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Diabetes is associated with reproductive impairment in the male reproductive system and causes complications such as decreased libido, fertility, spermatogenesis, sperm motility, and morphology. High levels of blood sugar may affect sperm quality and reduce the potential for male fertility. Increased levels of sperm DNA damage are often associated with reduced count and motility or abnormal morphology. This experimental study was conducted at Mashhad University of Medical Sciences. In this work, 40 mice (C57BL/6) were divided randomly into 4 groups: 1) Control, 2) Diabetic, 3) Diabetic + Insulin, and 4) Sham. After 35 days, the right epididymis of all specimens was used for Real-Time PCR and the left epididymis for evaluation of sperm parameters using Aniline blue, Toluidine blue, Papanicolaou, and immunohistochemical study. Also, testes were applied for immunohistochemical, TUNEL studies, and biochemical assay. Results of this study showed that chromatin integrity, morphology, cation channels of sperm (Catsper) expression, and biochemical factors level were significantly changed in diabetic mice in comparison to other groups (P

Programmed cell death protein-1 (PD-1), as an immune checkpoint molecule, attenuates T-cell activity and induces T-cell exhaustion. Although siRNA has a great potential in cancer immunotherapy, its delivery to target cells is the main limitation of using siRNA. This study aimed to prepare a liposomal formulation as a siRNA carrier to silence PD-1 expression in T cells and investigate it’s in vivo antitumor efficacy. The liposomal siRNA was prepared and characterized by size, zeta potential, and biodistribution. Following that, the uptake assay and mRNA silencing were evaluated in vitro at mRNA and protein levels. siRNA-PD-1 (siPD-1)-loaded liposome nanoparticles were injected into B16F0 tumor-bearing mice to evaluate tumor growth, tumor-infiltrating lymphocytes, and survival rate. Liposomal siPD-1 efficiently silenced PD-1 mRNA expression in T cells (P

This study compared the prophylactic effects from vaccines based on dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) by pulsing the cells in-vitro with p5 peptide. The different test groups of mice were injected with free peptide or with peptide pulsed with DCs or PBMCs. Two weeks after the last booster dose, immunological tests were performed on splenocyte suspensions from three mice in each group and the remaining mice (5/each group) were evaluated for tumor growth and survival time. The levels of IFN-γ, granzyme B, and IL-10 were detected in T cells. Additionally, IFN-γ and perforin as well as mRNA levels of some genes associated with immune responses were assessed after challenging the splenocytes with TUBO cells. A significant increase was observed in frequency of CD4+IFN-γ+, CD8+IFN-γ+, and CD8+granzyme B+T cells, and the perforin of supernatants from mice in the DC and PBMC treatment groups. Significant expression levels of Fas ligand (FasL) and forkhead box P3 (Foxp3) were observed in the DC and PBMC groups. These responses led to smaller tumors and longer survival time in our mouse model of breast cancer. The efficacy of the PBMC-based vaccine in improving the protective immune response makes it a simpler and less expensive candidate vaccine compared with DC-based vaccines.

This study compares the probiotic Lactobacillus strains isolated from dairy and Iranian traditional food products with those from human sources on intestinal microbiota using BALB/C mice model. First, Lactiplantibacillus plantarum (M11), Limosilactobacillus fermentum (19SH), Lactobacillus acidophilus (AC2), and Lactobacillus gasseri (52b) strains, isolated from either Iranian traditionally fermented products or human (healthy woman vaginal secretions), identified with molecular methods and selected based on the surface hydrophobicity, auto- and co-aggregation, were investigated for their probiotic properties and compared with their standard probiotic strains in vitro. The native strains and their mixtures (MIX) were then orally fed to five groups of female inbred BALB/C mice over the course of 38 days by gavage at 0.5 and 4 McFarland, respectively, equal to 1.5 × 10(8) and 1 × 10(9) cfu/ml. Feeding paused for 6 days to test the bacteria’s adhesion in vivo. According to the findings, the probiotic Lactobacillus strain isolated from human source (52b) exhibited the best in vitro and in vivo adhesion ability. Probiotic Lactobacillus strains isolated from Iranian traditional food products (19SH and AC2) had the most co-aggregation with Listeria monocytogenes (ATTC 7644), Salmonella enterica subsp. enterica (ATCC 13,076), and Escherichia coli (NCTC 12,900 O157:H7) in vitro. These strains produced the most profound decreasing effect on the mice intestinal microbiota and pathogens in vivo. The difference in the strains and their probiotic potential is related to the sources from which they are isolated as well as their cell walls. The results suggest that (19SH and 52b strains) are the best candidates to investigate the cell wall and its effect on the host immune system. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-022-00790-6.

Capacity and ability of Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus probiotic strains isolated from some human and food sources were evaluated for modulating and regulating the immune system against hypersensitivity type 1 (dependent on IgE). In this study, given the probiotic properties of the strains and the use of mouse models, the strains were orally administered (1×10

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.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-1β, and S100a9, were assessed with quantitative reverse transcriptase-polymerase chain reaction in the mice back skin.Application of anti-IL-17A aptamers significantly ameliorated IMQ-induced keratinocyte proliferation, psoriatic lesions cumulative PASI score, IL-17A, IL-β, and S100a9 inflammatory factors mRNA expression levels (p 0.05).According to our results, it seems that M2 in high concentration and M7 in low concentration can be appropriate candidates to alleviate psoriasis lesions.

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).

Allergen-specific sublingual immunotherapy (SLIT), a safe and efficient route for treating type I hypersensitivity disorders, requires high doses of allergens. SLIT is generally performed without adjuvants and delivery systems. Therefore, allergen formulation with appropriate presentation platforms results in improved allergen availability, targeting the immune cells, inducing regulatory immune responses, and enhancing immunotherapy's efficacy while decreasing the dose of the allergen. In this review, we discuss the adjuvants and delivery systems that have been applied as allergen-presentation platforms for SLIT. These adjuvants include TLRs ligands, 1α, 25-dihydroxy vitamin D3, galectin-9, probiotic and bacterial components that provoke allergen-specific helper type-1 T lymphocytes (TH1), and regulatory T cells (Tregs). Another approach is encapsulation or adsorption of the allergens into a particulate vector system to facilitate allergen capture by tolerogenic dendritic cells. Also, we proposed strategies to increasing the efficacy of SLIT via new immunopotentiators and carrier systems in the future.

