The role of serum circulating microbial toxins in severity and cytokine storm of COVID positive patients.

The emergence of Coronavirus disease 2019 (Covid-19) is a global problem nowadays, causing health difficulty with increasing mortality rates, which doesn't have a verified treatment. SARS-CoV-2 infection has various pathological and epidemiological characteristics, one of them is increased amounts of cytokine production, which in order activate an abnormal unrestricted response called "cytokine storm". This event contributes to severe acute respiratory distress syndrome (ARDS), which results in respiratory failure and pneumonia and is the great cause of death associated with Covid-19. Endotoxemia and the release of bacterial lipopolysaccharides (endotoxins) from the lumen into the bloodstream enhance proinflammatory cytokines. SARS-CoV-2 can straightly interplay with endotoxins via its S protein, leading to the extremely elevating release of cytokines and consequently increase the harshness of Covid-19. In this review, we will discuss the possible role of viral-bacterial interaction that occurs through the transfer of bacterial products such as lipopolysaccharide (LPS) from the intestine into the bloodstream, exacerbating the severity of Covid-19 and cytokine storms.

Staphylococcal enterotoxin B as DNA vaccine against breast cancer in a murine model.

Recently, many efforts have been made to treat cancer using recombinant bacterial toxins and this strategy has been used in clinical trials of various cancers. Therapeutic DNA cancer vaccines are now considered as a promising strategy to activate the immune system against cancer. Cancer vaccines could induce specific and long-lasting immune responses against tumors. This study aimed to evaluate the antitumor potency of the SEB DNA vaccine as a new antitumor candidate against breast tumors in vivo. To determine the effect of the SEB construct on inhibiting tumor cell growth in vivo, the synthetic SEB gene, subsequent codon optimization, and embedding the cleavage sites were sub-cloned to an expression vector. Then, SEB construct, SEB, and PBS were injected into the mice. After being vaccinated, 4T1 cancer cells were injected subcutaneously into the right flank of mice. Then, the cytokine levels of IL-4 and IFN-γ were estimated by the ELISA method to evaluate the antitumor activity. The spleen lymphocyte proliferation, tumor size, and survival time were assessed. The concentration of IFN-γ in the SEB-Vac group showed a significant increase compared to other groups. The production of IL-4 in the group that received the DNA vaccine did not change significantly compared to the control group. The lymphocyte proliferation increased significantly in the mice group that received SEB construct than PBS control group (p < 0.001). While there was a meaningful decrease in tumor size (p < 0.001), a significant increase in tumor tissue necrosis (p < 0.01) and also in survival time of the animal model receiving the recombinant construct was observed. The designed SEB gene construct can be a new model vaccine for breast cancer because it effectively induces necrosis and produces specific immune responses. This structure does not hurt normal cells and is a safer treatment than chemotherapy and radiation therapy. Its slow and long-term release gently stimulates the immune system and cellular memory. It could be applied as a new model for inducing apoptosis and antitumor immunity to treat cancer.

Immune response induced by recombinant pres2/S-protein and a pres2-S-protein fused with a core 18-27 antigen fragment of hepatitis B virus compared to conventional HBV vaccine.

