Perceived acceptability and appropriateness of a web-based program targeting risk for anxiety in young children and their parents.

OBJECTIVE: This mixed-methods study examined perceived acceptability and appropriateness of a novel digital mental health program targeting anxiety risk (i.e., perfectionism or error sensitivity) in 5-to-7-year-old children and their parents. METHODS: Parent-child dyads participated in a modular, web-based cognitive-behavioral program targeting negative overreactions to making mistakes. The program, "Making Mistakes", consisted of a 6-month series of short video clips, journaling activities, and weekly reminders, and modules were delivered to caregivers and children separately. 86 dyads completed self-report measures, 18 of whom participated in semi-structured interviews, following completion of the primary program module. A standard thematic analysis was used to elucidate themes from the parent and child interview content. RESULTS: Our quantitative and qualitative results were generally aligned. Children and parents viewed the novel digital mental health program as acceptable and appropriate, favoring the cognitive behavioral strategies such as modeling positive reactions to mistakes, responding positively to child mistakes, and emphasizing effort over outcome. Participants also provided helpful feedback related to program content, delivery, and engagement, as well as suggestions to enhance the program. CONCLUSIONS: Findings have implications for design and content features of parent-based and dyad-based programs, as well as digital mental health programs focused on reducing anxiety risk.

Prefrontal transcranial magnetic stimulation boosts response vigour during reinforcement learning in healthy adults.

A 10-Hz repetitive transcranial magnetic stimulation to the left dorsal lateral prefrontal cortex has been shown to increase dopaminergic activity in the dorsal striatum, a region strongly implicated in reinforcement learning. However, the behavioural influence of this effect remains largely unknown. We tested the causal effects of 10-Hz stimulation on behavioural and computational characteristics of reinforcement learning. A total of 40 healthy individuals were randomized into active and sham (placebo) stimulation groups. Each participant underwent one stimulation session (1500 pulses) in which stimulation was applied over the left dorsal lateral prefrontal cortex using a robotic arm. Participants then completed a reinforcement learning task sensitive to striatal dopamine functioning. Participants' choices were modelled using a reinforcement learning model (Q-learning) that calculates separate learning rates associated with positive and negative reward prediction errors. Subjects receiving active stimulation exhibited increased reward rate (number of correct responses per second of task activity) compared with those in sham. Computationally, although no group differences were observed, the active group displayed a higher learning rate for correct trials (αG) compared with incorrect trials (αL). Finally, when tested with novel pairs of stimuli, the active group displayed extremely fast reaction times, and a trend towards a higher reward rate. This study provided specific behavioural and computational accounts of altered striatal-mediated behaviour, particularly response vigour, induced by a proposed increase of dopamine activity by 10-Hz stimulation to the left dorsal lateral prefrontal cortex. Together, these findings bolster the use of repetitive transcranial magnetic stimulation to target neurocognitive disturbances attributed to the dysregulation of dopaminergic-striatal circuits.

Production of light-coloured, low heat-absorbing Holstein Friesian cattle by precise embryo-mediated genome editing.

CONTEXT: Genome editing enables the introduction of beneficial sequence variants into the genomes of animals with high genetic merit in a single generation. This can be achieved by introducing variants into primary cells followed by producing a live animal from these cells by somatic cell nuclear transfer cloning. The latter step is associated with low efficiencies and developmental problems due to incorrect reprogramming of the donor cells, causing animal welfare concerns. Direct editing of fertilised one-cell embryos could circumvent this issue and might better integrate with genetic improvement strategies implemented by the industry. METHODS: In vitro fertilised zygotes were injected with TALEN editors and repair template to introduce a known coat colour dilution mutation in the PMEL gene. Embryo biopsies of injected embryos were screened by polymerase chain reaction and sequencing for intended biallelic edits before transferring verified embryos into recipients for development to term. Calves were genotyped and their coats scanned with visible and hyperspectral cameras to assess thermal energy absorption. KEY RESULTS: Multiple non-mosaic calves with precision edited genotypes were produced, including calves from high genetic merit parents. Compared to controls, the edited calves showed a strong coat colour dilution which was associated with lower thermal energy absorbance. CONCLUSIONS: Although biopsy screening was not absolutely accurate, non-mosaic, precisely edited calves can be readily produced by embryo-mediated editing. The lighter coat colouring caused by the PMEL mutation can lower radiative heat gain which might help to reduce heat stress. IMPLICATIONS: The study validates putative causative sequence variants to rapidly adapt grazing cattle to changing environmental conditions.

The impact of punishment on error-related brain activity in children.

