Environmental health risk factors and cutaneous leishmaniasis (CL): A case-control study in northeastern Iran.

Background & objectives: Cutaneous leishmaniasis (CL) is one of the main causes of vector-born diseases in younger population. To evaluate the association of environmental health factors on the odds of CL incidence, a case-control study was conducted in northeastern Iran. Methods: This study was conducted within 2020-2021 based on individual and household data from a tertiary referral center. Cases were patients diagnosed with CL by PCR method; controls were selected among the patients' relatives, and information was obtained from a health registry system. Demographic and socioeconomic data of 1871 subjects, included age, sex, household information and environmental health factors. Multivariable models with environmental factors in various conditions and CL were separately fit by univariate and mixed multiple unconditional logistic regression. Results: Participants included 617 cases (mean [SD] age, 13.62[13.72] years; 58.20% male) and 1264 controls (mean [SD] age, 16.45[15.44] years; 50.40% male). Results revealed that the use of well-water sources compared to surface water is significantly associated with CL (odds ratio [OR]=0.204; 95%CI, 0.13-0.33;P<0.001). Muddy houses, ruined buildings or wastelands and stagnant water, canals and rivers near the houses were also associated with CL (OR=3.85; 95%CI, 1.66-8.89; P=.002; OR=2.47; 95%CI, 1.76-3.47; P<.001). Besides, existence of pine tree was found to be a risk factor (OR=3.25; 95%CI, 2.12-4.99; P<.001) and similarly for the use of waste collection system (OR=4.43; 95%CI, 3.32-7.51; P<.001). Interpretation & conclusion: Environmental factors related to houses were significantly associated with CL and may represent the modifiable risk factors of CL disease.

Naltrexone; as an efficient adjuvant in induction of Th1 immunity and protection against Fasciola hepatica infection.

Toxic effects of available therapeutics are major drawbacks for conventional management approaches in parasitic infections. Vaccines have provided a promising opportunity to obviate such unwanted complications. In present study, we examined immune augmenting capacities of an emerging adjuvant, Naltrexone, against Fasciola hepatica infection in BALB/c mice. Seventy BALB/c mice were divided into five experimental groups (14 mice per group) including 1- control (received PBS), 2- vaccine (immunized with F. hepatica E/S antigens), 3- Alum-vaccine (immunized with Alum adjuvant and E/S antigens), 4- NLT-vaccine (immunized with NLT adjuvant and E/S antigens), and 5- Alum-NLT-vaccine (immunized with mixed Alum-NLT adjuvant and E/S antigens). Lymphocyte stimulation index was assessed by MTT assay. Production of IFN-γ, IL-4, IgG2a and IgG1 was assessed by ELISA method. Results showed that NLT, either alone or in combination with alum, can induce immune response toward production of IFN-γ and IgG2a as representatives of Th1 immune response. Also, using this adjuvant in immunization experiment was associated with significantly high proliferative response of splenocytes/lymphocytes. Utilization of mixed Alum-NLT adjuvant revealed the highest protection rate (73.8%) in challenge test of mice infected with F. hepatica. These findings suggest the potential role of NLT as an effective adjuvant in induction of protective cellular and Th1 immune responses against fasciolosis.

Assessment of expression of interferon γ (IFN-G) gene and its antisense (IFNG-AS1) in breast cancer.

BACKGROUND: The role of long non-coding RNAs has been extensively appreciated in the contexts of cancer. Interferon γ-antisense RNA1 (IFNG-AS1) is an lncRNA located near to IFN-γ-encoding (IFNG) gene and regulates expression of IFNG in Th1 cells. METHODS: In the present study, we evaluated expression of IFNG and IFNG-AS1 in 108 breast samples including tumoral tissues and their adjacent non-cancerous tissues (ANCTs) using real-time PCR. IFNG-AS1 was significantly upregulated in tumoral tissues compared with ANCTs (expression ratio = 2.23, P = 0.03). RESULTS: Although the expression of IFNG was higher in tumoral tissues compared with ANCTs (relative expression = 1.89), it did not reach the level of significance (P = 0.07). IFNG expression was significantly higher in HER2-negative tumoral tissues compared with HER2-positive ones (P = 0.01) and in grade 1 samples compared with grade 2 ones (P = 0.03). No other significant difference was found in expressions of genes between other groups. CONCLUSION: Significant strong correlations were detected between expression of IFNG and IFNG-AS1 in both tumoral tissues and ANCTs. The present study provides evidences for participation of IFNG and IFNG-AS1 in the pathogenesis of breast cancer and warrants future studies to elaborate the underlying mechanism.

