Concurrent Ascaris infection modulates host immunity resulting in impaired control of Salmonella infection in pigs.

Ascaris is one of the most widespread helminth infections, leading to chronic morbidity in humans and considerable economic losses in pig farming. In addition, pigs are an important reservoir for the zoonotic salmonellosis, where pigs can serve as asymptomatic carriers. Here, we investigated the impact of an ongoing Ascaris infection on the immune response to Salmonella in pigs. We observed higher bacterial burdens in experimentally coinfected pigs compared to pigs infected with Salmonella alone. The impaired control of Salmonella in the coinfected pigs was associated with repressed interferon gamma responses in the small intestine and with the alternative activation of gut macrophages evident in elevated CD206 expression. Ascaris single and coinfection were associated with a rise of CD4-CD8α+FoxP3+ Treg in the lymph nodes draining the small intestine and liver. In addition, macrophages from coinfected pigs showed enhanced susceptibility to Salmonella infection in vitro and the Salmonella-induced monocytosis and tumor necrosis factor alpha production by myeloid cells was repressed in pigs coinfected with Ascaris. Hence, our data indicate that acute Ascaris infection modulates different immune effector functions with important consequences for the control of tissue-invasive coinfecting pathogens.IMPORTANCEIn experimentally infected pigs, we show that an ongoing infection with the parasitic worm Ascaris suum modulates host immunity, and coinfected pigs have higher Salmonella burdens compared to pigs infected with Salmonella alone. Both infections are widespread in pig production and the prevalence of Salmonella is high in endemic regions of human Ascariasis, indicating that this is a clinically meaningful coinfection. We observed the type 2/regulatory immune response to be induced during an Ascaris infection correlates with increased susceptibility of pigs to the concurrent bacterial infection.

Evaluation of copromicroscopy, multiplex-qPCR and antibody serology for monitoring of human ascariasis in endemic settings.

BACKGROUND: The standard diagnosis of Ascaris lumbricoides and other soil-transmitted helminth (STH) infections relies on the detection of worm eggs by copromicroscopy. However, this method is dependent on worm patency and shows only limited accuracy in low-intensity infection settings. We aimed to decipher the diagnostic accuracy of different antibodies using various Ascaris antigens in reference to copromicroscopy and quantitative PCR (qPCR), four months after national STH preventative chemotherapy among school children in western Kenya. METHODOLOGY: STH infection status of 390 school children was evaluated via copromicroscopy (Kato-Katz and mini-FLOTAC) and qPCR. In parallel, Ascaris-specific antibody profiles against larval and adult worm lysates, and adult worm excretory-secretory (ES) products were determined by enzyme-linked immunosorbent assay. Antibody cross-reactivity was evaluated using the closely related zoonotic roundworm species Toxocara cati and Toxocara canis. The diagnostic accuracy of each antibody was evaluated using receiver operating curve analysis and the correspondent area under the curve (AUC). PRINCIPAL FINDINGS: Ascaris was the predominant helminth infection with an overall prevalence of 14.9% (58/390). The sensitivity of mini-FLOTAC and Kato-Katz for Ascaris diagnosis reached only 53.5% and 63.8%, respectively compared to qPCR. Although being more sensitive, qPCR values correlated with microscopic egg counts (R = -0.71, P<0.001), in contrast to antibody levels. Strikingly, IgG antibodies recognizing the ES products of adult Ascaris worms reliably diagnosed active Ascaris infection as determined by qPCR and microscopy, with IgG1 displaying the highest accuracy (AUC = 0.83, 95% CI: 0.75-0.91). CONCLUSION: IgG1 antibody responses against adult Ascaris-ES products hold a promising potential for complementing the standard fecal and molecular techniques employed for monitoring Ascaris infections. This is of particular importance in the context of deworming programs as the antibody diagnostic accuracy was independent of egg counts.

Acute Ascaris infection impairs the effector functions of natural killer cells in single and Salmonella co-infected pigs.

