The Experts below are selected from a list of 31056 Experts worldwide ranked by ideXlab platform
M. Bishr Omary - One of the best experts on this subject based on the ideXlab platform.
-
Absence of Keratin 8 or 18 promotes antimitochondrial autoantibody formation in aging male mice
The FASEB Journal, 2015Co-Authors: Diana M. Toivola, Raymond Kwan, Aida Habtezion, Julia O. Misiorek, Linxing Zhang, Joel H. Nyström, Orr Sharpe, William H. Robinson, M. Bishr OmaryAbstract:Human mutations in Keratin 8 (K8) and Keratin 18 (K18), the intermediate filament proteins of hepatocytes, predispose to several liver diseases. K8-null mice develop chronic liver injury and fragile hepatocytes, dysfunctional mitochondria, and Th2-type colitis. We tested the hypothesis that autoantibody formation accompanies the liver damage that associates with K8/K18 absence. Sera from wild-type control, K8-null, and K18-null mice were analyzed by immunoblotting and immunofluorescence staining of cell and mouse tissue homogenates. Autoantibodies to several antigens were identified in 81% of K8-null male mice 8 mo or older. Similar autoantibodies were detected in aging K18-null male mice that had a related liver phenotype but normal colon compared with K8-null mice, suggesting that the autoantibodies are linked to liver rather than colonic disease. However, these autoantibodies were not observed in nontransgenic mice subjected to 4 chronic injury models. The autoantigens are ubiquitous and partition with mitochondria. Mass spectrometry and purified protein analysis identified, mitochondrial HMG-CoA synthase, aldehyde dehydrogenase, and catalase as the primary autoantigens, and glutamate dehydrogenase and epoxide hydrolase-2 as additional autoantigens. Therefore, absence of the hepatocyte Keratins results in production of anti-mitochondrial autoantibodies (AMA) that recognize proteins involved in energy metabolism and oxidative stress, raising the possibility that AMA may be found in patients with Keratin mutations that associate with liver and other diseases.
-
Keratin 8 absence down-regulates colonocyte HMGCS2 and modulates colonic ketogenesis and energy metabolism.
Molecular Biology of the Cell, 2015Co-Authors: Terhi O. Helenius, Aida Habtezion, Julia O. Misiorek, Joel H. Nyström, Lina E. Fortelius, Jian Liao, M. Nadeem Asghar, Haiyan Zhang, Salman Azhar, M. Bishr OmaryAbstract:Simple-type epithelial Keratins are intermediate filament proteins important for mechanical stability and stress protection. Keratin mutations predispose to human liver disorders, whereas their roles in intestinal diseases are unclear. Absence of Keratin 8 (K8) in mice leads to colitis, decreased Na/Cl uptake, protein mistargeting, and longer crypts, suggesting that Keratins contribute to intestinal homeostasis. We describe the rate-limiting enzyme of the ketogenic energy metabolism pathway, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), as a major down-regulated protein in the K8-knockout (K8(-/-)) colon. K8 absence leads to decreased quantity and activity of HMGCS2, and the down-regulation is not dependent on the inflammatory state, since HMGCS2 is not decreased in dextran sulfate sodium-induced colitis. Peroxisome proliferator-activated receptor α, a transcriptional activator of HMGCS2, is similarly down-regulated. Ketogenic conditions-starvation or ketogenic diet-increase K8(+/+) HMGCS2, whereas this response is blunted in the K8(-/-) colon. Microbiota-produced short-chain fatty acids (SCFAs), substrates in the colonic ketone body pathway, are increased in stool, which correlates with decreased levels of their main transporter, monocarboxylate transporter 1 (MCT1). Microbial populations, including the main SCFA-butyrate producers in the colon, were not altered in the K8(-/-). In summary, the regulation of the SCFA-MCT1-HMGCS2 axis is disrupted in K8(-/-) colonocytes, suggesting a role for Keratins in colonocyte energy metabolism and homeostasis.
-
Keratin 8 modulates β-cell stress responses and normoglycaemia.
