Main Ig Research Articles

Teleost swim bladder, an ancient air-filled organ that elicits mucosal immune responses

The air-filled organs (AOs) of vertebrates (lungs and swim bladders) have evolved unique functions (air-breathing or buoyancy control in water) to adapt to different environments. Thus far, immune responses to microbes in AOs have been described exclusively in the lungs of tetrapods. Similar to lungs, swim bladders (SBs) represent a mucosal surface, a feature that leads us to hypothesize a role for SB in immunity. In this study, we demonstrate that secretory IgT (sIgT) is the key SB immunoglobulin (Ig) responding to the viral challenge, and the only Ig involved in viral neutralization in that organ. In support of these findings, we found that the viral load of the SB from fish devoid of sIgT was much higher than that of control fish. Interestingly, similar to the lungs in mammals, the SB represents the mucosal surface in fish with the lowest content of microbiota. Moreover, sIgT is the main Ig class found coating their surface, suggesting a key role of this Ig in the homeostasis of the SB microbiota. In addition to the well-established role of SB in buoyancy control, our findings reveal a previously unrecognized function of teleost SB in adaptive mucosal immune responses upon pathogenic challenge, as well as a previously unidentified role of sIgT in antiviral defense. Overall, our findings indicate that despite the phylogenetic distance and physiological roles of teleost SB and mammalian lungs, they both have evolved analogous mucosal immune responses against microbes which likely originated independently through a process of convergent evolution.

Prevailing Role of Mucosal Igs and B Cells in Teleost Skin Immune Responses to Bacterial Infection.

The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent Ig in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, in this study, we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B cells and specific IgT titers, respectively, within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection and that, because of the large surface of the skin, its SALT probably represents a prominent IgT-inductive site in fish.

Immunogenicity study of a Streptococcus suis autogenous vaccine in preparturient sows and evaluation of passive maternal immunity in piglets

BackgroundStreptococcus suis is an important pathogen that causes severe diseases mostly in weaned piglets. Only available vaccines in the field are those composed of killed bacteria (bacterins) but data about their effectiveness are missing. We report here a field study on the immunological response induced by an autogenous vaccine applied in pre-parturient sows. Using a farm with recurrent S. suis serotype 7 problems, the study was divided in three experiments: (I) Sows received the vaccine at 7 and 3 weeks pre-farrowing. (II) Replacement gilts introduced to the herd received the vaccine at 4 and 7 weeks after their entry in quarantine and a boost 3 weeks pre-farrowing. (III) Gilts from experiment II received another boost 3 weeks pre-farrowing at their 3rd/4th parity. Levels, isotype profile and opsonophagocytosis capacity of the serum antibodies induced by vaccination were evaluated in sows and maternal immunity in piglets.ResultsIn sows (I), the vaccine induced a slight, albeit significant, increase in anti-S. suis total antibodies after 2 doses when compare to basal levels already present in the animals. These antibodies showed a high opsonic capacity in vitro, highlighting their potential protective capacity. A gilt vaccination program of 3 doses (II) resulted in a significant increase in anti-S. suis total antibodies. Levels of maternal immunity transferred to piglets were high at 7 days of age, but rapidly decreased by 18 days of age. A gilt vaccination program ensued a higher transfer of maternal immunity in piglets compared to control animals; nevertheless duration was not improved at 18 day-old piglets. The vaccine response in both gilts and sows was mainly composed of IgG1 subclass, which was also the main Ig transferred to piglets. IgG2 subclass was also found in piglets, but its level was not increased by vaccination. Finally, a recall IgG1 response was induced by another boost vaccination at 3rd/4th parity (III), indicating that the vaccine induced the establishment of a lasting memory response in the herd.ConclusionsOverall, an optimal gilt/sow vaccination program might result in increased antibody responses; nevertheless duration of maternal immunity would not last long enough to protect post-weaned piglets.

Immunoglobulins in teleosts.

