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Background & Aims: Transitioning into higher education (HE) impacts health behaviours. Poor dietary and lifestyle behaviours may correlate and increase risk of co-morbidities. The introduction of the Okanagan Charter detailed the important role of health promotion within a HE setting. The aim of this study was to assess the relationship between dietary quality and lifestyle behaviours of students attending HE., Methods: Full-time students, aged 18+, were eligible to participate in this online cross-sectional study. Self-reported questions were asked in relation to demographics, body mass index (BMI), smoking, and COVID-19. A food frequency questionnaire measured dietary quality along with tools assessing alcohol use, sleep quality, perceived stress, and physical activity. Statistical analyses were performed using chi-square, one-way ANOVA, independent sample t-tests, Pearson's correlation, and multivariate linear regression., Results: Evidence of a correlation between poor diet quality and having a higher BMI (p = 0.040), higher alcohol consumption (p = <0.001), poorer sleep quality (p = 0.003), higher stress levels (p = 0.006) and smoking (p = 0.001) was found. Low fruit and vegetable consumption were associated with higher BMI (p = 0.013), higher alcohol consumption (p = <0.001), lower physical activity levels (p = 0.006), higher stress levels (p = <0.001), smoking (p = <0.001) and being male (p = 0.002)., Conclusions: This study provides data on the association between dietary quality and lifestyle behaviours among HE students and will inform healthy campus initiatives., Competing Interests: Declaration of competing interest Authors state no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: The aim of the study was to identify components of the COM-B (capability, opportunity, motivation and behaviour) model that influences behaviour to modify dietary patterns in 40-55-year-olds living in the UK, in order to influence the risk of cognitive decline in later life., Design: This is a qualitative study using the COM-B model and theoretical domains framework (TDF) to explore beliefs to adopting the Mediterranean-DASH Intervention for Neurodegenerative delay (MIND) diet., Setting: Northern Ireland., Participants: Twenty-five participants were recruited onto the study to take part in either a focus group or an interview. Participants were men and women aged between 40 and 55 years. Participants were recruited via email, Facebook and face to face., Results: Content analysis revealed that the main perceived barriers to the adoption of the MIND diet were time, work environment, taste preference and convenience. The main perceived facilitators reported were improved health, memory, planning and organisation, and access to good quality food., Conclusions: This study provides insight into the personal, social and environmental factors that participants report as barriers and facilitators to the adoption of the MIND diet among middle-aged adults living in the UK. More barriers to healthy dietary change were found than facilitators. Future interventions that increase capability, opportunity and motivation may be beneficial. The results from this study will be used to design a behaviour change intervention using the subsequent steps from the Behaviour Change Wheel.

Background: The aim of the study was to identify and compare components of the COM-B (capability, opportunity, motivation and behaviour) model, that influences behaviour to modify dietary patterns in 40-55-year olds living in Northern Ireland (NI) and Italy, in order to reduce the risk of cognitive decline in later life., Methods: This was a qualitative study examining factors influencing Mediterranean-DASH (Dietary Approaches to Stop Hypertension) Intervention for Neurodegenerative Delay (MIND) diet behaviour. This study further elaborated the COM-B components into the 14 domains of the Theoretical Domains Framework to further understand behaviour. Twenty-five Northern Irish and Italian participants were recruited onto the study, to take part in either a focus group or an interview. Participants were both male and female aged between 40 and 55 years., Results: Thematic analysis revealed that the main barriers to the uptake of the MIND diet were; time, work environment (opportunity), taste preference and convenience (motivation). Culture (motivation), seasonal foods and lack of family support (opportunity) to be a barrier to the Italian sample only. The main facilitators reported were; improved health, memory, planning and organisation (motivation) and access to good quality food (opportunity). Cooking skills, knowledge (capability) and heathy work lunch (opportunity) reported as a facilitator to the Italian sample only., Conclusions: Cross-cultural differences in relation to psychosocial barriers and facilitators were found in both samples. More barriers than facilitators towards uptake of the MIND diet were found. There is a need for interventions that increase capability, opportunity, and motivation to aid behaviour change. The findings from this study will be used to design a behaviour change intervention using the subsequent steps from the Behaviour Change Wheel.