Journal of Cell Science, 2013Co-Authors: Catharina Alam, Jonas S. G. Silvander, Ebot N. Daniel, Sofie M. Kvarnström, Arno Hänninen, M. Bishr Omary, Parvez Alam, Diana M. ToivolaAbstract:Keratin intermediate filament (IF) proteins are epithelial cell cytoskeletal components that provide structural stability and protection from cell stress, among other cellular and tissue-specific functions. Numerous human diseases are associated with IF gene mutations, but the function of Keratins in the endocrine pancreas and their potential significance for glycaemic control are unknown. The impact of Keratins on β-cell organisation and systemic glucose control was assessed using Keratin 8 (K8) wild-type (K8+/+) and K8 knockout (K8−/−) mice. Islet β-cell Keratins were characterised under basal conditions, in streptozotocin (STZ)-induced diabetes and in non-obese diabetic (NOD) mice. STZ-induced diabetes incidence and islet damage was assessed in K8+/+ and K8−/− mice. K8 and K18 were the predominant Keratins in islet β-cells and K8−/− mice expressed only remnant K18 and K7. K8 deletion resulted in lower fasting glucose levels, increased glucose tolerance and insulin sensitivity, reduced glucose-stimulated insulin secretion and decreased pancreatic insulin content. GLUT2 localisation and insulin vesicle morphology were disrupted in K8−/− β-cells. The increased levels of cytoplasmic GLUT2 correlated with resistance to high-dose STZ-induced injury in K8−/− mice. However, K8 deletion conferred no long-term protection from STZ-induced diabetes and prolonged STZ-induced stress caused increased exocrine damage in K8−/− mice. β-cell Keratin upregulation occurred 2 weeks after treatments with low-dose STZ in K8+/+ mice and in diabetic NOD mice, suggesting a role for Keratins, particularly in non-acute islet stress responses. These results demonstrate previously unrecognised functions for Keratins in β-cell intracellular organisation, as well as for systemic blood glucose control under basal conditions and in diabetes-induced stress.
-
Glucose and SIRT2 reciprocally mediate the regulation of Keratin 8 by lysine acetylation.
Journal of Cell Biology, 2013Co-Authors: Natasha T. Snider, Raymond Kwan, Jessica M. Leonard, Nicholas W. Griggs, Liangyou Rui, M. Bishr OmaryAbstract:Lysine acetylation is an important posttranslational modification that regulates microtubules and microfilaments, but its effects on intermediate filament proteins (IFs) are unknown. We investigated the regulation of Keratin 8 (K8), a type II simple epithelial IF, by lysine acetylation. K8 was basally acetylated and the highly conserved Lys-207 was a major acetylation site. K8 acetylation regulated filament organization and decreased Keratin solubility. Acetylation of K8 was rapidly responsive to changes in glucose levels and was up-regulated in response to nicotinamide adenine dinucleotide (NAD) depletion and in diabetic mouse and human livers. The NAD-dependent deacetylase sirtuin 2 (SIRT2) associated with and deacetylated K8. Pharmacologic or genetic inhibition of SIRT2 decreased K8 solubility and affected filament organization. Inhibition of K8 Lys-207 acetylation resulted in site-specific phosphorylation changes of K8. Therefore, K8 acetylation at Lys-207, a highly conserved residue among type II Keratins and other IFs, is up-regulated upon hyperglycemia and down-regulated by SIRT2. Keratin acetylation provides a new mechanism to regulate Keratin filaments, possibly via modulating Keratin phosphorylation.
-
Keratin 8 phosphorylation regulates its transamidation and hepatocyte Mallory-Denk body formation
The FASEB Journal, 2012Co-Authors: Raymond Kwan, Shinichiro Hanada, Masaru Harada, Pavel Strnad, M. Bishr OmaryAbstract:Mallory-Denk bodies (MDBs) are hepatocyte inclusions that are associated with poor liver disease prognosis. The intermediate filament protein Keratin 8 (K8) and its cross-linking by transglutaminas...
Patrik Holm - One of the best experts on this subject based on the ideXlab platform.
