A New Class of Dimeric α-Interferons (IFN-α) Made by the Dock and Lock (DNL) Method for Improved Cancer Therapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1396-1396
Author(s):  
Edmund A. Rossi ◽  
William J. McBride ◽  
Diane L. Nordstrom ◽  
Preeti Trisal ◽  
Thomas M. Cardillo ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Renal Clearance ◽  
The Body ◽  
Myelogenous Leukemia ◽  
Saline Control ◽  
E Coli ◽  
New Class ◽  
Disease Therapy

Abstract Background: IFN-α2 is indicated for the therapy of hairy cell leukemia, chronic myelogenous leukemia, follicular lymphoma, and malignant melanoma. As is the case for most cytokines, the pharmacokinetic (PK) properties of IFN-α2 are critical for dosing and efficacy. In vivo, the protein is quickly degraded, diffuses widely throughout the body, and has a rapid rate of renal clearance. Pegylation of IFN-α2 significantly increases the serum half-life and reduces renal clearance, thus enhancing its efficacy. However, established pegylation of IFN-α2 results in a mixture of positional isomers and reduced in vitro activity, as known for PEGASYS (Roche) and PEG-INTRON (Schering-Plough). Methods: To improve the PK properties and the potency, the DNL method (Rossi et al. Proc. Natl. Acad. Sci. USA, 2006,103:6841) was used to generate novel agents having two copies of IFN-α2b conjugated to polyethylene glycol (PEG). Results: A fusion protein (DDD2-IFN-α2b) composed of IFN-α2b with a dimerization-and-docking domain (DDD2) and a six-His tag was expressed both in myeloma cells and in E. coli. Two PEG-based modules, each composed of a fluorescent molecule, an anchor domain (AD) and either a 20-kDa PEG (IMP362) or a 30-kDa PEG (IMP413), were synthesized. Combining DDD2-IFN-α2b and IMP362 or IMP413 under redox conditions resulted in the desirable DNL conjugates consisting of two copies of IFN-α2b and one PEG linked site-specifically via the DDD and AD interaction. The cytotoxic activity of DDD2-IFN-α2b on Daudi lymphoma cells was similar to that of commercially available recombinant IFN-α2 (rhIFN-α2). The purity and identity of the two DNL conjugates (α2b-413 and α2b-362) were demonstrated by SDS-PAGE, immunoblotting, and fluorescence imaging. Both also showed potent cytotoxic activity on Daudi cells in vitro and superior PK properties to PEG-INTRON. For example, the mean blood residence times for α2b-362 (10.3 h) and α2b-413 (21.7 h) were significantly longer than those of rhIFN-α2 (0.7 h) and PEG-INTRON (5.1 h). Initial studies in mice bearing Daudi xenografts showed a significant therapeutic advantage over PEG-INTRON for both α2b-362 and α2b-413. Animals given 14,000 IU of PEG-INTRON had a median survival (MS) of 32 days compared to 21 days for saline control, whereas those receiving α2b-362 at 14,000 IU, 7,000 IU and 3,500 IU resulted in MS of 45, 41 and 32 days, respectively. More remarkably, α2b-413 was the most effective, achieving MS of 46, >53, and >53 days with 3,500 IU, 7,000 IU and 14,000 IU, respectively, all statistically significant improvements (P< 0.0028) compared to PEG-INTRON given at each equivalent activity. Conclusions: The DNL method provides a novel pegylation strategy for generating a dimeric IFN-α2b that is linked site-specifically to a single PEG at the predetermined location. Since the resulting conjugates exhibit improved PK and efficacy in a Burkitt lymphoma model, they may represent a new class of interferons for use in cancer and infectious disease therapy.

Regulation of angiopoietin expression by bacterial lipopolysaccharide

2008 ◽  
Vol 294 (5) ◽  
pp. L955-L963 ◽  
Author(s):  
Mahroo Mofarrahi ◽  
Thamir Nouh ◽  
Salman Qureshi ◽  
Loic Guillot ◽  
Dominique Mayaki ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Inflammatory Responses ◽  
Cell Types ◽  
Vascular Leakage ◽  
Saline Control ◽  
E Coli ◽  
Protein Levels ◽  
Angiopoietin 2

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-κB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.

