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Objectives: To develop and practically test high-precision femtosecond laser ablation models for dental hard tissue that are useful for detailed planning of automated laser dental restorative treatment., Methods: Analytical models are proposed, derived, and demonstrated for practical calculation of ablation rates, ablation efficiency and ablated morphology of human dental enamel and dentin using femtosecond lasers. The models assume an effective optical attenuation coefficient for the irradiated material. To achieve ablation, it is necessary for the local energy density of the attenuated pulse in the hard tissue to surpass a predefined threshold that signifies the minimum energy density required for material ionization. A 1029 nm, 40 W carbide 275 fs laser was used to ablate sliced adult human teeth and generate the data necessary for testing the models. The volume of material removed, and the shape of the ablated channel were measured using optical profilometry., Results: The models fit with the measured ablation efficiency curve against laser fluence for both enamel and dentin, correctly capturing the fluence for optimum ablation and the volume of ablated material per pulse. The detailed shapes of a 400-micrometer wide channel and a single-pulse width channel are accurately predicted using the superposition of the analytical result for a single pulse., Conclusions: The findings have value for planning automated dental restorative treatment using femtosecond lasers. The measurements and analysis give estimates of the optical properties of enamel and dentin irradiated with an infrared femtosecond laser at above-threshold fluence and the proposed models give insight into the physics of femtosecond laser processing of dental hard tissue., (© 2024 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.)

We investigated the effect of femtosecond (fs) laser ablation of enamel and dentin for different pulse wavelengths: infrared (1030 nm), green (515 nm), and ultra-violet (343 nm) and for different pulse separations to determine the optimal irradiation conditions for the precise removal of dental hard tissues with the absence of structural and compositional damage. The ablation rates and efficiencies were established for all three laser wavelengths for both enamel and dentin at room temperature without using any irrigation or cooling system, and the surfaces were assessed with optical and scanning electron microscopy, optical profilometry, and Raman spectroscopy. We demonstrated that 515 nm fs irradiation provides the highest rate and efficiency for ablation, followed by infrared. Finally, we explored the temperature variations inside the dental pulp during the laser procedures for all three wavelengths and showed that the maximum increase at the optimum conditions for both infrared and green irradiations was 5.5 °C, within the acceptable limit of temperature increase during conventional dental treatments. Ultra-violet irradiation significantly increased the internal temperature of the teeth, well above the acceptable limit, and caused severe damage to tooth structures. Thus, ultra-violet is not a compatible laser wavelength for femtosecond teeth ablation., (© 2023. The Author(s).)

Streptococcus pneumoniae (Spn) remains a major cause of global mortality, with extensive antigenic diversity between capsular serotypes that poses an ongoing challenge for vaccine development. Widespread use of pneumococcal conjugate vaccines (PCVs) targeting Spn capsules has greatly reduced infections by vaccine-included serotypes but has led to increased infections by nonincluded serotypes. To date, high cost of PCVs has also limited their usefulness in low-income regions where disease burdens are highest. To overcome these limitations, serotype-independent vaccines are being actively researched. We have developed a whole-cell gamma-irradiated Spn vaccine (termed Gamma-PN) providing serotype-independent protection. We demonstrate that Gamma-PN immunization of mice or rabbits via the clinically relevant intramuscular route induces protein-specific antibodies able to bind numerous nonvaccine encapsulated serotypes, which mediate opsonophagocytic killing and protection against lethal challenges. Gamma-PN induced comparable or superior opsonophagocytic killing assay (OPKA) responses in rabbits to the licensed Prevnar 13 vaccine (PCV13) for vaccine-included serotypes, and a superior response to nonincluded serotypes, including emergent 22F and 35B. Additionally, despite a lower observed reactogenicity, administration of Gamma-PN without adjuvant resulted in higher OPKA responses and improved protection compared to adjuvanted Gamma-PN. To our knowledge, this has not been demonstrated previously for a whole-inactivated Spn vaccine. Eliminating the requirement for adjuvant comes with numerous benefits for clinical applications of this vaccine and poses interesting questions for the inclusion of adjuvant in similar vaccines in development. IMPORTANCE The target pathogen of this study, Streptococcus pneumoniae, kills over 300,000 children <5 years of age every single year, and is the leading cause of pneumonia-associated mortality globally. While the capsular polysaccharide (CPS)-based vaccine Prevnar13 prevents serious illness caused by 13 serotypes, ongoing Prevnar13 use has driven the emergence of nonincluded serotypes as major causes of infection and disease. To overcome this issue, we have developed a next-generation pneumococcal vaccine conferring serotype-independent protection. This vaccine shows equivalent or superior efficacy to Prevnar13, and performance was heightened when our vaccine was administered with no adjuvant. These findings should be considered for similar vaccines in development, as the benefit of adjuvant is often assumed and its automatic inclusion may be limiting product efficacy, resulting in potential abandonment of viable vaccine candidates, or prolonging their time to clinic.

High fluence focused femtosecond laser pulses were used to perform fast, high precision and minimally damaging cavity cutting of teeth at room temperature without using any irrigation or cooling system. The optimal ablation rates were established for both enamel and dentin, and the surfaces were assessed with optical and scanning electron microscopy, Raman spectroscopy and optical profilometry. No chemical change in the composition of enamel and dentin was observed. We explored temperature variations inside the dental pulp during the laser procedure and showed the maximum increase was 5.5°C, within the acceptable limit of temperature increase during conventional dental treatments., Competing Interests: Authors O.Z., A.H., T.H. and L.J.W. are affiliated with or employed by Dentroid (Emudent Technologies Pty Ltd). All other authors declare no competing financial interests., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Influenza A virus presents a constant pandemic threat due to the mutagenic nature of the virus and the inadequacy of current vaccines to protect against emerging strains. We have developed a whole-inactivated influenza vaccine using γ-irradiation (γ-Flu) that can protect against both vaccine-included strains as well as emerging pandemic strains. γ-irradiation is a widely used inactivation method and several γ-irradiated vaccines are currently in clinical or pre-clinical testing. To enhance vaccine efficacy, irradiation conditions should be carefully considered, particularly irradiation temperature. Specifically, while more damage to virus structure is expected when using higher irradiation temperatures, reduced radiation doses will be required to achieve sterility. In this study, we compared immunogenicity of γ-Flu irradiated at room temperature, chilled on ice or frozen on dry ice using different doses of γ-irradiation to meet internationally accepted sterility assurance levels. We found that, when irradiating at sterilising doses, the structural integrity and vaccine efficacy were well maintained in all preparations regardless of irradiation temperature. In fact, using a higher temperature and lower radiation dose appeared to induce higher neutralising antibody responses and more effective cytotoxic T cell responses. This outcome is expected to simplify irradiation protocols for manufacturing of highly effective irradiated vaccines., Competing Interests: MA is head of the Vaccine Research Group at the University of Adelaide and the Chief Scientific Officer of Gamma Vaccines Pty Ltd and TH is the Executive Chairman of Gamma Vaccines Pty Ltd. This does not alter adherence to policies on sharing data and materials. Gamma Vaccines Pty Ltd has no role in the study design, data collection and analysis, decision to publish, and preparation of the manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Singleton, Gates, David, Hirst, Davies and Alsharifi.)

