Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant clinical problem, given the lack of therapeutic options. The CRKP strains have emerged as an essential worldwide healthcare issue during the last 10 years. Global expansion of the CRKP has made it a significant public health hazard. We must consider to novel therapeutic techniques. Bacteriophages are potent restorative cases against infections with multiple drug-resistant bacteria. The Phages offer promising prospects for the treatment of CRKP infections. OBJECTIVE: In this study, a novel K. pneumoniae phage vB_KshKPC-M was isolated, characterized, and sequenced, which was able to infect and lyse Carbapenem-resistant K. pneumoniae host specifically. METHODS: One hundred clinical isolates of K. pneumoniae were collected from patients with COVID-19 associated with ventilator-associated acute pneumonia hospitalized at Shahid Beheshti Hospital, Kashan, Iran, from 2020 to 2021. Initially, all samples were cultured, and bacterial isolates identified by conventional biochemical tests, and then the ureD gene was used by PCR to confirm the isolates. The Antibiotic susceptibility test in the disc diffusion method and Minimum inhibitory concentrations for Colistin was done and interpreted according to guidelines. Phenotypic and molecular methods determined the Carbapenem resistance of isolates. The blaKPC, blaNDM, and blaOXA-23 genes were amplified for this detection. Biofilm determination of CRKP isolates was performed using a quantitative microtiter plate (MTP) method. The phage was isolated from wastewater during the summer season at a specific position from Beheshti Hospital (Kashan, Iran). The sample was processed and purified against the bacterial host, a CRKP strain isolated from a patient suffering from COVID-19 pneumoniae and resistance to Colistin with high potency for biofilm production. This isolate is called Kp100. The separated phages were diluted and titration by the double overlay agar plaque assay. The separate Phage is concentrated with 10% PEG and stored at -80 °C until use. The phage host range was identified by the spot test method. The purified phage morphology was determined using a transmission electron microscope. The phage stability tests (pH and temperature) were analyzed. The effect of cationic ions on phage adsorption was evaluated. The optimal titer of bacteriophage was determined to reduce the concentration of the CRKP strain. One-step growth assays were performed to identify the purified phage burst's latent cycle and size. The SDS-PAGE was used for phage proteins analysis. Phage DNA was extracted by chloroform technique, and the whole genome of lytic phage was sequenced using Illumina HiSeq technology (Illumina, San Diego, CA). For quality assurance and preprocessing, such as trimming, Geneious Prime 2021.2.2 and Spades 3.9.0. The whole genome sequence of the lytic phage is linked to the GenBank database accession number. RASTtk-v1.073 was used to predict and annotate the ORFs. Prediction of ORF was performed using PHASTER software. ResFinder is used to assess the presence of antimicrobial resistance and virulence genes in the genome. The tRNAs can-SE v2.0.6 is used to determine the presence of tRNA in the genome. Linear genome comparisons of phages and visualization of coding regions were performed using Easyfig 2.2.3 and Mauve 2.4.0. Phage lifestyles were predicted using the program PHACTS. Phylogenetic analysis and amino acid sequences of phage core proteins, such as the major capsid protein. Phylogenies were reconstructed using the Neighbor-Joining method with 1000 bootstrap repeat. HHpred software was used to predict depolymerase. In this study, GraphPad Prism version 9.1 was used for the statistical analysis. Student's t-test was used to compare the sets and the control sets, and the significance level was set at P ≤ 0.05. RESULTS: Phage vB_KshKPC-M is assigned to the Siphoviridae, order Caudovirales. It was identified as a linear double-stranded DNA phage of 54,378 bp with 50.08% G + C content, had a relatively broad host range (97.7%), a short latency of 20 min, and a high burst size of 260 PFU/cell, and was maintained stable at different pH (3-11) and temperature (45-65 °C). The vB_KshKPC-M genome contains 91 open-reading frames. No tRNA, antibiotic resistance, toxin, virulence-related genes, or lysogen-forming gene clusters were detected in the phage genome. Comparative genomic analysis revealed that phage vB_KshKPC-M has sequence similarity to the Klebsiella phages, phage 13 (NC_049844.1), phage Sushi (NC_028774.1), phage vB_KpnD_PeteCarol (OL539448.1) and phage PWKp14 (MZ634345.1). CONCLUSION: The broad host range and antibacterial activity make it a promising candidate for future phage therapy applications. The isolated phage was able to lyse most of the antibiotic-resistant clinical isolates. Therefore, this phage can be used alone or as a phage mixture in future studies to control and inhibit respiratory infections caused by these bacteria, especially in treating respiratory infections caused by resistant strains in sick patients.

