To see the other types of publications on this topic, follow the link: Immune serums – Purification.
Journal articles on the topic 'Immune serums – Purification'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Immune serums – Purification.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Kozlov, V. G., Yu Yu Ivin, and V. P. Grachev. "The use of human placenta for purification of normal and immune animal sera." Epidemiology and Infectious Diseases 20, no. 5 (October 15, 2015): 48–51. http://dx.doi.org/10.17816/eid40961.
Full textAbstract:
There was described and efficient and economical approach for the removal of toxic substances from normal and immune sera from various species of animals with the use of human placenta tissue. Purification brings about to perceptible losses of neither serum-specific activity nor the original volume. Being simple the method does not require any special equipment and can be used in conditions of low-volume or in laboratory production of serum preparations. There are considered as well possible origins of serum toxicity as mechanism of antitoxic activity of placenta.
2
Odiegwu C.N.C, Emenuga V. N., Ogamba S. E., Obi C. M., and Ejike C. E. "Microbial agglutination and lymphocyte blastogenesis potentials of isolated Achatina achatina snail lectin." World Journal of Advanced Research and Reviews 9, no. 1 (January 30, 2021): 104–13. http://dx.doi.org/10.30574/wjarr.2021.9.1.0505.
Full textAbstract:
Lectins are involved in recognition phenomena and their ability to bind particular Carbohydrate structures are the key to their biological functions. Bacteria typically attaches to prospective host cell membranes in receptors with lectin like sugar specificity. This is of great importance as the adherence of bacteria to host tissue surfaces is the initial event in bacterial infection. Lectins are also known to play important roles in immune system by recognizing carbohydrates that are found exclusively on pathogens, or that are inaccessible on host cells. This ability of lectins to selectively bind or agglutinate specific sugars have made them useful tools for the characterization of certain cell types or fragments, to detect cells in different states of development, to distinguish normal from tumour cells and to separate different cell types by affinity chromatography. A total of 120 samples of local Achatina achatina snail specie were collected, authenticated at the Zoology Department of the University of Nigeria, Nsukka and 80mls of pooled crude Lectin extract was obtained. Purifications were performed on 20mls of the crude extract in three steps viz, Ammonium sulphate precipitation and Dialysis (Partial purifications), Con A Sepharose 4B affinity chromatography column (Complete purification). The affinity purified lectin was used in all the tests conducted in this research. The crude, partially and complete/affinity purified lectin extracts were subjected to Haemagglutination and Protein Assay tests. The Molecular weight was deduced by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The microbial agglutination potentials of the lectin was assessed by testing typed bacterial organisms viz, Salmonella typhimurium, Escherichia coli, Lactobacilli acidophilus, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella aeruginosa and four typed fungal organisms: Aspergillus niger, Trichophyton mentagrophytes, Candida albicans and A. flavus. The lectin’s Lymphocyte blastogenesis activities was determined by its incubation with human lymphocytes for mitogenic stimulation assay. The results of the research showed as follows: On complete/affinity purification, 15mls of pure sample containing only the high molecular weight lectin was obtained. On standardization, the respective haemagglutination tests on the crude, partially and affinity purified lectin showed preferential agglutinations with Blood group A type. Only S. typhimurium (+++), E. coli (+) and L. acidophilus (+) reacted with the lectin but in different strengths. Incubation of the lectin with lymphocytes from human serum showed that it has the ability to stimulate lymphocytes to undergo mitosis. This research has therefore succeeded in assessing the Microbial agglutination and Lymphocyte blastogenesis potentials of the isolated and characterised A. achatina snail lectin.
3
Uchida, Kanji, Koh Nakata, Takuji Suzuki, Maurizio Luisetti, Masato Watanabe, Diana E. Koch, Carrie A. Stevens, et al. "Granulocyte/macrophage–colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects." Blood 113, no. 11 (March 12, 2009): 2547–56. http://dx.doi.org/10.1182/blood-2008-05-155689.
Full textAbstract:
High levels of granulocyte/macrophage–colony-stimulating factor (GM-CSF) autoantibodies are thought to cause pulmonary alveolar proteinosis (PAP), a rare syndrome characterized by myeloid dysfunction resulting in pulmonary surfactant accumulation and respiratory failure. Paradoxically, GM-CSF autoantibodies have been reported to occur rarely in healthy people and routinely in pharmaceutical intravenous immunoglobulin (IVIG) purified from serum pooled from healthy subjects. These findings suggest that either GM-CSF autoantibodies are normally present in healthy people at low levels that are difficult to detect or that serum pooled for IVIG purification may include asymptomatic persons with high levels of GM-CSF autoantibodies. Using several experimental approaches, GM-CSF autoantibodies were detected in all healthy subjects evaluated (n = 72) at low levels sufficient to rheostatically regulate multiple myeloid functions. Serum GM-CSF was more abundant than previously reported, but more than 99% was bound and neutralized by GM-CSF autoantibody. The critical threshold of GM-CSF autoantibodies associated with the development of PAP was determined. Results demonstrate that free serum GM-CSF is tightly maintained at low levels, identify a novel potential mechanism of innate immune regulation, help define the therapeutic window for potential clinical use of GM-CSF autoantibodies to treat inflammatory and autoimmune diseases, and have implications for the pathogenesis of PAP.
4
Menikou, Stephanie, Andrew J. McArdle, Ming-Shi Li, Myrsini Kaforou, Paul R. Langford, and Michael Levin. "A proteomics-based method for identifying antigens within immune complexes." PLOS ONE 15, no. 12 (December 23, 2020): e0244157. http://dx.doi.org/10.1371/journal.pone.0244157.
Full textAbstract:
A novel approach to recover and identify immune complexes (ICs) was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was applied to serum with artificially created immune complexes (ICs) comprising vaccine antigen (influenza) and antibody, which led to recovery and identification of influenza peptides within the recovered ICs. This approach was compared with the established method for IC detection and recovery, polyethylene glycol (PEG) precipitation, followed by LC MS/MS. Both approaches successfully enabled capture, recovery and characterization of immunoglobulins and influenza antigen(s) in complex with the immunoglobulins. However, PEG precipitation has the advantage of simplicity and is more suited for large scale studies.
5
Cawston, T. E., D. N. Noble, G. Murphy, A. J. Smith, C. Woodley, and B. Hazleman. "Rapid purification of tissue inhibitor of metalloproteinases from human plasma and identification as a γ-serum protein." Biochemical Journal 238, no. 3 (September 15, 1986): 677–82. http://dx.doi.org/10.1042/bj2380677.
Full textAbstract:
A rapid method is described for the purification of human tissue inhibitor of metalloproteinases (TIMP) from plasma which involves immuno-affinity chromatography and gel filtration. The purified plasma inhibitor is immunologically identical with the TIMP previously purified from human amniotic fluid, human synovial fluid and human fibroblast culture medium. It is proposed that this inhibitor is identical with the plasma inhibitor previously named ‘B1 anticollagenase’, although the plasma inhibitor was shown to migrate as a gamma-serum component.
6
Han, Sang Mi, Kwang Gill Lee, Joo Hong Yeo, Sung Jin Hwang, Chul Ho Jang, Peter J. Chenoweth, and Sok Cheon Pak. "Effects of Bee Venom Treatment on Growth Performance of Young Pigs." American Journal of Chinese Medicine 37, no. 02 (January 2009): 253–60. http://dx.doi.org/10.1142/s0192415x09006813.
Full textAbstract:
This study examined the effect of whole bee venom (BV) as a potential stimulant of the piglet immune system, on growth performance, blood parameters, plasma protein and immune globulin content of serum. Piglets (n = 97) received combinations of 0.5, 1.0, 1.5, 2.0 and 2.5 mg/kg of parenterally administered BV on 4 occasions between birth and Day 30. In the apipuncture group (n = 31), piglets were acupunctured with the worker honeybee. Two acupoints, GV-1 (Jiao-chao) and GV-20 (Bai-hui), were selected for apipuncture. All piglets (n = 128) in the treatment groups were treated 4 times throughout the study period of 60 days. The control piglets received no treatments. Blood was taken via jugular venipuncture on Day 30 after birth. Body weight and survivability were measured, and changes in hematological values were analyzed. Both the BV injection group and apipuncture group increased body weight and survivability by 26.6% and 21.8%, and 7.9% and 6.7% respectively compared to the controls. The numbers of leukocytes, erythrocytes, lymphocytes and monocytes were not influenced by treatments. However, a potential clinical benefit of high dose therapy was seen in increased populations of leukocytes, lymphocytes and monocytes compared with either the apipuncture or control groups. Other blood parameters such as total protein and albumin were not affected by treatment. However, IgG levels were generally higher in treated groups than in the controls. These findings indicate that BV might be useful to stimulate immuno-competence in pig production, possibly via the primary bioactive components of melittin, phospholipase A 2 and apamin. The administration of BV, either via injection or acupuncture, did not make any differences in growth performance of young pigs. These results would be useful for further purification and characterization of immune boosting agents from BV.
7
Law, R. D., and W. C. Plaxton. "Purification and characterization of a novel phosphoenolpyruvate carboxylase from banana fruit." Biochemical Journal 307, no. 3 (May 1, 1995): 807–16. http://dx.doi.org/10.1042/bj3070807.
