Possible diagnostic improvement for cutaneous leishmaniasis: Is it achievable?

The parasite, Leishmania, is the causative agent of the disease leishmaniasis and is highly endemic in 98 countries spread across the tropics, subtropics and Mediterranean Basin. It is a vector-borne disease transmitted by the bite of infected sand flies, and it appears in three clinical manifestations namely: cutaneous, mucocutaneous, and visceral leishmaniasis. It is known that at least 21 species are pathogenic to humans. To control the disease, there is no human vaccine, and the sole approach is through chemotherapy. Unfortunately, the available anti-leishmanial drugs are problematic due to their high toxicity, as well as increased parasite resistance. Therefore, making accurate diagnostic decisions for clinical treatment is highly important. Old World cutaneous leishmaniasis [CL] is mostly caused by two Leishmania species, named as L. major and L. tropica that produce skin ulcers. It is also important to note that the symptoms of CL can be confused with other skin diseases. Similar clinical symptoms such as sarcoidosis, lupus vulgaris, leprosy, and bacterial ulcer may have common clinical signs with CL ulcer. Therefore, the diagnostic confirmation of the parasite is mandatory before starting an appropriate treatment strategy

Antitumor Effect of IP-10 by Using Two Different Approaches: Live Delivery System and Gene Therapy / 한국유방암학회지

PURPOSE: Immunotherapy is one of the treatment strategies for breast cancer, the most common cancer in women worldwide. In this approach, the patient's immune system is stimulated to attack microscopic tumors and control metastasis. Here, we used interferon γ-induced protein 10 (IP-10), which induces and strengthens antitumor immunity, as an immunotherapeutic agent. We employed Leishmania tarentolae, a nonpathogenic lizard parasite that lacks the ability to persist in mammalian macrophages, was used as a live delivery system for carrying the immunotherapeutic agent. It has been already shown that arginase activity, and consequently, polyamine production, are associated with tumor progression. METHODS: A live delivery system was constructed by stable transfection of pLEXSY plasmid containing the IP-10-enhanced green fluorescent protein (IP-10-egfp) fusion gene into L. tarentolae. Then, the presence of the IP-10-egfp gene and the accurate integration location into the parasite genome were confirmed. The therapeutic efficacy of IP-10 delivered via L. tarentolae and recombinant pcDNA-(IP-10-egfp) plasmid was compared by determining the arginase activity in a mouse 4T1 breast cancer model. RESULTS: The pcDNA-(IP-10-egfp) group showed a significant reduction in tumor weight and growth. Histological evaluation also revealed that only this group demonstrated inhibition of metastasis to the lung tissue. The arginase activity in the tissue of the pcDNA-(IP-10-egfp) mice significantly decreased in comparison with that in normal mice. No significant difference was observed in arginase activity in the sera of mice receiving other therapeutic strategies. CONCLUSION: Our data indicates that IP-10 immunotherapy is a promising strategy for breast cancer treatment, as shown in the 4T1-implanted BALB/c mouse model. However, the L. tarentolae-(IP-10-EGFP) live delivery system requires dose modifications to achieve efficacy in the applied regimen (six injections in 3 weeks). Our results indicate that the arginase assay could be a good biomarker to differentiate tumoral tissues from the normal ones.

In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-gamma/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

HPV16 E7-CT [gp96] fusion protein: molecular cloning, expression and purification of a recombinant 6xHis-tagged protein in E. coli

The development of a therapeutic vaccine against human papillomavirus [HPV] is important for the control of cervical cancer. E7 is the major transforming protein produced in cervical cancers, and therefore represents potential tumor-specific antigen that could be the target of immunotherapy for cervical cancer. Among different vaccine strategies, protein-based vaccines are capable of generating CD8+ T cell responses in vaccinated animals and humans. Recently, development of novel strategies that enhance protein vaccine potency is important for generation of effective cancer vaccines and immunotherapies. Heat shock proteins [HSPs] including Gp96 have been shown to act as potent immuno-adjuvant to enhance antigen-specific tumor immunity. Therefore, the HSP-based protein vaccines can be administered by fusing antigens to HSPs, in vitro. It has been known that the HSP fragments [e.g., N-/or C-terminal regions] as mini-chaperones are better choice for immunization. The most straightforward method to produce large amounts of recombinant protein suitable for a vaccine is to clone the gene into a prokaryotic expression vector and produce the protein in Escherichia coli. In current study, we describe cloning of the HPV16 E7 gene linked to C-terminal fragment of gp96, identification and purification of the resultant E7-CT [gp96] fusion protein for next usage as a potential vaccine candidate protein against HPV in a pre-clinical trial. The recombinant E7-CT [gp96] migrated as a 51 kDa protein in SDS-PAGE. In Western blot experiment, the existence of a 51 kDa band for rE7-CT [gp96] was confirmed by rabbit anti-His as well as mouse anti-HPV16 E7 monoclonal antibodies. The protein of interest was both in the insoluble and the soluble fraction; therefore, purification was performed under denaturating and native conditions by affinity chromatography on Ni-NTA resin using 6xHis-tag