Background: The term 'whole dietary pattern' can be defined as the quantity, frequency, variety and combination of different foods and drinks typically consumed and a growing body of research supports the role of whole dietary patterns in influencing the risk of non-communicable diseases. For example, the 'Mediterranean diet', which compared to the typical Western diet is rich in fruits and vegetables, whole grains, and oily fish, is associated with reduced risk of cardiovascular disease and cancer. Social Cognition Models provide a basis for understanding the determinants of behaviour and are made up of behavioural constructs that interventions target to change dietary behaviour. The aim of this systematic review was to provide a comprehensive assessment of the effectiveness and use of psychological theory in dietary interventions that promote a whole dietary pattern., Methods: We undertook a systematic review using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis to synthesize quantitative research studies found in Embase, Medline, PsycInfo, CINAHL and Web of Science. The studies included were randomised and non-randomised trials published in English, involving the implementation of a whole dietary pattern using a Social Cognition Model to facilitate this. Two independent reviewers searched the articles and extracted data from the articles. The quality of the articles was evaluated using Black and Down quality checklist and Theory Coding Scheme., Results: Nine intervention studies met the criteria for inclusion. Data from studies reporting on individual food group scores indicated that dietary scores improved for at least one food group. Overall, studies reported a moderate application of the theory coding scheme, with poor reporting on fidelity., Conclusion: To our knowledge, this is the first review to investigate psychological theory driven interventions to promote whole dietary patterns. This review found mixed results for the effectiveness of using psychological theory to promote whole dietary pattern consumption. However, the studies in this review scored mostly moderate on the theory coding scheme suggesting studies are not rigorously applying theory to intervention design. Few studies reported high on treatment fidelity, therefore, translation of research interventions into practice may further impact on effectiveness of intervention. Further research is needed to identify which behaviour change theory and techniques are most salient in dietary interventions.

Purpose: Cognitive decline is commonly reported during the menopausal transition, with memory and attention being particularly affected. The aim of this study was to investigate the effects of a commercially available soy drink on cognitive function and menopausal symptoms in post-menopausal women., Methods: 101 post-menopausal women, aged 44-63 years, were randomly assigned to consume a volume of soy drink providing a low (10 mg/day; control group), medium (35 mg/day), or high (60 mg/day) dose of isoflavones for 12 weeks. Cognitive function (spatial working memory, spatial span, pattern recognition memory, 5-choice reaction time, and match to sample visual search) was assessed using CANTAB pre- and post-the 12 week intervention. Menopausal symptoms were assessed using Greene's Climacteric Scale., Results: No significant differences were observed between the groups for any of the cognitive function outcomes measured. Soy drink consumption had no effect on menopausal symptoms overall; however, when women were stratified according to the severity of vasomotor symptoms (VMS) at baseline, women with more severe symptoms at baseline in the medium group had a significant reduction (P = 0.001) in VMS post-intervention (mean change from baseline score: - 2.15 ± 1.73) in comparison to those with less severe VMS (mean change from baseline score: 0.06 ± 1.21)., Conclusions: Soy drink consumption had no effect on cognitive function in post-menopausal women. Consumption of ~ 350 ml/day (35 mg IFs) for 12 weeks significantly reduced VMS in those with more severe symptoms at baseline. This finding is clinically relevant as soy drinks may provide an alternative, natural, treatment for alleviating VMS, highly prevalent among western women.

Objective: Dietary soy may improve menopausal symptoms, and subsequently mediate mood. This novel study examines various doses of dietary soy drink on everyday mood stability and variability in postmenopausal women., Methods: Community-dwelling women (n = 101), within 7 years postmenopause, consumed daily either a low (10 mg, n = 35), medium (35 mg, n = 37), or high (60 mg, n = 29) dose of isoflavones, for 12 weeks. Menopausal symptoms and repeated measures of everyday mood (positive [PA] and negative [NA] affect) (assessed at four time points per day for 4 consecutive days, using The Positive and Negative Affect Schedule) were completed at baseline and follow-up., Results: The dietary soy intervention had no effect on everyday mood stability (for PA [F{2,70} = 0.95, P = 0.390] and NA [F{2,70} = 0.72, P = 0.489]) or variability (for PA [F{2,70} = 0.21, P = 0.807] and for NA [F{2,70} = 0.15, P = 0.864]), or on menopausal symptoms (for vasomotor [F{2,89} = 2.83, P = 0.064], psychological [F{2,88} = 0.63, P = 0.535], somatic [F{2,89} = 0.32, P = 0.729], and total menopausal symptoms [F{2,86} = 0.79, P = 0.458]). There were between-group differences with the medium dose reporting higher PA (low, mean 24.2, SD 6; and medium, mean 29.7, SD 6) and the low dose reporting higher NA (P = 0. 048) (low, mean 11.6, SD 2; and high, mean 10.6, SD 1) in mood scores. Psychological (baseline M = 18 and follow-up M = 16.5) and vasomotor (baseline M = 4.2 and follow-up M = 3.6) scores declined from baseline to follow-up for the overall sample., Conclusions: Soy isoflavones had no effect on mood at any of the doses tested. Future research should focus on the menopause transition from peri to postmenopause as there may be a window of vulnerability, with fluctuating hormones and increased symptoms which may affect mood.

