Advanced Pharmaceutical Bulletin
Volume:11 Issue: 3, May 2021

In this period of global pandemic caused by SARS-Cov-2, it is of paramount importance to recognize all risk factors that may increase the likelihood of infection. In addition to the risk factors known as pre-existing diseases and old age, risk factors could be drug treatments for chronic diseases, such as immunomodulating drugs that can alter immune defences and response to infectious agents. Antibodies that inhibit tumor necrosis factor (TNF) such as adalimumab infliximab etanercept and golimumab have been used for over 20 years in severe cases of autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease or ankylosing spondylitis. Due to their mechanism of action they reduce inflammation and can stop the progression of the disease by inhibiting a key factor of inflammation such as Tumor Necrosis Factor (TNF). In this article we want to examine the possible correlation between therapy with TNF inhibitors and the increased risk of SARS-CoV-2 infection, and the possible paradoxical therapeutic efficacy in patients with ongoing infection, especially in phase two and three. We express our opinion on this very complex and sensitive topic which is the subject of discussion among physicians and experts, based on current knowledge of the literature.

Glaucoma is an irreversible condition resulting from the increase in intraocular pressure (IOP); which leads to permanent loss of vision with the destruction of retinal ganglion cells (RGCs). The IOP elevations are controlled in normal by the physiological flow of aqueous humour. A population with age above 40 is more susceptible to glaucoma. Other factors like gender, genetics, race etc. plays major roles in the development of the disease. Current treatment methods available for the disease includes drugs come under the classes of beta receptor blockers, carbonic anhydrase inhibitors, cholinergic agonists, prostaglandins etc. N-methyl-D-aspartate (NMDA) antagonists, inducible nitric oxide synthase (iNOS) inhibition, cytoskeletal agents, Rho-kinase inhibitors etc are few novel targets sites which are in research focus for the treatment of the disease. Developments in nanomedicine are also being evaluated for their potential in treating the growing glaucomatous population. Nanosystems are suggested to avoid the difficulties in tackling the various ocular barriers to a limit, help to decrease the instillation frequency of topical medication and can provide drug delivery in a sustained or controlled manner. This review focuses on the current and emerging treatment methods for glaucoma along with some of the nanoformulations for ocular drug delivery.

The healthcare sector is considered to be one of the largest and fast-growing industries in the world. Innovations and novel approaches have always remained the prime aims in order to bring massive development. Before the emergence of technology, all the sectors, including the healthcare sector was dependant dependent on man power, which was time-consuming, and less accurate with lack of efficiency. With the recent advancements in machine learning, the condition is has been steadily revolutionizing. in the practice of the health care industry. Artificial Intelligence intelligence (AI) lies in the computer science department, which stresses on the intelligent machines’ creation, that work and react just like human beings. In simple words, AI is the capability of a computer program to think and learn, almost satisfying natural intelligence. It is the ability of a system to interpret the external data correctly, learn from it and finally use those learnings to execute some particular goals and tasks through adaptation. It utilizes multiple technologies to comprehend, act and understand from past experiences. Involving AI is not a science fiction that was once a very long time ago. It AI being an emerging technology has been adopted in various facets of healthcare ranging from drug discovery to patient monitoring. rapidly penetrated its wings developed itself into almost all the industries. Irrespective of the person’s background, whether he/she is a student, industry worker, an entrepreneur, or a scientist, having basic knowledge about the importance and applications of AI would be impactful. Currently, the applications of AI has have been expanding into those fields, which was once thought to be the only domain of human expertise such as health care sector. In this review article, we have shedthrown light on the present usage of AI in the healthcare sector, such as its working, and the way this system is being implemented in different domains, such as drug discovery, diagnosis of diseases, clinical trials, remote patient monitoring, and nanotechnology. We have also slightlybriefly touched upon its applications in touching other sectors as well. The public opinions have also been analyszed and discussed along with the future prospects.The main goals have been briefed. prospects. We have discussed the Along with the merits, we have also discussed about and the other side of AI, i.e. the disadvantages of this as wellin the last part of the manuscript.