In recent years there has been increasing advocacy for highly immunogenic gamma-irradiated vaccines, several of which are currently in clinical or pre-clinical trials. Importantly, various methods of mathematical modelling and sterility testing are employed to ensure sterility. However, these methods are designed for materials with a low bioburden, such as food and pharmaceuticals. Consequently, current methods may not be reliable or applicable to estimate the irradiation dose required to sterilize microbiological preparations for vaccine purposes, where bioburden is deliberately high. In this study we investigated the applicability of current methods to calculate the sterilizing doses for different microbes. We generated inactivation curves that demonstrate single-hit and multiple-hit kinetics under different irradiation temperatures for high-titre preparations of pathogens with different genomic structures. Our data demonstrate that inactivation of viruses such as Influenza A virus, Zika virus, Semliki Forest virus and Newcastle Disease virus show single-hit kinetics following exposure to gamma-irradiation. In contrast, rotavirus inactivation shows multiple-hit kinetics and the sterilizing dose could not be calculated using current mathematical methods. Similarly, Streptococcus pneumoniae demonstrates multiple-hit kinetics. These variations in killing curves reveal an important gap in current mathematical formulae to determine sterility assurance levels. Here we propose a simple method to calculate the irradiation dose required for a single log10 reduction in bioburden (D10) value and sterilizing doses, incorporating both single- and multiple-hit kinetics, and taking into account the possible existence of a resistance shoulder for some pathogens following exposure to gamma-irradiation., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Existing capsular polysaccharide-based vaccines against pneumococcal disease are highly effective against vaccine-included serotypes, but they are unable to combat serotype replacement. We have developed a novel pneumococcal vaccine that confers serotype-independent protection, and could therefore constitute a "universal" vaccine formulation. This preparation is comprised of whole un-encapsulated pneumococci inactivated with gamma irradiation (γ-PN), and we have previously reported induction of cross-reactive immunity after nonadjuvanted intranasal vaccination. To further enhance vaccine immunogenicity and safety, we modified the pneumococcal vaccine strain to induce a stressed state during growth. Specifically, the substrate binding component of the psaBCA operon for manganese import was mutated to create a pneumococcal surface antigen A (psaA) defective vaccine strain. psaA mutation severely attenuated the growth of the vaccine strain in vitro without negatively affecting pneumococcal morphology, thereby enhancing vaccine safety. In addition, antibodies raised against vaccine preparations based on the modified strain [γ-PN(ΔPsaA)] showed more diversified reactivity to wild-type pneumococcal challenge strains compared to those induced by the original formulation. The modified vaccine also induced comparable protective T H 17 responses in the lung, and conferred greater protection against lethal heterologous pneumococcal challenge. Overall, the current study demonstrates successful refinement of a serotype-independent pneumococcal vaccine candidate to enhance safety and immunogenicity., (© 2019 Australian and New Zealand Society for Immunology Inc.)

The upper respiratory tract is continuously exposed to a vast array of potentially pathogenic viruses and bacteria. Influenza A virus (IAV) has particular synergism with the commensal bacterium Streptococcus pneumoniae in this niche, and co-infection exacerbates pathogenicity and causes significant mortality. However, it is not known whether this synergism is associated with a direct interaction between the two pathogens. We have previously reported that co-administration of a whole-inactivated IAV vaccine (γ-Flu) with a whole-inactivated pneumococcal vaccine (γ-PN) enhances pneumococcal-specific responses. In this study, we show that mucosal co-administration of γ-Flu and γ-PN similarly augments IAV-specific immunity, particularly tissue-resident memory cell responses in the lung. In addition, our in vitro analysis revealed that S. pneumoniae directly interacts with both γ-Flu and with live IAV, facilitating increased uptake by macrophages as well as increased infection of epithelial cells by IAV. These observations provide an additional explanation for the synergistic pathogenicity of IAV and S. pneumoniae, as well as heralding the prospect of exploiting the phenomenon to develop better vaccine strategies for both pathogens.

Rotavirus (RV) causes significant morbidity and mortality in developing countries, where children and infants are highly susceptible to severe disease symptoms. While live attenuated vaccines are available, reduced vaccine efficacy in developing countries illustrates the need for highly immunogenic alternative vaccines. Here, we studied the possible inactivation of RV using gamma(γ)-irradiation, and assessed the sterility and immunogenicity of γ-irradiated RV (γ-RV) as a novel vaccine candidate. Interestingly, the inactivation curve of RV did not show a log-linear regression following exposure to increased doses of γ-rays, and consequently the radiation dose required to achieve the internationally accepted Sterility Assurance Level could not be calculated. Nonetheless, we performed sterility testing based on serial passages of γ-RV, and our data clearly illustrate the lack of infectivity of γ-RV preparations irradiated with 50 kGy. In addition, we tested the immunogenicity of 50 kGy γ-RV in mice and our data illustrate the induction of strong RV-specific neutralising antibody responses following administration of γ-RV without using adjuvant. Therefore, whilst γ-RV may not constitute a replacement for current RV vaccines, this study represents a proof-of-concept that γ-irradiation can be applied to inactivate RV for vaccine purposes. Further investigation will be required to address whether γ-irradiation can be applied to improve safety and efficacy of existing live attenuated vaccines., Competing Interests: Gamma-irradiation performed at ANSTO (Lucas Heights, New South Wales), supported by an Australian Institute of Nuclear Science and Engineering (AINSE) Research Award (ALNGRA15517; awarded to MA). The authors have no conflicting financial interests. MA is head of the vaccine research group at the University of Adelaide and the Chief Scientific Officer of Gamma Vaccines Pty Ltd. TRH is an affiliated professor at the University of Adelaide and the Executive Chairman of Gamma Vaccines Pty Ltd. This does not alter adherence to policies on sharing data and materials. Gamma Vaccines Pty Ltd has no role in the study design, data collection and analysis, decision to publish, and preparation of the manuscript.