Pyruvate kinase from the earthworm Allolobophora calliginosa: modification of the enzyme during anaerobiosis

1991 ◽  
Vol 69 (1) ◽  
pp. 251-254
Author(s):  
Athanasios I. Papadopoulos ◽  
Basile Michaelidis ◽  
Isidoros Beis
Keyword(s):  
Pyruvate Kinase ◽  
Deae Cellulose ◽  
Relative Activity ◽  
The Body ◽  
E Coli ◽  
In Vitro Incubation ◽  
Sigmoidal Kinetics ◽  
Independent Protein

The relative activity of pyruvate kinase from the body-wall muscle of the earthworm Allolobophora calliginosa was found to drop dramatically within 6 h of exposure to N2, whereas the opposite was observed during recovery. Two forms of pyruvate kinase (designated as peak I and peak II) were separated chromatographically on DEAE-cellulose and eluted at 50 and 150 mM of KCl, respectively. They displayed different kinetic behaviour with respect to substrate phosphoenolpyruvate; peak I exhibited Michaelis–Menten kinetics whereas peak II showed sigmoidal kinetics. The ratio of the enzyme units (peak I/peak II) decreased from 3.38 under normoxic conditions to 0.09 under anoxic conditions. In vitro incubation of the aerobic form of pyruvate kinase in the presence of ATP and Mg++ resulted in a reduction of the enzyme activity by 64%, suggesting the presence of an endogenous cyclic-nucleotide-independent protein kinase capable of phosphorylating pyruvate kinase. After in vitro incubation, alkaline phosphatase from E. coli increased the depressed activity of anaerobic pyruvate kinase, indicating that the enzyme molecule is phosphorylated in vivo during exposure to anoxia.

scholarly journals A novel Bursin-like peptide as a potential virus inhibitor and immunity regulator in SPF chickens infected with recombinant ALV

2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Yukun Zeng ◽  
Zuxin Gong ◽  
Binbin Wu ◽  
Wenchao Guan ◽  
Shenyi Yu ◽  
...  
Keyword(s):  
Growth Performance ◽  
Virus Titer ◽  
The Body ◽  
Bursa Of Fabricius ◽  
Virus Eradication ◽  
First Report ◽  
E Coli ◽  
Valuable Adjunct

Abstract Background Avian leukosis viruses (ALVs) are important contagious suppressive factors of chicken immunity and growth performance, resulted in enormous economic loss. Although virus eradication programs are applied in breeder flocks, ALVs are still widespread globally. Therefore, other valuable adjunct to reduce the negative effect of ALVs should be considered. Bursin-like peptide (BLP) showed remarkable immunomodulatory effects, whereas their influence on ALV-infected avian groups has not been reported. Here, a designed hybrid BLP was expressed in E. coli. The purified BLP was injected subcutaneously weekly in SPF chickens congenitally infected with a natural ALV strain. Then the influences of this BLP on the growth performance, immune response and virus titer of ALV-infected chickens were determined. Results This BLP injection significantly improved the body weights of ALV-infected birds (P < 0.05). BLP injection significantly enhanced organ index in the BF in ALV-infected birds (P < 0.05). The weekly injection of BLP significantly lengthened the maintenance time of antibodies against Newcastle disease virus (NDV) attenuated vaccine of ALV-infected birds (P < 0.05) and boosted the antibody titer against avian influenza virus (AIV) H5 inactive vaccine of mock chicken (P < 0.05). BLP injection in mock chickens enhanced the levels of serum cytokines (IL-2, IL-4 and interferon-γ) (P < 0.05). Surprisingly, the novel BLP significantly inhibited expression of the ALV gp85 gene in the thymus (P < 0.05), kidney (P < 0.05) and bursa of Fabricius (BF) (P < 0.01) of ALV-infected chickens. Both viral RNA copy number and protein level decreased significantly with BLP (50 μg/mL) inoculation before ALV infection in DF1 cells (P < 0.05). Conclusions This is the first report investigating the influence of BLP on the growth and immunity performance of chickens infected by ALV. It also is the first report about the antiviral effect of BLP in vivo and in vitro. This BLP expressed in E. coli showed potential as a vaccine adjuvant, growth regulator and antiretroviral drug in chickens to decrease the negative effects of ALV infection.

scholarly journals Biosynthesis of Ubiquinone Compounds with Conjugated Prenyl Side Chains