Full textAbstract:
Phosphoenolpyruvate carboxylase (PEPC) from ripened banana (Musa cavendishii L.) fruits has been purified 127-fold to apparent homogeneity and a final specific activity of 32 mumol of oxaloacetate produced/min per mg of protein. Non-denaturing PAGE of the final preparation resolved a single protein-staining band that co-migrated with PEPC activity. Polypeptides of 103 (alpha-subunit) and 100 (beta-subunit) kDa, which stain for protein with equal intensity and cross-react strongly with anti-(maize leaf PEPC) immune serum, were observed following SDS/PAGE of the final preparation. CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. The enzyme's native molecular mass was estimated to be about 425 kDa. These data indicate that in contrast to the homotetrameric PEPC from most other sources, the banana fruit enzyme exists as an alpha 2 beta 2 heterotetramer. Monospecific rabbit anti-(banana PEPC) immune serum effectively immunoprecipitated the activity of the purified enzyme. Immunoblotting studies established that the 100 kDa subunit did not arise via proteolysis of the 103 kDa subunit after tissue extraction, and that the subunit composition of banana PEPC remains uniform throughout the ripening process. PEPC displayed a typical pH activity profile with an alkaline optimum and activity rapidly decreasing below pH 7.0. Enzymic activity was absolutely dependent on the presence of a bivalent metal cation, with Mg2+ or Mn2+ fulfilling this requirement. The response of the PEPC activity to PEP concentration and to various effectors was greatly influenced by pH and glycerol addition to the assay. The enzyme was activated by hexose-monophosphates and potently inhibited by malate, succinate, aspartate and glutamate at pH 7.0, whereas the effect of these metabolites was considerably diminished or completely abolished at pH 8.0. The significance of metabolite regulation of PEPC is discussed in relation to possible functions of this enzyme in banana fruit metabolism.
8
Tanaka-Kawai, Hiroshi, and Satoshi Yomoda. "Molecular weight and substrate characteristics of human serum arylesterase following purification by immuno-affinity chromatography." Clinica Chimica Acta 215, no. 2 (June 1993): 127–38. http://dx.doi.org/10.1016/0009-8981(93)90120-s.
Full text
9
Doering, Christopher, Ernest Parker, John Healey, Heather Craddock, Rachel Barrow, and Pete Lollar. "Expression and Characterization of Recombinant Murine Factor VIII." Thrombosis and Haemostasis 88, no. 09 (2002): 450–58. http://dx.doi.org/10.1055/s-0037-1613237.
Full textAbstract:
SummaryHemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media. Subsequently, a two-step purification procedure resulted in 5,300-fold enrichment and a 70% yield. Highly purified recombinant murine fVIII had a specific coagulant activity of 660 units per nanomole. It underwent proteolytic processing by thrombin to yield an activated heterotrimer that demonstrated significantly greater stability than activated human fVIII. Recombinant murine fVIII was utilized to generate an anti-fVIII polyclonal antibody. Intravenous injection of recombinant murine fVIII into hemophilia A mice failed to induce a significant anti-fVIII immune response using a schedule that yielded high titer inhibitory antibodies to human fVIII. This may provide an important model for the study of immune tolerance to fVIII.
10
Radojković, J., and T. Ureta. "Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C." Biochemical Journal 242, no. 3 (March 15, 1987): 895–903. http://dx.doi.org/10.1042/bj2420895.
Full textAbstract:
The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.
11
Vajdy, Michael, Mark Selby, Angelica Medina-Selby, Doris Coit, John Hall, Laura Tandeske, David Chien, et al. "Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses." Journal of General Virology 87, no. 8 (August 1, 2006): 2253–62. http://dx.doi.org/10.1099/vir.0.81849-0.
Full textAbstract:
Although approximately 3 % of the world's population is infected with Hepatitis C virus (HCV), there is no prophylactic vaccine available. This study reports the design, cloning and purification of a single polyprotein comprising the HCV core protein and non-structural proteins NS3, NS4a, NS4b, NS5a and NS5b. The immunogenicity of this polyprotein, which was formulated in alum, oil-in-water emulsion MF59 or poly(dl-lactide co-glycolide) in the presence or absence of CpG adjuvant, was then determined in a murine model for induction of B- and T-cell responses. The addition of adjuvants or a delivery system to the HCV polyprotein enhanced serum antibody and T-cell proliferative responses, as well as IFN-γ responses, by CD4+ T cells. The antibody responses were mainly against the NS3 and NS5 components of the polyprotein and relatively poor responses were elicited against NS4 and the core components. IFN-γ responses, however, were induced against all of the individual components of the polyprotein. These data suggest that the HCV polyprotein delivered with adjuvants induces broad B- and T-cell responses and could be a vaccine candidate against HCV.
12
Ponda, Manish P., and Jan L. Breslow. "Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5." Proceedings of the National Academy of Sciences 113, no. 45 (October 24, 2016): E7059—E7068. http://dx.doi.org/10.1073/pnas.1615671113.
Full textAbstract:
Chemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.
13
Zeng, Yanhua, Xiaoxing You, Liangzhuan Liu, Jun He, Cuiming Zhu, Minjun Yu, Xiaohua Ma, and Yimou Wu. "The immune effects of multiple antigen peptides containing the mimic epitopes of the adhesion protein of Mycoplasma genitalium." Canadian Journal of Microbiology 59, no. 7 (July 2013): 479–84. http://dx.doi.org/10.1139/cjm-2013-0204.
Full textAbstract:
The purpose of this study was to investigate the humoral and cellular immune responses stimulated by multiple antigen peptides (MAPs) containing the mimic epitopes of Mycoplasma genitalium adhesion protein (MgPa). Three MAPs containing the mimic epitopes of MgPa were synthesized on a branched polylysine matrix. After purification and characterization, these MAPs were used to immunize BALB/c mice. The immunoglobulin G (IgG) antibody and the subtype of IgG antibody in the serum of the immunized mice were detected by indirect ELISA (enzyme-linked immunosorbent assay). The proliferation of the spleen lymphocyte was detected using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. The gamma interferon (IFN-γ) and interleukin-4 (IL-4) levels in the cultured supernatant of spleen lymphocytes were measured by ELISA. The 3 different MAPs were prepared with high purity. Levels of IgG, IgG1, and IgG2a antibodies were elevated in the mice serum immunized by all 3 MAPs. The major antibody isotype was IgG2a. Importantly, mice immunized with a mixture of the 3 MAPs produced significantly more antibodies than those immunized with a single MAP (p < 0.05). Moreover, these MAPs could stimulate the proliferation of spleen lymphocytes of immunized mice and induce the production of IFN-γ and IL-4. The IFN-γ and IL-4 levels stimulated by the mixed MAPs were significantly higher than those stimulated by a single MAP (p < 0.01). The 3 different MAPs could induce strong cellular and humoral immune responses. The immunoreactivity of the mixed MAPs was stronger than that of the single MAP.
14
Nabian, Sedigheh, Mohammad Taheri, Mohammad Mehdi Ranjbar, Alireza Sazmand, Parastou Youssefy, and Gholam Reza Nazaralipour. "Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatuslarvae." Revista Brasileira de Parasitologia Veterinária 23, no. 2 (June 2014): 187–93. http://dx.doi.org/10.1590/s1984-29612014036.
Full textAbstract:
Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatusis an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.
15
Edmondson, JC, RK Liem, JE Kuster, and ME Hatten. "Astrotactin: a novel neuronal cell surface antigen that mediates neuron-astroglial interactions in cerebellar microcultures." Journal of Cell Biology 106, no. 2 (February 1, 1988): 505–17. http://dx.doi.org/10.1083/jcb.106.2.505.
Full textAbstract:
A microculture system for mouse cerebellar cells has been used to identify an immune activity, raised in rabbits against postnatal cerebellar cells, that blocks neuron-glial interactions in vitro. In the presence of blocking antibodies, stable neuron-glial contacts did not form and neuronal induction of glial process outgrowth did not occur. Subsequently, neurons were randomly arranged in the cultures rather than organized along the arms of astroglia. We have named the immune activity that blocks neuron-astroglial interactions anti-astrotactin. Partial purification of the anti-astrotactin blocking antibodies was obtained by cellular absorption with PC12 cells, a clonal cell line which expresses both the N-CAM and NILE (Ng-CAM, L1) glycoproteins. Subsequent absorption with purified cerebellar granule cells, but not with astroglial cells, removed the blocking activity, suggesting that the antigen(s) bound by blocking antibodies are neuronal. Immunoprecipitation of [35S]methionine- or [3H]fucose-radiolabeled Triton extracts of early postnatal cerebellar cells showed that the unabsorbed antiserum recognized a large number of proteins. Among these were bands with apparent molecular masses of N-CAM (180 and 140 kD) and NILE (230 kD). After absorption of the immune serum with PC12 cells, the number of bands recognized by the antiserum was reduced to a prominent band at 100 kD and a diffuse smear of material between 80 and 90 kD. The prominent band at 100 kD was removed by subsequent absorption of the immune serum with granule cells, a step which removed the blocking activity in the cerebellar microculture assay. Further evidence suggests that the astrotactin activity is missing or defective on granule cells from the neurological mutant mouse weaver, an animal that suffers a failure of glial-guided neuronal migration. When anti-astrotactin Fab fragments were pre-absorbed with weaver cerebellar neurons and then tested in the functional assay of neuron-glial interactions, the immune blocking activity was not removed. In contrast, wild-type cerebellar neurons removed the anti-astrotactin blocking activity under the same conditions. Subsequently, when [3H]fucose-radiolabeled Triton extracts of weaver and normal cells were immunoprecipitated with whole or PC12-absorbed anti-astrotactin antiserum, the intensity of the band at 100 kD was reduced by 95% in weaver cells.
16
Alves, Rúbens Prince dos Santos, Lennon Ramos Pereira, Denicar Lina Nascimento Fabris, Felipe Scassi Salvador, Robert Andreata Santos, Paolo Marinho de Andrade Zanotto, Camila Malta Romano, Jaime Henrique Amorim, and Luís Carlos de Souza Ferreira. "Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen." Clinical and Vaccine Immunology 23, no. 6 (March 30, 2016): 460–69. http://dx.doi.org/10.1128/cvi.00081-16.
Full textAbstract:
ABSTRACTDengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed inEscherichia coliat large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses.
17
Giallongo, Cesarina, Nunziatina Parrinello, Daniele Tibullo, Piera La Cava, Alessandra Cupri, Annalisa Chiarenza, Fabio Stagno, Giuseppe A. Palumbo, and Francesco Di Raimondo. "Myeloid-Derived Suppressor Cells Increase in Chronic Myeloid Leukemia and Exert Immune Suppressive Activity." Blood 120, no. 21 (November 16, 2012): 2779. http://dx.doi.org/10.1182/blood.v120.21.2779.2779.