Gamma-irradiation, particularly an irradiation dose of 50kGy, has been utilised widely to sterilise highly pathogenic agents such as Ebola, Marburg Virus, and Avian Influenza H5N1. We have reported previously that intranasal vaccination with a gamma-irradiated Influenza A virus vaccine (γ-Flu) results in cross-protective immunity. Considering the possible inclusion of highly pathogenic Influenza strains in future clinical development of γ-Flu, an irradiation dose of 50kGy may be used to enhance vaccine safety beyond the internationally accepted Sterility Assurance Level (SAL). Thus, we investigated the effect of irradiation conditions, including high irradiation doses, on the immunogenicity of γ-Flu. Our data confirm that irradiation at low temperatures (using dry-ice) is associated with reduced damage to viral structure compared with irradiation at room temperature. In addition, a single intranasal vaccination with γ-Flu irradiated on dry-ice with either 25 or 50kGy induced seroconversion and provided complete protection against lethal Influenza A challenge. Considering that low temperature is expected to reduce the protein damage associated with exposure to high irradiation doses, we titrated the vaccine dose to verify the efficacy of 50kGy γ-Flu. Our data demonstrate that exposure to 50kGy on dry-ice is associated with limited effect on vaccine immunogenicity, apparent only when using very low vaccine doses. Overall, our data highlight the immunogenicity of influenza virus irradiated at 50kGy for induction of high titre antibody and cytotoxic T-cell responses. This suggests these conditions are suitable for development of γ-Flu vaccines based on highly pathogenic Influenza A viruses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Streptococcus pneumoniae and influenza are the world's foremost bacterial and viral respiratory pathogens. We have previously described a γ-irradiated influenza A virus (γ-FLU) vaccine that provides cross-protective immunity against heterosubtypic infections. More recently, we reported a novel non-adjuvanted γ-irradiated S pneumoniae (γ-PN) vaccine that elicits serotype-independent protection. Considering the clinical synergism of both pathogens, combination of a serotype-independent pneumococcal vaccine with a broad-spectrum influenza vaccine to protect against both infections would have a considerable clinical impact. In the present study, we co-immunized C57BL/6 mice intranasally (IN) with a mixture of γ-PN (whole inactivated cells) and γ-FLU (whole inactivated virions) and examined protective efficacy. Co-immunization enhanced γ-PN vaccine efficacy against virulent pneumococcal challenge, which was dependent on CD4 + T-cell responses. In contrast, vaccination with γ-PN alone, co-immunization enhanced pneumococcal-specific effector T-helper 17 cell (Th17) and Th1 memory cell, promoted development of CD4 + tissue-resident memory (TRM) cells and enhanced Pneumococcus-specific antibody responses. Furthermore, co-immunization elicited significant protection against lethal influenza challenge, as well as against co-infection with both influenza and S pneumoniae. This is the first report showing the synergistic effect of combining whole cell and whole virion vaccines to both S pneumoniae and influenza as a single vaccine to protect against individual and co-infection, without compromising pathogen-specific immunity., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Generating a pneumococcal vaccine that is serotype independent and cost effective remains a global challenge. γ-Irradiation has been used widely to sterilize biological products. It can also be utilized as an inactivation technique to generate whole-cell bacterial and viral vaccines with minimal impact on pathogen structure and antigenic determinants. In the present study, we utilized γ-irradiation to inactivate an un-encapsulated Streptococcus pneumoniae strain Rx1 with an unmarked deletion of the autolysin gene lytA and with the pneumolysin gene ply replaced with an allele encoding a non-toxic pneumolysoid (PdT) (designated γ-PN vaccine). Intranasal vaccination of C57BL/6 mice with γ-PN was shown to elicit serotype-independent protection in lethal challenge models of pneumococcal pneumonia and sepsis. Vaccine efficacy was shown to be reliant on B-cells and interleukin (IL)-17A responses. Interestingly, immunization promoted IL-17 production by innate cells not T helper 17 (Th17) cells. These data are the first to report the development of a non-adjuvanted intranasal γ-irradiated pneumococcal vaccine that generates effective serotype-independent protection, which is mediated by both humoral and innate IL-17 responses., (© 2016 Authors; published by Portland Press Limited.)

Escherichia coli's heat-labile enterotoxin (Etx) and its non-toxic B subunit (EtxB) have been characterized as adjuvants capable of enhancing T cell responses to co-administered antigen. Here, we investigate the direct effect of intravenously administered EtxB on the size of the dendritic and myeloid cell populations in spleen. EtxB treatment appears to enhance the development and turnover of dendritic and myeloid cells from precursors within the spleen. EtxB treatment also gives a dendritic cell (DC) population with higher viability and lower activation status based on the reduced expression of MHC-II, CD80 and CD86. In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide. In in vitro bone marrow cultures, EtxB treatment was also found to enhance the development of DC from precursors dependent on Flt3L. In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c(+) DC. In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen-presenting cells on encounter with T cells., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Endowing mucosal vaccines with ligands that target antigen to mucosal lymphoid tissues may improve immunization efficacy provided that the ligands withstand the proteolytic environment of the gastro-intestinal tract until they reach their destination. Our aim was to investigate whether and how three renowned ligands - Ulex europaeus agglutinin I and the B subunits of cholera toxin and E. coli heat-labile enterotoxin - master this challenge. We assessed the digestive power of natural murine intestinal fluid (natIF) using assays for trypsin, chymotrypsin and pancreatic elastase along with a test for nonspecific proteolysis. The natIF was compared with simulated murine intestinal fluid (simIF) that resembled the trypsin, chymotrypsin and elastase activities of its natural counterpart but lacked or contained albumins as additional protease substrates. The ligands were exposed to the digestive fluids and degradation was determined. The studies revealed that (i) the three pancreatic endoproteases constitute only one third of the total protease activity of natIF and (ii) the ligands resist proteolysis in natIF and protein-enriched simIF over 3 h but (iii) are partially destroyed in simIF that lacks additional protease substrate. We assume that the proteins of natIF are preferred substrates for the intestinal proteases and thus can protect vaccine-targeting ligands from destruction.

