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Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells’ transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication., Author summary The majority of metazoan cells release vesicles of different types and origins, such as exosomes and microvesicles, now collectively termed extracellular vesicles (EVs). EVs have gained much attention because they contain microRNAs (miRNAs) and thus could regulate their specific mRNA targets in recipient or acceptor cells that take up EVs. Using a novel fusion assay with superior sensitivity and specificity, we revisited this claim but found no convincing evidence for an efficient functional uptake of EVs in many different cell lines and primary human blood cells. Even EVs engineered to fuse and deliver their miRNA cargo to recipient cells had no measurable effect on target mRNAs in very carefully controlled, quantitative experiments. Our negative results clearly indicate that EVs do not act as vehicles for miRNA-based cell-to-cell communication.

Infections with the herpes simplex virus (HSV) and the human cytomegalovirus (HCMV) can lead to life-threatening diseases, particularly in immunosuppressed patients. Furthermore, HSV infections at birth (herpes neonatorum) can result in a disseminated disease associated with a fatal multiorgan failure. Congenital HCMV infections can result in miscarriage, serious birth defects or developmental disabilities. Antibody-based interventions with hyperimmunoglobulins showed encouraging results in clinical studies, but clearly need to be improved. The isolation of highly neutralizing monoclonal antibodies is a promising strategy to establish potent therapy options against HSV and HCMV infections. Monoclonal antibodies are commonly isolated from hybridomas or EBV-immortalized B-cell clones. The screening procedure to identify virus-specific cells from a cell mixture is a challenging step, since most of the highly neutralizing antibodies target complex conformational epitopes on the virus surface. Conventional assays such as ELISA are based on purified viral proteins and inappropriate to display complex epitopes. To overcome this obstacle, we have established two full-virus based methods that allow screening for cells and antibodies targeting complex conformational epitopes on viral surface antigens. The methods are suitable to detect surface antigen-specific cells from a cell mixture and may facilitate the isolation of highly neutralizing antibodies against HSV and HCMV.

The intraperitoneal route is favored for administration of inactivated and attenuated vaccines in Atlantic salmon. Nevertheless, the immune responses in the teleost peritoneal cavity (PerC) are still incompletely defined. In this study, we investigated the B cell responses after intraperitoneal Piscirickettsia salmonis (P. salmonis) challenge of Atlantic salmon, focusing on the local PerC response versus responses in the lymphatic organs: spleen and head kidney. We observed a major increase of leukocytes, total IgM antibody secreting cells (ASC), and P. salmonis-specific ASC in the PerC at 3- and 6-weeks post infection (wpi). The increase in ASC frequency was more prominent in the spleen and PerC compared to the head kidney during the observed 6 wpi. The serum antibody response included P. salmonis-specific antibodies and non-specific antibodies recognizing the non-related bacterial pathogen Yersinia ruckeri and the model antigen TNP-KLH. Finally, we present evidence that supports a putative role for the adipose tissue in the PerC immune response.

Current strategies for influenza virus vaccines primarily aim to elicit immune responses towards the globular head domain of the hemagglutinin (HA) protein so that binding of the virus to membrane receptors on the host cells is inhibited. In the present study, we show a novel strategy to generate immunity against the highly conserved region of the influenza virus. The globular head domain was replaced by different linkers to generate a headless HA (stalk domain) and then coexpressed with influenza M1 proteinin Tni insect cells. The expression was validated by western blot analysis, and stalk domain with peptides (GGGGS)4 linkers was identified to anchor in a stable way to the cell membrane. An immunoelectron microscope showed that stalk domain with (GGGGS)4 linkers were steadily incorporated to the surface of influenza virus-like particles (VLPs). Mice immunized with these VLPs exhibited enhanced systemic antibody responses with increased binding avidity and study found high titers of ADCC antibodies to the influenza virus, these VLPs also induced mucosal immune responses and produced antigen-specific IgG and IgA in nasal and lung washes. In addition, antigen-specific IgG antibody-secreting cells (ASCs) increased significantly in the spleen and lymph node. The results of this study suggest that the headless HA is a useful target in developing a universal vaccine against influenza virus.

Background CNS inflammation resulting from infection, injury, or neurodegeneration leads to accumulation of diverse B cell subsets. Although antibody secreting cells (ASC) within the inflamed CNS have been extensively examined, memory B cell (Bmem) characterization has been limited as they do not secrete antibody without stimulation. Moreover, unlike human Bmem, reliable surface markers for murine Bmem remain elusive. New method Using a viral encephalomyelitis model we developed a modified limiting dilution in vitro stimulation assay to convert CNS-derived virus specific Bmem into ASC. Comparison with existing methods Stimulation methods established for lymphoid tissue cells using prolonged stimulation with viral lysate resulted in substantial ASC loss and minimal Bmem to ASC conversion of CNS-derived cells. By varying stimulation duration, TLR activators, and culture supplements, we achieved optimal conversion by culturing cells with TLR7/8 agonist R848 in the presence of feeder cells for 2 days. Results Flow cytometry markers CD38 and CD73 characterizing murine Bmem from lymphoid tissue showed more diverse expression patterns on corresponding CNS-derived B cell subsets. Using the optimized TLR7/8 stimulation protocol, we compared virus-specific IgG Bmem versus pre-existing ASC within the brain and spinal cord. Increasing Bmem frequencies during chronic infection mirrored kinetics of ASC. However, despite initially similar Bmem and ASC accumulation, Bmem prevailed in the brain, but were lower than ASC in the spinal cord during persistence. Conclusion Simultaneous enumeration of antigen-specific Bmem and ASC using the Bmem assay optimized for CNS-derived cells enables characterization of temporal changes during microbial or auto-antigen induced neuroinflammation.

To establish a simple and widely accessible technique for rapidly selecting high producing Chinese hamster ovary (CHO) cells engineered to express a monoclonal antibody (mAb), we have exploited the transient display of recombinant protein on their cell surface. In combination with magnetic bead-based methods, we demonstrate the ability to select for cells of high productivity in the absence of any metabolic-based selection method. This technique is sufficient to obtain genetically stable engineered CHO cells via a single step of cell subcloning and yields sought-after stable, high IgG producing clonal cell lines. This technique may also be applied to other types of cells as well as polyclonal Ab cell pools.

The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs.

Background We have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC), which is the most common cause of bacterial diarrhea in children in developing countries and in travelers. Methods The vaccine was tested for safety and immunogenicity alone and together with double-mutant heat-labile toxin (dmLT) adjuvant in a double-blind, placebo-controlled Phase I study in 129 Swedish adults. The vaccine consists of four inactivated recombinant E. coli strains overexpressing the major ETEC colonization factors (CFs) CFA/I, CS3, CS5, and CS6 mixed with an LT B-subunit related toxoid, LCTB A . Volunteers received two oral doses of vaccine alone, vaccine plus 10 μg or 25 μg dmLT or placebo. Secretory IgA antibody responses in fecal samples and IgA responses in secretions from circulating intestine-derived antibody secreting cells were assessed as primary measures of vaccine immunogenicity. Results The vaccine was safe and well tolerated; adverse events were few and generally mild with no significant differences between subjects receiving placebo or vaccine with or without adjuvant. As many as 74% of subjects receiving vaccine alone and 83% receiving vaccine plus 10 μg dmLT showed significant mucosal IgA responses to all five primary vaccine antigens and about 90% of all vaccinees responded to at least four of the antigens. Subjects receiving vaccine plus 10 μg dmLT responded with significantly increased intestine-derived anti-CS6 responses compared to subjects receiving vaccine alone. Conclusions The vaccine was safe and broadly immunogenic. dmLT further enhanced mucosal immune responses to CF antigens present in low amounts in the vaccine. Based on these encouraging results, the vaccine will be tested for safety and immunogenicity in different age groups including infants in Bangladesh and for protective efficacy in travelers.