Although comprehensive vaccination is the cornerstone of public health programs to control hepatitis B virus (HBV) infections, 5% of people who receive the existing vaccine do not develop proper immunity against HBV. To overcome this challenge, researchers have tried using various protein fragments encoded by the virus genome to achieve better immunization rates. An important antigenic component of HBsAg called the preS2/S or M protein has also received much attention in this area. The gene sequences of preS2/S and Core18-27 peptide were extracted from the GenBank (NCBI). Final gene synthesis was conducted with pET28. Groups of BALB/c mice were immunized with 10 µg/ml of recombinant proteins and 1 µg/ml CPG7909 adjuvant. Serum levels of IF-γ, TNF-α, IL-2, IL-4, and IL-10 were measured by ELISA assay method on spleen cell cultures on day 45, and IgG1, IgG2a, and total IgG titers obtained from mice serum were quantified on days 14 and 45. Statistical analysis did not show any significant difference between the groups regarding IF-γ level. There were, however, significant differences in terms of IL-2 and IL-4 levels between the groups receiving preS2/S-C18-27 with and without adjuvant and the groups receiving both preS2/S and preS2/S-C18-27 (Plus Recomb-Plus Recomb: the group of mice that received both preS2/S and preS2/S-C18-27 simultaneously). The strongest total antibody production was induced by immunization with both recombinant proteins without CPG adjuvant. The groups that received both preS2/S and preS2/S-C18-27, whether with or without adjuvant, were significantly different from those that received the conventional vaccine considering most abundant interleukins. This difference suggested that higher levels of efficacy can be achieved by the use of multiple virus antigen fragments rather than using a single fragment.

Interaction Between SARS-CoV-2 and Pathogenic Bacteria.

The novel human coronavirus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which results in the coronavirus disease 2019 (COVID-19), has caused a serious threat to global public health. Therefore, many studies are performed on the causes and prevalence of this disease and the possible co-occurrence of the infection with other viral and bacterial pathogens is investigated. Respiratory infections predispose patients to co-infections and these lead to increased disease severity and mortality. Numerous types of antibiotics have been employed for the prevention and treatment of bacterial co-infection and secondary bacterial infections in patients with a SARS-CoV-2 infection. Although antibiotics do not directly affect SARS-CoV-2, viral respiratory infections often result in bacterial pneumonia. It is possible that some patients die from bacterial co-infection rather than virus itself. Therefore, bacterial co-infection and secondary bacterial infection are considered critical risk factors for the severity and mortality rates of COVID-19. In this review, we will summarize the bacterial co-infection and secondary bacterial infection in some featured respiratory viral infections, especially COVID-19.

Evaluating the Performance of PPE44, HSPX, ESAT-6 and CFP-10 Factors in Tuberculosis Subunit Vaccines.

Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen causing long-term infection in humans that mainly attacks macrophages and can escape from the immune system with the various mechanisms. The only FDA-approved vaccine against M. tuberculosis (MTB) is Mycobacterium bovis bacillus Calmette-Guérin (BCG). The protection of this vaccine typically lasts 10-15 years. Due to the increasing number of people becoming ill with MTB each year worldwide, the need to develop a new effective treatment against the disease has been increased. During the past two decades, the research budget for TB vaccine has quadrupled to over half a billion dollars. Most of these research projects were based on amplifying and stimulating the response of T-cells and developing the subunit vaccines. Additionally, these studies have demonstrated that secretory and immunogenic proteins of MTB play a key role in the pathogenesis of the bacteria. Therefore, these proteins were used to develop the new subunit vaccines. In this review, based on the use of these proteins in the successful new subunit vaccines, the PPE44, HSPX, CFP-10 and ESAT-6 antigens were selected and the role of these antigens in designing and developing new subunit vaccines against TB and for the prevention of TB were investigated.

The impact of the gut microbiome on toxigenic bacteria.

Millions of symbiotic and pathogenic microorganisms known as microbiota colonize the host body. The microbiome plays an important role in human health and colonizes hundreds of different species of multicellular organisms so that they are introduced as the metaorganisms. Changes in the microbial population of the gut microbiome may cause resistance to pathogenic bacteria-induced infection. Understanding the principles of Host-Microbiota Interactions (HMIs) is important because it clarifies our insight towards the mechanisms of infections established in the host. Interactions between the host and the microbiota help answer the question of how a microorganism can contribute to the health or disease of the host. Microbiota can increase host resistance to colonization of pathogenic species. Studying the HMIs network can in several ways delineate the pathogenic mechanisms of pathogens and thereby help to increase useful and novel therapeutic pathways. For example, the potentially unique microbial effects that target the distinct host or interfere with the endogenous host interactions can be identified. In addition, the way mutations in essential proteins in the host and/or in the microbes can influence the interactions between them may be determined. Furthermore, HMIs help in identifying host cell regulatory modules.