Full textAbstract:
Abstract Abstract 2779 Background: Tumor cells are able to develop immune evasion mechanisms which induce a state of immune tolerance and inactivate tumor-specific T cells. In this context, in some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. Aim: The aim of this study was to analyze MDSCs and investigate their involvement in T-cell anergy of CML patients. Methods: MDSCs were analyzed in peripheral blood (PB) of 13 CML patients (at diagnosis and during therapy) and healthy donors (HD; n=20) by cytofluorimetric analysis (CD14+DR- for monocytic MDSCs and CD11b+CD33+CD14-DR- for granulocytic MDSCs). Arginase 1 expression was assessed in PB of HD and CML patient using real time PCR. Purification of granulocytes, monocytes and lymphocytes from PB was performed by a positive magnetic separation kit (EasySep, STEMCELL Technologies). Arginase activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. To evaluate the activation of CD3+ T lymphocytes after incubation with phytoemagglutinin, we analyzed at 24, 48, 72 h the following markers: CD69+, CD71+, DR+. Microvesicles were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. Results: CML patients showed high levels of monocytic and granulocytic MDSCs at diagnosis in comparison to HD (63±8 and 83±12,2% respectively in CML vs 4,9±2,1 and 55,8±5,3% respectively in HD; p<0.001) while after 3–6 months of tyrosine kinase inhibitors (TKIs) therapy MDSC levels returned to normal values. Either in PB and in the purified granulocytes subpopulation, arginase1 expression showed a 30 fold increase in CML at diagnosis (CML vs HD: p<0.01) and decreased after therapy. We also evaluated arginase enzymatic activity in granulocytes and we found it increased in CML patients (n=4) compared to HD (n=5) (p<0.05). CML as well as HD T lymphocytes showed a normal activation in vitro which was significantly lost when they was incubated with CML serum (n=4). In addition, an increase of monocytic MDSCs in vitro was observed after incubation of HD monocytes with CML serum (39±6%; p<0.01) or microvescicles (9,2±1,2%; p<0.05) compared to control serum. Conclusions: Granulocytic and monocytic MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. Their levels also correlates with Arginase 1 expression and enzymatic activity in granulocytes. CML serum as well as CML microvesicles increase the percentage of HD monocytic MDSCs. Moreover, CML serum leads to anergy of T lymphocytes, probably by Arginase 1 secretion. Disclosures: Off Label Use: Eltrombopag is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura (ITP).
18
Lebrun, M., P. Filée, M. Galleni, J. G. Mainil, A. Linden, and B. Taminiau. "Purification of the recombinant beta2 toxin (CPB2) from an enterotoxaemic bovine Clostridium perfringens strain and production of a specific immune serum." Protein Expression and Purification 55, no. 1 (September 2007): 119–31. http://dx.doi.org/10.1016/j.pep.2007.04.021.
Full text
19
La Valle, Roberto, Silvia Sandini, Maria Jesus Gomez, Francesca Mondello, Giulia Romagnoli, Roberto Nisini, and Antonio Cassone. "Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response toCandida albicans." Infection and Immunity 68, no. 12 (December 1, 2000): 6777–84. http://dx.doi.org/10.1128/iai.68.12.6777-6784.2000.
Full textAbstract:
ABSTRACT A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designatedCaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His6-tagged protein (rCaMp65) was expressed inEscherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+ T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.
20
Hashinaka, Kazuya, Seiichi Hashida, Ichiro Nishikata, Akio Adachi, Shinichi Oka, and Eiji Ishikawa. "Recombinant p51 as Antigen in an Immune Complex Transfer Enzyme Immunoassay of Immunoglobulin G Antibody to Human Immunodeficiency Virus Type 1." Clinical Diagnostic Laboratory Immunology 7, no. 6 (November 1, 2000): 967–76. http://dx.doi.org/10.1128/cdli.7.6.967-976.2000.
Full textAbstract:
ABSTRACT An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of Escherichia coli using a rather long DNA fragment (3,012 bp) of the whole HIV-1 pol gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced inE. coli as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to β-d-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (log Y = 0.99 logX + 0.23; r = 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.
21
Holdom, M. D., B. Lechenne, R. J. Hay, A. J. Hamilton, and M. Monod. "Production and Characterization of RecombinantAspergillus fumigatus Cu,Zn Superoxide Dismutase and Its Recognition by Immune Human Sera." Journal of Clinical Microbiology 38, no. 2 (2000): 558–62. http://dx.doi.org/10.1128/jcm.38.2.558-562.2000.
Full textAbstract:
The Cu,Zn superoxide dismutase (SOD) of Aspergillus fumigatus has previously been purified and shown to be immunoreactive to the sera of patients with aspergillosis; however, the purification of large quantities of the enzyme for expanded immunological analysis is both difficult and time-consuming. Accordingly, a λEMBL3 A. fumigatus genomic library was screened with degenerate oligonucleotides based on N-terminal amino acid sequence data; from this initial screen a 1,400-bp fragment was identified, labelled, and used to screen an A. fumigatusλgt11 cDNA library. A full-length cDNA encoding Cu,Zn SOD was subsequently identified and cloned. The cDNA encodes a protein of 154 amino acids, which does not have a signal peptide. The A. fumigatus Cu,Zn SOD possesses the typical metal binding ligands of fungal Cu,Zn SODs (six histidines and one aspartic acid) and has significant overall homology with Cu,Zn SODs in general. A recombinantA. fumigatus Cu,Zn SOD has been expressed in Pichia pastoris, is enzymatically active, and has biochemical and biophysical properties that are similar to those of the native enzyme. A sheep polyclonal antibody raised against purified native A. fumigatus Cu,Zn SOD was reactive to the recombinant enzyme by immunoenzyme development of Western blots. Sixty percent of serum samples from patients with A. fumigatus infections were reactive against the recombinant Cu,Zn SOD via immunoenzyme development of Western blots, indicating that the recombinant protein may be useful in the serodiagnostic identification of A. fumigatusinfections.
22
Kamali, Ali N., Patricia Marín-García, Isabel G. Azcárate, Antonio Puyet, Amalia Diez, and José M. Bautista. "Experimental Immunization Based onPlasmodiumAntigens Isolated by Antibody Affinity." Journal of Immunology Research 2015 (2015): 1–11. http://dx.doi.org/10.1155/2015/723946.
Full textAbstract:
Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethalPlasmodium yoelii yoelii17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses ofP. yoelii yoelii17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites.
23
Oliveira, Rosane, Renan F. Domingos, Zenaide M. de Morais, Silvio A. Vasconcellos, Ivy J. Alves, Eliete C. Romero, and Ana L. T. O. Nascimento. "Intermediate and C-terminal regions of leptospiral adhesin Lsa66 are responsible for binding with plasminogen and extracellular matrix components." Journal of Medical Microbiology 63, no. 9 (September 1, 2014): 1119–30. http://dx.doi.org/10.1099/jmm.0.078378-0.
Full textAbstract:
Leptospirosis, a worldwide zoonotic infection, is an important human and veterinary health problem. We have previously identified a leptospiral multipurpose adhesin, Lsa66, capable of binding extracellular matrix (ECM) components and plasminogen (PLG). In this work, we report the cloning, expression, purification and characterization of three fragments derived from the full-length Lsa66: N-terminal, intermediate and C-terminal regions. We employed Escherichia coli BL21-SI as expression cells. The recombinant fragments tagged with N-terminal His6 were purified by metal-charged chromatography to major protein bands that were recognized by anti-His-tag mAbs. The recombinant fragments were evaluated for their capacity to attach to ECM components and to PLG. The intermediate region bound to laminin, plasma fibronectin and PLG. Laminin also bound to the C-terminal region. Antibodies in leptospirosis-positive serum samples recognized Lsa66, being the immune epitopes located at the N-terminal and intermediate fragments. The data confirm that Lsa66 is expressed during infection and that this protein might have a role in bacterial infection.
24
Alleman, A. Rick, Melanie G. Pate, John W. Harvey, Jack M. Gaskin, and Anthony F. Barbet. "Western Immunoblot Analysis of the Antigens ofHaemobartonella felis with Sera from Experimentally Infected Cats." Journal of Clinical Microbiology 37, no. 5 (1999): 1474–79. http://dx.doi.org/10.1128/jcm.37.5.1474-1479.1999.
Full textAbstract:
Cats were experimentally infected with a Florida isolate ofHaemobartonella felis in order to collect organisms and evaluate the immune response to H. felis. Cryopreserved organisms were thawed and injected intravenously into nonsplenectomized and splenectomized cats. Splenectomized animals were given 10 mg of methylprednisolone per ml at the time of inoculation. Blood films were evaluated daily for 1 week prior to infection and for up to 60 days postinfection (p.i.). Blood for H. felis purification was repeatedly collected from splenectomized animals at periods of peak parasitemias. Organisms were purified from infected blood by differential centrifugation, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes for immunoblot analysis. Serum was collected from nonsplenectomized animals prior to and for up to 60 days p.i. and was used on immunoblots to identify antigens. The combination of splenectomy and corticosteroid treatment resulted in marked, cyclic parasitemias without concurrent severe anemia, providing an opportunity to harvest organisms in a manner that was not lethal to the animals. Several antigens (150, 52, 47, 45, and 14 kDa) were identified. An antigen with a molecular mass of approximately 14 kDa appeared to be one of the most immunodominant and was consistently recognized by immune sera collected at various times during the course of infection. These data suggest that one or more of these antigens might be useful for the serologic diagnosis of H. felis infections in cats.
25
Jin, Sha, Charles J. Issel, and Ronald C. Montelaro. "Serological Method Using Recombinant S2 Protein To Differentiate Equine Infectious Anemia Virus (EIAV)-Infected and EIAV-Vaccinated Horses." Clinical Diagnostic Laboratory Immunology 11, no. 6 (November 2004): 1120–29. http://dx.doi.org/10.1128/cdli.11.6.1120-1129.2004.
