Substância Negra/patologia , Substância Negra/fisiopatologiaRESUMO
A number of neurotoxin- and gene-based rodent models of acute neurodegeneration of nigrostriatal dopamine (DA) neurons are used to study Parkinson's disease (PD). The rapid degeneration achieved by many of these current models limits the capacity of the model to develop pathogenic mechanisms and display the various stages of motor degradation representative of the human Parkinsonian condition. Chronic rodent models have been the only ones to reproduce these characteristics, yet do not show correlated progress of DA loss with multiple stepwise behavioral deficits as seen in humans. In the present study, we have developed a progressive model of increasing DA loss and motor dysfunction via progressively increased administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), in the C57Bl/6J mouse. Mice were administered a daily (5 d/wk) dose of MPTP that increased weekly over the course of 4 weeks (4 mg/kg, 8 mg/kg, 16 mg/kg and 32 mg/kg). Each treatment group was tested for exploratory and motor behavioral changes after every week leading up to their final dose, as well as changes in tyrosine hydroxylase immunoreactivity (TH-ir) of the substantia nigra pars compacta (SNpc) and caudate putamen (CPu). We detected a 24% decrease in the mean number of TH-ir SNpc neurons/section after 1 week, and a 62% decrease after 4 weeks as compared to the vehicle group. CPu TH-ir began at a 35% loss after 1 week and increased to a 74% loss after 4 weeks compared to the vehicle group. CPu DA content showed an initial decrease of 20% after 1 week, and a final decrease of 70% following week 4 versus the vehicle group. Free-standing rears (versus wall-assisted rears, in a cylinder), decreased from 35% to 8% of total rears as the dose of MPTP increased from 4 mg/kg to 32 mg/kg, respectively. However, motor impairment as measured by a Parallel Rod Activity Chamber test was not significant until week 4 at 32 mg/kg compared to the vehicle group. The present study is the first to show stepwise progression of behavioral deficits which correlate with gradual dopaminergic decline in the nigrostriatal pathway. This progressive lesioning regiment may be appropriate for future investigation of pathogenic mechanisms and various intervention therapies in PD.
Assuntos
Dopamina/metabolismo , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/fisiopatologia , Substância Negra/fisiopatologia , Animais , Western Blotting , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Transtornos Parkinsonianos/metabolismo , Substância Negra/metabolismo , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
BACKGROUND: The role of the orexigenic hormone ghrelin is of major interest in the altered appetite regulation of the elderly. METHODS: Basal and postprandial levels of active and total ghrelin were measured in 15 younger (mean age 35.4 years) and 19 older (80.7 years) participants following a carbohydrate-rich test meal. RESULTS: Our results showed that older participants felt postprandially less hungry and more full. Although basal levels were not significantly different, active and total ghrelin levels declined postprandially only in the younger study participants. Highly significant differences between the two age groups were shown for the changes of the area under the curve for active ghrelin (p = .024). CONCLUSIONS: Our study demonstrates for the first time that differences in hunger and satiety sensations in relation to age are paralleled by a substantially different response of acylated and total ghrelin, that is, the absence of a postprandial decline in ghrelin levels.
Assuntos
Envelhecimento/sangue , Regulação do Apetite/fisiologia , Grelina/sangue , Fome/fisiologia , Período Pós-Prandial/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-IdadeAssuntos
Cálcio/sangue , Bovinos/sangue , Fósforo/sangue , Potássio/sangue , Sódio/sangue , Animais , Cruzamento , MasculinoRESUMO
The multidisciplinary care, at different referral centers of cystic fibrosis, is aimed at monitoring and treating cystic fibrosis patients. Mortality attributed to this hereditary disease is high, since it affects the exocrine glands, involving multiple organs, and evolves in a chronic, progressive way. However, systemized care and the improved, shared understanding of gastroenterologists, nutritionists and pulmonologists, contribute to prolonged survival and abated morbimortality. The aim of this study is to describe the main aspects of clinical and nutritional intervention in cystic fibrosis patients so that monitoring by a multidisciplinary team is optimized and performed as early as possible. The review was carried out on articles indexed in the Medline, Lilacs, SciELO, Current Contents and Cochrane databases, finding 189 articles in Portuguese, English and Spanish, with emphasis on articles published between 2000 and 2011. Due to the scientific relevant contribution, some publications before 2000 were included totalized 77 related to the multidisciplinary care. The reviewed studies suggest that multidisciplinary care is essential for knowledge integration in order to impose permanent update of scientific information, thereby contributing to the development of intervention strategies that enhance survival and motivate the development of skills to cope with the complex treatment regimen that is necessary for cystic fibrosis treatment and prevention of related complications (AU)