Immunoglobulins are glycoproteins which are produced as membrane-bound receptors on B-cells or in a secreted form, known as antibodies. In teleosts, three immunoglobulin isotypes, IgM, IgT, and IgD, are present, each comprising two identical heavy and two identical light polypeptide chains. The basic mechanisms for generation of immunoglobulin diversity are similar in teleosts and higher vertebrates. The B-cell pre-immune repertoire is diversified by VDJ recombination, junctional flexibility, addition of nucleotides, and combinatorial association of light and heavy chains, while the post-immune repertoire undergoes somatic hypermutation during clonal expansion. Typically, the teleost immunoglobulin heavy chain gene complex has a modified translocon arrangement where the Dτ-Jτ-Cτ cluster of IgT is generally located between the variable heavy chain (VH) region and the Dμ/δ-Jμ/δ-Cμ-Cδ gene segments, or within the set of VH gene segments. However, multiple genome duplication and deletion events and loss of some individual genes through evolution has complicated the IgH gene organization. The IgH gene arrangement allows the expression of either IgT or IgM/IgD. Alternative splicing is responsible for the regulation of IgM/IgD expression and the secreted versus transmembrane forms of IgT, IgD, and IgM. The overall structure of IgM and IgT is usually conserved across species, whereas IgD has a large variety of structures. IgM is the main effector molecule in both systemic and mucosal immunity and shows a broad range of concentrations in different teleost species. Although IgM is usually present in higher concentrations under normal conditions, IgT is considered the main mucosal Ig.

State of humoral immunity in lactating sows and suckling piglets

The state of humoral immunity in lactating sows and its formation in suckling piglets in the conditions of a pig-breeding complex were studied. In sows after farrow, a high content of total immunoglobulins, the main classes of IgG, IgM and IgA, the concentration of which decreased in a day, followed by an increase in serum as lactation continued was detected in the serum and colostrum. In piglets at birth, the content of total immunoglobulins and the main Ig isotypes was insignificant, and at the age of twenty-four hours, as a result of absorption of colostral immunoglobulins in the small intestine, their amount in the serum was the highest, followed by a decrease in the concentration of IgG and IgA until the end of the suckling period, and IgM - up to day 14 with a further increase in its content, indicating the formation of a primary immune response. It has been detected that the dominant class of immunoglobulins in the serum and colostrum of sows and the serum of piglets is IgG, and in milk - IgA. An increase in the level of medium-dispersed circulating immune complexes (C4%) and their relation to giant (C3%) CICs was revealed, associated with an increase of the antigenic load in sows in the second half of lactation as a result of immunization, in milk - with a decrease in the content of the main classes of immunoglobulins, and in piglets, due to this, with a decrease in passive immunity.

Mediation of Mucosal Immunoglobulins in Buccal Cavity of Teleost in Antibacterial Immunity.

The buccal mucosa (BM) of vertebrates is a critical mucosal barrier constantly exposed to rich and diverse pathogens from air, water, and food. While mammals are known to contain a mucosal associated lymphoid tissue (MALT) in the buccal cavity which induces B-cells and immunoglobulins (Igs) responses against bacterial pathogens, however, very little is known about the evolutionary roles of buccal MALT in immune defense. Here we developed a bath infection model that rainbow trout experimentally exposed to Flavobacterium columnare (F. columnare), which is well known as a mucosal pathogen. Using this model, we provided the first evidence for the process of bacterial invasion in the fish BM. Moreover, strong pathogen-specific IgT responses and accumulation of IgT+ B-cells were induced in the buccal mucus and BM of infected trout with F. columnare. In contrast, specific IgM responses were for the most part detected in the fish serum. More specifically, we showed that the local proliferation of IgT+ B-cells and production of pathogen-specific IgT within the BM upon bacterial infection. Overall, our findings represent the first demonstration that IgT is the main Ig isotype specialized for buccal immune responses against bacterial infection in a non-tetrapod species.

Convergent Evolution of Mucosal Immune Responses at the Buccal Cavity of Teleost Fish.