Previously we have described studies on in vitro pentamer assembly of Escherichia coli (E. coli) derived heat-labile enterotoxin B subunit (EtxB) using conventional monoclonal antibodies (Amin et al., JBC 1995: 270, 20143-50 and Chung et al., JBC 2006: 281, 39465-70). To extend these studies further we have used phage-display to select single-chain Fragment variable (scFv) antibodies against different forms of the B-subunit. Two clones exhibiting strong and differential binding were chosen for detailed characterization. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions and a nonsense mutation present in one of these (scFv-B1.3.9) was corrected. Binding analysis showed that scFv-B1.3.9 bound in ELISA to both heat-denatured monomeric B-subunits (EtxB1) and also displayed cross-reactivity towards pentameric EtxB (EtxB5), although there was no reactivity towards monoganglioside (GM1) captured EtxB5. Another antibody (scFv-B5.2.14) had a different reactivity profile and, in ELISA, bound only to EtxB5 but not to EtxB1 or to EtxB5 captured via GM1. Surprisingly, in competition experiments, the assembled pentameric B-subunit inhibited binding of scFv-B5.2.14 to immobilized EtxB5 only weakly, whereas reduced, but not oxidized, monomeric EtxB1 was an efficient competitor. These results clearly demonstrate that B1.3.9 and B5.2.14 have different specificities for cryptic epitopes not accessible in the fully assembled GM1 bound pentameric form of EtxB. Taken together our results show that we were able to successfully isolate and characterize recombinant scFvs that differentially recognize diverse denatured forms or assembly intermediates of the heat-labile enterotoxin B subunit of E. coli.

A ganglioside GM1 probe bearing a dark-red fluorescent dye at the sphingosine moiety of the molecule was prepared by a convenient one-pot synthesis. The labeled GM1 permitted the detection of the natural ganglioside GM1 ligand Escherichia coli heat-labile enterotoxin subunit B (EtxB) in picomole quantities on a solid support. When an epitope mapping of several ganglioside binding proteins and protein fragments was performed by screening a cellulose membrane-bound synthetic library of 64 16mer peptides with the new probe, several peptides displaying ganglioside GM1 affinity could be identified. We consider the labeled glycolipid described herein a versatile tool for manifold biochemical investigations.

Heat-labile enterotoxin (Etx) produced by certain strains of Escherichia coli is a major virulence factor related to cholera toxin. Both are hexameric proteins comprising one A-subunit and five B-subunits. The pentameric B-subunit of E. coli has a high affinity for G(M1)-ganglioside receptors on gut epithelial cells and is directly responsible for toxin entry. The pentameric B-subunit (EtxB(5)) is an exceptionally stable protein, being able to maintain its quaternary structure over a wide pH range (2.0- 11.0). However, little is known about the formation of the pentameric structure (EtxB(5)) from newly synthesized B-subunit monomers (EtxB(1)). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB(1) (Amin, T., Larkins, A., James, R. F. L., and Hirst, T. R. (1995) J. Biol. Chem. 270, 20143-20150). Here we also describe a mAb (LDS16) with exquisite specificity for pentameric EtxB. In this study, we have used these two mAbs, in combination, to probe the in vitro assembly of EtxB(5) from EtxB(1). EtxB pentamers disassemble in highly acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH. Using this in vitro assembly model, it was found that at a molar ratio of 1:1; LDS47:EtxB, 50% of reassembly was inhibited, and that this inhibition increased to 90% at a ratio of 2:1. These results infer that the N-terminal decapeptide region (APQSITELCS) defined by the LDS47 antibody is crucial for competent pentameric B-subunit assembly and stabilization.

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by impaired antibody production, which is in many instances secondary to defective T-cell function (T-CVID). We have previously identified a subset of patients with T-CVID characterized by defective T-cell receptor (TCR)-dependent protein tyrosine phosphorylation. In these patients, ZAP-70 fails to be recruited to the TCR as the result of impaired CD3zeta phosphorylation, which is, however, not dependent on defective Lck expression or activity. Here we show that neither Fyn nor CD45 is affected in these patients. On the other hand, T-CVID T cells show dramatic defects in the Vav/Rac pathway controlling F-actin dynamics. A significant deficiency in Vav protein was indeed observed; in 3 of 4 patients with T-CVID, it was associated with reduced VAV1 mRNA levels. The impairment in Vav expression correlated with defective F-actin reorganization in response to TCR/CD28 co-engagement. Furthermore, TCR/CD28-dependent up-regulation of lipid rafts at the cell surface, which requires F-actin dynamics, was impaired in these patients. The actin cytoskeleton defect could be reversed by reconstitution of Vav1 expression in the patients' T cells. Results demonstrate an essential role of Vav in human T cells and strongly suggest Vav insufficiency in T-CVID.

The B-subunit component of Escherichia coli heat-labile enterotoxin (EtxB), which binds to cell surface GM1 ganglioside receptors, was recently shown to be a highly effective vehicle for delivery of conjugated peptides into the major histocompatibility complex (MHC) class I pathway. In this study we have investigated the pathway of epitope delivery. The peptides used contained the epitope either located at the C terminus or with a C-terminal extension. Pretreatment of cells with cholesterol-disrupting agents blocked transport of EtxB conjugates to the Golgi/endoplasmic reticulum, but did not affect EtxB-mediated MHC class I presentation. Under these conditions, EtxB conjugates entered EEA1-positive early endosomes where peptides were cleaved and translocated into the cytosol. Endosome acidification was required for epitope presentation. Purified 20 S immunoproteasomes were able to generate the epitope from peptides in vitro, but 26 S proteasomes were not. Only presentation from the C-terminal extended peptide was proteasome-dependent in cells, and this was found to be significantly slower than presentation from peptides with the epitope at the C terminus. These results implicate the proteasome in the generation of the correct C terminus of the epitope and are consistent with proteasome-independent N-terminal trimming. Epitope presentation was blocked in a TAP-deficient cell line, providing further evidence that conjugated peptides enter the cytosol as well as demonstrating a requirement for the peptide transporter. Our findings demonstrate the utility of EtxB-mediated peptide delivery for rapid and efficient loading of MHC class I epitopes in several different cell types. Conjugated peptides are released from early endosomes into the cytosol where they gain access to proteasomes and TAP in the "classical" pathway of class I presentation.

The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule that acts both as an adjuvant and to stimulate immune deviation processes, resulting in the suppression of Th1-associated inflammatory responses. The ability of EtxB to alter immune reactivity is dependent on its ability to modulate immune cell function through binding to cell surface molecules, the principal receptor of which is the ubiquitous GM1-ganglioside. EtxB activates B cells and antigen-presenting cells and induces the selective apoptosis of murine CD8+ T cells. We postulated that these effects are mediated by the induction of intracellular signaling pathways following EtxB-receptor interaction. We have previously shown that CD8+ T-cell apoptosis induced by EtxB results from the activation of the transcription factor NF-kappaB and caspases. Here we report that while caspase activity is required for apoptosis, additional features of cell death are caspase independent. EtxB induces a rapid loss of mitochondrial membrane potential and cell viability that are unaffected by caspase inhibitors. In addition, our data suggest that these processes are independent of the activity of Bax and Bcl-2 but are mediated by nitric oxide synthase.

Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (alpha2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles.

Cholera toxin (Ctx) from Vibrio cholerae and its closely related homologue, heat-labile enterotoxin (Etx) from Escherichia coli have become superb tools for illuminating pathways of cellular trafficking and immune cell function. These bacterial protein toxins should be viewed as conglomerates of highly evolved, multi-functional elements equipped to engage the trafficking and signalling machineries of cells. Ctx and Etx are members of a larger family of A-B toxins of bacterial (and plant) origin that are comprised of structurally and functionally distinct enzymatically active A and receptor-binding B sub-units or domains. Intoxication of mammalian cells by Ctx and Etx involves B pentamer-mediated receptor binding and entry into a vesicular pathway, followed by translocation of the enzymatic A1 domain of the A sub-unit into the target cell cytosol, where covalent modification of intracellular targets leads to activation of adenylate cyclase and a sequence of events culminating in life-threatening diarrhoeal disease. Importantly, Ctx and Etx also have the capacity to induce a wide spectrum of remarkable immunological processes. With respect to the latter, it has been found that these toxins activate signalling pathways that modulate the immune system. This review explores the complexities of the cellular interactions that are engaged by these bacterial protein toxins, and highlights some of the new insights to have recently emerged., (Copyright 2004 Taylor and Francis Ltd.)

PC12 cells undergo neuritogenesis upon nerve growth factor (NGF) activation of the TrkA receptor, an effect mimicked by the ganglioside GM1 binding B-subunit of cholera toxin (CTB). Modulation of neuritogenesis by a GM1 ligand indicates a possible pathway for pathophysiological actions of neuropathy-associated anti-GM1 antibodies. Here we examine the ability of GM1 binding toxins and antibodies to induce neuritogenesis, using a PC12 neurite outgrowth assay. Cholera toxin (CT) and commercially prepared CTB (sCTB, contaminated with traces of the adenyl cyclase activating CT A-subunit) were highly neuritogenic. Recombinant cholera toxin B-subunit (rCTB, free from CTA) induced a much smaller effect, suggesting that the potent effects of sCTB are largely due to contaminating CTA. The recombinant GM1 binding B-subunit of Escherichia coli heat-labile enterotoxin (rETxB) exhibited no neuritogenic activity, whilst rETx holotoxin, which activates adenyl cyclase, was highly neuritogenic. Monoclonal anti-GM1 IgM antibodies from human neuropathy subjects induced small neuritogenic effects. These data indicate that GM1/ligand interaction does not necessarily lead to neuritogenesis and suggest that a specialisation of CTB, not shared by anti-GM1 antibodies or rETxB, is required to activate TrkA. Our data also indicate that antibodies are unlikely to exert major modulatory effects on TrkA activity in patients with anti-GM1 antibody-associated peripheral neuropathies.

Receptor-mediated drug targeting via nanoengineered particulate delivery systems is an emerging field. However, little is known about how such magic bullets should be assembled to yield optimal targeting efficiency. Here we investigated the influence of targeting ligand flexibility on binding of ligand-coated microparticles to cell surface receptors. Using the ganglioside G(M1)-binding B subunit of cholera toxin as ligand and fluorescent microparticles as a model delivery system, conjugates with different numbers of linkages between ligand and particle were prepared and tested for their efficiency to bind to live fibroblast monolayers. Our results show that multiple bonds between ligand and particle reduce the targeting rate by up to 50% compared to constructs where ligands are attached via single aliphatic chains. Thus, for maximum performance, targeted particulate drug delivery systems should be assembled such that ligands are attached via single sigma bonds only, allowing the ligand molecules to adopt an optimal binding conformation.

Receptor-binding by the B-subunit of Escherichia coli heat-labile enterotoxin (EtxB) induces apoptosis of peripheral CD8(+), but not CD4(+) T-cells. Given that peripheral CD8(+) and CD4(+) T cells arise from a common developmental pathway in the thymus, we investigated the effects of EtxB on different thymocyte populations. We show that the acquisition of sensitivity to EtxB-mediated cell death arises following transition of CD4(+)CD8(+) double positive cells into the CD4(-)CD8(+) pathway. Maturation of T cells into CD4(-)CD8(+) single positive cells is associated with upregulated expression of receptors for EtxB.

The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinant Escherichia coli heat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.

Epstein-Barr virus (EBV) is associated with a number of important human cancers, including nasopharyngeal carcinoma, gastric carcinoma, and Hodgkin's lymphoma. These tumors express a viral nuclear antigen, EBV nuclear antigen 1 (EBNA1), which cannot be presented to T cells in a major histocompatibility complex class I context, and the viral latent membrane proteins (LMPs). Although the LMPs are expressed in these tumors, no effective immune response is made. We report here that exposure to the cholera-like enterotoxin B subunit (EtxB) in EBV-infected lymphoblastoid cell lines (LCLs) enhances their susceptibility to killing by LMP-specific CD8(+) cytotoxic T lymphocytes (CTLs) in a HLA class I-restricted manner. CTL killing of LCLs is dramatically increased through both transporter-associated protein-dependent and -independent epitopes after EtxB treatment. The use of mutant B subunits revealed that the enhanced susceptibility of LCLs to CTL killing is dependent on the B subunit's interaction with GM(1) but not its signaling properties. These important findings could underpin the development of novel approaches to treating EBV-associated malignancies and may offer a general approach to increasing the presentation of other tumor and viral antigens.

The homopentameric B-subunit components of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) possess the capacity to enter mammalian cells and to activate cell-signaling events in leukocytes that modulate immune cell function. Both properties have been attributed to the ability of the B subunits to bind to GM1-ganglioside receptors, a ubiquitous glycosphingolipid found in the plasma membrane. Here we describe the properties of EtxB(H57S), a mutant B subunit with a His-->Ser substitution at position 57. The mutant was found to be severely defective in inducing leukocyte signaling, as shown by failure to (i) trigger caspase 3-mediated CD8(+)-T-cell apoptosis, (ii) activate nuclear translocation of NF-kappaB in Jurkat T cells, (iii) induce a potent anti-B-subunit response in mice, or (iv) serve as a mucosal adjuvant. However, its GM1 binding, cellular uptake, and delivery functions remained intact. This was further validated by the finding that EtxB(H57S) was as effective as EtxB in delivering a conjugated model class I epitope into the major histocompatibility complex class I pathway of a dendritic cell line. These observations imply that GM1 binding alone is not sufficient to trigger the signaling events responsible for the potent immunomodulatory properties of EtxB. Moreover, they demonstrate that its signaling properties play no role in EtxB uptake and trafficking. Thus, EtxB(H57S) represents a novel tool for evaluating the complex cellular interactions and signaling events occurring after receptor interaction, as well as offering an alternative means of delivering attached peptides in the absence of the potent immunomodulatory signals induced by wild-type B subunits.