-
optimization of production of the anti Keratin 8 single chain fv ts1 218 in pichia pastoris using design of experiments
Microbial Cell Factories, 2011Co-Authors: Rozbeh Jafari, Birgitta Sundstrom, Patrik HolmAbstract:Background: Optimization of conditions during recombinant protein production for improved yield is a major goal for protein scientists. Typically this is achieved by changing single crucial factor settings one at a time while other factors are kept fixed through trial-and-error experimentation. This approach may introduce larger bias and fail to identify interactions between the factors resulting in failure of finding the true optimal conditions. Results: In this study we have utilized design of experiments in order to identify optimal culture conditions with the aim to improve the final yield of the anti-Keratin 8 scFv TS1-218, during expression in P. pastoris in shake flasks. The effect of: pH, temperature and methanol concentration on the yield of TS1-218 using buffered minimal medium was investigated and a predictive model established. The results demonstrated that higher starting pH and lower temperatures during induction significantly increased the yield of TS1-218. Furthermore, the result demonstrated increased biomass accumulation and cell viability at lower temperatures which suggested that the higher yield of TS1-218 could be attributed to lower protease activity in the culture medium. The optimal conditions (pH 7.1, temperature 11°C and methanol concentration 1.2%) suggested by the predictive model yielded 21.4 mg TS1-218 which is a 21-fold improvement compared to the yield prior to optimization. Conclusion: The results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that P. pastoris is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.
-
construction of divalent anti Keratin 8 single chain antibodies sc fv 2 expression in pichia pastoris and their reactivity with multicellular tumor spheroids
Journal of Immunological Methods, 2011Co-Authors: Rozbeh Jafari, Patrik Holm, Marco Piercecchi, Birgitta SundstromAbstract:Abstract Single-chain variable fragments (scFvs) are small monovalent recombinant antibody fragments that retain the specificity of their parent immunoglobulins. ScFvs are excellent building blocks for new and improved immunodiagnostic and therapeutic proteins. However, the monovalency and the rapid renal elimination of scFvs result in poor tumor accumulation and retention. Engineering divalent antibody fragments is an excellent way to address these shortcomings. In this study, covalent divalent single-chain variable fragments (sc(Fv)2s), were constructed from the monovalent anti-Keratin 8 scFvs, TS1-218 and its mutant, HE1-Q. The scFvs and sc(Fv)2s were expressed in the methylotrophic yeast Pichia pastoris, utilizing the alpha-factor secretion signal (α-factor) for extracellular secretion. The immunoreactivity and specificity of the antibody fragments were analyzed with enzyme-linked immunosorbent assay (ELISA) and the uptake and retention of the 125I labeled antibody fragments were evaluated using HeLa HEp-2 multicellular tumor spheroids (MCTSs). Analysis of the antibody fragments demonstrated that parts of the α-factor remained at the N-terminal of the antibody fragments. Despite incomplete processing of the α-factor, the antibody fragments were functional where the sc(Fv)2s gave a three-fold stronger signal in ELISA compared to their scFv counterparts and the mutant antibodies demonstrated a stronger signal than their initial wild types. In addition, the sc(Fv)2s DiTS1-218 and DiHE1-Q displayed an approximately two-fold higher uptake and were retained to a larger extent in the MCTS, demonstrating a 3.9 and 9.4-fold increase in half-life respectively compared to their corresponding scFvs. In conclusion, expression in P. pastoris improved the yield 20-fold and facilitated the purification of the antibody fragments. Furthermore, the sc(Fv)2s presented a higher functional affinity to K 8 both in ELISA and MCTS compared to the scFvs with DiHE1-Q being the best candidate for further studies.
-
evaluating targeting efficiency of anti Keratin 8 antibody fragments using multicellular tumor spheroids
38th International Society for Oncology and Biomarkers Munich Germany 2010, 2010Co-Authors: Birgitta Sundstrom, Patrik Holm, Rozbeh Jafari, Torgny StigbrandAbstract:Evaluating targeting efficiency of anti-Keratin 8 antibody fragments using multicellular tumor spheroids
-
anti Keratin 8 single chain antibody fragments and their evaluation using multicellular tumor spheroids
Cancerfondens planeringsgrupp för onkologisk nuklidterapi, 2010Co-Authors: Birgitta Sundstrom, Patrik Holm, Rozbeh Jafari, Torgny StigbrandAbstract:Anti-Keratin 8 single-chain antibody fragments and their evaluation using multicellular tumor spheroids
Diana M. Toivola - One of the best experts on this subject based on the ideXlab platform.
