Identification of new potential candidates to inhibit EGF via machine learning algorithm.

One of the cost-effective alternative methods to find new inhibitors has been the repositioning approach of existing drugs. The advantage of computational drug repositioning method is saving time and cost to remove the pre-clinical step and accelerate the drug discovery process. Hence, an ensemble computational-experimental approach, consisting of three steps, a machine learning model, simulation of drug-target interaction and experimental characterization, was developed. The machine learning type used here was a different tree classification method, which is one of the best randomize machine learning model to identify potential inhibitors from weak inhibitors. This model was trained more than one-hundred times, and forty top trained models were extracted for the drug repositioning step. The machine learning step aimed to discover the approved drugs with the highest possible success rate in the experimental step. Therefore, among all the identified molecules with more than 0.9 probability in more than 70% of the models, nine compounds, were selected. Besides, out of the nine chosen drugs, seven compounds have been confirmed to inhibit EGF in the published articles since 2019. Hence, two identified compounds, in addition to gefitinib, as a positive control, five weak-inhibitors and one neutral, were considered via molecular docking study. Finally, the eight proposed drugs, including gefitinib, were investigated using MTT assay and In-Cell ELISA to characterize the drugs' effect on A431 cell growth and EGF-signaling. From our experiments, we could conclude that salicylic acid and piperazine could play an EGF-inhibitor role like gefitinib.

Latent toxoplasmosis impairs learning and memory yet strengthens short-term and long-term hippocampal synaptic plasticity at perforant pathway-dentate gyrus, and Schaffer collatterals-CA1 synapses.

Investigating long-term potentiation (LTP) in disease models provides essential mechanistic insight into synaptic dysfunction and relevant behavioral changes in many neuropsychiatric and neurological diseases. Toxoplasma (T) gondii is an intracellular parasite causing bizarre changes in host's mind including losing inherent fear of life-threatening situations. We examined hippocampal-dependent behavior as well as in vivo short- and long-term synaptic plasticity (STP and LTP) in rats with latent toxoplasmosis. Rats were infected by T. gondii cysts. Existence of REP-529 genomic sequence of the parasite in the brain was detected by RT-qPCR. Four and eight weeks after infection, spatial, and inhibitory memories of rats were assessed by Morris water maze and shuttle box tests, respectively. Eight weeks after infection, STP was assessed in dentate gyrus (DG) and CA1 by double pulse stimulation of perforant pathway and Shaffer collaterals, respectively. High frequency stimulation (HFS) was applied to induce LTP in entorhinal cortex-DG (400 Hz), and CA3-CA1 (200 Hz) synapses. T. gondii infection retarded spatial learning and memory performance at eight weeks post-infection period, whereas inhibitory memory was not changed. Unlike uninfected rats that normally showed paired-pulse depression, the infected rats developed paired-pulse facilitation, indicating an inhibitory synaptic network disruption. T. gondii-infected rats displayed strengthened LTP of both CA1-pyramidal and DG-granule cell population spikes. These data indicate that T. gondii disrupts inhibition/excitation balance and causes bizarre changes to the post-synaptic neuronal excitability, which may ultimately contribute to the abnormal behavior of the infected host.

Positive effect of acellular amniotic membrane dressing with immobilized growth factors in skin wound healing.

The human amniotic membrane dressing has been shown to accelerate the wound healing process in the clinic. In this study, heparin was conjugated to a human Acellular Amniotic Membrane (hAAM) to provide affinity binding sites for immobilizing growth factors. To study the acceleration of the wound healing process, we bound epidermal growth factor and fibroblast growth factor 1 to heparinized hAAMs (GF-Hep-hAAMs). The heparinized hAAMs (Hep-hAAMs) were characterized by toluidine blue staining and infrared spectroscopy. The quality control of hAAM was performed by hematoxylin staining, swelling capacity test and biomechanical evaluation. The cytotoxicity, adhesion, and migration in vitro assays of GF-Hep-hAAMs on L-929 fibroblast cells were also studied by MTT assay, scanning electron microscopy, and scratch assay, respectively. Finally, in vivo skin wound healing study was performed to investigate the wound closure rate, re-epithelization, collagen deposition, and formation of new blood vessels. The results showed that GF-Hep-hAAMs enhance the rate of wound closure and epidermal regeneration in BALB/c mice. In conclusion, GF-Hep-hAAMs could accelerate the wound healing process, significantly in the first week.