To increase the efficiency of aptamers to their targets, a simple and novel method has been developed based on aptamer oligomerization. To this purpose, previously anti-human TNF-α aptamer named T1-T4 was trimerized through a trimethyl aconitate core for neutralization of in vitro and in vivo of TNF-α. At first, 54 mer T1-T4 aptamers with 5'-NH2 groups were covalently coupled to three ester residues in the trimethyl aconitate. In vitro activity of novel anti-TNF-α aptamer and its dissociation constant (Kd ) was done using the L929 cell cytotoxicity assay. In vivo anti-TNF-α activity of new oligomerized aptamer was assessed in a mouse model of cutaneous Shwartzman. Anchoring of three T1-T4 aptamers to trimethyl aconitate substituent results in formation of the 162 mer fragment, which was well revealed by gel electrophoresis. In vitro study indicated that the trimerization of T1-T4 aptamer significantly improved its anti-TNF-α activity compared to non-modified aptamers (P < 0.0001) from 40% to 60%. The determination of Kd showed that trimerization could effectively enhance Kd of aptamer from 67 nM to 36 nM. In vivo study showed that trimer aptamer markedly reduced mean scar size from 15.2 ± 1.2 mm to 1.6 ± 0.1 mm (P < 0.0001), which prevent the formation of skin lesions. In vitro and in vivo studies indicate that trimerization of anti-TNF-α aptamer with a novel approach could improve the anti-TNF-α activity and therapeutic efficacy. According to our findings, a new anti-TNF-α aptamer described here could be considered an appropriate therapeutic agent in treating several inflammatory diseases.

The imbalance between pro and anti-inflammatory cytokines plays an important role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Thymus vulgaris (thyme) as a traditional medicinal plant has been reported to exert antimicrobial, antioxidant, and anti-inflammatory effects. Therefore, this study evaluated the modulatory effects of Thymus vulgaris on the clinical symptoms, histopathological scores, and the production of some anti-inflammatory (TGF-β, IL-4, and IL-10) and pro-inflammatory (IFN-γ, IL-6 and IL-17) cytokines in EAE model. EAE was induced by MOG35–55 peptide and mice were treated intra-peritoneally (i.p) with phosphate buffered saline (PBS) in the control group or thyme extract (50 or 100 mg/kg of body weight, every other day) in thyme-treated EAE groups, from day 0 to +21 of post MOG immunization. Mice were sacrificed at day 22, and splenocytes were isolated and re-stimulated in vitro with MOG in order to measure the cytokine production and proliferation of re-stimulated cells by enzyme linked immunosorbent assay (ELISA) method and WST-1 reagent, respectively. The clinical symptoms and histopathological scores of the CNS were lower in thyme-treated than EAE control group. Furthermore, the production of IFN-γ and IL-6 by splenocytes was lower in thyme-treated EAE than in the control group. The production of IL-10 and TGF-β increased in mice treated with thyme extract compared to the control group. In this study, we showed for the first time that the immunomodulatory effects of Thymus vulgaris in EAE model. Thus, the possible therapeutic potential of thyme for treatment of MS could be considered in future research.

High expression of p32 in certain tumors makes it a potential target for immunotherapy. In the present study, the first goal was to design multi-epitope peptides from the P32 protein and the second goal was to compare the prophylactic effects of DCs- and PBMCs- based vaccines by pulsing them with designed peptides. For these purposes, 160 BALB/c mice were vaccinated in 5 different subgroups of each 4 peptides using PBS (F1-4a), F peptides alone (F1-4b), F peptides with CpG-ODN (F1-4c), F peptides with CpGODN and DCs (F1-4d), and F peptides with CpG-ODN and PBMCs (F1-4e). We found a significantly higher interferon-γ (IFN-γ) and granzyme B levels in T cells of F4d and F4e subgroups compared to control (p ≤ 0.05). The result of challenging spleen PBMCs of vaccinated mice with 4T1 cells showed significant up- and down- regulation of Fas ligand (FasL) and forkhead box P3 (Foxp3) gene expression between F4d and F4e subgroups with control, respectively. In addition, a significant change was seen in Caspase3 gene expression of F4d subgroup compared to control (p ≤ 0.05). Supernatant levels of IFN-γ and perforin were significantly increased in F4d and F4e subgroups compared to control. Consequently, significantly lower tumor sizes and prolonged survival time were detected in F4d and F4e subgroups compared to control after challenging mice with 4T1 cells. Accordingly, these results demonstrated that PBMCs pulsed F4 peptide-based vaccine could induce a protective immune response while it is a simple and less expensive vaccine.

This study was conducted to evaluate the effects of recombinant probiotic bacteria as a candidate for oral vaccine with the potential of treating allergy to Amaranthus retroflexus pollens. The main gene of this allergen, Ama r 2, was cloned into the food grade plasmid pNZ7025 and then was electrotransformed into the food grade Lactococcus lactis NZ1330. No expression was observed in the primary structure due to the distance between the ribosome binding site and the start codon. Therefore, the vector structure was corrected using the site-directed mutagenesis (SDM) technique. The cell extract of this strain was used for assessing the expression of the recombinant allergen in western blot analysis, and the existence of this protein with a molecular weight of 14.2 kDa was confirmed. To evaluate the efficacy of this strain in the treatment of allergies as an oral vaccine, probiotic ice cream was prepared. After the sensitization of mice, the treatment was performed by oral immunotherapy for 4 weeks, 4 to 5 times per week. 20 μl of functional ice cream with 1012 CFU/ml of r-L. lactis NZ1330 significantly reduced the serum IgE level. The levels of IFN-γ and TGF-β cytokines increased in the 20 μl ice cream treatment group as well as 40 μg/ml pure allergen compared with the PBS-treated group, and IL-4 cytokine levels decreased compared with the PBS-treated group. Overall, 20 μl ice cream with 1012 CFU/ml of the recombinant bacteria resulted in the best performance in terms of improving allergies to Th1 and Treg responses.

Allergy to non-specific lipidtransfer protein (nsLTP), the major allergen of grape (Vit v1), is considered as one of the most common fruit allergies in Iran. Therefore, a specific monoclonal antibody (mAb) can be used for the characterization and assessment of. Accordingly, this study aimed to generate and characterize a mAb against Vit v1 with a diagnostic purpose. To this end, Vit v1 allergen (9 kDa) was extracted using a modified Bjorksten extraction method. Natural Vit v1-immunized mouse splenocytes were fused with SP2/0Ag-14 myeloma cells for generating hybridoma cells. Specific antibody-secreting Hybridoma cells were selected using ELISA. Finally, anti-Vit v1 mAb was characterized by western blotting, ELISA, and isotyping methods. In the current study, a 9 kDa (Vit v1) protein was attained fromcrude and fresh juice of grape extracts and the isotype of desired anti-Vit v1 mAb was determined as IgM with k light chain. In addition, The ELISA results demonstrated that anti-Vit v1 mAb was specified against natural Vit v1 in the grape cultivar and related LTP allergens, such as Pla or 3 (p

