Combination of STAT3 inhibitor with Herceptin reduced immune checkpoints expression and provoked anti-breast cancer immunity: An in vitro study.

Breast cancer (BC) is the most prevalent diagnosed cancer among women. Herceptin blocks the effects of Her-2 and tumour cell growth. Despite many achievements using Herceptin in Her-2+ invasive BC treatment, there are treatment failures and resistances. The signal transducer and activator of transcription 3 (STAT3) is persistently activated in BC and is associated with immune suppression and tumour cell proliferation. We evaluated whether STAT3 inhibition could increase Herceptin impact on in vitro reduction of immune checkpoint inhibitors and polarize T cells to a protective immune response. We treated SK-BR-3 cells with Herceptin and the STAT3-inhibitor (FLLL32) and assessed the apoptosis and expression of apoptosis-related proteins, VEGF, Her-2 and apoptosis targets of STAT3. PBMCs were isolated from healthy donors and co-cultured with SK-BR-3 cells in the presence or absence of Herceptin and FLLL32. PD-L1, CTLA-4, TIM-3 and T-cell intracellular cytokines were then evaluated. Our results demonstrated that STAT3 inhibition and Herceptin increased SK-BR-3 cell apoptosis, significantly. STAT3 inhibition through combination treatment had a more significant effect on regulating PD-1, TIM-3 and CTLA-4 expression on PBMCs. Alternatively, the combination of FLLL32 and Herceptin promoted T helper-1 protective immune response. The combination of FLLL32 and Herceptin suppress the expression of immune checkpoints and provoke the T-helper1 immune response in lymphocytes. Our analysis indicates STAT3 as a promising target that improves Herceptin's role in breast cancer cell apoptosis.

Cutaneous leishmaniasis: multiomics approaches to unravel the role of immune cells checkpoints.

INTRODUCTION: Cutaneous leishmaniasis (CL) is the most frequent form of leishmaniases, associated with skin inflammation and ulceration. Understanding the interaction of different phagocytic cells in the recognition and uptake of different Leishmania species is critical for controlling the infection. Phagocytic cells have a pivotal role as professional antigen-presenting cells that bridge the innate and adaptive immunity and shape the outcome of the disease. AREAS COVERED: Here we reviewed new technologies with high-throughput data collection capabilities along with systems biology approaches which are recently being used to decode the paradox of CL immunology. EXPERT OPINION: We emphasized on the crosstalk between DC and T-cells while focusing on the immune checkpoints interactions between the human immune system and the Leishmania species. Further, we discussed omics technologies including bulk RNA sequencing, reverse transcriptase-multiplex ligation dependent probe amplification (RT-MLPA), and proximity extension assay (PEA) in studies on human blood or tissue-driven samples from CL patients in which we have so far been involved.

Cutaneous leishmaniasis in Iran: A systematic review and meta-analysis.

Cutaneous leishmaniasis as a public health concern that attracts many attentions in endemic area. There is no exact estimation of cutaneous leishmaniasis in Iran. This study aimed to assess the exact prevalence of disease and carried out in databases including: Pub Med, Google Scholar, Science Direct, Scopus, Web of Science, Magiran, Iran doc, Barakatkns and Scientific Information Database (SID) from 2000 to 2019. Totally 84 studies were eligible to be included in this systematic review and Meta-analysis study. Based on a random effect model the pooled prevalence of leishmaniasis was estimated 45% (95% CI: 39%-51%; I^2 = 99.8%P < 0.001). The highest prevalence of CL was related to Isfahan 66% (53%-78%), Golestan 64% (62%-65%) and Fars province 63% (38%-84%) and the lowest prevalence was estimated in Kermanshah province 4% (4%-5%), Hormozgan 10% (8%-11%), Bushehr 12% (1%-35%) and Kerman 15% (9%-22%) provinces respectively. The lowest prevalence was associated with L. trapica spices 23% (11%-38%) and the highest was associated with L. major spices 32% (21%-45%). The prevalence with both L. trapica and L. major spices was achieved 60% (48%-71%). It is essential for health authorities to take steps to control and prevent the epidemic by rapid treatment of patients, destroying gerbils and promotion of general and health education for the local population.

Immunogenic reactivity of recombinant PKF and AbOmpA proteins as serum resistance factors against sepsis of Acinetobacter baumannii.