Full textAbstract:
ABSTRACT We recently reported a highly protective attenuated live virus vaccine for equine infectious anemia virus (EIAV) based on a proviral construct (EIAVUKΔS2) with a genetically engineered mutation in the viral S2 gene that eliminates expression of this accessory protein. While the EIAVUKΔS2 vaccine provides protection from detectable infection by experimental challenge with highly virulent virus, the potential for commercial application of this vaccine is complicated by the fact that horses inoculated with the EIAVUKΔS2 vaccine strain become seropositive in various reference diagnostic assays based on detection of antibodies to virion core or envelope proteins. To address this issue, we describe here the development and optimization of a new serologic EIAV diagnostic enzyme-linked immunosorbent assay (ELISA) to detect serum antibodies to the EIAV S2 protein that are produced in infected horses but not in horses inoculated with the EIAVUKΔS2 vaccine virus. The test S2 protein antigen was developed using the S2 gene sequence from the EIAVUK strain of virus and a series of modifications to facilitate production and purification of the diagnostic antigen, designated HS2G. Using this HS2G as antigen, we describe the development of an affinity ELISA that provides a sensitive and specific detection of S2-specific serum antibodies in experimentally and field-infected horses (22 of 24), without detectable reactivity with immune serum from uninfected (12 of 12) or vaccinated (29 of 29) horses. These data indicate that the S2-based diagnostic ELISA has the potential to accurately differentiate horses infected with EIAV from horses inoculated with an attenuated EIAV vaccine strain with a mutant S2 gene.
26
Ritz, Danilo, Andreas Gloger, Dario Neri, and Tim Fugmann. "Purification of soluble HLA class I complexes from human serum or plasma deliver high quality immuno peptidomes required for biomarker discovery." PROTEOMICS 17, no. 1-2 (December 22, 2016): 1600364. http://dx.doi.org/10.1002/pmic.201600364.
Full text
27
Morita-Hoshi, Yuriko, Yuji Heike, Yuka Ohsaki, Mieko Shiwa, Kazuhiro Masuoka, Atsushi Wake, Shuichi Taniguchi, Kensei Tobinai, and Yoichi Takaue. "Serum Amyloid A (SAA) as a Molecular Marker for Pre-Engraftment Immune Reactions (PIR) after Cord Blood Transplantation." Blood 108, no. 11 (November 16, 2006): 3127. http://dx.doi.org/10.1182/blood.v108.11.3127.3127.
Full textAbstract:
Abstract 〈Background and Methods〉: High-grade fever prior to engraftment without any apparent signs of infection, mimicking hyperacute GVHD or engraftment syndrome, is often observed in patients who undergo cord blood transplantation (CBT). This “pre-engraftment immune reaction (PIR)” peaks at around day 9 of CBT, and is often accompanied by high-grade fever, skin rash and weight gain. Although PIR responds well to corticosteroid therapy, the prolonged use of steroid often causes an increased incidence of infectious complications, leading to significant treatment-related mortality, particularly in the elderly. It has been speculated that cytokines induced by the initial immune/inflammation reaction are the primary cause of PIR, but no confirming data are available. To clarify this point, we evaluated the protein expression profile of serum in CBT recipients using a surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF MS) system and found marker candidates in PIR after CBT. 〈Results〉: Thirty-eight blood samples were taken from 13 CBT recipients at 3 different occasions, i.e. afebrile period prior to PIR onset, onset of fever and resolution of fever, to compare the relative protein expression levels. Samples were analyzed in 56 spectra by changing the elution conditions with 4 kinds of chips (IMAC30, CM10, H50, Q10), 5 different rinse conditions and 3 kinds of energy absorption molecules. Six candidate protein peaks that commonly increase at the time of PIR with molecular masses of 8611Da, 8642Da, 11452Da, 11512Da, 11539Da and 11669Da were identified. The 11kDa protein peaks that bind to a Q10-anion exchange chip were selected for further analysis. Two 11kDa protein peaks were eluted at pH9 and pH4, but since the proteins eluted at pH4 included albumin which overlapped the candidate protein peak, the elution fraction at pH9 was used for purification and identification. Proteins eluted at pH9 were separated by SDS-PAGE. After in-gel digestion of the 11kDa band by trypsin, the digested peaks were analyzed by PCI Qstar MSMS and the protein was determined using the ProFound database. The results revealed that the 11kDa protein was an N-terminal portion of serum amyloid A (SAA). The SAA level was measured by ELISA in the same sample that was assessed by SELDI-TOF MS. The mean SAA level prior to fever onset was 14 (3–51) μg/mL, and this increased to 883 (40–2470) μg/mL at the time of PIR and decreased to 45 (8–126) μg/mL after resolution of the fever. 〈Conclusion〉: A specific marker for PIR after CBT was identified by an SELDI-TOF MS system. SAA increased by 10- to 100-fold at the time of PIR, and thus may become a useful tool for the diagnosis of PIR. Figure Figure
28
Bohrmann, J. "Antisera against a channel-forming 16 kDa protein inhibit dye-coupling and bind to cell membranes in Drosophila ovarian follicles." Journal of Cell Science 105, no. 2 (June 1, 1993): 513–18. http://dx.doi.org/10.1242/jcs.105.2.513.
Full textAbstract:
In Drosophila ovarian follicles, communication via gap junctions can be observed between the oocyte and its surrounding follicular epithelium. In the present study, the intercellular exchange of the fluorescent tracer Lucifer Yellow was analysed following pressure-injections of five different sera or protein solutions into the oocyte of stage-10 follicles. Three of the tested sera are directed against a channel-forming 16 kDa protein, which is a component of the vacuolar H(+)-ATPase and of Nephrops norvegicus gap junctions. When one of these antisera was injected 5–10 min prior to the dye, the percentage of follicles showing dye-coupling between oocyte and follicle cells was extremely small. On the other hand, injections of non-immune serum or of bovine serum albumin solution had only minor inhibitory effects. With indirect immunofluorescence, the three Nephrops antisera revealed a discrete punctate pattern at the membranes between neighbouring follicle cells as well as between follicle cells and oocyte. Most likely, this fluorescent pattern represents the distribution of gap junctions in the follicular epithelium. On immunoblots, the Nephrops antisera recognized a 29 kDa Drosophila ovary protein with high specificity. Affinity purification of one of these antisera against the 29 kDa protein revealed that this protein of Drosophila and the 16 kDa membrane-channel protein of Nephrops are immunologically related. Thus, the Nephrops antisera might help to reveal, in future injection experiments, the functional role of gap-junction mediated communication in Drosophila.
29
Damen, Carola W. N., Ellen J. B. Derissen, Jan H. M. Schellens, Hilde Rosing, and Jos H. Beijnen. "The bioanalysis of the monoclonal antibody trastuzumab by high-performance liquid chromatography with fluorescence detection after immuno-affinity purification from human serum." Journal of Pharmaceutical and Biomedical Analysis 50, no. 5 (December 2009): 861–66. http://dx.doi.org/10.1016/j.jpba.2009.04.031.
Full text
30
IGETEI, JOSEPH E., SUSAN LIDDELL, MARWA EL-FAHAM, and MICHAEL J. DOENHOFF. "Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase." Parasitology 143, no. 5 (February 29, 2016): 646–57. http://dx.doi.org/10.1017/s0031182016000184.
Full textAbstract:
SUMMARYA serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine β-naphthyl–ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or β-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host–parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion.
31
Jefferies, Meryem, Harunor Rashid, Grant A. Hill-Cawthorne, and Veysel Kayser. "A Brief History of Ebolavirus Disease: Paving the Way Forward by Learning from the Previous Outbreaks." Infectious Disorders - Drug Targets 20, no. 3 (July 20, 2020): 259–66. http://dx.doi.org/10.2174/1871526518666181001125106.
Full textAbstract:
In this review, Ebolavirus Disease (EVD) outbreaks have been comprehensively reviewed from their beginning until now. It chronologically discusses how each outbreak was tackled, national and international actions taken, diagnostic methods applied, the infection control procedures put in place, and the lessons learnt from each epidemic for the control of subsequent epidemics. Data for this review were obtained from literature published between 1967 and 2016 in key medical databases, the official websites of various governmental organisations, international public health agencies, and regulatory bodies. Despite major developments in the field of EVD, there has been little progress in its specific therapy or prevention. Historically, individuals who recovered from EVD acted as a source of fresh frozen plasma (containing IgG) that has been used to treat other acutely ill patients, however this therapeutic modality has limitations due to the risk of transmission of blood-borne infections. With the use of advanced and efficient purification methods the incidence of unwanted side effects following immune serum therapy has currently been greatly reduced. Creation of a safe plasma pool that covers immunoglobulins against all strains of EVD is now a research priority. Recommendations on how future EVD outbreaks can be better managed have been discussed.
32
Karan, Sweta, Pujarini Dash, Himani Kaushik, Pramoda K. Sahoo, Lalit C. Garg, and Aparna Dixit. "Structural and Functional Characterization of Recombinant Interleukin-10 from Indian Major CarpLabeo rohita." Journal of Immunology Research 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/3962596.
Full textAbstract:
Interleukin-10, an important regulator of both the innate and adaptive immune systems, is a multifunctional major cytokine. Though it is one of the major cytokines, IL-10 from the Indian major carp,Labeo rohita,has not yet been characterized. In the present study, we report large scale production and purification of biologically active recombinant IL-10 ofL. rohita(rLrIL-10) using a heterologous expression system and its biophysical and functional characterization. High yield (~70 mg/L) of soluble rLrIL-10 was obtained at shake flask level. The rLrIL-10 was found to exist as a dimer. Far-UV CD spectroscopy showed presence of predominantly alpha helices. The tertiary structure of the purified rLrIL-10 was verified by fluorescence spectroscopy. Two-dimensional gel analysis revealed the presence of six isoforms of the rLrIL-10. The rLrIL-10 was biologically active and its administration significantly reduced serum proinflammatory cytokines, namely, interleukin 1β, TNFα, and IL-8, and augmented the NKEF transcript levels in spleen ofL. rohita. Anti-inflammatory role of the rLrIL-10 was further established by inhibition of phagocytosis using NBT reduction assayin vitro.The data indicate that the dimeric alpha helical structure and function of IL-10 ofL. rohitaas a key regulator of anti-inflammatory response have remained conserved during evolution.