In murine systems, the B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulator capable of suppressing Th1-mediated autoimmune diseases. This results from its ability to bind cell surface receptors, principally GM1-ganglioside, and as a consequence down-regulate the pathological T helper type 1 (Th1) response. The capacity of EtxB to alter human T-cell responses has not been investigated. Here we show that EtxB, but not the receptor non-binding mutant EtxB (G33D), triggers the release of interleukin (IL)-10, IL-6 and tumour necrosis factor-alpha (TNF-alpha) by human monocytes. The production of IL-8, transforming growth factor-beta (TGF-beta) or IL-12 was not enhanced by EtxB. Indeed, EtxB was shown to inhibit IL-12 secretion in monocytes stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) by an IL-10-independent mechanism. When EtxB-treated monocytes were used as antigen presenting cells in an allogeneic mixed lymphocyte reaction (MLR), IL-10 and IFN-gamma production were increased in comparison to levels seen in cultures stimulated with untreated monocytes; proliferation was unaltered. This modulation of the T-cell response was not only evident in the primary MLR triggered by EtxB-treated monocytes, but also upon restimulation of the responding T cells with fresh untreated monocytes; indicating that presentation by EtxB-treated monocytes leads to altered T-cell differentiation. Sorting experiments showed that IL-10 secreting T cells from the MLR cultures were strong suppressors of T-cell proliferation following their addition into a fresh primary MLR.

The B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule capable of treating and preventing autoimmune disease. These properties result from its ability to bind to glycolipid receptors, principally G(M1) ganglioside, and modulate immune cell function. EtxB receptor binding causes B cell activation, modulates monocyte cytokine secretion and triggers apoptosis of CD8+ T cells. These wide-ranging effects suggest that B subunit receptor interaction triggers signaling events affecting cellular differentiation. We have investigated the processes by which EtxB induces CD8+ T cell apoptosis. We show that receptor interaction by EtxB activates caspase-3 in CD8+ but not in CD4+ T cells. Inhibition of caspase-3 blocks the apoptotic process. EtxB induces the activation of NF-kappaB in both CD8+ and CD4+ T cells. The findings that (i) SN50, a peptide inhibitor of NF-kappaB nuclear translocation, prevents caspase-3 activation and subsequent apoptosis, and (ii) CD8+CD4- thymocytes from transgenic mice expressing a dominant-negative form of the IkappaBalpha protein were markedly less susceptible to EtxB-induced apoptosis than cells from wild-type mice, indicate that NF-kappaB is important in the induction of the apoptotic pathway. Further investigations revealed that while caspase-8 activity is detected concomitant to caspase-3, caspase-9 activation, following mitochondrial cytochrome c release, is detectable later on. These observations are consistent with death receptor-mediated signaling, however, experiments using lpr/lpr and p55 TNFR -/- mice rule out the involvement of Fas and the p55 TNF receptor, respectively. The data therefore indicate that EtxB-mediated apoptosis occurs via a novel pathway involving NF-kappaB.

Current immunization strategies, using peptide or protein antigens, generally fail to elicit cytotoxic-T-lymphocyte responses, since these antigens are unable to access intracellular compartments where loading of major histocompatibility complex class I (MHC-I) molecules occurs. In an attempt to circumvent this, we investigated whether the GM1 receptor-binding B subunit of Escherichia coli heat-labile toxin (EtxB) could be used to deliver class I epitopes. When a class I epitope was conjugated to EtxB, it was delivered into the MHC-I presentation pathway in a GM1-binding-dependent fashion and resulted in the appearance of MHC-I-epitope complexes at the cell surface. Importantly, we show that the efficiency of EtxB-mediated epitope delivery could be strikingly enhanced by incorporating, adjacent to the class I epitope, a 10-amino-acid segment from the C terminus of the DNA polymerase (Pol) of herpes simplex virus. The replacement of this 10-amino-acid segment by a heterologous sequence or the introduction of specific amino acid substitutions within this segment either abolished or markedly reduced the efficiency of class I epitope delivery. If the epitope was extended at its C terminus, EtxB-mediated delivery into the class I presentation pathway was found to be completely dependent on proteasome activity. Thus, by combining the GM1-targeting function of EtxB with the 10-amino-acid Pol segment, highly efficient delivery of exogenous epitopes into the endogenous pathway of class I antigen processing and presentation can be achieved.

Objective: The receptor-binding B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a highly stable, nontoxic protein that is capable of modulating immune responses. This study was conducted to determine whether mucosal administration of EtxB can block collagen-induced arthritis (CIA) and to investigate the mechanisms involved., Methods: Clinical arthritis in DBA/1 mice was monitored following mucosal administration of EtxB on 4 occasions. The dependence of disease prevention on receptor binding by EtxB and the associated alterations to the immune response to type II collagen (CII) were assessed. Adoptive transfer experiments and lymph node cell cocultures were used to investigate the underlying mechanisms., Results: Both intranasal and intragastric delivery of EtxB were effective in preventing CIA; a 1-microg dose of EtxB was protective after intranasal administration. A non-receptor-binding mutant of EtxB failed to prevent disease. Intranasal EtxB lowered both the incidence and severity of arthritis when given either at the time of disease induction or 25 days later. EtxB markedly reduced levels of anti-CII IgG2a antibodies and interferon-gamma (IFNgamma) production while not affecting levels of IgG1, interleukin-4 (IL-4), or IL-10. Disease protection could be transferred by CD4+ T cells from treated mice, an effect that was abrogated upon depletion of the CD25+ population. In addition, CD4+CD25+ T cells from treated mice were able to suppress anti-CII IFNgamma production by CII-primed lymph node cells., Conclusion: Mucosal administration of EtxB can be used to prevent or treat CIA. Modulation of the anti-CII immune response by EtxB is associated with a reduction in Th1 cell reactivity without a concomitant shift toward Th2. Instead, EtxB mediates its effects through enhancing the activity of a population of CD4+ regulatory T cells.

Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB(5) (the B subunit pentamer of cholera toxin) is investigated. When CtxB(5) was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzyme-linked immunosorbent assay (t(12) approximately 8 min) and to migrate as SDS-stable pentamers (t(12) approximately 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of approximately 8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB(5) reassembly was generated that depended on trans to cis isomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking.

Feline immunodeficiency virus (FIV) is a lentivirus that causes feline acquired immunodeficiency syndrome. Infection can be transmitted experimentally via the vagina and rectum, making the cat a useful model for human immunodeficiency virus (HIV) infection. Some strains of FIV use the CXCR4 chemokine receptor in vitro to gain entry to feline cell lines, thymocytes and peripheral blood leucocytes (PBLs). In this study, the tissue expression of messenger ribonucleic acid (mRNA) encoding the CCR3, CXCR4 and CCR5 receptors was examined by reverse transcriptase polymerase chain reaction (RT-PCR). mRNA encoding each receptor was expressed by two feline T-cell lines (Mya-1 and FeTJ), a feline kidney fibroblast cell line (FKCU) and PBLs. Mesenteric lymph node, colon, rectum, uterus, cervix and vagina all expressed mRNA for CXCR4 and CCR5 whilst only lymph node expressed CCR3 mRNA. In order to locate this receptor mRNA expression, in-situ hybridization studies were performed with DNA probes specific for the chemokine receptor mRNAs. CCR5 and CXCR4 receptor mRNA was expressed by epithelial cells and some lamina propria cells of the colon and rectum. Epithelial cell expression of chemokine receptor mRNA was reduced in intensity towards the base of the crypts. Expression of CXCR4 receptor was also demonstrated immunohistochemically on some lamina propria and intraepithelial cells. The expression of these receptor molecules may be important in mucosal infection with FIV., (Copyright Elsevier Science Ltd. All rights reserved.)

When epithelial cells first encounter cholera toxin (Ctx) produced by Vibrio cholerae they secrete not only chloride ions responsible for causing diarrhoea, but also a number of cytokines that may contribute to the toxin's potent immunomodulatory properties. Much less is known about the ability of the heat-labile enterotoxin of Escherichia coli (Etx), a close homologue of Ctx, to elicit cytokine secretion by epithelial cells. This study shows that treatment of human intestinal epithelial T84 cells with Etx induces expression of IL-6, IL-10, IL-1R antagonist, as well as IL-1alpha and IL-1beta and low levels of IL-8. Such induction was totally dependent on the intrinsic ADP-ribosylating activity of the toxin A-subunit, and could be mimicked by cAMP-elevating agents, such as forskolin and dibutyryl cAMP. By comparison, neither an enzymically inactive mutant of Etx nor EtxB was able to induce cytokine secretion. The behaviour of Ctx and CtxB was very similar to that of Etx and EtxB, respectively. The spectrum of cytokines released by Etx and Ctx indicates that the toxins may create a local microenvironment that strongly biases the immune response towards an anti-inflammatory and a polarized Th2 response.

An ability to deliver macromolecules into the intracellular compartments of mammalian cells offers enormous potential for development of new therapeutics directed against intracellular targets. Unfortunately, most peptides or proteins are too large to enter the cell cytosol unaided, and any uptake that does occur primarily results in their entry into lysosomes for degradation. However, one group of proteins that possesses an inherent capacity to interact with and enter mammalian cells are bacterial toxins. These are being developed as efficient vehicles for the attachment and intracellular delivery of other macromolecules, including peptides, proteins and DNA. To date, most studies have concentrated on the delivery of immunological epitopes into the endogenous major histocompatibility class I (MHC-I) pathway for development of antiviral or anticancer vaccines. However, opportunities to use toxins to modulate inflammatory autoimmune disorders and cell-specific targeting of DNA for gene therapy illustrates the versatility of toxin molecules as delivery vehicles.

Cholera toxin and E. coli heat-labile enterotoxin are structurally homologous proteins comprised of an enzymatically active A-subunit and five B-subunits that bind with high affinity to GM1-ganglioside receptors found on the surface of mammalian cells. The B-subunits have long been thought of simply as trafficking vehicles that trigger entry and subsequent delivery of the 'toxic' A-subunit into cells. Indeed, such is the capacity of the B-subunits to enter cells, that they have been developed as generic carriers for attachment and delivery of a variety of peptides into mammalian cells. However, the B-subunits also appear to possess discrete 'signalling functions', that induce both transcription factor and cell activation. These are thought to be directly responsible for the potent immunomodulatory properties of the B-subunits, and have resulted in their use as adjuvants and as agents to suppress inflammatory immune disorders. The relationship between the signalling properties of the B-subunits and their capacity to act as trafficking vehicles has remained unclear. In an effort to understand the structural requirements for these two functions, a set of mutant B-subunits, with amino acid substitutions at position His-57, have been generated and studied. Importantly, such mutant B-subunits retain an ability to bind with high affinity to GM1 and to traffic into cells, but have entirely lost their capacity to activate immune cell populations. Thus, while binding via GM1 appears to be sufficient to trigger cellular uptake it is not sufficient to activate signal transduction. The His-57 region is therefore speculated to be actively engaged in triggering signalling events, possibly via cognate interaction with other cell surface molecules.

Entry of cholera toxin (CT) into target epithelial cells and the induction of toxicity depend on CT binding to the lipid-based receptor ganglioside G(M1) and association with detergent-insoluble membrane microdomains, a function of the toxin's B-subunit. The B-subunits of CT and related Escherichia coli toxins exhibit a highly conserved exposed peptide loop (Glu(51)-Ile(58)) that faces the cell membrane upon B-subunit binding to G(M1). Mutation of His(57) to Ala in this loop resulted in a toxin (CT-H57A) that bound G(M1) with high apparent affinity, but failed to induce toxicity. CT-H57A bound to only a fraction of the cell-surface receptors available to wild-type CT. The bulk of cell-surface receptors inaccessible to CT-H57A localized to detergent-insoluble apical membrane microdomains (lipid rafts). Compared with wild-type toxin, CT-H57A exhibited slightly lower apparent binding affinity for and less stable binding to G(M1) in vitro. Rather than being transported into the Golgi apparatus, a process required for toxicity, most of CT-H57A was rapidly released from intact cells at physiologic temperatures or degraded following its internalization. These data indicate that CT action depends on the stable formation of the CT B-subunit.G(M1) complex and provide evidence that G(M1) functions as a necessary sorting motif for the retrograde trafficking of toxin into the secretory pathway of target epithelial cells.