2008 ◽  
Vol 74 (22) ◽  
pp. 6908-6917 ◽  
Author(s):  
Pyung Cheon Lee ◽  
Christine Salomon ◽  
Benjamin Mijts ◽  
Claudia Schmidt-Dannert
Keyword(s):  
Antioxidant Properties ◽  
Side Chain ◽  
Nuclear Magnetic Resonance Analysis ◽  
Side Chains ◽  
Double Bonds ◽  
E Coli ◽  
New Class ◽  
Resonance Analysis

ABSTRACT Enzymatic steps from two different biosynthetic pathways were combined in Escherichia coli, directing the synthesis of a new class of biomolecules—ubiquinones with prenyl side chains containing conjugated double bonds. This was achieved by the activity of a C30 carotenoid desaturase, CrtN, from Staphylococcus aureus, which exhibited an inherent flexibility in substrate recognition compared to other carotenoid desaturases. By utilizing the known plasticity of E. coli's native ubiquinone biosynthesis pathway and the unusual activity of CrtN, modified ubiquinone structures with prenyl side chains containing conjugated double bonds were generated. The side chains of the new structures were confirmed to have different degrees of desaturation by mass spectrometry and nuclear magnetic resonance analysis. In vivo 14C labeling and in vitro activity studies showed that CrtN desaturates octaprenyl diphosphates but not the ubiquinone compounds directly. Antioxidant properties of conjugated side chain ubiquinones were analyzed in an in vitro β-carotene-linoleate model system and were found to be higher than the corresponding unmodified ubiquinones. These results demonstrate that by combining pathway steps from different branches of biosynthetic networks, classes of compounds not observed in nature can be synthesized and structural motifs that are functionally important can be combined or enhanced.

A Novel Anti-CD20 IgG-Interferon-α2b Fusion Protein with Potent in Vitro and in Vivo Anti-Lymphoma Activity Made by the Dock-and-Lock (DNL) Method.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1590-1590
Author(s):  
Edmund A Rossi ◽  
Chien-Hsing Chang ◽  
Thomas M Cardillo ◽  
Diane L Nordstrom ◽  
David M. Goldenberg
Keyword(s):  
Fusion Protein ◽  
Renal Clearance ◽  
Half Life ◽  
Tumor Targeting ◽  
Protein A ◽  
The Body ◽  
Anti Cd20 ◽  
Interferon Α2b