Background Sublingual immunotherapy (SLIT) was introduced to deliver allergens in an effective and non-invasive route, which can be considered as an alternative for allergen-specific subcutaneous immunotherapy (SCIT). On the other hand, the use of gold nanoparticles (AuNPs) in allergen delivery has beneficial effects on sublingual immunotherapy. In addition, the molecular targeting agents like aptamers (Apt), have been widely applied for targeted drug delivery. Therefore, the current study aimed to evaluate the effects of dendritic cells (DCs)-specific Aptamer-modified AuNPs coated with ovalbumin (OVA) on the improvement of the SLIT outcome in the mouse model of allergy. Material and methods AuNPs with approximately 15 nm diameter were prepared by citrate reduction of HAuCl4. Afterward, Apt-modified AuNP complex was prepared and OVA was then loaded onto this complex. Following sensitization of Balb/c mice to OVA, SLIT was performed with Apt-AuNPs containing 5 µg OVA twice a week for a 2-month period. Allergen-specific IgE in serum, as well as cytokines secretion of spleen cells, were analyzed using ELISA. Also, nasopharyngeal lavage Fluid (NALF) was collected for total and eosinophil counts. Moreover, the lungs were removed for histopathological examination. Results SLIT with Apt-modified AuNPs complex containing 5 μg OVA, decreased the IgE levels compared to the other groups. Also, IL-4 production has significantly decreased in spleen cells, while TGF-β and IFN-γ have significantly increased. The assessment of NALF in the group treated by this complex showed a decrease in total cell as well as in eosinophil count. Also, the examination of lung tissues revealed that, in the group treated by this complex, inflammation and perivascular infiltration were lesser than the other groups, which were observed in only one vessel of tissue. Conclusion It was shown that, Sublingual immunotherapy with DC specific Apt-modified AuNPs containing 5 μg OVA can improve the Th1 and Treg immunomodulatory responses.

Purpose Tuberculosis, a cost and life threatening disease, was being subjected for improving vaccine strategies beyond BCG. Thus, a novel particulate delivery system using alginate-coated chitosan nanoparticles including PPE17 protein and CpG were administered through intranasal (IN) and subcutaneous (SC) routes. Methods The encapsulated nanoparticles were first characterized for size, surface charge, encapsulation efficiency and in vitro release of PPE17 antigen. The nanoparticles were then administered intranasal and subcutaneously to evaluate the induction of systemic and/or mucosal immune responses in mice. Results According to our result, the mean size of nanoparticles was measured about 427 nm, and exhibited a negative zeta potential of −37 mV. Following subcutaneous and intranasal administration, the results from cytokines assay showed that an increasing in the level of IFN-γ, and adversely a decrease in the level of IL-4 (presumptive Th1 biased immune response) was happened and also a notable elicitation in IL-17 cytokine was observed. Conclusion In conclusion, our study demonstrated that alginate-coated chitosan nanoparticles showed to be an effective way to improve BCG efficiency as booster strategy for subcutaneous vaccine, and also can induce strong immune responses as prime strategy through intranasal vaccination.

Background Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Recent reports have shown that probiotics can induce immunomodulatory activity with promising effects in inflammatory diseases. This study was designed to reveal the molecular and cellular mechanisms underlying the effect of Lactobacillus plantarum A7, which comprises human commensal bacteria, and Bifidobacterium animalis, a potential probiotic strain, on alleviation of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Methods To evaluate the therapeutic effects of probiotic strains, female C57BL/6 mice (8–10 wks old) received Lactobacillus plantarum A7, Bifidobacterium animalis PTCC 1631or a mixture of both strains through oral administration daily for 22 days beginning simultaneous with induction of EAE. The clinical parameters were recorded daily. On Day 22, each mouse was bled, and their spinal cord was removed for histology analysis. The effects of the treatments on regulatory T (Treg) cells level were evaluated using flow cytometry, and T-cell proliferation was assessed using a BrdU incorporation assay. The supernatants of spleen and lymph nodes cultured and mononuclear cells were collected for quantification of different panel of pro and anti-inflammatory cytokines by ELISA. The analysis of gene expression was performed at RNA level for transcription factors by real-time PCR. Results The results showed that treatment with a mixture of the two strains caused a more significant delay in the time of disease onset and clinical score compared to when the strains were used alone. The pathological features of the disease, such as mononuclear infiltration into the CNS, were also inhibited more significantly by the combinational approach. The results also revealed that treatment with combination of both strains enhanced the population of CD4+CD25+Foxp3+-expressing T-cells in the lymph nodes and the spleen. Treatment with our probiotic strains markedly inhibited disease associated cytokines while increased anti-inflammatory cytokines. Additionally, L. plantarumA7 and B. animalis ameliorated EAE condition by favoring Th2 and Treg differentiation via up-regulation of Foxp3 and GATA3 in the brain and spleen as well as inhibited the differentiation of Th1 and Th17 cells. Conclusions The current research provided evidence that probiotic therapy with L. plantarum and B. animalis can effectively attenuate EAE progression as well as reinforce the polarization of regulatory T-cells.

Allergen-specific sublingual immunotherapy (SLIT) is well known as an effective and non-invasive route to induce allergy desensitization. The goal of this study was to investigate whether a TAT-fused recombinant allergen could enhance SLIT efficacy. BALB/c mice sensitized to the main allergen (Che a 3) of Chenopodium album pollen were treated sublingually either with rChe a 3 (100 μg/dose) or rTAT-Che a 3 (100 μg/dose), two times per week for eight weeks. SLIT with rTAT-Che a 3 led to significantly greater allergen-specific IgG2a than rChe a 3; however, neither rTAT-Che a 3 nor rChe a 3 affected allergen-specific IgE or IgG1 antibody levels. In addition, interleukin 4 (IL-4) levels in re-stimulated splenocytes from the rTAT-Che a 3 mice were significantly lower than in those from the rChe a 3 mice, while interferon-γ (IFN-γ) was significantly greater in the rChe a 3 mice than in the rTAT-Che a 3 mice. Furthermore, sublingual administration of rTAT-Che a 3 induced significantly greater TGF-β secretion in re-stimulated splenocytes than administration of rChe a 3. Accordingly, SLIT with rTAT-Che a 3 led to significantly greater expression of TGF-β- and Foxp3-specific mRNAs in the splenocytes than in those from the rChe a 3 mice. Our findings demonstrate that TAT-fused rChe a 3 suppressed the allergic response through preferential enhancement of systemic regulatory T-cell (Treg)-mediated immunity responses, likely by facilitating allergen capture and presentation by sublingual Langerhans-like dendritic cells.

