Parenteral and mucosal delivery of a novel multi-epitope M protein-based group A streptococcal vaccine construct: investigation of immunogenicity in mice

Primary vaccine strategies against group A streptococci (GAS) have focused on the M protein-the target of opsonic antibodies important for protective immunity. We have previously reported protection of mice against GAS infection following parenteral delivery of a multi-epitope vaccine construct, referred to as a heteropolymer. This current report has assessed mucosal (intranasal (i.n.) and oral) delivery of the heteropolymer in mice with regard to the induction and specificity of mucosal and systemic antibody responses, and compared this to parenteral delivery. GAS-specific IgA responses were detected in saliva and gut upon i.n. and oral delivery of the heteropolymer co-administered with cholera toxin B subunit, respectively. High titre serum IgG responses were elicited to the heteropolymer following all routes of delivery when administered with adjuvant. Moreover, as with parenteral delivery, serum IgG antibodies were detected to the individual heteropolymer peptides following i.n. but not oral delivery. These data support the potential of the i.n. route in the mucosal delivery of a GAS vaccine. (C) 2002 Elsevier Science Ltd. All rights reserved.

Systemic administration of interleukin-1 beta activates select populations of central amygdala afferents

The central nucleus of the amygdala (CeA) is activated robustly by an immune challenge such as the systemic administration of the proinflammatory cytokine interleukin-1beta (IL-1beta). Because IL-1beta is not believed to cross the blood-brain barrier in any significant amount, it is likely that IL-1beta elicits CeA cell recruitment by means of activation of afferents to the CeA. However, although many studies have investigated the origins of afferent inputs to the CeA, we do not know which of these also respond to IL-1beta. Therefore, to identify candidate neurons responsible for the recruitment of CeA cells by an immune challenge, we iontophoretically deposited a retrograde tracer, cholera toxin b-subunit (CTb), into the CeA of rats 7 days before systemic delivery of IL-1beta (1 mug/kg, i.a.). By using combined immunohistochemistry, we then quantified the number of Fos-positive CTb cells in six major regions known to innervate the CeA. These included the medial prefrontal cortex, paraventricular thalamus (PVT), ventral tegmental area, parabrachial nucleus (PB), nucleus tractus solitarius, and ventrolateral medulla. Our results show that after deposit of CTb into the CeA, the majority of double-labeled cells were located in the PB and the PVT, suggesting that CeA cell activation by systemic IL-1beta is likely to arise predominantly from cell bodies located in these regions. These findings may have significant implications in determining the central pathways involved in generating acute central responses to a systemic immune challenge. J. Comp. Neurol. 452:288-296, 2002. (C) 2002 Wiley-Liss, Inc.

Induction of immune response in broiler chickens immunized with recombinant FliC and challenged by Salmonella Typhimurium

The study examined (1) the immune response in broiler chickens after oral immunization with recombinant flagellin (rFliC) from Salmonella Typhimurium conjugated with sodium alginate microparticles, and the immune response enhancement in association with recombinant cholera toxin B subunit protein (rCTB) and pool of Lactobacillus spp. (PL). The immune responses were evaluated by dosage of IgY serum and IgA from intestinal fluid and immunostaining of CD8+ T lymphocytes in the cecum. The immunized animals were challenged with Salmonella Typhimurium (ST) 21 days after treatment. In all immunized groups, a significant increase (p<0.05) was observed in IgA levels (μg/mL), especially three weeks after immunization. The serum IgY levels (μg/mL) were little affected by the treatments and differed significantly among groups only in the second post-immunization week (p<0.05). After the challenge, the number of CD8+ T cells differed significantly between the treatments and negative control. Retrieval of Salmonella Typhimurium was not detected at 48 hours after the challenge in T2 (rFliC+rCTb), T3 (rFliC+PL) and T4 (rFliC+rCTB PL). The rFliC administered orally with or without rCTB and Lactobacillus spp. produces significant induction of humoral immune response, and the immunized chickens were more effective in eliminating Salmonella after challenge.

Evidence of reciprocal connections between the dorsal raphe nucleus and the retina in the monkey Cebus apella

Possible connections between the retina and the raphe nuclei were investigated in the monkey Cebus apella by intraocular injection of cholera toxin B subunit (CTb). CTb-positive fibers were seen in the lateral region of the dorsal raphe nucleus (DR) on the side contralateral to the injection, and a few labeled perikarya were observed in the lateral portion of the DR on the ipsilateral side. Our findings suggest that direct and reciprocal connections between the retina and DR may exist in Cebus apella. These connections might be part of an important pathway through which the light/dark cycle influences the Activity and/or functional status of raphe neurons, with potential effects on a broad set of neural and behavioral circuits. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Centros subcorticais dos sistemas visual primário e óptico acessório no mocó (kerodon rupestris): caracterização pela projeção retiniana e citoarquitetura.