Ultrasound-guided chemoradiotherapy of breast cancer using smart methotrexate-loaded perfluorohexane nanodroplets.

Chemoradiotherapy with controlled-release nanocarriers such as sono-sensitive nanodroplets (NDs) can enhance the anticancer activity of chemotherapy medicines and reduces normal tissue side effects. In this study, folic acid-functionalized methotrexate-loaded perfluorohexane NDs with alginate shell (FA-MTX/PFH@alginate NDs) were synthesized, characterized, and their potential for ultrasound-guided chemoradiotherapy of breast cancer was investigated in vitro and in vivo. The cancer cell (4T1) viabilities and surviving fractions after NDs and ultrasound treatments were significantly decreased. However, this reduction was much more significant for ultrasound in combination with X-ray irradiation. The in vitro and in vivo results confirmed that MTX-loaded NDs are highly biocompatible and they have no significant hemolytic activity and organ toxicity. Furthermore, the in vivo results indicated that the FA-MTX/PFH@alginate NDs were accumulated selectively in the tumor region. In conclusion, FA-functionalized MTX/PFH@alginate NDs have a great theranostic performance for ultrasound-controlled drug delivery in combination with radiotherapy of breast cancer.

Anti-leishmanial effects of resveratrol and resveratrol nanoemulsion on Leishmania major.

BACKGROUND: Leishmaniasis is a vector-borne disease that is endemic in the tropical and sub-tropical areas of the world. Low efficacy and high cytotoxicity of the current treatment regimens for leishmaniasis is one of the most important health problems. In this experimental study, anti-leishmanial effects of different concentrations of resveratrol and resveratrol nano-emulsion (RNE) were assessed. METHODS: RNE was prepared using the probe ultra-sonication method. The cytotoxicity was evaluated using the MTT technique on the L929 cell line. The anti-leishmanial activities on promastigotes of leishmania were assessed using vital staining and infected BALB/c mice were used to assess the in vivo anti-leishmanial effects. RESULTS: In vitro and in vivo assays revealed that all concentrations of resveratrol and RNE had valuable inhibitory effects against Leishmania major in comparison to the control group (P < 0.05). The half maximal inhibitory concentration (IC50) values were calculated as 16.23 and 35.71 µg/mL for resveratrol and RNE, respectively. Resveratrol and RNE showed no cytotoxicity against the L929 cell line. CONCLUSIONS: According to the potent in vitro and in vivo anti-leishmanial activity of RNE at low concentration against L. major, we suggest that it could be a promising anti-leishmanial therapeutic against L. major in the future.

Dual Stimuli-Responsive Pickering Emulsions from Novel Magnetic Hydroxyapatite Nanoparticles and Their Characterization Using a Microfluidic Platform.

Stimuli-responsive emulsifiers have emerged as a class of smart agents that can permit regulated stabilization and destabilization of emulsions, which is essential for food, cosmetic, pharmaceutical, and petroleum industries. Here, we report the synthesis of novel "smart" hydroxyapatite (HaP) magnetic nanoparticles and their corresponding stimuli-responsive Pickering emulsions and explore their movement under confined spaces using a microfluidic platform. Pickering emulsions prepared with our magnetic stearic acid-functionalized Fe2O3@HaP nanoparticles exhibited pronounced pH-responsive behavior. We observed that the diameter of emulsion droplets decreases with an increase in pH. Swift demulsification was achieved by lowering the pH, whereas the reformation of emulsions was achieved by increasing the pH; this emulsification-demulsification cycling was successful for at least ten cycles. We used a microfluidic platform to test the stability of the emulsions under flowing conditions and their response to a magnetic field. We observed that the emulsion stability was diminished and droplet coalescence was enhanced by the application of the magnetic field. The smart nanoparticles we developed and their HaP-based emulsions present promising materials for pharmaceutical and petroleum industries, where responsive emulsions with controlled stabilities are required.