Although dehydration of ≥ 2% body weight (BW) loss significantly impairs endurance performance, dehydration remains prevalent among athletes and may be owing to a lack of knowledge in relation to fluid requirements. The aim of this study was to assess the hydration status of university/club level athletes (n = 430) from a range of sports/activities (army officer cadet training; bootcamp training; cycling; Gaelic Athletic Association camogie, football and hurling; golf; hockey; netball; rugby; running (sprinting and endurance); Shotokan karate and soccer) immediately before and after training/competition and to assess their nutritional knowledge. Urine specific gravity (USG) was measured immediately before and after exercise and BW loss during exercise was assessed. Nutritional knowledge was assessed using a validated questionnaire. 31.9% of athletes commenced exercise in a dehydrated state (USG >1.020) with 43.6% of participants dehydrated posttraining/competition. Dehydration was particularly prevalent (>40% of cohort) among karateka, female netball players, army officer cadets, and golfers. Golfers that commenced a competitive 18 hole round dehydrated took a significantly higher number of strokes to complete the round in comparison with their euhydrated counterparts (79.5 ± 2.1 vs. 75.7 ± 3.9 strokes, p = .049). Nutritional knowledge was poor among participants (median total score [IQR]; 52.9% [46.0, 59.8]), albeit athletes who were euhydrated at the start of exercise had a higher overall score in comparison with dehydrated athletes (55.2% vs. 50.6%, p = .001). Findings from the current study, therefore, have significant implications for the education of athletes in relation to their individual fluid requirements around exercise.

Purpose: The Australian Early Development Census (AEDC) is a population-based measure of children's development across five domains in the first year of formal schooling. In this study, the AEDC data from two domains (Language and Cognitive Skills and Communication Skills and General Knowledge) were used to explore the extent and distribution of vulnerability in communication skills among children in Australian communities. Speech Pathology Australia (SPA) membership data were then used to explore the accessibility of services within those communities., Method: The 2012 AEDC data were accessed for 289,973 children, living in 577 communities across Australia. The number of children identified as "at risk" (10-25(th) percentile) or developmentally "vulnerable" (< 10(th) percentile) in each of the domains was calculated, then the location of communities with high proportions (> 20%) of these children was determined. These data were mapped against the location of paediatric speech-language pathologists (SLPs) to identify the number of communities with little to no access to speech-language pathology services., Result: Across Australia, there were 47,636 children (17.4%) identified as developmentally vulnerable/at risk in Language and Cognitive Skills and 69,153 children (25.3%) in Communication Skills and General Knowledge. There were 27 communities with > 20% of children identified as developmentally vulnerable/at risk in Language and Cognition in their first year of formal schooling. Of those, none had access to speech-language pathology services, according to current SPA membership data. There were also 27 local government areas with > 20% of children identified as developmentally vulnerable/at risk in the Communication Skills and General Knowledge domain. Of these, three had access to SLP(s) and these were in regional/metropolitan areas., Conclusion: The AEDC provides a means of identifying communities where children are performing well and communities which may benefit from population-based prevention or intervention. Given the number of communities within Australia without access to SLPs, there is a need to reconsider how such population-based services could be delivered, particularly in the communities with higher levels of vulnerability in communication development.

Background: The study aim was to determine if state and trait intra-individual measures of everyday affect predict cognitive functioning in healthy older community dwelling European adults (n = 387), aged 55-87 years., Methods: Participants were recruited from centres in France, Italy and Northern Ireland. Trait level and variability in positive and negative affect (PA and NA) were assessed using self-administered PANAS scales, four times a day for four days. State mood was assessed by one PANAS scale prior to assessment of recognition memory, spatial working memory, reaction time and sustained attention using the CANTAB computerized test battery., Results: A series of hierarchical regression analyses were carried out, one for each measure of cognitive function as the dependent variable, and socio-demographic variables (age, sex and social class), state and trait mood measures as the predictors. State PA and NA were both predictive of spatial working memory prior to looking at the contribution of trait mood. Trait PA and its variability were predictive of sustained attention. In the final step of the regression analyses, trait PA variability predicted greater sustained attention, whereas state NA predicted fewer spatial working memory errors, accounting for a very small percentage of the variance (1-2%) in the respective tests., Conclusion: Moods, by and large, have a small transient effect on cognition in this older sample.

