Evaluation of Kynu, Defb2, Camp, and Penk Expression Levels as Psoriasis Marker in the Imiquimod-Induced Psoriasis Model.

Background: Psoriasis is a noncontagious auto-inflammatory chronic skin disease. So far, some of the inflammatory genes were upregulated in mouse model of psoriasis. This study examined changes in skin mRNA expression of L-kynureninase (Kynu), cathelicidin antimicrobial peptide (Camp), beta-defensin 2 (Defb2), and proenkephalin (Penk) in a mouse model of imiquimod-induced psoriasis. Materials and Methods: Tree groups of C57BL/6 female mice were allocated. The imiquimod (IMQ) cream was administered to the mice dorsal skin of the two groups to induce psoriatic inflammation. In the treatment group, IMQ was administered 10 min after hydrogel-containing M7 anti-IL-17A aptamer treatment. Vaseline (Vas) was administered to the negative control group. The psoriatic skin lesions were evaluated based on the psoriasis area severity index (PASI) score, histopathology, and mRNA expression levels of Kynu, Camp, Defb2, and Penk using real-time PCR. In order to assess the systemic response, the spleen and lymph node indexes were also evaluated. Results: The PASI and epidermal thickness scores were 6.01 and 1.96, respectively, in the IMQ group, and they significantly decreased after aptamer administration to 1.15 and 0.90, respectively (P < 0.05). Spleen and lymph node indexes showed an increase in the IMQ group, followed by a slight decrease after aptamer treatment (P > 0.05). Additionally, the mRNA expression levels of Kynu, Defb2, Camp, and Penk genes in the IMQ-treated region showed a significant 2.70, 4.56, 3.29, and 2.61-fold increase relative to the Vas mice, respectively (P < 0.05). The aptamer-treated region exhibited a significant decrease in these gene expression levels (P < 0.05). A positive correlation was found between Kynu, Penk, and Camp expression levels and erythema, as well as Camp expression with PASI, scaling, and thickness (P < 0.05). Conclusion: According to our results, it seems that Kynu, Camp, and Penk can be considered appropriate markers for the evaluation of psoriasis in IMQ-induced psoriasis. Also, the anti-IL-17 aptamer downregulated these important genes in this mouse model.

Mannose targeting of poly(lactic-co-glycolic acid): a promising approach for improving sublingual allergen-specific immunotherapy.

OBJECTIVE: One of the most effective treatments for allergic respiratory diseases is allergen-specific sublingual immunotherapy (SLIT). While, mannose targeting has been applied in various immunostimulatory approaches, but it has not been investigated in sublingual allergen-specific immunosuppressive treatment. This study assesses mannose targeting for the ovalbumin (Ova) loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles(NPs). METHODS: The emulsion-solvent evaporation method was employed for the synthesis of PLGA NPs containing Ova, and subsequently they attached to D-mannose. Ova-sensitized mice underwent treatment in different ways: subcutaneous administration of 10 µg Ova, sublingual administration of 5 and 10 µg Ova loaded in PLGA NPs, 5 and 10 µg Ova loaded in mannose-targeted PLGA NPs, 10 µg Ova, and 10 µg Ova loaded in dendritic cell-specific aptamer-attached PLGA NPs. Serum Ova-specific IgE and IgG2a levels, as well as IFN-γ, IL-4, IL-10, and IL-17a levels in the supernatant of Ova-stimulated splenocytes were measured. Splenocyte proliferation was assessed using an MTT assay, and also lung histological examinations, and nasal lavage fluid cell counting were performed. RESULTS: Ova-specific IgE, IL-4, IL-17a levels, eosinophil cell count, and splenocyte proliferation were remarkably reduced in the mice treated with mannose or aptamer targeted NPs compared to other groups. Also, IL-10 and IFN-γ levels were remarkably increased in the targeted NPs groups. CONCLUSION: Our findings indicated that mannose targeting of PLGA NPs could decrease allergen dose and improve immunomodulatory effects of SLIT. However, this approach suggests an effective formulation for SLIT in the mice model, further studies with common allergens are needed for application in humans.

Application of CAR-T cell technology in autoimmune diseases and human immunodeficiency virus infection treatment.

Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy approach that has played a tremendous role in the battle against cancer for years. Since the CAR T lymphocytes are unrestricted-major histocompatibility complex T lymphocytes, they could identify more targets than natural T cells, resulting in practical and widespread functions. The good prospects of CAR T-cell therapy in oncology can be additionally applied to treat other diseases such as autoimmune and infectious diseases. CAR-T cell-derived immunotherapy for autoimmune disorders can be allocated to CAR-Tregs and chimeric autoantibody receptor T cells. Other generations of CARs target human immunodeficiency virus (HIV) proteins. In this review, we summarize CAR-T cell therapies in autoimmune disorders and HIV infection.

Topical anti-TNF-a ssDNA aptamer decreased the imiquimod induced psoriatic inflammation in BALB/c mice.

BACKGROUND: Tumor Necrosis Factor-α (TNF-α) is a pro-inflammatory factor that plays a pivotal role in psoriasis. Due to limitations of monoclonal antibody-based therapies, it is needed to discover new anti-TNF-α factors instead of usual anti-TNF-α monoclonal antibodies. Compared to antibodies, single-stranded DNA or RNA molecules named aptamers, have advantages such as time-saving, less risk for immunogenicity and cost-effectiveness. Therefore, the aim of the present study was to assess the therapeutic effects of T1-T4 dimer anti-TNF-ɑ ssDNA aptamer topical treatment in the imiquimod (IMQ)-induced psoriasis animal model. METHODS: 5% IMQ cream was prescribed on the right ear of BALB/c to induce psoriasis model. The hydrogel-containing anti-TNF-ɑ aptamer or treatment control aptamer (anti- Interleukin (IL)17A) was topically prescribed to the mice's ears 10 min before IMQ cream treatment. The psoriasis area severity index (PASI) score was used to evaluate psoriasis intensity. Histopathology analysis was done for mice ears sections. Mass, size, and cell number of mice spleens were measured. The IL-17 level was determined in culture supernatants of axillary lymph node cells using ELISA. The mRNA expression levels of IL-17A, IL-1ß, STAT3, and S100a9, were evaluated in mice treated ear with quantitative Real Time-PCR. RESULTS: The anti-TNF-ɑ ssDNA aptamer lower doses had significant decrease in IMQ-induced PASI score (p < 0.05). In addition, in these groups, the IL-17A, STAT3, and S100a9 mRNA levels were significantly lower than the IMQ group (p < 0.05). CONCLUSION: According to our findings, this aptamer seems to be a prospective candidate for treating psoriatic inflammation especially in lower concentrations.

Selection and characterization of a new human Interleukin-17A blocking DNA aptamer using protein-SELEX.

BACKGROUND: Interleukin-17A (IL-17A) is an important pro-inflammatory cytokine observed in the development of many disorders, such as psoriasis, rheumatoid arthritis, and multiple sclerosis. The anti-IL-17A biological drugs, including Secukinumab, Ixekizumab, and Brodalumab, are monoclonal antibodies approved for several disease treatments. Due to the disadvantages of biological therapies, including their immunogenicity, difficulties in scale generation, and high production costs and time, it is necessary to find new alternative anti- IL-17A agents for these monoclonal antibodies. Our study aimed to identify ssDNA aptamers that block IL-17A activity using the protein-SELEX procedure. METHODS: The hIL-17A was expressed in codon plus E. coli, and after 14 rounds of the SELEX process, monitoring of aptamer pools was done using the dot blot method. Three families of aptamers were obtained from the selected round 9 aptamer pool, and seven truncates were created. Inhibitory effects of aptamer truncate on IL-17-induced CCL20 expression in HaCaT keratinocytes were evaluated. RESULTS: All aptamer truncates had a significant inhibitory effect compared to the library, but the inhibitory effect of M2 and M7 truncates was more than 80%. Moreover, we evaluated the potential binding site of selected aptamers by ELISA. CONCLUSIONS: We introduced a new small 17-nucleotide DNA aptamer that efficiently binds and blocks hIL-17A with a 0.3 nM kd, a potential anti-IL-17A therapeutic agent.

Relationship between blood-based inflammatory indices and clinical score of schizophrenia patients: A cross-sectional study.