The role of Bax in the apoptosis of Leishmania-infected macrophages.

BACKGROUND: Leishmania is a protozoan parasite that nests in macrophages and is responsible for the Leishmaniasis disease. In spite of different defense pathways, last strategy of macrophage for killing parasite is apoptosis process. By permeableizing the mitochondrial outer membrane (MOM). As breaching MOM releases apoptogenic factors like cytochrome-c which activate caspases that result in the destruction of the cell. In this review, we summarized the appropriate manuscripts regarding the bax includes, its different types and the effect of bax on the apoptosis of Leishmania and parasite-infected macrophages. METHODS: Information about the role of BAX in the apoptosis of parasite-infected macrophage of recent articles were surveyed by searching computerized bibliographic database PubMed and Google Scholar entering the keywords BAX and leishmaniasis. RESULTS: The common studies revealed Leishmania use different survival strategies for inhibiting macrophage apoptosis. As Leishmania by preventing homooligomerization or upregulating the anti-apoptotic molecule Bcl-2 can prohibits proteins of host-cell apoptosis such as Bax that is required for mitochondrial permeabilisation during apoptosis. CONCLUSION: With regard to the supportive role of bax in apoptosis and the preventive role of Leishmania in its function, it seems that expression of bax gene in parasite by technologies like transgenic or down regulating of anti-apoptotic molecule Bcl-2 by miRNA could be prompted the apoptosis process of infected-macrophages and inhibited extensive spread of Leishmania and the resulting lesions.

Evaluation of transgenic Leishmania infantum expressing mLLO-BAX-SMAC in the apoptosis of the infected macrophages in vitro and in vivo.

BACKGROUND: Leishmaniasis is an important infectious disease that develops because of escaping parasite from the host immune system or preventing host macrophages apoptosis. Recently, the development of transgenic methods and the manipulation of the parasite genome has provided many advantages. So, in this study, the effect of the transgenic Leishmania infantum expressing mLLO-BAX-SMAC proteins was examined in accelerating host cell apoptosis. METHOD: The entire coding sequence of designed codon-optimized mLLO-Bax-Smac was cloned in the pLexyNeo2 vector and integrated downstream of the 18srRNA locus of L infantum genome by homologous recombination. Next, the expression of mLLO-BAX-SMAC fusion protein was evaluated by the Western blotting technique and the pathogenesis of transgenic parasite was surveyed in vitro and in vivo. RESULTS: The results of PCR and Western blot confirmed proper integration and expression of mLLO-Bax-Smac sequence into the 18srRNA locus of L infantum. Flow cytometry showed accelerating apoptosis of transgenic Leishmania-infected macrophages compared to wild-type parasite. Also, transgenic parasites were less virulent as a fewer parasitic burden was found in the spleen and liver of transgenic-infected mice compared to the control. CONCLUSION: The data suggested that the transgenic L infantum expressing BAX-SMAC can be used as an experimental model for developing vaccination against leishmaniasis.

A systematic review of Toxoplasma gondii antigens to find the best vaccine candidates for immunization.

At present, there is not any available accepted vaccine for prevention of Toxoplasma gondii (T. gondii) in human and animals. We conducted literature search through English (Google Scholar, PubMed, Science Direct, Scopus, EBSCO, ISI Web of Science) scientific paper databases to find the best vaccine candidates against toxoplasmosis among T. gondii antigens. Articles with information on infective stage, pathogenicity, immunogenicity and characterization of antigens were selected. We considered that the ideal and significant vaccines should include different antigens and been expressed in all infective stages of the parasite with a high pathogenicity and immunogenicity. Evaluation within this systematic review indicates that MIC 3, 4, 13, ROP 2, RON 5, GRA 1, 6, 8, 14 are expressed in all three infective stages and have pathogenicity and immunogenicity. MIC 5, ROM 4, GRA 2, 4, 15, ROP 5, 16, 17, 38, RON 4, MIC 1, GRA 10, 12, 16, SAG 3 are expressed in only tachyzoites and bradyzoites stages of T. gondii with pathogenicity/immunogenicity. Some antigens appeared to be expressed in a single stage (tachyzoites) but have high pathogenicity and induce immune response. They include enolase2 (ENO2), SAG 1, SAG5D, HSP 70, ROM 1, ROM 5, AMA 1, ROP 18, RON2 and GRA 24. In conclusion, current vaccination against T. gondii infection is not satisfactory, and with the increasing number of high-risk individuals, the development of an effective and safe specific vaccine is greatly valuable for toxoplasmosis prevention. This systematic review reveals prepare candidates for immunization studies.