Hyperforin an herbal compound, is commonly used in traditional medicine due to its anti-inflammatory activities. The aim of this study was to use a hyperforin loaded gold nanoparticle (Hyp-GNP) in the treatment of experimental autoimmune encephalomyelitis (EAE) an animal model of multiple sclerosis (MS). Hyp-GNP and hyperforin significantly reduced clinical severity of EAE, which was accompanied by a decrease in the number of inflammatory cell infiltration in the spinal cord. Additionally, treatment with Hyp-GNP significantly inhibited disease-associated cytokines as well as an increase in the anti-inflammatory cytokines in comparison to all groups including the free-hyp group. Furthermore, hyperforin and Hyp-GNP inhibited the differentiation of Th1 and Th17 cells while promoting Treg and Th2 cell differentiation via regulating their master transcription factors. The current study demonstrated the although, free-hyp improved clinical and laboratory data Hyp-GNP is significantly more efficient than free hyperforin in the treatment of EAE.

Previous studies have indicated that interleukin (IL)‑1β has an important role in the development of allergic diseases. Therefore, the present study aimed to investigate the upstream pathway underlying IL‑1β production in an experimental model of allergy. BALB/c mice (female, 6‑8 weeks old) were sensitized to recombinant (r)Che a 2 by intraperitoneal injection of rChe a 2 adsorbed onto an alum gel suspension on days 0, 7, 14 and 21. In the control group, mice received an injection of 20 mM phosphate‑buffered saline absorbed onto alum via the same route. The allergic status of the mice was confirmed serologically by measuring allergen‑specific immunoglobulin (Ig)E levels. The protein expression levels of IL‑1β and the mRNA expression levels of inflammasome compartments were measured by enzyme‑linked immunosorbent assay and semi‑quantitative reverse transcription polymerase chain reaction, respectively. In addition, caspase‑1 activity was determined by fluorometric assay. Sensitized mice exhibited significantly increased levels of specific IgE (P

The efficacy improvement of current sublingual immunotherapy (SLIT) for preventing and treating respiratory airway allergic diseases is the main purpose of many investigations. In this study, we aimed to assess whether ovalbumin (Ova) encapsulated poly (lactic-co-glycolic) acid nanoparticles (PLGA NPs) decorated with dendritic cells (DCs)-specific aptamer could be applied for this purpose.The nanoparticles containing Ova were synthesized by emulsion/solvent evaporation method and attached to DCs-specific aptamer. Ova-sensitized BALB/c mice have been treated in five ways: subcutaneously with free Ova (SCIT), sublingually either with free Ova, Ova-PLGA NPs (two doses), Apt-Ova-PLGA NPs (two doses) and placebo/control Apt-Ova-PLGA NPs. For assessment of immunologic responses, IL-4, IFN-γ, IL-17, IL10, and TGF-β and IgE antibody levels were measured by ELISA and T cell proliferation were evaluated by MTT. In addition, lung and nasal histological examinations, NALF cells counting were carried out. Results declared that the lowest IgE and IL- 4 levels were observed in Apt-Ova-PLGA NPs (both doses). In the other hands, Apt-Ova-PLGA NPs (high dose) showed the highest increase of IFN- γ and TGF- β, decrease of IL-17 levels, total cell count and T-cell proliferation. IL-10 levels showed more decrease in SCIT, Apt-Ova-PLGA NPs (high dose) and Ova-PLGA NPs (high dose) than other groups. Histopathological examinations also confirmed in vitro results. Our findings suggest SLIT with this functionalized delivery system could be a promising approach for promoting the SLIT efficiency by decreasing the required allergen doses through specific delivery of allergen to sublingual DCs and enhancing the suppression of allergic responses.

Recently, the main goal of many allergy epicutaneous immunotherapy (EPIT) studies is to enhance the allergen delivery through the intact skin. Therefore, applying new strategies for tackling this issue are inevitable. For this purpose, ten groups of Che a 2-sensitized BALB/c mice were epicutaneously treated for a 6-week period with the rChe a 2-GNPs-Aptamer, rChe a 2-GNPs-Aptamer + skin-penetrating peptides (SPPs), rChe a 2-GNPs, rChe a 2, GNPs, and PBS. Afterward, the serum IgE and IFN-γ, TGF-β, IL-10, IL-4, IL-17a cytokine production, NALF analysis, and lung/nasal histological examinations were performed. The present study results demonstrate that, EPIT in aptamer treated groups had a significant increase of IFN-γ, TGF-β, and IL-10 concentrations and a significant decrease of IgE, IL-4, and IL-17a concentrations as well as NALF infiltrated immune cell count compared to the non-targeted ones. In addition, SPPs led to more significant improvement of immunoregulatory parameters, especially IL-10 cytokine. Accordingly, the targeted-GNPs with DC-specific aptamers could act as an efficient approach for the improvement of EPIT efficacy compared to the free allergen. Moreover, the application of SPPs might be considered as a useful tool in achieving a successful EPIT with lower doses of allergen at a shorter duration of the treatment.

There is an evident relationship between the fertilizing capacity of sperm and the normal morphology, quality chromatin, and motility of sperm. It is well known that thyroid hormones are the important regulators of testicular function. A correlation was found between the hypothyroidism and sperm damages. The present study was conducted to investigate the effects of hypothyroidism on sperm morphology, chromatin quality, and motility. For this purpose, 20 male mice were divided into the control and the hypothyroid groups that received 0.05% 6-n-propyl-2-thiouracil (PTU) for 35 days. Sperm morphology with Papanicolaou staining and sperm chromatin quality with both Aniline Blue (AB) and Toluidine blue (TB) staining were assessed. Besides, immunohistochemistry and real-time PCR were performed to evaluate the changes of cation sperm channel (CatSper) genes. A significant increase in the sperm chromatin condensation was found in the hypothyroid mice compared to the control mice (p < 0.05). Furthermore, a significant decrease was observed in the morphology of normal sperm in hypothyroid mice compared to the controls (p < 0.05). The results showed that Hypothyroidism could downregulate the expression of CatSper genes. Immunohistochemical data confirmed the real time-PCR results. Furthermore, the results showed that hypothyroidism could adversely affect sperm morphology, sperm chromatin condensation, and CatSper gene expression in mice and these abnormalities may be related to the excessive production of reactive oxygen species (ROS) in a hypothyroid state.

The goal of this study was to investigate whether poly (lactic-co-glycolic) acid (PLGA) nanoparticles could enhance sublingual immunotherapy (SLIT) efficacy. BALB/c mice sensitized to rChe a 3 were treated sublingually either with soluble rChe a 3 (100μg/dose) or PLGA-encapsulated rChe a 3 (5, 25, or 50μg/dose). SLIT with PLGA-encapsulated rChe a 3 (equivalent to 25 and 50μg rChe a 3 per dose) led to significantly increased antigen-specific IgG2a, along with no effect on allergen-specific IgE and IgG1 antibody levels. In addition, interleukin 4 (IL-4) levels in restimulated splenocytes were significantly less, while interferon-γ (IFN-γ), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) levels, as well as Foxp3 expression, were significantly greater than in the control groups. Our findings suggest that PLGA nanoparticle-based vaccination may help rational development of sublingual immunotherapy through reduction of the needed allergen doses and also significantly enhanced systemic T regulatory (Treg) and T helper 1 (Th1) immune responses.