Elevated concentrations of the pro-inflammatory cytokines IL-1β and IL-6 have been associated with impaired cognitive performance. There are, however, few studies that have examined the relationship between cytokine production and specific aspects of cognition in healthy older individuals. Two-colour flow cytometry was used to determine intracellular cytokine production by activated monocytes, and neuropsychological tests were performed using the Cambridge Neuropsychological Test Automated Battery (CANTAB) in 93 apparently healthy men and women aged 55-70 years. A series of hierarchical regression analyses was carried out to examine the contribution of IL-1β and IL-6 (% expression and production (antibody binding capacity (ABC))) to recognition, attention and working memory, after controlling for socio-demographic variables (age, sex and social class). IL-1β% expression and IL-6 production predicted aspects of working memory. Recognition memory was found to be sensitive to the affects of age and social class. The current study suggests that higher intracellular cytokine production by activated monocytes may be predictive of lower cognitive performance in working memory in healthy older individuals. These findings indicate that utilization of models for in vivo cytokine production upon immune challenge may be useful in studying specific aspects of memory affected during inflammatory responses, for example in individuals at risk for cognitive decline owing to age-related inflammatory disorders., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Choline is an essential nutrient and can also be obtained by de novo synthesis via an oestrogen responsive pathway. Choline can be oxidised to the methyl donor betaine, with short-term supplementation reported to lower plasma total homocysteine (tHcy); however, the effects of longer-term choline supplementation are less clear. We investigated the effect of choline supplementation on plasma concentrations of free choline, betaine and tHcy and B-vitamin status in postmenopausal women, a group more susceptible to low choline status. We also assessed whether supplementation altered plasma lipid profiles. In this randomised, double-blinded, placebo-controlled study, forty-two healthy postmenopausal women received 1 g choline per d (as choline bitartrate), or an identical placebo supplement with their habitual diet. Fasting blood samples were collected at baseline, week 6 and week 12. Administration of choline increased median choline and betaine concentrations in plasma, with significant effects evident after 6 weeks of supplementation (P<0·001) and remaining significant at 12 weeks (P<0·001); no effect was observed on folate status or on plasma lipids. Choline supplementation induced a median (25th, 75th percentile) change in plasma tHcy concentration at week 6 of -0·9 (-1·6, 0·2) μmol, a change which, when compared to that observed in the placebo group 0·6 (-0·4, 1·9) μmol, approached statistical significance (P=0·058). Choline supplementation at a dose of 1 g/d significantly increases the circulating concentration of free choline, and can also significantly increase the concentration of the methyl donor, betaine, thereby potentially enhancing the betaine-homocysteine methyltransferase-mediated remethylation of tHcy.

That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-beta-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 h, results in an approximately 80% decrease in cell cholesterol, a fall in transepithelial electrical resistance, and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins-2, -3, and -7. There were negligible changes in levels of actin and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bound particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-beta-cyclodextrin for 30 min. In contrast caveolin-1 is detected only in the apical medium after adding methyl-beta-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-beta-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane-bound particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D did not diminish methyl-beta-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41- and 48-kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles., (Copyright 2009 Elsevier Inc. All rights reserved.)

Insight-Adherence-Abstinence focused treatment for first episode of schizophrenia and schizoaffective patients is described using examples from clinical practice with 68 patients, 30 of whom have recent or active cannabis misuse. The treatment model is based on the unique characteristics of first-episode patients, who have little insight or experience with the relapses of chronic patients, demonstrate a great deal of denial, and frequently attribute their illness to cannabis. Treatment focuses on building adherence, abstinence, and insight during the first year of treatment in order to prevent repeated relapse and to optimize recovery. Interventions recognize the many needs of cannabis-using first-episode patients and therefore include supportive, cognitive-behavioral, behavioral, and motivational therapies, as well as skill building and psychoeducation.

The function of occludin (Occ) in the tight junction is undefined. To gain insight into its role in epithelial cell biology, occludin levels in Madin-Darby canine kidney II cells were suppressed by stably expressing short interfering RNA. Suppression of occludin was associated with a decrease in claudins-1 and -7 and an increase in claudins-3 and -4. Claudin-2 levels were unaffected. The tight junction "fence" function was not impaired in suppressed Occ (Occ-) clones, as determined by BODIPY-sphingomyelin diffusion in the membrane. The most striking changes were those related to control of the cytoskeleton and the "gate" function of tight junctions. A reduced ability of Occ- clones to extrude apoptotic cells from the monolayers suggested that neighbors of apoptotic cells either failed to sense their presence or were unable to coordinate cytoskeletal activity necessary for their extrusion. To further test the extent to which actin cytoskeletal activity depends on the presence of occludin, Occ- and Occ+ monolayers were depleted of cholesterol. Previous studies showed that cholesterol depletion is associated with reorganization of the actin cytoskeleton and a fall in transepithelial electrical resistance. In contrast to control Occ (Occ+) cells, transepithelial electrical resistance did not fall significantly in cholesterol-depleted Occ- monolayers and they failed to generate Rho-GTP, one of the signaling molecules involved in regulating the actin cytoskeleton. While steady-state transepithelial electrical resistance was similar in all clones, tight junction permeability to mono- and divalent inorganic cations was increased in Occ- monolayers. In addition, there was a disproportionately large increase in permeability to monovalent organic cations, up to 6.96 A in diameter. Chloride permeability was unaffected and there was little change in mannitol flux. The data suggest that occludin transduces external (apoptotic cells) and intramembrane (rapid cholesterol depletion) signals via a Rho signaling pathway that, in turn, elicits reorganization of the actin cytoskeleton. Impaired signaling in the absence of occludin may also alter the dynamic behavior of tight junction strands, as reflected by an increase in permeability to large organic cations; the permeability of ion pores formed of claudins, however, is less affected.