SummaryThe buccal mucosa (BM) is a critical first line of defense in terrestrial animals. To gain further insights into the evolutionary origins and primordial roles of BM in teleosts here we show that rainbow trout, a teleost fish, contains a diffuse mucosal associated lymphoid tissue (MALT) within its buccal cavity. Upon parasite infection, a fish immunoglobulin specialized in mucosal immunity (sIgT) was induced to a high degree, and parasite-specific sIgT responses were mainly detected in the buccal mucus. Moreover, we show that the trout buccal microbiota is prevalently coated with sIgT. Overall our findings revealed that the MALT is present in the BM of a non-tetrapod species. As fish IgT and mucus-producing cells are evolutionarily unrelated to mammalian IgA and salivary glands, respectively, our findings indicate that mucosal immune responses in the BM of teleost fish and tetrapods evolved through a process of convergent evolution.

Dysregulation of B Cell Activity During Proliferative Kidney Disease in Rainbow Trout.

Proliferative kidney disease (PKD) is a widespread disease caused by the endoparasite Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea). Clinical disease, provoked by the proliferation of extrasporogonic parasite stages, is characterized by a chronic kidney pathology with underlying transcriptional changes indicative of altered B cell responses and dysregulated T-helper cell-like activities. Despite the relevance of PKD to European and North American salmonid aquaculture, no studies, to date, have focused on further characterizing the B cell response during the course of this disease. Thus, in this work, we have studied the behavior of diverse B cell populations in rainbow trout (Oncorhynchus mykiss) naturally infected with T. bryosalmonae at different stages of preclinical and clinical disease. Our results show a clear upregulation of all trout immunoglobulins (Igs) (IgM, IgD, and IgT) demonstrated by immunohistochemistry and Western blot analysis, suggesting the alteration of diverse B cell populations that coexist in the infected kidney. Substantial changes in IgM, IgD, and IgT repertoires were also identified throughout the course of the disease further pointing to the involvement of the three Igs in PKD through what appear to be independently regulated mechanisms. Thus, our results provide strong evidence of the involvement of IgD in the humoral response to a specific pathogen for the first time in teleosts. Nevertheless, it was IgT, a fish-specific Ig isotype thought to be specialized in mucosal immunity, which seemed to play a prevailing role in the kidney response to T. bryosalmonae. We found that IgT was the main Ig coating extrasporogonic parasite stages, IgT+ B cells were the main B cell subset that proliferated in the kidney with increasing kidney pathology, and IgT was the Ig for which more significant changes in repertoire were detected. Hence, although our results demonstrate a profound dysregulation of different B cell subsets during PKD, they point to a major involvement of IgT in the immune response to the parasite. These results provide further insights into the pathology of PKD that may facilitate the future development of control strategies.

Local induction of IgT responses to pathogens and microbiota in the gill of rainbow trout

Gas exchange structures are critical for acquiring oxygen, but they also represent portals for pathogen entry. Local mucosal immunoglobulin (Ig) responses against pathogens in specialized respiratory organs have only been described in tetrapods. We have previously shown that IgT is an Ig specialized in gut and skin mucosal immunity. Thus, we tested the hypothesis that IgT might play a pivotal role in mucosal immunity of teleost gills. In this study, we provide the first structural and functional characterization of all teleost Igs, including secreted IgD, IgM and IgT at a mucosal surface of a teleost fish. Here we show that IgT B cells represent the major B cell subset in the gill filaments. In contrast to reported results by others, we found that all gill B cells expressing surface IgM, also expressed surface IgD and that the percentage of B cells solely expressing either IgD or IgM was negligible. We found that the majority of bacterial microbiota in the gill mucosa is coated with IgT and, to a much lesser degree with IgM and IgD. More crucially, significant specific-IgT immune responses against Ichthyophthirius multifiliis (Ich) and Flavobacterium columnare were measured in the gill mucus, while pathogen-specific IgM responses were almost exclusively detected in the serum. Pathogen-specific IgD titers were absent both in gill mucus and serum. Importantly, we found significant IgT B-cell proliferative responses in the gill but not in the spleen or head kidney of fish that survived Ich infection. Moreover we also found that Ichand F. columnare-specific IgT titers were locally produced by gill explants of survivor fish while they were absent in the spleen or head kidney explants of the same animals. In addition to showing that IgT is the main Ig player in gill mucosal immunity, the observed generation of local IgT B cell proliferative and pathogen-specific IgT responses in the gills provides the first demonstration of locally induced B cell and secretory Ig responses in the mucosa of a teleost. Moreover, this represents the first study in which a bacterial pathogen is shown to induce dominant IgT responses in a fish mucosal site, thus strongly suggesting that IgT is induced by a variety of pathogens in addition to parasites. Our findings also have special relevance from an applied perspective as they may lead to the development of fish vaccines and immunostimulants that have the capacity to induce gill IgT mucosal immune responses.

