Non-toxic Mutant Research Articles

A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization

Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14 (Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-l-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. Together with earlier studies, these results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton.

Non-toxic conformer of amyloid β may suppress amyloid β-induced toxicity in rat primary neurons: Implications for a novel therapeutic strategy for Alzheimer’s disease

The 42-mer amyloid β-protein (Aβ42) oligomers cause neurotoxicity and cognitive impairment in Alzheimer’s disease (AD). We previously identified the toxic conformer of Aβ42 with a turn at positions 22–23 (“toxic” turn) to form oligomers and to induce toxicity in rat primary neurons, along with the non-toxic conformer with a turn at positions 25–26. G25P-Aβ42 and E22V-Aβ42 are non-toxic mutants that disfavor the “toxic” turn. Here we hypothesize that these non-toxic mutants of Aβ42 could suppress Aβ42-induced neurotoxicity, and examined their effects on the neurotoxicity, aggregation, and levels of the toxic conformer, which was evaluated by dot blotting using a monoclonal antibody (11A1) against the toxic conformer. G25P-Aβ42 and E22V-Aβ42 suppressed the neurotoxicity and aggregation of Aβ42 as well as the formation of the toxic conformer. The neurotoxicity induced by Aβ42 was also significantly reduced by the treatment of 11A1, but not of Aβ-sequence specific antibodies (6E10 and 4G8). Since recent studies indicate that Aβ oligomers contain parallel β-sheet, the present results suggest that the non-toxic mutants of Aβ42 without the “toxic” turn could prevent the propagation process of the toxic conformer of Aβ42 resulting in suppression of the formation of the toxic oligomers. This could be a promising strategy for AD therapeutics.

Escherichia coli Heat-Stable Toxin b Impairs Intestinal Epithelial Barrier Function by Altering Tight Junction Proteins

Escherichia coli heat-stable toxin b (STb) causes diarrhea in animals. STb binds to sulfatide, its receptor, and is then internalized. In the cytoplasm, through a cascade of events, STb triggers the opening of ion channels, allowing ion secretion and water loss and leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells, and some bacterial toxins are known to affect adversely TJs. The present study aimed at determining the effect of STb on TJs. T84 cells were treated for 24 h with purified STb and a nontoxic STb mutant (D30V). Transepithelial resistance (TER), paracellular flux marker, and confocal microscopy were used to analyze the effect of STb on TJs. Purified STb caused a significant reduction of TER parallel to an increase in paracellular permeability compared to the results seen in untreated cells or mutant D30V. The increased paracellular permeability was associated with a marked alteration of F-actin stress fibers. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. These changes were also observed following treatment of T84 cells with an 8-amino-acid peptide found in the STb sequence corresponding to a consensus sequence of Vibrio cholerae Zot toxin. These effects were not observed with a scrambled peptide or mutant D30V. Our findings indicate that STb induces epithelial barrier dysfunction through changes in TJ proteins that could contribute to diarrhea.

A novel approach to generate a recombinant toxoid vaccine against Clostridium difficile

The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.

Evaluation of Recombinant Forms of the Shiga Toxin Variant Stx2eB Subunit and Non-Toxic Mutant Stx2e as Vaccine Candidates against Porcine Edema Disease

ABSTRACTPorcine edema disease (ED) is a communicable disease of shoats caused by infection with Shiga toxin (Stx)-producing Escherichia coli. Stx2e is classified as a 1A5B-type toxin and is a decisive virulence determinant of ED. The single A subunit of Stx2e possesses enzymatic activity and is accompanied by a pentamer of B subunits, which binds to the host receptor and delivers the A subunit into the cell. In the present study, we used a mouse model to evaluate the immunogenicity of 3 ED vaccine candidates: a non-toxic mutant holotoxin mStx2e and 2 Stx2eB-based fusion proteins, Stx2eA2B-His and Stx2eB-His. Systemic inoculation of mice with mStx2e- and the Stx2eB-derived antigens induced anti-Stx2e IgG responses that were fully and partially capable of neutralizing Stx2e cellular cytotoxicity, respectively. Intranasal immunization with mStx2e protected the mice from subsequent intraperitoneal challenge with a lethal dose of Stx2e, whereas immunization with Stx2eA2B-His and Stx2eB-His afforded partial protection. Analysis of serum cytokines revealed that mStx2e, but not the Stx2eB-based antigens, was capable of inducing a Th2-type immune response. These results suggest that although the Stx2eB-based antigens elicited an immune response to Stx2e, they did so through a different mechanism to the Th2-type response induced by mStx2e.

