Brucella species circulating in rural and periurban dairy cattle farms: a comparative study in an endemic area.

Brucellosis is among the most important zoonotic infectious diseases worldwide affecting both humans and domestic animals. The present study aimed to determine and compare the seroprevalence of brucellosis among rural and periurban dairy cattle farms of four Iranian provinces from 2017 to 2019. We applied different serological tests, including RBT, SAT, and iELISA to evaluate the brucellosis prevalence among 2808 dairy cattle. Species-specific multiplex PCR and biotyping tests were also used to further identify the implicated Brucella species. Serological screening using RBT, SAT, and iELISA led to 157 (5.6%), 112 (3.9%), and 139 (4.9%) positive results among tested cattle, respectively. Brucella abortus biovars 1 (2 cases) and biovars 3 (42 cases) were identified by biotyping experiments and multiplex PCR in all 44 tested lymph node samples. Further, Cohen's kappa statistical analysis revealed that the best degree of agreement was seen between RBT and iELISA (99.4%), followed by SAT/iELISA (98.5%) and finally RBT/SAT (98.4%). Our results also showed a significantly lower seroprevalence of brucellosis in periurban dairy cattle when compared to rural dairy cattle population (p value= 0.01). These results reflect the need for better vaccine coverage using RB51 combined with an appropriate test-and-slaughter program in the rural dairy cattle population.

Comparison of different immunization routes on the immune responses induced by Mycobacterium tuberculosis ESAT-6/CFP-10 recombinant protein.

According to some difficulties against tuberculosis (TB) vaccination, development of new TB vaccines has been noted in recent years. Selection of proper route for vaccination is one of the most important factors for induction of good immune responses. Hence, in this study, the effects of different administration routes, including intranasal (I.N), subcutaneous (S.C) and intramuscular (I.M) on immune responses against Mycobacterium tuberculosis ESAT-6/CFP-10 recombinant protein has been considered. Recombinant ESAT-6/CFP-10 protein with or without adjuvant (MF59 or cholera toxin B (CTB)) was administered by three routes of I.M, I.N and S.C to mice for three times. Then, the levels of specific antibodies, lymphocyte proliferation and IFN-γ/IL-5 cytokine profile have been carried out to evaluate the humoral and cellular responses. The results showed that the titers of specific antibodies were quickly elevated in S.C and I.M groups after first immunization. Otherwise, the raise of antibody has delay in the I.N immunized animals. The levels of IFN-γ and lymphocyte proliferation have been increased in all of vaccinated groups. However, the I.N immunized mice have lower levels of IL-5 production. Based on our finding, the ESAT-6/CFP-10 recombinant protein is a potent stimulator of immune responses in all of three immunization strategies. However intranasal administration of this antigen has tended to reinforcement of cellular immune responses.

The Neuroprotective Effects of Curcumin Nanoparticles on The Cerebral Ischemia-Reperfusion Injury in The Rats-The Roles of The Protein Kinase RNA-Like ER Kinase/Extracellular Signal-Regulated Kinase and Transcription Factor EB proteins.

OBJECTIVE: Reduction of cerebral ischemia-reperfusion injury (IRI)/re-oxygenation injury, is defined as the paradoxical exacerbation of the cellular dysfunction and death, following restoration of the blood flow to previously ischemic tissues. The re-establishment of blood flow is essential to salvage the ischemic tissues. As a result, the treatment of IRI with novel therapies, which have fewer side effects, are of great importance. Therefore, this study aimed to investigate the effects of curcumin nanoparticle (CN) pre-treatment on the cerebral I/R rat model. MATERIALS AND METHODS: In this experimental study, CN was administered to rats orally five days before the bilateral common carotid artery occlusion (BCCAO) and continued for three days. The intensity of oxidative stress, the activities of antioxidant enzymes, glutathione (GSH) content, the activity of mitochondrial enzymes, including succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), curcumin bioavailability, pERK/ERK expression ratio and TFEB protein were studied. Data analysis was performed using Graphpad Prism V.8 software, one-way analysis of variance (ANOVA) with the statistical package for the social sciences (SPSS V.26 software). RESULTS: Cerebral IRI-damage significantly increased the oxidative stress (P=0.0008) and decreased the activity of the antioxidant enzymes including catalase (CAT) (P<0.001), super oxide dismutase (SOD) (P<0.001), reduced GSH (P<0.001), mitochondrial enzymes, pERK/ERK expression ratio (P=0.002) and TEFB protein (P=0.005) in rats' brains. In addition, the pre-treatment of the rats with CN resulted in a decrease in the reactive oxygen species (ROS), and an increase in the activities of antioxidants and mitochondrial enzymes. This in turn up-regulated the pERK/ERK expression ratio and TEFB expression. CONCLUSION: CN has neuroprotective effects on the cerebral IRI condition due to its antioxidant properties and is able to overexpress the pERK and TFEB proteins; thus, it can be considered as a suitable treatment option during and after the incidence of stroke.

