Human Antibodies - Volume Pre-press, issue Pre-press

Authors: Zandi, M. | Fallah Mehrabadi, J. | Mahdavi, M. | Irani, S.

Article Type: Research Article

Abstract: Uropathogenic E. coli is one of the major agents of urinary tract infection. Today, no effective treatment or vaccine against this infection is exist. Accordingly, in the present study, a genetic constrruct for inducing of cellular immune system was designed. At first, fimH gene from E. coli 35218 was amplified using PCR. PCR product inserted into pET23a expression vector and the recombinant vector was analysed by sequencing. The vector was transformed to E. coli strain Origami and the protein was expressed under the 1 mM IPTG. FimH was purified with Ni-NTA column and the purified protein was …used for immunization of BALB/c. Two weeks after the last injection, lymphocyte proliferation assay was carried out. In addition, IL-4 and IFN-γ cytokines, total antibody serum, IgG1 and IgG2a isotypes were quantified. Finally, protection ability of the vaccine in bladder and kidney infection of mice was evaluated. The results indicated that cellular immune response has a main protective role against UTI and FimH, as a vaccine candidate, significantly increase lymphocyte proliferation, IFN-γ response and total antibody amount. Immunization of mice with FimH conferred effective protection of kidney and bladder against urinary tract infection by uropathogenic E. coli (P < 0.002). It can be concluded that, the current FimH will be valuable for more trying to prepare a new vaccine against UTI. Show more

Keywords: Urinary tract infection, uropathogenic Escherichia coli, fimh, mouse model

DOI: 10.3233/HAB-200404

Citation: Human Antibodies, vol. Pre-press, no. Pre-press, pp. 1-10, 2020

Authors: Behring, Stefanie | Hänsch, Robert | Helmsing, Saskia | Schirrmann, Thomas | Schubert, Maren

Article Type: Research Article

Abstract: Intrabodies are antibodies that are not secreted but bind to their antigens inside the cell producing them. Intrabodies targeting antigens in the endoplasmatic reticulum were successfully used in vitro and in vivo . However, many target antigens interesting for research or therapy are located in the reducing environment of the cytosol, where correct folding and formation of disulfide bonds cannot be ensured. The majority of different scFv fragments, when expressed in the cytosol of the cell, do not fold correctly, are not stable or cannot bind their antigen. Such scFv antibodies are therefore not suited as intrabodies. In …this study, we evaluated fast and simple screening methods to identify scFv fragments that are stable and functional in the cytosol. We analyzed various phage display derived human scFv antibodies recognizing extracellular signal-regulated kinase 2 (Erk2) for stability and antigen binding under reducing and non-reducing conditions. Further, we developed an assay allowing to measure the interaction of the scFv intrabodies with their antigen in the cytosol of in living cells, by using a Split-Luciferase (Split-Luc) assay. ScFv fragments showing antigen binding in the cytosol could successfully be identified. Show more

Keywords: Immunoglobulin, scFv fragment, intrabody, phage display, Erk2, Luciferase

DOI: 10.3233/HAB-200402

Citation: Human Antibodies, vol. Pre-press, no. Pre-press, pp. 1-9, 2020

Authors: Sadeghi, Zahra | Maleki, Parichehr | Shahabi, Farzaneh | Bondarkhilli, Seyed Abolghasem Mohammadi | Masoumi, Maryam | Taheri, Mohammad | Mohammadi, Mehdi | Raheb, Jamshid

Article Type: Research Article

Abstract: Non-viral carriers based on nanoparticles are promising vectors for drug and gene delivery into the target cells. The data provided in this article are related to research entitled “Efficiency of surface modified SPION”. In this article the surface of superparamagnetic iron oxide (SPION) (core) coated with poly (ethylene glycol)-grafted polyethylenimine (mPEG-co-PEI) shell. PEI was used to increase gene transfection efficiency and poly (ethylene glycol) methyl ether was applied to reduce cytotoxicity of nanoparticles, because our goal is that two sets of mPEG-co-PEI coated SPIONs (mPEG-750 and 2000) were prepared as carrier for the purpose of gene delivery. Structure of the …mPEG-co-PEI product was elucidate by using 1H-NMR spectroscopy. Physicochemical features of the modified-SPIONs were evaluated by zeta-potential analysis. Cytotoxic effect of Nano carries were then assayed by MTT in NT2 cell line. Data analyzed by excel and p < 0.05 was considered significant. Finally siRNA absorption Ability of mPEG750-PEI-SPION and mPEG2000-PEI-SPION was tested by N/P ratio test (gel retardation assay). Our data shown that mPEG750-G-Pei-Spion and mPEG2000-G-Pei-Spion were non-toxic up to 100 μ g/ml in vitro for NT2 cell line. Show more

Keywords: Superparamagnetic iron oxide (SPION), NT2 cell line, mPEG2000-PEI-SPION, mPEG750-PEI-SPION, MTT assay