Acinetobacter baumannii is considered as a major cause of nosocomial infection worldwide. Various vaccine formulations have been mostly studied based on secreted or surface-exposed proteins of A. baumannii in murine models. Serum resistance proteins are critical virulence factors in bloodstream infections. AbOmpA and PKF are two major factors involved in serum resistance and could be considered as promising vaccine targets. In this study IgG1, IgG2c, Total-IgG concentrations, survival rates and spleen bacterial loads were studied in C57/BL mice model according to PKF, AbOmpA and AbOmpA + PKF vaccine formulations. The findings showed significant raises of IgG2c and Total-IgG in all three vaccinated groups in comparison with the control group. Whereas, there were low concentrations of IgG1 in all immunization plans. Colony counts of mice spleen showed the bacterial load of PKF plan had the most decrease of bacterial load (DBL = 5 log10 CFU/g). Taken together, this evaluation indicated that PKF vaccination plan induced a polarized Th1 response and rendered an effective protection against bloodstream infection caused by A. baumannii.

Mucosal and systemic immune responses elicited by recombinant Lactococcus lactis expressing a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis.

We constructed a food-grade expression system harboring a F1S1 fusion protein of Bordetella pertussis to be produced in Lactococcus lactis NZ3900 as a new oral vaccine model against whooping cough, caused by B. pertussis. F1S1 was composed of N-terminally truncated S1 subunit of pertussis toxin and type I immunodominant domain of filamentous hemagglutinin which are both known as protective immunogens against pertussis. The recombinant L. lactis was administered via oral or intranasal routes to BALB/c mice and the related specific systemic and mucosal immune responses were then evaluated. The results indicated significantly higher levels of specific IgA in the lung extracts and IgG in sera of mucosally-immunized mice, compared to their controls. It was revealed that higher levels of IgG2a, compared to IgG1, were produced in all mucosally-immunized mice. Moreover, immunized mice developed Th1 responses with high levels of IFN-γ production by the spleen cells. These findings provide evidence for L. lactis to be used as a suitable vehicle for expression and delivery of F1S1 fusion protein to mucosa and induction of appropriate systemic and mucosal immune responses against pertussis.

Subcutaneous administration CpG-ODNs acts as a potent adjuvant for an HIV-1-tat-based vaccine candidate to elicit cellular immunity in BALB/c mice.

OBJECTIVE: To evaluate the combined effects of CpG oligodeoxynucleotides (CpG-ODNs) adjuvant and subcutaneous injection route on efficacy of a HIV-1-tat DNA vaccine candidate using BALB/c mice as an animal model. RESULTS: Evaluation of cellular and humoral immunity of mice injected subcutaneously with HIV-1-tat gene cloned into a pcDNA3.1 vector indicated that significant levels of IFN-γ cytokine secretion (900 pg/ml), lymphocyte proliferation (2.5 stimulation index) and IgG2a (1.45 absorbance 450 nm) production could be achieved. These indicators of stimulated cellular immunity were elicited 2 weeks after the last injection (P < 0.05). CONCLUSIONS: Formulation of HIV-1-tat DNA vaccine candidate with CpG-ODNs as an adjuvant while administrated subcutaneously are a promising approach to induce effective cellular immunity responses against HIV-1 infection.

Nucleotide receptors control IL-8/CXCL8 and MCP-1/CCL2 secretions as well as proliferation in human glioma cells.

Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.

Oral treatment with zinc sulfate increases the expression of Th1 cytokines mRNA in BALB/c mice infected with Leishmania major.

Leishmaniases consist of a group of diseases caused by protozoan parasites of Leishmania genus. The outcome of the disease depends on the immune responses of the host as well as the pathogenicity of the strain of the parasite. In murine models, the inoculation of Leishmania major into resistant mice results in Th1 responses and recovery from the infection. However in the susceptible mice, the same inoculation leads to a profile of Th2 responses. Zinc (Zn) is an essential trace element which is required for the growth and development of the immune responses. In this study, the influence of Zn sulfate on mRNA expression of main cytokines of the immune response was studied in susceptible BALB/c mice infected with L. major. The inoculated mice were divided into 3 groups, namely the untreated (control), the zinc sulfate treated (weeks 2, 4 and 8), and the Glucantime-treated (weeks 4 and 8) mice. During different time points post-infection, the lesion sizes and the parasite burden were measured in all the groups. Moreover, the expression of Ifng, Il4, Il10 and Il12 mRNA levels in the draining lymph nodes of the treated mice were compared to the control mice using real-time PCR. Our data demonstrated significant decreases in lesion sizes and parasite loads in Zn sulfate treated group compared to the untreated group. Moreover, significant fold increases in expression of Ifng transcript were observed in mice treated with Zn sulfate compared to the control. The ratio of Ifng/Il4 mRNA was also higher in Zn sulfate-treated mice compared to Glucantime-treated animals. These results indicate that Zn Sulfate has the ability to induce strong Th1 responses in susceptible BALB/c mice inoculated with L. major.