Cancer is a complex mechanism involving a series of cellular events. The glycoproteins such as hyaluronan (HA) are a significant element of extracellular matrix (ECM), involve in the onset of cancer developmental process. The pivotal roles of HA in cancer progression depend on dysregulated expression in various cancer. HA, also gain attention due to consideration as a primary ligand of CD44 receptor. The CD44, complex transmembrane receptor protein, due to alternative splicing in the transcription process, various CD44 isoforms predominantly exist. The overexpression of distinct CD44 isoforms (CD44v) standard (CD44s) depends on the tumour type and stage. The receptor proteins, CD44 engage in a variety of biological processes, including cell growth, apoptosis, migration, and angiogenesis. HA-CD44 interaction trigger survival pathways that result in cell proliferation, invasion ultimately complex metastasis. The interaction and binding of ligand-receptor HA-CD44 regulate the downstream cytoskeleton pathways involve in cell survival or cell death. Thus, targeting HA, CD44 (variant and standard) isoform, and HA-CD44 binding consider as an attractive and useful approach towards cancer therapeutics. The use of various inhibitors of HA, hyaluronidase (HYALs), and utilizing targeted Nano-delivery of anticancer agents and antibodies against CD44, peptides gives promising results invitro and invivo. However, they are in clinical trials with favourable and unfavourable outcomes, which reflects the need for various modifications in targeting agents and a better understanding of potential targets in tumour progression pathways.

Timely diagnosis is the most important parameter for thedetection and hindrance with tissues (infected). Many conventional techniques are used for the determinationof the chronic disease like MRI, X-ray, mammography, ultrasound and other diagnosing methods. Nevertheless, they have some limitations. We epitomizebetween 4 and 34 % of all carcinogenic tissues are lacking because of weak,in adequate malignant/benign cancer tissue on the contrary. So,an effective alternative method is the validconcern in the field of medical right now. Imaging with the help of patch antenna to detect chronic disease like breast cancer, oxidative stress syndrome etc. it has been proved to be a suitable potential method, and there are many works in this area. All materials have different conductivity and permittivity. With the help of these antennas, a 3D tissue structure which has different conductivity and permittivity is modelled in high-frequency structure simulator (HFSS)through Finite Element Method (FEM) which resolves electromagnetic field values and a microstrip patch antenna operation process. As compared with conventional antennas, micro strip patch antennas have enhanced benefits and better prospects. An integrated Antenna plays an important or crucial role for supporting many applications in biomedical, commercial and in military fields. The Antenna designed for these applications should be wideband, not sensitive to the human body. In this present review,the precise application of the Antenna in different biomedical aspects is considered. Furthermore, the author has also discussed the analytical results using simulation models and experimental results for some of the significantdisease.

Cancer is one of the deadly diseases leading to approximately 7.6 million deaths worldwide, with the mortality rate of 13%, and the number of deaths is expected to increase to 13.1 million within the next 10 years. In controlled drug delivery systems (DDS), the drug is transported to the desired location. Thus, the influence of drugs on vital tissues and undesirable side effects can be minimised. Additionally, DDS protects the drug from rapid degradation or clearance and enhances drug concentration in target tissues, and therefore, minimise the required dose of drug. This modern form of therapy is particularly important when there is a discrepancy between the dose and concentration of a drug. Cell-specific targeting can be achieved by attaching drugs to individually designed carriers. Recent developments in nanotechnology have shown that nanoparticles (particles with diameter < 100 nm in at least one dimension) have great potential as drug carriers. Because of their small size, these nanostructures exhibit unique physicochemical and biological properties that make them a favourable material for biomedical applications. Therefore, in this review, we aimed to describe the importance and types of nanomedicines and efficient ways in which new drug delivery systems for the treatment of cancer can be developed.

