Elevated Extracellular cGMP Produced after Exposure to Enterotoxigenic Escherichia coli Heat-Stable Toxin Induces Epithelial IL-33 Release and Alters Intestinal Immunity.

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies identified heat-stable enterotoxin (ST)-producing ETEC as a prevalent diarrheal pathogen in children younger than 5 years. While many studies have evaluated the interaction of ETEC heat-labile enterotoxin (LT) with host epithelium and immunity, few investigations have attempted similar studies with ST. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. In addition to robust intracellular cGMP in T84 cells in the presence of phosphodiesterase inhibitors (PDEis) that prevent the breakdown of cyclic nucleotides, we found that prolonged ST intoxication induced extracellular cGMP accumulation in the presence or absence of PDEis. Further, ST intoxication induced luminal cGMP in vivo in mice, suggesting that secreted cGMP may have other cellular functions. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could also be induced by cell-permeative 8-Br-cGMP. Finally, when present during immunization, ST suppressed induction of antibodies to specific antigens. In conclusion, our studies indicate that ST modulates epithelial cell physiology and the interplay between the epithelial and immune compartments.

Intradermal or Sublingual Delivery and Heat-Labile Enterotoxin Proteins Shape Immunologic Responses to a CFA/I Fimbria-Derived Subunit Antigen Vaccine against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of infectious diarrhea in children, travelers, and deployed military personnel. As such, development of a vaccine would be advantageous for public health. One strategy is to use subunits of colonization factors combined with antigen/adjuvant toxoids as an ETEC vaccine. Here, we investigated the intradermal (i.d.) or sublingual (s.l.) delivery of CFA/I fimbrial antigens, including CfaEB and a CfaE-heat-labile toxin B subunit (LTB) chimera admixed with double mutant heat-labile toxin (LT) LT-R192G/L211A (dmLT). In addition, we compared dmLT with other LT proteins to better understand the generation of adjuvanted fimbrial and toxoid immunity as well as the influence on any local skin reactogenicity. We demonstrate that immunization with dmLT admixed with CfaEB induces robust serum and fecal antibody responses to CFA/I fimbriae and LT but that i.d. formulations are not optimal for s.l. delivery. Improved s.l. vaccination outcomes were observed when higher doses of dmLT (1 to 5 µg) were admixed with CfaEB or, even better, when a CfaE-LTB chimera antigen was used instead. Serum anti-CFA/I total antibodies, detected by enzyme-linked immunosorbent assay, were the best predictor of functional antibodies, based on the inhibition of red blood cell agglutination by ETEC. Immunization with other LT proteins or formulations with altered B-subunit binding during i.d. immunization (e.g., by addition of 5% lactose, LTA1, or LT-G33D) minimally altered the development of antibody responses and cytokine recall responses but reduced skin reactogenicity at the injection site. These results reveal how formulations and delivery parameters shape the adaptive immune responses to a toxoid and fimbria-derived subunit vaccine against ETEC.

Exposure-response analysis of blinatumomab in patients with relapsed/refractory acute lymphoblastic leukaemia and comparison with standard of care chemotherapy.

AIMS: The relationship between blinatumomab exposure and efficacy endpoints (occurrence of complete remission [CR] and duration of overall survival [OS]) or adverse events (occurrence of cytokine release syndrome [CRS] and neurological events) were investigated in adult patients with relapsed/refractory acute lymphoblastic leukaemia (r/r ALL) receiving blinatumomab or standard of care (SOC) chemotherapy to evaluate appropriateness of the blinatumomab dosing regimen. METHODS: Exposure, efficacy and safety data from adult patients (n = 646) with r/r ALL receiving stepwise (9 then 28 µg/day, 4-week cycle) continuous intravenous infusion (n = 537) of blinatumomab or SOC (n = 109) chemotherapy were pooled from phase 2 and 3 studies. The occurrence of CR, neurological and CRS events, and duration of OS were analysed using Cox proportional hazards models or logistic regression, as appropriate. Confounding factors were tested multivariately as needed. RESULTS: Blinatumomab steady-state concentration following 28 µg/day dosing was associated with the probability of achieving CR (odds ratio and 95% confidence interval: 1.073 [1.033-1.114]), and a longer duration of OS compared to SOC (hazard ratio and 95% confidence interval: 0.954 [0.936-0.973], P < .05) in multivariate analyses. The exposure-safety analyses indicated that blinatumomab steady-state concentration following the 9 or 28 µg/day dose was not associated with increased probability of CRS or neurological events, after accounting for blinatumomab treatment effect (P > .05). CONCLUSIONS: Blinatumomab step-dosing regimen of 9/28 µg/day provided treatment benefit in achieving CR and increasing the duration of OS over SOC and was appropriate in management of CRS and neurological events in patients with r/r ALL.

Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli.

Enterotoxigenic Escherichia coli(ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity.

Impact of detergent on biophysical properties and immune response of the IpaDB fusion protein, a candidate subunit vaccine against Shigella species.

Shigella spp. are causative agents of bacillary dysentery, a human illness with high global morbidity levels, particularly among elderly and infant populations. Shigella infects via the fecal-oral route, and its virulence is dependent upon a type III secretion system (T3SS). Two components of the exposed needle tip complex of the Shigella T3SS, invasion plasmid antigen D (IpaD) and IpaB, have been identified as broadly protective antigens in the mouse lethal pneumonia model. A recombinant fusion protein (DB fusion) was created by joining the coding sequences of IpaD and IpaB. The DB fusion is coexpressed with IpaB's cognate chaperone, IpgC, for proper recombinant expression. The chaperone can then be removed by using the mild detergents octyl oligooxyethelene (OPOE) or N,N-dimethyldodecylamine N-oxide (LDAO). The DB fusion in OPOE or LDAO was used for biophysical characterization and subsequent construction of an empirical phase diagram (EPD). The EPD showed that the DB fusion in OPOE is most stable at neutral pH below 55 °C. In contrast, the DB fusion in LDAO exhibited remarkable thermal plasticity, since this detergent prevents the loss of secondary and tertiary structures after thermal unfolding at 90 °C, as well as preventing thermally induced aggregation. Moreover, the DB fusion in LDAO induced higher interleukin-17 secretion and provided a higher protective efficacy in a mouse challenge model than did the DB fusion in OPOE. These data indicate that LDAO might introduce plasticity to the protein, promoting thermal resilience and enhanced protective efficacy, which may be important in its use as a subunit vaccine.

Characterization of heat-stable (STa) toxoids of enterotoxigenic Escherichia coli fused to double mutant heat-labile toxin peptide in inducing neutralizing Anti-STa antibodies.

A long-standing challenge in developing vaccines against enterotoxigenic Escherichia coli (ETEC), the most common bacteria causing diarrhea in children of developing countries and travelers to these countries, is to protect against heat-stable toxin type Ib (STa or hSTa). STa and heat-labile toxin (LT) are virulence determinants in ETEC diarrhea. LT antigens are often used in vaccine development, but STa has not been included because of its poor immunogenicity and potent toxicity. Toxic STa is not safe for vaccines, but only STa possessing toxicity is believed to be able to induce neutralizing antibodies. However, recent studies demonstrated that nontoxic STa derivatives (toxoids), after being fused to an LT protein, induced neutralizing antibodies and suggested that different STa toxoids fused to an LT protein might exhibit different STa antigenic propensity. In this study, we selected 14 STa toxoids from a mini-STa toxoid library based on toxicity reduction and reactivity to anti-native STa antibodies, and genetically fused each toxoid to a monomeric double mutant LT (dmLT) peptide for 14 STa-toxoid-dmLT toxoid fusions. These toxoid fusions were used to immunize mice and were characterized for induction of anti-STa antibody response. The results showed that different STa toxoids (in fusions) varied greatly in anti-STa antigenicity. Among them, STaN12S, STaN12T, and STaA14H were the top toxoids in inducing anti-STa antibodies. In vitro neutralization assays indicated that antibodies induced by the 3×STaN12S-dmLT fusion antigen exhibited the greatest neutralizing activity against STa toxin. These results suggested 3×STaN12S-dmLT is a preferred fusion antigen to induce an anti-STa antibody response and provided long-awaited information for effective ETEC vaccine development.

