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Past work has shown that a subset of effector T cells with unique characteristics could abrogate hapten- or antigen-induced tolerance, and the reconstitution of this immune response has been termed contrasuppression. We have studied contrasuppression in a model of oral tolerance (OT) in which adoptively transferred antigen-specific T contrasuppressor (Tcs) cells reverse OT and result in antibody responses to the eliciting antigen. In the present study, we show that murine intraepithelial lymphocytes (IELs) from mice orally immunized with sheep red blood cells (SRBC) contain T cells that exhibit Tcs cell activity. This effect was mediated by CD3+ gamma/delta T cell receptor-positive (TCR+), but not alpha/beta TCR+ T cells, and gamma/delta TCR+ Tcs cells were associated with both the CD4-,CD8+ and CD4-,CD8- (double-negative) IEL fractions. The CD4-,CD8+ gamma/delta TCR+ IELs were further separated into Vicia villosa-adherent and -nonadherent fractions. Adoptive transfer of V. villosa-adherent gamma/delta TCR+ T cells to mice with OT to SRBC resulted in splenic IgA, IgM, and IgG subclass anti-SRBC responses, while V. villosa-nonadherent gamma/delta TCR+ T cells were without activity. The gamma/delta TCR+ IELs did not support in vitro antibody responses in B cell cultures, while alpha/beta TCR+ IELs were effective T helper cells. Further, cytokine production by the gamma/delta TCR+ IELs was examined, and the gamma/delta TCR+ V. villosa-adherent fraction, which possessed contrasuppressor function, contained low levels of IL-5 mRNA and small numbers of IL-5-producing cells when compared with alpha/beta TCR+ IELs and V. villosa-nonadherent gamma/delta TCR+ IELs. Our results now show that mouse IELs contain two distinct types of T cells that function in the immune response, e.g., alpha/beta TCR+ T cells that produce IL-5 and function as helper cells, and gamma/delta TCR+ T cells that restore antibody responses in mice that had been orally tolerized with antigen.

To evaluate how Advanced Placement (AP) courses affect college-going, we randomly assigned the offer of enrollment into an AP science course to over 1,800 students in twenty-three schools that had not previously offered the course. We find no AP course effects on students' college entrance exam scores (SAT/ACT). As expected, AP course-takers are substantially more likely to take the AP exam than their control group counterparts. At the same time, treatment group students opt out of the exam at very high rates and most do not earn a passing score on the AP exam. Though less precisely estimated, the results also suggest that taking the AP course increases students' aspirations to attend higher-quality colleges but does not lead to enrollment in such institutions. [ABSTRACT FROM AUTHOR]

We describe the conceptualization, design, development, validation, and testing of a summative instrument that measures high school students' ability to analyze and evaluate data, construct scientific explanations, and formulate scientific arguments in biology and chemistry disciplinary contexts. Data from 1,405 students were analyzed to evaluate the properties of the instrument. Student measurement separation reliability was 0.71 with items showing satisfactory fit to the Partial Credit Model. The use of the Evidence-Centered Design framework during the design and development process provided a strong foundation for the validity argument. Additional evidence for validation were also gathered. The strengths of the instrument lie in its relatively brief time for administration and a unique approach that integrates science practice and disciplinary knowledge, while simultaneously seeking to decouple their measurement. This research models how to design assessments that align to the National Research Council's framework and informs the design of Next Generation Science Standards-aligned assessments. [ABSTRACT FROM AUTHOR]

Procedures introduced here make it possible, first, to show that background (piecemeal) extinction is recorded throughout geologic stages and substages (not all extinction has occurred suddenly at the ends of such intervals); second, to separate out background extinction from mass extinction for a major crisis in earth history; and third, to correct for clustering of extinctions when using the rarefaction method to estimate the percentage of species lost in a mass extinction. Also presented here is a method for estimating the magnitude of the Signor-Lipps effect, which is the incorrect assignment of extinctions that occurred during a crisis to an interval preceding the crisis because of the incompleteness of the fossil record. Estimates for the magnitudes of mass extinctions presented here are in most cases lower than those previously published. They indicate that only ~81% of marine species died out in the great terminal Permian crisis, whereas levels of 90-96% have frequently been quoted in the literature. Calculations of the latter numbers were incorrectly based on combined data for the Middle and Late Permian mass extinctions. About 90 orders and more than 220 families of marine animals survived the terminal Permian crisis, and they embodied an enormous amount of morphological, physiological, and ecological diversity. Life did not nearly disappear at the end of the Permian, as has often been claimed. [ABSTRACT FROM AUTHOR]

