Application of the Leishmania infantum 21-kDa recombinant protein for the development of an immunochromatographic test.

BACKGROUND: Visceral leishmaniasis (VL), caused by Leishmania infantum, is a systemic parasitic disease and presents a global health problem which can be fatal if not diagnosed and treated. Dogs are the main hosts and provide reservoirs for the transmission of the disease to humans. METHODS: In this study, the gene encoding a 21-kDa protein was cloned and expressed as a fusion protein in Escherichia coli strain BL21 (DE3) for developing a rapid immunochromatographic test (ICT) to identify infected dogs. The expression of the recombinant 21-kDa protein (r21) was investigated using SDS-PAGE and Western blot methods. The purified r21-kDa protein was spotted onto ICT strips and tested by sera from experimentally infected, naturally infected and uninfected dogs. RESULTS: The SDS-PAGE and Western blot methods showed the successful expression of r21-kDa protein. The ICT strip test revealed that the r21-kDa protein was detected by the sera of experimentally and naturally infected dogs. The specificity tests also confirmed no cross-reactivity with animals infected with Trypanosoma cruzi, Toxoplasma gondii and Ehrlichia canis. CONCLUSIONS: Based on these findings, the new r21-kDa protein may be a suitable target for developing a new simple, specific and rapid serological method to detect VL in infected dogs.

Production and purification of polyclonal antibody against attenuated and wild type Leishmania infantum in dogs.

Antibodies are still widely used in several programs including early research, imaging, Targeting drug delivery system, Affinity chromatography, flowcytometry technic, diagnosis and treatment. Purification of antibody is a standard approach for detection of infection agent in different species. The reservoir hosts for Leishmania infantum are Dogs and they have active role in the transmission of leishmania to humans by the bite of a sand fly belonging to genus Phlebotomus and Lutzomiya. Consequently, elimination of dogs in endemic areas and vaccination of dogs contributes to reduction of the human and canine VL cases. Serological antibody tests such as IFAT (Indirect Fluorescent Antbody Test), DFAT (Direct Fluorescent Antbody Test), ELISA (Enzyme-Linked Immunosorbent Assay), PCR (Polymerase chain Reaction Assay) have been extensively used to investigate canine infection with L. infantum. In this study we produced and purified polyclonal antibody against attenuated and wild type leishmania infantum in dogs. Anti-leishmania in dog serums precipitated with ammonium sulphate. The IgG recovered from ammonium sulphate precipitation was subject to ion exchange chromatography (IEC) and the purity of IgG was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced condition. The purity of proteins were above 95% and then purified IgG was conjugated with FITC. We determined optimum titer of dog IgG by observation parasites under fluorescent microscope. The optimum dilution of prepared FITC conjugated dog IgG was 1: 400. This polyclonal antibody can be used for other applications in research, diagnosis and clinic.

Sodium Butyrate as a Histone Deacetylase Inhibitor Affects Toll-Like Receptor 4 Expression in Colorectal Cancer Cell Lines.

We assessed the effect of sodium butyrate (SB) as a histone deacetylase inhibitor (HDACi) on Toll-like receptor 4 (TLR4) gene expression levels, in low TLR4 expressing (HCT116) and high TLR4 expressing (SW480) colorectal cancer cells. The cytotoxic effect of SB was assessed by culturing SW480 and HCT116 cell lines using a broad spectrum of times and concentrations of SB. The MTT assay was done to check the cytotoxic properties of different SB concentrations. Gene expression levels of TLR4 was then evaluated for non-cytotoxic SB concentrations. Morphological analysis and MTT assay confirmed that SB concentrations equal to or less than 5mM were not cytotoxic for both cell lines. At 5mM concentration of SB in SW480 cell line and 1mM concentration of SB in HCT116 cell line, TLR4 gene expression level significantly increased from 24 to 48 hrs and decreased significantly from 48 to 72 hrs with an "early increased and late decreased pattern". At 1mM concentration of SB in SW480 cell line and 5mM concentration of SB in HCT116 cell line, TLR4 expression had a "gradually increased pattern". This study focuses on the dose-time-effect of SB in the pathogenesis of colorectal cancer. SB alters the expression level of TLR4 in colorectal cancer cells. This effect may depend on the cell type, treatment duration and SB concentration. The alterations in TLR4 expression may be due to the direct effect of SB on TLR4 and/or the expression changes of in other genes which may indirectly affect the TLR4 expression. Abbreviations: TLR4: Toll-like receptor 4; HDACi: histone deacetylase inhibitor; SB: sodium Butyrate; CRC: colorectal cancer; SCFA: short-chain fatty acid; hrs: hours.