Vaccination with Plasmid DNA Encoding a Mutant Toxic Shock Syndrome Toxin-1 Ameliorates Toxin-induced Lethal Shock in Mice

Staphylococcal toxic shock syndrome toxin-1 (TSST-1), a superantigenic toxin produced by Staphylococcus (S.) aureus, is a major cause of septic shock and toxic shock syndrome. To investigate whether vaccination with a plasmid DNA encoding a non-toxic mutant TSST-1 (mTSST-1) can protect mice against wild-type TSST-1-induced lethal shock, the mice were intranasally immunized with the plasmid DNA (named pcDNA-mTSST-1) plus a mucosal adjuvant, a non-toxic mutant labile toxin (mLT). After the immunization, the mice were challenged with TSST-1 and lipopolysaccharide (LPS). The survival rate of mice immunized with pcDNA-mTSST-1 plus mLT was higher than that of the control mice immunized with PBS alone, mLT alone, pcDNA-mTSST-1 alone, or a parent plasmid plus mLT. The titers of interferon-γ (IFN-γ) in the sera of mice immunized with pcDNA-mTSST-1 plus mLT were significantly lower than those of the mLT control mice. Immunization with pcDNA-mTSST-1 plus mLT increased the serum levels of TSST-1-specific antibodies, especially immunoglobulin G1 (IgG1) and IgG2a subclasses. Furthermore, the sera obtained from mice immunized with pcDNA-mTSST-1 plus mLT significantly inhibited the TSST-1-induced secretion of IFN-γ and tumor necrosis factor-α (TNF-α) in murine spleen cells in vitro. These results indicate that immunization with pcDNA-mTSST-1 plus mLT provides protection against the lethal toxic shock of mice induced by wild-type TSST-1. The protective effect could be due to TSST-1-specific neutralizing antibodies as well as the inhibition of IFN-γ and TNF-α secretions. Since TSST-1 is commonly released by invasive S. aureus, the pcDNA-mTSST-1 should be useful in preventing toxin-induced shock resulting from S. aureus infection.

A novel monoclonal antibody reveals a conformational alteration shared by amyotrophic lateral sclerosis‐linked SOD1 mutants

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by the selective loss of upper and lower motoneurons. Although >100 different Cu, Zn superoxide dismutase (SOD1) mutations have been identified in ALS patients, it remains controversial whether all of them are disease-causative mutations. Therefore, it is necessary to develop molecular mechanism-based diagnosis and treatment of ALS caused by SOD1 mutations. We previously reported that 3 pathogenic mutations of SOD1 cause chronic endoplasmic reticulum (ER) stress by inducing the binding of SOD1 to Derlin-1, a component of the ER homeostatic machinery. Here, we systematically analyzed 132 SOD1 mutants and found that most have a constitutively exposed Derlin-1-binding region (DBR) that is occluded in the wild-type protein. To develop the novel molecular mechanism-based antibody that can specifically recognize the aberrant structure of toxic SOD1 mutants, we generated the monoclonal antibody against the DBR. MS785, a monoclonal antibody generated against the DBR, distinguished most ALS-causative SOD1 mutants from both wild-type and nontoxic mutants. Moreover, MS785 recognized endogenous SOD1 in B lymphocytes derived from 14 ALS patients carrying SOD1 mutations but not from 11 healthy controls. This is the first study to address the common property of all ALS-causative SOD1 mutants. MS785 is the first molecular mechanism-based antibody that was shown to be able to distinguish ALS-linked toxic SOD1 mutants from both wild-type and nontoxic mutants. MS785 may thus become an innovative tool for the diagnosis of ALS.