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins.

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj)]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

Induction of prominent Th1 response in C57Bl/6 mice immunized with an E. coli-expressed multi T-cell epitope EgA31 antigen against Echinococcus granulosus.

First step in developing an epitope-based vaccine is to predict peptide binding to the major histocompatibility complex (MHC) molecules. We performed computational analysis of unique available EgA31 sequence to locate appropriate antigenic propensity positions. T-cell epitopes with best binding affinity values of < 50% inhibitory concentration were selected using different available servers (Propred and IEDB). Peptides with 100% population coverage were selected. A DNA fragment corresponding to the furin linker enriched in Golgi apparatus was inserted sequentially between each epitope sequences in a synthetic DNA in order to cleave the chimeric protein into four separated peptides. Subsequently, the synthetic DNA was cloned into the pGEX4T-1 and pEGFP-N1 vectors and GST-ChEgA31 was expressed in E. coli strain BL21-DE3. The recombinant protein was detected by western blotting using an HRP-conjugated polyclonal anti-GST antibody. Fusion protein purified by affinity chromatography was used to raise antisera in rabbits. Results in agar gel immunodiffusion assay indicated induction of specific antibodies against multiepitope antigen in the tested rabbits. Cytokine assay was carried out in C57Bl/6 mice and the levels of cytokines were analyzed by sandwich ELISA. Interestingly, production of specific IFN-gamma was prominently higher in mice immunized with GST-ChEgA31 and pEGFP-ChEgA31 (650-1300 pg/ml) compared to control groups. No difference was observed in the level of IL-10 and IL-4 in immunized and GST control group. Challenge study with 500 live protoscolices of Echinococcus granulosus on immunized mice demonstrated protectivity level (50-60%). Based on our results, it appeared that the chimeric protein in the study was able to stimulate T-helper cell-1 (Th1) development and high level of cell mediated immunity in mice.

Improvement the expression and purification of Loa22: a lipoprotein with OmpA domain from pathogenic Leptospira serovars.

Background and Objectives: One of the highly conserved outer membrane proteins expressed only by pathogenic Leptospires is Loa22. The study aims is to achieve the optimum conditions for high expression of recombinant Loa22 (rLoa22) protein. Materials and Methods: Complete coding sequence of loa22 gene sub-cloned into a pET32a (+) expression vector. BL21 competent E. coli (pLysS) used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers. Then expression conditions optimized by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration. Results: SDS-PAGE analysis showed that high production of rLoa22 protein obtained when post induction incubation, IPTG concentration, and duration of induction were 37°C, 0.1 M and 5 h in 2×TY medium respectively. The purification of rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration. Conclusion: The findings of the study led to high production of pure Loa22 protein, which can form the basis for future investigation on the design of rapid diagnostic tests and more effective vaccine candidates for leptospirosis.

A Combination of Recombinant HA1-and Nucleoprotein-Based Chitosan Nanoparticles Induces Early and Potent Immune Responses Against the H9N2 Influenza Virus.