Simultaneous exposure to Escherichia coli heat-labile and heat-stable enterotoxins increases fluid secretion and alters cyclic nucleotide and cytokine production by intestinal epithelial cells.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrheal disease and death, especially in children in developing countries. ETEC causes disease by colonizing the small intestine and producing heat-labile toxin (LT), heat-stable toxin (ST), or both LT and ST (LT+ST). The majority of ETEC strains produce both ST and LT. Despite the prevalence of LT+ST-producing organisms, few studies have examined the physiologic or immunologic consequences of simultaneous exposure to these two potent enterotoxins. In the current report, we demonstrate that when LT and ST are both present, they increase water movement into the intestinal lumen over and above the levels observed with either toxin alone. As expected, cultured intestinal epithelial cells increased their expression of intracellular cyclic GMP (cGMP) when treated with ST and their expression of intracellular cyclic AMP (cAMP) when treated with LT. When both toxins were present, cGMP levels but not cAMP levels were synergistically elevated compared with the levels of expression caused by the corresponding single-toxin treatment. Our data also demonstrate that the levels of inflammatory cytokines produced by intestinal epithelial cells in response to LT are significantly reduced in animals exposed to both enterotoxins. These findings suggest that there may be complex differences between the epithelial cell intoxication and, potentially, secretory outcomes induced by ETEC strains expressing LT+ST compared with strains that express LT or ST only. Our results also reveal a novel mechanism wherein ST production may reduce the hosts' ability to mount an effective innate or adaptive immune response to infecting organisms.

Characterization of a novel fusion protein from IpaB and IpaD of Shigella spp. and its potential as a pan-Shigella vaccine.

Shigellosis is an important disease in the developing world, where about 90 million people become infected with Shigella spp. each year. We previously demonstrated that the type three secretion apparatus (T3SA) proteins IpaB and IpaD are protective antigens in the mouse lethal pulmonary model. In order to simplify vaccine formulation and process development, we have evaluated a vaccine design that incorporates both of these previously tested Shigella antigens into a single polypeptide chain. To determine if this fusion protein (DB fusion) retains the antigenic and protective capacities of IpaB and IpaD, we immunized mice with the DB fusion and compared the immune response to that elicited by the IpaB/IpaD combination vaccine. Purification of the DB fusion required coexpression with IpgC, the IpaB chaperone, and after purification it maintained the highly α-helical characteristics of IpaB and IpaD. The DB fusion also induced comparable immune responses and retained the ability to protect mice against Shigella flexneri and S. sonnei in the lethal pulmonary challenge. It also offered limited protection against S. dysenteriae challenge. Our results show the feasibility of generating a protective Shigella vaccine comprised of the DB fusion.

The A subunit of Escherichia coli heat-labile enterotoxin functions as a mucosal adjuvant and promotes IgG2a, IgA, and Th17 responses to vaccine antigens.

Enterotoxigenic Escherichia coli (ETEC) produces both heat-labile (LT) and heat-stable (ST) enterotoxins and is a major cause of diarrhea in infants in developing countries and in travelers to those regions. In addition to inducing fluid secretion, LT is a powerful mucosal adjuvant capable of promoting immune responses to coadministered antigens. In this study, we examined purified A subunit to further understand the toxicity and adjuvanticity of LT. Purified A subunit was enzymatically active but sensitive to proteolytic degradation and unable to bind gangliosides, and even in the presence of admixed B subunit, it displayed low cyclic AMP (cAMP) induction and no enterotoxicity. Thus, the AB5 structure plays a key role in protecting the A subunit from proteolytic degradation and in delivering the enzymatic signals required for secretion. In contrast, the A subunit alone was capable of activating dendritic cells and enhanced immune responses to multiple antigens following intranasal immunization; therefore, unlike toxicity, LT adjuvanticity is not dependent on the AB5 holotoxin structure or the presence of the B subunit. However, immune responses were maximal when signals were received from both subunits either in an AB5 structure or with A and B admixed. Furthermore, the quality of the immune response (i.e., IgG1/IgG2 balance and mucosal IgA and IL-17 secretion) was determined by the presence of an A subunit, revealing for the first time induction of Th17 responses with the A subunit alone. These results have important implications for understanding ETEC pathogenesis, unraveling immunologic responses induced by LT-based adjuvants, and developing new mucosal vaccines.

Broadly protective Shigella vaccine based on type III secretion apparatus proteins.

Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine.

A clade C HIV-1 vaccine protects against heterologous SHIV infection by modulating IgG glycosylation and T helper response in macaques.

