The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BackgroundMutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear.ObjectiveWe sought to define the functional consequences of the C104R mutation on B-cell function.MethodsWe performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge.ResultsC104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis.ConclusionsThese data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.

Viral superantigen drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Mouse mammary tumor virus (MMTV[SW]) encodes a superantigen expressed by infected B cells. It evokes an antibody response specific for viral envelope protein, indicating selective activation of antigen-specific B cells. The response to MMTV(SW) in draining lymph nodes was compared with the response to haptenated chicken gamma globulin (NP-CGG) using flow cytometry and immunohistology. T cell priming occurs in both responses, with T cells proliferating in association with interdigitating dendritic cells in the T zone. T cell proliferation continues in the presence of B cells in the outer T zone, and B blasts then undergo exponential growth and differentiation into plasma cells in the medullary cords. Germinal centers develop in both responses, but those induced by MMTV(SW) appear later and are smaller. Most T cells activated in the T zone and germinal centers in the MMTV(SW) response are superantigen specific and these persist for weeks in lymph nodes draining the site MMTV(SW) injection: this contrasts with the selective loss of superantigen-specific T cells from other secondary lymphoid tissues. The results indicate that this viral superantigen, when expressed by professional antigen-presenting cells, drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production.

Glyoxysomal malate-dehydrogenase and malate synthase from Soybean cotyledons (Glycine max. L.): Enzyme association, antibody-production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD(+) oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M(r)) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M, 510 000 and of gMDH as a dimer of M, 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X(6))HL motif also found in plant and mammalian thiolases.

Development of T-B cell collaboration in neonatal mice.

The neonatal immune response is impaired during the first weeks after birth. To obtain a better understanding of this immaturity, we investigated the development of T cell interactions with B cells in mice. For this purpose, we analyzed the immune response to three T-dependent antigens in vivo: (i) the polyclonal antibody response induced by vaccinia virus; (ii) the production of polyclonal and specific antibodies following immunization with hapten-carrier conjugates; (iii) the mouse mammary tumor virus superantigen (sAg) response involving an increase in sAg-reactive T cells and induction of polyclonal antibody production. After vaccinia virus injection into neonates, the polyclonal antibody response was similar to that observed in adult mice. The antibody response to hapten-carrier conjugates, however, was delayed and reduced. Injection with sAg-expressing B cells from neonatal or adult mice allowed us to determine whether B cells, T cells or both were implicated in the reduced immune response. In these sAg responses, neonatal T cells were stimulated by both neonatal and adult sAg-presenting B cells but only B cells from adult mice differentiated into IgM- and IgG-secreting plasma cells in the neonatal environment in vivo. Injecting neonatal B cells into adult mice did not induce antibody production. These results demonstrate that the environment of the neonatal lymph node is able to support a T and B cell response, and that immaturity of B cells plays a key role in the reduced immune response observed in the neonate.