Abstract BACKGROUND: Interferon-α2b (IFN-α2b) is active alone and in combination with other agents in the therapy of a variety of cancers, including hairy cell leukemia, chronic myelocytic leukemia, follicular lymphoma, and malignant melanoma. As for most cytokines, the pharmacokinetics are a major factor affecting schedule and efficacy. The protein is rapidly degraded, diffuses widely throughout the body, and has a rapid rate of renal clearance. Commercially available IFNs that are pegylated, such as PEG-INTRON and PEGASYS, have increased serum half-life and reduced renal clearance, which augment their biological activity. For therapy of lymphoma and other cancers, fusing IFN-α2 to tumor-targeting antibodies could increase serum half-life and target the IFN-α2 to the tumor, conceivably allowing less frequent and lower dosing with improved therapeutic efficacy and reduced side effects. METHODS: The modular DNL method exploits a pair of distinct protein domains involved in the natural binding between protein kinase A (PKA) and A-kinase anchoring proteins (AKAP), whereby the dimerization-and-docking domain (DDD) of PKA and the anchoring domain (AD) of an interactive AKAP are each fused to a biological entity, resulting in respective DDD- and AD-modules that are readily combined to quantitatively generate stably-tethered structures of defined composition with retained bioactivity. We have selectively combined recombinant DDD-modules comprising IFN-α2b with recombinant AD-modules derived from the anti-CD20 humanized mAb, veltuzumab, and other humanized mAbs to generate complexes comprising four copies of IFN-α2b site-specifically linked to the bivalent IgG. RESULTS: The IgG-AD2 and IFN-α2b-DDD2 modules were expressed in separate myeloma cell cultures and purified from culture broths by Protein A and IMAC, respectively. Combining an IgG-AD2 module with slightly more than 2 molar equivalents of the cytokine-DDD2 module under mild redox conditions resulted in the formation of a covalent complex comprising one IgG and 4 IFN-α2b via the docking of each of the two AD2 domains on IgG with a dimer of IFN-α2b-DDD2, and subsequent formation of disulfide bonds (locking) between DDD2 and AD2. The 255-kDa conjugates, which were purified by Protein A, were readily detected by size-exclusion HPLC and non-reducing SDS-PAGE, and retained the biological functions of IFN-α2b in vitro. The IgG-IFN-α2b constructs exhibited potent anti-viral activity in vitro, with specific activities approaching that of recombinant human IFN-α2b. Additionally, the constructs all showed highly potent in vitro cytotoxicity against Burkitt lymphoma cell lines. Notably, the CD20-targeted IFN-α2b construct (20-2b) was 30-fold more potent than a control, non-targeting IgG-IFN-α2b. The enhanced cytotoxicity of 20-2b was not reproduced when non-targeting IgG-IFN-α2b was used in combination with veltuzumab, suggesting that IFN-α2b must be physically linked to achieve maximal potency. IgG-IFN-α2b fusion proteins, including 20-2b, induced significantly more potent ADCC compared to their parental MAbs. The targeting properties of 20-2b were comparable to veltuzumab, and its serum half-life was significantly longer than PEG-INTRON and PEGASYS. In the human Daudi xenograft model, 20-2b showed superior anti-tumor efficacy compared to both veltuzumab and other IgG-IFN-α2 agents. The median survival time (MST) for mice treated with single-dose 170 ng 20-2b was 101.5 days, whereas those treated with an equivalent dose of veltuzumab and untreated mice survived 39 and 28 days, respectively (P&lt;0.0005). Lower 20-2b doses of 80, 17 and 8 ng resulted in MST of 97.5, 56.5 and 48 days, respectively, with the lowest dose still significantly better than the highest dose of veltuzumab (P=0.0434). Using a single 170-ng dose, a CD22-targeting IFN-α2b (22-2b) also increased MST significantly to 47 days (P =0.0119), while a non-targeting IgG-IFN-α2b (734-2b) did not. CONCLUSIONS: The DNL method provided an IFN-α2-targeting mAb fusion protein that showed improved anti-tumor efficacy over the mAb by itself, based on improved pharmacokinetics, ADCC, and tumor targeting, as well as reduced systemic toxicity. Thus, DNL provides a modular approach to efficiently tether cytokines to targeting antibodies, resulting in higher in vivo potency than the original cytokines or mAbs.

scholarly journals Mechanism of action of oxazolidinones: effects of linezolid and eperezolid on translation reactions.

1997 ◽  
Vol 41 (10) ◽  
pp. 2132-2136 ◽  
Author(s):  
D L Shinabarger ◽  
K R Marotti ◽  
R W Murray ◽  
A H Lin ◽  
E P Melchior ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Pathogenic Bacteria ◽  
Coli Strain ◽  
In Vitro Translation ◽  
E Coli ◽  
Initiation Phase ◽  
New Class ◽  
Bacterial Translation

The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

scholarly journals JUSTIFICATION OF THE APPLICATION OF THE METHOD OF THREE-DIMENSIONAL PHOTODYNAMIC THERAPY OF DISEASES AND PROCESSES OF MICROBIAL AND NEOPLASTIC NATURE IN CLINICAL GYNECOLOGY

2017 ◽  
Vol 4 (4) ◽  
pp. 194-200
Author(s):  
M. T Aleksandrov ◽  
Vladimir M. Zuev ◽  
Yu. I Pimancheva ◽  
E. P Pashkov ◽  
G. E Bagramova
Keyword(s):  
Photodynamic Therapy ◽  
Experimental Studies ◽  
Three Dimensional ◽  
The Body ◽  
Myelogenous Leukemia ◽  
Ehrlich Carcinoma ◽  
Neoplastic Processes ◽  
Medical Diagnostic

There were executed experimental studies on test subjects of microbial (Staphylococcus aureus and Pseudomonas aeruginosa) and neoplastic nature (in vitro - suspension of cells of the line of chronic myelogenous leukemia K562 in a volume of 60 μl and in an amount of 60 ± 1 × 103, in vivo - mice infected with Ehrlich carcinoma) on the substantiation the use of chlorophyll-containing drugs activated for photodynamic therapy (PDT) outside the biological object. No additional PDT activation of the drug was performed.The high bactericidal (on the test objects of microbes) and anti-tumor PDT efficacy of chlorophyll-containing preparations activated outside the organism was substantiated, with their subsequent administration per os and accumulation in practically all organs and tissues of the body was validated. The developed medical diagnostic technology in its clinical application has proved its effectiveness in women with inflammatory and/or neoplastic processes of the pelvic organs. The used equipment and preparation are approved for clinical use.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Репрограммированые in vitro на М3 фенотип макрофаги останавливают рост солидной карциномы in vivo