Background IgG 4 -related disease (IgG 4 -RD) is a poorly understood, multiorgan, chronic inflammatory disease characterized by tumefactive lesions, storiform fibrosis, obliterative phlebitis, and accumulation of IgG 4 -expressing plasma cells at disease sites. Objective The role of B cells and IgG 4 antibodies in IgG 4 -RD pathogenesis is not well defined. We evaluated patients with IgG 4 -RD for activated B cells in both disease lesions and peripheral blood and investigated their role in disease pathogenesis. Methods B-cell populations from the peripheral blood of 84 patients with active IgG 4 -RD were analyzed by using flow cytometry. The repertoire of B-cell populations was analyzed in a subset of patients by using next-generation sequencing. Fourteen of these patients were longitudinally followed for 9 to 15 months after rituximab therapy. Results Numbers of CD19 + CD27 + CD20 − CD38 hi plasmablasts, which are largely IgG4 + , are increased in patients with active IgG 4 -RD. These expanded plasmablasts are oligoclonal and exhibit extensive somatic hypermutation, and their numbers decrease after rituximab-mediated B-cell depletion therapy; this loss correlates with disease remission. A subset of patients relapse after rituximab therapy, and circulating plasmablasts that re-emerge in these subjects are clonally distinct and exhibit enhanced somatic hypermutation. Cloning and expression of immunoglobulin heavy and light chain genes from expanded plasmablasts at the peak of disease reveals that disease-associated IgG 4 antibodies are self-reactive. Conclusions Clonally expanded CD19 + CD27 + CD20 − CD38 hi plasmablasts are a hallmark of active IgG 4 -RD. Enhanced somatic mutation in activated B cells and plasmablasts and emergence of distinct plasmablast clones on relapse indicate that the disease pathogenesis is linked to de novo recruitment of naive B cells into T cell–dependent responses by CD4 + T cells, likely driving a self-reactive disease process.

Introduction The key feature of heparin-induced thrombocytopenia (HIT) is the production of antibodies (Ab) against the platelet factor 4 (PF4)/heparin complex. These Ab are directed against neoepitopes of the PF4 tetramer, which are induced by the complex formation with heparin. To study this humoral immune response in greater detail, either in a murine immunization model or in human blood samples, reliable and specific immune assays to detect specifically Ab against the PF4/heparin complexes, but not PF4 alone are required. Materials and Methods We established fluid-phase enzyme-immunoassays in which the soluble biotinylated antigen, PF4/heparin, is firstly captured by specific Ab, and secondly directly detected with enzyme-conjugated streptavidin. Results The use of this fluid-phase principle allowed a higher specificity than the traditional solid-phase enzyme-immunoassays in terms of Ab binding to murine PF4/heparin compared to murine PF4 alone. This fluid-phase approach applied to the detection of specific murine PF4/heparin Ab-secreting cells (ASC) identified the spleen as the main lymphatic organ that contributes to the PF4/heparin Ab response in mice. IgG ASC specific for PF4/heparin are very transiently detectable in mice, which might explain why anti-PF4/heparin IgG Ab typically disappear within 100 days in humans. Furthermore, this fluid-phase approach was successfully transferred to detect human PF4/heparin-specific Ab. Conclusion The fluid-phase principle for the specific detection of anti-PF4/heparin IgG and IgM Ab enables new and improved assays for HIT research in men and mice. At least in mice PF4/heparin antibodies are produced by transient B cells.

Humoral immunity depends on efficient activation of B-cells and their subsequent differentiation to antibody secreting cells (ASCs). The transcription factor NFκB cRel is critical for B-cell proliferation, but incorporating its known regulatory interactions into a mathematical model of the ASC differentiation circuit prevented ASC generation in simulations. Indeed, in experimental studies cRel was dynamically repressed during ASC differentiation, and ectopic cRel expression blocked ASC differentiation by inhibiting the transcription factor Blimp1. Conversely, after Blimp1 is induced in ASCs, it represses cRel by binding the Rel locus. Including this bi-stable circuit of mutual cRel-Blimp1 antagonism into a multi-scale model revealed that dynamic repression of cRel controls the switch from B-cell proliferation to ASC generation phases and hence the respective cell population dynamics. Our studies provide a mechanistic explanation of how dysregulation of this bi-stable circuit may result in pathologic B-cell population phenotypes and present avenues for diagnostic stratification and treatment. SUGGESTED REVISION: Precise regulation of transcription factor NFkB mediates efficient activation of B-cells and their subsequent differentiation to antibody secreting cells (ASCs). To obtain a quantitative understanding of how specific NFκB dimers control ASC differentiation, we developed a mathematical model that investigated NFkB subunits cRel and RelA as distinct regulators. This model predicted that cRel inhibits ASC generation. Indeed, cRel was dynamically repressed during ASC differentiation, and ectopic cRel expression blocked ASC differentiation by inhibiting the transcription factor Blimp1. Conversely, Blimp1 inhibited cRel expression by binding the Rel locus. Including this bi-stable circuit of mutual cRel-Blimp1 antagonism into a multi-scale model revealed that dynamic repression of cRel controls the switch from B-cell proliferation to ASC generation phases and hence the respective cell population dynamics. Our studies provide a mechanistic explanation of how dysregulation of this bi-stable circuit may result in pathologic B-cell population phenotypes and present avenues for diagnostic stratification and treatment.

Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.

B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax®23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax®23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax®23.

Oral tolerance is defined as an inhibition of specific immune responsiveness to a previously ingested antigen. Paradoxically, we found an increased lymphocyte activity in tolerant mice alongside the specific inhibition. Orally-tolerant mice presented higher number of immunoglobulin secreting cells (ISC) in spleen and bone marrow; showed a greater variety of Ig classes being produced: IgM and IgA in the spleen and IgG and IgM in the bone marrow. ISC from immunized mice produced mainly IgG. Despite having the same number of regulatory and activated T cells in the spleen after immunization, these cells appeared earlier in tolerant mice, right after the primary immunization. Also, tolerant mice showed a prompt expression of regulatory cytokines (TGF-β and IL-10) and a transient expression of effector cytokines (IL-2 and IFN-γ). Thus, in addition to an inhibited specific responsiveness, orally-tolerant mice displayed an early and widespread mobilization of activated and regulatory lymphocytes.

A previous study showed that increases in chemokine expression and recruitment of antibody secreting cells (ASCs) in the CNS after intracerebral immunization contributed to the suppression of a neurotropic virus. In this study, intracerebral chemokine injection was used to investigate the usefulness of chemokines for controlling neurotropic viruses. Both CXCL12 and a cocktail chemokine (a mixture of CXCL9, 10, 12 and 13) attracted antigen-specific ASCs more strongly than CXCL9, 10 and 13 in an in vitro chemotaxis assay and in vivo intracerebral chemokine injection experiments. Mice pre-treated intracerebrally with CXCL12 and the cocktail chemokine showed an increased survival rate after intracerebral infection with rabies virus. These results suggest that intracerebral CXCL12 injection induces the migration of ASCs and suppresses the neuropathogenicity of rabies virus in the CNS.