Rational Design and Production of Bioactive Analogs of Recombinant Human Keratinocyte Growth Factor (rhKGF) with Reduced Aggregation Propensity.

Recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The present study aimed to reduce aggregation propensity of rhKGF by engineering the aggregation hotspots. Initially, 21 mutants were designed based on the previously-identified aggregation-prone regions (APRs) and then four of them including mutants No. 4 (L91K, I119K), 7 (V13S, L91K), 14 (L91D, I119D), and 21 (A51E) were selected based on molecular dynamics (MD) simulations for further experimental studies. The recombinantly produced rhKGF and mutants were analyzed regarding secondary structure, thermal stability, aggregation propensity, and biological activity. Far-UV CD spectroscopy showed that the mutants have similar secondary structure with rhKGF. A51E mutant showed enhanced stability and decreased monomer loss under heat stress suggesting its reduced aggregation propensity compared to rhKGF. Mutant No. 14 showed higher stability and less aggregation tendency than mutant No. 4 indicating that only mutations decreasing pI of rhKGF are effective in reducing its aggregation tendency. All of the mutants were at least as potent as rhKGF in stimulating proliferation of MCF-7 epithelial cells. Our results identified A51E as an equally potent, more stable, and less aggregation-prone analog of rhKGF which could be a promising alternative drug candidate for the commercially available rhKGF (Palifermin).

In silico design of fusion keratinocyte growth factor containing collagen-binding domain for tissue engineering application.

Keratinocyte growth factor (KGF) is a potential therapeutic factor in wound healing. However, its applications have been restricted due to its low stability, short half-life, and limited target specificity. We aimed to immobilize KGF on collagen-based biomaterials for long-lasting and targeted therapy by designing fusion forms of KGF with collagen-binding domains (CBD) from natural origins. Twelve fusion proteins were designed consisting of KGF and CBDs with different lengths and amino acid compositions. Three-dimensional (3D) structures of the fusions were predicted by homology modeling. Physiochemical properties and secondary structure of the fusions were evaluated by bioinformatics tools. Moreover, the effect of the CBDs on the 3D structure and dynamic behavior of the fusions was investigated by molecular dynamics (MD) simulation. The binding affinity of the fusions to collagen, KGF receptor, and heparin was assessed using docking tools. Our results demonstrated that fusions with small CBDs like CBD of mammalian collagenase and decapeptide CBD of von Willebrand factor (VWF) were more stable and properly folded than those with larger CBDs. On the other hand, the insertion of bulky CBDs, including Fibronectin CBD and CBD of Clostridium histolyticum collagenase, into KGF resulted in stronger binding to collagen. Therefore, very small or large CBDs are inappropriate for constructing KGF fusions because they suffer from low collagen affinity or poor stability. By comparing the results of MD simulation and docking, this study proposed that CBDs belonging to Vibrio mimicus metalloprotease and A3 domain of VWF would be good candidates to produce stable fusions with proper affinities toward collagen and KGF receptors. Moreover, the secondary structure analysis showed that the overall structure of KGF and CBDs was better preserved when CBDs were inserted at the C-terminal of KGF. This computational information about novel KGF fusions may help find the best constructs for experimental studies.

Enhancement of keratinocyte growth factor potential in inducing adipose-derived stem cells differentiation into keratinocytes by collagen-targeting.

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials.

Quality by Design in Downstream Process Development of Romiplostim

Background: Background: Downstream processing of therapeutic recombinant proteins expressed as the inclusion bodies (IBs) in E. coli is quite challenging. This study aimed to use the quality by design approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim. Methods: Methods: For development of a successful downstream process, risk analysis and experimental designs were used to characterize the most critical quality attributes (CQAs) and effects of process parameters on these quality attributes. Results: Results: The solubilization of IBs was optimized by design of experiment on three parameters with a focus on solubility yield, which resulted in >75% increase of the target protein solubilization. The pH of sample was identified as CQA in anion exchange chromatography that might have an impact on achieving >85% host cell proteins removal and >90% host cell DNA reduction. In the refolding step, process parameters were screened. Cystine/cysteine ratio, pH, and incubation time identified as CPPs were further optimized using Box-Behnken analysis, which >85% of the target protein was refolded. The design space for further purification step by HIC was mapped with a focus on high molecular weight impurities. After polishing by gel filtration, the final product's biological activity showed no statistically significant differences among the groups received romiplostim and Nplate®, as the reference product. Conclusions: Conclusion: This research presents a precise and exhaustive model for mapping the design space in order to describe and anticipate the link between the yield and quality of romiplostim and its downstream process parameters.

