Iranian Biomedical Journal
Volume:26 Issue: 1, Jan 2022

Leishmaniasis is caused by protozoan Leishmania parasites. The disease is predominantly endemic to the tropics and has been reported in more than 98 countries with an estimated increase of approximately 1.5 million per year and more than 12 million infected cases. Key management of control and treatment relies mainly on the detection of infected cases that have been disrupted due to the toxicity of drugs, side effects, and the emergence of drug resistance in parasites. Control of reservoirs and vectors is complex in most involved countries due to operational problems and lack of financial resources. Therefore, it is highly desirable to establish an effective and reasonable immunized agent against leishmaniasis. There have been advances to understand underlying immune mechanisms using a variety of candidate antigens, including attenuated live parasites, crude antigens, pure or recombinant Leishmania proteins, Leishmania genes encoding protective proteins, also, immune system activators from the saliva of flies. However, there is still no protective vaccine against different types of human leishmaniasis. In recent years, more focus on leishmaniasis vaccine studies has been forced on cutaneous leishmaniasis. In this review, we will take a look at the works done or being done concerning this issue. Previous studies have shown that different ways the vaccine enters the body play an important role in making protective immunity. The process of identifying articles was developed using the following electronic databases in which the immune response and vaccines of leishmaniasis were assayed: PubMed, SciELO, ScienceDirect, Scopus, Google Scholar, and Web of Science databases.

Gastric cancer (GC) and chronic gastritis are induced by Helicobacter pylori (H.pylori) in most cases. Pattern recognition receptors (PRRs), especially Toll-like receptors (TLRs), as the first line of defense for pathogen detection, were found to be associated with H. pylori infection and gastric cancer. However, the expression levels of TLRs, especially TLR2 and TLR4 as the main receptors sensed H. pylori, still remain largely ambiguous. Herein, we aimed to investigate the patterns of key transcripts of TLR2 and TLR4 in 100 GC transcriptome data. Additionally, we evaluated TLR2 and TLR4 gene expressions in GC samples obtained from Iranian GC biopsy specimen, in order to validate RNA-seq outputs.

100 runs of GC samples and controls were processed and analyzed using map read to reference test by CLC genomics workbench package.  DGE method was used to distinguish between GC and Ctrl samples in the expression of TLRs and other innate immune molecules. Also using qRT-PCR assay and Pffafle method, transcripts of TLRs molecules for 15 GC and 15 Ctrl samples were analyzed based on analysis of variance and least significant differences. 

The results clearly showed that all signaling pathways molecules  of TLR4, especially, TLR4(P-value = 0.019), NF-κB (P-value = 0.047), IL-1β( P-value = 0.0096), and TNF-α( P-value= 0.048), have up-regulated  in a cancerous condition in different parts and at various stages of gastric cancers.

Totally, this finding confirmed that molecules involved in inflammation including TLR4 and its related pro-inflammatory cytokines play a critical role in the development and progression of GCs.. Accordingly, the controlling of.  H.pylori infection consequently reduce inflammation in the gastric system can play an important role in preventing gastrointestinal disorders.

A mouse model of lipopolysaccharide (LPS)-induced inflammation was used to investigate pharmacological inhibition of nuclear enzyme poly (ADP-ribose) polymerase-1 (PARP-1) on oocyte maturation, apoptotic and necrotic death as well as DNA integrity of follicular cells. In addition, the relative expression of cumulus genes (HAS2, COX2 and GREM1) associated with oocyte developmental competence was assessed.

Mice were treated with PARP-1 inhibitor, 4-hydroxyquinazoline (4-HQN), 1h before LPS administration. After 24h oocyte in vitro maturation was detected. Granulosa cell DNA damage was determined by the alkaline comet assay. Live, necrotic and apoptotic cells were identified  usingdouble vital staining by fluorescent dyes Hoechst 33342 and propidium iodide. The expression levels of cumulus genes were assessed using reverse transcriptase polymerase chain reaction.

The administration of 4-HQN to LPS-treated mice ameliorated oocyte meiotic maturation and exerted a significant cytoprotective effect. 4-HQN attenuated LPS-induced DNA damage and favored cell survival by decreasing necrosis and apoptosis in granulosa cells. Exposure to 4-HQN increased mRNA expression levels for HAS2, COX2 and GREM1 in cumulus cells.

 The obtained results indicated the involvement of PARP-1 in the pathogenesis of ovarian dysfunction caused by LPS. We suppose that this enzyme can be an attractive target for the therapy of inflammatory disorders in ovary. The protective action of PARP-1 inhibition could be at least partly associated with the diminish of necrotic death of follicular cells as well as in other cells. However, the detailed mechanisms of favorable effect of PARP inhibitors on endotoxin-induced ovarian disorders  need to be further explored.

Single nucleotide polymorphisms result in dysregulation of the proto-oncogene TCF3 gene, which is associated with the development, metastasis, and chemoresistance of different malignancies.

GSE10810 microarray dataset and GEPIA2 online software were used to find differentially expressed genes and the TCF3 status in BC and GC, respectively. Plots and figures of microarray analysis were prepared by ggplot2 and pheatmap packages. Differentially expressed genes were obtained by the Bioconductor limma package. In silico analysis was used to predict the functions of rs72618599. BC (n = 123), GC (n = 132) and healthy age and gender matched controls (n = 184) were genotyped, using the high-resolution melting technique.