The inhibitory impacts of Lactobacillus rhamnosus GG-derived extracellular vesicles on the growth of hepatic cancer cells.

Bacterial extracellular vesicles (EVs) have come forth into notice as possible important agent to mediate host-pathogen interactions. In this scientific research, the authors have tried to find out the effect of EVs derived from Lactobacillus rhamnosus GG (LDEVs) on the apoptosis induction in HepG2 cell line. The EVs were purified from the conditioned medium of Lactobacillus rhamnosus GG using ultrafiltration and confirmed by transmission electron microscopy (TEM). The HepG2 cells were treated with different concentrations of purified LDEVs and the cytotoxicity and their effects on the expression of bcl-2 and bax genes were assessed by the MTT assay and semi-quantitative RT-PCR, respectively. The MTT assay showed that only 100 µg/ml of LDEVs had a significant cytotoxic effect on cancer cells (p < 0.05). The apoptotic index (bax/bcl2 expression ratio) was significantly increased after treating with 50 and 100 µg/ml LDEVs (p < 0.05). Increased bax/bcl-2 ratio was led to cancer cell death.

Macrophage cell-derived exosomes/staphylococcal enterotoxin B against fibrosarcoma tumor.

Targeted immune therapies are a modern approach to harness the immunity to treat cancer patients. Exosomes (EXOs) are nano-vesicles used for drug delivery in cancer treatment. We aimed to assess the effectiveness of novel designed EXO structures for immunotherapy alone and in combination with other components in animal models. EXO derived from untreated macrophage (EXO), WEHI-164 cell lysate treated EXO (EXOLys), HSP70 enriched WEHI-164 cell lysate treated EXO (EXOHSP70), Naloxone (NLX) treated EXO (EXONLX), Propranolol (PRP) treated EXO (EXOPRP) and staphylococcal enterotoxin B (SEB) anchored to three kinds of EXOs designated as EXO/SEB, EXOLys/SEB, EXOHSP70/SEB were purified from J774 cell line. To determine the therapeutic effect of these novel constructed nano-vesicles, the animals were immunized with different types of EXOs at weekly intervals for three consecutive weeks and in the fourth week the WEHI-164 tumor cells were injected. Finally, the splenocyte proliferation was examined by MTT assay and tumor growth was also determined in each group. We observed that EXOHSP was more effective than EXO and EXOLys to decrease the number of tumor cells and to stimulate immune responses in animal models (P < 0.05). In SEB-anchored EXO group, EXOHSP70/SEB has the potency to stimulate immune responses more efficiently than EXO/SEB and EXOLys/SEB and the tumor was not palpable until 28th day which may refer to synergistic effect of HSP70 and SEB on immunity. In EXONLX treated mice proliferative response decreased significantly compared to control group (P > 0.05) and the tumor number was constant within a period of 28 days and EXOPRP may delay the occurrence of the fibrosarcoma tumor; After development of fibrosarcoma the number of tumors diminished over the studied period of time. Our results demonstrate that HSP70 enriched EXO is an effective immunoadjuvant in cancer immunotherapy and causes tumor regression in animal model.