Comparison between MDCK and MDCK-SIAT1 cell lines as preferred host for cell culture-based influenza vaccine production.

OBJECTIVES: To evaluate MDCK and MDCK-SIAT1 cell lines for their ability to produce the yield of influenza virus in different Multiplicities of Infection. RESULTS: Yields obtained for influenza virus H1N1 grown in MDCK-SIAT1 cell was almost the same as MDCK; however, H3N2 virus grown in MDCK-SIAT1 had lower viral titers in comparison with MDCK cells. The optimized MOIs to infect the cells on plates and microcarrier were selected 0.01 and 0.1 for H1N1 and 0.001 and 0.01 for H3N2, respectively. CONCLUSIONS: MDCK-SIAT1 cells may be considered as an alternative mean to manufacture cell-based flu vaccine, especially for the human strains (H1N1), due to its antigenic stability and high titer of influenza virus production.

Leishmania major strains isolated from distinct endemic areas show diverse cytokine mRNA expression levels in C57BL/6 mice: Toward selecting an ideal strain for the vaccine studies.

Leishmania major, the causative agent of zoonotic cutaneous leishmaniasis shows heterogeneity and diverse clinical manifestations in different areas of infection and experimental models. Such polymorphism may cause difficulties in selection of reliable strains for development of prophylaxes. Hence, the aim of this study was to identify an ideal strain of L. major, capable of inducing protective and long-lasting Th1 responses in an animal model that mimics the human response to L. major infection. The isolates were from patients residing in 4 endemic areas of L. major in Iran, namely Damghan (north), Kashan (center), Dehloran (west) and Shiraz (south) which their heterogeneity had been previously confirmed in BALB/c mice. In this study, the same isolates as well as the Iranian reference strain of L. major were inoculated to C57BL/6 mice to evaluate their pathogenicity and changes in expression of key cytokine genes from lymph nodes of the mice in different time points, in order to evaluate their ability to control leishmaniasis by development of Th1 responses. Our results showed the lowest and highest parasite burden in lymph nodes of mice infected with all strains at weeks 3 and 8 post-infection, respectively. However, the Damghan strain (DA39) showed comparatively lower number of viable parasite than other strains at week 8 post-infection. Furthermore, DA39 showed higher expression of Ifng and Il12 mRNA at week 8 post-infection while the ratio of its Ifng/Il4 mRNA expressions was higher than other strains. In conclusion, DA39 among the studied strains appears to induce strong and lasting Th1 cytokine gene expressions with minimum virulence, making it a suitable candidate strain for vaccine studies in leishmaniasis.

Purine-metabolizing ectoenzymes control IL-8 production in human colon HT-29 cells.

Interleukin-8 (IL-8) plays key roles in both chronic inflammatory diseases and tumor modulation. We previously observed that IL-8 secretion and function can be modulated by nucleotide (P2) receptors. Here we investigated whether IL-8 release by intestinal epithelial HT-29 cells, a cancer cell line, is modulated by extracellular nucleotide metabolism. We first identified that HT-29 cells regulated adenosine and adenine nucleotide concentration at their surface by the expression of the ectoenzymes NTPDase2, ecto-5'-nucleotidase, and adenylate kinase. The expression of the ectoenzymes was evaluated by RT-PCR, qPCR, and immunoblotting, and their activity was analyzed by RP-HPLC of the products and by detection of Pi produced from the hydrolysis of ATP, ADP, and AMP. In response to poly (I:C), with or without ATP and/or ADP, HT-29 cells released IL-8 and this secretion was modulated by the presence of NTPDase2 and adenylate kinase. Taken together, these results demonstrate the presence of 3 ectoenzymes at the surface of HT-29 cells that control nucleotide levels and adenosine production (NTPDase2, ecto-5'-nucleotidase and adenylate kinase) and that P2 receptor-mediated signaling controls IL-8 release in HT-29 cells which is modulated by the presence of NTPDase2 and adenylate kinase.