Background There are no vaccines against paratyphoid fever in clinical use. The disease has become more wide-spread and there is a growing problem of antibiotic resistance among the strains. Previous reports suggest that the oral live Salmonella Typhi Ty21a-vaccine confers protection against paratyphoid B fever. Data on efficacy against paratyphoid A fever are somewhat contentious. The present study investigated the immunological basis for such efficacy reports at a single-cell level: plasmablasts (identified as antibody-secreting cells, ASC) were studied for secretion of antibodies cross-reactive with Salmonella Paratyphi in the circulation of patients with enteric fever and of volunteers vaccinated with Ty21a. Materials and methods Thirty volunteers immunized with Ty21a and five patients with enteric fever were investigated for Salmonella Typhi and Salmonella Paratyphi A/B/C-specific circulating plasmablasts. PBMC were sorted by their expression of homing receptors (HR) for the intestine (α4β7), peripheral lymph node ( l -selectin) and skin (CLA) and typhoid- and paratyphoid-specific plasmablasts were enumerated with ELISPOT. Results Before vaccination, no cross-reactive ASC were found in the volunteers. In addition to the Salmonella Typhi-specific response, a significant cross-reactive immune response was mounted against Salmonella Paratyphi A and B both in the patients and the vaccinees. The magnitude of the response increased in the order Salmonella Paratyphi A (median 30 ASC/10 6 PBMC) → Salmonella Paratyphi B (median 81) → Salmonella Typhi (median 301) in the vaccinees. Both in patients and in vaccinees, the homing receptor (HR) selection favored homing to the gut, indicating a humoral intestinal immune response. Conclusions These immunological data provide evidence consistent with previous reports describing certain levels of cross-protective efficacy of Ty21a against paratyphoid fever. Controlled studies are needed to evaluate cross-protective efficacy. In the current situation where paratyphoid fever is emerging and no vaccines are available, any level of cross-protective capacity is valuable.

Prolonged survival of long-lived antibody-secreting cells in the BM has been implicated as a key component of long-term humoral immunity. The current study was designed to uncover the extrinsic signals required for the generation and maintenance of ASC in several niches (peritoneum, spleen and bone-marrow). Our results show that protein mixture of the Thalassophryne nattereri venom induced a chronic Th2 humoral response that is characterized by splenic hyperplasia with GC formation and venom retention by follicular DCs. Retention of B1a in the BM were observed. In the late phase (120 d) of chronic venom-response the largest pool of ASC into the peritoneal cavity consisted of B220 neg CD43 high phenotype; the largest pool of ASC into spleen was constituted by B220 positive cells (B220 high and B220 low ), whereas the largest pool of ASC into in the BM was constituted by the B220 high CD43 low phenotype; and finally, terminally differentiated cells (B220 neg CD43 high ) were only maintained in the inflamed peritoneal cavity in late phase. After 120 d a sustained production of cytokines (KC, IL-5, TNF-α, IL-6, IL-17A and IL-23) and leukocytes recruitment (eosinophils, mast cells, and neutrophils) were induced. IL-5- and IL-17A-producing CD4+ CD44+ CD40L+ Ly6C+ effector memory T cells were also observed in peritoneal cavity. Finally, treatment of venom-mice with anti-IL-5- and anti-IL17A-neutralizing mAbs abolished the synthesis of specific IgE, without modifying the splenic hyperplasia or GC formation. In addition, IL-5 and IL-17A negatively regulated the expansion of B1a in peritoneal cavity and BM, and promoted the differentiation of these cells in spleen. And more, IL-5 and IL-17A are sufficient for the generation of ASC B220 neg in the peritoneal cavity and negatively regulate the number of ASC B220 pos , confirming that the hierarchical process of ASC differentiation triggered by venom needs the signal derived from IL-5 and IL-17A.

B lymphocytes, like all mammalian cells, are equipped with the unfolded protein response (UPR), a complex signaling system allowing for both pro- and mal-adaptive responses to increased demands on the endoplasmic reticulum (ER). The UPR is comprised of three signaling pathways initiated by the ER transmembrane stress sensors, IRE1α/β, PERK and ATF6α/β. Activation of IRE1 yields XBP1(S), a transcription factor that directs expansion of the ER and enhances protein biosynthetic and secretory machinery. XBP1(S) is essential for the differentiation of B lymphocytes into antibody-secreting cells. In contrast, the PERK pathway, a regulator of translation and transcription, is dispensable for the generation of antibody-secreting cells. Functioning as a transcription factor, ATF6α can augment ER quality control processes and drive ER expansion, but the potential role of this UPR pathway in activated B cells has not been investigated. Here, we report studies of ATF6α-deficient B cells demonstrating that ATF6α is not required for the development of antibody-secreting cells. Thus, when B cells are stimulated to secrete antibody, a specialized UPR relies exclusively on the IRE1-XBP1 pathway to remodel the ER and expand cellular secretory capacity.

With the advent of enzyme replacement therapy (ERT) with alglucosidase alfa (rhGAA, Myozyme®) for Pompe disease, the clinical course of the disease has changed. We have previously described the poor outcome in cross reactive immunologic material (CRIM)-negative and high-titer CRIM-positive (HTCP) patients secondary to high sustained antibody titers (HSAT) which effectively neutralize ERT efficacy. Various immunomodulation strategies are being explored to diminish the immune response to ERT. However, once HSAT are formed, tolerization therapy has uniformly failed to lower antibody titers. Here we describe a case in which immunomodulation over a prolonged period of 28 months with cyclophosphamide, intravenous immunoglobulin, plasmapheresis, increased doses of rhGAA and rituximab failed to lower antibody titers and resulted in continued clinical decline in an infantile Pompe disease patient treated with ERT. Thus, it appears that the failure to target the antibody-secreting plasma cells responsible for HSAT led to a failure of tolerance induction. This is the first report using this combination of agents over a very extensive period of time with no success.

Immunoglobulin E (IgE) antibodies are major contributors to the pathology of atopic and allergic diseases as well as to immune response to helminth infections. Development of an adequate immunoglobulin G (IgG) immune response against infectious agents and vaccine antigens is considered in most cases as crucial for protection from disease. In vivo and in vitro production of IgE and IgG depends on cytokines and other soluble factors. Recently it has been shown that IgG antibody secreting cells (ASCs) can be generated by in vitro maturation of blood cells with Interleukin- (IL-)15 and CpG DNA or other stimulation cocktails, while IgE-ASCs develop upon cultivation with anti-CD40 and IL-4. In the present study we employed an enzyme linked immunospot assay (ELISPOT) to assess the capacity of individuals to develop into either IgE-ASCs or IgG-ASCs upon stimulation with different combinations of stimulation cocktails in order to investigate the influence of cytokines that are dysregulated in IgE-mediated immune reactions on ASC generation. Furthermore, we modified the method to assess IgG- and IgE-ASCs specific for two model antigens causing allergic rhinitis in humans. We demonstrate that IL-15, which is important for development of IgG-ASCs, decreases the number of IgE-ASCs when added to media commonly used for in vitro development of IgE-ASCs. We show that our method is suitable for the detection of specific and non-specific IgE-ASCs and IgG-ASCs and allows the investigation of the interplay between IgG-ASCs and IgE-ASCs in different populations.