A review on strategies for decreasing E. coli O157:H7 risk in animals.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that younger children are most prone to this microorganism. Hemolytic Uremic Syndrome (HUS) caused by EHEC, leads to the destruction of red blood cells and kidney failure. The virulence of E.coli O157:H7 is attributed to fimbriae, that facilitate colonization of bacteria within the colon and verotoxins (VT) or Shiga toxins (Stx) that are released into the blood. Although, in most cases, the infection is self-limitedin young children and aged population, it may cause HUS. Therefore, several investigations are performed in order to offer effective therapies and vaccines, which can prevent and treat the infection in appropriate time. As the pathogenesis of this infection is complicated, a multi-targeted strategy is required. Since cattle are the most important reservoir of EHEC and the root of contamination, reducing E. coli O157:H7 at the farm level should decrease the risk of human illness. Several vaccine approaches have been employed with different proper outcomes in animal models, including recombinant proteins (virulence factors such as; Stx1/2, intimin, EspA, fusion proteins of A and B Stx subunits), avirulent ghost cells of EHEC O157:H7, live attenuated bacteria expressing recombinant proteins, recombinant fimbrial proteins. In addition to protein-based vaccines, DNA vaccines have provided proper prevention in the laboratory animal model. This review paper summarizes the previous studies, current status and future perspective of different immunization strategies for eradicating Enterohemorrhagic Escherichia coli O157:H7.

Bacterial toxin's DNA vaccine serves as a strategy for the treatment of cancer, infectious and autoimmune diseases.

DNA vaccination -a third generation vaccine-is a modern approach to stimulate humoral and cellular responses against different diseases such as infectious diseases, cancer and autoimmunity. These vaccines are composed of a gene that encodes sequences of a desired protein under control of a proper (eukaryotic or viral) promoter. Immune response following DNA vaccination is influenced by the route and the dose of injection. In addition, antigen presentation following DNA administration has three different mechanisms including antigen presentation by transfected myocytes, transfection of professional antigen presenting cells (APCs) and cross priming. Recently, it has been shown that bacterial toxins and their components can stimulate and enhance immune responses in experimental models. A study demonstrated that DNA fusion vaccine encoding the first domain (DOM) of the Fragment C (FrC) of tetanus neurotoxin (CTN) coupled with tumor antigen sequences is highly immunogenic against colon carcinoma. DNA toxin vaccines against infectious and autoimmune diseases are less studied until now. All in all, this novel approach has shown encouraging results in animal models, but it has to go through adequate clinical trials to ensure its effectiveness in human. However, it has been proven that these vaccines are safe, multifaceted and simple and can be used widely in organisms which may be of advantage to public health in the near future. This paper outlines the mechanism of the action of DNA vaccines and their possible application for targeting infectious diseases, cancer and autoimmunity.

Regulation of immune responses to infection through interaction between stem cell-derived exosomes and toll-like receptors mediated by microRNA cargoes.

Infectious diseases are among the factors that account for a significant proportion of disease-related deaths worldwide. The primary treatment approach to combat microbial infections is the use of antibiotics. However, the widespread use of these drugs over the past two decades has led to the emergence of resistant microbial species, making the control of microbial infections a serious challenge. One of the most important solutions in the field of combating infectious diseases is the regulation of the host's defense system. Toll-like receptors (TLRs) play a crucial role in the first primary defense against pathogens by identifying harmful endogenous molecules released from dying cells and damaged tissues as well as invading microbial agents. Therefore, they play an important role in communicating and regulating innate and adaptive immunity. Of course, excessive activation of TLRs can lead to disruption of immune homeostasis and increase the risk of inflammatory reactions. Targeting TLR signaling pathways has emerged as a new therapeutic approach for infectious diseases based on host-directed therapy (HDT). In recent years, stem cell-derived exosomes have received significant attention as factors regulating the immune system. The regulation effects of exosomes on the immune system are based on the HDT strategy, which is due to their cargoes. In general, the mechanism of action of stem cell-derived exosomes in HDT is by regulating and modulating immunity, promoting tissue regeneration, and reducing host toxicity. One of their most important cargoes is microRNAs, which have been shown to play a significant role in regulating immunity through TLRs. This review investigates the therapeutic properties of stem cell-derived exosomes in combating infections through the interaction between exosomal microRNAs and Toll-like receptors.

Host and Pathogen-Directed Therapies against Microbial Infections Using Exosome- and Antimicrobial Peptide-derived Stem Cells with a Special look at Pulmonary Infections and Sepsis.

