A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary fo... more A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary for specific transcription of a 5 S RNA gene was separated into fractions required for specific transcription initiation and an additional fraction needed in the reconstituted system to produce the 3' end characteristic of mature 5 S RNA. The latter fraction contained a novel processing activity characterized by an exonuclease specific for highly structured RNAs, including 5 S RNA. An intact helical stem formed between the 5' and 3' ends of the 5 S RNA precursor determines the 3' nucleotide. In addition, the presence of ATP is required for specific processing. However, the possibility has not been ruled out that ATP inhibits a nonspecific ribonuclease in the extract since processing proceeds into the helical stem in its absence.
In bacterial infections, mononuclear and polymorphonuclear phagocytes are key components of host ... more In bacterial infections, mononuclear and polymorphonuclear phagocytes are key components of host defenses. Recent investigations have indicated that chemokines are able to recruit and activate phagocytes. In particular, interleukin-8 (IL-8) attracts polymorphonuclear leukocytes (PMNs), while monocyte chemoattractant protein-1 (MCP-1) is selective for cells of the monocyte/macrophage lineage. In this investigation, we analyzed the in situ expression of IL-8 and MCP-1 mRNAs in human periodontal infections. Specific mRNA was detected by in situ hybridization using 35S-labeled riboprobes in frozen tissue sections. Phagocytes (PMNs and macrophages) were specifically detected as elastase-positive or CD68+ cells by a three-stage immunoperoxidase technique. Results indicated that expression of phagocyte-specific cytokines was confined to selected tissue locations and, in general, paralleled phagocyte infiltration. In particular, IL-8 expression was maximal in the junctional epithelium adjac...
African trypanosomes are the causative agents of many medically and economically important diseas... more African trypanosomes are the causative agents of many medically and economically important diseases of man and domestic animals. The cell body of these blood-dwelling protozoa is enveloped with a dense layer of pellicular microtubules, which confer both motility and mechanical stability on these cells; microtubules are also important components of the flagellum. The major structural components of the microtubuli are two related proteins, alpha- and beta-tubulin. We have analyzed the genomic organization of alpha- and beta-tubulin genes in Trypanosoma brucei. In this organism, the majority of these genes are arranged in a single, tightly packed cluster of alternating alpha- and beta-tubulin genes with a basic repeat length together of 3.6 kilobase pairs. A genomic library of T. brucei was constructed by using the phage vector A 1059, and recombinant phages carrying tubulin genes were isolated by screening the library with heterologous chicken tubulin cDNA probes. The results of restr...
The present invention relates to human monoclonal antibodies specific for P. aeruginosa serotype ... more The present invention relates to human monoclonal antibodies specific for P. aeruginosa serotype IATS O6, hybridomas producing them, nucleic acids encoding them and host cells transfected with the nucleic acids. The present invention further relates to a method for producing the monoclonal antibody. The invention also relates to a pharmaceutical composition comprising at least one antibody or at least one nucleic acid encoding said antibody.
The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) ... more The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG-related hemolytic events. We present in this work a modification of the manufacturing process of Privigen-a 10% l-proline-stabilized IVIG product-that allows extensive reduction of isoagglutinin concentrations in the final product. An additional immunoaffinity chromatography (IAC) step was introduced toward the end of the manufacturing process of Privigen. Isoagglutinin titers were measured using the indirect agglutination method and a published flow cytometry-based binding assay. Quality attributes, such as microorganism counts and concentration of endotoxins, IgG, IgA, IgM, aggregates, and so forth were measured using standardized procedures. The introduction of an IAC step in the manufacturing process of Privigen resulted in an 88% to 90% reduction in isoagglutinins between the feed of the chromatography column and the flow-through fraction. All other product quality attributes measured were nearly identical before and after IAC. This process modification resulted in a three-titer-step reduction in isoagglutinin levels in the final IgG product compared to Privigen lots produced by the unmodified process. Introducing an isoagglutinin-specific IAC step in the manufacturing process of Privigen is an efficient strategy for reduction of anti-A and anti-B titers. Such reductions might help minimize the risk of hemolytic events in patients receiving IVIG therapy.