Unresponsiveness to meglumine antimoniate in anthroponotic cutaneous leishmaniasis field isolates: analysis of resistance biomarkers by gene expression profiling.

BACKGROUND: Resistance to antimonials is a fundamental determinant of treatment failure in anthroponotic cutaneous leishmaniasis (ACL). Detection of reliable molecular markers to distinguish unresponsive and responsive parasites is critical for consolidating strategies to monitor drug efficacy. METHODS: We analysed the expression of five major antimony resistance-associated genes that is aquaglyceroporin1 (AQP1), γ-glutamylcysteine synthetase (γ-GCS), multidrug resistance protein A (MRPA), trypanothione reductase (TR) and thiol-dependent reductase 1 (TDR1), in unresponsive and responsive Leishmania tropica field isolates by quantitative real-time PCR in comparison with sensitive and resistant reference strains. RESULTS: Gene expression analysis showed the down-regulation of AQP1, γ-GCS and TDR1 by a factor of 1.9, 1.7 and 3.55, respectively, in unresponsive isolates vs. responsive ones. The average RNA expression level of MRPA increased by a factor of 1.9 in the unresponsive group. Isolates exhibited a strong positive linear correlation between gene expression of AQP1 and γ-GCS. A negative correlation between the AQP1 and γ-GCS expression level and lesion duration in responsive patients indicated the potential role in diagnosing drug-unresponsive parasites in endemic areas of ACL. CONCLUSION: In cases of inconclusive outcomes of resistance tests in clinical isolates, expression analysis of a set of influential genes can be beneficial to identify distinctive biomarkers between antimony-unresponsive and responsive parasites.

Host-parasite Responses Outcome Regulate the Expression of Antimicrobial Peptide Genes in the Skin of BALB/c and C57BL/6 Murine Strains Following Leishmania major MRHO/IR/75/ER Infection.

BACKGROUND: Different background of immunity responses determine resistance or susceptibility of certain mouse strains to Leishmania major infection. Some have been well known previously. Antimicrobial peptides (AMPs) such as cathelicidins and defensins are unique fragments of innate immunity system with well-known effects against the invasion pathogens. Despite their outstanding roles and being of extensive cases of cutaneous leishmaniasis (CL) caused by L. major, they have been less studied in Leishmania fields. The aim of present study was to determine whether these components play a role in the protection of skin against Leishmania infections. METHODS: The animal model of Leishmania infection was established by the subcutaneous inoculation of 5×106(parasites/ml) from the stationary phase of L. major promastigotes to BALB/c and C57BL/6 mice from January 2016 to August 2016 in Kerman Province, southeast of Iran. After 1, 3 and 7 d of post-infection (PI), the samples needed for detecting of the mRNA levels of mouse beta-defensin (mBD)-1, mBD2, mBD3, mBD4, mBD6, cathlin-related antimicrobial peptide (CRAMP), interleukin (IL)-10, IL-12 and parasite load were taken under standard methods. RESULTS: The findings related to cytokines profiles in BALB/c (↑IL-10, ↓IL-12) and C57BL/6 mouse strains (↓IL-10, ↑IL-12) demonstrated that immunity system has been accurately activated during CL caused by L. Major parasites. We also observed a significant up-regulation of all aforementioned AMPs genes in BALB/c mice at selected times compared to another strain. CONCLUSION: CL occurred in BALB/c mice in spite of the fact that the expression of AMPs was higher than the other strain. AMPs genes are well expressed to provide defense against the parasites that have increased and escaped from immunity system but cannot create an absolute protection.