Microbial diseases are a great threat to global health and cause considerable mortality and extensive economic losses each year. The medications for treating this group of diseases (antibiotics, antiviral, antifungal drugs, etc.) directly attack the pathogenic agents by recognizing the target molecules. However, it is necessary to note that excessive use of any of these drugs can lead to an increase in microbial resistance and infectious diseases. New therapeutic methods have been studied recently using emerging drugs such as mesenchymal stem cell-derived exosomes (MSC-Exos) and antimicrobial peptides (AMPs), which act based on two completely different strategies against pathogens including Host-Directed Therapy (HDT) and Pathogen-Directed Therapy (PDT), respectively. In the PDT approach, AMPs interact directly with pathogens to interrupt their intrusion, survival, and proliferation. These drugs interact directly with the cell membrane or intracellular components of pathogens and cause the death of pathogens or inhibit their replication. The mechanism of action of MSC-Exos in HDT is based on immunomodulation and regulation, promotion of tissue regeneration, and reduced host toxicity. This review studies the potential of mesenchymal stem cell-derived exosomes/ATPs therapeutic properties against microbial infectious diseases especially pulmonary infections and sepsis.

Evaluation of UVB light efficacy for inducing apoptosis in Candida albicans cultures.

BACKGROUND: Candida albicans is known as part of human's skin normal flora; but simultaneously, is the most common opportunistic fungal pathogen of human which can cause a variety of infections including cutaneous candidiasis. Because of the importance of superficial infections like cutaneous candidiasis, we tried to use Ultraviolet B light as a method of phototherapy for inducing apoptosis in irradiated colonies of Candida albicans. MATERIALS AND METHODS: The Ultraviolet B with the wavelength of 302 nanometer was used for irradiating the colonies of Candida albicans. For detecting the eventual apoptotic reactions, the DNA of irradiated colonies as well as control colonies were extracted and then were run in 1% agarose gel electrophoresis containing ethidium bromide to observe luminescent DNA bands. RESULTS: Despite irradiating of Ultraviolet B to yeast cells, no abnormalities including DNA laddering bands or smears were detected in bands formed by total genomic DNA. DISCUSSION: The applied irradiation protocol in this investigation was not successful to induce apoptotic reactions in Ultraviolet-exposed colonies. Maybe, Heat shock proteins as the important part of fungal protein pool, inhibit the inducing pathway of apoptosis in irradiated colonies of Candida albicans.

Comparison of Immune Response in Mice Immunized with Recombinant PreS2/S-C18-27 Protein Derived from Hepatitis B Virus with Commercial Vaccine.

Background & Objective: The vaccine available to prevent Hepatitis B virus disease is ineffective in 5% of people due to the use of HBsAg as a weak immunogenic factor. In the present study, PreS2/S fused to C18-27 peptide fragment as an effective antigen and is proposed as a promising vaccine candidate compared with the conventional vaccine prescribed in the vaccination program. Methods: After the synthesis of PreS2/S genes and C18-27 peptide fragment in pET28a, the recombinant protein was confirmed by Western blotting. The efficacy of the PreS2/S-C18-27 protein was compared with the conventional vaccine injected into five groups of rats. Finally, the cytokine level of IF-r, IL-2, IL-4, IL-10, TNF-a, IgG1, and IgG2a were measured using the ELISA method. Results: This study showed no significant difference between the recombinant vaccine group and PBS control group in the IF-r test, but there was a significant difference between groups testing IL-2 and IL-10. In addition, the group receiving the recombinant vaccine with CPG adjuvant at a dilution of 1/10 in the IgG total test on days 14 and 45 after the first injection showed a significant difference in comparison with other groups. Conclusion: This study showed no statistically significant difference between the recombinant protein vaccine group and the conventional vaccine group. The Th1- mediated immune responses obtained from recombinant proteins with and without CPG performed better than conventional vaccines, possibly due to the functional deficiency of the available vaccines.