The primary and accessory optic systems comprise two set of retinorecipient neural clusters. In this study, these visual related centers in the rock cavy were evaluated by using the retinal innervations pattern and Nissl staining cytoarchigtecture. After unilateral intraocular injection of cholera toxin B subunit and immunohistochemical reaction of coronal and sagittal sections from the diencephalon and midbrain region of rock cavy. Three subcortical centres of primary visual system were identified, superior colliculus, lateral geniculate complex and pretectal complex. The lateral geniculate complex is formed by a series of nuclei receiving direct visual information from the retina, dorsal lateral geniculate nucleus, intergeniculate leaflet and ventral lateral geniculate nucleus. The pretectal complex is formed by series of pretectal nuclei, medial pretectal nucleus, olivary pretectal nucleus, posterior pretectal nucleus, nucleus of the optic tract and anterior pretectal nucleus. In the accessory optic system, retinal terminals were observed in the dorsal terminal, lateral terminal and medial terminal nuclei as well as in the interstitial nucleus of the superior fasciculus, posterior fibres. All retinorecipient nuclei received bilateral input, with a contralateral predominance. This is the first study of this nature in the rock cavy and the results are compared with the data obtained for other species. The investigation represents a contribution to the knowledge regarding the organization of visual optic systems in relation to the biology of species.

Evidence of reciprocal connections between the dorsal raphe nucleus and the retina in the monkey Cebus apella

Possible connections between the retina and the raphe nuclei were investigated in the monkey Cebus apella by intraocular injection of cholera toxin B subunit (CTb). CTb-positive fibers were seen in the lateral region of the dorsal raphe nucleus (DR) on the side contralateral to the injection, and a few labeled perikarya were observed in the lateral portion of the DR on the ipsilateral side. Our findings suggest that direct and reciprocal connections between the retina and DR may exist in Cebus apella. These connections might be part of an important pathway through which the light/dark cycle influences the Activity and/or functional status of raphe neurons, with potential effects on a broad set of neural and behavioral circuits. (c) 2007 Elsevier B.V. All rights reserved.

Haemagglutination induced by Bordetella pertussis filamentous haemagglutinin adhesin (FHA) is inhibited by antibodies produced against FHA(430-873) fragment expressed in Lactobacillus casei

Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough.

Induction of immune response in broiler chickens immunized with recombinant FliC and challenged by Salmonella Typhimurium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat

The mesopontine rostromedial tegmental nucleus (RMTg) is a mostly ?-aminobutyric acid (GABA)ergic structure believed to be a node for signaling aversive events to dopamine (DA) neurons in the ventral tegmental area (VTA). The RMTg receives glutamatergic inputs from the lateral habenula (LHb) and sends substantial GABAergic projections to the VTA, which also receives direct projections from the LHb. To further specify the topography of LHb projections to the RMTg and VTA, small focal injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin were aimed at different subdivisions of the LHb. The subnuclear origin of LHb inputs to the VTA and RMTg was then confirmed by injections of the retrograde tracer cholera toxin subunit b into the VTA or RMTg. Furthermore, we compared the topographic position of retrogradely labeled neurons in the RMTg resulting from VTA injections with that of anterogradely labeled axons emerging from the LHb. As revealed by anterograde and retrograde tracing, LHb projections were organized in a strikingly topographic manner, with inputs to the RMTg mostly arising from the lateral division of the LHb (LHbL), whereas inputs to the VTA mainly emerged from the medial division of the LHb (LHbM). In the RMTg, profusely branched LHb axons were found in close register with VTA projecting neurons and were frequently apposed to the latter. Overall, our findings demonstrate that LHb inputs to the RMTg and VTA arise from different divisions of the LHb and provide direct evidence for a disynaptic pathway that links the LHbL to the VTA via the RMTg. J. Comp. Neurol. 520:12781300, 2012. (C) 2011 Wiley Periodicals, Inc.