Animal Cell Technology: Products from Cells, Cells as Products, 1999
ABSTRACT Hemolytic Disease of the Newborn (HDN) can be a life threatening condition in Rh D posit... more ABSTRACT Hemolytic Disease of the Newborn (HDN) can be a life threatening condition in Rh D positive babies born from Rh D negative mothers. For several decades, prophylactic treatment with immunoglobulins derived from human blood plasma from immunized donors has been available. Because of concerns for donor safety and limited supply of raw material, alternatives for production of specific immunoglobulins were investigated. Since 1994 ZLB has, in collaboration with a group at the University Hospital in Bern, started to develop recombinant anti-D antibodies. In 1997, a multidisciplinary team with expertise ranging from molecular biology to biochemical engineering for large scale operations was constituted, co-funded by the Swiss National Science Foundation and ZLB, as the industrial partner. The aims of this team are to establish the prophylactic principle and the manufacturing process for anti-D therapy based on recombinant antibody preparations produced from Chinese hamster ovary (CHO) cells.
A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary fo... more A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary for specific transcription of a 5 S RNA gene was separated into fractions required for specific transcription initiation and an additional fraction needed in the reconstituted system to produce the 3' end characteristic of mature 5 S RNA. The latter fraction contained a novel processing activity characterized by an exonuclease specific for highly structured RNAs, including 5 S RNA. An intact helical stem formed between the 5' and 3' ends of the 5 S RNA precursor determines the 3' nucleotide. In addition, the presence of ATP is required for specific processing. However, the possibility has not been ruled out that ATP inhibits a nonspecific ribonuclease in the extract since processing proceeds into the helical stem in its absence.
S U N D A Y 311 Selective Exclusion of High Titer Donor Plasma and Immunoadsorption Chromatograph... more S U N D A Y 311 Selective Exclusion of High Titer Donor Plasma and Immunoadsorption Chromatography As Steps to Reduce Isoagglutinin Titers in IVIG Liane Hoefferer, Ibrahim El Menyawi, Brigitte Siani, Martin Imboden, Annette Gaida, Isabelle Glauser, Reinhard Bolli, Katharina Willimann, Sandra Wymann, A. Adriano Marques, Eleanora Widmer; CSL Behring, Berne, Switzerland. RATIONALE: Hemolysis is a rare but potentially serious complication of high-dose IVIG therapy. Isoagglutinins originating in donor plasma are believed to play a major role in these reactions, but host factors are also important. We determined the effects of excluding plasma from donors with high isoagglutinin titers from the pools used to prepare IVIG, and of specific anti-A/B immunoaffinity chromatography (IAC), on the anti-A and anti-B titers of IVIG products prepared using the process for Privigen/ Hizentra. METHODS: A high-throughput assay was used to identify plasma donors with the highest anti-A titers. The effects of addition of IAC with A/Btrisaccharide-coupled resins into the manufacturing process were also studied. Anti-A/B isoagglutinin titers in the resulting IVIG preparations were measured by indirect agglutination and flow cytometry. RESULTS: Exclusion of the 5% of plasma donors with the highest anti-A titers reduced both anti-A and anti-B isoagglutinins in the final product by approx. 50% (a single 2-fold dilution step, i.e. reduction of the titer from 1:16 to 1:8, and 1:8 to 1:4, respectively. N530 lots). Specific IAC reduced anti-A and anti-B titers by at least two 2-fold dilution steps in the final product (>80% reduction shown by flow cytometry on standard red cells), while the content of antibodies against common microbial antigens remained unchanged. CONCLUSIONS: Anti-A/B isoagglutinin reduction in IVIG products is feasible using screening and exclusion of a small percentage of high anti-A titer donors andwith specific IAC. These approachesmay reduce the risk of hemolysis in IgG therapy.
