Levels Of Antigen-specific IgG1 Research Articles

Evaluation of mRNA lipoplexes prepared using modified ethanol injection method as a tumour vaccine

We have previously demonstrated that messenger RNA (mRNA) lipoplexes composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and polyethylene glycol-cholesteryl ether (PEG-Chol) exhibited high protein expression in the lungs and spleen of mice after intravenous injection and induced high levels of antigen-specific IgG1 upon immunisation. In this study, we optimised PEG modification in mRNA lipoplexes to reduce mRNA accumulation in the lungs and evaluated the suppression of tumour growth in mice bearing mouse lymphoma E.G7-ovalbumin (OVA) tumours by immunising them with an intravenous injection of OVA mRNA lipoplexes. PEGylation of mRNA lipoplexes with 3 mol% PEG-Chol (LP-DC-1-16-3PCL) prevented agglutination of erythrocytes and reduced accumulation in the lungs. Intravenous injection of LP-DC-1-16-3PCL lipoplexes containing OVA mRNA into mice induced high levels of anti-OVA IgG1 (83,000 mU/mL) in serum, and exhibited a high cytotoxic activity (97%) against E.G7-OVA cells by the splenocytes of mice. Furthermore, immunisation with LP-DC-1-16-3PCL lipoplexes containing OVA mRNA suppressed E.G7-OVA tumour growth compared to control mRNA. Based on these results, LP-DC-1-16-3PCL lipoplexes may be an effective mRNA vaccine for inducing antibody- and cytotoxic cell-mediated immune responses to tumours through intravenous injection.

Recombinant bacteriophage T4 displaying key epitopes of the foot-and-mouth disease virus as a novel nanoparticle vaccine

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that has caused significant economic losses in the livestock industry. Peptide vaccines engineered with the protective epitopes of FMDV have provided a safer alternative for disease prevention than the traditional inactivated vaccines. However, the immunogenicity of the peptide is usually poor and therefore an adjuvant is required. Here, we showed that recombinant T4 phages displaying the B-cell epitope of the FMDV VP1 protein (VP1130–158), without additional adjuvants, induced similar levels of antigen-specific IgG1 but higher levels of IgG2a compared to the peptide vaccine. Incorporation of a CD4+ T cell epitope, either 3A21–35 of FMDV 3A protein or P2830–844 of tetanus toxoid, further enhanced the immunogenicity of VP1-T4 phage nanoparticles. Interestingly, the extrinsic adjuvant cannot enhance the immunogenicity of the nanoparticles, indicating the intrinsic adjuvant activities of T4 phage. Furthermore, the recombinant T4 phage can be produced on a large scale within a short period of time at a relatively low-cost using Escherichia coli, heralding its potential in the development of a safe and effective FMDV vaccine.

Efficient mRNA Delivery with mRNA Lipoplexes Prepared Using a Modified Ethanol Injection Method.

Messenger RNA (mRNA)-based therapies are a novel class of therapeutics used in vaccination and protein replacement therapies for monogenic diseases. Previously, we developed a modified ethanol injection (MEI) method for small interfering RNA (siRNA) transfection, in which cationic liposome/siRNA complexes (siRNA lipoplexes) were prepared by mixing a lipid-ethanol solution with a siRNA solution. In this study, we applied the MEI method to prepare mRNA lipoplexes and evaluated the in vitro and in vivo protein expression efficiencies. We selected six cationic lipids and three neutral helper lipids to generate 18 mRNA lipoplexes. These were composed of cationic lipids, neutral helper lipids, and polyethylene glycol-cholesteryl ether (PEG-Chol). Among them, mRNA lipoplexes containing N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16) or 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl) propan-2-yl) amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12) with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and PEG-Chol exhibited high protein expression in cells. Furthermore, mRNA lipoplexes composed of DC-1-16, DOPE, and PEG-Chol exhibited high protein expression in the lungs and spleen of mice after systemic injection and induced high antigen-specific IgG1 levels upon immunization. These results suggest that the MEI method can potentially increase the efficiency of mRNA transfection, both in vitro and in vivo.

Vaccine delivery by zwitterionic polysaccharide-based hydrogel microparticles showing enhanced immunogenicity and suppressed foreign body responses.