-
Absence of Keratin 8 or 18 promotes antimitochondrial autoantibody formation in aging male mice
The FASEB Journal, 2015Co-Authors: Diana M. Toivola, Raymond Kwan, Aida Habtezion, Julia O. Misiorek, Linxing Zhang, Joel H. Nyström, Orr Sharpe, William H. Robinson, M. Bishr OmaryAbstract:Human mutations in Keratin 8 (K8) and Keratin 18 (K18), the intermediate filament proteins of hepatocytes, predispose to several liver diseases. K8-null mice develop chronic liver injury and fragile hepatocytes, dysfunctional mitochondria, and Th2-type colitis. We tested the hypothesis that autoantibody formation accompanies the liver damage that associates with K8/K18 absence. Sera from wild-type control, K8-null, and K18-null mice were analyzed by immunoblotting and immunofluorescence staining of cell and mouse tissue homogenates. Autoantibodies to several antigens were identified in 81% of K8-null male mice 8 mo or older. Similar autoantibodies were detected in aging K18-null male mice that had a related liver phenotype but normal colon compared with K8-null mice, suggesting that the autoantibodies are linked to liver rather than colonic disease. However, these autoantibodies were not observed in nontransgenic mice subjected to 4 chronic injury models. The autoantigens are ubiquitous and partition with mitochondria. Mass spectrometry and purified protein analysis identified, mitochondrial HMG-CoA synthase, aldehyde dehydrogenase, and catalase as the primary autoantigens, and glutamate dehydrogenase and epoxide hydrolase-2 as additional autoantigens. Therefore, absence of the hepatocyte Keratins results in production of anti-mitochondrial autoantibodies (AMA) that recognize proteins involved in energy metabolism and oxidative stress, raising the possibility that AMA may be found in patients with Keratin mutations that associate with liver and other diseases.
-
Keratin 8 modulates β-cell stress responses and normoglycaemia.
Journal of Cell Science, 2013Co-Authors: Catharina Alam, Jonas S. G. Silvander, Ebot N. Daniel, Sofie M. Kvarnström, Arno Hänninen, M. Bishr Omary, Parvez Alam, Diana M. ToivolaAbstract:Keratin intermediate filament (IF) proteins are epithelial cell cytoskeletal components that provide structural stability and protection from cell stress, among other cellular and tissue-specific functions. Numerous human diseases are associated with IF gene mutations, but the function of Keratins in the endocrine pancreas and their potential significance for glycaemic control are unknown. The impact of Keratins on β-cell organisation and systemic glucose control was assessed using Keratin 8 (K8) wild-type (K8+/+) and K8 knockout (K8−/−) mice. Islet β-cell Keratins were characterised under basal conditions, in streptozotocin (STZ)-induced diabetes and in non-obese diabetic (NOD) mice. STZ-induced diabetes incidence and islet damage was assessed in K8+/+ and K8−/− mice. K8 and K18 were the predominant Keratins in islet β-cells and K8−/− mice expressed only remnant K18 and K7. K8 deletion resulted in lower fasting glucose levels, increased glucose tolerance and insulin sensitivity, reduced glucose-stimulated insulin secretion and decreased pancreatic insulin content. GLUT2 localisation and insulin vesicle morphology were disrupted in K8−/− β-cells. The increased levels of cytoplasmic GLUT2 correlated with resistance to high-dose STZ-induced injury in K8−/− mice. However, K8 deletion conferred no long-term protection from STZ-induced diabetes and prolonged STZ-induced stress caused increased exocrine damage in K8−/− mice. β-cell Keratin upregulation occurred 2 weeks after treatments with low-dose STZ in K8+/+ mice and in diabetic NOD mice, suggesting a role for Keratins, particularly in non-acute islet stress responses. These results demonstrate previously unrecognised functions for Keratins in β-cell intracellular organisation, as well as for systemic blood glucose control under basal conditions and in diabetes-induced stress.
-
Absence of Keratin 8 confers a paradoxical microflora-dependent resistance to apoptosis in the colon
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Aida Habtezion, Diana M. Toivola, M. Nadeem Asghar, Eugene C. Butcher, Greg S. Kronmal, Jacqueline D. Brooks, M. Bishr OmaryAbstract:Keratin 8 (K8) is a major intermediate filament protein present in enterocytes and serves an antiapoptotic function in hepatocytes. K8-null mice develop colonic hyperplasia and colitis that are reversed after antibiotic treatment. To investigate the pathways that underlie the mechanism of colonocyte hyperplasia and the normalization of the colonic phenotype in response to antibiotics, we performed genome-wide microarray analysis. Functional annotation of genes that are differentially regulated in K8−/− and K8+/+ isolated colon crypts (colonocytes) identified apoptosis as a major altered pathway. Exposure of K8−/− colonocytes or colon organ (“organoid”) cultures, but not K8−/− small intestine organoid cultures, to apoptotic stimuli showed, surprisingly, that they are resistant to apoptosis compared with their wild-type counterparts. This resistance is not related to inflammation per se because T-cell receptor α-null (TCR-α−/−) and wild-type colon cultures respond similarly upon induction of apoptosis. Following antibiotic treatment, K8−/− colonocytes and organ cultures become less resistant to apoptosis and respond similarly to the wild-type colonocytes. Antibiotics also normalize most differentially up-regulated genes, including survivin and β4-integrin. Treatment of K8−/− mice with anti–β4-integrin antibody up-regulated survivin, and induced phosphorylation of focal adhesion kinase with decreased activation of caspases. Therefore, unlike the proapoptotic effect of K8 mutation or absence in hepatocytes, lack of K8 confers resistance to colonocyte apoptosis in a microflora-dependent manner.