Schizophrenia is a severe mental disorder that may involve inflammation. Inflammatory indices, such as the neutrophil to lymphocyte ratio (NLR), the monocyte to lymphocyte ratio (MLR), the platelet to lymphocyte ratio (PLR), and the systemic inflammation index (SII), are simple and inexpensive measures of inflammation that have been associated with various diseases. However, few studies have compared these indices and their relationships with clinical symptoms in schizophrenia. We conducted a cross-sectional study of 121 schizophrenia patients (101 males, 20 females). We measured the blood-based inflammatory indices (NLR, MLR, PLR, and SII) and assessed the clinical symptoms of schizophrenia using the Positive and Negative Syndrome Scale (PANSS). Statistical analyses were performed to examine the correlations and effects of the inflammatory indices on PANSS scores. We found that NLR, MLR, PLR, and SII were positively correlated with PANSS total score, PANSS positive score, PANSS negative score, and general psychopathology score (adjusted P < 0.02 for all correlations). Subgroup analysis showed that correlations between inflammatory indices and the clinical scores differed by gender. In males, all inflammatory indices were positively correlated with all clinical scores. On the other hand, in females, only NLR and SII were positively correlated with all clinical scores. After adjusting for confounders, we also found that NLR was a predictor of PANSS total score (ß = 23, adjusted P < 0.02), PANSS positive score (ß = 2.6, adjusted P = 0.03), PANSS negative score (ß = 6.8, adjusted P < 0.02), and PANSS general psychopathology score (ß = 13.6, adjusted P < 0.02), while SII was only a predictor for PANSS total score (ß = -0.00003, adjusted P = 0.01) and general psychopathology scores (ß = -0.00002, adjusted P < 0.02). These findings suggest that inflammation is involved in the pathophysiology and clinical manifestations of schizophrenia, and that blood-based inflammatory indices may serve as screening tools or indicators for the inflammatory status and severity of symptoms of schizophrenia patients.

In vitro identification of single-stranded DNA aptamers targeting human IL-23 using the protein-SELEX strategy.

Interleukin (IL)-23 inhibitor monoclonal antibodies shown significant efficacy in treating autoimmune diseases. DNA or RNA aptamers exhibit comparable specificity to antibodies, are cost-effective, non-immunogenic, and do not have batch to batch variation. This study aimed to characterize a single-stranded DNA (ssDNA) aptamer targeting human IL-23. The alpha subunit of IL-23 (P19) and intact IL-23 were cloned, expressed, and the proteins finally were purified through Ni2+-iminodiacetic acid affinity chromatography. The selection and characterization of ssDNA aptamer against P19 were conducted using the protein-systematic evolution of ligands by exponential enrichment (SELEX). Dot blot assay was carried out to monitor binding of the aptamer output of SELEX rounds, to P19 protein. The dissociation constant (Kd) of aptamers with positive results in dot blot assay, determined based on their binding to IL-23 using an ELISA method. Recombinant P19 and IL-23 proteins were 26 and 72 kDa, respectively, observed on SDS-PAGE .12 %. The aptamers output from 7, 8, 9, 10, 11, and 12 rounds of the SELEX was monitored by dot blot assay, revealing that the aptamer from the round 8 has stronger luminescent signal and was selected for TA-cloning. After analyzing the biotinylated aptamers from clones, positive clones in dot blot assay and ELISA were sequenced. Finally, the Kd calculation revealed three aptamers with high affinity, named A23P3, A23P6, and A23P15 with Kd values of 1.37, 2.139, and 2.88 nM, respectively. Results of this study introduced three specific anti-IL-23 ssDNA aptamers with high affinity, which could be utilized for therapeutic and diagnostic purposes.

Development of fast-dissolving sublingual nanofibers containing allergen and curcumin for immune response modulation in a mouse model of allergic rhinitis.