The effectiveness of inactivated H9N2 influenza vaccines is doubtful due to changes in antigenic regions of the virus hemagglutinin (HA) protein. One strategy for the development of the efficacious vaccine is the use of nanoparticles that display more immunogenic regions of the influenza virus. In this study, chitosan (CS)-based nanoparticles were developed as a delivery system for intranasal immunization using recombinant H9N2 virus HA1 and nucleoprotein (NP), for the induction of humoral and cellular responses. CS-HA1 and CS-NP nanoparticles were prepared by the ionic gelation method and characterized for their physicochemical properties and shape. The immunogenicity and the protective efficacy were evaluated by measuring antibody titers, T cell proliferation response, CD4+/CD8+ ratio, and quantitative real-time RT-PCR following intranasal administration of the prepared nanoparticles alone or in combination in chickens compared to an inactivated H9N2 vaccine. The average size, surface charge, and spherical structure of the synthesized nanoparticles showed high quality. Serologic analysis revealed that the immunization of inactivated vaccine groups resulted in strong influenza antibodies, which were significantly (p < 0.05) higher compared to the other groups. The vaccinated chickens with CS-HA1+CS-NP developed higher specific anti-influenza antibodies than in those vaccinated with each of rHA1 and rNP. Administration of a combination of the protein-based nanoparticles has stimulated the activation of both CD4+ and CD8+T cells and induced a significantly higher T cell proliferation. The viral shedding was significantly lower in CS-HA1+CS-NP and inactivated vaccine groups compared with other challenged groups. The data demonstrate the potential of CS-HA1+CS-NP nanoparticles for eliciting specific influenza antibodies and conferring protection in chickens.

Designing of novel chimeric PvpA-pMGA protein of Mycoplasma gallisepticum, applicable for indirect ELISA.

BACKGROUND: Mycoplasma gallisepticum is the primary agent of chronic respiratory disease in chickens creating important economic losses in poultry industry. pMGA and pvpA genes encode major surface proteins in M. gallisepticum containing pathogenic, antigenic, and immune evasion characteristics. The objective of the present study was to design, express, and purify the recombinant chimeric PvpA-pMGA protein from M.gallisepticum for using in serological diagnostic test. METHODS: Antigenic regions of PvpA and pMGA proteins were predicted for designing chimeric pvpA-pMGA gene construct. The codon optimized sequence was cloned into the expression vector pET32a+ and transformed into the Escherichia coli strain BL21 (DE3). The pET32a-PvpA-pMGA recombinant plasmid was expressed and confirmed by SDS-PAGE and immunoblotting. PvpA-pMGA recombinant protein (20µg and 50µg), ts-11 vaccine strain, and S6 strain that formulated by montanide adjuvant and two control groups (PBS and adjuvant) were injected subcutaneously to six groups of chickens. RESULTS: High yield of protein was purified amount 138 mg/L by affinity batch formation method. Indirect ELISA showed the levels of antibodies in rPvpA-pMGA was significantly higher than ts-11 and S6 groups (p<0.05). The results indicated that antigen-specific response was successfully elicited by the rpMGA-PvpA in chickens. The result of the ELISA with sera collected from ts-11 and S6 groups showed that indirect PvpA-pMGA-ELISA is appropriate candidate for detection of specific antibodies against M. gallisepticum with 100% sensitivity and specificity. CONCLUSIONS: The rPvpA-pMGA is a highly candidate immunogenic protein which induced high amount of humoral immune response. Novel rPvpA-pMGA protein could be useful for evaluation of antibody level in vaccinated poultry flocks.

Modulation of Immune Responses Against HA1 Influenza Vaccine Candidate by B-lymphocyte Stimulator Cytokine in Mice.