We propose a new ecological ranking of the major Phanerozoic biodiversity crises in which the Serpukhovian biodiversity crisis is ranked fifth in ecological impact, lesser than the Late Devonian but greater than the end-Ordovician, and the end-Ordovician mass extinction is ranked sixth. It is interesting that both the end-Ordovician mass extinction and the Serpukhovian biodiversity crisis were triggered by glaciations. Other than that common trigger, the two events were very different. Glaciation in the Ordovician triggered an enormous jump in the extinction rate of marine organisms and was taxonomically very severe, yet the ecological impact of those extinctions was minimal. Glaciation in the Serpukhovian triggered a precipitous drop in the speciation rate but only moderate diversity losses, yet the ecological impact of those diversity losses and ecosystem restructuring was an ecological level of magnitude larger than that seen in the end-Ordovician mass extinction. [ABSTRACT FROM AUTHOR]

A geometric analysis of lophophore shape and arrangement in the fenestrate dendroid graptolite genus Dictyonema reveals that the shape of the zooid domain in the majority of Dictyonema species colonies is highly elliptical, with the long axis of the ellipse perpendicular to the proximodistal axis of the stipe. A complex lophophore, bilaterally symmetrical and consisting of two tentaculated arms that are laterally directed and perpendicular to the stipe axis, provides the best geometric solution to completely filling the elliptical zooid domain seen in the majority of Dictyonema species. Working under the assumption that fenestrate graptolites, like fenestrate bryozoans, needed to form a continuous filtering surface with contact between adjacent lophophores, two optimum close-parking models exist for lophophores with elliptical zooid domains: either a proximodistal-row arrangement or a lateral-row arrangement of the lophophores. Of the two possible geometries, the most probable close-packing arrangement of hypothetical biplumed lophophores within the fenestrate graptolite meshwork is in proximodistal rows. [ABSTRACT FROM AUTHOR]

Oral administration of large doses of protein antigen generally induces a state of systemic unresponsiveness currently termed mucosally induced tolerance. In this study, we used human milk protein (HMP) without casein as a multi-protein antigen for the study of mucosally induced tolerance. The HMP utilized in this study mainly contained secretory (S) IgA, lactoferrin (LO and a-lactalbumin (Lact). When mice were given 1 or 25 mg of HMP orally 3 times or 25 mg orally four consecutive weeks prior to systemic immunization, antigen-specific serum IgG responses to HMP were induced by subsequent parenteral immunization with 100 μg of HMP. Analysis of IgG subclasses revealed that IgG1 followed by IgG2b accounted for the IgG responses noted. When both HMP and ovalbumin (OVA) were fed to mice, tolerance developed to OVA but not to HMP. To further investigate the nature of immune responses seen following oral gavage of HMP, we examined responses to individual protein of HMP. Brisk serum IgG1 and IgG2b responses to both S-IgA and Lf were induced by oral followed by systemic immunization with HMP. Analysis of splenic CD4+ T cells from mice given oral HMP revealed production of Th2- but not Thl-type cytokines. These results show that oral administration of HMP preferentially induces exclusive Th2-type immune responses, which may prevent the development of HMP (S-IgA and Lf)-specific mucosally induced tolerance. [ABSTRACT FROM PUBLISHER]

The article presents the material of geometric models as an addendum to the research published in the March 2003 issue of the periodical "Journal of Paleontology." The said models will allow other researchers to assess the geometry of lophophore shapes and arrangements in the construction of such extinct modular organisms.

The Advanced Placement (AP) program has undergone two major reforms in recent decades: the first aimed at increasing access and the second at increasing relevance. Both initiatives are partially designed to increase the number of high school students from low-income backgrounds who have access to college-level coursework. Yet critics argue that schools in less-resourced communities are unable to implement AP at the level expected by its founders. We offer the first model of the components inherent in a well-implemented AP science course and the first evaluation of AP implementation with a focus on public schools newly offering the inquiry-based version of AP Biology and Chemistry courses. We find that these frontier schools were able to implement most, but not all, of the key components of an AP science course.