33
Jacobs, Dirk, Martine Vercammen, and Eric Saman. "Evaluation of Recombinant Dense Granule Antigen 7 (GRA7) of Toxoplasma gondii for Detection of Immunoglobulin G Antibodies and Analysis of a Major Antigenic Domain." Clinical Diagnostic Laboratory Immunology 6, no. 1 (January 1, 1999): 24–29. http://dx.doi.org/10.1128/cdli.6.1.24-29.1999.
Full textAbstract:
ABSTRACT Dense granule protein 7 (GRA7) of Toxoplasma gondii was expressed in Escherichia coli as a fusion protein. The leader peptide contained a 25-amino-acid mouse tumor necrosis factor fragment and six histidyl residues. After purification by metal chelate affinity chromatography, the antigen was evaluated in an enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG). For two sets of IgG-positive human serum samples, obtained from routine screening, an overall sensitivity of 81% was obtained. For chronic-phase sera, the sensitivity of detection was 79%, but chronic-phase sera with low titers were more difficult to detect (65% sensitivity for sera with immunofluorescence titer of 1/64). When GRA7 was combined with Tg34AR (rhoptry protein 2 C-terminal fragment), the sensitivity rose to 96%. For a set of acute-phase serum samples tested on GRA7, the sensitivity of detection was 94%, and high-titer IgM-positive sera were detected at an especially high rate. In contrast, when Tg34AR was used, the sensitivity was only 85% for this latter set of serum samples. Three truncated GRA7 fragments containing the same leader peptide as that of recombinant GRA7 were produced. The shortest fragment (97 N-terminal amino acids) was not reactive with human sera or with a specific anti-GRA7 monoclonal antibody, while the two larger fragments were reactive. The most important antigenic domain of GRA7 for human sera was localized between residues 97 and 146. The epitope for the specific monoclonal antibody could be further narrowed down by the use of synthetic peptides, but this epitope is not recognized by sera from T. gondii-infected humans. These results indicate that GRA7 may be considered as an additional tool for studying the immune response to T. gondii.
34
Belinin, G. Ju, V. I. Vasiliev, E. E. Efremov, L. A. Gorgidze, N. I. Zozulya, T. N. Moiseeva, L. S. Al-Radi, and S. A. Vasiliev. "Аpplication of plasma proteins cryoheparinoprecipitation method in the treatment of patients with cryoglobulinemia." Meditsinskiy sovet = Medical Council, no. 11 (August 8, 2020): 210–18. http://dx.doi.org/10.21518/2079-701x-2020-11-210-218.
Full textAbstract:
Introduction. The term “cryoglobulinemia” is currently used to identify immunoglobulins in vitro in the blood serum that precipitate at temperatures below 37 °C; in vivo they form immune complexes that can be deposited in small vessels and activate the complement system with the development of leukocytoclastic vasculitis. Cryoglobulinemia may develop in various lymphoproliferative, autoimmune and infectious diseases. Aim of study. To develop the technique of plasma proteins cryofraction (selective plasmapheresis with the use of heparin as a stimulant of fibronectin opsonic activity and purified autoplasma to compensate for the removed volume), to evaluate the effectiveness and tolerability of the developed technique in the treatment of patients with cryoglobulinemia. Materials and methods. 159 patients were treated (120 women and 39 men aged 21 to 83 years). Research results. Heparinocryofraction technique is a highly effective method of extracorporeal blood purification, which allows to selectively remove from the patients’ plasma such pathological components as cryoglobulins (up to 100% of the initial content), adhesive proteins (up to 84% of the initial content), fibronectin and immune complexes (up to 7% of the initial content). It is possible to reduce significantly and reliably the level of cryoglobulins, circulating immune complexes, non-specific markers of inflammation, daily proteinuria, as well as to normalize the initially reduced concentration of complement components and hemoglobin in the blood of patients with cryoglobulinemia before and after the procedure of cryofractionation. Purified by the proposed method autoplasma is a solution of albumin and normal immunoglobulins, which allows to use it for plasma substitution during a course of cryofractionation procedures, on average 7 procedures with an interval of 1–2 days. Conclusion. The technique of cryofractionation using heparin and purified autoplasma can and should be widely used in the complex treatment of patients with cryoglobulinemia. Carrying out 6–-7 sessions of plasma cryofractionation allows to remove cryoglobulins from plasma effectively and selectively. Application of purified autoplasma allows to avoid using of blood preparations in plasmapheresis. The proposed method allows to significantly improve the efficiency and tolerance of medication therapy and increase the duration of disease remission.
35
Elbagir, S., A. Sohrabian, A. Elshafie, E. M. Elagib, N. A. Mohammed, M. A. M. Nur, E. Svenungsson, I. Gunnarsson, and J. Rönnelid. "AB0127 ACCUMULATION OF ANTI-NUCLEAR ASSOCIATED AUTOANTIBODIES IN CIRCULATING IMMUNE COMPLEXES IS MORE PROMINENT IN SLE PATIENTS FROM SUDAN COMPARED TO SWEDEN." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1364–65. http://dx.doi.org/10.1136/annrheumdis-2020-eular.3089.
Full textAbstract:
Background:Systemic Lupus Erythematosus (SLE) is a systemic immune complex (IC)-mediated disease associated with autoantibodies targeting multiple nuclear specificities (ANA). SLE has an aggressive nature among African populations. The role of SLE-related autoantibodies in the formation of IC has only been studied to a limited extent, and not at all in African SLE patients.Objectives:To quantify ANA specificities present in circulating IC in Sudanese and Swedish SLE sera and to compare to corresponding serum levelsMethods:93 Sudanese and 337 Swedish SLE patients who fulfilled the 1982 ACR classification criteria were included. IC were captured using magnetic microparticles coated with purified human C1q, then separated by a two-step elusion procedure (Figure 1). ANA-associated autoantibodies against dsDNA, Sm, the Sm/U1RNP complex, U1RNP, SSA/Ro52, SSA/Ro60, SSB/La, ribosomal P antigen, proliferating cell nuclear antigen (PCNA) and histones were quantified in sera and corresponding IC using a bead-based multiplex immunoassay. The IC purification technique has been developed and validated in our laboratory (Sohrabian ARD 2018), and previously used to study treatment responses to belimumab in Swedish SLE patients (Sohrabian ART 2019). Occurrence of ANA specificities in serum was determined using manufacturer’s suggested cutoffs. These cutoffs were used in regression formulas to determine cutoffs for ANA levels detected in the corresponding IC.Results:Swedish patients had higher serum levels of anti-Sm, anti-dsDNA and anti-ribosomal P antibodies compared to Sudanese patients. On the contrary, IC levels of all ANA specificities except anti-SSA/Ro52 and anti-SSA/Ro60 were higher in Sudanese patients (Table 1 and Figure 2). Sudanese patients were more often positive for anti-Sm, anti- Sm/U1RNP, anti-dsDNA and anti-histone antibodies in IC, whereas a borderline statistical significance was found for only anti-Sm in corresponding serum samples (Table 1).Table 1.Levels median/meanSudanserumSweden serumPSudanICSwedenICPSSA/Ro5216/52.117/52.30.82.9/14.42.4/10.70.07SSA/Ro602.0/47.23.0/48.70.21.5/14.61.3/12.50.7SSB/La2.0/222.0/220.61.1/4.20.8/5.50.02Sm1.0/21.71.0/11.40.00080.5/4.20.3/1.3<0.0001SmRNP1.0/20.51.0/160.23.7/8.62.0/4.6<0.0001U1RNP6.0/43.87.0/40.20.91.0/3.50.6/2.20.0005dsDNA14.0/124.528.0/198.30.00132.1/50.812.4/25.4<0.0001Ribosomal P1.0/9.32.0/11.70.00061.7/3.31.4/2.90.005Histone5.0/20.56.0/19.30.24.0/5.91.9/3.4<0.0001PCNA5.0/8.45.0/8.90.611.1/14.27.0/11<0.0001Occurrence n(%)serumserumPICICPSSA/Ro5230(32.2)105(31.7)0.918(19.3)55(16.6)0.5SSA/Ro6034(36.6)122(36.9)0.927(29)89(26.8)0.7SSB/La15(16.1)64(19.3)0.59(9.7)38(11.4)0.3Sm12(12.9)21(6.3)0.046(6.4)5(1.5)0.008SmRNP15(16.1)42(12.7)0.417(18.3)33(9.9)0.03U1RNP20(21.5)69(20.8)0.922(23.7)55(16.6)0.1dsDNA31(33.3)147(44.4)0.05564(68.8)105(31.6)<0.0001Ribosomal P4(4.3)20(6)0.52(2.1)9(2.7)0.8Histone10(10.7)34(10.3)0.934(36.6)58(17.5)<0.0001PCNA1(1.1)10(3)0.320(21.5)45(13.5)0.06Conclusion:ANA-associated autoantibodies are more accumulated in circulating IC from Sudanese than Swedish SLE patients. The clinical significance of these findings is yet to be investigated in our upcoming analysesDisclosure of Interests:None declared
36
Chen, Changlin, Andrew F. Rowley, Russell P. Newton, and Norman A. Ratcliffe. "Identification, purification and properties of a β-1,3-glucan-specific lectin from the serum of the cockroach, Blaberus discoidalis which is implicated in immune defence reactions." Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 122, no. 3 (March 1999): 309–19. http://dx.doi.org/10.1016/s0305-0491(99)00020-6.
Full text
37
Zancopé-Oliveira, Rosely M., Errol Reiss, Timothy J. Lott, Leonard W. Mayer, and George S. Deepe. "Molecular Cloning, Characterization, and Expression of the M Antigen of Histoplasma capsulatum." Infection and Immunity 67, no. 4 (April 1, 1999): 1947–53. http://dx.doi.org/10.1128/iai.67.4.1947-1953.1999.