Reducing the undesirable systemic effect of photodynamic therapy (PDT) can be achieved by incorporating a photosensitizer in microparticles (MPs). This study is devoted to the preparation of biocompatible biodegradable MPs with the inclusion of the natural photosensitizer Radachlorin (RС) and an assessment of the possibility of their use for PDT.

RC-containing MPs (RС MPs) with poly(lactic-co-glycolic acid) copolymer (PLGA) matrix were prepared by a double emulsion solvent evaporation methods. The size and morphology of RC MPs were surveyed using scanning electron microscopy, confocal laser scanning microscopy, and dynamic light scattering. The content of RC, its release from RC MPs, and singlet oxygen generation were evaluated by the optical spectroscopy. Cellular uptake and cytotoxic photodynamic effect of RC MPs were investigated with in vitro assays.

The average diameter of the prepared RC MPs was about 2-3 μm. The RC MPsprepared by the water/oil/oil method had a significantly higher inclusion of RC (1.74 μg/mg) then RC MPs prepared by the water/oil/water method (0.089 μg/mg). Exposure of the prepared RC MPs to PDT light radiation was accompanied by the singlet oxygen generation and a cytotoxic effect for tumor cells. The release of the RC from the RC MPs was prolonged and lasted at least two weeks.

PLGA RC MPs were found to cause a photoactivated cytotoxic effect for tumor cells and can be used for local application in PDT of tumors.

The development biosensing technologies capable of delivering fast and reliable analysis is a growing trend in drug quality control. Considering the emerging use of plant-based polyphenol oxidases (PPO) as biological component of electrochemical biosensors, this work reports the first Solanum lycocarpum PPO biosensor and its use in the pharmaceutical analysis of paracetamol in tablet formulations.

The biosensor was optimized regarding fruit maturation (immature and mature-ripe), vegetal extract volume to be used in biosensor construction as well as optimal pH of electrochemical cell fluid.

Results evidenced that the extract which rendered the biosensor with best analytical performance was from immature fruits, and the biosensor produced using 100 µL of crude plant extract promoted better faradaic signal gathering. Moreover, when neutral pH media was used in the electrochemical cell, the biosensor showcased best faradaic signal output from the used redox probe (catechol), suggesting thence that the method presents high sensibility for phenolic compounds detection. Furthermore, the biosensor was able to quantify paracetamol in a linear range from 50 to 300 μM, showcasing LoD and LoQ of 3 μM and 10 μM, respectively.

after careful evaluation, this biosensor might be a low-cost alternative for conventional pharmaceutical quality control methods.

In last decades, by increasing multi-drug resistant microbial pathogens an urgent demand was felt in the development of novel antimicrobial agents.

Promising nanocomposites composed of clay/alginate/imidazolium-based ionic liquid, have been developed via intercalation of calcium alginate and ionic liquid by ion-exchange method. These tailored nanocomposites were used as nanocarriers to simultaneously deliver methotrexate (MTX), and ciprofloxacin (CIP), as anticancer and antibacterial agents, respectively to MCF-7 breast cancer cells. Nanocomposites were fully characterized by SEM, XRD, FTIR spectroscopy, and TGA methods. The in vitro antimicrobial potential of the mentioned nanocomposites in free and dual-drug loaded form was investigated on Pseudomonas aeruginosa and Escherichia coli bacteria. The antitumor activity of nano-formulations was evaluated by both MTT assay and cell cycle arrest.

The dual drug-loaded nanocomposites with exceptionally high loading efficiency (MTX: 99 ±0.4% and CIP: 98 ±1.2%) and mean particle size of 70 nm were obtained with obvious pH-responsive MTX and CIP release (both drugs release rate was increased at pH 5.8 compared to 7.4). The antibacterial activity of CIP-loaded nanocomposites was significantly higher in comparison with free CIP (p <0.001). The antitumor activity results revealed that MTX cytotoxicity on MCF-7 cells was significantly higher in nano-formulations compared to free MTX (p <0.001). Both MTX-loaded nanocomposites caused S-phase arrest in MCF-7 cells compared to non-treated cells (P˂0.001).