Age-associated alterations in the mucosal immune system are generally termed mucosal immunosenescence. The major change seen in the aged mucosa is a failure to elicit an antigen-specific secretory IgA (SIgA) antibody response, which is a central player for host defense from various pathogens at mucosal surfaces. In this regard, it would be a first priority to compensate for mucosal dysregulation in the elderly in order to maintain their health in aging. We have successfully established antigen-specific SIgA antibody responses in aged (2 years old) mice, which provide protective immunity from Streptococcus pneumoniae and influenza virus infections, by using a new adjuvant system consisting of a plasmid encoding Flt3 ligand (pFL) and CpG ODN. In order to explore possible use of current mucosal vaccine strategies for the elderly, we have adoptively transferred adipose tissue-derived mesenchymal stem cells (AMSCs) to aged mice prior to mucosal vaccination. This immune therapy successfully resulted in protective antigen-specific antibody responses in the intestinal mucosa of aged mice that were comparable to those seen in young adult mice. In this regard, we postulate that adoptively transferred AMSCs could augment dendritic cell functions in aged mice. The potential cellular and molecular mechanisms whereby AMSCs restore mucosal immunity in immunosenescence are discussed in this short review. A stem cell transfer system could be an attractive and effective immunologic intervention strategy to reverse mucosal immunosenescence., (© The Japanese Society for Immunology. 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Convergent evolution reveals to us that the number of possibilities available for contingent events is limited, that historically contingent evolution is constrained to occur within a finite number of limited pathways, and that contingent evolution is thus probabilistic and predictable. That is, the phenomenon of convergence proves that truly contingent evolutionary processes can repeatedly produce the same, or very similar, organic designs in nature and that evolution is directional in these cases. For this reason it is argued in this paper that evolution can be directional without being teleological, and that the dichotomy that evolution must either be directionless and unpredictable or directional and predetermined (teleological) is false., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into adipocytes, chondrocytes and osteoblasts. Several clinical trials have shown the ability of AMSCs to regenerate these differentiated cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier during agingthan is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>18 mo old) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT) three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA- and CT-B-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. Similar results were also seen aged mice which received AMSCs at one year of age. When cytokine production was examined, OVA-stimulated Peyer's patch CD4+ T cells produced increased levels of IL-4. Further, CD4+ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA- or CT-B-specific mucosal SIgA or plasma IgG Ab or cytokine responses. Of importance, fecal extracts from AMSC transferred aged mice showed neutralization activity to CT intoxication. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice.

Limits in the evolution of biological form can be empirically demonstrated by using theoretical morphospace analyses, and actual analytic examples are given for univalved ammonoid shell form, bivalved brachiopod shell form and helical bryozoan colony form. Limits in the evolution of form in these animal groups can be shown to be due to functional and developmental constraints on possible evolutionary trajectories in morphospace. Future evolutionary-limit research is needed to analyse the possible existence of temporal constraint in the evolution of biological form on Earth, and in the search for the possible existence of functional alien life forms on Titan and Triton that are developmentally impossible for Earth life.

Streptococcus pneumoniae (the pneumococcus) causes a major upper respiratory tract infection often leading to severe illness and death in the elderly. Thus, it is important to induce safe and effective mucosal immunity against this pathogen in order to prevent pnuemocaccal infection. However, this is a very difficult task to elicit protective mucosal IgA antibody responses in older individuals. A combind nasal adjuvant consisting of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligonucleotide (CpG ODN) successfully enhanced S. pneumoniae-specific mucosal immunity in aged mice. In particular, a pneumococcal surface protein A-based nasal vaccine given with pFL and CpG ODN induced complete protection from S. pneumoniae infection. These results show that nasal delivery of a combined DNA adjuvant offers an attractive potential for protection against the pneumococcus in the elderly.

We assessed the role of CCR5(+)/CCR6(+)/CD11b(+)/CD11c(+) dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5(+)/CCR6(+)/CD11b(+)/CD11c(+) DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b(+)/CD11c(+) DCs were markedly decreased in both CCR5(-/-) and CCR6(-/-) mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5(-/-) or CD11c-DTR and CCR6(-/-) mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5(+)CCR6(+) DCs play an important role in the induction of Ag-specific SIgA Ab responses.

