RESUMO
Synthetic tripalmitoyl-S-glycerylcysteinyl (Pam3Cys) peptides are derived from the N-terminal part of bacterial lipoprotein and constitute polyclonal B-lymphocyte and macrophage activators. In order to elucidate the primary events of leukocyte activation, we investigated the biophysical interaction of lipopeptides containing spin labels or fluorescent markers with phosphatidylcholine vesicles or immune cells. Utilizing fluorescence microscopy and FACS analysis we found, that the surface of cells, after incubation with a fluorescein-labelled lipopeptide, was highly fluorescent. In addition, capping and patching was observed. Furthermore, fluorescence quenching experiments and electron paramagnetic resonance studies using vesicles incubated with lipopeptides suggested, that the peptide moiety and other more polar molecules linked to the lipo-amino acid are exposed to the hydrophilic compartment. These results show that in lipopeptide conjugates the Pam3Cys moiety acts as an efficient membrane anchor for molecules covalently coupled to it. The sequestering of the fatty-acid chains of the lipopeptide within the membrane is an early step of interaction, which might induce the uptake of the lipopeptide into the cell and the stimulation of immunocompetent cells.
Assuntos
Linfócitos B/metabolismo , Proteínas da Membrana Bacteriana Externa/farmacologia , Proteínas de Transporte/farmacologia , Membrana Celular/metabolismo , Proteínas de Escherichia coli , Mitógenos/farmacologia , Peptídeos/farmacologia , Sequência de Aminoácidos , Linfócitos B/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Transporte/química , Cisteína/análogos & derivados , Cisteína/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Ácidos Graxos/metabolismo , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Sonicação , Células Tumorais CultivadasRESUMO
Xylanpoly-(hydrogen sulphate) disodium salt with a molecular weight of about 6000 daltons (HOE/BAY 946) completely inhibited syncytium formation induced by the infection of T lymphocytes with HIV as well as viral replication at concentrations above 25 micrograms/ml. This dose was found to be inhibitory for several strains of HIV-1 and HIV-2. Low molecular weight fractions of the compound were less active against HIV, and high molecular derivatives were as active as HOE/BAY 946. A direct influence of the drug on the infectivity of the virus could not be demonstrated. The drug inhibited the reverse transcriptase of HIV. Treatment of permanently HIV-infected U937 cells resulted in a drastic reduction of virus particles released into the supernatant and points to an additional mode of action. A therapeutic effect of HOE/BAY 946 against retroviruses in vivo could be demonstrated in Friend leukaemia virus-infected mice. A clinical pilot study with the compound was started recently in Germany with AIDS patients who did not tolerate or refused to take zidovudine and with asymptomatic virus carriers.
Assuntos
HIV/efeitos dos fármacos , Polissacarídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Medula Óssea/efeitos dos fármacos , Células da Medula Óssea , Sobrevivência Celular/efeitos dos fármacos , Feminino , HIV/enzimologia , HIV/fisiologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-2/efeitos dos fármacos , HIV-2/fisiologia , Humanos , Técnicas In Vitro , Interleucina-1/biossíntese , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Oxigênio/metabolismo , Poliéster Sulfúrico de Pentosana , Inibidores da Transcriptase ReversaRESUMO
A novel immunoassay technique using synthetic lipopeptide (Pam3Cys-Ser) linked to immunodominant peptide domains of HIV-1 and HIV-2 envelope proteins as an antigen adsorbent has been developed. Attachment of peptides to microtiter plates can be considerably improved with this method by employing the hydrophobic properties of lipopeptide. From the sera of 121 HIV-1 infected patients 117 reacted with Pam3Cys-Ser-[HIV-1(598-609)cyclic disulfide]. Five of 5 HIV-2 positive sera were positive with Pam3Cys-Ser-[HIV-2(593-603)cyclic disulfide]. Control sera failed to react with these conjugates.