Visceral Leishmaniasis in Southeastern Iran: A Narrative Review.

BACKGROUND: Visceral leishmaniasis (VL) has strong links with poverty, substantial medical and veterinary impacts. This review aimed to focus in studies published during 1994-2016 on VL in southeastern Iran. METHODS: The present review is based on expert knowledge and historical studies published during the past 23 yr (1994-2016) on VL in southeastern Iran. In addition, related literature found in PubMed by using the keywords such as visceral leishmaniasis, kala-azar, and Leishmania infantum are included. RESULTS: Overall, 118 children aged 4.2 yr were detected as infected with human VL (HVL). The majority of the cases were from Orzoieh district (37.1%) in southwest of Kerman Province, followed by Sirjan (15.7%), Jiroft (14.8%), Kahnuj (9.3%) and to lesser extent from other areas. The male to female ratio was 1.7. The three most frequent clinical features were represented by fever (100.0%), anemia (95.0%) and splenomegaly (91.5%). Altogether, 42.0% of the VL cases developed secondary bacterial infections, the overall case-fatality rate was 3.4%, and majorities (88.0%) of the VL patients were undernourished. Overall, 733 dogs and wild canines were examined by different techniques with various seroprevalence ranges. CONCLUSION: In southeastern Iran, VL is endemic in Orzoieh district in Kerman Province. While the dogs are implicated as the main domestic reservoir of VL, wide range of wild canines can serve as a secondary potential reservoir host.

Association between interleukin-1 receptor associated kinase 1 rs3027898 A/C gene polymorphism and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease predominantly involving the synovial joints and affects up to 1% of adults worldwide. The aim of the present study was to determine whether the interleukin-1 receptor (IL1R)-associated kinase (IRAK1) rs3027898 gene polymorphism confers susceptibility to RA in a sample of patients from Iran. This gene encodes IRAK1, one of two putative serine/threonine kinases that associates with IL1R upon stimulation. IRAK1 is partially responsible for IL-1-induced upregulation of the transcription factor, nuclear factor-κB. The present case-control study was performed on 120 patients with RA and 120 healthy individuals. Genomic DNA was extracted from whole blood, and the gene polymorphism was evaluated using a tetra-primer amplification refractory mutation system-polymerase chain reaction method. The results demonstrated that there was no association between IRAK1 rs3027898 CA genotype and the risk of RA in women (odds ratio=0.72, 95% confidence interval=0.41-1.49; P=0.446). Further studies with larger sample sizes and different ethnicities are required to validate the present findings.

Lectin, galactoside-binding, soluble, 3 rs4652 A/C gene variation and the risk for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a complex genetic disease. The lectin, galactoside-binding, soluble, 3 (LGALS3) gene, encodes a member of the galectin family of carbohydrate binding proteins, and is one of the best examples of a non-human leukocyte antigen gene associated with a risk for RA in various populations. In the current study, the association between LGALS3 rs4652 gene polymorphism and RA was examined. This case-control study was performed on the 120 patients with RA and 120 healthy subjects. Genomic DNA was extracted from whole blood, and gene polymorphism was tested using a tetra-primer amplification refractory mutation system-polymerase chain reaction. The results demonstrated that LGALS3 rs4652 AC genotype increased the risk of RA (OR=11.622, 95% CI=4.473-28.656; P=0.001) when compared with the AA genotype. However, the CC genotype and the C allele were not associated with RA. These findings indicated an association between LGALS3 rs4652 variation and the risk of RA in a sample of Iranian individuals. Further studies with larger sample sizes and populations of different ethnicities are required to validate our findings.

Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid.

Clostridium perfringens type D infects ruminants and causes the enterotoxemia disease by ε-toxin. A mutated ε-toxin gene lacking toxicity was designed, synthesized, and cloned into the pT1NX vector and electroporated into Lactobacillus casei competent cells to yield LC-pT1NX-ε recombinant strain. BALB/c mice, immunized orally with this strain, highly induced mucosal, humoral, and cell-mediated immune responses and developed a protection against 200 MLD/ml of the activated ε-toxin. This study showed that the LC-pT1NX-ε could be a promising vaccine candidate against the enterotoxemia disease.