The rising global HIV-1 burden urgently requires vaccines capable of providing heterologous protection. Here, we developed a clade C HIV-1 vaccine consisting of priming with modified vaccinia Ankara (MVA) and boosting with cyclically permuted trimeric gp120 (CycP-gp120) protein, delivered either orally using a needle-free injector or through parenteral injection. We tested protective efficacy of the vaccine against intrarectal challenges with a pathogenic heterologous clade C SHIV infection in rhesus macaques. Both routes of vaccination induced a strong envelope-specific IgG in serum and rectal secretions directed against V1V2 scaffolds from a global panel of viruses with polyfunctional activities. Envelope-specific IgG showed lower fucosylation compared with total IgG at baseline, and most of the vaccine-induced proliferating blood CD4+ T cells did not express CCR5 and α4ß7, markers associated with HIV target cells. After SHIV challenge, both routes of vaccination conferred significant and equivalent protection, with 40% of animals remaining uninfected at the end of six weekly repeated challenges with an estimated efficacy of 68% per exposure. Induction of envelope-specific IgG correlated positively with G1FB glycosylation, and G2S2F glycosylation correlated negatively with protection. Vaccine-induced TNF-α+ IFN-γ+ CD8+ T cells and TNF-α+ CD4+ T cells expressing low levels of CCR5 in the rectum at prechallenge were associated with decreased risk of SHIV acquisition. These results demonstrate that the clade C MVA/CycP-gp120 vaccine provides heterologous protection against a tier2 SHIV rectal challenge by inducing a polyfunctional antibody response with distinct Fc glycosylation profile, as well as cytotoxic CD8 T cell response and CCR5-negative T helper response in the rectum.

Prophylactic administration of bacterially derived immunomodulators improves the outcome of influenza virus infection in a murine model.

Prophylactic or therapeutic immunomodulation is an antigen-independent strategy that induces nonspecific immune system activation, thereby enhancing host defense to disease. In this study, we investigated the effect of prophylactic immunomodulation on the outcome of influenza virus infection using three bacterially derived immune-enhancing agents known for promoting distinct immunological profiles. BALB/c mice were treated nasally with either cholera toxin (CT), a mutant form of the CT-related Escherichia coli heat-labile enterotoxin designated LT(R192G), or CpG oligodeoxynucleotide. Mice were subsequently challenged with a lethal dose of influenza A/PR/8/34 virus 24 h after the last immunomodulation treatment and either monitored for survival or sacrificed postchallenge for viral and immunological analysis. Treatment with the three immunomodulators prevented or delayed mortality and weight loss, but only CT and LT(R192G) significantly reduced initial lung viral loads as measured by plaque assay. Analysis performed 4 days postinfection indicated that prophylactic treatments with CT, LT(R192G), or CpG resulted in significantly increased numbers of CD4 T cells, B cells, and dendritic cells and altered costimulatory marker expression in the airways of infected mice, coinciding with reduced expression of pulmonary chemokines and the appearance of inducible bronchus-associated lymphoid tissue-like structures in the lungs. Collectively, these results suggest that, despite different immunomodulatory mechanisms, CT, LT(R192G), and CpG induce an initial inflammatory process and enhance the immune response to primary influenza virus challenge while preventing potentially damaging chemokine expression. These studies provide insight into the immunological parameters and immune modulation strategies that have the potential to enhance the nonspecific host response to influenza virus infection.

Drug-disease interaction: Crohn's disease elevates verapamil plasma concentrations but reduces response to the drug proportional to disease activity.

AIM: Inflammation is involved in the pathogenesis of cardiovascular diseases that includes reduced response to pharmacotherapy due to altered pharmacokinetics and pharmacodynamics. It is not known if these effects exist in general in all inflammatory conditions. It also remains unknown whether in a given population the effect is a function of disease severity. We investigated whether pharmacokinetics and pharmacodynamics of a typical calcium channel inhibitor are influenced by Crohn's disease (CD), a disease for which the disease severity can be readily ranked. METHODS: We administered 80 mg verapamil orally to (i) healthy control subjects (n= 9), (ii) patients with clinically quiescent CD (n= 22) and (iii) patients with clinically active CD (n= 14). Serial analysis of verapamil enantiomers (total and plasma unbound), blood pressure and electrocardiograms were recorded over 8 h post dose. The severity of CD was measured using the Harvey-Bradshaw Index. RESULTS: CD substantially and significantly increased plasma verapamil concentration and in a stereoselective fashion (S, 9-fold; R, 2-fold). The elevated verapamil concentration, however, failed to result in an increased verapamil pharmacodynamic effect so that the patients with elevated verapamil concentration demonstrated no significant increase in response measured as PR interval and blood pressure. Instead, the greater the disease severity, the lower was the drug potency to prolong PR interval (r= 0.86, P < 0.0006), CONCLUSIONS: CD patients with severe disease may not respond to cardiovascular therapy with calcium channel blockers. Reducing the severity increases response despite reduced drug concentration. This observation may have therapeutic implication beyond the disease and the drug studies herein.