Assuntos
Síndrome da Imunodeficiência Adquirida/diagnóstico , Infecções por Deltaretrovirus/diagnóstico , Ensaio de Imunoadsorção Enzimática , HIV-1 , HIV-2 , Epitopos , Antígenos HIV , Humanos , Peptídeos Cíclicos , Proteínas dos Retroviridae/imunologia , Software , Proteínas do Envelope Viral/imunologiaRESUMO
The paper describes the production of a prothrombin complex concentrate (PCC) with high virus safety and a well-balanced content of vitamin K-dependent clotting factors and inhibitors. Solid-phase extraction is followed in a second step by optimized anion exchange chromatography using a radial column. A step for virus removal by nanofiltration is introduced in addition to the solvent/detergent step. By speeding up the chromatographic step, the period of time required for production is reduced considerably. The activities of the four vitamin K-dependent clotting factors II, VII, IX and X are in ratios of about 1:1:1:1. Protein C, Protein S, and Protein Z are also present in therapeutically effective concentrations. The product shows no thrombogenicity, in either in vivo nor in vitro models. Clinical investigations show that the PCC is a safe and efficient preparation for the substitutive treatment of FIX or FVII in patients suffering from the respective deficiencies. All bleeding episodes have been efficiently controlled with relatively low doses of the concentrate. The surgical procedures have been conducted without any problems in severely FIX and FVIII deficient patients.
Assuntos
Protrombina/isolamento & purificação , Animais , Humanos , Protrombina/farmacologia , Protrombina/uso terapêutico , Ratos , Trombose/prevenção & controleRESUMO
OBJECTIVE: A validation study of the viral safety of a new polyvalent intravenous immunoglobulin (OCTAGAM) according to EU-guideline III/8115/89-EN and the requirements of the Federal Agency for Sera and Vaccines in Germany was undertaken in May 1994. The following processing steps were analyzed: Cohn-Oncley fractionation, solvent/detergent (SD) treatment, pH 4 exposure, storage of the final product at low pH and immune neutralisation. METHODS: The following virus reduction factors were obtained: Cohn-Oncley fractionation: HIV-1 > 5.50; sindbis virus > 6.36; pseudorabies virus > 7.28; coxsackievirus-B6 2.70; poliovirus-1 > 3.80; SV40 > 5.51. Solvent/Detergent treatment: HIV-1 > 6.03; sindbis virus > 7.80; pseudorabies virus > 8.38. pH 4 exposure: HIV-1 > 8.60; sindbis virus > 8.94; pseudorabies virus > 5.95; coxsackievirus-B6 2.72; SV40 1.15. Immune neutralisation: coxsackievirus-B6 > 4.98, polio-virus-1 > 5.14, HAV > 3.44, HSV-1 > 5.92. The following virus reduction factors were calculated for the final product: HIV-1 > 20.13; sindbis virus > 23.10; pseudorabies virus > 21.61; coxsackievirus-B6 > 10.4; poliovirus-1: > 8.94; HAV > 3.44; SV40 > 6.66. CONCLUSION: The results of our validation studies demonstrated that in addition to Cohn fractionation and immune neutralization, the two additional steps of solvent/detergent treatment and pH 4 exposure, mainly contribute to the safety of OCTAGAM with respect to both enveloped and non-enveloped viruses.
Assuntos
Produtos Biológicos/normas , Sangue/virologia , Imunoglobulinas Intravenosas/normas , Viroses/prevenção & controle , Detergentes , Alemanha , Órgãos Governamentais/normas , Guias como Assunto , HIV-1/efeitos dos fármacos , Humanos , Programas de Rastreamento , Controle de Qualidade , Reprodutibilidade dos Testes , Sindbis virus/efeitos dos fármacos , Viroses/transmissãoRESUMO
The inactivation of both transfusion-relevant and model viruses by modified pasteurization has been evaluated following the established guidelines of the European Union Committee for Proprietary Medical Products Ad Hoc Working Party on Biotechnology/Pharmacy. This heat treatment in solution for 10 h at 63 degrees C was introduced into the manufacturing process of OCTAVI, a very high purity factor VIII concentrate stabilized by von Willebrand factor. It could be demonstrated that both enveloped (human immunodeficiency virus, herpes simplex virus, pseudorabies virus) and non-enveloped viruses (poliovirus, coxsackievirus, hepatitis A virus) were inactivated by this heating step with an efficacy of greater than 4.5 log10 TCID50. The combination of the solvent/detergent step already used in the manufacture with this modified pasteurization leads to a double virus-inactivated factor VIII concentrate (OCTAVI SDPlus) with a viral safety distinctly superior to monoinactivated products.