Natural killer (NK) cells play a key role in defense against Salmonella infections during the early phase of infection. Our previous work showed that the excretory/secretory products of Ascaris suum repressed NK activity in vitro. Here, we asked if NK cell functionality was influenced in domestic pigs during coinfection with Ascaris and Salmonella enterica serotype Typhimurium. Ascaris coinfection completely abolished the IL-12 and IL-18 driven elevation of IFN-γ production seen in CD16 + CD8α + perforin + NK cells of Salmonella single-infected pigs. Furthermore, Ascaris coinfection prohibited the Salmonella-driven rise in NK perforin levels and CD107a surface expression. In line with impaired effector functions, NK cells from Ascaris-single and coinfected pigs displayed elevated expression of the inhibitory KLRA1 and NKG2A receptors genes, contrasting with the higher expression of the activating NKp46 and NKp30 receptors in NK cells during Salmonella single infection. These differences were accompanied by the highly significant upregulation of T-bet protein expression in NK cells from Ascaris-single and Ascaris/Salmonella coinfected pigs. Together, our data strongly indicate a profound repression of NK functionality by an Ascaris infection which may hinder infected individuals from adequately responding to a concurrent bacterial infection.

Th2-biased immune responses to body migrating Ascaris larvae in primary infection are associated with pathology but not protection.

Helminth infections lead to an overdispersion of the parasites in humans as well as in animals. We asked whether early immune responses against migrating Ascaris larvae are responsible for the unequal distribution of worms in natural host populations and thus investigated a susceptible versus a resistant mouse strain. In mice, the roundworm larvae develop until the lung stage and thus early anti-Ascaris immune responses against the migrating larvae in the liver and lung can be deciphered. Our data show that susceptible C57BL/6 mice respond to Ascaris larval migration significantly stronger compared to resistant CBA mice and the anti-parasite reactivity is associated with pathology. Increased eosinophil recruitment was detected in the liver and lungs, but also in the spleen and peritoneal cavity of susceptible mice on day 8 post infection compared to resistant mice. In serum, eosinophil peroxidase levels were significantly higher only in the susceptible mice, indicating functional activity of the recruited eosinophils. This effect was associated with an increased IL-5/IL-13 production by innate lymphoid cells and CD4+ T cells and a pronounced type 2 macrophage polarization in the lungs of susceptible mice. Furthermore, a comparison of wildtype BALB/c and eosinophil-deficient dblGATA-1 BALB/c mice showed that eosinophils were not essential for the early control of migrating Ascaris larvae. In conclusion, in primary infection, a strong local and systemic type 2 immune response during hepato-tracheal helminth larval migration is associated with pathology rather than protection.

Ascaris suum infection in juvenile pigs elicits a local Th2 response in a setting of ongoing Th1 expansion.

Ascaris spp. undergo extensive migration within the body before establishing patent infections in the small intestinal tract of humans and pigs. However, whether larval migration is critical for inducing efficient type 2 responses remains poorly understood. Therefore, we investigated systemic versus local adaptive immune responses along the hepato-tracheal migration of Ascaris suum during primary, single infections in conventionally raised pigs. Neither the initial invasion of gut tissue nor migration through the liver resulted in discernable Th2 cell responses. In contrast, lung-stage larvae elicited a Th2-biased pulmonary response, which declined after the larvae had left the lungs. In the small intestine, we observed an accumulation of Th2 cells upon the arrival of fourth-stage larvae (L4) to the small intestinal lumen. In parallel, we noticed robust and increasing Th1 responses in circulation, migration-affected organs, and draining lymph nodes. Phenotypic analysis of CD4+ T cells specifically recognizing A. suum antigens in the circulation and lung tissue of infected pigs confirmed that the majority of Ascaris-specific T cells produced IL-4 (Th2) and, to a much lesser extent, IL-4/IFN-g (Th2/1 hybrids) or IFN-g alone (Th1). These data demonstrate that lung-stage but not the early liver-stage larvae lead to a locally restricted Th2 response. Significant Th2 cell accumulation in the small intestine occurs only when L4 complete the body migration. In addition, Th2 immunity seems to be hampered by the concurrent, nonspecific Th1 bias in growing pigs. Together, the late onset of Th2 immunity at the site of infection and the Th1-biased systemic immunity likely enable the establishment of intestinal infections by sufficiently large L4 stages and pre-adult worms, some of which resist expulsion mechanisms.