To develop protective immune responses against mucosal pathogens, the delivery route and adjuvants for vaccination are important. The host, however, strives to maintain mucosal homeostasis by responding to mucosal antigens with tolerance, instead of immune activation. Thus, induction of mucosal immunity through vaccination is a rather difficult task, and potent mucosal adjuvants, vectors or other special delivery systems are often used, especially in the elderly. By taking advantage of the common mucosal immune system, the targeting of mucosal dendritic cells and microfold epithelial cells may facilitate the induction of effective mucosal immunity. Thus, novel routes of immunization and antigen delivery systems also show great potential for the development of effective and safe mucosal vaccines against various pathogens. The purpose of this review is to introduce several recent approaches to induce mucosal immunity to vaccines, with an emphasis on mucosal tissue targeting, new immunization routes and delivery systems. Defining the mechanisms of mucosal vaccines is as important as their efficacy and safety, and in this article, examples of recent approaches, which will likely accelerate progress in mucosal vaccine development, are discussed.

An intricate network of innate and immune cells and their derived mediators function in unison to protect us from toxic elements and infectious microbial diseases that are encountered in our environment. This vast network operates efficiently by use of a single cell epithelium in, for example, the gastrointestinal (GI) and upper respiratory (UR) tracts, fortified by adjoining cells and lymphoid tissues that protect its integrity. Perturbations certainly occur, sometimes resulting in inflammatory diseases or infections that can be debilitating and life threatening. For example, allergies in the eyes, skin, nose, and the UR or digestive tracts are common. Likewise, genetic background and environmental microbial encounters can lead to inflammatory bowel diseases (IBDs). This mucosal immune system (MIS) in both health and disease is currently under intense investigation worldwide by scientists with diverse expertise and interests. Despite this activity, there are numerous questions remaining that will require detailed answers in order to use the MIS to our advantage. In this issue of PLOS Biology, a research article describes a multi-scale in vivo systems approach to determine precisely how the gut epithelium responds to an inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), given by the intravenous route. This article reveals a previously unknown pathway in which several cell types and their secreted mediators work in unison to prevent epithelial cell death in the mouse small intestine. The results of this interesting study illustrate how in vivo systems biology approaches can be used to unravel the complex mechanisms used to protect the host from its environment., Competing Interests: The authors have declared that no competing interests exist.

We explore cellular and molecular mechanisms of nasal adjuvant of a combination of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligodeoxynucleotides (CpG ODN). The double DNA adjuvant given with OVA maintained prolonged OVA-specific secretory IgA (S-IgA) Ab responses in external secretions for more than 25 weeks after the final immunization. Further, both Th1- and Th2-type cytokine responses were induced by this combined adjuvant regimen. The frequencies of plasmacytoid DCs (pDCs) and CD8(+) DCs were significantly increased in nasopharyngeal-associated lymphoreticular tissue (NALT) of mice given the combined adjuvant. Importantly, when we examined adjuvanticity of pFL plus CpG ODN in 2-year-old mice, significant levels of mucosal IgA Ab responses were also induced. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for the development of an effective mucosal vaccine for the elderly.

Background & Aims: The follicle-associated epithelium (FAE) plays key roles in antigen uptake and subsequent induction of mucosal immunity. In this study, we examined whether M-cell targeting using a protein antigen (Ag) delivery system would induce oral tolerance instead of enhancement of Ag-specific mucosal antibody (Ab) responses., Methods: Mice were fed different doses of a recombinant protein sigma 1 of reovirus genetically conjugated to ovalbumin (OVA-psigma1), psigma1 only, or phosphate-buffered saline (PBS) before oral challenge with OVA plus cholera toxin as mucosal adjuvant. OVA-specific Ab and CD4-positive (CD4(+)) T-cell responses were determined., Results: A low dose of OVA-psigma1 reduced anti-OVA Ab and CD4(+) T-cell responses in both mucosal and systemic lymphoid tissues. OVA/MHC I-A(d) tetramer staining showed that the numbers of OVA-specific CD4(+) T cells were significantly reduced in lamina propria of mice fed OVA-psigma1 than those fed psigma1 only or PBS only. In fact, Foxp3 expressing CD25(+) CD4(+) T cells were markedly increased in this tissue. Nonetheless, CD25(+) CD4(+) T cells from the spleen, mesenteric lymph nodes, and Peyer's patches of orally tolerized mice showed increased transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) production compared with nontolerized mice., Conclusions: These results show that an FAE M-cell targeting protein Ag delivery system facilitates oral tolerance induction because of a reduction in Ag-specific CD4(+) T cells and increased levels of TGF-beta1 and IL-10 producing, CD25(+) CD4(+) regulatory T cells in both systemic and mucosal lymphoid tissues.

Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4(+) Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4(+) T cell proliferative responses and OVA-induced IFN-gamma and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b(+)CD11c(+) DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b(+)CD11c(+) DCs that migrate to effector sites for subsequent CD4(+) Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.

Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein sigma1 (psigma1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via psigma1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus psigma1, termed OVA-psigma1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-psigma1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-psigma1 could be adoptively transferred using cervical lymph node CD4(+) T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-beta1, with greatly reduced IL-17 and IFN-gamma. The induced IL-10 was derived predominantly from FoxP3(+)CD25(+)CD4(+) T cells. No FoxP3(+)CD25(+)CD4(+) T cells were induced in OVA-psigma1-dosed IL-10-deficient (IL-10(-/-)) mice, and despite showing increased TGF-beta1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using psigma1 as a mucosal delivery platform specifically for low-dose tolerance induction.

Most current animal models focus on eosinophil-mediated asthma, despite compelling evidence that a neutrophil-mediated disease occurs in some asthma patients. Using intranasal challenge of mice sensitized either orally or nasally with whole peanut protein extract in the presence of cholera toxin, we developed mouse models of eosinophil- and neutrophil-mediated asthma, respectively. In this study, mice deficient in Th1 (IL-12 and IFN-gamma) or Th2 (IL-4 and IL-13) pathways were used to characterize the role played by Th1 and Th2 cytokines during the initial priming phase in the two models. Antigen-specific Ab responses were controlled primarily by Th2 cytokines in mice sensitized by the oral route, whereas Th1 cytokines appeared to play a predominant role in mice sensitized by the nasal route. Furthermore, the absence of key Th1 or Th2 cytokines during the initial phase of priming reduced lung reactivity in both mouse models of airway inflammation.

In this study, we examine whether native cholera toxin (nCT) as a mucosal adjuvant can support trinitrophenyl (TNP)-LPS-specific mucosal immune responses. C57BL/6 mice were given nasal TNP-LPS in the presence or absence of nCT. Five days later, significantly higher levels of TNP-specific mucosal IgA Ab responses were induced in the nasal washes, saliva, and plasma of mice given nCT plus TNP-LPS than in those given TNP-LPS alone. High numbers of TNP-specific IgA Ab-forming cells were also detected in mucosal tissues such as the nasal passages (NPs), the submandibular glands (SMGs), and nasopharyngeal-associated lymphoreticular tissue of mice given nCT. Flow cytometric analysis showed that higher numbers of surface IgA+, CD5+ B cells (B-1a B cells) in SMGs and NPs of mice given nasal TNP-LPS plus nCT than in those given TNP-LPS alone. Furthermore, increased levels of IL-5R alpha-chain were expressed by B-1a B cells in SMGs and NPs of mice given nasal TNP-LPS plus nCT. Thus, CD4+ T cells from these mucosal effector lymphoid tissues produce high levels of IL-5 at both protein and mRNA levels. When mice were treated with anti-IL-5 mAb, significant reductions in TNP-specific mucosal IgA Ab responses were noted in external secretions. These findings show that nasal nCT as an adjuvant enhances mucosal immune responses to a T cell-independent Ag due to the cross-talk between IL-5Ralpha+ B-1a B cells and IL-5-producing CD4+ T cells in the mucosal effector lymphoid tissues.

The gastrointestinal immune system consists of immune cells in organized gut-associated lymphoreticular tissues (GALT) and diffuse lamina propria, which give rise to mucosal secretory IgA antibody responses. A recent study showed that the retinoic acid produced by GALT dendritic cells (DCs) imprints B cells for gut homing. Surprisingly, GALT DCs, together with interleukin-5 (IL-5) and IL-6, also provided a milieu for both B cell switching to IgA and IgA synthesis.

Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a beta-trefoil (Hcbetatre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hcbetatre or Hcbetatre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hcbetatre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hcbetatre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hcbetatre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hcbetatre-dosed mice. Mice immunized with adjuvanted Hcbetatre-Ad2F or Hcbetatre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hcbetatre-Ad2F or Hcbetatre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hcbetatre Ab titers even in the absence of adjuvant. This study shows that the Hcbetatre structure can confer protective immunity and that use of Hcbetatre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.

Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.

The traditional view of HIV-1 infection characterized by the slow decline of CD4+ T cells has radically changed in light of recent observations in rhesus macaques and humans of rapid and extensive infection and removal of memory CD4+ T cells in mucosal tissues within the first three weeks of infection. This initial strike to the immune system seems to be the distinguishing feature of HIV-1 pathogenesis and its extent sets the overall course of the ensuing infection. Qualitatively different mechanisms of CD4+ T-cell depletion prevail during the acute, chronic and advanced phases of infection depending on the availability of the target-cell population and competence of the immune system. The elimination of CD4+ T cells in mucosal lymphoid tissues results in the absence of important regulatory and effector functions that these cells normally perform in controlling immune responses to environmental antigens and pathogens. Ablation of acute HIV-1 viremia limits the initial damage to the CD4+ T-cell compartment and helps to establish a state of equilibrium between the replicating virus, the availability of the target-cell population and the immune control characteristic of long-term non-progression.

Anthrax edema toxin (EdTx) is an AB-type toxin that binds to anthrax toxin receptors on target cells via the binding subunit, protective Ag (PA). Edema factor, the enzymatic A subunit of EdTx, is an adenylate cyclase. We found that nasal delivery of EdTx enhanced systemic immunity to nasally coadministered OVA and resulted in high OVA-specific plasma IgA and IgG (mainly IgG1 and IgG2b). The edema factor also enhanced immunity to the binding PA subunit itself and promoted high levels of plasma IgG and IgA responses as well as neutralizing PA Abs. Mice given OVA and EdTx also exhibited both PA- and OVA-specific IgA and IgG Ab responses in saliva as well as IgA Ab responses in vaginal washes. EdTx as adjuvant triggered OVA- and PA-specific + T cells which secreted IFN-gamma and selected Th2-type cytokines. The EdTx up-regulated costimulatory molecule expression by APCs but was less effective than cholera toxin for inducing IL-6 responses either by APCs in vitro or in nasal washes in vivo. Finally, nasally administered EdTx did not target CNS tissues and did not induce IL-1 mRNA responses in the nasopharyngeal-associated lymphoepithelial tissue or in the olfactory bulb epithelium. Thus, EdTx derivatives could represent an alternative to the ganglioside-binding enterotoxin adjuvants and provide new tools for inducing protective immunity to PA-based anthrax vaccines.

Purpose: To compare magnetic resonance imaging (MRI) and computed tomography (CT) with each other and to International Federation of Gynecology and Obstetrics (FIGO) clinical staging in the pretreatment evaluation of early invasive cervical cancer, using surgicopathologic findings as the reference standard., Patients and Methods: This prospective multicenter clinical study was conducted by the American College of Radiology Imaging Network and the Gynecologic Oncology Group from March 2000 to November 2002; 25 United States health centers enrolled 208 consecutive patients with biopsy-confirmed cervical cancer of FIGO stage > or = IB who were scheduled for surgery based on clinical assessment. Patients underwent FIGO clinical staging, helical CT, and MRI. Surgicopathologic findings constituted the reference standard for statistical analysis., Results: Complete data were available for 172 patients; surgicopathologic findings were consistent with FIGO stages IA to IIA in 76% and stage > or = IIB in 21%. For the detection of advanced stage (> or = IIB), sensitivity was poor for FIGO clinical staging (29%), CT (42%), and MRI (53%); specificity was 99% for FIGO clinical staging, 82% for CT, and 74% for MRI; and negative predictive value was 84% for FIGO clinical staging, 84% for CT, and 85% for MRI. MRI (area under the receiver operating characteristic curve [AUC], 0.88) was significantly better than CT (AUC, 0.73) for detecting cervical tumors (P = .014). For 85% of patients, FIGO clinical staging forms were submitted after MRI and/or CT was performed., Conclusion: CT and MRI performed similarly; both had lower staging accuracy than in prior single-institution studies. Accuracy of FIGO clinical staging was higher than previously reported. The temporal data suggest that FIGO clinical staging was influenced by CT and MRI findings.