Newly developed smart nanocomposites are potentially effective pH-sustainable delivery systems for enhanced tumor therapy.

The aim of the present investigation was to formulate protein nanoparticles loaded suppositories for colon targeting of metronidazole (MZ), to achieve sustain release effect.

Protein nanoparticles were formulated via desolvation technique by utilizing 2 3 factorial design which results into eight formulations. The synthesized protein nanoparticles were characterized for different physicochemical and in vitro parameters viz. particle size, surface morphology, entrapment efficiency and zeta potential, drug- excipients compatibility studies.

The formulated protein nanoparticles were found to be spherical in shape and have an average size in the range of 300.7 nm to 504.8 nm. Based on the results obtained, F7 was found to be the optimized formulation that was loaded into the suppository base. Furthermore, suppositories were also characterized for several parameters like content uniformity, weight variation and liquefaction time.

Resultant, suppositories were free from pits, fissures and cracks. The in-vitro release data of metronidazole protein nanoparticles (MZ-PNPs) loaded suppositories were compared with the suppositories loaded with active ingredient only i.e. metronidazole. Screening against Pheretima posthuma was also conducted. The results of in vitro drug release testing proved that protein nanoparticle loaded suppositories is a better approach, compared to pure metronidazole loaded suppositories. Release kinetic study concluded that the formulation follows higuchi’s equation i.e. having a biphasic release pattern. The efficiency of the formulated dosage form was evaluated using Indian earthworms, Pheretima posthuma (P. posthuma).

In this study, leaf/flower aqueous extract of medicinal plant species Mentha pulegium was used to synthesize ZnO and CuO nanoparticles (NPs) as a cost-effective, one-step, and eco-friendly method.

Physicochemical properties of both metal oxide NPs (MONPs) were determined by UV-Vis spectroscopy, X-Ray Diffraction (XRD), Fourier-Transform Infra-Red (FTIR) spectroscopy, Scanning Electron Microscope (SEM) and Energy Dispersive X-ray (EDX) techniques.

Phytofabricated ZnONPs and CuNPs illustrated 65.02±7.55 and 26.92±4.7 nm with antibacterial activities against antibiotic-resistant Escherichia coli and Staphylococcus aureus. Higher antibacterial activities were observed for CuONPs compared with ZnONPs.

Large surface area and more reactivity resulted from smaller size as well as higher production of reactive oxygen species (ROS) were considered to antibacterial efficiency of CuONPs against antibiotic-resistant E. coli and S. aureus.

Multiple sclerosis (MS) is a debilitating neuroinflammatory disorder of the central nervous system. It is believed to result from an impaired immune response against myelin components especially myelin oligodendrocyte glycoprotein (MOG). Some efforts have been made to bioconjugate the MOG peptides to tolerogenic particles like poly (lactic-co-glycolic acid) (PLGA) for treating animal models of autoimmune disorders. Accordingly, we aimed to elucidate the tolerogenic effects of MOG-PLGA particles on experimental autoimmune encephalomyelitis (EAE).

PGLA nanoparticles were synthesized using water/oil/water procedure. Next, the MOG or ovalbumin (OVA) peptides covalently linked to the PLGA particles. These particles were then intravenously or subcutaneously administered to nine groups of C57BL/6 mice before and after EAE induction. The brain tissues were assessed for the infiltration of immune cells. The Tolerogenic effect of the vaccine was also assessed on the quantity of the Treg cells. Moreover, the amount of interferon-γ (IFN-γ), interleukin-10 (IL-10), and interleukin-17 levels produced by splenic lymphocytes were then quantified by ELISA.

Intravenous administration of PLGA500-MOG35-55 nanoparticles before EAE induction ameliorated EAE clinical scores as well as infiltration of immune cells into the brain. In the spleen, the treatment increased CD4+CD25+FoxP3+ Treg population and restored the homeostasis of IFN-γ, IL-10, and IL-17 (all p values <0.0001) among splenocytes.

