Tumor and organ uptake of (64)Cu-labeled MORAb-009 (amatuximab), an anti-mesothelin antibody, by PET imaging and biodistribution studies.

PubMed

Lee, Jae-Ho; Kim, Heejung; Yao, Zhengsheng; Lee, Sung-Jin; Szajek, Lawrence P; Grasso, Luigi; Pastan, Ira; Paik, Chang H

2015-11-01

To investigate the effect of the injection dose of MORAb-009 (amatuximab, an anti-mesothelin monoclonal antibody), the tumor size and the level of shed mesothelin on the uptake of the antibody in mesothelin-positive tumor and organs by biodistribution (BD) and positron emission tomography (PET) imaging studies. 2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was conjugated to amatuximab and labeled with (64)CuCl2 in 0.25 M acetate buffer, pH4.2. The resulting (64)Cu-NOTA-amatuximab was purified with a PD 10 column. To investigate the dose effect or the effect of tumor size, the BD was performed in groups of nude mice (n=5) with mesothelin-expressing A431/H9 tumors (range, 80-300 mm(3)) one day after iv injection of (64)Cu-NOTA-amatuximab (10 μCi) containing a total amatuximab dose of 2, 30, or 60 μg. The BD and PET imaging were also investigated 3, 24 and 48 h after injecting a total dose of 30 μg (10 μCi for BD), and 2 or 60 μg (300 μCi for PET), respectively. Comparing the results of the BDs from three different injection doses, the major difference was shown in the uptake (%ID/g) of the radiolabel in tumor, liver and blood. The tumor uptake and blood retention from 30 and 60 μg doses were greater than those from 2 μg dose, whereas the liver uptake was smaller. The BD studies also demonstrated a positive correlation between tumor size (or the level of shed mesothelin in blood) and liver uptake. However, there was a negative correlation between tumor size (or the shed mesothelin level) and tumor uptake and between tumor size and blood retention. These findings were confirmed by the PET imaging study, which clearly visualized the tumor uptake with the radiolabel concentrated in the tumor core and produced a tumor to liver ratio of 1.2 at 24h post-injection with 60 μg amatuximab, whereas the injection of 2 μg amatuximab produced a tumor to liver ratio of 0.4 at 24h post-injection. Our studies using a nude mouse

Two Saporin-Containing Immunotoxins Specific for CD20 and CD22 Show Different Behavior in Killing Lymphoma Cells

PubMed Central

Polito, Letizia; Mercatelli, Daniele; Bortolotti, Massimo; Maiello, Stefania; Djemil, Alice; Battelli, Maria Giulia; Bolognesi, Andrea

2017-01-01

Immunotoxins (ITs) are hybrid proteins combining the binding specificity of antibodies with the cytocidal properties of toxins. They represent a promising approach to lymphoma therapy. The cytotoxicity of two immunotoxins obtained by chemical conjugation of the plant toxin saporin-S6 with the anti-CD20 chimeric antibody rituximab and the anti-CD22 murine antibody OM124 were evaluated on the CD20-/CD22-positive cell line Raji. Both ITs showed strong cytotoxicity for Raji cells, but the anti-CD22 IT was two logs more efficient in killing, probably because of its faster internalization. The anti-CD22 IT gave slower but greater caspase activation than the anti-CD20 IT. The cytotoxic effect of both immunotoxins can be partially prevented by either the pan-caspase inhibitor Z-VAD or the necroptosis inhibitor necrostatin-1. Oxidative stress seems to be involved in the cell killing activity of anti-CD20 IT, as demonstrated by the protective role of the H2O2 scavenger catalase, but not in that of anti-CD22 IT. Moreover, the IT toxicity can be augmented by the contemporary administration of other chemotherapeutic drugs, such as PS-341, MG-132, and fludarabine. These results contribute to the understanding of the immunotoxin mechanism of action that is required for their clinical use, either alone or in combination with other drugs. PMID:28556822

Two Saporin-Containing Immunotoxins Specific for CD20 and CD22 Show Different Behavior in Killing Lymphoma Cells.

PubMed

Polito, Letizia; Mercatelli, Daniele; Bortolotti, Massimo; Maiello, Stefania; Djemil, Alice; Battelli, Maria Giulia; Bolognesi, Andrea

2017-05-30

Immunotoxins (ITs) are hybrid proteins combining the binding specificity of antibodies with the cytocidal properties of toxins. They represent a promising approach to lymphoma therapy. The cytotoxicity of two immunotoxins obtained by chemical conjugation of the plant toxin saporin-S6 with the anti-CD20 chimeric antibody rituximab and the anti-CD22 murine antibody OM124 were evaluated on the CD20-/CD22-positive cell line Raji. Both ITs showed strong cytotoxicity for Raji cells, but the anti-CD22 IT was two logs more efficient in killing, probably because of its faster internalization. The anti-CD22 IT gave slower but greater caspase activation than the anti-CD20 IT. The cytotoxic effect of both immunotoxins can be partially prevented by either the pan-caspase inhibitor Z-VAD or the necroptosis inhibitor necrostatin-1. Oxidative stress seems to be involved in the cell killing activity of anti-CD20 IT, as demonstrated by the protective role of the H₂O₂ scavenger catalase, but not in that of anti-CD22 IT. Moreover, the IT toxicity can be augmented by the contemporary administration of other chemotherapeutic drugs, such as PS-341, MG-132, and fludarabine. These results contribute to the understanding of the immunotoxin mechanism of action that is required for their clinical use, either alone or in combination with other drugs.

Differential cellular internalization of anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity.

PubMed

Du, Xing; Beers, Richard; Fitzgerald, David J; Pastan, Ira

2008-08-01

B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two immunotoxins, FMC63(Fv)-PE38-targeting CD19 and RFB4(Fv)-PE38 (BL22)-targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, approximately 7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for immunotoxins and probably for other immunoconjugates that act inside cells.

Antiviral Immunotoxin Against Bovine herpesvirus-1: Targeted Inhibition of Viral Replication and Apoptosis of Infected Cell

PubMed Central

Xu, Jian; Li, Xiaoyang; Jiang, Bo; Feng, Xiaoyu; Wu, Jing; Cai, Yunhong; Zhang, Xixi; Huang, Xiufen; Sealy, Joshua E.; Iqbal, Munir; Li, Yongqing

2018-01-01

Bovine herpesvirus 1 (BoHV-1) is a highly contagious viral pathogen which causes infectious bovine rhinotracheitis in cattle worldwide. Currently, there is no antiviral prophylactic treatment available capable of mitigating the disease impact and facilitating recovery from latent infection. In this study, we have engineered a novel recombinant anti-BoHV-1 immunotoxin construct termed “BoScFv-PE38” that consists of a single-chain monoclonal antibody fragment (scFv) fused with an active domain of Pseudomonas exotoxin A as a toxic effector (PE38). The recombinant BoScFv-PE38 immunotoxin expressed in a prokaryotic expression system has specific binding affinity for BoHV-1 glycoprotein D (gD) with a dissociation constant (Kd) of 12.81 nM and for BoHV-1 virus particles with a Kd value of 97.63 nM. We demonstrate that the recombinant BoScFv-PE38 is internalized into MDBK cell compartments that inhibit BoHV-1 replication with a half-maximal inhibitory concentration (IC50) of 4.95 ± 0.33 nM and a selective index (SI) of 456 ± 31. Furthermore, the BoScFv-PE38 exerted a cytotoxic effect through the induction of ATP and ammonia, leading to apoptosis of BoHV-1-infected cells and the inhibition of BoHV-1 replication in MDBK cells. Collectively, we show that BoScFv-PE38 can potentially be employed as a therapeutic agent for the treatment of BoHV-1 infection. PMID:29670605

Plasma Mesothelin as a Novel Diagnostic and Prognostic Biomarker in Colorectal Cancer

PubMed Central

Li, Shuwei; Xie, Lisheng; He, Lei; Fan, Zhimin; Xu, Junhua; Xu, Kaili; Zhu, Lingjun; Ma, Gaoxiang; Du, Mulong; Chu, Haiyan; Zhang, Zhengdong; Ni, Min; Wang, Meilin

2017-01-01

Objective Mesothelin is a cell surface protein and overexpressed in many cancers. However, the potential value of mesothelin as plasma biomarker in colorectal cancer has not been explored. The purpose of this study was to identify whether plasma mesothelin is a suitable diagnostic and prognostic biomarker for colorectal cancer. Methods We performed a two-stage case-control study to evaluate plasma mesothelin levels in colorectal cancer using enzyme-linked immunosorbent assay (ELISA). Preoperative and postoperative plasma were collected to examine the level changes influenced by surgery. Receiver operating characteristic (ROC) curves were applied to identify the diagnostic value of plasma mesothelin. We also conducted univariate Kaplan-Meier survival analysis and Cox regression analysis of patients with survival information. Results We found that the plasma mesothelin levels in colorectal cancer patients were significantly higher than that in the controls (P < 0.001) with an AUC value of 0.690 (95% CI = 0.625 to 0.752). Individuals with lower mesothelin level had a longer survival time (adjusted HR = 4.43, 95% CI = 1.93-10.15, P < 0.001). Furthermore, Patients had slightly decreased mesothelin levels in postoperative plasma than preoperative plasma, although the alteration was not statistically significant (P = 0.052). Conclusion Our findings highlight the correlative relationship between plasma mesothelin levels and the presence and progression of colorectal cancer. Plasma mesothelin may be a potential diagnostic and, or prognostic biomarker for colorectal cancer. PMID:28638449

Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding

PubMed Central

Sirois, Allison R.; Deny, Daniela A.; Baierl, Samantha R.; George, Katia S.

2018-01-01

Mesothelin is a cell surface protein that is overexpressed in numerous cancers, including breast, ovarian, lung, liver, and pancreatic tumors. Aberrant expression of mesothelin has been shown to promote tumor progression and metastasis through interaction with established tumor biomarker CA125. Therefore, molecules that specifically bind to mesothelin have potential therapeutic and diagnostic applications. However, no mesothelin-targeting molecules are currently approved for routine clinical use. While antibodies that target mesothelin are in development, some clinical applications may require a targeting molecule with an alternative protein fold. For example, non-antibody proteins are more suitable for molecular imaging and may facilitate diverse chemical conjugation strategies to create drug delivery complexes. In this work, we engineered variants of the fibronectin type III domain (Fn3) non-antibody protein scaffold to bind to mesothelin with high affinity, using directed evolution and yeast surface display. Lead engineered Fn3 variants were solubly produced and purified from bacterial culture at high yield. Upon specific binding to mesothelin on human cancer cell lines, the engineered Fn3 proteins internalized and co-localized to early endosomes. To our knowledge, this is the first report of non-antibody proteins engineered to bind mesothelin. The results validate that non-antibody proteins can be engineered to bind to tumor biomarker mesothelin, and encourage the continued development of engineered variants for applications such as targeted diagnostics and therapeutics. PMID:29738555

The current status of immunotoxins: an overview of experimental and clinical studies as presented at the Third International Symposium on Immunotoxins.

PubMed

Uckun, F M; Frankel, A

1993-02-01

The Third International Symposium on Immunotoxins was held on June 19-21, 1992 in Orlando, Florida. This symposium was sponsored by NATO, NIH, Pierce Chemical Company, Walt Disney Cancer Institute at Florida Hospital, Duke Comprehensive Cancer Center, Xoma, Immunogen, Seragen, Bristol-Myers Squibb, Chiron, Ortho Biotech, Upjohn, Merck Sharp & Dohme Research Laboratories, Abbot Laboratories, Lilly Research Laboratories, and Evans & Sutherland. The Pierce Immunotoxin Award which recognizes outstanding contributions to immunotoxin research and development, was presented to Drs David FitzGerald, Fatih Uckun, David Eisenberg, and Ira Wool, for their contributions to the immunotoxin field.

B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

PubMed Central

Vooijs, W. C.; Otten, H. G.; van Vliet, M.; van Dijk, A. J.; de Weger, R. A.; de Boer, M.; Bohlen, H.; Bolognesi, A.; Polito, L.; de Gast, G. C.

1997-01-01

In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkin's disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration. Images Figure 1 PMID:9365164

Serum Mesothelin for Diagnosing Malignant Pleural Mesothelioma: An Individual Patient Data Meta-Analysis

PubMed Central

Hollevoet, Kevin; Reitsma, Johannes B.; Creaney, Jenette; Grigoriu, Bogdan D.; Robinson, Bruce W.; Scherpereel, Arnaud; Cristaudo, Alfonso; Pass, Harvey I.; Nackaerts, Kristiaan; Rodríguez Portal, José A.; Schneider, Joachim; Muley, Thomas; Di Serio, Francesca; Baas, Paul; Tomasetti, Marco; Rai, Alex J.; van Meerbeeck, Jan P.

2012-01-01

Purpose Mesothelin is currently considered the best available serum biomarker of malignant pleural mesothelioma. To examine the diagnostic accuracy and use of serum mesothelin in early diagnosis, we performed an individual patient data (IPD) meta-analysis. Methods The literature search identified 16 diagnostic studies of serum mesothelin, measured with the Mesomark enzyme-linked immunosorbent assay. IPD of 4,491 individuals were collected, including several control groups and 1,026 patients with malignant pleural mesothelioma. Mesothelin levels were standardized for between-study differences and age, after which the diagnostic accuracy and the factors affecting it were examined with receiver operating characteristic (ROC) regression analysis. Results At a common diagnostic threshold of 2.00 nmol/L, the sensitivities and specificities of mesothelin in the different studies ranged widely from 19% to 68% and 88% to 100%, respectively. This heterogeneity can be explained by differences in study population, because type of control group, mesothelioma stage, and histologic subtype significantly affected the diagnostic accuracy. The use of mesothelin in early diagnosis was evaluated by differentiating 217 patients with stage I or II epithelioid and biphasic mesothelioma from 1,612 symptomatic or high-risk controls. The resulting area under the ROC curve was 0.77 (95% CI, 0.73 to 0.81). At 95% specificity, mesothelin displayed a sensitivity of 32% (95% CI, 26% to 40%). Conclusion In patients suspected of having mesothelioma, a positive blood test for mesothelin at a high-specificity threshold is a strong incentive to urge further diagnostic steps. However, the poor sensitivity of mesothelin clearly limits its added value to early diagnosis and emphasizes the need for further biomarker research. PMID:22412141

78 FR 21131 - Prospective Grant of An Exclusive Evaluation Option License: Pre-clinical Evaluation of Anti...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-09

... immunotoxins for the treatment of human ROR1 expressing cancers, wherein the immunotoxin comprises an anti-ROR1... human ROR1 expressing cancers. The immunotoxin will comprise a chimeric mouse anti-human receptor... Exclusive Evaluation Option License: Pre- clinical Evaluation of Anti-tyrosine Kinase-like Orphan Receptor 1...

Significance of serum mesothelin in an asbestos-exposed population in the Czech Republic.

PubMed

Jakubec, Petr; Pelclova, Daniela; Smolkova, Petra; Kolek, Vitezslav; Nakladalova, Marie

2015-09-01

Pleural mesothelioma is a highly aggressive and difficult-to-treat form of cancer induced by asbestos in 80-90% of cases. The population group most at risk of the condition are asbestos-exposed workers. Mesothelin or soluble mesothelin-related protein (SMRP) is studied as a potential marker of mesothelioma in the at-risk population. The study comprised 239 subjects with a mean duration of occupational exposure to asbestos of 19.9 years. In all of them, a complete medical history was taken, focused on exposure duration and a physical examination, a chest X-ray or other imaging investigations and a lung function test were performed. Their serum SMRP levels were measured and biopsy samples were taken to diagnose pleural disease. Based on the above examinations, the subjects were classified into subgroups and serum SMRP concentrations were statistically analyzed with respect to individual parameters. In asbestos-exposed individuals, mesothelin levels were significantly higher in those with pathological X-ray findings than in those with normal X-ray results (0.78 ± 0.63 vs. 0.50 ± 0.35, P<0.0001). The group of patients with benign disease had statistically significantly higher mesothelin levels than those with normal X-ray findings (0.755 ± 0.543 vs. 0.50 ± 0.35, P<0.001). In the group with present malignant processes, mesothelin levels were higher than in individuals with benign disease (1.19 ± 0.89 vs. 0.76 ± 0.54, P=0.015). Only a weak correlation was found between mesothelin levels and asbestos exposure duration. There were relatively high sensitivity and high specificity (75% and 90.6%, respectively) of serum mesothelin for pleural mesothelioma. However, given the small number of mesothelioma cases in the group, the results cannot be considered as statistically significant. In persons followed up for asbestos exposure, increased mesothelin levels signalize pathological processes in the chest and correlate with severity of the disease. The study suggests that

Immunotoxins: magic bullets or misguided missiles?

PubMed

Vitetta, E S; Thorpe, P E; Uhr, J W

1993-05-01

Thirteen years have passed since specific in vitro and in vivo killing of tumour cells by immunotoxins was first described. Why, then, has it taken so long to determine whether these drugs will have a major impact on the treatment of cancer, AIDS and autoimmune disease? The answer is that the transfer of basic discoveries to the clinic is a slow, multistep, interdisciplinary process. Thus, immunotoxin molecules must be designed and redesigned by the basic scientist depending on the efficacy and toxicity shown in vitro and in relevant experimental models. Next, each version must be evaluated by clinicians in humans through a lengthy process (1-3 years) in which the dose regimen is optimized and in which new problems and issues frequently emerge. These problems must again be modelled and studied in animals before additional clinical trials are initiated. In this article, Ellen Vitetta and colleagues discuss both basic and clinical aspects of the development of immunotoxin therapy.

Safety and biodistribution of 111In-amatuximab in patients with mesothelin expressing cancers using Single Photon Emission Computed Tomography-Computed Tomography (SPECT-CT) imaging

PubMed Central

Adler, Stephen; Mena, Esther; Kurdziel, Karen; Maltzman, Julia; Wallin, Bruce; Hoffman, Kimberly; Pastan, Ira; Paik, Chang Hum; Choyke, Peter; Hassan, Raffit

2015-01-01

Amatuximab is a chimeric high-affinity monoclonal IgG1/k antibody targeting mesothelin that is being developed for treatment of mesothelin-expressing cancers. Considering the ongoing clinical development of amatuximab in these cancers, our objective was to characterize the biodistribution, and dosimetry of 111Indium (111In) radiolabelled amatuximab in mesothelin-expressing cancers. Between October 2011 and February 2013, six patients including four with malignant mesothelioma and two with pancreatic adenocarcinoma underwent Single Photon Emission Computed Tomography-Computed Tomography (SPECT/CT) imaging following administration of 111In amatuximab. SPECT/CT images were obtained at 2–4 hours, 24–48 hours and 96–168 hours after radiotracer injection. In all patients, tumor to background ratios (TBR) consistently met or exceeded an uptake of 1.2 (range 1.2–62.0) which is considered the minimum TBR that can be visualized. TBRs were higher in tumors of patients with mesothelioma than pancreatic adenocarcinoma. 111In-amatuximab uptake was noted in both primary tumors and metastatic sites. The radiotracer dose was generally well-tolerated and demonstrated physiologic uptake in the heart, liver, kidneys and spleen. This is the first study to show tumor localization of an anti-mesothelin antibody in humans. Our results show that 111In-amatuximab was well tolerated with a favorable dosimetry profile. It localizes to mesothelin expressing cancers with a higher uptake in mesothelioma than pancreatic cancer. PMID:25756664

Overexpression of periostin and distinct mesothelin forms predict malignant progression in a rat cholangiocarcinoma model

PubMed Central

Manzanares, Miguel Á.; Campbell, Deanna J.W.; Maldonado, Gabrielle T.

2017-01-01

Periostin and mesothelin have each been suggested to be predictors of poor survival for patients with intrahepatic cholangiocarcinoma, although the clinical prognostic value of both of these biomarkers remains uncertain. The aim of the current study was to investigate these biomarkers for their potential to act as tumor progression factors when assessed in orthotopic tumor and three‐dimensional culture models of rat cholangiocarcinoma progression. Using our orthotopic model, we demonstrated a strong positive correlation between tumor and serum periostin and mesothelin and increasing liver tumor mass and associated peritoneal metastases that also reflected differences in cholangiocarcinoma cell aggressiveness and malignant grade. Periostin immunostaining was most prominent in the desmoplastic stroma of larger sized more aggressive liver tumors and peritoneal metastases. In comparison, mesothelin was more highly expressed in the cholangiocarcinoma cells; the slower growing more highly differentiated liver tumors exhibited a luminal cancer cell surface immunostaining for this biomarker, and the rapidly growing less differentiated liver and metastatic tumor masses largely showed cytoplasmic mesothelin immunoreactivity. Two molecular weight forms of mesothelin were identified, one at ∼40 kDa and the other, a more heavily glycosylated form, at ∼50 kDa. Increased expression of the 40‐kDa mesothelin over that of the 50 kDa form predicted increased malignant progression in both the orthotopic liver tumors and in cholangiocarcinoma cells of different malignant potential in three‐dimensional culture. Moreover, coculturing of cancer‐associated myofibroblasts with cholangiocarcinoma cells promoted overexpression of the 40‐kDa mesothelin, which correlated with enhanced malignant progression in vitro. Conclusion: Periostin and mesothelin are useful predictors of tumor progression in our rat desmoplastic cholangiocarcinoma models. This supports their relevance to

Essential role for Bim in mediating the apoptotic and antitumor activities of immunotoxins.

PubMed

Antignani, A; Segal, D; Simon, N; Kreitman, R J; Huang, D; FitzGerald, D J

2017-08-31

Protein synthesis is crucial for regulating cell homeostasis and, when unrestricted, it can lead to tumorigenesis. Immunotoxins derived from Pseudomonas exotoxin are antibody-toxin fusion proteins that inhibit protein synthesis of mammalian cells via ADP-ribosylation of the eukaryotic elongation factor-2. Here we investigate the role of the Bcl-2 family proteins in the response of cancer cells to immunotoxin challenge. Besides the well-known reduction of the prosurvival Bcl-2 family member, Mcl-1, following inhibition of protein synthesis, we show for the first time that immunotoxins also reduce the levels of selected proapoptotic BH-3-only proteins. Among these, only Bim protein levels correlated with the ability of immunotoxins to induce an apoptotic response. To support our findings, we verified that a Bim knockout completely abolished immunotoxin-mediated apoptosis. Further, mice bearing either wild-type or Bid knockout tumors responded to immunotoxin treatment with a decrease in growth kinetics, whereas mice engrafted with Bim knockout tumors showed no reduction in tumor size or prolongation of survival following immunotoxin treatment. From these results, we conclude that Bim expression is a major susceptibility factor for tumor cell death and, as such, constitutes a potential biomarker that could be evaluated before immunotoxin treatment. In support of this hypothesis, clinically, we analyzed patient cells before immunotoxin treatment and report that samples of hairy cell leukemia with high levels of Bim protein responded with a greater decrease in leukemic cell count compared with those samples expressing a low level of Bim.

A Multifunctional Mesothelin Antibody-tagged Microparticle Targets Human Mesotheliomas

PubMed Central

Macura, Sherrill L.; Hillegass, Jedd M.; Steinbacher, Jeremy L.; MacPherson, Maximilian B.; Shukla, Arti; Beuschel, Stacie L.; Perkins, Timothy N.; Butnor, Kelly J.; Lathrop, Melissa J.; Sayan, Mutlay; Hekmatyar, Khan; Taatjes, Douglas J.; Kauppinen, Risto A.; Landry, Christopher C.

2012-01-01

Pleural and peritoneal mesotheliomas (MMs) are chemoresistant tumors with no effective therapeutic strategies. The authors first injected multifunctional, acid-prepared mesoporous spheres (APMS), microparticles functionalized with tetraethylene glycol oligomers, intraperitoneally into rodents. Biodistribution of APMS was observed in major organs, peritoneal lavage fluid (PLF), and urine of normal mice and rats. After verification of increased mesothelin in human mesotheliomas injected into severe combined immunodeficient (SCID) mice, APMS were then functionalized with an antibody to mesothelin (APMS-MB) or bovine serum albumin (BSA), a nonspecific protein control, and tumor targeting was evaluated by inductively coupled plasma mass spectrometry and multifluorescence confocal microscopy. Some APMS were initially cleared via the urine over a 24 hr period, and small amounts were observed in liver, spleen, and kidneys at 24 hr and 6 days. Targeting with APMS-MB increased APMS uptake in mesenteric tumors at 6 days. Approximately 10% to 12% of the initially injected amount was observed in both spheroid and mesenteric MM at this time point. The data suggest that localized delivery of APMS-MB into the peritoneal cavity after encapsulation of drugs, DNA, or macromolecules is a novel therapeutic approach for MM and other tumors (ovarian and pancreatic) that overexpress mesothelin. PMID:22723527

Expression of nestin, mesothelin and epithelial membrane antigen (EMA) in developing and adult human meninges and meningiomas.

PubMed

Petricevic, Josko; Forempoher, Gea; Ostojic, Ljerka; Mardesic-Brakus, Snjezana; Andjelinovic, Simun; Vukojevic, Katarina; Saraga-Babic, Mirna

2011-11-01

The spatial and temporal pattern of appearance of nestin, epithelial membrane antigen (EMA) and mesothelin proteins was immunohistochemically determined in the cells of normal developing and adult human meninges and meningiomas. Human meninges developed as two mesenchymal condensations in the head region. The simple squamous epithelium on the surface of leptomeninges developed during mesenchymal to epithelial transformation. Nestin appeared for the first time in week 7, EMA in week 8, while mesothelin appeared in week 22 of development. In the late fetal period and after birth, nestin expression decreased, whereas expression of EMA and mesothelin increased. EMA appeared in all surface epithelial cells and nodules, while mesothelin was found only in some of them. In adult meninges, all three proteins were predominantly localized in the surface epithelium and meningeal nodules. In meningothelial meningiomas (WHO grade I), EMA was detected in all tumor cells except in the endothelial cells, mesothelin characterized nests of tumor cells, while nestin was found predominantly in the walls of blood vessels. The distribution pattern of those proteins in normal meningeal and tumor cells indicates that nestin might characterize immature cells, while EMA and mesothelin appeared in maturing epithelial cells. Neoplastic transformation of these specific cell lineages contributes to the cell population in meningiomas. Copyright © 2010 Elsevier GmbH. All rights reserved.

Effect of Chelator Conjugation Level and Injection Dose on Tumor and Organ Uptake of 111In Labeled MORAb-009, an Anti-mesothelin Antibody

PubMed Central

Shin, I. S.; Lee, S.-M.; Kim, H. S.; Yao, Z.; Regino, C.; Sato, N.; Cheng, K. T.; Hassan, R.; Campo, M. F.; Albone, E. F.; Choyke, P. L.; Pastan, I.; Paik, C. H.

2012-01-01

Introduction Radiolabeling of a monoclonal antibody (mAb) with a metallic radionuclide requires the conjugation of a bifunctional chelator to the mAb. The conjugation, however, can alter the physical and immunological properties of the mAb, consequently affecting its tumor targeting pharmacokinetics. In this study, we investigated the effect of the amount of 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriamine-pentaacetic acid (CHX-A″) conjugated to MORAb-009, a mAb directed against mesothelin and the effect of MORAb dose on the biodistribution of 111In labeled MORAb-009. Methods We used nude mice bearing A431/K5 tumor as a mesothelin-positive tumor model and A431 tumor as a mesothelin-negative control. To find the optimal level of CHX-A″ conjugation, CHX-A″-MORAb-009 conjugates with 2.4, 3.5, and 5.5 CHX-A″ molecules were investigated. To investigate the effect of injected MORAb-009 dose on neutralizing the shed-mesothelin in the circulation, the biodistribution studies were performed after the i.v. co-injection of the 111In labeled MORAb-009 (2.4 CHX-A″/MORAb-009) with three different doses, 0.2, 2, and 30 μg of MORAb-009. Results The tumor uptake in A431/K5 tumor was 4 times higher than that in A431 tumor, indicating that the tumor uptake in A431/K5 was mesothelin-mediated. The conjugate with 5.5 CHX-A″ showed a lower isoelectric point (pI) and lower immunoreactivity (IR) than the 2.4 CHX-A″ conjugate. These differences were reflected in biodistribution of the 111In label. The 111In labeled MORAb-009 conjugated with 2.4 CHX-A″ produced higher tumor uptake, and lower liver and spleen uptakes than the 5.5 CHX-A″ conjugate. The biodistribution studies also revealed that the tumor uptake was significantly affected by the injected MORAb-009 dose and tumor size. The 30 μg dose produced higher tumor uptake than the 0.2 and 2 μg doses whereas the 30 μg dose produced lower liver and spleen uptakes than the 0.2 μg dose. Conclusion This study

Effect of chelator conjugation level and injection dose on tumor and organ uptake of 111In-labeled MORAb-009, an anti-mesothelin antibody.

PubMed

Shin, In Soo; Lee, Sang-Myung; Kim, Hyung Sub; Yao, Zhengsheng; Regino, Celeste; Sato, Noriko; Cheng, Kenneth T; Hassan, Raffit; Campo, Melissa F; Albone, Earl F; Choyke, Peter L; Pastan, Ira; Paik, Chang H

2011-11-01

Radiolabeling of a monoclonal antibody (mAb) with a metallic radionuclide requires the conjugation of a bifunctional chelator to the mAb. The conjugation, however, can alter the physical and immunological properties of the mAb, consequently affecting its tumor-targeting pharmacokinetics. In this study, we investigated the effect of the amount of 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriamine-pentaacetic acid (CHX-A″) conjugated to MORAb-009, a mAb directed against mesothelin, and the effect of MORAb dose on the biodistribution of (111)In-labeled MORAb-009. We used nude mice bearing the A431/K5 tumor as a mesothelin-positive tumor model and the A431 tumor as a mesothelin-negative control. To find the optimal level of CHX-A″ conjugation, CHX-A″-MORAb-009 conjugates with 2.4, 3.5 and 5.5 CHX-A″ molecules were investigated. To investigate the effect of injected MORAb-009 dose on neutralizing the shed mesothelin in the circulation, biodistribution studies were performed after the intravenous co-injection of (111)In-labeled MORAb-009 (2.4 CHX-A″/MORAb-009) with three different doses: 0.2, 2 and 30 μg of MORAb-009. The tumor uptake in A431/K5 tumor was four times higher than that in A431 tumor, indicating that the tumor uptake in A431/K5 was mesothelin mediated. The conjugate with 5.5 CHX-A″ showed a lower isoelectric point (pI) and lower immunoreactivity (IR) than the 2.4 CHX-A″ conjugate. These differences were reflected in the biodistribution of the (111)In label. The (111)In-labeled MORAb-009 conjugated with 2.4 CHX-A″ produced higher tumor uptake and lower liver and spleen uptakes than the 5.5 CHX-A″ conjugate. The biodistribution studies also revealed that the tumor uptake was significantly affected by the injected MORAb-009 dose and tumor size. The 30-μg dose produced higher tumor uptake than the 0.2- and 2-μg doses, whereas the 30-μg dose produced lower liver and spleen uptakes than the 0.2-μg dose. This study demonstrates that the

ImmunoPET with Anti-Mesothelin Antibody in Patients with Pancreatic and Ovarian Cancer before Anti-Mesothelin Antibody-Drug Conjugate Treatment.

PubMed

Lamberts, Laetitia E; Menke-van der Houven van Oordt, Catharina W; ter Weele, Eva J; Bensch, Frederike; Smeenk, Michiel M; Voortman, Johannes; Hoekstra, Otto S; Williams, Simon P; Fine, Bernard M; Maslyar, Daniel; de Jong, Johan R; Gietema, Jourik A; Schröder, Carolien P; Bongaerts, Alphons H H; Lub-de Hooge, Marjolijn N; Verheul, Henk M W; Sanabria Bohorquez, Sandra M; Glaudemans, Andor W J M; de Vries, Elisabeth G E

2016-04-01

Mesothelin (MSLN) is frequently overexpressed in pancreatic and ovarian cancers, making it a potential drug target. We performed an (89)Zr-PET imaging study with MMOT0530A, a MSLN antibody, in conjunction with a phase I study with the antibody-drug conjugate DMOT4039A, containing MMOT0530A bound to MMAE. The aim was to study antibody tumor uptake, whole-body distribution, and relation between uptake, response to treatment, and MSLN expression. Before DMOT4039A treatment, patients received 37 MBq (89)Zr-MMOT0530A followed by PET/CT imaging 2, 4, and 7 days postinjection. Tracer uptake was expressed as standardized uptake value (SUV). MSLN expression was determined with immunohistochemistry (IHC) on archival tumor tissue. Eleven patients were included, 7 with pancreatic and 4 with ovarian cancer. IHC MSLN expression varied from absent to strong. Suitable tracer antibody dose was 10 mg MMOT0530A and optimal imaging time was 4 and 7 days postinjection. Tumor tracer uptake occurred in 37 lesions with mean SUVmax of 13.1 (±7.5) on PET 4 days postinjection, with 11.5 (±7.5) in (N= 17) pancreatic and 14.5 (±8.7) in (N= 20) ovarian cancer lesions. Within patients, a mean 2.4-fold (±1.10) difference in uptake between tumor lesions existed. Uptake in blood, liver, kidneys, spleen, and intestine reflected normal antibody distribution. Tracer tumor uptake was correlated to IHC. Best response to DMOT4039A was partial response in one patient. With (89)Zr-MMOT0530A-PET, pancreatic and ovarian cancer lesions as well as antibody biodistribution could be visualized. This technique can potentially guide individualized antibody-based treatment. ©2015 American Association for Cancer Research.

New clinical trial combines LMB-100 with nab-paclitaxel for metastatic or locally advanced pancreatic cancer | Center for Cancer Research

Cancer.gov

Christine Alewine, Lasker Scholar, in the Laboratory of Molecular Biology is conducting a clinical study for patients with pancreatic cancer using a new immunotoxin drug, called LMB-100. It is a targeted immunotherapy that delivers a toxin to cancer cells that make a protein called mesothelin that is made by most pancreatic cancers.

Anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22-positive hematologic malignancies of childhood: preclinical studies and phase I clinical trial.

PubMed

Wayne, Alan S; Kreitman, Robert J; Findley, Harry W; Lew, Glen; Delbrook, Cynthia; Steinberg, Seth M; Stetler-Stevenson, Maryalice; Fitzgerald, David J; Pastan, Ira

2010-03-15

Although most children with B-lineage acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma are cured, new agents are needed to overcome drug resistance and reduce toxicities of chemotherapy. We hypothesized that the novel anti-CD22 immunotoxin, RFB4(dsFv)-PE38 (BL22, CAT-3888), would be active and have limited nonspecific side effects in children with CD22-expressing hematologic malignancies. We conducted the first preclinical and phase I clinical studies of BL22 in that setting. Lymphoblasts from children with B-lineage ALL were assessed for CD22 expression by flow cytometry and for BL22 sensitivity by in vitro cytotoxicity assay. BL22 was evaluated in a human ALL murine xenograft model. A phase I clinical trial was conducted for pediatric subjects with CD22+ ALL and non-Hodgkin lymphoma. All samples screened were CD22+. BL22 was cytotoxic to blasts in vitro (median IC(50), 9.8 ng/mL) and prolonged the leukemia-free survival of murine xenografts. Phase I trial cohorts were treated at escalating doses and schedules ranging from 10 to 40 microg/kg every other day for three or six doses repeated every 21 or 28 days. Treatment was associated with an acceptable safety profile, adverse events were rapidly reversible, and no maximum tolerated dose was defined. Pharmacokinetics were influenced by disease burden consistent with rapid drug binding by CD22+ blasts. Although no responses were observed, transient clinical activity was seen in most subjects. CD22 represents an excellent target and anti-CD22 immunotoxins offer therapeutic promise in B-lineage hematologic malignancies of childhood.

Hyaluronan and N-ERC/mesothelin as key biomarkers in a specific two-step model to predict pleural malignant mesothelioma.

PubMed

Mundt, Filip; Nilsonne, Gustav; Arslan, Sertaç; Csürös, Karola; Hillerdal, Gunnar; Yildirim, Huseyin; Metintas, Muzaffer; Dobra, Katalin; Hjerpe, Anders

2013-01-01

Diagnosis of malignant mesothelioma is challenging. The first available diagnostic material is often an effusion and biochemical analysis of soluble markers may provide additional diagnostic information. This study aimed to establish a predictive model using biomarkers from pleural effusions, to allow early and accurate diagnosis. Effusions were collected prospectively from 190 consecutive patients at a regional referral centre. Hyaluronan, N-ERC/mesothelin, C-ERC/mesothelin, osteopontin, syndecan-1, syndecan-2, and thioredoxin were measured using ELISA and HPLC. A predictive model was generated and validated using a second prospective set of 375 effusions collected consecutively at a different referral centre. Biochemical markers significantly associated with mesothelioma were hyaluronan (odds ratio, 95% CI: 8.82, 4.82-20.39), N-ERC/mesothelin (4.81, 3.19-7.93), CERC/mesothelin (3.58, 2.43-5.59) and syndecan-1 (1.34, 1.03-1.77). A two-step model using hyaluronan and N-ERC/mesothelin, and combining a threshold decision rule with logistic regression, yielded good discrimination with an area under the ROC curve of 0.99 (95% CI: 0.97-1.00) in the model generation dataset and 0.83 (0.74-0.91) in the validation dataset, respectively. A two-step model using hyaluronan and N-ERC/mesothelin predicts mesothelioma with high specificity. This method can be performed on the first available effusion and could be a useful adjunct to the morphological diagnosis of mesothelioma.

Targeting malignant B cells with an immunotoxin against ROR1

PubMed Central

Baskar, Sivasubramanian; Wiestner, Adrian; Wilson, Wyndham H.; Pastan, Ira; Rader, Christoph

2012-01-01

The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the VH and VL fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC50 = 16 pM–16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers. PMID:22531447

Aberrant Expression of Calretinin, D2-40 and Mesothelin in Mucinous and Non-Mucinous Colorectal Carcinomas and Relation to Clinicopathological Features and Prognosis.

PubMed

Foda, Abd AlRahman Mohammad; El-Hawary, Amira Kamal; Hamed, Hazem

2016-10-01

CRC is a heterogeneous disease in terms of morphology, invasive behavior, metastatic capacity, and clinical outcome. Recently, many so-called mesothelial markers, including calretinin, D2-40, WT1, thrombomodulin, mesothelin, and others, have been certified. The aim of this study was to assess the immunohistochemical expression of calretinin and other mesothelial markers (D2-40 and mesothelin) in colorectal mucinous adenocarcinoma (MA) and non mucinous adenocarcinoma (NMA) specimens and relation to clinicopathological features and prognosis using manual tissue microarray technique. We studied tumor tissue specimens from 150 patients with colorectal MA and NMA who underwent radical surgery from January 2007 to January 2012. High-density manual tissue microarrays were constructed using a modified mechanical pencil tip technique, and paraffin sections were submitted for immunohistochemistry using Calretinin, D2-40 and mesothelin expressions. We found that NMA showed significantly more calretinin and D2-40 expression than MA In contrast, no statistically significant difference between NMA and MA was detected in mesothelin expression. There were no statistically significant relations between any of the clinicopathological or histological parameters and any of the three markers. In a univariate analysis, neither calretinin nor D2-40 expressions showed any significant relations to DFS or OS. However, mesothelin luminal expression was significantly associated with worse DFS. Multivariate Cox regression analysis proved that luminal mesothelin expression was an independent negative prognostic factor in NMA. In conclusion, Calretinin, D2-40 and mesothelin are aberrantly expressed in a proportion of CRC cases with more expression in NMA than MA. Aberrant expression of these mesothelial markers was not associated with clinicopathological or histological features of CRCs. Only mesothelin expression appears to be a strong predictor of adverse prognosis.

The novel multispecies Fc-specific Pseudomonas exotoxin A fusion protein α-Fc-ETA' enables screening of antibodies for immunotoxin development.

PubMed

Klausz, Katja; Kellner, Christian; Derer, Stefanie; Valerius, Thomas; Staudinger, Matthias; Burger, Renate; Gramatzki, Martin; Peipp, Matthias

2015-03-01

Immunoconjugates that deliver cytotoxic payloads to cancer cells represent a promising class of therapeutic agents which are intensively investigated in various clinical applications. Prerequisites for the generation of effective immunoconjugates are antibodies which efficiently deliver the respective cytotoxic payload. To facilitate the selection of human or mouse antibodies that display favorable characteristics as immunotoxins, we developed a novel Pseudomonas exotoxin A (ETA)-based screening protein. The α-Fc-ETA' consists of a multispecies-specific Fc-binding domain antibody genetically fused to a truncated ETA version (ETA'). α-Fc-ETA' non-covalently bound to human and mouse antibodies but did not form immune complexes with bovine immunoglobulins. In combination with antibodies harboring human or mouse Fc domains α-Fc-ETA' inhibited proliferation of antigen-expressing tumor cells. The cytotoxic effects were strictly antibody dependent and were observed with low α-Fc-ETA' concentrations. Mouse antibodies directed against CD7 and CD317/HM1.24 that previously had been used for the generation of functional recombinant immunotoxins, also showed activity in combination with α-Fc-ETA' by inhibiting growth of antigen-positive myeloma and leukemia cell lines. In contrast, α-kappa-ETA', a similarly designed human kappa light chain-specific fusion protein, was only specifically active in combination with antibodies containing a human kappa light chain. Thus, the novel α-Fc-ETA' fusion protein is broadly applicable in screening antibodies and Fc-containing antibody derivatives from different species to select for candidates with favorable characteristics for immunotoxin development. Copyright © 2015 Elsevier B.V. All rights reserved.

Complete suppression of in vivo growth of human leukemia cells by specific immunotoxins: nude mouse models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hara, H.; Seon, B.K.

1987-05-01

In this study, immunotoxins containing monoclonal anti-human T-cell leukemia antibodies are shown to be capable of completely suppressing the tumor growth of human T-cell leukemia cells in vivo without any overt undersirable toxicity. These immunotoxins were prepared by conjugating ricin A chain (RA) with our monoclonal antibodies, SN1 and SN2, directed specifically to the human T-cell leukemia cell surface antigens TALLA and GP37, respectively. The authors have shown that these monoclonal antibodies are highly specific for human T-cell leukemia cells and do not react with various normal cells including normal T and B cells, thymocytes, and bone marrow cells. Asciticmore » and solid human T-cell leukemia cell tumors were generated in nude mice. The ascitic tumor was generated by transplanting Ichikawa cells (a human T-cell leukemia cell) i.p. into nude mice, whereas the solid tumor was generated by transplanting s.c. MOLT-4 cells (a human T-cell leukemia cell line) and x-irradiated human fibrosarcoma cells into x-irradiated nude mice. To investigate the efficacy of specific immunotoxins in suppression the in vivo growth of the ascitic tumor, they divided 40 nude mice that were injected with Ichikawa cells into four groups. None of the mice in group 4 that were treated with SN1-RA and SN2-RA showed any signs of a tumor or undesirable toxic effects for the 20 weeks that they were followed after the transplantation. Treatment with SN1-RA plus SN2-RA completely suppressed solid tumor growth in 4 of 10 nude mice carrying solid tumors and partially suppressed the tumor growth in the remaining 6 nude mice. These results strongly suggest that SN1-RA and SN2-RA may be useful for clinical treatment.« less

[Study on the anti-NTHi infection of Hap recombinant protein in vivo].

PubMed

Li, Wan-yi; Wang, Bao-ning; Zuo, Feng-qiong; Zeng, Wei; Feng, Feng; Kuang, Yu; Jiang, Zhong-hua; Li, Ming-yuan

2010-07-01

To observe the immune effect of Hap recombinant protein on murine model of bronchopneumonia infected with NTHi, and explore the mechanism about the anti-NTHi infection. The C57BL/6 mice intranasally immunized with purified Hap recombinant protein and CT-B were challenged by NTHi encased in agar beads. The immunifaction of anti-infection was observed through encocyte counting of BALF, bacteria detection of lung and the pathologyical change of lung tissue. In the challenge with NTHi experiment, the inflammatory exudation of the infected murine and pathological change of lung tissue was relieved by combined immunization of Hap recombinant protein and CT-B, and quantity of NTHi in lung of the infected murine was reduced obviously. The Hap recombinant protein also had good ability of anti-NTHi infection in the murine model of NTHi bronchopneumonia. This study could offer the oretical and experimental basis for development of new vaccine against NTHi.

A novel far-red fluorescent xenograft model of ovarian carcinoma for preclinical evaluation of HER2-targeted immunotoxins

PubMed Central

Zdobnova, Tatiana; Sokolova, Evgeniya; Stremovskiy, Oleg; Karpenko, Dmitry; Telford, William; Turchin, Ilya; Balalaeva, Irina; Deyev, Sergey

2015-01-01

We have created a novel fluorescent model of a human ovarian carcinoma xenograft overexpressing receptor HER2, a promising molecular target of solid tumors. The model is based on a newly generated SKOV-kat cell line stably expressing far-red fluorescent protein Katushka. Katushka is most suitable for the in vivo imaging due to an optimal combination of high brightness and emission in the “window of tissue transparency”. The relevance of the fluorescent model for the in vivo monitoring of tumor growth and response to treatment was demonstrated using a newly created HER2-targeted recombinant immunotoxin based on the 4D5scFv antibody and a fragment of the Pseudomonas exotoxin A. PMID:26436696

Recombinant platelet factor 4: a therapeutic, anti-neoplastic chimera?

PubMed

Lippi, Giuseppe; Favaloro, Emmanuel J

2010-07-01

Angiogenesis plays a pivotal role in many serious and life-threatening disorders (e.g., cancer, atherosclerosis, diabetes, arthritis, psoriasis, nephropathy, and retinopathy) and is regulated by a delicate equilibrium between a variety of pro- and anti-angiogenic factors. Although recombinant platelet factor 4 (PF4) was originally developed and evaluated as a clinical alternative to protamine for heparin neutralization, the current scientific evidence supports a role for this protein and derivative peptides in inhibiting tumor growth and spread, by suppression of tumor-induced neovascularization in many different types of solid tumors. As a heparin-binding tetramer, recombinant PF4 interferes with several steps of endothelial cell proliferation, migration, and angiogenesis, regulates apoptotic death through activation of distinct signal transduction pathways, inhibits growth factor receptor binding, amplifies the inflammatory response of natural killer cells through regulation of cytokines production, and induces and maintains a nonspecific immune response to cancer cells. These biological evidences paved the way for the development and marketing of novel PF4-based angiostatic agents characterized by reduced toxicity and improved bioavailability, thus raising the possibility of an alternative approach for preventing and treating growth and metastasis of tumors. Some PF4-derived molecules such as carboxyl-terminal fragments of recombinant human PF4 and modified and chimeric peptides have already been developed that exhibit stronger anti-angiogenic properties than the parent molecule and may serve as leads for further therapeutic developments. Newer means of delivering of this anti-angiogenic agent are also being attempted, including PF4-bearing polymeric microspheres, vector-mediated PF4 transduction, transgene transfection into oncolytic viruses, and molecular targeting therapy against PF4 and rHuPF4 conjugates. These delivery systems aim to produce high

The Use of Plant-Derived Ribosome Inactivating Proteins in Immunotoxin Development: Past, Present and Future Generations

PubMed Central

Rust, Aleksander; Partridge, Lynda J.; Davletov, Bazbek

2017-01-01

Ribosome inactivating proteins (RIPs) form a class of toxins that was identified over a century ago. They continue to fascinate scientists and the public due to their very high activity and long-term stability which might find useful applications in the therapeutic killing of unwanted cells but can also be used in acts of terror. We will focus our review on the canonical plant-derived RIPs which display ribosomal RNA N-glycosidase activity and irreversibly inhibit protein synthesis by cleaving the 28S ribosomal RNA of the large 60S subunit of eukaryotic ribosomes. We will place particular emphasis on therapeutic applications and the generation of immunotoxins by coupling antibodies to RIPs in an attempt to target specific cells. Several generations of immunotoxins have been developed and we will review their optimisation as well as their use and limitations in pre-clinical and clinical trials. Finally, we endeavour to provide a perspective on potential future developments for the therapeutic use of immunotoxins. PMID:29076988

Purification and characterization of a novel type i ribosome inactivating protein, pachyerosin, from Pachyrhizus erosus seeds, and preparation of its immunotoxin against human hepatoma cells.

PubMed

Guo, Jin-Lin; Cheng, Yuan-Liu; Qiu, Yi; Shen, Cai-Hong; Yi, Bin; Peng, Cheng

2014-07-01

Pachyrhizus erosus seeds have a high protein content and are used in China due to their cytotoxic effect. Here we report the biological and pharmacological activity of the protein extracts from P. erosus seeds. A novel ribosome-inactivating protein, pachyerosin, from P. erosus seeds was successively purified to homogeneity using ammonium sulfate precipitation, DEAE-sepharose FF, and Sephacryl S-200. Pachyerosin showed to be a type I ribosome-inactivating protein with a molecular mass of 29 kDa and an isoelectric point of 9.19. It strongly inhibited protein synthesis of rabbit reticulocyte lysate with an IC50 of 0.37 ng/mL and showed N-glycosidase activity on rat liver ribosomes with an EC50 of 85.9 pM. The N-terminal 27 amino acids of pachyerosin revealed a 60.71% sequence identity with abrin A from the seeds of Abrus precatorius. With the aim of targeting the delivery of pachyerosin, immunotoxin was prepared by conjugating pachyerosin with anti-human AFP monoclonal antibodies SM0736. The immunotoxin pachyerosin-SM0736 efficiently inhibited the growth of the human hepatoma cell line HuH-7 with an IC50 of 0.050 ± 0.004 nM, 2360 times lower than that of pachyerosin and 430 times lower than that of the immunotoxin against human gastric cancer cell line SGC7901. These results imply that pachyerosin may be used as a new promising anticancer agent. Georg Thieme Verlag KG Stuttgart · New York.

Chimpanzees Immunized with Recombinant Soluble CD4 Develop Anti-Self CD4 Antibody Responses with Anti-Human Immunodeficiency Virus Activity

NASA Astrophysics Data System (ADS)

Watanabe, Mamoru; Boyson, Jonathan E.; Lord, Carol I.; Letvin, Norman L.

1992-06-01

In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.

Production of a pokeweed antiviral protein (PAP)-containing immunotoxin, B43-PAP, directed against the CD19 human B lineage lymphoid differentiation antigen in highly purified form for human clinical trials.

PubMed

Myers, D E; Irvin, J D; Smith, R S; Kuebelbeck, V M; Uckun, F M

1991-02-15

We describe a standardized method for the preparation and purification of a potent immunotoxin against B-lineage leukemia/lymphoma cells, constructed with the ribosome inhibitory single chain plant toxin pokeweed antiviral protein (PAP) and a murine IgG1 monoclonal antibody (MoAb) specific for the human B lineage differentiation antigen CD19 for human clinical trials. PAP was prepared from spring leaves of Phytolacca americana plants by ammonium sulfate precipitation and purified to homogeneity by successive steps of ion exchange chromatography. B43 MoAb was produced in vitro by hollow fiber technology and purified to homogeneity by affinity chromatography. PAP toxin and B43 MoAb were modified via their free amino groups prior to their intermolecular conjugation. 2-iminothiolane was used to introduce reactive sulfhydryl groups into PAP and N-succinimidyl 3-(2-pyridyldithio) propionate was used to introduce 2-pyridyl disulfide bonds into B43 MoAb. Modified PAP was reacted with modified B43 MoAb resulting in a sulfhydryl-disulfide exchange reaction and yielding disulfide linked PAP-B43 MoAb conjugates, which we refer to as B43-PAP immunotoxin. B43-PAP immunotoxin was subjected to preparative gel filtration chromatography and cation exchange chromatography to obtain a highly purified, sterile, and pyrogen-free immunotoxin preparation with less than 5% free antibody contamination and less than 0.5% free PAP contamination. The final product displayed a high affinity for and a very potent anti-leukemic activity against B lineage leukemia cells. With slight modifications, the procedures detailed in this report should be generally applicable to preparation of other PAP-MoAb conjugates for treatment of cancer or AIDS.

Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity

PubMed Central

Adusumilli, Prasad S.; Cherkassky, Leonid; Villena-Vargas, Jonathan; Colovos, Christos; Servais, Elliot; Plotkin, Jason; Jones, David R.; Sadelain, Michel

2015-01-01

Translating the recent success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies to solid tumors will necessitate overcoming several obstacles, including inefficient T cell tumor infiltration and insufficient functional persistence. Taking advantage of an orthotopic model that faithfully mimics human pleural malignancy, we evaluated two routes of administration of mesothelin-targeted T cells using the M28z CAR. We found that intra-pleurally administered CAR T cells vastly out-performed systemically infused T cells, requiring 30-fold fewer M28z T cells to induce long-term complete remissions. Following intrapleural T cell administration, prompt in vivo antigen-induced T cell activation allowed robust CAR T cell expansion and effector differentiation, resulting in enhanced anti-tumor efficacy and functional T cell persistence for 200 days. Regional T cell administration also promoted efficient elimination of extrathoracic tumor sites. This therapeutic efficacy was dependent on early CD4+ T cell activation associated with a higher intra-tumoral CD4/CD8 cell ratios and CD28-dependent CD4+ T cell-mediated cytotoxicity. In contrast, intravenously delivered CAR T cells, even when accumulated at equivalent numbers in the pleural tumor, did not achieve comparable activation, tumor eradication or persistence. The remarkable ability of intrapleurally administered T cells to circulate and persist supports the concept of delivering optimal CAR T cell therapy through “regional distribution centers.” Based on these results, we are opening a phase I clinical trial to evaluate the safety of intrapleural administration of mesothelin-targeted CAR T cells in patients with primary or secondary pleural malignancies. PMID:25378643

Use of Ribosome-Inactivating Proteins from Sambucus for the Construction of Immunotoxins and Conjugates for Cancer Therapy

PubMed Central

Ferreras, José M.; Citores, Lucía; Iglesias, Rosario; Jiménez, Pilar; Girbés, Tomás

2011-01-01

The type 2 ribosome-inactivating proteins (RIPs) isolated from some species belonging to the Sambucus genus, have the characteristic that although being even more active than ricin inhibiting protein synthesis in cell-free extracts, they lack the high toxicity of ricin and related type 2 RIPs to intact cells and animals. This is due to the fact that after internalization, they follow a different intracellular pathway that does not allow them to reach the cytosolic ribosomes. The lack of toxicity of type 2 RIPs from Sambucus make them good candidates as toxic moieties in the construction of immunotoxins and conjugates directed against specific targets. Up to now they have been conjugated with either transferrin or anti-CD105 to target either transferrin receptor- or endoglin-overexpressing cells, respectively. PMID:22069717

Chimeric Antigen Receptors that Recognize Mesothelin for Cancer Immunotherapy | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the NCI have developed chimeric antigen receptors (CARs) with a high affinity for mesothelin to be used as an immunotherapy to treat pancreatic cancer, ovarian cancer, and mesothelioma. Cells that express CARs, most notably T cells, are highly reactive against their specific tumor antigen in an MHC-unrestricted manner to generate an immune response that promotes robust tumor cell elimination when infused into cancer patients.

Detection of anti-PL-12 autoantibodies by ELISA using a recombinant antigen; study of the immunoreactive region

PubMed Central

García-Lozano, J R; González-Escribano, M F; Rodríguez, R; Rodriguez-Sanchez, J L; Targoff, I N; Wichmann, I; Núñez-Roldán, A

1998-01-01

Autoantibodies to aminoacyl-tRNA synthetases are highly associated with myositis and detection is important in clinical diagnosis; however, current methods of screening limit its clinical utility. In the present study, alanyl-tRNA synthetase (PL-12) recombinant protein was obtained by immunological screening of a HeLa expression library and used in an ELISA with 22 anti-PL-12 sera, 200 autoimmune sera negative for PL-12 and 100 healthy individual sera. Sensitivity of the method was 95% (21/22) and specificity 100%. Mapping of the immunoreactive region was carried out using three anti-PL-12 sera and different recombinant protein-derived peptides. Results show that the same conformational epitope located within amino acids 730–951 of the PL-12 antigen outside the catalytic region was recognized by the three anti-PL-12 sera tested. We conclude that ELISA using recombinant protein is an effective and useful method for routine screening for anti-PL-12 autoantibodies. PMID:9822271

Development of a GMP Phase III purification process for VB4-845, an immunotoxin expressed in E. coli using high cell density fermentation.

PubMed

Premsukh, Arjune; Lavoie, Joelle M; Cizeau, Jeannick; Entwistle, Joycelyn; MacDonald, Glen C

2011-07-01

VB4-845 is a recombinant immunotoxin comprised of an anti-epithelial cell adhesion molecule (EpCAM) scFv fused to a truncated form of the bacterial toxin, Pseudomonas exotoxin A. VB4-845, purified from TB fed-batch fermentation, showed clinical efficacy when administered locally to treat non-muscle invasive bladder cancer (NMIBC) and squamous cell carcinomas of the head and neck (SCCHN). Here, we describe the implementation of an Escherichia coli high cell density (HCD) cultivation and purification process for VB4-845. HCD cultivation was a prerequisite for achieving higher yields necessary for Phase III clinical trials and commercialization. Using this process, the VB4-845 titer in the supernatant was increased by 30-fold over the original TB fed-batch cultivation. To obtain clinical grade material, a process involving a five-step column purification procedure was implemented and led to an overall recovery of ∼ 40%. VB4-845 purity of >97% was achieved after the first three columns following the removal of low-molecular weight product-related impurities and aggregates. Endotoxins were effectively separated from VB4-845 on the Q-columns and by washing the Ni-column with a detergent buffer while host cell proteins were removed using ceramic hydroxyapatite. Comparability studies demonstrated that the purified product from the Phase III process was identical to the Phase II reference standard produced using TB fed-batch fermentation. Copyright © 2011 Elsevier Inc. All rights reserved.

Immunotoxins recognising a new epitope on the neural cell adhesion molecule have potent cytotoxic effects against small cell lung cancer.

PubMed

Zangemeister-Wittke, U; Collinson, A R; Frösch, B; Waibel, R; Schenker, T; Stahel, R A

1994-01-01

The present study describes a comparison of two potent immunotoxins which utilise an identical targeting component, a monoclonal antibody (SEN7) specific for small cell lung cancer (SCLC), conjugated to two different effector components, blocked ricin (bR) and Pseudomonas exotoxin A (PE). SEN7 recognises a novel epitope on the neural cell adhesion molecule (NCAM) which is highly associated with SCLC. The immunotoxins SEN7-PE and SEN7-bR were selectively and potently active against a number of SCLC cell lines, of both classic and variant morphologies, inhibiting the incorporation of [3H]leucine with IC50 values ranging between 22 pM and 85 pM and between 7 pM and 62 pM for SEN7-PE and SEN7-bR respectively. Intoxication by both immunotoxins proceeded rapidly following short 2 h lag phases; the initial rates of protein synthesis inhibition occurred with t50 values of 6.5 h for SEN7-PE and 5.5 h for SEN7-bR. Monensin drastically enhanced the cytotoxic activity of the weakly active SEN7-ricin A-chain by 2,100-fold and of SEN7-bR by 80-fold but had no effect on SEN7-PE. In limiting dilution assays, four and more than 4.5 logs of clonogenic SW2 tumour cells were selectively eliminated from the cultures during continuous exposure to the immunotoxins SEN7-PE and SEN7-bR respectively, while antigen-negative cells required up to 1,000-fold more drug for a similar cell kill. SW2 cells surviving SEN7-bR treatment in the cultures did not express NCAM and consequently were not selectively killed by SEN7 immunotoxins. SW2 cells surviving continuous exposure to SEN7-PE showed no alteration in NCAM expression but were more resistant to intoxication mediated by PE. These cells were still sensitive to SEN7-bR.

Engineering and biological characterization of VB6-845, an anti-EpCAM immunotoxin containing a T-cell epitope-depleted variant of the plant toxin bouganin.

PubMed

Cizeau, Jeannick; Grenkow, Danielle M; Brown, Jennifer G; Entwistle, Joycelyn; MacDonald, Glen C

2009-01-01

The clinical development of immunotoxins in the treatment of solid tumors has been impeded in part, by the induction of an immune response directed primarily against the toxin moiety. Bouganin, a type I ribosome inactivating protein isolated from the leaf of Bougainvillea spectabilis Willd, was mutated to remove the T-cell epitopes while preserving the biological activity of the wild-type molecule. The T-cell epitope-depleted variant of bouganin (de-bouganin) was genetically linked to an anti-epithelial cell adhesion molecule (EpCAM) Fab moiety via a peptidic linker containing a furin proteolytic site to create the fusion construct VB6-845. To determine the optimal construct design for VB6-845, several dicistronic units where de-bouganin was genetically linked to either the N-terminal or C-terminal of either the heavy or light chain were engineered. Only the C-terminal variants expressed the full-length molecule. An in vitro assessment of the biological activity of VB6-845 showed that it bound and selectively killed EpCAM-positive cell lines with a greater potency than many commonly used chemotherapeutic agents. In vivo efficacy was demonstrated using an EpCAM-positive human tumor xenograft model in SCID mice with the majority of the mice treated being tumor free at the end of the study.

Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses.

PubMed

Rasala, Beth A; Mayfield, Stephen P

2015-03-01

Recombinant proteins are widely used for industrial, nutritional, and medical applications. Green microalgae have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins for a number of reasons. These photosynthetic eukaryotic microorganisms are safe, scalable, easy to genetically modify through transformation, mutagenesis, or breeding, and inexpensive to grow. Many microalgae species are genetically transformable, but the green alga Chlamydomonas reinhardtii is the most widely used host for recombinant protein expression. An extensive suite of molecular genetic tools has been developed for C. reinhardtii over the last 25 years, including a fully sequenced genome, well-established methods for transformation, mutagenesis and breeding, and transformation vectors for high levels of recombinant protein accumulation and secretion. Here, we review recent successes in the development of C. reinhardtii as a biomanufacturing host for recombinant proteins, including antibodies and immunotoxins, hormones, industrial enzymes, an orally-active colostral protein for gastrointestinal health, and subunit vaccines. In addition, we review the biomanufacturing potential of other green algae from the genera Dunaliella and Chlorella.

[Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].

PubMed

Gao, Guohui; Chen, Chong; Yang, Yanmei; Yang, Han; Wang, Jindan; Zheng, Yi; Huang, Qidi; Hu, Xiaoqu

2012-08-01

To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2

Coupling Gd-DTPA with a bispecific, recombinant protein anti-EGFR-iRGD complex improves tumor targeting in MRI

PubMed Central

XIN, XIAOYAN; SHA, HUIZI; SHEN, JINGTAO; ZHANG, BING; ZHU, BIN; LIU, BAORUI

2016-01-01

Recombinant anti-epidermal growth factor receptor-internalizing arginine-glycine-aspartic acid (anti-EGFR single-domain antibody fused with iRGD peptide) protein efficiently targets the EGFR extracellular domain and integrin αvβ/β5, and shows a high penetration into cells. Thus, this protein may improve penetration of conjugated drugs into the deep zone of gastric cancer multicellular 3D spheroids. In the present study, a novel tumor-targeting contrast agent for magnetic resonance imaging (MRI) was developed, by coupling gadolinium-diethylene triamine pentaacetate (Gd-DTPA) with the bispecific recombinant anti-EGFR-iRGD protein. The anti-EGFR-iRGD protein was extracted from Escherichia coli and Gd was loaded onto the recombinant protein by chelation using DTPA anhydride. Single-targeting agent anti-EGFR-DTPA-Gd, which served as the control, was also prepared. The results of the present study showed that anti-EGFR-iRGD-DTPA-Gd exhibited no significant cyto toxicity to human gastric carcinoma cells (BGC-823) under the experimental conditions used. Compared with a conventional contrast agent (Magnevist), anti-EGFR-iRGD-DTPA-Gd showed higher T1 relaxivity (10.157/mM/sec at 3T) and better tumor-targeting ability. In addition, the signal intensity and the area under curve for the enhanced signal time in tumor, in vivo, were stronger than Gd-DTPA alone or the anti-EGFR-Gd control. Thus, Gd-labelled anti-EGFR-iRGD has potential as a tumor-targeting contrast agent for improved MRI. PMID:27035336

Improved Formulation of a Recombinant Ricin A-chain Vaccine Increases its Stability and Effective Antigenicity

DTIC Science & Technology

2007-03-26

Adjuvant adsorption; Toxin . Introduction Ricin, a highly potent toxin derived from the castor bean, ills human cells by depurinating a specific...moieties n the cell surface. Antibodies elicited against either the ricin (RTA) or B-chain can neutralize the toxin, although anti- Abbreviations: RTA...human B- cell epitope for RTA has been identified by astelletti et al. [6] from cancer patients treated with a ricin- onjugate immunotoxin, and lies

Anti-melanoma activity of the 9.2.27PE immunotoxin in dacarbazine resistant cells.

PubMed

Risberg, Karianne; Fodstad, Oystein; Andersson, Yvonne

2010-04-01

We have earlier shown that the 9.2.27 Pseudomonas Exotoxin A (PE) immunotoxin (IT) efficiently kills melanoma cells through inhibition of protein synthesis followed by some morphologic and biochemical features of apoptosis, a different cell killing mechanism than the one caused by Dacarbazine (DTIC), a chemotherapeutic drug used to treat malignant melanoma. To examine whether induced DTIC resistance also is a determining factor for the effectiveness of 9.2.27PE IT, we developed a DTIC resistant subline, FEMX-200DR, from the DTIC sensitive cell line FEMX. The cell variants were treated with 9.2.27PE, an IT binding to the high molecular weight-melanoma associated antigen (HMW-MAA) expressed on most malignant melanoma cells. The IT was equally effective in killing the FEMX-200DR and the FEMX cells, and the cell death was primarily caused by inhibition of protein synthesis. The DNA repair enzyme and apoptotic marker PARP, a substrate of caspase-3, was inactivated, although we observed only a minor activation of caspase-3 and caspase-8, intracellular proteases involved in apoptosis. In addition to being DTIC resistant, the FEMX-200DR cells were also more resistant to apoptosis than the parent cells as a 3 times higher concentration of the apoptotic inducer Staurosporine was needed to obtain IC50. Furthermore, in early passage malignant melanoma cell lines established from lymph node metastases, the 9.2.27PE caused a time-dependent and dose-dependent decrease in cell viability independent of their DTIC sensitivity. These findings show that the 9.2.27PE IT efficiently can cause cell death in malignant melanoma cells independent of their level of resistance to apoptosis and DTIC.

Assay of anti-HBs antibodies using a recombinant antigen and latex particle counting: comparison with five commercial tests.

PubMed

Galanti, L M; Cornu, C; Masson, P L; Robert, A R; Becheanu, D; Lamy, M E; Cambiaso, C L

1991-05-01

An assay of anti-HBs antibodies based on agglutination of latex particles coated with recombinant HBs-antigen was compared with Abbott radioimmunoassay (Abbott-RIA), which uses a human plasma-derived antigen. The population examined consisted of 76 Abbott-RIA anti-HBs-negative prevaccinated subjects and 1044 serum samples anti-HBs found positive by Abbott-RIA, including 283 samples of subjects vaccinated either with a human plasma-derived vaccine (group A; n = 180) or with a recombinant vaccine (group B; n = 103). Correlation coefficients between the two techniques were respectively r = 0.89 for the whole population (n = 1044), r = 0.98 in group A and r = 0.74 in group B. Anti-HBs titres were higher with latex than with RIA in group B as shown by the regression slopes: latex = 508 + 1.11 RIA in group A and latex = -1138 + 3.97 RIA in group B, suggesting that some vaccinated subjects from group B produced antibodies against epitopes proper to the recombinant antigen. In the prevaccinated population and in group A, the latex results were compared with those of radioimmunoassays (Abbott, Sorin) and enzyme immunoassays (Behring, Roche, Pasteur). Only the Roche-EIA detected anti-HBs in the prevaccinated subjects. The correlation between the various immunoassays was r greater than 0.96 only for values higher than 100 IU/l.

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

PubMed

Li, Ming; Nie, Yao; Mu, Xiao Qing; Zhang, Rongzhen; Xu, Yan

2016-07-03

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

Paclitaxel synergizes with exposure time adjusted CD22-targeting immunotoxins against B-cell malignancies.

PubMed

Müller, Fabian; Stookey, Stephanie; Cunningham, Tyler; Pastan, Ira

2017-05-09

CD22-targeted recombinant immunotoxins (rIT) are active in hairy cell leukemia or acute lymphoblastic leukemia (ALL), but not in mantle cell lymphoma (MCL) patients. The goal was to enhance rIT efficacy in vivo and to define a strong combination treatment. Activity of Moxetumomab pasudotox (Moxe) and LR combined with paclitaxel was tested against MCL cell lines in vitro and as bolus doses or continuous infusion in xenograft models. In the KOPN-8 ALL xenograft, Moxe or paclitaxel alone was active, but all mice died from leukemia; when combined, 60% of the mice achieved a sustained complete remission. Against MCL cells in vitro, LR was more active than Moxe and the cells had to be exposed to rIT for more than 24 hours for them to die. To maintain high blood levels in vivo, LR was administered continuously by 7-day pumps achieving a well-tolerated steady plasma concentration of 45 ng/ml. In JeKo-1 xenografts, continuously administered LR was 14-fold more active than bolus doses and the combination with paclitaxel additionally improved responses by 135-fold. Maintaining high rIT-plasma levels greatly improves responses in the JeKo-1 model and paclitaxel substantially enhances bolus and continuously infused rIT, supporting a clinical evaluation against B-cell malignancies.

Homologous recombination deficiency and host anti-tumor immunity in triple-negative breast cancer.

PubMed

Telli, M L; Stover, D G; Loi, S; Aparicio, S; Carey, L A; Domchek, S M; Newman, L; Sledge, G W; Winer, E P

2018-05-07

Triple-negative breast cancer (TNBC) is associated with worse outcomes relative to other breast cancer subtypes. Chemotherapy remains the standard-of-care systemic therapy for patients with localized or metastatic disease, with few biomarkers to guide benefit. We will discuss recent advances in our understanding of two key biological processes in TNBC, homologous recombination (HR) DNA repair deficiency and host anti-tumor immunity, and their intersection. Recent advances in our understanding of homologous recombination (HR) deficiency, including FDA approval of PARP inhibitor olaparib for BRCA1 or BRCA2 mutation carriers, and host anti-tumor immunity in TNBC offer potential for new and biomarker-driven approaches to treat TNBC. Assays interrogating HR DNA repair capacity may guide treatment with agents inducing or targeting DNA damage repair. Tumor infiltrating lymphocytes (TILs) are associated with improved prognosis in TNBC and recent efforts to characterize infiltrating immune cell subsets and activate host anti-tumor immunity offer promise, yet challenges remain particularly in tumors lacking pre-existing immune infiltrates. Advances in these fields provide potential biomarkers to stratify patients with TNBC and guide therapy: induction of DNA damage in HR-deficient tumors and activation of existing or recruitment of host anti-tumor immune cells. Importantly, these advances provide an opportunity to guide use of existing therapies and development of novel therapies for TNBC. Efforts to combine therapies that exploit HR deficiency to enhance the activity of immune-directed therapies offer promise. HR deficiency remains an important biomarker target and potentially effective adjunct to enhance immunogenicity of 'immune cold' TNBCs.

A novel immunotoxin reveals a new role for CD321 in endothelial cells

PubMed Central

Kim, Jia; Hokaiwado, Shintaro; Nawa, Makiko; Okamoto, Hayato; Kogiso, Tomohiko; Watabe, Tetsuro; Hattori, Nobutaka

2017-01-01

There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy. PMID:29028806

Distribution and co-localization of choline acetyltransferase and p75 neurotrophin receptors in the sheep basal forebrain: implications for the use of a specific cholinergic immunotoxin.

PubMed

Ferreira, G; Meurisse, M; Tillet, Y; Lévy, F

2001-01-01

The basal forebrain cholinergic system is involved in different forms of memory. To study its role in social memory in sheep, an immunotoxin, ME20.4 immunoglobulin G (IgG)-saporin, was developed that is specific to basal forebrain cholinergic neurons bearing the p75 neurotrophin receptor. The distribution of sheep cholinergic neurons was mapped with an antibody against choline acetyltransferase. To assess the localization of the p75 receptor on basal forebrain cholinergic neurons, the distribution of p75 receptor-immunoreactive neurons with ME20.4 IgG was examined, and a double-labeling study with antibodies against choline acetyltransferase and p75 receptor was undertaken. The loss of basal forebrain cholinergic neurons and acetylcholinesterase fibers in basal forebrain projection areas was assessed in ewes that had received intracerebroventricular injections of the immunotoxin (50, 100 or 150 microg) alone, as well as, in some of the ewes treated with the highest dose, with bilateral immunotoxin injections in the nucleus basalis (11 microg/side). Results indicated that choline acetyltransferase- and p75 receptor-immunoreactive cells had similar distributions in the medial septum, the vertical and horizontal limbs of the band of Broca, and the nucleus basalis. The double-labeling procedure revealed that 100% of the cholinergic neurons are also p75 receptor positive in the medial septum and in the vertical and horizontal limbs of the band of Broca, and 82% in the nucleus basalis. Moreover, 100% of the p75 receptor-immunoreactive cells of these four nuclei were cholinergic. Combined immunotoxin injections into ventricles and the nucleus basalis produced a near complete loss (80-95%) of basal forebrain cholinergic neurons and acetylcholinesterase-positive fibers in the hippocampus, olfactory bulb and entorhinal cortex. This study provides the first anatomical data concerning the basal forebrain cholinergic system in ungulates. The availability of a selective

Safety and Survival With GVAX Pancreas Prime and Listeria Monocytogenes–Expressing Mesothelin (CRS-207) Boost Vaccines for Metastatic Pancreatic Cancer

PubMed Central

Le, Dung T.; Wang-Gillam, Andrea; Picozzi, Vincent; Greten, Tim F.; Crocenzi, Todd; Springett, Gregory; Morse, Michael; Zeh, Herbert; Cohen, Deirdre; Fine, Robert L.; Onners, Beth; Uram, Jennifer N.; Laheru, Daniel A.; Lutz, Eric R.; Solt, Sara; Murphy, Aimee Luck; Skoble, Justin; Lemmens, Ed; Grous, John; Dubensky, Thomas; Brockstedt, Dirk G.; Jaffee, Elizabeth M.

2015-01-01

Purpose GVAX pancreas, granulocyte-macrophage colony-stimulating factor–secreting allogeneic pancreatic tumor cells, induces T-cell immunity to cancer antigens, including mesothelin. GVAX is administered with low-dose cyclophosphamide (Cy) to inhibit regulatory T cells. CRS-207, live-attenuated Listeria monocytogenes–expressing mesothelin, induces innate and adaptive immunity. On the basis of preclinical synergy, we tested prime/boost vaccination with GVAX and CRS-207 in pancreatic adenocarcinoma. Patients and Methods Previously treated patients with metastatic pancreatic adenocarcinoma were randomly assigned at a ratio of 2:1 to two doses of Cy/GVAX followed by four doses of CRS-207 (arm A) or six doses of Cy/GVAX (arm B) every 3 weeks. Stable patients were offered additional courses. The primary end point was overall survival (OS) between arms. Secondary end points were safety and clinical response. Results A total of 90 patients were treated (arm A, n = 61; arm B, n = 29); 97% had received prior chemotherapy; 51% had received ≥ two regimens for metastatic disease. Mean number of doses (± standard deviation) administered in arms A and B were 5.5 ± 4.5 and 3.7 ± 2.2, respectively. The most frequent grade 3 to 4 related toxicities were transient fevers, lymphopenia, elevated liver enzymes, and fatigue. OS was 6.1 months in arm A versus 3.9 months in arm B (hazard ratio [HR], 0.59; P = .02). In a prespecified per-protocol analysis of patients who received at least three doses (two doses of Cy/GVAX plus one of CRS-207 or three of Cy/GVAX), OS was 9.7 versus 4.6 months (arm A v B; HR, 0.53; P = .02). Enhanced mesothelin-specific CD8 T-cell responses were associated with longer OS, regardless of treatment arm. Conclusion Heterologous prime/boost with Cy/GVAX and CRS-207 extended survival for patients with pancreatic cancer, with minimal toxicity. PMID:25584002

Safety and survival with GVAX pancreas prime and Listeria Monocytogenes-expressing mesothelin (CRS-207) boost vaccines for metastatic pancreatic cancer.

PubMed

Le, Dung T; Wang-Gillam, Andrea; Picozzi, Vincent; Greten, Tim F; Crocenzi, Todd; Springett, Gregory; Morse, Michael; Zeh, Herbert; Cohen, Deirdre; Fine, Robert L; Onners, Beth; Uram, Jennifer N; Laheru, Daniel A; Lutz, Eric R; Solt, Sara; Murphy, Aimee Luck; Skoble, Justin; Lemmens, Ed; Grous, John; Dubensky, Thomas; Brockstedt, Dirk G; Jaffee, Elizabeth M

2015-04-20

GVAX pancreas, granulocyte-macrophage colony-stimulating factor-secreting allogeneic pancreatic tumor cells, induces T-cell immunity to cancer antigens, including mesothelin. GVAX is administered with low-dose cyclophosphamide (Cy) to inhibit regulatory T cells. CRS-207, live-attenuated Listeria monocytogenes-expressing mesothelin, induces innate and adaptive immunity. On the basis of preclinical synergy, we tested prime/boost vaccination with GVAX and CRS-207 in pancreatic adenocarcinoma. Previously treated patients with metastatic pancreatic adenocarcinoma were randomly assigned at a ratio of 2:1 to two doses of Cy/GVAX followed by four doses of CRS-207 (arm A) or six doses of Cy/GVAX (arm B) every 3 weeks. Stable patients were offered additional courses. The primary end point was overall survival (OS) between arms. Secondary end points were safety and clinical response. A total of 90 patients were treated (arm A, n = 61; arm B, n = 29); 97% had received prior chemotherapy; 51% had received ≥ two regimens for metastatic disease. Mean number of doses (± standard deviation) administered in arms A and B were 5.5 ± 4.5 and 3.7 ± 2.2, respectively. The most frequent grade 3 to 4 related toxicities were transient fevers, lymphopenia, elevated liver enzymes, and fatigue. OS was 6.1 months in arm A versus 3.9 months in arm B (hazard ratio [HR], 0.59; P = .02). In a prespecified per-protocol analysis of patients who received at least three doses (two doses of Cy/GVAX plus one of CRS-207 or three of Cy/GVAX), OS was 9.7 versus 4.6 months (arm A v B; HR, 0.53; P = .02). Enhanced mesothelin-specific CD8 T-cell responses were associated with longer OS, regardless of treatment arm. Heterologous prime/boost with Cy/GVAX and CRS-207 extended survival for patients with pancreatic cancer, with minimal toxicity. © 2015 by American Society of Clinical Oncology.

[Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

PubMed

Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

2015-10-01

To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

PubMed

Loutre, Romuald; Heckel, Anne-Marie; Jeandard, Damien; Tarassov, Ivan; Entelis, Nina

2018-01-01

Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion

A protein in neuroblastoma could be a target of immunotoxins or immunotherapy | Center for Cancer Research

Cancer.gov

A cell surface protein, glycoprotein glypican-2 (GPC2), has been found to be an effective therapeutic target in cell cultures and mouse models that mimic childhood neuroblastoma.  The CCR scientists who made this discovery, reported July 24, 2017, in PNAS, have also produced immunotoxins and chimeric antigen receptor (CAR) T cells, a type of immunotherapy, that have shown

Ligand-free method to produce the anti-angiogenic recombinant Galectin-3 carbohydrate recognition domain.

PubMed

Wiecikowski, Adalgisa; Cabral, Katia Maria Dos Santos; Almeida, Marcius da Silva; Carvalho, Renato Sampaio

2018-04-01

Galectin-3 (Gal3) is involved in many physiological processes related to tumor growth, such as promoting angiogenesis, cell migration/invasion, resistance to apoptosis and immune response modulation. Usually the overexpression of Gal3 is a poor prognostic marker for cancer patients. Recombinant Gal3 carbohydrate domain (Gal3C) has been proposed as a useful tool to inhibit angiogenesis. So far, all production protocols reported for Gal3C production have used proteolytic cleavage of full length Gal3 and/or affinity-based purification. This involves dialysis, a time consuming step used to eliminate the elution ligand, usually lactose. In this report, we describe an alternative method to produce human recombinant Gal3C in E. coli, purified with cationic exchange and size exclusion chromatography. The recombinant protein was characterized using circular dichroism and nuclear magnetic resonance, showing a beta sheet enriched well-folded globular structure. The average yield obtained was 26 mg/L of broth and the purity was above 99%. The anti-angiogenic activity was assessed in vitro and showed a reduction of 70% and 77% in endothelial cells tubule formation upon treatment with 10 and 20 μg/mL, respectively and also had no impact on cell viability. The method described here is more suitable for both laboratory and industrial production of the potential anti-tumor Gal3C. Copyright © 2017 Elsevier Inc. All rights reserved.

A protein in neuroblastoma could be a target of immunotoxins or immunotherapy | Center for Cancer Research

Cancer.gov

A cell surface protein, glycoprotein glypican-2 (GPC2), has been found to be an effective therapeutic target in cell cultures and mouse models that mimic childhood neuroblastoma.  The CCR scientists who made this discovery, reported July 24, 2017, in PNAS, have also produced immunotoxins and chimeric antigen receptor (CAR) T cells, a type of immunotherapy, that have shown promise against this solid tumor. Read more...

A deimmunised form of the ribotoxin, α-sarcin, lacking CD4+ T cell epitopes and its use as an immunotoxin warhead

PubMed Central

Jones, Tim D.; Hearn, Arron R.; Holgate, Robert G.E.; Kozub, Dorota; Fogg, Mark H.; Carr, Francis J.; Baker, Matthew P.; Lacadena, Javier; Gehlsen, Kurt R.

2016-01-01

Fungal ribotoxins that block protein synthesis can be useful warheads in the context of a targeted immunotoxin. α-Sarcin is a small (17 kDa) fungal ribonuclease produced by Aspergillus giganteus that functions by catalytically cleaving a single phosphodiester bond in the sarcin–ricin loop of the large ribosomal subunit, thus making the ribosome unrecognisable to elongation factors and leading to inhibition of protein synthesis. Peptide mapping using an ex vivo human T cell assay determined that α-sarcin contained two T cell epitopes; one in the N-terminal 20 amino acids and the other in the C-terminal 20 amino acids. Various mutations were tested individually within each epitope and then in combination to isolate deimmunised α-sarcin variants that had the desired properties of silencing T cell epitopes and retention of the ability to inhibit protein synthesis (equivalent to wild-type, WT α-sarcin). A deimmunised variant (D9T/Q142T) demonstrated a complete lack of T cell activation in in vitro whole protein human T cell assays using peripheral blood mononuclear cells from donors with diverse HLA allotypes. Generation of an immunotoxin by fusion of the D9T/Q142T variant to a single-chain Fv targeting Her2 demonstrated potent cell killing equivalent to a fusion protein comprising the WT α-sarcin. These results represent the first fungal ribotoxin to be deimmunised with the potential to construct a new generation of deimmunised immunotoxin therapeutics. PMID:27578884

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning

PubMed Central

Klimka, A; Matthey, B; Roovers, R C; Barth, S; Arends, J-W; Engert, A; Hoogenboom, H R

2000-01-01

In various clinical studies, Hodgkin’s patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the non-human therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics. © 2000 Cancer Research Campaign PMID:10901379

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

Long term persistence of anti-HBs protective levels in young patients with type 1 diabetes after recombinant hepatitis B vaccine.

PubMed

Marseglia, G; Alibrandi, A; d'Annunzio, G; Gulminetti, R; Avanzini, M A; Marconi, M; Tinelli, C; Lorini, R

2000-11-22

The aim of the present study was to evaluate the persistence of anti-hepatitis B protective levels in young patients with type 1 diabetes, successfully immunised with a recombinant hepatitis B vaccine. We re-evaluated, after a 4 year follow-up, 54 patients and 70 age and sex-matched healthy subjects. Protective antibodies levels were found in 50/54 (92%) patients and in 67/70 (96%) controls. Moreover, anti-HBs levels were similar in diabetic patients and controls (means of log-titre and (sd); 1.95 (0.88) and 2.18 (0.64) patients and controls, respectively; P=0.11). No cases of clinical hepatitis were reported and all patients and controls remained HBc negative. These data demonstrate the persistence of anti-HBs levels in children, adolescents and young patients with type 1 diabetes after recombinant hepatitis B vaccine showing evidence of longterm immunogenity and protective effect.

[Bacterial recombinant L-asparaginases: properties, structure and anti-proliferative activity].

PubMed

Sokolov, N N; Eldarov, M A; Pokrovskaya, M V; Aleksandrova, S S; Abakumova, O Yu; Podobed, O V; Melik-Nubarov, N S; Kudryashova, E V; Grishin, D V; Archakov, A I

2015-01-01

For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.

Overexpression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo.

PubMed

Mahmoud, Salma; Ibrahim, Mohammed; Hago, Ahmed; Huang, Yuhong; Wei, Yuanyi; Zhang, Jun; Zhang, Qingqing; Xiao, Yu; Wang, Jingwen; Adam, Munkaila; Guo, Yu; Wang, Li; Zhou, Shuting; Xin, Boyi; Xuan, Wei; Tang, Jianwu

2016-11-15

Lymphatic vessels function as transport channels for tumor cells to metastasize from the primary site into the lymph nodes. In this experiment we evaluated the effect of Sulfatase-1 (Sulf-1) on metastasis by upregulating it in murine hepatocarcinoma cell line Hca-F with high lymph node metastatic rate of >75%. The study in vitro showed that up regulation of Sulf-1 in Hca-F cells significantly reduced cell proliferation, migration and invasion (p<0.05). Also, the forced expression of Sulf-1 down regulated Mesothelin (Msln) at both the protein and mRNA levels. The experiment in vivo further showed that up-regulation of Sulf-1 with the attendant downregulation of mesothelin delayed tumor growth and decreased lymph node metastasis. In conclusion, our findings show that Sulf-1 is an important tumor suppressor gene in hepatocellular carcinoma (HCC), and its over expression downregulates Msln and results in a decrease in HCC cell proliferation, migration, invasion, and lymphatic metastasis. This functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy.

Overexpression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo

PubMed Central

Mahmoud, Salma; Ibrahim, Mohammed; Hago, Ahmed; Huang, Yuhong; Wei, Yuanyi; Zhang, Jun; Zhang, Qingqing; Xiao, Yu; Wang, Jingwen; Adam, Munkaila; Guo, Yu; Wang, Li; Zhou, Shuting; Xin, Boyi; Xuan, Wei; Tang, Jianwu

2016-01-01

Lymphatic vessels function as transport channels for tumor cells to metastasize from the primary site into the lymph nodes. In this experiment we evaluated the effect of Sulfatase-1 (Sulf-1) on metastasis by upregulating it in murine hepatocarcinoma cell line Hca-F with high lymph node metastatic rate of >75%. The study in vitro showed that upregulation of Sulf-1 in Hca-F cells significantly reduced cell proliferation, migration and invasion (p<0.05). Also, the forced expression of Sulf-1 downregulated Mesothelin (Msln) at both the protein and mRNA levels. The experiment in vivo further showed that up-regulation of Sulf-1 with the attendant downregulation of mesothelin delayed tumor growth and decreased lymph node metastasis. In conclusion, our findings show that Sulf-1 is an important tumor suppressor gene in hepatocellular carcinoma (HCC), and its overexpression downregulates Msln and results in a decrease in HCC cell proliferation, migration, invasion, and lymphatic metastasis. This functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy. PMID:27626699

5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin.

PubMed

Lund, Kaja; Olsen, Cathrine Elisabeth; Wong, Judith Jing Wen; Olsen, Petter Angell; Solberg, Nina Therese; Høgset, Anders; Krauss, Stefan; Selbo, Pål Kristian

2017-12-19

Development of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma. 5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS 2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS 2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS 2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin. The 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS 2a , altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105

Enhanced Eradication of Lymphoma by Tumor-Specific Cytotoxic T Cells Secreting and Engineered Tumor-Specific Immunotoxin

DTIC Science & Technology

2009-06-01

target delivery of an immunotoxin, the CD22 -Pseudomonas exotoxin A ( CD22 -PEA), which has already been used in a clinical setting . The toxin portion...contains the transloc ating and ADP-ribosylat ing dom ains of PEA, and the native cell-binding portion is replaced with a CD22 scFv that directs...targeting to B lymphocytes. CD22 -PEA was tested in a Phase I trial in B-cell malignancies, but tumor responses, particularly in hairy cell leukemia

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

PubMed Central

Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

2017-01-01

Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors.

PubMed

Wels, Winfried; Biburger, Markus; Müller, Tina; Dälken, Benjamin; Giesübel, Ulrike; Tonn, Torsten; Uherek, Christoph

2004-03-01

Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cells' potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.

Anti-tumor immunotherapy by blockade of the PD-1/PD-L1 pathway with recombinant human PD-1-IgV.

PubMed

Zhang, C; Wu, S; Xue, X; Li, M; Qin, X; Li, W; Han, W; Zhang, Y

2008-01-01

Blockade of the programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway can delay tumor growth and prolong the survival of tumor-bearing mice. The extracellular immunoglobulin (Ig) V domain of PD-1 is important for the interaction between PD-1 and PD-L1, suggesting that PD-1-IgV may be a potential target for anti-tumor immunotherapy. The extracellular sequence of human PD-1-IgV (hPD-1-IgV) was expressed in Escherichia coli and purified. The anti-tumor effect of hPD-1-IgV on tumor-bearing mice was tested. hPD-1-IgV recombinant protein could bind PD-L1 at molecular and cellular levels and enhance Cytotoxic T Lymphocyte (CTL) activity and anti-tumor effect on tumor-bearing mice in vivo. The percentage of CD4(+)CD25(+) T cells in tumor-bearing mice was decreased compared with control mice after administration of the recombinant protein. Our results suggest that inhibition of the interaction between PD-1 and PD-L1 by hPD-1-IgV may be a promising strategy for specific tumor immunotherapy.

Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma

PubMed Central

Li, Nan; Fu, Haiying; Hewitt, Stephen M.; Dimitrov, Dimiter S.

2017-01-01

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma. PMID:28739923

Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

PubMed

Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

2017-08-08

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers: A Lethal Cocktail with Tumor Specific Efficacy

PubMed Central

Gilabert-Oriol, Roger; Weng, Alexander; von Mallinckrodt, Benedicta; Melzig, Matthias F; Fuchs, Hendrik; Thakur, Mayank

2014-01-01

The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success. PMID:25341935

Sulfatase-1 knockdown promotes in vitro and in vivo aggressive behavior of murine hepatocarcinoma Hca-P cells through up-regulation of mesothelin.

PubMed

Mahmoud, Salma Abdi; Ibrahim, Mohammed Mohammed; Musa, Ahmed Hago; Huang, Yuhong; Zhang, Jun; Wang, Jingwen; Wei, Yuanyi; Wang, Li; Zhou, Shunting; Xin, Boyi; Xuan, Wei; Tang, Jianwu

2017-12-23

Our previous study (Oncotarget 2016; 7:46) demonstrated that the over-expression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line (a murine HCC cell with lymph node metastatic [LNM] rate of >75%) downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo. In current work, we investigated the effects of Sulf-1 knockdown on mesothelin (Msln) and it's effects on the in vitro cell proliferation, migration, invasion, and in vivo tumor growth and LNM rate for Hca-P cells (a murine HCC cell with LNM rate of <25%). Western blotting and qRT-PCR assay indicated that both in vitro and in vivo Sulf-1 was down-regulated by 75% and 68% and led to up regulation of Msln by 55% in shRNA-transfected-Sulf-1-Hca-P cells compared with Hca-P and nonspecific sequence control plasmid transfected Hca-P cell (shRNA-Nc-Hca-P). The in vitro proliferation, migration and invasion potentials were significantly enhanced following Sulf-1 stable down-regulation. In addition, Sulf-1 knock-down significantly promoted tumor growth and increased LNM rates of shRNA-Sulf-1-Hca-P-transplanted mice by 78.6% (11 out of 14 lymph nodes were positive of cancer). Consistent with our previous work, we confirmed that Sulf-1 plays an important role in hepatocarcinoma cell proliferation, migration, invasion and metastasis. The interaction between Sulf-1 and Msln is a potential therapeutic target in the development of liver cancer therapy.

A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells.

PubMed

Gulce Iz, Sultan; Inevi, Muge Anil; Metiner, Pelin Saglam; Tamis, Duygu Ayyildiz; Kisbet, Nazli

2018-01-01

Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid

Biotechnical paving of recombinant enterocin A as the candidate of anti-Listeria agent.

PubMed

Hu, Xiaoyuan; Mao, Ruoyu; Zhang, Yong; Teng, Da; Wang, Xiumin; Xi, Di; Huang, Jianzhong; Wang, Jianhua

2014-08-28

Enterocin A is a classic IIa bacteriocin isolated firstly from Enterococcus faecium CTC492 with selective antimicrobial activity against Listeria strains. However, the application of enterocin A as an anti-Listeria agent has been limited due to its very low native yield. The present work describes high production of enterocin A through codon optimization strategy and its character study. The gene sequence of enterocin A was optimized based on preferential codon usage in Pichia pastoris to increase its expression efficiency. The highest anti-Listeria activity reached 51,200 AU/ml from 180 mg/l of total protein after 24 h of induction in a 5-L fermenter. Recombinant enterocin A (rEntA), purified by gel filtration chromatography, showed very strong activity against Listeria ivanovii ATCC 19119 with a low MIC of 20 ng/ml. In addition, the rEntA killed over 99% of tested L. ivanovii ATCC19119 within 4 h when exposed to 4 × MIC (80 ng/ml). Moreover, it showed high stability under a wide pH range (2-10) and maintained full activity after 1 h of treatment at 80°C within a pH range of 2-8. Its antimicrobial activity was enhanced at 25 and 50 mM NaCl, while 100-400 mM NaCl had little effect on the bactericidal ability of rEntA. The EntA was successfully expressed in P. pastoris, and this feasible system could pave the pre-industrial technological path of rEntA as a competent candidate as an anti-Listeria agent. Furthermore, it showed high stability under wide ranges of conditions, which could be potential as the new candidate of anti-Listeria agent.

RECOMBINATION OF ANTIBODY POLYPEPTIDE CHAINS IN THE PRESENCE OF ANTIGEN

PubMed Central

Metzger, Henry; Mannik, Mart

1964-01-01

Conditions were developed by which the separated H and L chains of gamma2 globulins recombined to form four-chained molecules in good yields. In the absence of antigen, anti-2,4-dinitrophenyl (anti-DNP) H chains randomly reassociated with a mixture of antibody and non-specific gamma2 globulin L chains. In the presence of a specific hapten, however, the antibody H chains preferentially interacted with the anti-DNP L chains. Antibody H chain-antibody L chain recombinants formed in the presence of hapten were more active than the corresponding recombinants formed in the absence of hapten. Speculations are made regarding the possible mechanisms and biological significance of these effects. PMID:14247718

Cytokinetic study of MCF-7 cells treated with commercial and recombinant bromelain.

PubMed

Fouz, Nour; Amid, Azura; Hashim, Yumi Zuhanis Has-Yun

2014-01-01

Breast cancer is a leading cause of death in women. The available chemotherapy drugs have been associated with many side effects. Bromelain has novel medicinal qualities including anti-inflammatory, anti-thrombotic, fibrinolytic and anti-cancer functions. Commercially available bromelain is obtained through tedious methods; therefore, recombinant bromelain may provide a cheaper and simpler choice with similar quality. This study aimed to assess the effects of commercial and recombinant bromelain on the cytokinetic behavior of MCF-7 breast cancer cells and their potential as therapeutic alternatives in cancer treatment. Cytotoxic activities of commercial and recombinant bromelain were determined using (sulforhodamine) SRB assay. Next, cell viability assays were conducted to determine effects of commercial and recombinant bromelain on MCF-7 cell cytokinetic behavior. Finally, the established growth kinetic data were used to modify a model that predicts the effects of commercial and recombinant bromelain on MCF-7 cells. Commercial and recombinant bromelain exerted strong effects towards decreasing the cell viability of MCF-7 cells with IC50 values of 5.13 μg/mL and 6.25 μg/mL, respectively, compared to taxol with an IC50 value of 0.063 μg/mL. The present results indicate that commercial and recombinant bromelain both have anti-proliferative activity, reduced the number of cell generations from 3.92 to 2.81 for commercial bromelain and to 2.86 for recombinant bromelain, while with taxol reduction was to 3.12. Microscopic observation of bromelain-treated MCF-7 cells demonstrated detachment. Inhibition activity was verified with growth rates decreased dynamically from 0.009 h-1 to 0.0059 h-1 for commercial bromelain and to 0.0063 h-1 for recombinant bromelain. Commercial and recombinant bromelain both affect cytokinetics of MCF-7 cells by decreasing cell viability, demonstrating similar strength to taxol.

Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

PubMed

Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P

2012-05-01

Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.

Capture of mesothelioma cells with 'universal' CTC-chip.

PubMed

Yoneda, Kazue; Chikaishi, Yasuhiro; Kuwata, Taiji; Ohnaga, Takashi; Tanaka, Fumihiro

2018-02-01

Malignant mesothelioma (MM) is a highly aggressive malignant tumor, predominantly associated with job-related exposure to asbestos. Development of effective and non-invasive modalities for diagnosis is an important issue in occupational medicine. Circulating tumor cells (CTCs), which are tumor cells that are shed from primary tumors and circulate in the peripheral blood, may be detected at an earlier stage than malignant tumors, and detection of CTCs may provide a novel insight into the diagnosis of MM. In a previous study evaluating clinical utility of CTCs, detected with a widely used system 'CellSearch', the authors indicated a significant however insufficient capability in the diagnosis of MM, suggesting need for a more sensitive system. Accordingly, the authors developed a novel microfluidic system to capture CTCs (CTC-chip), and demonstrated that the CTC-chip effectively captured MM cells (ACC-MESO-4) spiked in the blood by conjugating an anti-podoplanin antibody. The results of the present study demonstrated that the CTC-chip coated with the anti-podoplanin antibody captured another MM cell (ACC-MESO-1). However, the capture efficiencies were lower than those for ACC-MESO-4. In addition, an anti-mesothelin antibody was used to capture CTCs, however the CTC-chip coated with the anti-mesothelin antibody failed to effectively capture MM cells, possibly due to low mesothelin expression. Overall, the CTC-chip may capture specific types of CTCs by conjugating any antibody against an antigen expressed on CTCs, and may be a useful system for the diagnosis of malignant tumors, including MM.

A novel capture-ELISA for detection of anti-neutrophil cytoplasmic antibodies (ANCA) based on c-myc peptide recognition in carboxy-terminally tagged recombinant neutrophil serine proteases.

PubMed

Lee, Augustine S; Finkielman, Javier D; Peikert, Tobias; Hummel, Amber M; Viss, Margaret A; Specks, Ulrich

2005-12-20

Testing for antineutrophil cytoplasmic antibodies (ANCA) reacting with proteinase 3 (PR3) is part of the routine diagnostic evaluation of patients with small vessel vasculitis. For PR3-ANCA detection, capture ELISAs are reported to be superior to direct ELISAs. Standard capture ELISAs, in which PR3 is anchored by anti-PR3 monoclonal antibodies (moAB), have two potential disadvantages. First, the capturing moAB may compete for epitopes recognized by some PR3-ANCA, causing occasional false-negative results. Second, the capture of recombinant PR3 mutant molecules becomes unpredictable as modifications of specific conformational epitopes may not only affect the binding of PR3-ANCA, but also the affinity of the capturing anti-PR3 moAB. Here, we describe a new capture ELISA, and its application for PR3-ANCA detection. This new assay is based on the standardized capture of a variety of different carboxy-terminally c-myc tagged recombinant ANCA target antigens using anti-c-myc coated ELISA plates. Antigen used include c-myc tagged human rPR3 variants (mature and pro-form conformations), mouse mature rPR3 and human recombinant neutrophil elastase. This new anti-c-myc-capture ELISA for PR3-ANCA detection has an intra- and inter-assay coefficient of variation of 3.6% to 7.7%, and 15.8% to 18.4%, respectively. The analytical sensitivity and specificity for PR3-ANCA positive serum samples were 93% and 100%, respectively when rPR3 with mature conformation was used as target antigen, and 83% and 100% when the pro-enzyme conformation was employed. In conclusion, this new anti-c-myc capture ELISA compares favorably to our standard capture ELISA for PR3-ANCA detection, enables the unified capture of different ANCA target antigens through binding to a c-myc tag, and allows capture of rPR3 mutants necessary for PR3-ANCA epitope mapping studies.

Enhanced targeting of triple-negative breast carcinoma and malignant melanoma by photochemical internalization of CSPG4-targeting immunotoxins.

PubMed

Eng, M S; Kaur, J; Prasmickaite, L; Engesæter, B Ø; Weyergang, A; Skarpen, E; Berg, K; Rosenblum, M G; Mælandsmo, G M; Høgset, A; Ferrone, S; Selbo, P K

2018-05-16

Triple-negative breast cancer (TNBC) and malignant melanoma are highly aggressive cancers that widely express the cell surface chondroitin sulfate proteoglycan 4 (CSPG4/NG2). CSPG4 plays an important role in tumor cell growth and survival and promotes chemo- and radiotherapy resistance, suggesting that CSPG4 is an attractive target in cancer therapy. In the present work, we applied the drug delivery technology photochemical internalization (PCI) in combination with the novel CSPG4-targeting immunotoxin 225.28-saporin as an efficient and specific strategy to kill aggressive TNBC and amelanotic melanoma cells. Light-activation of the clinically relevant photosensitizer TPCS2a (fimaporfin) and 225.28-saporin was found to act in a synergistic manner, and was superior to both PCI of saporin and PCI-no-drug (TPCS2a + light only) in three TNBC cell lines (MDA-MB-231, MDA-MB-435 and SUM149) and two BRAFV600E mutated malignant melanoma cell lines (Melmet 1 and Melmet 5). The cytotoxic effect was highly dependent on the light dose and expression of CSPG4 since no enhanced cytotoxicity of PCI of 225.28-saporin compared to PCI of saporin was observed in the CSPG4-negative MCF-7 cells. The PCI of a smaller, and clinically relevant CSPG4-targeting toxin (scFvMEL-rGel) validated the CSPG4-targeting concept in vitro and induced a strong inhibition of tumor growth in the amelanotic melanoma xenograft A-375 model. In conclusion, the combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.

76 FR 71048 - Sixth Annual Philip S. Chen, Jr. Distinguished Lecture on Innovation and Technology Transfer

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-16

..., Jr. Distinguished Lecture on Innovation and Technology Transfer AGENCY: National Institutes of Health... sixth annual Philip S. Chen, Jr., Ph.D. Distinguished Lecture on Innovation and Technology Transfer... present ``Treatment of Cancer with Recombinant Immunotoxins: From Technology Transfer to the Patient.'' Dr...

Recombinant expression of the alternate reading frame protein (ARFP) of hepatitis C virus genotype 4a (HCV-4a) and detection of ARFP and anti-ARFP antibodies in HCV-infected patients.