TLR 2 modulates inflammation in zymosan-induced arthritis in mice

Résumé L'objectif de cette étude est la compréhension des mécanismes sous-jacents à l'inflammation articulaire dans un modèle murin d'arthrite induite par le zymosan (ZIA). En particulier, la participation du récepteur Toll 2 (TLR2) et du complément C3 a été recherchée. L'inflammation articulaire a été quantifiée par l'accumulation de Technetium (Tc) in vivo, et par histologie des articulations arthritiques. Les réponses humorales et cellulaires induites par le zymosan ont été quantifiées par la prolifération lymphocytaire in vitro et par la mesure de la production d'anticorps dirigés contre le zymosan in vivo. L'inflammation associée à l'arthrite induite au zymosan est, d'après le Tc-uptake, d'aspect biphasique, avec un pic après 1 jour, puis une deuxième phase plus tardive. La deuxième phase persiste jusqu'au 24 ème jour et est associée au développement d'une immunité spécifique contre le zymosan. Les souris déficientes pour TLR-2 présentent une réduction significative de l'inflammation articulaire précoce (jour 1) et tardive (jour 24), ainsi qu'une nette diminution de l'infiltrat inflammatoire dans la membrane synoviale. De plus, la prolifération de cellules du ganglion lymphatique ainsi que le taux d'IgG dirigés contre le zymosan sont diminués de façon significative après 25 jour d'arthrite chez les souris déficientes en TLR2 par rapport aux souris sauvages contrôles. Par contraste, chez les souris déficientes pour C3 on n'observe pas de différence dans l'uptake de Tc ou le scoring histologique par rapport à la lignée sauvage. Ces résultats montrent que l'arthrite induite au zymosan n'est pas seulement un modèle d'inflammation aigue, mais que l'inflammation synoviale persiste même après 25 jours. Ce modèle implique à la fois des mécanismes d'immunité innée et acquise. Le signalling via TLR 2 semble jouer in rôle dans l'immunité au zymosan et pourrait être responsable de la nature biphasique de ce modèle d'arthrite. Abstract The interplay between the innate and acquired immune systems in chronic inflammation is not well documented. We have investigated the mechanisms of inflammation in murine zymosan-induced arthritis (ZIA) in the light of recent data on the roles of Toll-like receptor 2 (TLR2) and Dentin-1 in the activation of monocyte/macrophages by zymosan. The severity of inflammation, joint histology, lymphocyte proliferation and antibody production in response to zymosan were analyzed in mice deficient in TLR2 and complement C3, and the effects of Dentin-1 inhibition by laminarin were studied. In comparison with wild-type animals, TLR2-deficient mice showed a significant decrease in the early (day 1) and late phases (day 24) of joint inflammation. C3-deficient mice showed no differences in technetium uptake or histological scoring. TLR2-deficient mice also showed a significant decrease in lymph node cell proliferation in response to zymosan and a lower IgG antibody response to zymosan at day 25 in comparison with wild-type controls, indicating that TLR2 signalling has a role in the development of acquired immune responses to zymosan. Although laminarin, a soluble β-glucan, was able to significantly inhibit zymosan uptake by macrophages in vitro, it had no effect on ZIA in vivo. These results show that ZIA is more prolonged than was originally described and involves both the innate and acquired immune pathways. C3 does not seem to have a major role in this model of joint inflammation.

Strength and cost of an induced immune response are associated with a heritable melanin-based colour trait in female tawny owls.

1. Melanin pigments provide the most widespread source of coloration in vertebrates, but the adaptive function of such traits remains poorly known. 2. In a wild population of tawny owls (Strix aluco), we investigated the relationships between plumage coloration, which varies continuously from dark to pale reddish, and the strength and cost of an induced immune response. 3. The degree of reddishness in tawny owl feather colour was positively correlated with the concentration of phaeomelanin and eumelanin pigments, and plumage coloration was highly heritable (h(2) = 0.93). No carotenoids were detected in the feathers. 4. In mothers, the degree of melanin-based coloration was associated with antibody production against a vaccine, with dark reddish females maintaining a stronger level of antibody for a longer period of time compared to pale reddish females, but at a cost in terms of greater loss of body mass. 5. A cross-fostering experiment showed that, independent of maternal coloration, foster chicks reared by vaccinated mothers were lighter than those reared by nonvaccinated mothers. Hence, even though dark reddish mothers suffered a stronger immune cost than pale reddish mothers, this asymmetric cost was not translated to offspring growth. 6. Our study suggests that different heritable melanin-based colorations are associated with alternative strategies to resist parasite attacks, with dark reddish individuals investing more resources towards the humoral immune response than lightly reddish conspecifics.

Developmental, metabolic and immunological costs of flea infestation in the common vole

Parasites use resources from their hosts, which can indirectly affect a number of host functions because of trade-offs in resource allocation. In order to get a comprehensive view of the costs imposed by blood sucking parasites to their hosts, it is important to monitor multiple components of the development and physiology of parasitized hosts over long time periods. The effect of infestation by fleas on body mass, body length growth, haematocrit, resistance to oxidative stress, resting metabolic rate and humoral immune response were experimentally evaluated. During a 3-month period, male common voles, Microtus arvalis, were either parasitized by rat fleas (Nosopsyllus fasciatus), which are naturally occurring generalist ectoparasites of voles, or reared without fleas. Then voles were challenged twice by injecting Keyhole Limpet Haemocyanin (KLH) to assess whether the presence of fleas affects the ability of voles to produce antibodies against a novel antigen. During the immune challenge we measured the evolution of body mass, haematocrit, resistance to oxidative stress and antibody production. Flea infestation negatively influenced the growth of voles. Moreover, parasitized voles had reduced haematocrit, higher resting metabolic rate and lower production of antibodies against the KLH. Resistance to oxidative stress was not influenced by the presence of fleas. During the immune challenge with KLH, body mass decreased in both groups, while the resistance to oxidative stress remained stable. In contrast, the haematocrit decreased only in parasitized voles. Our experiment shows that infestation by a haematophageous parasite negatively affects multiple traits like growth, energy consumption and immune response. Fleas may severely reduce the survival probability and reproductive success of their host in natural conditions.