DOI: 10.3233/HAB-200403

Citation: Human Antibodies, vol. Pre-press, no. Pre-press, pp. 1-9, 2020

Authors: Astaneh, Maliheh | Ghafouri-Fard, Soudeh | Fazeli, Zahra | Taherian-Esfahani, Zahra | Dashti, Sepideh | Motevaseli, Elahe

Article Type: Research Article

Abstract: BACKGROUND: Recent studies have highlighted the role of natural elements in reduction of cancer cell growth and apoptosis. Koenimbine, a natural product isolated from Murraya koenigii (L) Spreng is a substance with cytotoxic effects on cancer cells. AIM: The effects of koenimbine on HT-29 and SW48 colon cancer cells were evaluated by MTT and Annexin V assays. Expression levels of Wnt/β -catenin pathway genes were quantified by real time PCR. RESULTS: The IC50 values of koenimbine in HT-29 and SW48 was calculated to be 50 μ g/ml based on the …results of MTT assay. This value was 75 μ g/ml in IEC-18 cells which were used as normal control. Annexin V assays revealed induction of cell apoptosis and necrosis in HT-29 and SW48 cells but not IEG18 cells by koenimbine. Koenimbin treatment resulted in significant down-regulation of CYCLD1 expression in SW48 cell line, but up-regulation of this gene in HT29 cell line. Expression of TBLR1, DKK1, GSK3B and β -catenin was significantly decreased after koenimbin treatment in HT-19 cell line. Moreover, expression of DKK1 and GSK3B was significantly decreased after koenimbin treatment in SW-40 cell line. TCF4 expression was not detected in any of cell lines either before or after treatment with koenimbin. CONCLUSION: The current in vitro study showed the cytotoxic effects of koenimbin on two colon cancer cell lines and the effects of this substance on expression of selected genes from Wnt-β catenin pathway. Future in vivo studies are needed before suggestion of this substance as an anti-cancer drug. Show more

Keywords: Koenimbine, colon cancer, Wnt-β catenin

DOI: 10.3233/HAB-200405

Citation: Human Antibodies, vol. Pre-press, no. Pre-press, pp. 1-6, 2020

Authors: Fadiloglu, Erdem | Unal, Canan | Tanacan, Atakan | Cagan, Murat | Beksac, Mehmet Sinan

Article Type: Research Article

Abstract: OBJECTIVE: To demonstrate the effect of preconceptional complement levels on perinatal outcomes of pregnancies with autoimmune disorders. METHODS: Pregnant women with autoimmune disorders (autoimmune disease and/or autoimmune antibody positivity) who were screened for complement levels (C3 and C4) prior to their pregnancies were enrolled in a special antenatal care program. These patients were administered low-dose low-molecular-weight heparin (enoxaparine, 1 × 2000 Anti-XA IU/0.2 mL/day), low-dose salysilic acid (100 mg/day) and low-dose corticosteroid (methylprednisolone, 1 × 4 mg/day orally) as soon as their pregnancies were confirmed according to the institutional protocol. We have …compared hypo- and normocomplement pregnancies with autoimmune disorders in terms of their obstetric and perinatal outcomes. We have also used Beksac Obstetric Index (BOI) which is “[living child + (π /10)]/gravidity” for the comparison of their previous obstetric histories. RESULTS: Obstetric and neonatal outcomes showed no significant difference between hypocomplement patients (n = 38) and control group (n = 157) (p > 0.05). “Composite obstetric and perinatal adverse outcome” rates were 26.2% and 27.3% in study and control groups, respectively (p > 0.05). BOI was significantly lower in hypocomplement patients (p : 002). Then, we have classified hypocomplement patients into 3 subgroups according to the type of complement (C3, C4 or both). Comparison inbetween these groups revealed no statistical significance in any of the analyzed parameters (p > 0.05). CONCLUSION: Low complement levels in pregnant women with autoimmune disorders may be associated with gestational problems and poor obstetric history. Immunomodulatory treatment modalities such as ours may be beneficial for improving the obstetric and neonatal outcomes. Show more

Keywords: Hypocomplementemia, complement activation, obstetric outcome, neonatal outcome, immunesuppression

DOI: 10.3233/HAB-200401

Citation: Human Antibodies, vol. Pre-press, no. Pre-press, pp. 1-6, 2020

Authors: Kolivand, Sedighe | Nazari, Mahboobeh | Modarressi, Mohammad Hossein | Najafabadi, Mohammad Reza Hosseini | Hemati, Atefeh | Ghafouri-Fard, Soudeh | Motevaseli, Elahe