The controlled release of antigens from injectable depots has been actively pursued to achieve long-lasting immune responses in vaccine development. Nonetheless, subcutaneous depots are often susceptible to foreign body responses (FBRs) dominated by macrophage clearance and fibrotic encapsulation, resulting in limited antigen delivery to target dendritic cells (DCs) that bridge innate and adaptive immunity. Here, we aim to develop a long-term antigen depot that can bypass FBR and engage DCs to mature and migrate to lymph nodes to activate antigen-specific T-cells. Leveraging the immunomodulatory properties of exogenous polysaccharides and the anti-fouling characteristics of zwitterionic phosphorylcholine (PC) polymers, we developed a PC functionalized dextran (PCDX) hydrogel for long-term antigen delivery. We observed that PCDX in both injectable scaffold and microparticle (MP) forms could effectively evade FBR as the anionic carboxymethyl DX (CMDX) in vitro and in vivo. Meanwhile, PCDX provided slower and longer release of antigens than CMDX, resulting in local enrichment of CD11c+ DCs at the MP injection sites. DC cultured on PCDX exhibited stronger immunogenic activation with higher CD86, CD40, and MHC-I/peptide complex than CMDX. PCDX also generated DC with greater propensity in migration to lymph nodes, as well as antigen presentations to trigger both CD4+ and CD8+ arms of T-cell responses, as compared to other charge derivatives of DX. Besides cellular responses, PCDX could also induce more durable and potent humoral responses, with higher levels of antigen specific IgG1 and IgG2a by day 28, as compared to other treatment groups. In conclusion, PCDX can incorporate the benefits of both immunogenic DX and anti-fouling properties of zwitterionic PC and thus, shows great promise in providing long-term delivery of antigens for vaccine development.

A Vaccine Based on Kunitz-Type Molecule Confers Protection Against Fasciola hepatica Challenge by Inducing IFN-γ and Antibody Immune Responses Through IL-17A Production.

Fasciola hepatica is helminth parasite found around the world that causes fasciolosis, a chronic disease affecting mainly cattle, sheep, and occasionally humans. Triclabendazole is the drug of choice to treat this parasite. However, the continuous use of this drug has led to the development of parasite resistance and, consequently, the limitation of its effectiveness. Hence, vaccination appears as an attractive option to develop. In this work, we evaluated the potential of F. hepatica Kunitz-type molecule (FhKTM) as an antigen formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate ester (Coa-ASC16) and the synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) during an experimental model of fasciolosis in mice, and we further dissected the immune response associated with host protection. Our results showed that immunization of mice with FhKTM/CpG-ODN/Coa-ASC16 induces protection against F. hepatica challenge by preventing liver damage and improving survival after F. hepatica infection. FhKTM/CpG-ODN/Coa-ASC16-immunized mice elicited potent IFN-γ and IL-17A with high levels of antigen-specific IgG1, IgG2a, and IgA serum antibodies. Strikingly, IL-17A blockade during infection decreased IgG2a and IgA antibody levels as well as IFN-γ production, leading to an increase in mortality of vaccinated mice. The present study highlights the potential of a new vaccine formulation to improve control and help the eradication of F. hepatica infection, with potential applications for natural hosts such as cattle and sheep.

Innate signalling molecules as genetic adjuvants do not alter the efficacy of a DNA-based influenza A vaccine.

In respect to the heterogeneity among influenza A virus strains and the shortcomings of current vaccination programs, there is a huge interest in the development of alternative vaccines that provide a broader and more long-lasting protection. Gene-based approaches are considered as promising candidates for such flu vaccines. In our study, innate signalling molecules from the RIG-I and the NALP3 pathways were evaluated as genetic adjuvants in intramuscular DNA immunizations. Plasmids encoding a constitutive active form of RIG-I (cRIG-I), IPS-1, IL-1β, or IL-18 were co-administered with plasmids encoding the hemagglutinin and nucleoprotein derived from H1N1/Puerto Rico/8/1934 via electroporation in BALB/c mice. Immunogenicity was analysed in detail and efficacy was demonstrated in homologous and heterologous influenza challenge experiments. Although the biological activities of the adjuvants have been confirmed by in vitro reporter assays, their single or combined inclusion in the vaccine did not result in superior vaccine efficacy. With the exception of significantly increased levels of antigen-specific IgG1 after the co-administration of IL-1β, there were only minor alterations concerning the immunogenicity. Since DNA electroporation alone induced substantial inflammation at the injection site, as demonstrated in this study using Mx2-Luc reporter mice, it might override the adjuvants´ contribution to the inflammatory microenvironment and thereby minimizes the influence on the immunogenicity. Taken together, the DNA immunization was protective against subsequent challenge infections but could not be further improved by the genetic adjuvants analysed in this study.