The error-related negativity (ERN) is sensitive to individual differences relating to anxiety and is modulated by manipulations that increase the threat-value of committing errors. In adults, the ERN magnitude is enhanced when errors are followed by punishment, especially among anxious individuals. Punitive parenting is related to an elevated ERN in children; however, the effects of task-based punishment on the ERN in children have yet to be understood. Furthermore, there is a need to assess developmental periods wherein the ERN might be especially prone to modulation by punishment. We examined the impact of punishment on the ERN in a sample of children and assessed whether the impact of punishment on the ERN was moderated by age and anxiety. Punishment potentiated the ERN in children, especially among higher trait-anxious individuals; the punishment potentiation of the ERN was also associated with older age. The interaction between child age and anxiety symptoms did not significantly predict the punishment potentiation of the ERN; however, both child age and anxiety symptoms uniquely predicted the punishment potentiation of the ∆ERN. Anxious children may be especially prone to punishment-related alterations in error monitoring, and the impact of punishment on the ERN may become more pronounced as children age.

The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis.

BACKGROUND: Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. RESULTS: No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations. CONCLUSIONS: We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.

Identification of Targeting Peptides for Mucosal Delivery in Sheep and Mice.

In this study we identified and characterized a novel cyclic peptide that facilitates the rapid transportation of conjugated molecules across the epithelial layer of the small intestine. The peptide was initially selected from phage display libraries using a large animal experimental model, which employed consecutive in vitro and in vivo panning. The procedure was designed to enrich for peptides that facilitated transcytosis across the intestinal epithelium into the intestinal afferent lymphatic system. A small set of peptides was repeatedly isolated using this selection method; however, the cyclic nonamer CTANSSAQC, 13C, dominated. The activity of the putative targeting peptide 13C was then verified using a mouse model. These experiments showed that the 13C peptide as well as macromolecules conjugated to it were rapidly transported across the intestinal mucosa into distinct subsets of epithelial cells and CD11c+ cells located in the lamina propria and Peyer's Patches. Significant amounts of intact protein could be delivered into the systemic circulation after rectal and nasal application. Thus, peptide 13C is regarded as an attractive carrier candidate for mucosal delivery of large molecules. The preferential targeting to distinct intestinal cells may be utilized to deliver active biological drugs for the effective control of diseases of the gut.

Correlates of alcohol use and alcohol use disorder among individuals with DSM-5 social anxiety disorder: A population based study.

Alcohol use disorder (AUD) commonly cooccurs with social anxiety disorder (SAD). With changes to diagnostic criteria of psychiatric disorders in the DSM-5, the present study sought to observe the associations between daily alcohol use, AUD, and social anxiety in a large sample of individuals with lifetime (N = 1255) and past-year (N = 908) SAD. The sample was derived from a large nationally representative study of adults in the United States. Of note, we found that at the symptom level, daily alcohol use and AUD were significantly related to panic attacks due to social anxiety, subjective distress, and impairment in relationships. Daily alcohol use and AUD were significantly associated with lifetime SAD severity; however, only past-year daily alcohol use was significantly related to past-year SAD severity. We also found that AUD was significantly related to greater treatment-seeking for SAD, and both AUD and daily alcohol use were significantly associated with lifetime history of suicide attempts even after covarying for SAD severity. The present study provides an updated investigation of alcohol use in individuals with DSM-5 SAD, and it underscores the significance of daily alcohol use as an important factor to consider in individuals with SAD.

Relational victimization prospectively predicts increases in error-related brain activity and social anxiety in children and adolescents across two years.

Recent research has focused on identifying neural markers associated with risk for anxiety, including the error-related negativity (ERN). An elevated ERN amplitude has been observed in anxious individuals from middle childhood onward and has been shown to predict risk for future increases in anxiety development. The ERN is sensitive to environmental influences during development, including interpersonal stressors. Of note, one particular type of interpersonal stressor, relational victimization, has been related to increases in anxiety in adolescents. We tested whether relational victimization predicts increases in the ERN and social anxiety symptoms across two years in a sample of 152 child and adolescent females (ages 8 - 15). Results indicated that children and adolescents' baseline ERN was positively related to the ERN two years later. Furthermore, greater relational victimization at baseline predicted greater increases in the ERN two years later, controlling for baseline ERN. Moreover, relational victimization at baseline predicted increases in social anxiety, and this relationship was mediated by increases in the ERN. These results suggest that relational victimization impacts the developmental trajectory of the neural response to errors and thereby impacts increases in social anxiety among children and adolescents.

Cytoplasmic Injection of Zygotes to Genome Edit Naturally Occurring Sequence Variants Into Bovine Embryos.