Synthetic Optical Coherence Tomography Angiographs for Detailed Retinal Vessel Segmentation Without Human Annotations.

Optical coherence tomography angiography (OCTA) is a non-invasive imaging modality that can acquire high-resolution volumes of the retinal vasculature and aid the diagnosis of ocular, neurological and cardiac diseases. Segmenting the visible blood vessels is a common first step when extracting quantitative biomarkers from these images. Classical segmentation algorithms based on thresholding are strongly affected by image artifacts and limited signal-to-noise ratio. The use of modern, deep learning-based segmentation methods has been inhibited by a lack of large datasets with detailed annotations of the blood vessels. To address this issue, recent work has employed transfer learning, where a segmentation network is trained on synthetic OCTA images and is then applied to real data. However, the previously proposed simulations fail to faithfully model the retinal vasculature and do not provide effective domain adaptation. Because of this, current methods are unable to fully segment the retinal vasculature, in particular the smallest capillaries. In this work, we present a lightweight simulation of the retinal vascular network based on space colonization for faster and more realistic OCTA synthesis. We then introduce three contrast adaptation pipelines to decrease the domain gap between real and artificial images. We demonstrate the superior segmentation performance of our approach in extensive quantitative and qualitative experiments on three public datasets that compare our method to traditional computer vision algorithms and supervised training using human annotations. Finally, we make our entire pipeline publicly available, including the source code, pretrained models, and a large dataset of synthetic OCTA images.

NAD(P)H Fluorescence Lifetime Imaging for the Metabolic Analysis of the Murine Intestine and Parasites During Nematode Infection.

Parasites generally have a negative effect on the health of their host. They represent a huge health burden, as they globally affect the health of the infested human or animal in the long term and, thus, impact agricultural and socio-economic outcomes. However, parasite-driven immune-regulatory effects have been described, with potential therapeutic relevance for autoimmune diseases. While the metabolism in both the host and parasites contributes to their defense and is the basis for nematode survival in the intestine, it has remained largely understudied due to a lack of adequate technologies. We have developed and applied NAD(P)H fluorescence lifetime imaging to explanted murine intestinal tissue during infection with the natural nematode Heligmosomoides polygyrus to study the metabolic processes in both the host and parasites in a spatially resolved manner. The exploitation of the fluorescence lifetime of the co-enzymes nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), hereafter NAD(P)H, which are preserved across species, depends on their binding status and the binding site on the enzymes catalyzing metabolic processes. Focusing on the most abundantly expressed NAD(P)H-dependent enzymes, the metabolic pathways associated with anaerobic glycolysis, oxidative phosphorylation/aerobic glycolysis, and NOX-based oxidative burst, as a major defense mechanism, were distinguished, and the metabolic crosstalk between the host and parasite during infection was characterized.

Th2 and metabolic responses to nematodes are independent of prolonged host microbiota abrogation.

Antibiotic treatment can lead to elimination of both pathogenic bacteria and beneficial commensals, as well as to altered host immune responses. Here, we investigated the influence of prolonged antibiotic treatment (Abx) on effector, memory and recall Th2 immune responses during the primary infection, memory phase and secondary infection with the small intestinal nematode Heligmosomoides polygyrus. Abx treatment significantly reduced gut bacterial loads, but neither worm burdens, nor worm fecundity in primary infection were affected, only worm burdens in secondary infection were elevated in Abx treated mice. Abx mice displayed trends for elevated effector and memory Th2 responses during primary infection, but overall frequencies of Th2 cells in the siLP, PEC, mLN and in the spleen were similar between Abx treated and untreated groups. Gata3+ effector and memory Th2 cytokine responses also remained unimpaired by prolonged Abx treatment. Similarly, the energy production and defence mechanisms of the host tissue and the parasite depicted by NAD(P)H fluorescence lifetime imaging (FLIM) did not change by the prolonged use of antibiotics. We show evidence that the host Th2 response to intestinal nematodes, as well as host and parasite metabolic pathways are robust and remain unimpaired by host microbiota abrogation.