Potential aggregation hot spots in recombinant human keratinocyte growth factor: a computational study.

The recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The high aggregation liability of rhKGF is manifested by loss of the monomeric state, and accumulation of the aggregated species even at moderate temperatures. Here, we analyzed the rhKGF for its vulnerability toward aggregation by detection of aggregation-prone regions (APRs) using several sequence-based computational tools including TANGO, ZipperDB, AGGRESCAN, Zyggregator, Camsol, PASTA, SALSA, WALTZ, SODA, Amylpred, AMYPDB, and structure-based tools including SolubiS, CamSol structurally corrected, Aggrescan3D and spatial aggregation propensity (SAP) algorithm. The sequence-based prediction of APRs in rhKGF indicated that they are mainly located at positions 10-30, 40-60, 61-66, 88-120, and 130-140. Mapping on the rhKGF structure revealed that most of these residues including F16-R25, I43, E45, R47-I56, F61, Y62, N66, L88-E91, E108-F110, A112, N114, T131, and H133-T140 are surface-exposed in the native state which can promote aggregation without major unfolding event, or the conformational change may occur in the oligomers. The other regions are buried in the native state and their contribution to non-native aggregation is mediated by a preceding unfolding event. The structure-based prediction of APRs using the SAP tool limited the number of identified APRs to the dynamically-exposed hydrophobic residues including V12, A50, V51, L88, I89, L90, I118, L135, and I139 mediating the native-state aggregation. Our analysis of APRs in rhKGF identified the regions determining the intrinsic aggregation propensity of the rhKGF which are the candidate positions for engineering the rhKGF to reduce its aggregation tendency.Communicated by Ramaswamy H. Sarma.

Co-delivery of PLGA nanoparticles loaded with rSAG1 antigen and TLR ligands: An efficient vaccine against chronic toxoplasmosis.

Although vaccination is a promising approach for the control of toxoplasmosis, there is currently no commercially available human vaccine. Adjuvants such as delivery vehicles and immunomodulators are critical components of vaccine formulations. In this study, Poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles were applied to serve as delivery system for both surface antigen-1 (SAG1), a candidate vaccine against toxoplasmosis and two TLR ligands, monophosphoryl lipid A (MPL) and imiquimod (IMQ), respectively. Compared to rSAG1 alone, CBA/J mice immunized with rSAG1-PLGA produced higher anti-SAG1 IgG antibodies titers. This response was increased by the co-administration of IMQ-PLGA (p < 0.01). Compared to IMQ-PLGA co-administration, MPL-PLGA co-administration further increased the humoral response (p < 0.01) and potentiated the Th1 humoral response. Compared to rSAG1 alone, rSAG1-PLGA, or rSAG1-PLGA mixed with IMQ-PLGA or MPL-PLGA similarly enhanced the cellular response characterized by the production of IFN-γ, IL-2, TNF-α and low levels of IL-5, indicating a Th1-biased immunity. The induced immune responses, led to significant brain cyst reductions (p < 0.01) after oral challenge with T. gondii cysts in mice immunized with either rSAG1-PLGA, rSAG1-PLGA + IMQ-PLGA, rSAG1-PLGA + MPL-PLGA formulations. Taken together the results indicated that PLGA nanoparticles could serve as a platform for dual-delivery of antigens and immunomodulators to provide efficacious vaccines against toxoplasmosis.

Valproic Acid Inhibits Chronic Toxoplasma Infection and Associated Brain Inflammation in Mice.

Individuals infected with Toxoplasma gondii are prone to psychobehavioral disorders, most notably schizophrenia and bipolar disorder. Valproic acid reportedly inhibits the proliferation of T. gondii tachyzoites in vitro. However, animals treated with the drug neither lived longer during acute infection nor had fewer brain cysts upon chronic infection. In this study, a quantitative real-time PCR (qPCR) method was applied to quantify copy numbers of BAG1 (a bradyzoite-specific protein), REP529 DNA (a repetitive DNA fragment of the parasite), and SAG1 (a highly expressed tachyzoite-specific surface protein) in the brains of chronically infected mice treated with valproic acid. The treatment inhibited the infection and decreased BAG1, SAG1, and REP529 copy numbers in mice brains (P < 0.0001), comparable to the effects of trimethoprim-sulfamethoxazole (TMP-SMZ), the common medication for toxoplasmosis treatment. Moreover, valproic acid decreased brain tumor necrosis factor alpha (TNF-α) expression (P < 0.0001) comparably to TMP-SMZ. Histological examination of mouse brains showed marked reductions in cyst establishment, perivascular infiltration of lymphocytes, and glial nodules to the same levels as those in the TMP-SMZ group. Our results provide direct evidence for the efficacy of valproic acid, a mood-stabilizing and antipsychotic drug, against chronic Toxoplasma infection. These results might help modulate therapeutic regimens for neuropsychiatric patients and aid in the design of more effective anti-Toxoplasma drugs.