Are RIG-1 and MDA5 Expressions Associated with Chronic HBV Infection?

Melanoma differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene 1 (RIG-1) as the pattern recognition receptors play important roles in viral mRNA recognition. Chronic HBV-infected (CHB) patients are unable to properly respond to hepatitis B virus (HBV). Therefore, the aim of the present study was to evaluate the mRNA levels of MDA5 and RIG-1 in the peripheral blood immune cells of CHB patients in comparison to healthy controls. In this cross-sectional study, the mRNA levels of MDA5 and RIG-1 were examined in 60 CHB patients and 60 healthy controls using the real-time polymerase chain reaction (PCR) technique. Our results showed that mRNA levels of MDA5 and RIG-1 were significantly decreased and increased, respectively, in CHB patients when compared to healthy controls. Our results also revealed that mRNA levels of MDA5 and RIG-1 were not altered among CHB patients with various states of e-antigen of hepatitis B and HBV-DNA viral loads. According to the results presented here, it may be concluded that downregulation of MDA5 may be a responsible mechanism from several reasons, which leads to HBV persistence in CHB patients.

Decreased Expressions of STING but not IRF3 Molecules in Chronic HBV Infected Patients.

CONTEXT: The stimulator of interferon genes (STING) induces the activation of interferon regulatory factor 3 (IRF3) in response to intracellular viral double-stranded (ds) DNA. The aim of this study was to evaluate mRNA levels of STING and its downstream transcription factor, IRF3, in the isolated peripheral blood mononuclear cells (PBMCs) of patients with chronic HBV (CHB) infection. METHODS: This study was performed on 60 healthy controls and 60 CHB patients. The mRNA levels of STING and IRF3 were determined using Real-Time polymerase chain reaction (PCR) techniques. The SPSS software version 18 was used to analyze raw data. RESULTS: The results revealed that mRNA levels of STING were significantly decreased in CHB patients in comparison to healthy controls (P = 0.013). Our results also revealed that expression levels of IRF3 were not different between CHB patients and healthy controls (P = 0.828). CONCLUSIONS: In the present study, we found that CHB patients were unable to express appropriate levels of STING. Thus, it may result in impairment of HBV-DNA recognition and subsequently disruption of immune responses. These results suggest a plausible mechanism which may partially define the fact that immune responses are impaired in CHB patients.

In vitro antileishmanial activity of methanolic and aqueous extracts of Eucalyptus camaldulensis against Leishmania major.

Pentavalent antimony compounds are expensive, toxic and drug resistance is prevalent, whereas the plant extract derivatives are safe. In the present study, the effect of methanolic and aqueous extracts of Eucalyptus camaldulensis on the promastigotes of Leishmania major was evaluated. The methanolic and aqueous extracts of E. camaldulensis leaves were prepared. The compounds were dried and powdered. Serial dilutions of the extracts and control drugs in phosphate buffer solution were prepared. The stationary phase promastigotes of L. major were incubated to the methanolic and aqueous extractions in vitro. Tartar emetic was used as the positive control drug. After 72 h of incubation the activity of the extracts was measured, using MTT method. The IC50 values (50 % inhibitory concentration) were 586.2 ± 47.6 and 1,108.6 ± 51.9 µg/ml for methanolic and aqueous extracts, respectively, whereas it was 32.5 ± 6.8 µg/ml for tartar emetic. The results indicated that the methanolic extract was more effective than aqueous extract, although there was no significant difference. The extracts were less effective as compared to the control drug. Further investigation is required to evaluate these extracts on clinical stage in macrophage-amastigote model.

Evaluation of the circulating levels of IL-12 and IL-33 in patients with breast cancer: influences of the tumor stages and cytokine gene polymorphisms.