PubMed

Shehat, Michael G; Bahey-El-Din, Mohammed; Kassem, Mervat A; Farghaly, Faten A; Abdul-Rahman, Medhat H; Fanaki, Nourhan H

2015-08-01

HCV is a single-stranded RNA virus with a single open reading frame (ORF) that is translated into a polyprotein that is then processed to form 10 viral proteins. An additional eleventh viral protein, the alternative reading frame protein (ARFP), was discovered relatively recently. This protein results from a translational frameshift in the core region during the expression of the viral proteins. Recombinant expression of different forms of ARFP was previously done for HCV genotypes 1 and 2, and more recently, genotype 3. However, none of the previous studies addressed the expression of ARFP of HCV genotype 4a, which is responsible for 80 % of HCV infections in the Middle East and Africa. Moreover, the direct detection of the ARFP antigen in HCV-infected patients was never studied before for any HCV genotype. In the present study, recombinant ARFP derived from HCV genotype 4a was successfully expressed in E. coli and purified using metal affinity chromatography. The recombinant ARFP protein and anti-ARFP antibodies were used for detection of ARFP antigen in patients' sera, employing competitive enzyme-linked immunosorbent assay (ELISA) procedures. Furthermore, the recombinant antigen was also used to detect and quantify anti-ARFP antibodies in HCV-infected Egyptian patients at different stages of pegylated interferon/ribavirin therapy, using an ELISA assay. The ARFP antigen was detectable in 69.4 % of RNA-positive sera, indicating that ARFP antigen is produced during the natural course of HCV infection. In addition, significant levels of anti-ARFP antibodies were present in 41 % of the serum samples tested. The important diagnostic value of the recombinant ARFP antigen was also demonstrated.

Evolution of collagen arthritis in mice is arrested by treatment with anti-tumour necrosis factor (TNF) antibody or a recombinant soluble TNF receptor.

PubMed Central

Piguet, P F; Grau, G E; Vesin, C; Loetscher, H; Gentz, R; Lesslauer, W

1992-01-01

Immunization of DBA/1 mice with type II collagen within complete Freund's adjuvant leads to arthritis, lasting more than 3 months. Injection of anti-tumour necrosis factor (TNF) IgG, 2 and 3 weeks after immunization prevented the development of arthritis in the following months. This treatment had no effect when started 2 months after induction of the disease. A soluble form of the human recombinant TNF receptor type-beta (rsTNFR-beta), continuously infused at a rate of 20 micrograms/day during the second and third week after immunization, also had a long-term protective effect. Anti-TNF antibody had no effect upon the production of anti-type II collagen antibodies. These results indicate that TNF is critically involved in an early phase of this arthritis. Images Figure 1 Figure 2 PMID:1337334

Anti-Tumor Activity of Cytotoxic T Lymphocytes Elicited with Recombinant and Synthetic Forms of a Model Tumor-Associated Antigen

PubMed Central

Wang, Michael; Chen, Pauline W.; Bronte, Vincenzo; Rosenberg, Steven A.; Restifo, Nicholas P.

2008-01-01

Summary The recent cloning of tumor-associated antigens (TAAs) recognized by CD8 + T lymphocytes (TCD8−) has made it possible to use recombinant and synthetic forms of TAAs to generate TCD8− with anti-tumor activity. To explore new therapeutic strategies in a mouse model, we retrovirally transduced the experimental murine tumor CT26 (H-2d), with the lacZ gene encoding our model TAA, (β-galactosidase (β-gal). The transduced cell line, CT26.CL25, grew as rapidly and as lethally as the parental cell line in normal, immuno-competent animals. In an attempt to elicit TCD8+ directed against our model TAA by using purely recombinant and synthetic forms of our model TAA, we synthesized a nine-amino-acid long immunodominant peptide of (β-gal (TPH-PARIGL), corresponding to amino acid residues 876–884, which was known to be presented by the Ld major histocompatibility complex (MHC) class I molecule, and a recombinant vaccinia virus encoding the full-length β-gal protein (VJS6). Splenocytes obtained from naïve mice and co-cultured with (β-gal peptide could not be expanded in primary ex vivo cultures. However, mice immunized with VJS6, but not with a control recombinant vaccinia virus, yielded splenocytes that were capable of specifically lysing CT26.CL25 in vitro after co-culture with (β-gal peptide. Most significantly, adoptive transfer of these cells could effectively treat mice bearing 3-day-old established pulmonary metastases. These observations show that therapeutic TCD8+ directed against a model TAA could be generated by using purely recombinant and synthetic forms of this antigen. These findings point the way to a potentially useful immunotherapeutic strategy, which has been made possible by the recent cloning of immunogenic TAAs that are expressed by human malignancies. PMID:8770769

Photochemical internalization (PCI) of immunotoxins targeting CD133 is specific and highly potent at femtomolar levels in cells with cancer stem cell properties.

PubMed

Bostad, Monica; Berg, Kristian; Høgset, Anders; Skarpen, Ellen; Stenmark, Harald; Selbo, Pål K

2013-06-28

CD133 is a putative cancer stem cell (CSC) marker for a number of different cancers and is suggested to be a therapeutic target. Since also normal stem cells express CD133 it is of paramount importance that targeting strategies provide a specific and efficient delivery of cytotoxic drugs in only CD133-positive CSCs. In this study, we have employed photochemical internalization (PCI), a minimally invasive method for light-controlled, specific delivery of membrane-impermeable macromolecules from endocytic vesicles to the cytosol, to specifically target CD133-positive cancer cells. We demonstrate that PCI increases the cytotoxic effect of an immunotoxin (IT) targeting CD133-expressing cancer cells of colon (WiDr and HCT116) and pancreas (BxPC-3) origin. The IT consisted of the mAb CD133/1 (AC133) bound to the ribosome inactivating plant toxin saporin (anti-CD133/1-sap). We show that TPCS2a-PCI of anti-CD133/1-sap is specific, and highly cytotoxic at femto-molar concentrations. Specific binding and uptake of CD133/1, was shown by fluorescence microscopy and co-localization with TPCS2a in endosomes/lysosomes was determined by confocal microscopy. CD133(high) WiDr cells, isolated by fluorescence activated cell sorting, had a 7-fold higher capacity to initiate spheroids than CD133(low) cells (P<0.001) and were resistant to photodynamic therapy (PDT). However, PDT-resistance was bypassed by the PCI strategy. Tumor initiation and aggressive growth in athymic nude mice was obtained with only 10 CD133(high) cells in contrast to CD133(low) cells where substantially higher cell numbers were needed. The excellent high efficacy and selectivity of eliminating CD133-expressing cells by PCI warrant further pre-clinical evaluations of this novel therapeutic approach. Copyright © 2013 Elsevier B.V. All rights reserved.

A novel recombinant anti-epidermal growth factor receptor peptide vaccine capable of active immunization and reduction of tumor volume in a mouse model.

PubMed

Asadi-Ghalehni, Majid; Rasaee, Mohamad Javad; RajabiBazl, Masoumeh; Khosravani, Masood; Motaghinejad, Majid; Javanmardi, Masoud; Khalili, Saeed; Modjtahedi, Helmout; Sadroddiny, Esmaeil

2017-12-01

Over-expression of epidermal growth factor receptor (EGFR) has been reported in a number of human malignancies. Strong expression of this receptor has been associated with poor survival in many such patients. Active immunizations that elicit antibodies of the desired type could be an appealing alternative to conventional passive immunization. In this regard, a novel recombinant peptide vaccine capable of prophylactic and therapeutic effects was constructed. A novel fusion recombinant peptide base vaccine consisting of L2 domain of murine extra-cellular domain-EGFR and EGFR mimotope (EM-L2) was constructed and its prophylactic and therapeutic effects in a Lewis lung carcinoma mouse (C57/BL6) model evaluated. Constructed recombinant peptide vaccine is capable of reacting with anti-EGFR antibodies. Immunization of mice with EM-L2 peptide resulted in antibody production against EM-L2. The constructed recombinant peptide vaccine reduced tumor growth and increased the survival rate. Designing effective peptide vaccines could be an encouraging strategy in contemporary cancer immunotherapy. Investigating the efficacy of such cancer immunotherapy approaches may open exciting possibilities concerning hyperimmunization, leading to more promising effects on tumor regression and proliferation. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Cloning and expression of recombinant, functional ricin B chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, M.S.; Russell, D.W.; Uhr, J.W.

1987-08-01

The cDNA encoding the B chain of the plant toxin ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with (/sup 35/S)methionine and (/sup 35/S)-cysteine and demonstrating the secretion of a protein with a M/sub r/ of 30,000-32,000 that was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B-chain antibody and the amount of recombinant B chain secreted by the COS-M6 cells was determined by a radioimmunoassay. Virtually all of the recombinant B chain formed active ricinmore » when mixed with native A chain; it could also bind to the galactose-containing glycoprotein asialofetuin as effectively as native B chain.These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function« less

Detection of potential (anti)progestagenic endocrine disruptors using a recombinant human progesterone receptor binding and transactivation assay.

PubMed

Viswanath, Gunda; Halder, Sujata; Divya, Gunda; Majumder, Chandrajeet B; Roy, Partha

2008-11-25

The present work describes the identification of (anti)progestin endocrine disrupting chemicals (EDC) using a two step screening system. In the first step a competitive binding assay was developed using recombinant human progesterone receptor (hPR). The tested chemicals were of various classes like insecticides, their metabolites, industrial chemicals and waste water treatment plant (WWTP) effluents. All the tested chemicals demonstrated a high affinity binding for hPR. The average IC50 values of the test chemicals were within the range of 1-25microM. In the second step of screening, a mammalian cell-based hPR transactivation assay was developed where HEK 293 cells were co-transfected with hPR and luciferase reporter gene under the control of progesterone-response element. Stimulation of the cells with progesterone resulted in about 25-fold up regulation of luciferase activity, with EC50 value of 4nM. Potent anti-progesterone, RU486, significantly inhibited progesterone-induced transactivation and non-progestagenic steroids failed to transactivate hPR till 1microM concentrations. The chemicals showing high binding affinities in competitive binding assays were then tested in transactivation assay and all of them were found to be anti-progestative except WWTP effluents. Transactivation assays using extracted water samples from five different WWTP effluents showed that it was rich in progestative compounds. The levels of induction caused by these effluents were in the range of 15-25% of induction by progesterone and they represented about 6ng/l equivalent progesterone activities. In conclusion, we demonstrated that this two step assay provides an efficient screening tool for the detection of (anti)progestative EDC in various samples.

Recombinant Listeria monocytogenes as a Live Vaccine Vehicle for the Induction of Protective Anti-Viral Cell-Mediated Immunity

NASA Astrophysics Data System (ADS)

Shen, Hao; Slifka, Mark K.; Matloubian, Mehrdad; Jensen, Eric R.; Ahmed, Rafi; Miller, Jeff F.

1995-04-01

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2L^d-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8^+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8^+ T cells.

Improved Therapy by Pretargeted Radioimmunotherapy of Non-Hodgkin Lymphoma with a New Recombinant, Trivalent, Anti-CD20, Bispecific Antibody

PubMed Central

Sharkey, Robert M.; Karacay, Habibe; Litwin, Samuel; Rossi, Edmund A.; McBride, William J.; Chang, Chein-Hsing; Goldenberg, David M.

2008-01-01

We examined whether a pretargeting method using a new, recombinant anti-CD20 bispecific antibody (bsMAb) followed by 90Y-DOTA-peptide could reduce hematological toxicity yet improve therapeutic responses, compared to conventional 90Y-anti-CD20 IgG and a chemically-conjugated bsMAb. TF4, a humanized, tri-Fab, bsMAb with 2 Fabs binding CD20 and 1 Fab binding HSG (histamine-succinyl-glycine), developed by the Dock-and-Lock (DNL) method, was tested in nude mice with Ramos B-cell lymphomas. Optimal pretargeting required a 29-h interval between TF4 and 90Y-DOTA-HSG, and 20-fold more moles of TF4. TF4 cleared more rapidly from the blood than anti-CD20 IgG, with early processing in the liver, spleen and kidneys. At 24 h, TF4 improved tumor uptake of 111In-HSG-peptide 2.6-fold (13% vs 5% injected-dose/g) and enhanced tumor/blood ratios more than 45-fold (770 vs 17), compared to an anti-CD20 Fab x anti-HSG Fab chemical conjugate, and by 1.6-fold (9.0% vs 5.6% injected-dose/g) and 1600-fold (522 vs 0.32), respectively, compared to radiolabeled anti-CD20 IgG. A severe (≥90%) and prolonged reduction of white blood cells was observed at the maximum dose of 90Y-anti-CD20 IgG, whereas pretargeting resulted in a ≤60% transient drop. TF4-pretargeting resulted in highly significant improvement in survival, curing 33-90% of the animals, even at relatively low doses, while most tumors progressed quickly without cures with 90Y-anti-CD20 IgG. These results indicate an improved therapeutic index with pretargeting radioimmunotherapy using a DNL-constructed tri-Fab, bsMAb, as compared to conventional therapy with directly-radiolabeled antibody or with a chemically-conjugated bsMAb. These encouraging results prompt testing of these constructs for pretargeting radioimmunotherapy in patients. PMID:18593929

In vivo anti-tumour activity of recombinant MVM parvoviral vectors carrying the human interleukin-2 cDNA.

PubMed

El Bakkouri, Karim; Servais, Charlotte; Clément, Nathalie; Cheong, Siew Chiat; Franssen, Jean-Denis; Velu, Thierry; Brandenburger, Annick

2005-02-01

The natural oncotropism and oncotoxicity of vectors derived from the autonomous parvovirus, minute virus of mice (prototype strain) [MVM(p)], combined with the immunotherapeutic properties of cytokine transgenes, make them interesting candidates for cancer gene therapy. The in vivo anti-tumour activity of a recombinant parvoviral vector, MVM-IL2, was evaluated in a syngeneic mouse melanoma model that is relatively resistant in vitro to the intrinsic cytotoxicity of wild-type MVM(p). In vitro infection of the K1735 melanoma cells prior to their injection resulted in loss of tumorigenicity in 70% of mice (7/10). Tumour-free mice were protected against a challenge with non-infected parental cells. In addition, MVM-IL2-infected tumour cells induced an anti-tumour activity on parental cells injected at a distant location. These non-infected tumour cells were injected either at the same time or 7 days before the injection of MVM-IL2-infected cells. In the latter setting, which mimics a therapeutic model for small tumours, 4/10 mice were still tumour-free after 4 months. Our results show that (i) the MVM-IL2 parvoviral vector efficiently transduces tumour cells; and (ii) the low multiplicity of infection (MOI = 1) used in our experiments was sufficient to elicit an anti-tumour effect on distant cells, which supports further studies on this vector as a new tool for cancer gene therapy. Copyright (c) 2004 John Wiley & Sons, Ltd.

In vivo characterization of fusion protein comprising of A1 subunit of Shiga toxin and human GM-CSF: Assessment of its immunogenicity and toxicity.

PubMed

Oloomi, Mana; Bouzari, Saeid; Shariati, Elaheh

2010-10-01

Most cancer cells become resistant to anti-cancer agents. In the last few years, a new approach for targeted therapy of human cancer has been developed using immunotoxins which comprise both the cell targeting and the cell killing moieties. In the present study, the recombinant Shiga toxin A1 subunit fused to human granulocyte-macrophage colony stimulating factor (A1-GM-CSF), previously produced in E. coli, was further characterized. The recombinant protein could cause 50% cytotoxicity and induced apoptosis in cells bearing GM-CSF receptors. The non-specific toxicity of the fusion protein was assessed in C57BL/6 and BALB/c mice. No mortality was observed in either group of mice, with different concentration of fusion protein. The lymphocyte proliferation assay, induction of specific IgG response and a mixed (Th1/Th2) response were observed only in BALB/c mice. The mixed response in BALB/c mice (Th1/Th2) could be explained on the basis of the two components of the fusion protein i.e. A1 and GM-CSF.

Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities.

PubMed

Niesen, Judith; Stein, Christoph; Brehm, Hannes; Hehmann-Titt, Grit; Fendel, Rolf; Melmer, Georg; Fischer, Rainer; Barth, Stefan

2015-12-01

The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. EGFR-specific monoclonal antibodies (mAbs), such as cetuximab and panitumumab, have been approved for the treatment of colorectal and head and neck cancer. To increase tissue penetration, we constructed single-chain fragment variable (scFv) antibodies derived from these mAbs and evaluated their potential for targeted cancer therapy. The resulting scFv-based EGFR-specific immunotoxins (ITs) combine target specificity of the full-size mAb with the cell-killing activity of a toxic effector domain, a truncated version of Pseudomonas exotoxin A (ETA'). The ITs and corresponding imaging probes were tested in vitro against four solid tumor entities (rhabdomyosarcoma, breast, prostate and pancreatic cancer). Specific binding and internalization of the ITs scFv2112-ETA' (from cetuximab) and scFv1711-ETA' (from panitumumab) were demonstrated by flow cytometry and for the scFv-SNAP-tag imaging probes by live cell imaging. Cytotoxic potential of the ITs was analyzed in cell viability and apoptosis assays. Binding of the ITs was proofed ex vivo on rhabdomyosarcoma, prostate and breast cancer formalin-fixed paraffin-embedded biopsies. Both novel ITs showed significant pro-apoptotic and anti-proliferative effects toward the target cells, achieving IC50 values of 4 pM (high EGFR expression) to 460 pM (moderate EGFR expression). Additionally, rapid internalization and specific in vitro and ex vivo binding on patient tissue were confirmed. These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA'-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes.

T-cell receptor BV gene usage in colorectal carcinoma patients immunised with recombinant Ep-CAM protein or anti-idiotypic antibody.

PubMed

Mosolits, Szilvia; Markovic, Katja; Fagerberg, Jan; Frödin, Jan-Erik; Rezvany, Mohammad-Reza; Kiaii, Shahryar; Mellstedt, Håkan; Jeddi-Tehrani, Mahmood

2005-06-01

The tumour-associated antigen, Ep-CAM, is over-expressed in colorectal carcinoma (CRC). In the present study, a recombinant Ep-CAM protein or a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM, either alone or in combination, was used for vaccination of CRC patients (n=9). GM-CSF was given as an adjuvant cytokine. A cellular immune response was assessed by measuring anti-Ep-CAM lymphoproliferation, IFN-gamma production (ELISPOT) and by analysing the TCR BV gene usage within the CD4+ and CD8+ T-cell subsets followed by CDR3 fragment analysis. A proliferative and/or IFN-gamma T-cell response was induced against the Ep-CAM protein in eight out of nine patients, and against Ep-CAM-derived peptides in nine out of nine patients. Analysis of the TCR BV gene usage showed a significantly higher usage of BV12 family in CD4+ T cells of patients both before and after immunisation than in those of healthy control donors (p<0.05). In the CD8+ T-cell subset, a significant (p<0.05) increase in the BV19 usage was noted in patients after immunisation. In individual patients, a number of TCR BV gene families in both CD4+ and CD8+ T cells were over-expressed mainly in post-immunisation samples. Analysis of the CDR3 length polymorphism revealed a higher degree of clonality in post-immunisation samples than in pre-immunisation samples. In vitro stimulation with Ep-CAM protein confirmed the expansion of anti-Ep-CAM T-cell clones. The results indicate that immunisation with the Ep-CAM protein and/or anti-Id entails the induction of an anti-Ep-CAM T-cell response in CRC patients, and suggest that BV19+ CD8+ T cells might be involved in a vaccine-induced immune response.

When No Response Is a Good Thing | Center for Cancer Research

Cancer.gov

Custom-designed therapies that target cell-surface antigens or receptors represent a promising immunological approach in cancer therapy. Antibodies that bind these targets are the starting point.  Potent toxins can then be added to them by fusing antibody fragments to powerful bacterial toxins such as Pseudomonas exotoxin (PE). This recombinant immunotoxin combines antibody

Secapin, a bee venom peptide, exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities.

PubMed

Lee, Kwang Sik; Kim, Bo Yeon; Yoon, Hyung Joo; Choi, Yong Soo; Jin, Byung Rae

2016-10-01

Bee venom contains a variety of peptide constituents that have various biological, toxicological, and pharmacological actions. However, the biological actions of secapin, a venom peptide in bee venom, remain largely unknown. Here, we provide the evidence that Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. The recombinant mature AcSecapin-1 peptide was expressed in baculovirus-infected insect cells. AcSecapin-1 functions as a serine protease inhibitor-like peptide that has inhibitory effects against plasmin, elastases, microbial serine proteases, trypsin, and chymotrypsin. Consistent with these functions, AcSecapin-1 inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products, thus indicating the role of AcSecapin-1 as an anti-fibrinolytic agent. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi and gram-positive and gram-negative bacteria. Taken together, our data demonstrated that the bee venom peptide secapin has multifunctional roles as an anti-fibrinolytic agent during fibrinolysis and an anti-microbial agent in the innate immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anti-influenza M2e antibody

DOEpatents

Bradbury, Andrew M.

2013-04-16

Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

Anti-influenza M2e antibody

DOEpatents

Bradbury, Andrew M [Santa Fe, NM

2011-12-20

Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

Autoantibody detection in type 2 autoimmune hepatitis using a chimera recombinant protein.

PubMed

Vitozzi, Susana; Lapierre, Pascal; Djilali-Saiah, Idriss; Alvarez, Fernando

2002-04-01

Autoantibodies against cytochrome P450 2D6 (CYP2D6), known as anti-liver/kidney microsome type 1 (LKM1) and/or anti-human formiminotransferase cyclodeaminase, formally known as anti-liver cytosol type 1 (LC1) define type 2 autoimmune hepatitis (AIH). The aims of this work are to develop a sensitive and specific test to detect anti-LKM1 and/or anti-LC1 autoantibodies and to establish the prevalence of anti-LC1. Sera from children with type 2 AIH (n=48) and those from a control group (n=100) were evaluated for anti-LKM1 and anti-LC1 by Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. Each serum sample was assayed for reactivity against formiminotransferase cyclodeaminase and CYP2D6 alone or as part of a recombinant chimera protein. By ELISA with recombinant chimera protein, 50 serum samples were positive, 48 from patients with type 2 AIH and 2 from patients with chronic hepatitis C. Twenty-five of 48 (52%) patients studied were positive for both CYP2D6 and LC1 autoantibodies. Anti-LC1, either as the only marker or associated with anti-LKM1, was positive in 34/48 (71%). By Western blotting, anti-LC1 was found in 27/48 (56%) patients. This ELISA technique has proven to be antigen-specific and more sensitive than Western blot for the detection of anti-LC1 and anti-LKM1 autoantibodies. The prevalence of anti-LC1 (71%) confirms it as an important immunomarker in type 2 AIH.

Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.

PubMed Central

Bae, Y S; Kawasaki, I; Ikeda, H; Liu, L F

1988-01-01

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes. Images PMID:2832845

Role of mesothelin in carbon nanotube-induced carcinogenic transformation of human bronchial epithelial cells

PubMed Central

He, Xiaoqing; Despeaux, Emily; Stueckle, Todd A.; Chi, Alexander; Castranova, Vincent; Dinu, Cerasela Zoica; Wang, Liying

2016-01-01

Carbon nanotubes (CNTs) have been likened to asbestos in terms of morphology and toxicity. CNT exposure can lead to pulmonary fibrosis and promotion of tumorigenesis. However, the mechanisms underlying CNT-induced carcinogenesis are not well defined. Mesothelin (MSLN) is overexpressed in many human tumors, including mesotheliomas and pancreatic and ovarian carcinomas. In this study, the role of MSLN in the carcinogenic transformation of human bronchial epithelial cells chronically exposed to single-walled CNT (BSW) was investigated. MSLN overexpression was found in human lung tumors, lung cancer cell lines, and BSW cells. The functional role of MSLN in the BSW cells was then investigated by using stably transfected MSLN knockdown (BSW shMSLN) cells. MSLN knockdown resulted in significantly decreased invasion, migration, colonies on soft agar, and tumor sphere formation. In vivo, BSW shMSLN cells formed smaller primary tumors and less metastases. The mechanism by which MSLN contributes to these more aggressive behaviors was investigated by using ingenuity pathway analysis, which predicted that increased MSLN could induce cyclin E expression. We found that BSW shMSLN cells had decreased cyclin E, and their proliferation rate was reverted to nearly that of untransformed cells. Cell cycle analysis showed that the BSW shMSLN cells had an increased G2 population and a decreased S phase population, which is consistent with the decreased rate of proliferation. Together, our results indicate a novel role of MSLN in the malignant transformation of bronchial epithelial cells following CNT exposure, suggesting its utility as a potential biomarker and drug target for CNT-induced malignancies. PMID:27422997

Transient expression of CCL21as recombinant protein in tomato.

PubMed

Beihaghi, Maria; Marashi, Hasan; Bagheri, Abdolreza; Sankian, Mojtaba

2018-03-01

The main goal of this study was to investigate the possibility of expressing recombinant protein of C-C chemokine ligand 21 (CCL21) in Solanum lycopersicum via agroinfiltration. CCL21 is a chemokine can be used for anti-metastatic of cancer cell lines. To examine the expression of CCL21 protein in S. lycopersicum , the construct of ccl21 was synthesized. This construct was cloned into pBI121 and the resulting CCL21 plasmid was agro-infiltrated into S. lycopersicum leaves. Within three days after infiltration, Expression of the foreign gene was confirmed by quantitative Real-time PCR. A recombinant CCL21 protein was immunogenically detected by western blot, dot blot and ELISA assay. And results showed that the foreign gene was expressed in the transformed leaves in high level. Also scratch assay was used to investigate the role of this protein in anti-metastatic function. The results demonstrated anti-metastatic of cancer cells in the presence of this protein.

Membrane-proximal TRAIL species are incapable of inducing short circuit apoptosis signaling: Implications for drug development and basic cytokine biology.

PubMed

Tatzel, Katharina; Kuroki, Lindsay; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Goedegebuure, S Peter; Powell, Matthew A; Mutch, David G; Hawkins, William G; Spitzer, Dirk

2016-03-03

TRAIL continues to garner substantial interest as a recombinant cancer therapeutic while the native cytokine itself serves important tumor surveillance functions when expressed in membrane-anchored form on activated immune effector cells. We have recently developed the genetically stabilized TRAIL platform TR3 in efforts to improve the limitations associated with currently available drug variants. While in the process of characterizing mesothelin-targeted TR3 variants using a single chain antibody (scFv) delivery format (SS-TR3), we discovered that the membrane-tethered cytokine had a substantially increased activity profile compared to non-targeted TR3. However, cell death proceeded exclusively via a bystander mechanism and protected the mesothelin-positive targets from apoptosis rather than leading to their elimination. Incorporation of a spacer-into the mesothelin surface antigen or the cancer drug itself-converted SS-TR3 into a cis-acting phenotype. Further experiments with membrane-anchored TR3 variants and the native cytokine confirmed our hypothesis that membrane-proximal TRAIL species lack the capacity to physically engage their cognate receptors coexpressed on the same cell membrane. Our findings not only provide an explanation for the "peaceful" coexistence of ligand and receptor of a representative member of the TNF superfamily but give us vital clues for the design of activity-enhanced TR3-based cancer therapeutics.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Hitoshi; Akazawa, Daisuke; Toray Industries, Inc., Kanagawa

2010-05-14

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K.more » Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.« less

A reevaluation of CD22 expression in human lung cancer.

PubMed

Pop, Laurentiu M; Barman, Stephen; Shao, Chunli; Poe, Jonathan C; Venturi, Guglielmo M; Shelton, John M; Pop, Iliodora V; Gerber, David E; Girard, Luc; Liu, Xiao-yun; Behrens, Carmen; Rodriguez-Canales, Jaime; Liu, Hui; Wistuba, Ignacio I; Richardson, James A; Minna, John D; Tedder, Thomas F; Vitetta, Ellen S

2014-01-01

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B-cell receptor and its coreceptor CD19. Recent reports indicate that most human lung cancer cells and cell lines express CD22, making it an important new therapeutic target for lung cancer. The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by quantitative real-time PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200 to 60,000-fold lower than those observed in the human CD22(+) Burkitt lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by either CD22 antibodies or our highly potent anti-CD22 immunotoxin. In contrast, CD22(+) Daudi cells expressed high levels of CD22 mRNA and protein, and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from more than 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells, and that these cells cannot be killed by anti-CD22 immunotoxins.

A Re-evaluation of CD22 Expression by Human Lung Cancer

PubMed Central

Pop, Laurentiu M.; Barman, Stephen; Shao, Chunli; Poe, Jonathan C.; Venturi, Guglielmo M.; Shelton, John M.; Pop, Iliodora V.; Gerber, David E.; Girard, Luc; Liu, Xiao-yun; Behrens, Carmen; Rodriguez-Canales, Jaime; Liu, Hui; Wistuba, Ignacio I.; Richardson, James A.; Minna, John D.; Tedder, Thomas F.; Vitetta, Ellen S.

2014-01-01

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B cell receptor and its co-receptor CD19. Recently it was reported that most human lung cancer cells and cell lines express CD22 making it an important new lung cancer therapeutic target (Can Res 72:5556, 2012). The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by qRT-PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200–60,000- fold lower than those observed in the human CD22+ Burkitt’s lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by CD22 antibodies or our highly potent anti-CD22 immunotoxin. By contrast, CD22+ Daudi cells expressed high levels of CD22 mRNA and protein and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from over 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells and that these cells can not be killed by anti-CD22 immunotoxins. PMID:24395821

IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8⁺ T cell avidity following HIV-1 recombinant pox viral vaccination.

PubMed

Wijesundara, Danushka K; Jackson, Ronald J; Tscharke, David C; Ranasinghe, Charani

2013-09-23

We have shown that mucosal HIV-1 recombinant pox viral vaccination can induce high, avidity HIV-specific CD8(+) T cells with reduced interleukin (IL)-4 and IL-13 expression compared to, systemic vaccine delivery. In the current study how these cytokines act to regulate anti-viral CD8(+) T, cell avidity following HIV-1 recombinant pox viral prime-boost vaccination was investigated. Out of a panel of T cell avidity markers tested, only CD8 expression levels were found to be enhanced on, KdGag197-205 (HIV)-specific CD8(+) T cells obtained from IL-13(-/-), IL-4(-/-) and signal transducer and, activator of transcription of 6 (STAT6)(-/-) mice compared to wild-type (WT) controls following, vaccination. Elevated CD8 expression levels in this instance also correlated with polyfunctionality, (interferon (IFN)-γ, tumour necorsis factor (TNF)-α and IL-2 production) and the avidity of HIVspecific CD8(+) T cells. Furthermore, mucosal vaccination and vaccination with the novel adjuvanted IL-13 inhibitor (i.e. IL-13Rα2) vaccines significantly enhanced CD8 expression levels on HIV-specific CD8(+), T cells, which correlated with avidity. Using anti-CD8 antibodies that blocked CD8 availability on CD8(+), T cells, it was established that CD8 played an important role in increasing HIV-specific CD8(+) T cell avidity and polyfunctionality in IL-4(-/-), IL-13(-/-) and STAT6(-/-) mice compared to WT controls, following vaccination. Collectively, our data demonstrate that IL-4 and IL-13 dampen CD8 expression levels on anti-viral CD8(+) T cells, which can down-regulate anti-viral CD8(+) T cell avidity and, polyfunctionality following HIV-1 recombinant pox viral vaccination. These findings can be exploited to, design more efficacious vaccines not only against HIV-1, but many chronic infections where high, avidity CD8(+) T cells help protection. Copyright © 2013 Elsevier Ltd. All rights reserved.

Recombinant anti-tenascin antibody constructs

DOE Office of Scientific and Technical Information (OSTI.GOV)

ZALUTSKY, MICHAEL R

2006-08-29

The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploitmore » our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr -particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti

When No Response Is a Good Thing | Center for Cancer Research

Cancer.gov

Custom-designed therapies that target cell-surface antigens or receptors represent a promising immunological approach in cancer therapy. Antibodies that bind these targets are the starting point.  Potent toxins can then be added to them by fusing antibody fragments to powerful bacterial toxins such as Pseudomonas exotoxin (PE). This recombinant immunotoxin combines antibody selectivity with toxin cell-killing potency.

Recombinant follicle-stimulating hormone: new biotechnology for infertility.

PubMed

Prevost, R R

1998-01-01

The frequency of infertility in developed countries is approximately 8-10%. New drugs are available for assisted reproduction techniques. Two recombinant follicle-stimulating hormone (FSH) products, follitropin-beta (Follistim in the United States, Puregon in Europe) and follitropin-alpha (Gonal-F), join compounds derived through transfecting nonhuman cell lines with genetic material capable of replicating identical amino acid sequences to human compounds. The cell line used for recombinant (r)-FSH production is the Chinese hamster ovary (CHO). Previously, the only agents that showed benefit in controlled ovulatory stimulation were derived from the urine of menopausal women. Those compounds contain additional substances, such as urinary proteins and various amounts of luteininzing hormone. The amino acid sequence of r-FSH is identical to that of human FSH, but the two recombinant products exist in many different isoforms and differ from each other and from human FSH due to varied carbohydrate side chains. Due to variation in the carbohydrate side chains, follitropin-beta in solution has a higher pH than urine-derived FSH, which enhances receptor affinity and therefore is a greater inducer of folliculogenesis. Follitropin-beta does not cause endogenous production of anti-CHO or anti-FSH antibodies, and is well tolerated.

A Novel PET Imaging Using 64Cu-Labeled Monoclonal Antibody against Mesothelin Commonly Expressed on Cancer Cells

PubMed Central

Kobayashi, Kazuko; Sasaki, Takanori; Takenaka, Fumiaki; Yakushiji, Hiromasa; Fujii, Yoshihiro; Kishi, Yoshiro; Kita, Shoichi; Shen, Lianhua; Kumon, Hiromi; Matsuura, Eiji

2015-01-01

Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions. PMID:25883990

X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEG 3350 in crystallization.

PubMed

Valjakka, J; Hemminki, A; Teerinen, T; Takkinen, K; Rouvinen, J

2000-02-01

Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 A. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.

Novel Chemokine-Based Immunotoxins for Potent and Selective Targeting of Cytomegalovirus Infected Cells

PubMed Central

Spiess, Katja; Jeppesen, Mads G.; Malmgaard-Clausen, Mikkel; Krzywkowski, Karen

2017-01-01

Immunotoxins as antiviral therapeutics are largely unexplored but have promising prospective due to their high selectivity potential and their unparalleled efficiency. One recent example targeted the virus-encoded G protein-coupled receptor US28 as a strategy for specific and efficient treatment of human cytomegalovirus (HCMV) infections. US28 is expressed on virus-infected cells and scavenge chemokines by rapid internalization. The chemokine-based fusion-toxin protein (FTP) consisted of a variant (F49A) of CX3CL1 specifically targeting US28 linked to the catalytic domain of Pseudomonas exotoxin A (PE). Here, we systematically seek to improve F49A-FTP by modifications in its three structural domains; we generated variants with (1) altered chemokine sequence (K14A, F49L, and F49E), (2) shortened and elongated linker region, and (3) modified toxin domain. Only F49L-FTP displayed higher selectivity in its binding to US28 versus CX3CR1, the endogenous receptor for CX3CL1, but this was not matched by a more selective killing of US28-expressing cells. A longer linker and different toxin variants decreased US28 affinity and selective killing. Thereby, F49A-FTP represents the best candidate for HCMV treatment. Many viruses encode internalizing receptors suggesting that not only HCMV but also, for instance, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus may be targeted by FTPs. PMID:28251165

Novel Chemokine-Based Immunotoxins for Potent and Selective Targeting of Cytomegalovirus Infected Cells.

PubMed

Spiess, Katja; Jeppesen, Mads G; Malmgaard-Clausen, Mikkel; Krzywkowski, Karen; Kledal, Thomas N; Rosenkilde, Mette M

2017-01-01

Immunotoxins as antiviral therapeutics are largely unexplored but have promising prospective due to their high selectivity potential and their unparalleled efficiency. One recent example targeted the virus-encoded G protein-coupled receptor US28 as a strategy for specific and efficient treatment of human cytomegalovirus (HCMV) infections. US28 is expressed on virus-infected cells and scavenge chemokines by rapid internalization. The chemokine-based fusion-toxin protein (FTP) consisted of a variant (F49A) of CX 3 CL1 specifically targeting US28 linked to the catalytic domain of Pseudomonas exotoxin A (PE). Here, we systematically seek to improve F49A-FTP by modifications in its three structural domains; we generated variants with (1) altered chemokine sequence (K14A, F49L, and F49E), (2) shortened and elongated linker region, and (3) modified toxin domain. Only F49L-FTP displayed higher selectivity in its binding to US28 versus CX 3 CR1, the endogenous receptor for CX 3 CL1, but this was not matched by a more selective killing of US28-expressing cells. A longer linker and different toxin variants decreased US28 affinity and selective killing. Thereby, F49A-FTP represents the best candidate for HCMV treatment. Many viruses encode internalizing receptors suggesting that not only HCMV but also, for instance, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus may be targeted by FTPs.

Immunotoxin – a new treatment option in patients with relapsed and refractory Hodgkin lymphoma

PubMed Central

Novakovic, Barbara Jezersek

2015-01-01

Background Even though Hodgkin lymphoma is a highly curable disease, some of the patients have either a refractory disease or experience a relapse following a successful primary therapy. Durable responses and remissions in patients with relapsed or refractory disease may be achieved in approximately one-half with salvage chemotherapy followed by high dose chemotherapy (HDT) and autologous hematopoietic cell rescue (SCT). On the other hand, patients who relapse after HDT and autologous SCT or those who have failed at least two prior multi-agent chemotherapy regimens and are not candidates for HDT have limited treatment options. Conclusions A new treatment option in this population is an immunotoxin Brentuximab vedotin composed of a CD30 directed antibody linked to the antitubulin agent monomethyl auristatin E. It has demonstrated a substantial effectiveness and an acceptable toxicity. In the pivotal study, the overall response rate was 75% with 34% of complete remissions. The median durations of response were 20.5 and 6.7 months for those with complete remission and all responding patients, respectively. The median overall survival was 40.5 months (3-years overall survival 54%) and the median progression-free survival 9.3 months. The most common non-hematologic toxicities were peripheral sensory neuropathy, nausea, and fatigue while the most common severe side effects were neutropenia, thrombocytopenia, anemia, and peripheral sensory neuropathy. PMID:26834516

Over-expression and purification strategies for recombinant multi-protein oligomers: a case study of Mycobacterium tuberculosis σ/anti-σ factor protein complexes.

PubMed

Thakur, Krishan Gopal; Jaiswal, Ravi Kumar; Shukla, Jinal K; Praveena, T; Gopal, B

2010-12-01

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the σ factor/anti-σ factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of σ factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems. Copyright © 2010 Elsevier Inc. All rights reserved.

[Prokaryotic expression of Nanog gene and preparation of anti-Nanog antibody].

PubMed

Li, Jun; Wang, Xiao-min; Dou, Zhong-ying; Li, Yong

2012-07-01

To express Nanog fusion protein in Escherichia coli ( E.coli), and to prepare rabbit anti-mouse polyclonal antibodies to the Nanog fusion protein. Mouse Nanog gene was amplified from the pNA992 recombinant plasmid and inserted into pET-32a vector to construct a recombinant expression vector pET-32a-Nanog. The recombinant vector was transfected into E.coli BL21 and induced by IPTG to express in them. The acquired Nanog fusion protein was purified with HisTrap affinity column and injected as an antigen into rabbits for preparing polyclonal antibodies. At last, the titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA, Western blotting and immunocytochemical staining, respectively. The recombinant expression vector pET-32a-Nanog was successfully prepared, transfected and induced to obtain the high expression of the Nanog fusion protein in a form of inclusion bodies in E.coli. After purification, its purity was up to 97%. The titer of anti-Nanog antibodies was 1:32 000 in the immunized rabbit serum, and exhibited a high specificity to Nanog protein. The rabbit anti-mouse polyclonal antibodies have been prepared successfully with a high titer and specificity to the Nanog fusion protein.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody produced from multiple clones for the selection of a master cell bank candidate.

PubMed

Wetzel, Hanna N; Webster, Rose P; Saeed, Fatima O; Kirley, Terence L; Ball, William J; Norman, Andrew B

2017-06-03

We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 min after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [ 3 H] cocaine binding assay. All three antibodies had long elimination half-lives, 2-5 nM Kd for cocaine, and prevented cocaine's entry into the brain by sequestering it in the plasma. Pharmacokinetic and radioligand binding assays supported designation of the highest producing clone 85 as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy. Copyright © 2017 Elsevier Inc. All rights reserved.

Successful ovulation induction, conception, and normal delivery after chronic therapy with etanercept: a recombinant fusion anti-cytokine treatment for rheumatoid arthritis.

PubMed

Sills, E S; Perloe, M; Tucker, M J; Kaplan, C R; Palermo, G D

2001-11-01

Etanercept (Enbrel; Wyeth-Ayerst/Immunex Inc, Seattle, WA, USA) is a subcutaneously administered novel fusion protein consisting of the extracellular ligand-binding domain of the 75 kD receptor for tumor necrosis factor-alpha (anti-TNFalpha) and the Fc portion of human IgG1. The agent is synthesized by plasmid transfection of a Chinese hamster ovary cell line, utilizing recombinant DNA technology. Etanercept was approved by the US FDA for treatment of multi-drug resistant rheumatoid arthritis in 1998, but no human data exist regarding the impact of anti-TNFalpha therapy on human reproductive function or its use before ovulation induction. As TNFalpha potentiates collagenolysis via matrix metalloproteinase gene expression (thereby facilitating ovulation), there exists a theoretical risk that TNFalpha-inhibition could exert an undesirable effect on ovulation and pregnancy. In this report, we describe the first case of ovulation induction, intrauterine insemination, normal pregnancy and singleton delivery of a healthy infant following chronic ( > 1 year) pre-ovulatory TNFalpha-inhibitor therapy for rheumatoid arthritis. Reproductive endocrinologists and obstetrician-gynecologists should be familiar with etanercept therapy in the context of severe rheumatic disease, and offer appropriate reassurance regarding its safe use for infertility patients planning ovulation induction.

75 FR 41873 - Prospective Grant of Exclusive License: The Development of Human Therapeutics for the Treatment...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-19

... preferentially expressed on several types of hematological cancer cells, the anti-CD22 antibody binding fragment... Exclusive License: The Development of Human Therapeutics for the Treatment of Cancer AGENCY: National... Immunotoxin in Which All B-Cell Epitopes Have Been Removed and Which Has High Cytotoxic Activity'' [HHS Ref. E...

Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus.

PubMed

Abdelwahab, Mohamed; Loa, Chien Chang; Wu, Ching Ching; Lin, Tsang Long

2015-06-01

Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks. Copyright © 2015 Elsevier B.V. All rights reserved.

[Immune Response of Recombinant Pseudorabies Virus rPRV-VP2 Expressing VP2 Gene of Porcine Parvovirus in Mice].

PubMed

Fu, Pengfei; Pan, Xinlong; Han, Qiao; Yang, Xingwu; Zhu, Qianlei; Guo, Xiaoqing; Zhang, Yu; Chen, Hongying

2016-03-01

In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.

Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

PubMed

Shibaei, Naeimeh; Majidi, Jafar; Razavi, Khadijeh; Karkhane, Ali Asghar; Sokhandan-Bashir, Nemat; Aghebati-Maleki, Leili

2017-02-01

There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

Anti-Rh(c), "little c," isoimmunization: the role of rHuEpo in preventing late anemia.

PubMed

Zuppa, Antonio A; Cardiello, Valentina; Alighieri, Giovanni; Cota, Francesco; D'Antuono, Annamaria; Riccardi, Riccardo; Catenazzi, Piero; Romagnoli, Costantino

2013-08-01

The overall prevalence of non-Rh-D isoimmunization seems to lie between 0.15% and 1.1%. Anti-Rh(c) alloimmunization, "little c," occurs in 0.07% of pregnancies and shows a quite broad clinical presentation. Late anemia is a frequent problem occurring in the setting of isoimmunization. It occurs more frequently after intrauterine blood transfusions or exsanguinotransfusion, and it can be thought as a hyporegenerative anemia. The authors describe the use of human recombinant erythropoietin in preventing late anemia in a case of anti-Rh(c) isoimmunization. The use of human recombinant erythropoietin is a valid tool for preventing late-onset anemia due to either anti-Rh-D or non-anti-Rh-D isoimmunization.

Recombinant dengue 2 virus NS3 protein conserves structural antigenic and immunological properties relevant for dengue vaccine design.

PubMed

Ramírez, Rosa; Falcón, Rosabel; Izquierdo, Alienys; García, Angélica; Alvarez, Mayling; Pérez, Ana Beatriz; Soto, Yudira; Muné, Mayra; da Silva, Emiliana Mandarano; Ortega, Oney; Mohana-Borges, Ronaldo; Guzmán, María G

2014-10-01

The NS3 protein is a multifunctional non-structural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a His-tagged fusion protein in Escherichia coli. The pNS3 protein was purified by two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-α in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques. The successfully purified recombinant protein was able to preserv the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.

Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

PubMed Central

Grinberg, Yehudit; Benhar, Itai

2017-01-01

Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called “human cytotoxic fusion proteins”, in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies. PMID:28574434

Purification and biological characterization of soluble, recombinant mouse IFNβ expressed in insect cells.

PubMed

Stifter, Sebastian A; Gould, Jodee A; Mangan, Niamh E; Reid, Hugh H; Rossjohn, Jamie; Hertzog, Paul J; de Weerd, Nicole A

2014-02-01

Interferon β (IFNβ) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNβ utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNβ. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNβ purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNβ than any other reported eukaryotic-based expression system. Recombinant murine IFNβ produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNβ activity in vivo. Recombinant IFNβ also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNβ. Copyright © 2013 Elsevier Inc. All rights reserved.

Recombinant vaccine for canine parvovirus in dogs.

PubMed

López de Turiso, J A; Cortés, E; Martínez, C; Ruiz de Ybáñez, R; Simarro, I; Vela, C; Casal, I

1992-05-01

VP2 is the major component of canine parvovirus (CPV) capsids. The VP2-coding gene was engineered to be expressed by a recombinant baculovirus under the control of the polyhedrin promoter. A transfer vector that contains the lacZ gene under the control of the p10 promoter was used in order to facilitate the selection of recombinants. The expressed VP2 was found to be structurally and immunologically indistinguishable from authentic VP2. The recombinant VP2 shows also the capability to self-assemble, forming viruslike particles similar in size and appearance to CPV virions. These viruslike particles have been used to immunize dogs in different doses and combinations of adjuvants, and the anti-CPV responses have been measured by enzyme-linked immunosorbent assay, monolayer protection assays, and an assay for the inhibition of hemagglutination. A dose of ca. 10 micrograms of VP2 was able to elicit a good protective response, higher than that obtained with a commercially available, inactivated vaccine. The results indicate that these viruslike particles can be used to protect dogs from CPV infection.

Recombinant vaccine for canine parvovirus in dogs.

PubMed Central

López de Turiso, J A; Cortés, E; Martínez, C; Ruiz de Ybáñez, R; Simarro, I; Vela, C; Casal, I

1992-01-01

VP2 is the major component of canine parvovirus (CPV) capsids. The VP2-coding gene was engineered to be expressed by a recombinant baculovirus under the control of the polyhedrin promoter. A transfer vector that contains the lacZ gene under the control of the p10 promoter was used in order to facilitate the selection of recombinants. The expressed VP2 was found to be structurally and immunologically indistinguishable from authentic VP2. The recombinant VP2 shows also the capability to self-assemble, forming viruslike particles similar in size and appearance to CPV virions. These viruslike particles have been used to immunize dogs in different doses and combinations of adjuvants, and the anti-CPV responses have been measured by enzyme-linked immunosorbent assay, monolayer protection assays, and an assay for the inhibition of hemagglutination. A dose of ca. 10 micrograms of VP2 was able to elicit a good protective response, higher than that obtained with a commercially available, inactivated vaccine. The results indicate that these viruslike particles can be used to protect dogs from CPV infection. Images PMID:1313899

Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin

PubMed Central

Kornberger, Petra; Skerra, Arne

2014-01-01

We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography—utilizing the Strep-tag II appended to gelonin—and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins. PMID:24492291

A recombinant adenovirus bicistronically expressing porcine interferon-α and interferon-γ enhances antiviral effects against foot-and-mouth disease virus.

PubMed

Kim, Su-Mi; Kim, Se-Kyung; Park, Jong-Hyeon; Lee, Kwang-Nyeong; Ko, Young-Joon; Lee, Hyang-Sim; Seo, Min-Goo; Shin, Yeun-Kyung; Kim, Byounghan

2014-04-01

Foot-and-mouth disease (FMD) is a virulent and economically costly disease in domestic livestock. Since the current vaccine available against FMD provides no protection until 7days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is by the application of anti-viral agents. The combination of recombinant adenovirus expressing type I interferon (IFN-α) and adenovirus expressing type II IFN (IFN-γ) has been reported to be an effective anti-viral treatment strategy against FMDV. Nevertheless, the recombinant adenovirus mixture may be inefficient because of the low anti-viral efficiency of IFN-γ compared to that of IFN-α. In this study, we generated a recombinant adenovirus co-expressing porcine IFN-α and IFN-γ in tandem using an FMDV 2A sequence to mediate effective cleavage of the two proteins (referred to as Ad-porcine IFN-αγ). We demonstrated that both recombinant porcine IFN-α and IFN-γ were expressed and interferon stimulated gene (ISG)s related with IFN-α and IFN-γ were induced in porcine kidney (IBRS-2) cells infected with Ad-porcine IFN-αγ. Additionally, the anti-viral effects of Ad-porcine IFN-αγ against FMDV were enhanced both in IBRS-2 cells and in CD-1 (ICR) suckling mice compared to that of adenovirus expressing only a single protein. We propose that Ad-porcine IFN-αγ could be a rapid, highly efficient, convenient anti-viral agent against FMDV. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression, Purification and Characterization of Recombinant Canine FGF21 in Escherichia coli.

PubMed

Zheng, Zhong; Yang, Chengjun; Yin, Ruofeng; Jiang, Jinxi; He, Haiting; Wang, Xinxin; Kan, Mujie; Xiao, Yechen

2016-01-01

The canine metabolic diseases, such as obesity and diabetes, have become a worldwide problem. Fibroblast growth factor 21 (FGF21) is a potent regulator which has many biological functions relative to metabolism regulation. It suggests that FGF21 plays important roles in regulating canine metabolic diseases. To acquire the recombinant canine FGF21 (rcFGF21) in Escherichia coli, the recombinant bacteria were induced by 0.5 mM IPTG for 16 hours at 16 °C, and the rcFGF21 protein was purified by Ni-NTA. 8 mg rcFGF21 was acquired from one liter bacteria. The rcFGF21 protein has specific immunoblot reactivity against anti-FGF21 and anti-His antibody. The in vivo experimental result showed that rcFGF21 can significantly reduce plasma glucose of STZ-induced diabetic mice.

Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate.

PubMed

Kaiser, Gitte S; Germann, Susanne M; Westergaard, Tine; Lisby, Michael

2011-08-01

Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced genetic recombination as well as DNA double-strand break repair during mating-type interconversion. Treatment with PBA is accompanied by a dramatic reduction in histone H4 lysine 8 acetylation. Live cell imaging of homologous recombination proteins indicates that repair of CPT-induced DNA damage is redirected to a non-recombinogenic pathway in the presence of PBA without loss in cell viability. In contrast, the suppression of MMS-induced recombination by PBA is accompanied by a dramatic loss in cell viability. Taken together, our results demonstrate that PBA inhibits DNA damage-induced homologous recombination likely by mediating changes in chromatin acetylation. Moreover, the combination of PBA with genotoxic agents can lead to different cell fates depending on the type of DNA damage inflicted. 2011 Elsevier B.V. All rights reserved.

PE38KDEL-loaded anti-HER2 nanoparticles inhibit breast tumor progression with reduced toxicity and immunogenicity.