The adjuvant effect of methylvinylether-co-maleic anhydride (Gantrez®AN) nanoparticles was investigated during oral vaccination of mice with F4 adhesins of F4-positive Escherichia coli. To differentiate whether the adjuvant effect originated from a nanoparticle effect or a polymer effect, 20 μg F4 was administered as slightly crosslinked F4-containing nanoparticles (g(F4)(0.01)) or as F4 mixed with slightly crosslinked pure nanoparticles (F4+g(0.01)). The F4-specific immune response was reduced using F4-containing nanoparticles due to complete shielding of F4, whereas oral administration of F4+g(0.01) increased the level of F4-specific antibody-secreting cells (ASC) in the spleen. When repeating the vaccination study after 6months using freshly prepared nanoparticles, the adjuvant effect of F4+g(0.01) was lost due to an altered polymer reactivity caused by partial hydrolysis of anhydride groups of Gantrez®AN. Combining F4 with nanoparticles stabilised with a higher crosslinker amount during nanoparticle synthesis (F4+g(0.22)) could overcome the effect of partial polymer hydrolysis, as higher levels of ASC were detected. Hence, an in-depth characterisation of the Gantrez®AN polymer is required as stability issues can alter its biological effect during oral vaccination.

Background Shigella flexneri 2a lipopolysaccharide 50 is a nasally delivered subunit vaccine consisting of a macromolecular complex composed of LPS, IpaB, IpaC and IpaD. The current study examined vaccine safety and immunogenicity across a dose range and the clinical performance of a new intranasal delivery device. Methods Volunteers ( N = 36) were randomized to receive vaccine via the Dolphin™ (Valois of America, Congers, New York) intranasal spray device at one of three doses (240, 480, and 690 μg) on days 0, 14, and 28. Another group ( N = 8) received the 240 μg dose via pipette. Vaccine safety was actively monitored and antigen-specific humoral and mucosal immune responses were determined. Results There were no serious adverse events and the majority of adverse events (98%) were mild. Antibody secreting cells (ASC), plasma, and mucosal immune responses to Shigella antigens were detected at all three dose levels with the 690 μg dose inducing the highest magnitude and frequency of responses. Vaccination with comparable doses of Invaplex 50 via the Dolphin™ resulted in higher plasma and ASC immune responses as compared to pipette delivery. Conclusion In this trial the S. flexneri 2a Invaplex 50 vaccine was safe, well-tolerated and induced robust levels of antigen-specific intestinal IgA and ASC responses. The spray device performed well and offered an advantage over pipette intranasal delivery.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterised by hypogammaglobulinaemia and antibody deficiency to T dependent and independent antigens. Patients suffer from recurrent respiratory infections and poor response to vaccination. Although the underlying molecular defect is unknown, most CVID patients show impaired late B cell differentiation. We investigated B cell differentiation and immunoglobulin secretion induced by two different stimuli: TLR9 specific ligand (CpG-ODN) and anti-CD40 combined with IL21. The contribution of BCR signalling (anti-IgM stimulation) was also evaluated. B cells from CVID patients produced low levels of IgG and IgA in response to both kinds of stimuli that was not restored by anti-IgM. Production of IgM was conserved when cells were stimulated with anti-CD40 and IL21. These results point to a wide signalling defect in B lymphocytes from CVID patients that may be related to their hypogammaglobulinaemia and poor response to vaccination.

Group A bovine rotavirus (BRV) is the major cause of neonatal calf diarrhea worldwide. As a preventive strategy, we evaluated the protection and immunomodulation in two groups of BRV-inoculated calves. All calves received control colostrum (CC; VN = 65,536; IgG1 = 16,384) prior to gut closure followed by the milk supplemented with immune colostrum (VN = 1,048,576; IgG1 = 262,144), twice a day, for 14 days. Calves received milk supplemented with 0.8% immune colostrum [(Gp 1) VN = 16,384; IgG1 = 4096] or milk supplemented with 0.4% immune colostrum [(Gp 2) VN = 1024; IgG1 = 1024]. Calves receiving CC or colostrum deprived calves (CD) fed antibody (Ab) free milk served as controls (Gp 3 and 4). Calves were inoculated with virulent BRV IND at 2 days of age. Group 1 calves (milk IgG1 4096) showed 80% protection against BRV diarrhea and significantly reduced virus shedding. At 21 post-inoculation days (PID), the antibody secreting cell (ASC) responses of Gp 1 calves were limited mainly to duodenal and jejunal lamina propria (LP) with limited or no responses in systemic sites (spleen and PBL) and mesenteric lymph nodes. The profile of serum and fecal Ab responses as well as the ASC responses was also modulated by the presence of passive IgG1 Abs and probably other colostrum components, toward higher titers of IgA Ab in serum and feces and a greater number of IgA ASC in the proximal intestine, reflecting positive modulation by colostrum toward this isotype associated with optimal protection of the intestinal mucosa. After challenge, at PID 21, all calves in Gp 1 and 2 were fully protected against diarrhea and only 1 of 5 calves in Gp 1 shed virus asymptomatically, indicating that the passive Ab treatment for 14 days was effective in protecting most of the animals after a first and a second virus exposure. The final outcome was a positive modulation of the mucosal immune responses and a high protection rate against diarrhea and virus shedding during the period of peak susceptibility to BRV infection.

Upon vaccination, B cells differentiate into antibody secreting cells (ASCs) that migrate via the circulation to tissues. The kinetics of this response and the relationship of circulating ASCs to protective antibody titers have not been completely explored. Methods Influenza-specific and total-IgG ASCs were enumerated by Elispot and flow cytometry daily in the blood in 6 healthy adults after trivalent influenza vaccination (TIV). Results Peak H1-specific IgG ASC frequencies occurred variably from day 5 to 8 and correlated with the fold-rise rise in hemagglutination inhibition (HAI titers); r = 0.91, p = 0.006. H3-specific IgG ASC frequencies correlated less well, perhaps due to a mismatch of the H3 protein in the vaccine and that used in the Elispot assay. Peak frequencies of vaccine-specific and total-IgG ASCs were 0.3% and 0.8%, respectively, of peripheral blood mononuclear cells (PBMC). Peak TIV-, H1-, H3-, and total-IgG ASC frequencies were 1736 ± 1133, 626 ± 520, 592 ± 463, and 4091 ± 2019 spots/106 PBMC, respectively. Peak TIV-, H1-, and H3-specific IgG ASC of total-IgG ASC frequencies constituted 63% ± 21, 26% ± 10, 22% ± 17, respectively. Conclusion After immunization with inactivated influenza vaccine the peak in influenza-specific ASC frequencies is variable but correlates well with the magnitude of protective HAI responses.