-
Keratin 8 overexpression promotes mouse Mallory body formation
Journal of Cell Biology, 2005Co-Authors: Ikuo Nakamichi, Helene Baribault, Diana M. Toivola, Pavel Strnad, Robert G. Oshima, Sara A. Michie, M. Bishr OmaryAbstract:Keratins 8 and 18 (K8/18) are major constituents of Mallory bodies (MBs), which are hepatocyte cytoplasmic inclusions seen in several liver diseases. K18-null but not K8-null or heterozygous mice form MBs, which indicates that K8 is important for MB formation. Early stages in MB genesis include K8/18 hyperphosphorylation and overexpression. We used transgenic mice that overexpress K8, K18, or K8/18 to test the importance of K8 and/or K18 in MB formation. MBs were induced by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Livers of young K8 or K8/K18 overexpressors had no histological abnormalities despite increased Keratin protein and phosphorylation. In aging mice, only K8-overexpressing livers spontaneously developed small “pre-MB” aggregates. Only K8-overexpressing young mice are highly susceptible to MB formation after short-term DDC feeding. Thus, the K8 to K18 ratio, rather than K8/18 overexpression by itself, plays an essential role in MB formation. K8 overexpression is sufficient to form pre-MB and primes animals to accumulate MBs upon DDC challenge, which may help explain MB formation in human liver diseases.
-
Keratin-8-deficient mice develop chronic spontaneous Th2 colitis amenable to antibiotic treatment
Journal of Cell Science, 2005Co-Authors: Aida Habtezion, Diana M. Toivola, Eugene C. Butcher, M. Bishr OmaryAbstract:Keratin 8 (K8) is the major intermediate filament protein present in intestinal epithelia. Depending on the mouse genetic background, absence of K8 causes embryonic lethality or colonic hyperplasia and colitis. We studied disease progression, the inflammatory responses, and role of luminal bacteria in K8-null mice in order to characterize the intestinal pathology of K8-associated colitis. Colon lymphocytes were isolated for analysis of their phenotype and cytokine production, and vascular and lymphocyte adhesion molecule expression in K8–/– mice of varying ages. K8–/– mice had a marked increase in TCR β -positive/CD4-positive T cells infiltrating the colon lamina propria, in association with enhanced Th2 cytokine (IL-4, IL-5 and IL-13) production. K8–/– mice show early signs of inflammation even prior to weaning, that increases with age, and their epithelial cells overexpress MHC class II antigens. The chronic colitis is related to increased CD4-positive infiltrating T cells displaying memory and naive phenotypes, and an altered vascular endothelium with aberrant expression of peripheral node addressin. Analysis of normal gut-specific homing molecules, reveals an increased number of α 4 β 7 -positive cells and vascular mucosal addressin cell adhesion molecule-1 in K8-null colons. Antibiotic treatment markedly decreased colon inflammation and ion transporter AE1/2 mistargeting, indicating that luminal bacteria play an important role in the observed phenotype. Therefore, K8-null mice develop chronic spontaneous Th2-type colitis due to a primary epithelial rather than immune cell defect, which is amenable to antibiotic therapy. These mice provide a model to investigate epithelial-leukocyte and epithelial-microbial cross-talk.
Rozbeh Jafari - One of the best experts on this subject based on the ideXlab platform.