When sampling inhaled antigens, dendritic cells (DC) must penetrate the tight junction (TJ) barrier while maintaining the TJ seal. In matrix metalloproteinase (MMP)-9-deficient mice, in vivo experiments suggest that migration of DC into air spaces is impaired. To examine the underlying mechanisms, we established a well-defined in vitro model using mouse tracheal epithelial cells and mouse bone marrow DC (BMDC). Transmigration was elicited with either macrophage inflammatory protein (MIP)-1alpha or MIP-3beta in a time-dependent manner. Control MMP-9(+/+) BMDC cultured with granulocyte macrophage-colony-stimulating factor for 7 d showed a 30-fold greater transepithelial migration toward MIP-3beta than MIP-1alpha, indicating a more mature DC phenotype. MMP-9(-/-) BMDC as well as MMP-9(+/+) BMDC in the presence of the MMP inhibitor GM6001, although showing a similar preference for MIP-3beta, were markedly impaired in their ability to traverse the epithelium. Expression levels of CCR5 and CCR7, however, were similar in both MMP-9(-/-) and MMP-9(+/+) BMDC. Expression of the integral TJ proteins, occludin and claudin-1, were examined in BMDC before and after transepithelial migration. Interestingly, occludin but not claudin-1 was degraded following transepithelial migration in both MMP-9(-/-) and control BMDC. In addition, there was a > 2-fold increase in claudin-1 expression in MMP-9(-/-) as compared with control BMDC. These observations indicate that occludin and claudin-1 are differentially regulated and suggest that the lack of MMP-9 may affect claudin-1 turnover.

Intratracheal (IT) administration of heat-killed Listeria monocytogenes (HKL) results in an influx of macrophage and dendritic cell (DC) precursors into the lung interstitium. Low-density, FcR+, interstitial lung cells isolated from rats instilled 24 hr before with HKL or vehicle alone, were > 90% Mar1+. After culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 3 days, up to 24% of the loosely adherent cells were DC that stimulated allogeneic T-cell proliferation in an mixed lymphocyte reaction (MLR) assay. After only an overnight incubation with GM-CSF, however, the capacity of interstitial Mar1+ cells to stimulate HKL immune T-cell proliferation without exogenous antigen was low. By contrast, when DC were isolated as major histocompatibility complex (MHC) class II+ cells from rat lungs at 1, 3, 7 and 14 days after HKL instillation and cultured overnight with GM-CSF, their antigen presentation capacity without added exogenous antigen was robust, but declined over the 2-week period. Interestingly, hilar lymph node DC maintained their HKL antigen-presenting capacity for up to 2 weeks after instillation of HKL. Following IT administration of PKH-26 labelled HKL, fluorescent or immunolabelled organisms were detected in OX62+ DC in airway epithelium, lung interstitium and hilar lymph nodes in situ and in MHC class II+ DC isolated from these sites. We conclude that newly immigrated Mar1+ lung DC precursors, while efficient in endocytosing particulate antigens, are incapable of eliciting a significant proliferative response from HKL-sensitized T cells. By contrast, MHC class II+ DC isolated from lungs and incubated overnight with GM-CSF induce vigorous antigen-specific T-cell proliferation. Antigen-loaded lung DC in hilar lymph nodes maintain their antigen presentation capacity for up to 2 weeks.

Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.

Quality control (QC) in laboratories performing molecular histocompatibility class II typing often includes a polymerase chain reaction (PCR) approach for monitoring DNA contamination. An oligonucleotide primer set was designed, (RBQBf/RBQBr), which is specific for nonpolymorphic regions of the DR-B, DQ-B, and DP-B consensus sequences with an expected PCR product size of 81 bp. RBQBf/RBQBr detected genomic DNA from reference cell lines LWAGS and BM21 (50 to 100 picograms) as well as DR-B, DP-B, and DQ-B amplicon (1 copy). Additionally, RBQBf/RBQBr detected SSP products from routine DR-B and DQ-B typings. Validation studies employing controlled DNA contamination of laboratory surfaces revealed that increasing amounts of wipe test samples (5% to 20% v/v) were inhibitory to the wipe test PCR, whereas lower amounts (1% to 2%), or alternatively, a diluted wipe test sample, increased the sensitivity of the test and optimized the results. Collectively, this study describes a primer set, RBQBf/RBQBr, which detects both genomic DNA and DR-B, DQ-B, or DP-B amplicon and furthermore illustrates the necessity of routine testing for potential inhibitory factors that may be introduced into the wipe test PCR.