The ontogeny of IgM-producing cells was studied in juvenile mandarin fish Simperca chuatsi, an important fish in China's aquaculture sector. The IgM-producing cells were localised through in situ hybridisation with a probe complementary to the Ig mu-chain in lymphoid-related tissues, including head kidney, spleen, thymus, intestine and gills. In head kidney, transcripts of Ig mu were first detected at 20 days post-hatching (dph) with a few positive signals. and the number of IgM-producing cells increased obviously from 39 dph onwards. At 136 dph, a large amount of positive cells were observed in the entire organ with clusters of these cells located around the blood vessels. In spleen, IgM-producing cells were found from 26 dph onwards, followed by an increase until 67 dph: clusters of positive cells were also detected around blood vessels at 102 dph. In thymus, IgM-producing cells were first observed at 39 dph; thereafter, no obvious increase was detected until 78 dph. The positive cells in thymus were distributed mainly in the outer zone of thymus. A few IgM-producing cells were still observed in thymus of 1-year-old mandarin fish. IgM-producing cells were not detected in the intestine until 87 dph, with several discrete positively stained cells distributed in the lamina propria. IgM-producing cells, scattered mainly in primary gill filaments around blood vessels, were detected in gills from 90 dph. As in other teleosts, these results indicated that the head kidney appears to be the primary organ for IgM production in mandarin fish, and IgM-producing cells exist in all organs examined in the present study, implying their lymphoid role in fish. In addition, it is suggested that vaccination after 20 dph may be much more effective in mandarin fish. (C) 2009 Elsevier B.V. All rights reserved.

Cancer vaccines efficacy may improve inducing a rapid and persistent immune response, early at diagnosis along with standard therapies. EGF chemically conjugated to the carrier protein P64k from Neisseria meningitidis in montanide ISA 51 adjuvant is under evaluation, aiming to stimulate a B-cell response. High-dose cyclophosphamide and doxorubicin after priming enhanced the long-term frequency of EGF-specific antibody-forming cells (AFC) of IgM and IgG isotypes, but not the P64k response. Resulting combination, limitedly operational in Btk deficient xid mice, suggests that preferential B-cell lymphocyte space promoted by cyclophosphamide facilitates remaining EGF-specific AFC undergo homeostatic proliferation driven by boosting, amplifying the response.

The rise in influenza-specific neutralizing antibody levels is proceeded by a burst of antigen-specific antibody secreting cells (ASC) or plasmablasts identified in peripheral blood approximately 5-10 days post immunization. Blood antigen-specific ASC may function as an immune marker of vaccine responses in comparison to the pre- and post-neutralizing titers; however, some have questioned whether there is adequate survival of ASC isolated from peripheral blood after freezing, making multi-center vaccine trials difficult. Here, we demonstrate similar frequencies of influenza-specific ASC from fresh and frozen peripheral blood mononuclear cells (PBMC). Influenza Hemagglutinin (HA) H1, H3, and H7-specific ASC IgG ELISpots frequencies were compared from the same fresh and frozen PBMC 7 days after 2006 Trivalent Influenza Vaccine (TIV) in 10 young healthy subjects. H1-, H3-, and H7-specific IgG ASC spots/10(6) from fresh PBMC on day 7 were 229+/-341, 98+/-90, and 6+/-11 respectively. Total IgG ASC spots/million PBMC pre- and 7-day post-vaccination were 290+/-188 (0.029% PBMC) and 1691+/-836 (0.17% PBMC) respectively. There was no difference in the H1 -H3-, and total specific ASC IgG ELISpot frequencies from the fresh versus frozen PBMC on day 7 (p=0.43, 0.28, 0.28 respectively). These results demonstrate feasibility of testing whether antigen-specific ASC from frozen PBMC are an early biomarker of long-term antibody responses in multi-center vaccine trials.

Most antibody-secreting cells (ASCs) in mucosal tissues produce immunoglobulin A (IgA), the most abundant immunoglobulin in the body and the main class of antibody found in secretions. IgA-ASCs differentiate in the mucosal-associated lymphoid tissues and are usually considered as a homogeneous population of cells. However, IgA-ASCs that travel to the small intestine have unique characteristics in terms of their migratory requirements. These IgA-ASCs require the homing molecules alpha4beta7 and CCR9 to interact with their ligands, mucosal addressin cell adhesion molecule-1 and CCL25, which are constitutively expressed in the small intestine. Indeed, recent work has shown that IgA-ASCs specific for the small bowel are generated under different conditions as compared with IgA-ASCs in other mucosal compartments. Moreover, the mechanisms inducing IgA class switching may also vary according to the tissue where IgA-ASCs differentiate. Here we describe the mechanisms involved in the differentiation of IgA-ASCs in mucosal compartments, in particular those involved in the generation of gut-homing IgA-ASCs.

Antibodies in cutaneous mucus and skin of teleosts play a critical role in the protective immune response against infection. We demonstrate by ELISPOT that antibody-secreting cells (ASC), which include LPS-inducible B cells (plasmablasts) and non-replicating plasma cells, reside in low numbers in the skin of channel catfish. Following immunization against the protozoan parasite Ichthyophthirius multifiliis, which infects skin and gills, the number of ASC in skin increased 20-fold, indicating that the number of ASC in skin is dynamic and increases in response to parasite infection. The number of ASC in skin remained elevated for at least 17 weeks after the last parasite exposure. Cutaneous ASC included I. multifiliis-specific ASC, which undoubtedly serve as the primary source of cutaneous antibodies that confer long-term humoral immunity against reinfection. Our demonstration that skin contains B cells and plasma cells suggests that it is an integral component of the teleost immune system.

The potential of sublingual (s.l.) delivery of vaccine was examined in mice. We show the existence of a dense network of dendritic cells (DCs) in the s.l. epithelium and a rapid and transient increase in the frequency of s.l. DCs after topical application of cholera toxin (CT) adjuvant under the tongue. S.l. immunization with ovalbumin and CT induced vigorous systemic and mucosal antibody responses. Such treatment promoted mixed Th1 and Th2 cytokine responses and induced cytotoxic CD8(+) T cells in lung tissues and in systemic lymphoid organs. S.l. immunization was comparable to intranasal immunization and was superior to oral immunization regarding the magnitude and anatomic dissemination of the induced immune responses. S.l. administration of live influenza virus at a dose lethal by the nasal route was well tolerated and did not redirect virus to the olfactory bulb. These features underscore the potential of the s.l. mucosa to serve as an alternative vaccine delivery route.

As reported previously, the lack of the transcriptional co-activator OBF-1 prevented development of autoimmunity in Aiolos knockout mice. To further investigate the role and mechanism of OBF-1 in autoimmunity, we crossed OBF-1 null mice with MRL- lpr mice and generated OBF-1-deficent MRL- lpr mice. OBF-1 deletion abrogated all autoantibodies in the MRL- lpr mice, including anti-dsDNA Ab and anti-Sm Ab. The failure to produce autoantibodies was not related to development of immature or mature B cells, but correlated with severely reduced antibody-secreting cells (ASCs). The loss of OBF-1 protected against hypergammaglobulinemia, immune complex deposition, glomerulonephritis, and early mortality in MRL- lpr mice. In addition, accumulation of CD4 − CD8 − B220 + CD3 + T cells that characteristically develop in Fas mutation mice were markedly reduced in MRL- lpr mice without OBF-1. These results identify OBF-1 as a critical gene in the development of autoantibodies and reveal an essential role for OBF-1 in the generation of antibody/autoantibody-secreting cells in vivo .