Blood transcriptome profile induced by an efficacious vaccine formulated with salivary antigens from cattle ticks

Ticks cause massive damage to livestock and vaccines are one sustainable alternative for the acaricide poisons currently heavily used to control infestations. An experimental vaccine adjuvanted with alum and composed by four recombinant salivary antigens mined with reverse vaccinology from a transcriptome of salivary glands from Rhipicephalus microplus ticks was previously shown to present an overall efficacy of 73.2% and cause a significant decrease of tick loads in artificially tick-infested, immunized heifers; this decrease was accompanied by increased levels of antigen-specific IgG1 and IgG2 antibodies, which were boosted during a challenge infestation. In order to gain insights into the systemic effects induced by the vaccine and by the tick challenge we now report the gene expression profile of these hosts’ whole-blood leukocytes with RNA-seq followed by functional analyses. These analyses show that vaccination induced unique responses to infestations; genes upregulated in the comparisons were enriched for processes associated with chemotaxis, cell adhesion, T-cell responses and wound repair. Blood transcriptional modules were enriched for activation of dendritic cells, cell cycle, phosphatidylinositol signaling, and platelets. Together, the results indicate that by neutralizing the tick’s salivary mediators of parasitism with vaccine-induced antibodies, the bovine host is able to mount normal homeostatic responses that hinder tick attachment and haematophagy and that the tick otherwise suppresses with its saliva.

Immunoadjuvant activity in mice of polysaccharides isolated from the leaves of Panax ginseng C.A. Meyer

Our previous study showed polysaccharide (GS-P) isolated from the leaves of Panax ginseng C.A. Meyer possessed anti-tumor metastatic activity in mouse model. In this study, we evaluated the immunoadjuvant effect of GS-P on the induction of humoral and cellular immune responses against ovalbumin (OVA) in mice. When mice were immunized subcutaneously with OVA admixed with or without GS-P, the OVA+GS-P group showed significantly higher antibody production than the group immunized with OVA alone. This suggests that GS-P has the ability to enhance the adaptive immune response. In addition, the OVA+GS-P+FIA (Freund’s incomplete adjuvant) group induced higher levels of antigen-specific IgG1 and IgG2b antibodies than the OVA+FIA group. The culture supernatant obtained from the splenocytes of mice immunized with OVA+GS-P+FIA showed higher levels of OVA-specific Th1-type (IL-2, IFN-γ, GM-CSF) and Th2-type (IL-10) cytokines. Following in vitro analysis of T cell proliferation, the splenocytes of mice treated with OVA+GS-P+FIA showed significantly more proliferation than those treated with OVA+FIA. Further, the production of IgE antibody was dramatically reduced when OVA+GS-P+FIA was used to immunize mice rather than OVA+FIA or OVA+FCA (Freund’s complete adjuvant). Collectively, these results suggest that GS-P may possess adjuvant activity that potentially enhances humoral as well as cellular immune responses.

A TNFRSF14-FcɛRI-mast cell pathway contributes to development of multiple features of asthma pathology in mice.

Asthma has multiple features, including airway hyperreactivity, inflammation and remodelling. The TNF superfamily member TNFSF14 (LIGHT), via interactions with the receptor TNFRSF14 (HVEM), can support TH2 cell generation and longevity and promote airway remodelling in mouse models of asthma, but the mechanisms by which TNFSF14 functions in this setting are incompletely understood. Here we find that mouse and human mast cells (MCs) express TNFRSF14 and that TNFSF14:TNFRSF14 interactions can enhance IgE-mediated MC signalling and mediator production. In mouse models of asthma, TNFRSF14 blockade with a neutralizing antibody administered after antigen sensitization, or genetic deletion of Tnfrsf14, diminishes plasma levels of antigen-specific IgG1 and IgE antibodies, airway hyperreactivity, airway inflammation and airway remodelling. Finally, by analysing two types of genetically MC-deficient mice after engrafting MCs that either do or do not express TNFRSF14, we show that TNFRSF14 expression on MCs significantly contributes to the development of multiple features of asthma pathology.

