PRMT5 is essential for B cell development and germinal center dynamics.

Mechanisms regulating B cell development, activation, education in the germinal center (GC) and differentiation, underpin the humoral immune response. Protein arginine methyltransferase 5 (Prmt5), which catalyzes most symmetric dimethyl arginine protein modifications, is overexpressed in B cell lymphomas but its function in normal B cells is poorly defined. Here we show that Prmt5 is necessary for antibody responses and has essential but distinct functions in all proliferative B cell stages in mice. Prmt5 is necessary for B cell development by preventing p53-dependent and p53-independent blocks in Pro-B and Pre-B cells, respectively. By contrast, Prmt5 protects, via p53-independent pathways, mature B cells from apoptosis during activation, promotes GC expansion, and counters plasma cell differentiation. Phenotypic and RNA-seq data indicate that Prmt5 regulates GC light zone B cell fate by regulating transcriptional programs, achieved in part by ensuring RNA splicing fidelity. Our results establish Prmt5 as an essential regulator of B cell biology.

Proteomic analysis of Mecistocirrus digitatus and Haemonchus contortus intestinal protein extracts and subsequent efficacy testing in a vaccine trial.

BACKGROUND: Gastrointestinal nematode infections, such as Haemonchus contortus and Mecistocirrus digitatus, are ranked in the top twenty diseases affecting small-holder farmers' livestock, yet research into M. digitatus, which infects cattle and buffalo in Asia is limited. Intestine-derived native protein vaccines are effective against Haemonchus, yet the protective efficacy of intestine-derived M. digitatus proteins has yet to be determined. METHODOLOGY/PRINCIPAL FINDINGS: A simplified protein extraction protocol (A) is described and compared to an established method (B) for protein extraction from H. contortus. Proteomic analysis of the H. contortus and M. digitatus protein extracts identified putative vaccine antigens including aminopeptidases (H11), zinc metallopeptidases, glutamate dehydrogenase, and apical gut membrane polyproteins. A vaccine trial compared the ability of the M. digitatus extract and two different H. contortus extracts to protect sheep against H. contortus challenge. Both Haemonchus fractions (A and B) were highly effective, reducing cumulative Faecal Egg Counts (FEC) by 99.19% and 99.89% and total worm burdens by 87.28% and 93.64% respectively, compared to the unvaccinated controls. There was no effect on H. contortus worm burdens following vaccination with the M. digitatus extract and the 28.2% reduction in cumulative FEC was not statistically significant. However, FEC were consistently lower in the M. digitatus extract vaccinates compared to the un-vaccinated controls from 25 days post-infection. CONCLUSIONS/SIGNIFICANCE: Similar, antigenically cross-reactive proteins are found in H. contortus and M. digitatus; this is the first step towards developing a multivalent native vaccine against Haemonchus species and M. digitatus. The simplified protein extraction method could form the basis for a locally produced vaccine against H. contortus and, possibly M. digitatus, in regions where effective cold chains for vaccine distribution are limited. The application of such a vaccine in these regions would reduce the need for anthelmintic treatment and the resultant selection for anthelmintic resistant parasites.

Expression of sheep interleukin 23 (IL23A, alpha subunit p19) in two distinct gastrointestinal diseases.

This paper reports the sequence of sheep interleukin 23A (p19), and shows that it shares 98% identity with bovine IL23A, 85% with human and 76% with mouse IL23A. It also reports the existence of two allelic variants that differ largely within the region encoding the amino terminal polypeptide signal sequence. An optimized RT-qPCR assay was used to quantify IL23A transcripts in sheep infected with two common gastrointestinal pathogens, the intracellular bacterium Mycobacterium avium subspecies paratuberculosis and the parasitic nematode Teladorsagia circumcincta. No differential expression of IL23A was detected in the mesenteric lymph node of sheep with the different pathogenic forms of paratuberculosis, however significantly high levels of IL23A were detected in the ileal mucosa of the paucibacillary form in comparison with the asymptomatic or multibacillary forms. Similarly, significantly high levels were present in the gastric lymph node draining T. circumcincta-infected abomasum in susceptible sheep. High levels of IL23A seem to be associated with lymphocytic infiltration and inflammation in both diseases but not with the macrophage infiltrate of multibacillary paratuberculosis.

Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization.

BACKGROUND: The extensive similarities between helminth proteins and allergens are thought to contribute to helminth-driven allergic sensitization. OBJECTIVE: The objective of this study was to investigate the cross-reactivity between a major glutathione-S transferase allergen of cockroach (Bla g 5) and the glutathione-S transferase of Wuchereria bancrofti (WbGST), a major lymphatic filarial pathogen of humans. METHODS: We compared the molecular and structural similarities between Bla g 5 and WbGST by in silico analysis and by linear epitope mapping. The levels of IgE, IgG, and IgG(4) antibodies were measured in filarial-infected and filarial-uninfected patients. Mice were infected with Heligmosomoides bakeri, and their skin was tested for cross-reactive allergic responses. RESULTS: These 2 proteins are 30% identical at the amino acid level with remarkable similarity in the N-terminal region and overall structural conservation based on predicted 3-dimensional models. Filarial infection was associated with IgE, IgG, and IgG(4) anti-Bla g 5 antibody production, with a significant correlation between antibodies (irrespective of isotype) to Bla g 5 and WbGST (P< .0003). Preincubation of sera from cockroach-allergic subjects with WbGST partially depleted (by 50%-70%) anti-Bla g 5 IgE, IgG, and IgG(4) antibodies. IgE epitope mapping of Bla g 5 revealed that 2 linear N-terminal epitopes are highly conserved in WbGST corresponding to Bla g 5 peptides partially involved in the inhibition of WbGST binding. Finally, mice infected with H bakeri developed anti-HbGST IgE and showed immediate-type skin test reactivity to Bla g 5. CONCLUSION: These data demonstrate that helminth glutathione-S transferase and the aeroallergen Bla g 5 share epitopes that can induce allergic cross-sensitization.

A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses?

A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling.

Cytokine expression in naïve and previously infected lambs after challenge with Teladorsagia circumcincta.

Infection of sheep with Teladorsagia circumcincta triggers an immune response with predominantly type-2 (Th2) characteristics, including local eosinophila, mastocytosis and increased mucus production. In order to better understand the protective immune responses elicited, we used RT-PCR assays to define the changes in expression levels of a range of cytokine transcripts in lymph nodes draining the ovine abomasum following a challenge infection with T. circumcincta. This study compared the changes in cytokine expression in the abomasal lymph node following challenge with T. circumcincta in naïve sheep (Group 2) and sheep immunised by a previous trickle infection (Group 3), in comparison to unchallenged naive sheep (Group 1). There was a significant up-regulation of interleukin-4 (IL-4), IL-5 and IL-13 in both the challenged groups compared to naïve individuals. There was also an up-regulation of IL-1beta, IL-6, IL-10, IL-18, transforming growth factor-beta1 (TGFbeta1) and tumour necrosis factor-alpha (TNFalpha) by day 5 after infection. IL-12p40 was found to be increased in the previously infected Group 3 animals by day 5 following challenge. By contrast, transcription of this cytokine was found to be reduced by day 10 following infection of Group 2 animals. Expression of IL-2 and Interferon-gamma (IFNgamma) did not significantly differ between the three groups.

Eosinophil and IgA responses in sheep infected with Teladorsagia circumcincta.

Infections of sheep with the gastrointestinal nematode Teladorsagia circumcincta are characterised by increased concentrations of IgA, eosinophilia and mastocytosis but the interactions between these immune responses are unclear. We investigated the kinetics of the parasite-specific IgA and eosinophil responses in controlled infections in lambs to determine if there were any associations with subsequent worm growth and survival. IgA and eosinophil responses had very similar kinetics and variation in both responses accounted for more of the variation in adult worm length than either trait alone suggesting that IgA and eosinophils interact in regulating the growth of T. circumcincta. Curiously, those animals with higher peak eosinophil responses had longer worms at slaughter emphasising the intriguing complexity of the immune responses to these parasitic infections.