Locked nucleic acid -anti- let-7a induces apoptosis and necrosis in macrophages infected with Leishmania major.

BACKGROUND: Protozoan parasites of the genus Leishmania are etiologic agents which are intracellular pathogens of vertebrates and replicate inside infected macrophages. Leishmania have developed complex strategies to reverse host immune responses in favor of it. One of the major species causing cutaneous involvements is Leishmania major. MicroRNAs (miRNA) are non-coding small RNAs encoding 22-nucleotide (nt) long RNAs. miRNAs affect diverse biological processes, including cell cycle, proliferation, differentiation, growth and development, metabolism, aging, apoptosis, gene expression and immune regulation. This study aimed at evaluating apoptosis and necrosis after transfection locked nucleic acid (LNA) inhibitor of let-7a in the human macrophages miRNAs upon infectionwith L. major. MATERIALS AND METHODS: Inhibition of let-7a in macrophages was derived originally from the human monocytes (MDM), using locked nucleic acid (LNA) antagomir. The total cellular RNA was extracted 24 and 48 h post transfection. The levels o Let-7a expression was measured by qPCR Real Time using specific primer. Annexin-V/Propidium Iodide staining method was performed to detect apoptosis and necrosis in the MDM cells. Data were analyzed using the Kruskal-Wallis and Mann-Whitney tests. RESULTS: Let-7a inhibition increased the MDM cells apoptosis and necrosis using flow cytometry method. CONCLUSIONS: The results suggested that inhibition of let-7a could be a new approach in treatment of leishmaniasis.

miR-20a inhibition using locked nucleic acid (LNA) technology and its effects on apoptosis of human macrophages infected by Toxoplasma gondii RH strain.

Toxoplasma gondii is a ubiquitous and infectious parasite that multiplies in any nucleated cell of warm-blooded animals and humans worldwide. This parasite has intricate mechanisms to reciprocate host-cell apoptosis to exist in the host cell. So far, the details of the parasite interactions with host cells are not well known. MicroRNAs (miRNAs) are one of the small noncoding RNAs that are now considered as a key mechanism of gene regulation. They are important in physiological and pathological processes such as apoptosis. In this study a Real Time quantitative PCR technique was used to evaluate the levels of miR-20a of miRNAs family in human macrophage during T. gondii infection to determine the role of miR-20a in apoptosis. Then, the inhibition of miR-20a function through interaction with transfection of Locked Nucleic Acid (LNA) antisense oligomer was studied. Furthermore, it was examined whether miR-20a is involved in apoptosis of human macrophages with T. gondii infected cells using flow cytometry. We found that miR-20a expression is up-regulated in human macrophages following T. gondii infection. After LNA anti miR-20a oligomer transfection, miR-20a inhibition was evaluated by quantitative reverse transcriptase polymerase chain reaction. Flow cytometry results showed that LNA anti-miR20a oligomer increased apoptosis. In agreement with this result, we found that specific LNA oligonucleotides prevent the functional activity of miR-20a and promotion of human macrophages apoptosis with T. gondii infection by inhibition of this miRNAs gene. Also, the results support the concept that LNA oligomer antisense may be used as a therapeutic implement for blocking detrimental miRNAs overexpressed in infections.

Contributions of HLA haplotypes, IL8 level and Toxoplasma gondii infection in defining celiac disease's phenotypes.