La atención multidisciplinaria, en diferentes centros de referencia de la fibrosis quística, tiene por objeto el seguimiento y el tratamiento de pacientes con fibrosis quística. La mortalidad atribuidas a esta enfermedad hereditaria es alto, ya que afecta las glándulas exocrinas, afecta múltiples órganos, y evoluciona de manera crónica y progresiva. Sin embargo, la atención sistematizada y la mejor comprensión compartida de los gastroenterólogos, nutricionistas y neumólogos, contribuir a la prolongación de la supervivencia y la morbi-mortalidad disminuyó. El objetivo de este estudio es describir los principales aspectos de la intervención clínica y nutricional en pacientes con fibrosis quística, para que la supervisión de un equipo multidisciplinario se optimiza y realiza lo más pronto posible. La revisión se llevó a cabo en artículos indexados en el Medline, Lilacs, SciELO, Current Contents y bases de datos Cochrane, la búsqueda de 189 artículos en Portugués, Inglés y Español, con énfasis en los artículos publicados entre 2000 y 2011. Debido a la destacada contribución científica, algunas publicaciones antes de 2000 se incluyeron totalizado 77 relacionados con la atención multidisciplinaria. Los estudios revisados sugieren que la atención multidisciplinaria es esencial para la integración del conocimiento con el fin de imponer actualización permanente de información científica, contribuyendo así al desarrollo de estrategias de intervención que mejoren la supervivencia y motivar el desarrollo de habilidades para hacer frente a la pauta de tratamiento complejo que es necesario para tratamiento de la fibrosis quística y la prevención de las complicaciones relacionadas (AU)
Assuntos
Humanos , Fibrose Cística/complicações , Pneumopatias/epidemiologia , Distúrbios Nutricionais/epidemiologia , Equipe de Assistência ao Paciente/organização & administração , Estado Nutricional , Espirometria , Triagem NeonatalAssuntos
Doença Iatrogênica/prevenção & controle , Acidentes por Quedas/prevenção & controle , Acidentes por Quedas/estatística & dados numéricos , Idoso , Idoso de 80 Anos ou mais , Delírio/induzido quimicamente , Delírio/epidemiologia , Delírio/prevenção & controle , Humanos , Doença Iatrogênica/epidemiologia , Período Perioperatório , Polimedicação , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/prevenção & controle , Psicotrópicos/efeitos adversos , Fatores de RiscoRESUMO
The effectiveness of health educational measures increases with the time spent for them. A health education which motivates the patients towards personal activity is unequivocally superior to passive forms of health education such as information, instruction and demonstration.
Assuntos
Educação em Saúde Bucal/normas , Higiene Bucal , Placa Dentária/diagnóstico , Relações Dentista-Paciente , Alemanha Oriental , Humanos , Motivação , Folhetos , Coloração e Rotulagem , Escovação DentáriaRESUMO
High transgene stabilities of 1 year and more have been reported in immunodeficient hosts after adenovirus-mediated gene transfer. Transgene persistence of this duration could be due to inherently high stability of the episomal viral vector DNA. An alternative explanation would be limited 'autoreplication' of transgenic vector DNA, just sufficient to counteract slow but continuous degradation within the host cells. Autoreplication could occur in the absence of any production of infectious virus particles, based on residual activity of the adenoviral DNA replication system only. To test this hypothesis, a series of DNA metabolic labeling studies in non-permissive cells cultures transfected with different vectors was conducted. Due to extensive E1 region deletions none of the vectors was able to produce viral progeny in non-permissive cells. Vectors fell into two categories, however, with respect to their autoreplication potential. Neosynthesis of vector DNA in non-permissive vector-transfected cells was readily detectable in 'type A', but not in 'type B' vectors. In addition to their different transgene expression cassettes, vector DNA sequencing showed a less extensive E1 deletion in type A (nucleotides 453-3333 of wild-type virus) as compared to type B vectors (nucleotides 325-3523). Autoreplication was also associated with high transcriptional activity of several viral genes (E1B-14k, adenoviral DNA polymerase, single-strand DNA-binding protein, E4-25k), in contrast to type B vectors. In addition to these 'wild-type' transcripts, 'irregular' recombinant transcripts were detected in autoreplication vectors which contained the transgenic cDNA in conjunction with adenoviral vector sequences. Exogenous or cryptic promotors may (under certain conditions) enhance the transcriptional activity of a vector in such a way that autoreplication occurs. Conditions determining the level of transcriptional enhancement (extent of E1 deletion, type of promoter and transgene, etc) need to be further defined before rational design of adenovectors with high autoreplication capacity becomes possible. In summary, we have shown autoreplication to be a novel feature of certain E1-deleted adenovectors with likely relevance for their stability in vivo, but also with possibly adverse consequences for target cell function or vector immunogenicity. Full characterization of adenoviral vector systems should therefore include a description of their autoreplication capacity.
