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Avian influenza viruses (AIVs) are a major economic burden to the poultry industry and pose serious zoonotic risks, with human infections being reported every year. To date, the vaccination of birds remains the most important method for the prevention and control of AIV outbreaks. Most national vaccination strategies against AIV infection use whole virus-inactivated vaccines, which predominantly trigger a systemic antibody-mediated immune response. There are currently no studies that have examined the antibody repertoire of birds that were infected with and/or vaccinated against AIV. To this end, we evaluate the changes in the H9N2-specific IgM and IgY repertoires in chickens subjected to vaccination(s) and/or infectious challenge. We show that a large proportion of the IgM and IgY clones were shared across multiple individuals, and these public clonal responses are dependent on both the immunisation status of the birds and the specific tissue that was examined. Furthermore, the analysis revealed specific clonal expansions that are restricted to particular H9N2 immunisation regimes. These results indicate that both the nature and number of immunisations are important drivers of the antibody responses and repertoire profiles in chickens following H9N2 antigenic stimulation. We discuss how the repertoire biology of avian B-cell responses may affect the success of AIV vaccination in chickens, in particular the implications of public versus private clonal selection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dascalu, Sealy, Sadeyen, Flammer, Fiddaman, Preston, Dixon, Bonsall, Smith and Iqbal.)

Background: Colorectal cancers (CRCs) from people with biallelic germline likely pathogenic/pathogenic variants in MUTYH or NTHL1 exhibit specific single base substitution (SBS) mutational signatures, namely combined SBS18 and SBS36 (SBS18+SBS36), and SBS30, respectively. The aim was to determine if adenomas from biallelic cases demonstrated these mutational signatures at diagnostic levels., Methods: Whole-exome sequencing of FFPE tissue and matched blood-derived DNA was performed on 9 adenomas and 15 CRCs from 13 biallelic MUTYH cases, on 7 adenomas and 2 CRCs from 5 biallelic NTHL1 cases and on 27 adenomas and 26 CRCs from 46 non-hereditary (sporadic) participants. All samples were assessed for COSMIC v3.2 SBS mutational signatures., Results: In biallelic MUTYH cases, SBS18+SBS36 signature proportions in adenomas (mean±standard deviation, 65.6%±29.6%) were not significantly different to those observed in CRCs (76.2%±20.5%, p-value =0.37), but were significantly higher compared with non-hereditary adenomas (7.6%±7.0%, p-value =3.4×10 -4 ). Similarly, in biallelic NTHL1 cases, SBS30 signature proportions in adenomas (74.5%±9.4%) were similar to those in CRCs (78.8%±2.4%) but significantly higher compared with non-hereditary adenomas (2.8%±3.6%, p-value =5.1×10 -7 ). Additionally, a compound heterozygote with the c.1187G>A p.(Gly396Asp) pathogenic variant and the c.533G>C p.(Gly178Ala) variant of unknown significance (VUS) in MUTYH demonstrated high levels of SBS18+SBS36 in four adenomas and one CRC, providing evidence for reclassification of the VUS to pathogenic., Conclusions: SBS18+SBS36 and SBS30 were enriched in adenomas at comparable proportions observed in CRCs from biallelic MUTYH and biallelic NTHL1 cases, respectively. Therefore, testing adenomas may improve the identification of biallelic cases and facilitate variant classification, ultimately enabling opportunities for CRC prevention., Competing Interests: Robert C. Grant received a scholarship from Pfizer and provided consulting or advisory roles for Astrazeneca, Tempus, Eisai, Incyte, Knight Therapeutics, Guardant Health, and Ipsen. All other authors have no relevant financial or non-financial interests to disclose.