Ascaris suum excretory/secretory products differentially modulate porcine dendritic cell subsets.

Helminths produce excretory/secretory products (E/S) which can modulate the immune responses of their hosts. Dendritic cells (DC) are essential for initiating the host T cell response and are thus potential targets for modulation by helminth E/S. Here we study immunomodulation of porcine peripheral blood DC subsets following ex vivo stimulation with E/S from Ascaris suum, a common helminth of pigs with considerable public health and economic importance. Our data showed that the relative frequencies of DC subsets in porcine blood differ, with plasmacytoid DC (pDC) being the most prominent in healthy 6-month-old pigs. pDC are an important cytokine source, and we found that A. suum E/S suppressed production of the type 1 cytokines IL-12p40 and TNF-α by this subset following toll-like receptor (TLR) ligation. In contrast, conventional DC (cDC) are more efficient antigen presenters, and the expression of CD80/86, costimulatory molecules essential for efficient antigen presentation, were modulated differentially by A. suum E/S between cDC subsets. CD80/86 expression by type 1 cDC (cDC1) following TLR ligation was greatly suppressed by the addition of A. suum E/S, while CD80/86 expression by type 2 cDC (cDC2) was upregulated by A. suum E/S. Further, we found that IFN-γ production by natural killer (NK) cells following IL-12 and IL-18 stimulation was suppressed by A. suum E/S. Finally, in the presence of E/S, IFN-γ production by CD4+ T cells co-cultured with autologous blood-derived DC was significantly impaired. Together, these data provide a coherent picture regarding the regulation of type 1 responses by A. suum E/S. Responsiveness of pDC and cDC1 to microbial ligands is reduced in the presence of E/S, effector functions of Th1 cells are impaired, and cytokine-driven IFN-γ release by NK cells is limited.

Comparison of Sources and Methods for the Isolation of Equine Adipose Tissue-Derived Stromal/Stem Cells and Preliminary Results on Their Reaction to Incubation with 5-Azacytidine.

Physiological particularities of the equine heart justify the development of an in vitro model suitable for investigations of the species-specific equine cardiac electrophysiology. Adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point from which to develop such a cardiomyocyte (CM)-like cell model. Therefore, we compared abdominal, retrobulbar, and subcutaneous adipose tissue as sources for the isolation of ASCs applying two isolation methods: the collagenase digestion and direct explant culture. Abdominal adipose tissue was most suitable for the isolation of ASCs and both isolation methods resulted in comparable yields of CD45-/CD34-negative cells expressing the mesenchymal stem cell markers CD29, CD44, and CD90, as well as pluripotency markers, as determined by flow cytometry and real-time quantitative PCR. However, exposure of equine ASCs to 5-azacytidine (5-AZA), reportedly inducing CM differentiation from rats, rabbits, and human ASCs, was not successful in our study. More precisely, neither the early differentiation markers GATA4 and NKX2-5, nor the late CM differentiation markers TNNI3, MYH6, and MYH7 were upregulated in equine ASCs exposed to 10 µM 5-AZA for 48 h. Hence, further work focusing on the optimal conditions for CM differentiation of equine stem cells derived from adipose tissue, as well as possibly from other origins, are needed.

Age-dependent rise in IFN-γ competence undermines effective type 2 responses to nematode infection.