The conjugation of MOG peptides to the PLGA nanoparticles significantly recovered clinical symptoms and the autoimmune response of EAE. The MOG-PGLA particles are potentially valuable for further evaluations, hopefully progressing toward an optimal approach that can be translated to the clinic.

In this research, electrospinning method was employed to fabricate food-grade nanofibers (NFs) from chitosan-gelatin combination for stigmasterol encapsulation. The spinnability of mixed chitosan and gelatin solutions was investigated at different polymer ratios, and the physicochemical properties of the NFs were evaluated.

The mixture solution of chitosan (1.5 % w/v) and gelatin (20 % w/v) in acetic acid indicated spinnability under the following conditions: the ratio of 25:75, voltage of 17 kV, and 15 cm capillary collector distance with a flow rate of 0.2 mL/min. Stigmasterol (0.04 % w/v) was incorporated into NFs of chitosan-gelatin at a respective ratio of 25:75.

Encapsulation efficiency (EE) of loaded stigmasterol was found to be 87 ± 5 %. The antioxidant ability of loaded stigmasterol was considerably higher than that observed for free stigmasterol. Scanning electron microscopy (SEM) results demonstrated the formation of the ultrathin fibers with no bead (with diameters of 217 ± 43 nm). The concentration of polymeric solution and viscosity had a notable effect on the electrospinning efficiency of the chitosan-gelatin-based nanofibers. The thermal stability of chitosan and gelatin fibers was more than that of native gelatin and chitosan. The in vitro stigmasterol release from these nanofibers followed a controlled-release pattern. The released phytosterol from chitosan formula was less than from those without chitosan formula (46 ± 3 % and 96 ± 4 % respectively).

The obtained results suggested that gelatin had a high potential for enhancing the spinnability of chitosan under acidic conditions at optimized concentrations.

Hydrophilic drugs are extensively applied in clinical applications. Inadequate dermal penetration of these drugs is a great challenge. Incorporation of drugs into nano-carrier systems overcomes lower penetration drawbacks. Invasomes are novel nano-carrier systems which enhance transdermal penetration by using terpene and ethanol in their structures. buprenorphine and bupivacaine hydrochlorides are two potent analgesic drugs that are loaded simultaneously in the nano-invasome structure as opioid and non-opioid drugs.

The full factorial experimental design was used for planning and estimating optimum formulations of invasome systems. Three influential factors like terpene type, terpene concentration and preparation method were comprehensively analyzed for achieving high encapsulation efficiency and optimum size.

The mean sizes of designed invasomes were in the range of 0.39-5.86 µm and high values of EE and LC were reported as 98.77 and 19.75 for buprenorphine-loaded invasome, respectively. Zeta potential measurements confirmed that the obtained high value of EE might be as a result of reversible ionic interactions between positively charged drugs and negatively charged phospholipidic part of invasome structure. Another characterization of the prepared formulations was carried out by FTIR, XRD and DLS technique.

The satisfactory obtained results of formulations encourage researchers to get optimum topical analgesic formulations with potent and rapid onset time properties required in invasive cutaneous procedures.

MDMA (methylenedioxymethamphetamine) is a synthetic compound, which is a structurally derivative of amphetamine. Also, it acts like an amphetamine, structurally, and functionally. MDMA uses mechanism-based inhibition, to inhibit isoenzyme CYP2D6. It can also inhibit other isoenzymes contributing to its metabolism, including CYP3A4 which is the most important member of the cytochrome P450 superfamily. Since more than 50% of drugs are metabolized by CYP3A4, its inhibition may cause harmful and even lethal drug interactions. Tramadol, as an opioid-like analgesic, is mainly metabolized into O-desmethyl tramadol (M1), by CYP2D6 and undergoes N-demethylation to M2, by CYP2B6 and CYP3A4. Due to the significant potential of abusing tramadol, either alone or in combination with MDMA, the rate of its toxicity and side effects may increase following possible MDMA relevant enzyme inhibition.