Background: This study aimed to investigate clinicopathological and molecular tumour features associated with intratumoral pks + Escherichia coli (pks + E.coli + ), pks + E.coli - (non-E.coli bacteria harbouring the pks island), Enterotoxigenic Bacteroides fragilis (ETBF) and Fusobacterium nucleatum (F. nucleatum)., Methods: We screened 1697 tumour-derived DNA samples from the Australasian Colorectal Cancer Family Registry, Melbourne Collaborative Cohort Study and the ANGELS study using targeted PCR., Results: Pks + E.coli + was associated with male sex (P < 0.01) and APC:c.835-8 A > G somatic mutation (P = 0.03). The association between pks + E.coli + and APC:c.835-8 A > G was specific to early-onset CRCs (diagnosed<45years, P = 0.02). The APC:c.835-A > G was not associated with pks + E.coli - (P = 0.36). F. nucleatum was associated with DNA mismatch repair deficiency (MMRd), BRAF:c.1799T>A p.V600E mutation, CpG island methylator phenotype, proximal tumour location, and high levels of tumour infiltrating lymphocytes (Ps < 0.01). In the stratified analysis by MMRd subgroups, F. nucleatum was associated with Lynch syndrome, MLH1 methylated and double MMR somatic mutated MMRd subgroups (Ps < 0.01)., Conclusion: Intratumoral pks + E.coli + but not pks + E.coli - are associated with CRCs harbouring the APC:c.835-8 A > G somatic mutation, suggesting that this mutation is specifically related to DNA damage from colibactin-producing E.coli exposures. F. nucleatum was associated with both hereditary and sporadic MMRd subtypes, suggesting the MMRd tumour microenvironment is important for F. nucleatum colonisation irrespective of its cause., (© 2024. The Author(s).)

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. Lynch syndrome specific tumor features were evaluated for their ability to support the ACMG/InSiGHT framework in classifying variants of uncertain clinical significance (VUS) in the MMR genes. Twenty-eight CRC or EC tumors from 25 VUS carriers (6x MLH1 , 9x MSH2 , 6x MSH6 , 4x PMS2 ), underwent targeted tumor sequencing for the presence of microsatellite instability/MMR-deficiency (MSI-H/dMMR) status and identification of a somatic MMR mutation (second hit). Immunohistochemical testing for the presence of dMMR crypts/glands in normal tissue was also performed. The ACMG/InSiGHT framework reclassified 7/25 (28%) VUS to likely pathogenic (LP), three (12%) to benign/likely benign, and 15 (60%) VUS remained unchanged. For the seven re-classified LP variants comprising nine tumors, tumor sequencing confirmed MSI-H/dMMR (8/9, 88.9%) and a second hit (7/9, 77.8%). Of these LP reclassified variants where normal tissue was available, the presence of a dMMR crypt/gland was found in 2/4 (50%). Furthermore, a dMMR endometrial gland in a carrier of an MSH2 exon 1-6 duplication provides further support for an upgrade of this VUS to LP. Our study confirmed that identifying these Lynch syndrome features can improve MMR variant classification, enabling optimal clinical care.

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. However, mosaic variants in the MMR genes have been rarely described. We identified a likely de novo mosaic MSH6:c.1135&#95;1139del p.Arg379* pathogenic variant in a patient diagnosed with suspected Lynch syndrome/Lynch-like syndrome. The patient developed MSH6-deficient EC and CRC at 54 and 58 years of age, respectively, without a detectable germline MMR pathogenic variant. Multigene panel sequencing of tumor and blood-derived DNA identified an MSH6 somatic mutation (MSH6:c.1135&#95;1139del p.Arg379*) common to both the EC and CRC, raising suspicion of mosaicism. A droplet digital polymerase chain reaction (ddPCR) assay detected the MSH6 variant at 5.34% frequency in normal colonic tissue, 3.49% in saliva and 1.64% in blood DNA, demonstrating the presence of the MSH6 variant in all three germ layers. This study highlights the utility of tumor sequencing to guide sensitive ddPCR testing to detect low-level mosaicism in the MMR genes. Further investigation of the prevalence of MMR mosaicism is needed to inform routine diagnostic approaches and genetic counselling., (© 2023. The Author(s).)

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations., (© 2023. The Author(s).)

Microbial colonisation is paramount to the normal development of the immune system, particularly at mucosal sites. However, the relationships between the microbiome and the adaptive immune repertoire have mostly been explored in rodents and humans. Here, we report a high-throughput sequencing analysis of the chicken TCRβ repertoire and the influences of microbial colonisation on tissue-resident TCRβ+ cells. The results reveal that the microbiome is an important driver of TCRβ diversity in both intestinal tissues and the bursa of Fabricius, but not in the spleen. Of note, public TCRβ sequences (shared across individuals) make a substantial contribution to the repertoire. Additionally, different tissues exhibit biases in terms of their V family and J gene usage, and these effects were influenced by the gut-associated microbiome. TCRβ clonal expansions were identified in both colonised and germ-free birds, but differences between the groups were indicative of an influence of the microbiota. Together, these findings provide an insight into the avian adaptive immune system and the influence of the microbiota on the TCRβ repertoire., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dascalu, Preston, Dixon, Flammer, Fiddaman, Boyd, Sealy, Sadeyen, Kaspers, Velge, Iqbal, Bonsall and Smith.)

