Elrashdy M. Redwan

Camel Lactoferrin Markedly Inhibits Hepatitis C Virus Genotype 4 Infection of Human Peripheral Blood Leukocytes

Abstract Hepatitis C virus (HCV) is a serious worldwide health risk and, to date, no effective treatments to prevent progression to chronic infection have been discovered. To combat the disease, Egyptian patients often use traditional medicines, for instance, camel milk, which contains lactoferrin. Currently, lactoferrin is one of the primary biopharmaceutical drug candidates against HCV infection. Camel lactoferrin (cLf) purification and biochemical and immunological characterization have shown its similarity to human and bovine lactoferrin, and crossreacts with the anti‐human lactoferrin antibody. Incubation of human leukocytes with cLf then infected with HCV did not prevent the HCV entry into the cells, while the direct interaction between the HCV and cLf leads to a complete virus entry inhibition after seven days incubation. Our results suggest that the cLf may be one of the camel milk components having antiviral activity. In conclusion, we have demonstrated the potential for cLf to inhibit HCV entry into human leukocytes with more efficiency than human or bovine lactoferrin.

Cell factories for insulin production

The rapid increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to escalate the demand for recombinant insulin in near future. Current manufacturing technologies would be unable to meet the growing demand of affordable insulin due to limitation in production capacity and high production cost. Manufacturing of therapeutic recombinant proteins require an appropriate host organism with efficient machinery for posttranslational modifications and protein refolding. Recombinant human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in human. We would focus in this review, on various approaches that can be exploited to increase the production of a biologically active insulin and its analogues in E. coli and yeast. Transgenic plants are also very attractive expression system, which can be exploited to produce insulin in large quantities for therapeutic use in human. Plant-based expression system hold tremendous potential for high-capacity production of insulin in very cost-effective manner. Very high level of expression of biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost technology for both injectable as well as oral delivery of proinsulin.

Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives

Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, cost-effectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.

Animal-Derived Pharmaceutical Proteins

Abstract Livestock animals have made a significant contribution to human health and well-being throughout humankinds history. A significant contribution of farm animals to human health are the longstanding use of bovine and porcine for production of insulin (for treatment of diabetes), gelatin (for pharmaceutical and other purposes), as well as horse and sheep antibody against natural venoms, toxins, drugs and microbial peptides. Gelatin being the biggest animal protein consumed in human health, follows with antibodies fragments. The chronic problem of animal-derived therapeutics, especially those of high molecular weight, is the immunogenicity induction in addition to their biosafety. However, the invertebrates and lower vertebrates donate the human being a several crucial emergency saving life small-peptides or their analogs such as RefludanR, PrialtR, ExendinR. Not only, but the farm animals are enormously using as models for novel surgical strategies, testing of biodegradable implants and sources of tissue replacements, such as skin and heart valves. Recently, they are being harnessing as bioreactor for production of biopharmaceutical related products through gene farming with efficiency far greater than any conventional microbial or cell-culture production systems. Only 16 transgenic cows would be covering the worldwide needs from human growth hormone. The transgenic, especially animal, technology would be solving a several biopharmaceutical products disadvantages, such as cost, biosafety, immunogenicity and the availability dimensions.

Oyster mushroom laccase inhibits hepatitis C virus entry into peripheral blood cells and hepatoma cells.

There is no protective vaccine or effective drug against hepatitis C virus (HCV). Sustained virological response to INF/ribavirin treatment regimen has an efficiency of about 50%. Many patients worldwide have used traditional medicines and herbal medicine in particular. A laccase has been purified from oyster mushroom (Pleurotus ostreatus) to homogeneity by DEAE Affi-gel blue gel, CM-Sephadex G-50 and Sephadex G-100. The molecular weight of the laccase was about 58 kDa in SDS-PAGE. The optimum pH and temperature of the laccase activity were pH 4.0 and 60 degrees C, respectively. The activity of the enzyme increased steadily from 20 to 40 degrees C, then very slowly from 40 degrees to 60 degrees C, while the enzyme activity decreased to 9% at 90 degrees C. The activity of the laccase changed gradually over the pH range 2.0-4.0. However, the enzyme activity was totally abrogated at the pH 8 and above. Incubation of peripheral blood cells PBCs and hepatoma HepG2 cells with laccase which were then infected with HCV did not protect the cells from HCV attack and entry, while direct interaction between HCV and the laccase at the concentrations of 2.0 and 2.5 mg/ml led to a complete inhibition of virus entry after seven days of incubation. Meantime, the laccase at the concentrations of 1.0 and 1.5 mg/ml did not display any blocking activity. The potential activity of the laccase on intracellular HCV replication in infected HepG2 cells has been examined. The laccase was capable of inhibiting HCV replication at the concentrations of 1.25 and 1.5 mg/ml after first dose of treatment for four days and at the concentrations of 0.75, 1.0, 1.25 and 1.5 mg/ml after the second dose of treatment for another four days.