Despite structural and functional differences between the initial sites of contact with allergens in the gastrointestinal and nasal tracts, few animal models have examined the influence of the mucosal routes of sensitization on host reactivity to food or environmental antigens. We compared the oral and nasal routes of peanut sensitization for the development of a mouse model of allergy. Mice were sensitized by administration of peanut proteins in the presence of cholera toxin as adjuvant. Antibody and cytokine responses were characterized, as well as airway reactivity to nasal challenge with peanut or unrelated antigens. Oral sensitization promoted higher levels of IgE, but lower IgG responses, than nasal sensitization. Both orally and nasally sensitized mice experienced airway hyperreactivity on nasal peanut challenge. The peanut challenge also induced lung eosinophilia and type 2 helper T-cell-type cytokines in orally sensitized mice. In contrast, peanut challenge in nasally sensitized mice promoted neutrophilia and higher levels of lung MAC-1(+) I-A(b low) cells and inflammatory cytokines. In addition, nasal but not oral, sensitization promoted lung inflammatory responses to unrelated antigens. In summary, both oral and nasal peanut sensitization prime mice for airway hyperreactivity, but the initial mucosal route of sensitization influences the nature of lung inflammatory responses to peanut and unrelated allergens.

The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1beta or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.

The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 x 10(3) LD50 of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD50 was determined. When mice were given an oral challenge of 5 microg (2 x oral LD50) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.

Age-associated dysregulation of the immune system of the gastrointestinal (GI) tract has been well documented for both secretory (S)-IgA immunity and oral tolerance. Thus, impaired antigen-specific Ab responses in aged animals and the elderly have been reported. Further, it has been shown that gut-associated lymphoreticular tissue (GALT) mediated immune responses are more susceptible to aging than are lymphoid tissues involved in peripheral immunity. Aging also impairs oral tolerance, which may be of central importance for maintaining GI homeostasis. Thus, as early as 6-8-month-old mice failed to establish systemic unresponsiveness to orally introduced antigens. Despite these studies, the precise mechanisms for impaired GI tract immune system responses remain unclear. The evidence of reduced sizes of Peyer's patches through aging suggests that age-associated mucosal dysregulation may be the result of mucosal inductive tissue dysfunction. Indeed, the frequencies of naive CD4+ T cells and dendritic cells (DCs) in Peyer's patches of aged mice were reduced and this led to a lack of essential cytokine synthesis for the induction of either S-IgA immunity or oral tolerance.

Chemokines constitute a large family of structurally related proteins that play a role in leukocyte migration and differentiation. Indeed, the early expression of human CXC chemokine receptor 1 (hCXCR1) and hCXCR2 [homologous to mouse interleukin (IL)-8Rbeta] ligands by the epithelium is a hallmark of the mucosal host defense. Mice lack IL-8; however, granulocyte chemotactic protein-2 (GCP-2)/lipopolysaccharide-induced CXC chemokine, a murine homologue of human GCP-2, has 32% and 61% sequence identity to human IL-8 and GCP-2, respectively, and binds hCXCR1, hCXCR2, and mouse IL-8Rbeta. To better understand the role of GCP-2 in adaptive immunity and as a nasal adjuvant, we characterized the exogenous effects of this CXC chemokine on cellular and humoral mucosal immune responses. GCP-2 significantly enhanced serum immunoglobulin G (IgG) and mucosal IgA antibodies through increased cytokine secretion by CD4+ T cells. These alterations in humoral and cellular responses were preceded by an increase in the number of B cells in the nasal tract, a decrease in the number of CD4+ T cells in the nasal tract as well as cervical lymph nodes, and an increase in the number of neutrophils in the nasal tract 12 h after GCP-2 immunization. This chemokine also modulated CD28 expression by CD4+ T cells during CD3epsilon stimulation of wild-type mice. GCP-2 increased CD80 and CD86 expression on B cells during in vitro stimulation in a dose-dependent manner. In contrast, cytokine and costimulatory molecule enhancement by GCP-2 was not induced by lymphocytes from IL-8Rbeta-/- mice, suggesting that GCP-2 modulates cellular immunity in part through IL-8Rbeta interactions.