NTPDase1 controls IL-8 production by human neutrophils.

The ectonucleotidase NTPDase1 (CD39) terminates P2 receptor activation by the hydrolysis of extracellular nucleotides (i.e., the P2 receptor ligands). In agreement with that role, exacerbated inflammation has been observed in NTPDase1-deficient mice. In this study, we extend these observations by showing that inhibition of NTPDase1 markedly increases IL-8 production by TLR-stimulated human neutrophils. First, immunolabeling of human blood neutrophils and neutrophil-like HL60 cells displayed the expression of NTPDase1 protein, which correlated with the hydrolysis of ATP at their surface. NTPDase1 inhibitors (e.g., NF279 and ARL 67156) as well as NTPDase1-specific small interfering RNAs markedly increased IL-8 production in neutrophils stimulated with LPS and Pam(3)CSK(4) (agonists of TLR4 and TLR1/2, respectively) but not with flagellin (TLR5) and gardiquimod (TLR7 and 8). This increase in IL-8 release was due to the synergy between TLRs and P2 receptors. Indeed, ATP was released from neutrophils constitutively and accumulated in the medium upon NTPDase1 inhibition by NF279. Likewise, both human blood neutrophils and neutrophil-like HL60 cells produced IL-8 in response to exogenous nucleotides, ATP being the most potent inducer. In agreement, P2Y(2) receptor knockdown in neutrophil-like HL60 cells markedly decreased LPS- and Pam(3)CSK(4)-induced IL-8 production. In line with these in vitro results, injection of LPS in the air pouches of NTPDase1-deficient mice triggered an increased production of the chemokines MIP-2 and keratinocyte-derived chemokine (i.e., the rodent counterparts of human IL-8) compared with that in wild-type mice. In summary, NTPDase1 controls IL-8 production by human neutrophils via the regulation of P2Y(2) activation.

Evaluation of the Immunogenicity of Recombinant Espb, Espc Proteins from Mycobacterium Tuberculosis and the Fusion Espc/Espb Protein in BALB/C Mice.

Background: Two newly identified proteins, EspB and EspC are involved in the pathogenesis of Mycobacterium tuberculosis. The objective of the present study was to evaluate the immunogenicity of recombinant EspC, EspB, and EspC/EspB fusion proteins in mice. Methods: BALB/c mice were immunized subcutaneously with recombinant EspC, EspB, and fusion EspC/EspB proteins, three times with along with Quil-A as an adjuvant. The cellular and humoral immune responses were evaluated by quantifying IFN-γ, IL-4, IgG, IgG1, and IgG2a antibodies against the antigens. Results: The results showed that the mice immunized with recombinant EspC, EspB, and EspC/EspB proteins did not produce IL-4, whereas IFN-γ was secreted in response to all three proteins. EspC/EspB group produced significant amounts of IFN-γ in response to stimulation with all the three recombinant proteins (P<0.001). In mice immunized with EspC, high levels of IFN-γ were detected in response to EspC/EspB, and EspC (P<0.0001); while mice immunized with EspB produced lower levels of IFN-γ in response to EspC/EspB, and EspB (P<0.05).Mice immunized with recombinant EspC, EspB, and EspC/EspB proteins exhibited significantly high levels of IgG and IgG2a/IgG1 ratio (P< 0.001). Moreover, high levels of IgG and IgG2a were detected in the sera of mice immunized with EspC/EspB fusion protein. Conclusions: All the three recombinant proteins induced Th1-type immune responses in mice against EspB and EspC; however, EspC/EspB protein is more desirable due to the presence of epitopes from both EspC and EspB proteins and the production of immune responses against both.

Molecular identification of Phlebotomus kandelakii apyrase and assessment of the immunogenicity of its recombinant protein in BALB/c mice.