-
optimization of production of the anti Keratin 8 single chain fv ts1 218 in pichia pastoris using design of experiments
Microbial Cell Factories, 2011Co-Authors: Rozbeh Jafari, Birgitta Sundstrom, Patrik HolmAbstract:Background: Optimization of conditions during recombinant protein production for improved yield is a major goal for protein scientists. Typically this is achieved by changing single crucial factor settings one at a time while other factors are kept fixed through trial-and-error experimentation. This approach may introduce larger bias and fail to identify interactions between the factors resulting in failure of finding the true optimal conditions. Results: In this study we have utilized design of experiments in order to identify optimal culture conditions with the aim to improve the final yield of the anti-Keratin 8 scFv TS1-218, during expression in P. pastoris in shake flasks. The effect of: pH, temperature and methanol concentration on the yield of TS1-218 using buffered minimal medium was investigated and a predictive model established. The results demonstrated that higher starting pH and lower temperatures during induction significantly increased the yield of TS1-218. Furthermore, the result demonstrated increased biomass accumulation and cell viability at lower temperatures which suggested that the higher yield of TS1-218 could be attributed to lower protease activity in the culture medium. The optimal conditions (pH 7.1, temperature 11°C and methanol concentration 1.2%) suggested by the predictive model yielded 21.4 mg TS1-218 which is a 21-fold improvement compared to the yield prior to optimization. Conclusion: The results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that P. pastoris is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.
-
construction of divalent anti Keratin 8 single chain antibodies sc fv 2 expression in pichia pastoris and their reactivity with multicellular tumor spheroids
Journal of Immunological Methods, 2011Co-Authors: Rozbeh Jafari, Patrik Holm, Marco Piercecchi, Birgitta SundstromAbstract:Abstract Single-chain variable fragments (scFvs) are small monovalent recombinant antibody fragments that retain the specificity of their parent immunoglobulins. ScFvs are excellent building blocks for new and improved immunodiagnostic and therapeutic proteins. However, the monovalency and the rapid renal elimination of scFvs result in poor tumor accumulation and retention. Engineering divalent antibody fragments is an excellent way to address these shortcomings. In this study, covalent divalent single-chain variable fragments (sc(Fv)2s), were constructed from the monovalent anti-Keratin 8 scFvs, TS1-218 and its mutant, HE1-Q. The scFvs and sc(Fv)2s were expressed in the methylotrophic yeast Pichia pastoris, utilizing the alpha-factor secretion signal (α-factor) for extracellular secretion. The immunoreactivity and specificity of the antibody fragments were analyzed with enzyme-linked immunosorbent assay (ELISA) and the uptake and retention of the 125I labeled antibody fragments were evaluated using HeLa HEp-2 multicellular tumor spheroids (MCTSs). Analysis of the antibody fragments demonstrated that parts of the α-factor remained at the N-terminal of the antibody fragments. Despite incomplete processing of the α-factor, the antibody fragments were functional where the sc(Fv)2s gave a three-fold stronger signal in ELISA compared to their scFv counterparts and the mutant antibodies demonstrated a stronger signal than their initial wild types. In addition, the sc(Fv)2s DiTS1-218 and DiHE1-Q displayed an approximately two-fold higher uptake and were retained to a larger extent in the MCTS, demonstrating a 3.9 and 9.4-fold increase in half-life respectively compared to their corresponding scFvs. In conclusion, expression in P. pastoris improved the yield 20-fold and facilitated the purification of the antibody fragments. Furthermore, the sc(Fv)2s presented a higher functional affinity to K 8 both in ELISA and MCTS compared to the scFvs with DiHE1-Q being the best candidate for further studies.
-
evaluating targeting efficiency of anti Keratin 8 antibody fragments using multicellular tumor spheroids
38th International Society for Oncology and Biomarkers Munich Germany 2010, 2010Co-Authors: Birgitta Sundstrom, Patrik Holm, Rozbeh Jafari, Torgny StigbrandAbstract:Evaluating targeting efficiency of anti-Keratin 8 antibody fragments using multicellular tumor spheroids
-
anti Keratin 8 single chain antibody fragments and their evaluation using multicellular tumor spheroids
Cancerfondens planeringsgrupp för onkologisk nuklidterapi, 2010Co-Authors: Birgitta Sundstrom, Patrik Holm, Rozbeh Jafari, Torgny StigbrandAbstract:Anti-Keratin 8 single-chain antibody fragments and their evaluation using multicellular tumor spheroids
Birgitta Sundstrom - One of the best experts on this subject based on the ideXlab platform.