In the presence of Ca2+, application of trypsin to the basolateral surface of confluent MDCK cell monolayers with formed tight junctions (TJ), induces the formation of basolaterally oriented aberrant TJ strands. Induction of aberrant TJ strands is accompanied by an increase in transepithelial electrical resistance (TER), up to 90%, which upon addition of trypsin inhibitor is maintained for up to 1 h. Thereafter TER returns slowly to baseline values. Under similar conditions, application of trypsin to the apical surface has little or no effect on either TER or the number of aberrant TJ strands. Confocal microscopy of monolayers, immunostained for ZO-1, revealed that this TJ associated cytoplasmic protein, extended below the TJ along the basolateral surface following brief exposure to trypsin. Removing Ca2+ after treatment of the monolayer with basolaterally applied trypsin resulted, after 20 min, in the increased partitioning of TJ particles onto the E fracture face, of both normal and aberrant TJ strands. Like the TJ strands themselves, therefore, aberrant strands may be linked to cytoskeletal elements. Aberrant TJ strands do not form when monolayers, maintained in low Ca2+ medium, are exposed to trypsin, suggesting that under these conditions TJ precursors, and/or trypsin-sensitive proteins regulating TJ strand assembly, are sequestered in a vesicular compartment that is inaccessible to exogenous trypsin. Prolonged exposure of the apical surface of an established, polarized epithelium with intact TJ to trypsin, had little effect on TJ integrity and did not induce aberrant strands.

Using mAb and magnetic and fluorescence-activated cell sorting, together with culturing in semisolid culture medium, we show that essentially all splenic macrophage progenitors are Ly-6ChiMac-1-. By contrast, bone marrow macrophage progenitors were more heterogeneous in Ly-6C expression, the majority being restricted to the Ly-6CdimMac-1- and the Ly-6C-Mac-1- subsets. These findings provide immunophenotype evidence that macrophages in the spleen and bone marrow arise from different progenitors.

Approximately one-third of mouse splenic macrophage (M theta) progenitors yield progeny that constitutively present MHC class I alloantigen to naive T cells, a response that is restricted to CD8+ T cells and is elicited in a CD4+ Th cell-independent manner. In addition, both the alloantigen-presenting (alloAP+) and nonpresenting (alloAP-) M theta subsets constitutively express similar levels of MHC class I molecules, and their alloAP phenotypes are unaffected by IFN-gamma, which enhances the expression of both class I and II MHC molecules. We therefore postulated the restricted expression of costimulator molecules to account for the alloAP+ phenotype. Using cytokine-specific antibodies, recombinant mouse cytokines, and polymerase chain reaction analyses (to detect specific cytokine mRNA transcripts), we identified the putative costimulators as IL-1 alpha, IL-6, and TNF-alpha. TNF-alpha transcripts were present in both the alloAP+ and alloAP- M theta subsets, but IL-1 alpha and IL-6 were not constitutively expressed by the alloAP+ subset of M theta; rather, they were induced by IFN-gamma, which was released from naive CD8+ T cells only during coculture with alloAP+ M theta. Although IFN-gamma induced IL-6 gene transcription in both alloAP+ and alloAP- M theta subsets, it induced IL-1 alpha transcripts only in the alloAP+ subset. Finally, CD8+ T cells exposed to alloAP- M theta were unresponsive when subsequently cultured with alloAP+ M theta. We conclude that the ability of some M theta to elicit IFN-gamma from CD8+ T cells and to respond to this cytokine by producing IL-1 alpha defines the alloAP phenotype of the cell population, and that alloAP- M theta induce a state of alloantigen-specific tolerance in naive CD8+ T cells.

Anti-myosin antibodies are found in acute rheumatic fever (ARF), a sequela of group A streptococcal infection. An antiidiotypic serum was produced that was specific for idiotopes expressed by anti-myosin antibodies in ARF (anti-My1). Studies indicated that idiotypic determinants detected with this serum were present in anti-myosin antibodies and absent from normal human immunoglobulins that lacked specificity for myosin. Anti-My1 was tested against sera from patients with other types of autoimmune diseases as well as uncomplicated streptococcal infections. Sera from systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), and poststreptococcal acute glomerulonephritis patients demonstrated idiotypic reactivity with anti-My1. Affinity-purified anti-myosin antibodies from SLE, SS, and ARF sera also reacted strongly with anti-My1, indicating that immunoglobulins produced in these diseases share idiotypic determinants. The data demonstrated an association of the My1 idiotype with poststreptococcal sequelae and the two autoimmune diseases SLE and SS.