BACKGROUND: It is not clear why some patients with coeliac disease (CD) present with severe symptoms and small intestinal mucosal damages while others present with milder symptoms and no frank enteropathy. There is no study to assess the associated factors with mild/severe symptoms and enteropathy. The terminologies like latent, silent and potential are difficult to use and are unrepresentative. In the present study we describe coeliac disease's phenotypes based on HLA haplotypes, IL8 production and past infection with Toxoplasma gondii (T. gondii) infection. METHODS: In this case-control study, sera originating from 150 healthy subjects and 150 patients diagnosed with CD during the years 2013-14 were analyzed for the presence of antibodies specific T. gondii of the IgG and IgM subclasses. The level of IL8 were measured and HLA-DQ2 and HLA-DQ8 alleles were genotyped. The correlation between these parameters and the damages in intestinal mucosal were assessed using an accepted histopathological classification. RESULTS: High levels of IgG antibodies against T. gondii were found in the sera of control group compared to the CD group (52.6% vs. 39.4%, P = 0.02). Mean serum levels of IL8 was significantly higher in CD patients compared with control (P ≤ 0.05). By comparing the level of anti- T. gondii IgG and mucosal damage in celiac disease, we found a significant relationship between the severity of mucosal damages and anti- T. gondii IgG level (P = 0.02). No correlation was detected between Toxoplasma gondii infection and types of HLA (P > 0.05). However, patients with severely abnormal histology carried HLA-DQ2 risk alleles (92 patients (61%)) more often than the controls and those with mild histological abnormalities. CONCLUSIONS: CD patients with severe histological changes had more often Toxoplasma gondii infection than those affected with mild histological features. This suggests that CD's phenotypes are correlated to additional factors like infections and to particular HLA DQ2 alleles that may need additional investigations and potentially will require additional treatment.

A lentiviral vaccine expressing KMP11-HASPB fusion protein increases immune response to Leishmania major in BALB/C.

Hydrophilic acylated surface protein B (HASPB) is an immunogenic Leishmania-specific protein that antibodies are produced against it in the sera of Leishmania-infected individuals. Kinetoplastid membrane protein 11 (KMP11) is another Leishmania antigen and considered as the suitable candidate for vaccine development Leishmaniasis. It is a highly conserved surface protein expressed in both promastigotes and amastigotes. In this study, KMP11 and HASPB coding sequences were cloned into a pCDH-cGFP lentiviral vector as a fusion protein to be used as a DNA vaccine against L. major. The KMP11-HASPB fusion protein was successfully expressed as evidenced by RT-PCR and Western blot assays. The effect of the vaccine was determined by evaluating the level of IFN-γ, IL-10, IgG1, and IgG2a performed using ELISA as well as determining the parasite load after challenge with L. major in vaccinated mice. The results revealed that IFN-γ, IL-10, IgG1, and IgG2a significantly increased after vaccination using KMP11-HASPB-expressing lentiviruses in BALB/c mice. It is noteworthy that the level of IFN-γ and IgG2a was higher than that of IL-10 and IgG1, respectively, which indicates the activation Th1 cells, macrophages, and cellular immunity. Moreover, the parasite load in the spleen and lymph node of vaccinated mice after challenge was significantly lower than that of controls.

Immunology and Genetic of Leishmania infantum: The Role of Endonuclease G in the Apoptosis.

Leishmania infantum is the causative agent of infantile visceral leishmaniasis (VL) in the Mediterranean region. Despite developing protective responses, the disease progresses due to many of factors. These include the action of suppressive cytokines, exhaustion of specific T cells, loss of lymphoid tissue, and defective humoral response. Genetic changes that occur inside the genome of alienated or parasite cells, along with immune responses, play an important role in controlling or progressing the disease. Proapoptotic proteins such as Smac/DIABLO, EndoG, AIF (apoptosis-inducing factor), and cytochrome C are effective in apoptosis. EndoG is a mitochondrion-specific nuclease that translocates to the nucleus during apoptosis. Once released from mitochondria, endoG cleaves chromatin DNA into nucleosomal fragments independently of caspases. Therefore, endoG represents a caspase-independent apoptotic pathway initiated from the mitochondria. A comprehensive understanding of the immune and genetic events that occur during VL is very important for designing immunotherapy strategies and developing effective vaccines for disease prevention. In this review which explained the immunological responses and also the important factors that can contribute to parasite apoptosis and are used in subsequent studies as a target for the preparation of drugs or recombinant vaccines against parasites are briefly reviewed.