Analysis of the protective immune response following intramuscular vaccination of calves against the intestinal parasite Cooperia oncophora

Recently we reported the successful vaccination of calves against Cooperia oncophora with a double domain activation-associated secreted protein, purified from the excretory-secretory material of adult stage parasites. In an attempt to elucidate the immune mechanisms involved in protection, the humoral and cell-mediated immune responses following vaccination and infection were compared with non-vaccinated control animals. Antigen-specific IgG1, IgG2 and IgA levels were significantly increased in sera of vaccinated animals post vaccination, whereas no effect was observed for IgM. Antigen-specific intestinal IgG1 levels were significantly increased in the vaccinated animals, whereas no differences were observed for antigen-specific IgA, IgM and IgG2 levels. Upon re-stimulation in vitro with the vaccine antigen, a significant proliferation of both αβ- and γδ-T cells, and B cells, collected from mesenteric lymph nodes, was only observed in vaccinated animals. RNA-seq analysis of intestinal tissue yielded a list of 67 genes that were differentially expressed in vaccinated animals following challenge infection, amongst which were several cell adhesion molecules, lectins and glycosyl transferases. A correlation analysis between all immunological and parasitological parameters indicated that intestinal anti-double domain activation-associated secreted protein IgG1 levels correlated negatively with cumulative faecal egg counts and positively with the proportion of L4s and L5s. The proportion of immature stages was also positively correlated with the proliferation of αβ T cells. Worm length was negatively correlated with the transcript levels of several lectins and cell adhesion molecules. Overall, the results indicate that intramuscular administration of the vaccine resulted in an immune memory response particularly characterised by increased antigen-specific IgG1 levels in the intestinal mucosa.

IgG1 as a potential biomarker of post-chemotherapeutic relapse in visceral leishmaniasis, and adaptation to a rapid diagnostic test.

BackgroundVisceral leishmaniasis (VL), caused by protozoa of the Leishmania donovani complex, is a widespread parasitic disease of great public health importance; without effective chemotherapy symptomatic VL is usually fatal. Distinction of asymptomatic carriage from progressive disease and the prediction of relapse following treatment are hampered by the lack of prognostic biomarkers for use at point of care.Methodology/Principal FindingsAll IgG subclass and IgG isotype antibody levels were determined using unpaired serum samples from Indian and Sudanese patients with differing clinical status of VL, which included pre-treatment active VL, post-treatment cured, post-treatment relapsed, and post kala-azar dermal leishmaniasis (PKDL), as well as seropositive (DAT and/or rK39) endemic healthy controls (EHCs) and seronegative EHCs. L. donovani antigen-specific IgG1 levels were significantly elevated in relapsed versus cured VL patients (p<0.0001). Using paired Indian VL sera, consistent with the known IgG1 half-life, IgG1 levels had not decreased significantly at day 30 after the start of treatment (p = 0.8304), but were dramatically decreased by 6 months compared to day 0 (p = 0.0032) or day 15 (p<0.0001) after start of treatment. Similarly, Sudanese sera taken soon after treatment did not show a significant change in the IgG1 levels (p = 0.3939). Two prototype lateral flow immunochromatographic rapid diagnostic tests (RDTs) were developed to detect IgG1 levels following VL treatment: more than 80% of the relapsed VL patients were IgG1 positive; at least 80% of the cured VL patients were IgG1 negative (p<0.0001).Conclusions/SignificanceSix months after treatment of active VL, elevated levels of specific IgG1 were associated with treatment failure and relapse, whereas no IgG1 or low levels were detected in cured VL patients. A lateral flow RDT was successfully developed to detect anti-Leishmania IgG1 as a potential biomarker of post-chemotherapeutic relapse.

Inducible Deletion of CD28 Prior to Secondary Nippostrongylus brasiliensis Infection Impairs Worm Expulsion and Recall of Protective Memory CD4+ T Cell Responses

IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28−/− mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28−/loxCre+/−+TM) to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28−/− mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28−/loxCre+/− mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28−/− mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5+ TFH cell population. Furthermore, total number of CD4+ T cells and B220+ B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28−/− mice and tamoxifen treated CD28−/loxCre+/− mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4+ T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28−/loxCre+/− mice. Therefore, it can be concluded that CD28 costimulation is essential for conferring host protection during secondary N. brasiliensis infection.

Protection against experimental melioidosis following immunisation with a lipopolysaccharide-protein conjugate.

