The pattern of expression of CD147/neurothelin during human T-cell ontogeny as defined by the monoclonal antibody 8D6

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66424/1/j.1399-0039.1997.tb02853.x.pd

CD99 tumor associated antigen is a potential target for antibody therapy of T-cell acute lymphoblastic leukemia

Monoclonal antibodies (mAbs) are an effective drug for targeted immunotherapy in several cancer types. However, so far, no antibody has been successfully developed for certain types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). T-ALL is an aggressive hematologic malignancy. T-ALL patients who are treated with chemotherapeutic drugs frequently relapse and become drug resistant. Therefore, antibody-based therapy is promising for T-ALL treatment. To successfully develop an antibody-based therapy for T-ALL, antibodies that induce death in malignant T cells but not in nonmalignant T cells are required to avoid the induction of secondary T-cell immunodeficiency. In this review, CD99 tumor associated antigen, which is highly expressed on malignant T cells and lowly expressed on nonmalignant T cells, is proposed to be a potential target for antibody therapy of T-ALL. Since certain clones of anti-CD99 mAbs induce apoptosis only in malignant T cells, these anti-CD99 mAbs might be a promising antibody drug for the treatment of T-ALL with high efficiency and low adverse effects. Moreover, over the past 25 years, many clones of anti-CD99 mAbs have been studied for their direct effects on T-ALL. These outcomes are gathered here

Generation of functional scFv intrabody to abate the expression of CD147 surface molecule of 293A cells

<p>Abstract</p> <p>Background</p> <p>Expression of intracellular antibodies (intrabodies) has become a broadly applicable technology for generation of phenotypic knockouts <it>in vivo</it>. The method uses surface depletion of cellular membrane proteins to examine their biological function. In this study, we used this strategy to block the transport of cell surface molecule CD147 to the cell membrane. Phage display technology was introduced to generate the functional antibody fragment to CD147, and we subsequently constructed a CD147-specific scFv that was expressed intracellularly and retained in the endoplasmic reticulum by adenoviral gene transfer.</p> <p>Results</p> <p>The recombinant antibody fragments, Fab and scFv, of the murine monoclonal antibody (clone M6-1B9) reacted specifically to CD147 by indirect enzyme-linked immunosorbent assays (ELISA) using a recombinant CD147-BCCP as a target. This indicated that the Fab- and scFv-M6-1B9 displaying on phage surfaces were correctly folded and functionally active. We subsequently constructed a CD147-specific scFv, scFv-M6-1B9-intrabody, in 293A cells. The expression of CD147 on 293A cell surface was monitored at 36 h after transduction by flow cytometry and demonstrated remarkable reduction. Colocalization of scFv-M6-1B9 intrabody with CD147 in the ER network was depicted using a 3D deconvolution microscopy system.</p> <p>Conclusion</p> <p>The results suggest that our approach can generate antibody fragments suitable for decreasing the expression of CD147 on 293A cells. This study represents a step toward understanding the role of the cell surface protein, CD147.</p

Simplified approaches for the development of an ELISA to detect circulating autoantibodies to p53 in cancer patients

<p>Abstract</p> <p>Background</p> <p>The recognition that human tumors stimulate the production of autoantibodies has initiated the use of this immune response as serological markers for the early diagnosis and management of cancer. The enzyme-linked immunosorbent assay (ELISA) is the most common method used in detecting autoantibodies, which involves coating the microtiter plate with the tumor associated antigen (TAA) of interest and allowing serum antibodies to bind. The patient's sample is directly in contact with the coating antigen so the protein used for coating must be pure to avoid non-specific binding. In this study, a simplified method to selectively and specifically immobilize TAAs onto microtiter plates in order to detect circulating autoantibodies in cancer patients without prior purification process was described. Wild type full-length p53 protein was produced in fusion with biotin carboxyl carrier peptide (BCCP) or hexahistidine [(His)6] using pAK400 and pET15b(+) vectors, respectively. The recombinant p53 fusion protein produced was then subjected to react with either a commercial p53 monoclonal antibody (mAb) or sera from lung cancer patients and healthy volunteers in an enzyme-linked immunosorbent assay (ELISA) format.</p> <p>Results</p> <p>Both of the immobilized p53 fusion proteins as well as the purified (His)6-p53 fusion protein had a similar dose response of detection to a commercial p53 mAb (DO7). When the biotinylated p53-BCCP fusion protein was used as an antigen to detect p53 autoantibodies in clinical samples, the result showed that human serum reacted strongly to avidin-coated microwell even in the absence of the biotinylated p53-BCCP fusion protein, thus compromised its ability to differentiate weakly positive sera from those that were negative. In contrast, the (His)6-p53 protein immobilized directly onto Ni+ coated microplate was able to identify the p53 autoantibody positive serum. In addition, its reactivity to clinical serum samples highly correlated with those obtained from using purified p53 as an antigen (R = 0.9803, p < 0.0001). Moreover, this directly immobilized p53 antigen can clearly differentiate p53 autoantibody positive sera in cancer patients from healthy volunteers' sera.</p> <p>Conclusion</p> <p>A method of coating directly and specifically TAAs onto a microtiter plate without the purification processes was developed in this study. Although in this study only one tumor antigen was examined, the simplicity and the ability of coated antigens to identify p53 specific autoantibodies in serum accurately might enable a larger panel of TAAs specific autoantibodies to be explored as serological markers for cancer.</p