Assessment of the duration of protection in Campylobacter jejuni experimental infection in humans.

A human Campylobacter jejuni infection model provided controlled exposure to assess vaccine efficacy and investigate protective immunity for this important diarrheal pathogen. A well-characterized outbreak strain, C. jejuni 81-176, was investigated using a volunteer experimental infection model to evaluate the dose range and duration of protection. Healthy Campylobacter-seronegative adults received C. jejuni strain 81-176 via oral inoculation of 10(5), 10(7), or 10(9) CFU (5 adults/dose), which was followed by clinical and immunological monitoring. Based on dose range clinical outcomes, the 10(9)-CFU dose (n = 31) was used to assess homologous protection at 28 to 49 days (short-term veterans [STV]; n = 8) or 1 year (long-term veterans [LTV]; n = 7) after primary infection. An illness dose effect was observed for naïve subjects (with lower doses, 40 to 60% of the subjects were ill; with the 10(9)-CFU dose, 92% of the subjects were ill) along with complete protection for the STV group and attenuated illness for the LTV group (57%). Partial resistance to colonization was seen in STV (25% of the subjects were not infected; 3-log-lower maximum excretion level). Systemic and mucosal immune responses were robust in naïve subjects irrespective of the dose or the severity of illness. In contrast, in STV there was a lack of circulating antibody-secreting cells (ASC), reflecting the local mucosal effector responses. LTV exhibited comparable ASC responses to primary infection, and anamnestic fecal IgA responses likely contributed to self-resolving illness prior to antibiotic treatment. Campylobacter antigen-dependent production of gamma interferon by peripheral blood mononuclear cells was strongly associated with protection from illness, supporting the hypothesis that TH1 polarization has a primary role in acquired immunity to C. jejuni. This study revealed a C. jejuni dose-related increase in campylobacteriosis rates, evidence of complete short-term protection that waned with time, and immune response patterns associated with protection.

Kinetics of immune responses to influenza virus-like particles and dose-dependence of protection with a single vaccination.

The format of influenza virus-like particles (VLPs) as a nonreplicating particulate vaccine candidate is a promising alternative to conventional egg-based vaccines. In this study, we have investigated the detailed kinetics of immune responses and protective efficacy after a single intranasal immunization with different doses of VLPs alone or in the presence of an Escherichia coli mutant heat-labile enterotoxin [mLT(R192G)] or cholera toxin subunit B as adjuvants. Analysis of immune responses showed differential kinetics in a VLP antigen dose-dependent manner and dynamic changes in the ratios of antibody immunoglobulin G isotypes over the time course. Protection against lethal challenge was observed with a single immunization with influenza VLPs even without adjuvant. The addition of adjuvant showed significant antigen-sparing effects with improved protective efficacy. The protective immune responses, efficacies of protection, and antigen-sparing effects were significantly improved by a second immunization as determined by the levels of neutralizing antibodies, morbidity postchallenge, lung viral titers, and inflammatory cytokines. Our results are informative for a better understanding of the protective immunity induced by a single dose or two doses of influenza VLPs, which is dependent on antigen dosage and the presence of adjuvant, and will provide insights into designing effective vaccines based on VLPs.

Enterotoxigenic Escherichia coli Heat-Stable Toxin Increases the Rate of Zinc Release from Metallothionein and Is a Zinc- and Iron-Binding Peptide.