Different doses of MDMA (1-10 mg/Kg) were intraperitoneally administered to Wistar male rats of both control and treatment groups. Then, after one hour, their isolated livers were perfused by perfusion buffer containing tramadol (1 µg/mL). Afterward, perfusate samples were collected. They were analyzed by HPLC to determine the concentrations of tramadol and its metabolites.

MDMA administration in treatment groups reduced M1 production. On the other hand, by following the treatment with different MDMA doses, the M2 metabolic ratio increased by 46 to 101%.

it seems that the regular doses of MDMA cannot inhibit the CYP3A4 activity.

Effective and selective T-cell activation and proliferation during the T-cell expansion phase of a cellular adoptive immunotherapy method, challenging because recent studies revealed the importance of each subtype of T-cells in different immunologic strategies against tumors, like CAR-T cell therapies. Artificial Antigen Presenting Cells regarded as a natural way to manipulate T-cell subtypes activation and specific proliferation. In the current study, we utilized K562 cells based aAPC method expressing the ICOSL molecule, to evaluate T-cell subtypes differentiation rate and functional status.

CD3+T-cells isolated and, co-cultured with ICOSL expressing K562 cells. After 4, 6, and 10 days selective CD markers of T-cell subtypes and each subtype’s activity-related genes levels evaluated by qPCR methods.

During the culture period, CD4+ Th related phenotype reduced continuously, and in day 10th of culture CD4+ T-cell’s population significantly reduced (p=0.029). In contrast, the CD8+ population ratio was ascending during the study period but was not statistically significant. FoxP3+CD25-, Treg population ratio was significantly increased during the time in comparison with the control group, as well as memory T-cell phenotypic marker, CD127+, expressing cells ratio. T-cell subpopulations activity-related genes expression levels evaluated too, and the Th1 related IL-2 and INF-γ reductions observed alongside regulatory T-cells gene (IL-10) and Cytotoxic T-cell’s related gene (Geranzym-A) elevations.

We concluded that the K562-ICOSL based aAPC system is working and effective in T-cell short to medium culture periods, and this approach preparing relatively selective milieu for CD8+ T-Cell differentiation and much less Treg differentiation.

Biosurfactants are applied in drug formulations to improve drug solubility and in some cases, treat diseases. This study is focused on generating, extracting, purifying and then characterizing biosurfactants from bacterial isolates of palm oil wastes and abattoir soil origins.

Eight (8) bacteria were isolated from the soil and sludge samples, out of which four (50 %) were found to produce biosurfactants. Bacillus subtilis (37.5 %) and Pseudomonas aeruginosa (50%) were isolated and identified from these samples using mineral salt medium, nutrient agar and Cetrimide agar. Mutant isolates of Bacillus subtilis BS3 and Pseudomonas aeruginosa PS2 were used to produce biosurfactants using mineral salt medium as enrichment medium and extraction was done using membrane filter.

The mutant strains Bacillus subtilis BS3 and Pseudomonas aeruginosa PS2generated biosurfactants that displayed significant solubility and dissolution properties by enhancing the percentage solubility of piroxicam to 62.86 and 54.29 % respectively, and achieved 51.71 and 48.71 % dissolution of the drug in 0.1N HCl.

From the results obtained, the produced biosurfactants could serve as a better alternative to conventional surfactants. Notably, the study indicated that the biosurfactant produced by mutant strain of Bacillus subtilis produced more potent activities (surface tension reduction ability, high emulsification) than those of Pseudomonas aeruginosa. Keywords: Biosurfactant production; Bacillus subtilis; Pseudomonas aeruginosa; Microbial Biotechnology; Palm oil wastes; Abattoir soil

DOF (DNA-binding with One Finger) proteins are plant-specific transcription factors which mediate numerous biological processes. The purpose of the current study is to report new naturally occurring mutations in the gene encoding for one of the members of DOF proteins named DOF 4.2.