Innate immune response to poxvirus vectors

Summary : A large body of evidence indicates that the innate immune system plays a key role in host response to viral infection. Recently, Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptor receptors (NLRs) have emerged as key innate immune sensors of microbial products, eliciting intracellular signaling and leading to the production of chemokines, cytokines and interferons (IFNs) that shape innate immune responses and coordinate the development of adaptive immunity. Poxviruses are currently developed as vaccines vectors for infectious diseases such as HIV, tuberculosis and malaria. Modified vaccinia virus Ankara (MVA) and New York vaccinia virus (NWAC) are attenuated, replication deficient strains of poxvirus. The mechanisms underlying innate immune responses to MVA and NYVAC are poorly characterized. Thus, the objectives of the project were to determine the innate immune profile stimulated by poxviruses in innate immune cells and to evaluate the impact of modifications in the viral genome on MVA and NYVAC immunogenicity. MVA stimulated the production of abundant amounts of chemokines and IFNß but low levels of cytokines by human macrophages. In contrast, NYVAC weakly stimulated the production of all mediators. Interestingly, MVA and NYVAC strongly stimulated innate immune responses in vivo and in human whole blood, suggesting that a soluble factors}, possibly a complement component, was required for optimal activation of innate immune cells by poxviruses. Modified MVA and NYVAC produced by single or multiple deletions of viral genes targeting crucial pathways of host innate immunity, and mutant poxviruses with limited replication capacity, increased the production of pro-inflammatory molecules by human whole blood. Gene expression profiling in human macrophages confirmed the increased immunologic stimulatory capacity of modified poxviruses. The pathways activated by MVA and NYVAC in innate immune cells were described by analysing the response of knockdown or shRNA transduced macrophages with impaired expression of TLRs and their adaptors (MyD8$ and TRIF), RLRs (RIG-I, MDA-5 and the adaptor IPS-1) and the NALP3 inflammasome composed óf the NLR NALP3, caspase-1 and ASC. These experiments revealed a critical role for TLR2-TLR6-MyD88 in the production of tFNß-independent chemokines and of MDA-5-IPS-1 in the production of IFNß and IFNßdependent chemokines. The transcription of the iL1b gene encoding for the IL-1ß cytokine was initiated through TLR2-MyD88, whereas the maturation and the secretion of IL-1ß were controlled by the NALP3 inflammasome. Finally, we analyzed the role of macrophage migration inhibitory factor (MIF), a mediator of inflammation and innate immune responses, in MVA infection. We observed that MVA infection increased MIF production by innate immune cells and that MIF deficiency impaired macrophage and dendritic cell responses (ie migration, maturation, cytokine and IFN production) to MVA infection in vitro and in vivo. Moreover, MIF-deficiency resulted in delayed anti-MVA specific antibody production in mice immunized with the virus. In conclusion, we demonstrate. that poxviruses can be modified genetically to improve their immunogenicity. We also report the first comprehensive analysis of poxvirus sensing by innate immune cells, showing that the TLR, RLR and NLR pathways play specific and coordinated roles in regulating cytokine, chemokine and IFN response to poxvirus infection. Finally, we show that MIF is an integral host component involved in innate and adaptive immune responses to MVA infection. The present findings provide important information relevant to the study of the pathogenesis of poxvirus infections and allow a better understanding of the immunogenic potential of vaccine vectors, which is required for the development of optimized modìfied pox-vaccine vectors.

Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome.