Sublethal Staphylococcal Enterotoxin B Challenge Model in Pigs To Evaluate Protection following Immunization with a Soybean-Derived Vaccine

In an effort to develop a sustainable platform for manufacturing protein-based vaccine candidates, we expressed a triple mutant of staphylococcal enterotoxin B carrying the L45R, Y89A, and Y94A modifications in transgenic soybean seeds (soy-mSEB). Soy-mSEB possessed no detectable superantigen activity in vitro. We found that this soybean-derived, nontoxic mutant of SEB could be stably expressed, stored in seeds for extended periods at room temperature without degradation, and easily purified from contaminating soy proteins. Vaccination of pigs with purified soy-mSEB, or the identical triple mutant expressed in Escherichia coli (E. coli-mSEB), resulted in high antibody titers against the native toxin in immunized animals. In fact, titers were indistinguishable regardless of the immunogen used, demonstrating the equivalence of soy-mSEB and E. coli-mSEB vaccinations. Antisera from either immunized group were able to block native SEB superantigen activity in an in vitro neutralization assay. Similar results were obtained when immunized animals were challenged with a sublethal dose of native toxin. Significant reductions in toxin-induced serum cytokine levels were observed in soy-mSEB- and E. coli-mSEB-immunized pigs compared to control animals. The reductions in SEB-induced cytokine responses were similar regardless of the immunogen used for vaccination. Surprisingly, however, some clinical symptoms, such as prostration, lethargy, emesis, and/or diarrhea, were still observed in all immunized animals. These studies demonstrate the potential for soybean-derived proteins as a platform technology for sustainable vaccine manufacturing and the usefulness of a sublethal challenge model in pigs for evaluating the efficacy of potential SEB vaccine candidates.

Opsonic and protective properties of antibodies raised to conjugate vaccines targeting six Staphylococcus aureus antigens.

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections for which a vaccine is greatly desired. Antigens found on the S. aureus outer surface include the capsular polysaccharides (CP) of serotype 5 (CP5) or 8 (CP8) and/or a second antigen, a β-(1→6)-polymer of N-acetyl-D-glucosamine (PNAG). Antibodies specific for either CP or PNAG antigens have excellent in vitro opsonic killing activity (OPKA), but when mixed together have potent interference in OPKA and murine protection. To ascertain if this interference could be abrogated by using a synthetic non-acetylated oligosaccharide fragment of PNAG, 9GlcNH2, in place of chemically partially deacetylated PNAG, three conjugate vaccines consisting of 9GlcNH2 conjugated to a non-toxic mutant of alpha-hemolysin (Hla H35L), CP5 conjugated to clumping factor B (ClfB), or CP8 conjugated to iron-surface determinant B (IsdB) were used separately to immunize rabbits. Opsonic antibodies mediating killing of multiple S. aureus strains were elicited for all three vaccines and showed carbohydrate antigen-specific reductions in the tissue bacterial burdens in animal models of S. aureus skin abscesses, pneumonia, and nasal colonization. Carrier-protein specific immunity was also shown to be effective in reducing bacterial levels in infected lungs and in nasal colonization. However, use of synthetic 9GlcNH2 to induce antibody to PNAG did not overcome the interference in OPKA engendered when these were combined with antibody to either CP5 or CP8. Whereas each individual vaccine showed efficacy, combining antisera to CP antigens and PNAG still abrogated individual OPKA activities, indicating difficulty in achieving a multi-valent vaccine targeting both the CP and PNAG antigens.

The Adjuvant LT-K63 Can Restore Delayed Maturation of Follicular Dendritic Cells and Poor Persistence of Both Protein- and Polysaccharide-Specific Antibody-Secreting Cells in Neonatal Mice