This study evaluated antibody production against sheep red blood cells (SRBC) in hyperthyroid rats during treatment with triiodothyronine (T 3 ). The immune response was evaluated by measuring plaque forming cells (PFC) in the spleen and by enzyme-linked immunosorbent assay (ELISA) in serum of male Wistar rats (180 ± 10 g) treated with 25 μg/day of triiodotironine (T 3 ) during 7–12 days and immunized with SRBC at the 8th day of treatment. The results showed that anti-SRBC antibody production was significantly decreased in animals treated for 12 days when compared to normal rats immunized with the same antigen, as evaluated by the two assays. These results show that in this experimental model hyperthyroidism decreases antibody response. We previously observed the opposite effect, that is, an increase in this response in hypothyroid rats resulting from the treatment with propylthyouracil, a blocker of thyroid hormone biosynthesis. It is suggested that antibody production is affected by thyroid hormone levels.

Campylobacter jejuni is a common cause of enteritis worldwide. The mechanisms by which C. jejuni causes disease are unclear. Challenge studies in humans are currently considered unethical due to the possibility of severe complications, such as Guillain-Barre syndrome. Campylobacter infection in non-human primates closely mimics the disease and immune response, seen in humans. In this study, we attempted to determine the minimal dose of a pathogenic C. jejuni 81-176 strain required for clinical signs and symptoms of disease (> or = 80% attack rate) in Macaca mulatta monkeys using an escalating dosage (three doses for three monkey groups: 10(7), 10(9) and 10(11) cfu). Eighty percent of the monkeys challenged with highest dose (10(11) cfu) had mild disease, but the 80% attack rate (moderate diarrhea in 80% of the monkeys) was not achieved. However, 100% of monkeys showed IgA seroconversions (three-fold over pre-challenge titers). The elicited immune response was challenge dose-dependent. Campylobacter antigen specific fecal s-IgA responses were observed in all challenged groups but the response was not dose-dependent. Only IgM antibody secreting cells response was observed against Campylobacter antigens. The elicited immune response in three groups of rhesus monkeys was dose-dependent, indicating this monkey model can be used for pre-clinical evaluation of Campylobacter candidate vaccines, however these adult rhesus monkeys are less prone to Campylobacter infection.

A recently developed oral immunisation model in pigs in which F4 (K88) fimbriae of enterotoxigenic Escherichia coli are administered to induce a protective intestinal immunity, was used to determine the optimal inductive sites of the F4-specific intestinal immune response. Hereto, pigs were immunised with F4 orally, in the lumen of the mid-jejunum, ileum or mid-colon. Throughout the small intestine, the highest number of ASC was found following jejunal immunisation, followed by ileal, oral and colonic immunisation. To determine the signifance of Peyer's patches in the induced immune response, F4 was injected into the jejunal Peyer's patches (JPP), lamina propria (LP) and ileal Peyer's patches (IPP). Immunisation in the JPP induced the highest number ASC in the small intestine, whereas immunisation in the LP and IPP resulted in lower intestinal antibody responses. In conclusion, we have shown that the JPP are the major inductive sites of the F4-specific intestinal antibody response. This knowledge could be important when using the pig as an animal model for vaccination studies.

The eustachian tube (ET) plays an important role in the pathogenesis of otitis media (OM). To better understand its biology and to develop a nasal vaccine for preventing OM, mucosal lymphocytes in the ET were analyzed, and the ET's immunological function was investigated. Mononuclear cells were isolated from murine ET, and lymphocyte subsets were analyzed by flow cytometry. Antibody-producing cells were determined by enzyme-linked immunospot assay. The expression of cytokine mRNA in ET CD4(+) T cells was determined by RT-PCR. Results in naive mice showed that the ET contained many immunocompetent cells, including a relative large number of IgA-producing cells and Th2 cytokine-expressing T cells. Next, we investigated antigen-specific immune responses in the ET. Mice were immunized intranasally with the P6 outer membrane of nontypeable Haemophilus influenzae (NTHi) and cholera toxin (CT), and P6-specific immune responses in the ET were examined. P6-specific IgA producing cells markedly increased in the ET. Moreover, in vitro stimulation with P6 of purified CD4(+) T cells from immunized mice resulted in the proliferation of CD4(+) T cells that expressed Th2 cytokine mRNA. These results indicate that the ET might be characterized as a mucosal effector site and that antigen-specific IgA and Th2 immune responses could be induced in the ET by intranasal immunization. These findings suggest that the ET might be a key immunological organ in the pathogenesis of OM, and in the development of a nasal vaccine.

To show that an anti-human chorionic gonadotrophin-beta (hCGbeta) antibody response can be induced by inoculating Lb. expressing hCGbeta through different mucosal pathways in mice of two strains, female BALB/c and C57BL/6 mice were immunized via vaginal, oral or nasal routes with 10(8), 10(9), and 10(10)Lb.hCGbeta (a recombinant Lactobacillus expressing hCGbeta). The mice were immunized twice with a booster in study week 3. An indirect ELISA was used to determine anti-hCGbeta IgG and IgA antibodies in vaginal lavage and serum, obtained from the 2nd to 8th week after the primary immunization. Flow cytometry was used to analyze the lymphocyte proliferation from these tissues, 1 week after the primary immunization. The hCGbeta antigen-specific antibody-secreting cells of spleen, uterus, and vagina were evaluated by enzyme-linked immunospot assay (ELISpot), 2 weeks after the booster. The analysis showed that 10(9) and 10(10)Lb.hCGbeta inoculations induced similar anti-hCGbeta antibody responses, while the three mucosal pathways induced similar antibody responses. The antiserum obtained after boosters with 10(9) and 10(10)Lb. hCGbeta was able to neutralize more than 100 ng/ml hCG antigen, both in BALB/c and C57BL/6 mice. The highest antibody titer induced by vaginal mucosal immunization was stronger than that obtained via the other mucosal pathways. The B cells in the vagina appeared to proliferate after vaginal immunization (P

Objective To assess impact of probiotics and breastfeeding on gut microecology. Study design Mothers were randomized to receive placebo or Lactobacillus rhamnosus GG before delivery, with treatment of the infants after delivery. We assessed gut microbiota, humoral immune responses, and measured soluble cluster of differentiation 14 (sCD14) in colostrum in 96 infants. Results Fecal Bifidobacterium and Lactobacillus/Enterococcus counts were higher in breastfed than formula-fed infants at 6 months; P P =.01, respectively. At 3 months, total number of immunoglobulin (Ig)G-secreting cells in breastfed infants supplemented with probiotics exceeded those in breastfed infants receiving placebo; P =.05, and their number correlated with concentration of sCD14 in colostrum. Total numbers of IgM-, IgA-, and IgG-secreting cells at 12 months were higher in infants breastfed exclusively for at least for 3 months and supplemented with probiotics as compared with breastfed infants receiving placebo; P =.005, P =.03 and P =.04, respectively. Again, sCD14 in colostrum correlated with numbers of IgM and IgA cells; P =.05 in both. Conclusions We found an interaction between probiotics and breastfeeding on number of Ig-secreting cells, suggesting that probiotics during breastfeeding may positively influence gut immunity.

