Expression of phosphorylcholine-specific B cells during murine development

The TEPC 15 (T15) clonotype, a putatively germline antibody specificity, does not appear in the neonatal B-cell repertoire until approximately 1 wk of age. This report extends this observation by the demonstration that (a) the T15 clonotype follows similar kinetics of appearance in germfree as well as conventionally-reared mice; (b) maternal influences and genetic background play a minor role in the development of the T15 clonotype since CBFI neonates raised by C57BL/6 or BALB/c mothers acquire the T15 clonotype at the same time in ontogeny as BALB/c neonates; (c) the lack of phosphorylcholine (PC)-specific B cells shortly after birth is reflected in a dearth of PC-binding cells in the neonate as well; and (d) no PC-specifc B cells are found in 19-day fetal liver or in bone marrow until 7 days of life, coincident with their appearance in the spleen. These findings, along with a previous report that PC-specific splenic B cells are tolerizable as late as day 10 after birth, confirm the invariant, late occurrence of the T15 clonotype and support a highly- ordered, rigorously predetermined mechanism for the acquisition of the B- cell repertoire. The results are discussed in light of other studies on the ontogeny of B-cell specificity, and in terms of the implications on the mechanism by which antibody diversity is generated

Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis

A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 °C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 °C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin

CpG Immunotherapy in Chenopodium album sensitized mice: The comparison of IFN-gamma, IL-10 and IgE responses in intranasal and subcutaneous administrations

<p>Abstract</p> <p>Background</p> <p>Mucosal-based immunotherapy has been already used as an alternative form of allergen delivery. In asthma, the poor success rate of immune modulation could be a consequence of inadequate immune modulation in the airways. Previously, we have found that subcutaneous (S.C) co-administration of a homemade allergenic extract from Chenopodium album (Ch.a) pollen and Guanine-Cytosine containing deoxynucleotides (CpG-ODNs) is effective to prevent the inflammatory responses in mouse. In this study we used CpG/Ch.a for immunotherapy of Ch.a-induced asthma and compared the intranasal (I.N) and S.C routes of administration concerning IFN-γ, IL-10 and total IgE responses.</p> <p>Methods</p> <p>Ch.a sensitized mice were treated intranasaly or subcutaneously using CpG and Ch.a. extract. IFN-γ, IL-10 and total IgE were measured in supernatant culture of splenocytes and bronchoalveolor lavage (BAL) fluids by ELISA. Student's t test was used in the analysis of the results obtained from the test and control mice.</p> <p>Results</p> <p>We found that I.N administration of CpG/Ch.a in sensitized mice significantly increased the production of systemic and mucosal IFN-γ and IL-10 compared to phosphate buffered saline (PBS), Ch.a alone and control ODNs treated sensitized mice (P ≤ 0.001). On the other hand, S.C. route induced the systemic and mucosal IFN-γ in the lower levels than in I.N one, and failed to increase systemic IL-10 induction (P = 0.06). Total serum IgE in CpG/Ch.a treated mice in both routes showed significant decreases compared to three control groups (P ≤ 0.01). The amounts of IgE in BAL fluids were not measurable in all groups.</p> <p>Conclusion</p> <p>According to the results of this experiment we concluded that immunotherapy via the I.N co-administration of CpG/Ch.a in comparison with S.C route is more effective to stimulate the mucosal and regulatory responses in Ch.a induced asthma.</p

Patterns of Evolution and Host Gene Mimicry in Influenza and Other RNA Viruses

It is well known that the dinucleotide CpG is under-represented in the genomic DNA of many vertebrates. This is commonly thought to be due to the methylation of cytosine residues in this dinucleotide and the corresponding high rate of deamination of 5-methycytosine, which lowers the frequency of this dinucleotide in DNA. Surprisingly, many single-stranded RNA viruses that replicate in these vertebrate hosts also have a very low presence of CpG dinucleotides in their genomes. Viruses are obligate intracellular parasites and the evolution of a virus is inexorably linked to the nature and fate of its host. One therefore expects that virus and host genomes should have common features. In this work, we compare evolutionary patterns in the genomes of ssRNA viruses and their hosts. In particular, we have analyzed dinucleotide patterns and found that the same patterns are pervasively over- or under-represented in many RNA viruses and their hosts suggesting that many RNA viruses evolve by mimicking some of the features of their host's genes (DNA) and likely also their corresponding mRNAs. When a virus crosses a species barrier into a different host, the pressure to replicate, survive and adapt, leaves a footprint in dinucleotide frequencies. For instance, since human genes seem to be under higher pressure to eliminate CpG dinucleotide motifs than avian genes, this pressure might be reflected in the genomes of human viruses (DNA and RNA viruses) when compared to those of the same viruses replicating in avian hosts. To test this idea we have analyzed the evolution of the influenza virus since 1918. We find that the influenza A virus, which originated from an avian reservoir and has been replicating in humans over many generations, evolves in a direction strongly selected to reduce the frequency of CpG dinucleotides in its genome. Consistent with this observation, we find that the influenza B virus, which has spent much more time in the human population, has adapted to its human host and exhibits an extremely low CpG dinucleotide content. We believe that these observations directly show that the evolution of RNA viral genomes can be shaped by pressures observed in the host genome. As a possible explanation, we suggest that the strong selection pressures acting on these RNA viruses are most likely related to the innate immune response and to nucleotide motifs in the host DNA and RNAs