Many currently used and candidate vaccine adjuvants are particulate in nature, but their mechanism of action is not well understood. Here, we show that particulate adjuvants, including biodegradable poly(lactide-co-glycolide) (PLG) and polystyrene microparticles, dramatically enhance secretion of interleukin-1beta (IL-1beta) by dendritic cells (DCs). The ability of particulates to promote IL-1beta secretion and caspase 1 activation required particle uptake by DCs and NALP3. Uptake of microparticles induced lysosomal damage, whereas particle-mediated enhancement of IL-1beta secretion required phagosomal acidification and the lysosomal cysteine protease cathepsin B, suggesting a role for lysosomal damage in inflammasome activation. Although the presence of a Toll-like receptor (TLR) agonist was required to induce IL-1beta production in vitro, injection of the adjuvants in the absence of TLR agonists induced IL-1beta production at the injection site, indicating that endogenous factors can synergize with particulates to promote inflammasome activation. The enhancement of antigen-specific antibody production by PLG microparticles was independent of NALP3. However, the ability of PLG microparticles to promote antigen-specific IL-6 production by T cells and the recruitment and activation of a population of CD11b(+)Gr1(-) cells required NALP3. Our data demonstrate that uptake of microparticulate adjuvants by DCs activates the NALP3 inflammasome, and this contributes to their enhancing effects on innate and antigen-specific cellular immunity.

Sudden weather deterioration but not brood size affects baseline corticosterone levels in nestling Alpine swifts.

While evidence is accumulating that stress-induced glucocorticoid responses help organisms to quickly adjust their physiology and behaviour to life-threatening environmental perturbations, the function and the ecological factors inducing variation in baseline glucocorticoid levels remain poorly understood. In this study we investigated the effects of brood size by experimentally manipulating the number of nestlings per brood and the effect of weather condition on baseline corticosterone levels of nestling Alpine swifts (Apus melba). We also examined the potential negative consequences of an elevation of baseline corticosterone on nestling immunity by correlating corticosterone levels with ectoparasite intensity and the antibody production towards a vaccine. Although nestlings reared in enlarged broods were in poorer condition than nestlings reared in reduced broods, they showed similar baseline corticosterone levels. In contrast, nestling baseline corticosterone levels were higher immediately after cold and rainy episodes with strong winds. Neither nestling infestation rate by ectoparastic flies nor nestling antibody production against a vaccine was correlated with baseline corticosterone levels. Thus, our results suggest that altricial Alpine swift nestlings can quickly modulate baseline corticosterone levels in response to unpredictable variations in meteorological perturbation but not to brood size which may be associated with the degree of sibling competition. Apparently, short-term elevations of baseline corticosterone have no negative effects on nestling immunocompetence.

APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa.

The bone marrow constitutes a favorable environment for long-lived antibody-secreting plasma cells, providing blood-circulating antibody. Plasma cells are also present in mucosa-associated lymphoid tissue (MALT) to mediate local frontline immunity, but how plasma cell survival there is regulated is not known. Here we report that a proliferation-inducing ligand (APRIL) promoted survival of human upper and lower MALT plasma cells by upregulating expression of the antiapoptotic proteins bcl-2, bcl-xL, and mcl-1. The in situ localization of APRIL was consistent with such a prosurvival role in MALT. In upper MALT, tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils recruited from the blood infiltrated the crypt epithelium. Heparan sulfate proteoglycans (HSPGs) retained secreted APRIL in the subepithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL, giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. Furthermore, we found that mucosal humoral immunity in APRIL-deficient mice is less persistent than in WT mice. Hence, production of APRIL by inflammation-recruited neutrophils may create plasma cell niches in MALT to sustain a local antibody production.

Acute Lyme Neuroborreliosis With Transient Hemiparesis and Aphasia.

Nervous system involvement in Lyme disease often mimics other conditions and thus represents a diagnostic challenge, especially in an emergency department setting. We report a case of a female teenager presenting with sudden-onset aphasia and transient right-sided faciobrachial hemiplegia, along with headache and agitation. Ischemia, vasculitis, or another structural lesion was excluded by brain imaging. Toxicologic evaluation results were negative. Cerebral perfusion computed tomography and electroencephalography showed left parietotemporal brain dysfunction. Lumbar puncture result, although atypical, suggested bacterial infection and intravenous ceftriaxone was initiated. Finally, microbiological cerebrospinal fluid analysis revealed Lyme neuroborreliosis, showing specific intrathecal antibody production and high level of C-X-C motif chemokine 13. The patient rapidly recovered. To our knowledge, this report for the first time illustrates that acute-onset language and motor symptoms may be directly related to Lyme neuroborreliosis. Neuroborreliosis may mimic other acute neurologic events such as stroke and should be taken into diagnostic consideration even in the absence of classic symptoms and evolution.

Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.

Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehyde-fixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.