Utilizing subunit vaccines is one of the strategies to address influenza infection. Recent innovations have focused on high doses of vaccine antigens and immune enhancers or adjuvant to induce more robust and long-lasting immune responses. Here, an effect of the B cell-activating factor receptor (BAFF-R) to increase the magnitude and durability of immune responses of the recombinant HA1 (rHA1) protein against the H1N1 influenza virus was studied. The HA1 protein and the effector domain of BAFF-R were expressed in the pET-28a (+) vector. Eight-week-old BALB/c mice were equally grouped into five groups (n=20). The 15 and 25 µg/µL of rHA1 were mixed with 2 µg/µL of rBAFF-R and injected three times for vaccinated groups. Three control groups were received normal saline and two concentrations of rHA1. The ability of rBAFF-R in eliciting HA-specific antibody response and stimulating T lymphocyte proliferation to induce the cell-mediated immunity was assayed. Induction of protection was evaluated following the challenge with PR8 strain. Analysis of immune responses showed that the co-administration of rBAFF-R with rHA1 boosted HI responses to the antigen in mice, whilst it was not able to promote the T cell proliferation responses against influenza. Compared to rHA1alone, the rBAFF-R/rHA1 generated efficient protection for the animals. There were no significant differences in eliciting the immune responses in mice immunized with the lower dose of rHA1 than that with the higher dose. The data indicate the rBAFF-R can enhance the primary and memory immune responses to protect against influenza infection.

Active immunotherapy against pathogenic Cis pT231-tau suppresses neurodegeneration in traumatic brain injury mouse models.

Traumatic brain injury (TBI), characterized by acute neurological impairment, is associated with a higher incidence of neurodegenerative diseases, particularly chronic traumatic encephalopathy (CTE), Alzheimer's disease (AD), and Parkinson's disease (PD), whose hallmarks include hyperphosphorylated tau protein. Recently, phosphorylated tau at Thr231 has been shown to exist in two distinct cis and trans conformations. Moreover, targeted elimination of cis P-tau by passive immunotherapy with an appropriate mAb that efficiently suppresses tau-mediated neurodegeneration in severe TBI mouse models has proven to be a useful tool to characterize the neurotoxic role of cis P-tau as an early driver of the tauopathy process after TBI. Here, we investigated whether active immunotherapy can develop sufficient neutralizing antibodies to specifically target and eliminate cis P-tau in the brain of TBI mouse models. First, we explored the therapeutic efficacy of two different vaccines. C57BL/6 J mice were immunized with either cis or trans P-tau conformational peptides plus adjuvant. After rmTBI in mice, we found that cis peptide administration developed a specific Ab that precisely targeted and neutralized cis P-tau, inhibited the development of neuropathology and brain dysfunction, and restored various structural and functional sequelae associated with TBI in chronic phases. In contrast, trans P-tau peptide application not only lacked neuroprotective properties, but also contributed to a number of neuropathological features, including progressive TBI-induced neuroinflammation, widespread tau-mediated neurodegeneration, worsening functional deficits, and brain atrophy. Taken together, our results suggest that active immunotherapy strategies against pathogenic cis P-tau can halt the process of tauopathy and would have profound clinical implications.

Influenza A viruses: why focusing on M2e-based universal vaccines.

The threat of highly virulent avian influenza, such as H5N1 and swine-origin H1N1 influenza viruses, bring out an urgent need to develop a universal influenza vaccine, which may provide cross-protection against different strain of influenza A viruses. The extra-domain of influenza M2 protein (M2e), which is almost completely conserved among all subtypes of influenza A viruses, is considered as a promising candidate target for the development of a broad-spectrum recombinant influenza A vaccine. The results of several preclinical studies with M2e protein, with or without carriers, have already proved the successful protection of M2e-based vaccinated animal model against lethal challenge of heterologous and homologous influenza A viruses. Recently, the results of Phase I/II clinical trail studies with M2e-based vaccines have raised hopes for considering these vaccines against seasonal and pandemic influenza A strains. Hence, it is expected that more and more effective and safe universal influenza vaccines based on M2e will be developed for prevention of seasonal and pandemic influenza in the near future.

Cloning, expression, and immunogenicity of novel fusion protein of Mycobacterium tuberculosis based on ESAT-6 and truncated C-terminal fragment of HSP70.