The effects of 6-Gingerol on reproductive improvement, liver functioning and Cyclooxygenase-2 gene expression in estradiol valerate - Induced polycystic ovary syndrome in Wistar rats.

6-Gingerol is the major pungent ingredient of ginger with anti-inflammatory and antioxidant properties. In this study, we evaluate the effects of 6-gingerol on the biochemical parameters and ovarian histological improvements in estradiol valerate (EV) induced PCOS rats. Thirty six female Wistar rats were divided into 4 groups: control, received normal diet, PCOS control, received 4 mg/kg EV injection for 28 days and two experimental groups, received an EV injection for 28 days and followed by 6-gingerol (200 µg/kg and 400 µg/kg) for 14 days. The administration of EV led to increase body and ovarian weights, abnormality in serum sex steroid profile, decrease in antioxidant activity and increase in COX-2 gene expression. 6-gingerol treatments, particularly the 400 µg/kg dose, markedly attenuated these alterations. 6-gingerol showed beneficial effects in the EV induced PCOS rats via decreased expression of COX-2, restored biochemical parameters to normal and decreased of cysts in the ovaries.

Aquaglyceroporin1 gene expression in antimony resistance and susceptible Leishmania major isolates.

BACKGROUND & OBJECTIVES: The mechanism of antimony resistance in Leishmania has been studied extensively, in connection with decreased influx and/or increased eflux of the drug. Aquaporin 1 (AQP1) protein has been shown to mediate the uptake of trivalent antimony. This study was aimed to find the expression level of AQP1 gene in resistant versus non-resistant clinical isolates of Leishmania major in Iranian patients. METHODS: Clinical isolates were obtained from 16 considered patients referred to Navab Safavi Clinical Center, Isfahan, Iran from October 2014 to December 2015. After diagnosis of cutaneous leishmaniasis using microscopic observation, biopsy was performed from lesion(s) of each patient and stored inside RNAlater solution at -20΀C. Written informed consent was obtained from all the patients to participate in the study before recording their information and sampling based on Helsinki declaration. Each patient was treated with Glucantime and followed for three months. All sensitive and resistance isolates were considered and compared with AQP1 gene expression using real time PCR that was analyzed with delta-delta Ct. RESULTS: Out of 16 clinical isolates, four patients were resistant and 12 were non-resistant. The AQP1 gene expression in resistant isolates was significantly higher than the one in response failure isolates (p = 0.001). INTERPRETATION & CONCLUSION: The significant over expression (0.5 fold) of AQP1 gene in resistant versus non- resistant isolates suggests different mechanism of drug resistance such as mutations. Mutations may change the physiological function of the Aquaporin 1 protein that might affect its expression level.

SEROPREVALENCE OF TOXOPLASMA GONDII INFECTION AMONG PATIENTS ADMITTED TO AL-ZAHRA HOSPITAL, ISFAHAN, IRAN.

BACKGROUND: Toxoplasma gondii (T. gondii) infection is one of the most common parasitic infections among humans and other warm-blooded animals worldwide. The aim of this study was to evaluate toxoplasmosis status in patients admitted to Al-Zahra hospital, Isfahan, Iran. METHODS: This cross-sectional study was conducted from October 2012 to January 2015. During this period, 716 patients referred to AI-Zahra hospital in Isfahan city, Iran, were studied to investigate the IgG and IgM antibodies against T. gondii using ELISA kit. The data were analysed by Chi-square and Fisher's exact tests. In addition, the relation of data with age and sex were also examined. RESULTS: Among 716 patients, 21 patients (2.9%) had positive IgM and 288 patients (40.2%) had positive IgG titer against T. gondii. Data analysis by Chi-square and Fisher's exact tests revealed that there was no significant relationship between IgG titer and age (p > 0.05). Additionally, there was no relationship between IgM titer and age (p > 0.05). The data showed that there was no relationship between IgG and IgM antibody titer and sex (p > 0.05). CONCLUSION: The prevalence of toxoplasmosis in Isfahan inhabitants seems fairly high but it can be concluded that the rate of seropositive patient is moderate comparing to other regions of country. Accordingly, the authors propose that all sensitive patients have to be tested for T. gondii antibody in order to prevent the consequences of disease.