DNA Immunization as an efficient strategy for vaccination

The field of vaccinology provides excellent promises to control different infectious and non-infectious diseases. Genetic immunization as a new tool in this area by using naked DNA has been shown to induce humoral as well as cellular immune responses with high efficiency. This demonstrates the enormous potential of this strategy for vaccination purposes. DNA vaccines have been widely used to develop vaccines against various pathogens as well as cancer, autoimmune diseases and allergy. However, despite their successful application in many pre-clinical disease models, their potency in human clinical trials has been insufficient to provide protective immunity. Several strategies have been applied to increase the potency of DNA vaccine. Among these strategies, the linkage of antigens to Heat Shock Proteins [HSPs] and the utilization of different delivery systems have been demonstrated as efficient approaches for increasing the potency of DNA vaccines. The uptake of DNA plasmids by cells upon injection is inefficient. Two basic delivery approaches including physical delivery to achieve higher levels of antigen production and formulation with microparticles to target Antigen-Presenting Cells [APCs] are effective in animal models. Alternatively, different regimens called prime-boost vaccination are also effective. In this regimen, naked DNA is utilized to prime the immune system and either recombinant viral vector or purified recombinant protein with proper adjuvant is used for boosting. In this review, we discuss recent advances in upgrading the efficiency of DNA vaccination in animal models

Comparison of two delivery system efficiency by using polyethylenimine [PEI] for plasmid HPV16E7 DNA transfection into COS-7 cells

DNA vaccines have been widely used to develop immunity against various pathogens including parasites and viruses. The potential of DNA vaccine to induce an effective immune response is related to the expression levels of the encoded protein in eukaryotic cells. Therefore, optimization of plasmid DNA delivery system is a major concern in protein expression in order to make an efficient DNA vaccination. Non-viral vectors such as polymers and cationic peptides have been recently known as efficient gene delivery systems into eukaryotic cells. In this study, transfection efficiency of HPV16E7 gene was evaluated by two non-viral delivery systems in vitro. DNA construct encoding HPV16E7 [pEGFP-E7] was prepared in large scale with high purity. Then, two delivery systems including polymer PEI 25 kDa and polymer-peptide hybrid as PEI600-Tat conjugate were used to compare their efficiency for HPV16E7 DNA transfection in vitro. Our data demonstrated that both delivery systems including PEI 25 kDa and PEI600-Tat in vitro, but its toxicity was obstacle in vivo. Therefore, with regard to low toxicity of PEI600-Tat delivery system and its potent plasmid DNA delivery, it is critical issue to study its potency as new delivery system in vivo

Construction, expression and preliminary immunological evaluation of a DNA plasmid encoding the GRA2 protein of toxoplasma gondii

Toxoplasmosis is a worldwide infection which is commonly asymptomatic but can cause serious medical problems in immunocompromised individuals and fetus. The infection also causes considerable economic loss because of abortion in livestock, mostly in sheep and goats. DNA vaccination may be a powerful approach against intracellular parasites such as Toxoplasma gondii. The goal of this study was to construct and evaluate the functionality of an eukaryotic expression plasmid pRC/CMV-GRA2, harboring dense granule antigen-2 [GRA2] gene of T. gondii and to perform preliminary studies on its immunogenicity in a mouse model. The GRA2 complete cDNA was inserted in PCR2.1 plasmid, sequenced, then cut and inserted in pRC/CMV plasmid, to produce the recombinant plasmid pRC/CMV-GRA2 [pGRA2]. To verify that the plasmid construct pGRA2 was capable of expressing GRA2 in mammalian cells, it was transfected into 293-T cells, an embryonic kidney cell line. Western-blot analysis of the transfected cells using a monoclonal antibody specific for GRA2 indicated specific expression of GRA2 protein. CBA/J mice were subcutaneously immunized three times with 100 micro g of pGRA2 plasmid. The obtained sera recognized GRA2 that is shown by Western-blotting. These findings indicate that pGRA2 plasmid directs high-level expression of antigenic GRA2 protein in mammalian cells and is immunogenic in CBA/J mice

High level expression of recombinant ribosomal protein [L7/L12] from Brucella abortus and its reaction with infected human sera

Brucellosis, caused by Brucella spp., is an important zoonotic disease that causes abortion and infertility in cattle and undulant fever in humans. Various studies have examined cell-free native and recombinant proteins as candidate protective antigens in animal models. Among Brucella immunogenes, antigen based on ribosomal preparation has been widely investigated. In this study, the immunogenic ribosomal protein L7/L12 gene from Brucella abortus, S19, was amplified by PCR and sub-cloned to prokaryotic expression vector pET28a. Escherichia coli BL21 [DE3] pLysS was transformed with pET28a-L7/L12 and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography with Ni-NTA resin. The concentration of purified recombinant protein calculated to 8 mg/L of initial culture. The integrity of product was confirmed by Western-blot analysis using a standard rabbit anti Brucella abortus ribosomal protein L7/L12 antibody. Sera reactivity of five infected individual were further analyzed against the recombinant ribosomal L7/L12 protein. Data indicated that recombinant ribosomal L7/L12 protein from Brucella abortus was recognized by patient sera