A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary fo... more A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary for specific transcription of a 5 S RNA gene was separated into fractions required for specific transcription initiation and an additional fraction needed in the reconstituted system to produce the 3' end characteristic of mature 5 S RNA. The latter fraction contained a novel processing activity characterized by an exonuclease specific for highly structured RNAs, including 5 S RNA. An intact helical stem formed between the 5' and 3' ends of the 5 S RNA precursor determines the 3' nucleotide. In addition, the presence of ATP is required for specific processing. However, the possibility has not been ruled out that ATP inhibits a nonspecific ribonuclease in the extract since processing proceeds into the helical stem in its absence.
In bacterial infections, mononuclear and polymorphonuclear phagocytes are key components of host ... more In bacterial infections, mononuclear and polymorphonuclear phagocytes are key components of host defenses. Recent investigations have indicated that chemokines are able to recruit and activate phagocytes. In particular, interleukin-8 (IL-8) attracts polymorphonuclear leukocytes (PMNs), while monocyte chemoattractant protein-1 (MCP-1) is selective for cells of the monocyte/macrophage lineage. In this investigation, we analyzed the in situ expression of IL-8 and MCP-1 mRNAs in human periodontal infections. Specific mRNA was detected by in situ hybridization using 35S-labeled riboprobes in frozen tissue sections. Phagocytes (PMNs and macrophages) were specifically detected as elastase-positive or CD68+ cells by a three-stage immunoperoxidase technique. Results indicated that expression of phagocyte-specific cytokines was confined to selected tissue locations and, in general, paralleled phagocyte infiltration. In particular, IL-8 expression was maximal in the junctional epithelium adjac...
African trypanosomes are the causative agents of many medically and economically important diseas... more African trypanosomes are the causative agents of many medically and economically important diseases of man and domestic animals. The cell body of these blood-dwelling protozoa is enveloped with a dense layer of pellicular microtubules, which confer both motility and mechanical stability on these cells; microtubules are also important components of the flagellum. The major structural components of the microtubuli are two related proteins, alpha- and beta-tubulin. We have analyzed the genomic organization of alpha- and beta-tubulin genes in Trypanosoma brucei. In this organism, the majority of these genes are arranged in a single, tightly packed cluster of alternating alpha- and beta-tubulin genes with a basic repeat length together of 3.6 kilobase pairs. A genomic library of T. brucei was constructed by using the phage vector A 1059, and recombinant phages carrying tubulin genes were isolated by screening the library with heterologous chicken tubulin cDNA probes. The results of restr...
The present invention relates to human monoclonal antibodies specific for P. aeruginosa serotype ... more The present invention relates to human monoclonal antibodies specific for P. aeruginosa serotype IATS O6, hybridomas producing them, nucleic acids encoding them and host cells transfected with the nucleic acids. The present invention further relates to a method for producing the monoclonal antibody. The invention also relates to a pharmaceutical composition comprising at least one antibody or at least one nucleic acid encoding said antibody.
The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) ... more The passive transfer of antibodies specific to blood groups A and B (also called isoagglutinins) contained in immunoglobulin (Ig)G products for intravenous administration (IVIG) is believed to be largely responsible for rare but sometimes serious IVIG-related hemolytic events. We present in this work a modification of the manufacturing process of Privigen-a 10% l-proline-stabilized IVIG product-that allows extensive reduction of isoagglutinin concentrations in the final product. An additional immunoaffinity chromatography (IAC) step was introduced toward the end of the manufacturing process of Privigen. Isoagglutinin titers were measured using the indirect agglutination method and a published flow cytometry-based binding assay. Quality attributes, such as microorganism counts and concentration of endotoxins, IgG, IgA, IgM, aggregates, and so forth were measured using standardized procedures. The introduction of an IAC step in the manufacturing process of Privigen resulted in an 88% to 90% reduction in isoagglutinins between the feed of the chromatography column and the flow-through fraction. All other product quality attributes measured were nearly identical before and after IAC. This process modification resulted in a three-titer-step reduction in isoagglutinin levels in the final IgG product compared to Privigen lots produced by the unmodified process. Introducing an isoagglutinin-specific IAC step in the manufacturing process of Privigen is an efficient strategy for reduction of anti-A and anti-B titers. Such reductions might help minimize the risk of hemolytic events in patients receiving IVIG therapy.