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies have identified heat-stable enterotoxin (ST)-producing ETEC as one of the major diarrhea-causing pathogens in children younger than five years. In this study, we examined iron and zinc binding by both human and porcine ST variants and determined how host metallothionein could detoxify ST. We found that ST purified from ETEC culture supernatants eluted as a doublet during C18 reverse-phase chromatography. Leading edge fractions of the ST doublet were found to be devoid of iron, while trailing edge fractions of the ST doublet were found to contain measurable iron. Next, we found that purified ST could be reconstituted with iron under reducing and anaerobic conditions, and iron-bound ST attenuated the induction of cGMP in T84 epithelial cells. Moreover, we demonstrated that supernatants of ETEC 214-4 grown under increasing iron concentrations were only able to induce cGMP at iron concentrations greater than 5 µM. In vitro studies also demonstrated that ST binds zinc, and once bound, zinc removal from ST required denaturing conditions. Zinc-bound ST also failed to induce cGMP. We found that ST contributes disulfide bonds to the perceived oxidized glutathione pool, increases the rate of zinc release from metallothionein, and can be detoxified by metallothionein. Lastly, we showed ST induces transcriptional changes in genes previously shown to be regulated by deferoxamine. These studies demonstrate ST ETEC pathogenesis may be tied intimately to host mucosal metal status.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries, deployed military personnel, and travelers to regions of endemicity. The heat-stable toxin (ST) is a small nonimmunogenic secreted peptide with 3 disulfide bonds. It has been appreciated that dietary disulfides modulate intestinal redox potential and that ST could be detoxified using exogenous reductants. Using biochemical and spectroscopic approaches, we demonstrated that ST can separately bind iron and zinc under reducing conditions, thereby reducing ST toxicity. Moreover, we demonstrated that ST modulates the glutathione (GSH)/oxidized glutathione (GSSG) ratio and that ST should be considered a toxin oxidant. ST can be detoxified by oxidizing zinc-loaded metallothionine, causing free zinc to be released. These studies help lay a foundation to understand how diarrheal pathogens modulate intestinal redox potential and may impact how we design therapeutics and/or vaccines for the pathogens that produce them.

Comparative Evaluation of Prophylactic SIV Vaccination Modalities Administered to the Oral Cavity.

Attempts to develop a protective human immunodeficiency virus (HIV) vaccine have had limited success, especially in terms of inducing protective antibodies capable of neutralizing different viral strains. As HIV transmission occurs mainly via mucosal surfaces, HIV replicates significantly in the gastrointestinal tract, and the oral route of vaccination is a very convenient one to implement worldwide, we explored three SIV vaccine modalities administered orally and composed of simian immunodeficiency virus (SIV) DNA priming with different boosting immunogens, with the goal of evaluating whether they could provide lasting humoral and cellular responses, including at mucosal surfaces that are sites of HIV entry. Twenty-four Cynomolgus macaques (CyM) were primed with replication-incompetent SIV DNA provirus and divided into three groups for the following booster vaccinations, all administered in the oral cavity: Group 1 with recombinant SIV gp140 and Escherichia coli heat-labile toxin adjuvant dmLT, Group 2 with recombinant SIV-Oral Poliovirus (SIV-OPV), and Group 3 with recombinant SIV-modified vaccinia ankara (SIV-MVA). Cell-mediated responses were measured using blood, lymph node, rectal and vaginal mononuclear cells. Significant levels of systemic and mucosal T-cell responses against Gag and Env were observed in all groups. Some SIV-specific plasma IgG, rectal and salivary IgA antibodies were generated, mainly in animals that received SIV DNA + SIV-MVA, but no vaginal IgA was detected. Susceptibility to infection after SIVmac251 challenge was similar in vaccinated and nonvaccinated animals, but acute infection viremia levels were lower in the group that received SIV DNA + SIV-MVA. Nonvaccinated CyM maintained central memory and total CD4+ T-cell levels in the normal range during the 5 months of postinfection follow-up as did the vaccinated animals, precluding evaluation of vaccine impact on disease progression. We conclude that the oral cavity vaccination tested in these regimens can stimulate cell-mediated immunity systemically and mucosally, but humoral response stimulation was limited with the doses and the vaccine platforms used.

Automated threshold detection for auditory brainstem responses: comparison with visual estimation in a stem cell transplantation study.