To clarify the origin and function of the microglia residing in the central nervous system, we cloned brain cells from newborn and adult mice in soft agar containing the macrophage-specific growth factor, colony-stimulating factor-1 and expanded the cells from individual colonies in liquid culture medium. The results of molecular, immunophenotypic and functional analyses showed that the clones consisted of microglia derived from the macrophage family of cells. For instance, the microglia contain mRNA transcripts for the receptor for colony-stimulating factor-1 and truncated CD4 transcripts similar to those found in mouse macrophages but not T helper cells. About a third of the microglial progenitors gave rise to progeny that constitutively induced the selective proliferation of naive allogeneic CD8+ T cells in a CD4+ T cell-independent manner, a response that was inhibited by monoclonal antibodies to major histocompatibility complex (MHC) class I molecules on the microglia. Since all microglia expressed similar levels of MHC class I molecules, the basis for the alloantigen presentation likely resides in the ability of some clones of microglia to synthesize co-stimulator molecules that are required for CD8+ T cell proliferation. Thus, at least some microglia in mouse brain arise from endogenous progenitors and appear capable of specialized functions.

A panel of seven mouse splenic macrophage cell lines, derived from cloned progenitors, was compared for their ability to present antigen to Th1 or Th2 helper T cell lines and hybridomas, as well as to naive T cells, and to provide accessory cell function for the synthesis of antibody from primed B cells. One of the cell lines expressed MHC class II molecules and was the only line with constitutive antigen-presenting activity for Th1 cells. It may represent a subset of splenic macrophages responsible for the activation of naive Th1 helper cells in situ. The remaining six cell lines responded to INF-gamma by up-regulating their class II expression and acquiring Th1 antigen-presenting activity. They may represent cells which, in situ, lack constitutive antigen-presenting activity but are promoted to presenting status by Th1-derived INF-gamma. Five of the cell lines provided accessory cell function to Th2 cells, as indicated by antibody synthesis in suspensions of spleen cells from primed mice depleted of their antigen-presenting cells. One of the cell lines lacking accessory cell activity had constitutive antigen-presenting activity for Th1 cells. This reciprocal expression of antigen-presenting activity supports the idea that Th1 and Th2 helper cells are activated by different antigen-presenting cells. Finally, the cell lines differed in their ability to constitutively induce an allogeneic response; a response that was limited to CD8+ T cells occurred in a CD4+ helper cell-independent manner and was unaffected by the addition of INF-gamma. The alloantigen-presenting macrophage cell lines also possessed the most efficient accessory cell activity for antibody synthesis. These cell lines, which represent a spectrum of antigen-presenting activities in the spleen afford models for defining the roles of macrophages in the induction of immune responses and for resolving issues concerning their development.

About a third of mouse splenic macrophage (M phi) progenitors give rise to cloned progeny that constitutively induce the selective proliferation of naive allogeneic CD8+ T cells in a CD4+ helper cell-independent manner--a response that is inhibited by mAb to the MHC class I molecules present on the M phi. Colony-mixing experiments indicated that the failure of most M phi clones to present allo-Ag was not due to their suppression of the ability of CD8+ cells to respond, nor did the nonpresenting clones interfere with the activity of the allo-Ag presenting M phi. The allo-Ag presenting phenotypes were found to be a stable characteristic in a panel of cell lines derived from individual clones of M phi. Analysis of the cell lines revealed that the differential expression of allo-APC activity could not be attributed to the levels of MHC class I molecules; rather, the cell lines and the primary M phi clones differ in their expression of a cell-associated costimulator molecule that likely functions to induce the expression of the IL-2R on and the secretion of IL-2 from the T cells.

We describe a nonviral transformation strategy for the establishment of permanent cell lines derived from the progeny of individual mouse splenic macrophage (M phi) progenitors. These colony stimulating factor-1 (CSF-1)-dependent cell lines possess many features of mature M phi s, including antibody-dependent phagocytic and cellular cytotoxic activities, ability to secrete lysozyme, and expression of the Mac-1 antigen and mRNA for the CSF-1 receptor. It was also possible to immortalize selected clones of splenic M phi s differing in their constitutive antigen-presenting activities with the retention of the antigen-presenting phenotype in the resultant cell lines. The approach described in this report should be useful in obtaining additional cell lines of M phi s expressing other phenotypes of interest.

The structure of intercellular tight junctions of rat airway submucosal glands was examined by freeze fracture techniques and their permeability assessed by the use of colloidal lanthanum. The submucosal glands were organized into three distinct regions: a) serous tubules and b) mucous tubules lined, respectively, by serous and mucous cells, and c) ducts lined by cuboidal epithelial cells, containing few secretory granules, and some ciliated cells. The mean number of parallel fibrils constituting the tight junctions between serous cells was 3.6 +/- 0.4, which was significantly smaller than those between any of the other cell types. Colloidal lanthanum permeated the tight junctions between serous cells up to the level of the acinar lumen. There was a progressive increase in the mean number of parallel fibrils of tight junctions between mucous (5.1 +/- 0.6), ductal (5.4 +/- 0.5), and ciliated cells (8.5 +/- 0.7); none of these junctions was permeated by colloidal lanthanum. These results imply that tight junctions between serous cells are more permeable to small water-soluble solutes than those present in the more proximal portions of the gland. Gap junctions were observed between serous cells and between mucous cells, suggesting that these secretory cells may be electotronically and metabolically coupled.