Genome editing provides opportunities to improve current cattle breeding strategies through targeted introduction of natural sequence variants, accelerating genetic gain. This can be achieved by harnessing homology-directed repair mechanisms following editor-induced cleavage of the genome in the presence of a repair template. Introducing the genome editors into zygotes and editing in embryos has the advantage of uncompromised development into live animals and alignment with contemporary embryo-based improvement practices. In our study, we investigated the potential to introduce sequence variants, known from the pre-melanosomal protein 17 (PMEL) and prolactin receptor (PRLR) genes, and produce non-mosaic, edited embryos, completely converted into the precision genotype. Injection of gRNA/Cas9 editors into bovine zygotes to introduce a 3 bp deletion variant into the PMEL gene produced up to 11% fully converted embryos. The conversion rate was increased to up to 48% with the use of TALEN but only when delivered by plasmid. Testing three gRNA/Cas9 editors in the context of several known PRLR sequence variants, different repair template designs and delivery as DNA, RNA or ribonucleoprotein achieved full conversion rates up to 8%. Furthermore, we developed a biopsy-based screening strategy for non-mosaic embryos which has the potential for exclusively producing non-mosaic animals with intended precision edits.

Megakaryopoiesis impairment through acute innate immune signaling activation by azacitidine.

Thrombocytopenia, prevalent in the majority of patients with myeloid malignancies, such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), is an independent adverse prognostic factor. Azacitidine (AZA), a mainstay therapeutic agent for stem cell transplant-ineligible patients with MDS/AML, often transiently induces or further aggravates disease-associated thrombocytopenia by an unknown mechanism. Here, we uncover the critical role of an acute type-I interferon (IFN-I) signaling activation in suppressing megakaryopoiesis in AZA-mediated thrombocytopenia. We demonstrate that megakaryocytic lineage-primed progenitors present IFN-I receptors and, upon AZA exposure, engage STAT1/SOCS1-dependent downstream signaling prematurely attenuating thrombopoietin receptor (TPO-R) signaling and constraining megakaryocytic progenitor cell growth and differentiation following TPO-R stimulation. Our findings directly implicate RNA demethylation and IFN-I signal activation as a root cause for AZA-mediated thrombocytopenia and suggest mitigation of TPO-R inhibitory innate immune signaling as a suitable therapeutic strategy to support platelet production, particularly during the early phases of AZA therapy.

Resting posterior alpha power and adolescent major depressive disorder.

For several decades, resting electroencephalogram (EEG) alpha oscillations have been used to characterize neurophysiological alterations related to major depressive disorder. Prior research has generally focused on frontal alpha power and asymmetry despite resting alpha being maximal over posterior electrode sites. Research in depressed adults has shown evidence of hemispheric asymmetry for posterior alpha power, however, the resting posterior alpha-depression link among adolescents remains unclear. To clarify the role of posterior alpha among depressed adolescents, the current study acquired eyes-closed 128-channel resting EEG data from 13 to 18 year-old depressed (n = 31) and healthy (n = 35) female adolescents. Results indicated a significant group by hemisphere interaction, as depressed adolescents exhibited significantly larger posterior alpha (i.e., lower brain activity) over the right versus left hemisphere, whereas healthy adolescents showed no hemispheric differences. Relatively greater alpha over the right versus left hemisphere correlated with depression symptoms, anhedonia symptoms, rumination, and self-criticism. Further, depressed adolescents had reduced overall posterior alpha compared to healthy youth; though, no associations with symptoms and related traits emerged. Resting posterior alpha may be a promising neurophysiological index of adolescent depression, and more broadly, may relate to risk factors characterized by enhanced perseveration.

Neurophysiological predictors of gaze-contingent music reward therapy among adults with social anxiety disorder.

Social anxiety disorder (SAD) is associated with fear of negative evaluation and heightened performance monitoring. The best-established treatments help only a subset of patients, and there are no well-established predictors of treatment response. The current study investigated whether individual differences in processing errors might predict response to gaze-contingent music reward therapy (GC-MRT). At baseline, healthy control subjects (HC; n = 20) and adults with SAD (n = 29), ages 19-43 years, completed the Flanker Task while electroencephalography (EEG) data were recorded. SAD participants then received up to 12 sessions over 8 weeks of GC-MRT, designed to train participants' attention away from threatening and toward neutral faces. Clinical assessments were completed 9- (post-treatment) and 20-weeks (follow-up) after initiating the treatment. At baseline, compared to HC, SAD performed the task more accurately and exhibited increased error-related negativity (ERN) and delta power to error commission. After controlling for age and baseline symptoms, more negative ERN and increased frontal midline theta (FMT) predicted reduced self-reported social anxiety symptoms at post-treatment, and FMT also predicted clinician-rated and self-reported symptom reduction at the follow-up assessment. Hypervigilance to error is characteristic of SAD and warrants further research as a predictor of treatment response for GC-MRT.