Diabetes is a disease associated with impairment of the male reproductive system that causes complications such as decreased testosterone, the diameter of the seminiferous tubule, libido, and fertility. Extracellular matrix (ECM) molecules are involved in testicular development and spermatogenesis. Laminin and collagen are key proteins in seminiferous tubule basement membrane and play an important role in spermatogenesis. The present study was conducted to investigate the effect of diabetes on collagen IV and laminin α5 changes in mice testis. In this experimental study, 40 mice (C57BL/6) were divided randomly into 4 groups: 1) Control group: without intervention, 2) Diabetic group: treated mice with 50 mg/kg streptozotocin (STZ), 3) Diabetic + Insulin group: treated mice with STZ and insulin, and 4) Sham group: received citrate buffer. After 35 days, the left testes of all specimens were used for Real-Time PCR while their right testes were applied for immunohistochemical study and Periodic acid–Schiff (PAS) staining. This study showed that gene expression and immunoreactivity of laminin α5 and collagen IV were significantly increased in diabetic mice compared to other groups (P

Diabetes during pregnancy impairs brain development in offspring, leading to behavioral problems, motor dysfunction and learning deficits. Insulin and insulin-like growth factor-1 (IGF-1) are important regulators of developmental and cognitive functions in the central nervous system. Aim of the present study was to examine the effects of maternal diabetes on insulin receptor (InsR) and IGF-1 receptor (IGF-1R) expression in the developing rat cerebellum. Wistar female rats were maintained diabetic from a week before pregnancy through parturition and male offspring was killed at P0, P7, and P14, an active neurogenesis period in brain development equivalent to the third trimester in human. The expression of InsR and IGF-1R in cerebelli was evaluated using real-time PCR and western blot analysis. We found a significant upregulation of both IGF-1R and InsR transcripts in cerebellum of pups born to diabetic mothers at P0, compared to controls. However, at the same time point, the results of western blot analysis revealed only a slight change in their protein levels. In contrast to InsR, which does not show any difference, there was a markedly reduction in cerebellar expression of IGF-1R mRNA and protein level in the diabetic group of newborns at P7. Moreover, 2 weeks after birth, mRNA expression and protein levels of both InsR and IGF-1R in cerebellum of the diabetic group was significantly downregulated. Compared to controls, we did not find any difference in cerebellar InsR or IGF-1R mRNA and protein levels in the insulin treated group. The present study revealed that diabetes during pregnancy strongly influences the regulation of both InsR and IGF-1R in the developing cerebellum. Furthermore, optimal maternal glycaemia control by insulin administration normalized these effects.

Tuberculosis (TB) remains a major global health threat despite chemotherapy and Bacilli Calmette-Guerin (BCG) vaccination. Therefore, a safer and more effective vaccine against TB is urgently needed. This study evaluated the immunogenicity of a recombinant fusion protein consisting of early secreted antigenic target protein 6 kDa (ESAT-6), culture filtrate protein 10 kDa (CFP-10) and the Fc-domain of mouse IgG2a as a novel subunit vaccine. The recombinant expression vectors (pPICZαA-ESAT-6:CFP-10:Fcγ2a and pPICZαA-ESAT-6:CFP-10:His) were transferred into Pichia pastoris. After SDS-PAGE and immunoblotting, the immunogenicity of the recombinant proteins was evaluated in mice. When both recombinant proteins (ESAT-6:CFP-10:Fcγ2a and ESAT-6:CFP-10:His) were used for vaccination, Th1-type cellular responses were induced producing high levels of IFN-γ and IL-12. However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a small increase in IL-4 as compared to the BCG and ESAT-6:CFP-10:His groups. Moreover, mice primed with BCG and then supplemented with ESAT-6:CFP-10:Fcγ2a produced the highest levels of IFN-γ and IL-12 in immunized groups. The findings indicate that when Fcγ2a is fused to the ESAT-6:CFP-10 complex, as a delivery vehicle, there could be an increase in the immunogenicity of this type of subunit vaccine. Therefore, additional investigations are necessary for the development of appropriate Fc-based tuberculosis vaccines.

Targeting of dendritic cells (DCs) by aptamers increases antigen capture and presentation to the immune system. Our aim was to produce aptamers against DC molecules using the cell-SELEX procedure. For this purpose, 18 rounds of cell-SELEX were performed on mouse macrophage J774A.1 and CT26 as target and control cells, respectively. The selected aptamers were truncated and their binding to mouse macrophages, and immature and mature DCs analyzed. Two macrophage-specific aptamers, Seq6 and Seq7, were identified. A truncated form of Seq7, Seq7-4, 33 nucleotides in length and containing the G-quadruplex, bound macrophages and immature DCs with KD values in the nanomolar range. We anticipate that Seq7-4 has potential as a therapeutic tool in targeting of mouse macrophages and immature DCs to efficiently improve different immunotherapy approaches.

To prevent the spread of HTLV-I (Human T-lymphotropic virus type 1), a safe and effective vaccine is required. To increase immune responses against the peptide antigens can be potentiated with polymer-based nanoparticles, like chitosan (CHT) and trimethylchitosan (TMC), as delivery system/adjuvant. CHT and TMC nanoparticles loaded with recombinant proteins (env23 & env13) of gp46 were prepared by direct coating of antigens with positively charged polymers. The size of CHT and TMC nanoparticles (NPs) loaded with each antigen was about 400 nm. The physical stability of NPs was followed for 4 weeks. Both formulations showed to be stable for about 15 days. The immunogenicity of NPs loaded with antigens was studied after nasal and subcutaneous immunization in mice. Three immunizations (7.5 μg antigen) were performed with 2 weeks intervals. Two weeks after the last booster dose, sera IgG subtypes were measured. After subcutaneous administration, for both nanoparticulate antigens, serum IgG1 and IgGtotal levels were higher than antigen solution (P

Air pollutants and their interaction with environmental allergens have been considered as an important reason for the recent increase in the prevalence of allergic diseases. The aim of this study was to investigate the traffic pollution effect, as a stressor, on Platanus orientalis pollen allergens messenger RNA (mRNA) and protein expression. P. orientalis pollen grains were collected along main streets of heavy traffic and from unpolluted sites in Mashhad city, in northeast Iran. The pollen samples were examined by scanning electron microscopy. To assess the abundance of pollen allergens (Pla or 1, Pla or 2, and Pla or 3) from polluted and unpolluted sites, immunoblotting was performed. Moreover, the sequences encoding P. orientalis allergens were amplified using real-time PCR. Scanning electron microscopy showed a number of particles of 150–550 nm on the surface of pollen from polluted sites. Also, protein and gene expression levels of Pla or 1 and Pla or 3 were considerably greater in pollen samples from highly polluted areas than in pollen from unpolluted areas (p