PubMed

Gao, Jie; Kou, Geng; Wang, Hao; Chen, Huaiwen; Li, Bohua; Lu, Ying; Zhang, Dapeng; Wang, Shuhui; Hou, Sheng; Qian, Weizhu; Dai, Jianxin; Zhao, Jian; Zhong, Yanqiang; Guo, Yajun

2009-05-01

The clinical use of Pseudomonas exotoxin A (PE)-based immunotoxins is limited by the toxicity and immunogenicity of PE. To overcome the limitations, we have developed PE38KDEL-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles conjugated with Fab' fragments of a humanized anti-HER2 monoclonal antibody (rhuMAbHER2). The PE38KDEL-loaded nanoparticles-anti-HER2 Fab' bioconjugates (PE-NP-HER) were constructed modularly with Fab' fragments of rhuMAbHER2 covalently linked to PLGA nanoparticles containing PE38KDEL. Compared with nontargeted nanoparticles that lack anti-HER2 Fab', PE-NP-HER specifically bound to and were sequentially internalized into HER2 overexpressing breast cancer cells, which result in significant cytotoxicity in vitro. In HER2 overexpressing tumor xenograft model system, administration of PE-NP-HER showed a superior efficacy in inhibiting tumor growth compared with PE-HER referring to PE38KDEL conjugated directly to rhuMAbHER2. Moreover, PE-NP-HER was well tolerated in mice with a higher LD(50) (LD(50) of 6.86 +/- 0.47 mg/kg vs. 2.21 +/- 0.32 mg/kg for PE-NP-HER vs. PE-HER (mean +/- SD); n = 3), and had no influence on the plasma level of plasma alanine aminotransferase (ALT) of animals when injected at a dose of 1 mg/kg where PE-HER caused significant increase of serum ALT in the treated mice. Notably, PE-NP-HER was of low immunogenicity in development of anti-PE38KDEL neutralizing antibodies and was less susceptible to inactivation by anti-PE38KDEL antibodies compared with PE-HER. This novel bioconjugate, PE-NP-HER, may represent a useful strategy for cancer treatment.

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

PubMed

Crusius, Kerstin; Finster, Silke; McClary, John; Xia, Wei; Larsen, Brent; Schneider, Douglas; Lu, Hong-Tao; Biancalana, Sara; Xuan, Jian-Ai; Newton, Alicia; Allen, Debbie; Bringmann, Peter; Cobb, Ronald R

2006-10-01

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

Recombinant Plants Provide a New Approach to the Production of Bacterial Polysaccharide for Vaccines

PubMed Central

Smith, Claire M.; Fry, Stephen C.; Gough, Kevin C.; Patel, Alexandra J. F.; Glenn, Sarah; Goldrick, Marie; Roberts, Ian S.; Andrew, Peter W.

2014-01-01

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections. PMID:24498433

Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

PubMed

Kirley, Terence L; Norman, Andrew B

2015-01-01

Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

Anti-viral activity of galectin-1 from flounder Paralichthys olivaceus.

PubMed

Liu, Shousheng; Hu, Guobin; Sun, Chen; Zhang, Shicui

2013-06-01

Galectins are a family of Ca(2+)-independent soluble lectins characterized by their affinity to β-galactosides. Mammalian galectins have been shown to play a defense role against certain bacteria, fungi and viruses. However, the immunological functions of galectins in fish is poorly characterized. Here we demonstrated that the expression of galectin-1 gene from the flounder Paralichthys olivaceus was decreased in the initial 8 h after challenge with poly I:C, then increased markedly from 24 h onwards, and the recombinant galectin-1 was able to neutralize the lymphocystis disease virus (LCDV), inhibiting the formation of cytopathic effects. In addition, the recombinant galectin had a potential anti-inflammatory activity against infection by LCDV, and was able to restrain the overexpression of the anti-viral protein gene mx against virus infection. These results indicate that flounder galectin-1 has an anti-viral activity, capable of reducing LCDV pathogenicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity

PubMed Central

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane. PMID:19531344

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity.

PubMed

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.

A mathematical model of a recombinant humanized anti-cocaine monoclonal antibody's effects on cocaine pharmacokinetics in mice.

PubMed

Wetzel, Hanna N; Zhang, Tongli; Norman, Andrew B

2017-09-01

A recombinant humanized anti-cocaine monoclonal antibody (mAb), h2E2, is at an advanced stage of pre-clinical development as an immunotherapy for cocaine abuse. It is hypothesized that h2E2 binds to and sequesters cocaine in the blood. A three-compartment model of the effects of h2E2 on cocaine's distribution was constructed. The model assumes that h2E2 binds to cocaine and that the h2E2-cocaine complex does not enter the brain but distributes between the central and peripheral compartments. Free cocaine is eliminated from both the central and peripheral compartments, and h2E2 and the h2E2-cocaine complex are eliminated from the central compartment only. This model was tested against a new dataset measuring cocaine concentrations in the brain and plasma over 1h in the presence and absence of h2E2. The mAb significantly increased plasma cocaine concentrations with a concomitant significant decrease in brain concentration. Plasma concentrations declined over the 1-hour sampling period in both groups. With a set of parameters within reasonable physiological ranges, the three-compartment model was able to qualitatively and quantitatively simulate the increased plasma concentration in the presence of the antibody and the decreased peak brain concentration in the presence of antibody. Importantly, the model explained the decline in plasma concentrations over time as distribution of the cocaine-h2E2 complex into a peripheral compartment. This model will facilitate the targeting of ideal mAb PK/PD properties thus accelerating the identification of lead candidate anti-drug mAbs. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus.

PubMed Central

Jouanneau, Y; Duport, C; Meyer, C; Gaillard, J

1992-01-01

The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter. The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E. coli cell-free extracts by anti-FdII serum. The purified recombinant ferredoxin was indistinguishable from R. capsulatus FdII on the basis of its molecular, redox and spectroscopic properties. These results indicate that the [3Fe-4S] and [4Fe-4S] clusters were correctly inserted into the recombinant ferredoxin. Images Fig. 2. PMID:1325780

Conjugation of gold nanoparticles and recombinant human endostatin modulates vascular normalization via interruption of anterior gradient 2-mediated angiogenesis.

PubMed

Pan, Fan; Yang, Wende; Li, Wei; Yang, Xiao-Yan; Liu, Shuhao; Li, Xin; Zhao, Xiaoxu; Ding, Hui; Qin, Li; Pan, Yunlong

2017-07-01

Several studies have revealed the potential of normalizing tumor vessels in anti-angiogenic treatment. Recombinant human endostatin is an anti-angiogenic agent which has been applied in clinical tumor treatment. Our previous research indicated that gold nanoparticles could be a nanoparticle carrier for recombinant human endostatin delivery. The recombinant human endostatin-gold nanoparticle conjugates normalized vessels, which improved chemotherapy. However, the mechanism of recombinant human endostatin-gold nanoparticle-induced vascular normalization has not been explored. Anterior gradient 2 has been reported to be over-expressed in many malignant tumors and involved in tumor angiogenesis. To date, the precise efficacy of recombinant human endostatin-gold nanoparticles on anterior gradient 2-mediated angiogenesis or anterior gradient 2-related signaling cohort remained unknown. In this study, we aimed to explore whether recombinant human endostatin-gold nanoparticles could normalize vessels in metastatic colorectal cancer xenografts, and we further elucidated whether recombinant human endostatin-gold nanoparticles could interrupt anterior gradient 2-induced angiogenesis. In vivo, it was indicated that recombinant human endostatin-gold nanoparticles increased pericyte expression while inhibit vascular endothelial growth factor receptor 2 and anterior gradient 2 expression in metastatic colorectal cancer xenografts. In vitro, we uncovered that recombinant human endostatin-gold nanoparticles reduced cell migration and tube formation induced by anterior gradient 2 in human umbilical vein endothelial cells. Treatment with recombinant human endostatin-gold nanoparticles attenuated anterior gradient 2-mediated activation of MMP2, cMyc, VE-cadherin, phosphorylation of p38, and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in human umbilical vein endothelial cells. Our findings demonstrated recombinant human endostatin-gold nanoparticles might normalize

Immunogenicity assessment of recombinant human c1-inhibitor: an integrated analysis of clinical studies.

PubMed

Hack, C Erik; Mannesse, Maurice; Baboeram, Aartie; Oortwijn, Beatrijs; Relan, Anurag

2012-10-01

Recombinant human C1-inhibitor (rhC1INH) is used to treat acute angioedema attacks in hereditary angioedema (HAE) due to a genetic C1INH deficiency. Recombinant proteins in general may induce antibody responses and therefore evaluation of such responses in the target population is an essential step in the clinical development program of a recombinant protein. Here we report the assessment of the immunogenicity of rhC1INH in symptomatic HAE patients. Blood samples collected before and after administration of rhC1INH were tested for antibodies against plasma-derived (pd) or rhC1INH, or against host-related impurities (HRI). Above cut-off screening results were confirmed with displacement assays, and also tested for neutralizing anti-C1INH antibodies. Finally, the relation of antibodies to clinical efficacy and safety of rhC1INH was analyzed. Data from 155 HAE patients who received 424 treatments with rhC1INH were analyzed. 1.5% of all pre-exposure tests and 1.3% of all post-exposure tests were above the cut-off level in the screening assay for anti-C1INH antibodies. Six patients (3.9%) had anti-rhC1INH antibodies positive in the confirmatory assay. In two patients, confirmed antibodies were pre-existing with no increase post-exposure; in three patients, the antibodies occurred on a single occasion post-exposure; and in one patient, on subsequent occasions post-exposure. Neutralizing anti-pdC1INH antibodies were not found. Anti-HRI antibodies in the screening assay occurred in <0.7% of the tests before exposure to rhC1INH, in <1.9% after first exposure and in <3.1% after repeat treatment with rhC1INH. Five patients had anti-HRI antibodies positive in the confirmatory assay. In one patient, the antibodies were pre-existing, whereas in three of the 155 rhC1INH-treated patients (1.9%), confirmed anti-HRI antibodies occurred at more time points. Antibody findings were not associated with altered efficacy of rhC1INH or adverse events. These results indicate a reassuring

Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Deepti; Rawat, Surender; Waseem, Mohd

The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, K{sub m} values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative propertymore » against BSA and Cu{sup 2+}/H{sub 2}O{sub 2} model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies. - Highlights: • Laccase from Yersinia enterocolitica strain 7 was expressed in Escherichia coli BL21 (DE3). • Recombinant laccase was found to be thermostable and alkali tolerant. • The in silico and experimental studied proves the biotransformation of NSAIDs. • Laccase binds to ligands differentially under different protonation state. • Laccase also possesses free radical scavenging property.« less

Pancreatic cancer therapy with combined mesothelin-redirected chimeric antigen receptor T cells and cytokine-armed oncolytic adenoviruses

PubMed Central

Watanabe, Keisuke; Luo, Yanping; Da, Tong; Scholler, John; Keith, Brian; Young, Regina M.; Sorsa, Suvi; Siurala, Mikko; Havunen, Riikka; Tähtinen, Siri; Hemminki, Akseli

2018-01-01

Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. Mesothelin-redirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA. PMID:29618658

SPAR1/RTEL1 maintains genomic stability by suppressing homologous recombination

PubMed Central

Barber, Louise J.; Youds, Jillian L.; Ward, Jordan D.; McIlwraith, Michael J.; O’Neil, Nigel J.; Petalcorin, Mark I.R.; Martin, Julie S.; Collis, Spencer J.; Cantor, Sharon B.; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C.; Rose, Ann M.; Boulton, Simon J.

2013-01-01

SUMMARY Inappropriate homologous recombination (HR) can cause gross chromosomal rearrangements that in mammalian cells may lead to tumorigenesis. In yeast, the Srs2 protein is an anti-recombinase that eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has proven to be elusive. In this work, we identify C. elegans SPAR-1 as a functional analogue of Srs2 and describe its vertebrate counterpart, SPAR1/RTEL1, which is required for genome stability and tumour avoidance. We find that spar-1 mutant worms and SPAR1 knockdown human cells share characteristic phenotypes with yeast srs2 mutants, including inviability upon deletion of the sgs1/BLM homologue, hyper-recombination, and DNA damage sensitivity. In vitro, purified human SPAR1 antagonises HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control following deregulation of SPAR1/RTEL1 may be a critical event that drives genome instability and cancer. PMID:18957201

High cancer-specific expression of mesothelin (MSLN) is attributable to an upstream enhancer containing a transcription enhancer factor dependent MCAT motif.

PubMed

Hucl, Tomas; Brody, Jonathan R; Gallmeier, Eike; Iacobuzio-Donahue, Christine A; Farrance, Iain K; Kern, Scott E

2007-10-01

Identification of genes with cancer-specific overexpression offers the potential to efficiently discover cancer-specific activities in an unbiased manner. We apply this paradigm to study mesothelin (MSLN) overexpression, a nearly ubiquitous, diagnostically and therapeutically useful characteristic of pancreatic cancer. We identified an 18-bp upstream enhancer, termed CanScript, strongly activating transcription from an otherwise weak tissue-nonspecific promoter and operating selectively in cells having aberrantly elevated cancer-specific MSLN transcription. Introducing mutations into CanScript showed two functionally distinct sites: an Sp1-like site and an MCAT element. Gel retardation and chromatin immunoprecipitation assays showed the MCAT element to be bound by transcription enhancer factor (TEF)-1 (TEAD1) in vitro and in vivo. The presence of TEF-1 was required for MSLN protein overexpression as determined by TEF-1 knockdown experiments. The cancer specificity seemed to be provided by a putative limiting cofactor of TEF-1 that could be outcompeted by exogenous TEF-1 only in a MSLN-overexpressing cell line. A CanScript concatemer offered enhanced activity. These results identify a TEF family member as a major regulator of MSLN overexpression, a fundamental characteristic of pancreatic and other cancers, perhaps due to an upstream and highly frequent aberrant cellular activity. The CanScript sequence represents a modular element for cancer-specific targeting, potentially suitable for nearly a third of human malignancies.

[Vaccination of rhesus monkeys with recombinant antigen fragments and protection from hepatitis E virus infection].

PubMed

Ma, Yan-bing; Xie, Tian-hong; Zhang, Guang-ming; Li, Chun-hong; Dai, Xie-Jie; Dai, Chang-bai; Sun, Mao-sheng; Lu, Jian; Bi, Sheng-li

2002-12-01

To observe anti-HEV IgG response to vaccination of recombinant antigen fragments and evaluate its protection from Hepatitis E Virus infection in rhesus monkeys (Macaca mulatta). Twelve monkeys were divided into three groups and immunized respectively with three different recombinant antigens: namely Ag1 (carboxyl terminal 431 amino acids of ORF2), Ag2 (128aa fragment at the carboxyl terminal of ORF2), and Ag3 (full length ORF3 ligated with two ORF2 fragments encoded by 6743-7126nt and 6287-6404nt). The monkeys were challenged intravenously with fecal suspension from experimentally infected rhesus monkeys, and the other three monkeys served as the placebo group for challenge with HEV. The dynamic changes of the levels of ALT and anti-HEV IgG were examined. Pathological changes of liver tissue were observed by light microscope. Excretion of virus was detected by RT-nPCR. Hepatic histopathology of two monkeys in the placebo group was consistent with acute viral hepatitis, and ALT was elevated 3-4 weeks after inoculated with virus, up to 10-20 times higher than normal level. The liver tissue of monkeys immunized with antigen kept normal, ALT in several monkeys elevated mildly, and anti-HEV IgG conversation occurred at 1-2 weeks after vaccination, with the titer reaching 1:12,800. The virus RNA could be detected by RT-nPCR from days 7 to 50 in monkeys of control group, and from days 7 to 21 in vaccinated monkeys after challenged with virus. The recombinant antigens could induce the production of anti-HEV IgG, which protected rhesus monkeys from acute Hepatitis symptoms related to HEV infection.

Recombinant latcripin 11 of Lentinula edodes C91-3 suppresses the proliferation of various cancer cells.

PubMed

Gao, Yifan; Padhiar, Arshad Ahmed; Wang, Jia; Zhang, Wei; Zhong, Mintao; Liu, Ben; Kang, Zhijie; Wang, Xiaoli; Li, Xingyun; Huang, Min

2018-02-05

Lentinula edodes C91-3 is an edible mushroom that has demonstrated a remarkable anti-tumor effect in various cancer cells both in vitro and in vivo. In the present study, we report the ability of recombinant thioredoxin-like latcripin 11 (LP-11) of Lentinula edodes C91-3 to suppress the proliferation of various cancer cells. The LP-11 gene of Lentinula edodes C91-3 was cloned in the pET-32a(+) expression vector and expressed in a prokaryotic system. The expressed protein was refolded by gradual dialysis and purified by affinity gel filtration chromatography. The antioxidant activity of LP-11 was tested by 1,1-dipheny l-2-picrylhydrazyl (DPPH) assay. The anti-tumor activity of recombinant LP-11 was tested in eight kinds of tumor cell lines by CCK-8 assay. Recombinant LP-11 significantly suppressed the proliferation of various cancer cells, but not normal human umbilical vein endothelial cells. Human lymphoma U937 cells exhibited the most sensitivity to LP-11 protein. U937 cell apoptosis was assessed by Annexin V staining coupled with flow cytometry, and mitochondrial morphology was analyzed by light and electron microscopy. It was revealed that recombinant LP-11 induced apoptosis in human leukemic monocyte lymphoma U937 cells. Our findings suggest that recombinant LP-11 is a promising agent for the treatment of lymphoma. Copyright © 2017. Published by Elsevier B.V.

Specific Genetic Immunotherapy Induced by Recombinant Vaccine Alpha-Fetoprotein-Heat Shock Protein 70 Complex

NASA Astrophysics Data System (ADS)

Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, P<0.01; 156.32±10.42 μg/mL vs 66.52±7.35 μg/mL, 5.73±2.89 μg/mL, P<0.01). The tumor volume in mAFP/HSP70 group was significantly smaller than that in mAFP and HSP70 groups (42.44±7.14 mm3 vs 392.23±12.46 mm3, 838.63±13.84 mm3, P<0.01). Conclusions: The study further confirmed the function of heat shock protein 70's immune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

Toll-like receptor 9 agonist enhances anti-tumor immunity and inhibits tumor-associated immunosuppressive cells numbers in a mouse cervical cancer model following recombinant lipoprotein therapy.

PubMed

Chang, Li-Sheng; Leng, Chih-Hsiang; Yeh, Yi-Chen; Wu, Chiao-Chieh; Chen, Hsin-Wei; Huang, Hai-Mei; Liu, Shih-Jen

2014-03-19

Although cytotoxic T lymphocytes (CTLs) play a major role in eradicating cancer cells during immunotherapy, the cancer-associated immunosuppressive microenvironment often limits the success of such therapies. Therefore, the simultaneous induction of cancer-specific CTLs and reversal of the immunosuppressive tumor microenvironment may be more effectively achieved through a single therapeutic vaccine. A recombinant lipoprotein with intrinsic Toll-like receptor 2 (TLR2) agonist activity containing a mutant form of E7 (E7m) and a bacterial lipid moiety (rlipo-E7m) has been demonstrated to induce robust CTL responses against small tumors. This treatment in combination with other TLR agonists is able to eliminate large tumors. Mouse bone marrow-derived dendritic cells (DCs) were employed to determine the synergistic production of pro-inflammatory cytokines upon combination of rlipo-E7m and other TLR agonists. Antigen-specific CTL responses were investigated using immunospots or in vivo cytolytic assays after immunization in mice. Mice bearing various tumor sizes were used to evaluate the anti-tumor effects of the formulation. Specific subpopulations of immunosuppressive cells in the tumor infiltrate were quantitatively determined by flow cytometry. We demonstrate that a TLR9 agonist (unmethylated CpG oligodeoxynucleotide, CpG ODN) enhances CTL responses and eradicates large tumors when combined with rlipo-E7m. Moreover, combined treatment with rlipo-E7m and CpG ODN effectively increases tumor infiltration by CTLs and reduces the numbers of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment. These findings suggest that the dramatic anti-tumor effects of the recombinant lipoprotein together with CpG ODN may reflect the amplification of CTL responses and the repression of the immunosuppressive environment. This promising approach could be applied for the development of additional

Characterization of recombinant Raccoonpox Vaccine Vectors in Chickens

USGS Publications Warehouse

Hwa, S.-H.; Iams, Keith P.; Hall, Jeffrey S.; Kingstad, B.A.; Osorio, Jorge E.

2010-01-01

Raccoonpox virus (RCN) has been used as a recombinant vector against several mammalian pathogens but has not been tested in birds. The replication of RCN in chick embryo fibroblasts (CEFs) and chickens was studied with the use of highly pathogenic avian influenza virus H5N1 hemagglutinin (HA) as a model antigen and luciferase (luc) as a reporter gene. Although RCN replicated to low levels in CEFs, it efficiently expressed recombinant proteins and, in vivo, elicited anti-HA immunoglobulin yolk (IgY) antibody responses comparable to inactivated influenza virus. Biophotonic in vivo imaging of 1-wk-old chicks with RCN-luc showed strong expression of the luc reporter gene lasting up to 3 days postinfection. These studies demonstrate the potential of RCN as a vaccine vector for avian influenza and other poultry pathogens. ?? American Association of Avian Pathologists 2010.

Characterization of anti-liver-kidney microsome antibody (anti-LKM1) from hepatitis C virus-positive and -negative sera.

PubMed

Yamamoto, A M; Cresteil, D; Homberg, J C; Alvarez, F

1993-06-01

Hepatitis C virus-related antibodies were found in sera positive for antibodies to liver/kidney microsome antibody, usually considered a marker of autoimmune hepatitis. The aim of this study was to analyze the specificity of this autoantibody in sera from patients with and without hepatitis C virus infection. Fifteen anti-hepatitis C virus- and anti-liver kidney microsome-positive sera were compared with 11 sera from patients with autoimmune hepatitis, for reactivity against rat and human liver microsomal proteins, P450IID6 recombinant proteins, and various synthetic peptides spanning the 241-429 amino acids sequence of the P450IID6. Ten of 11 sera from patients with autoimmune hepatitis bound to recombinant proteins spanning the P450IID6 region between amino acids 72 and 458. These sera bound to the 254-271 peptide, and some also recognized the 321-351, 373-389 and 410-429 peptides. Four of 15 antihepatitis C virus recognized the fusion protein coded by the full-length P450IID6 complementary DNA; 3 of them also reacted with the P450IID6 region between amino acids 72-456. Only 1 sera recognized the 321-351 peptide. P450IID6 antigenic sites recognized by anti-hepatitis C virus-positive sera were different from those recognized by sera from patients with autoimmune hepatitis.

Detection of anti-Yta antibodies using a sensitive and specific enzyme-linked immunosorbent assay.

PubMed

Geen, J; Hullin, D A; Hogg, S I

1999-01-01

A specific, sensitive and semi-quantitative enzyme-linked immunosorbent assay (ELISA) is described to detect anti-Yta antibodies in human serum. Recombinant acetylcholinesterase (AChE E.C.3.1.1.7) was employed as the coating antigen in the microtitre plate and horseradish peroxidase (HRP)-conjugated specific antibody (IgG) was used as the secondary antibody. The method developed showed excellent sensitivity, detecting a titre > 1 in 600,000 (3.5 ng/mL mouse IgG protein) for mouse monoclonal (mMAb) anti-AChE antibody. No cross-reaction was seen with other common blood group antibodies, confirming the specificity of the method. The recombinant antigen's AChE phenotype was confirmed as Yta, as no reaction was detected with anti-Ytb-positive sera. The ELISA method correlated closely with the established serological grading system used routinely in blood transfusion laboratories.

Binding specificity of anti-HNK-1 IgM M-protein in anti-MAG neuropathy: possible clinical relevance.

PubMed

Hamada, Yukihiro; Hirano, Makito; Kuwahara, Motoi; Samukawa, Makoto; Takada, Kazuo; Morise, Jyoji; Yabuno, Keiko; Oka, Shogo; Kusunoki, Susumu

2015-02-01

Anti-myelin-associated-glycoprotein (MAG) neuropathy is an intractable autoimmune polyneuropathy. The antigenic region of MAG is the human natural killer-1 (HNK-1) carbohydrate. We and others previously suggested that the extension of antibody reactivities to HNK-1-bearing proteins other than MAG was associated with treatment resistance, without statistical analyses. In this study, we established an ELISA method with recombinant proteins to test binding specificities of currently available monoclonal antibodies to MAG and another HNK-1-bearing protein, phosphacan. Using this system, we found the distinct binding specificities of anti-MAG antibody in 19 patients with anti-MAG neuropathy. Their clinical relevance was then determined retrospectively with the adjusted 10-points INCAT disability score (0 = normal and 10 = highly disable). The results showed that strong reactivities of anti-MAG antibodies to phosphacan were significantly associated with treatment resistance or progressive clinical courses, indicating a possible clinical relevance of the binding specificities. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep

PubMed Central

Faburay, Bonto; Wilson, William; McVey, D. Scott; Drolet, Barbara S.; Weingartl, Hana; Madden, Daniel; Young, Alan; Ma, Wenjun

2013-01-01

Abstract The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). PMID:23962238

Vaccine platform recombinant measles virus.

PubMed

Mühlebach, Michael D

2017-10-01

The classic development of vaccines is lengthy, tedious, and may not necessarily be successful as demonstrated by the case of HIV. This is especially a problem for emerging pathogens that are newly introduced into the human population and carry the inherent risk of pandemic spread in a naïve population. For such situations, a considerable number of different platform technologies are under development. These are also under development for pathogens, where directly derived vaccines are regarded as too complicated or even dangerous due to the induction of inefficient or unwanted immune responses causing considerable side-effects as for dengue virus. Among platform technologies are plasmid-based DNA vaccines, RNA replicons, single-round infectious vector particles, or replicating vaccine-based vectors encoding (a) critical antigen(s) of the target pathogens. Among the latter, recombinant measles viruses derived from vaccine strains have been tested. Measles vaccines are among the most effective and safest life-attenuated vaccines known. Therefore, the development of Schwarz-, Moraten-, or AIK-C-strain derived recombinant vaccines against a wide range of mostly viral, but also bacterial pathogens was quite straightforward. These vaccines generally induce powerful humoral and cellular immune responses in appropriate animal models, i.e., transgenic mice or non-human primates. Also in the recent first clinical phase I trial, the results have been quite encouraging. The trial indicated the expected safety and efficacy also in human patients, interestingly independent from the level of prevalent anti-measles immunity before the trial. Thereby, recombinant measles vaccines expressing additional antigens are a promising platform for future vaccines.

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c.

PubMed

Kahlert, H; Cromwell, O; Fiebig, H

2003-09-01

The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.

CAT-8015: A Second-generation Pseudomonas Exotoxin A-Based Immunotherapy Targeting CD22 -Expressing Hematological Malignancies

PubMed Central

Alderson, Ralph F.; Kreitman, Robert J.; Chen, Tianling; Yeung, Peter; Herbst, Ronald; Fox, Judy A.; Pastan, Ira

2009-01-01

Purpose To compare the in vitro and in vivo efficacy of CAT-8015, a second-generation recombinant immunotoxin composed of disulfide linked affinity matured VH and VL chains of the mouse anti-CD22 monoclonal antibody RFB4 fused to PE38, to the parental compound CAT-3888. Experimental Design The biological activity of CAT-8015 was examined in vitro using B cell tumor lines and in vivo in a JD38-based subcutaneous tumor model in NCr athymic mice. Pharmacokinetics and interspecies scaling of CAT-8015 were evaluated in mice, rats, and Cynomologus monkeys. The potential toxicity of CAT-8015 was assessed in monkeys in a toxicological study and compared to CAT-3888. Results The IC50s of CAT-8015 in vitro using the EHEB, MEC1, Daudi, CA46, and JD38 cell lines ranged from 0.3 - 8.6 ng/mL. Pharmacokinetic studies with CAT-8015 were conducted in mouse, rat and Cynomolgus monkey. The T1/2 was calculated to be 0.42, 0.61, and 0.79 hr and the Vss was 1.37, 5.57, and 140.3 mL in mouse, rat, and monkey, respectively. In vivo, when JD38 tumor-bearing animals were treated with CAT-8015 at doses ≥ 75 μg/kg at 48 hr intervals for a total of 3 doses, a rapid reduction in tumor volume and in some cases complete remission in tumor growth was observed. The comparative toxicological study showed comparable clinical and anatomical pathology changes for CAT-8015 and CAT-3888. Conclusions CAT-8015 is a CD22-targeting immunotoxin that, in preclinical studies, has greatly improved efficacy as compared to CAT-3888. PMID:19188153

Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin by photochemical internalization - A minimally invasive cancer stem cell-targeting strategy.

PubMed

Bostad, Monica; Olsen, Cathrine Elisabeth; Peng, Qian; Berg, Kristian; Høgset, Anders; Selbo, Pål Kristian

2015-05-28

The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin (PCIAC133-saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133-saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133-saporin. PCI of picomolar levels of AC133-saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133-saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133-saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133-saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133-saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133-saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133+ cells in localized primary tumors or metastasis. Copyright © 2015

A recombinant chimeric Ad5/3 vector expressing a multi-stage Plasmodium antigen induces protective immunity in mice using heterologous prime-boost immunization regimens1

PubMed Central

Cabrera-Mora, Monica; Fonseca, Jairo Andres; Singh, Balwan; Zhao, Chunxia; Makarova, Natalia; Dmitriev, Igor; Curiel, David T.; Blackwell, Jerry; Moreno, Alberto

2016-01-01

An ideal malaria vaccine should target several stages of the parasite life cycle and induce anti-parasite and anti-disease immunity. We have reported a Plasmodium yoelii chimeric multi-stage recombinant protein (PyLPC/RMC), engineered to express several autologous T cell epitopes and sequences derived from the circumsporozoite protein (CSP) and the merozoite surface protein 1 (MSP-1). This chimeric protein elicits protective immunity, mediated by CD4+ T cells and neutralizing antibodies. However, experimental evidence from pre-erythrocytic vaccine candidates and irradiated sporozoites has shown that CD8+ T cells play a significant role in protection. Recombinant viral vectors have been used as a vaccine platform to elicit effective CD8+ T cell responses. The human adenovirus serotype 5 (Ad5) has been tested in malaria vaccine clinical trials with excellent safety profile. Nevertheless, a major concern for the use of Ad5 is the high prevalence of anti-vector neutralizing antibodies in humans, hampering its immunogenicity. To minimize the impact of anti-vector pre-existing immunity we developed a chimeric Ad5/3 vector in which the knob region of Ad5 was replaced with that of Ad3, conferring partial resistance to anti-Ad5 neutralizing antibodies. Furthermore, we implemented heterologous adenovirus/protein immunization regimens which include a single immunization with recombinant Ad vectors. Our data show that immunization with the recombinant Ad5/3 vector induces protective efficacy indistinguishable from that elicited by Ad5. Our study also demonstrate that the dose of the Ad vectors has an impact on the memory profile and protective efficacy. The results support further studies with Ad5/3 for malaria vaccine development. PMID:27574299

Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli.

PubMed

Natsume, Tohru; Taoka, Masato; Manki, Hiroshi; Kume, Shouen; Isobe, Toshiaki; Mikoshiba, Katsuhiko

2002-09-01

We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates. Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip. The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip. The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies. This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

PubMed

Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

2016-02-01

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

PubMed

Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

2016-04-01

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and cDNA cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per a litter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide-binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48-1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

Immunization with recombinant V10 protects cynomolgus macaques from lethal pneumonic plague.

PubMed

Cornelius, Claire A; Quenee, Lauriane E; Overheim, Katie A; Koster, Frederick; Brasel, Trevor L; Elli, Derek; Ciletti, Nancy A; Schneewind, Olaf

2008-12-01

Vaccine and therapeutic strategies that prevent infections with Yersinia pestis have been sought for over a century. Immunization with live attenuated (nonpigmented) strains and immunization with subunit vaccines containing recombinant low-calcium-response V antigen (rLcrV) and recombinant F1 (rF1) antigens are considered effective in animal models. Current antiplague subunit vaccines in development for utilization in humans contain both antigens, either as equal concentrations of the two components (rF1 plus rLcrV) or as a fusion protein (rF1-rLcrV). Here, we show that immunization with either purified rLcrV (a protein at the tip of type III needles) or a variant of this protein, recombinant V10 (rV10) (lacking amino acid residues 271 to 300), alone or in combination with rF1, prevented pneumonic lesions and disease pathogenesis. In addition, passive immunization studies showed that specific antibodies of macaques immunized with rLcrV, rV10, or rF1, either alone or in combination, conferred protection against bubonic plague challenge in mice. Finally, we found that when we compared the reactivities of anti-rLcrV and anti-rV10 immune sera from cynomolgus macaques, BALB/c mice, and brown Norway rats with LcrV-derived peptides, rV10, but not rLcrV immune sera, lacked antibodies recognizing linear LcrV oligopeptides.

Genetically Engineered Immunotherapy for Advanced Cancer

Cancer.gov

In this trial, doctors will collect T lymphocytes from patients with advanced mesothelin-expressing cancer and genetically engineer them to recognize mesothelin. The gene-engineered cells will be multiplied and infused into the patient to fight the cancer

Comparative study of immunological and structural properties of two recombinant vaccine candidates against botulinum neurotoxin type E.

PubMed

Rostamian, Mosayeb; Mousavy, Seyed Jafar; Ebrahimi, Firouz; Ghadami, Seyyed Abolghasem; Sheibani, Nader; Minaei, Mohammad Ebrahim; Arefpour Torabi, Mohammad Ali

2012-01-01

Recently, botulinum neurotoxin (BoNT)-derived recombinant proteins have been suggested as potential botulism vaccines. Here, with concentrating on BoNT type E (BoNT/E), we studied two of these binding domain-based recombinant proteins: a multivalent chimer protein, which is composed of BoNT serotypes A, B and E binding subdomains, and a monovalent recombinant protein, which contains 93 amino acid residues from recombinant C-terminal heavy chain of BoNT/E (rBoNT/E-HCC). Both proteins have an identical region (48 aa) that contains one of the most important BoNT/E epitopes (YLTHMRD sequence). The recombinant protein efficiency in antibody production, their structural differences, and their BoNT/E-epitope location were compared by using ELISA, circular dichroism, computational modeling, and hydrophobicity predictions. Immunological studies indicated that the antibody yield against rBoNT/E-HCC was higher than chimer protein. Cross ELISA confirmed that the antibodies against the chimer protein recognized rBoNT/E-HCC more efficiently. However, both antibody groups (anti-chimer and anti-rBoNT/E-HCC antibodies) were able to recognize other proteins. Structural studies with circular dichroism showed that chimer proteins have slightly more secondary structures than rBoNT/E-HCC. The immunological results suggested that the above-mentioned identical region in rBoNT/E-HCC is more exposed. Circular dichroism, computational protein modeling and hydrophobicity predictions indicated a more exposed location for the identical region in rBoNT/E-HCC than the chimer protein, which is strongly in agreement with immunological results.

Photoionization and Recombination

NASA Technical Reports Server (NTRS)

Nahar, Sultana N.

2000-01-01

Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

Recombinant egg drop syndrome subunit vaccine offers an alternative to virus propagation in duck eggs.

PubMed

Gutter, B; Fingerut, E; Gallili, G; Eliahu, D; Perelman, B; Finger, A; Pitcovski, J

2008-02-01

Egg drop syndrome (EDS) virus vaccines are routinely produced in embryonated duck eggs (Solyom et al., 1982). This procedure poses the risk of dissemination of pathogens, such as avian influenza virus, as the eggs used are not from specific pathogen free birds. To address this problem, the knob and part of the shaft domain of the fibre protein of the EDS virus (termed knob-s) were expressed in Escherichia coli and assessed as a subunit vaccine. A single vaccination with the recombinant protein induced the production of anti-EDS virus antibodies, as detected by haemagglutination inhibition, enzyme-linked immunosorbent assay and virus neutralization tests, for at least 20 weeks. A positive correlation was demonstrated between these three assays. A dose-response assessment showed that the vaccine was effective over the range of 2 to 64 microg protein per dose. Two vaccinations with the recombinant protein, administered before the onset of lay, induced high haemagglutination inhibition antibody titres, comparable with those induced by an inactivated whole-virus vaccine. The vaccine did not have any adverse effects on egg production, quality or weight. The present study has shown that two vaccinations with the recombinant knob-s protein elicited high neutralizing antibody titres that persisted for more than 50 weeks of lay.

Detection of antibodies reacting with the antithetical duffy blood group antigens Fy(a) and Fy(b) using recombinant fusion proteins containing the duffy extracellular domain.

PubMed

Sheffield, William P; Bhakta, Varsha; Branch, Donald R; Denomme, Gregory A

2006-12-01

Detecting blood group-specific antibodies in patient sera is essential to the management of blood transfusions or pregnancies. We produced the antithetical forms of the 65 amino acid extracellular domain (ECD) of the Duffy (Fy) blood group protein fused to glutathione sulfotransferase (GST): GST-Fy(a); and GST-Fy(b), differing only in Gly or Asp at position 44, respectively. The purified recombinant proteins were recognized more effectively by reference polyclonal or monoclonal antibodies than the antithetical Fy specificity by either ELISA or immunoblotting. Combined immunoblot and ELISA tests performed at 1:200 dilutions of sera using the recombinant proteins gave results in agreement with undiluted sera and agglutination for 17/19 alloimmunized patients. At 1:200, agglutination detected anti-Fy(a) or anti-Fy(b) in only three of 12 samples that were positive by ELISA. Recombinant ECD-Fy proteins are suitable and sensitive reagents for the detection of anti-Fy that use technology amenable to automation and/or miniaturization and avoid the need for intact red cells.

Respiratory syncytial virus subunit vaccine based on a recombinant fusion protein expressed transiently in mammalian cells.

PubMed

Nallet, Sophie; Amacker, Mario; Westerfeld, Nicole; Baldi, Lucia; König, Iwo; Hacker, David L; Zaborosch, Christiane; Zurbriggen, Rinaldo; Wurm, Florian M

2009-10-30

Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.

Safety and immunogenicity of a combined Tetanus, Diphtheria, recombinant acellular Pertussis vaccine (TdaP) in healthy Thai adults.

PubMed

Sirivichayakul, Chukiat; Chanthavanich, Pornthep; Limkittikul, Kriengsak; Siegrist, Claire-Anne; Wijagkanalan, Wassana; Chinwangso, Pailinrut; Petre, Jean; Hong Thai, Pham; Chauhan, Mukesh; Viviani, Simonetta

2017-01-02

An acellular Pertussis (aP) vaccine containing recombinant genetically detoxified Pertussis Toxin (PTgen), Filamentous Hemagglutinin (FHA) and Pertactin (PRN) has been developed by BioNet-Asia (BioNet). We present here the results of the first clinical study of this recombinant aP vaccine formulated alone or in combination with tetanus and diphtheria toxoids (TdaP). A phase I/II, observer-blind, randomized controlled trial was conducted at Mahidol University in Bangkok, Thailand in healthy adult volunteers aged 18-35 y. The eligible volunteers were randomized to receive one dose of either BioNet's aP or Tetanus toxoid-reduced Diphtheria toxoid-acellular Pertussis (TdaP) vaccine, or the Tdap Adacel® vaccine in a 1:1:1 ratio. Safety follow-up was performed for one month. Immunogenicity was assessed at baseline, at 7 and 28 d after vaccination. Anti-PT, anti-FHA, anti-PRN, anti-tetanus and anti-diphtheria IgG antibodies were assessed by ELISA. Anti-PT neutralizing antibodies were assessed also by CHO cell assay. A total of 60 subjects (20 per each vaccine group) were enrolled and included in the safety analysis. Safety laboratory parameters, incidence of local and systemic post-immunization reactions during 7 d after vaccination and incidence of adverse events during one month after vaccination were similar in the 3 vaccine groups. One month after vaccination, seroresponse rates of anti-PT, anti-FHA and anti-PRN IgG antibodies exceeded 78% in all vaccine groups. The anti-PT IgG, anti-FHA IgG, and anti-PT neutralizing antibody geometric mean titers (GMTs) were significantly higher following immunization with BioNet's aP and BioNet's TdaP than Adacel® (P< 0.05). The anti-PRN IgG, anti-tetanus and anti-diphtheria GMTs at one month after immunization were comparable in all vaccine groups. All subjects had seroprotective titers of anti-tetanus and anti-diphtheria antibodies at baseline. In this first clinical study, PTgen-based BioNet's aP and TdaP vaccines showed a

Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

PubMed Central

Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G.; Court, Donald L.

2009-01-01

Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about a week's time. PMID:19180090

Immunization with a streptococcal multiple-epitope recombinant protein protects mice against invasive group A streptococcal infection.

PubMed

Kuo, Chih-Feng; Tsao, Nina; Hsieh, I-Chen; Lin, Yee-Shin; Wu, Jiunn-Jong; Hung, Yu-Ting

2017-01-01

Streptococcus pyogenes (group A Streptococcus; GAS) causes clinical diseases, including pharyngitis, scarlet fever, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. A number of group A streptococcus vaccine candidates have been developed, but only one 26-valent recombinant M protein vaccine has entered clinical trials. Differing from the design of a 26-valent recombinant M protein vaccine, we provide here a vaccination using the polyvalence epitope recombinant FSBM protein (rFSBM), which contains four different epitopes, including the fibronectin-binding repeats domain of streptococcal fibronectin binding protein Sfb1, the C-terminal immunogenic segment of streptolysin S, the C3-binding motif of streptococcal pyrogenic exotoxin B, and the C-terminal conserved segment of M protein. Vaccination with the rFSBM protein successfully prevented mortality and skin lesions caused by several emm strains of GAS infection. Anti-FSBM antibodies collected from the rFSBM-immunized mice were able to opsonize at least six emm strains and can neutralize the hemolytic activity of streptolysin S. Furthermore, the internalization of GAS into nonphagocytic cells is also reduced by anti-FSBM serum. These findings suggest that rFSBM can be applied as a vaccine candidate to prevent different emm strains of GAS infection.

Immunization with a streptococcal multiple-epitope recombinant protein protects mice against invasive group A streptococcal infection

PubMed Central

Kuo, Chih-Feng; Tsao, Nina; Hsieh, I-Chen; Lin, Yee-Shin; Wu, Jiunn-Jong; Hung, Yu-Ting

2017-01-01

Streptococcus pyogenes (group A Streptococcus; GAS) causes clinical diseases, including pharyngitis, scarlet fever, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. A number of group A streptococcus vaccine candidates have been developed, but only one 26-valent recombinant M protein vaccine has entered clinical trials. Differing from the design of a 26-valent recombinant M protein vaccine, we provide here a vaccination using the polyvalence epitope recombinant FSBM protein (rFSBM), which contains four different epitopes, including the fibronectin-binding repeats domain of streptococcal fibronectin binding protein Sfb1, the C-terminal immunogenic segment of streptolysin S, the C3-binding motif of streptococcal pyrogenic exotoxin B, and the C-terminal conserved segment of M protein. Vaccination with the rFSBM protein successfully prevented mortality and skin lesions caused by several emm strains of GAS infection. Anti-FSBM antibodies collected from the rFSBM-immunized mice were able to opsonize at least six emm strains and can neutralize the hemolytic activity of streptolysin S. Furthermore, the internalization of GAS into nonphagocytic cells is also reduced by anti-FSBM serum. These findings suggest that rFSBM can be applied as a vaccine candidate to prevent different emm strains of GAS infection. PMID:28355251

Functional genomic mRNA profiling of a large cancer data base demonstrates mesothelin overexpression in a broad range of tumor types.

PubMed

Lamberts, Laetitia E; de Groot, Derk Jan A; Bense, Rico D; de Vries, Elisabeth G E; Fehrmann, Rudolf S N

2015-09-29

The membrane bound glycoprotein mesothelin (MSLN) is a highly specific tumor marker, which is currently exploited as target for drugs. There are only limited data available on MSLN expression by human tumors. Therefore we determined overexpression of MSLN across different tumor types with Functional Genomic mRNA (FGM) profiling of a large cancer database. Results were compared with data in articles reporting immunohistochemical (IHC) MSLN tumor expression. FGM profiling is a technique that allows prediction of biologically relevant overexpression of proteins from a robust data set of mRNA microarrays. This technique was used in a database comprising 19,746 tumors to identify for 41 tumor types the percentage of samples with an overexpression of MSLN compared to a normal background. A literature search was performed to compare the FGM profiling data with studies reporting IHC MSLN tumor expression. FGM profiling showed MSLN overexpression in gastrointestinal (12-36%) and gynecological tumors (20-66%), non-small cell lung cancer (21%) and synovial sarcomas (30%). The overexpression found in thyroid cancers (5%) and renal cell cancers (10%) was not yet reported with IHC analyses. We observed that MSLN amplification rate within esophageal cancer depends on the histotype (31% for adenocarcinomas versus 3% for squamous-cell carcinomas). Subset analysis in breast cancer showed MSLN amplification rates of 28% in triple-negative breast cancer (TNBC) and 33% in basal-like breast cancer. Further subtype analysis of TNBCs showed the highest amplification rate (42%) in the basal-like 1 subtype and the lowest amplification rate (9%) in the luminal androgen receptor subtype.

Insect cell-produced recombinant protein subunit vaccines protect against Zika virus infection.

PubMed

Qu, Panke; Zhang, Wei; Li, Dapeng; Zhang, Chao; Liu, Qingwei; Zhang, Xueyang; Wang, Xuesong; Dai, Wenlong; Xu, Yongfen; Leng, Qibin; Zhong, Jin; Jin, Xia; Huang, Zhong

2018-06-01

Infection with Zika virus (ZIKV) may lead to severe neurologic disorders. It is of significant importance and urgency to develop safe and effective vaccines to prevent ZIKV infection. Here we report the development of ZIKV subunit vaccines based on insect cell-produced recombinant proteins. The N-terminal approximately 80% region (designated as E80) and the domain III (designated as EDIII) of ZIKV envelope (E) protein were efficiently produced as secreted proteins in a Drosophila S2 cell expression system. Both E80 and EDIII could inhibit ZIKV infection in vitro, suggesting that they may have folded properly to display native conformations. Immunization studies demonstrated that both E80 and EDIII vaccines were able to trigger antigen-specific antibody and T-cell responses in mice. The resulting anti-E80 and anti-EDIII sera could potently neutralize ZIKV infection in vitro. More importantly, passive transfer of either anti-E80 or anti-EDIII sera protected recipient mice against lethal ZIKV challenge. It is worth noting that the anti-EDIII sera possessed higher neutralizing titers and conferred more complete protection than the anti-E80 sera, indicating that the S2 cell-produced EDIII is a superior ZIKV vaccine candidate compared with the E80. These data support further preclinical and clinical development of a ZIKV subunit vaccine based on S2 cell-produced EDIII. Copyright © 2018 Elsevier B.V. All rights reserved.

Effectiveness of serum megakaryocyte potentiating factor in evaluating the effects of chrysotile and its heated products on respiratory organs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takata, Ayako; Yamauchi, Hiroshi, E-mail: hyama@kitasato-u.ac.jp; Toya, Tadao

Chrysotile (CH), the most common form of asbestos, is rendered less toxic by heating it at 1000 {sup o}C and converting it to forsterite (FO-1000). However, further safety tests are needed to evaluate human health risk of these materials. It has been reported that serum concentrations of megakaryocyte potentiating factor N-ERC/mesothelin become elevated in patients with mesotheliomas caused by asbestos exposure. In this study, a single 2 mg dose of CH or FO-1000 was intratracheally administered to rats. Within 180 days after the administrations, serum N-ERC/mesothelin concentrations, levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in lung tissues and pathological changes in respiratory organsmore » were determined. In the CH group, a significant increase in serum N-ERC/mesothelin concentrations was observed immediately after intratracheal administration, and the elevation lasted for 30 days. In lung tissues, positive staining for 8-OHdG in bronchioles, alveolar epithelium, inflammatory cells, and granulomas was evidence of a marked DNA oxidative damage. Furthermore, measurements of 8-OHdG in lung tissues based on the HPLC-ECD method suggested that serum N-ERC/mesothelin concentrations tended to increase when there are significant DNA damages in lung tissues. In contrast, in the FO-1000 group, a marked rise in serum N-ERC/mesothelin concentrations occurred only in the early phase (1-7 days) after intratracheal administration. Similarly, FO-1000 induced elevation of 8-OHdG in lung tissues was transient and modest compared with those of the CH-treated animals. In both the CH and FO-1000 groups, we observed significant correlations between serum N-ERC/mesothelin concentrations and lung 8-OHdG concentrations (r = 0.559, p = 0.001 for the CH group; r = 0.516, p = 0.01 for the FO-1000 group). In summary, we demonstrated the possibility of using serum N-ERC/mesothelin concentrations as a useful biomarker for early phase exposure to either CH or FO-1000.« less

The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

PubMed

Jin, Qingwen; Chen, Hong; Wang, Xingxia; Zhao, Liandong; Xu, Qingchen; Wang, Huijuan; Li, Guanyu; Yang, Xiaofan; Ma, Hongming; Wu, Haoquan; Ji, Xiaohui

2015-01-01

Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

Quantitative anti-F1 and anti-V IgG ELISAs as serological correlates of protection against plague in female Swiss Webster mice.

PubMed

Little, S F; Webster, W M; Wilhelm, H; Fisher, D; Norris, S L W; Powell, B S; Enama, J; Adamovicz, J J

2010-01-22

A recombinant fusion protein composed of Yersinia pestis fraction 1 capsule (F1) and virulence-associated V antigen (V) (F1-V) has been developed as the next-generation vaccine against plague. In this study, female Swiss Webster mice received a single intramuscular vaccination with one of eight doses of the F1-V vaccine and exposed 4 weeks later to either Y. pestis CO92 or C12 organisms by the subcutaneous or aerosol routes of infection. Quantitative anti-F1 and anti-V immunoglobulin G (IgG) ELISAs were used to examine the relationship between survival outcome and antibody titers to F1 and V. Results suggested that each 1log(10) increase in week 4 quantitative anti-F1 and anti-V IgG ELISA titers were associated with a 1.7-fold (p=0.0051) and 2.5-fold (p=0.0054) increase in odds of survival, respectively, against either bubonic or pneumonic plague and may serve as serological correlates of protection.

Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators.

PubMed Central

Shimomura, O; Inouye, S; Musicki, B; Kishi, Y

1990-01-01

Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+. PMID:2400391

Dengue Type-2 Virus Envelope Protein Made Using Recombinant Baculovirus Protects Mice Against Virus Challenge

DTIC Science & Technology

1994-01-01

Spodoptera frugiperda (Sf9) cells, approximately I mg of recombinant E antigen was made per 10’ cells. This antigen reacted with polyclonal, anti...entry by fusion at acidic pH with host cell mem- in Spodoptera frugiperda (Sf9) cells brane.Ř The E antigen contains both T and B cell epitopes that

A novel anti-CD22 scFv-apoptin fusion protein induces apoptosis in malignant B-cells.

PubMed

Agha Amiri, Solmaz; Shahhosseini, Soraya; Zarei, Najmeh; Khorasanizadeh, Dorsa; Aminollahi, Elahe; Rezaie, Faegheh; Zargari, Mehryar; Azizi, Mohammad; Khalaj, Vahid

2017-12-01

CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv-apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv-apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.

Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas

PubMed Central

Souza, Ana Paula; Andrade, Bruno Bezerril; Aquino, Dorlene; Entringer, Petter; Miranda, José Carlos; Alcantara, Ruan; Ruiz, Daniel; Soto, Manuel; Teixeira, Clarissa R.; Valenzuela, Jesus G.; de Oliveira, Camila Indiani; Brodskyn, Cláudia Ida; Barral-Netto, Manoel; Barral, Aldina

2010-01-01

Background Leishmania is transmitted by female sand flies and deposited together with saliva, which contains a vast repertoire of pharmacologically active molecules that contribute to the establishment of the infection. The exposure to vector saliva induces an immune response against its components that can be used as a marker of exposure to the vector. Performing large-scale serological studies to detect vector exposure has been limited by the difficulty in obtaining sand fly saliva. Here, we validate the use of two sand fly salivary recombinant proteins as markers for vector exposure. Methodology/principal findings ELISA was used to screen human sera, collected in an area endemic for visceral leishmaniasis, against the salivary gland sonicate (SGS) or two recombinant proteins (rLJM11 and rLJM17) from Lutzomyia longipalpis saliva. Antibody levels before and after SGS seroconversion (n = 26) were compared using the Wilcoxon signed rank paired test. Human sera from an area endemic for VL which recognize Lu. longipalpis saliva in ELISA also recognize a combination of rLJM17 and rLJM11. We then extended the analysis to include 40 sera from individuals who were seropositive and 40 seronegative to Lu. longipalpis SGS. Each recombinant protein was able to detect anti-saliva seroconversion, whereas the two proteins combined increased the detection significantly. Additionally, we evaluated the specificity of the anti-Lu. longipalpis response by testing 40 sera positive to Lutzomyia intermedia SGS, and very limited (2/40) cross-reactivity was observed. Receiver-operator characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for the prediction of anti-SGS positivity. These ROC curves evidenced the superior performance of rLJM17+rLJM11. Predicted threshold levels were confirmed for rLJM17+rLJM11 using a large panel of 1,077 serum samples. Conclusion Our results show the possibility of substituting Lu. longipalpis SGS for two

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

PubMed Central

Iacono-Connors, L C; Schmaljohn, C S; Dalrymple, J M

1990-01-01

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response. Images PMID:2105271

Immunoconjugates in the management of hairy cell leukemia

PubMed Central

Kreitman, Robert J.; Pastan, Ira

2015-01-01

Hairy cell leukemia (HCL) is an indolent B-cell malignancy effectively treated but not often cured by purine analog therapy; after multiple courses of purine analogs, patients can become purine analog resistant and in need of alternative therapies. Complete remission to single-agent purine analog is often accompanied by minimal residual disease (MRD), residual HCL cells detectable by immunologic methods, considered a risk factor for eventual relapse. Several different non-chemotherapy approaches are being used to target relapsed and refractory HCL, including inhibitors of BRAF, but so far only monoclonal antibody (MAb)-based approaches have been reported to eliminate MRD in a high percentage of patients. One of the MAb-based options for HCL currently under clinical investigation involves recombinant immunotoxins, containing a fragment of a MAb and a bacterial toxin. The bacterial toxin, a highly potent fragment from Pseudomonas exotoxin, catalytically ADP-ribosylates elongation factor 2 (EF2), resulting in protein synthesis inhibition and apoptotic cell death. Recombinant immunotoxins tested in HCL patients include LMB-2, targeting CD25, and BL22, targeting CD22. An affinity matured version of BL22, termed moxetumomab pasudotox (formerly HA22 or CAT-8015) achieved high CR rates in phase I, and is currently undergoing multicenter Phase 3 testing. Phase I testing was without dose-limiting toxicity, although 2 patients had grade 2 hemolytic uremic syndrome (HUS) with transient grade 1 abnormalities in platelets and creatinine. Preclinical work is underway to identify residues on moxetumomab pasudotox leading to immunogenicity. Moxetumomab pasudotox is undergoing pivotal testing for relapsed and refractory HCL. PMID:26614902

Genome-wide recombination rate variation in a recombination map of cotton.

PubMed

Shen, Chao; Li, Ximei; Zhang, Ruiting; Lin, Zhongxu

2017-01-01

Recombination is crucial for genetic evolution, which not only provides new allele combinations but also influences the biological evolution and efficacy of natural selection. However, recombination variation is not well understood outside of the complex species' genomes, and it is particularly unclear in Gossypium. Cotton is the most important natural fibre crop and the second largest oil-seed crop. Here, we found that the genetic and physical maps distances did not have a simple linear relationship. Recombination rates were unevenly distributed throughout the cotton genome, which showed marked changes along the chromosome lengths and recombination was completely suppressed in the centromeric regions. Recombination rates significantly varied between A-subgenome (At) (range = 1.60 to 3.26 centimorgan/megabase [cM/Mb]) and D-subgenome (Dt) (range = 2.17 to 4.97 cM/Mb), which explained why the genetic maps of At and Dt are similar but the physical map of Dt is only half that of At. The translocation regions between A02 and A03 and between A04 and A05, and the inversion regions on A10, D10, A07 and D07 indicated relatively high recombination rates in the distal regions of the chromosomes. Recombination rates were positively correlated with the densities of genes, markers and the distance from the centromere, and negatively correlated with transposable elements (TEs). The gene ontology (GO) categories showed that genes in high recombination regions may tend to response to environmental stimuli, and genes in low recombination regions are related to mitosis and meiosis, which suggested that they may provide the primary driving force in adaptive evolution and assure the stability of basic cell cycle in a rapidly changing environment. Global knowledge of recombination rates will facilitate genetics and breeding in cotton.

Genome-wide recombination rate variation in a recombination map of cotton

PubMed Central

Shen, Chao; Li, Ximei; Zhang, Ruiting

2017-01-01

Recombination is crucial for genetic evolution, which not only provides new allele combinations but also influences the biological evolution and efficacy of natural selection. However, recombination variation is not well understood outside of the complex species’ genomes, and it is particularly unclear in Gossypium. Cotton is the most important natural fibre crop and the second largest oil-seed crop. Here, we found that the genetic and physical maps distances did not have a simple linear relationship. Recombination rates were unevenly distributed throughout the cotton genome, which showed marked changes along the chromosome lengths and recombination was completely suppressed in the centromeric regions. Recombination rates significantly varied between A-subgenome (At) (range = 1.60 to 3.26 centimorgan/megabase [cM/Mb]) and D-subgenome (Dt) (range = 2.17 to 4.97 cM/Mb), which explained why the genetic maps of At and Dt are similar but the physical map of Dt is only half that of At. The translocation regions between A02 and A03 and between A04 and A05, and the inversion regions on A10, D10, A07 and D07 indicated relatively high recombination rates in the distal regions of the chromosomes. Recombination rates were positively correlated with the densities of genes, markers and the distance from the centromere, and negatively correlated with transposable elements (TEs). The gene ontology (GO) categories showed that genes in high recombination regions may tend to response to environmental stimuli, and genes in low recombination regions are related to mitosis and meiosis, which suggested that they may provide the primary driving force in adaptive evolution and assure the stability of basic cell cycle in a rapidly changing environment. Global knowledge of recombination rates will facilitate genetics and breeding in cotton. PMID:29176878

DNA prime–protein boost increased the titer, avidity and persistence of anti-Aβ antibodies in wild-type mice

PubMed Central

Davtyan, H; Mkrtichyan, M; Movsesyan, N; Petrushina, I; Mamikonyan, G; Cribbs, DH; Agadjanyan, MG; Ghochikyan, A

2010-01-01

Recently, we reported that a DNA vaccine, composed of three copies of a self B cell epitope of amyloid-β (Aβ42) and the foreign T-cell epitope, Pan DR epitope (PADRE), generated strong anti-Aβ immune responses in wild-type and amyloid precursor protein transgenic animals. Although DNA vaccines have several advantages over peptide–protein vaccines, they induce lower immune responses in large animals and humans compared with those in mice. The focus of this study was to further enhance anti-Aβ11 immune responses by developing an improved DNA vaccination protocol of the prime–boost regimen, in which the priming step would use DNA and the boosting step would use recombinant protein. Accordingly, we generated DNA and recombinant protein-based epitope vaccines and showed that priming with DNA followed by boosting with a homologous recombinant protein vaccine significantly increases the anti-Aβ antibody responses and do not change the immunoglobulin G1 (IgG1) profile of humoral immune responses. Furthermore, the antibodies generated by this prime–boost regimen were long-lasting and possessed a higher avidity for binding with an Aβ42 peptide. Thus, we showed that a heterologous prime–boost regimen could be an effective protocol for developing a potent Alzheimer’s disease (AD) vaccine. PMID:19865176

Fine-Scale Recombination Maps of Fungal Plant Pathogens Reveal Dynamic Recombination Landscapes and Intragenic Hotspots

PubMed Central

Stukenbrock, Eva H.; Dutheil, Julien Y.

2018-01-01

Meiotic recombination is an important driver of evolution. Variability in the intensity of recombination across chromosomes can affect sequence composition, nucleotide variation, and rates of adaptation. In many organisms, recombination events are concentrated within short segments termed recombination hotspots. The variation in recombination rate and positions of recombination hotspot can be studied using population genomics data and statistical methods. In this study, we conducted population genomics analyses to address the evolution of recombination in two closely related fungal plant pathogens: the prominent wheat pathogen Zymoseptoria tritici and a sister species infecting wild grasses Z. ardabiliae. We specifically addressed whether recombination landscapes, including hotspot positions, are conserved in the two recently diverged species and if recombination contributes to rapid evolution of pathogenicity traits. We conducted a detailed simulation analysis to assess the performance of methods of recombination rate estimation based on patterns of linkage disequilibrium, in particular in the context of high nucleotide diversity. Our analyses reveal overall high recombination rates, a lack of suppressed recombination in centromeres, and significantly lower recombination rates on chromosomes that are known to be accessory. The comparison of the recombination landscapes of the two species reveals a strong correlation of recombination rate at the megabase scale, but little correlation at smaller scales. The recombination landscapes in both pathogen species are dominated by frequent recombination hotspots across the genome including coding regions, suggesting a strong impact of recombination on gene evolution. A significant but small fraction of these hotspots colocalize between the two species, suggesting that hotspot dynamics contribute to the overall pattern of fast evolving recombination in these species. PMID:29263029

Fine-Scale Recombination Maps of Fungal Plant Pathogens Reveal Dynamic Recombination Landscapes and Intragenic Hotspots.

PubMed

Stukenbrock, Eva H; Dutheil, Julien Y

2018-03-01

Meiotic recombination is an important driver of evolution. Variability in the intensity of recombination across chromosomes can affect sequence composition, nucleotide variation, and rates of adaptation. In many organisms, recombination events are concentrated within short segments termed recombination hotspots. The variation in recombination rate and positions of recombination hotspot can be studied using population genomics data and statistical methods. In this study, we conducted population genomics analyses to address the evolution of recombination in two closely related fungal plant pathogens: the prominent wheat pathogen Zymoseptoria tritici and a sister species infecting wild grasses Z. ardabiliae We specifically addressed whether recombination landscapes, including hotspot positions, are conserved in the two recently diverged species and if recombination contributes to rapid evolution of pathogenicity traits. We conducted a detailed simulation analysis to assess the performance of methods of recombination rate estimation based on patterns of linkage disequilibrium, in particular in the context of high nucleotide diversity. Our analyses reveal overall high recombination rates, a lack of suppressed recombination in centromeres, and significantly lower recombination rates on chromosomes that are known to be accessory. The comparison of the recombination landscapes of the two species reveals a strong correlation of recombination rate at the megabase scale, but little correlation at smaller scales. The recombination landscapes in both pathogen species are dominated by frequent recombination hotspots across the genome including coding regions, suggesting a strong impact of recombination on gene evolution. A significant but small fraction of these hotspots colocalize between the two species, suggesting that hotspot dynamics contribute to the overall pattern of fast evolving recombination in these species. Copyright © 2018 Stukenbrock and Dutheil.

Recombination monitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, S. Y.; Blaskiewicz, M.

This is a brief report on LEReC recombination monitor design considerations. The recombination produced Au 78+ ion rate is reviewed. Based on this two designs are discussed. One is to use the large dispersion lattice. It is shown that even with the large separation of the Au 78+ beam from the Au 79+ beam, the continued monitoring of the recombination is not possible. Accumulation of Au 78+ ions is needed, plus collimation of the Au79+ beam. In another design, it is shown that the recombination monitor can be built based on the proposed scheme with the nominal lattice. From machinemore » operation point of view, this design is preferable. Finally, possible studies and the alternative strategies with the basic goal of the monitor are discussed.« less

Production and characterization of egg yolk antibody (IgY) against recombinant VP8-S2 antigen.

PubMed

Nasiri, K; Nassiri, M R; Tahmoorespur, M; Haghparast, A; Zibaee, S

2016-01-01

Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. VP8 subunit of rotavirus is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Studies showed that immunoglobulin of egg yolk (IgY) from immunized hens has been identified to be a convenient source for specific antibodies for using in immunotherapy and immunodiagnostic to limit the infections. In this study, chimeric VP8-S2 gene was designed using by computational techniques. The chimeric VP8-S2 gene was cloned and sub-cloned into pGH and pET32a (+) vectors. Then, recombinant pET32a-VP8-S2 vector was transferred into E. coli BL21 CodonPlus (DE3). The expressed protein was purified by Ni-NTA chromatography column. Hens were immunized with the purified VP8-S2 protein three times. IgY was purified from egg yolks using polyethylene glycol precipitation method. Activity and specificity of anti-VP8-S2 IgY were detected by dot-blotting, Western-blotting and indirect ELISA. We obtained anti-VP8-S2 IgY by immunizing hens with the recombinant VP8-S2 protein. The anti-VP8-S2 IgY was showed to bind specifically to the chimeric VP8-S2 protein by dot-blotting, Western-blotting analyses and indirect ELISA. The result of this study indicated that such construction can be useful to investigate as candidates for development of detection methods for simultaneous diagnosis of both infections. Specific IgY against the recombinant VP8-S2 could be recommended as a candidate for passive immunization against bovine rotavirus and bovine coronavirus.

Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

PubMed Central

Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

2013-01-01

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×103 and 95.4×103 CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×105 and 622.2×105 cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals. PMID:23799010

Anti-bacterial activity of recombinant human β-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

PubMed

Liu, Jun; Luo, Yan; Ge, Hengtao; Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

2013-01-01

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.

[Construction and immunogenicity of recombinant bacteriophage T7 vaccine expressing M2e peptides of avian influenza virus].

PubMed

Xu, Hai; Wang, Yi-Wei; Tang, Ying-Hua; Zheng, Qi-Sheng; Hou, Ji-Bo

2013-06-01

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.

First identification of a recombinant form of hepatitis C virus in Austrian patients by full-genome next generation sequencing

PubMed Central

Haas, Bernhard; Bauer, Bernd; Zhang, Sherry; Fiss, Ellen H.; Hillman, Grantland; Hamilton, Aaron T.; Mehta, Rochak; Heil, Marintha L.; Marins, Ed G.; Santner, Brigitte I.; Kessler, Harald H.

2017-01-01

Hepatitis C virus (HCV) intergenotypic recombinant forms have been reported for various HCV genotypes/subtypes in several countries worldwide. In a recent study, four patients living in Austria had been identified to be possibly infected with a recombinant HCV strain. To clarify results and determine the point of recombination, full-genome next-generation sequencing using the Illumina MiSeq v2 300 cycle kit (Illumina, San Diego, CA, USA) was performed in the present study. Samples of all of the patients contained the recombinant HCV strain 2k/1b. The point of recombination was found to be within the HCV NS2 gene between nucleotide positions 3189–3200 based on H77 numbering. While three of four patients were male and had migration background from Chechnya (n = 2) and Azerbaijan (n = 1), the forth patient was a female born in Austria. Three of the four patients including the female had intravenous drug abuse as a risk factor for HCV transmission. While sequencing techniques are limited to a few specialized laboratories, a genotyping assay that uses both ends of the HCV genome should be employed to identify patients infected with a recombinant HCV strain. The correct identification of recombinant strains also has an impact considering the tailored choice of anti-HCV treatment. PMID:28742818

First identification of a recombinant form of hepatitis C virus in Austrian patients by full-genome next generation sequencing.

PubMed

Stelzl, Evelyn; Haas, Bernhard; Bauer, Bernd; Zhang, Sherry; Fiss, Ellen H; Hillman, Grantland; Hamilton, Aaron T; Mehta, Rochak; Heil, Marintha L; Marins, Ed G; Santner, Brigitte I; Kessler, Harald H

2017-01-01

Hepatitis C virus (HCV) intergenotypic recombinant forms have been reported for various HCV genotypes/subtypes in several countries worldwide. In a recent study, four patients living in Austria had been identified to be possibly infected with a recombinant HCV strain. To clarify results and determine the point of recombination, full-genome next-generation sequencing using the Illumina MiSeq v2 300 cycle kit (Illumina, San Diego, CA, USA) was performed in the present study. Samples of all of the patients contained the recombinant HCV strain 2k/1b. The point of recombination was found to be within the HCV NS2 gene between nucleotide positions 3189-3200 based on H77 numbering. While three of four patients were male and had migration background from Chechnya (n = 2) and Azerbaijan (n = 1), the forth patient was a female born in Austria. Three of the four patients including the female had intravenous drug abuse as a risk factor for HCV transmission. While sequencing techniques are limited to a few specialized laboratories, a genotyping assay that uses both ends of the HCV genome should be employed to identify patients infected with a recombinant HCV strain. The correct identification of recombinant strains also has an impact considering the tailored choice of anti-HCV treatment.

Comparative efficacy of two dosages of recombinant hepatitis B vaccine in healthy adolescents in India.

PubMed

Velu, Vijayakumar; Nandakumar, Subhadra; Shanmugam, Saravanan; Shankar, Esaki Muthu; Thangavel, Sundararajan; Kulkarni, Prasad Suryakant; Thyagarajan, Sadras Panchatcharam

2007-11-01

Inclusion of hepatitis B vaccine in the Universal Programme of Immunization of all Asian and African countries is hampered by the economic burden on the health budget because of the cost of hepatitis B vaccines. Here we evaluated the immunogenicity, safety, efficacy, and the persistence of antibody to hepatitis B surface antigen (anti-HBs) titers of a new and a low cost recombinant hepatitis B vaccine GeneVac B, with 2 different dosages in healthy adolescents in India. GeneVac-B, a recombinant hepatitis B vaccine (Serum Institute of India, Pune, India), was administered in 10 or 20 microg dose intramuscularly to 2 groups of 100 healthy school-going adolescents at 0-, 1-, and 6-month intervals, who were followed up for 1 year. Group I received 20 mug doses whereas Group II received 10 mug doses. Blood samples were collected 1 month after each dose and 1 year after the third dose. The anti-HBs titers were assayed using commercially available kits to assess the immunogenicity of the 2 dosage schedules. Safety studies were also carried out. The geometric mean titer value of the anti-HBs titer 1 month after the third dose was 2629 (mlU/mL) in Group I and 1373 mlU/mL for Group II subjects. One year after the third dose, the persistence of anti-HBs in those who had received 20 mug was 2262 mlU/mL whereas it was 1039 mlU/mL in the group receiving 10 microg doses. All the subjects in both the groups were seroprotected at 1 year after vaccination. None of the vaccinees exhibited serious adverse reactions throughout the study period. The study demonstrated the immunogenicity of the recombinant hepatitis B vaccine, and confirms that the 0.5 mL (10 microg) dose of GeneVac B can be administered with satisfactory safety and immunogenicity to adolescents up to 19 years of age, reducing the cost to less than U.S. $1.00 per dose making it acceptable for the Universal Programme of Immunization of developing and under developed countries.

Enhancement of anti-stokes sensitized luminescence in AgCl(I) crystals in the presence of silver nanoparticles

NASA Astrophysics Data System (ADS)

Chung, Nguyen Thi Kim; Egorushina, E. A.; Latyshev, A. N.; Ovchinnikov, O. V.; Smirnov, M. S.; Suvorova, T. I.

2012-01-01

We have observed a significant increase in the intensity with anti-Stokes excitation of recombination luminescence in AgCl(I) microcrystals sensitized by methylene blue molecules in the presence of silver nanoparticles.

Genetic Recombination

ERIC Educational Resources Information Center

Whitehouse, H. L. K.

1973-01-01

Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

Overlapping but distinct specificities of anti-liver-kidney microsome antibodies in autoimmune hepatitis type II and hepatitis C revealed by recombinant native CYP2D6 and novel peptide epitopes

PubMed Central

Klein, R; Zanger, U M; Berg, T; Hopf, U; Berg, P A

1999-01-01

Anti-liver-kidney microsome antibodies (anti-LKM) occur in autoimmune hepatitis (AIH) type II and in a subset of patients with hepatitis C. Anti-LKM1 in AIH are directed against cytochrome P4502D6 (CYP2D6), but conflicting data exist concerning the specificity of anti-LKM in hepatitis C. The aim of this study was to evaluate binding specificities of anti-LKM antibodies in both diseases using novel test antigens as well as their inhibitory capacity on CYP2D6 enzyme activity. Sera from 22 patients with AIH type II and 17 patients with hepatitis C being anti-LKM-positive in the immunofluorescence test were investigated for binding to native recombinant CYP2D6 and liver microsomes by ELISA and immunoblotting, and to synthetic peptides covering the region 254–339 (254–273, 257–269, 270–294, 291–310, 307–324, 321–339, 373–389) as well as the novel peptide 196–218 by ELISA. Furthermore, all sera were tested for inhibition of CYP2D6-dependent bufuralol 1′-hydroxylase activity. Twenty of the 22 AIH type II sera (91%) and nine of the 17 hepatitis C sera (53%) were positive for CYP2D6 by ELISA and/or immunoblotting. The previously described major peptide epitope comprising CYP2D6 amino acids 257–269 was recognized by 16 of the 22 AIH sera but by only one hepatitis C serum. A further epitope, 196–218, could be defined for the first time as another immunodominant epitope for AIH because it was recognized by 15 of the 22 AIH (68%) but only three of the 17 hepatitis C sera (18%). With the exception of the peptide 254–273, the other peptides showed no significant reactivity. Analysing the inhibitory properties of anti-LKM antibodies it emerged that 95% of AIH sera and 88% of hepatitis C sera inhibited enzyme function. These data indicate that anti-LKM antibodies in AIH and hepatitis C react with CYP2D6, as shown by their inhibitory activity, and that besides the known epitope 257–269 a further immunodominant epitope exists on CYP2D6 which is recognized

Field-enhanced route to generating anti-Frenkel pairs in HfO2

NASA Astrophysics Data System (ADS)

Schie, Marcel; Menzel, Stephan; Robertson, John; Waser, Rainer; De Souza, Roger A.

2018-03-01

The generation of anti-Frenkel pairs (oxygen vacancies and oxygen interstitials) in monoclinic and cubic HfO2 under an applied electric field is examined. A thermodynamic model is used to derive an expression for the critical field strength required to generate an anti-Frenkel pair. The critical field strength of EaFcr˜101GVm-1 obtained for HfO2 exceeds substantially the field strengths routinely employed in the forming and switching operations of resistive switching HfO2 devices, suggesting that field-enhanced defect generation is negligible. Atomistic simulations with molecular static (MS) and molecular dynamic (MD) approaches support this finding. The MS calculations indicated a high formation energy of Δ EaF≈8 eV for the infinitely separated anti-Frenkel pair, and only a decrease to Δ EaF≈6 eV for the adjacent anti-Frenkel pair. The MD simulations showed no defect generation in either phase for E <3 GVm-1 , and only sporadic defect generation in the monoclinic phase (at E =3 GVm-1 ) with fast (trec<4 ps ) recombination. At even higher E but below EaFcr both monoclinic and cubic structures became unstable as a result of field-induced deformation of the ionic potential wells. Further MD investigations starting with preexisting anti-Frenkel pairs revealed recombination of all pairs within trec<1 ps , even for the case of neutral vacancies and charged interstitials, for which formally there is no electrostatic attraction between the defects. In conclusion, we find no physically reasonable route to generating point-defects in HfO2 by an applied field.

Development of ELISA using recombinant antigens for specific detection of mouse parvovirus infection.

PubMed

Kunita, Satoshi; Chaya, Miyuki; Hagiwara, Kozue; Ishida, Tomoko; Takakura, Akira; Sugimoto, Tatsuya; Iseki, Hiroyoshi; Fuke, Kumiko; Sugiyama, Fumihiro; Yagami, Ken-ichi

2006-04-01

Nucleotide sequences of mouse parvovirus (MPV) isolate, named MPV/UT, and mouse minute virus (MMV) were analyzed and used for expressing recombinant proteins in E. coli. ELISA tests using recombinant major capsid protein (rVP2) and recombinant major non-structural protein (rNS1) as antigens were developed and their performance in serologic detection of rodent parvovirus infection was assessed. MPV-rVP2 and MMV-rVP2 ELISAs reacted specifically with anti-MPV and anti-MMV mouse sera, respectively. MMV-rNS1 antigen had a wide reaction range with antisera to rodent parvoviruses including MPV, MMV, Kilham rat virus (KRV) and H-1 virus. All mice oronasally infected with MPV were seropositive at 4 weeks post-infection in screening by ELISAs using MPV-rVP2 and MMV-rNS1 antigens, but were negative by conventional ELISA using whole MMV antigen. A contact transmission experiment revealed that transmission of MPV occurred up to 4 weeks post-infection, and all cage mates were seropositive in screening with MPV-rVP2 and MMV-rNS1 ELISAs. These results indicate that MPV-rVP2 and MMV-rVP2 are specific ELISA antigens which distinguish between MPV and MVM infection, while MMV-rNS1 antigen can be used in generic ELISA for a variety of rodent parvoviruses. The higher sensitivity of MPV-rVP2 ELISA than conventional ELISA for detecting seroconversion to MPV in oronasally infected mice as well as in cage mates suggests the usefulness of MPV-rVP2 ELISA in quarantine and microbiological monitoring of MPV infection in laboratory mice.

Advanced and recurring thymic carcinoma is target of new clinical trial | Center for Cancer Research

Cancer.gov

Adults diagnosed with thymic carcinoma who overexpress the protein mesothelin may be eligible to participate in a new clinical trial at the NIH Clinical Center. The study will look at the safety and effectiveness of an investigational drug, anetumab ravtansine, developed by Bayer HealthCare Pharmaceuticals. The drug works by binding to mesothelin, therefore overexpression of

Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting-out.

PubMed

Bian, Liujiao; Ji, Xu; Hu, Wei

2014-07-01

In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine-tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni-chelated Sepharose Fast-Flow affinity column, ammonium sulfate salting-out and Sephadex G-75 size-exclusion column in turn. The purity analysis by SDS-PAGE, high-performance size-exclusion and reversed-phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti-angiogenic activity under the effective range of 5.0-25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%. Copyright © 2013 John Wiley & Sons, Ltd.

Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

PubMed

Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

2004-10-01

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites

PubMed Central

Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

2016-01-01

Background Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Methodology/Principal Findings Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Conclusions/Significance Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

PubMed

Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

2016-03-01

Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

Detection of rabies-specific antigens by egg yolk antibody (IgY) to the recombinant rabies virus proteins produced in Escherichia coli.

PubMed

Motoi, Yurie; Inoue, Satoshi; Hatta, Hajime; Sato, Kozue; Morimoto, Kinjiro; Yamada, Akio

2005-04-01

We obtained rabies-specific egg yolk antibodies (IgY) by immunizing hens with recombinant His-tagged nucleoprotein and phosphoprotein (rN, rP) of the rabies virus (CVS-11 strain) expressed in Escherichia coli. The anti-rN and rP IgY were shown to bind specifically to the respective proteins of the CVS-11 strain of rabies virus by Western blotting, immune fluorescent assay and immunohistochemistry, indicating that IgY to rabies recombinant proteins could serve as a reagent for diagnosis of rabies virus infection.

Recombinant raccoon pox vaccine protects mice against lethal plague

USGS Publications Warehouse

Osorio, J.E.; Powell, T.D.; Frank, R.S.; Moss, K.; Haanes, E.J.; Smith, S.R.; Rocke, T.E.; Stinchcomb, D.T.

2003-01-01

Using a raccoon poxvirus (RCN) expression system, we have developed new recombinant vaccines that can protect mice against lethal plague infection. We tested the effects of a translation enhancer (EMCV-IRES) in combination with a secretory (tPA) signal or secretory (tPA) and membrane anchoring (CHV-gG) signals on in vitro antigen expression of F1 antigen in tissue culture and the induction of antibody responses and protection against Yersinia pestis challenge in mice. The RCN vector successfully expressed the F1 protein of Y. pestis in vitro. In addition, the level of expression was increased by the insertion of the EMCV-IRES and combinations of this and the secretory signal or secretory and anchoring signals. These recombinant viruses generated protective immune responses that resulted in survival of 80% of vaccinated mice upon challenge with Y. pestis. Of the RCN-based vaccines we tested, the RCN-IRES-tPA-YpF1 recombinant construct was the most efficacious. Mice vaccinated with this construct withstood challenge with as many as 1.5 million colony forming units of Y. pestis (7.7×104 LD50). Interestingly, vaccination with F1 fused to the anchoring signal (RCN-IRES-tPA-YpF1-gG) elicited significant anti-F1 antibody titers, but failed to protect mice from plague challenge. Our studies demonstrate, in vitro and in vivo, the potential importance of the EMCV-IRES and secretory signals in vaccine design. These molecular tools provide a new approach for improving the efficacy of vaccines. In addition, these novel recombinant vaccines could have human, veterinary, and wildlife applications in the prevention of plague.

Anti-inflammatory and anti-fibrinolytic effects of thrombomodulin alfa through carboxypeptidase B2 in the presence of thrombin.

PubMed

Tawara, Shunsuke; Sakai, Takumi; Matsuzaki, Osamu

2016-11-01

Thrombomodulin (TM) alfa, a recombinant human soluble TM, enhances activation of pro-carboxypeptidase B2 (pro-CPB2) by thrombin. Activated pro-CPB2 (CPB2) exerts anti-inflammatory and anti-fibrinolytic activities. Therefore, TM alfa may also have anti-inflammatory and anti-fibrinolytic effects through CPB2. However, these effects of TM alfa have not been elucidated. In the present study, we investigated the effects of TM alfa on inactivation of complement component C5a as an anti-inflammatory effect and prolongation of clot lysis time as an anti-fibrinolytic effect via CPB2 in vitro. CPB2 activity and tissue factor-induced thrombin generation was examined by a chromogenic assay. C5a inactivation was evaluated by C-terminal cleavage of C5a and inhibition of C5a-induced human neutrophil migration. Clot lysis time prolongation was examined by a tissue-type plasminogen activator-induced clot lysis assay. CPB2 activity in human plasma was increased by TM alfa and thrombin in a concentration-dependent manner. TM alfa inhibited tissue factor-induced thrombin generation and enhanced pro-CPB2 activation in human plasma simultaneously. The mass spectrum of C5a treated with TM alfa, thrombin, and pro-CPB2 was decreased at 156m/z, indicating that TM alfa enhanced the processing of C5a to C-terminal-cleaved C5a, an inactive form of C5a. C5a-induced human neutrophil migration was decreased after C5a treatment with TM alfa, thrombin, and pro-CPB2. TM alfa prolonged the clot lysis time in human plasma, and this effect was completely abolished by addition of a CPB2 inhibitor. TM alfa exerts anti-inflammatory and anti-fibrinolytic effects through CPB2 in the presence of thrombin in vitro. Copyright © 2016 Elsevier Ltd. All rights reserved.

Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (Apis cerana) venom.

PubMed

Yang, Jie; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Yoon, Hyung Joo; Jia, Jingming; Jin, Byung Rae

2017-10-01

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

Promise and problems associated with the use of recombinant AAV for the delivery of anti-HIV antibodies

PubMed Central

Fuchs, Sebastian P; Desrosiers, Ronald C

2016-01-01

Attempts to elicit antibodies with potent neutralizing activity against a broad range of human immunodeficiency virus (HIV) isolates have so far proven unsuccessful. Long-term delivery of monoclonal antibodies (mAbs) with such activity is a creative alternative that circumvents the need for an immune response and has the potential for creating a long-lasting sterilizing barrier against HIV. This approach is made possible by an incredible array of potent broadly neutralizing antibodies (bnAbs) that have been identified over the last several years. Recombinant adeno-associated virus (rAAV) vectors are ideally suited for long-term delivery for a variety of reasons. The only products made from rAAV are derived from the transgenes that are put into it; as long as those products are not viewed as foreign, expression from muscle tissue may continue for decades. Thus, use of rAAV to achieve long-term delivery of anti-HIV mAbs with potent neutralizing activity against a broad range of HIV-1 isolates is emerging as a promising concept for the prevention or treatment of HIV-1 infection in humans. Experiments in mice and monkeys that have demonstrated protective efficacy against AIDS virus infection have raised hopes for the promise of this approach. However, all published experiments in monkeys have encountered unwanted immune responses to the AAV-delivered antibody, and these immune responses appear to limit the levels of delivered antibody that can be achieved. In this review, we highlight the promise of rAAV-mediated antibody delivery for the prevention or treatment of HIV infection in humans, but we also discuss the obstacles that will need to be understood and solved in order for the promise of this approach to be realized. PMID:28197421

A Recombinant Humanized Anti-Cocaine Monoclonal Antibody Inhibits the Distribution of Cocaine to the Brain in Rats

PubMed Central

Gooden, Felicia C. T.; Tabet, Michael R.; Ball, William J.

2014-01-01

The monoclonal antibody (mAb), h2E2, is a humanized version of the chimeric human/murine anti-cocaine mAb 2E2. The recombinant h2E2 protein was produced in vitro from a transfected mammalian cell line and retained high affinity (4 nM Kd) and specificity for cocaine over its inactive metabolites benzoylecgonine (BE) and ecgonine methyl ester. In rats, pharmacokinetic studies of h2E2 (120 mg/kg i.v.) showed a long terminal elimination half-life of 9.0 days and a low volume of distribution at steady state (Vdss) of 0.3 l/kg. Pretreatment with h2E2 produced a dramatic 8.8-fold increase in the area under the plasma cocaine concentration-time curve (AUC) and in brain a concomitant decrease of 68% of cocaine’s AUC following an i.v. injection of an equimolar cocaine dose. Sequestration of cocaine in plasma by h2E2, shown via reduction of cocaine’s Vdss, indicates potential clinical efficacy. Although the binding of cocaine to h2E2 in plasma should inhibit distribution and metabolism, the elimination of cocaine remained multicompartmental and was still rapidly eliminated from plasma despite the presence of h2E2. BE was the major cocaine metabolite, and brain BE concentrations were sixfold higher than in plasma, indicating that cocaine is normally metabolized in the brain. In the presence of h2E2, brain BE concentrations were decreased and plasma BE was increased, consistent with the observed h2E2-induced changes in cocaine disposition. The inhibition of cocaine distribution to the brain confirms the humanized mAb, h2E2, as a lead candidate for development as an immunotherapy for cocaine abuse. PMID:24733787

Recombinant Scorpine Produced Using SUMO Fusion Partner in Escherichia coli Has the Activities against Clinically Isolated Bacteria and Inhibits the Plasmodium falciparum Parasitemia In Vitro

PubMed Central

Gu, Yaping; Zhou, Huayun; Cao, Jun; Gao, Qi

2014-01-01

Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni2+–NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+–NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future. PMID:25068263

Diffusion controlled initial recombination

NASA Astrophysics Data System (ADS)

Christen, T.; Büttiker, M.

1998-08-01

This work addresses nucleation rates in systems with strong initial recombination. Initial (or ``geminate'') recombination is a process where a dissociated structure (anion, vortex, kink, etc.) recombines with its twin brother (cation, antivortex, antikink) generated in the same nucleation event. Initial recombination is important if there is an asymptotically vanishing interaction force instead of a generic saddle-type activation barrier. At low temperatures, initial recombination strongly dominates homogeneous recombination. In a first part, we discuss the effect in one-, two-, and three-dimensional diffusion controlled systems with spherical symmetry. Since there is no well-defined saddle, we introduce a threshold which is to some extent arbitrary but which is restricted by physically reasonable conditions. We show that the dependence of the nucleation rate on the specific choice of this threshold is strongest for one-dimensional systems and decreases in higher dimensions. We also discuss the influence of a weak driving force, and show that the transport current is directly determined by the imbalance of the activation rate in the direction of the field and the rate against this direction. In a second part, we apply the results to the overdamped sine-Gordon system at equilibrium. It turns out that diffusive initial recombination is the essential mechanism which governs the equilibrium kink nucleation rate. We emphasize analogies between the single particle problem with initial recombination and the multidimensional kink-antikink nucleation problem.

Antibody treatment of human tumor xenografts elicits active anti-tumor immunity in nude mice

PubMed Central

Liebman, Meredith A.; Roche, Marly I.; Williams, Brent R.; Kim, Jae; Pageau, Steven C.; Sharon, Jacqueline

2007-01-01

Athymic nude mice bearing subcutaneous tumor xenografts of the human anti-colorectal cancer cell line SW480 were used as a preclinical model to explore anti-tumor immunotherapies. Intratumor or systemic treatment of the mice with murine anti-SW480 serum, recombinant anti-SW480 polyclonal antibodies, or the anti-colorectal cancer monoclonal antibody CO17-1A, caused retardation or regression of SW480 tumor xenografts. Interestingly, when mice that had regressed their tumors were re-challenged with SW480 cells, these mice regressed the new tumors without further antibody treatment. Adoptive transfer of spleen cells from mice that had regressed their tumors conferred anti-tumor immunity to naïve nude mice. Pilot experiments suggest that the transferred anti-tumor immunity is mediated by T cells of both γδ and αβ lineages. These results demonstrate that passive anti-tumor immunotherapy can elicit active immunity and support a role for extra-thymic γδ and αβ T cells in tumor rejection. Implications for potential immunotherapies include injection of tumor nodules in cancer patients with anti-tumor antibodies to induce anti-tumor T cell immunity. PMID:17920694

Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

PubMed Central

Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

2015-01-01

Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

Oral immunization of BALB/c mice with Giardia duodenalis recombinant cyst wall protein inhibits shedding of cysts.

PubMed

Larocque, R; Nakagaki, K; Lee, P; Abdul-Wahid, A; Faubert, G M

2003-10-01

The process of encystation is a key step in the Giardia duodenalis life cycle that allows this intestinal protozoan to survive between hosts during person-to-person, animal-to-person, waterborne, or food-borne transmission. The release of cysts from infected persons and animals is the main contributing factor to contamination of the environment. Genes coding for cyst wall proteins (CWPs), which could be used for developing a transmission-blocking vaccine, have been cloned. Since the immunogenicity of recombinant Giardia CWP is unknown, we have investigated the immunogenicity of recombinant CWP2 (rCWP2) and its efficacy in interfering with the phenomenon of encystation taking place in the small bowels of BALB/c mice vaccinated with the recombinant protein. Here we report that the immunization of BALB/c mice with rCWP2 stimulated the immune system in a manner comparable to that for a live infection with Giardia muris cysts. Fecal and serum anti-rCWP2 immunoglobulin A (IgA) antibodies were detected in the immunized mice. In addition, anti-rCWP2 IgG1 and IgG2a antibodies were detected in the serum. mRNAs coding for Th1 and Th2 types of cytokines were detected in spleen and Peyer's patch cells from immunized mice. When the vaccinated mice were challenged with live cysts, the animals shed fewer cysts. We conclude that rCWP2 is a possible candidate antigen for the development of a transmission-blocking vaccine.

Synthesis, evaluation and modeling of some triazolothienopyrimidinones as anti-inflammatory and antimicrobial agents.

PubMed

Bekhit, Adnan A; Farghaly, Ahmed M; Shafik, Ragab M; Elsemary, Mona Ma; El-Shoukrofy, Mai S; Bekhit, Alaa El-Din A; Ibrahim, Tamer M

2017-06-01

New triazolotetrahydrobenzothienopyrimidinone derivatives were synthesized. Their structures were confirmed, and their anti-inflammatory, antimicrobial activities and ulcerogenic potentials were evaluated. Compounds 7a, 10a and 11a showed minimal ulcerogenic effect and high selectivity toward human recombinant COX-2 over COX-1 enzyme with IC 50 values of 1.39, 1.22 and 0.56 μM, respectively. Their docking outcome correlated with their biological activity and confirmed the high selectivity binding toward COX-2. Compound 12b displayed antimicrobial activity comparable to that of ampicillin against Escherichia coli while compounds 6 and 11c were similar to ampicillin against Staphylococcus aureus. In addition, compounds 7a, 9a, 10b and 11c showed dual anti-inflammatory/antimicrobial activities. This work represents a promising matrix for developing new potential anti-inflammatory, antimicrobial and dual antimicrobial/anti-inflammatory candidates. [Formula: see text].

Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli.

PubMed

Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

2016-08-01

Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans.

Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

PubMed Central

Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

2016-01-01

Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila.

PubMed

Smukowski Heil, Caiti S; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A F

2015-10-01

Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human-chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Therapeutic Recombinant Monoclonal Antibodies

ERIC Educational Resources Information Center

Bakhtiar, Ray

2012-01-01

During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin.

PubMed Central

Panicali, D; Davis, S W; Weinberg, R L; Paoletti, E

1983-01-01

Recombinant vaccinia viruses containing the cloned hemagglutinin (HA) gene from influenza virus were constructed. The biological activity of these poxvirus vectors was demonstrated both in vitro and in vivo. Expression of HA in cells infected with recombinant vaccinia was detected by using specific anti-HA antiserum and 125I-labeled protein A, showing that HA synthesized under the regulation of vaccinia virus was antigenic. Immunization of rabbits with these recombinant poxviruses resulted in the production of antibodies reactive with authentic influenza HA as detected by radioimmunoassay, by inhibition of HA erythrocyte agglutination, and by neutralization of influenza virus infectivity. The production of antibodies directed against influenza HA suggested that the HA gene expressed in vaccinia is immunogenic. These data indicate the potential of genetically engineered poxviruses for use as generic live vaccine vehicles that have both human and veterinary applications. Images PMID:6310573

Advanced and recurring thymic carcinoma is target of new clinical trial | Center for Cancer Research

Cancer.gov

Adults diagnosed with thymic carcinoma who overexpress the protein mesothelin may be eligible to participate in a new clinical trial at the NIH Clinical Center. The study will look at the safety and effectiveness of an investigational drug, anetumab ravtansine, developed by Bayer HealthCare Pharmaceuticals. The drug works by binding to mesothelin, therefore overexpression of the protein could be useful for targeting cancer cells. Read more...

Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

PubMed

Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

2017-02-01

Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

5-Aminolevulinic acid-based photochemical internalization of the immunotoxin MOC31-gelonin generates synergistic cytotoxic effects in vitro.

PubMed

Selbo, P K; Kaalhus, O; Sivam, G; Berg, K

2001-08-01

Photochemical internalization (PCI) is a novel method for the endosomal or lysosomal release of membrane-impermeable molecules into the cytosol of target cells. This novel technology is based on the photodynamically induced rupture of endocytic vesicles preloaded with molecules of therapeutic interest. PCI of the ribosome-inactivating plant toxin gelonin and the immunotoxin monoclonal antibody 31 (MOC31) gelonin has been performed previously by the use of the endocytic vesicle-localizing photosensitizers TPPS2a and AIPcS2a and light, demonstrating synergistic toxicity against the more than 20 different cell lines tested, most of them of neoplastic origin. In this study we demonstrate that 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) is also capable of inducing PCI of MOC31-gelonin in the human colon adenocarcinoma cell line WiDr. The cells were incubated with 1 mM 5-ALA for up to 8 h in serum-free medium and from 24 to 96 h in serum-containing medium. Fluorescence microscopical studies indicate a partial plasma membrane localization of PpIX when 5-ALA was applied under serum-free conditions. This plasma membrane localization was not seen when 5-ALA was given in the presence of serum. There was a granular component of the PpIX localization in addition to a diffuse cytoplasmic localization. The granular component resembled the localization of the fluorescent dye conjugate Alexa-gelonin and the lysosomal localizing dye acridine orange. Our present results provide evidence for an endocytic vesicle-associated fraction of PpIX after 5-ALA incubation of the WiDr cells. We demonstrate that PCI, by combining 5-ALA, MOC31-gelonin and light, induces a synergistic cytotoxic effect against the WiDr cells.

Ricin A chain reaches the endoplasmic reticulum after endocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Qiong; Department of Biochemistry and Molecular Biology, Ningbo University Medical School, Ningbo 315211; Zhan Jinbiao

Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purifiedmore » by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.« less

[Double-antigen sandwich ELISA for detecting Aspergillus fumigatus anti-Afmp1cr and Afmp2cr antibodies].

PubMed

Yang, Mei; Wang, Zhuoya; Hao, Wei; Wang, Yanfang; Huang, Li; Cai, Jianpiao; Jiang, Lingxiao; Che, Xiaoyan; Zhong, Xiaozhu; Yu, Nan

2014-05-01

To establish two double-antigen sandwich ELISA systems to detect anti-Afmp1cr and Afmp2cr antibodies of Aspergillus fumigatus. Recombinant Afmp1cr and Afmp2cr proteins of A.fumigatus expressed in Pichia pastoris were obtained. Double-antigen sandwich ELISA systems for detecting specific anti-Afmp1cr and anti-Afmp2cr antibodies were developed after chessboard titrating to determine the appropriate concentrations of the recombinant proteins and HRP-labeled proteins. The sensitivity of the assay was evaluated using serum samples of rabbits immunized with Afmp1cr and Afmp2cr. The specificity of the assay was evaluated by detecting serum samples from healthy donors and patients with other pathogenic fungal and baterial infections. The performance of the two ELISA kits was furthered evaluated using serum samples from patients with suspected Aspergillus infection. The established ELISA kits were capable of detecting anti-Afmp1cr and anti-Afmp2cr antibodies in immunized rabbit serum at the maximum dilutions of 800 and 3200, respectively. No cross-reactivity was observed in detecting serum from patients with other pathogenic fungal or bactetial infections. Both of the two kits yielded positive results in sera from two established Aspergillus-infected cases and a suspected case. Two antibody-capture ELISA kits were developed for the laboratory diagnosis of A.fumigatus infection and can be potentially useful in the clinical diagnosis of Aspergillosis infections.

Overexpression of a Mycobacterium ulcerans Ag85B-EsxH Fusion Protein in Recombinant BCG Improves Experimental Buruli Ulcer Vaccine Efficacy.

PubMed

Hart, Bryan E; Lee, Sunhee

2016-12-01

Buruli ulcer (BU) vaccine design faces similar challenges to those observed during development of prophylactic tuberculosis treatments. Multiple BU vaccine candidates, based upon Mycobacterium bovis BCG, altered Mycobacterium ulcerans (MU) cells, recombinant MU DNA, or MU protein prime-boosts, have shown promise by conferring transient protection to mice against the pathology of MU challenge. Recently, we have shown that a recombinant BCG vaccine expressing MU-Ag85A (BCG MU-Ag85A) displayed the highest level of protection to date, by significantly extending the survival time of MU challenged mice compared to BCG vaccination alone. Here we describe the generation, immunogenicity testing, and evaluation of protection conferred by a recombinant BCG strain which overexpresses a fusion of two alternative MU antigens, Ag85B and the MU ortholog of tuberculosis TB10.4, EsxH. Vaccination with BCG MU-Ag85B-EsxH induces proliferation of Ag85 specific CD4+ T cells in greater numbers than BCG or BCG MU-Ag85A and produces IFNγ+ splenocytes responsive to whole MU and recombinant antigens. In addition, anti-Ag85A and Ag85B IgG humoral responses are significantly enhanced after administration of the fusion vaccine compared to BCG or BCG MU-Ag85A. Finally, mice challenged with MU following a single subcutaneous vaccination with BCG MU-Ag85B-EsxH display significantly less bacterial burden at 6 and 12 weeks post-infection, reduced histopathological tissue damage, and significantly longer survival times compared to vaccination with either BCG or BCG MU-Ag85A. These results further support the potential of BCG as a foundation for BU vaccine design, whereby discovery and recombinant expression of novel immunogenic antigens could lead to greater anti-MU efficacy using this highly safe and ubiquitous vaccine.

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Huanyu; Wei, Na; Wang, Qian

Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particlesmore » (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.« less

A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model.

PubMed

Mukherjee, Jean; Tremblay, Jacqueline M; Leysath, Clinton E; Ofori, Kwasi; Baldwin, Karen; Feng, Xiaochuan; Bedenice, Daniela; Webb, Robert P; Wright, Patrick M; Smith, Leonard A; Tzipori, Saul; Shoemaker, Charles B

2012-01-01

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V(H) (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.

Designing Trojan Horses | Center for Cancer Research

Cancer.gov

Waging battle against cancer cells without inflicting damage on normal tissue has long been a goal for cancer treatment. A new type of drug called immunotoxins may help make this goal a reality. Much like the Greeks used a wooden horse to get soldiers inside the gates of Troy, immunotoxins use clever genetic engineering to get a lethal toxin inside cancer cells. Each immunotoxin consists of two components an antibody and a toxin that are fused together. The custom-designed antibody acts as a homing signal, seeking out a specific target present on the surface of cancer cells. When the antibody binds its target, the whole immunotoxin is brought inside the cell. Unwittingly, the cancer cell has exposed itself to a powerful poison, a mistake that will likely condemn it to death.

[Construction and prokaryotic expression of recombinant gene EGFRvIII HBcAg and immunogenicity analysis of the fusion protein].

PubMed

Duan, Xiao-yi; Wang, Jian-sheng; Guo, You-min; Han, Jun-li; Wang, Quan-ying; Yang, Guang-xiao

2007-01-01

To construct recombinant prokaryotic expression plasmid pET28a(+)/c-PEP-3-c and evaluate the immunogenicity of the fusion protein. cDNA fragment encoding PEP-3 was obtained from pGEM-T Easy/PEP-3 and inserted into recombinant plasmid pGEMEX/HBcAg. Then it was subcloned in prokaryotic expression vector and transformed into E.coli BL21(DE3). The fusion protein was expressed by inducing IPTG and purified by Ni(2+)-NTA affinity chromatography. BALB/c mice were immunized with fusion protein and the antibody titre was determined by indirect ELISA. The recombinant gene was confirmed to be correct by restriction enzyme digestion and DNA sequencing. After prokaryotic expression, fusion protein existed in sediment and accounted for 56% of all bacterial lysate. The purified product accounted for 92% of all protein and its concentration was 8 g/L. The antibody titre in blood serum reached 1:16 000 after the fourth immunization and reached 1:2.56x10(5) after the sixth immunization. The titre of anti-PEP-3 antibody reached 1:1.28x10(5) and the titre of anti-HBcAg antibody was less than 1:4x10(3). Fusion gene PEP-3-HBcAg is highly expressed in E.coli BL21. The expressed fusion protein can induce neutralizing antibody with high titer and specificity, which lays a foundation for the study of genetically engineering vaccine for malignant tumors with the high expression of EGFRvIII.

Integrin-mediated human glioblastoma cells adhesion, migration and invasion by native and recombinant phospholipases of Scorpio maurus venom glands.

PubMed

Krayem, Najeh; Abdelkefi-Koubaa, Zaineb; Gargouri, Youssef; Luis, José

2018-05-01

Integrins are a large family of cell surface receptors mediating the interaction of cells with their microenvironment and they play an important role in glioma biology. In the present work, we reported the anti-tumor effect of Sm-PLGV a phospholipase A 2 from Tunisian scorpion venom glands-as well as its recombinant forms expressed in Escherichia coli-through interference with integrin receptor function in malignant glioma cells U87. These phospholipases inhibited in a dose dependent manner the adhesion, migration and invasion onto fibrinogen and fibronectin without any cytotoxicity. We showed that Sm-PLGV and its recombinant constructs blocked U87 migration by reducing their velocity and directional persistence. The inhibitory effect was related to a blockage of the integrins αvβ3 and α5β1 function. Inactivation of the enzymatic activity of Sm-PLGV by chemical modification with p-bromophenacyl bromide did not affect its anti-tumor properties, suggesting the presence of 'pharmacological sites' distinct from the catalytic site in scorpion venom phospholipases A 2 . Copyright © 2018 Elsevier Inc. All rights reserved.

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

PubMed

Battersby, J E; Snedecor, B; Chen, C; Champion, K M; Riddle, L; Vanderlaan, M

2001-08-24

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli.

PubMed

Verdon, Julien; Girardin, Nicolas; Marchand, Adrienne; Héchard, Yann; Berjeaud, Jean-Marc

2013-06-01

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for (15)N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.

Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

PubMed

Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained*

PubMed Central

Zheng, Hongying; Nagaraja, Ganachari M.; Kaur, Punit; Asea, Edwina E.; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72bv (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72bv enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72bv in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72bv can now be used to unlock the important role Hsp72 plays in modulating immune function. PMID:19861412

Evolution via recombination: Cell-to-cell contact facilitates larger recombination events in Streptococcus pneumoniae.