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is refractory to antibiotic treatment and there is currently no licensed vaccine. In this report we detail the construction and protective efficacy of a polysaccharide-protein conjugate composed of B. pseudomallei lipopolysaccharide and the Hc fragment of tetanus toxin. Immunisation of mice with the lipopolysaccharide-conjugate led to significantly reduced bacterial burdens in the spleen 48 hours after challenge and afforded significant protection against a lethal challenge with B. pseudomallei. The conjugate generated significantly higher levels of antigen-specific IgG1 and IgG2a than in lipopolysaccharide-immunised mice. Immunisation with the conjugate also demonstrated a bias towards Th1 type responses, evidenced by high levels of IgG2a. In contrast, immunisation with unconjugated lipopolysaccharide evoked almost no IgG2a demonstrating a bias towards Th2 type responses. This study demonstrates the effectiveness of this approach in the development of an efficacious and protective vaccine against melioidosis.

Antiasthmatic effects of schizandrae fructus extract in mice with asthma.

Background:Schizandrae fructus (SF), the fruit of Schisandra chinensis, has been used for the treatment of cough, wheezing, dry mouth, hepatitis, cardiovascular disease, and as a tonic and astringent in China, Japan, and Korea.Objective:Investigation of the antiasthmatic effects of SF.Materials and Methods:We investigated the effects of SF on airway hyperresponsiveness (AHR) to methacholine, production levels of antigen-specific antibodies, and histopathological changes in the lung tissue in a mouse model (Balb/c) of asthma induced by repeated intranasal instillation of an antigen.Results:SF lowered AHR to methacholine (P < 0.05), antigen-specific immunoglobulin E (IgE) level (P < 0.01), and immune cell infiltration in mice with asthma. Prednisolone (PD) effectively decreased AHR (P < 0.01), total antibody (P < 0.01) and IgE (P < 0.01) levels, and immune cell infiltration. SF and PD did not affect the levels of antigen-specific IgG1 and IgG2a antibodies.Conclusion:Our data suggest that SF has possible application as an antiasthmatic drug. We also suggest that SF could be used as a complementary or alternative medicine to glucocorticoids.

Renal Ischemia-Reperfusion Injury Amplifies the Humoral Immune Response

Renal transplant recipients who experience delayed graft function have increased risks of rejection and long-term graft failure. Ischemic damage is the most common cause of delayed graft function, and although it is known that tissue inflammation accompanies renal ischemia, it is unknown whether renal ischemia affects the production of antibodies by B lymphocytes, which may lead to chronic humoral rejection and allograft failure. Here, mice immunized with a foreign antigen 24-96 hours after renal ischemia-reperfusion injury developed increased levels of antigen-specific IgG1 compared with sham-treated controls. This amplified IgG1 response did not follow unilateral ischemia, and it did not occur in response to a T-independent antigen. To test whether innate immune activation in the kidney after ischemia affects the systemic immune response to antigen, we repeated the immunization experiment using mice deficient in factor B that lack a functional alternative pathway of complement. Renal ischemia-reperfusion injury did not cause amplification of the antigen-specific antibodies in these mice, suggesting that the increased immune response requires a functional alternative pathway of complement. Taken together, these data suggest that ischemic renal injury leads to a rise in antibody production, which may be harmful to renal allografts, possibly explaining a mechanism underlying the link between delayed graft function and long-term allograft failure.

Interleukin-18 enhances a Th2 biased response and susceptibility to Leishmania mexicana in BALB/c mice

Interleukin-18 deficient mice on a BALB/c background display increased resistance to cutaneous infection with Leishmania mexicana, with reduced lesion progression and reduced parasite burdens compared with wild-type mice. Infected IL-18 −/− mice had lower antigen specific IgG1 levels and total IgE levels and conversely higher antigen specific IgG2a levels than similarly infected wild-type mice. Splenocytes isolated from infected IL-18 −/− mice produced significantly lower levels of antigen induced IL-4 and higher levels of IFN-γ than wild-type animals. Consequently IL-18 during L. mexicana infection of BALB/c mice promotes a Th2 biased response and thereby has a disease exacerbating role.

A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-γ and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H 37Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.