Allergen specific immunotherapy is the only remedy to prevent the progression of allergic diseases. Nowadays, using of recombinant allergens with reduced IgE-binding capacity is an ideal tool for allergen immunotherapy. Therefore, in this study we focused on a hybrid molecule (HM) production with reduced IgE reactivity from Chenopodium album pollen allergens. By means of genetic engineering, a head to tail structure of the three allergens of the C. album pollen was designed. The resulting DNA construct coding for a 46 kDa HM was inserted into an expression vector and expressed as hexahistidine tagged fusion protein in Escherichia coli . IgE reactivity of the HM was evaluated by western blotting, inhibition ELISA and in vivo skin prick test and its immunogenic property was tested by proliferation assay. The recombinant HM was expressed and purified by nickel-affinity chromatography. Comparison of the recombinant HM with a mixture of three recombinant allergens, as well as natural allergens in the whole C. album pollen extract via immunological experiments revealed that it has a much lower potential of IgE reactivity. Furthermore, in vivo skin prick tests showed that it has a significantly lower potency to induce cutaneous reactions than the mixture of recombinant wild type allergens and whole extract while, it had been preserved immunogenic properties. Our results have demonstrated that assembling three allergens of C. album in a hybrid molecule can reduce its IgE reactivity.

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175-764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl beta-D-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.

Multiple sclerosis (MS) is a central nervous system disorder mainly characterized by inflammation, demyelination and axonal injury. Anti-inflammatory agents can be used to ameliorate the disease process. Hypericum perforatum L or St. John's wort is widely used as an anti-depressant and anti-inflammatory remedy in traditional and herbal medicine. Based on St. John's wort properties, the therapeutic potentials of an H. perforatum extract (HPE) and a single component, hyperforin were evaluated for effectiveness against MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. Female C57BL/6 mice were immunized with specific antigen MOG35-55 and then administered different doses of hyperforin or HPE post-immunization. Clinical symptoms/other relevant parameters were assessed daily. Histological analysis of the spinal cord was performed. T-cell proliferative activity was also evaluated using a BrdU assay. The effect of hyperforin on regulatory T-cells (Treg cells) was assessed using flow cytometry. The results indicate hyperforin and HPE reduced the incidence and severity of EAE, an outcome that closely correlated with an inhibition of pathological features (leukocyte infiltration and demyelination) and antigen-specific T-cell proliferation. The study also showed that hyperforin caused increased Treg cell levels in the spleen. These results indicated that hyperforin and HPE could attenuate EAE autoimmune responses by inhibiting immune cell infiltration and expansion of Treg cell and could eventually be considered as a potential candidate for use in the treatment of MS.

Tuberculosis (TB) remains a major health problem worldwide. Currently, the Bacilli Calmette-Guerin (BCG) is the only available licensed TB vaccine, which has low efficacy in protection against adult pulmonary TB. Therefore, the development of a safe and effective vaccine against TB needs global attention. In the present study, a novel multi-stage subunit vaccine candidate from culture filtrate protein-10 (CFP-10) and heat shock protein X (HspX) of Mycobacterium tuberculosis fused to the Fc domain of mouse IgG2a as a selective delivery system for antigen-presenting cells (APCs) was produced and its immunogenicity assessed. The optimized gene constructs were introduced into pPICZαA expression vectors, and the resultant plasmids (pPICZαA-CFP-10:Hspx:Fcγ2a and pPICZαA-CFP-10:Hspx:His) were transferred into Pichia pastoris by electroporation. The identification of both purified recombinant fusion proteins was evaluated by SDS-PAGE and immunoblotting. Then the immunogenicity of the recombinant proteins with and without BCG was evaluated in BALB/c mice by assessing the level of IFN-γ, IL-12, IL-4, IL-17 and TGF-β cytokines. Both multi-stage vaccines (CFP-10:HspX:Fcγ2a and CFP-10:HspX:His) induced Th1-type cellular responses by producing high level of IFN-γ (272 pg/mL, p

Aims Cardiac complications are common in carbon monoxide (CO) poisoning and associated with high morbidity and mortality. We have previously shown that erythropoietin (EPO) could reduce CO-induced cardiac ischemia in rat. In the current study, the anti-apoptotic effect of EPO during CO cardiotoxicity was investigated in order to elucidate the mechanism of EPO anti-ischemic action. Main methods Wistar rats were exposed to CO (250, 1000 and 3000 ppm). EPO (5000 IU/kg) was administered to all groups by intraperitoneal injection at the end of CO exposure period. TUNEL and caspase-3 activity levels were assessed to investigate the effects of CO exposure and subsequent EPO administration on myocardial apoptosis. The changes of mitochondrial membrane potential (MMP) were also assessed with sensitive lipophilic dye JC-1 by flow cytometry. The roles of Bcl2 and Bax in EPO protective effect were investigated by Western blotting. Key findings Myocardial apoptosis was observed following CO exposure. Moreover, mitochondrial membrane depolarization and significant reduction in Bcl2/Bax ratio were shown following CO poisoning especially at 3000 ppm. On the other hand, EPO administration could effectively suppress apoptosis in myocardial cells. Also, EPO significantly prevented the CO-induced depolarization of MMP (p Significance EPO reduces myocardial injury due to CO intoxication. Thus EPO could be suggested as a possible candidate for the management of CO cardiotoxicity with clinical applications.

Erythropoietin (EPO) plays a critical role in the development of the nervous system. In this study, the effects of EPO in carbon monoxide (CO) neurotoxicity were examined. Rats were exposed to 3000 ppm CO for 1 h and then different doses of EPO were administrated intraperitoneally. After 24 h, glial fibrillary acidic protein (GFAP) levels in the serum were determined and water content of brain and the extravasation of a tracer (Evans blue) were measured. Brain lipid peroxidation, myeloperoxidase activity Myelin basic protein (MBP) and BAX/BcL2 protein relative expressions were determined. Cation exchange chromatography was used to evaluate MBP alterations. Seven days after exposure, pathological assessment was performed after Kluver-Barrera staining. EPO reduced malondialdehyde levels at all doses (2500, 5000 and 10,000 u/kg). Lower doses of EPO (625, 1250, 2500 u/kg) significantly decreased the elevated serum levels of GFAP. EPO could not reduce the water content of the edematous poisoned brains. However, at 5000 and 10,000 u/kg it protected the blood brain barrier against integrity loss as a result of CO. EPO could significantly decrease the MPO activity. CO-mediated oxidative stress caused chemical alterations in MBP and EPO could partially prevent these biochemical changes. Fewer vacuoles and demyelinated fibers were found in the EPO-treated animals. EPO (5000 u/kg) could restore the MBP density. CO increased brain BAX/Bcl-2 ratio 38.78%. EPO reduced it 38.86%. These results reveal that EPO could relatively prevent different pathways of neurotoxicity by CO poisoning and thus has the potential to be used as a novel approach to manage this poisoning.