DNA oligonucleotides and plasmids perform equally as donors for targeted gene conversion.

Site-specific gene modifications in cells are initiated by the introduction of exogenous DNA. We used a recently established cell assay to compare the ability of DNA donors to induce a single point mutation that converts a target gene encoding blue fluorescent protein (BFP) into expressing green fluorescent protein (GFP). In a chromosomal assay with cells stably expressing BFP, we showed that fluorescently labeled single-stranded oligonucleotides and a donor plasmid cotranscribing a red fluorescent protein provide similar efficiencies in triggering BFP-GFP conversions. In transient cotransfections, an isogenic donor plasmid comprising a nonfunctional GFP gene yielded a greater efficiency for the conversion of the BFP target gene than a nonisogenic donor, and all plasmid donors were superior to oligonucleotides.

Cognitive resources are distributed among the entire auditory landscape in auditory scene analysis.

Although attention has been shown to enhance neural representations of selected inputs, the fate of unselected background sounds is still debated. The goal of the current study was to understand how processing resources are distributed among attended and unattended sounds during auditory scene analysis. We used a three-stream paradigm with four acoustic features uniquely defining each sound stream (frequency, envelope shape, spatial location, tone quality). We manipulated task load by having participants perform a difficult auditory task and an easy movie-viewing task with the same set of sounds in separate conditions. The mismatch negativity (MMN) component of event-related brain potentials (ERPs) was measured to evaluate sound processing in both conditions. We found no effect of task demands on unattended sound processing: MMNs were elicited by unattended deviants during both low- and high-load task conditions. A key factor of this result was the use of unique tone feature combinations to distinguish each of the three sound streams, strengthening the segregation of streams. In the auditory task, the P3b component demonstrates a two-stage process of target evaluation. Thus, these results, in conjunction with results of previous studies, suggest that stimulus-driven factors that strengthen stream segregation can free up processing capacity for higher-level analyses. The results illustrate the interactive nature of top-down and stimulus-driven processes in stream formation, supporting a distributive theory of attention that balances the strength of the bottom-up input with perceptual goals in analyzing the auditory scene.

Transgenic goats producing an improved version of cetuximab in milk.

Therapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10 g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

Long-Term Evaluation of Women Referred to a Breast Cancer Family History Clinic (Manchester UK 1987-2020).

Clinics for women concerned about their family history of breast cancer are widely established. A Family History Clinic was set-up in Manchester, UK, in 1987 in a Breast Unit serving a population of 1.8 million. In this review, we report the outcome of risk assessment, screening and prevention strategies in the clinic and propose future approaches. Between 1987-2020, 14,311 women were referred, of whom 6.4% were from known gene families, 38.2% were at high risk (≥30% lifetime risk), 37.7% at moderate risk (17-29%), and 17.7% at an average/population risk who were discharged. A total of 4168 (29.1%) women were eligible for genetic testing and 736 carried pathogenic variants, predominantly in BRCA1 and BRCA2 but also other genes (5.1% of direct referrals). All women at high or moderate risk were offered annual mammographic screening between ages 30 and 40 years old: 646 cancers were detected in women at high and moderate risk (5.5%) with a detection rate of 5 per 1000 screens. Incident breast cancers were largely of good prognosis and resulted in a predicted survival advantage. All high/moderate-risk women were offered lifestyle prevention advice and 14-27% entered various lifestyle studies. From 1992-2003, women were offered entry into IBIS-I (tamoxifen) and IBIS-II (anastrozole) trials (12.5% of invitees joined). The NICE guidelines ratified the use of tamoxifen and raloxifene (2013) and subsequently anastrozole (2017) for prevention; 10.8% women took up the offer of such treatment between 2013-2020. Since 1994, 7164 eligible women at ≥25% lifetime risk of breast cancer were offered a discussion of risk-reducing breast surgery and 451 (6.2%) had surgery. New approaches in all aspects of the service are needed to build on these results.

Cattle with a precise, zygote-mediated deletion safely eliminate the major milk allergen beta-lactoglobulin.