PubMed

Cowley, Lauren A; Petersen, Fernanda C; Junges, Roger; Jimson D Jimenez, Med; Morrison, Donald A; Hanage, William P

2018-06-01

Homologous recombination in the genetic transformation model organism Streptococcus pneumoniae is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent 'target' cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs. Experiments designed to explore the effect of environment on large scale recombination events used saturating purified donor DNA, short-term cell assemblages on Millipore filters, and mature biofilm mixed cultures. WGS of 22 recombinants for each environment mapped all SNPs that were identical between the recombinant and the donor but not the recipient. The mean recombination event size was found to be significantly larger in cell-to-cell contact cultures (4051 bp in filter assemblage and 3938 bp in biofilm co-culture versus 1815 bp with saturating DNA). Up to 5.8% of the genome was transferred, through 20 recombination events, to a single recipient, with the largest single event incorporating 29,971 bp. We also found that some recombination events are clustered, that these clusters are more likely to occur in cell-to-cell contact environments, and that they cause significantly increased linkage of genes as far apart as 60,000 bp. We conclude that pneumococcal evolution through homologous recombination is more likely to occur on a larger scale in environments that permit cell-to-cell contact.

Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants.

PubMed

Routsias, John G; Touloupi, Evgenia; Dotsika, Eleni; Moulia, Avrilia; Tsikaris, Vassilios; Sakarellos, Constantinos; Sakarellos-Daitsiotis, Maria; Moutsopoulos, Haralampos M; Tzioufas, Athanasios G

2002-06-01

Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a

Native and recombinant phospholipases A2 of Scorpio maurus venom glands impair angiogenesis by targeting integrins α5β1 and αvβ3.

PubMed

Krayem, Najeh; Abdelkefi-Koubaa, Zaineb; Marrakchi, Naziha; Gargouri, Youssef; Luis, José

2018-04-30

We recently purified an heterodimeric phospholipase A 2 named Sm-PLGV from the venom glands of scorpion Scorpio maurus containing a Long chain, a penta-peptide insertion, which is cut out during the maturation, followed by a short chain. Three recombinant forms of Sm-PLGV were produced in Escherichia coli: rPLA 2 (+5) containing the full-length sequence including the penta-peptide insert, rPLA 2 (-5) a fused continuous chain of the Long and the short chains without the penta-peptide and the Long chain alone without the short one. In this study, we showed that Sm-PLGV, rPLA 2 (+5) and rPLA 2 (-5) displayed more potent anti-angiogenic properties than the recombinant Long chain and the short chain obtained by chemical synthesis. These phospholipases A 2 inhibited in a dose-dependent manner adhesion, migration and invasion of human microvascular endothelial cells through the alteration of α5β1 and αvβ3 integrins function. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrated that Sm-PLGV, rPLA 2 (+5) and rPLA 2 (-5) significantly inhibited both in vitro and in vivo angiogenesis. We also showed a clear dissociation of the anti-angiogenic effect of Sm-PLGV and its catalytic activity. This is the first study describing an anti-angiogenic effect for recombinant scorpion venom enzymes. Copyright © 2018 Elsevier B.V. All rights reserved.

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

PubMed

Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

2014-01-01

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Regulation of Meiotic Recombination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gregory p. Copenhaver

Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diversemore » as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a

Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice.

PubMed

Jo, Dong Hyun; Park, Sung Wook; Cho, Chang Sik; Powner, Michael B; Kim, Jin Hyoung; Fruttiger, Marcus; Kim, Jeong Hun

2015-01-01

Anti-vascular endothelial growth factor (VEGF) agents are the mainstay treatment for various angiogenesis-related retinal diseases. Currently, bevacizumab, a recombinant humanized anti-VEGF antibody, is trailed in retinopathy of prematurity, a vasoproliferative retinal disorder in premature infants. However, the risks of systemic complications after intravitreal injection of anti-VEGF antibody in infants are not well understood. In this study, we show that intravitreally injected anti-VEGF antibody is transported into the systemic circulation into the periphery where it reduces brown fat in neonatal C57BL/6 mice. A considerable amount of anti-VEGF antibody was detected in serum after intravitreal injection. Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondria-related genes, Ppargc1a and Ucp1, all of which are characteristics of "whitening" of brown fat. With increasing age and body weight, brown fat restored its morphology and vascularity. Our results show that there is a transient, but significant impact of intravitreally administered anti-VEGF antibody on brown adipose tissue in neonatal mice. We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants.

Intravitreally Injected Anti-VEGF Antibody Reduces Brown Fat in Neonatal Mice

PubMed Central

Powner, Michael B.; Kim, Jin Hyoung; Fruttiger, Marcus; Kim, Jeong Hun

2015-01-01

Anti-vascular endothelial growth factor (VEGF) agents are the mainstay treatment for various angiogenesis-related retinal diseases. Currently, bevacizumab, a recombinant humanized anti-VEGF antibody, is trailed in retinopathy of prematurity, a vasoproliferative retinal disorder in premature infants. However, the risks of systemic complications after intravitreal injection of anti-VEGF antibody in infants are not well understood. In this study, we show that intravitreally injected anti-VEGF antibody is transported into the systemic circulation into the periphery where it reduces brown fat in neonatal C57BL/6 mice. A considerable amount of anti-VEGF antibody was detected in serum after intravitreal injection. Furthermore, in interscapular brown adipose tissue, we found lipid droplet accumulation, decreased VEGF levels, loss of vascular network, and decreased expression of mitochondria-related genes, Ppargc1a and Ucp1, all of which are characteristics of “whitening” of brown fat. With increasing age and body weight, brown fat restored its morphology and vascularity. Our results show that there is a transient, but significant impact of intravitreally administered anti-VEGF antibody on brown adipose tissue in neonatal mice. We suggest that more attention should be focused on the metabolic and developmental significance of brown adipose tissue in bevacizumab treated retinopathy of prematurity infants. PMID:26226015

Recombination of cluster ions

NASA Technical Reports Server (NTRS)

Johnsen, Rainer

1993-01-01

Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

PubMed

Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

2017-12-01

Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a

Updates in the Development of ImmunoRNases for the Selective Killing of Tumor Cells

PubMed Central

Jordaan, Sandra; Akinrinmade, Olusiji A.; Nachreiner, Thomas; Cremer, Christian; Naran, Krupa; Chetty, Shivan; Barth, Stefan

2018-01-01

Targeted cancer therapy includes, amongst others, antibody-based delivery of toxic payloads to selectively eliminate tumor cells. This payload can be either a synthetic small molecule drug composing an antibody-drug conjugate (ADC) or a cytotoxic protein composing an immunotoxin (IT). Non-human cytotoxic proteins, while potent, have limited clinical efficacy due to their immunogenicity and potential off-target toxicity. Humanization of the cytotoxic payload is essential and requires harnessing of potent apoptosis-inducing human proteins with conditional activity, which rely on targeted delivery to contact their substrate. Ribonucleases are attractive candidates, due to their ability to induce apoptosis by abrogating protein biosynthesis via tRNA degradation. In fact, several RNases of the pancreatic RNase A superfamily have shown potential as anti-cancer agents. Coupling of a human RNase to a humanized antibody or antibody derivative putatively eliminates the immunogenicity of an IT (now known as a human cytolytic fusion protein, hCFP). However, RNases are tightly regulated in vivo by endogenous inhibitors, controlling the ribonucleolytic balance subject to the cell’s metabolic requirements. Endogenous inhibition limits the efficacy with which RNase-based hCFPs induce apoptosis. However, abrogating the natural interaction with the natural inhibitors by mutation has been shown to significantly enhance RNase activity, paving the way toward achieving cytolytic potency comparable to that of bacterial immunotoxins. Here, we review the immunoRNases that have undergone preclinical studies as anti-cancer therapeutic agents. PMID:29510557

Updates in the Development of ImmunoRNases for the Selective Killing of Tumor Cells.

PubMed

Jordaan, Sandra; Akinrinmade, Olusiji A; Nachreiner, Thomas; Cremer, Christian; Naran, Krupa; Chetty, Shivan; Barth, Stefan

2018-03-05

Targeted cancer therapy includes, amongst others, antibody-based delivery of toxic payloads to selectively eliminate tumor cells. This payload can be either a synthetic small molecule drug composing an antibody-drug conjugate (ADC) or a cytotoxic protein composing an immunotoxin (IT). Non-human cytotoxic proteins, while potent, have limited clinical efficacy due to their immunogenicity and potential off-target toxicity. Humanization of the cytotoxic payload is essential and requires harnessing of potent apoptosis-inducing human proteins with conditional activity, which rely on targeted delivery to contact their substrate. Ribonucleases are attractive candidates, due to their ability to induce apoptosis by abrogating protein biosynthesis via tRNA degradation. In fact, several RNases of the pancreatic RNase A superfamily have shown potential as anti-cancer agents. Coupling of a human RNase to a humanized antibody or antibody derivative putatively eliminates the immunogenicity of an IT (now known as a human cytolytic fusion protein, hCFP). However, RNases are tightly regulated in vivo by endogenous inhibitors, controlling the ribonucleolytic balance subject to the cell's metabolic requirements. Endogenous inhibition limits the efficacy with which RNase-based hCFPs induce apoptosis. However, abrogating the natural interaction with the natural inhibitors by mutation has been shown to significantly enhance RNase activity, paving the way toward achieving cytolytic potency comparable to that of bacterial immunotoxins. Here, we review the immunoRNases that have undergone preclinical studies as anti-cancer therapeutic agents.

Protective activity and immunogenicity of two recombinant anthrax vaccines for veterinary use.

PubMed

Fasanella, A; Tonello, F; Garofolo, G; Muraro, L; Carattoli, A; Adone, R; Montecucco, C

2008-10-23

In this study, the efficacy of two experimental vaccines against Bacillus anthracis toxinaemia was evaluated in the rabbit model. A recombinant Protective Antigen (rPA) mutant and a trivalent vaccine (TV) composed by the rPA, a inactive mutant of Lethal Factor (mLF-Y728A; E735A) and a inactive mutant of Edema Factor (mEF-K346R), both emulsified with mineral oils, were evaluated for their immunogenicity and protective activity in New Zealand white rabbits. Rabbits vaccinated subcutaneously with rPA and TV rapidly produced high level of anti-PA, anti-LF and anti-EF antibodies, which were still present 6 months later. In the efficacy test, these vaccines protected 100% of rabbits challenged with B. anthracis virulent strain 0843 one week after the vaccination. Moreover, all animals vaccinated twice with rPA and TV, resisted B. anthracis infection 6 months later. Our data indicate that rPA and TV could be good vaccine candidates for inducing protection against B. anthracis infection in target animal host. They could successfully be used in an emergency with simultaneous long-acting antibiotics to halt incubating infections or during an anthrax epidemic.

The effects of a repeated dose of a recombinant humanized anti-cocaine monoclonal antibody on cocaine self-administration in rats.

PubMed

Wetzel, Hanna N; Tsibulsky, Vladimir L; Norman, Andrew B

2016-11-01

Immunotherapy has shown potential as a treatment for cocaine abuse. The humanized recombinant anti-cocaine monoclonal antibody (mAb) with the preclinical designation h2E2 has been shown to decrease cocaine concentrations in the brain in rats, but its effects on cocaine self-administration behavior have never been tested. The amount of cocaine needed to reinstate self-administration behavior (priming threshold) was calculated and the inter-injection intervals at unit doses of 0.3μmol/kg and 3μmol/kg during maintained self-administration were measured over a five-week baseline period. Rats trained to self-administer cocaine were infused with two doses of h2E2 (120mg/kg i.v.) 35days apart. Priming threshold and inter-injection intervals were measured for 35days after both injections. After both injections of h2E2, priming thresholds were significantly increased (3-fold) compared to expected baseline and then gradually declined over 35days. A significant decrease (15-33%) in inter-injection intervals during maintained self-administration was also observed following both h2E2 infusions at the lower dose, and after the first injection at the higher dose. No significant decreases in body weight were observed after either injection, indicating a lack of toxicity following a second injection. These data predict that the safety and effectiveness of h2E2 will be maintained after multiple treatments of this potential immunotherapy for cocaine abuse. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The effects of a repeated dose of a recombinant humanized anti-cocaine monoclonal antibody on cocaine self-administration in rats

PubMed Central

Wetzel, Hanna N.; Tsibulsky, Vladimir L.; Norman, Andrew B.

2016-01-01

Background Immunotherapy has shown potential as a treatment for cocaine abuse. The humanized recombinant anti-cocaine monoclonal antibody (mAb) with the preclinical designation h2E2 has been shown to decrease cocaine concentrations in the brain in rats, but its effects on cocaine self-administration behavior have never been tested. Methods The amount of cocaine needed to reinstate self-administration behavior (priming threshold) was calculated and the inter-injection intervals at unit doses of 0.3 μmol/kg and 3 μmol/kg during maintained self-administration were measured over a five-week baseline period. Rats trained to self-administer cocaine were infused with two doses of h2E2 (120 mg/kg i.v.) 35 days apart. Priming threshold and inter-injection intervals were measured for 35 days after both injections. Results After both injections of h2E2, priming thresholds were significantly increased (3-fold) compared to expected baseline and then gradually declined over 35 days. A significant decrease (15–33%) in inter-injection intervals during maintained self-administration was also observed following both h2E2 at the lower dose, and after the first injection at the higher dose. No significant decreases in body weight were observed after either injection, indicating a lack of toxicity following a second injection. Conclusions These data predict that the safety and efficacy of h2E2 will be maintained after multiple treatments of this potential immunotherapy for cocaine abuse. PMID:27736682

Expression of biologically active recombinant porcine interleukin-12 from Escherichia coli.

PubMed

Morris, K R; Bruce, M P; Janardhana, V; Thomas, J D; Bean, A G D; Strom, D G

2008-12-15

The control of viral infections is of critical importance to livestock industries worldwide and is highlighted by costly infection outbreaks, such as that seen with foot and mouth disease virus. To ameliorate the impact of increasing problems with viral infections, new vaccine and anti-viral strategies are required and a greater understanding of the anti-viral response is essential. Furthermore, in pigs, evidence is still being gathered on the components of a defined anti-viral immune response. However, this has been greatly improved by the recent cloning and expression of critical cytokines involved in the anti-viral response. To assess the use of recombinant porcine interleukin-12 (rPoIL-12) as an immunotherapeutic and immunomodulator of swine, we have cloned and expressed rPoIL-12 as a single-chain fusion protein from Esherichia coli (E. coli). The fusion encodes the p40 and p35 subunits, linked by a glycine-serine linker and expressed as a C-terminal 6xHis tagged protein. rPoIL-12 stimulated the proliferation of human lymphoblasts and its activity on porcine cells was demonstrated by the ability of rPoIL-12 to increase the mRNA expression of porcine interleukin-18 receptor-alpha (poIL-18Ralpha) from porcine peripheral blood mononuclear cells (PoPMBCs). This data supports the inclusion of E. coli produced rPoIL-12 in immunomodulation strategies in the pig.

A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model

PubMed Central

Leysath, Clinton E.; Ofori, Kwasi; Baldwin, Karen; Feng, Xiaochuan; Bedenice, Daniela; Webb, Robert P.; Wright, Patrick M.; Smith, Leonard A.; Tzipori, Saul; Shoemaker, Charles B.

2012-01-01

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant ‘targeting agent’ that binds a toxin at two unique sites and a ‘clearing Ab’ that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab VH (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit. PMID:22238680

Bacteriophage recombination systems and biotechnical applications.

PubMed

Nafissi, Nafiseh; Slavcev, Roderick

2014-04-01

Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

Development of a recombinant toxin fragment vaccine for Clostridium difficile infection.

PubMed

Karczewski, Jerzy; Zorman, Julie; Wang, Su; Miezeiewski, Matthew; Xie, Jinfu; Soring, Keri; Petrescu, Ioan; Rogers, Irene; Thiriot, David S; Cook, James C; Chamberlin, Mihaela; Xoconostle, Rachel F; Nahas, Debbie D; Joyce, Joseph G; Bodmer, Jean-Luc; Heinrichs, Jon H; Secore, Susan

2014-05-19

Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability. Copyright © 2014 Elsevier Ltd. All rights reserved.

Routinely used immunoassays do not detect circulating anti-GBM antibodies against native NC1 hexamer and EA epitope of the α3 chain of type IV collagen.

PubMed

Clavarino, Giovanna; Gauthier, Arnaud; Hellmark, Thomas; Carron, Pierre-Louis; Giovannini, Diane; Colliard, Sophie; Dragon-Durey, Marie-Agnès; Segelmark, Mårten; Cesbron, Jean-Yves; Dumestre-Pérard, Chantal

2018-04-12

Detection of circulating anti-GBM antibodies has a key role for the diagnosis of Goodpasture syndrome but immunoassays using purified or recombinant alpha3(IV)NC1 as antigen do not recognize all anti-GBM antibodies. We show that anti-GBM antibodies directed against epitopes in their native conformation or cryptic epitopes are detected by indirect immunofluorescence. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

PubMed Central

Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

2016-01-01

Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

IL-2 Enhances the Function of Recombinant Poxvirus-Based Vaccines in the Treatment of Established Pulmonary Metastases

PubMed Central

Bronte, Vincenzo; Tsung, Kangla; Rao, Jay B.; Chen, Pauline W.; Wang, Michael; Rosenberg, Steven A.; Restifo, Nicholas P.

2007-01-01

Neoplastic cells are generally poor immunogens. Transfection of the murine tumor CT-26 with β-galactosidase (β-gal), a proteinfrom Escherichia coli, did not alter its growth rate in vivo, or its lethality, and did not elicit a measurable anti-β-gal immune response. Immunization with β-gal-expressing recombinant vaccinia viruses (rVV) elicited specific anti-β-gal cytolytic T lymphocytes, but rVV-β-gal was only marginally therapeutic when given to tumor-bearing mice. With the aim of expanding the immune response against β-gal, used here as a model tumor Ag, we gave mice exogenous IL-2 starting 12 h after the poxvirus. The therapeutic effectiveness of the combination of poxvirus and IL-2 was far greater than either of these treatments alone. When the cDNA for IL-2 was inserted into the viral genome of the rVV construct to make a double recombinant (drVV), antitumor activity was further augmented. One mechanism of action may be the enhanced activation or expansion of cytotoxicT cells, because a marked increase in primary cytotoxic responses against vaccinia determinants was observed. Interestingly, other cytokines (mGM-CSF, mTNF-α, and mIFN-γ) inserted into the rVV genome did not modify the efficacy of the rVV constructs. The increase in specific CTL responses against β-gal by drVV expressing the tumor-associated Ags (TAA) and IL-2 was morepronounced inmice bearing the lacZ-transduced tumor than in those bearing the parental cell line, suggesting that the TAA presented by growing tumor cells can either pre-activate or otherwise amplify the immune response induced by the rVV. Unfortunately, in several long-term surviving mice, tumor recurred that no longer expressed β-gal. These results indicate that treatment of disseminated tumors by using recombinant viruses expressing TAA can be enhanced by IL-2 provided exogenously, or encoded within the recombinant virus. PMID:7730632

Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

PubMed

Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

2015-05-01

Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the

Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashizume, Tomomi; Momoi, Fumiki; Kurita-Ochiai, Tomoko

2007-08-24

In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-{alpha}-deficient (LT{alpha}{sup -/-}) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer's patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF.more » Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LT{alpha}{sup -/-} mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.« less

Ethanol production by recombinant hosts

DOEpatents

Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

1996-01-01

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Ethanol production by recombinant hosts

DOEpatents

Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

1995-01-01

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

PubMed

Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D

2016-07-01

Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

Protective efficacy of six immunogenic recombinant proteins of Vibrio anguillarum and evaluation them as vaccine candidate for flounder (Paralichthys olivaceus).

PubMed

Xing, Jing; Xu, Hongsen; Wang, Yang; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

2017-06-01

Vibrio anguillarum is a severe bacterium that causes terminal haemorrhagic septicaemia in freshwater and marine fish. Virulence-associated proteins play an important role in bacterial pathogenicity and could be applied for immunoprophylaxis. In this study, six antigenic proteins from V. anguillarum were selected and the immune protective efficacy of their recombinant proteins was investigated. VirA, CheR, FlaC, OmpK, OmpR and Hsp33 were recombinantly produced and the reactions of recombinant proteins to flounder-anti-V. anguillarum antibodies (fV-ab) were detected, respectively. Then the recombinant proteins were injected to fish, after immunization, the percentages of surface membrane immunoglobulin-positive (sIg+) cell in lymphocytes, total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were analyzed, respectively. The results showed that all the recombinant proteins could react to fV-ab, proliferate sIg + cells in lymphocytes and induce production of total antibodies, specific antibodies against V. anguillarum or the recombinant proteins; the RPS of rVirA, rCheR, rFlaC, rOmpK, rOmpR and rHsp33 against V. anguillarum was 70.27%, 27.03%, 16.22%, 62.16%, 45.95% and 81.08%, respectively. The results revealed that rHsp33, rVirA and rOmpK have good protections against V. anguillarum and could be vaccine candidates against V. anguillarum. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

PubMed

Kim, Ji-Hun; Sim, Dae-Won; Park, Dongsun; Jung, Tai-Geun; Lee, Seonghwan; Oh, Taeheun; Ha, Jong-Ryul; Seok, Seung-Hyeon; Seo, Min-Duk; Kang, Ho Chul; Kim, Young Pil; Won, Hyung-Sik

2016-12-01

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose.

PubMed

Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo; Wang, Junwei

2011-05-27

Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs. Copyright © 2011 Elsevier Inc. All rights reserved.

Anomalous Abundances in Gaseous Nebulae From Recombination and Collisional Lines: Improved Photoionization and Recombination Studies

NASA Astrophysics Data System (ADS)

Pradhan, Anil Kumar; Nahar, S. N.; Eissner, W. B.; Montenegro, M.

2011-01-01

A perplexing anomaly arises in the determination of abundances of common elements in gaseous nebulae, as derived from collisionally excited lines (CEL) as opposed to those from Recombination Lines (RCL). The "abundance discrepancy factors" can range from a factor of 2 to an order of magnitude or more. That has led to quite different interpretation of the physical structure and processes in gaseous nebulae, such as temperature fluctuations across the object, or metal-rich concentrations leading to a dual-abundnace scenario. We show that the problem may lie in inaccuracies in photoionization and recombination models neglecting low-energy resonance phenomena due to fine structure. Whereas the atomic physics of electron impact excitation of forbidden lines is well understood, and accurate collision strengths have long been available, that is not generally the case for electron-ion recombination cross sections. A major problem is the inclusion of relativisitic effects as it pertains to the existence of very low-energy fine structure resonances in photoionization cross sections. We carry out new relativistic calculations for photoionization and recombination cross sections using a recently extended version of the Breit-Pauli R-matrix codes, and the unified electron-ion recombination method that subsumes both the radiative and the dielectronic recombination (RR and DR) processes in an ab initio and self-consistent manner. We find that near-thresold resonances manifest themselves within fine structure levels of the ground state of ions, enhancing low-temperature recombination rate coefficients at 1000-10,000 K. The resulting enahncement in level-specific and total recombination rate coefficients should therefore lead to reduced abundances derived from RCL, and in accordance with those from CEL. We present results for photoionization of O II into, and recombination from, O III. Theoretical cross sections are benchmarked against high-resolution measurements from synchrotron

Hapten mediated display and pairing of recombinant antibodies accelerates assay assembly for biothreat countermeasures.

PubMed

Sherwood, Laura J; Hayhurst, Andrew

2012-01-01

A bottle-neck in recombinant antibody sandwich immunoassay development is pairing, demanding protein purification and modification to distinguish captor from tracer. We developed a simple pairing scheme using microliter amounts of E. coli osmotic shockates bearing site-specific biotinylated antibodies and demonstrated proof of principle with a single domain antibody (sdAb) that is both captor and tracer for polyvalent Marburgvirus nucleoprotein. The system could also host pairs of different sdAb specific for the 7 botulinum neurotoxin (BoNT) serotypes, enabling recognition of the cognate serotype. Inducible supE co-expression enabled sdAb populations to be propagated as either phage for more panning from repertoires or expressed as soluble sdAb for screening within a single host strain. When combined with streptavidin-g3p fusions, a novel transdisplay system was formulated to retrofit a semi-synthetic sdAb library which was mined for an anti-Ebolavirus sdAb which was immediately immunoassay ready, thereby speeding up the recombinant antibody discovery and utilization processes.

Auger recombination in sodium iodide

NASA Astrophysics Data System (ADS)

McAllister, Andrew; Kioupakis, Emmanouil; Åberg, Daniel; Schleife, André

2014-03-01

Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

Chemokines cooperate with TNF to provide protective anti-viral immunity and to enhance inflammation.

PubMed

Alejo, Alí; Ruiz-Argüello, M Begoña; Pontejo, Sergio M; Fernández de Marco, María Del Mar; Saraiva, Margarida; Hernáez, Bruno; Alcamí, Antonio

2018-05-03

The role of cytokines and chemokines in anti-viral defense has been demonstrated, but their relative contribution to protective anti-viral responses in vivo is not fully understood. Cytokine response modifier D (CrmD) is a secreted receptor for TNF and lymphotoxin containing the smallpox virus-encoded chemokine receptor (SECRET) domain and is expressed by ectromelia virus, the causative agent of the smallpox-like disease mousepox. Here we show that CrmD is an essential virulence factor that controls natural killer cell activation and allows progression of fatal mousepox, and demonstrate that both SECRET and TNF binding domains are required for full CrmD activity. Vaccination with recombinant CrmD protects animals from lethal mousepox. These results indicate that a specific set of chemokines enhance the inflammatory and protective anti-viral responses mediated by TNF and lymphotoxin, and illustrate how viruses optimize anti-TNF strategies with the addition of a chemokine binding domain as soluble decoy receptors.

Dissociative recombination in aeronomy

NASA Technical Reports Server (NTRS)

Fox, J. L.

1989-01-01

The importance of dissociative recombination in planetary aeronomy is summarized, and two examples are discussed. The first is the role of dissociative recombination of N2(+) in the escape of nitrogen from Mars. A previous model is updated to reflect new experimental data on the electronic states of N produced in this process. Second, the intensity of the atomic oxygen green line on the nightside of Venus is modeled. Use is made of theoretical rate coefficients for production of O (1S) in dissociative recombination from different vibrational levels of O2(+).

Recombination phenomena in high efficiency silicon solar cells

NASA Technical Reports Server (NTRS)

Sah, C. T.

1985-01-01

The dominant recombination phenomena which limit the highest efficiency attainable in silicon solar cells under terrestrial sunlight are reviewed. The ultimate achievable efficiency is limited by the two intrinsic recombination mechanisms, the interband Auger recombination and interband Radiative recombination, both of which occur in the entire cell body but principally in the base layer. It is suggested that an optimum (26%) cell design is one with lowly doped 50 to 100 micron thick base, a perfect BSF, and zero extrinsic recombination such as the thermal mechanism at recombination centers the Shockley-Read-Hall process (SRH) in the bulk, on the surface and at the interfaces. The importance of recombination at the interfaces of a high-efficiency cell is demonstrated by the ohmic contact on the back surface whose interface recombination velocity is infinite. The importance of surface and interface recombination is demonstrated by representing the auger and radiative recombination losses by effective recombination velocities. It is demonstrated that the three highest efficiency cells may all be limited by the SRH recombination losses at recombination centers in the base layer.

Development of a chimeric recombinant disintegrin as a cost-effective anticancer agent with promising translational potential

PubMed Central

Minea, Radu; Helchowski, Corey; Rubino, Barbara; Brodmann, Kyle; Swenson, Stephen; Markland, Francis

2011-01-01

Vicrostatin (VCN) is a chimeric recombinant disintegrin generated in Origami B (DE3) E. coli as a genetic fusion between the C-terminal tail of a viperid disintegrin echistatin and crotalid disintegrin contortrostatin (CN). The therapeutic modulation of multiple integrin pathways via soluble disintegrins was previously shown by us and others to elicit potent anti-angiogenic and anti-metastatic effects in several animal cancer models. Despite these favorable attributes, these polypeptides are notoriously difficult to produce recombinantly in significant quantity due to their structure which requires the correct pairing of multiple disulfide bonds for biological activity. In this report, we show that VCN can be reliably produced in large amounts (yields in excess of 200mg of active purified disintegrin per liter of bacterial culture) in Origami B (DE3), an E. coli expression strain engineered to support the folding of disulfide-rich heterologous proteins directly in its oxidative cytoplasmic compartment. VCN retains the integrin binding specificity of both parental molecules it was derived from, but with a different binding affinity profile. While competing for the same integrin receptors that are preferentially upregulated in the tumor microenvironment, VCN exerts a potent inhibitory effect on endothelial cell (EC) migration and tube formation in a dose-dependent manner, by forcing these cells to undergo significant actin cytoskeleton reorganization when exposed to this agent in vitro. Moreover, VCN has a direct effect on breast cancer cells inhibiting their in vitro motility. In an effort to address our main goal of developing a clinically relevant delivery method for recombinant disintegrins, VCN was efficiently packaged in liposomes (LVCN) and evaluated in vivo in an animal breast cancer model. Our data demonstrate that LVCN is well tolerated, its intravenous administration inducing a significant delay in tumor growth and an increase in animal survival, results

Helicases as Prospective Targets for Anti-Cancer Therapy

PubMed Central

Gupta, Rigu; Brosh, Robert M.

2008-01-01

It has been proposed that selective inactivation of a DNA repair pathway may enhance anti-cancer therapies that eliminate cancerous cells through the cytotoxic effects of DNA damaging agents or radiation. Given the unique and critically important roles of DNA helicases in the DNA damage response, DNA repair, and maintenance of genomic stability, a number of strategies currently being explored or in use to combat cancer may be either mediated or enhanced through the modulation of helicase function. The focus of this review will be to examine the roles of helicases in DNA repair that might be suitably targeted by cancer therapeutic approaches. Treatment of cancers with anti-cancer drugs such as small molecule compounds that modulate helicase expression or function is a viable approach to selectively kill cancer cells through the inactivation of helicase-dependent DNA repair pathways, particularly those associated with DNA recombination, replication restart, and cell cycle checkpoint. PMID:18473724

Mutation of the Kunitz-type proteinase inhibitor domain in the amyloid β-protein precursor abolishes its anti-thrombotic properties in vivo.

PubMed

Xu, Feng; Davis, Judianne; Hoos, Michael; Van Nostrand, William E

2017-07-01

Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid β-protein precursor (AβPP) inhibit cerebral thrombosis. KPI domain-lacking forms of AβPP are abundant in brain. Regions of AβPP other than the KPI domain may also be involved with regulating cerebral thrombosis. To determine the contribution of the KPI domain to the overall function of AβPP in regulating cerebral thrombosis we generated a reactive center mutant that was devoid of anti-thrombotic activity and studied its anti-thrombotic function in vitro and in vivo. To determine the extent of KPI function of AβPP in regulating cerebral thrombosis we generated a recombinant reactive center KPI R13I mutant devoid of anti-thrombotic activity. The anti-proteolytic and anti-coagulant properties of wild-type and R13I mutant KPI were investigated in vitro. Cerebral thrombosis of wild-type, AβPP knock out and AβPP/KPI R13I mutant mice was evaluated in experimental models of carotid artery thrombosis and intracerebral hemorrhage. Recombinant mutant KPI R13I domain was ineffective in the inhibition of pro-thrombotic proteinases and did not inhibit the clotting of plasma in vitro. AβPP/KPI R13I mutant mice were similarly deficient as AβPP knock out mice in regulating cerebral thrombosis in experimental models of carotid artery thrombosis and intracerebral hemorrhage. We demonstrate that the anti-thrombotic function of AβPP primarily resides in the KPI activity of the protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeting vector construction through recombineering.

PubMed

Malureanu, Liviu A

2011-01-01

Gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. Conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific PCR amplification of repetitive DNA sequences, and cloning of large DNA fragments. Recombineering is a technique that exploits the highly efficient homologous recombination function encoded by λ phage in Escherichia coli. Bacteriophage-based recombination can recombine homologous sequences as short as 30-50 bases, allowing manipulations such as insertion, deletion, or mutation of virtually any genomic region. The large availability of mouse genomic bacterial artificial chromosome (BAC) libraries covering most of the genome facilitates the retrieval of genomic DNA sequences from the bacterial chromosomes through recombineering. This chapter describes a successfully applied protocol and aims to be a detailed guide through the steps of generation of targeting vectors through recombineering.

Anti-soluble liver antigen (SLA) antibodies in chronic HCV infection.

PubMed

Vitozzi, Susana; Lapierre, Pascal; Djilali-Saiah, Idriss; Marceau, Gabriel; Beland, Kathie; Alvarez, Fernando

2004-05-01

Hepatitis C infection is associated with autoimmune disorders, such as the production of autoantibodies. Anti-LKM1 and anti-LC1, immunomarkers of type 2 autoimmune hepatitis, have been previously associated with a HCV infection. Anti-Soluble-Liver-Antigen autoantibodies (SLA) are specifically associated with type 1 and type 2 autoimmune hepatitis and more closely related to patients who relapse after steroid therapy. The recent molecular cloning of the soluble liver antigen provides the opportunity to develop more specific tests for the detection of antibodies against it. The aim of this work is to characterize anti-soluble-liver autoantibodies in sera from patients chronically infected by HCV. A recombinant cDNA from activated Jurkat cells coding for the full length tRNP(Ser)Sec/SLA antigen was obtained. ELISA, Western Blot and immunoprecipitation tests were developed and used to search for linear and conformational epitopes recognized by anti-SLA antibodies in sera from patients chronically infected by HCV. Anti-soluble liver antigen antibodies were found in sera from 10.4% of HCV-infected patients. The prevalence was significantly increased to 27% when anti-LKM1 was also present. Most anti-SLA reactivity was directed against conformational epitopes on the antigen. The means titers by ELISA were lower than those obtained in type 2 AIH. The result of autoantibody isotyping showed a subclass restriction to IgG1 and also IgG4. This study shows the presence of anti-SLA antibodies in approximately 10% of HCV infected patients. The prevalence of SLA autoantibodies in HCV infected patients increases when LKM1 autoantibodies are also present. The relationship between the prevalence of this characteristic autoimmune hepatitis autoantibody and the implication of an autoimmune phenomenon in the liver injury of patients chronically infected by HCV needs further investigation.

Intradermal vaccination of adults with three low doses (2 micrograms) of recombinant hepatitis B vaccine. II. Persistence of immunity and induction of immunologic memory.

PubMed

Elisbão, Maria do Carmo M; Baldy, José Luís da S; Bonametti, Ana Maria; Reiche, Edna Maria V; Morimoto, Helena K; Pontello, Rubens; Matsuo, Tiemi; Ferelle, Antônio; Neves, Jayme

2003-12-01

Of the 110 dentists who had presented seroconversion 50 days after the intradermal application of three 2 micrograms doses of the Belgian recombinant vaccine against hepatitis B (HB), administered eight years before at an interval of one month between the 1st and 2nd doses and of five months between the 2nd and 3rd doses, 51 were included for the assessment of the persistence of immunity. None of the dentists had hepatitis or had received HB vaccine during this period. All subjects were submitted to serological tests for the detection of the following markers of hepatitis B virus (HBV) infection: HBsAg, anti-HBc, HBeAg, anti-HBe, and anti-HBs, with no HBsAg, anti-HBc, HBeAg or anti-HBe being detected. A microparticle enzyme immunoassay (MEIA) revealed the presence of anti-HBs at protective titers (> or = 10 mIU/ml) in 42 dentists (82.4%), with the anti-HBs titer being higher than 100 mIU/ml in 36 of them (70.6%) (good responders), between 10 and 100 mIU/ml in 6 (11.8%) (poor responders), and lower than 10 mIU/ml in 9 (17.6%) (non-responders). According to clinical data and serological tests, none of the dentists had presented disease or latent HBV infection during the eight years following the first vaccination. A 2 micrograms booster dose was administered intradermally to eight dentists with anti-HBs titers lower than 10 mIU/ml (non-responders) and to six dentists with titers ranging from 10 to 100 mIU/ml (poor responders); the determination of anti-HBs one month later demonstrated the occurrence of seroconversion in the eight non-responders and an increase in anti-HBs titer in the six poor responders. In summary, the present results demonstrated the prolonged persistence of protection against HBV infection and the development of immunologic memory provided by vaccination against HB--with intradermal application of three 2 micrograms doses of the Belgian recombinant vaccine at 0, 1, and 6 months--carried out eight years before in 51 dentists.

Superoxide dismutase recombinant Lactobacillus fermentum ameliorates intestinal oxidative stress through inhibiting NF-κB activation in a trinitrobenzene sulphonic acid-induced colitis mouse model.

PubMed

Hou, C L; Zhang, J; Liu, X T; Liu, H; Zeng, X F; Qiao, S Y

2014-06-01

Superoxide dismutase (SOD) can prevent and cure inflammatory bowel diseases by decreasing the amount of reactive oxygen species. Unfortunately, short half-life of SOD in the gastrointestinal tract limited its application in the intestinal tract. This study aimed to investigate the treatment effects of recombinant SOD Lactobacillus fermentum in a colitis mouse model. In this study, we expressed the sodA gene in Lact. fermentum I5007 to obtain the SOD recombinant strain. Then, we determined the therapeutic effects of this SOD recombinant strain in a trinitrobenzene sulphonic acid (TNBS)-induced colitis mouse model. We found that SOD activity in the recombinant Lact. fermentum was increased by almost eightfold compared with that in the wild type. Additionally, both the wild type and the recombinant Lact. fermentum increased the numbers of lactobacilli in the colon of mice (P < 0·05). Colitis mice treated with recombinant Lact. fermentum showed a higher survival rate and lower disease activity index (P < 0·05). Recombinant Lact. fermentum significantly decreased colonic mucosa histological scoring for infiltration of inflammatory cells, lipid peroxidation, the expression of pro-inflammatory cytokines and myeloperoxidase (P < 0·05) and inhibited NF-κB activity in colitis mice (P < 0·05). SOD recombinant Lact. fermentum significantly reduced oxidative stress and inflammation through inhibiting NF-κB activation in the TNBS-induced colitis model. This study provides insights into the anti-inflammatory effects of SOD recombinant Lact. fermentum, indicating the potential therapeutic effects in preventing and curing intestinal bowel diseases. © 2014 The Society for Applied Microbiology.

Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

PubMed

Barucca, A; Capitani, M; Cesca, M; Tomassoni, D; Kazmi, U; Concetti, F; Vincenzetti, L; Concetti, A; Venanzi, F M

2014-11-01

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

DOEpatents

Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

2001-04-03

Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

Recombinant Hepatitis E virus like particles can function as RNA nanocarriers.

PubMed

Panda, Subrat Kumar; Kapur, Neeraj; Paliwal, Daizy; Durgapal, Hemlata

2015-06-24

Assembled virus-like particles (VLPs) without genetic material, with structure similar to infectious virions, have been successfully used as vaccines. We earlier described in vitro assembly, characterisation and tissue specific receptor dependent Clathrin mediated entry of empty HEV VLPs, produced from Escherichia coli expressed HEV capsid protein (pORF2). Similar VLP's have been described as a potential candidate vaccine (Hecolin) against HEV. We have attempted to use such recombinant assembled Hepatitis E virus (HEV) VLPs as a carrier for heterologous RNA with protein coding sequence fused in-frame with HEV 5' region (containing cap and encapsidation signal) and investigated, if the relevant protein could be expressed and elicit an immune response in vivo. In vitro transcribed red fluorescent protein (RFP)/Hepatitis B virus surface antigen (HBsAg) RNA, fused to 5'-HEV sequence with cap and encapsidation signal (1-249 nt), was packaged into the recombinant HEV-VLPs and incubated with five different cell lines (Huh7, A549, Vero, HeLa and SiHa). The pORF2-VLPs could specifically transfer exogenous coding RNA into Huh7 and A549 cells. In vivo, Balb/c mice were immunized (intramuscular injections) with 100 µg pORF2-VLP encapsidated with 5'-methyl-G-HEV (1-249 nt)-HBsAg RNA, blood samples were collected and screened by ELISA for anti-pORF2 and anti-HBsAg antibodies. Humoral immune response could be elicited in Balb/c mice against both HEV capsid protein and cargo RNA encoded HBsAg protein. These findings suggest that other than being a possible vaccine, HEV pORF2-VLPs can be used as a promising non-replicative tissue specific gene delivery system.

HIV-1 Neutralization Profile and Plant-Based Recombinant Expression of Actinohivin, an Env Glycan-Specific Lectin Devoid of T-Cell Mitogenic Activity

PubMed Central

Matoba, Nobuyuki; Husk, Adam S.; Barnett, Brian W.; Pickel, Michelle M.; Arntzen, Charles J.; Montefiori, David C.; Takahashi, Atsushi; Tanno, Kazunobu; Omura, Satoshi; Cao, Huyen; Mooney, Jason P.; Hanson, Carl V.; Tanaka, Haruo

2010-01-01

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH's anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1′s AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide. PMID:20559567

Recombinant spider silk genetically functionalized with affinity domains.

PubMed

Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

2014-05-12

Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1.

PubMed

Min, Won-Ki; Cho, Young-Jin; Park, Jun-Bock; Bae, Yi-Hyun; Kim, Eun-Jeong; Park, Kyungmoon; Park, Yong-Cheol; Seo, Jin-Ho

2010-01-01

Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb.

Toxoplasma gondii Recombinant Antigens as Tools for Serodiagnosis of Human Toxoplasmosis: Current Status of Studies

PubMed Central

2013-01-01

Toxoplasma gondii is a parasitic protozoan which is the cause of toxoplasmosis. Although human toxoplasmosis in healthy adults is usually asymptomatic, serious disease can occur in the case of congenital infections and immunocompromised individuals. Furthermore, despite the exact recognition of its etiology, it still presents a diagnostic problem. Diagnosis of toxoplasmosis is mainly based on the results of serological tests detecting anti-T. gondii-specific antibodies in the patient's serum sample. The specificities and sensitivities of serology tests depend mostly on the diagnostic antigen(s) used. Most of the commercial serological kits currently available are based on Toxoplasma lysate antigens (TLAs). In recent years, many studies showed that recombinant antigenic proteins of T. gondii may be an alternative source of antigens which are very useful for the serodiagnosis of toxoplasmosis. This article presents a review of current studies on the application and usefulness of different T. gondii recombinant antigens in serological tests for the diagnosis of human toxoplasmosis. PMID:23784855

Genomic correlates of recombination rate and its variability across eight recombination maps in the western honey bee (Apis mellifera L.).

PubMed

Ross, Caitlin R; DeFelice, Dominick S; Hunt, Greg J; Ihle, Kate E; Amdam, Gro V; Rueppell, Olav

2015-02-21

Meiotic recombination has traditionally been explained based on the structural requirement to stabilize homologous chromosome pairs to ensure their proper meiotic segregation. Competing hypotheses seek to explain the emerging findings of significant heterogeneity in recombination rates within and between genomes, but intraspecific comparisons of genome-wide recombination patterns are rare. The honey bee (Apis mellifera) exhibits the highest rate of genomic recombination among multicellular animals with about five cross-over events per chromatid. Here, we present a comparative analysis of recombination rates across eight genetic linkage maps of the honey bee genome to investigate which genomic sequence features are correlated with recombination rate and with its variation across the eight data sets, ranging in average marker spacing ranging from 1 Mbp to 120 kbp. Overall, we found that GC content explained best the variation in local recombination rate along chromosomes at the analyzed 100 kbp scale. In contrast, variation among the different maps was correlated to the abundance of microsatellites and several specific tri- and tetra-nucleotides. The combined evidence from eight medium-scale recombination maps of the honey bee genome suggests that recombination rate variation in this highly recombining genome might be due to the DNA configuration instead of distinct sequence motifs. However, more fine-scale analyses are needed. The empirical basis of eight differing genetic maps allowed for robust conclusions about the correlates of the local recombination rates and enabled the study of the relation between DNA features and variability in local recombination rates, which is particularly relevant in the honey bee genome with its exceptionally high recombination rate.

Recombinant allergens

PubMed Central

Jutel, Marek; Solarewicz-Madejek, Katarzyna; Smolinska, Sylwia

2012-01-01

Allergen specific immunotherapy (SIT) is the only known causative treatment of allergic diseases. Recombinant allergen-based vaccination strategies arose from a strong need to both to improve safety and enhance efficacy of SIT. In addition, new vaccines can be effective in allergies including food allergy or atopic dermatitis, which poorly respond to the current treatment with allergen extracts. A number of successful clinical studies with both wild-type and hypoallergenic derivatives of recombinant allergens vaccines have been reported for the last decade. They showed high efficacy and safety profile as well as very strong modulation of T and B cell responses to specific allergens. PMID:23095874

Higher cytotoxicity of divalent antibody-toxins than monovalent antibody-toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Won, JaeSeon; Nam, PilWon; Lee, YongChan

2009-04-24

Recombinant antibody-toxins are constructed via the fusion of a 'carcinoma-specific' antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G{sub 4}S) between the Fabmore » fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38]{sub 2}) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.« less

On the conservative nature of intragenic recombination

PubMed Central

Drummond, D. Allan; Silberg, Jonathan J.; Meyer, Michelle M.; Wilke, Claus O.; Arnold, Frances H.

2005-01-01

Intragenic recombination rapidly creates protein sequence diversity compared with random mutation, but little is known about the relative effects of recombination and mutation on protein function. Here, we compare recombination of the distantly related β-lactamases PSE-4 and TEM-1 to mutation of PSE-4. We show that, among β-lactamase variants containing the same number of amino acid substitutions, variants created by recombination retain function with a significantly higher probability than those generated by random mutagenesis. We present a simple model that accurately captures the differing effects of mutation and recombination in real and simulated proteins with only four parameters: (i) the amino acid sequence distance between parents, (ii) the number of substitutions, (iii) the average probability that random substitutions will preserve function, and (iv) the average probability that substitutions generated by recombination will preserve function. Our results expose a fundamental functional enrichment in regions of protein sequence space accessible by recombination and provide a framework for evaluating whether the relative rates of mutation and recombination observed in nature reflect the underlying imbalance in their effects on protein function. PMID:15809422

On the conservative nature of intragenic recombination.

PubMed

Drummond, D Allan; Silberg, Jonathan J; Meyer, Michelle M; Wilke, Claus O; Arnold, Frances H

2005-04-12

Intragenic recombination rapidly creates protein sequence diversity compared with random mutation, but little is known about the relative effects of recombination and mutation on protein function. Here, we compare recombination of the distantly related beta-lactamases PSE-4 and TEM-1 to mutation of PSE-4. We show that, among beta-lactamase variants containing the same number of amino acid substitutions, variants created by recombination retain function with a significantly higher probability than those generated by random mutagenesis. We present a simple model that accurately captures the differing effects of mutation and recombination in real and simulated proteins with only four parameters: (i) the amino acid sequence distance between parents, (ii) the number of substitutions, (iii) the average probability that random substitutions will preserve function, and (iv) the average probability that substitutions generated by recombination will preserve function. Our results expose a fundamental functional enrichment in regions of protein sequence space accessible by recombination and provide a framework for evaluating whether the relative rates of mutation and recombination observed in nature reflect the underlying imbalance in their effects on protein function.

Electron-Beam Recombination Lasers

NASA Astrophysics Data System (ADS)

Rhoades, Robert Lewis

1992-01-01

The first known instance of electron-beam pumping of the 546.1 nm mercury laser is reported. This has been achieved using high-energy electrons to create intense ionization in a coaxial diode chamber containing a mixture of noble gases with a small amount of mercury vapor. Also reported are the results of a study of the 585.3 nm neon laser in He:Ne:Ar mixtures under similar experimental conditions. Both of these lasers are believed to be predominantly pumped by recombination. For the mercury laser, kinetic processes in the partially ionized plasma following the excitation pulse of high-energy electrons should favor the production of atomic mercury ions and molecular ions containing mercury. Subsequent recombination with electrons heavily favors the production of the 7^3S and 6^3 D states of Hg, of which 7^3S is the upper level of the reported laser. For the neon laser, the dominant recombining ion has been previously shown to be Ne_2^{+}. One of the dominant roles of helium in recombination lasers is inferred from the data for the neon laser at low helium concentrations. Helium appears to be necessary for the rapid relaxation of the electron energy which then increases the reaction rates for all known recombination processes thus increasing the pump rate into the upper state.

Prevalence of IgG antibodies to human parvovirus B19 in haemophilia children treated with recombinant factor (F)VIII only or with at least one plasma-derived FVIII or FIX concentrate: results from the French haemophilia cohort.

PubMed

Gaboulaud, Valérie; Parquet, Armelle; Tahiri, Cedric; Claeyssens, Ségolène; Potard, Valérie; Faradji, Albert; Peynet, Jocelyne; Costagliola, Dominique

2002-02-01

Human parvovirus B19 (B19) has been transmitted by some brands of virally attenuated plasma-derived factor VIII (FVIII) or IX (FIX) concentrates. To quantify the differences of human parvovirus B19 risk transmission between albumin-stabilized recombinant factor and plasma-derived factor, we studied the prevalence of IgG antibodies to B19 (anti-B19) in 193 haemophiliac children between 1 and 6-years of age who had previously been treated with albumin-stabilized recombinant FVIII only (n = 104), and in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates (n = 89). Association between the prevalence of anti-B19 and the treatment group was analysed using multivariate logistic regression. Age, severity and type of haemophilia, number of cumulative days of exposure to factor VIII or IX, previous history of red blood cells or plasma transfusion were considered as potential confounding variables. A higher prevalence of anti-B19 was found in children previously treated with solvent/detergent high-purity non-immunopurified and non-nanofiltered FVIII or IX concentrates than in children treated with albumin- stabilized recombinant FVIII only (OR: 22.3; CI: 7.9-62.8), independently of the other factors studied.

Site-specific genetic recombination: hops, flips, and flops.

PubMed

Sadowski, P D

1993-06-01

Genetic recombination plays a key role in the life of organisms as diverse as bacteriophages and humans. Contrary to our idea that chromosomes are stable structures, studies of recombination over the past few decades have shown that in fact DNA replicons are remarkably plastic, undergoing frequent recombination-induced rearrangements. This review summarizes our recent knowledge of the biochemistry of the two major classes of site-specific recombination: 1) transpositional recombination, and 2) conservative site-specific recombination.

Human alpha-enolase from endothelial cells as a target antigen of anti-endothelial cell antibody in Behçet's disease.

PubMed

Lee, Kwang Hoon; Chung, Hae-Shin; Kim, Hyoung Sup; Oh, Sang-Ho; Ha, Moon-Kyung; Baik, Ja-Hyun; Lee, Sungnack; Bang, Dongsik

2003-07-01

To identify and recombine a protein of the human dermal microvascular endothelial cell (HDMEC) that specifically reacts with anti-endothelial cell antibody (AECA) in the serum of patients with Behçet's disease (BD), and to evaluate the usefulness of this protein in BD. The proteomics technique, with 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, was used to identify and recombine HDMEC antigen. Western blotting and enzyme-linked immunosorbent assay (ELISA) of recombinant protein isolated by gene cloning were performed on serum from healthy controls, patients with BD, and patients with other rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus, and Wegener's granulomatosis). Eighteen of 40 BD patients had serum IgM antibody to HDMEC antigen. The purified protein that reacted with AECA in BD patient sera was found to be alpha-enolase by 2-dimensional gel electrophoresis followed by immunoblotting and MALDI-TOF mass spectrometry. Recombinant alpha-enolase protein was isolated and refined by gene cloning. On Western blots, AECA-positive IgM from the sera of patients with active BD reacted strongly with recombinant human alpha-enolase. BD patient sera positive for anti-alpha-enolase did not react with human gamma-enolase. On dot-blotting, reactivity to human alpha-enolase was detected only in the IgM-positive group. Fifteen of the 18 AECA-positive sera that were positive for the HDMEC antigen showed reactivity to recombinant alpha-enolase IgM antibody by ELISA. The alpha-enolase protein is the target protein of serum AECA in BD patients. This is the first report of the presence of IgM antibodies to alpha-enolase in endothelial cells from the serum of BD patients. Although further studies relating this protein to the pathogenesis of BD will be necessary, alpha-enolase and its antibody may prove useful in the development of new diagnostic and treatment modalities in BD.