Assuntos
Antivirais , Bancos de Sangue/normas , Doadores de Sangue , Fator VIII/isolamento & purificação , Reação Transfusional , Detergentes , União Europeia , Guias como Assunto , Temperatura Alta , Humanos , Reprodutibilidade dos Testes , SolventesRESUMO
This study summarises the biochemical and functional properties of a new generation plasma-derived, double virus inactivated von Willebrand Factor/Factor VIII (VWF/FVIII) concentrate, Wilate, targeted for the treatment of both von Willebrand disease (VWD) and haemophilia A. The manufacturing process comprises two chromatographic steps based on different performance principles, ensuring a high purity of the concentrate (mean specific activity in 15 consecutive production batches: 122 IU FVIII:C/mg total protein) and, thus, minimising the administered protein load to the patient (specification: < or = 15 mg total protein per 900 IU Wilate). The optimised solvent/detergent (S/D) treatment and prolonged terminal dry-heat (PermaHeat) treatment of the lyophilised product at a specified residual moisture (RM) provide two mechanistically independent, effective and robust virus inactivation procedures for enveloped viruses and one step for non-enveloped viruses. These process steps are aggressive enough to inactivate viruses efficiently, but yet gentle enough to maintain the structural integrity and function of the VWF and FVIII molecules, as proven by state-of-the-art assays covering the diverse features of importance. The VWF multimeric pattern is close to the one displayed by normal plasma, with a consistent content of more than 10 multimers, but a relatively lower portion of the very high multimers. The multimeric triplet structure is normal, underlining the gentle and effective manufacturing process, which does not require the addition of protein stabilisers at any step. The balanced activity ratio of VWF to FVIII is close to that of plasma from healthy subjects, rendering Wilate suitable also for the safe and effective treatment of patients with VWD.
Assuntos
Fator VIII/isolamento & purificação , Fator VIII/fisiologia , HIV-1/fisiologia , Inativação de Vírus , Fator de von Willebrand/isolamento & purificação , Fator de von Willebrand/fisiologia , Proteínas Sanguíneas/fisiologia , Fator VIII/química , HIV-1/isolamento & purificação , Humanos , Segurança , Fator de von Willebrand/químicaRESUMO
BACKGROUND AND OBJECTIVES: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by aberrantly folded cellular proteins (PrP(Sc); prions) that are generally resistant to conventional pathogen-inactivation techniques. To ensure effective decontamination and inactivation of prions that could be present in source material, we investigated critical factors that influence prion inactivation by NaOH. MATERIALS AND METHODS: A decrease in prion infectivity correlates with the disappearance of the protease-resistant core of PrPSc (PrPRES) observed in biochemical assays. To model prion inactivation, hamster scrapie (strain 263K) brain homogenate (SBH) was incubated for specific periods of time in 0.1 m NaOH at 4 or 18 degrees C, with or without detergent. Neutralized samples were subjected to limited digestion with proteinase K (PK) and then analysed using an endpoint dilution western blot assay and antibody 3F4. Structural changes in prions exposed to NaOH were examined using differential immunoprecipitation. RESULTS: Treatment of SBH with 0.1 m NaOH for 15 min, in the absence of detergent, at 4 and 18 degrees C caused a reduction in the PrP(RES) signal of 3.5 and 4.0 log10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60-min incubation in NaOH further enhanced PrPRES reduction to > or = 4.5 log10 units (i.e. below the limit of detection). NaOH treatment induced conformational changes in PrP that resulted in the exposure of a hidden epitope and enabled prion immunoprecipitation by antibody 3F4. CONCLUSIONS: The use of NaOH can effectively reduce prion levels in an in vitro inactivation assay. After pretreatment of SBH with detergent, NaOH completely eliminates the PrPRES signal. Detergent may liberate lipid membrane-protected PrPSc to improve access to NaOH, which can then inactivate PrPSc by altering its structure. In cases of unidentified exposure to PrPSc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.
