The role of intercellular junction proteins in the penetration resistance of Drosophila larvae to avermectin.

Insecticide resistance has become an increasingly serious challenge for agriculture in the world. To reveal the mechanisms of insecticide resistance, majority of studies have been carried out on the insensitivity of insecticide targets and the metabolism of insecticides. However, the mechanism of the insecticide penetration resistance in insects remains unclear. This study aimed to reveal the mechanism underlying the penetration resistance of Drosophila larvae to insecticide avermectin (AVM). Levels of intercellular junction proteins (IJPs) in the larvae were determined by Western blotting analysis and immunofluorescence assay. The result showed that the expression of IJPs septate junction and adherens junction proteins increased in the AVM-resistant insects compared with those in the AVM-susceptible ones, and the upregulation of the IJPs was mediated by the activation of protein kinase C (PKC) pathway. That AVM induced the activation of PKC was found not only in the Drosophila larvae but also in Drosophila S2 cells. These findings revealed that AVM could activate PKC pathway in Drosophila larvae, which mediated the upregulation of the IJPs and then led to the resistance to AVM, suggesting that the chemicals that can disrupt PKC activation may potentially be used to circumvent the resistance to AVM in insects.

Safety and immunogenicity of a primary series and booster dose of the meningococcal serogroup B-factor H binding protein vaccine (MenB-FHbp) in healthy children aged 1-9 years: two phase 2 randomised, controlled, observer-blinded studies.

BACKGROUND: The meningococcal serogroup B-factor H binding protein vaccine (MenB-FHbp) is licensed for use in children aged 10 years or older for protection against invasive serogroup B meningococcal disease. Because young children are at increased risk of invasive meningococcal disease, MenB-FHbp clinical data in this population are needed. METHODS: We conducted two phase 2 randomised, controlled, observer-blinded studies including healthy toddlers (age 12-23 months) across 26 Australian, Czech, Finnish, and Polish centres, and older children (age 2-9 years) across 14 Finnish and Polish centres. Exclusion criteria included previous vaccinations against serogroup B meningococcus or hepatitis A virus (HAV), and chronic antibiotic use. Toddlers were randomly allocated (2:1) via an interactive response technology system to receive either 60 µg or 120 µg MenB-FHbp or HAV vaccine and saline (control). Older children were randomly allocated (3:1) to receive 120 µg MenB-FHbp or control, with stratification by age group (2-3 years and 4-9 years). All vaccinations were administered as three doses (0, 2, and 6 months, with only saline given at 2 months in the control group). Toddlers who received 120 µg MenB-FHbp could receive a 120 µg booster dose 24 months after the end of the primary series. The percentages of participants with serum bactericidal activity using human complement (hSBA) titres at or above the lower limit of quantification (LLOQ; all greater than the 1:4 correlate of protection) against four test strains of serogroup B meningococcus 1 month after the third dose (primary immunogenicity endpoint) were measured in the evaluable immunogenicity populations (participants who received the vaccine as randomised, had available and determinate hSBA results, and had no major protocol violations). Not all participants were tested against all strains because of serum sample volume constraints. The frequencies of reactogenicity and adverse events after each dose were recorded in the safety population (all participants who received at least one dose and had safety data available). These studies are registered with ClinicalTrials.gov (NCT02534935 and NCT02531698) and are completed. FINDINGS: Between Aug 31, 2015, and Aug 22, 2016, for the toddler study and between Aug 27, 2015, and March 7, 2016, for the older children study, we enrolled and randomly allocated 396 toddlers (60 µg MenB-FHbp group n=44; 120 µg MenB-FHbp group n=220; control group n=132) and 400 older children (120 µg MenB-FHbp group n=294; control group n=106). 1 month after the third dose, the proportions of participants with hSBA titres at or above the LLOQ ranged across test strains from 85·0% (95% CI 62·1-96·8; 17 of 20 participants) to 100·0% (82·4-100·0; 19 of 19) in toddlers receiving 60 µg MenB-FHbp, and from 71·6% (61·4-80·4; 68 of 95) to 100·0% (96·2-100·0; 95 of 95) in toddlers receiving 120 µg MenB-FHbp, and from 79·1% (71·2-85·6; 106 of 134) to 100·0% (97·4-100·0; 139 of 139) in children aged 2-9 years receiving 120 µg MenB-FHbp. hSBA titres peaked at 1 month after the third primary dose of MenB-FHbp and then declined over time. 24 months after the third dose in the toddler study, the proportions with hSBA titres at or above the LLOQ ranged from 0·0% (0·0-17·6; 0 of 19 participants) to 41·2% (18·4-67·1; seven of 17) in those who received 60 µg MenB-FHbp and from 3·7% (0·8-10·4; three of 81) to 22·8% (14·1-33·6; 18 of 79) in those who received 120 µg MenB-FHbp. 1 month after the booster dose in toddlers, the proportions with hSBA titres at or above the LLOQ were higher than at 1 month after the primary series. MenB-FHbp reactogenicity was mostly transient and of mild to moderate severity. Adverse event frequency was similar between the MenB-FHbp and control groups and less frequent following MenB-FHbp booster than following primary doses. Two participants from the toddler study (both from the 120 µg MenB-FHbp group) and four from the older children study (three from the 120 µg MenB-FHbp group and one from the control group) were withdrawn from the study because of adverse events. INTERPRETATION: MenB-FHbp was well tolerated and induced protective immune responses in a high proportion of participants. These findings support a favourable MenB-FHbp immunogenicity and reactogenicity profile in young children, a population at increased risk of adverse invasive meningococcal disease outcomes. FUNDING: Pfizer.