Perioperative recombinant human granulocyte colony-stimulating factor (Filgrastim) treatment prevents immunoinflammatory dysfunction associated with major surgery.

PubMed

Schneider, Christian; von Aulock, Sonja; Zedler, Siegfried; Schinkel, Christian; Hartung, Thomas; Faist, Eugen

2004-01-01

To examine the effects of perioperative rhG-CSF administration on immune function in patients subjected to major surgery. Severe trauma, such as major surgery, initiates acute immunodysfunction which predisposes the patient towards infectious complications. Sixty patients undergoing elective surgery received either recombinant human granulocyte colony-stimulating factor/rh G-CSF (Filgrastim) or a placebo perioperatively. At several time points before and after the surgical intervention immunofunctional parameters were assessed. RESULTS Leukocyte counts and serum levels of anti-inflammatory mediators (IL-1ra and TNF-R) were increased in Filgrastim-treated patients, while the post-operative acute phase response was attenuated. Monocyte deactivation (reduced TNF-alpha release and HLA-DR expression) and lymphocyte anergy (impaired mitogenic proliferation and reduced TH1 lymphokine release) were blunted and the incidence and severity of infectious complications were reduced. These results suggest that Filgrastim treatment reinforces innate immunity, enabling better prevention of infection. Thus, this unique combination of hematopoietic, anti-inflammatory and anti-infectious effects on the innate immune system warrants further study of clinical efficacy and sepsis prophylaxis.

Perioperative Recombinant Human Granulocyte Colony-Stimulating Factor (Filgrastim) Treatment Prevents Immunoinflammatory Dysfunction Associated With Major Surgery

PubMed Central

Schneider, Christian; von Aulock, Sonja; Zedler, Siegfried; Schinkel, Christian; Hartung, Thomas; Faist, Eugen

2004-01-01

Objective: To examine the effects of perioperative rhG-CSF administration on immune function in patients subjected to major surgery. Summary Background Data: Severe trauma, such as major surgery, initiates acute immunodysfunction which predisposes the patient towards infectious complications. Methods: Sixty patients undergoing elective surgery received either recombinant human granulocyte colony-stimulating factor/rh G-CSF (Filgrastim) or a placebo perioperatively. At several time points before and after the surgical intervention immunofunctional parameters were assessed. Results: Leukocyte counts and serum levels of anti-inflammatory mediators (IL-1ra and TNF-R) were increased in Filgrastim-treated patients, while the post-operative acute phase response was attenuated. Monocyte deactivation (reduced TNF-α release and HLA-DR expression) and lymphocyte anergy (impaired mitogenic proliferation and reduced TH1 lymphokine release) were blunted and the incidence and severity of infectious complications were reduced. Conclusions: These results suggest that Filgrastim treatment reinforces innate immunity, enabling better prevention of infection. Thus, this unique combination of hematopoietic, anti-inflammatory and anti-infectious effects on the innate immune system warrants further study of clinical efficacy and sepsis prophylaxis. PMID:14685103

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

PubMed

Joan, Stella Siaw Xiu; Pui-Fong, Jee; Song, Adelene Ai-Lian; Chang, Li-Yen; Yusoff, Khatijah; AbuBakar, Sazaly; Rahim, Raha Abdul

2016-05-01

An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

[Requirement of standardizing anti-HBs assay methods in Japan for HBV infection-preventing strategy--discrepancy of anti-HBs measurements among three different kits widely used in Japan].

PubMed

Ogata, Norio

2006-09-01

The strategy to eliminate hepatitis B virus (HBV) infection by administrating an HB vaccine is changing worldwide; however, this is not the case in Japan. An important concern about the HBV infection-preventing strategy in Japan may be that the assay methods for the antibody to hepatitis B surface antigen (anti-HBs) are not standardized. The minimum protective anti-HBs titer against HBV infection has been established as 10 mIU/ml by World Health Organization (WHO) -standardized assay methods worldwide, but that is still determined as a "positive" test result by the passive hemagglutination (PHA) method in Japan. We compared anti-HBs measurements in given samples among PHA(Mycell II, Institute of Immunology), chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse, Fujirebio), and chemiluminescent immunoassay (CLIA) (Architect, Abbott), all of which are currently in wide use in Japan. First, anti-HBs measurements in serum from individuals who received a yeast-derived recombinant HB vaccine composed of the major surface protein of either subtype adr or subtype ayw were compared. The results clearly showed that in subtype adr-vaccinees CLIA underestimated the anti-HBs amount compared with CLEIA and PHA, but in ayw-vaccinees, the discordance in the measurements among the three kits was not prominent. Second, anti-HBs measurements in standard or calibration solutions of each assay kit were compared. Surprisingly, CLEIA showed higher measurements in all three kit-associated standard or calibration solutions than CLIA. Thus, the anti-HBs titer of 10 mIU/ml is difficult to introduce in Japan as the minimum protective level against HBV infection. Efforts to standardize anti-HBs assay methods are expected to share international evidence about the HBV infection-preventing strategy.

[Recombinant expression of Schistosoma japonicum fructose-1, 6-bisphos- phate aldolase and its expression in different developmental stages of S. japonicum].

PubMed

Yan, Ke; Zhong, Zheng-rong; Xu, Yun-xia; Ding, Shu-qin; Hu, Jian-guo; Xu, Yuan-hong; Luo, Qing-lie; Shen, Ji-long

2015-06-01

To clone, express and purify Schistosoma japonicum fructose-1, 6-bisphosphate aldolase (SjFBPA) in E. coli and observe its expression in different developmental stages of S. japonicum. FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid, and inducibly expressed with IPTG in E. coli BL21. SDS-PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA (rSjFBPA). Then, rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS- PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore, SjFBPA mRNA was ana- lyzed in different developmental stages of S. japonicum by RT-PCR. SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro- tein could specifically reactive to the anti-His-tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT-PCR showed that SjFBPA mRNA was expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum. SjFBPA is successfully recombined and expressed in a prokaryotic system, and SjFBPA mRNA is expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum.

Experimental evolution across different thermal regimes yields genetic divergence in recombination fraction but no divergence in temperature associated plastic recombination.

PubMed

Kohl, Kathryn P; Singh, Nadia D

2018-04-01

Phenotypic plasticity is pervasive in nature. One mechanism underlying the evolution and maintenance of such plasticity is environmental heterogeneity. Indeed, theory indicates that both spatial and temporal variation in the environment should favor the evolution of phenotypic plasticity under a variety of conditions. Cyclical environmental conditions have also been shown to yield evolved increases in recombination frequency. Here, we use a panel of replicated experimental evolution populations of D. melanogaster to test whether variable environments favor enhanced plasticity in recombination rate and/or increased recombination rate in response to temperature. In contrast to expectation, we find no evidence for either enhanced plasticity in recombination or increased rates of recombination in the variable environment lines. Our data confirm a role of temperature in mediating recombination fraction in D. melanogaster, and indicate that recombination is genetically and plastically depressed under lower temperatures. Our data further suggest that the genetic architectures underlying plastic recombination and population-level variation in recombination rate are likely to be distinct. © 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.

Anti-Stokes Luminescence in High Quality Quantum Wells

NASA Astrophysics Data System (ADS)

Vinattieri, A.; Bogani, F.; Miotto, A.; Ceccherini, S.

1997-11-01

We present a detailed investigation of the anti-Stokes (AS) luminescence which originates from exciton recombination when below gap excitation is used, in a set of high quality quantum well structures. We observe strong excitonic resonances in the AS signal as measured from photoluminescence and photoluminescence excitation spectra. We demonstrate that neither the electromagnetic coupling between the wells nor the morphological disorder can explain this up-conversion effect. Time-resolved luminescence data after ps excitation and fs correlation spectroscopy results provide clear evidence of the occurrence of a two-step absorption which is assisted by the exciton population resonantly excited by the first photon.

Immunization of mice with baculovirus-derived recombinant SV40 large tumour antigen induces protective tumour immunity to a lethal challenge with SV40-transformed cells.

PubMed Central

Shearer, M H; Bright, R K; Lanford, R E; Kennedy, R C

1993-01-01

In this study, we examined the humoral immune responses and in vivo tumour immunity induced by baculovirus recombinant simian virus 40 (SV40) large tumour antigen (rSV40 T-ag). BALB/c mice immunized with rSV40 T-ag produced antibody responses that recognized SV40 large tumour antigen (T-ag) by ELISA. Analysis of these anti-SV40 T-ag responses indicated that the antibodies recognized epitopes associated with both the carboxy and amino terminus of SV40 T-ag. This pattern of SV40 T-ag epitope recognition was similar to that observed in anti-SV40 T-ag responses induced by inoculation with irradiated SV40-transformed cells. Mice immunized with either rSV40 T-ag or with the inactivated transformed cells were protected from a subsequent in vivo lethal tumour challenge with live SV40-transformed cells. These studies suggest that humoral immune responses induced by rSV40 T-ag are similar in epitope specificity to that induced by inactivated SV40-transformed cells. In addition, recombinant tumour-specific antigens from papovaviruses, such as SV40, can be used to induce tumour immunity which protects from a subsequent lethal tumour challenge. This study may provide insight into the use of recombinant tumour antigens as putative tumour vaccines and in the development of active immunotherapeutic strategies for treating virus-induced cancers. PMID:7679059

Selections that isolate recombinant mitochondrial genomes in animals

PubMed Central

Ma, Hansong; O'Farrell, Patrick H

2015-01-01

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germline and revealed somatic recombination. When the recombination partner was a diverged Drosophila melanogaster genome or a genome from a different species such as Drosophila yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome. DOI: http://dx.doi.org/10.7554/eLife.07247.001 PMID:26237110

Immunogenicity of Recombinant Proteins Consisting of Plasmodium vivax Circumsporozoite Protein Allelic Variant-Derived Epitopes Fused with Salmonella enterica Serovar Typhimurium Flagellin

PubMed Central

Leal, Monica Teixeira Andrade; Camacho, Ariane Guglielmi Ariza; Teixeira, Laís Helena; Bargieri, Daniel Youssef; Soares, Irene Silva; Tararam, Cibele Aparecida

2013-01-01

A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax. PMID:23863502

Heterologous mitochondrial DNA recombination in human cells.

PubMed

D'Aurelio, Marilena; Gajewski, Carl D; Lin, Michael T; Mauck, William M; Shao, Leon Z; Lenaz, Giorgio; Moraes, Carlos T; Manfredi, Giovanni

2004-12-15

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

Age-Dependent Recombination Rates in Human Pedigrees

PubMed Central

Hussin, Julie; Roy-Gagnon, Marie-Hélène; Gendron, Roxanne; Andelfinger, Gregor; Awadalla, Philip

2011-01-01

In humans, chromosome-number abnormalities have been associated with altered recombination and increased maternal age. Therefore, age-related effects on recombination are of major importance, especially in relation to the mechanisms involved in human trisomies. Here, we examine the relationship between maternal age and recombination rate in humans. We localized crossovers at high resolution by using over 600,000 markers genotyped in a panel of 69 French-Canadian pedigrees, revealing recombination events in 195 maternal meioses. Overall, we observed the general patterns of variation in fine-scale recombination rates previously reported in humans. However, we make the first observation of a significant decrease in recombination rates with advancing maternal age in humans, likely driven by chromosome-specific effects. The effect appears to be localized in the middle section of chromosomal arms and near subtelomeric regions. We postulate that, for some chromosomes, protection against non-disjunction provided by recombination becomes less efficient with advancing maternal age, which can be partly responsible for the higher rates of aneuploidy in older women. We propose a model that reconciles our findings with reported associations between maternal age and recombination in cases of trisomies. PMID:21912527

Monitoring Recombination During Meiosis in Budding Yeast.

PubMed

Owens, Shannon; Tang, Shangming; Hunter, Neil

2018-01-01

Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization. © 2018 Elsevier Inc. All rights reserved.

Oral Immunization with Recombinant Lactobacillus acidophilus Expressing the Adhesin Hp0410 of Helicobacter pylori Induces Mucosal and Systemic Immune Responses

PubMed Central

Hongying, Fan; Xianbo, Wu; Fang, Yu; Yang, Bai

2014-01-01

Helicobacter pylori infection is relatively common worldwide and is closely related to gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic gastritis, and stomach ulcers. Therefore, a safe and effective method for preventing H. pylori infection is urgently needed. Given that developing an effective vaccine against H. pylori is one of the best alternatives, H. pylori adhesin Hp0410 was expressed in the food-grade bacterium Lactobacillus acidophilus. The recombinant live bacterial vaccine was then used to orally vaccinate mice, and the immunoprotective effects of Hp0410-producing strains were investigated. H. pylori colonization in the stomach of mice immunized with the recombinant L. acidophilus was significantly reduced, in comparison with that in control groups. Furthermore, mucosal secretory IgA antibodies were elicited in the mucosal tissue of mice immunized with the recombinant bacteria, and specific anti-Hp0410 IgG responses were also detected in mouse serum. There was a significant increase in the level of protection against gastric Helicobacter infection following a challenge with H. pylori Sydney strain 1 (SS1). Our results collectively indicate that adhesin Hp0410 is a promising candidate vaccine antigen, and recombinant L. acidophilus expressing Hp0410 is likely to constitute an effective, low-cost, live bacterial vaccine against H. pylori. PMID:24285819

Recombination of Globally Circulating Varicella-Zoster Virus

PubMed Central

Depledge, Daniel P.; Kundu, Samit; Atkinson, Claire; Brown, Julianne; Haque, Tanzina; Hussaini, Yusuf; MacMahon, Eithne; Molyneaux, Pamela; Papaevangelou, Vassiliki; Sengupta, Nitu; Koay, Evelyn S. C.; Tang, Julian W.; Underhill, Gillian S.; Grahn, Anna; Studahl, Marie; Breuer, Judith; Bergström, Tomas

2015-01-01

ABSTRACT Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the

Expression, purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe.

PubMed

Hu, Lulu; Gao, Chen-Hui; Hong, Chao; Zhong, Qiao; Dong, Hong-Liang; Gao, Xiao-Ming

2016-01-01

Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343-682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.

Detecting and Analyzing Genetic Recombination Using RDP4.

PubMed

Martin, Darren P; Murrell, Ben; Khoosal, Arjun; Muhire, Brejnev

2017-01-01

Recombination between nucleotide sequences is a major process influencing the evolution of most species on Earth. The evolutionary value of recombination has been widely debated and so too has its influence on evolutionary analysis methods that assume nucleotide sequences replicate without recombining. When nucleic acids recombine, the evolution of the daughter or recombinant molecule cannot be accurately described by a single phylogeny. This simple fact can seriously undermine the accuracy of any phylogenetics-based analytical approach which assumes that the evolutionary history of a set of recombining sequences can be adequately described by a single phylogenetic tree. There are presently a large number of available methods and associated computer programs for analyzing and characterizing recombination in various classes of nucleotide sequence datasets. Here we examine the use of some of these methods to derive and test recombination hypotheses using multiple sequence alignments.

Recombinant microorganisms for increased production of organic acids

DOEpatents

Yi, Jian [East Lansing, MI; Kleff, Susanne [East Lansing, MI; Guettler, Michael V [Holt, MI

2012-02-21

Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

Recombinant microorganisms for increased production of organic acids

DOEpatents

Yi, Jian; Kleff, Susanne; Guettler, Michael V

2013-04-30

Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

Immunoglobulin class switch recombination is impaired in Atm-deficient mice.

PubMed

Lumsden, Joanne M; McCarty, Thomas; Petiniot, Lisa K; Shen, Rhuna; Barlow, Carrolee; Wynn, Thomas A; Morse, Herbert C; Gearhart, Patricia J; Wynshaw-Boris, Anthony; Max, Edward E; Hodes, Richard J

2004-11-01

Immunoglobulin class switch recombination (Ig CSR) involves DNA double strand breaks (DSBs) at recombining switch regions and repair of these breaks by nonhomologous end-joining. Because the protein kinase ataxia telengiectasia (AT) mutated (ATM) plays a critical role in DSB repair and AT patients show abnormalities of Ig isotype expression, we assessed the role of ATM in CSR by examining ATM-deficient mice. In response to T cell-dependent antigen (Ag), Atm-/- mice secreted substantially less Ag-specific IgA, IgG1, IgG2b, and IgG3, and less total IgE than Atm+/+ controls. To determine whether Atm-/- B cells have an intrinsic defect in their ability to undergo CSR, we analyzed in vitro responses of purified B cells. Atm-/- cells secreted substantially less IgA, IgG1, IgG2a, IgG3, and IgE than wild-type (WT) controls in response to stimulation with lipopolysaccharide, CD40 ligand, or anti-IgD plus appropriate cytokines. Molecular analysis of in vitro responses indicated that WT and Atm-/- B cells produced equivalent amounts of germline IgG1 and IgE transcripts, whereas Atm-/- B cells produced markedly reduced productive IgG1 and IgE transcripts. The reduction in isotype switching by Atm-/- B cells occurs at the level of genomic DNA recombination as measured by digestion-circularization PCR. Analysis of sequences at CSR sites indicated that there is greater microhomology at the mu-gamma1 switch junctions in ATM B cells than in wild-type B cells, suggesting that ATM function affects the need or preference for sequence homology in the CSR process. These findings suggest a role of ATM in DNA DSB recognition and/or repair during CSR.

Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells

PubMed Central

Rahimi, Roghayeh; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Mostafaie, Ali; Mahdavi, Mehdi

2015-01-01

Objective(s): Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs) are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of recombinant proteins, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env recombinant protein (HIVtop4) expression by E. coli and conjugation of purified protein to anti DEC-205 monoclonal antibody as candidate vaccine. Materials and Methods: In this study, expression was induced in BL21 (DE3) E. coli cells by optimization of induction condition, post induction incubation time, temperature and culture medium formula. Some culture mediums were used for cell culture, and isopropyl-beta-D-thiogalactopyranoside was used for induction of expression. Protein was purified by Ni-NTA column chromatography and confirmed against anti-His antibody in western-blotting. To exploit DCs properties for immunization purposes, recombinant protein chemically coupled to αDEC-205 monoclonal antibody and confirmed against anti-His antibody in western-blotting. Results: The optimum condition for expression was 1 mM IPTG during 4 hr cultures in 2XYT medium, and final protein produced in soluble form. Conjugation of purified protein to αDEC-205 antibody resulted in smears of protein: antibodies conjugate in different molecular weights. Conclusion: The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. The final concentration of purified protein was 500 µg/ml. PMID:25810888

Hapten Mediated Display and Pairing of Recombinant Antibodies Accelerates Assay Assembly for Biothreat Countermeasures

PubMed Central

Sherwood, Laura J.; Hayhurst, Andrew

2012-01-01

A bottle-neck in recombinant antibody sandwich immunoassay development is pairing, demanding protein purification and modification to distinguish captor from tracer. We developed a simple pairing scheme using microliter amounts of E. coli osmotic shockates bearing site-specific biotinylated antibodies and demonstrated proof of principle with a single domain antibody (sdAb) that is both captor and tracer for polyvalent Marburgvirus nucleoprotein. The system could also host pairs of different sdAb specific for the 7 botulinum neurotoxin (BoNT) serotypes, enabling recognition of the cognate serotype. Inducible supE co-expression enabled sdAb populations to be propagated as either phage for more panning from repertoires or expressed as soluble sdAb for screening within a single host strain. When combined with streptavidin-g3p fusions, a novel transdisplay system was formulated to retrofit a semi-synthetic sdAb library which was mined for an anti-Ebolavirus sdAb which was immediately immunoassay ready, thereby speeding up the recombinant antibody discovery and utilization processes. PMID:23150778

SequenceLDhot: detecting recombination hotspots.

PubMed

Fearnhead, Paul

2006-12-15

There is much local variation in recombination rates across the human genome--with the majority of recombination occurring in recombination hotspots--short regions of around approximately 2 kb in length that have much higher recombination rates than neighbouring regions. Knowledge of this local variation is important, e.g. in the design and analysis of association studies for disease genes. Population genetic data, such as that generated by the HapMap project, can be used to infer the location of these hotspots. We present a new, efficient and powerful method for detecting recombination hotspots from population data. We compare our method with four current methods for detecting hotspots. It is orders of magnitude quicker, and has greater power, than two related approaches. It appears to be more powerful than HotspotFisher, though less accurate at inferring the precise positions of the hotspot. It was also more powerful than LDhot in some situations: particularly for weaker hotspots (10-40 times the background rate) when SNP density is lower (< 1/kb). Program, data sets, and full details of results are available at: http://www.maths.lancs.ac.uk/~fearnhea/Hotspot.

Recombination device for storage batteries

DOEpatents

Kraft, H.; Ledjeff, K.

1984-01-01

A recombination device including a gas-tight enclosure connected to receive the discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

Recombination device for storage batteries

DOEpatents

Kraft, Helmut; Ledjeff, Konstantin

1985-01-01

A recombination device including a gas-tight enclosure connected to receive he discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

Controlled Release from Recombinant Polymers

PubMed Central

Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

2014-01-01

Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori).

PubMed

Tada, Minoru; Tatematsu, Ken-ichiro; Ishii-Watabe, Akiko; Harazono, Akira; Takakura, Daisuke; Hashii, Noritaka; Sezutsu, Hideki; Kawasaki, Nana

2015-01-01

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO- and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.

Integrated Development of Serum Molecular Markers for Early Diagnosis of Breast Cancer

DTIC Science & Technology

2006-09-01

Fas, FasL, Cyfra 21-1, TPA/TPS, IGFBP1, S100, angiostatin, SSC, ULBP1,2,3, βHCG, MICA , HE4, SMRP, mesothelin, SAA, and TTR. The procedure we used for... MICA , S-100, DR5, mesothelin, and myeloperoxidase (MPO), ULBP-1,2, S100, angiostatin. Our results have demonstrated that the expression of CA 125, EGF...4066. (8) Adam, B. L.; Qu , Y.; Davis, J. W.; Ward, M. D.; Clements, M. A.; Cazares, L. H.; Semmes, O. J.; Schellhammer, P. F.; Yasui, Y.; Feng, Z

RNAi and heterochromatin repress centromeric meiotic recombination

PubMed Central

Ellermeier, Chad; Higuchi, Emily C.; Phadnis, Naina; Holm, Laerke; Geelhood, Jennifer L.; Thon, Genevieve; Smith, Gerald R.

2010-01-01

During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis. PMID:20421495

Recombination properties of dislocations in GaN

NASA Astrophysics Data System (ADS)

Yakimov, Eugene B.; Polyakov, Alexander Y.; Lee, In-Hwan; Pearton, Stephen J.

2018-04-01

The recombination activity of threading dislocations in n-GaN with different dislocation densities and different doping levels was studied using electron beam induced current (EBIC). The recombination velocity on a dislocation, also known as the dislocation recombination strength, was calculated. The results suggest that dislocations in n-GaN giving contrast in EBIC are charged and surrounded by a space charge region, as evidenced by the observed dependence of dislocation recombination strength on dopant concentration. For moderate (below ˜108 cm-2) dislocation densities, these defects do not primarily determine the average diffusion length of nonequilibrium charge carriers, although locally, dislocations are efficient recombination sites. In general, it is observed that the effect of the growth method [standard metalorganic chemical vapor deposition (MOCVD), epitaxial lateral overgrowth versions of MOCVD, and hydride vapor phase epitaxy] on the recombination activity of dislocations is not very pronounced, although the average diffusion lengths can widely differ for various samples. The glide of basal plane dislocations at room temperature promoted by low energy electron irradiation does not significantly change the recombination properties of dislocations.

Production of Chicken Egg Yolk Antibody (IgY) Against Recombinant Cholera Toxin B Subunit and Evaluation of Its Prophylaxis Potency in Mice.

PubMed

Barati, Babak; Ebrahimi, Firouz; Nazarian, Shahram

2018-03-01

Cholera toxin (CT), responsible for the harmful effects of cholera infection, is made up of one A subunit (enzymatic), and five B subunits (cell binding). The release of cholera toxin is the main reason for the debilitating loss of intestinal fluid. Inhibition of the B subunit (CTB) may block CT activity. To determine the effect of anti CTB-IgY against oral challenge with V. cholera in suckling infant mice. The binding domain of cholera toxin was amplified and ligated into pET28a vector. The pET28a (+)/ctb expression vector was confirmed by endonuclease digestion and sequence analysis. The expression of recombinant CTB in E. coli was performed by induction with IPTG. After immunizing the chickens with recombinant CTB, IgY was purified by water dilution method and NaCl precipitation and analyzed by SDS-PAGE. Moreover, the activity and specificity of the IgY antibody were assessed by ELISA. The SDS-PAGE and western blot techniques showed that CTB protein was successfully expressed and specifically recognized by polyclonal antibodies against the cholera toxin. The oral administration of anti- (V. cholera+CTB) in infant mice in challenge with active V. cholera bacterium demonstrated high rate of survival. The increase in the number of antibiotic resistant bacteria implies the necessity of finding novel antibiotics. Our results suggest the possibility of passive protection from purified IgY, hence implying that anti CTB-IgY may be useful in the treatment of cholera infections.

Annealing Vs. Invasion in Phage λ Recombination

PubMed Central

Stahl, M. M.; Thomason, L.; Poteete, A. R.; Tarkowski, T.; Kuzminov, A.; Stahl, F. W.

1997-01-01

Genetic recombination catalyzed by λ's Red pathway was studied in rec(+) and recA mutant bacteria by examining both intracellular λ DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec(+) cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of RecA and the presence of non-allelic DNA ends, the Red pathway of λ catalyzes recombination primarily by annealing. PMID:9383045

A human recombinant monoclonal antibody to cocaine: Preparation, characterization and behavioral studies

PubMed Central

Eubanks, Lisa M.; Ellis, Beverly A.; Cai, Xiaoqing; Schlosburg, Joel E.; Janda, Kim D.

2014-01-01

Cocaine abuse remains prevalent worldwide and continues to be a major health concern; nonetheless, there is no effective therapy. Immunopharmacothery has emerged as a promising treatment strategy by which anti-cocaine antibodies bind to the drug blunting its effects. Previous passive immunization studies using our human monoclonal antibody, GNCgzk, resulted in protection against cocaine overdose and acute toxicity. To further realize the clinical potential of this antibody, a recombinant IgG form of the antibody has been produced in mammalian cells. This antibody displayed a high binding affinity for cocaine (low nanomolar) in line with the superior attributes of the GNCgzk antibody and reduced cocaine-induced ataxia in a cocaine overdose model. PMID:25205191

Oligonucleotide recombination enabled site-specific mutagenesis in bacteria

USDA-ARS?s Scientific Manuscript database

Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligo recombination is one type of recombineering that uses ssDNA olig...

Anti-signal recognition particle autoantibody ELISA validation and clinical associations.

PubMed

Aggarwal, Rohit; Oddis, Chester V; Goudeau, Danielle; Fertig, Noreen; Metes, Ilinca; Stephens, Chad; Qi, Zengbiao; Koontz, Diane; Levesque, Marc C

2015-07-01

The aim of this study was to develop and validate a quantitative anti-signal recognition particle (SRP) autoantibody serum ELISA in patients with myositis and longitudinal association with myositis disease activity. We developed a serum ELISA using recombinant purified full-length human SRP coated on ELISA plates and a secondary antibody that bound human IgG to detect anti-SRP binding. Protein immunoprecipitation was used as the gold standard for the presence of anti-SRP. Serum samples from three groups were analysed: SRP(+) myositis subjects by immunoprecipitation, SRP(-) myositis subjects by immunoprecipitation and non-myositis controls. The ELISA's sensitivity, specificity, positive predictive value and negative predictive value were evaluated. Percentage agreement and test-retest reliability were assessed. Serial samples from seven SRP immunoprecipitation-positive subjects were also tested, along with serum muscle enzymes and manual muscle testing. Using immunoprecipitation, we identified 26 SRP(+) myositis patients and 77 SRP(-) controls (including 38 patients with necrotizing myopathy). Non-myositis control patients included SLE (n = 4) and SSc (n = 7) patients. Anti-SRP positivity by ELISA showed strong agreement (97.1%) with immunoprecipitation (κ = 0.94). The sensitivity, specificity, positive predictive value, and negative predictive value of the anti-SRP ELISA were 88, 100, 100 and 96, respectively. The area under the curve was 0.94, and test-retest reliability was strong (r = 0.91, P < 0.001). Serial samples showed that anti-SRP levels paralleled changes in muscle enzymes and manual muscle testing. We developed a quantitative ELISA for detecting serum anti-SRP autoantibodies and validated the assay in myositis. Longitudinal assessment of SRP levels by ELISA may be a useful biomarker for disease activity. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions

Genetic recombination of the hepatitis C virus: clinical implications.

PubMed

Morel, V; Fournier, C; François, C; Brochot, E; Helle, F; Duverlie, G; Castelain, S

2011-02-01

Genetic recombination is a well-known feature of RNA viruses that plays a significant role in their evolution. Although recombination is well documented for Flaviviridae family viruses, the first natural recombinant strain of hepatitis C virus (HCV) was identified as recently as 2002. Since then, a few other natural inter-genotypic, intra-genotypic and intra-subtype recombinant HCV strains have been described. However, the frequency of recombination may have been underestimated because not all known HCV recombinants are screened for in routine practice. Furthermore, the choice of treatment regimen and its predictive outcome remain problematic as the therapeutic strategy for HCV infection is genotype dependent. HCV recombination also raises many questions concerning its mechanisms and effects on the epidemiological and physiopathological features of the virus. This review provides an update on recombinant HCV strains, the process that gives rise to recombinants and clinical implications of recombination. © 2010 Blackwell Publishing Ltd.

Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii.

PubMed

Tomás, A L; Cardoso, F; Esteves, F; Matos, O

2016-11-08

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis.

Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii

PubMed Central

Tomás, A. L.; Cardoso, F.; Esteves, F.; Matos, O.

2016-01-01

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis. PMID:27824115

Widespread recombination in published animal mtDNA sequences.

PubMed

Tsaousis, A D; Martin, D P; Ladoukakis, E D; Posada, D; Zouros, E

2005-04-01

Mitochondrial DNA (mtDNA) recombination has been observed in several animal species, but there are doubts as to whether it is common or only occurs under special circumstances. Animal mtDNA sequences retrieved from public databases were unambiguously aligned and rigorously tested for evidence of recombination. At least 30 recombination events were detected among 186 alignments examined. Recombinant sequences were found in invertebrates and vertebrates, including primates. It appears that mtDNA recombination may occur regularly in the animal cell but rarely produces new haplotypes because of homoplasmy. Common animal mtDNA recombination would necessitate a reexamination of phylogenetic and biohistorical inference based on the assumption of clonal mtDNA transmission. Recombination may also have an important role in producing and purging mtDNA mutations and thus in mtDNA-based diseases and senescence.

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

PubMed Central

Zieg, J; Maples, V F; Kushner, S R

1978-01-01

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products. PMID:350859

Marker-Dependent Recombination in T4 Bacteriophage. IV. Recombinational Effects of Antimutator T4 DNA Polymerase

PubMed Central

Shcherbakov, V. P.; Plugina, L. A.; Kudryashova, E. A.

1995-01-01

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3' -> 5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49) -> 3'->5' exonuclease (gp43) -> DNA polymerase (gp43) -> DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates. PMID:7635281

Recombination Modulates How Selection Affects Linked Sites in Drosophila

PubMed Central

McGaugh, Suzanne E.; Heil, Caiti S. S.; Manzano-Winkler, Brenda; Loewe, Laurence; Goldstein, Steve; Himmel, Tiffany L.; Noor, Mohamed A. F.

2012-01-01

One of the most influential observations in molecular evolution has been a strong association between local recombination rate and nucleotide polymorphisms across the genome. This is interpreted as evidence for ubiquitous natural selection. The alternative explanation, that recombination is mutagenic, has been rejected by the absence of a similar association between local recombination rate and nucleotide divergence between species. However, many recent studies show that recombination rates are often very different even in closely related species, questioning whether an association between recombination rate and divergence between species has been tested satisfactorily. To circumvent this problem, we directly surveyed recombination across approximately 43% of the D. pseudoobscura physical genome in two separate recombination maps and 31% of the D. miranda physical genome, and we identified both global and local differences in recombination rate between these two closely related species. Using only regions with conserved recombination rates between and within species and accounting for multiple covariates, our data support the conclusion that recombination is positively related to diversity because recombination modulates Hill–Robertson effects in the genome and not because recombination is predominately mutagenic. Finally, we find evidence for dips in diversity around nonsynonymous substitutions. We infer that at least some of this reduction in diversity resulted from selective sweeps and examine these dips in the context of recombination rate. PMID:23152720

Function-blocking antibodies to human vascular adhesion protein-1: a potential anti-inflammatory therapy.

PubMed

Kirton, Christopher M; Laukkanen, Marja-Leena; Nieminen, Antti; Merinen, Marika; Stolen, Craig M; Armour, Kathryn; Smith, David J; Salmi, Marko; Jalkanen, Sirpa; Clark, Michael R

2005-11-01

Human vascular adhesion protein-1 (VAP-1) is a homodimeric 170-kDa sialoglycoprotein that is expressed on the surface of endothelial cells and functions as a semicarbazide-sensitive amine oxidase and as an adhesion molecule. Blockade of VAP-1 has been shown to reduce leukocyte adhesion and transmigration in in vivo and in vitro models, suggesting that VAP-1 is a potential target for anti-inflammatory therapy. In this study we have constructed mouse-human chimeric antibodies by genetic engineering in order to circumvent the potential problems involved in using murine antibodies in man. Our chimeric anti-VAP-1 antibodies, which were designed to lack Fc-dependent effector functions, bound specifically to cell surface-expressed recombinant human VAP-1 and recognized VAP-1 in different cell types in tonsil. Furthermore, the chimeric antibodies prevented leukocyte adhesion and transmigration in vitro and in vivo. Hence, these chimeric antibodies have the potential to be used as a new anti-inflammatory therapy.

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

PubMed Central

Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

2009-01-01

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

[AntiEGFRnano inhibites proliferation and migration of estrogen-dependent Ishikawa cells of human endometrial cancer cell line].

PubMed

Diao, Zhen-yu; Lu, Wu-guang; Cao, Peng; Hu, Yun-long; Zhou, Xing; Xue, Ping-ping; Shen, Li; Sun, Hai-xiang

2012-10-01

Nanobody is a kind of antibody from camel, which misses light chain. Nanobody has the same antigen binding specificity and affinity as mAb. Moreover, because of its small molecular weight, high stability and easy preparation, nanobody has great value of biomedical applications. In this study, we successfully prepared highly pure antiEGFR nanobody in E.coli using genetic engineering techniques. Cell proliferation assay (CCK-8 assay) and migration experiments (cell scratch test and Transwell assay) indicated that the recombinant antiEGFRnano can significantly inhibit the proliferation and migration of endometrial cancer cells. These results provide a new way of thinking and methods for EGFR-targeted therapy of endometrial cancer.

Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

PubMed Central

Miller, A M; Savinelli, E A; Couture, S M; Hannigan, G M; Han, Z; Selden, R F; Treco, D A

1993-01-01

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8367472

Evolution of meiotic recombination genes in maize and teosinte.

PubMed

Sidhu, Gaganpreet K; Warzecha, Tomasz; Pawlowski, Wojciech P

2017-01-25

Meiotic recombination is a major source of genetic variation in eukaryotes. The role of recombination in evolution is recognized but little is known about how evolutionary forces affect the recombination pathway itself. Although the recombination pathway is fundamentally conserved across different species, genetic variation in recombination components and outcomes has been observed. Theoretical predictions and empirical studies suggest that changes in the recombination pathway are likely to provide adaptive abilities to populations experiencing directional or strong selection pressures, such as those occurring during species domestication. We hypothesized that adaptive changes in recombination may be associated with adaptive evolution patterns of genes involved in meiotic recombination. To examine how maize evolution and domestication affected meiotic recombination genes, we studied patterns of sequence polymorphism and divergence in eleven genes controlling key steps in the meiotic recombination pathway in a diverse set of maize inbred lines and several accessions of teosinte, the wild ancestor of maize. We discovered that, even though the recombination genes generally exhibited high sequence conservation expected in a pathway controlling a key cellular process, they showed substantial levels and diverse patterns of sequence polymorphism. Among others, we found differences in sequence polymorphism patterns between tropical and temperate maize germplasms. Several recombination genes displayed patterns of polymorphism indicative of adaptive evolution. Despite their ancient origin and overall sequence conservation, meiotic recombination genes can exhibit extensive and complex patterns of molecular evolution. Changes in these genes could affect the functioning of the recombination pathway, and may have contributed to the successful domestication of maize and its expansion to new cultivation areas.

Mitigating Mitochondrial Genome Erosion Without Recombination.

PubMed

Radzvilavicius, Arunas L; Kokko, Hanna; Christie, Joshua R

2017-11-01

Mitochondria are ATP-producing organelles of bacterial ancestry that played a key role in the origin and early evolution of complex eukaryotic cells. Most modern eukaryotes transmit mitochondrial genes uniparentally, often without recombination among genetically divergent organelles. While this asymmetric inheritance maintains the efficacy of purifying selection at the level of the cell, the absence of recombination could also make the genome susceptible to Muller's ratchet. How mitochondria escape this irreversible defect accumulation is a fundamental unsolved question. Occasional paternal leakage could in principle promote recombination, but it would also compromise the purifying selection benefits of uniparental inheritance. We assess this tradeoff using a stochastic population-genetic model. In the absence of recombination, uniparental inheritance of freely-segregating genomes mitigates mutational erosion, while paternal leakage exacerbates the ratchet effect. Mitochondrial fusion-fission cycles ensure independent genome segregation, improving purifying selection. Paternal leakage provides opportunity for recombination to slow down the mutation accumulation, but always at a cost of increased steady-state mutation load. Our findings indicate that random segregation of mitochondrial genomes under uniparental inheritance can effectively combat the mutational meltdown, and that homologous recombination under paternal leakage might not be needed. Copyright © 2017 by the Genetics Society of America.

Lessons learnt from many years of experience using anti-D in humans for prevention of RhD immunization and haemolytic disease of the fetus and newborn

PubMed Central

Kumpel, B M

2008-01-01

For 40 years prophylactic anti-D has been given to D-negative women after parturition to prevent haemolytic disease of the fetus and newborn. Monoclonal or recombinant anti-D may provide alternatives to the current plasma-derived polyclonal IgG anti-D, although none of them have yet proved as effective in phase 1 clinical trials. The variation in efficacy of the antibodies may have been influenced by heterogeneity in glycosylation of anti-D produced from different cell lines. Some aspects of the conduct of the human studies, most notably the use of low doses of anti-D and target D positive red cells in vivo, may aid the design of the clinical development of other immunomodulatory drugs in order to minimize adverse effects. PMID:18727626

EXAMINATION OF Zr AND Ti RECOMBINER LOOP SPECIMENS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rittenhouse, P.L.

1958-12-19

Cold-worked specimens of iodide zirconium, Zircaloy-2, iodide titanium, and A-55 titanium were tested in a high-pressure recombiner loop in an attempt to duplicate anomalous results obtained in a prior recombiner loop. Hydrogen analyses and metallographic examinations were made on all specimens. The titanium materials and Zircaloy-2 picked up major amounts of hydrogen in the cell section. None of the materials tested showed appreciable hydrogen absorption in the recombiner section. Complete recrystallization occurred in all cell specimens while only Zircaloy-2, of the recombiner specimens, showed any degree of recrystallization. No explanation for this behavior can be given. A survnnary of themore » data obtained in previous recombiner loops is compared with the results of this loop. Conclusions were based on the results of three recombiner loops. Primarlly because of the hydrogen absorption data obtained in all three recombiner loops it is recommended that the zirconium and titunium materials tested not be used in environments similar to those encountered in high pressure recombiner loops. (auth)« less

Mitochondrial recombination increases with age in Podospora anserina.

PubMed

van Diepeningen, Anne D; Goedbloed, Daniël J; Slakhorst, S Marijke; Koopmanschap, A Bertha; Maas, Marc F P M; Hoekstra, Rolf F; Debets, Alfons J M

2010-05-01

With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer of organelles takes place, making it possible to study mitochondrial recombination when introduced mitochondria contain different markers. A survey of wild-type isolates from a local population of the filamentous fungus Podospora anserina for the presence of seven optional mitochondrial introns indicated that mitochondrial recombination does take place in nature. Moreover the recombination frequency appeared to be correlated with age: the more rapidly ageing fraction of the population had a significantly lower linkage disequilibrium indicating more recombination. Direct confrontation experiments with heterokaryon incompatible strains with different mitochondrial markers at different (relative) age confirmed that mitochondrial recombination increases with age. We propose that with increasing mitochondrial damage over time, mitochondrial recombination - even within a homoplasmic population of mitochondria - is a mechanism that may restore mitochondrial function. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus.

PubMed

Golden, Allison; Stevens, Eric J; Yokobe, Lindsay; Faulx, Dunia; Kalnoky, Michael; Peck, Roger; Valdez, Melissa; Steel, Cathy; Karabou, Potochoziou; Banla, Méba; Soboslay, Peter T; Adade, Kangi; Tekle, Afework H; Cama, Vitaliano A; Fischer, Peter U; Nutman, Thomas B; Unnasch, Thomas R; de los Santos, Tala; Domingo, Gonzalo J

2016-01-01

Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests. A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011-2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions. The recombinant human anti-Ov16 IgG4 antibody-based positive

Anti-Inflammatory Thioredoxin Family Proteins for Medicare, Healthcare and Aging Care.

PubMed

Yodoi, Junji; Matsuo, Yoshiyuki; Tian, Hai; Masutani, Hiroshi; Inamoto, Takashi

2017-09-29

Human thioredoxin (TRX) is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys-, which is induced by biological stress due to oxidative damage, metabolic dysfunction, chemicals, infection/inflammation, irradiation, or hypoxia/ischemia-reperfusion. Our research has demonstrated that exogenous TRX is effective in a wide variety of inflammatory diseases, including viral pneumonia, acute lung injury, gastric injury, and dermatitis, as well as in the prevention and amelioration of food allergies. Preclinical and clinical studies using recombinant TRX (rhTRX) are now underway. We have also identified substances that induce the expression of TRX in the body, in vegetables and other plant ingredients. Skincare products are being developed that take advantage of the anti-inflammatory and anti-allergic action of TRX. Furthermore, we are currently engaged in the highly efficient production of pure rhTRX in several plants, such as lettuce, grain and rice.

Complexity of genetic mechanisms conferring nonuniformity of recombination in maize.

PubMed

Pan, Qingchun; Deng, Min; Yan, Jianbing; Li, Lin

2017-04-26

Recombinations occur nonuniformly across the maize genome. To dissect the genetic mechanisms underlying the nonuniformity of recombination, we performed quantitative trait locus (QTL) mapping using recombinant inbred line populations. Genome-wide QTL scan identified hundreds of QTLs with both cis-prone and trans- effects for recombination number variation. To provide detailed insights into cis- factors associated with recombination variation, we examined the genomic features around recombination hot regions, including density of genes, DNA transposons, retrotransposons, and some specific motifs. Compared to recombination variation in whole genome, more QTLs were mapped for variations in recombination hot regions. The majority QTLs for recombination hot regions are trans-QTLs and co-localized with genes from the recombination pathway. We also found that recombination variation was positively associated with the presence of genes and DNA transposons, but negatively related to the presence of long terminal repeat retrotransposons. Additionally, 41 recombination hot regions were fine-mapped. The high-resolution genotyping of five randomly selected regions in two F 2 populations verified that they indeed have ultra-high recombination frequency, which is even higher than that of the well-known recombination hot regions sh1-bz and a1-sh2. Taken together, our results further our understanding of recombination variation in plants.

Development of immunodetection system for botulinum neurotoxin type B using synthetic gene based recombinant protein

PubMed Central

Jain, Swati; Ponmariappan, S.; Kumar, Om

2011-01-01

Background & objectives: Botulinum neurotoxins (A-G) are among most poisonous substances in the world, produced by obligate anaerobic bacteria Clostridum botulinum. Among the seven serotypes A, B, E and F are of human importance. In India, the prevalence of C. botulinum as well as botulism outbreaks have been reported. Due to its extreme toxicity it has been classified in the Category A of biological warfare agent. So far, there is no commercial detection system available in India to detect botulism. The present study aims to develop an immuno detection system for botulinum neurotoxin serotype B using synthetic gene approach. Methods: The truncated fragment of the botulinum neurotoxin type B from amino acid 1-450 was synthesized using PCR overlap primers; the constructed gene was cloned in the pQE30UA vector and transformed to Escherichia coli SG 13009. The recombinant protein expression was optimized using various concentration of isopropylthiogalactoside (IPTG) induction, further the expression was confirmed by Western blot analysis using anti-His antibody. Recombinant protein was purified under denatured condition using Ni-NTA affinity chromatography. Antibody was generated against the recombinant protein using alum adjuvant in BALB/c mice and tested for cross reactivity with other serotypes of C. botulinum as well as closely related clostridia. An ELISA test was developed for the detection of botulinum neurotoxin and the minimum detection limit was also estimated. Results: The recombinant protein was expressed at maximum yield at 4.3 h of post-induction with 0.5 mM IPTG concentration. The recombinant protein was purified using Ni-NTA affinity chromatography up to the homogeneity level. The polyclonal antibodies were raised in mice with a titre of 1:2048000. The developed antibody was highly specific with a sensitivity of detecting approximately 15 ng/ml of recombinant protein and not showing any cross-reactivity with other serotypes. Interpretation

Anti-high mobility group box-1 antibody therapy for traumatic brain injury.

PubMed

Okuma, Yu; Liu, Keyue; Wake, Hidenori; Zhang, Jiyong; Maruo, Tomoko; Date, Isao; Yoshino, Tadashi; Ohtsuka, Aiji; Otani, Naoki; Tomura, Satoshi; Shima, Katsuji; Yamamoto, Yasuhiko; Yamamoto, Hiroshi; Takahashi, Hideo K; Mori, Shuji; Nishibori, Masahiro

2012-09-01

High mobility group box-1 (HMGB1) plays an important role in triggering inflammatory responses in many types of diseases. In this study, we examined the involvement of HMGB1 in traumatic brain injury (TBI) and evaluated the ability of intravenously administered neutralizing anti-HMGB1 monoclonal antibody (mAb) to attenuate brain injury. Traumatic brain injury was induced in rats or mice by fluid percussion. Anti-HMGB1 mAb or control mAb was administered intravenously after TBI. Anti-HMGB1 mAb remarkably inhibited fluid percussion-induced brain edema in rats, as detected by T2-weighted magnetic resonance imaging; this was associated with inhibition of HMGB1 translocation, protection of blood-brain barrier (BBB) integrity, suppression of inflammatory molecule expression, and improvement of motor function. In contrast, intravenous injection of recombinant HMGB1 dose-dependently produced the opposite effects. Experiments using receptor for advanced glycation end product (RAGE)(-/-) , toll-like receptor-4 (TLR4)(-/-) , and TLR2(-/-) mice suggested the involvement of RAGE as the predominant receptor for HMGB1. Anti-HMGB1 mAb may provide a novel and effective therapy for TBI by protecting against BBB disruption and reducing the inflammatory responses induced by HMGB1. Copyright © 2012 American Neurological Association.

BAY 81-8973, a full-length recombinant factor VIII: Human heat shock protein 70 improves the manufacturing process without affecting clinical safety.

PubMed

Maas Enriquez, Monika; Thrift, John; Garger, Stephen; Katterle, Yvonne

2016-11-01

BAY 81-8973 is a full-length, unmodified recombinant human factor VIII (FVIII) approved for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as the currently marketed sucrose-formulated recombinant FVIII (rFVIII-FS) product and is produced using additional advanced manufacturing technologies. One of the key manufacturing advances for BAY 81-8973 is introduction of the gene for human heat shock protein 70 (HSP70) into the rFVIII-FS cell line. HSP70 facilitates proper folding of proteins, enhances cell survival by inhibiting apoptosis, and potentially impacts rFVIII glycosylation. HSP70 expression in the BAY 81-8973 cell line along with other manufacturing advances resulted in a higher-producing cell line and improvements in the pharmacokinetics of the final product as determined in clinical studies. HSP70 protein is not detected in the harvest or in the final BAY 81-8973 product. However, because this is a new process, clinical trial safety assessments included monitoring for anti-HSP70 antibodies. Most patients, across all age groups, had low levels of anti-HSP70 antibodies before exposure to the investigational product. During BAY 81-8973 treatment, 5% of patients had sporadic increases in anti-HSP70 antibody levels above a predefined threshold (cutoff value, 239 ng/mL). No clinical symptoms related to anti-HSP70 antibody development occurred. In conclusion, addition of HSP70 to the BAY 81-8973 cell line is an innovative technology for manufacturing rFVIII aimed at improving protein folding and expression. Improved pharmacokinetics and no effect on safety of BAY 81-8973 were observed in clinical trials in patients with hemophilia A. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli

NASA Astrophysics Data System (ADS)

Jiang, Shan; Liu, Mei; Wang, Baojie; Jiang, Keyong; Wang, Lei

2011-11-01

Anti-lipopolysaccharide factors (ALFs) are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In this study, we optimized the production of a recombinant ALF by expressing E. sinensis ALF genes in Escherichia coli maintained in shake-flasks. In particular, we focused on optimization of both the medium composition and the culture condition. Various medium components were analyzed by the Plackett-Burman design, and two significant screened factors, (NH4)2SO4 and KH2PO4, were further optimized via the central composite design (CCD). Based on the CCD analysis, we investigated the induction start-up time, the isopropylthio-D-galactoside (IPTG) concentration, the post-induction time, and the temperature by response surface methodology. We found that the highest level of ALF fusion protein was achieved in the medium containing 1.89 g/L (NH4)2SO4 and 3.18 g/L KH2PO4, with a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7°C, and a post-induction time of 4 h. Applying the whole optimization strategy using all optimal factors improved the target protein content from 6.1% (without optimization) to 13.2%. We further applied the optimized medium and conditions in high cell density cultivation, and determined that the soluble target protein constituted 10.5% of the total protein. Our identification of the economic medium composition, optimal culture conditions, and details of the fermentation process should facilitate the potential application of ALF for further research.

Recombination rate plasticity: revealing mechanisms by design

PubMed Central

Sefick, Stephen; Rushton, Chase

2017-01-01

For over a century, scientists have known that meiotic recombination rates can vary considerably among individuals, and that environmental conditions can modify recombination rates relative to the background. A variety of external and intrinsic factors such as temperature, age, sex and starvation can elicit ‘plastic’ responses in recombination rate. The influence of recombination rate plasticity on genetic diversity of the next generation has interesting and important implications for how populations evolve. Further, many questions remain regarding the mechanisms and molecular processes that contribute to recombination rate plasticity. Here, we review 100 years of experimental work on recombination rate plasticity conducted in Drosophila melanogaster. We categorize this work into four major classes of experimental designs, which we describe via classic studies in D. melanogaster. Based on these studies, we highlight molecular mechanisms that are supported by experimental results and relate these findings to studies in other systems. We synthesize lessons learned from this model system into experimental guidelines for using recent advances in genotyping technologies, to study recombination rate plasticity in non-model organisms. Specifically, we recommend (1) using fine-scale genome-wide markers, (2) collecting time-course data, (3) including crossover distribution measurements, and (4) using mixed effects models to analyse results. To illustrate this approach, we present an application adhering to these guidelines from empirical work we conducted in Drosophila pseudoobscura. This article is part of the themed issue ‘Evolutionary causes and consequences of recombination rate variation in sexual organisms’. PMID:29109222

Designing Trojan Horses | Center for Cancer Research

Cancer.gov

Waging battle against cancer cells without inflicting damage on normal tissue has long been a goal for cancer treatment. A new type of drug called immunotoxins may help make this goal a reality. Much like the Greeks used a wooden horse to get soldiers inside the gates of Troy, immunotoxins use clever genetic engineering to get a lethal toxin inside cancer cells. Each

The Dissociative Recombination of OH(+)

NASA Technical Reports Server (NTRS)

Guberman, Steven L.