Sand fly salivary proteins have immunomodulatory and anti-inflammatory features; hence, they are proven to perform important roles in the early establishment of Leishmania parasite in the vertebrate host. Among them, salivary apyrase with anti-hemostatic properties has a crucial role during the blood meal process. In the present study, a Genome-Walking method was used to characterize a full-length nucleotide sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot and ELISA results indicated that both recombinant apyrases could induce antibodies in BALB/c. Moreover, a partial cross-reactivity between Pkapy and Ppapy was found. In vitro stimulation of splenocytes from immunized mice with the recombinant proteins indicated cross-reactive T cell proliferative responses. Cytokine analysis revealed significant production of IFN-γ (p < 0.001) and IL-10 (p < 0.01) in response to Pkapy. In conclusion, the full-length nucleotide sequence and molecular characteristics of Pkapy were identified for the first time. Immunologic analyses indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show partial cross-reactive responses. The immunity to Pkapy was found to be a Th1-dominant response that highlights its potential as a component for an anti-Leishmania vaccine.

Dr. Abolghasem Bahrami (1894­1950): Physician, Pasteurian, and a Pioneer of Microbiology and Public Health Planning in Iran

Dr. Abolghasem Bahrami was among the generation of Iranian scientists in the early twentieth century who gained most of their knowledge through resources available inside the country. Educated at Dar-ul-Funun Medical School, he was a physician with a great talent in learning, especially self-teaching natural sciences and European languages. He joined the Pasteur Institute of Iran (IPI) at the early days of its foundation and became an integral contributor to this institution during the first twenty-five years of its mission. One of his first assignments at IPI was to help initiating an anti-rabies department by bringing back the rabies vaccine and its manufacturing equipment from Institut Pasteur of Paris. During his IPI years, aside from managerial tasks, he actively participated in upgrading the medical treatments and protocols used for controlling many infectious diseases. He functioned twice as the provisional director of IPI (1925-1926 and 1937-1946) and is considered as the first Iranian director of the Institute. Meanwhile, Dr. Bahrami was a significant contributor to the public health system and assumed several responsibilities such as Chief Quarantine Medical Officer, Chief of Public Health, and the Head of Public Health Administration, in order to improve public health planning throughout the country.

A Historic Review of the Role of CD4+ T-Cell Subsets in Development of the Immune Responses against Cutaneous and Visceral Leishmaniases

The heterogeneity of CD4+ T cells has been investigated since the late 1970s, when their Th1 and Th2 subsets were coined. Later studies on the cutaneous form of the Leishmaniasis were focused on the experimental models of Leishmania major infection using the susceptible BALB/c and the resistant C57BL/6 mice. At the early 21st century, the regulatory T-cells subpopulation was introduced and its role in concomitant immunity, responsible for lifelong resistance of the host to the reinfection was proposed. Subsequent studies, mainly focused on the visceral form of the infection pointed to the role of IL-17, produced by Th17 subset of CD4+ T cells that along the neutrophils were shown to have important yet equivocal functions in protection against or exacerbation of the infection. Altogether, the current knowledge indicates that the above four subsets could orchestrate the immune, the regulatory and the inflammatory responses of the host against different forms of leishmaniases.

An overview of the sand fly salivary proteins in vaccine development against leishmaniases.

Leishmaniases are a group of vector-borne parasitic diseases transmitted through the infected sand flies. Leishmania parasites are inoculated into the host skin along with sand fly saliva. The sand fly saliva consists of biologically active molecules with anticoagulant, anti-inflammatory, and immunomodulatory properties. Such properties help the parasite circumvent the host's immune responses. The salivary compounds support the survival and multiplication of the parasite and facilitate the disease progression. It is documented that frequent exposure to uninfected sand fly bites produces neutralizing antibodies against specific salivary proteins and further activates the cellular mechanisms to prevent the establishment of the disease. The immune responses due to sand fly saliva are highly specific and depend on the composition of the salivary molecules. Hence, thorough knowledge of these compounds in different sand fly species and information about their antigenicity are paramount to designing an effective vaccine. Herein, we review the composition of the sand fly saliva, immunomodulatory properties of some of its components, immune responses to its proteins, and potential vaccine candidates against leishmaniases.

Immunoinformatics Evaluation of a Fusion Protein Composed of Leishmania infantum LiHyV and Phlebotomus kandelakii Apyrase as a Vaccine Candidate against Visceral Leishmaniasis.