Persistence of hSBA titers elicited by the meningococcal serogroup B vaccine menB-FHbp for up to 4 years after a 2- or 3-dose primary series and immunogenicity, safety, and tolerability of a booster dose through 26 months.

BACKGROUND: To demonstrate extended protection against meningococcal serogroup B (MenB) disease after MenB-FHbp (bivalent rLP2086) vaccination, this study evaluated immunopersistence through 26 months following MenB-FHbp boosting after 2 or 3 primary doses in adolescents. STUDY DESIGN: This phase 3, open-label study was an extension of 3 phase 2 studies with participants aged 11-18 years randomized to receive primary MenB-FHbp vaccination following 1 of 5 dosing schedules or control. A booster dose was administered 48 months after the primary series. Immunopersistence through 48 months after the last primary dose (persistence stage) and 26 months postbooster (booster stage) was determined by serum bactericidal assays using human complement (hSBAs) against 4 vaccine-heterologous test strains. Safety evaluations included adverse events (AEs) and local and systemic reactions. RESULTS: Overall, 698 and 304 subjects enrolled in the persistence and booster stages, respectively. hSBA titers declined in all groups during 12 months postprimary vaccination, then remained stable through 48 months. One month postbooster, 93.4-100.0% of subjects achieved hSBA titers ≥ lower limit of quantitation against each test strain; percentages at 12 and 26 months postbooster were higher than at similar time points following primary vaccination. Primary and booster MenB-FHbp vaccinations were well tolerated, with ≤ 12.5% of subjects reporting AEs during each stage. The most common local (reported by 84.4-93.8% of subjects) and systemic (68.8-76.6%) reactions to the booster were injection site pain and fatigue and headache, respectively; ≤ 3.7% of subjects reported severe systemic events. CONCLUSION: Protective hSBA titers initially declined but were retained by many subjects for 4 years irrespective of primary MenB-FHbp vaccination schedule. Boosting at 48 months after primary vaccination was safe, well tolerated, and induced immune responses indicative of immunological memory that persisted through 26 months. Booster vaccination during late adolescence may prolong protection against MenB disease.

Broad vaccine coverage predicted for a bivalent recombinant factor H binding protein based vaccine to prevent serogroup B meningococcal disease.

Factor H binding proteins (fHBP), are bacterial surface proteins currently undergoing human clinical trials as candidate serogroup B Neisseria meningitidis (MnB) vaccines. fHBP protein sequences segregate into two distinct subfamilies, designated A and B. Here, we report the specificity and vaccine potential of mono- or bivalent fHBP-containing vaccines. A bivalent fHBP vaccine composed of a member of each subfamily elicited substantially broader bactericidal activity against MnB strains expressing heterologous fHBP than did either of the monovalent vaccines. Bivalent rabbit immune sera tested in serum bactericidal antibody assays (SBAs) against a diverse panel of MnB clinical isolates killed 87 of the 100 isolates. Bivalent human immune sera killed 36 of 45 MnB isolates tested in SBAs. Factors such as fHBP protein variant, PorA subtype, or MLST were not predictive of whether the MnB strain could be killed by rabbit or human immune sera. Instead, the best predictor for killing in the SBA was the level of in vitro surface expression of fHBP. The bivalent fHBP vaccine candidate induced immune sera that killed MnB isolates representing the major MLST complexes, prevalent PorA subtypes, and fHBP variants that span the breadth of the fHBP phylogenetic tree. Importantly, epidemiologically prevalent fHBP variants from both subfamilies were killed.