1995-01-01

Theoretical quantum chemical calculations of the cross sections and rates for the dissociative recombination of the upsilon = 0 level of the ground state of OH(+) show that recombination occurs primarily along the 2 (2)Pi diabatic route. The products are 0((1)D) and a hot H atom with 6.1 eV kinetic energy. The coupling to the resonances is very small and the indirect recombination mechanism plays only a minor role. The recommended value for the rate coefficient is (6.3 +/- 0.7) x 10(exp -9)x (T(e)/1300)(exp -0.48) cu.cm/s for 10 less than T(e) less than 1000 K.

Connecting theory and data to understand recombination rate evolution.

PubMed

Dapper, Amy L; Payseur, Bret A

2017-12-19

Meiotic recombination is necessary for successful gametogenesis in most sexually reproducing organisms and is a fundamental genomic parameter, influencing the efficacy of selection and the fate of new mutations. The molecular and evolutionary functions of recombination should impose strong selective constraints on the range of recombination rates. Yet, variation in recombination rate is observed on a variety of genomic and evolutionary scales. In the past decade, empirical studies have described variation in recombination rate within genomes, between individuals, between sexes, between populations and between species. At the same time, theoretical work has provided an increasingly detailed picture of the evolutionary advantages to recombination. Perhaps surprisingly, the causes of natural variation in recombination rate remain poorly understood. We argue that empirical and theoretical approaches to understand the evolution of recombination have proceeded largely independently of each other. Most models that address the evolution of recombination rate were created to explain the evolutionary advantage of recombination rather than quantitative differences in rate among individuals. Conversely, most empirical studies aim to describe variation in recombination rate, rather than to test evolutionary hypotheses. In this Perspective, we argue that efforts to integrate the rich bodies of empirical and theoretical work on recombination rate are crucial to moving this field forward. We provide new directions for the development of theory and the production of data that will jointly close this gap.This article is part of the themed issue 'Evolutionary causes and consequences of recombination rate variation in sexual organisms'. © 2017 The Author(s).

Exceptionally high levels of recombination across the honey bee genome.

PubMed

Beye, Martin; Gattermeier, Irene; Hasselmann, Martin; Gempe, Tanja; Schioett, Morten; Baines, John F; Schlipalius, David; Mougel, Florence; Emore, Christine; Rueppell, Olav; Sirviö, Anu; Guzmán-Novoa, Ernesto; Hunt, Greg; Solignac, Michel; Page, Robert E

2006-11-01

The first draft of the honey bee genome sequence and improved genetic maps are utilized to analyze a genome displaying 10 times higher levels of recombination (19 cM/Mb) than previously analyzed genomes of higher eukaryotes. The exceptionally high recombination rate is distributed genome-wide, but varies by two orders of magnitude. Analysis of chromosome, sequence, and gene parameters with respect to recombination showed that local recombination rate is associated with distance to the telomere, GC content, and the number of simple repeats as described for low-recombining genomes. Recombination rate does not decrease with chromosome size. On average 5.7 recombination events per chromosome pair per meiosis are found in the honey bee genome. This contrasts with a wide range of taxa that have a uniform recombination frequency of about 1.6 per chromosome pair. The excess of recombination activity does not support a mechanistic role of recombination in stabilizing pairs of homologous chromosome during chromosome pairing. Recombination rate is associated with gene size, suggesting that introns are larger in regions of low recombination and may improve the efficacy of selection in these regions. Very few transposons and no retrotransposons are present in the high-recombining genome. We propose evolutionary explanations for the exceptionally high genome-wide recombination rate.

The evolution of recombination in a heterogeneous environment.

PubMed Central

Lenormand, T; Otto, S P

2000-01-01

Most models describing the evolution of recombination have focused on the case of a single population, implicitly assuming that all individuals are equally likely to mate and that spatial heterogeneity in selection is absent. In these models, the evolution of recombination is driven by linkage disequilibria generated either by epistatic selection or drift. Models based on epistatic selection show that recombination can be favored if epistasis is negative and weak compared to directional selection and if the recombination modifier locus is tightly linked to the selected loci. In this article, we examine the joint effects of spatial heterogeneity in selection and epistasis on the evolution of recombination. In a model with two patches, each subject to different selection regimes, we consider the cases of mutation-selection and migration-selection balance as well as the spread of beneficial alleles. We find that including spatial heterogeneity extends the range of epistasis over which recombination can be favored. Indeed, recombination can be favored without epistasis, with negative and even with positive epistasis depending on environmental circumstances. The selection pressure acting on recombination-modifier loci is often much stronger with spatial heterogeneity, and even loosely linked modifiers and free linkage may evolve. In each case, predicting whether recombination is favored requires knowledge of both the type of environmental heterogeneity and epistasis, as none of these factors alone is sufficient to predict the outcome. PMID:10978305

Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn.

PubMed

Tacket, Carol O; Pasetti, Marcela F; Edelman, Robert; Howard, John A; Streatfield, Stephen

2004-10-22

Previous clinical studies have demonstrated the feasibility of using edible transgenic plants to deliver protective antigens as new oral vaccines. Transgenic corn is particularly attractive for this purpose since the recombinant antigen is stable and homogeneous, and corn can be formulated in several edible forms without destroying the cloned antigen. Transgenic corn expressing 1 mg of LT-B of Escherichia coli without buffer was fed to adult volunteers in three doses, each consisting of 2.1 g of plant material. Seven (78%) of nine volunteers developed rises in both serum IgG anti-LT and numbers of specific antibody secreting cells after vaccination. Four (44%) of nine volunteers also developed stool IgA. Transgenic plants represent a new vector for oral vaccine antigens.

Activated recombinant adenovirus proteinases

DOEpatents

Anderson, C.W.; Mangel, W.F.

1999-08-10

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

Activated recombinant adenovirus proteinases

DOEpatents

Anderson, Carl W.; Mangel, Walter F.

1999-08-10

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

Nonmutagenic carcinogens induce intrachromosomal recombination in dividing yeast cells.

PubMed

Schiestl, R H

1993-12-01

A large number of animal and human carcinogens without apparent genotoxic activity exist (nonmutagenic carcinogens) that are difficult or impossible to detect with the currently used short-term tests. Because of the association of carcinogenesis with genome rearrangement, a system selecting for intrachromosomal recombination (DEL recombination) that results in genome rearrangement has been constructed in the yeast Saccharomyces cerevisiae. Because DEL recombination is under different genetic control than interchromosomal recombination and meiotic recombination, it is probably due to a different mechanism. It has been found that DEL recombination is readily inducible by 10 mutagenic carcinogens and 17 nonmutagenic carcinogens that are not detectable (false negatives) with the Ames assay. In addition, three out of four mutagens that do not cause cancer (false positives in the Ames assay) do not induce DEL recombination. DEL recombination is inducible by UV only in dividing cells but not in cells synchronized in the G1 or G2 phase of the cell cycle. Interchromosomal recombination, on the other hand, is inducible in G1 but not in G2. The nonmutagenic carcinogens induce DEL recombination only in actively growing cells, which may give some indication as to their mechanism. Further characterization of the mechanism involved in induction of DEL recombination may contribute to the understanding of the biological activity of nonmutagenic carcinogens.

Evaluation of a new automated enzyme fluoroimmunoassay using recombinant plasmid dsDNA for the detection of anti-dsDNA antibodies in SLE.

PubMed

Villalta, D; Bizzaro, N; Corazza, D; Tozzoli, R; Tonutti, E

2002-01-01

ELISA methods to detect anti-double-stranded DNA (anti-dsDNA) antibodies are highly sensitive, but are less specific for the diagnosis of SLE than the immunofluorescence test on Crithidia luciliae (CLIFT) and the Farr assay because they also detect low-avidity antibodies. This study evaluated the specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of a new automated fluoroimmunoassay (EliA dsDNA; Pharmacia, Freiburg, Germany). We compared the results with those obtained using a commercial CLIFT and an in-house anti-dsDNA IgG ELISA method, and verified its putative ability to detect only high-avidity anti-dsDNA antibodies. Sera from 100 SLE patients and 120 controls were studied. The control group included 20 healthy donors, 70 patients with other rheumatic diseases (32 systemic sclerosis (SSc); 18 primary Sjögren syndrome (pSS), 20 rheumatoid arthritis (RA)), and 30 patients with various infectious diseases (ID). Anti-dsDNA avidity was estimated using an ELISA method based upon the law of mass action, and a simplified Scatchard plot analysis for data elaboration; the apparent affinity constant (Kaa) was calculated and expressed as arbitrary units (L/U). Sensitivity, specificity, PPV, and NPV for SLE were 64%, 95.8%, 93.8% and 72.7%, respectively, for the EliA anti-dsDNA assay; 55%, 99.2%, 98.5%, and 68.8%, respectively, for the CLIFT; and 64%, 93.3%, 90.6%, and 72.3%, respectively, for the in-house ELISA. Although EliA anti-dsDNA was positive mainly in SLE patients with high- (Kaa>80 L/U) and intermediate- (Kaa 30-80 L/U) avidity antibodies (45.3% and 49.9%, respectively), it was also positive in five (7.8%) SLE patients with low-avidity anti-dsDNA antibodies, and five controls (three SSc, one pSS, and one ID) (mean Kaa = 16.4 +/- 9.04 L/U). In conclusion, EliA anti-dsDNA assay showed a higher sensitivity than the CLIFT, and a good specificity and PPV for SLE. Its putative ability to detect only high-avidity anti

Evolution of recombination in a constant environment

PubMed Central

Feldman, Marcus W.; Christiansen, Freddy B.; Brooks, Lisa D.

1980-01-01

The theory of evolution at a selectively neutral locus that controls the recombination between two major loci that are under selection is studied. If the major loci are at a stable equilibrium in linkage disequilibrium under selection and recombination, then a mutation at the modifier locus will increase in frequency when rare if and only if it decreases the recombination fraction. If the major loci are in disequilibrium at a balance between selection against deleterious alleles and mutation towards them, then two new phenomena are observed. First, a recombination increasing mutation will succeed if the disequilibrium is negative and the modifier is sufficiently tightly linked to the major loci. Second, depending on the strength of selection, even if the disequilibrium is negative, recombination reduction may occur for looser linkage between the major and modifier loci. PMID:16592864

Rapid one-step recombinational cloning

PubMed Central

Fu, Changlin; Wehr, Daniel R.; Edwards, Janice; Hauge, Brian

2008-01-01

As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restriction endonucleases and ligase, have been widely used for high-throughput (HTP) procedures. We have developed a recombination cloning method, which uses truncated recombination sites to clone PCR products directly into destination/expression vectors, thereby bypassing the requirement for first producing an entry clone. Cloning efficiencies in excess of 80% are obtained providing a highly efficient method for directional HTP cloning. PMID:18424799

Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors.

PubMed

Tan, Janice G L; Lee, Yih Yean; Wang, Tianhua; Yap, Miranda G S; Tan, Tin Wee; Ng, Say Kong

2015-05-01

CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Induction of homologous recombination in Saccharomyces cerevisiae.

PubMed

Simon, J R; Moore, P D

1988-09-01

We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

Spectrum Recombination.

ERIC Educational Resources Information Center

Greenslade, Thomas B., Jr.

1984-01-01

Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

Prisoner's dilemma posed by fitness-associated recombination strategies.

PubMed

Wexler, Ydo; Rokhlenko, Oleg

2007-07-07

Genetic recombination is a central and repeated topic of study in the evolution of life. However, along with the influence of recombination on evolution, we understand surprisingly little of how selection shapes the nature of recombination. One explanation for recombination is that it allows organisms to escape from perilous situations where they experience very low fitness. As a corollary, it has been suggested that selection should favor recombination at low fitness and not at high fitness (fitness-associated recombination, FAR), and theory suggests that such strategies can indeed be selected. Here we develop models to further investigate the evolution of FAR. Consistent with previous works, we find that FAR can invade and dominate over a strategy of uniform recombination that is independent of fitness. However, our simulation results suggest that extreme FAR strategies, known as group-elitism, are not necessarily superior to other FAR strategies. Moreover, we argue that FAR domination will often occur with a net loss of mean population fitness. Interestingly, this suggests that the strategy of not recombining at high fitness will sometimes be analogous to a defector strategy from the famous "prisoner's dilemma" game: a selfish strategy that is selected but leads to a loss of mean fitness for all players.

Recombinant Allergens in Structural Biology, Diagnosis, and Immunotherapy

PubMed Central

Tscheppe, Angelika; Breiteneder, Heimo

2017-01-01

The years 1988–1995 witnessed the beginning of allergen cloning and the generation of recombinant allergens, which opened up new avenues for the diagnosis and research of human allergic diseases. Most crystal and solution structures of allergens have been obtained using recombinant allergens. Structural information on allergens allows insights into their evolutionary biology, illustrates clinically observed cross-reactivities, and makes the design of hypoallergenic derivatives for allergy vaccines possible. Recombinant allergens are widely used in molecule-based allergy diagnosis such as protein microarrays or suspension arrays. Recombinant technologies have been used to produce well-characterized, noncontaminated vaccine components with known biological activities including a variety of allergen derivatives with reduced IgE reactivity. Such recombinant hypoallergens as well as wild-type recombinant allergens have been used successfully in several immunotherapy trials for more than a decade to treat birch and grass pollen allergy. As a more recent application, the development of antibody repertoires directed against conformational epitopes during immunotherapy has been monitored by recombinant allergen chimeras. Although much progress has been made, the number and quality of recombinant allergens will undoubtedly increase and keep improving our knowledge in basic scientific investigations, diagnosis, and therapy of human allergic diseases. PMID:28467993

Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

PubMed Central

Thor, Sharmi W.; Hilt, Deborah A.; Kissinger, Jessica C.; Paterson, Andrew H.; Jackwood, Mark W.

2011-01-01

Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV) isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus. PMID:21994806

Selfish genes, pleiotropy and the origin of recombination.

PubMed Central

Hey, J

1998-01-01

If multiple linked polymorphisms are under natural selection, then conflicts arise and the efficiency of natural selection is hindered relative to the case of no linkage. This simple interaction between linkage and natural selection creates an opportunity for mutations that raise the level of recombination to increase in frequency and have an enhanced chance of fixation. This important finding by S. Otto and N. Barton means that mutations that raise the recombination rate, but are otherwise neutral, will be selectively favored under fairly general circumstances of multilocus selection and linkage. The effect described by Otto and Barton, which was limited to neutral modifiers, can also be extended to include all modifiers of recombination, both beneficial and deleterious. Computer simulations show that beneficial mutations that also increase recombination have an increased chance of fixation. Similarly, deleterious mutations that also decrease recombination have an increased chance of fixation. The results suggest that a simple model of recombination modifiers, including both neutral and pleiotropic modifiers, is a necessary explanation for the evolutionary origin of recombination. PMID:9691060

Hepatitis B vaccine booster dose: low-dose recombinant hepatitis B vaccines as a booster dose.

PubMed

Bryan, J P; MacArthy, P; Rudock, A; Fogarty, J P; Dowd, H; Legters, L J; Perine, P L

1997-06-01

The timing and best regimen for a booster dose of hepatitis B vaccine have not been determined. Two studies were conducted to determine the response to a booster dose of 5 micrograms recombinant hepatitis B vaccine. In the first study, a 5 micrograms (0.5 ml) dose of Recombivax HB was administered intramuscularly 38 months after the initial dose to 71 volunteers. In a second study, we offered a 5 micrograms dose recombinant hepatitis B vaccine, either Recombivax HB (0.5 ml) or Engerix B (0.25 ml), to students who had previously been immunized with three doses of vaccine. In the first study, among the 44 persons for whom postbooster sera were available, the geometric mean concentration of anti-hepatitis B surface antigens increased from 42 to 2090 mIU/ml after the 5 micrograms (0.5 ml) dose of Recombivax. In the second study, after a 5 micrograms (0.5 ml) dose of Recombivax, the geometric mean concentration increased from 43 to 990 mIU/ml (n = 48), and in the group that received a 5 micrograms (0.25 ml) dose of Engerix B, the concentration increased from 83 to 2337 mIU/ml (n = 45) (p = 0.18 for postdose concentrations). A 5 micrograms dose of recombinant vaccine results in an excellent booster response at a cost one fourth to one half that of a full 1 ml dose of vaccine.

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

PubMed

Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

2004-10-27

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

Phylogenetic study of recombinant strains of Potato virus Y

USDA-ARS?s Scientific Manuscript database

Potato virus Y (PVY) exists as a complex of strains, including a growing number of recombinants. Evolution of PVY proceeds through accumulation of mutations and more rapidly through recombination. Here, the role of recombination in PVY evolution and the origin of common PVY recombinants were studied...

DNA recombination activity in soybean mitochondria.

PubMed

Manchekar, Medha; Scissum-Gunn, Karyn; Song, Daqing; Khazi, Fayaz; McLean, Stephanie L; Nielsen, Brent L

2006-02-17

Mitochondrial genomes in higher plants are much larger and more complex as compared to animal mitochondrial genomes. There is growing evidence that plant mitochondrial genomes exist predominantly as a collection of linear and highly branched DNA molecules and replicate by a recombination-dependent mechanism. However, biochemical evidence of mitochondrial DNA (mtDNA) recombination activity in plants has previously been lacking. We provide the first report of strand-invasion activity in plant mitochondria. Similar to bacterial RecA, this activity from soybean is dependent on the presence of ATP and Mg(2+). Western blot analysis using an antibody against the Arabidopsis mitochondrial RecA protein shows cross-reaction with a soybean protein of about 44 kDa, indicating conservation of this protein in at least these two plant species. mtDNA structure was analyzed by electron microscopy of total soybean mtDNA and molecules recovered after field-inversion gel electrophoresis (FIGE). While most molecules were found to be linear, some molecules contained highly branched DNA structures and a small but reproducible proportion consisted of circular molecules (many with tails) similar to recombination intermediates. The presence of recombination intermediates in plant mitochondria preparations is further supported by analysis of mtDNA molecules by 2-D agarose gel electrophoresis, which indicated the presence of complex recombination structures along with a considerable amount of single-stranded DNA. These data collectively provide convincing evidence for the occurrence of homologous DNA recombination in plant mitochondria.

Anti-Inflammatory Thioredoxin Family Proteins for Medicare, Healthcare and Aging Care

PubMed Central

Matsuo, Yoshiyuki; Tian, Hai; Masutani, Hiroshi; Inamoto, Takashi

2017-01-01

Human thioredoxin (TRX) is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys-, which is induced by biological stress due to oxidative damage, metabolic dysfunction, chemicals, infection/inflammation, irradiation, or hypoxia/ischemia-reperfusion. Our research has demonstrated that exogenous TRX is effective in a wide variety of inflammatory diseases, including viral pneumonia, acute lung injury, gastric injury, and dermatitis, as well as in the prevention and amelioration of food allergies. Preclinical and clinical studies using recombinant TRX (rhTRX) are now underway. We have also identified substances that induce the expression of TRX in the body, in vegetables and other plant ingredients. Skincare products are being developed that take advantage of the anti-inflammatory and anti-allergic action of TRX. Furthermore, we are currently engaged in the highly efficient production of pure rhTRX in several plants, such as lettuce, grain and rice. PMID:28961169

Mechanisms and Factors that Influence High Frequency Retroviral Recombination

PubMed Central

Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga; Mens, Helene; Pathak, Vinay K.; Hu, Wei-Shau

2011-01-01

With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development. PMID:21994801

mtDNA recombination in a natural population.

PubMed

Saville, B J; Kohli, Y; Anderson, J B

1998-02-03

Variation in mtDNA has been used extensively to draw inferences in phylogenetics and population biology. In the majority of eukaryotes investigated, transmission of mtDNA is uniparental and clonal, with genotypic diversity arising from mutation alone. In other eukaryotes, the transmission of mtDNA is biparental or primarily uniparental with the possibility of "leakage" from the minority parent. In these cases, heteroplasmy carries the potential for recombination between mtDNAs of different descent. In fungi, such mtDNA recombination has long been documented but only in laboratory experiments and only under conditions in which heteroplasmy is ensured. Despite this experimental evidence, mtDNA recombination has not been to our knowledge documented in a natural population. Because evidence from natural populations is prerequisite to understanding the evolutionary impact of mtDNA recombination, we investigated the possibility of mtDNA recombination in an organism with the demonstrated potential for heteroplasmy in laboratory matings. Using nucleotide sequence data, we report here that the genotypic structure of mtDNA in a natural population of the basidiomycete fungus Armillaria gallica is inconsistent with purely clonal mtDNA evolution and is fully consistent with mtDNA recombination.

Recombinant gp19 as a potential antigen for detecting anti-Ehrlichia canis antibodies in dog sera.

PubMed

Oliveira, Rômulo Silva de; Cunha, Rodrigo Casquero; Moraes-Filho, Jonas; Gonçales, Relber Aguiar; Lara, Ana Paula de Souza Stori de; Avila, Luciana Farias da Costa de; Labruna, Marcelo Bahia; Leite, Fábio Pereira Leivas

2015-01-01

The canine monocytic ehrlichiosis, caused by Ehrlichia canis, is endemic in several regions of Brazil. Some serological diagnostic techniques using immunodominant proteins of E. canis as antigens are available, but their specificities and sensitivities are questionable. Based on this, the objective of this study was to test the antigenic potential of the recombinant gp19 protein (rGP19) for subsequent use in diagnostic tests. The rGP19 expressed in the Escherichia coli strain BL21 (DE3) C41 was recognized in the sera from experimentally infected dogs using ELISA and Western blotting. Thus, it was possible to obtain a promising antigen with the ability to differentiate between E. canis-positive and -negative animals, even 1 week after infection.

Genome-wide variation in recombination rate in Eucalyptus.

PubMed

Gion, Jean-Marc; Hudson, Corey J; Lesur, Isabelle; Vaillancourt, René E; Potts, Brad M; Freeman, Jules S

2016-08-09

Meiotic recombination is a fundamental evolutionary process. It not only generates diversity, but influences the efficacy of natural selection and genome evolution. There can be significant heterogeneity in recombination rates within and between species, however this variation is not well understood outside of a few model taxa, particularly in forest trees. Eucalypts are forest trees of global economic importance, and dominate many Australian ecosystems. We studied recombination rate in Eucalyptus globulus using genetic linkage maps constructed in 10 unrelated individuals, and markers anchored to the Eucalyptus reference genome. This experimental design provided the replication to study whether recombination rate varied between individuals and chromosomes, and allowed us to study the genomic attributes and population genetic parameters correlated with this variation. Recombination rate varied significantly between individuals (range = 2.71 to 3.51 centimorgans/megabase [cM/Mb]), but was not significantly influenced by sex or cross type (F1 vs. F2). Significant differences in recombination rate between chromosomes were also evident (range = 1.98 to 3.81 cM/Mb), beyond those which were due to variation in chromosome size. Variation in chromosomal recombination rate was significantly correlated with gene density (r = 0.94), GC content (r = 0.90), and the number of tandem duplicated genes (r = -0.72) per chromosome. Notably, chromosome level recombination rate was also negatively correlated with the average genetic diversity across six species from an independent set of samples (r = -0.75). The correlations with genomic attributes are consistent with findings in other taxa, however, the direction of the correlation between diversity and recombination rate is opposite to that commonly observed. We argue this is likely to reflect the interaction of selection and specific genome architecture of Eucalyptus. Interestingly, the differences amongst

Ion recombination correction in carbon ion beams.

PubMed

Rossomme, S; Hopfgartner, J; Lee, N D; Delor, A; Thomas, R A S; Romano, F; Fukumura, A; Vynckier, S; Palmans, H

2016-07-01

In this work, ion recombination is studied as a function of energy and depth in carbon ion beams. Measurements were performed in three different passively scattered carbon ion beams with energies of 62 MeV/n, 135 MeV/n, and 290 MeV/n using various types of plane-parallel ionization chambers. Experimental results were compared with two analytical models for initial recombination. One model is generally used for photon beams and the other model, developed by Jaffé, takes into account the ionization density along the ion track. An investigation was carried out to ascertain the effect on the ion recombination correction with varying ionization chamber orientation with respect to the direction of the ion tracks. The variation of the ion recombination correction factors as a function of depth was studied for a Markus ionization chamber in the 62 MeV/n nonmodulated carbon ion beam. This variation can be related to the depth distribution of linear energy transfer. Results show that the theory for photon beams is not applicable to carbon ion beams. On the other hand, by optimizing the value of the ionization density and the initial mean-square radius, good agreement is found between Jaffé's theory and the experimental results. As predicted by Jaffé's theory, the results confirm that ion recombination corrections strongly decrease with an increasing angle between the ion tracks and the electric field lines. For the Markus ionization chamber, the variation of the ion recombination correction factor with depth was modeled adequately by a sigmoid function, which is approximately constant in the plateau and strongly increasing in the Bragg peak region to values of up to 1.06. Except in the distal edge region, all experimental results are accurately described by Jaffé's theory. Experimental results confirm that ion recombination in the investigated carbon ion beams is dominated by initial recombination. Ion recombination corrections are found to be significant and cannot be

Metal binding proteins, recombinant host cells and methods

DOEpatents

Summers, Anne O.; Caguiat, Jonathan J.

2004-06-15

The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice

PubMed Central

Bagherpour, Ghasem; Ghasemi, Hosnie; Zand, Bahare; Zarei, Najmeh; Roohvand, Farzin; Ardakani, Esmat M.; Azizi, Mohammad; Khalaj, Vahid

2018-01-01

Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins. PMID

Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice.

PubMed

Bagherpour, Ghasem; Ghasemi, Hosnie; Zand, Bahare; Zarei, Najmeh; Roohvand, Farzin; Ardakani, Esmat M; Azizi, Mohammad; Khalaj, Vahid

2018-01-01

Saccharomyces boulardii , a subspecies of Saccharomyces cerevisiae , is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi ® ) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3 - S. boulardii . To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group ( P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group ( P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii , as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.

Construction and characterization of a recombinant invertebrate iridovirus.

PubMed

Ozgen, Arzu; Muratoglu, Hacer; Demirbag, Zihni; Vlak, Just M; van Oers, Monique M; Nalcacioglu, Remziye

2014-08-30

Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-Δ157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol. Copyright © 2014 Elsevier B.V. All rights reserved.

Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

PubMed Central

Kempf, Brian J.; Peersen, Olve B.

2016-01-01

ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a

Genome-wide recombination dynamics are associated with phenotypic variation in maize.

PubMed

Pan, Qingchun; Li, Lin; Yang, Xiaohong; Tong, Hao; Xu, Shutu; Li, Zhigang; Li, Weiya; Muehlbauer, Gary J; Li, Jiansheng; Yan, Jianbing

2016-05-01

Meiotic recombination is a major driver of genetic diversity, species evolution, and agricultural improvement. Thus, an understanding of the genetic recombination landscape across the maize (Zea mays) genome will provide insight and tools for further study of maize evolution and improvement. Here, we used c. 50 000 single nucleotide polymorphisms to precisely map recombination events in 12 artificial maize segregating populations. We observed substantial variation in the recombination frequency and distribution along the ten maize chromosomes among the 12 populations and identified 143 recombination hot regions. Recombination breakpoints were partitioned into intragenic and intergenic events. Interestingly, an increase in the number of genes containing recombination events was accompanied by a decrease in the number of recombination events per gene. This kept the overall number of intragenic recombination events nearly invariable in a given population, suggesting that the recombination variation observed among populations was largely attributed to intergenic recombination. However, significant associations between intragenic recombination events and variation in gene expression and agronomic traits were observed, suggesting potential roles for intragenic recombination in plant phenotypic diversity. Our results provide a comprehensive view of the maize recombination landscape, and show an association between recombination, gene expression and phenotypic variation, which may enhance crop genetic improvement. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Selection of an anti-solvent for efficient and stable cesium-containing triple cation planar perovskite solar cells.

PubMed

Xiao, Meng; Zhao, Li; Geng, Min; Li, Yanyan; Dong, Binghai; Xu, Zuxun; Wan, Li; Li, Wenlu; Wang, Shimin

2018-06-19

The perovskite layer is a crucial component influencing high-performance perovskite solar cells (PSCs). In the one-step solution method, anti-solvents are important for obtaining smooth and uniform perovskite active layers. This work explored the effect of various anti-solvents on the preparation of triple cation perovskite active layers. In general, anti-solvents with low dielectric constants, low polarity, and low boiling point are suitable for the preparation of perovskite films. Microstructural and elemental analyses of the perovskite films were systematically conducted by scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy. The photoelectric properties, carrier transfer, and recombination process in the PSCs were investigated using photocurrent-voltage characteristic curves and electrochemical impedance spectroscopy. Optimum performance was obtained when the anti-solvent was diethyl ether (DEE) and the ratio of the optimum amount of DEE to the volume of the precursor was 1 : 10. Meanwhile, we found that the partial replacement of formamidinium/methylammonium by cesium could increase the stability of the PSCs and enhance the power conversion efficiency from 15.49% to over 17.38%.

Temporally-Controlled Site-Specific Recombination in Zebrafish

PubMed Central

Hans, Stefan; Kaslin, Jan; Freudenreich, Dorian; Brand, Michael

2009-01-01

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms. PMID:19247481

Algae-based oral recombinant vaccines

PubMed Central

Specht, Elizabeth A.; Mayfield, Stephen P.

2014-01-01

Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for “molecular pharming” in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae could be poised to become the next candidate in recombinant subunit vaccine production, as they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and systemic immune reactivity. PMID:24596570

Recombinant Incretin-Secreting Microbe Improves Metabolic Dysfunction in High-Fat Diet Fed Rodents.

PubMed

Ryan, Paul M; Patterson, Elaine; Kent, Robert M; Stack, Helena; O'Connor, Paula M; Murphy, Kiera; Peterson, Veronica L; Mandal, Rupasri; Wishart, David S; Dinan, Timothy G; Cryan, John F; Seeley, Randy J; Stanton, Catherine; Ross, R Paul

2017-10-19

The gut hormone glucagon-like peptide (GLP)-1 and its analogues represent a new generation of anti-diabetic drugs, which have also demonstrated propensity to modulate host lipid metabolism. Despite this, drugs of this nature are currently limited to intramuscular administration routes due to intestinal degradation. The aim of this study was to design a recombinant microbial delivery vector for a GLP-1 analogue and assess the efficacy of the therapeutic in improving host glucose, lipid and cholesterol metabolism in diet induced obese rodents. Diet-induced obese animals received either Lactobacillus paracasei NFBC 338 transformed to express a long-acting analogue of GLP-1 or the isogenic control microbe which solely harbored the pNZ44 plasmid. Short-term GLP-1 microbe intervention in rats reduced serum low-density lipoprotein cholesterol, triglycerides and triglyceride-rich lipoprotein cholesterol substantially. Conversely, extended GLP-1 microbe intervention improved glucose-dependent insulin secretion, glucose metabolism and cholesterol metabolism, compared to the high-fat control group. Interestingly, the microbe significantly attenuated the adiposity associated with the model and altered the serum lipidome, independently of GLP-1 secretion. These data indicate that recombinant incretin-secreting microbes may offer a novel and safe means of managing cholesterol metabolism and diet induced dyslipidaemia, as well as insulin sensitivity in metabolic dysfunction.

Construction and characterization of human rotavirus recombinant VP8* subunit parenteral vaccine candidates.

PubMed

Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka

2012-09-21

Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines. Published by Elsevier Ltd.

Construction and Characterization of Human Rotavirus Recombinant VP8* Subunit Parenteral Vaccine Candidates

PubMed Central

Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W.; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka

2012-01-01

Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in E. coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., (P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines. PMID:22885016

Analysis of the Functions of Recombination-Related Genes in the Generation of Large Chromosomal Deletions by Loop-Out Recombination in Aspergillus oryzae

PubMed Central

Ogawa, Masahiro; Koyama, Yasuji

2012-01-01

Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70, ligD, rad52, rad54, and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae. The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δku70 and Δku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the ΔligD, Δku70-rad52, and Δku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD, rad52, and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure. PMID:22286092

A broad survey of recombination in animal mitochondria.

PubMed

Piganeau, Gwenaël; Gardner, Michael; Eyre-Walker, Adam

2004-12-01

Recombination in mitochondrial DNA (mtDNA) remains a controversial topic. Here we present a survey of 279 animal mtDNA data sets, of which 12 were from asexual species. Using four separate tests, we show that there is widespread evidence of recombination; for one test as many as 14.2% of the data sets reject a model of clonal inheritance and in several data sets, including primates, the recombinants can be identified visually. We show that none of the tests give significant results for obligate clonal species (apomictic pathogens) and that the sexual species show significantly greater evidence of recombination than asexual species. For some data sets, such as Macaca nemestrina, additional data sets suggest that the recombinants are not artifacts. For others, it cannot be determined whether the recombinants are real or produced by laboratory error. Either way, the results have important implications for how mtDNA is sequenced and used.

Bimolecular recombination quenching in Langmuir Blodgett multilayers

NASA Astrophysics Data System (ADS)

Elliott, J. E.; Jeong, I. S.; Scott, K.; Donovan, K. J.; Wilson, E. G.

2000-11-01

A model is developed that describes bimolecular recombination of photogenerated carriers in two dimensional systems. Carriers are free to diffuse in two dimensions and undergo bimolecular recombination, while drifting under the influence of an electric field in the third dimension. The model describes a competition between carrier loss due to transiting and loss due to bimolecular recombination. This model of recombination quenching is then used to obtain information on microscopic parameters associated with photogeneration efficiency and charge transport in organic quantum wells formed from Langmuir Blodgett films of conjugated molecules. The ratio of the intralayer to interlayer tunneling rates is found along with the quantum efficiency for photocarrier generation for two bis-phthalocyanine amphiphilic molecules.

Passive transfer of growth-inhibitory antibodies raised against yeast-expressed recombinant Plasmodium falciparum merozoite surface protein-1(19).

PubMed

Gozalo, A; Lucas, C; Cachay, M; Wellde, B T; Hall, T; Bell, B; Wood, J; Watts, D; Wooster, M; Lyon, J A; Moch, J K; Haynes, J D; Williams, J S; Holland, C; Watson, E; Kester, K E; Kaslow, D C; Ballou, W R

1998-12-01

Purified rabbit immunoglobulin raised against yeast-expressed recombinant FVO or 3D7 Plasmodium falciparum merozoite surface protein-1 (MSP-1) 19k-D C terminal fragment (MSP-1(19)) was transfused into malaria-naive Aotus nancymai monkeys that were immediately challenged with FVO asexual stage malaria parasites. Control monkeys received rabbit immunoglobulin raised against the sexual stage antigen Pfs25 or Aotus hyperimmune serum obtained from monkeys immunized by P. falciparum infection and drug cure. Passive transfer of rabbit anti-MSP-1(19) failed to protect against homologous or heterologous challenge and, when compared with negative controls, there were no differences in prepatent periods or time to treatment. Interestingly, rabbit anti-MSP-1(19), but not anti-Pfs25, immunoglobulin, and immune monkey serum prevented the development of antibodies directed against MSP-1(19) fragment by infected monkeys, indicating that the antibodies were reactive with native MSP-1(19) antigen in vivo. The prepatent period and time to treatment was greatly delayed in the two monkeys that received Aotus immune serum, both of which developed a chronic intermittent low level infection. In vitro parasite growth inhibition assays (GIAs) confirmed the presence of inhibitory activity (40% maximum inhibition) in concentrated anti-MSP-1(19) immunoglobulin (4.8 mg/ml), but the peak concentrations we achieved in vivo (1 mg/ml) were not inhibitory in vitro. Subinhibitory levels of anti-MSP-1(19) antibodies achieved by passive transfer were not protective against P. falciparum challenge.

[Vaccine application of recombinant herpesviruses].

PubMed

Yokoyama, N; Xuan, X; Mikami, T

2000-04-01

Recently, genetic engineering using recombinant DNA techniques has been applied to design new viral vaccines in order to reduce some problems which the present viral vaccines have. Up to now, many viruses have been investigated for development of recombinant attenuated vaccines or live viral vectors for delivery of foreign genes coding immunogenic antigens. In this article, we introduced the new vaccine strategy using genetically engineered herpesviruses.

Dissociative Recombination without a Curve Crossing

NASA Technical Reports Server (NTRS)

Guberman, Steven L.

1994-01-01

Ab initio calculations show that a curve crossing is not always needed for a high dissociative- recombination cross section. For HeH(+), in which no neutral states cross the ion potential curve, dissociative recombination is driven by the nuclear kinetic-energy operator on adiabatic potential curves. The kinetic-energy derivative operator allows for capture into repulsive curves that are outside of the classical turning points for the nuclear motion. The dominant dissociative route is the C (2)Sigma(+) state leading to H(n = 2) atoms. An analogous mechanism is proposed for the dissociative recombination of H3(+).

Evidence of reduced recombination rate in human regulatory domains.

PubMed

Liu, Yaping; Sarkar, Abhishek; Kheradpour, Pouya; Ernst, Jason; Kellis, Manolis

2017-10-20

Recombination rate is non-uniformly distributed across the human genome. The variation of recombination rate at both fine and large scales cannot be fully explained by DNA sequences alone. Epigenetic factors, particularly DNA methylation, have recently been proposed to influence the variation in recombination rate. We study the relationship between recombination rate and gene regulatory domains, defined by a gene and its linked control elements. We define these links using expression quantitative trait loci (eQTLs), methylation quantitative trait loci (meQTLs), chromatin conformation from publicly available datasets (Hi-C and ChIA-PET), and correlated activity links that we infer across cell types. Each link type shows a "recombination rate valley" of significantly reduced recombination rate compared to matched control regions. This recombination rate valley is most pronounced for gene regulatory domains of early embryonic development genes, housekeeping genes, and constitutive regulatory elements, which are known to show increased evolutionary constraint across species. Recombination rate valleys show increased DNA methylation, reduced doublestranded break initiation, and increased repair efficiency, specifically in the lineage leading to the germ line. Moreover, by using only the overlap of functional links and DNA methylation in germ cells, we are able to predict the recombination rate with high accuracy. Our results suggest the existence of a recombination rate valley at regulatory domains and provide a potential molecular mechanism to interpret the interplay between genetic and epigenetic variations.

Dissociation of recombinant prion autocatalysis from infectivity.

PubMed

Noble, Geoffrey P; Supattapone, Surachai

2015-01-01

Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication--that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain. (1) We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor. (1) In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.

Recombination rate predicts inversion size in Diptera.

PubMed Central

Cáceres, M; Barbadilla, A; Ruiz, A

1999-01-01

Most species of the Drosophila genus and other Diptera are polymorphic for paracentric inversions. A common observation is that successful inversions are of intermediate size. We test here the hypothesis that the selected property is the recombination length of inversions, not their physical length. If so, physical length of successful inversions should be negatively correlated with recombination rate across species. This prediction was tested by a comprehensive statistical analysis of inversion size and recombination map length in 12 Diptera species for which appropriate data are available. We found that (1) there is a wide variation in recombination map length among species; (2) physical length of successful inversions varies greatly among species and is inversely correlated with the species recombination map length; and (3) neither the among-species variation in inversion length nor the correlation are observed in unsuccessful inversions. The clear differences between successful and unsuccessful inversions point to natural selection as the most likely explanation for our results. Presumably the selective advantage of an inversion increases with its length, but so does its detrimental effect on fertility due to double crossovers. Our analysis provides the strongest and most extensive evidence in favor of the notion that the adaptive value of inversions stems from their effect on recombination. PMID:10471710

RNA Recombination In Vivo in the Absence of Viral Replication

PubMed Central

Gallei, Andreas; Pankraz, Alexander; Thiel, Heinz-Jürgen; Becher, Paul

2004-01-01

To study fundamental aspects of RNA recombination, an in vivo RNA recombination system was established. This system allowed the efficient generation of recombinant cytopathogenic pestiviruses after transfection of synthetic, nonreplicatable, subgenomic transcripts in cells infected with a replicating noncytopathogenic virus. Studies addressing the interplay between RNA recombination and replication revealed that cotransfection of noninfected cells with various pairs of nonreplicatable RNA derivatives also led to the emergence of recombinant viral genomes. Remarkably, homologous and nonhomologous recombination occurred between two overlapping transcripts, each lacking different essential parts of the viral RNA-dependent RNA polymerase (RdRp) gene. Apart from the generally accepted viral replicative copy choice recombination, our results prove the existence of a viral RdRp-independent mechanism of RNA recombination that occurs in vivo. It appears likely that such a mechanism not only contributes to the evolution of RNA viruses but also leads to the generation of recombinant cellular RNAs. PMID:15163720

Evaluation and characterization of anti-estrogenic and anti-androgenic activities in soil samples along the Second Songhua River, China.

PubMed

Li, Jian; Wang, Yafei; Kong, Dongdong; Wang, Jinsheng; Teng, Yanguo; Li, Na

2015-11-01

In the present study, re-combined estrogen receptor (ER) and androgen receptor (AR) gene yeast assays combined with a novel approach based on Monte Carlo simulation were used for evaluation and characterization of soil samples collected from Jilin along the Second Songhua River to assess their antagonist/agonist properties for ER and AR. The results showed that estrogenic activity only occurred in the soil samples collected in the agriculture area, but most soil samples showed anti-estrogenic activities, and the bioassay-derived 4-hydroxytamoxifen equivalents ranged from N.D. to 23.51 μg/g. Hydrophilic substance fractions were determined as potential contributors associated with anti-estrogenic activity in these soil samples. Moreover, none of the soil samples exhibited AR agonistic potency, whereas 54% of the soil samples exhibited AR antagonistic potency. The flutamide equivalents varied between N.D. and 178.05 μg/g. Based on Monte Carlo simulation-related mass balance analysis, the AR antagonistic activities were significantly correlated with the media polar and polar fractions. All of these results support that this novel calculation method can be adopted effectively to quantify and characterize the ER/AR agonists and antagonists of the soil samples, and these data could help provide useful information for future management and remediation efforts.

Host Immunization with Recombinant Proteins to Screen Antigens for Tick Control.

PubMed

Galay, Remil Linggatong; Miyata, Takeshi; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

2016-01-01

Ticks (Parasitiformes: Ixodida) are known for their obligate blood feeding habit and their role in transmitting pathogens to various vertebrate hosts. Tick control using chemical acaricides is extensively used particularly in livestock management, but several disadvantages arise from resistance development of many tick species, and concerns on animal product and environmental contamination. Vaccination offers better protection and more cost-effective alternative to application of chemical acaricides, addressing their disadvantages. However, an ideal anti-tick vaccine targeting multiple tick species and all the tick stages is still wanting. Here, we describe the procedures involved in the evaluation of a vaccine candidate antigen against ticks at the laboratory level, from the preparation of recombinant proteins, administration to the rabbit host and monitoring of antibody titer, to tick infestation challenge and determination of the effects of immunization to ticks.

Theoretical studies of dissociative recombination

NASA Technical Reports Server (NTRS)

Guberman, S. L.

1985-01-01

The calculation of dissociative recombination rates and cross sections over a wide temperature range by theoretical quantum chemical techniques is described. Model calculations on electron capture by diatomic ions are reported which illustrate the dependence of the rates and cross sections on electron energy, electron temperature, and vibrational temperature for three model crossings of neutral and ionic potential curves. It is shown that cross sections for recombination to the lowest vibrational level of the ion can vary by several orders of magnitude depending upon the position of the neutral and ionic potential curve crossing within the turning points of the v = 1 vibrational level. A new approach for calculating electron capture widths is reported. Ab initio calculations are described for recombination of O2(+) leading to excited O atoms.

Recombinant DNA production of spider silk proteins

PubMed Central

Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

2013-01-01

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

Risk-managed production of bioactive recombinant proteins using a novel plant virus vector with a helper plant to complement viral systemic movement.

PubMed

Fukuzawa, Noriho; Ishihara, Takeaki; Itchoda, Noriko; Tabayashi, Noriko; Kataoka, Chiwa; Masuta, Chikara; Matsumura, Takeshi

2011-01-01

A plant viral vector has the potential to efficiently produce recombinant proteins at a low cost in a short period. Although recombinant proteins can be also produced by transgenic plants, a plant viral vector, if available, may be more convenient when urgent scale-up in production is needed. However, it is difficult to use a viral vector in open fields because of the risk of escape to the environment. In this study, we constructed a novel viral vector system using a movement-defective Cucumber mosaic virus (CMV) vector, which is theoretically localized in the inoculated cells but infects systemically only with the aid of the transgenic helper plant that complements viral movement, diminishing the risk of viral proliferation. Interestingly, the helper plant systemically infected with the vector gave strong cross-protection against challenge inoculation with wild-type CMVs. Using CMV strains belonging to two discrete CMV groups (subgroups I and II), we also improved the system to prevent recombination between the vector and the transgene transcript in the helper plant. We here demonstrate the expression of an anti-dioxin single chain variable fragment (DxscFv) and interleukin-1 receptor antagonist (IL1-Ra) in Nicotiana benthamiana by this viral vector confinement system, which is applicable for many useful high-quality recombinant proteins. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd.

Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

NASA Astrophysics Data System (ADS)

Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

1995-02-01

The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

CATALYTIC RECOMBINER FOR A NUCLEAR REACTOR

DOEpatents

King, L.D.P.

1960-07-01

A hydrogen-oxygen recombiner is described for use with water-boiler type reactors. The catalyst used is the wellknown platinized alumina, and the novelty lies in the structural arrangement used to prevent flashback through the gas input system. The recombiner is cylindrical, the gases at the input end being deflected by a baffle plate through a first flashback shield of steel shot into an annular passage adjacent to and extending the full length of the housing. Below the baffle plate the gases flow first through an outer annular array of alumina pellets which serve as a second flashback shield, a means of distributing the flowing gases evenly and as a means of reducing radiation losses to the walls. Thereafter the gases flow inio the centrally disposed catalyst bed where recombination is effected. The steam and uncombined gases flow into a centrally disposed cylindrical passage inside the catalyst bod and thereafter out through the exit port. A high rate of recombination is effected.

A new subtype (subgenotype) Ac (A3) of hepatitis B virus and recombination between genotypes A and E in Cameroon.

PubMed

Kurbanov, Fuat; Tanaka, Yasuhito; Fujiwara, Kei; Sugauchi, Fuminaka; Mbanya, Dora; Zekeng, Leopold; Ndembi, Nicaise; Ngansop, Charlotte; Kaptue, Lazare; Miura, Tomoyuki; Ido, Eiji; Hayami, Masanori; Ichimura, Hiroshi; Mizokami, Masashi

2005-07-01

Blood samples (n=544) from two different populations (Pygmies and Bantus) in Cameroon, West Africa, were analysed. Serological tests indicated that the anti-hepatitis C virus (HCV) prevalence in Bantus (20.3 %) was higher than that in Pygmies (2.3 %, P<0.0001), whereas the distribution of hepatitis B virus (HBV) serological markers was equally high in both populations: in total, 9.4, 17.3 and 86.8 % for HBsAg, anti-HBs and anti-HBc, respectively. HBV genotype A (HBV/A) and HBV/E were predominant (43.5 % each) in both populations, and HBV/D was found in a minority (13 %). The preS/S region was sequenced in nine cases (five HBV/A and four HBV/E) and the complete genome in six cases (four HBV/A and two HBV/E). Subsequent phylogenetic analysis revealed that the HBV/A strains were distinct from the subtypes (subgenotypes) described previously, Ae (A2) and Aa (A1), and in the preS/S region they clustered with previously reported sequences from Cameroon. Based on the nucleotide difference from Aa (A1) and Ae (A2), more than 4 % in the complete genome, the Cameroonian strains were suggested to represent a new subtype (subgenotype), designated HBV/Ac (A3). A high (3.9 %) nucleotide divergence in HBV/Ac (A3) strains suggested that the subtype (subgenotype) has a long natural history in the population of Cameroon. One of the HBV/Ac (A3) strains was found to be a recombinant with an HBV/E-specific sequence in the polymerase reverse transcriptase domain. Further cohort studies will be required to assess detailed epidemiological, virological and clinical characteristics of HBV/Ac (A3), as well as its recombinant form.

Same-period emission and recombination in nonsequential double-recombination high-order-harmonic generation

NASA Astrophysics Data System (ADS)

Hansen, Kenneth K.; Madsen, Lars Bojer

2016-05-01

Nonsequential double-recombination (NSDR) high-order-harmonic generation (HHG) is studied in a molecular model system. We observe a unique molecular two-electron effect with a characteristic cutoff in the HHG spectrum at higher energies than what was previously seen for NSDR HHG in atoms. The effect is corroborated with a classical model where it is found that the effect is sensitive to the molecular potential and originates from same-period emission and recombination (SPEAR) of two electrons. The effect persists for intermediate nuclear distances of R ≳8.0 a.u.

Site directed recombination

DOEpatents

Jurka, Jerzy W.

1997-01-01

Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

Recombination rate variation in mice from an isolated island.

PubMed

Wang, Richard J; Gray, Melissa M; Parmenter, Michelle D; Broman, Karl W; Payseur, Bret A

2017-01-01

Recombination rate is a heritable trait that varies among individuals. Despite the major impact of recombination rate on patterns of genetic diversity and the efficacy of selection, natural variation in this phenotype remains poorly characterized. We present a comparison of genetic maps, sampling 1212 meioses, from a unique population of wild house mice (Mus musculus domesticus) that recently colonized remote Gough Island. Crosses to a mainland reference strain (WSB/EiJ) reveal pervasive variation in recombination rate among Gough Island mice, including subchromosomal intervals spanning up to 28% of the genome. In spite of this high level of polymorphism, the genomewide recombination rate does not significantly vary. In general, we find that recombination rate varies more when measured in smaller genomic intervals. Using the current standard genetic map of the laboratory mouse to polarize intervals with divergent recombination rates, we infer that the majority of evolutionary change occurred in one of the two tested lines of Gough Island mice. Our results confirm that natural populations harbour a high level of recombination rate polymorphism and highlight the disparities in recombination rate evolution across genomic scales. © 2016 John Wiley & Sons Ltd.

Recombination rate variation in mice from an isolated island

PubMed Central

Wang, Richard J.; Gray, Melissa M.; Parmenter, Michelle D.; Broman, Karl W.; Payseur, Bret A.

2016-01-01

Recombination rate is a heritable trait that varies among individuals. Despite the major impact of recombination rate on patterns of genetic diversity and the efficacy of selection, natural variation in this phenotype remains poorly characterized. We present a comparison of genetic maps, sampling 1,212 meioses, from a unique population of wild house mice (Mus musculus domesticus) that recently colonized remote Gough Island. Crosses to a mainland reference strain (WSB/EiJ) reveal pervasive variation in recombination rate among Gough Island mice, including sub-chromosomal intervals spanning up to 28% of the genome. In spite of this high level of polymorphism, the genome-wide recombination rate does not significantly vary. In general, we find that recombination rate varies more when measured in smaller genomic intervals. Using the current standard genetic map of the laboratory mouse to polarize intervals with divergent recombination rates, we infer that the majority of evolutionary change occurred in one of the two tested lines of Gough Island mice. Our results confirm that natural populations harbor a high level of recombination rate polymorphism and highlight the disparities in recombination rate evolution across genomic scales. PMID:27864900

G-CSF/anti-G-CSF antibody complexes drive the potent recovery and expansion of CD11b+Gr-1+ myeloid cells without compromising CD8+ T cell immune responses

PubMed Central

2013-01-01

Background Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo. Methods We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice. Results We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b+Gr-1+ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8+ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation. Conclusions Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF. PMID:24279871

Population-specific recombination sites within the human MHC region.

PubMed

Lam, T H; Shen, M; Chia, J-M; Chan, S H; Ren, E C

2013-08-01

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.

Recombinant DNA encoding a desulfurization biocatalyst