OBJECTIVES: IL-12 as an anti-tumor cytokine and IL-33 a novel identified cytokine with both pro- or anti-tumor activities, play important roles in response against tumor cells. Our aim was to evaluate the IL-12 and IL-33 levels and single nucleotide polymorphisms (SNP) in their genes in patients with breast cancer. MATERIALS AND METHODS: Blood samples were collected from 100 patients with breast cancer, and 100 healthy women were controls. The serum IL-12 and IL-33 levels were measured by ELISA. The SNP rs3212227 (in IL-12 gene) and rs1929992 (in IL-33 gene) were determined using PCR-RFLP. RESULTS: The IL-12 levels similarly expressed in patients and controls. IL-12 levels in patients at stage I were significantly lower than in the healthy group (P<0.05). IL-33 levels and the IL-33/IL-12 ratio were significantly higher in patients than the control group (P<0.001). The IL-33 levels and IL-33/IL-12 ratio in stage IV patients were significantly higher than other stages and controls (P<0.0001 and P<0.001, respectively). There were no significant differences in the frequencies of genotypes in rs3212227 and rs1929992 between patients and the control group. No significant differences were observed between subjects with various genotypes at rs3212227 and rs1929992 with respect to related cytokine levels. CONCLUSION: These results indicate that the diminished IL-12 production may contribute to the tumor establishment. The higher IL-33 levels and IL-33/IL-12 ratio in patients also indicate an imbalance in Th1/Th2 responses that may contribute to tumor development. Thus, correcting the imbalance of Th1/Th2 could be an important strategy for cancer immunotherapy.

Gentamicin-attenuated Leishmania infantum vaccine: protection of dogs against canine visceral leishmaniosis in endemic area of southeast of Iran.

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A vaccine trial was conducted using 103 naive dogs from a leishmaniosis non-endemic area (55 vaccinated and 48 unvaccinated) brought into an endemic area of southeast Iran. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination. The efficacy of the vaccine was evaluated after 24 months (4 sandfly transmission seasons) by serological, parasitological analyses and clinical examination. In western blot analysis of antibodies to L. infantum antigens, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs which were seropositive at >100, recognized the 21 kDa antigen of L. infantum wild-type (WT). Nine out of 31 (29%) unvaccinated dogs, but none of vaccinated dogs, were positive for the presence of Leishmania DNA. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. These results suggest that gentamicin-attenuated L. infantum induced a significant and strong protective effect against canine visceral leishmaniosis in the endemic area.

Important roles played by TGF-ß in hepatitis B infection.

Hepatitis B virus (HBV) which includes, fulminant, acute, chronic, asymptomatic, and occult HBV infection is the most prevalent virus that leads to human liver diseases. Chronic, asymptomatic, and occult infection can induce further sever diseases such as hepatocellular carcinoma (HCC) and cirrhosis of the liver. The underlying mechanisms that allow progression of the prolonged forms of the infection and subsequent HCC or cirrhosis of the liver are yet to be clarified. However, many researchers have suggested that immunological and genetic parameters may play important roles in the etiology of hepatitis B. Transforming growth factor beta (TGF-ß) is an important cytokine with dual regulatory functions in the immune system and in the responses against viral infections. However, the pathways and mechanisms controlling these are not fully understood. The crucial roles of TGF-ß in the development of Th17 and T regulatory lymphocytes, the main cell types involved in autoimmunity and destructive immune related diseases, have been documented and this provides insights into TGF-ß function during hepatitis infection and subsequent HCC and cirrhosis of the liver. Recent findings also confirm that TGF-ß directly alters hepatocyte function during hepatitis B, hence, the aim of this review is to address the current data regarding the association and status of TGF-ß with hepatitis B infection and its related disorders including HCC and cirrhosis of the liver.

Decreased expression of toll like receptor signaling molecules in chronic HBV infected patients.