Assuntos
Bioensaio , Descontaminação , Endopeptidase K/química , Proteínas PrPSc/química , Doenças Priônicas/prevenção & controle , Sarcosina/análogos & derivados , Hidróxido de Sódio/química , Animais , Cricetinae , Proteínas PrPSc/patogenicidade , Sarcosina/químicaRESUMO
SARS-coronavirus (SARS-CoV) is a newly emerged, highly pathogenic agent that caused over 8000 human infections with nearly 800 deaths between November 2002 and September 2003. While direct person-to-person transmission via respiratory droplets accounted for most cases, other modes have not been ruled out. SARS-CoV viraemia does not seem to reach high titres, however, it has to be excluded that virus transmission may occur via blood transfusion or application of therapeutic plasma products, e.g. fresh-frozen plasma or single components derived thereof. Manufacturing processes of all plasma derivatives are required to comprise dedicated virus inactivation/removal steps. Treatment with a mixture of solvent and detergent (SD) has successfully been applied to inactivate the most members of the transfusion-relevant viruses without affecting therapeutic properties of the products. The SD treatment irreversibly disrupts the lipid envelope of viruses such as HIV, HBV, HCV, HGV and CMV. In this study we evaluated the manufacturing process of an immunoglobulin preparation (OCTAGAM, manufactured by Octapharma Pharmazeutika Produktionsges.m.b.H., Vienna, Austria) for its capacity to inactivate the SARS-CoV. Our results demonstrate that SARS-CoV was completely inactivated below the limit of detection. This was found to occur within 1 min of SD treatment.
Assuntos
Detergentes/farmacologia , Imunoglobulinas , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Solventes/farmacologia , Inativação de Vírus , HumanosRESUMO
OCTAPLAS is a cell-free standardized blood group specific human coagulation active plasma for transfusion. The viral safety is mainly based on the treatment with solvent/detergent. SD is known to irreversibly inactivate the lipid enveloped viruses including HIV 1 + 2, HBV and HCV and has been recommended both by European guidelines and the FDA. The potential limitation of each inactivation method should be validated according to the current EU-guidelines CPMP/BWP/268/95 and CPMP/BWP/269/95. Our studies demonstrate that the SD method inactivates lipid enveloped viruses within a few minutes to below the limit of detection. The concentration of the SD reagents as well as several other process parameters chosen for the virus inactivation step of plasma pools of different protein and lipid composition result in a high safety margin towards the clinically relevant viruses.
Assuntos
Detergentes , Troca Plasmática/efeitos adversos , Plasma/virologia , Solventes , Esterilização/métodos , Patógenos Transmitidos pelo Sangue , Guias como Assunto , Humanos , Retroviridae/crescimento & desenvolvimento , Segurança , Viroses/prevenção & controle , Viroses/transmissãoRESUMO
Due to the closer virus-host interactions viral diseases are more difficult to control by drugs than most of the bacterial infections. Moreover, the search for antiviral compounds against HIV is an exceptional challenge. The enormous genetic and biological variability, the life-long persistence and the invasion of the brain are only some of the aspects which complicate a fast solution. In principal there are three different strategies to develop new chemotherapeutics. Most of the drugs so far tested in clinical trials were identified by routine screening, others were modified by specific chemical synthesis. After the three-dimensional structure of some HIV proteins became known, rational drug design by computer modeling resulted in compounds exhibiting excellent anti-HIV activity in vitro. The reverse transcriptase inhibitor AZT has been used for HIV-infected individuals for several years. However, side effects and the development of AZT-resistant virus strains force to combine AZT with other drugs and to develop additional lead compounds.
Assuntos
Antivirais , Infecções por HIV/tratamento farmacológico , HumanosRESUMO
Membrane proteins from murine lymphoblasts enriched by the Triton-X114 procedure were analysed by 2-dimensional gel electrophoresis to reveal proteins differentially expressed in mitogen-reactive subpopulations of B cells. The protein patterns from C57BL/6 normal and nude mice and from the B10.Sc.Cr LPS-non-responder strain, activated with lipopolysaccharide (LPS) or an analogue of lipoprotein, were virtually identical when run under strictly parallel conditions. These experiments raise the question as to whether mitogen receptors, serologically and functionally found to be membrane-bound, do exist as membrane proteins.