Campylobacter from contaminated poultry meat is a major source of human gastroenteritis worldwide. To date, attempts to control this zoonotic infection with on-farm biosecurity measures have been inconsistent in outcome. A cornerstone of these efforts has been the detection of chicken infection with microbiological culture, where Campylobacter is generally not detectable until birds are at least 21 days old. Using parallel sequence-based bacterial 16S profiling analysis and targeted sequencing of the porA gene, Campylobacter was identified at very low levels in all commercial flocks at less than 8 days old that were tested from the United Kingdom, Switzerland, and France. These young chicks exhibited a much greater diversity of porA types than older birds testing positive for Campylobacter by culture or quantitative PCR (qPCR). This suggests that as the bacteria multiply sufficiently to be detected by culture methods, one or two variants, as indicated by porA type, dominate the infection. The findings that (i) most young chicks carry some Campylobacter and (ii) not all flocks become Campylobacter positive by culture suggest that efforts to control infection, and therefore avoid contamination of poultry meat, should concentrate on how to limit Campylobacter to low levels by the prevention of the overgrowth of single strains. IMPORTANCE Our results demonstrate the presence of Campylobacter DNA among fecal samples from a range of commercially reared meat chicks that are less than 8 days of age, consistent across 3 European countries. The recently developed, sensitive detection method indicates that infection occurs on commercial farms much earlier and more widely than previously thought, which opens up new opportunities to control Campylobacter contamination at the start of the food chain and reduce the unacceptably high levels of human disease.

Background: Despite increasing interest in γδ T cells and their non-classical behaviour, most studies focus on animals with low numbers of circulating γδ T cells, such as mice and humans. Arguably, γδ T cell functions might be more prominent in chickens where these cells form a higher proportion of the circulatory T cell compartment. The TCR repertoire defines different subsets of γδ T cells, and such analysis is facilitated by well-annotated TCR loci. γδ T cells are considered at the cusp of innate and adaptive immunity but most functions have been identified in γδ low species. A deeper understanding of TCR repertoire biology in γδ high and γδ low animals is critical for defining the evolution of the function of γδ T cells. Repertoire dynamics will reveal populations that can be classified as innate-like or adaptive-like as well as those that straddle this definition., Results: Here, a recent discrepancy in the structure of the chicken TCR gamma locus is resolved, demonstrating that tandem duplication events have shaped the evolution of this locus. Importantly, repertoire sequencing revealed large differences in the usage of individual TRGV genes, a pattern conserved across multiple tissues, including thymus, spleen and the gut. A single TRGV gene, TRGV3.3, with a highly diverse private CDR3 repertoire dominated every tissue in all birds. TRGV usage patterns were partly explained by the TRGV-associated recombination signal sequences. Public CDR3 clonotypes represented varying proportions of the repertoire of TCRs utilising different TRGVs, with one TRGV dominated by super-public clones present in all birds., Conclusions: The application of repertoire analysis enabled functional annotation of the TCRG locus in a species with a high circulating γδ phenotype. This revealed variable usage of TCRGV genes across multiple tissues, a pattern quite different to that found in γδ low species (human and mouse). Defining the repertoire biology of avian γδ T cells will be key to understanding the evolution and functional diversity of these enigmatic lymphocytes in an animal that is numerically more reliant on them. Practically, this will reveal novel ways in which these cells can be exploited to improve health in medical and veterinary contexts., (© 2021. The Author(s).)