Methoxychlor, a chlorinated hydrocarbon pesticide, is a persistent environmental contaminant that has been identified in human reproductive tissues. Methoxychlor has been shown to be estrogenic in both in vivo and in vitro studies. As an endocrine disrupter, it may have the potential to adversely affect endocrine, reproductive, and immune systems in animals. The present study evaluated methoxychlor's immunotoxic potential in F0 (dams) and F1 generations of Sprague Dawley rats exposed to an isoflavone-free diet containing methoxychlor at concentrations of 10, 100, and 1000 ppm. In dams, exposure to methoxychlor from gestation day 7 to postpartum day 51 (65 days total exposure) produced a significant increase in the NK activity (1000 ppm) and the percentages of T cells (1000 ppm), helper T cells (1000 ppm) and macrophages (100 and 1000 ppm). In contrast, a decrease in the numbers of splenocytes and B cells was observed at the 100 and 1000 ppm concentrations. In F1 males, exposure to methoxychlor gestationally, lactationally and through feed from postnatal day 22–64 (78 days total exposure) produced an increase in the spleen IgM antibody-forming cell response to sheep red blood cells (100 and 1000 ppm) and the activity of NK cells (1000 ppm). However, there was a decrease in the terminal body weight (1000 ppm), spleen weight (1000 ppm), thymus weight (100 and 1000 ppm), and the numbers of splenocytes (1000 ppm), B cells (100 and 1000 ppm), cytotoxic T cells (1000 ppm) and NK cells (100 and 1000 ppm). In F1 females, exposure to methoxychlor produced a decrease in the terminal body weight (1000 ppm) and the percentages of cytotoxic T cells (10, 100 and 1000 ppm). These results demonstrate that developmental and adult dietary exposure to methoxychlor modulates immune responses in Sprague Dawley rats. Immunological changes were more pronounced in the F1 generation male rats that were exposed during gestation and postpartum, when compared to the F0 and F1 generation females. Increases in antibody-forming cell response and NK cell activity, and altered spleen cell subpopulation numbers were observed in the F1 generation male rats, without similar changes to the F1 generation females.

The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterized, and specific immunological tools are needed. To investigate both the humoral and cellular immune response, we used ovalbumin (OVA) and cholera toxin B (CTB) as an antigenic model to set-up ELISA and ELISPOT antibodies secreting cells (ASC) assays in the fox model. Identification of antibodies that cross-react with fox immunoglobulin was performed by Western blot, and their use was adapted for both the ELISA and ELISPOT ASC assay. The humoral and cellular specific immune responses were assessed after intra-muscular or intra-nasal immunization. Intra-muscular immunization resulted in the development of both cellular and humoral anti-OVA and anti-CTB responses in peripheral blood mononuclear cells (PBMCs). Immunization via the intra-nasal route resulted in the development of a cellular and humoral response against CTB in PBMCs. This immune response was confirmed using splenocytes from immunized animals by ELISPOT assay at euthanasia. Females immunized via the intra-nasal route developed specific anti-CTB IgM, IgA and IgG in vaginal fluids after the initial boost (day 26) showing that mucosal immunization produces a vaginal immune response in foxes. These immunological tools developed here are now available to be adapted to other antigenic models to facilitate further immune studies in foxes.

Previous clinical studies have demonstrated the feasibility of using edible transgenic plants to deliver protective antigens as new oral vaccines. Transgenic corn is particularly attractive for this purpose since the recombinant antigen is stable and homogeneous, and corn can be formulated in several edible forms without destroying the cloned antigen. Transgenic corn expressing 1 mg of LT-B of Escherichia coli without buffer was fed to adult volunteers in three doses, each consisting of 2.1 g of plant material. Seven (78%) of nine volunteers developed rises in both serum IgG anti-LT and numbers of specific antibody secreting cells after vaccination. Four (44%) of nine volunteers also developed stool IgA. Transgenic plants represent a new vector for oral vaccine antigens.

Nontypeable Haemophilus influenzae (NTHi) is one of the leading pathogens in sinusitis. One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is an attractive candidate for a subunit bacterial vaccine. In this study, we characterized normal sinus mucosa (SM) and investigated the potential of intranasal immunization with P6 and cholera toxin (CT) for induction of mucosal protective immunity against NTHi in the maxillary sinuses of rats. Intranasal immunization induced P6-specific sinus mucosal and systemic immunological responses, mainly of the IgA and IgG isotype. The protective effect of intranasal immunization was demonstrated by enhancement of sinus clearance of NTHi. The present study showed that unilateral intranasal immunization has a capacity to induce protective immunity against NTHi in the bilateral maxillary sinuses. Systemic administration of the vaccine did not affect sinus clearance of NTHi. These findings suggest that a nasal vaccine might be useful for preventing sinusitis.

We analysed whether hepatitis C virus (HCV)-specific antibody producing B lymphocytes can be induced in vitro with HCV-derived lipopeptides containing different T helper cell epitopes. HCV-specific antibody producing B cells were detected by ELISPOT at the single cell level. HCV-derived lipopeptides, but not their corresponding unlipidated peptides, induced B lymphocytes, which produced antibodies mainly reacting with the HCV-derived lipopeptides. The number of antigen-specific B cells was dependent on the number of added autologous T helper lymphocytes during the incubation period. Thus, HCV lipopeptides are more immunogenic than unmodified peptides and can induce HCV-reactive B lymphocytes in antigen-naïve lymphocytes.

The isotype-specific antibody responses of sheep immunised with keyhole limpet hemocyanin by a peripheral route (intramuscular (i.m.) injection) were compared to those induced by immunisation via different mucosal routes: (1) intra-nasal spray; (2) rectal deposition with cholera toxin; (3) injection into the mucosa of the small intestine or rectum. Antigen-specific IgG1 antibodies were induced in the i.m., intra-intestinal and intra-rectal injection groups and in a proportion of the cholera toxin immunised sheep, but not in the intra-nasal immunisation group. IgA was the only antibody isotype detected in serum collected from the intra-nasal immunisation group. No significant differences in serum IgA levels were detected in any of the mucosal immunisation groups as compared to the i.m. injection group. In contrast, analysis of the in vitro antibody profiles secreted by circulating antibody-secreting cells (ASC) revealed significantly higher IgA responses in the supernatants from all mucosal immunisation groups. This suggests that the measurement of antibodies secreted by circulating ASCs may be a better correlate of local mucosal responses in ruminants, as has been previously demonstrated in human studies. In addition to IgG1 and IgA responses, immunisation by direct injection of antigen formulations into the intestinal and rectal mucosa were the only groups to induce consistently high IgG2 antibodies in serum and ASC cultures.

To understand the mechanisms involved in maintaining long-term immunological memory following mucosal immunizations, we determined the quality of serum hapten-specific immunoglobulins (Ig) and localized Ig-secreting cells (SC) of various isotypes in acute, persistent/resting memory and effector memory phases following oral versus intra-muscular (IM) immunizations. In the acute phase, both oral and IM immunizations induced high avidity Ig. However, in the persistent/resting memory phase, oral immunizations induced low avidity Ig while IM immunizations induced high avidity Ig. Following oral immunizations, in the persistent/resting memory phase, hapten-specific IgM titers in serum and IgM-SC in bone marrow (BM) dominated the immune response, suggesting an important role for IgM in the maintenance of memory.