Liposome–protamine–DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen’s immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.

Smad ubiquitination regulatory factor 2 (Smurf2) consists of multiple WW domains which can interact with Smad7 molecule and inhibit signaling of transforming growth factor-beta (TGF-β) cytokine. Arginase I (ArgI) is one of the main products of TGF-β signaling that plays important roles in tumor escape and airway tissue fibrosis and remodeling in asthma. In this study, the effects of TAT fused to WW2/WW3 (TAT-WW2/WW3) recombinant protein on TGF-β signaling and ArgI gene expression were evaluated on J774A.1 cell culture. For this purpose, interaction of TAT-WW2/WW3 with Smad7, mRNA expression of ArgI, and phosphorylated Smad3 (P-Smad3) were analyzed in TAT-WW2/WW3-treated J774A.1 cell. The results showed interaction of TAT-WW2/WW3 with Smad7, downregulation of ArgI gene expression (P 0.05), and higher amount of P-Smad3 in the TAT-WW2/WW3-treated cells. In conclusion, we suggest that TAT-WW2/WW3 could interfere with TGF-β signaling and reduce ArgI gene expression. Since, ArgI has important effects on tissue remodeling in asthma and cancer progression, so these findings could be used to develop a new approach in the treatment of asthma and cancers.

Introduction Most of pathogenesis related (PR) proteins possess complicated structures; hence their active recombinant forms are usually produced in eukaryotic systems. In this study, we employed an insect cell line to express a recombinant form of a previously identified grape PR3 allergen categorised as class IV chitinase. Methods Grape chitinase cDNA was amplified by RT-PCR and inserted into pFastBacHTA using restriction enzymes. The recombinant pFastBacHTA was applied for the transformation of Escherichia coli DH10Bac cells. The purified recombinant bacmid was used for transfection of Sf9 cells. Finally, the IgE-immunoreactivity of purified recombinant protein was evaluated using grape allergic patient's sera. Moreover, polyclonal anti-6His-tag and monoclonal anti-chitinase antibodies were used for further assessment of recombinant protein. Results SDS–PAGE analysis of the transfected Sf9 cells showed expression of a monomeric 25 kDa and a dimeric 50 kDa recombinant protein. Western blotting revealed considerable IgE reactivity of the recombinant protein with grape allergic patients’ sera. Furthermore, confirmatory assays showed specific reactivity of the recombinant protein with anti-His tag and anti-chitinase antibodies. Conclusion This study showed that, in contrast to E. coli, insect cells are suitable hosts for the production of a soluble and IgE-reactive recombinant form of grape class IV chitinase. This recombinant allergen could be used for component resolved diagnosis of grape allergy or other immunodiagnostic purposes.

The aim of this study is to assess the therapeutic potential of a recombinant hybrid molecule (rHM) alongside an allergenic cocktail from recombinant wild-type allergens as well as pollen extract on Chenopodium album allergy, using a BALB/c mouse model.The BALB/c mice had already been sensitized to C. album via intraperitoneal injections of alum-adsorbed allergenic cocktail and immunotherapy procedure was followed by subcutaneous injections of the rHM, allergenic cocktail and pollen extract at weekly intervals. Humoral immune responses were determined via measurement of specific antibodies in serum. Splenocytes of immunized mice were stimulated in vitro and then proliferation responses, cytokine secretion and mRNA expression of genes involved in immunotherapy were examined by ELISA and real-time PCR.Sensitized mice were identified with high specific IgE against allergenic cocktail when compared with healthy mice. Immunotherapy with the rHM induced the highest ratio of the IgG2a/IgG1 levels compared to allergenic cocktail or C. album pollen extract. The rHM was able to induce proliferative responses as well as the allergenic cocktail in cultured splenocytes. Immunotherapy with the rHM significantly improved secretion of IFN-γ and IL-10, while secretion of IL-13 rapidly diminished. Interestingly, mRNA expression of GATA3 was strongly decreased in rHM-treated mice whereas mRNA expression of T-bet and Foxp3 was significantly increased.Our results prove that immunotherapy with the rHM effectively controlled allergic responses by shifting from a Th2-like immune response to a Th1-dominated immune response.

The insulin and insulin-like growth factor-1 (IGF-1) are considered to play important roles in brain development; and their cognate receptors -InsR and IGF-1R- localized within distinct brain regions including cerebellum. Using Real-Time PCR and western blot analysis, we compared the expression of InsR and IGF-1R in male and female developing rat cerebellum at P0, P7, and P14. At all time points studied, the cerebellar expression of IGF-1R, both at mRNA and protein levels was higher than that of InsR. The lowest InsR and IGF-1R mRNA and protein levels were measured in the neonate cerebellum, independent of gender. In males, the highest InsR and IGF-1R mRNA and protein expression were found at P7. InsR and IGF-1R expression increased significantly between P0 and P7, followed by a marked downregulation at P14. In contrast, in females, mRNA and protein levels of InsR and IGF-1R remain unchanged between P0 and P7, and are upregulated at P14. Therefore, peaked InsR and IGF-1R expression in female cerebelli occurred at P14. Interestingly, changes in mRNA expression and in protein levels followed the same developmental pattern, indicating that InsR and IGF-1R transcription is not subject to modulatory effects during the first 2 weeks of development. These findings indicate that there are prominent sexual differences in InsR and IGF-1R expression in the developing rat cerebellum, suggesting a probable mechanism for the control of gender differences in development and function of the cerebellum.

Diabetes during pregnancy causes neurodevelopmental and neurocognitive abnormalities in offspring. Insulin and insulin-like growth factor-1 (IGF-1) are important regulators of developmental and cognitive functions in the central nervous system. We examined the effects of maternal diabetes on insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (InsR) expression in the developing rat hippocampus. Female rats were maintained diabetic from a week before pregnancy through parturition and male offspring was killed at P0, P7, and P14. We found a significant bilateral upregulation of both IGF-1R and InsR transcripts in the hippocampus of pups born to diabetic mothers at P0, as compared to controls. However, at the same time point, the results of western blot analysis revealed only a slight change in their protein levels. At P7, there was a marked bilateral reduction in mRNA expression and protein levels of IGF-1R, although not of InsR in the diabetic group. We also found a downregulation in IGF1-R transcripts, especially in left hippocampus of the diabetic group at P14. Moreover, at the same time point, InsR expression was significantly decreased in both hippocampi of diabetic newborns. When compared with controls, we did not find any difference in hippocampal IGF-1R or InsR mRNA and protein levels in the insulin-treated group. The present study revealed that diabetes during pregnancy strongly influences the regulation of both IGF-1R and InsR in the right/left developing hippocampi. Furthermore, the rigid control of maternal glycaemia by insulin administration normalized these effects.