The efficient induction of type 2 immune responses is central to the control of helminth infections. Previous studies demonstrated that strong Th1 responses driven by intracellular pathogens as well as a bias for type 1 activity in senescent mice impedes the generation of Th2 responses and the control of intestinal nematode infections. Here, we show that the spontaneous differentiation of Th1 cells and their expansion with age restrains type 2 immunity to infection with the small intestinal nematode H. polygyrus much earlier in life than previously anticipated. This includes the more extensive induction of IFN-γ competent, nematode-specific Th2/1 hybrid cells in BALB/c mice older than three months compared to younger animals. In C57BL/6 mice, Th1 cells accumulate more rapidly at steady state, translating to elevated Th2/1 differentiation and poor control of parasite fitness in primary infections experienced at a young age. Blocking of early IFN-γ and IL-12 signals during the first week of nematode infection leads to sharply decreased Th2/1 differentiation and promotes resistance in both mouse lines. Together, these data suggest that IFN-γ competent, type 1 like effector cells spontaneously accumulating in the vertebrate host progressively curtail the effectiveness of anti-nematode type 2 responses with rising host age.

Whip- and pinworm infections elicit contrasting effector and distinct regulatory responses in wild house mice.

Infections with high doses of intestinal nematodes result in protective immunity based on robust type 2 responses in most mouse lines under laboratory conditions. Here, we report on cellular responses of wild house mice from northern Germany. We detected robust Th1 responses in wild house mice naturally infected with the whipworm Trichuris muris. In contrast, mice infected with pinworms (Syphacia, Aspiculuris) reported type-2 activity by elevated IgG1 levels and eosinophil counts, but also harbored high frequencies of Foxp3+ regulatory T cells, suggesting that natural whip- and pinworm infections induce distinct immunoregulatory as well as effector profiles.

NAD(P)H fluorescence lifetime imaging of live intestinal nematodes reveals metabolic crosstalk between parasite and host.

Infections with intestinal nematodes have an equivocal impact: they represent a burden for human health and animal husbandry, but, at the same time, may ameliorate auto-immune diseases due to the immunomodulatory effect of the parasites. Thus, it is key to understand how intestinal nematodes arrive and persist in their luminal niche and interact with the host over long periods of time. One basic mechanism governing parasite and host cellular and tissue functions, metabolism, has largely been neglected in the study of intestinal nematode infections. Here we use NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) fluorescence lifetime imaging of explanted murine duodenum infected with the natural nematode Heligmosomoides polygyrus and define the link between general metabolic activity and possible metabolic pathways in parasite and host tissue, during acute infection. In both healthy and infected host intestine, energy is effectively produced, mainly via metabolic pathways resembling oxidative phosphorylation/aerobic glycolysis features. In contrast, the nematodes shift their energy production from balanced fast anaerobic glycolysis-like and effective oxidative phosphorylation-like metabolic pathways, towards mainly anaerobic glycolysis-like pathways, back to oxidative phosphorylation/aerobic glycolysis-like pathways during their different life cycle phases in the submucosa versus the intestinal lumen. Additionally, we found an increased NADPH oxidase (NOX) enzymes-dependent oxidative burst in infected intestinal host tissue as compared to healthy tissue, which was mirrored by a similar defense reaction in the parasites. We expect that, the here presented application of NAD(P)H-FLIM in live tissues constitutes a unique tool to study possible shifts between metabolic pathways in host-parasite crosstalk, in various parasitic intestinal infections.

Novel protocol for the isolation of highly purified neonatal murine microglia and astrocytes.