We investigated aberrant DNA methylation (DNAm) changes and the contribution of ageing-associated methylomic drift and age acceleration to early-onset colorectal cancer (EOCRC) carcinogenesis. Genome-wide DNAm profiling using the Infinium HM450K on 97 EOCRC tumour and 54 normal colonic mucosa samples was compared with: (1) intermediate-onset CRC (IOCRC; diagnosed between 50-70 years; 343 tumour and 35 normal); and (2) late-onset CRC (LOCRC; >70 years; 318 tumour and 40 normal). CpGs associated with age-related methylation drift were identified using a public dataset of 231 normal mucosa samples from people without CRC. DNAm-age was estimated using epiTOC2. Common to all three age-of-onset groups, 88,385 (20% of all CpGs) CpGs were differentially methylated between tumour and normal mucosa. We identified 234 differentially methylated genes that were unique to the EOCRC group; 13 of these DMRs/genes were replicated in EOCRC compared with LOCRCs from TCGA. In normal mucosa from people without CRC, we identified 28,154 CpGs that undergo ageing-related DNAm drift, and of those, 65% were aberrantly methylated in EOCRC tumours. Based on the mitotic-based DNAm clock epiTOC2, we identified age acceleration in normal mucosa of people with EOCRC compared with normal mucosa from the IOCRC, LOCRC groups ( p = 3.7 × 10 -16 ) and young people without CRC ( p = 5.8 × 10 -6 ). EOCRC acquires unique DNAm alterations at 234 loci. CpGs associated with ageing-associated drift were widely affected in EOCRC without needing the decades-long accrual of DNAm drift as commonly seen in intermediate- and late-onset CRCs. Accelerated ageing in normal mucosa from people with EOCRC potentially underlies the earlier age of diagnosis in CRC carcinogenesis.

Helminth infections are among the World Health Organization's top neglected diseases with significant impact in many Less Economically Developed Countries. Despite no longer being endemic in Europe, the widespread presence of helminth eggs in archaeological deposits indicates that helminths represented a considerable burden in past European populations. Prevalence of infection is a key epidemiological feature that would influence the elimination of endemic intestinal helminths, for example, low prevalence rates may have made it easier to eliminate these infections in Europe without the use of modern anthelminthic drugs. To determine historical prevalence rates we analysed 589 grave samples from 7 European sites dated between 680 and 1700 CE, identifying two soil transmitted nematodes (Ascaris spp. and Trichuris trichiura) at all locations, and two food derived cestodes (Diphyllobothrium latum and Taenia spp.) at 4 sites. The rates of nematode infection in the medieval populations (1.5 to 25.6% for T. trichiura; 9.3-42.9% for Ascaris spp.) were comparable to those reported within modern endemically infected populations. There was some evidence of higher levels of nematode infection in younger individuals but not at all sites. The genetic diversity of T. trichiura ITS-1 in single graves was variable but much lower than with communal medieval latrine deposits. The prevalence of food derived cestodes was much lower (1.0-9.9%) than the prevalence of nematodes. Interestingly, sites that contained Taenia spp. eggs also contained D. latum which may reflect local culinary practices. These data demonstrate the importance of helminth infections in Medieval Europe and provide a baseline for studies on the epidemiology of infection in historical and modern contexts. Since the prevalence of medieval STH infections mirror those in modern endemic countries the factors affecting STH decline in Europe may also inform modern intervention campaigns., Competing Interests: The authors have declared that no competing interests exist.

Chicken meat represents an important source of Campylobacter infections of humans world-wide. A better understanding of Campylobacter epidemiology in commercial chicken flocks will facilitate the development of more effective intervention strategies. We developed a gene-specific parallel sequencing approach that efficiently indicated genetic diversity in farm-derived samples and revealed Campylobacter genotypes that would not be detected using microbiological culture. Parallel sequencing of the porA nucleotide fragment identified a different pattern of diversity in broiler flocks compared with broiler-breeder flocks at both individual bird and flock levels. Amongst the flocks tested, broiler flocks and individual birds were dominated by one or two porA fragment types whereas co-dominance with up to six porA fragment types was evident in breeder birds. A high proportion (83.6-93.3%) of porA variants were shared between broiler and breeder flocks. The porA-based diversity profiling could be a useful addition to the repertoire of tools employed to attribute potential sources of contamination for broiler flocks, including the environment, wild animals or other chickens. This approach can be extended to include other loci within Campylobacter and developed for molecular epidemiology studies of other bacterial species.