Comparison of immune regulatory factors in acute and chronic lesions of cutaneous leishmaniasis due to Leishmania major.

BACKGROUND: Adaptive immune response is an important factor in the healing process and development of protection in cutaneous leishmaniasis (CL). Little information is available in human CL about the importance of the balance between effector and regulatory immune responses. Therefore, the aim of this study was to asses messenger ribonucleic acid (mRNA) expression of interleukin-10 (IL-10), IL-4, transforming growth factor-ß1 (TGF-ß1), interferon-g (IFN-γ), and forkhead box P3 (Foxp3) (as a marker of regulatory T cells) in acute and chronic CL lesions caused by Leishmania major compared with normal skin samples. MATERIALS AND METHODS: Thirty biopsies were obtained from CL patients with acute lesions (AL, n = 13), chronic lesions (CH, n = 11) and healthy volunteers (n = 6). Relative expressions of target genes were determined by means of reverse transcription real time polymerase chain reaction and were compared with the controls. RESULTS: Expression of Foxp3, IL-4, and IFN-γ were significantly more in CH than AL group of patients (Foxp3: Median 0.48, inter-quartile range 0.32-0.76 [arbitrary units] for AL, and 0.97 (0.75-1.30) for CH, P = 0.006; IFN-γ: 45.98 (33.39-173.48) for AL, and 200.53 (97.49-361.76) for CH, P = 0.023; IL-4: 0.49 (0.34-2.16) for AL, and 2.14 (1.30-7.11) for CH, P = 0.021). Expression of TGF-ß was not significantly different between groups. CONCLUSION: The results indicate that IL-4 secretion at the site of L. major infection rather than low IFN-γ production might have a role in prolongation of disease. Despite a moderate increase of Foxp3 expression in chronic lesions, function of Tregs in persistent infection is not clear.

Molecular docking and synthesis of N-alkyl-isatin-3-imino aromatic amine derivatives and their antileishmanial and cytotoxic activities.

Background and purpose: Isatin derivatives have excited attention due to their biological attractions, especially, anticancer properties. Isatin analogs such as semaxanib and sunitinib were exposed to tyrosine kinase inhibitory properties. N-substituted isatins were reported to show cytotoxic activity. On the other, the extension of impressive and cost-effective agents against leishmaniasis is necessary in third-world countries. The capability of isatin derivatives to create novel anticancer and anti-leishmanial compounds has been identified in medicinal chemistry research. The current study aimed to synthesize N-alkyl-isatin-3-imino aromatic amine compounds and evaluate their biological effects. Experimental approach: Synthesis started with the formation of 2-chloro-N-phenylacetamide derivatives by the reaction of aniline derivatives with chloroacetyl chloride. N-alkylation of isatin was performed in the presence of K2CO3 in N, N-dimethylformamide. Final products were prepared via the condensation of N-alkyl isatin derivatives with aromatic amines. Cell viability was checked out by using the MTT assay against cancer cells. Final compounds were screened for anti-leishmanial activity. The molecules were docked in the active sites of the epidermal growth factor receptor tyrosine kinase to define the possible interactions. Findings/Results: Compounds 5c and 4d with IC50 value of 50 µΜ showed cytotoxic activity on the MCF-7 cell line. Compound 5b presented anti-leishmanial activity against promastigote form after 48 h (IC50:59 µΜ) and 72 h (IC50: 41 µΜ) incubations. The highest docking score was -7.33 kcal/mol for compound 4d. Conclusions and implications: The nature of substitution in the N1 region of isatin seems to be able to influence the cytotoxic activity. Based on the obtained results of docking and cytotoxic tests, compound 4d seems to be a good compound for further investigations.

Echinococcus granulosus sheep strain (G1) as the predominant genotype in definitive host (dogs) isolates in northeastern Iran.