Clinical and immunological research of qualities of antimicrobial disepidermic cryolyophylized xenoderm grafts

Nazarchuk Serhii, Sukhodolia Anatolii, Nazarchuk Oleksandr. Clinical and immunological research of qualities of antimicrobial disepidermic cryolyophylized xenoderm grafts. Journal of Education, Health and Sport. 2015;5(8):434-442. ISSN 2391-8306. DOI 10.5281/zenodo.31322 http://dx.doi.org/10.5281/zenodo.31322 http://ojs.ukw.edu.pl/index.php/johs/article/view/2015%3B5%288%29%3A434-442 https://pbn.nauka.gov.pl/works/632549 Formerly Journal of Health Sciences. ISSN 1429-9623 / 2300-665X. Archives 2011–2014 http://journal.rsw.edu.pl/index.php/JHS/issue/archive Deklaracja. Specyfika i zawartośc merytoryczna czasopisma nie ulega zmianie. Zgodnie z informacją MNiSW z dnia 2 czerwca 2014 r., ze w roku 2014 nie bedzie przeprowadzana ocena czasopism naukowych; czasopismo o zmienionym tytule otrzymuje tyle samo punktow co na wykazie czasopism naukowych z dnia 31 grudnia 2014 r. The journal has had 5 points in Ministry of Science and Higher Education of Poland parametric evaluation. Part B item 1089. (31.12.2014). © The Author (s) 2015; This article is published with open access at Licensee Open Journal Systems of Kazimierz Wielki University in Bydgoszcz, Poland and Radom University in Radom, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 26.08.2015. Revised 05.09.2015. Accepted: 24.09.2015. UDC: 616+615.47.014.47-616.5-089-74 CLINICAL AND IMMUNOLOGICAL RESEARCH OF QUALITIES OF ANTIMICROBIAL DISEPIDERMIC CRYOLYOPHYLIZED XENODERM GRAFTS Serhii Nazarchuk 1 , Anatolii Sukhodolia 2 , Oleksandr Nazarchuk 3 1 Khmelnytskyi Regional Clinical Oncological Di s pensary, Department of Abdominal Surgery 2 Vinnytsya National Pirogov Memorial Medical University, Department of Surgery of the Faculty of Postgraduate Study 3 Vinnytsya National Pirogov Memorial Medical University, Department of Microbiology, Virology and Immunology Abstract In the article the results of the research of immunological changes after the using of antimicrobial disepidermic cryolyophilized xenoderm grafts in the model of intestinal anastomosis forming; clinical effectiveness of their use for prevention intestinal sutures’ and anastomosis’ insolvency in patients with oncological pathology are presented. In experiment, it is shown, that while using DCXG the essential changes of local immune reactivity have place. It is proved that the tendency of unevenly and not proportionally increasing of cells with main classes Ig A, Ig M, Ig G, Ig E shows highly active and non-stable local immunologic reactions of intestinal mucosa on using DCXG; the intensity of local immune reactions significantly decreases by the 25 th day and is nearby to normal ones. DCXG were used in 50 patients, who underwent surgery in Khmelnytskyi Regional Clinical Oncological Dispensary and Khmelnytskyi Regional Clinical Hospital in 2009–2013 years. The incidence of purulent-inflammatory complications of intestinal anastomosis’ insolvency in the main group of patients was above 4,3 % and 16 % in control one. Key words: sutures’ insolvency, anastomosis, xenoderm grafts, antimicrobial materials.