A novel adjuvant, the mixture of alum and naltrexone, augments vaccine-induced immunity against Plasmodium berghei.

We previously showed that the mixture of naltrexone (NLT), a general opioid antagonist, and alum, acts as an effective adjuvant in enhancing vaccine-induced T helper 1 (TH1) humoral immune responses against Toxoplasma gondii. Here, we tested the efficacy of the mixture of NLT and alum in the induction of immunity in response to blood stages of Plasmodium berghei (BSPb) as a model vaccine. BALB/c mice were divided into five vaccination groups. Mice in the experimental groups received the BSPb vaccine alone or in combination with the adjuvant alum, NLT or the alum-NLT mixture. Mice in the control group received PBS. All mice were immunized on days 0, 7 and 14. Two weeks after the last immunization, immune responses to Plasmodium berghei were assessed. Our results indicated that including the alum-NLT mixture as an adjuvant during vaccination increased the ability of the BSPb vaccine to enhance lymphocyte proliferation, shifted the immune response towards a TH1 profile and increased Plasmodium berghei-specific IgG2a. This resulted in improved protective immunity against Plasmodium berghei. In conclusion, administering alum-NLT mixture in combination with the BSPb vaccine enhanced the vaccine-induced immunity, and shifted the immune response toward TH1 pattern.

Seroepidemiology of Toxoplasma gondii Infection in Patients with Liver Disease in Southeastern Iran.

INTRODUCTION: Recent research has been concentrated on investigating the involvement of Toxoplasma gondii (T. gondii) in the progression of liver disorders. This study aims to investigate the prevalence of T. gondii infection in patients with non-alcoholic fatty liver disease (NAFLD), as well as the effects of toxoplasmosis infection on biological biomarkers such as aspirate transaminase (AST), alanine transaminase (ALT), cholesterol (Chol), and triglyceride (Tg) levels in Sistan, southeast Iran. METHODS: A case-control study was conducted between December 2021 and September 2022. The study included 225 patients diagnosed with NAFLD as the case group and 225 healthy blood donors as the control group. The controls were selected from the same region and were matched with the patients based on gender and age. We collected serum samples from all patients and utilized an enzyme-linked immunosorbent assay to analyze them for the existence of anti-T. gondii IgG antibodies. A questionnaire and medical records were utilized to gather data on the patient's demographic factors. RESULTS: The prevalence of anti-T. gondii IgG antibodies were 68 (30.2%) in patients with NAFLD, whereas it was 11 (4.88%) in the control group. The seroprevalence of T. gondii in NAFLD patients increased in correlation with age (P < 0.001). The prevalence of NAFLD was significantly greater in the seropositive group compared to the seronegative group (P < 0.001). In addition, the levels of the metabolic markers Chol and Tg were significantly higher in T. gondii seropositive NAFLD patients compared to T. gondii seronegative NAFLD patients. CONCLUSION: The findings of this study indicate a high seroprevalence of T. gondii in patients with NAFLD. Further studies are warranted to investigate the underlying mechanisms and potential therapeutic implications of this association.

Naloxone Effects Against Cutaneous Leishmaniasis Caused by Leishmania major Strain MRHO/IR/75/ER in the BALB/c Mice.