Echinococcus granulosus sensu lato (E. granulosus s.l.) is a zoonotic parasite, causing cystic echinococcosis in humans. In the present study, prevalence and genotypes of E. granulosus s.l. was assessed in stools collected from 244 dogs including 138 stray and 106 domestic animals using high resolution melting curve (HRM) method. Initially, to detect taeniid eggs in feces, all samples were examined using the formalin-ether techniques. Genomic DNA was extracted from the positive samples and E. granulosus s.l. was differentiated from other Taeniidae parasites using SSU-rDNA gene and E. granulosus s.l. was analyzed for genotyping using HRM based on the cox1 gene. In total, 12.7% (31/244) of the samples were positive for Taeniidae eggs. In addition, among the positive samples, 77.4% (24/31) were positive for E. granulosus s.l.. In details, 11.3% (12/106) of the domestic dogs and 8.7% (12/138) of the stray dogs were positive for E. granulosus s.l.. The results of HRM analysis showed that all E. granulosus s.l. isolates were G1 strain. Findings of the present study indicated a considerable prevalence of E. granulosus G1 among dogs in the northeast of Iran and imply a serious risk of transmitting to humans and livestock.

Chitosan-Imidazolium Core-Shell Nanoparticles of Gd-Mn-Mo Polyoxometalate as Novel Potential MRI Nano-Agent for Breast Cancer Detection.

Polyoxometalates (POMs) are mineral nanoclusters with many advantages in various diagnostic fields, in particular cancer detection. This study aimed to synthesize and evaluate the performance of gadolinium-manganese-molybdenum polyoxometalate (Gd-Mn-Mo; POM) nanoparticles coated with chitosan-imidazolium (POM@CSIm NPs) for detecting 4T1 breast cancer cells by magnetic resonance imaging in vitro and in vivo. The POM@Cs-Im NPs were fabricated and characterized by FTIR, ICP-OES, CHNS, UV-visible, XRD, VSM, DLS, Zeta potential, and SEM. Cytotoxicity, cellular uptake, and MR imaging in vivo and in vitro of L929 and 4T1 cells were also assessed. The efficacy of nanoclusters was demonstrated using MR images of BALB/C mice bearing a 4T1 tumor in vivo. The evaluation of the in vitro cytotoxicity of the designed NPs showed their high biocompatibility. In fluorescence imaging and flow cytometry, NPs had a higher uptake rate by 4T1 than L929 (p < 0.05). Furthermore, NPs significantly increased the signal strength of MR images, and its relaxivity (r1) was calculated as 4.71 mM-1 s-1. MR imaging also confirmed the attachment of nanoclusters to cancer cells and their selective accumulation in the tumor region. Overall, the results showed that fabricated POM@CSIm NPs have considerable potential as an MR imaging nano-agent for early 4T1 cancer detection.

Enhanced in vivo anti-tumor efficacy of whole tumor lysate in combination with whole tumor cell-specific polyclonal antibody.

Background and purpose: Despite the widespread utilization of cancer vaccines with specified antigens, the use of whole tumor cell lysates in tumor immunotherapy would be a very promising approach that can overcome several significant obstacles in vaccine production. Whole tumor cells provide a broad source of tumor-associated antigens and can activate cytotoxic T lymphocytes and CD4+ T helper cells concurrently. On the other hand, as an effective immunotherapy strategy, recent investigations have shown that the multi-targeting of tumor cells with polyclonal antibodies, which are also more effective than monoclonal antibodies at mediating effector functions for target elimination, might minimize the escape variants. Experimental approach: We prepared polyclonal antibodies by immunizing rabbits with the highly invasive 4T1 breast cancer cell line. Findings/Results: In vitro investigation indicated that the immunized rabbit serum inhibited cell proliferation and induced apoptosis in target tumor cells. Moreover, in vivo analysis showed enhanced anti-tumor efficacy of whole tumor cell lysate in combination with tumor cell-immunized serum. This combination therapy proved beneficial in significant inhibition of the tumor growth and the established tumor was entirely eradicated in treated mice. Conclusion and implications: Serial intravenous injections of tumor cell immunized rabbit serum significantly inhibited tumor cell proliferation and induced apoptosis in vitro and in vivo in combination with whole tumor lysate. This platform could be a promising method for developing clinical-grade vaccines and open up the possibility of addressing the effectiveness and safety of cancer vaccines.