Full textAbstract:
ABSTRACT The major diagnostic antigens of Histoplasma capsulatumare the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to H. capsulatum are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic H. capsulatum DNA. This fragment was used to screen an H. capsulatum genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2,187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from Aspergillus fumigatus, Aspergillus niger, and Eimericella nidulans. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of H. capsulatum is a presumptive catalase, and the recombinant protein retains serodiagnostic activity.
38
Hess, David A., Krysta D. Levac, Francis N. Karanu, Michael Rosu-Myles, Martin J. White, Lisa Gallacher, Barbara Murdoch, et al. "Functional analysis of human hematopoietic repopulating cells mobilized with granulocyte colony-stimulating factor alone versus granulocyte colony-stimulating factor in combination with stem cell factor." Blood 100, no. 3 (August 1, 2002): 869–78. http://dx.doi.org/10.1182/blood.v100.3.869.
Full textAbstract:
Abstract Using in vitro progenitor assays, serum-free in vitro cultures, and the nonobese diabetic/severe combined immune-deficient (NOD/SCID) ecotropic murine virus knockout xenotransplantation model to detect human SCID repopulating cells (SRCs) with multilineage reconstituting function, we have characterized and compared purified subpopulations harvested from the peripheral blood (PB) of patients receiving granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF). Mobilized G-CSF plus SCF PB showed a 2-fold increase in total mononuclear cell content and a 5-fold increase in CD34-expressing cells depleted for lineage-marker expression (CD34+Lin−) as compared with patients treated with G-CSF alone. Functionally, G-CSF plus SCF–mobilized CD34+CD38−Lin−cells contained a 2-fold enhancement in progenitor frequency as compared with G-CSF–mobilized subsets. Despite enhanced cellularity and progenitor capacity, G-CSF plus SCF mobilization did not increase the frequency of SRCs as determined by limiting dilution analysis by means of unfractionated PB cells. Purification of SRCs from these sources demonstrated that as few as 1000 CD34+CD38−Lin− cells from G-CSF–mobilized PB contained SRC capacity while G-CSF plus SCF–mobilized CD34+CD38−Lin−cells failed to repopulate at doses up to 500 000 cells. In addition, primitive CD34−CD38−AC133+Lin−cells derived from G-CSF plus SCF–mobilized PB were capable of differentiation into CD34-expressing cells, while the identical subfractions from G-CSF PB were unable to produce CD34+cells in serum-free cultures. Our study defines qualitative and quantitative distinctions among subsets of primitive cells mobilized by means of G-CSF plus SCF versus G-CSF alone, and therefore has implications for the utility of purified repopulating cells from these sources.
39
Nardi, Michael, and Simon Karpatkin. "Antiidiotype Antibody against Platelet Anti-Gpiiia Contributes to the Regulation of Thrombocytopenia in HIV-1–Itp Patients." Journal of Experimental Medicine 191, no. 12 (June 12, 2000): 2093–100. http://dx.doi.org/10.1084/jem.191.12.2093.
Full textAbstract:
Patients with human immunodeficiency virus 1–associated immunological thrombocytopenia (HIV-1–ITP) have markedly elevated platelet-bound immunoglobulin (Ig)G, IgM, and C3C4, as well as serum circulating immune complexes (CICs) composed of the same. Affinity purification of IgGs from their CICs with fixed platelets reveals high-affinity antibody (Ab) against platelet glycoprotein (GP)IIIa 49–66, which correlates inversely with their platelet count. However, sera from these patients have little to no anti-GPIIIa activity. To investigate this, we assayed serum, purified serum IgG, and CIC-Ig from these patients. This revealed ∼150-fold greater Ab activity in purified serum IgG, and ∼4,000-fold greater reactivity in CIC-IgG. This was shown to be associated with the presence of antiidiotype Ab2 (both IgG and IgM) sequestered in the CIC-IgG. The IgM antiidiotype was predominantly blocking Ab, as demonstrated by specificity for F(ab′)2 fragments of anti–GPIIIa 49–66 of HIV-1–ITP patients and inhibition of reactivity with peptide GPIIIa 49–66, not with a control peptide. The IgM antiidiotype was not polyreactive. Similar measurements were made in nonthrombocytopenic HIV-1–infected patients. Their serum reactivity was not measurable, but serum Ig and CIC-IgG against platelet GPIIIa 49–66 was present, although considerably lower than that found in HIV-1–ITP patients (26- and 35-fold lower, respectively). In addition, their IgM antiidiotype reactivity was 12-fold greater than that found in HIV-1–ITP patients. The IgM antiidiotype Ab titer of both cohorts correlated with in vivo platelet count (r = 0.7, P = 0.0001, n = 32). To test the in vivo effectiveness of the IgM antiidiotype, thrombocytopenia was induced in mice with 25 μg of affinity-purified anti–GPIIIa 49–66 (mouse GPIIIa has 83% homology with human GPIIIa and Fc receptors for human IgG1). Maximum effect was obtained at 4–6 h after intraperitoneal injection into Balb/c mice with a platelet count of ∼30% baseline value. Preincubation of the anti-GPIIIa Ab with control IgM at molar ratios of IgM/IgG of 1:7 before intraperitoneal injection had no effect on the in vivo platelet count, whereas preincubation with patient IgM antiidiotype improved the platelet count to 50–80% of normal. Thrombocytopenia could be reversed after addition of IgM antiidiotype 4 h after induction of thrombocytopenia. Thus, CICs of HIV-1–infected patients contain IgM antiidiotype Ab against anti-GPIIIa, which appears to regulate their serum reactivity in vitro and their level of thrombocytopenia in vivo.
40
Takami, Akiyoshi, Takanori Teshima, Koshin Ushizaki, Takumi Taniguchi, Tohru Endo, Hiroshi Sakurai, Yukio Kondo, et al. "A Novel Strategy for the Treatment of Acute Graft-Versus-Host Disease Using the Adsorbent To Remove Excessive Inflammatory Cytokines." Blood 110, no. 11 (November 16, 2007): 5001. http://dx.doi.org/10.1182/blood.v110.11.5001.5001.
Full textAbstract:
Abstract Acute graft-versus-host disease (aGVHD) remains the major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Previous studies show that inflammatory cytokines such as tumor necrosis factor alfa (TNFα), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-18 (IL-18) are involved in the pathogenesis of aGVHD, and that the excess of these cytokines is associated with severity and mortality of aGVHD. We hypothesized that removal of these excessive cytokines from patients’ blood at the onset of aGVHD might improve the treatment outcome. A novel absorbent CTR can effectively adsorb small- to middle-sized proteins like cytokines and enterotoxins in vitro. In view of future exploitation of extracorporeal treatment using CTR column, we tested whether CTR could remove these inflammatory cytokines from blood. When the serum containing a mix of recombinant cytokines was incubated with a CTR adsorbent for 2 hrs, 55% of TNFα, 81% of IL-6, 83% of IL-8, and 22% of IL-18 were successfully removed. Next, we measured TNFα, soluble TNFα receptor 1 (TNFR1), IL-6, IL-8, and IL-18 levels in serum samples obtained from 5 patients (median age 38y, range 26–63y) who underwent myeloablative SCT in 4 and non-myeloablative SCT in 1. AGVHD developed in 2 with grade 3 and in 3 with grade 2. When cytokine levels in patients were expressed as a ratio to the mean cytokine level in control serum samples obtained from three healthy individuals, the mean ratios of TNFα, TNFR1, IL-6, IL-8, and IL-18 at the onset of aGVHD were 6.0 (range, 1.2–12.0), 6.5 (2.5–9.0), 274 (3.5–651), 48.3 (11.3–75.2), and 6.7 (3.2–10.8). The CTR adsorption considerably reduced the concentrations of these cytokines except for IL-18 (Figure 1). The adsorption rates of these cytokines were 64% for TNFα, 48% for TNFR1, 59% for IL-6, more than 94% for IL-8, and 0% for IL-18. The efficient removal of inflammatory cytokines suggests that extracorporeal blood purification with CTR column may be effective in the treatment of aGVHD. This treatment strategy may be promising because it essentially has no deleterious effects on immune functions of SCT recipients unlike other GVHD treatments. Figure Figure
41
MOIO, LUIGI, CRISTINA MARCHISANO, and FRANCESCO ADDEO. "Isolation of specific oligoclonal antibodies against bovine αs1-casein by FPLC tandem immunoaffinity of the polyclonal antibodies." Journal of Dairy Research 65, no. 3 (August 1998): 515–20. http://dx.doi.org/10.1017/s002202999800291x.
Full textAbstract:
Polyclonal antibodies specifically directed towards native casein fractions have been recently used to solve some analytical problems such as localization of casein antigenic sites (Otani et al. 1985; Ametani et al. 1987), identification of casein variants (Moio et al. 1989a; Chianese et al. 1992), detection of bovine casein adulterating goats', ewes' and water-buffalo milk and cheese (Elbertzhagen & Wenzel, 1982; Bernhauer et al. 1983; Aranda et al. 1988; Moio et al. 1992; Rolland et al. 1993, 1995; Addeo et al. 1995b), evaluation of the efficiency of chromatographic fractionation of casein (Addeo et al. 1992) and detection of casein proteolysis in cheese (Addeo et al. 1995a). However, the use of polyclonal antibodies for immune analysis is often limited owing to nonspecific binding. The use of monoclonal antibodies may overcome this problem to some extent. Although the binding affinity of a hyperimmune serum is seldom attainable with a monoclonal antibody, an alternative approach consists of the isolation of specific antibodies by affinity chromatography (Johnstone & Thorpe, 1982). In a single step 1000–10000-fold purification has been achieved.In this paper a fast protein liquid chromatography (FPLC) tandem immunoaffinity is used to enhance the specificity of bovine αs1-casein (CN) polyclonal antiserum. In the first column a caprine whole casein lacking αs1-CN was bound to a solid phase matrix and in the second column bovine αs1-CN was bound. After elution of macromolecular contaminants by a washing step, the two columns were detached and the purified bovine αs1-CN antibodies were eluted from the second column by a simple pH change.