The Role of Recombinant Fibroblast Growth Factor 1 in Enhancing the Angiogenesis in Random Cutaneous Flaps in Animal Model of Rat.

BACKGROUND: Randomized skin flaps have been used as a basic treatment modality for covering skin defects for a long time but they have always been in the risk of an inherent ischemia. Fibroblast growth factor 1 is a known angiogenic factor in in vitro studies which has shown conflicting results in in vivo investigation. We aimed to determine the effect of recombinant fibroblast growth factor on the angiogenesis rate of random cutaneous flap in animal model of rats. METHODS: This experimental study was conducted on 24 adult male rats randomized to 2 groups. In the first group FGF1 was injected subdermally in equally divided doses and distances of random flap surface in days 1, 3 and 5. In second group, normal saline was injected as control. Flap surgery was done on day 21 after first injection. The extent of necrosis and angiogenesis (mean vessel density) were assessed in day 14 after surgery. RESULTS: The mean percentage of clinically apparent necrosis was 35.2% (±10.5) in intervention (FGF1) group and 38.1% (±8.7) in control (normal saline), respectively. Mean vessel density was 86.20±5.6/mm2 in control group and 90.17±5.5/mm2 in intervention group, which showed no statistically significant difference. CONCLUSION: Mean vessel density and mean percentage of clinically apparent necrosis area were similar in 2 groups of rats with random cutaneous flaps receiving FGF1 or normal saline.

Recombinant pebulin protein, a type 2 ribosome-inactivating protein isolated from dwarf elder (Sambucus ebulus L.) shows anticancer and antifungal activities in vitro.

In this study, encoding sequence of a new type 2 RIP (pebulin) was isolated and cloned from dwarf elder (Sambucus ebulus L.) native to the northern regions of Iran. The nucleotide sequence of pebulin was ligated to the pET-28a(+) expression plasmid and cloned into the E. coli strain BL21 (DE3) in order to express heterologously of recombinant protein. The recombinant pebulin protein was mainly produced in the form of insoluble inclusion bodies probably because to absence of N-glycosylation process in E. coli. Therefore, in order to increase the expression of recombinant protein in soluble form, co-expression of the target protein with the pG-Tf2 chaperone plasmid and incubation of bacterial culture under low temperature were used to enhance solubility and accumulation of recombinant protein. After purification of the recombinant protein using affinity chromatography method, the bioactivity of pebulin was analyzed by hemagglutination, anticancer, and antifungal assays. The results of the hemagglutination assay showed that purified pebulin agglutinated erythrocytes in all human blood groups. In addition, pebulin considerably inhibited the proliferation of cancer cell lines MCF-7 and HT-29 in a time- and dose-dependent manner and indicated remarkably growth-inhibiting effect against the plant pathogenic fungi such as Alternaria solani and Fusarium oxysporum.

Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples.

BACKGROUND: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples. METHODS: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction). RESULTS: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant. CONCLUSION: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.

Isolation and characterization of nanobodies against epithelial cell adhesion molecule as novel theranostic agents for cancer therapy.

Epithelial cell adhesion molecule (EpCAM) plays an important role in tumorigenesis. Camelids produce functional antibodies composed of heavy chains only that bind to their antigens via a single domain variable fragment known as nanobody. Nanobodies show multiple advantages over traditional monoclonal antibodies. Isolation of functional anti-EpCAM nanobodies (Nbs) was the main aim of this study. An immune nanobody library containing 108 members was constructed previously. Anti -EpCAM nanobodies were isolated from camel immune library using phage display. Four consecutive rounds of biopanning were performed on immobilized EpCAM. Four nanobodies (Nb4, Nb5, Nb22, and Nb23) with highest signal intensity in monoclonal phage ELISA were selected. Affinity of these selected nanobodies for EpCAM was in the nanomolar range. Selected nanobodies significantly inhibited proliferation of MCF-7 cells. The in vivo study revealed that a significant reduction in tumor size occurred when treated with nanobodies Nb4 and Nb5, after 14 days monitoring. Our data revealed that nanobodies Nb4 and Nb5 could be considered as attractive theranostic agents for EpCAM overexpressing cancers.