Assuntos
Adenoviridae/genética , Genes Virais/genética , Replicação Viral/genética , Células Cultivadas , Deleção de Genes , Vetores Genéticos/genética , Genoma Viral , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNARESUMO
During the past few years great efforts have been made to construct and to test human factor VIII (hFVIII) and IX (hFIX) vectors suitable for haemophilia gene therapy in vivo. However, little is known about the molecular mechanisms of persistence and shut-off of transgene expression in the target organs after gene transfer using recombinant adenoviral vectors. To evaluate low transgene mRNA levels in different tissues, especially at long times after the gene transfer, the common northern blot method is often not sensitive enough. For this reason we developed a new, highly sensitive and species-specific method for hFIX mRNA quantification and employed it in mice treated with an adenoviral vector (Ad5CMVFIX) expressing human FIX. In addition to its very high sensitivity (lowest detection level=1 fg RNA), the method was shown to be strictly species-specific, since hFIX mRNA signals were never detected in untreated mice. In a long-term study of 18 vector-treated mice we compared the human FIX:Ag levels in the mouse plasma, the human FIX mRNA levels and human FIX vector DNA concentrations in the mouse liver. We found that a slow but continuous decrease of hFIX:Ag levels in mouse plasma was associated with a corresponding decrease of hFIX mRNA levels in the liver. However, the Ad5CMVFIX vector DNA levels did not decrease to a comparable degree, suggesting that the decrease of human FIX:Ag levels in mouse plasma is, to a significant extent, also caused by CMV promotor shut-off and only to a minor degree by loss of vector DNA.
Assuntos
Vetores Genéticos/análise , Transgenes/genética , Adenoviridae/genética , Animais , Fator IX/genética , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Transient expression of full-length wild-type (wt) and a new B-domain truncated (deltaB) FVIII has been investigated in three eukaryotic cell lines (HEK-293, COS, CHO). When expressed in CHO cells, both FVIII proteins reached the same peak antigen levels, whereas in HEK-293 and COS cells those of FVIII/deltaB were up to sixfold those of FVIII/wt. Investigation of specific activity of the recombinant FVIII proteins using a chromogenic and a one-stage assay in addition to the FVIII-antigen ELISA revealed large variations: In HEK-293 cells specific activity of FVIII/deltaB measured with both assays was higher than that of FVIII/wt. In COS cells specific activity of both FVIII proteins was higher measured in the one-stage assay than in the chromogenic assay. In CHO cells both FVIII proteins had similar specific activity in each assay. In summary, expression kinetics and specific activity of conditioned medium vary depending on cell type used.
Assuntos
Fator VIII/genética , Expressão Gênica , Animais , Células CHO , Células COS , Linhagem Celular , Cricetinae , Embrião de Mamíferos , Fator VIII/química , Fator VIII/metabolismo , Humanos , Rim , Cinética , Engenharia de Proteínas , Proteínas Recombinantes , Deleção de Sequência , Transfecção , Fator de von Willebrand/metabolismoRESUMO
Complex interactions between replication deficient adenoviral vectors (Ad5) and the immune system of the host influence the stability of transgenes in vivo. Vector-infected cells are attacked by diverse cellular immune mechanisms which limit transgene persistence. On the other hand, the products of several E3 region genes of wild-type adenovirus can suppress host immune reactions by interference with the expression of MHC class I molecules and by other mechanisms. We have developed an adenoviral vector for human factor IX (Ad5E3+FIX) which carries the E3 region of wild-type adenovirus, and an E3-deleted vector of otherwise similar structure (ad5 delta E3FIX). Intravenous injection of Ad5E3+FIX in C57BI/6 mice resulted in expression levels up to 6000 ng/ml of recombinant human factor IX in the mouse plasma and in enhanced transgene stability as compared with the vector Ad5 delta E3FIX. Whereas expression from E3-deleted vectors was essentially turned off 8 weeks after the gene transfer, the vector Ad5E3+FIX3+FIX supported transgene expression with therapeutic levels of human factor IX in the mouse plasma for > 4 months. The enhanced stability of the vector Ad5E3+FIX appears to be a consequence of efficient E3 region-mediated suppression of the host's antivector immune response. As an additional approach to improving transgene stability the influence of transient CD4+ T cell depletion of the host was investigated. CD4+ cytotoxic T lymphocytes contribute to the clearance of adenovirus-infected cells and play a pivotal role in the activation of CD8+ cytotoxic T cells and as helper T cells in the formation of human adenovirus neutralizing antibodies (HANA). Transient anti-CD4 treatment of the host limited to the time of vector injection resulted in a significant prolongation of transgene expression from the factor IX vector Ad5E3+FIX and a luciferase vector Ad5Luc. The combination of transient anti-CD4 treatment of the host and integration of a complete E3 region in an adenoviral vector resulted in markedly improved transgene stability after gene transfer to the liver (therapeutic factor IX levels for > 6 months).