Natural Killer Cell Deficiencies in a Consecutive Series of Children with Herpetic Encephalitis

Natural killer (NK) cells play a fundamental role in innate and early phases of adaptive immunity against viral infections, both in humans and in animal models. To date, NK cell deficiencies in patients with severe herpetic infections have been reported in single cases, and their role as predisposing factor is still controversial. Five children affected by herpetic encephalitis were consecutively admitted to the Anna Meyer Children's Hospital in Florence (Italy) between 2003 and 2005. We therefore investigated the presence of NK cell deficiencies in a consecutive series of children with herpetic encephalitis. Five healthy children were included in the study as controls. Differential WBC counts, main Ig and IgE class serum analysis, cytofluorimetric analysis of circulating T, B and NK cells were performed on our study population. Sequencing of a selected region of CD16A gene transcript was carried out in two patients. All patients resulted to be affected by deficiencies related to NK cells in respect to controls. One patient was also affected by lymphopenia, while no other significant deficits of immunity were detected in the study population. To date, this is the first survey that demonstrates isolated NK cell deficiencies in a cohort of consecutive patients affected by severe herpes simplex infections. These findings suggest a role for NK cell deficiencies as a predisposing factor for increased susceptibility and severe course of disease in these patients.

Serum but not secreted Ag-specific IgA inhibits oral Ag induced anaphylaxis. (97.6)

Abstract Epidemiologic evidence suggests that IgA, the main mucosal Ig isotype, inhibits allergy, but it is not known whether it suppresses anaphylaxis. We used mouse models to evaluate whether IgA can suppress systemic anaphylactic responses to ingested allergens by neutralizing them before or after their systemic absorption. Passive systemic anaphylaxis (PSA), measured as hypothermia, was induced by sensitizing mice with IgE anti-TNP mAb, then challenging them orally (og) with TNP-OVA. The ability of IgA to inhibit PSA by neutralizing Ag prior to or after systemic absorption was evaluated by pretreating mice with IgA anti-TNP mAb either og or iv. TNP-OVA was detectible in serum by 5 min after og administration. PSA developed within 5 min and was inhibited by iv, but not og IgA. Extent of inhibition was proportional to the IgA dose and Fcα/μR- and FcγRIIb-independent. Intestinal anaphylaxis (IA), defined as diarrhea <60 minutes after OVA challenge, was induced by priming mice ip with OVA/alum, then challenging them og 3x/wk with OVA. IA development was delayed in J-chain- and PIgR-deficient mice, which have markedly increased serum IgA and decreased intestinal IgA. Thus, Ag-specific IgA acts in the blood but not the gut to inhibit systemic and intestinal anaphylaxis. This indicates that ingested antigen must be absorbed systemically to induce systemic anaphylaxis and suggests that immunotherapy should aim at increasing blood, rather than gut levels of non-IgE Abs to food allergens.

From criticality to final collapse: Evolution of the “ b-value” from 1.5 to 1.0

Extensive research and studies on concrete fracture and failure by means of the acoustic emission (AE) technique have shown that fracture and damage growth can be characterized through a single synthetic parameter, namely the b-value, which changes systematically during the different stages of the failure process, as shown by several AE tests carried out from the specimen to the structural scale [Sammonds PR, Meredith PG, Murrel SAF, Main IG. Modelling the damage evolution in rock containing porefluid by acoustic emission. In: Proceedings of the Eurock’94; 1994; Colombo S, Main IG, Forde MC. Assessing damage of reinforced concrete beam using “ b-value” analysis of acoustic emission signals. J Mater Civil Eng ASCE 2003;15:280–6; Carpinteri A, Lacidogna G, Niccolini G. Critical behaviour in concrete structures and damage localisation by Acoustic Emission. Key Eng Mater 2006;312:305–10]. This parameter can be linked to the value of the exponent α of the power-law distribution of the crack size in a damaged structure. In this paper, we propose a statistical interpretation for the variation of the b-value during the evolution of damage, based on a treatment originally proposed by [Carpinteri A. Mechanical damage and crack growth in concrete: plastic collapse to brittle fracture. Dordrecht: Martinus Nijhoff Publishers; 1986; Carpinteri A. Decrease of apparent tensile and bending strength with specimen size: two different explanations based on fracture mechanics. Int J Solid Struct 1989;25:407–29; Carpinteri A. Scaling laws and renormalization groups for strength and toughness of disordered materials. Int J Solid Struct 1994;31:291–302]. The proposed model captures the transition from the condition of criticality, in which α = 3, to that of imminent failure, characterized by α = 2, in terms of damage localisation.