42
Anderson, Jenna, Emmanuel Bréard, Karin Lövgren Bengtsson, Kjell-Olov Grönvik, Stéphan Zientara, Jean-Francois Valarcher, and Sara Hägglund. "Purification, Stability, and Immunogenicity Analyses of Five Bluetongue Virus Proteins for Use in Development of a Subunit Vaccine That Allows Differentiation of Infected from Vaccinated Animals." Clinical and Vaccine Immunology 21, no. 3 (January 22, 2014): 443–52. http://dx.doi.org/10.1128/cvi.00776-13.
Full textAbstract:
ABSTRACTBluetongue virus (BTV) causes bluetongue disease, a vector-borne disease of ruminants. The recent northerly spread of BTV serotype 8 in Europe resulted in outbreaks characterized by clinical signs in cattle, including unusual teratogenic effects. Vaccination has been shown to be crucial for controlling the spread of vector-borne diseases such as BTV. With the aim of developing a novel subunit vaccine targeting BTV-8 that allows differentiation of infected from vaccinated animals, five His-tagged recombinant proteins, VP2 and VP5 of BTV-8 and NS1, NS2, and NS3 of BTV-2, were expressed in baculovirus orEscherichia coliexpression systems for further study. Optimized purification protocols were determined for VP2, NS1, NS2, and NS3, which remained stable for detection for at least 560 to 610 days of storage at +4°C or −80°C, and Western blotting using sera from vaccinated or experimentally infected cattle indicated that VP2 and NS2 were recognized by BTV-specific antibodies. To characterize murine immune responses to the four proteins, mice were subcutaneously immunized twice at a 4-week interval with one of three protein combinations plus immunostimulating complex ISCOM-Matrix adjuvant or with ISCOM-Matrix alone (n= 6 per group). Significantly higher serum IgG antibody titers specific for VP2 and NS2 were detected in immunized mice than were detected in controls. VP2, NS1, and NS2 but not NS3 induced specific lymphocyte proliferative responses upon restimulation of spleen cells from immunized mice. The data suggest that these recombinant purified proteins, VP2, NS1, and NS2, could be an important part of a novel vaccine design against BTV-8.
43
Funke, Christina, Donald P. King, Rory M. Brotheridge, Dieter Adelung, and Jeffrey L. Stott. "Harbor seal (Phoca vitulina) C-reactive protein (C-RP): purification, characterization of specific monoclonal antibodies and development of an immuno-assay to measure serum C-RP concentrations." Veterinary Immunology and Immunopathology 59, no. 1-2 (October 1997): 151–62. http://dx.doi.org/10.1016/s0165-2427(97)00059-7.
Full text
44
Cavassani, Karen Angelica, Sungyong You, Rebecca Meza, Chintda Santiskulvong, Helen Goodridge, and Edwin M. Posadas. "Defining the monocyte subset transcriptional signature associated with progression during androgen-target therapy in prostate cancer patients." Journal of Clinical Oncology 38, no. 6_suppl (February 20, 2020): 157. http://dx.doi.org/10.1200/jco.2020.38.6_suppl.157.
Full textAbstract:
157 Background: Myeloid-derived circulating monocytes are emerging as cells of interest in cancer biology. The role of circulating monocytes in human Prostate Cancer (PCa) is poorly understood. Here we asked what is the association of monocyte-specific subsets and their transcriptional signatures with PCa progression during (AR)-target therapy. Methods: Single-cell RNAseq analysis were performed in blood monocytes from 4 patients at two specific time points over their clinical, course: T1)responding to next generation androgen receptor signaling inhibitors (e.g. abiraterone or enzalutamide) as reflected by a decline in serum PSA and/or radiographic response, and T2)progressing through treatment as detected by increases in the serum PSA concentration and/or radiographic signs of progression .PBMC were subjected to Ficoll-purification, and FACS sorted to exclude dead cells and cells expressing B- and T- lineage markers. Samples were then pooled using a BD™ Single-Cell Multiplexing Kit. The samples for scRNA-seq analysis included monocytes defined as classical CD14++CD16−, intermediate CD14+CD16+, and non-classical CD14low/-CD16+++. Results: We have observed nine (9) transcriptionally-defined clusters.Clusters 0, 1, 4, 5, and 8 mapped closely to that of classical monocytes with high expression of CD14and low expression of FCG3RA (CD16). Clusters 2 and 3 mapped closely to that of NK cells with high expression of FCG3RA(CD16) and KLRB1(CD161), and clusters 6 and 7 corresponded to CD14low FCG3RAhigh SIGLEC10+ monocytes. Importantly, our preliminary data revealed a decrease in the percentage of cells in clusters 2 and 6 and an increase in the percentage of cells in cluster 4 in progressing patients (T2). At the transcriptional level, the three main clusters (classical, non-classical, NK-like) were distinguished by 32, 33, and 72 genes, respectively. Our preliminary findings show that progression was associated with several innate immune transcripts while cytotoxic genes were associated with response to Enzalutamide. Conclusions: These data suggest that monocytes transcriptional signature may be reshaped by PCa. Further study of monocytes in PCa progression are warranted.
45
Gallo, Pasquale, Floriana Vinci, Giovanna Fusco, and Luigi Serpe. "An analytical strategy for identification of a somatotropin-like bioactive peptide by ion trap liquid chromatography/electrospray ionization tandem mass spectrometry after immuno-affinity purification from buffalo serum." Rapid Communications in Mass Spectrometry 23, no. 3 (February 15, 2009): 395–402. http://dx.doi.org/10.1002/rcm.3888.
Full text
46
Dechant, Michael, Stefan Lohse, Stefanie Derer, Thomas Beyer, Katja Klausz, Matthias Peipp, Jeanette H. W. Leusen, Jan G. J. van de Winkel, and Thomas Valerius. "Recombinant Dimeric IgA Antibodies as Tumor-Specific Agents." Blood 116, no. 21 (November 19, 2010): 1488. http://dx.doi.org/10.1182/blood.v116.21.1488.1488.
Full textAbstract:
Abstract Abstract 1488 Dimeric IgA antibodies contribute significantly to the humoral part of the mucosal immune system. However, their potential as immunotherapeutic agent has hardly been explored. Here, we describe the production, purification and functional evaluation of recombinant dimeric IgA, exemplarily directed against the epidermal growth factor receptor (EGF-R). Human J-chain-containing IgA was produced under serum-free conditions by transfecting non-adherent CHO-K1 cells expressing EGF-R specific monomeric IgA with a vector coding for the His-tagged human J-chain. For purification of J-chain-containing dimeric IgA two different affinity (anti-human-kappa and anti-His-tag) and one size exclusion chromatography were combined, resulting in a homogenous preparation of highly pure IgA dimers, as determined by gel electrophoresis under denaturing and native conditions using silver stain, Coomassie blue or Western blots for protein detection, respectively. Functional studies demonstrated dimeric IgA (dIgA) to be at least as effective as monomeric IgA (mIgA) in triggering ADCC of A431 tumor cells by isolated monocytes (EC50 0.35 and 0.30 μg/ml for mIgA and dIgA, 0.23 μg/ml for the corresponding IgG1; maximal killing 51.2 vs 43.3 vs 56.3 %), by isolated PMN (EC50 0.96, 1.20 and 0.8 μg/ml for mIgA, dIgA and IgG1; maximal killing 52.6 vs 50.6 vs 19.9 %) and in human whole blood assays (EC50 0.32 and 0.32 μg/ml for mIgA and dIgA; maximal killing 7.9 vs 12.5 %; IgG1: maximal killing 0.7 %, EC50 not determinable). Importantly, dimeric IgA was more effective in F(ab)-mediated mechanisms: inhibition of binding of FITC-labelled EGF to A431 cells by dimeric IgA was achieved at significantly lower concentrations than by monomeric IgA (EC50 7.1 vs 18.1 μg/ml, respectively). In addition, growth of EGF-R expressing DiFi colon carcinoma cells was inhibited at significantly lower concentrations by dimeric than by monomeric IgA (EC50 1.6 vs 3.7 μg/ml). Both IgA isoforms and their IgG1 variant were similarly effective in triggering apoptosis of DiFi cells, as analyzed by PARP cleavage. Furthermore, only dimeric but not monomeric IgA or IgG was directionally transported by the polymeric immunoglobulin receptor (pIgR) through an epithelial cell monolayer, as analyzed by an experimental transcytosis assay with polarized MDCK cells stably transfected with human pIgR. Together, these studies demonstrate that recombinant dimeric IgA antibodies recruit a distinct repertoire of effector functions compared to monomeric IgA or IgG1 antibodies, trigger important biological functions and constitute an interesting antibody format for future tumor therapies. Disclosures: van de Winkel: Genmab: Employment, Membership on an entity's Board of Directors or advisory committees. Valerius:Genmab: Consultancy, Research Funding.
47
Morton, H., AC Cavanagh, S. Athanasas-Platsis, KA Quinn, and BE Rolfe. "Early pregnancy factor has immunosuppressive and growth factor properties." Reproduction, Fertility and Development 4, no. 4 (1992): 411. http://dx.doi.org/10.1071/rd9920411.