We applied precise  zygote-mediated genome editing to eliminate beta-lactoglobulin (BLG), a major allergen in cows' milk. To efficiently generate LGB knockout cows, biopsied embryos were screened to transfer only appropriately modified embryos. Transfer of 13 pre-selected embryos into surrogate cows resulted in the birth of three calves, one dying shortly after birth. Deep sequencing results confirmed conversion of the genotype from wild type to the edited nine bp deletion by more than 97% in the two male calves. The third calf, a healthy female, had in addition to the expected nine bp deletion (81%), alleles with an in frame 21 bp deletion (<17%) at the target site. While her milk was free of any mature BLG, we detected low levels of a BLG variant derived from the minor deletion allele. This confirmed that the nine bp deletion genotype completely knocks out production of BLG. In addition, we showed that the LGB knockout animals are free of any TALEN-mediated off-target mutations or vector integration events using an unbiased whole genome analysis. Our study demonstrates the feasibility of generating precisely biallelically edited cattle by zygote-mediated editing for the safe production of hypoallergenic milk.

Antisense STAT3 inhibitor decreases viability of myelodysplastic and leukemic stem cells.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Transcriptomic analysis of stem and progenitor populations in MDS and AML demonstrated overexpression of STAT3 that was validated in an independent cohort. STAT3 overexpression was predictive of a shorter survival and worse clinical features in a large MDS cohort. High STAT3 expression signature in MDS CD34+ cells was similar to known preleukemic gene signatures. Functionally, STAT3 inhibition by a clinical, antisense oligonucleotide, AZD9150, led to reduced viability and increased apoptosis in leukemic cell lines. AZD9150 was rapidly incorporated by primary MDS/AML stem and progenitor cells and led to increased hematopoietic differentiation. STAT3 knockdown also impaired leukemic growth in vivo and led to decreased expression of MCL1 and other oncogenic genes in malignant cells. These studies demonstrate that STAT3 is an adverse prognostic factor in MDS/AML and provide a preclinical rationale for studies using AZD9150 in these diseases.

Cytokine and antibody subclass responses in the intestinal lymph of sheep during repeated experimental infections with the nematode parasite Trichostrongylus colubriformis.

The expression of interleukin (IL)-4, IL-5, IL-10, IL-13, TNF-alpha and IFN-gamma genes, and parasite-specific IgM, IgG1, IgG2, IgA and total IgE levels, were monitored daily in intestinal lymph of sheep infected repeatedly with the nematode parasite Trichostrongylus colubriformis. Host genotype had a significant influence on IL-13 gene activity, with resistant-line (R) sheep consistently expressing higher levels of mRNA than susceptible-line (S) sheep. Mean gene expression of IL-13, IL-4 and IFN-gamma did not differ significantly between the first and second nematode challenge. Field-primed R and S as well as field-primed R and naïve S sheep had lower mean gene expression of IL-5 and IL-10, respectively, during the second when compared to primary challenge. Genes for IL-13 and IL-5 were transiently and strongly up-regulated after nematode infection, particularly in animals with previous exposure to nematodes. Genes for TNF-alpha and IFN-gamma were also transiently up-regulated, but to a lesser extent and more typically after primary challenge. Naïve sheep of both genotypes produced relatively little antibody response after primary challenge. A second nematode challenge resulted in large increases in the lymphatic levels of all antibody sub-classes which were significant for adult antigen-specific IgA and larval antigen-specific IgG1. In naïve S line sheep, the larval-specific IgA and IgG2 response appeared delayed when compared to the R line animals. Field-primed R and S line sheep had relatively high lymphatic IgG1 levels prior to experimental infection and these did not change significantly afterwards. These results demonstrate that during nematode infections, the intestinal micro-environment of sheep is transiently skewed towards Th2 cytokine dominance, although IFN-gamma gene expression continues. This response is accompanied by increases of nematode-specific IgG1, IgA, IgG2 and IgM, as well as of total IgE in lymph plasma.

Long-term collection and characterization of afferent lymph from the ovine small intestine.

Reliable methods for long-term collection of afferent lymph draining from the small intestine of sheep are described and validated. The procedure was used successfully in normal sheep, in animals infected experimentally with the parasitic intestinal nematode Trichostrongylus colubriformis and in animals infected naturally with Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. Our approach enabled afferent lymph draining from the small intestine to be collected continuously for up to 4 months, without any detrimental effects on the animals. Based on cytokine gene expression profiles of afferent intestinal lymph cells, the two infections induced contrasting regional immune responses, namely, Th2-type immunity in the case of T. colubriformis infection and Th1-type immunity in natural cases of Johne's disease. Some immune parameters differed markedly between the two disease models, highlighting the potential value of this approach to gain real-time insights into distinctive host-pathogen interactions as they occur in vivo within the regional immune system of the gastrointestinal tract.