Detection of LP2086 on the cell surface of Neisseria meningitidis and its accessibility in the presence of serogroup B capsular polysaccharide.

The outer membrane protein LP2086, a human factor H binding protein, is undergoing clinical trials as a vaccine against invasive serogroup B meningococcal (MnB) disease. As LP2086 is a surface protein, expression of capsular polysaccharide could potentially limit accessibility of anti-LP2086 antibodies to LP2086 expressed on the surface of bacteria. To determine whether variability in expression levels of the serogroup B capsule (Cap B) might interfere with accessibility of anti-LP2086 antibody binding to LP2086, we evaluated the ability of anti-Cap B and anti-LP2086 antibodies to bind to the surface of 1263 invasive clinical MnB strains by flow cytometry. One of the anti-LP2086 monoclonal antibodies used recognizes virtually all LP2086 sequence variants. Our results show no correlation between the amount of Cap B expressed and the binding of anti-LP2086 antibodies. Furthermore, the susceptibility of MnB bacteria to lysis by anti-LP2086 immune sera was independent of the level of Cap B expressed. The data presented in this paper demonstrates that Cap B does not interfere with the binding of antibodies to LP2086 expressed on the outer membrane of MnB clinical isolates.

Early bacterial dependent induction of inducible nitric oxide synthase (iNOS) in epithelial cells upon transfer of CD45RB(high) CD4(+) T cells in a model for experimental colitis.

BACKGROUND: Both the role of inducible nitric oxide synthase (iNOS) in the development of inflammatory bowel disease (IBD) as well as the molecular details governing its mucosal induction remain unclear. METHODS: In the present study we evaluated the role of the residing intestinal microflora in the induction of epithelial iNOS upon transfer of CD45RB(high) CD4(+) T cells to SCID mice. CB-17 SCID mice were reared with conventional flora (CNV) or germfree CB-17 SCID mice were monoassociated with Helicobacter muridarum, act A(-) mutant Listeria monocytogenes, segmented filamentous bacteria (SFB), or Ochrobactrum anthropi. RESULTS: Within 2 weeks CNV SCID mice injected with CD45RB(high) CD4(+) T cells showed a focal, epithelial iNOS expression on the apical site of villi that preceded the infiltration of CD4(+) T cells and cytokine production followed by extension of this expression to the entire surface along the whole crypt axis as the colitis progressed. SCID mice monoassociated with H. muridarum developed a severe colitis and showed high epithelial iNOS expression. CNV-SCID mice without T cells and SCID mice monoassociated with SFB did not show any iNOS expression, whereas SCID mice monoassociated with act A(-) mutant L. monocytogenes and O. anthropi showed some scattered epithelial iNOS staining on the apical site of a few villi, but none of these mice developed colitis. CONCLUSIONS: These findings demonstrate that the expression of epithelial iNOS is highly bacterium-specific and correlates with the severity of disease, suggesting an important role for this enzyme in the development of IBD.

Recent immune status determines the source of antigens that drive homeostatic T cell expansion.

Homeostatic proliferation of naive T cells transferred to T cell-deficient syngeneic mice is driven by low-affinity self-MHC/peptide ligands and the cytokine IL-7. In addition to homeostatic proliferation, a subset of naive T cells undergoes massive proliferation in chronically immunodeficient hosts, but not in irradiated normal hosts. Such rapid T cell proliferation occurs largely independent of homeostatic factors, because it was apparent in the absence of IL-7 and in T cell-sufficient hosts devoid of functional T cell immunity. Strikingly, immunodeficient mice raised under germfree conditions supported only slow homeostatic proliferation, but not the marked T cell proliferation observed in conventionally raised immunodeficient mice. Thus, polyclonal naive T cell expansion in T cell-deficient hosts can be driven predominantly by either self-Ags or foreign Ags depending on the host's previous state of T cell immunocompetency.

Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts.