Production of High-Value Proteins under Stringent Cost Constraints—The Case of Hollow Fiber Technology for Cell Culture

For decades, the benefits of utilizing hollow fiber bioreactors for continuous cell culture to produce monoclonal antibodies have been widely recognized. However, the suitability of this technology for laboratories or centers with limited resources and expertise seeking to expand their production capacity is uncertain, mainly due to unknown cost-effectiveness. In this study, a hollow fiber bioreactor with a 4.7-mL culture volume was used to culture a hybridoma clone producing immunoglobulin G antibody specific to hemoglobin F (HbF). The antibody reached a maximum concentration of 1.22 g/L and totaled 21 mg over a 44-day culture period. This preliminary production data was used to estimate the cost of consumables required for using the hollow fiber bioreactor to produce 130 mg of monoclonal antibodies, which was found to be THB35.8k (£880). The cost was slightly more expensive than batch cultivation in typical culture dishes, which ranged from THB27.8 to 30.2k (£680 to 740). Despite the advantages in terms of reduced hands-on time, shorter production duration, and highly concentrated products, the primary challenges associated with using hollow fiber bioreactors were the cost and availability of the cartridges

Secreted NS1 of Dengue Virus Attaches to the Surface of Cells via Interactions with Heparan Sulfate and Chondroitin Sulfate E

Dengue virus (DENV) nonstructural protein-1 (NS1) is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection

Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy

Aim: The present study aims to generate chimeric mouse single-chain variable fragment (scFv) and immunoglobulin G1 (IgG1) crystallizable fragment (Fc) antibody against disialoganglioside (GD2) for the treatment of neuroblastoma (NB). The generated scFv-IgG Fc antibody, lacking first constant domain of heavy chain (CH1), is of a smaller size than the natural antibody and has anti-tumor activity. Methods: Vector for scFv-IgG Fc antibody was constructed and scFv-IgG Fc antibody was expressed in human embryonic kidney 293T (HEK293T) cell line. Purification of scFv-IgG Fc antibody from the culture supernatant of transfected HEK293T cells was performed by Protein G affinity chromatography. The structure and binding activity of scFv-IgG Fc antibody were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting (WB), and immunofluorescence techniques. Anti-tumor activities by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were determined. Results: Using plasmid fusion-human IgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2), a plasmid vector encoding chimeric mouse scFv and hIgG1 Fc antibody against GD2 was successfully constructed. This vector was transfected into human HEK293T cells to produce scFv-IgG Fc antibody. The transfected HEK293T cells could produce chimeric scFv-IgG Fc antibody against GD2, which lacks the IgG heavy chain CH1 domain but carries CH2 and CH3 domains. The chimeric antibodies could be purified from the culture supernatant of the transfected HEK293T culture in the presence of zeocin drug. The produced GD2 scFv-IgG Fc antibodies, which are smaller in size than the intact antibody, could trigger the killing of GD2 expressed NB cell line SH-SY5Y by ADCC and ADCP mechanisms. Conclusions: The results indicate that chimeric scFv-hIgG Fc antibody, lacking heavy chain CH1 domain, could mediate antibody induced anti-tumor activities. The small size of this type of chimeric antibody may be employed as anti-GD2 antibody for NB therapy

Human γδ T cells recognize CD1b by two distinct mechanisms

γδ T cells form an abundant part of the human cellular immune system, where they respond to tissue damage, infection, and cancer. The spectrum of known molecular targets recognized by Vδ1-expressing γδ T cells is becoming increasingly diverse. Here we describe human γδ T cells that recognize CD1b, a lipid antigen-presenting molecule, which is inducibly expressed on monocytes and dendritic cells. Using CD1b tetramers to study multiple donors, we found that many CD1b-specific γδ T cells use Vδ1. Despite their common use of Vδ1, three CD1b-specific γδ T cell receptors (TCRs) showed clear differences in the surface of CD1b recognized, the requirement for lipid antigens, and corecognition of butryophilin-like proteins. Several Vγ segments were present among the CD1b-specific TCRs, but chain swap experiments demonstrated that CD1b specificity was mediated by the Vδ1 chain. One of the CD1b-specific Vδ1+ TCRs paired with Vγ4 and shows dual reactivity to CD1b and butyrophilin-like proteins. αβ TCRs typically recognize the peptide display platform of MHC proteins. In contrast, our results demonstrate the use of rearranged receptors to mediate diverse modes of recognition across the surface of CD1b in ways that do and do not require carried lipids