Ab responses in early life are low and short-lived; therefore, induction of protective immunity requires repeated vaccinations. One of the major limitations in early-life immunity is delayed maturation of follicular dendritic cells (FDCs), which play a central role in mediating the germinal center (GC) reaction leading to production of Ab-secreting cells (AbSCs). We assessed whether a nontoxic mutant of Escherichia coli heat-labile enterotoxin (LT-K63) and CpG1826 as model adjuvants could accelerate FDC maturation and immune response in neonatal mice, using a pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (Pnc1-TT) as a model vaccine. In neonatal NMRI mice, a single dose of Pnc1-TT coadministered with LT-K63 enhanced Pnc1-TT-induced GC reaction. In contrast, CpG1826 had no effect. Accordingly, LT-K63, but not CpG1826, accelerated the maturation of FDC networks, detected by FDC-M2(+) staining, characteristic for adult-like FDCs. This coincided with migration of MOMA-1(+) macrophages into the GCs that can enhance GC reaction and B cell activation. The FDC-M2(+) FDC networks colocalized with enhanced expression of TNF-α, which is critical for the maintenance of mature FDCs and is poorly expressed in neonates. The accelerated maturation of FDC networks correlated with increased frequency and prolonged persistence of polysaccharide- and protein-specific IgG(+) AbSCs in spleen and bone marrow. Our data show for the first time, to our knowledge, that an adjuvant (LT-K63) can overcome delayed maturation of FDCs in neonates, enhance the GC reaction, and prolong the persistence of vaccine-specific AbSCs in the BM. These properties are attractive for parenteral vaccination in early life.

Immunization with the conjugate vaccine Vi-CRM197 against Salmonella Typhi induces Vi-specific mucosal and systemic immune responses in mice

Typhoid fever is a public health problem, especially among young children in developing countries. To address this need, a glycoconjugate vaccine Vi-CRM197, composed of the polysaccharide antigen Vi covalently conjugated to the non-toxic mutant of diphtheria toxin CRM197, is under development. Here, we assessed the antibody and cellular responses, both local and systemic, following subcutaneous injection of Vi-CRM197. The glycoconjugate elicited Vi-specific serum IgG titers significantly higher than unconjugated Vi, with prevalence of IgG1 that persisted for at least 60 days after immunization. Vi-specific IgG, but not IgA, were present in intestinal washes. Lymphocytes proliferation after restimulation with Vi-CRM197 was observed in spleen and mesenteric lymph nodes. These data confirm the immunogenicity of Vi-CRM197 and demonstrate that the vaccine-specific antibody and cellular immune responses are present also in the intestinal tract, thus strengthening the suitability of Vi-CRM197 as a promising candidate vaccine against Salmonella Typhi.

Effects of CRM197, a specific inhibitor of HB-EGF, in oral cancer

CRM197, a nontoxic mutant of diphtheria toxin, is a specific inhibitor of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), which belongs to the EGF family that has been implicated in the increased progression, proliferation, and metastasis of oral cancer. In this study, we analyzed the antitumor effects of CRM197, which represent possible chemotherapeutic agents for oral cancer. In the experiment, we used the oral squamous cell carcinoma cell lines HSC3 and SAS. Cells treated with CRM197 were analyzed based on cell viability, MTT assay, invasion assay, Western blot, and zymography. HSC3 cells were injected subcutaneously into female BALB/c nu/nu mice at 5 weeks of age. CRM197 and/or CDDP were injected intraperitoneally into tumor-bearing mice, and tumor volume was measured over time. HB-EGF expression in HSC3 and SAS cells treated with CRM197 was significantly reduced and cell proliferation was inhibited. The invasiveness of CRM197-treated cells was relatively low. MMP-9 and VEGF were suppressed in HSC3 treated with CRM197 on zymography and Western blot. Further, tumor growth in xenografted mice was suppressed by CRM197 or CDDP at 1 mg/kg/day. Also, the coadministration of CDDP and CRM197 at 1 mg/kg/day completely inhibited tumor formation. These results suggest that HB-EGF is a target for oral cancer and that CRM197 is effective in oral cancer therapy.

A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast

BackgroundThe cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified. MethodsAtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined. ResultsAtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA∆ (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA∆ was confirmed by co-immunoprecipitation analysis. ConclusionThe plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates. General significanceOur studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD.

Regulation of cellular growth, apoptosis, and Akt activity in human U251 glioma cells by a combination of cisplatin with CRM197