Tuberculosis (TB) remains as a major public health problem worldwide. Identification and selection of immunodominant antigens of Mycobacterium tuberculosis (MTB), capable of efficiently inducing a protective immune response is the ultimate goal of TB vaccine development studies. Accordingly, this study was designed to produce a novel M. tuberculosis fusion protein consisted of MTB ESAT-6 (early secreted antigenic target-6 kDa), as a potent immunogenic protein, fused to C-terminus of MTB HSP70 (HSP70(359-610)), as an appropriate carrier and adjuvant. The constructed gene was inserted into a prokaryotic expression vector (pQE30); consequently, the recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M15. Inclusion bodies from bacterial cell lysates were solubilized and the recombinant fusion protein was easily purified by Ni-NTA affinity chromatography under denaturing conditions followed by urea gradient dialysis. The purified and refolded protein was then applied for immunization of mice that resulted in the detection of high titers of specific antibodies, high level of IFN-γ and cell proliferation. The results of our study could confirm the capability of E6H70C fusion protein, as a potential tuberculosis vaccine candidate, for the efficient induction of specific immune responses in a mouse model. However, further investigation need to evaluate the protectivity of this recombinant protein in host model.

Study of infection with an Iranian field-isolated H9N2 avian influenza virus in vaccinated and unvaccinated Japanese quail.

In the present study, we examined the mortality rate, egg production, and clinical signs of quail experimentally infected with a field isolate of A/Chicken/Iran/339/02 (H9N2) avian influenza virus obtained from an infected commercial layer farm with severe morbidity and mortality. A total of 120 quail at 14 days old were randomly divided into four groups of vaccinated (B and C) and unvaccinated (A and D) birds. Vaccination was done on days 20 and 32, and viral inoculation of birds in groups C and D was then carried out on day 43. For evaluation of viral transmission, at 24 hr postinoculation additional unvaccinated birds were placed in direct contact with challenged birds. All the birds were evaluated for clinical signs, egg production, antibody production, viral titration in lung homogenates, and viral transmission following inoculation. All unvaccinated-challenged birds were infected and showed clinical signs, whereas the infection rate along with clinical signs of vaccinated-challenged birds reached 30%-40%. Although vaccination induced high antibody titers, reduction in food and water consumption was evident in this vaccinated-challenged group compared with the unchallenged control group. These results could indicate that inactivated vaccine did not fully prevent the infection, although it was capable of protecting birds against clinical signs and significantly decreased viral titers in lungs after intranasal challenge.

Cloning, expression and purification of the influenza A (H9N2) virus M2e antigen and truncated Mycobacterium tuberculosis HSP70 as a fusion protein in Pichia pastoris.

Due to its conservation, the extracellular domain of the influenza A M2 protein (M2e) has the potential for being applied as a recombinant vaccine candidate against a wide range of strains, though its immunogenicity may need to be improved. The occurrence of several post-translational modifications within the structure of M2 protein may affect its immunopotency for the induction of humoral immune response. Herein, to construct a recombinant M2e-based vaccine candidate with the appropriate structural conformation and immunogenicity the corresponding nucleotide sequence from an H9N2 influenza strain was fused to the N-terminus of the truncated Mycobacterium tuberculosis HSP70(359-610), as a potent adjuvant, and following its cloning into the pPICZ alpha A plasmid the fusion gene was expressed in Pichia pastoris KM71H yeast. The secreted protein was then easily purified from the culture media, based on the presence of polyhistidine tag and used for the production of rabbit polyclonal antisera. This raised antisera could recognize the native M2e protein on the surface of H9N2 influenza virus-infected MDCK cells at a comparable level with the commercial H2N2-specific anti-M2 antibody, which was evidenced with immunofluorescence and cell-ELISA assays. These results not only re-emphasized on the conservancy of the M2e antigen, but also pointed towards the applicability of the M2e-HSP70(359-610) fusion protein for the induction of specific antibodies capable of binding to the native M2e antigen on the infected cells. Collectively, this study implied that purified M2e-HSP70(359-610) represents a promising vaccine candidate; however, its in vivo potency for the induction of protection remains to be evaluated.

Heterologous expression, purification and characterization of the influenza A virus M2e gene fused to Mycobacterium tuberculosis HSP70(359-610) in prokaryotic system as a fusion protein.