Effectiveness of human, camel, bovine and sheep lactoferrin on the hepatitis C virus cellular infectivity: comparison study

PurposeThe prevalence of HCV infection has increased during recent years and the incidence reach 3% of the worlds population, and in some countries like Egypt, may around 20%. The developments of effective and preventive agents are critical to control the current public health burden imposed by HCV infection. Lactoferrin in general and camel lactoferrin specifically has been shown to have a compatitive anti-viral activity against hepatitis C virus (HCV). The purpose of this study was to examine and compare the anti-infectivity of native human, camel, bovine and sheep lactoferrin on continuous of HCV infection in HepG2 cells.Material and methodsUsed Lfs were purified by Mono S 5/50 GL column and Superdex 200 5/150 column. The purified Lfs were evaluated in two ways; 1. the pre-infected cells were treated with the Lfs to inhibit intracellular replication at different concentrations and time intervals, 2. Lfs were directly incubated with the virus molecules then used to cells infection. The antiviral activity of the Lfs were determined using three techniques; 1. RT-nested PCR, 2. Real-time PCR and 3. Flowcytometric.ResultsHuman, camel, bovine and sheep lactoferrin could prevent the HCV entry into HepG2 cells by direct interaction with the virus instead of causing significant changes in the target cells. They were also able to inhibit virus amplification in HCV infected HepG2 cells. The highest anti-infectivity was demonstrated by the camel lactoferrin.ConclusioncLf has inhibitory effect on HCV (genotype 4a) higher than human, bovine and sheep lactoferrin.

EXAMINATION OF THE ACTIVITY OF CAMEL MILK CASEIN AGAINST HEPATITIS C VIRUS (GENOTYPE-4A) AND ITS APOPTOTIC POTENTIAL IN HEPATOMA AND HELA CELL LINES

Background Hepatitis C is a global health concern that represents a major cause of liver disease and socioeconomic burden. Currently, there is no vaccine that protects against this infection or drug that treats it effectively. The current treatment for hepatitis C virus (HCV) infection does not produce a sustained virologic response. Therefore,discovery and identification of a new drug for HCV treatment is a high priority.Camel milk is a traditional medicine that could improve the control of HCV. Objectives To assess the potential effect of casein purified from camel milk on HCV cellular infectivity in a tissue culture model. Materials and Methods Casein was purified from defatted camel milk to electrophoretic homogeneity. PBMCs and HepG2 and HeLa cell lines were used. Three kinds of experiments were conducted. HCV was directly interacted with casein and then mixed with different cell types, casein was incubated with the cells and then exposed to HCV, and the HCV pre-infected cells were treated with casein at different concentrations and time intervals. Non-infected cells were used to assess cytotoxicity and the apoptosis effect of casein. Results Direct interaction of casein (with or without α-lactalbumin) with neither the virus nor the cells prevented HCV cell entry. However, casein with α-lactalbumin induced a cytotoxic effect in HepG2 and HeLa cell lines but not in human naïve leukocytes. At all concentrations tested, casein with α-lactalbumin could induce apoptosis in both infected and non-infected HepG2 cells. Conclusions Camel milk casein (with or without α-lactalbumin) did not demonstrate any anti-HCV activity. However, the cellular apoptotic cascade was initiated in HepG2 and HeLa cells treated with casein (with α-lactalbumin) but not in naïve leukocytes.

Ovine anti-rabies antibody production and evaluation.

In view of the disadvantages of human and equine rabies immunoglobulin still there is urgent needs for safe and cost-control anti-rabies immunoglobulins especially for person who have been severely exposed (categories III) to the virus. Our attempt to produce a less immunogenic and cheaper anti-rabies immunoglobulin affordable for those people living in developing countries, has been harnessed the ovine as a bioreactor instead the horse. The animals have been intramuscular immunized, and the plasma processed with 5% caprylic acid to yield IgG with purity of 95%. Moreover, antibody apparently indicated that the titer and neutralizing indexes were harmonized, especially at higher antibody dilution. The results showed that three immunized sheep were produced about 7000 IU of purified anti-rabies antibody. Sheeps IgG has low immunogenic effect than human and horse antibodies when injected into the mouse. Pure concentrated ovine antibody may serve as a possible alternative to currently available anti-rabies human or equine immunoglobulin.

Synthesis of the Human Insulin Gene: Protein Expression, Scaling Up and Bioactivity

Abstract Optimized Synthetic human insulin gene was preferred to easy of cloning, plasmid stability, and protein expression away from the native sequence and its rare codons. Two steps to obtain the insulin, so we assembled the gene of 293 bp using a battery of overlapped synthetic oligos, then cloned into pET101directional TOPO expression vector downstream to the T7 promoter. The proinsulin products were produced as inclusion bodies in E. coli at a level of 10%. The batch cultivation of the strain yielded 6 g/L, while the high cell density of fed‐batch cultivation yielded 46 g/L. The proinsulin purification yielded 110 mg/gram cell weight, and 1.3 mg/gram of a bioactive insulin. The native insulin was generated by enzymatic conversion of chemically processed proinsulin. The produced insulin was matched with that of a commercial aqueous version at a level of enzyme immunoassys, SDS‐PAGE, RP‐HPLC, and bioactivity. The present results showed that the produced insulin has a comparable biochemical and potency similar to that of commercial one.

Antimicrobial potentials and structural disorder of human and animal defensins

Defensins are moonlighting peptides which are broadly distributed throughout all the living kingdoms. They play a multitude of important roles in human health and disease, possessing several immunoregulatory functions and manifesting broad antimicrobial activities against viruses, bacteria, and fungi. Based on their patterns of intramolecular disulfide bridges, these small cysteine-rich cationic proteins are divided into three major types, α-, β-, and θ-defensins, with the α- and β-defensins being further subdivided into a number of subtypes. The various roles played by the defensins in the innate (especially mucosal) and adoptive immunities place these polypeptides at the frontiers of the defense against the microbial invasions. Current work analyzes the antimicrobial activities of human and animal defensins in light of their intrinsic disorder propensities.