Evaluation of Triple Fragment Vaccine HSPX (Rv2031c) + PPE44 (Rv2770c) + Mouse IgG1 (Fcγ2a) with Auxiliary Adjuncts IL-22 in Comparison with BCG Vaccine.

Background & Objective: Despite the vaccination with the BCG vaccine, tuberculosis (TB) remains one of the major health problems in the world. The aim of this study was to evaluate our newly designed vaccine using IL-22 as an adjuvant in comparison with the common BCG vaccine. Methods: The gene constructs were cloned into the expression vector of pET28a and then into the recombinant vector of PET28a - HSPX, and PPE44 was transformed into Escherichia coli BL21 (DE3). Finally, the immunogenicity of recombinant proteins with and without BCG and IL-22 in BALB/c mice was investigated. Results: The key cytokines INF-γ and TNF-α were elevated more greatly in BCG immunized group than in PHF immunized group. Immunization with PHF showed a significant increase in IL-4 levels versus the BCG group. Adding IL-22 to the vaccine formulations indicated a tiny increase in IL-4 levels compared to their related vaccine groups.Specific total IgG1 in the experimental groups showed an increase in comparison with control groups, but in the vaccinated groups, no significant differences were observed, and the presence of IL-22 in the vaccine formulations indicated a slight decrease compared with the related mere vaccine groups. Results of specific total IgG2a in the experimental groups revealed that only in the PHF group formulated with IL-22 a significant increase occurs compared with all other experimental groups. Conclusion: It seems that BCG, as the only licensed vaccine for TB infection, could be more potent than a recombinant vaccine in the induction of cellular and humoral immune responses.

Diagnostic Accuracy and Validity of Serological and Molecular Tests for Hepatitis B and C.

INTRODUCTION: Hepatitis B and C viruses are one of the leading causes of health problems in the world and early diagnosis and treatment of them are very important. Thereby, this study aimed to evaluate the validity and reliability of usable diagnostic tests for the detection of hepatitis B and C viruses in the clinical setting and to compare them with each other. MATERIALS AND METHODS: In this review article, we have searched major online databases, including PubMed and EMBASE. 42 retrieved articles were published between January 2000 and January 2020, which are summarized in this review. RESULTS: Immunoassay approaches are general techniques for the identification of pathogenic agents, among which ELISA is the gold standard for the detection of HBsAg. While serological techniques are not conclusive, molecular assays are really important because of the high sensitivity to detect chronic hepatitis B without HBeAg, in which viral loads are very low. Biosensors have more elevated selectivity and sensitivity and faster responses compared to other methods. CONCLUSION: This study suggests that all of the molecular, serological, and biotechnological assays have advantages and disadvantages for diagnosing hepatitis B and C viruses which are dependent on the condition, so we should choose one of them in regards to the time, cost, and laboratory equipment along with the clinical symptoms.

Evaluation of the Role of Probiotics As a New Strategy to Eliminate Microbial Toxins: a Review.

Probiotics are living microorganisms that have favorable effects on human and animal health. The most usual types of microorganisms recruited as probiotics are lactic acid bacteria (LAB) and bifidobacteria. To date, numerous utilizations of probiotics have been reported. In this paper, it is suggested that probiotic bacteria can be recruited to remove and degrade different types of toxins such as mycotoxins and algal toxins that damage host tissues and the immune system causing local and systemic infections. These microorganisms can remove toxins by disrupting, changing the permeability of the plasma membrane, producing metabolites, inhibiting the protein translation, hindering the binding to GTP binding proteins to GM1 receptors, or by preventing the interaction between toxins and adhesions. Here, we intend to review the mechanisms that probiotic bacteria use to eliminate and degrade microbial toxins.