Curcumin (CUR) has been considered a potential therapeutic agent for allergic reactions due to its antioxidant and anti-inflammatory activities. Nanofibers have attracted increasing attention in drug delivery. The aim of this study was to investigate the combined therapeutic effects of curcumin and allergen in nanofiber-based treatments in order to increase the effectiveness of sublingual immunotherapy (SLIT) efficacy in a mouse model of allergic rhinitis. Nanofibers containing CUR (1.25% and 2.5%) and ovalbumin 2% (OVA) as an allergen were prepared via electrospinning and characterized. BALB/c mice were sensitized with OVA to the induced allergic rhinitis model. SLIT with free and/or nanofibers was carried out. IL-4, INF-γ, and IgE serum levels were measured using ELISA. Splenocyte proliferation was evaluated by the MTT assay. Lung and nasal histological examinations and nasal lavage fluid (NALF) cell counting were carried out. Nanofibers containing 1.25% CUR and 2% OVA were chosen as the optimal formulations. SLIT treatment with the CUR and OVA nanofiber co-administration led to a significantly decreased serum IgE. Nanofiber containing 2.5 µg of CUR/mouse combined with OVA nanofiber showed a significant decrease in IL-4 and an increase in IFN-γ compared to other groups. NALF assessment showed a significant decrease in specific cell and eosinophil counts in the treated nanofiber groups. The histopathological results of NAL in the optimal formulations were near normal, with diminished cellular infiltration and inflammation. Our findings suggest that co-sublingual administration of allergen and CUR nanofibers can be considered as potential immunomodulatory agents.

An epicutaneous therapeutic pollen-allergen extract delivery system in an allergic rhinitis mouse model: based on allergen loading on DC-specific aptamers conjugated nanogolds.

BACKGROUND: Gold nanoparticles (GNPs) have previously been suggested as appropriate carriers for allergen-specific immunotherapy (AIT). In this study, we assessed efficacy of GNPs and dendritic cells (DC)-specific aptamer-modified GNPs (Apts-GNP) for epicutaneous immunotherapy (EPIT) in the case of pollen allergen extracts containing a variety of allergenic and non-allergenic components. METHODS: BALB/c mice were sensitized to the total protein extract of Platanus orientalis pollen and epicutaneously treated in different groups either with free P. orientalis total pollen extract, naked GNPs, total extract loaded GNPs, and total extract loaded Apts-GNPs with and without skin-penetrating peptides (SPPs). Then, the specific IgE level (sIgE), total IgE concentration (tIgE) in the serum sample, IL-4, IL-17a, IFN-γ, and IL-10 cytokine concentrations in re-stimulated splenocytes with the total extract and mixture of recombinant allergens, nasopharyngeal lavage fluid (NALF) analysis, and histopathological analysis of lung tissue were evaluated. RESULTS: This study indicated the total extract-loaded GNPs, especially Pla. ext (50 µg)-GNPs, significantly decreased sIgE, tIgE, IL-17a, and IL-4 concentrations, immune cells and eosinophils infiltration in NALF, and increased IL-10 and IFN-γ concentrations compared with the PBS-treated group. In addition, the histopathological analysis of lung tissue showed a significant decrease in allergic inflammation and histopathological damage. The DC-targeted group revealed the most significant improvement in allergic-related immune factors with no histopathological damage compared with the same dose without aptamer. CONCLUSION: Loading total protein extract on the GNPs and the Apt-modified GNPs could be an effective approach to improve EPIT efficacy in a pollen-induced allergic mouse model.

Polyvinyl Alcohol can Stabilize FITC Conjugated Recombinant Annexin V for Apoptotic Cells Detection.

BACKGROUND: Annexin V, a member of calcium-dependent phospholipid-binding proteins, selectively binds to the exposed phosphatidylserine, which can be used for in vitro apoptosis detection. Simultaneous staining of cells with annexin V-fluorescein isothiocyanate (FITC) and the non-vital dye propidium iodide (PI) enables the detection of apoptotic and necrotic cells. OBJECTIVE: Our study aimed to express, purify, and stabilize the recombinant annexin V. METHODS: The recombinant annexin V was cloned and expressed in E. coli bacteria and was purified using Ni-IDA resin. The FITC conjugation was performed, and apoptosis detection of HaCaT cells by FITC-labeled annexin V was evaluated by flow cytometry. Then, the stability of FITC-labeled annexin in various conditions, including polyvinyl alcohol (PVA), glycerol, and trehalose, was evaluated. RESULTS: The results showed that annexin V was appropriately expressed and purified. After FITC conjugation, it could perfectly detect the cell death of HaCat cells in different apoptosis percentages. FITC-labeled annexin had more stability with PVA than glycerol and trehalose. CONCLUSION: It seems that PVA has an acceptable effect on FITC-labeled annexin V stability in concentrations lower than 1 mg mL-1 without interfering with fluorescent intensity.