Oral-tolerance induction in diet-induced obese mice

Objective: It is known that immune functions are altered in various ways by obesity. However, changes in the intestinal immune system resulting from obesity remain poorly understood. Oral tolerance is a system that suppresses antigen specific immune responses to orally administrated antigens. The intestinal immune system is intimately associated with the oral tolerance system, that acts to prevent allergic and inflammatory diseases. In this study we investigated the effect of obesity on induction of oral tolerance to ovalbumin (OVA) in an animal model of obesity. Research methods and procedures: Obese mice induced by a high fat diet and control mice were allowed free access for 3 days to a 1%-ovalbumin (OVA) solution in drinking water. After continuous feeding of the antigen, all the mice were immunized by two intraperitoneal injections of OVA administered 7 days apart. Results: In the control mice, induction of oral tolerance caused an increase in antigen specific IgG1 levels and a decrease in IgG2a levels. In contrast, the IgG1/IgG2a ratio was reversed in obese mice. OVA-specific IL-2 production was suppressed by antigen feeding in both the control and obese mice; however, suppression of OVA-specific IL-10 was observed only in the control mice. Although OVA-specific IgA and IgM were not affected by antigen feeding, the obese groups of mice had significantly lower titers of antibodies. Discussion: These findings suggest that obesity may affect induction of oral tolerance following antigen feeding and that these changes may be related to the inflammatory reaction.

Endothelial cells present antigens in vivo

BackgroundImmune recognition of vascular endothelial cells (EC) has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli β-galactosidase (β-gal) in two lines of transgenic mice that express β-gal exclusively in their EC. TIE2-lacZ mice express β-gal in all EC and VWF-lacZ mice express β-gal in heart and brain microvascular EC.ResultsTransgenic and congenic wild type FVB mice immunized with β-gal expression vector DNA or β-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express β-gal, and their blood vessels showed no histological abnormalities. In response to β-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-γ. Infection with recombinant vaccinia virus encoding β-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express β-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost β-gal+ EC and the hosts developed β-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained β-gal+ EC and no antisera developed, suggesting a tolerant host immune system.ConclusionResting, β-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against β-gal expressed by EC within transplanted skin. We conclude that EC effectively present intracellular "self" proteins to the immune system. However, antigen presentation by EC does not delete or anergize a large population of specific lymphocytes that respond to the same protein following conventional immunization with protein or expression vector DNA. These results clearly demonstrate striking context sensitivity in the immune recognition of EC, a subtlety that must be better understood in order to treat immune diseases and complications involving the vasculature.

Antibody responses in pregnancy-induced transmammary transmission of Ancylostoma caninum hookworm larvae

Third stage larvae of the Ancylostoma caninum hookworm nematode have the capacity to infect a dog, abort the normal maturation pathway to become blood-feeding intestinal worms, and instead distribute throughout the body in a developmentally arrested state that is relatively resilient to most chemotherapeutic agents. During pregnancy, a percentage of the arrested larvae reactivate and transmit via the mammary glands to infect the nursing puppies with resulting iron-deficiency anemia and potential mortality. To determine if the suppression of parasite-specific antibody responses during pregnancy facilitates the reactivation and transmammary transfer of hookworm larvae, a murine model of A. caninum infection was used to compare the infected versus uninfected animals that were either bred or not bred. Initial comparisons of genetically divergent BALB/c versus C57BL/6 mice showed that both the strains mounted strong Th2 biased IgG1and IgE antibody responses to A. caninum infection. Using the BALB/c strain for the breeding analyses, it was confirmed that larval transfer to the mouse pups only occurred during the post-partum lactational period. In the dams, levels of total and antigen-specific IgG1 and total IgE were highly correlated with parasite burden. During most phases of pregnancy and lactation, infected dams had lower total IgG1, IgG2a and IgE levels as compared to unbred mice at comparable times post-infection; this downward modulation of antibody responses supports the established dogma of a generalized immunosuppression associated with pregnancy. However, at parturition and post-partum lactation, antigen-specific IgG1 levels measured at 1 : 5000 serum dilutions were comparable between bred and unbred mice, and antigen-specific IgG2a levels at 1 : 100 serum dilutions were also not significantly different except for a marginal reduction in the bred mice at the lactational timepoint. The comparable anti- A. caninum IgG1 levels between bred and unbred mice, and low correlation between IgG2a levels and larval burden suggest that parasite-specific antibody responses do not play a major role in the pregnancy-associated transmammary transmission of A. caninum larvae. This conclusion does not rule out the possibility that underlying fluxes in the levels of specific cytokines associated with pregnancy and infection may be involved in the process of larval reactivation and transmission.