INTRODUCTION: Toll like receptors (TLRs) and their signaling molecules play important roles in microbe recognition and induction of immune responses, including production of inflammatory cytokines, against viral infections. Therefore, the aim of this study was to examine expression levels of TLR signaling molecules (IRAK1, IRAK4, TRAF3, and IRF7) and the pro-inflammatory cytokines, IL-12 and IL-6 in chronic HBV infected (CHB) patients. DESIGN: This study was performed on 60 CHB patients and 60 healthy controls and the expression of IRAK1, IRAK4, TRAF3, and IRF7 and their downstream inflammatory cytokines (IL-12 and IL-6) were evaluated by Real-Time PCR and ELISA techniques. RESULTS: The results demonstrated that expression of IRAK4, TRAF3, and IRF7 were significantly decreased in PBMCs of CHB patients in comparison to healthy controls. Serum levels of IL-12 were significantly increased, while, IL-6 were not differ between patients and controls. CONCLUSIONS: Based on the results presented here it seems that CHB patients do not express appropriate levels of the genes in the TLRs pathway which may lead to impaired immune responses against HBV infection which is seen in the patients.

Alteration in mononuclear cell subpopulations in dogs immunized with gentamicin-attenuated Leishmania infantum.

The impact of immunization with gentamicin-attenuated Leishmania infantum (H-line) on the immunophenotypic profile of popliteal lymph node (PLN) and peripheral blood mononuclear cells (PBMCs) of dogs was assessed by flow cytometry and immunohistochemistry. Compared with the dogs infected with L. infantum wild-type (Group WT), there was a significantly higher percentage of CD4+, CD44+ T cells and CD14+, MHC-II+ cells and a lower percentage of CD4+ CD25+ regulatory T cells in PLN of the immunized dogs with L. infantum H-line (Group H). The percentage of CD4+ and CD8+ T cells in PBMCs of immunized dogs was higher than that in dogs of Group WT. The CD4:CD8 ratio in PLN of dogs of Group H was significantly higher than that in dogs of Group WT. A significantly higher percentage of CD21+ B cells and a lower percentage of CD79b+ cells were found in PLN of the immunized dogs compared with dogs of Group WT. Immunohistochemical investigation showed no parasites in the PLN of immunized dogs whereas there were parasites in the PLN of 60% of dogs infected with L. infantum WT. In this study, the immunophenotypic profile of mononuclear cells of the immunized dogs correlates with cellular immunity.

Preparation and evaluation of niosomes containing autoclaved Leishmania major: a preliminary study.

In this study, different positively charged niosomal formulations containing sorbitan esters, cholesterol and cetyl trimethyl ammonium bromide were prepared by film hydration method for the entrapment of autoclaved Leishmania major (ALM). Size distribution pattern and stability of niosomes were investigated by laser light scattering method and ALM encapsulation per cent was measured by the bicinchoninic acid method. Finally, the selected formulation was used for the induction of the immune response against cutaneous leishmaniasis in BALB/c mice. Size distribution curves of all the formulations followed a log-normal pattern and the mean volume diameter was in the range 7.57-15.80 µm. The mean volume diameters were significantly increased by adding Tween to Span formulations (p < 0.05). The percentage of ALM entrapped in all formulations varied between 14.88% and 36.65%. In contrast to ALM, in vivo studies showed that the niosomes containing ALM have a moderate effect in the prevention of cutaneous leishmaniasis in BALB/c mice.

Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiol-redox metabolism.

We have previously described an attenuated line of Leishmania infantum (H-line), selected by culturing promastigotes in vitro in the presence of gentamicin. To elucidate the molecular basis for this attenuation, we undertook a comparative proteomic analysis using multiplex 2-dimensional (2D) difference gel electrophoresis. Eighteen proteins that showed significant and reproducible changes in expression were identified. Many of these were components of the thiol-redox control system in Leishmania and this observation, validated by Western blot, prompted us to investigate the sensitivity of the attenuated line to oxidative stress. The attenuated line was found to be significantly more susceptible to hydrogen peroxide, a change which may explain the loss of virulence. In a direct assay of trypanothione-dependent peroxidase activity, hydrogen peroxide metabolism in the H-line was significantly lower than in wild type. Furthermore, trypanothione reductase activity was significantly lower in the H-line, suggesting that gentamicin selection may result in pleiotropic affects on thiol metabolism in Leishmania. A putative RNA-binding protein was very strongly up-regulated in the attenuated line, suggesting a possible target for gentamicin in Leishmania.