Anti-IL-17A ssDNA aptamer ameliorated psoriasis skin lesions in the imiquimod-induced psoriasis mouse model.

OBJECTIVES: IL-17 is an important player in the psoriasis pathogenesis, which recruits inflammatory cells to the psoriatic lesions, induced keratinocyte proliferation and plaque formation. Three monoclonal antibodies that block IL-17 have been approved for psoriasis treatment in the last decade. Compared to monoclonal antibodies, aptamers which are single-stranded DNA or RNA, bind with high affinity to proteins or other molecules and are more cost-effective. We previously showed that M2 and M7 anti-IL17A ssDNA aptamers could block IL-17 in vitro. The current study evaluated the therapeutic effects of M2 and M7 anti-IL17A ssDNA aptamers in the imiquimod (IMQ)-induced psoriasis mouse model. METHODS: IMQ cream and Vaseline (Vas) were administered on the back skin of C57BL/6 mice as IMQ-induced psoriasis and Vas control groups, respectively. In addition, hydrogel-containing aptamers were topically administered on the back skin of the mice, 10 min before IMQ treatment. Psoriatic lesions were evaluated by histology, clinical factors, and psoriasis area severity index (PASI) score. The mRNA expression levels of inflammatory factors, including IL-17A, IL-1ß, and S100a9, were assessed with quantitative reverse transcriptase-polymerase chain reaction in the mice back skin. RESULTS: Application of anti-IL-17A aptamers significantly ameliorated IMQ-induced keratinocyte proliferation, psoriatic lesions cumulative PASI score, IL-17A, IL-ß, and S100a9 inflammatory factors mRNA expression levels (p < 0.05). CONCLUSION: According to our results, it seems that M2 in high concentration and M7 in low concentration can be appropriate candidates to alleviate psoriasis lesions.

Plaque-type psoriasis inhibitors.

Psoriasis is a common inflammatory skin disorder, which is mediated by the immune system and affects 1-4% of the world's population. Psoriasis is caused by a complex interaction between the immune system, autoantigens, psoriasis-associated genetic factors, and various environmental factors. As a chronic disease requiring long-term treatment, psoriasis is associated with follow-up costs and an economic burden on the patients, their families, and healthcare systems. The current treatments for moderate-to-severe plaque psoriasis include topical therapy, phototherapy, and systemic drugs consisting of biological/non-biological drugs. Within the past two decades, recent biological therapies for psoriasis have rapidly advanced. Moreover, new bispecific agents have the potential for better disease control, while small molecule drugs offer a future alternative to biological drugs and the more cost-effective, long-term treatment of the disease. The present study aimed to review updated data regarding the inhibitors used to improve plaque psoriasis that contain biologics, bispecific agents, small molecules, and aptamers (either approved or in the research phase).

In Vitro evaluation of immediate cytotoxicity of resterilised orthodontic bands on HGF-1 cell line.

OBJECTIVES: The aim of this study was to evaluate the immediate cytotoxic effects of orthodontic molar bands, on HGF-1 cell line, after multiple times of sterilization following size selection procedure. MATERIAL AND METHODS: 48 stainless steel orthodontic molar bands were divided into 4 groups according to times of sterilization (1, 2, 4 and 8 times). A liquid extract containing the ions released from each band was prepared and the HGF-1 cell line was exposed to the extracts. 2 control groups (positive and negative) were designated. An MTT assay was performed, and the absorbance was read at 492nm in a microplate reader (Antos 2020, Austria). RESULTS: There was no significant difference in pure optical density (OD) among the 4 groups (P=0.749) however a statistically significant difference was seen between the positive control group and other 4 groups (P<0.001). CONCLUSION: The stainless-steel orthodontic bands used in this study were inert as manufactured and even multiple times of sterilization did not decrease the biocompatibility of these bands for clinical use. The present study shows that clinicians can sterilize the tried-in molar bands for at least 8 times without any risk of cytotoxicity for patients.

Chenopodium Botrys Essential Oil as A Source of Sesquiterpenes to Induce Apoptosis and G1 Cell Cycle Arrest in Cervical Cancer Cells.