BACKGROUND: The crosstalk and reactivity of the cell type glia, especially microglia and astrocytes, have progressively gathered research attention in understanding proper brain function regulated by the innate immune response. Therefore, methods to isolate highly viable and pure glia for the analysis on a cell-specific level are indispensable. NEW METHOD: We modified previously established techniques: Animal numbers were reduced by multiple microglial harvests from the same mixed glial culture, thereby maximizing microglial yields following the principles of the 3Rs (replacement, reduction, and refinement). We optimized Magnetic-activated cell sorting (MACS®) of microglia and astrocytes by applying cultivated primary glial cell suspensions instead of directly sorting dissociated single cell suspension. RESULTS: We generated highly viable and pure microglia and astrocytes derived from a single mixed culture with a purity of ~99%, as confirmed by FACS analysis. Field emission scanning electron microscopy (FESEM) demonstrated integrity of the MACS-purified glial cells. Tumor necrosis factor (TNF) and Interleukin-10 (IL-10) ELISA confirmed pro- and anti-inflammatory responses to be functional in purified glia, but significantly weakened compared to non-purified cells, further highlighting the importance of cellular crosstalk for proper immune activation. COMPARISON WITH EXISTING METHOD(S): Unlike previous studies that either isolated a single type of glia or displayed a substantial proportion of contamination with other cell types, we achieved isolation of both microglia and astrocytes at an increased purity (99-100%). CONCLUSIONS: We have created an optimized protocol for the efficient purification of both primary microglia and astrocytes. Our results clearly demonstrate the importance of purity in glial cell cultivation in order to examine immune responses, which particularly holds true for astrocytes. We propose the novel protocol as a tool to investigate the cell type-specific crosstalk between microglia and astrocytes in the frame of CNS diseases.

TLR9 signalling inhibits Plasmodium liver infection by macrophage activation.

Recognition of pathogen-associated molecular patterns (PAMPs) through Toll-like receptors (TLRs) plays a pivotal role in first-line pathogen defense. TLRs are also likely triggered during a Plasmodium infection in vivo by parasite-derived components. However, the contribution of innate responses to liver infection and to the subsequent clinical outcome of a blood infection is not well understood. To assess the potential effects of enhanced TLR-signalling on Plasmodium infection, we systematically examined the effect of agonist-primed immune responses to sporozoite inoculation in the P. berghei/C57Bl/6 murine malaria model. We could identify distinct stage-specific effects on the course of infection after stimulation with two out of four TLR-ligands tested. Priming with a TLR9 agonist induced killing of pre-erythrocytic stages in the liver that depended on macrophages and the expression of inducible nitric oxide synthase (iNOS). These factors have previously not been recognized as antigen-independent effector mechanisms against Plasmodium liver stages. Priming with TLR4 and -9 agonists also translated into blood stage-specific protection against experimental cerebral malaria (ECM). These insights are relevant to the activation of TLR signalling pathways by adjuvant systems of antimalaria vaccine strategies. The protective role of TLR4-activation against ECM might also explain some unexpected clinical effects observed with pre-erythrocytic vaccine approaches.

Early Immune Initiation by Porcine Cells following Toxoplasma gondii Infection versus TLR Ligation.

Containment of acute Toxoplasma gondii infection is dependent on an efficient interferon gamma response. However, the earliest steps of immune response initiation immediately following exposure to the parasite have not been previously characterized in pigs. Murine and human myeloid cells produce large quantities of interleukin (IL)-12 during early T. gondii infection. We therefore examined IL-12 expression by porcine peripheral blood monocytes and dendritic cell (DC) subsets following toll-like receptor (TLR) ligation and controlled T. gondii tachyzoite infection. We detected IL-12p40 expression by porcine plasmacytoid DC, but not conventional or monocyte-derived DC following TLR ligation. Unexpectedly, we also observed considerable IL-12p40 production by porcine CD3- NKp46+ cells-a classical natural killer cell phenotype-following TLR ligation. However, in response to T. gondii exposure, no IL-12 production was observed by either DC or CD3- NKp46+ cells. Despite this, IL-18 production by DC-enriched peripheral blood mononuclear cells was detected following live T. gondii tachyzoite exposure. Only combined stimulation of porcine peripheral blood mononuclear cells with recombinant IL-12p70 and IL-18 induced innate interferon gamma production by natural killer cells, while T cells and myeloid cells did not respond. Therefore, porcine CD3- NKp46+ cells serve as important IL-12 producers following TLR ligation, while IL-18 likely plays a prominent role in early immune response initiation in the pig following T. gondii infection.

Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents.

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

Plasma cell free DNA methylation markers for hepatocellular carcinoma surveillance in patients with cirrhosis: a case control study.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of death in patients with cirrhosis, primarily due to failed early detection. HCC screening is recommended among individuals with cirrhosis using biannual abdominal ultrasound, for earlier tumor detection, administration of curative treatment, and improved survival. Surveillance by imaging with or without biomarkers such as alpha-fetoprotein (AFP) remains suboptimal for early stage HCC detection. Here we report on the development and assessment of methylation biomarkers from liquid biopsies for HCC surveillance in cirrhotic patients. METHODS: DNA methylation markers including the HCCBloodTest (Epigenomics AG) and a DNA-methylation panel established by next generation sequencing (NGS) were assessed using a training/testing design. The NGS panel algorithm was established in a training study (41 HCC patients; 46 cirrhotic non-HCC controls). For testing, plasma samples were obtained from cirrhotic patients (Child class A or B) with (60) or without (103) early stage HCC (BCLC stage 0, A, B). The assays were then tested using blinded sample sets and analyzed by preset algorithms. RESULTS: The HCCBloodTest and the NGS panel exhibited 76.7% and 57% sensitivities at 64.1% and 97% specificity, respectively. In a post-hoc analysis, a combination of the NGS panel with AFP (20 ng/mL) achieved 68% sensitivity at 97% specificity (AUC = 0.9). CONCLUSIONS: Methylation biomarkers in cell free plasma DNA provide a new alternative for HCC surveillance. Multiomic panels comprising DNA methylation markers with other biological markers, such as AFP, provide an option to further increase the overall clinical performance of surveillance via minimally invasive blood samples. TRIAL REGISTRATION: Test set study-ClinicalTrials.gov (NCT03804593) January 11, 2019, retrospectively registered.

Eosinophils are dispensable for the regulation of IgA and Th17 responses in Giardia muris infection.

AIMS: IgA and Th17 responses are pivotal for the control of Giardia infections. Eosinophils support IgA class switching, the survival of intestinal IgA+ plasma cells at steady state and can control Th17 activity in the small intestine. To see whether eosinophils regulate adaptive immune responses during giardiasis, we investigated Giardia muris infections in wild-type BALB/c and eosinophil-deficient ∆dblGATA-1 mice. METHODS AND RESULTS: Infected ∆dblGATA-1 mice did not differ markedly in parasite control from wild-type mice. Confirming previous studies, naive ∆dblGATA-1 mice displayed diminished IgA+ B cell frequencies in Peyer's patches. However, IgA class switching and intestinal IgA secretion in response to G muris infection were comparable in wild-type BALB/c and ∆dblGATA-1 mice. Both strains displayed similarly low intestinal Th17 responses, accompanied by a mild expansion of type 3 innate lymphoid cells (ILC3). CONCLUSIONS: Contrasting previous reports on overt small intestinal Th17 activity in eosinophil-deficient mice, IL-17A production is kept in check in the absence of eosinophils during Giardia infection. Suboptimal homeostatic IgA responses in the absence of eosinophils are transiently fostered in infected mice and the maintenance of IgA+ plasma cells appears to be restored during persisting Giardia infection.

Trilateral Relationship: Ascaris, Microbiota, and Host Cells.

Ascariasis is a globally spread intestinal nematode infection of humans and a considerable concern in pig husbandry. Ascaris accomplishes a complex body migration from the intestine via the liver and lung before returning to the intestine. Tissue migration and the habitat shared with a complex microbial community pose the question of how the nematode interacts with microbes and host cells from various tissues. This review addresses the current knowledge of the trilateral relationship between Ascaris, its microbial environment, and host cells, and discusses novel approaches targeting these interactions to combat this widespread infection of livestock and man.