TLR9 expression in glioma tissues correlated to glioma progression and the prognosis of GBM patients

<p>Abstract</p> <p>Background</p> <p>Our study aims to evaluate the expression of TLR9 in glioma tissues, examine the association between TLR9 expression, clinicopathological variables, and glioma patient outcome, we further characterized the direct effects of TLR9 agonist CpG ODN upon the proliferation and invasion of glioma cells <it>in vitro</it>.</p> <p>Methods</p> <p>RT-PCR and immunofluorescence were used to determine the expression of TLR9 in glioma cell lines and clinical glioma samples. Tissue microarry and immunohistochemistry were applied to evaluated TLR9 expression in 292 newly diagnosed glioma and 13 non-neoplastic brain tissues. We further investigated the effect of CpG ODN on the proliferation and invasion of glioma cells <it>in vitro </it>with MTT assays and matrigel transwell assay respectively.</p> <p>Results</p> <p>RT-PCR showed that TLR9 expressed in all the glioma samples and glioma cell lines we examined. The tissue array analysis indicated that TLR9 expression is correlated with malignancy of glioma (p < 0.01). Multivariate Cox regression analysis revealed that TLR9 expression is an independent prognostic factor for PFS of GBM patients(P = 0.026). TLR9 agonist CpG ODN has no significant effect on glioma proliferation, but matrigel transwell analysis showed that TLR9 agonist CpG ODN can significantly enhance glioma invasion <it>in vitro</it>.</p> <p>Conclusions</p> <p>Our data indicated that TLR9 expression increases according to the histopathological grade of glioma, and the TLR9 expression level is related to the PFS of GBM patients. In addition, our findings warrant caution in the directly injection of TLR9 agonist CpG ODN into glioma tissues for the glioma immunotherapy.</p

C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis with an annual incidence of approximately 2 million cases and is endemic in 88 countries, including Iran. CL's continued spread, along with rather ineffectual treatments and drug-resistant variants emergence has increased the need for advanced preventive strategies. We studied Type II cysteine proteinase (CPA) and Type I (CPB) with its C-terminal extension (CTE) as cocktail DNA vaccine against murine and canine leishmaniasis. However, adjuvants' success in enhancing immune responses to selected antigens led us to refocus our vaccine development programs. Herein, we discuss cationic solid lipid nanoparticles' (cSLN) ability to improve vaccine-induced protective efficacy against CL and subsequent lesion size and parasite load reduction in BALB/c mice. For this work, we evaluated five different conventional as well as novel parasite detection techniques, i.e., footpad imaging, footpad flowcytometry and lymph node flowcytometry for disease progression assessments. Vaccination with cSLN-cpa/cpb-CTE formulation showed highest parasite inhibition at 3-month post vaccination. Immunized mice showed reduced IL-5 level and significant IFN-ã increase, compared to control groups. We think our study represents a potential future and a major step forward in vaccine development against leishmaniasis

Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage

DNA vaccination elicits humoral and cellular immune responses and has been shown to confer protection against several viral, bacterial, and parasitic pathogens. Here we report that optimized codon usage of an injected DNA sequence considerably increases both humoral and cellular immune responses. We recently generated a synthetic human immunodeficiency virus type 1 gp120 sequence in which most wild-type codons were replaced with codons from highly expressed human genes (syngp120). In vitro expression of syngp120 is considerably increased in comparison to that of the respective wild-type sequence. In BALB/c mice, DNA immunization with syngp120 resulted in significantly increased antibody titers and cytotoxic T-lymphocyte reactivity, suggesting a direct correlation between expression levels and the immune response. Moreover, syngp120 is characterized by rev-independent expression and a low risk of recombination with viral sequences. Thus, synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy and safety of DNA vaccination

Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine

Liposomes are versatile (sub)micron-sized membrane vesicles that can be used for a variety of applications, including drug delivery and in vivo imaging but they also represent excellent models for artificial membranes or cells. Several studies have demonstrated that in vitro transcription and translation can take place inside liposomes to obtain compartmentalized production of functional proteins within the liposomes (Kita et al. in Chembiochem 9(15):2403–2410, 2008; Moritani et al.in FEBS J, 2010; Kuruma et al. in Methods Mol Biol 607:161–171, 2010; Murtas et al. in Biochem Biophys Res Commun 363(1):12–17, 2007; Sunami et al. in Anal Biochem 357(1):128–136, 2006; Ishikawa et al. in FEBS Lett 576(3):387–390, 2004; Oberholzer et al. in Biochem Biophys Res Commun 261(2):238–241, 1999). Such a minimal artificial cell-based model is ideal for synthetic biology based applications. In this study, we propose the use of liposomes as artificial microbes for vaccination. These artificial microbes can be genetically programmed to produce specific antigens at will. To show proof-of-concept for this artificial cell-based platform, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen β-galactosidase were entrapped inside multilamellar liposomes. Vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (AnExILs) elicited higher specific humoral immune responses against the produced antigen (β-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal β-galactosidase or pDNA encoding β-galactosidase). In conclusion, AnExILs present a new platform for DNA-based vaccines which combines antigen production, adjuvanticity and delivery in one system and which offer several advantages over existing vaccine formulations