The aberrantly activated antiapoptotic phospatidyl-3-inositol-kinase (PI3K)/Akt signaling induced by cisplatin limits the effectiveness of chemotherapy; inhibition of this pathway may augment the sensitivity of tumor cells to cisplatin-induced toxicity and promote apoptosis. Cross-reacting material 197 (CRM197), the nontoxic mutant of diphtheria toxin, could act as an heparin-binding epidermal growth factor inhibitor and has been shown to have some anticancer effects, but the effect of CRM197 on glioma cells remains unclear. The aim of this study was to investigate the effects of a combination of cisplatin with CRM197 on the growth and apoptosis of human U251 glioma cells and the possible mechanism. In this study, we demonstrated that cisplatin or CRM197 induced a dose-dependent growth inhibition in U251 cells, but cisplatin at 5 µg/ml and CRM197 at 1 µg/ml did not affect the viability of human astrocytes. Cisplatin induced a time-dependent growth inhibition in U251 cells, whereas the growth-inhibitory effects induced by CRM197 alone or combined with cisplatin reached a peak at 24 h after treatment. Compared with the administration of cisplatin or CRM197 alone, CRM197 combined with cisplatin significantly enhanced U251 cell growth inhibition and apoptosis. Cisplatin induced sustained activation of Akt, whereas CRM197 markedly suppressed the Akt phosphorylation induced by cisplatin. The effects of growth inhibition and apoptosis were markedly enhanced after a combination of cisplatin with CRM197 plus the PI3K inhibitor LY294002 or wortmannin. Therefore, CRM197 combined with cisplatin could enhance growth inhibition and apoptosis of glioma cells by inhibiting the cisplatin-induced PI3K/Akt pathway.

Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi

A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM(197) via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammonium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA(+) counter ion with Na(+). The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials.

Increasing oxygen radicals and water temperature select for toxic Microcystis sp.

Pronounced rises in frequency of toxic cyanobacterial blooms are recently observed worldwide, particularly when temperatures increase. Different strains of cyanobacterial species vary in their potential to produce toxins but driving forces are still obscure. Our study examines effects of hydrogen peroxide on toxic and non-toxic (including a non-toxic mutant) strains of M. aeruginosa. Here we show that hydrogen peroxide diminishes chlorophyll a content and growth of cyanobacteria and that this reduction is significantly lower for toxic than for non-toxic strains. This indicates that microcystins protect from detrimental effects of oxygen radicals. Incubation of toxic and non-toxic strains of M. aeruginosa with other bacteria or without (axenic) at three temperatures (20, 26 and 32°C) reveals a shift toward toxic strains at higher temperatures. In parallel to increases in abundance of toxic (i.e. toxin gene possessing) strains and their actual toxin expression, concentrations of microcystins rise with temperature, when amounts of radicals are expected to be enhanced. Field samples from three continents support the influence of radicals and temperature on toxic potential of M. aeruginosa. Our results imply that global warming will significantly increase toxic potential and toxicity of cyanobacterial blooms which has strong implications for socio-economical assessments of global change.

Critical appraisal of a quadrivalent CRM197 conjugate vaccine against meningococcal serogroups A, C W-135 and Y (Menveo®) in the context of treatment and prevention of invasive disease

Worldwide, invasive meningococcal disease affects about 500,000 people annually. Case fatality in developed countries averages 10%, and higher rates are reported in less prosperous regions. According to the World Health Organization, the most important pathogenic serogroups are A, B, C, W-135, X, and Y. Clinical features of invasive meningococcal disease make diagnosis and management difficult. Antibiotic measures are recommended for prophylaxis after exposure and for treatment of invasive meningococcal disease cases; however, resistant strains may be emerging. Vaccines are generally regarded as the best preventative measure for invasive meningococcal disease. Polysaccharide vaccines against serogroups A, C, W-135, and Y using protein conjugation technology have clear advantages over older plain polysaccharide formulations without a protein component. The first quadrivalent meningococcal conjugate vaccine (MenACWY-D) was licensed in the US in 2005. More recently, MenACWY-CRM (Menveo®) was licensed in Europe, the US, the Middle East, and Latin America. MenACWY-CRM uses cross-reactive material 197, a nontoxic mutant of diphtheria toxin, as the carrier protein. MenACWY-CRM offers robust immunogenicity in all age groups, with a tolerability profile similar to that of a plain polysaccharide vaccine. Given its potential for protecting persons from infancy to old age, MenACWY-CRM offers the opportunity to protect broad populations against invasive meningococcal disease. The most optimal strategy for use of the vaccine has to be assessed country by country on the basis of local epidemiology, individual health care systems, and need.