Multi-Laboratory Evaluation of a Lateral Flow Rapid Test for Detection of Amebic Liver Abscess.

Independent evaluations of XEh Rapid®, an IgG4-based rapid dipstick test, were performed to assess its diagnostic performance to detect amebic liver abscess (ALA) using 405 samples at seven laboratories in four countries. The test showed high diagnostic specificity (97-100%) when tested with samples from healthy individuals (n = 100) and patients with other diseases (n = 151). The diagnostic sensitivity was tested with a total of 154 samples, and the results were variable. It was high in three laboratories (89-94%), and moderate (72%) and low (38%) in two other laboratories. Challenges and issues faced in the evaluation process are discussed. Nevertheless, XEh Rapid is promising to be developed into a point-of-care test in particular for resource-limited settings, and thus merits further confirmation of its diagnostic sensitivity.

Size effect of human epidermal growth factor-conjugated polystyrene particles on cell proliferation.

Conjugation of growth factors to a carrier is a favorable method to improve their efficacy as therapeutic molecules. Here, we report the carrier size effect on bioactivity of human epidermal growth factor (hEGF) conjugated to polystyrene particles. BALB/3T3 cells were treated with hEGF-conjugated particles (hEGF-conjs) sized from 20 to 1000 nm. At hEGF concentrations less than 0.5 ng ml-1, free hEGF was more potent than the hEGF-conjs at inducing cell proliferation. However, cell proliferation was size-dependent at higher concentrations of hEGF i.e. hEGF-conjs sized equal to or less than 200 nm displayed lower cell proliferation, compared to free hEGF, but larger particles showed increased cell proliferation. This is in agreement with previous studies showing accumulation of activated-EGFRs in early endosomes triggers apoptosis of A431 and HeLa cells. The confocal microscopy and co-localization fluorescence staining showed the 500 and 1000 nm hEGF-conjs exclusively remained on the cell surface, probably enabling them to activate EGF receptors for a longer time. Conversely, smaller particles were mostly inside the cells, indicating their rapid endocytosis. Similarly, A431 cells treated with 20 nm hEGF-conj, endocytosed the particles and experienced decreased cell proliferation, while the 500 and 1000 nm hEGF-conjs were not internalized, and induced partial cell proliferation. Moreover, we showed multivalency of hEGF-conjs is not the cause of enhanced cell proliferation by large particles, as the degree of EGFR phosphorylation by free EGF was higher, compared to hEGF-conjs. Our results suggest the potential of micron-sized particles as a carrier for hEGF to enhance cell proliferation, which could be explored as a promising approach for topical application of growth factors for accelerating wound healing.

Molecular cloning and in-depth bioinformatics analysis of type II ribosome-inactivating protein isolated from Sambucus ebulus.

Plant ribosome-inactivating proteins (RIPs) are N-glycosidases which inhibit protein synthesis through depurination of the ribosomal RNA sequence. Type II RIPs are heterodimer proteins which can bind to cell surfaces. The cytotoxicity of these RIPs is different. Sambucus spp. are a rich source of RIP proteins with different properties. In the present study, a type II RIP was isolated from S. ebulus plant that grows widely in the north of Iran, and different bioinformatics tools were used for the evaluation of physicochemical, functional and 3D protein characteristics. The results showed significant differences among isolated RIP and other Sambucus RIP proteins. The study of these differences can not only expand our insight into the functioning mechanisms of plant RIPs but also provide information about a novel RIP protein with potential biological applications.

Evaluation of Immunogenic Effect of Toxoplasma gondii Recombinant SAG-1 Antigen with Propranolol as Adjuvant in BALB/c Mice.