Vaccination of young lambs against infection with Nematodirus battus using gamma irradiated larvae.

Helminthologically naIve 6-week-old Suffolk lambs were given 1-3 doses of 20000 gamma-irradiated infective larvae (L3) of the nematode Nematodirus battus at weekly intervals. Following an anthelmintic drench they were challenged with 50000 viable L3 at 10 weeks of age. Nematode worm burdens 14 days post-challenge showed a significant (P<0.01) 66% reduction in the single vaccine dose group. The two and three dose groups had mean worm burdens which were 30 and 42% lower than controls, respectively, although these were not statistically significant. There was little measurable stimulation of the immune system in the vaccinated lambs, suggesting that the repeatedly dosed animals may have developed immunological unresponsiveness to the parasite.

Haemopoietic cell responses in the blood and bone marrow of sheep infected with the abomasal nematode Telodorsagia circumcincta.

The generation of bone marrow and blood haemopoietic progenitor colony-forming cells (CFCs) in sheep given primary or challenge infections with the nematode parasite Telodorsagia circumcincta is described. Ten days after a primary infection, the frequency of early multipotential-CFCs, eosinophil-CFCs, macrophage-CFCs and mast cell or basophil-CFCs was greater than in controls. These frequencies then declined to pre-infection levels by day 21. Blood CFCs (mainly macrophage-CFCs and eosinophil-CFCs) also increased after infection, indicating a migration of CFCs, presumably to the site of infection. Ten days after challenge infection there was less marked myelopoiesis than in the primary infection on day 10, though both eosinophil-CFCs and mast cell or basophil-CFCs were significantly above control values. Blood CFC output (mainly macrophage-CFCs and eosinophil-CFCs) reached a peak 2-6 days after challenge, evidence of rapid recruitment to the site of infection. Telodorsagia circumcincta infection is therefore associated with an increase in myelopoiesis, particularly for the cell types characteristic of the local inflammatory response to abomasal nematodes. There was no correlation between any of the haemopoietic cell responses measured and worm burdens in individual animals after either primary or challenge infection.

Characterization, cloning and host-protective activity of a 30-kilodalton glycoprotein secreted by the parasitic stages of Trichostrongylus colubriformis.

The helminth Trichostrongylus colubriformis is a parasitic nematode infecting the small intestine of sheep. We report the isolation and characterization of a 30-kDa glycoprotein capable of partially protecting guinea-pigs against the parasite. This glycoprotein is secreted by the L4 and adult parasitic stages of the worm. The sequence of three separate cDNA clones predicts the polypeptide to be about 15 kDa, with four N-linked carbohydrate chains and an internal disulphide bond. The clones also indicate the existence of sequence variability in this antigen. Limited sequence homology to a porcine intestinal peptide suggests an influence on host gut physiology.

Relationship between cytologic changes in bronchoalveolar lavage fluid and weight gain in calves with gastrointestinal nematodes and lungworms.

Two of three groups of 10 calves each were infected with either 100,000 infective larvae (L3) of Ostertagia spp. and 100,000 L3 of Cooperia spp. or with 4000 L3 of Dictyocaulus viviparus, respectively, at the age of 14 weeks. The third group was not infected. After treatment with an anthelminthic five calves from each group were challenged with either 100,000 L3 of Ostertagia spp. and 100,000 L3 of Cooperia spp. or 4000 L3 of Dictyocaulus at the age of 20 weeks. The calves were 25 weeks old when slaughtered. Total and differential cell counts were determined in bronchoalveolar lavage fluid and showed that neutrophils were the most frequent and eosinophils the least frequent cell present. There was a significant negative relationship between eosinophil levels and weight gain of the calves.

Demonstration of IgA-containing cells in the small intestine of lambs infected with Nematodirus battus.