A study on apoptosis inducing effects of UVB irradiation in Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an important bacterial pathogen which causes different infectious diseases such as wound and skin lesion infections. The main goal of this study was to induce eventual apoptotic reactions in ultraviolet-exposed colonies of Pseudomonas aeruginosa. MATERIALS AND METHODS: The colonies of Pseudomonas aeruginosa were irradiated by UVB light; then, the DNA molecules of control and UVB-exposed colonies were extracted. Eventually, the extracted DNA molecules mixed in loading dye were run in 1% agarose gel containing ethidium bromide. RESULTS: No unusual pattern like DNA laddering bands or smear, were detected upon the 1% agarose gel. DISCUSSION: Through the applied protocol in this survey, the UVB radiation is not able to trigger apoptosis pathway in UV light exposed colonies of Pseudomonas aeruginosa. It seems that the cytoprotective property of Heat shock proteins inhibit the inducing effect of UVB light in irradiated colonies of Pseudomonas aeruginosa.

Potent in vitro antitumor activity of B-subunit of Shiga toxin conjugated to the diphtheria toxin against breast cancer.

Immunotoxins are protein-based drugs consist of a target-specific binding domain and a cytotoxic domain to eliminate target cells. Such compounds are potentially therapeutic to combat diseases such as cancer. Generally, the B-subunit of Shiga toxin (STXB) receptor, globotriaosylceramide (Gb3), is expressed in high amounts on a number of human tumors cancer cells. In this study, we evaluated a new antitumor candidate called DT389-STXB chimeric protein, which genetically fused the DT to B-subunit of Shiga-like toxin (STXB). First a chimeric protein, encoding DT389-STXB was synthesized. The optimized chimeric protein expressed in E.coli BL21 (DE3) and confirmed by anti-His Western blot analysis. T47D, SKBR3, 4T1 and MCF7 cell lines were treated separately with purified DT389-STXB recombinant protein and functional activity of DT389-STXB was analyzed by the cell enzyme-linked immunosorbentassay (ELISA), MTT, ICC, Western blot and apoptosis tests. The results indicated that the recombinant DT389-STXB fusion protein with a molecular weight of 53 kDa was successfully expressed in E.coli BL21 (DE3) and the anti-His western-blot was used to confirm the presence of the protein. The DT389-STXB fusion protein attached to T47D, SKBR3 and 4T1 cell lines with the proper affinity and induced dose-dependent cytotoxicity against GB3-expressing cancer cells in vitro. Our results showed that DT389-STXB fusion protein may be a promising candidate for antitumor therapy agent against breast cancer; however, further studies are required to explore its efficacy in vivo for therapeutic applications.

Breast cancer targeted/ therapeutic with double and triple fusion Immunotoxins.

Target-specific transport of therapeutic agents holds promise to increase the efficacy of cancer treatment by decreasing injury to normal tissues and post treatment problems. HER2 is a tumor cell surface marker that is expressed in 25-30 % of breast cancer patients. The significant role of HER2 in cancer development and its biological feature makes it a highly appealing goal for the therapeutic treatment of cancer targeted therapy using HER2 monoclonal antibody. This approach is currently used as a special treatment against breast cancer in some research. In the present study, HER2 monoclonal antibody (mAb), (Herceptin) fused to PE38 by recombinant DNA technology and a new recombinant IT was developed. The scFv(Herceptin)-PE-STXA and scFv(Herceptin)-PE fusions cloned in pET28a and recombinant protein expression was carried out and then the proteins were purified. MCF-7 and SKBR-3 cells were used as HER2-negative and HER2-positive breast cancer cells, respectively. The cytotoxicity of its evaluated using MTT assay. The cell ELISA was used to determine the binding ability of immunotoxins (ITs) to the cell receptor and internalization and apoptosis were also assessed. The results revealed that cell cytotoxicity occurred in SKBR-3 cells in a dose-dependent manner but not in MCF-7 cells. It is possible that this ITs can attach to HER2-positive breast cancer cells and then, internalize and eradicate cancer cells by apoptosis. Here, we concluded that the recombinant ITs have therapeutic potential against HER2-positive breast cancer.