Purpose: Propranolol as a novel adjuvant, was used to evaluate the immunogenic effect of three doses of recombinant SAG-1 (rSAG-1) antigen of Toxoplasma gondii in BALB/c mice for finding the optimal dose, and was compared with efficacy of tachyzoite lysate antigen (TLA). Methods: Eight different groups of 15 BALB/c mice received different volumes of the immunogenic material (three doses of r SAG-1 and one dose of TLA antigens), with or without propranolol adjuvant, subcutaneously. The control group mice received only PBS. Three weeks after the last immunization, the serum levels of IgG2a, IgG1 and IgG total antibodies against TLA, splenic interleukin-5 (IL-5) and Interferon-gamma (IFN-γ) (produced against TLA) and the splenic lymphocyte proliferation after adding TLA were measured to evaluate humoral and cellular immune responses. Challenge test was performed by subcutaneously injection of 1000 alive and active tachyzoites in to five mice per each group and survival days for each group of mice were recorded. Results: The mice group that received propranolol adjuvant and 20 µg of r SAG-1 antigen per dose of injection showed significantly more IFN-γ production, more proliferation of splenic lymphocytes and higher anti-TLA-specific IgG2a production (three main indexes for cell mediated immunity) in comparison with other groups. Moreover, in the challenge test, this group of mice had a significantly increased survival time, indicating the positive effect of propranolol in the more stimulating of cellular immunity that is necessary for toxoplasmosis prevention or suppress. Conclusion: Our results showed that T. gondii rSAG-1 antigen in combination with propranolol as adjuvant (which can induce Th1 related responses) are good candidates for further study to a vaccine design.

Prevalence of Toxoplasma gondii Antibodies and DNA in Iranian HIV Patients.

BACKGROUND & OBJECTIVE: Toxoplasma gondii infection has public health importance and can lead to serious diseases in immunosuppressed patients, such as HIV cases. Appropriate control of T. gondii infection in HIV patients requires information about the prevalence of T. gondii antibodies and DNA in different population. In this study, we aimed to determine the prevalence of Toxoplasma gondii antibodies and DNA in HIV patients in Tehran, Iran. METHODS: A total of 149 HIV patients from the Iranian Research Center for HIV/AIDS, Tehran, Iran were enrolled in the study. Anti-Toxoplasma IgG and IgM were detected by ELISA and T. gondii DNA was evaluated by PCR and quantita- tive real-time PCR. IgG positive samples were also assessed for their avidity. RESULTS: Anti-Toxoplasma IgG and IgM were positive in 46.3% and 2.7% of cases respectively. 92.7% of our patients showed past infection and 4.3% revealed recently acquired toxoplasmosis based on their IgG avidity test. T. gondii DNA was not detected by PCR but real-time PCR results showed DNA in 4.7% of total patients and 13.1% of the IgG seropositive cases. CONCLUSION: Our findings indicated that latent toxoplasmosis was relatively prevalent in our study population, but new T. gondii infection had low prevalence. Almost half of our patients were IgG negative and at risk of acquiring toxoplasma infection. Low copy numbers of DNA were detected in 4.7% of the cases without any clinical manifestation. Therefore, detection and monitoring of anti-Toxoplasma antibodies and DNA in HIV patients is substantial to estimate the risk of reactivation and new infection.

In silico analysis of different signal peptides for the secretory production of recombinant human keratinocyte growth factor in Escherichia coli.

BACKGROUND: The recombinant human truncated Keratinocyte growth factor (Palifermin) is the only FDA approved medicine for the treatment of oral mucositis. The Keratinocyte growth factor is a fairly unstable protein due to its high aggregation propensity and therefore its expression as a secretory protein may results in the production of a protein with more stability, higher solubility, better folding, enhanced biological activity, N-terminal authenticity and simplified downstream processing. OBJECTIVE: The aim of this study was in silico evaluation of 31 different secretory signal peptides to determine the best theoretical candidates for the secretory production of recombinant truncated human KGF in E. coli. METHODS: Thirty different prokaryotic signal peptides experimentally shown to be capable of recombinant protein secretion in E.coli, along with the native KGF signal peptide were selected for further investigations. The signal peptide sequences were retrieved from the UniProt database. The ability of SPs to act as a secretory leader peptide for rhKGF and the location of cleavage sites were predicted by SignalP 4.1. Physicochemical properties of the signal peptides, which may influence protein secretion, were analyzed by ProtParam and PROSOII. Furthermore, the mRNA secondary structure and Gibbs free energy profile of the selected SPs were analyzed in the fusion state with the rhKGF using Visual Gene Developer package. RESULTS AND CONCLUSION: Computational analysis of the physicochemical properties affecting protein secretion identified Sec-B dependent OmpC, Bla, and StaI and SRP dependent TolB signal peptides as the best theoretical candidates for the secretory production of recombinant truncated human KGF in E.coli.