Reorganization of CA3 area of the mouse hippocampus after pilocarpine induced temporal lobe epilepsy with special reference to the CA3-septum pathway

We showed that when CA3 pyramidal neurons in the caudal 80% of the dorsal hippocampus had almost disappeared completely, the efferent pathway of CA3 was rarely detectable. We used the mouse pilocarpine model of temporal lobe epilepsy (TLE), and injected iontophoretically the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L) into gliotic CA3, medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei, or the retrograde tracer cholera toxin B subunit (CTB) into gliotic CA3 area of hippocampus. In the afferent pathway, the number of neurons projecting to CA3 from medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei increased significantly. In the hippocampus, where CA3 pyramidal neurons were partially lost, calbindin, calretinin, parvalbumin immunopositive back-projection neurons from CA1-CA3 area were observed. Sprouting of Schaffer collaterals with increased number of large boutons in both sides of CA1 area, particularly in the stratum pyramidale, was found. When CA3 pyramidal neurons in caudal 80% of the dorsal hippocampus have almost disappeared completely, surviving CA3 neurons in the rostral 20% of the dorsal hippocampus may play an important role in transmitting hyperactivity of granule cells to surviving CA1 neurons or to dorsal part of the lateral septum. We concluded that reorganization of CA3 area with its downstream or upstream nuclei may be involved in the occurrence of epilepsy.

Passive transfer of IgG anti-GM1 antibodies impairs peripheral nerve repair.

Anti-GM1 antibodies are present in some patients with autoimmune neurological disorders. These antibodies are most frequently associated with acute immune neuropathy called Guillain-Barré syndrome (GBS). Some clinical studies associate the presence of these antibodies with poor recovery in GBS. The patients with incomplete recovery have failure of nerve repair, particularly axon regeneration. Our previous work indicates that monoclonal antibodies can inhibit axon regeneration by engaging cell surface gangliosides (Lehmann et al., 2007). We asked whether passive transfer of human anti-GM1 antibodies from patients with GBS modulate axon regeneration in an animal model. Human anti-GM1 antibodies were compared with other GM1 ligands, cholera toxin B subunit and a monoclonal anti-GM1 antibody. Our results show that patient derived anti-GM1 antibodies and cholera toxin beta subunit impair axon regeneration/repair after PNS injury in mice. Comparative studies indicated that the antibody/ligand-mediated inhibition of axon regeneration is dependent on antibody/ligand characteristics such as affinity-avidity and fine specificity. These data indicate that circulating immune effectors such as human autoantibodies, which are exogenous to the nervous system, can modulate axon regeneration/nerve repair in autoimmune neurological disorders such as GBS.

Medial prefrontal cortex control of the Paraventricular hypothalamic nucleus response to psychological stress: Possible role of the bed nucleus of the stria terminalis

The medial prefrontal cortex (mPFC) has been strongly implicated in control of the paraventricular nucleus of the hypothalamus (PVN) response to stress. Because of the paucity of direct projections from the mPFC to the PVN, we sought to investigate possible brain regions that might act as a relay between the two during psychological stress. Bilateral ibotenic acid lesions of the rat mPFC enhanced the number of Fos-immunoreactive cells seen in the PVN after exposure to the psychological stressor, air puff. Altered neuronal recruitment was seen in only one of the candidate relay populations examined, the ventral bed nucleus of the stria terminalis (vBNST). Furthermore, bilateral ibotenic acid lesions of the BNST caused a significant attenuation of the PVN response to air puff. To better characterize the structural relationships between the mPFC and PVN, retrograde tracing studies were conducted examining Fos expression in cells retrogradely labeled with cholera toxin b subunit (CTb) from the PVN and the BNST. Results obtained were consistent with an important role for both the mPFC and BNST in the mpPVN CRF cell response to air puff. We suggest a set of connections whereby a direct PVN projection from the ipsilateral vBNST is involved in the mpPVN response to air puff and this may, in turn, be modulated by an indirect projection from the mPFC to the BNST. (C) 2004 Wiley-Liss, Inc.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection.

BACKGROUND & AIMS: Infection with Helicobacter induces a T helper type 1 response in mice and humans. Mice can be cured or protected from infection with Helicobacter by mucosal immunization with recombinant H. pylori urease B subunit (rUreB). This study characterizes the immune response of infected mice immunized with rUreB. METHODS: BALB/c mice were infected with H. felis. Two weeks later, they were orally immunized four times with rUreB and cholera toxin (CT) at weekly intervals. Controls were only infected or sham-immunized with CT. Animals were killed at various times after immunization. Splenic CD4(+) cells were obtained and cultured in vitro with rUreB to evaluate antigen-specific proliferation and induction of interferon gamma and interleukin 4 secretion. RESULTS: All rUreB-immunized mice (n = 8) were cured from infection 3 weeks after the fourth immunization. Immunization induced a proliferative response of splenic CD4(+) cells, a progressive decrease in interferon gamma secretion, and a concomitant increase in interleukin 4 secretion after each immunization. A simultaneous increase in rUreB specific serum immunoglobulin G1 levels was observed in infected/immunized mice. CONCLUSIONS: In BALB/c mice, therapeutic mucosal immunization with rUreB induces progressively a Th2 CD4(+) T cell response resulting in the elimination of the pathogen.

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the eft operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the eft operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.