INTRODUCTION: Cutaneous leishmaniasis (CL) is a serious health problem in some parts of the world, such as Iran. Since the use of pentavalent antimonial compounds such as meglumine antimoniate (Glucantime, MA) for the treatment of CL has side effects, naloxone as a new treatment in the footpad of Leishmania major (L. major)-infected BALB/c mice was investigated by evaluating the lesion size and the parasite burden. METHOD: The animals were infected with L. major (MRHO/IR/75/ER). 40 BALB/c mice were divided into 4 groups (10/group), and were treated as follows 39 days after L. major infection: Group 1 treated with intraperitoneal injections of MA (100 mg/kg, positive control group) daily for six weeks; Group 2 received a 100 µl injection of PBS (negative control group); Group 3 received subcutaneous (SC) injections of naloxone (10 mg/kg) daily for six weeks (Naloxone1), and Group 4 was SC injected with naloxone (10 mg/kg) weekly for six weeks (Naloxone2). The lesion size was measured using a digital caliper. RESULT: After the end of treatment, the lesion parasite burden was evaluated. As compared to the negative control group, the groups that received MA and naloxone (groups 1, 3, and 4) showed fewer parasites. Also, the naloxone-treated mice showed significantly smaller lesion sizes than the negative control group (p˂0.05), but they did not differ significantly from the MA-treated mice. CONCLUSION: Taken together, the results suggest that naloxone might be a promising and alternative treatment for CL.

Seroepidemiology of Toxocara canis in Children under 14 Years Referring to Laboratories of Sistan and Baluchestan Province in Southeast of Iran.

BACKGROUND: The aim of the present survey was to assess thr seroepidemiologic and parasitological aspects of Toxocara canis infection in children under 14 yr old. METHODS: Overall, 963 sera were collected from children in the Sistan and Baluchistan Province, Southeast of Iran during the period from Sep 2015 to Jun 2016. IgG antibody against T. canis in the subjects' sera was evaluated using the commercial ELISA kit. RESULTS: Anti-Toxocara IgG were detected in the serum of 17 (1.7%) of the participants. In the examined children, the highest presence of anti-Toxocara antibodies was 2.1% (9/418) in 6-10-yr olds, which was higher than other age groups (P<0.05). Anti-Toxocara antibodies were significantly higher in males (2.4% or 12/492) than in females (1.1% or 5/471) (P<0.03). Highest serological prevalence of T. canis occurred in tribes (5.5% or 4/69), followed by rural areas (0.9% or 7/757), while in the urban area it was 0.1% (6/163) (P<0.01). A significant association was seen between the serological prevalence of T. canis and laboratory findings such as eosinophilia (P=0.001) and red blood cell count (P=0.02). CONCLUSION: Seroprevalence of Toxocara infection is high among children living in the poor regions of southeast Iran.

Identification and Phylogenetic Classification of Fasciola species Isolated from Sheep and Cattle by PCR-RFLP in Zabol, in Sistan and Baluchistan Province, Southeast Iran.

BACKGROUND: The detection of Fasciola species in various geographical regions is essential for health policymaking. Here, we aimed to identify livestock (cattle and sheep) related Fasciola genotypes by restriction fragment length polymorphism PCR. METHODS: Seventy adult Fasciola flukes were collected from 70 infected livers of 35 cattle and 35 sheep slaughtered in Zabol abattoir, south-east Iran (Jan-Jul 2017). Fasciola species were determined based on molecular features. For molecular detection, Fasciola ITS1 region was amplified and sequenced. A 700 bp fragment was amplified. These were digested with RasΙ enzyme. F. hepatica specific fragments were 47, 59, 68, 104, and 370, while those related to F. gigantica had 45, 55, 170, 370. RESULTS: The two main species of F. hepatica and F. gigantica are responsible for fasciolosis in sheep and cattle in our region. From 35 Fasciola isolated from cattle, 3 and 32 were F. hepatica and F. giagantica respectively. From 35 Fasciola isolated from sheep, 4 were F. hepatica and 31 were F. gigantica. CONCLUSION: All Seventy Fasciola samples from two different hosts (cattle and sheep) were identified as either F. hepatica or F. gigantica by PCR-RFLP. Genotypic variability of Fasciola species was high in our region. It is recommended to assess molecular variation of Fasciola isolates in other host livestock.

Beta-Secretase 1 (BACE1) Is Down-Regulated in Invasive Ductal Carcinoma of Breast.