2018 ◽  
pp. 41-46
Author(s):  
А.А. Раецкая ◽  
С.В. Калиш ◽  
С.В. Лямина ◽  
Е.В. Малышева ◽  
О.П. Буданова ◽  
...  
Keyword(s):  
Solid Tumor ◽  
Antitumor Effect ◽  
Tumor Development ◽  
Significant Inhibition ◽  
The Body ◽  
Ehrlich Carcinoma ◽  
Solid Carcinoma ◽  
Ec Cells

Цель исследования. Доказательство гипотезы, что репрограммированные in vitro на М3 фенотип макрофаги при введении в организм будут существенно ограничивать развитие солидной карциномы in vivo . Методика. Рост солидной опухоли инициировали у мышей in vivo путем подкожной инъекции клеток карциномы Эрлиха (КЭ). Инъекцию макрофагов с нативным М0 фенотипом и с репрограммированным M3 фенотипом проводили в область формирования солидной КЭ. Репрограммирование проводили с помощью низких доз сыворотки, блокаторов факторов транскрипции STAT3/6 и SMAD3 и липополисахарида. Использовали две схемы введения макрофагов: раннее и позднее. При раннем введении макрофаги вводили на 1-е, 5-е, 10-е и 15-е сут. после инъекции клеток КЭ путем обкалывания макрофагами с четырех сторон область развития опухоли. При позднем введении, макрофаги вводили на 10-е, 15-е, 20-е и 25-е сут. Через 15 и 30 сут. после введения клеток КЭ солидную опухоль иссекали и измеряли ее объем. Эффект введения макрофагов оценивали качественно по визуальной и пальпаторной характеристикам солидной опухоли и количественно по изменению ее объема по сравнению с группой без введения макрофагов (контроль). Результаты. Установлено, что M3 макрофаги при раннем введении от начала развития опухоли оказывают выраженный антиопухолевый эффект in vivo , который был существенно более выражен, чем при позднем введении макрофагов. Заключение. Установлено, что введение репрограммированных макрофагов M3 ограничивает развитие солидной карциномы в экспериментах in vivo . Противоопухолевый эффект более выражен при раннем введении М3 макрофагов. Обнаруженные в работе факты делают перспективным разработку клинической версии биотехнологии ограничения роста опухоли, путем предварительного программирования антиопухолевого врожденного иммунного ответа «в пробирке». Aim. To verify a hypothesis that macrophages reprogrammed in vitro to the M3 phenotype and injected into the body substantially restrict the development of solid carcinoma in vivo . Methods. Growth of a solid tumor was initiated in mice in vivo with a subcutaneous injection of Ehrlich carcinoma (EC) cells. Macrophages with a native M0 phenotype or reprogrammed towards the M3 phenotype were injected into the region of developing solid EC. Reprogramming was performed using low doses of serum, STAT3/6 and SMAD3 transcription factor blockers, and lipopolysaccharide. Two schemes of macrophage administration were used: early and late. With the early administration, macrophages were injected on days 1, 5, 10, and 15 following the injection of EC cells at four sides of the tumor development area. With the late administration, macrophages were injected on days 10, 15, 20, and 25. At 15 and 30 days after the EC cell injection, the solid tumor was excised and its volume was measured. The effect of macrophage administration was assessed both qualitatively by visual and palpation characteristics of solid tumor and quantitatively by changes in the tumor volume compared with the group without the macrophage treatment. Results. M3 macrophages administered early after the onset of tumor development exerted a pronounced antitumor effect in vivo , which was significantly greater than the antitumor effect of the late administration of M3 macrophages. Conclusion. The observed significant inhibition of in vivo growth of solid carcinoma by M3 macrophages makes promising the development of a clinical version of the biotechnology for restriction of tumor growth by in vitro pre-programming of the antitumor, innate immune response.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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