-
optimization of production of the anti Keratin 8 single chain fv ts1 218 in pichia pastoris using design of experiments
Microbial Cell Factories, 2011Co-Authors: Rozbeh Jafari, Birgitta Sundstrom, Patrik HolmAbstract:Background: Optimization of conditions during recombinant protein production for improved yield is a major goal for protein scientists. Typically this is achieved by changing single crucial factor settings one at a time while other factors are kept fixed through trial-and-error experimentation. This approach may introduce larger bias and fail to identify interactions between the factors resulting in failure of finding the true optimal conditions. Results: In this study we have utilized design of experiments in order to identify optimal culture conditions with the aim to improve the final yield of the anti-Keratin 8 scFv TS1-218, during expression in P. pastoris in shake flasks. The effect of: pH, temperature and methanol concentration on the yield of TS1-218 using buffered minimal medium was investigated and a predictive model established. The results demonstrated that higher starting pH and lower temperatures during induction significantly increased the yield of TS1-218. Furthermore, the result demonstrated increased biomass accumulation and cell viability at lower temperatures which suggested that the higher yield of TS1-218 could be attributed to lower protease activity in the culture medium. The optimal conditions (pH 7.1, temperature 11°C and methanol concentration 1.2%) suggested by the predictive model yielded 21.4 mg TS1-218 which is a 21-fold improvement compared to the yield prior to optimization. Conclusion: The results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that P. pastoris is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.
-
construction of divalent anti Keratin 8 single chain antibodies sc fv 2 expression in pichia pastoris and their reactivity with multicellular tumor spheroids
Journal of Immunological Methods, 2011Co-Authors: Rozbeh Jafari, Patrik Holm, Marco Piercecchi, Birgitta SundstromAbstract:Abstract Single-chain variable fragments (scFvs) are small monovalent recombinant antibody fragments that retain the specificity of their parent immunoglobulins. ScFvs are excellent building blocks for new and improved immunodiagnostic and therapeutic proteins. However, the monovalency and the rapid renal elimination of scFvs result in poor tumor accumulation and retention. Engineering divalent antibody fragments is an excellent way to address these shortcomings. In this study, covalent divalent single-chain variable fragments (sc(Fv)2s), were constructed from the monovalent anti-Keratin 8 scFvs, TS1-218 and its mutant, HE1-Q. The scFvs and sc(Fv)2s were expressed in the methylotrophic yeast Pichia pastoris, utilizing the alpha-factor secretion signal (α-factor) for extracellular secretion. The immunoreactivity and specificity of the antibody fragments were analyzed with enzyme-linked immunosorbent assay (ELISA) and the uptake and retention of the 125I labeled antibody fragments were evaluated using HeLa HEp-2 multicellular tumor spheroids (MCTSs). Analysis of the antibody fragments demonstrated that parts of the α-factor remained at the N-terminal of the antibody fragments. Despite incomplete processing of the α-factor, the antibody fragments were functional where the sc(Fv)2s gave a three-fold stronger signal in ELISA compared to their scFv counterparts and the mutant antibodies demonstrated a stronger signal than their initial wild types. In addition, the sc(Fv)2s DiTS1-218 and DiHE1-Q displayed an approximately two-fold higher uptake and were retained to a larger extent in the MCTS, demonstrating a 3.9 and 9.4-fold increase in half-life respectively compared to their corresponding scFvs. In conclusion, expression in P. pastoris improved the yield 20-fold and facilitated the purification of the antibody fragments. Furthermore, the sc(Fv)2s presented a higher functional affinity to K 8 both in ELISA and MCTS compared to the scFvs with DiHE1-Q being the best candidate for further studies.
-
evaluating targeting efficiency of anti Keratin 8 antibody fragments using multicellular tumor spheroids
38th International Society for Oncology and Biomarkers Munich Germany 2010, 2010Co-Authors: Birgitta Sundstrom, Patrik Holm, Rozbeh Jafari, Torgny StigbrandAbstract:Evaluating targeting efficiency of anti-Keratin 8 antibody fragments using multicellular tumor spheroids
-
anti Keratin 8 single chain antibody fragments and their evaluation using multicellular tumor spheroids
Cancerfondens planeringsgrupp för onkologisk nuklidterapi, 2010Co-Authors: Birgitta Sundstrom, Patrik Holm, Rozbeh Jafari, Torgny StigbrandAbstract:Anti-Keratin 8 single-chain antibody fragments and their evaluation using multicellular tumor spheroids