IFN‐γ, But Not Nitric Oxide or Specific IgG, is Essential for the In vivo Control of Low‐virulence Sylvio X10/4 Trypanosoma cruzi Parasites

Highly virulent strains of Trypanosoma cruzi are frequently used as murine models of Chagas' disease. However, these strains do not fully represent the spectrum of parasites involved in the human infection. In this paper, we analysed parasitaemia, mortality, tissue pathology and parasite-specific IgG serum levels in immune-deficient mice infected with Sylvio X10/4 parasites, a T. cruzi derived from a chagasic patient that yields very low parasitaemias and in C3H/HePAS mice induces a chronic cardiopathy resembling the human disease. IFN-gamma was identified as a crucial element for parasite control as its absence determined a drastic increase in parasitaemia, tissue parasitism, leukocyte infiltrates at the heart and striated muscles and mortality. The lack of IFN-gamma or IL-12p40, a molecule shared by IL-12 and IL-23, also resulted in spinal cord lesions and a progressive paralysis syndrome. Whereas IgG2a was the main Ig isotype in infected C57BL/6 mice, IL-12p40-KO mice produced IgG2a and IgG1 and IFN-gamma-KO mice produced only IgG1. The IFN-gamma-protective effect was not essentially mediated by nitric oxide (NO), inasmuch as infected iNOS-KO mice showed no parasitaemia and low tissue damage. Mice deficient in CD4(+) or CD8(+) T cells showed an intermediate phenotype with increased mortality and tissue pathology but no parasitaemia. Interestingly, CD28-KO mice were unable to produce anti-T. cruzi IgG antibodies but presented moderate tissue pathology and managed to control the infection. Thus, differently from infections with high virulence parasites, neither IgG, NO nor CD28-mediated signalling are essential for the non-sterile control of Sylvio X10/4 parasites.

Humoral and cellular immune responses to Trypanosoma cruzi-derived paraflagellar rod proteins in patients with Chagas' disease.

Sera and peripheral blood mononuclear cells (PBMC) from patients displaying different clinical symptoms as well as from normal uninfected individuals (NI) were used to evaluate the humoral and cellular responses of Chagas' disease patients to Trypanosoma cruzi-derived paraflagellar rod proteins (PFR). Our results show that sera from both asymptomatic Chagas' disease patients (ACP) and cardiac Chagas' disease patients (CCP) have higher levels of antibodies to PFR than sera from NI. Immunoglobulin G1 (IgG1) and IgG3 were the main Ig isotypes that recognized PFR. We also tested three recombinant forms of PFR, named rPAR-1, rPAR-2, and rPAR-3, by Western blot analysis. Sera from seven out of eight patients with Chagas' disease recognized one of the three rPAR forms. Sera from 75, 50, and 37.5% of Chagas' disease patients tested recognized rPAR-3, rPAR-2, and rPAR-1, respectively. PFR induced proliferation of 100 and 70% of PBMC from ACP and CCP, respectively. Further, stimulation of cells from Chagas' disease patients with PFR enhanced the frequencies of both small and large CD4(+) CD25(+) and CD4(+) CD69(+) lymphocytes, as well as that of small CD8(+) CD25(+) lymphocytes. Finally, we evaluated the ability of PFR to elicit the production of gamma interferon (IFN-gamma) by PBMC from patients with Chagas' disease. Fifty percent of the PBMC from ACP as well as CCP produced IFN-gamma upon stimulation with PFR. PFR enhanced the percentages of IFN-gamma-producing cells in both CD3(+) and CD3(-) populations. Within the T-cell population, large CD4(+) T lymphocytes were the main source of IFN-gamma.