Full textAbstract:
Early pregnancy factor (EPF) was first described as a pregnancy-associated substance, although recent studies suggest a more general link with cell development. It is a product of actively dividing cells and its apparent functional importance to them suggests its potential as a regulator of cell proliferation. The recent discovery of EPF in platelets has provided a comparatively rich and readily available source of EPF. The purification procedures employed to isolate EPF from this source have also been applied to pregnancy serum and urine, medium conditioned by oestrous mouse ovaries (stimulated with prolactin and embryo-conditioned medium), medium conditioned by tumour cells, and serum from rats 24 h after partial hepatectomy (PH). In all instances, biological activity followed the same pattern throughout. Furthermore, the final active reversed-phase high-performance liquid chromatography fraction from all sources was bound specifically by immobilized anti-EPF monoclonal antibodies (MAbs), indicating that the active fractions produced from these diverse sources are very closely related, if not identical. Some differences have been observed in the behaviour of EPF in various conditions. EPF is produced by proliferating tumour cells and by liver cells post-PH, and passive immunization studies with anti-EPF MAbs have shown that these cells need EPF for survival. In contrast, EPF has not been detected as a product of the pre-embryo, and addition of anti-EPF MAbs to embryo cultures does not adversely affect development from the 2-cell to the blastocyst stage. Although the pre-embryo is not dependent on EPF for its development in vitro, neutralization of EPF in vivo by anti-EPF MAbs retards its development. Thus, EPF appears to play an indirect role in maintaining the pre-embryo. By virtue of its ability to suppress the delayed-type hypersensitivity reaction, it has been suggested that EPF might act as an immunological response modifier of the maternal immune system. Alternatively, the effect of EPF on lymphocytes may be to reduce the expression of all or some cytokines and this could inhibit development. Whether or not EPF acts more directly as an autocrine growth factor from around the time of implantation, when the embryo first begins synthesis of EPF, is not known and remains to be investigated.
48
Chepurnov, A. A., K. A. Sharshov, E. I. Kazachinskaya, Yu V. Kononova, E. A. Kazachkova, O. P. Khripko, K. S. Yurchenko, A. Yu Alekseev, M. I. Voevoda, and A. M. Shestopalov. "Antigenic properties of sARs-CoV-2/human/RUs/nsk-FRCFtM-1/2020 coronavirus isolate from a patient in novosibirsk." Journal Infectology 12, no. 3 (August 2, 2020): 42–50. http://dx.doi.org/10.22625/2072-6732-2020-12-3-42-50.
Full textAbstract:
Objective: isolation of coronavirus SARS-CoV-2 from clinical sample of patient with COVID-19 in Novosibirsk; obtaining a purified and inactivated viral antigen and study of its antigenic properties. Materials and methods: virus isolation was carried out in Vero cell culture from nasopharyngeal swab positive on SARS-CoV-2 RNA. The efficiency of SARSCoV-2 replication in cell culture was assessed on the appearance of cytopathic effect (CPE) and the presence of viral RNA in cultural medium with reverse transcription – polymerase chain reaction (RT-PCR). Purification, concentration and inactivation of the viral preparation were carried out according to standard methods. The purity of the purified preparation and the profile of viral proteins were determined by electrophoresis in 10% polyacrylamide gel (PAG) with the addition of sodium dodecyl sulfate (SDS). The presence and specificity of viral proteins were detected using COVID-19 convalescent’s sera with enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results: SARS-CoV-2/human/ RUS/Nsk-FRCFTM-1/2020 isolate was obtained after passage on Vero cells from a virus-containing clinical sample. A purified, concentrated, inactivated, whole-virion antigen was obtained. It contains three structural proteins: glycoprotein S (approximately 200 kDa), nucleoprotein N (48 kDa), and matrix protein M (20-25 kDa). All viral proteins were detected with serum antibodies of COVID-19 convalescents. Conclusion: SARS-CoV-2 coronavirus can be isolated in Vero cell culture. The antigenic specificity of the three structural viral proteins (S, N, and M) is preserved in the purified inactivated viral preparation. The inactivated whole-virion antigen of SARS-CoV-2/human/RUS/Nsk-FRCFTM-1/2020 isolate can be used to study the antigenic immunomodulating properties of viral proteins, to obtain immune sera of laboratory animals, and also as a component of test systems for the detection of specific antibodies with ELISA and immunoblotting.
49
Turecek, Peter, Susanne Vejda, Katalin Varadi, Hanspeter Rottensteiner, Ernst Boehm, Manfred Reiter, Martin Kaliwoda, Wolfgang Mundt, Hartmut J. Ehrlich, and Friedrich Scheiflinger. "Biochemical Characterization of a Recombinant FIX Drug Candidate for Treatment of Hemophilia B." Blood 116, no. 21 (November 19, 2010): 4658. http://dx.doi.org/10.1182/blood.v116.21.4658.4658.
Full textAbstract:
Abstract Abstract 4658 Human coagulation factor IX (FIX) is a vitamin-K-dependent coagulation factor whose absence or dysfunction causes hemophilia B. Treatment of hemophilia B is based on replacement therapy using highly purified FIX concentrates. Baxter has developed a recombinant factor IX for treating hemophilia B patients that is produced in a CHO cell-line using a serum and protein-free fermentation technology. The purification process avoids the use of immune-affinity chromatography and includes two viral reduction steps. The final drug product is formulated in the absence of proteins of animal or human origin. Baxter's recombinant FIX resembles commercially available rFIX in most characteristics with the exception of a significantly lower FIXa content, which might improve standardization compared to commercial rFIX products. Preclinical and clinical lots of rFIX were characterized with respect to their hemostatic potency, efficiency of activation by FXIa and FVIIa in the presence of tissue factor, and capacity to bind to phospholipid vesicles. Three lots of commercial rFIX with different potencies and one lot of a plasma-derived FIX product were included in the study. Similarity could be shown between the preclinical and clinical lots of rFIX in all these assays. Furthermore, the functional and biochemical characterization of Baxter's recombinant FIX showed that it resembles the recombinant comparator product. The phase I clinical trial which has been initiated will now have to show whether Baxter's rFIX can become an alternative drug candidate product for treating patients suffering from hemophilia B. Disclosures: Turecek: Baxter Innovations GmbH: Employment. Vejda:Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Boehm:Baxter Innovations GmbH: Employment. Reiter:Baxter Innovations GmbH: Employment. Kaliwoda:Baxter Innovations GmbH: Employment. Mundt:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.
50
Driscoll, James J., Irim Aslam, and Ehsan Malek. "Counteracting PD-L1/PD-L2 Immune Suppression in Multiple Myeloma through Kinase Inhibition." Blood 128, no. 22 (December 2, 2016): 3295. http://dx.doi.org/10.1182/blood.v128.22.3295.3295.
Full textAbstract:
Abstract Introduction: Multiple myeloma (MM) is clonal plasma cell malignancy that remains incurable. Checkpoint inhibitors represent a revolutionary form of cancer therapy that empowers the immune system to defeat cancer. Programmed death-1 (PD-1) is an inhibitory receptor expressed on immune cells, particularly cytotoxic T cells, that interacts with two ligands, PD-ligand 1 (PD-L1) and PD-L2 expressed on tumor cells. PD-L1 and PD-L2 engage PD-1 on the T cell surface to negatively modulate the magnitude of T-cell-mediated responses. Such negative feedback is critically important in maintaining homeostasis of the immune response to prevent autoimmunity during infection or inflammation in normal tissue. However, in cancers, it presents a major problem in blocking cellular antitumor responses. PD-L1/PD-L2 ligation with PD-1 provides a mechanism of immune escape for tumor cells by turning off the cytotoxic T cells. Currently available monoclonal antibodies (mAbs) that disrupt the PD-1/PD-L1 interaction have exhibited remarkable responses in selected patients and tumor types. However, mAbs demonstrate many drawbacks that include a lack of tumor cell specificity, low response rates in unselected patient populations and induction of de novoautoimmune disease that excludes many patients from therapy. We hypothesize that small molecule checkpoint inhibitors can provide greater specificity, shortened half-lives to diminish autoimmune or other adverse events, increased oral bioavailability, enhanced bio-efficacy, and higher stability at ambient temperature facilitating purification during production. Here, we identified novel, small molecule PI3K inhibitors that reduce PD-L1/PD-L2 levels on MM cells and enhance the antimyeloma activity of autologous T cells. Methods: To determine the effect of proteasome inhibitors on PD-L1/PD-L2 surface expression, RPMI8226 cells were incubated with bortezomib (BTZ), carfilzomib (CFZ) or ixazomib (IXZ) (1nM) for 36 h. Cells were then incubated in PBS/10% normal goat serum followed by antibodies (1/100) for 30 min at 4ºC and then treated with Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2. RPMI8226 cells were also incubated with the small molecule PI3K inhibitor DT97 (500nM) alone or combined with BTZ, CFZ, or IXZ. Cells were similarly stained using Alexa-647-conjugated anti-PD-L1 or FITC-conjugated anti-PD-L2 and a BD LSRFortessa™ cell analyzer was used for multicolor flow cytometry to acquire >10,000 events/sample. To determine the effect of DT97 on T cell-mediated cytolysis, bone marrow biopsy was performed on an MM patient and CD138+ cells were isolated by Miltenyi positive selection and CD4+ T cells by MojoSort™ CD4+ Human T Cell isolation. CD138+ cells were incubated with CD4+ T cells, proteasome inhibitors, or DT97 as indicated for 16 h at 37ºC. CD138+cells were then affinity-isolated, incubated with a FITC-conjugated anti-Annexin-V antibody and quantitated using a BD LSRFortessa™ cell analyzer. Results: Treatment of MM cells with BTZ, CFZ, or IXZ significantly increased the surface expression of PD-L1 and PD-L2 (Fig. 1). In contrast, treatment with the PI3K inhibitor DT97 suppressed the expression of PD-L1 and PD-L2 on MM cells (Fig. 2). Importantly, DT97 co-treatment with either BTZ, CFZ, or IXZ suppressed the induction of PD-L1 and PD-L2 seen after treatment with proteasome inhibitors alone. Treatment with the proteasome inhibitors alone or combined with T cells did not promote significant killing of MM cells (Fig. 3). However, DT97 treatment significantly enhanced autologous T cell-mediated MM death and the effect was further enhanced by addition of proteasome inhibitors. Conclusions: The results presented here establish the proof-of-principle that small molecule PI3K inhibitors reduce PD-L1/PD-L2 levels on tumor cells and enhance the anti-myeloma effect of autologous T cells. Small molecules checkpoint inhibitors represent a safe therapeutic alternative that can avoid the problems associated with antibodies, while retaining their functionality. Taken together, our studies indicate the promise of small molecules checkpoint inhibitors and support further translational and clinical development as a transformative form of cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.