Heat-labile enterotoxin from enterotoxinogenic Escherichia coli is not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8(+) T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8(+) T cells but not of CD4(+) T cells triggers the specific up-regulation of the transcription factor c-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-myc antisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IkappaBalpha turnover, suggesting that elevated Myc expression may be dependent on NF-kappaB. When CD8(+) T cells were pretreated with inhibitors of IkappaBalpha turnover and NF-kappaB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Haemolytic uraemic syndrome (HUS), which is caused by Shiga toxin (Stx)-producing Escherichia coli, is the commonest cause of acute renal failure in childhood. It is widely believed that HUS develops following the release of Stx, an AB5 toxin that inhibits protein synthesis and has a direct toxic effect on the kidney endothelium. There remains, however, a mismatch between the current understanding of the pathogenesis of HUS and the evolution of the clinical signs of the disease. Our hypothesis is that Stx-mediated immune cell activation in the gut is the missing link in the pathogenesis of this condition, initiating the characteristic renal pathology of HUS either alone or in synergy with Stx. Validation of this hypothesis could lead to a targeted anti-inflammatory approach aimed at modulating immune cell function in HUS.

Although cholera toxin (Ctx) and Escherichia coli heat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits. Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB. EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes. These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants.

Cholera toxin (Ctx) and the closely related Escherichia coli heat-labile enterotoxin (Etx) not only act as mediators of diarrhoeal disease but also exert potent immunomodulatory properties on mammalian immune systems. The toxins normally exert their diarrhoeagenic effects by initiating receptor-mediated uptake into vesicles that enter a retrograde trafficking pathway, circumventing degradative compartments and targeting them to the trans-Golgi network (TGN) and endoplasmic reticulum. Here, we examine whether receptor-mediated binding and cellular entry by the toxin B-subunits also lead to concomitant changes in uptake and trafficking of exogenous antigens that could contribute to the potent immunomodulatory properties of these toxins. Treatment of the macrophage (J774.2) cell line with Etx B-subunit (EtxB) resulted in EtxB transport to the TGN and also led to the formation of large, translucent, non-acidic, EtxB-devoid vacuoles. When exogenous antigens were added, EtxB-treated cells were found to be proficient in both internalization of ovalbumin (OVA) and phagocytosis of bacterial particles. However, the internalized OVA, instead of trafficking along a lysosome-directed endocytic pathway via acidified endosomes, persisted in a non-acidic, light-density compartment that was distinct from the translucent vacuoles. The rerouted OVA did not co-localize with the endosomal markers rab5 or rab11, nor with EtxB, but was retained in a transferrin receptor-positive compartment. The failure of OVA to enter the late endosomal/lysosomal compartments correlated with a striking inhibition of OVA peptide processing and presentation to OVA-responsive CD4+ T-cells. CtxB also modulated OVA trafficking and inhibited antigen presentation. These findings demonstrate that the B-subunits of Ctx and Etx alter the progression of exogenous antigens along the endocytic processing pathway, and prevent or delay efficient epitope presentation and T-cell stimulation. The formation of such 'antigen depots' could contribute to the immunomodulatory properties of these bacterial virulence determinants.

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.

In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and likely endoplasmic reticulum (ER) (Lencer et al., J. Cell Biol. 131, 951-962 (1995)). We have recently found that the toxin's apical membrane receptor ganglioside GM1 acts specifically in this signal transduction pathway, likely by coupling CT with caveolae or caveolae-related membrane domains (lipid rafts) (Wolf et al., J. Cell Biol. 141, 917-927 (1998)). Work in progress shows that 1) cholesterol depletion uncouples the CT-GM1 receptor complex from signal transduction, a characteristic of lipid rafts; 2) the GM1 acyl chains rather than the carbohydrate head groups appear to account for the structural basis of ganglioside specificity in toxin trafficking; and 3) intestinal epithelial cells obtained from normal adult humans exhibit lipid rafts which differentiate between CT-GM1 and LTIIb-GD1a complexes and which contain caveolin 1.

Collagen-binding protein (CNBP) synthesized by Aeromonas veronii is located conserved within the subcellular fraction. The results of this study show that 98% of the total CNBP produced by Aer. veronii is present in the extracellular medium, and that the remaining CNBP is distributed either on the cell surface, within the periplasm or anchored on the outer membrane. CNBP is specifically secreted from Aer. veronii into the culture medium, because all the beta-lactamase activity was located in the cells and could be released by polymixin B extraction of periplasmic proteins. CNBP was produced at growth temperatures from 12 degrees C to 42 degrees C, but not at 4 degrees C. The findings indicate that the level of CNBP in the medium increases during the exponential growth phase and reaches a maximum during the early stationary phase. There was less CNBP production in poor nutrient MMB medium than in the rich LB nutrient medium. CNBP secretion, in contrast to aerolysin secretion, was unaffected by the exeA mutation of Aer. hydrophila. It is concluded that CNBP secretion from Aer. veronii must be achieved by a mechanism different from that reported for aerolysin secretion.

Cholera toxin (Ctx) from Vibrio cholerae and the closely related Escherichia coli heat-labile enterotoxin (Etx) are the primary virulence factors responsible for causing cholera and traveller's diarrhea, respectively. Studies on the mode of action of these toxins on gut epithelial cells have revealed important insights into the mechanisms of toxin uptake and trafficking in eukaryotic cells. However, of perhaps even greater fascination have been the discoveries that Ctx and Etx exhibit remarkable immunological properties. When either of these toxins is administered via mucosal routes, it triggers a potent mucosal and systemic anti-toxin immune response. By contrast, local or systemic immunization with other soluble protein antigens usually stimulates only a meagre immune response, or results in a state of immunological tolerance. Even more striking are the findings that when Ctx or Etx are mixed with heterologous antigens, they function as adjuvants, leading to stimulation of mucosal responses to the admixed antigen, and the abrogation of oral tolerance. In addition, recent observations have shown that the receptor-binding component of these toxins can down-regulate inflammatory diseases associated with the induction of autoimmune disorders such as rheumatoid arthritis, diabetes, and multiple sclerosis. While the underlying mechanisms responsible for these remarkable properties have yet to be resolved, it is clear that the toxins' ability to bind to cell surface receptors plays an important role in their potent immunogenicity, adjuvanticity, and immunotherapeutic properties. This review provides an overview of the latest developments within the Ctx/Etx field, with a special emphasis on the cell entry mechanisms and immunomodulatory action of Ctx/Etx and their component subunits.