The expression of zinc finger domain of DOF 4.2 (DOF 4.2-ZF) was investigated by first synthesis of cDNA library using different parts of Arabidopsis thaliana plant. Then the coding sequence for zinc finger domain of DOF 4.2 protein was prepared using nested PCR experiment and cloned into pGEX-6P-1 expression vector. Finally, the prepared construct was used for protein expression. Furthermore, molecular dynamics (MD) simulation was carried out to predict DNA binding affinity of DOF 4.2-ZF using AMBER package.

For the first time a new variant of DOF 4.2-ZF protein with three mutations was detected. One of the mutations is silent while the other two mutations lead to amino acid replacement (S18G) as well as introduction of a stop codon ultimately resulting in a truncated protein production. In order to investigate whether the truncated form is able to recognize DNA binding motif, MD simulations were carried out and the results showed that the chance of binding of DOF 4.2-ZF protein to cognate DNA in its truncated form is very low.

The findings demonstrated that the observed mutations adversely affect the DNA binding ability of the truncated form of DOF4.2 if it is expressed in the mutant variant of A. thaliana used in this study.

Ocriplasmin (Jetrea TM) is a FDA approved recombinant enzyme utilized in the treatment of vitreomacular adhesion (VMA). This is a recombinant C-terminal fragment of human plasmin produced using yeast Pichia pastoris. Since ocriplasmin does not contain any O-or N-glycosylation or some other post-translational modifications, bacterial expression systems such as Escherichia coli could be considered as an economical host for recombinant expression. In the present study, we aimed to evaluate the efficiency of E. coli expression system for high-level expression of recombinant ocriplasmin.

The gene coding for ocriplasmin was cloned and expressed in E. coli BL21. The bacterial cells were cultured on large scale and the expressed recombinant protein was purified using Ni-NTA chromatography. Refolding of denatured ocriplasmin to active enzyme was carried out by the stepwise removal of denaturant. The identity of recombinant ocriplasmin was confirmed using western blotting and ELISA assays. The presence of the active ocriplasmin was monitored by the hydrolytic activity assay against the chromogenic substrate S-2403.

The final yield of E. coli BL21-produced ocriplasmin was approximately 1 mg/mL which was greater than that of P. pastoris. Using western blotting and ELISA assay, the identity of recombinant ocriplasmin was confirmed. The hydrolysis of chromogenic substrate S-2403 verified the functional activity of E. coli produced ocriplasmin.

The results of this study indicated that E. coli could be used for high level expression of ocriplasmin. Although the recombinant protein was expressed as inclusion body, the stepwise refolding leads to the biologically active proteins.

The introduction of novel adjuvants is an important step in attempts to develop a safe and more efficient vaccine. The present study was performed to determine whether the use of a mixed beta-adrenergic receptor antagonist propranolol (PRP) and aluminum (alum), as an adjuvant, have efficacy for Toxoplasma gondii (T. gondii) vaccine to induce protective immunity in a mouse model.

Female BALB/c mice divided into five groups were immunized with excretory-secretory antigens (ESA) vaccine, alum-ESA vaccine, PRP-ESA vaccine, and alum-PRP ESA vaccine, as well as with phosphate buffered saline (PBS), as a negative control group. The immune responses were evaluated by lymphocyte proliferation assay for measuring delayed-type hypersensitivity (DTH) response and by cytokine assay for evaluating IFN-γ and IL-5 levels. The survival rate of mice in all groups was assessed during a three-week monitoring period after an intraperitoneal challenge with T. gondii tachyzoites.

The results showed that mice immunized with PRP, as an adjuvant, could secret a higher level of IFN-γ, which was significant in comparison to other groups. However, mice vaccinated with alum-precipitated ESA antigen had ability to produce an elevated level of IL-5 compared to other mouse groups (p≤0.05). Moreover, alum-PRP co-administration together with ESA vaccine resulted in the longer survival of mice.

The findings of this study revealed that the combination of alum-PRP adjuvants and ESA vaccine of T. gondii elicits both humoral and cellular immune responses, which are comparable to either alum or PRP alone.