Cystic echinococcosis (hydatidosis) is a zoonotic helminthic disease of human and other intermediated hosts wherein infection is caused by the larval stages of tapeworm Echinococcus granulosus. Growth of the larval stage is formed throughout the internal organs, the liver and lung, causing their destruction. Important pathways are unknown about suppression and survival of cysts in human body. In this study, apoptotic bifunctional effects are evaluated in relationship between host and parasite in cystic echinococcosis. Human lymphocytes were treated with hydatid fluid (HF). After 6 h of exposure, caspase-3 activity was measured by fluorometric assay in the HF-treated lymphocytes and control cells. Also, the expression of Bax (as pro-apoptotic protein) and Bcl-2 (an anti-apoptotic protein) mRNA was assessed by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) after 12 h of exposure. For surveying of apoptosis-inducing ligands TNF-related apoptosis-inducing ligand and Fas-L, germinal layer and accompaniment peripheral tissues as healthy control were separated by scalpel from each cyst in sterile condition, then were assess by semiquantitative RT-PCR method in mRNA expression. Both the ratio of Bax/Bcl-2 mRNA expression and caspase-3 activity were higher in the fertile fluid-treated lymphocytes relative to infertile fluid-treated lymphocytes and control group versus the expression level of apoptosis-inducing ligands having a relatively high level in germinal layer of infertile cyst in comparison to fertile cyst and healthy tissue. Apoptosis of germinal layer of fertile cysts is possibly one of the suppression mechanisms in hydatidosis patients, in contrast to lymphocytes apoptosis by modulator of hydatid fluid, one of the hydatid cyst survival mechanisms.

Sex differences and laterality of rat hippocampus with respect to insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (InsR) expression as two important contributors to/regulators of developmental and cognitive functions were examined using real-time PCR and western blot analysis at P0, P7 and P14. Expression of the IGF-1R gene was lowest at P0 in all studied hippocampi. In males, we found the highest expression at P7 in the right hippocampus, and at P14 in the left one. In contrast, the peaked IGF-1R expression occurred at P7 in female hippocampi independent of laterality. Hippocampal InsR expression in males decreased significantly between P0 and P7, followed by a marked upregulation at P14. Conversely, the expression of InsR in females peaked at P7 and then decreased again significantly at P14. We found significant interhemispheric differences in IGF-1R mRNA levels in both male and female hippocampi at different time points. In contrast, we only found significant interhemispheric differences in InsR mRNA expression in P14 male rats, with higher values in the left hippocampus. Interestingly, changes in mRNA expression and in protein levels followed the same developmental pattern, indicating that IGF-1R and InsR transcription is not subject to modulatory effects during the first two weeks of development. These findings indicate that there are prominent interhemispheric and sex differences in IGF-1R and InsR expression in the developing rat hippocampus, suggesting a probable mechanism for the control of gender and laterality differences in development and function of the hippocampus.

The goal of study was first to design multi-epitope peptides from the rat HER2/neu (rHER2/neu) oncogene and then to evaluate the effectiveness of these peptides encapsulated in liposome-polycation-DNA(LPD) nanoparticles (NPs) for the induction of immune response in BALB/c mice. Four multi-epitope peptides derived from the rHER2/neu were designed and different groups of mice were vaccinated with free peptides or peptides encapsulated in NPs. Two of the four tested peptides (p5 and p435), as well as their combinations with the LPD NPs induced a significantly higher IFN-γ and CTLresponses in comparison with the control groups. Consequently, these responses led to lower tumor sizes and longer survival time in TUBO tumor mice model. Our results demonstrate that rHER2/neu-peptides (p5 and p435) and their encapsulation can induce an antigen-specific immunity. This study also presents the first attempt to evaluate the effectiveness of natural rHER2/neu-peptides containing CTL multi-epitope and encapsulated in LPD NPs. From the Clinical Editor This study represents the first attempt to evaluate the effectiveness of natural rHER2/neu-peptides containing CTL multi-epitope encapsulated in LPD NPs, demonstrating that rHER2/neu-peptides (p5 and p435) and their encapsulation can induce tumor antigen-specific immunity.

Allergy to Saffron (Crocus sativus) pollen has been described in people involved in processing of saffron flower stamens. Profilins have been identified as a pan-allergen in different plant pollens and foods. This molecule is an actin-binding protein with a molecular weight of 12-16 kDa found in eukaryotic species.The aim of this study was to generate monoclonal antibody against Cro s 2 in order to characterize this major allergen of saffron pollen.BALB/c mice were immunized to obtain adequate humoral response. Splenocytes were prepared from the immunized animals, mixed with the P3-X63-Ag8.653 myeloma cells and fused by means of PEG 1500. After two weeks of culturing in HAT-containing media, the supernatant from those wells growing hybridomas were screened by ELISA using plates coated with Cro s 2. Cells from positive wells were cloned at least 3 times by limiting dilution. Specificity and cross-reactivity of the mAbs were determined by Western blot analysis and sandwich ELISA.Two stable hybridoma clones secreting mAbs against Cro s 2 were obtained and expanded. The anti-Cro s 2 mAbs were also found to cross-react with other plant profilins. Isotype of this mAb was identified as micro heavy chain and k light chain.The anti-Cro s 2 mAb could be a useful tool for characterization and standardization of many pollen and fruit-derived profilins.

Aims Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study. Methods Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-xL, and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-xL, and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Results Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-xL proteins expression. The Bax/Bcl-2 and Bax/Bcl-xL ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-xL proteins expression. The Bcl-2/Bax and Bcl-xL/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity. Conclusions Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.

We assessed the expression of Bcl-2 family members at both mRNA and protein levels as well as the Caspase-3 activity, in order to investigate the occurrence of apoptosis in hippocampus of STZ-induced diabetic rats. We selected twenty-four Wistar rats; half of them were made diabetic by intraperitoneal injection of a single 60 mg/kg dose of streptozotocin (STZ, IP), while the others received normal saline and served as controls. The expressions of Bcl-2,Bcl-xL, and Bax mRNA and proteins were measured using RT-PCR and western blotting, respectively. Caspases-3 activity was determined by using the Caspase-3/CPP32 Fluorometric Assay Kit. The result showed that mRNA and protein levels of Bcl-2 andBcl-xLwere lower in hippocampus of diabetic group than that of the control group, whereas expressions of Bax in hippocampus of diabetic rats were higher than that of controls at both mRNA and protein levels (P<.01). Hyperglycemia was found to raise 6.9-fold hippocampal caspase-3 activity in diabetic group compared with control group (P<.001). Therefore, the induction of diabetes is associated with increased ratios of Bax/Bcl-2, Bax/Bcl-xL, and increased caspase-3 activity in hippocampus which shows that apoptosis is favored in hippocampal region.