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (V(H)) genes by H-CDR3 spectrotyping, cloning, and sequencing of V(H) genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of V(H) genes and multiple clonally related sequences. The restricted usage of V(H) genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed V(H) gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer's patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.

Bacterial colonization leads to the colonic secretion of RELMbeta/FIZZ2, a novel goblet cell-specific protein.

BACKGROUND & AIMS: Goblet cells are highly polarized exocrine cells found throughout the small and large intestine that have a characteristic morphology due to the accumulation of apical secretory granules. These granules contain proteins that play important physiologic roles in cellular protection, barrier function, and proliferation. A limited number of intestinal goblet cell-specific proteins have been identified. In this study, we investigate the expression and regulation of RELMbeta, a novel colon-specific gene. METHODS: The regulation of RELMbeta messenger RNA expression was determined in LS174T, Caco-2, and HT-29 cell lines in response to stimulation with interleukin 13 and lipopolysaccharide. Quantitative reverse-transcription polymerase chain reaction, immunoblots, and immunohistochemistry were used to examine the expression of RELMbeta in BALB/c and C.B17.SCID mice housed in conventional, germ-free, and gnotobiotic environments. RESULTS: Messenger RNA for RELMbeta is restricted to the undifferentiated, proliferating colonic epithelium. Immunohistochemistry shows that this protein is expressed in goblet cells located primarily in the distal half of the colon and cecum with lower levels detectable in the proximal colon. High levels of RELMbeta can be detected in the stool of mice and humans, where it exists as a homodimer under nonreducing conditions. Interestingly, the secretion of RELMbeta is dramatically reduced in germ-free mice. Furthermore, introduction of germ-free mice into a conventional environment results in enhanced expression and robust secretion of RELMbeta within 48 hours. CONCLUSIONS: These studies define a new goblet cell-specific protein and provide the first evidence that colon-specific gene expression can be regulated by colonization with normal enteric bacteria.

Distinct mechanisms for cross-protection of the upper versus lower respiratory tract through intestinal priming.

A main feature of the common mucosal immune system is that lymphocytes primed in one mucosal inductive site may home to distant mucosal effector sites. However, the mechanisms responsible for such cross-protection remain elusive. To address these we have used a model of local mucosal infection of mice with reovirus. In immunocompetent mice local duodenal priming protected against subsequent respiratory challenge. In the upper respiratory tract this protection appeared to be mainly mediated by specific IgA- and IgG2a-producing B cells, whereas ex vivo active effector memory CTL were found in the lower respiratory tract. In accordance with these findings, clearance of reovirus from the lower respiratory tract, but not from the upper respiratory tract, of infected SCID mice upon transfer of gut-primed lymphocytes depended on the presence of T cells. Taken together this study reveals that intestinal priming leads to protection of both the upper and lower respiratory tracts, however through distinct mechanisms. We suggest that cross-protection in the common mucosal immune system is mediated by trafficking of B cells and effector memory CTL.

Monoassociation of SCID mice with Helicobacter muridarum, but not four other enterics, provokes IBD upon receipt of T cells.

BACKGROUND & AIMS: Recently, a number of animal models for different aspects of inflammatory bowel disease (IBD) have been developed. The aim of this study was to use one of these to determine whether particular, ostensibly innocuous, intestinal bacteria could provoke or exacerbate IBD. METHODS: Conventionally reared C.B17 SCID mice were compared with germ-free and gnotobiotic mice, monoassociated with 1 of 5 intestinal bacteria, after transfer of CD45RB(high) CD4(+) T cells from conventionally reared congenic BALB/c mice. Recipient mice were monitored over 7-12 weeks for clinical signs of IBD, and tissues were analyzed by histology/flow cytometry for abnormal inflammation and CD4(+) T cell outgrowth. RESULTS: Neither germ-free mice nor mice monoassociated with segmented filamentous bacteria, Ochrobactrum anthropi, a nonpathogenic mutant of Listeria monocytogenes, or Morganella morganii developed any signs of IBD. In contrast, mice monoassociated with Helicobacter muridarum displayed an accelerated development of IBD in 5-6 weeks compared with 8-12 weeks observed in conventionally reared mice. The outgrowth of CD4(+) T cells in spleen and large intestine of H. muridarum monoassociated mice, as well as in conventionally reared mice was significantly higher than that in the other monoassociated mice. CONCLUSIONS: Among the intestinal bacteria tested, H. muridarum can serve as a provocateur of IBD in this model.