Assuntos
Linfócitos B/análise , Lipopolissacarídeos/farmacologia , Lipoproteínas/farmacologia , Proteínas de Membrana/análise , Animais , Feminino , Lipopeptídeos , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligopeptídeos/farmacologiaRESUMO
Lipoprotein from the outer membrane of Escherichia coli constitutes a potent mitogen and polyclonal activator for B lymphocytes of different species. The binding of lipoprotein to murine spleen cells was investigated using water-soluble 125I-labelled citraconylated lipoprotein from E. coli B/r. Our results indicate that the binding of this B-cell mitogen to splenocytes is a saturable, time- and dose-dependent, reversible process; about 9.7 X 10(8) lipoprotein molecules were bound to each cell. The mechanism of the binding of lipoprotein to lymphocytes was investigated by using the synthetic analogue of its N-terminal part, S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-( S)-seryl- (S)-seryl-(S)-asparaginyl-(S)-alanine (tripalmitoyl pentapeptide). This compound had been shown by us previously to be the molecular part of lipoprotein responsible for mitogenicity and exhibited, in all experiments performed, a stimulatory activity towards B lymphocytes comparable, or even superior, to native lipoprotein. Binding proteins for the synthetic N-terminus were enriched by affinity chromatography, using an affinity column prepared by coupling the mitogenic compound to CPG-aminopropyl controlled-pore glass beads by the carbodiimide method. [3H]Leucine-labelled murine spleen cells were solubilized by the nonionic detergent NP40 and applied to the affinity adsorbent. Proteins bound to the column were selectively eluted by a solution of tripalmitoyl pentapeptide, and the fractions were analyzed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and autoradiography. Our results indicate the presence of a major binding protein of Mr 35000 on mouse primary lymphocytes for the biologically active N-terminal structure of lipoprotein, which might play a role as membrane receptor in mitogenic B lymphocyte activation.
Assuntos
Linfócitos B/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Mitógenos/metabolismo , Oligopeptídeos/metabolismo , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Lipopeptídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Fotofluorografia , Baço/metabolismoRESUMO
A virus removal system based on tangential flow filtration was introduced into a Factor IX production process. Beside the intended virus reduction potency of filter membranes, an additional purification effect could be achieved. This purification effect was evaluated in detail by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion HPLC. High-molecular-mass impurities were retained by the membrane, thus increasing the specific activity of the product.
Assuntos
Patógenos Transmitidos pelo Sangue/isolamento & purificação , Fator IX/isolamento & purificação , Ultrafiltração/métodos , Vírus/isolamento & purificação , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Humanos , ImunodifusãoRESUMO
Lipoprotein from the outer membrane of Escherichia coli and a synthetic analogue of its N-terminal lipopeptide part, tripalmitoyl-pentapeptide, constitute potent mitogens and polyclonal activators of murine B-lymphocytes in vitro. When entering the circulation after intravenous administration in experimental animals, they interact with the humoral and cellular elements of the blood, which results in splenomegaly and B-lymphocyte activation in vivo. We investigated lipopeptide-binding proteins in normal mouse serum and on splenocytes. By affinity chromatography using an affinity adsorbent prepared by coupling the lipoprotein analogue to CPG-aminopropyl derivatized glass beads, we could enrich one major binding protein for tripalmitoyl-pentapeptide from mouse serum, which was identified as albumin. Binding proteins on lymphocytes were determined as follows: Spleen cells of C3H/HeJ mice were activated by the B cell mitogen lipoprotein, biosynthetically labelled with [3H]leucine, and solubilized by the nonionic detergent Nonidet P40. From the cell lysate, binding proteins were isolated by affinity chromatography: As analysed by polyacrylamide gel electrophoresis and autoradiography, proteins with molecular masses of 24, 27, 33, 45, 53, 61 and 71 kDa were eluted from the tripalmitoyl-pentapeptide adsorbent. The eluted material was further enriched for glycoproteins by Lens culinaris lectin affinity chromatography, and immunoprecipitation studies were performed with the glycoprotein fractions using alloantisera specific for class I and class II gene products of the H-2k haplotype. We could show that both class I and class II MHC glycoproteins could be enriched on the tripalmitoyl-pentapeptide column. This finding might suggest that, among other proteins, MHC-encoded proteins are involved in lymphocyte activation by a mitogenic lipopeptide.