Background: Visceral leishmaniasis (VL) is a lethal parasitic disease, transmitted by sand fly vectors. Immunomodulatory properties of sand fly saliva proteins and their protective effects against Leishmania infection in pre-exposed animals suggest that a combination of an antigenic salivary protein along with a Leishmania antigen can be considered for designing a vaccine against leishmaniasis. Methods: Three different fusion forms of L. infantum hypothetical protein (LiHyV) in combination with Phlebotomus kandelakii salivary apyrase (PkanAp) were subjected to insilico analyses. Major Histocompatibility Complex (MHC) class I and II epitopes in both humans and BALB/c mice were predicted. Antigenicity, immunogenicity, epitope conservancy, toxicity, and population coverage were also evaluated. Results: Highly antigenic promiscuous epitopes consisting of truncated LiHyV (10-285) and full-length PkanAp (21-329) were identified in human and was named Model 1. This model contained 25 MHC-I and 141 MHC-II antigenic peptides which among them, MPANSDIRI and AQSLFDFSGLALDSN were fully conserved. LALDSNATV, RCSSALVSI, ALVSINVPL, SAVESGALF of MHC-I epitopes, and 28 MHC-II binding epitopes showed 60% conservancy among various clades. A population coverage with a rate of >75% in the Iranian population and >70% in the whole world was also identified. Conclusion: Based on this in-silico approach, the predicted Model 1 could potentially be used as a vaccine candidate against VL.

Blood transcriptional profiles distinguish different clinical stages of cutaneous leishmaniasis in humans.

Cutaneous leishmaniasis (CL) is a neglected tropical disease with severe morbidity and socioeconomic sequelae. A better understanding of underlying immune mechanisms that lead to different clinical outcomes of CL could inform the rational design of intervention measures. While transcriptomic analyses of CL lesions were recently reported by us and others, there is a dearth of information on the expression of immune-related genes in the blood of CL patients. Herein, we investigated immune-related gene expression in whole blood samples collected from individuals with different clinical stages of CL along with healthy volunteers in an endemic CL region where Leishmania (L.) tropica is prevalent. Study participants were categorized into asymptomatic (LST+) and healthy uninfected (LST-) groups based on their leishmanin skin test (LST). Whole blood PAXgene samples were collected from volunteers, who had healed CL lesions, and patients with active L. tropica cutaneous lesions. Quality RNA extracted from 57 blood samples were subjected to Dual-color reverse-transcription multiplex ligation-dependent probe amplification (dcRT-MLPA) assay for profiling 144 immune-related genes. Results show significant changes in the expression of genes involved in interferon signaling pathway in the blood of active CL patients, asymptomatics and healed individuals. Nonetheless, distinct profiles for several immune-related genes were identified in the healed, the asymptomatic, and the CL patients compared to the healthy controls. Among others, IFI16 and CCL11 were found as immune transcript signatures for the healed and the asymptomatic individuals, respectively. These results warrant further exploration to pinpoint novel blood biomarkers for different clinical stages of CL.

High resolution melting assay in discrimination of the main etiologic agents of leishmaniasis in Iran.

BACKGROUND AND OBJECTIVES: The three old world Leishmania species i.e., L. major, L. tropica, and L. infantum are considered as potential etiological agents of the various clinical forms of leishmaniasis in Iran. Different species co-exist in some areas. Accurate differentiation between the species is essential for choosing an appropriate therapy. Conventional and gold standard methods for the detection and characterization of parasites are time-consuming, laborious, and have low sensitivity. A polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis has been employed for detection and species identification. Most of the studies suffer from the use of multiple targets and/or requiring more than one reaction to identify a single sample. The present study aimed to design a PCR method based on the amplification of kinetoplast DNA minicircles (kDNA) and HRM analysis of the amplicons for rapid discrimination of the three mentioned species. MATERIALS AND METHODS: DNA from reference strains including L. major, L. tropica, and L. infantum and fifty-eight strains subjected to PCR-HRM analysis targeting kDNA. All the samples were also analyzed by conventional kDNA-PCR. RESULTS: The PCR-HRM analysis allowed discrimination between the three Old World species. The normalized HRM curves for the amplicons of kDNA indicated a unique and repeatable melting plot for each species, even in combination with human and mouse genomic DNA. Conventional kDNA-PCR could not properly discriminate L. tropica from L. infantum. CONCLUSION: PCR-HRM analysis of kDNA proved to be fast and accurate for discrimination of L. major, L. tropica, and L. infantum.