Animal Cell Technology: Products from Cells, Cells as Products, 1999
ABSTRACT Hemolytic Disease of the Newborn (HDN) can be a life threatening condition in Rh D posit... more ABSTRACT Hemolytic Disease of the Newborn (HDN) can be a life threatening condition in Rh D positive babies born from Rh D negative mothers. For several decades, prophylactic treatment with immunoglobulins derived from human blood plasma from immunized donors has been available. Because of concerns for donor safety and limited supply of raw material, alternatives for production of specific immunoglobulins were investigated. Since 1994 ZLB has, in collaboration with a group at the University Hospital in Bern, started to develop recombinant anti-D antibodies. In 1997, a multidisciplinary team with expertise ranging from molecular biology to biochemical engineering for large scale operations was constituted, co-funded by the Swiss National Science Foundation and ZLB, as the industrial partner. The aims of this team are to establish the prophylactic principle and the manufacturing process for anti-D therapy based on recombinant antibody preparations produced from Chinese hamster ovary (CHO) cells.
A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary fo... more A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary for specific transcription of a 5 S RNA gene was separated into fractions required for specific transcription initiation and an additional fraction needed in the reconstituted system to produce the 3' end characteristic of mature 5 S RNA. The latter fraction contained a novel processing activity characterized by an exonuclease specific for highly structured RNAs, including 5 S RNA. An intact helical stem formed between the 5' and 3' ends of the 5 S RNA precursor determines the 3' nucleotide. In addition, the presence of ATP is required for specific processing. However, the possibility has not been ruled out that ATP inhibits a nonspecific ribonuclease in the extract since processing proceeds into the helical stem in its absence.
S U N D A Y 311 Selective Exclusion of High Titer Donor Plasma and Immunoadsorption Chromatograph... more S U N D A Y 311 Selective Exclusion of High Titer Donor Plasma and Immunoadsorption Chromatography As Steps to Reduce Isoagglutinin Titers in IVIG Liane Hoefferer, Ibrahim El Menyawi, Brigitte Siani, Martin Imboden, Annette Gaida, Isabelle Glauser, Reinhard Bolli, Katharina Willimann, Sandra Wymann, A. Adriano Marques, Eleanora Widmer; CSL Behring, Berne, Switzerland. RATIONALE: Hemolysis is a rare but potentially serious complication of high-dose IVIG therapy. Isoagglutinins originating in donor plasma are believed to play a major role in these reactions, but host factors are also important. We determined the effects of excluding plasma from donors with high isoagglutinin titers from the pools used to prepare IVIG, and of specific anti-A/B immunoaffinity chromatography (IAC), on the anti-A and anti-B titers of IVIG products prepared using the process for Privigen/ Hizentra. METHODS: A high-throughput assay was used to identify plasma donors with the highest anti-A titers. The effects of addition of IAC with A/Btrisaccharide-coupled resins into the manufacturing process were also studied. Anti-A/B isoagglutinin titers in the resulting IVIG preparations were measured by indirect agglutination and flow cytometry. RESULTS: Exclusion of the 5% of plasma donors with the highest anti-A titers reduced both anti-A and anti-B isoagglutinins in the final product by approx. 50% (a single 2-fold dilution step, i.e. reduction of the titer from 1:16 to 1:8, and 1:8 to 1:4, respectively. N530 lots). Specific IAC reduced anti-A and anti-B titers by at least two 2-fold dilution steps in the final product (>80% reduction shown by flow cytometry on standard red cells), while the content of antibodies against common microbial antigens remained unchanged. CONCLUSIONS: Anti-A/B isoagglutinin reduction in IVIG products is feasible using screening and exclusion of a small percentage of high anti-A titer donors andwith specific IAC. These approachesmay reduce the risk of hemolysis in IgG therapy.
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Papers by Martin Imboden