Injury to the liver results in rapid induction of transforming growth factor-beta1 (TGF-beta(1)) consistent with a role for TGF-beta(1) in repairing damaged tissue. In addition to its ubiquitous role in injury repair, TGF-beta(1) is also well established as a critical regulator of immune homeostasis; however, its mechanisms of action remain enigmatic. We have previously demonstrated that the hepatotoxic chlorinated hydrocarbon, carbon tetrachloride, suppresses helper T-lymphocyte function in a TGF-beta(1)-dependent manner. Here, we report that, in opposition to its immunosuppressive effects at picomolar concentrations, femtomolar concentrations of TGF-beta(1) augment T cell-dependent anti-sRBC IgM antibody forming cell (AFC) and T cell-independent DNP-Ficoll-induced AFC responses. These data support a concentration-dependent bifunctional effect by TGF-beta(1) on humoral immune responses in vitro. We further investigated a putative mechanistic role for Smad3, an intracellular mediator of TGF-beta(1) signaling, in propagating the inhibitory effects of TGF-beta(1) on humoral immune responses. Relative to wild type littermates, splenocytes from mice homologous for a null mutation in the gene encoding the TGF-beta receptor-activated Smad3 (Smad3(Exon8-/-)) were less sensitive to inhibition by TGF-beta(1) following anti-sRBC- and LPS-sensitization in vitro. In agreement, inhibition of IgM protein production by TGF-beta(1) was also dampened in LPS-sensitized Smad3(Exon8-/-) splenic B cells. Moreover, stimulation of IgA by TGF-beta(1) was abrogated in LPS-sensitized Smad3(Exon8-/-) splenocytes suggesting an additional role for Smad3 in regulating IgA production in vitro. Our results suggest that the effects of TGF-beta(1) on humoral immune responses fundamentally differ in a concentration-dependent manner and are mediated, in part, through Smad3 signaling.

The tonsils, consisting of the adenoid, tubal, palatine and pharyngeal tonsils, form a ring like structure in humans called Waldeyer's ring. The ring of tonsils is rich in lymphocytes and may play an important role as a reservoir of memory and immune competent cells serving the respiratory tract. The tonsils may also function as an activating and effector site for immune responses against respiratory pathogens. In this study, we have examined histological tissue sections from palatine tonsils for influenza specific antibody secreting cells (ASC) and several cell surface markers, from non-vaccinated and influenza vaccinated subjects. We found an increase in the number of influenza specific ASC in the tonsils of the influenza vaccinated subjects. These ASC was found scattered inside and surrounding the germinal centres, indicating that they may have homed to the tonsils. In addition, we observed a significant decrease in CD4 positive cells in tonsils of vaccinated subjects. Similar trends were also detected for CD45RA and CD45RO positive cells, which were significantly reduced in the vaccinated tonsils. The number of macrophages bearing the CD68 surface marker increased in numbers in vaccinated subjects. This shows that dynamic changes takes place in the tonsils after parenteral influenza vaccination, which may point to an important role of the tonsils in combating respiratory pathogens.

Cyclophosphamide (CP) is an anti-tumor drug commonly used in the chemotherapy of human cancer and autoimmune diseases. In our previous studies, we have demonstrated that lactoferrin (LF), given orally to CP-immunosuppressed mice, could reconstitute a T cell mediated immune response by the renewal of the T cell population. The aim of this present study was to evaluate the effects of LF on humoral responses in mice treated with cyclophosphamide. We demonstrate that a single, sublethal dose of cyclophosphamide (400 mg/kg body weight) profoundly inhibited the humoral immune response of CBA mice to sheep red blood cells (SRBC), as measured by the number of antibody forming cells (AFC) in the spleen after 5 weeks following CP treatment. Administration of 0.5% bovine LF in drinking water for 5 weeks partially reconstituted the AFC number (30–40% of the control values, but 7–10× more than in CP-treated controls). Determination of T and B cell levels in the spleens by flow cytometry revealed that the content of CD3 + and CD4 + as well as Ig + splenocytes was elevated in the immunocompromised mice treated with LF. In addition, the number of peritoneal macrophages was partially restored following LF treatment. Evaluation of the proliferative response to concanavalin A (ConA) and pokeweed mitogen (PWM) demonstrated that the diminished reactivity of splenocytes from CP-treated mice was significantly enhanced by LF. In summary, we conclude that the prolonged, oral treatment of immunocompromised mice with LF led to partial reconstitution of the humoral response, associated with elevation of T and B cell and macrophage content and the proliferative response of splenocytes to mitogens.

Serum antibody and antibody secretory cell (ASC) responses to the cold-adapted (CA) live attenuated influenza A donor strain A/Leningrad/134/17/57 in BALB/c mice were determined in the lungs and mediastinal lymph nodes after administration by the intranasal, subcutaneous and intramuscular routes. Both types of response were greatest when an inoculum consisting of 10(6.5) 50% egg infectious doses (EID(50)) was administered twice intranasally at an interval of 3 weeks. Serum responses by the intramuscular route were much higher than by the subcutaneous route but, at doses of 10(6.5-7.5) EID(50), were still lower than that obtained with two doses of an intranasal inoculum of 10(6.5) EID(50). Virus-specific ASC responses for IgA and IgG2a were obtained in the lungs and mediastinal lymph nodes of mice inoculated with 10(6.5) EID(50) by the intranasal route. However, ASC responses after inoculation by either the subcutaneous or intramuscular routes were barely detectable, even at doses as high as 10(7.5) EID(50). These results confirm that intranasal administration of live vaccines induces far higher virus-specific IgA and IgG2a responses in the respiratory tract of mice than can be achieved by parenteral administration and that serum antibody levels induced by parenteral vaccination are unrelated to the respiratory ASC response.

Bullous pemphigoid is a subepidermal blistering disease characterized by the synthesis of autoantibodies against the 180 kDa and the 230 kDa bullous pemphigoid antigens. Whether autoimmunity is also reflected by the presence of circulating autoantigen-specific memory B cells is still a matter of debate. We used a new assay combining two-step immunomagnetic enrichment with multiparameter flow cytometry to detect and characterize bullous pemphigoid 180 kDa-specific IgG+ B cells in blood of bullous pemphigoid patients. In a first magnetic separation, B cells were isolated from peripheral blood mononuclear cells using releasable microbeads conjugated to a CD19 antibody. From pre-enriched B cells, bullous pemphigoid 180 kDa-specific cells were then positively selected using microbeads directly conjugated with a recombinant N-terminal fragment of the bullous pemphigoid 180 kDa ectodomain, containing the noncollagenous 16A domain, which was recently shown to harbor major epitopes of autoantibodies in bullous pemphigoid sera. Noncollagenous 16A domain-specific IgG+ B cells were detectable in blood of most, if not all patients with serum autoantibodies against the noncollagenous 16A domain. The specificity of the cells was confirmed by in vitro differentiation into antibody-forming cells and analysis of the culture supernatant for the presence of noncollagenous 16A domain-specific IgG antibodies. All noncollagenous 16A domain-specific IgG+ B cells showed a clear memory immunophenotype. Noncollagenous 16A domain-specific IgG+ memory B cells may be crucial for continuous noncollagenous 16A domain-specific autoantibody production and/or play a part as antigen-presenting cells for priming and restimulation of bullous pemphigoid 180 kDa-specific T helper cells.