BACKGROUND: Auditory brainstem responses (ABRs) are used to study auditory acuity in animal-based medical research. ABRs are evoked by acoustic stimuli, and consist of an electrical signal resulting from summated activity in the auditory nerve and brainstem nuclei. ABR analysis determines the sound intensity at which a neural response first appears (hearing threshold). Traditionally, threshold has been assessed by visual estimation of a series of ABRs evoked by different sound intensities. Here we develop an automated threshold detection method that eliminates the variability and subjectivity associated with visual estimation. RESULTS: The automated method is a robust computational procedure that detects the sound level at which the peak amplitude of the evoked ABR signal first exceeds four times the standard deviation of the baseline noise. Implementation of the procedure was achieved by evoking ABRs in response to click and tone stimuli, under normal and experimental conditions (adult stem cell transplantation into cochlea). Automated detection revealed that the threshold shift from pre- to post-surgery hearing levels was similar in mice receiving stem cell transplantation or sham injection for click and tone stimuli. Visual estimation by independent observers corroborated these results but revealed variability in ABR threshold shifts and significance levels for stem cell-transplanted and sham-injected animals. CONCLUSION: In summary, the automated detection method avoids the subjectivity of visual analysis and offers a rapid, easily accessible http://axograph.com/source/abr.html approach to measure hearing threshold levels in auditory brainstem response.

Norepinephrine transporter is involved in down-regulation of beta1-adrenergic receptors caused by adjuvant arthritis.

PURPOSE: Inflammation in forms of rheumatoid and experimental arthritis cause not only joint pain but also excessive cardiovascular mortality. The condition also reduces response to calcium channel and beta-adrenergic (beta1-AR) antagonists. For calcium channel inhibitors, the reduced response is shown to be due to the reduced expression of target proteins. Hydroxymethylglutaryl CoA reductase inhibitors (statins) restore response to propranolol and verapamil. We tested the effect of adjuvant arthritis on the norepinephrine (NE) transporter (NET) density since altered sympathetic nervous system innervation has been observed in rheumatoid arthritis. METHODS: Male Sprague-Dawley rats were divided into the following groups: Healthy/Placebo, Healthy/Statin, Pre-AA/Placebo, and Pre-AA/Statin (n=7-8/group). On Day 0, to the Pre-AA and Healthy groups, was injected Mycobacterium butyricum or saline, respectively. On Days 4-8, Statin and Placebo groups received either pravastatin (6 mg/kg) or placebo twice daily, respectively. On day 8, heart and blood samples were collected. The density of NET and 1-AR in heart homogenate; NE in plasma and heart and inflammatory mediators (nitrite and interferon-gamma) in serum were determined. RESULTS: Inflammation was associated with a significant reduction in both beta1-AR and NET density with a positive correlation between the two proteins (r=0.978, p<0.0001). The down-regulating effect of inflammation was not reversed by pravastatin. Inflammation had no significant effect on the plasma or heart NE concentration. CONCLUSION: The close relations of NET and beta1-AR implicates altered sympathetic innervation and/or local NE handling in pharmacotherapeutic desensitization observed in arthritis.

Intradermal vaccination with a Pseudomonas aeruginosa vaccine adjuvanted with a mutant bacterial ADP-ribosylating enterotoxin protects against acute pneumonia.

Respiratory infections are a leading cause of morbidity and mortality globally. This is partially due to a lack of effective vaccines and a clear understanding of how vaccination route and formulation influence protective immunity in mucosal tissues such as the lung. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing acute pulmonary infections and is a leading cause of hospital-acquired and ventilator-associated pneumonia. With multidrug-resistant P. aeruginosa infections on the rise, the need for a vaccine against this pathogen is critical. Growing evidence suggests that a successful P. aeruginosa vaccine may require mucosal antibody and Th1- and Th17-type CD4+ T cells to prevent pulmonary infection. Intradermal immunization with adjuvants, such as the bacterial ADP-Ribosylating Enterotoxin Adjuvant (BARE) double mutant of E. coli heat-labile toxin (dmLT), can direct protective immune responses to mucosal tissues, including the lungs. We reasoned that intradermal immunization with P. aeruginosa outer membrane proteins (OMPs) adjuvanted with dmLT could drive neutralizing antibodies and migration of CD4+ T cells to the lungs and protect against P. aeruginosa pneumonia in a murine model. Here we show that mice immunized with OMPs and dmLT had significantly more antigen-specific IgG and Th1- and Th17-type CD4+ memory T cells in the pulmonary environment compared to control groups of mice. Furthermore, OMPs and dmLT immunized mice were significantly protected against an otherwise lethal lung infection. Protection was associated with early IFN-γ and IL-17 production in the lungs of immunized mice. These results indicate that intradermal immunization with dmLT can drive protective immunity to the lung mucosa and may be a viable vaccination strategy for a multitude of respiratory pathogens.