Assuntos
Antígenos H-2/isolamento & purificação , Mitógenos/metabolismo , Oligopeptídeos/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Antígenos H-2/genética , Técnicas In Vitro , Lipopeptídeos , Lipoproteínas/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C3H , Baço/imunologia , Baço/metabolismoRESUMO
The inactivation of both transfusion-relevant and model viruses by modified pasteurisation (10 hours at 63 degrees C in solution) has been evaluated following the established guidelines of the EU CPMP Ad Hoc Working Party on Biotechnology/Pharmacy. This heat treatment was introduced into the manufacturing process of OCTAVI, a very high purity factor VIII concentrate stabilized only by von Willebrand factor, in the presence of a proprietary mixture of low molecular weight stabilizers. Both enveloped (human immunodeficiency virus, Sindbis virus, herpes simplex virus, pseudorabies virus) and nonenveloped viruses (poliovirus, Coxsackievirus, hepatitis A virus) were inactivated by this heating step by more than 4.7 log10. The combination of the solvent/detergent step used in the manufacture of OCTAVI with this modified pasteurization leads to a double virus-inactivated factor VIII concentrate (OCTATE) with a viral safety distinctly superior to monoinactivated products.
Assuntos
Contaminação de Medicamentos , Fator VIII , Calefação , Esterilização , Vírus , Animais , Linhagem Celular , Química Farmacêutica/normas , Qualidade de Produtos para o Consumidor , Detergentes , HumanosRESUMO
The component compositions of the DNA-dependent RNA polymerases of the extremely thermophilic, anaerobic sulfur-respiring archaebacteria Thermoproteus tenax and Desulfurococcus mucosus strongly resemble each other but also that of the RNA polymerase of Sulfolobus acidocaldarius suggesting that both organisms belong to the same novel order Thermoproteales, which together with the order represented by Sulfolobus, forms the thermoacidophilic branch of archaebacteria. The component pattern of the RNA polymerase of Thermoplasma acidophilum, which does not belong to this branch, also appears homologous. The archaebacterial type of the DNA-dependent RNA polymerase is thus characterized by 9-10 components yielding a characteristic pattern which resembles that of yeast RNA polymerase A(I). In contrast to the alpha subunit of eubacterial RNA polymerases, the third largest component of archaebacterial RNA polymerases, although similar in size, is present only one per enzyme monomer. The polymerases of T. tenax and D. mucosus, like those previously isolated from other archaebacteria, are completely resistant against 100 microgram/ml rifampicin and streptolydigin. The RNA polymerases of both organisms are highly thermostable. The enzyme from D. mucosus transcribes selectively and almost completely the H strand of phase T7 DNA.
Assuntos
Archaea/enzimologia , Bactérias/enzimologia , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Fenômenos Químicos , Química , Peso Molecular , Temperatura , Transcrição GênicaRESUMO
Product development and process validation are shown in the case of several products obtained from human plasma. These are virus-inactivated plasma, intravenous immunoglobulins and the clotting factors VIII and IX. Different analytical methods are presented, which are used for product control and in-process control. For the production of virus-inactivated human plasma a down-scale protocol is presented, allowing a simulation of the production on a laboratory scale. Virus validation has shown that the reduction of transfusion-relevant viruses in the process was higher than six log steps. Determination of leachables from the RP-column, which was used in this production, proved that they appear in the final product in quantities below the detection limits only. It was also shown that the chemicals used for virus inactivation could be quantitatively removed from the product. For the isolation of other products, here intravenous gamma globulins and the clotting factors VIII and IX, similar validation steps had to be taken. In the case of clotting factor VIII the following data were determined, the reduction of viruses, the amount of leachables from the column, the residues of chemicals from the solvent/detergent treatment for virus inactivation. Virus reduction was successfully performed as well as the removal of chemicals used for virus inactivation. The amount of leachables from the columns used for chromatographic purification was found to be far below the permissible levels.
