Indomethacin attenuates neuroinflammation and memory impairment in an STZ-induced model of Alzheimer's like disease.

Objective: Non-steroidal anti-inflammatory drugs (NSAIDs) exposure might be considerably associated with a decreased risk of Alzheimer's disease (AD). Therefore, we conducted an experiment to investigate the impact of indomethacin (IND) on inflammasome as a key player of neuroinflammation.Methods: The Alzheimer's-like condition was induced by streptozotocin (STZ) in rats. IND was injected intraperitoneally 1 d prior to STZ administration and resumed with 2 d interval up to 60 d. Morris water maze (MWM) was utilized to assess learning and memory. The expression level of genes that contribute to the inflammasome pathway was measured using real-time polymerase chain reaction (PCR). To authenticate the obtained outcomes, immunostaining for caspase-1, interleukin-1ß (IL-1ß), and phosphorylated tau (p-Tau) protein was conducted.Results: Behavioral experiments indicated that IND treatment was able to improve learning and memory performance (p<.05). A significant decrease in C-terminal caspase recruitment domain [CARD] domain-containing protein 4 (NLRC4), nucleotide-binding oligomerization domain [NOD]-like receptor protein 3 (NLRP3), IL-1ß, and apoptosis-associated speck-like protein containing CARD (ASC) mRNA expression was recorded in IND administered group compared with the STZ group (p<.05). Furthermore, expression levels of IL-18 and caspase-1 in the hippocampus of IND-treated group tended to decrease. Immunostaining evaluations showed that few positive cells for caspase-1, IL-1ß, and p-Tau protein in IND treated animals, whereas the number of positive cells was considerably increased in STZ treated animals (p<.05).Conclusion: It could be deduced that IND improves neuroinflammation and memory impairment in AD through decreasing IL-1ß and caspase-1 that are associated with suppression of NLRC4 and NLRP3 inflammasome genes. This holds the potential to introduce valuable targets in the field for successful combat against AD.

Novel Computational Approaches to Developing Potential STAT4 Silencing siRNAs for Immunomodulation of Atherosclerosis.

BACKGROUND: Small interfering RNAs (siRNAs) are known as commonly used targeting mRNAs tools for suppressing gene expression. Since Signal Transducer and Activator of Transcription 4 (STAT4) is considered as a significant transcription factor for generation and differentiation of Th1 cells during vascular dysfunction and atherosclerosis, suppressing STAT4 could represent novel immunomodulatory therapies against atherosclerosis. OBJECTIVE: Therefore, the current study was conducted to design efficient siRNAs specific for STAT4 and to evaluate different criteria affecting their functionality. METHODS: In the present study, all related sequences of STAT4 gene were retrieved from Gen Bank database. Multiple sequence alignment was carried out to recognize Open Reading Frame (ORF) and conserved region. Then, siDirect 2.0 server was applied for the development of candidate siRNA molecules and confirmation of predicted molecules was performed using Dharma siRNA technology and GeneScript siRNA targetfinder. In addition, BLAST tool was used against whole Genebank databases to identify potential off-target genes. DNA/RNA GC content calculator and mfold server were used to calculate GC content and secondary structure prediction of designed siRNA, respectively. Finally, IntaRNA program was used to study the thermodynamics of interaction between predicted siRNA and target gene. RESULTS: Based on the obtained results, three efficient siRNA molecules were designed and validated for STAT4 gene silencing using computational methods, which may result in suppressing STAT4 gene expression. CONCLUSION: According to our results, this study shows that siRNA targeting STAT4 can be considered as a therapeutic agent in many Th1-mediated pathologic conditions specially atherosclerosis.

Involvement of NLRC4 inflammasome through caspase-1 and IL-1ß augments neuroinflammation and contributes to memory impairment in an experimental model of Alzheimer's like disease.

Inflammatory response through interleukin-1ß (IL-1ß) plays a key role in the pathogenesis of Alzheimer's disease (AD). However, the molecular mechanism of pro-IL-1ß processing in AD is not clearly defined. The current study was designed to investigate which of the inflammasome complexes are critical for IL-1ß production in AD. An experimental model for Alzheimer like disease was induced in male Wistar rats and Morris Water Maze was used to evaluate the function of learning and memory. The expression of genes involved in inflammasome complex including NLRP1, NLRP3, NLRC4, AIM2, ASC, IL18, IL-1ß and caspase-1 was determined via Real-time PCR. Hematoxylin and Eosin (H&E) staining and Immunohistochemistry (IHC) for CD45 was applied to assess inflammatory cells infiltration. Furthermore, caspase-1, IL-1ß and phosphorylated tau (p-Tau) protein expressing cells were investigated in the lesion area using immunofluorescence staining technique. The behavioral study revealed that streptozotocin (STZ) injection significantly impaired learning and memory function. In addition, the infiltration of inflammatory cells was confirmed in the hippocampus region of STZ-treated animals. Furthermore, a significant increase in the expression level of NLRC4 inflammasome, ASC and IL-1ß was identified in STZ-treated animals. In contrast, no significant difference was observed in other inflammasome components including NLRP1, NLRP3, AIM2, IL-18 and caspase-1 in STZ-treated group compared with the control group. Moreover, the number of caspase-1, IL-1ß and p-Tau protein positive cells were remarkably increased in STZ-treated animals. Based on the obtained results, it can be concluded that increased production of IL-1ß, caspase-1 and p-Tau through association with NLRC4 inflammasome may be involved in neuroinflammation and memory impairment in AD, which creates a new horizon in this regard. Hence, strategies targeting NLRC4 inflammasome could be beneficial for the treatment of AD.

Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches.

Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7-L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7-L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis.

In silico analyses of heat shock protein 60 and calreticulin to designing a novel vaccine shifting immune response toward T helper 2 in atherosclerosis.

Recent experiments demonstrated that atherosclerosis is a Th1 dominant autoimmune condition, whereas Th2 cells are rarely detected within the atherosclerotic lesions. Several studies have indicated that Th2 type cytokines could be effective in the reduction and stabilization of atherosclerotic plaque. Therefore, the modulation of the adaptive immune response by shifting immune responses toward Th2 cells by a novel vaccine could represent a promising approach to prevent from progression and thromboembolic events in coronary artery disease. In the present study, an in silico approach was applied to design a novel multi-epitope vaccine to elicit a desirable immune response against atherosclerosis. Six novel IL-4 inducing epitopes were selected from HSP60 and calreticulin proteins. To enhance epitope presentation, IL-4 inducing epitopes were linked together by AAY and HEYGAEALERAG linkers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Moreover, cholera toxin B (CTB) was employed as an adjuvant. A multi-epitope construct was designed based on predicted epitopes which was 320 residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this chimeric protein were analyzed using bioinformatics tools and servers. Based on bioinformatics analysis, a soluble, and non-allergic protein with 35.405kDa molecular weight was designed. Expasy ProtParam classified this chimeric protein as a stable protein. In addition, predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell mediated immune response and shift immune responses toward protective Th2 immune response. Various in silico analyses indicate that this vaccine is a qualified candidate for improvement of atherosclerosis by inducing immune responses toward T helper 2.