BACKGROUND: The enzyme beta-secretase 1 (BACE1) and its antisense transcript (BACE1-AS) have been implicated in the pathogenesis of Alzheimer's disease. Moreover, several lines of evidence point to their contribution in tumorigenesis. METHODS: In the present study, we evaluated expression of BACE1 mRNA (BACE1) and BACE1-AS in 54 breast cancer tissues and 54 adjacent non-cancerous tissues (ANCTs) from the same patients using quantitative real-time PCR. RESULTS: BACE1 was significantly down-regulated in tumoral tissues compared with ANCTs, while BACE1- AS expression was not significantly different between tumoral tissues and ANCTs. The Bayesian Multilevel model showed a significant difference in BACE1 expression between stage 1 and 2 cancers after age-effect adjustments. BACE1-AS expression was significantly greater in ER-positive than in ER-negative samples (P=0.01). BACE1 and BACE1-AS expression were not correlated with patient ages in any sample sets. CONCLUSION: Significant correlations were detected between expression of these genes in both tumoral tissues and ANCTs. The current study provides evidence for differential BACE1 expression in breast tissues and suggests further assessment of the role of BACE1 in the pathogenesis of cancer.

Intra-Species Diversity of Leishmania major and L. tropica from Clinical Isolates of Cutaneous Leishmaniasis in Southwest Iran Inferred by ITS1-rDNA.

BACKGROUND: Cutaneous leishmaniasis (CL) as a public health concern is increasingly circulating by causative agents of Leishmania tropica and L. major in Iran. As regard to recent treatment failure and controlling problems, the accurate elucidation of heterogeneity traits and taxonomic status of Leishmania spp. should be broadly addressed by policymakers. This study was designed to determine the genetic variability and molecular characterization of L. major and L. tropica from Iranian CL patients. METHODS: One hundred positive Giemsa-stained slides were taken from clinical isolates of CL at Pol-e-Dokhtar County, Southwest Iran, from May 2014 to Sep 2016. DNAs were directly extracted and amplified by targeting ribosomal internal transcribed spacer (ITS) gene following microscopic observation. To identify Leishmania spp. amplicons were digested by restriction enzyme HaeIII subsequent PCR-RFLP technique. To reconfirm, the isolates were directly sequenced to conduct diversity indices and phylogenetic analysis. RESULTS: Based upon the RFLP patterns, 84 and 16 isolates were explicitly identified to L. tropica and L. major respectively. No co-infection was found in clinical isolates. The high genetic diversity of L. tropica (Haplotype diversity 0.9) was characterized compared to L. major isolates (Hd 0.476). The intra-species diversity for L. tropica and L. major isolates corresponded to 3%-3.9% and 0%-0.4%, respectively. CONCLUSION: Findings indicate the L. tropica isolates with remarkable heterogeneity than L. major are predominantly circulating at Pol-e-Dokhtar County. Occurrence of high genetic variability of L. tropica may be noticed in probable treatment failure and/or emerging of new haplotypes; however, more studies are warranted from various geographic regions of Southwest Iran, using large sample size.

Super Infection of Cutaneous Leishmaniasis Caused by Leishmania major and L. tropica to Crithidia fasciculata in Shiraz, Iran.

BACKGROUND: The aims of the current study were to determined present status of CL in Shiraz City, identify the causative species of Leishmania and conduct phylogenetic evaluations in detected parasites. METHODS: This study was conducted on 70 individuals with suspected CL that referred to the major health centers of Shiraz (Valfajr), Fars province, Iran, from Sep 2016 to Jul 2017. DNA was extracted from cultured Leishmania promastigotes and PCR-RFLP were performed using ITS1-rDNA gene. RESULTS: Overall, 39 male (55.70%) and 31 (44.30%) female were found to be positive microscopically. All of direct examined positive samples were confirmed to be positive for Leishmania spp. DNA. Based upon the PCRRFLP patterns and phylogenetic analysis, 46 (65.72%), 17 (24.28%) and 7 (10%) isolates were clearly identified as L. major, L. tropica and C. fasciculata, respectively. CONCLUSION: The dominat detected species in Shiraz City was L. major and L. tropica, respectively. CL has high prevalence in Shiraz City; therefore, more studies on leishmaniasis in the natural vectors and also reservoirs infection in this region is exceedingly recommended. Skin leisons due to C. fasciculata, was described for the first time in Iran (Shiraz City).