Assuntos
Adenoviridae , Proteínas E3 de Adenovirus/genética , Anticorpos Monoclonais/farmacologia , Antígenos CD4/imunologia , Fator IX/biossíntese , Fator IX/genética , Expressão Gênica , Vetores Genéticos , Animais , Formação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Primers do DNA , Endotélio Vascular , Genes MHC Classe I , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Subpopulações de Linfócitos T/imunologia , Transfecção , Veias UmbilicaisRESUMO
Recombinant adenoviral vectors are broadly applied in gene therapy protocols. However, adenovector-mediated gene transfer has limitations in vivo. One of these is the low gene transfer rate into organs other than the liver after systemic intravenous vector injection. Local direct injection into the target organ has been used as one possible solution, but increases necessary equipment and methodology and is traumatic to the target. Wild-type adenovirus infection as well as adenovector-mediated gene transfer depends on virus interaction with the Coxsackie adenovirus receptor (CAR) mediating virus attachment to the cell surface, and on interaction with alphavbeta3 and alphavbeta5 integrins mediating virus entry into the cell. In order to assess the receptor-associated potential of different tissues to act as adenovector targets, we have therefore determined CAR and alphav-integrin expression in multiple organs from different species. In addition, we have newly determined several human, rat, pig and dog CAR-mRNA sequences. Sequence comparison and structural analyses of known and of newly determined sequences suggests a potential adenovirus binding site between amino acids 29 and 128 of the CAR. With respect to the virus receptor expression patterns we found that CAR-mRNA expression was extremely variable between different tissues, with the highest levels in the liver, whereas alphav-integrin expression was far more homogenous among different organs. Both CAR and alphav-integrin showed similar expression patterns among different species. There was no correlation, however, between the adenovector expression patterns after intravenous, intracardiac and aortic root injection, respectively, and the virus receptor patterns. In summary, many organs carry both receptors required to make them potential adenovector targets. In sharp contrast, their actual targeting clearly indicates that adenovirus receptor expression is necessary but not sufficient for vector transfer after systemic injection. The apparently very important role of anatomical barriers, in particular the endothelium, requires close attention when developing non-traumatic, organ-specific gene therapy protocols.
Assuntos
Adenoviridae/genética , Antígenos CD/metabolismo , Endotélio/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Receptores Virais , Animais , Northern Blotting , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Cães , Expressão Gênica , Terapia Genética/métodos , Proteínas de Fluorescência Verde , Humanos , Integrina alfa5 , Rim/metabolismo , Fígado/metabolismo , Luciferases/genética , Proteínas Luminescentes/genética , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miocárdio/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Suínos , TransgenesRESUMO
BACKGROUND: Recombinant human G-CSF is widely used to mobilize PBPCs in healthy donors for allogeneic transplantation. There have been concerns about donor safety because of splenic ruptures during G-CSF application. To address this problem, changes in splenic size in 91 healthy donors during G-CSF mobilization of allogeneic PBPCs were investigated. STUDY DESIGN AND METHODS: For mobilization, G-CSF in a dosage of 7.5 microg per kg per day was administered for 5 days and PBPC collection started Day 5. Splenic size was determined by ultrasound before G-CSF application was started and on the day of the first apheresis. RESULTS: The mean increase in splenic length was 11 mm (range, 0-28 mm; p<0.0001), whereas a mean increase of 5 mm in width (range, 0-14 mm; p<0.0001) was measured. No major side effects could be observed. There was no significant correlation between the increase in splenic size and the hematologic values, or the age and body-mass index. In a multivariant analysis, no independent risk factor for the development of a spleen enlargement over 19 mm in length and 9 mm in thickness was found in 20 percent of investigated donors. CONCLUSION: In this prospective trial, a significant spleen enlargement was observed in healthy donors during G-CSF mobilization of allogeneic PBPCs. Further investigations are needed to define the degree of spleen enlargement with higher G-CSF dosages to improve donor safety.