Gene Expression Profile of Human Colon Cancer Cells Treated with Cross-Reacting Material 197, a Diphtheria Toxin Non-Toxic Mutant

Cross-Reacting Material 197 (CRM197) is a diphtheria toxin non-toxic mutant that has shown antitumor activity in mice and humans. It is still unclear whether this anti-tumorigenic effect depends on its strong inflammatory-immunological property, its ability to inhibit heparin-binding epidermal growth factor (HB-EGF), or even its possible weak toxicity. CRM197 is utilized as a specific inhibitor of HB-EGF that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. In this study we evaluate the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible influence on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment (MTT modified assay), or changes in cell cycle distribution (flow cytometry), in EGFR localization, phospho-EGFR detected signals (immunohistochemistry) or in morphology (scanning electron microscopy, SEM) they show a change in the gene expression profile by microarray analysis (cDNA microarray SS-H19k8). The overexpression of genes like protein phosphatase 2, catalytic subunit, alpha isozyme (PPP2CA), guanine nucleotide-binding protein G subunit alpha-1(GNAI1) and butyrophilin, subfamily 2, member A1 (BTN2A1) has been confirmed with real-time-qPCR. This is the first study where the CRM197 treatment on HT-29 shows a possible scarce implication of endogenous HB-EGF on EGFR expression and cancer cell development. At the same time, our results show the alteration of a specific and selected number of genes.

Sublingually administered Bacillus subtilis cells expressing tetanus toxin C fragment induce protective systemic and mucosal antibodies against tetanus toxin in mice

Sublingual (SL) immunization against infectious agents or bacterial toxins is not a common route for antigen delivery. However, in our continued search for a needle-free platform for vaccine administration, we evaluated the efficacy of SL immunization with Bacillus subtilis engineered to express tetanus toxin fragment C (TTFC). We compared the results obtained with those for intranasal (IN) immunization with the same vaccine, which we recently reported to induce complete protection in mice against a 2×LD100 challenge of tetanus toxin (Lee et al., Vaccine 28:6658–65). Groups of animals received 3–4 immunizations of 109B. subtilis vegetative cells expressing TTFC given IN or SL. Other SL immunized groups received either purified recombinant TTFC (rTTFC) or B. subtilis placebo. A non-toxic mutant of Escherichia coli heat labile enterotoxin (mLT) was included as adjuvant in some of the studies. Mice inoculated by either IN or SL administration developed protective IgG antibodies against tetanus toxin challenge. Similar of higher IgA levels in saliva, vaginal wash and feces were detected in animals immunized SL with B. subtilis cells expressing TTFC compared with IN-immunized mice or mice immunized SL with rTTFC. SL immunization promoted a mixed Th1/Th2 response, based on cytokine analysis (IL-2, IL-4, IL-10 and INFγ). Antigen-stimulated tissues (lung, intestine, spleen and lymph nodes) revealed a dramatic increase in the density of MHC class II+ expressing cells compared to all other groups. The antibody response to TTFC was superior when the adjuvant mLT was excluded from IN and SL immunizations. However, SL administration of mLT induced strong systemic and mucosal antibody responses, indicating that successful use of this route of immunization is not specific to tetanus toxin. We conclude that SL immunization is a promising, effective, safe, non-invasive and convenient method for mucosal delivery of B. subtilis cells expressing tetanus vaccine and, potentially, other immunogens. SL immunization appears to induce both systemic and mucosal immune responses.

Essential Role for Retinoic Acid in the Promotion of CD4+ T Cell Effector Responses via Retinoic Acid Receptor Alpha

Vitamin A and its metabolite, retinoic acid (RA) are implicated in the regulation of immune homeostasis via the peripheral induction of regulatory Tcells. Here we showed RA was also required to elicit proinflammatory CD4(+) helper Tcell responses to infection and mucosal vaccination. Retinoic acid receptor alpha (RARα) was the critical mediator of these effects. Antagonism of RAR signaling and deficiency in RARα (Rara(-/-)) resulted in a cell-autonomous CD4(+) Tcell activation defect, which impaired intermediate signaling events, including calcium mobilization. Altogether, these findings reveal a fundamental role for the RA-RARα axis in the development of both regulatory and inflammatory arms of adaptive immunity and establish nutritional status as a broad regulator of adaptive Tcell responses.