The development of a safe and effective mucosal adjuvant is a crucial step toward a mucosal HIV/AIDS vaccine. This study seeks to determine the promise of a nontoxic mutant of cholera toxin (mCT; E112K) as a mucosal adjuvant in nonhuman primates. HIV-1 gp120 was nasally administered together with mCT E112K or native CT (nCT) as adjuvant on five to six occasions over a 6- to 8-wk period to groups of four rhesus macaques and alone to two monkeys that acted as controls. Macaques given nasal gp120 with either mCT E112K or nCT showed elevated gp120-specific IgG and IgA Ab responses with virus-neutralizing activity in both their plasma and mucosal external secretions, as well as higher numbers of gp120-specific IgA Ab-forming cells in their mucosal and peripheral lymphoid tissues and of IL-4-producing Th2-type CD4-positive (CD4(+)) T cells than did controls. Even though significant mucosal adjuvanticity was seen with both mCT E112K and nCT, neuronal damage was observed only in the nCT-treated, but not in the control or mCT E112K-treated groups. These results clearly show that mCT E112K is an effective and safe mucosal adjuvant for the development of a nasal HIV/AIDS vaccine.

We have analyzed the gastrointestinal inflammation which develops following adoptive transfer of IL-4 gene knockout (IL-4(-/-)) CD4(+)CD45RB(Hi) (RB(Hi)) T cells to severe combined immunodeficient (SCID) or to T cell-deficient, T cell receptor beta and delta double knockout (TCR(-/-)) mice. Transfer of IL-4(-/-) RB(Hi) T cells induced a similar type of colitis to that seen in SCID or TCR(-/-) recipients of wild-type (wt) RB(Hi) T cells as reported previously. Interestingly, transfer of both wt and IL-4(-/-) RB(Hi) T cells to TCR(-/-) but not to SCID mice induced inflammation in the gastric mucosa. Notably, TCR(-/-) recipients of IL-4(-/-) RB(Hi) T cells developed a more severe gastritis with erosion, apoptosis of the antral epithelium, and massive infiltration of macrophages. This gastritis was partially dependent on the indigenous microflora. Recipients of both wt and IL-4(-/-) RB(Hi) T cells developed duodenitis with multinuclear giant cells, expansion of mucosal macrophages, and dendritic cells. Full B cell responses were reconstituted in TCR(-/-) recipients of RB(Hi) T cells; however, anti-gastric autoantibodies were not detected. We have now developed and characterized a novel model of chronic gastroduodenitis in mice, which will help in our understanding of the mechanisms involved in chronic inflammation in the upper gastrointestinal tract of humans.

This study assessed the roles of the postnatal lymphotoxin-beta receptor (LTbetaR)-mediated signals in the gut-associated lymphoreticular tissues of mice for subsequent regulation of Ag-specific intestinal IgA responses. Blockade of LTbetaR-dependent events by postnatal administration of the fusion protein of LTbetaR and IgG Fc (LTbetaR-Ig) reduced both the size and numbers of Peyer's patches (PP) without influencing the PP microarchitecture. Interestingly, inhibition of LTbetaR-dependent signaling revealed significant reductions in the formation of follicular dendritic cell clusters in mesenteric lymph nodes (MLN). Furthermore, these postnatal signaling events controlled the development of isolated lymphoid follicles (ILF) because treatment with LTbetaR-Ig eliminated the formation of ILF. LTbetaR-Ig-treated mice with altered microarchitecture of MLN and lacking ILF were still able to produce significant Ag-specific mucosal IgA responses after oral immunization; however, the levels were significantly lower than those seen in control mice. These results imply the importance of ILF for Ag-specific intestinal immunity. However, mice treated with both TNFR55-Ig and LTbetaR-Ig in utero, which lack PP and MLN, but retain intact ILF, failed to induce Ag-specific IgA responses after oral immunization. These findings demonstrate that ILF are not essential for induction of intestinal IgA Ab responses to orally administered Ag. Furthermore, the induction of intestinal IgA Ab responses requires the proper maintenance of the MLN microarchitecture, including a follicular dendritic cell network.