We studied the effect of highly purified recombinant interleukin 2 from Escherichia coli (rIL-2) on antibody production by our human hybridomas. Increasing concentrations of rIL-2 were directly related to increased production of immunoglobulin, which reached peak levels of 13-25 micrograms/ml, a 10- to 20-fold increase over untreated hybridomas. We were unable to demonstrate large quantities of the IL-2 receptor on the hybridomas as measured by an anti-TAC monoclonal antibody. The data suggest that the antibody enhancement phenomenon is mediated by IL-2 and that is a new, effective way to achieve higher levels of immunoglobulin from human hybridomas.

Soluble extracts of Cryptococcus neoformans were examined for their ability to induce a macrophage-regulatory T-suppressor cell known to appear in the spleens of mice infected with cryptococci. Suppressor cells were induced by injection of extracts of encapsulated or thinly encapsulated strains of cryptococci. Dose-response analysis showed that as little as 25 micrograms of soluble capsular polysaccharide antigen could induce significant suppressor cell activity, with maximum suppression occurring at a dose of 100 micrograms. The suppressor cells appeared within 1 week of injection of antigen and persisted for at least 2 months. Suppressor cells were induced in animals given tolerogenic doses of levan, human gamma globulin, and soluble capsular polysaccharide antigen. When these same antigens were administered in immunogenic form, no suppressor cell activity was detected. Therefore, the suppressive mechanism was common to models of immunologic tolerance and was not unique to cryptococcal disease or cryptococcal capsular polysaccharide antigen. The phagocytosis-inhibiting lymphokine produced by the suppressor cell population completely inhibited the phagocytic activity of only a portion of peritoneal exudate cells. Other macrophages in the population were not totally inhibited but exhibited a reduction in the number of yeast cells engulfed.

Molecular mimicry or epitope similarity between group A streptococcal M proteins and myosin may contribute to the presence of heart reactive antibodies in acute rheumatic fever. In our study overlapping synthetic peptides copying the entire sequence of PepM5 protein were used to map the myosin cross-reactive epitopes of streptococcal M protein recognized by mouse and human mAb and affinity purified myosin-specific antibodies from acute rheumatic fever and rheumatic heart disease sera. Overlapping M protein peptides SM5(164-197)C and SM5(184-197)C inhibited the murine mAb reactions with PepM5 protein. The human mAb and affinity purified myosin-specific antibodies reacted exclusively with SM5(184-197)C. However, one of the five different purified myosin-specific antibodies not only reacted with SM5(184-197)C but also reacted with SM5(84-116)C. The synthetic subpeptides SM5(175-184)C and SM5(188-197C) did not react with any of the antibodies to PepM5 and myosin demonstrating a requirement of the 184-188 amino acid sequence for antibody recognition. A heptapeptide containing the sequence SM5(183-189) was also found to inhibit selected human myosin-specific antibodies and a human antimyosin mAb. Therefore, the majority of mouse and human myosin crossreactive antibodies recognized an epitope within the 14 residue carboxy terminus of PepM5 which appeared to involve the GLN-LYS-SER-LYS-GLN sequence.

Human mAb were produced from tonsillar or PBL of normal individuals or patients infected with group A streptococci. Lymphocytes were purified on Ficoll-Hypaque gradients and stimulated in vitro with purified group A streptococcal membranes or M protein extracts. The mAb were selected for study based on their reaction with group A streptococci, pep M5 protein, and/or M6 Escherichia coli protein. Further analysis by Western immunoblot or competitive inhibition ELISA revealed that there were two types of antibodies: one type that reacted with myosin and DNA and the other type that reacted with myosin, keratin, and/or actin. The specificities of these human mAb are similar to specificities observed in our previous studies of murine mAb reactive with group A streptococci and heart Ag. For comparison, anti-myosin antibodies were affinity purified from the sera of infected or acute rheumatic fever patients and were shown to react with myosin and DNA as well as with group A streptococci and M protein. To affinity purify these antibodies from normal sera, five times the amount of sera was required to obtain detectable quantities. These data suggest that the human mAb reactive with group A streptococci and myosin reflect the antibodies seen in sera from infected patients or acute rheumatics and that the B lymphocyte clones capable of producing these cross-reactive antibodies are also present in normal individuals.