Immunostimulatory effect of Bacillus firmus on mouse lymphocytes.

Bacillus firmus (a Gram-positive nonpathogenic and harmless bacterium), was shown to be a strong polyclonal activator of mouse B lymphocytes as estimated by ELISA testing of Ig concentrations in culture supernatants after incubation of BALB/c mouse splenocytes with inactivated bacillus. Synthesis of all main Ig classes and all IgG subclasses was stimulated in vitro, the considerable effect on IgA formation being the most interesting feature. B cell stimulation was T cell dependent, as was demonstrated by the effect of B. firmus on all Ig isotypes and by comparison of lymphocyte response of nu/nu mice and heterozygous nu/+ mice. The effect of B. firmus on splenocyte proliferation was stimulatory or suppressive depending on the dose of the bacterium. Increased synthesis of IFN-gamma and IL-10 (detected by ELISA in splenocyte culture supernatants) showed probable stimulation of Th1 and Th2 subpopulations. Considering the stimulatory effect on IgA formation and macrophage stimulation, B. firmus seems to be a prospective mucosal adjuvant and/or probiotic.

Human immature dendritic cells efficiently bind and take up secretory IgA without the induction of maturation.

Immature dendritic cells (DC) reside in peripheral tissues, where they pick up and process incoming pathogens via scavenger receptors or FcR such as FcgammaR and FcepsilonR. At mucosal surfaces, IgA is the main Ig to protect the body from incoming pathogens. In addition, DC are present in high numbers at these sites. We detected expression of FcalphaR (CD89) on the CD14+ population of CD34+ progenitor-derived DC and on monocyte-derived DC (MoDC). However, CD89 expression was strongly decreased upon differentiation from monocyte to DC. We found only minimal binding of serum IgA to MoDC but strong binding of secretory IgA (SIgA). The SIgA binding to MoDC could not be blocked by anti-CD89 blocking Abs. DC efficiently internalized SIgA, but not serum IgA, and uptake of SIgA could be blocked by specific sugars or partially by Ab reactive with mannose receptor. Importantly, binding and uptake of SIgA was not accompanied by signs of DC maturation, such as increased expression of CD86 and CD83 or induction of cytokine secretion. These data indicate that SIgA can interact with DC not via CD89, but via carbohydrate-recognizing receptors like mannose receptor and suggest that uptake of SIgA-containing immune complexes by immature DC may be a mechanism to modulate mucosal immune responses.

Regulation of immunoglobulin secretion by plasma cells infiltrating nasal polyps.

To learn more about the role of plasma cells infiltrating nasal polyps in the pathogenesis of nasal polyposis, we examined their function by analyzing immunoglobulin (Ig) production and the factors implicated in the secretion. A series of 19 consecutive nasal polyp tissue samples and, as a control, peripheral blood samples from the same patients, were studied by histopathological and immunological examination. Hematoxylin-eosin and immunohistochemical staining was carried out to identify plasma cells infiltrating nasal polyps. Nasal polyp mononuclear cells (NPMNCs) were purified from nasal polyp tissue samples, and Ig-secreting cells were identified in cytospin preparations stained with fluorescein isothiocyanate-conjugated antibodies against IgA, IgG, IgM, and IgE. Purified NPMNCs were cultured in basal conditions and after the addition of several stimuli. Ig secreted into the culture supernatants was evaluated by an enzyme-linked immunosorbent assay. Plasma cells accounted for an important fraction of the inflammatory infiltrate. The main Ig isotype synthesized by these cells was IgA, whereas little IgE was detected. In vitro cultures demonstrated that the plasma cells actively secreted Ig for a short period. When cytokine dependence was analyzed, interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) were shown to be partially responsible for the Ig production. Dependence on CD95-mediated apoptosis was not observed. Nasal polyp-infiltrating plasma cells are mainly IgA-secreting cells, the latter property being related to the mucosal immune system. The IgA production is partly dependent on IL-10 and TNF-alpha. The absence of IgE-secreting cells in most of the samples suggests that a type I hypersensitivity reaction is not essential for the development of nasal polyp.