A Survey on the Adjuvant Role of Naloxone Alone or Combined with Alum in Vaccination Against Fasciolosis in BALB/c Mice.

BACKGROUND: Fasciolosis is a zoonotic parasitic disease imposing a heavy load of livestock losses worldwide. PURPOSE: We aimed to evaluate immune-stimulatory effects of naloxone (NLX), an opioid receptor antagonist, in combination with alum in mice vaccinated with excretory-secretory antigens (E/S) of Fasciola hepatica. METHODS: 8-week-old female BALB/c mice were subcutaneously vaccinated using E/S antigens of F. hepatica. Experimental groups (14 mice per group) included: vaccine (E/S antigen), alum vaccine (E/S antigen plus alum), NLX vaccine (E/S antigen plus NLX), and alum-NLX vaccine (E/S antigen plus a mixture of alum-NLX). The control group was infused with PBS. Lymphocyte proliferation and the levels of IFN-γ, IL-4, IgG2a, IgG1, and total IgG were measured. RESULTS: Mice vaccinated with NLX or alum-NLX adjuvants showed significantly higher rates of lymphocyte proliferation, IFN-γ, total IgG, and IgG2a levels. The mice that were injected with alum showed a significantly higher concentration of IL-4. Ratios of IFN-γ/Il-4 and IgG2a/IgG1 were significantly higher in the NLX and alum-NLX groups in comparison with the groups vaccinated either with alum or without any adjuvant. A significantly higher protection rate (62.5%) was seen in mice vaccinated with the alum-NLX adjuvant compared to the other groups. CONCLUSION: NLX can be effective in conferring cellular immunity and protection against F. hepatica. It is recommended to consider this agent as a potential adjuvant in vaccines against fasciolosis.

High-Resolution Melting Analysis as an Appropriate Method to Differentiate between Fasciola hepatica and F. gigantica.

BACKGROUND: Fasciolosis is a shared disease between humans and livestock caused by hepatic trematodes; Fasciola hepatica and F. gigantica. Differentiate between the two species of this genus is essential. High-Resolution Melting (HRM) Analysis represents a new approach to this issue. This method can be performed right after termination of Real-Time PCR. This technique has not been used for identification of adult F. hepatica and F. gigantica genotypes. The aim of this study was to determine Fasciola species by using HRM in isolates taken from Iran, respectively. METHODS: Ninety-three Fasciola spp. samples were collected from infected slaughtered animals in different regions of Iran, including North West (Ardebil Province) and South East (Zahedan Province) during 2016. Genomic DNA from the samples was extracted using a DNA extraction kit and then after Real-Time PCR amplification, HRM was done. RESULTS: Overall, 59 and 34 isolates were identified as F. hepatica and F. gigantica, respectively. The percentages of each species from animals were as follows: sheep (F. hepatica, 80.39% and F. gigantica, 19.61%), cattle (F. hepatica, 42.85% and F. gigantica, 57.15%). CONCLUSION: HRM technique developed in the present study is a powerful, rapid and sensitive technique for epidemiological survey and molecular identification between F. hepatica and F. gigantica.

Molecular and Morphometrical Characterization of Fasciola Species Isolated from Domestic Ruminants in Ardabil Province, Northwestern Iran.