One of the concerns about influenza A vaccine based on M2e protein is their limited potency; hence, optimal approaches to enhance immunogenicity of M2e protein immunization remain to be established. It seems by linking this M2e-peptide to an appropriate carrier such as mycobacterium tuberculosis C-terminal 28-kDa domain of HSP70 (HSP70(359-610)), we can render it very immunogenic. According to previous reports, this study was designed to produce a novel influenza A virus recombinant fusion protein consisted of M2e, a potent immunogenic protein from influenza A virus, fused to C-terminal domain of mycobacterium tuberculosis HSP70, HSP70(359-610), as a carrier and adjuvant. We fused the genes of M2e and HSP70 ( 359-610 ) then inserted in pQE-60, prokaryotic expression vector. This recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M-15. The recombinant fusion protein was purified by Ni-NTA affinity chromatography under denaturing conditions, followed by urea gradient dialysis. The purified fusion protein was analyzed on SDS-PAGE. Western blot assay was used to examine the immunoreaction of the expressed protein using commercial penta-His HRP conjugate antibody. The antigenicity and biological activity of the recombinant protein was also qualitatively detected on the infected MDCK cells surface by immunofluorescence and cell-ELISA assay using rabbit's immunized antiserum. This observation suggest that the expressed fusion protein is useful as a universal recombinant vaccine for overcoming highly mutational influenza virus, but more immunological study in animal lab remains to be evaluated.

Prokaryotic expression and characterization of avian influenza A virus M2 gene as a candidate for universal recombinant vaccine against influenza A subtypes; specially H5N1 and H9N2.

The conserved M2 protein of influenza A virus is considered as a promising candidate target for a broad-spectrum, recombinant influenza A vaccine. In the present study, the open reading frame (ORF) of avian influenza A/chicken/Iran/101/1998 (H9N2) M2 gene was amplified then cloned in pAED4, prokaryotic expression vector. M2 protein was produced through the expression of this recombinant expression vector (pAED4-M2) in E. coli BL21 (DE3) strain. The expressed M2 protein was analyzed on SDS-PAGE. Western blot assay was used to examine the immunoreaction of the expressed protein using commercial polyclonal anti-M2 antibody. The antigenicity and biological activity of the recombinant protein was also qualitatively detected on infected MDCK cells surface by immunofluorescence assay using rabbit's immunized antiserum. So, according to the sequence alignment based on the mentioned isolate and the result of immunoassay reaction, it seems recombinant vaccine based on A/chicken/Iran/101/1998(H9N2) M2 protein isolate might cover majority of influenza A virus strains specially H5 and H9 circulating in Iran and neighbor regions significantly.

Invasive aspergillosis promotes tumor growth and severity in a tumor-bearing mouse model.

Invasive aspergillosis increases in chronic immunosuppressive diseases such as cancer. There is little information about the mechanisms by which Aspergillus infection affects the immune regulation and microenvironment of cancer cells. Hence, this study was aimed at investigating the effect of invasive aspergillosis on immunosurveillance, metastasis, and prognosis of cancer in tumor-bearing mice. After implantation of mouse mammary tumor in BALB/c mice, they were infected with Aspergillus conidia intravenously. For comparison, groups of mice were experimentally infected with Aspergillus conidia or implanted with tumor cells separately. Seven days after Aspergillus infection, the serum levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by ELISA, and subsequently regulatory T lymphocytes were analyzed by flow cytometry. The survival of animals and mean tumor size were then determined. Our results indicated that tumor sizes in mice increased significantly after infection with Aspergillus conidia. Moreover, invasive aspergillosis enhanced the population of regulatory lymphocytes and level of TIMP-1. This study supports the idea that massive Aspergillus infection could stimulate tumor growth and increases the possibility of a bad prognosis. As a result, treatment of Aspergillus infection could be considered an important issue for efficient cancer therapy.

Physico-chemical and cytotoxic analysis of a novel large molecular weight bacteriocin produced by Lactobacillus casei TA0021.