Based on the allelic comparison study, C allele of rs72618599 was associated with the BC tumor stage IV (66.1%, 78/120, p < 0.0001) and grade III (52.4%, 55/72, p < 0.0001), while the T allele was associated with metastasis (84.2%, 10/162, p < 0.0001). However, in GC patients, the C allele was significantly correlated with H. pylori infection (51.7%, 30/58, p = 0.008), stage III of primary tumors (47.7%, 62/88, p=0.017), stage II of lymph node status (35.5%, 44/74, p = 0.017), and metastasis (52.9%, 90/132, p = 0.044). In silico analysis predicted that rs72618599 leads to the creation of a binding site for hsa-miR526b-5p in the 3′-UTR of TCF3 transcript.

Regarding the rs72618599 SNP, the C allele, is associated with poor prognosis of BC and GC. Furthermore, rs72618599 may be associated with cancer progression by altering the regulatory affinity of hsa-miR526b-5p to 3′-UTR of TCF3.

Long noncoding RNAs (LncRNAs), as critical regulators, have attracted more attention from researchers for diagnostic, prognostic, and therapy in human carcinogenesis via interfering with mRNAs such as Enhancer of zeste homolog 2 (EZH2). Nevertheless, the potent roles and molecular mechanisms of these RNAs are not clearly known in colorectal cancer (CRC). In this study, the tissue expression of lncRNA MINCR and EZH2 were compared between colorectal tumors and polyps with the adjacent normal tissues collected from 114 Iranian patients using Real-time PCR method. Furthermore, expression levels correlation of MINCR and EZH2 and other clinical parameters was also evaluated in patients. Finally, we showed that expressing MINCR and EZH2 has been significantly overexpressed in the CRC-tissues (P<0.0001). Also, this was confirmed that these expression enhancements were associated with the pathological stage of CRC patients. In short, we concluded that the expression of MINCR has been significantly altered during CRC development and it can be identified as a potential biomarker for the detection of this cancer.

Colorectal cancer is the third most common cancer worldwide. Microsatellite instability (MSI) is a molecular marker of a deficient mismatch repair (MMR) system and happens in almost 15% of colorectal cancers. Because of The wide frequency of MSI+ colorectal cancer in Iran compared to other parts of the world, the importance of screening for this type of cancer is highlighted. The most common MSI detection techniques is a fluorescent PCR-based in which fragments are analyzed by capillary electrophoresis (CE). This technique is very time-consuming, difficult, and expensive. Here, we sought to develop and evaluate a proper method with high accuracy, specificity and sensitivity to screen the MSI+ colorectal cancer. A high-resolution melting (HRM) analysis procedure is relying on the analysis of the melting curve attributes. Low cost, feasibility, high specificity and sensitivity are outstanding attributes of HRM analysis.  To this end five mononucleotide microsatellite markers including, BAT-26, BAT-25, NR-24, NR-21, and NR-27 in 25 archival CRC tumor tissue samples were compared with normal tissue adjacent using high resolution melting (HRM) method. The specificity and sensitivity of BAT25 with HRM method were 100 % in comparison with CE while other markers lower sensitivity. However, when all the markers were considered together, the sensitivity and specificity became 100%. The number of MSI+ samples was 56%, which shows a higher ratio than previous Iranian studies. The highest microsatellite instability was related to BAT26, (52%). The HRM method is much simpler and more cost-effective than current MSI techniques, and its sensitivity and accuracy are comparable, and can be replaced in cases where capillary electrophoresis is not available.

Gastric cancer (GC) is the fourth most common human malignancy and the second reason for cancer morbidity worldwide. Long non-coding RNA HOTAIR has recently emerged as a promoter of metastasis in various cancer types, including GC, through EMT process. However, the exact mechanism of HOTAIR in promoting of EMT has been unknown. Aberrant expression of the miR-200 family has been also linked to the occurrence and development of various types of malignant tumors. This study investigates the correlation between the HOTAIR and miR-200 family genes expression patterns in GC cell lines. We investigated the miR-200 and HOTAIR due to their common molecular features in EMT process.

We transfected the AGS and MKN45 cell lines with siRNA targeting the HOTAIR along with a negative control. We analyzed the effect of HOTAIR knock down on cell viability as well as expression of miR-200 family members including miR-200a, 200b, and 200c in cell lines using qRT-PCR. Statistical analysis was performed to find the potential correlation between the expression level of HOTAIR and miRs.

Our results showed significantly increased miR-200 family expression level in transfected AGS and MKN45 GC cells (Fold changes>2, P-value <0.001). Moreover, we observed a negative correlation between HOTAIR and miR-200 expression levels in GC cell lines (P-value<0.05).

Our findings showed a significant association between miR-200 family and HOTAIR expression levels in GC cell lines. Taken together, the HOTAIR-miR-200 axis seems to play a vital role in human GC, suggesting a potential therapeutic target in future GC treatment.

 Interferon alpha-2b is a vital biotherapeutic produced using the recombinant DNA technology in Escherichia coli which forms inclusion bodies.

 In this study Interferon alpha-2b was produced as inclusion body in Escherichia coli.  Purification of Interferon alpha-2b from solubilized IB was performed using 2-phase extraction. To optimize refolding conditions, the effects of pH and different additives including cysteine, cystine, Urea, Glycerol, Triton x-100, NaCl, and Arginine were investigated.  Optimal refolding buffer (0.64 mM urea, 5.57 mM cysteine ​​, and 1.8 mM cystine) was obtained using RSM. The refolding process was carried out with optimized refolding buffer in the dilution and fed-batch refolding method at different protein concentrations (25-1000 µg/mL of protein). 

  Fed-batch refolding in the protein concentration of 500 µg/mL resulted in biological activity of 2.24×108IU/mg was twofold the biological activity obtained from dilution method .

 This method resulted in the biologically active IFN-α2b with high purity, which can be used for research and industrial manufacturing.