Throughout history, humans have been afflicted by parasitic worms, and eggs are readily detected in archaeological deposits. This study integrated parasitological and ancient DNA methods with a large sample set dating between Neolithic and Early Modern periods to explore the utility of molecular archaeoparasitology as a new approach to study the past. Molecular analyses provided unequivocal species-level parasite identification and revealed location-specific epidemiological signatures. Faecal-oral transmitted nematodes ( Ascaris lumbricoides and Trichuris trichiura ) were ubiquitous across time and space. By contrast, high numbers of food-associated cestodes ( Diphyllobothrium latum and Taenia saginata ) were restricted to medieval Lübeck. The presence of these cestodes and changes in their prevalence at approximately 1300 CE indicate substantial alterations in diet or parasite availability. Trichuris trichiura ITS-1 sequences grouped into two clades; one ubiquitous and one restricted to medieval Lübeck and Bristol. The high sequence diversity of T.t ITS-1 detected in Lübeck is consistent with its importance as a Hanseatic trading centre. Collectively, these results introduce molecular archaeoparasitology as an artefact-independent source of historical evidence., (© 2018 The Authors.)

Two crystal structures of Japanin, an 18 kDa immune-modulatory lipocalin from the Brown Ear Tick (Rhipicephalus appendiculatus), have been determined at 2.2 and 2.4 Å resolution. In both crystal forms the protein is in complex with cholesterol, which sits in a closed pocket at the centre of the lipocalin barrel. Both crystal forms are dimers, which are also observed in solution. Molecular modelling suggests that previously-described members of a tick protein family bearing high sequence homology to Japanin are also likely to bind cholesterol or cholesterol derivatives.

The European badger is recognised as a wildlife reservoir for bovine tuberculosis (bTB); the control of which is complex, costly and controversial. Despite the importance of badgers in bTB and the well-documented role for macrophages as anti-mycobacterial effector cells, badger macrophage (bdMφ) responses remain uncharacterised. Here, we demonstrate that bdMφ fail to produce nitric oxide (NO) or upregulate inducible nitric oxide synthase (iNOS) mRNA following Toll-like receptor (TLR) agonist treatment. BdMφ also failed to make NO after stimulation with recombinant badger interferon gamma (bdIFNγ) or a combination of bdIFNγ and lipopolysaccharide. Exposure of bdMφ to TLR agonists and/or bdIFNγ resulted in upregulated cytokine (IL1β, IL6, IL12 and TNFα) mRNA levels indicating that these critical pathways were otherwise intact. Although stimulation with most TLR agonists resulted in strong cytokine mRNA responses, weaker responses were evident after exposure to TLR9 agonists, potentially due to very low expression of TLR9 in bdMφ. Both NO and TLR9 are important elements of innate immunity to mycobacteria, and these features of bdMφ biology would impair their capacity to resist bTB infection. These findings have significant implications for the development of bTB management strategies, and support the use of vaccination to reduce bTB infection in badgers.

Lynch syndrome is an inherited cancer-predisposing disorder caused by germline mutations in the DNA mismatch repair (MMR) genes but there is a high degree of variability in cancer risk observed among carriers, suggesting the existence of modifying factors. Our aim was to investigate variants within the hTERT gene as a potential colorectal cancer (CRC) risk modifier for MMR gene mutation carriers. We identified 1098 MMR gene mutation carriers (420 MLH1, 481 MSH2, 126 MSH6, 53 PMS2 and 18 EPCAM) from 330 families recruited from either family cancer clinics or population cancer registries of the Australasian Colorectal Cancer Family Registry between 1997 and 2012. Using weighted Cox regression after adjusting for ascertainment bias, we estimated associations between 23 SNPs within the hTERT gene and CRC risk. During 46,836 person-years observation, 392 (36%) carriers were diagnosed with CRC at a mean age of 42.2 (standard deviation 11.4) years. There was no evidence of association between any of the hTERT SNPs and CRC risk, overall and stratified by sex and MMR gene mutated, after adjustment for multiple testing. Our findings suggest no evidence for clinical utility of the SNPs within the hTERT gene in Lynch syndrome.

Objectives: To review the state of the art and assess future potential in the use of inorganic particulates as vaccine adjuvants., Key Findings: An adjuvant is an entity added to a vaccine formulation to ensure that robust immunity to the antigen is inculcated. The inclusion of an adjuvant is typically vital for the efficacy of vaccines using inactivated organisms, subunit and DNA antigens. With increasing research efforts being focused on subunit and DNA antigens because of their improved safety profiles, the development of appropriate adjuvants is becoming ever more crucial. Despite this, very few adjuvants are licensed for use in humans (four by the FDA, five by the European Medicines Agency). The most widely used adjuvant, alum, has been used for nearly 90 years, yet its mechanism of action remains poorly understood. In addition, while alum produces a powerful antibody Th2 response, it does not provoke the cellular immune response required for the elimination of intracellular infections or cancers. New adjuvants are therefore needed, and inorganic systems have attracted much attention in this regard., Summary: In this review, the inorganic adjuvants currently in use are considered, and the efforts made to date to understand their mechanisms of action are summarised. We then move on to survey the literature on inorganic particulate adjuvants, focusing on the most interesting recent developments in this area and their future potential., (© 2014 Royal Pharmaceutical Society.)