BACKGROUND AND OBJECTIVES: Antimicrobial peptides produced by lactic acid bacteria have gained enormous attention owing to their health benefits. This study aimed to isolate, purify and characterize the antibacterial protein produced by autochthonous Lactobacillus casei TA0021 strain. MATERIALS AND METHODS: The antagonistic activity of L. casei TA0021 against a number of pathogenic bacteria was tested by agar well diffusion assay. The antimicrobial agent in the neutralized supernatant fluids was subjected to the action of proteolytic enzymes, catalase, lipase and lysozyme, and their tolerance to variable pH and temperature was estimated. The proteinaceous antagonistic compound was precipitated by 60% w/v ammonium sulphate, desalted and subjected to cation exchange and gel filtration chromatography. Approximate molecular weight of Lactocin was determined by SDS-PAGE and non-denaturing gel electrophoresis. Hemoglobin release assay and cytotoxicity effect of Lactocin TA0021 was determined. The results were statistically analyzed. RESULTS: The antagonistic agent active against Salmonella Typhimurium and Shigella flexneri appeared resistant to catalase and lipase treatments, while sensitive to the tested proteolytic enzymes. Lactocin TA0021 resisted acidic pH values of 3.0, while alkaline pH values of >9 completely destroyed the activity. The antibacterial peptide was approximately 68 KDa and heat labile as lost its activity at 100°C after 5 minutes. The bacteriocin was non-toxic to MRC-5 cell lines and non-hemolytic. Purification method lead to increase in antibacterial activity while, subsequent decrease in recovery and yield was observed with increasing purification fold. CONCLUSION: The purified antimicrobial protein from L. casei TA0021 might be used for application in medicinal and food products.

Bordetella pertussis antigens encapsulated into N-trimethyl chitosan nanoparticulate systems as a novel intranasal pertussis vaccine.

The mucosal immune system serves as the first line of defense against Bordetella pertussis. Intranasal vaccination, due to its potential to induce systemic and mucosal immune responses, appears to prevent the initial adherence and colonization of the bacteria at the first point of contact. In the present study, two B. pertussis antigens, pertussis Toxoid (PTd) and Filamentous hemagglutinin (FHA), which play a very significant role in virulence and protection against pertussis, were encapsulate into N-trimethyl chitosan (TMC) nanoparticulate systems. After preparation of TMC nanoparticles (NPs), the NPs were characterized and their ability to induce efficient immune responses against B. pertussis was studied in a mouse model. Our findings showed that PTd + FHA-loaded TMC NPs have strong ability to induce IL-4, IL-17, IFN-γ, IgG, and IgA in the mouse model. Results from this study suggest that nasal administration of the PTd + FHA-loaded TMC NPs induced not only a systemic immune response but also a local mucosal response, which may improve the efficacy of pertussis prevention through respiratory tract transmission.

Nasal vaccination with r4M2e.HSP70c antigen encapsulated into N-trimethyl chitosan (TMC) nanoparticulate systems: Preparation and immunogenicity in a mouse model.

In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of the r4M2e.HSP70c, as an M2e-based universal recombinant influenza virus vaccine candidate, was investigated in mice. The anti-M2e specific cellular and humoral immune responses were assessed and the protective efficacy against a 90% lethal dose (LD90) of influenza A/PR/8/34 (H1N1) in a mice model was evaluated. Our results showed that the intranasal immunization of mice with r4M2e.HSP70c+TMC rather than the control groups, r4M2e+TMC, r4M2e and PBS (Phosphate buffer saline), significantly elevated both longevity and serum level of the total M2e-specific IgG antibody with a significant shift in the IgG2a/IgG1 ratio toward IgG2a, induced a Th1 skewed humoral and cellular immune responses, increased IFN-γ, IgG, and IgA in the bronchoalveolar lavage fluid (BALF), and promoted the proliferation of peripheral blood lymphocytes with lower morbidity and mortality rate against viral challenge. In conclusion, based on evidence to our finding, nasal vaccination with r4M2e.HSP70c antigen encapsulated into N-Trimethyl Chitosan (TMC) nanoparticulate system showed to induce a long lasting M2e-specific humoral and cellular immune responses and also provided full protection against a 90% lethal dose (LD90) of the influenza virus A/PR/8/34 (H1N1). It seems, protective immunity following intranasal administration of r4M2e could be resulted by the cooperation of both adjuvants, TMC and HSP70c.