Article Type: Research Article

Abstract: BACKGROUND: Inhibin A, a member of TGF-β superfamily, consists of α and β subunits. These subunits contain several cysteine residues in amino acid sequence that forms inter- and intra-subunits disulfide bonds. Due to the reducing environment of the bacterial cytoplasm, disulfide bonds formation in E.coli cytoplasm is not possible. Therefore, this can cause misfolding, aggregation and inclusion bodies formation during protein expression. As a result, the expression of inhibin subunits in E.coli produces inclusion bodies OBJECTIVE: We aimed at identification of an optimized protocol for …expression and recovery of inhibin α -subunit from inclusion bodies. METHOODS: Two vectors, four different E.coli strains, and six solubilization conditions for were used for the optimization of inhibin α -subunit production. Then, the solubilized proteins were purified through Ni-NTA affinity chromatography, characterized by SDS-PAGE and Western blotting (WB) using anti-his tag antibody, and refolded by dilution. RESULTS: The results showed that inhibin α -subunits were successfully expressed in both vectors and the pET22b+ inhibin α -subunit in Shuffle TM T7 strain had the highest expression; however, most of the expression was in an insoluble form. Among solubilization buffers examined, a buffer containing 2M urea with pH 12 was the best buffer to dissolve the insoluble protein. The high purity of protein was confirmed by SDS-PAGE and WB. Non-reducing SDS-PAGE demonstrating inhibin α -subunit refolded well. Show more

DOI: 10.3233/HAB-190399

Citation: Human Antibodies, vol. Pre-press, no. Pre-press, pp. 1-9, 2019

Authors: Fallah, Hamid | Akbari, Mohammad Taghi | Mirzajani, Sara | Ranjbaran, Fatemeh | Mehdizadeh, Bijan | Sayad, Arezou | Taheri, Mohammad

Article Type: Research Article

Abstract: Celiac disease (CD) is a common autoimmune disease that is manifested by inflammation of the small intestine and varying extra intestinal symptoms, also considered to be associated with human HLA-DQ genes. In this study, 40 patients of CD and 40 healthy control samples were genotyped for HLA-DQB1 and 14 patients of CD and 14 healthy control samples were genotyped for HLA-DQA1genes using the SSP-PCR technique and a commercial kit. The DQA1*05 allele had the highest frequency among the patient group (42.86%). The frequency of this allele was 28.57% in healthy controls, and there was no statistically significant difference in …this case (p = 0.771). The DQB1*02 allele was the most common in patients (33.75%) followed by the DQB1*03 allele (31.25%). The difference in frequency of the HLA-DQB1*02 allele in the patient and control groups was statistically significant (P = 0.0002, OR = 4.72). The remarkable differences in the distribution of HLA-DQ2 in Iranian patients compared to controls and relative risks signified the role of these alleles in the development of CD in Iranian patients and confirmed the likelihood of using HLA-DQ typing in the substantiation of the disease. Show more

Keywords: Celiac disease, HLA, immune response

DOI: 10.3233/HAB-190398

Citation: Human Antibodies, vol. Pre-press, no. Pre-press, pp. 1-7, 2019

Authors: Barabas, Arpad Z. | Cole, Chad D. | Kovacs, Zoltan B. | Kovacs, Erno I. | Lafreniere, Rene | Weir, Donald Mackay

Article Type: Research Article

Abstract: In spite of enormous efforts there have been no solutions to date for preventing/terminating certain acute and chronic disorders of humans by vaccination or drugs. Yet it is well understood that if the target antigen (ag) could be presented appropriately to the cells of the immune system then solutions could be found. Recently, the Barabas research group has introduced and described the third vaccination method – called modified vaccination technique (MVT) – which has the ability to provide a corrective immune response in experimental animals with an autoimmune kidney disease. Injections of immune complexes – made up of the target …ag and specific non-pathogenic IgM antibodies directed against the target ag – achieved downregulation of pathogenic immune responses and tolerance to self was regained. Utilizing the immune system’s natural abilities to respond to corrective information, the MVT technique was able to prevent an autoimmune kidney disease from occurring (prophylactic effect) in experimental animals, and when present, terminating it (therapeutic effect) specifically and without measurable side effects. It is predicted that the application of the MVT will have the potential in the future to revolutionize the preventative and therapeutic options for dealing with chronic disorders in humans (such as autoimmune disease, cancer and acute chronic infections) and achieve cures. Show more

Keywords: Autoimmune disease, modified vaccination technique, slowly progressive Heymann nephritis, membranous glomerulonephritis, autoantibody

DOI: 10.3233/HAB-190396

Citation: Human Antibodies, vol. Pre-press, no. Pre-press, pp. 1-11, 2019

Authors: Storz, Ulrich

Article Type: Research Article

Abstract: Epitope-based antibody claims have emerged to become one of the most important claim species in antibody patents. At the same time, they are heavily disputed. This article strives to give an overview about the as is situation both in Europe and the United States.

Keywords: Patent, antibody, epitope, target, competition

DOI: 10.3233/HAB-190395

Citation: Human Antibodies, vol. Pre-press, no. Pre-press, pp. 1-22, 2019