BACKGROUND: We aimed to describe morphological and morphometrical characteristics of Fasciola spp. in livestock from Ardabil Province, Northwest Iran. METHODS: Forty adult flukes were collected from different definitive hosts (cattle and sheep). Previously specimens were identified as F. hepatica or F. gigantica based on PCR-RFLP of the ITS-1 region with RsaI enzyme. We identified Fasciola spp. based on morphological and metric assessment of external features of fresh adults, morphological and metric assessment of internal anatomy of stained mounted worms. Statistical analysis was conducted using the Student's t-test implemented in SPSS 15.0 (SPSS, Chicago, Illinois). Then the morphometric criteria of Fasciola samples were compared with PCR-RFLP data. The results of PCR-RFLP were confirmed by COI gene sequence. RESULTS: The differences between the body length, area of the body, peripheral of the body, succer area, cone length, cone width, in two species were significant (P < 0.05). Based on Morphological characterizations, 6 specimens had the intermediate morphological features and 19 and 15 specimens had morphological features of F. hepatica and F. gigantica, respectively. In contrast, RFLP results showed, F. hepatica was present in 20 of the isolates, and F. gigantica in 20 isolates. No hybrid forms were detected. CONCLUSION: PCR-RFLP method can be used for differentiation of Fasciola species, which is more reliable method than morphology. Using morphology methods, merely, is not efficient for determination of genetic diversity.

Modulation of the Immune Response to DNA Vaccine Encoding Gene of 8-kDa Subunit of Echinococcus granulosus Antigen B Using Murine Interleukin-12 Plasmid in BALB/c Mice.

BACKGROUND: The current study was designed to evaluate immune responses induced by DNA vaccines encoding 8-kDa subunit of antigen B (HydI) of Echinococcus granulosus and murine interleukin 12 (IL-12) as genetic adjuvants in BALB/c mice. METHODS: Expression plasmid pcDNA3.1 containing HydI (pcHyd1) as vaccine along with the murine interleukin 12 (pcMIL12) as adjuvant were used. Thirty-five mice in the five experimental groups received PBS, empty pcDNA3.1, pcHydІ, pcMIL-12, and pcHydІ+ pcMIL-12 in days zero, 14th and 28th. Two weeks after the last immunization, evaluation of the immune response was performed by evaluating the proliferation of splenic lymphocytes, IFN-γ and IL-4, determination of IgG isotyping titer. RESULTS: Mice that received the pcHydI+pcMIL12 exhibited higher levels of lymphocyte proliferation compared to mice that received the pcHydI alone (P<0.001), and produced significantly more IFN-γ in comparison to other groups (P< 0.001). In addition, they produced significantly less IL-4 than mice receiving the PBS and the empty plasmid (P<0.023). The IgG2a levels were clearly higher in pcHydI+pcMIL12 group in comparison with the groups of pcHydI alone, empty plasmid, and PBS. In contrast, IgG1 was elevated in the group of pcHydI. CONCLUSION: Co-delivery of IL-12 with DNA encoding 8-kDa subunit of antigen B was effective significantly in inducing the immune response in mice.

Molecular Cloning and Expression an 8-kDa Subunit of Antigen B from G1 strain of Echinococcus granulosus.

BACKGROUND: Echinococcosis or hydatidosis is a chronic, zoonotic worldwide infection caused by the larval stage of the dog taeniid tapeworm Echinococcus granulosus. Vaccination has been considered as one of the ways to prevent of hydatidosis in recent decades. The aim of this study was to construct a pcDNA3.1 eukaryotic expression vector containing the subunit 8-kDa antigen B (Hyd1) of E. granulosus (G1 strain) and investigate its capability to induce protein expression in mammalian cell line, as a basis toward developing a DNA vaccine against hydatidosis. METHODS: The coding sequence of HydI was amplified by PCR with the specific PCR primers from pQE/HydI, and then was sub-cloned into pcDNA3.1 plasmid as expression vector. The pcHyd1 plasmid was digested by restriction enzymes and amplified with the specific PCR primers to confirm cloning of this gene in pcDNA3 plasmid. In last step, the sub-cloned gene was expressed in mammalian cell line (NIH 3T3 cells). RESULT: The subunit 8-kDa antigen B (Hyd1) was successfully sub-cloned in pcDNA3.1 and Hyd1 protein was expressed in eukaryotic cell confirmed by SDS-PAGE and Western blot. CONCLUSION: Recombinant plasmid of pcDNA3.1 was successfully constructed and express of recombinant Hyd1 protein was confirmed. That is promising step for forthcoming measures on providing vaccine against human and animal hydatidosis.