Lethargy

The production of ECTV stocks for infection of mice and the determination of titers in stocks and organs were done as described previously [8 (link)]. To generate ECTV 189898-p7.5-EGFP, we adapted the method described by Johnston and McFadden [60 (link)]. Briefly, a construct containing the ECTV Moscow fragment 189543–189897, the VACV early/late promoter p7.5, the sequence of EGFP, and the ECTV Moscow fragment 189950–190297, in that order, was made by recombinant PCR and cloned into plasmid Bluescript II SK+ to generate the targeting vector pBS-EVM189898-p7.5-EGFP. This targeting vector was used to transfect mouse A9 cells using Lipofectamine 2000 as per manufacturer's instructions (Invitrogen). The transfected cells were infected with wild-type ECTV (Moscow strain, 0.3 pfu/cell) in 6-well plates. 2 d later, transfected/infected A9 cells were harvested using a rubber policeman, frozen and thawed, and different dilutions of cell lysates were used to infect BSC-1 cells in 6-well plates. 2 h after infection, the cells were overlaid with media containing 0.5% agarose. 4 d later, green-fluorescent plaques were picked with a pipette tip and used to infect a new set of cells. The purification procedure was iterated five times until all plaques were fluorescent. The resulting virus, ECTV 189898-p7.5-EGFP, carries EGFP in a non-coding region and is as pathogenic as wild-type ECTV Moscow (not shown). For preparation of ECTV stock for infection of different cell lines, A9 cells were infected with 0.2 pfu ECTV/cell, and incubated at 37 °C, 5% CO₂. After 5 d, the cells were collected, frozen and thawed three times, and then sonicated in a water-bath sonicator. The solid material was pelleted by centrifugation, and the supernatant was stored in aliquots at −80 °C. The DAP10-deficient mice [61 (link),62 (link)] (generously provided by Dr. Joe Phillips) and DAP12-deficient mice [63 (link)] on the C57BL/6 background were bred at UCSF. All the other mice were bred at the Fox Chase Cancer Center Laboratory Animal Facility in specific pathogen-free rooms or were purchased from Jackson Laboratories. IFN-γ-deficient C57BL/6 mice were generously provided by Dr. Glenn Rall. For infections, sex-matched animals 8–12 wk old were transferred to a biosafety level 3 room. For ECTV infection, anesthetized mice were infected in the left footpad with 25 μl PBS containing 3 × 10³ pfu ECTV. Following infections, mice were observed daily for signs of disease (lethargy, ruffled hair, weight loss, skin rash, and eye secretions) and imminent death (unresponsiveness to touch and lack of voluntary movements). Moribund mice were euthanized by halothane inhalation. All of the experimental protocols involving animals were approved by the Fox Chase Cancer Center Institutional Animal Care and Use Committee.
For ECTV infection of cells, 3–5 × 10⁵ cells were plated in 6-well plates and cultured overnight to allow cells to adhere. The cells were then infected with 0.5 pfu ECTV/cell for 18 h, collected, washed, stained, and analyzed for surface expression of various markers. For ECTV infection of peritoneal cells, the mice were euthanized by halothane inhalation and injected i.p. with PBS, the abdomen massaged gently, and the peritoneal cells were collected by aspiration and washed.