There is currently no paradigm in immunology that enables an accurate prediction of how the immune system will respond to any given agent. Here we show that the immunological responses induced by members of a broad class of inorganic crystalline materials are controlled purely by their physicochemical properties in a highly predictable manner. We show that structurally and chemically homogeneous layered double hydroxides (LDHs) can elicit diverse human dendritic cell responses in vitro. Using a systems vaccinology approach, we find that every measured response can be modeled using a subset of just three physical and chemical properties for all compounds tested. This correlation can be reduced to a simple linear equation that enables the immunological responses stimulated by newly synthesized LDHs to be predicted in advance from these three parameters alone. We also show that mouse antigen-specific antibody responses in vivo and human macrophage responses in vitro are controlled by the same properties, suggesting they may control diverse responses at both individual component and global levels of immunity. This study demonstrates that immunity can be determined purely by chemistry and opens the possibility of rational manipulation of immunity for therapeutic purposes., (© 2014 Williams et al.)

Hard ticks subvert the immune responses of their vertebrate hosts in order to feed for much longer periods than other blood-feeding ectoparasites; this may be one reason why they transmit perhaps the greatest diversity of pathogens of any arthropod vector. Tick-induced immunomodulation is mediated by salivary components, some of which neutralise elements of innate immunity or inhibit the development of adaptive immunity. As dendritic cells (DC) trigger and help to regulate adaptive immunity, they are an ideal target for immunomodulation. However, previously described immunoactive components of tick saliva are either highly promiscuous in their cellular and molecular targets or have limited effects on DC. Here we address the question of whether the largest and globally most important group of ticks (the ixodid metastriates) produce salivary molecules that specifically modulate DC activity. We used chromatography to isolate a salivary gland protein (Japanin) from Rhipicephalus appendiculatus ticks. Japanin was cloned, and recombinant protein was produced in a baculoviral expression system. We found that Japanin specifically reprogrammes DC responses to a wide variety of stimuli in vitro, radically altering their expression of co-stimulatory and co-inhibitory transmembrane molecules (measured by flow cytometry) and their secretion of pro-inflammatory, anti-inflammatory and T cell polarising cytokines (assessed by Luminex multiplex assays); it also inhibits the differentiation of DC from monocytes. Sequence alignments and enzymatic deglycosylation revealed Japanin to be a 17.7 kDa, N-glycosylated lipocalin. Using molecular cloning and database searches, we have identified a group of homologous proteins in R. appendiculatus and related species, three of which we have expressed and shown to possess DC-modulatory activity. All data were obtained using DC generated from at least four human blood donors, with rigorous statistical analysis. Our results suggest a previously unknown mechanism for parasite-induced subversion of adaptive immunity, one which may also facilitate pathogen transmission.

The capacity of different lung parenchymal cells to accumulate putrescine was investigated by incubating slices of rat lung in a medium containing the tritiated compound. Quantitative examination of autoradiographs, by electron microscopy, indicated that accumulation of putrescine occurred in both the Type I and Type II cells of the alveolar epithelium. Putrescine uptake was abolished by the addition of spermidine to the medium or by incubating at 0 degrees C. Lung samples from rats dosed with the pneumotoxin O,S,S-trimethyl phosphorodithioate (OSSMeO), which selectively damages Type I pneumocytes, showed a large reduction in the uptake of label by both Type I and Type II cells. This treatment also resulted in an increase in the labeling of alveolar macrophages. Control samples, from undosed rats, were incubated in medium containing tritiated 5-hydroxytryptamine; this compound did not accumulate in epithelial cells but it was concentrated in the endothelium of the alveolar capillaries and in the blood cells within these vessels. The demonstration of putrescine uptake by both Type I and Type II pneumocytes, together with its reduction by dosing with OSSMeO, has vindicated the use of this activity, in lung slices, as an index of damage to the alveolar epithelium.