Voltage gating of Shaker K+ channels. The effect of temperature on ionic and gating currents.

Ionic (Ii) and gating currents (Ig) from noninactivating Shaker H4 K+ channels were recorded with the cut-open oocyte voltage clamp and macropatch techniques. Steady state and kinetic properties were studied in the temperature range 2-22 degreesC. The time course of Ii elicited by large depolarizations consists of an initial delay followed by an exponential rise with two kinetic components. The main Ii component is highly temperature dependent (Q10 > 4) and mildly voltage dependent, having a valence times the fraction of electric field (z) of 0.2-0.3 eo. The Ig On response obtained between -60 and 20 mV consists of a rising phase followed by a decay with fast and slow kinetic components. The main Ig component of decay is highly temperature dependent (Q10 > 4) and has a z between 1.6 and 2.8 eo in the voltage range from -60 to -10 mV, and approximately 0.45 eo at more depolarized potentials. After a pulse to 0 mV, a variable recovery period at -50 mV reactivates the gating charge with a high temperature dependence (Q10 > 4). In contrast, the reactivation occurring between -90 and -50 mV has a Q10 = 1.2. Fluctuation analysis of ionic currents reveals that the open probability decreases 20% between 18 and 8 degreesC and the unitary conductance has a low temperature dependence with a Q10 of 1.44. Plots of conductance and gating charge displacement are displaced to the left along the voltage axis when the temperature is decreased. The temperature data suggests that activation consists of a series of early steps with low enthalpic and negative entropic changes, followed by at least one step with high enthalpic and positive entropic changes, leading to final transition to the open state, which has a negative entropic change.

Evolution of alloantibodies and suppressor cells in allografted mice treated for passive enhancement

The kinetics and quality of the alloimmune reaction were studied in CBA (H-2 k) mice treated for passive enhancement of tumor allografts (Sa 1 indigeneous of A/J (H-2 a or H-2 k/d) mice). Serum samples of treated animals were tested for their biological properties relevant to different antibody isotypes in vitro (hemagglutination, complement-dependent cytotoxicity, and anaphylaxis, i.e., mast cell degranulation involving all main Ig isotypes; IgM, IgG2, and IgG1, IgE, respectively) as well as in vivo (allograft enhancement). Spleen cells from these treated animals were examined for their capacity to interfere with the rejection of tumor allografts by adoptive transfers into syngeneic recipients. In vitro, 51Cr release cytolysis assays were performed in order to test their cytolytic and regulatory activities in comparison to rejecting control animals. It has been shown that: (1) grafted mice, pretreated for passive enhancement, kept their grafts longer and synthetized anaphylactic antibodies (mainly IgG1) earlier and at higher titers than normal serum controls, which rejected the same Sa 1 allografts. Mice with enhanced tumors synthetized cytotoxic antibodies (mainly IgG2) later than rejecting controls. (2) Serum samples from treated and control animals, harvested 10 days (early sera) and 30 days (late sera) after grafting, were injected with a “normal dose” (0.2 ml) and a “high” dose (0.4 ml) to new CBA recipients grafted with Sa 1. Early immune sera were only enhancing at high doses when derived from animals previously treated for enhancement (at the low dose both immune sera were enhancing). Late sera, presenting both complement-fixing, cytotoxic (predominantly IgG2), and IgG1 anaphylactic alloantibodies in the two groups, induced enhancement in all cases, but more strongly when derived from the group treated for Sa 1 enhancement. (3) Adoptive transfer of spleen cells from animals treated for passive enhancement were able either to inhibit the accelerated rejection (Day 10) or to promote enhancement of Sa 1 allogeneic cells (Day 30) while similar cells taken (Day 10 and Day 30) from control graft-rejecting mice transferred accelerated rejection. Among the transferred T-cell subpopulations, the suppressive effect was mediated by Lyt 2 T cells. (4) In vitro, these spleen cells showed a weaker cytolytic activity than those of allograft-rejecting mice. Moreover, they were able to regulate the cytolytic activity of cytotoxic effector cells from specifically immunized CBA mice.