The social isolation scale used by the UK Biobank was constructed from three questions: (1) “Including yourself, how many people are living together in your household? Include those who usually live in the house such as students living away from home during term time, partners in the armed forces or professions such as pilots” (1 point for living alone); (2) “How often do you visit friends or family or have them visit you?” (1 point for friends and family visit less than once a month); and (3) “Which of the following [leisure/social activities] do you engage in once a week or more often? You may select more than one” (1 point for no participation in social activities at least weekly). Thus, individuals could score a total of 0–3; an individual was defined as socially isolated if he or she scored 2 or 3; those who scored 0 or 1 were classified as not isolated. Similar scales have been used previously in other UK studies.¹² (link)
Loneliness was assessed with two questions: “Do you often feel lonely?” (no=0, yes=1) and “How often are you able to confide in someone close to you?” (0=almost daily to once every few months; 1=never or almost never). An individual was defined as lonely if he or she scored 2, and not lonely if he or she scored 0 or 1. Similar questions are included in scales such as the revised UCLA Loneliness Scale.¹³ (link)
Follow-up for all deaths irrespective of cause started at inclusion in the UK Biobank study (from national death registers) and ended on Aug 14, 2015, or upon death, for all participants. The cause-specific-mortality International Classification of Diseases (ICD) codes were as follows: neoplasms (C00–D48), diseases of the circulatory system (I05–I89), and other diseases (all remaining ICD-10 codes).
Details of the assessments of participants' variables are publicly available.¹⁴ Briefly, participants completed several touch-screen computer-based questionnaires, and then had a face-to-face interview with a trained researcher. The information collected included basic demographics (sex and age), ethnic origin (white vs other), socioeconomic factors (educational attainment, household income, and postcode of residence with the corresponding Townsend deprivation index score), and chronic diseases (diabetes, cardiovascular disease, cancer, and other long-standing illness, disability, or infirmity). The Townsend deprivation index is an integrated neighbourhood-level measure of unemployment, non-car ownership, non-home ownership, and household overcrowding across the UK.¹⁵ (link)
To assess biological factors, trained data collectors measured height and weight in all participants during clinic attendance using standard operating procedures, and the body-mass index (BMI) was subsequently calculated. Procedures for measuring systolic and diastolic blood pressure and handgrip strength are reported in the UK Biobank protocol, which is available online.¹¹ (link) Behavioural factors, including cigarette smoking (current smoker [yes or no]; ex-smoker [yes or no]), physical activity (moderate and vigorous), and alcohol intake frequency (at least three times a week vs twice a week or less) were self-reported on a questionnaire.
Psychological factors comprised current depressive symptoms and general cognitive capacity. Depressive symptoms were measured using the frequency of four items from the Patient Health Questionnaire (PHQ):16 (link), 17 (link) (1) depressed mood, (2) disinterest or absence of enthusiasm, (3) tenseness or restlessness, and (4) tiredness or lethargy in the previous 2 weeks. General cognitive capacity (numeric memory, verbal–numerical reasoning, reaction time, and visual memory) was assessed by use of a touch-screen application.¹⁸ (link) Self-rated health was assessed using the following question answered on a four-point scale (1=poor; 4=excellent): “In general, how would you rate your overall health?”

Four symptoms of sickness behavior (piloerection, ptosis, lethargy, and huddling; Kelley et al., 2003 (link)) were monitored and recorded each hour for 6 h following the administration of LPS (1.0 mg/kg) and vehicle in 8 rats per treatment condition (four per phenotype). The researchers observing sickness behavior were blinded to the phenotype. At each time point, the presence of absence of these four symptoms was recorded. Piloerection was marked when the animals’ body hair stood up, eyelid drooping was scored as ptosis, lethargy was scored if a rat did not approach the front of the home cage when approached by the researcher, and hunched or curled body posture was marked as huddling.

In addition to the determination of brain CHT ubiquitination levels and concentrations of cytokines and chemokines (below) in STs and GTs, the effects of activation of the innate immune system by the bacterial endotoxin lipopolysaccharide (LPS) were assessed. LPS, from Escherichia coli (serotype O111:B4), was obtained from Millipore Sigma and suspended in 0.9% saline (Teknova 0.9% sterile saline solution; Fisher Scientific) vortexed, allocated to 1.0 mg/ml, and stored in 1.5 ml Eppendorf tubes at −20°C until used. Preparation and injections of LPS were conducted in a chemical fume hood and using procedures approved by the University of Michigan Environment, Health and Safety Department.
STs and GTs were randomly assigned to the administration of LPS (1.0 or 5.0 mg/kg in 0.2 ml saline, i.p.) or 0.9% saline. Sickness behaviors, including piloerection, ptosis, lethargy, and huddling were monitored each hour for 6 h following the treatment injection. A lethal dose of sodium pentobarbital was administered (1.5 g/kg, i.p.) 6 h after injections, followed by transcardiac perfusion with 0.9% saline. The right frontal cortex and right striatum were dissected out and synaptosomal pellets were prepared for determination of CHT ubiquitination levels as described above. Furthermore, spleen, left frontal cortex, and left striatum were harvested, flash-frozen in 2-methyl butane, and stored at −80°C for subsequent determination of cytokine and chemokine levels.