Conducting cell apoptosis pathways is a novel strategy in cancer treatment. This study aimed to explain that C. botrys essential oil could induce apoptosis and arrest the cell cycle in HeLa cells. Cytotoxic and apoptogenic effects of the essential oil of Jerusalem-oak (Chenopodium botrys L.), which was obtained from the aerial parts of the plant, were evaluated in HeLa cells. Cell viability was assessed by MTT and LDH assays, and the mechanism of cell apoptosis was investigated using flow cytometry. Expression of the apoptosis-related genes was assessed using real-time polymerase chain reaction (PCR). GC-MS analysis of the herbal essential oil revealed 37 components. The major components were α-Eudesmol (16.81%), Elemol acetate (13.2%), Elemol (9.0%), and α-Chenopodiol-6-acetate (7.9%). The essential oil inhibited the growth of HeLa cells and increased the expression of p21 and p53. In addition, essential oil treatment increased the sub-G1 DNA content and induced apoptosis due to the increased Bax/Bcl-2 ratio and up-regulation of caspase-3 gene expression. According to the results, C. botrys essential oil exhibited anticancer effects through intrinsic apoptosis pathways and arresting cell proliferation.

CD8+ T Lymphocyte Subsets in Bladder Tumor Draining Lymph Nodes.

BACKGROUND: Cytotoxic CD8+ T cells, as essential parts of the adaptive immune system, play pivotal roles in anti-tumor immune responses. It is well documented that cytokine expression profiles and activation status of these cells during anti-tumor immune responses affect the outcome of host-tumor interaction. OBJECTIVE: To investigate the percentages of CD8+ lymphocytes and their subsets in tumor draining lymph nodes of patients with bladder cancer. METHODS: Forty-five patients with bladder cancer, candidate for radical cystectomy, were recruited. Mononuclear cells were isolated from draining lymph nodes using Ficoll-Hypaque gradient centrifugation, and were activated by PMA/Ionomycin in the presence of Golgi inhibitors. The cells were then permeabilized and stained with appropriate flourochrome conjugated antibodies against CD3, CD8, IFN-γ, IL-17 and IL-4 molecules. Data were collected on a four-color flow cytometer and analyzed by CellQuestPro software. RESULTS: Despite no difference in the frequency of IL-17 producing CD8+ (Tc17) lymphocytes, the mean expression of IL-17 in this subset was significantly elevated in high-grade patients (p=0.011). The percentage of double positive IFN-γ/IL-17 CD8+ lymphocytes was also significantly increased in node positive patients compared to node negative ones (p=0.046). Our results also demonstrated that the percentage of IFN-γ producing CD8+ (Tc1) lymphocytes was significantly increased in the patients with higher histological grade compared to those with lower ones (p=0.038). CONCLUSION: IFN-γ and IL-17 producing CD8+ T cells may increase in advanced stages of bladder cancer, but their correlation with tumor prognosis remains to be investigated.

Uterine Natural Killer Cell and Human Leukocyte Antigen-G1 and Human Leukocyte Antigen-G5 Expression in Vaginal Discharge of Threatened-Abortion Women: A Case-Control Study.

The immunotolerant human leukocyte antigen-G (HLA-G) molecules have a major role in fetal-maternal tolerance during pregnancy. Interaction between these molecules and uterine natural killer (uNK) cells inhibitory receptors prevents NK cell invasion against fetus trophoblast cells. The aim of this study was to evaluate the percentages of uNK cells and HLA-G1 and HLA-G5 isoforms expression in vaginal discharge of threatened-abortion women in comparison with control. In a case-control study, we investigated 30 threatened-abortion women with bleeding or spotting less than 20 weeks of pregnancy as compared to 30 normal pregnant women. uNK cells percentage was assessed by flow cytometry. Furthermore, we evaluated HLA-G1 and HLA-G5 isoforms expression by Real-Time PCR in these groups. The results of this study showed that threatened-abortion women had increased uNK cells and decreased T cells percentage in vaginal discharge in comparison with normal pregnant women (p = 0.01, p = 0.003, resp.). In addition, HLA-G1 isoform had lower expression in threatened-abortion women in comparison with control group (p = 0.0001). The increase of uNK cells level with the decrease of HLA-G expression in vaginal discharge of threatened-abortion pregnant women is an indicator of mother's immune dysregulation. It is concluded that HLA-G expression level with uNK cells percentage can be determined as a diagnostic marker for threatened-abortion women.