The Pupils\u27 Opinion of the Examination and Grading Process in the Classroom

U članku se oslikava kompleksnost nastavnog provjeravanja i ocjenjivanja učenika kroz eksplikaciju značenja i zadataka ove etape nastavnog procesa te isticanje poteškoća koje se pojavljuju prilikom njene realizacije. Naznačeni su i zakonski okviri provedbe provjeravanja i ocjenjivanja učenika u osnovnim i srednjim školama.Praksa nastavnog provjeravanja i ocjenjivanja prikazana je kroz analizu i interpretaciju rezultata ispitivanja mišljenja skupine osnovnoškolskih i srednjoškolskih učenika (N=147) o navedenoj problematici. Utvrđeni su najčešći i preferirani oblici te mišljenja učenika o ne/korektnosti i transparentnosti provjeravanja i ocjenjivanja znanja i sposobnosti učenika u nastavnoj praksii prilikama za samoevaluaciju. Prikupljeni podaci upućuju na to da se u provođenju navedene etape nastavnog procesa ostvaruje nedovoljna raznovrsnost u oblicima njene realizacije, da učenicima nisu uvijek pružene adekvatne povratne informacije o rezultatima njihova rada te da je provjeravanje i ocjenjivanje dominantno nastavnikova aktivnost prilikom koje učenici najčešće nisu u prilici da samostalno ocijene svoj napredak u učenju i ostvarivanju zadataka nastave. Iz navedenog logično proizlazi često prisutan dojam učenika da su nekorektno ocijenjeni. Nastavno bi provjeravanje i ocjenjivanje trebalo uvažavati kompleksnost učenikove osobnosti i njen originalni doprinos u nastavi. Dijelom je to moguće ostvariti uporabom različitih oblika realizacije, pružanjem jasnih informacija o rezultatima učenja te većim stupnjem uključenosti učenika u ocjenjivanje svojeg napretka u učenju.The article portrays the complexity of classroom examination and grading of pupils via theexplication of the meaning and tasks of this stage of the teaching process and by stressingthe difficulties that arise during its realization. The legal framework for the conducting of theexamination and grading of pupils in elementary and high schools is also outlined.The practice of classroom examination and grading is shown through the analysis andinterpretation of the results of a survey of a group of elementary and highschool pupils (N=147)about the said process. The most common and the preferred types and the pupils\u27 opinion of thein/correctness and transparency of the testing and grading of pupils\u27 knowledge and abilities inclassroom practice were determined, as well as the opportunities for self-evaluation. The gathereddata indicate a lack of variety in the ways of conducting this stage of the teaching process, thepupils aren\u27t always given adequate feedback information about the results of their work andthe examination is predominantly a teachers activity during which the pupils most often aren\u27tgiven a chance to independently evaluate their progress in learning and carrying out classroomassignments. The above mentioned logically leads to the impression, commonly present amongpupils, that they were incorrectly graded.Classroom examination and grading should take into consideration the complexity of the pupil\u27spersonality and her original contribution in class. It is partially possible to achieve that by usingdifferent methods of realization, by giving clear information about the results of their studying,and by a greater degree of inclusion of the pupils in evaluating the progress of their studying.</p

Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses

<p>Abstract</p> <p>Background</p> <p>CD99, a leukocyte surface glycoprotein, is broadly expressed in many cell types. On the cell surface, CD99 is expressed as two distinct isoforms, a long form and a short form. CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T cell activation. However, the molecular mechanisms by which CD99 participates in such processes are unclear. As CD99 contains a short cytoplasmic tail, it is unlikely that CD99 itself takes part in its multi-functions. Association of CD99 with other membrane proteins has been suggested to be necessary for exerting its functions.</p> <p>Results</p> <p>In this study, we analyzed the association of CD99 with other cell surface molecules involved in T cell activation. We demonstrate the association of MHC class I, MHC class II and tetraspanin CD81 with CD99 molecules on the cell surface. Association of CD99 with its partners was observed for both isoforms. In addition, we determined that CD99 is a lipid raft-associated membrane protein and is recruited into the immunologic synapse during T cell activation. The implication of CD99 on T cell activation was investigated. Inhibition of anti-CD3 induced T cell proliferation by an anti-CD99 monoclonal antibody was observed.</p> <p>Conclusions</p> <p>We provide evidence that CD99 directly interact and form the complex with the MHC class I and II, and tetraspanin CD81, and is functionally linked to the formation of the immunologic synapse. Upon T cell activation, CD99 engagement can inhibit T cell proliferation. We speculate that the CD99-MHC-CD81 complex is a tetraspanin web that plays an important role in T cell activation.</p