Assuntos
Produtos Biológicos/isolamento & purificação , Proteínas Sanguíneas/isolamento & purificação , Plasma/virologia , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Detergentes , Contaminação de Medicamentos , Humanos , Octoxinol , Reprodutibilidade dos Testes , Segurança , Viroses/transmissão , Vírus/isolamento & purificaçãoRESUMO
Sequential virus isolates from an HIV-1-infected woman treated orally with 3'-azido-3'-deoxythymidine (AZT) for over two years showed a 10-fold reduced sensitivity for AZT after 8 months and a 100-fold resistance after 24-32 months of drug therapy. These AZT-resistant mutants were totally sensitive in vitro to other reverse transcriptase (RT)-inhibitors like the AZT-analogue 3'-fluoro-3'-deoxythymidine (FdT) or the chemically less related nucleoside analogue 2',3'-dideoxycytosine (ddC). Even the benzodiazepin derivative 4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)-imidazo [4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), a new drug specific for HIV-1 RT, was inhibitory for these virus strains. Moreover, compounds with different modes of action, e.g. polysulfated polyxylan, exhibited full antiviral activity as well. Thus, AZT resistance seems to be highly specific and should allow to develop further drugs to be used when AZT resistance has emerged. 5.9 kb fragments of the 5'-genomic halves of these sequential HIV-isolates were amplified by PCR and cloned. DNA sequence analysis revealed that the RT gene of the two highly AZT-resistant isolates carried two of the mutations described by Larder et al. [Science 246, (1989)], the Lys 70----Arg and the Thr 215----Tyr transitions. The isolate obtained after 32 months of AZT-therapy in addition contained a third mutation at position 67 (Asp----Asn); in contrast to Larder's report, no mutation was found at position 219. Thus, although these virus isolates showed at least a 100-fold reduced susceptibility for AZT in vitro, the four mutations postulated to be relevant for highly resistant strains were only partially confirmed.
Assuntos
Resistência Microbiana a Medicamentos/genética , HIV/genética , Replicação Viral/efeitos dos fármacos , Zidovudina/farmacologia , Sequência de Aminoácidos , Antivirais/farmacologia , Sequência de Bases , Células Cultivadas , Clonagem Molecular , DNA Viral/genética , HIV/efeitos dos fármacos , HIV/fisiologia , Humanos , Monócitos , Reação em Cadeia da PolimeraseRESUMO
The polysulfated polyxylan HOE/BAY946, which has been tested in two pilot studies in ARC/AIDS patients and in asymptomatic HIV carries in Germany, was believed to act by inhibiting virus attachment to the cell. However, the drug was also found to reduce the amount of HIV particles released from infected peripheral blood mononuclear cells (PBMC) in vitro. Furthermore, preincubation of PBMC with the drug led to a partial inhibition of a following HIV infection, suggesting that the drug also affects virus entry. Electron Paramagnetic Resonance (EPR) measurements on uninfected human lymphocytes using 5-proxyl-nonane as spin label demonstrated smaller hyperfine coupling constant (aN) values in the presence of HOE/BAY946 or dextran sulfate 5000. Accordingly, h-1p/h-1H ratios were decreased, indicating increased plasma membrane hydrophobicity and a membrane-stabilizing effect of the drugs. Culture of the chronically HIV-infected monocytic cell line U937/HIV-2D194 in the presence of HOE/BAY946 specifically and drastically reduced the release of virions and the intracellular synthesis of viral proteins as determined by radioimmunoprecipitation and reverse transcriptase assays. In conclusion, although the EPR studies showed a physico-chemical effect on membrane polarity, HOE/BAY946 and dextran sulfate clearly affect processes beyond the cell membrane. Thus, in contrast to previous reports suggesting that polysulfated sugars affect HIV only by inhibiting virus binding to uninfected cells, they clearly inhibit HIV in infected cells as well and appear to have a pleiotropic mode of action. Such drugs may be less likely to result in viral resistance after prolonged application than substances acting only on one step in the life cycle of the virus.