A peroxidase anti-peroxidase immunohistochemical technique was used to demonstrate IgA-containing cells in the small intestine of lambs infected with Nematodirus battus. These cells were more numerous in the infected lambs than in the uninfected animals. The difference was greatest for the first three sites, 1 to 3 m distal to the pylorus, where the preponderance of the N battus infection is found. It is suggested that future immunohistochemical studies on the relationships between IgA and resistance to N battus should be directed to this area.

Analysis of the mechanism of immunodepression following heterologous antigenic stimulation during concurrent infection with Nematospiroides dubius.

The suppression of immune responsiveness to heterologous antigenic stimulation during concurrent infection with Nematospiroides dubius was reproduced using soluble antigens derived from adult parasites. Immunosuppression appeared to be selective in that the administration of equivalent quantities of an irrelevant heterogeneous antigen had no immunosuppressive effect, and suppression was transferable using spleen cells from parasite antigen-treated donors. The differential immunomodulatory activity of parasite antigens from a variety of nematode species suggested that a correlation might exist between suppressor activity and chronicity of infection. A role for suppressor T cell activity in the infected host was implicated by the restorative effect of 2'deoxyguanosine treatment on the immune response, and non-specific suppressor cell activity was detected in splenocyte populations from infected mice. It is suggested that a parasite-induced defect in antigen processing led to the induction of suppressor cell activity in the infected host and that this may be one mechanism of parasite survival. The relevance of these observations to vaccination against chronic gastrointestinal nematode infections is discussed.

Changes in the adenylate energy charge of Nippostrongylus brasiliensis and Nematodirus battus during the development of immunity to these nematodes in their host.

Infection of rats with 2000 infective juveniles of Nippostrongylus brasiliensis and of lambs with 60 000 infective juveniles of Nematodirus battus results in a well-marked immunity to these nematodes in their respective host. There is a fall in the adenylate energy charge value of these nematodes during the course of these infections, reaching values of 0.37 in males and 0.27 in females of N. brasiliensis, and 0.31 in males and 0.23 in females of N. battus towards the end of the infections. In hosts given relatively small numbers of infective juveniles, the values for the nematodes removed from the hosts late in the infection remain at a relatively high level. These results indicate that the immune response of the host may affect the energy status of these nematodes, and this could help to explain their subsequent expulsion from the immune host.

Studies of the responses of basophil and eosinophil leucocytes and mast cells to the nematode Trichostrongylus colubriformis. II. Changes in cell numbers following infection of thymectomised and adoptively or passively immunised guinea-pigs.

The role of the immune response in the generation of the basophilia and eosinophilia found during expulsion of the intestinal nematode parasite, Trichostrongylus colubriformis, by guinea-pigs was investigated by studying cell numbers in animals whose immune responsiveness had been modified by thymectomy and adoptive or passive immunisation. Basophilia, but not eosinophilia, was depressed in thymectomised guinea-pigs. Bone marrow basophil numbers were significantly increased in T. colubriformis-infected guinea-pigs following the infection of mesenteric lymph-node cells from both normal and T. colubriformis-immune syngeneic donors. Bone marrow basophil counts were also increased following the injection of immune lymph-node cells into uninfected recipients. Small intestine eosinophil numbers in adoptively immunised guinea-pigs showed a pronounced increase following infection with T. colubriformis. A smaller increase followed infection of passively immunised guinea-pigs. These results, and other work with this system, suggest that basophilia and eosinophilia during T. colubriformis infection, although associated with the immune response, might not be fully explained as direct consequences of the interaction of parasitic antigens and sensitised lymphocytes or antibodies.

Studies of the responses of basophil and eosinophil leucocytes and mast cells to the nematode Trichostrongylus colubriformis. I. Observations during the expulsion of first and second infections by guinea-pigs.

During expulsion by guniea-pigs of both first and second infections with the intestinal nematode parasite Trichostrongylus colubriformis, increased numbers of basophils and eosinophils were present in the bone marrow and blood. In addition, increased numbers of basophils, mast cells and eosinophils, many possessing reduced numbers of cytoplasmic granules, accumulated at the sites of infection. Blood eosinophil counts, however, did not show significant changes during the early part of a first infection.