Responsiveness and meaningful thresholds of PROMIS pain interference, fatigue, and physical function forms in adults with idiopathic inflammatory myopathies: Report from the OMERACT Myositis Working Group.

BACKGROUND: A series of qualitative studies conducted by the OMERACT Myositis Working Group identified pain interference, fatigue, and physical function as highly important life impact domains for adults with idiopathic inflammatory myositis (IIM). In this study, our goal was to assess the responsiveness and minimal important difference of PROMIS pain interference (6a), fatigue (7a), and physical function (8b). METHODS: Adults with IIM from USA, Netherlands, Korea, Sweden, and Australia with two "clinical" visits were enrolled in this prospective study. Anchor questions on a Likert scale were collected at baseline, and manual muscle testing (MMT), physician and patient reported global disease activity, and PROMIS instruments were collected at both visits. Responsiveness was assessed with i) ANOVA, ii) paired t-test, effect size and standardized response mean, and iii) Pearson correlation. Minimal important difference (MID), minimal important change (MIC) and minimal detectable change (MDC) values were calculated. RESULTS: 114 patients with IIM (median age 60, 60 % female) completed both visits. Changes in PROMIS instruments were significantly different among anchor categories. Patients who reported improvement had a significant improvement in their PROMIS scores with at least medium effect size, while patients who reported worsening and stability did not show a significant change with weak effect size. PROMIS instruments had weak to moderate correlations with MMT, patient and physician global disease activity. MID was approximately 2-3 points for Pain Interference and 3-4 points for Fatigue and Physical Function forms based on the method used. MIC was approximately 4-5 for improvement of all the instruments, while MDC was 1.7-2 points for Pain Interference and Physical Function and 3.2-3.9 for Fatigue. CONCLUSION: This study provides evidence towards the responsiveness of the PROMIS instruments in a large international prospective cohort of adults with IIM supporting their use as PROMs in adult myositis.

Detecting host responses to microbial stimulation using primary epithelial organoids.

The intestinal epithelium is constantly exposed to microbes residing in the lumen. Traditionally, the response to microbial interactions has been studied in cell lines derived from cancerous tissues, e.g. Caco-2. It is, however, unclear how the responses in these cancer cell lines reflect the responses of a normal epithelium and whether there might be microbial strain-specific effects. To address these questions, we derived organoids from the small intestine from a cohort of healthy individuals. Culturing intestinal epithelium on a flat laminin matrix induced their differentiation, facilitating analysis of microbial responses via the apical membrane normally exposed to the luminal content. Here, it was evident that the healthy epithelium across multiple individuals (n = 9) demonstrates robust acute both common and strain-specific responses to a range of probiotic bacterial strains (BB-12Ⓡ, LGGⓇ, DSM33361, and Bif195). Importantly, parallel experiments using the Caco-2 cell line provide no acute response. Collectively, we demonstrate that primary epithelial cells maintained as organoids represent a valuable resource for assessing interactions between the epithelium and luminal microbes across individuals, and that these models are likely to contribute to a better understanding of host microbe interactions.

An organoid-based CRISPR-Cas9 screen for regulators of intestinal epithelial maturation and cell fate.

Generation of functionally mature organs requires exquisite control of transcriptional programs governing cell state transitions during development. Despite advances in understanding the behavior of adult intestinal stem cells and their progeny, the transcriptional regulators that control the emergence of the mature intestinal phenotype remain largely unknown. Using mouse fetal and adult small intestinal organoids, we uncover transcriptional differences between the fetal and adult state and identify rare adult-like cells present in fetal organoids. This suggests that fetal organoids have an inherent potential to mature, which is locked by a regulatory program. By implementing a CRISPR-Cas9 screen targeting transcriptional regulators expressed in fetal organoids, we establish Smarca4 and Smarcc1 as important factors safeguarding the immature progenitor state. Our approach demonstrates the utility of organoid models in the identification of factors regulating cell fate and state transitions during tissue maturation and reveals that SMARCA4 and SMARCC1 prevent precocious differentiation during intestinal development.

Transcriptional and epigenomic profiling identifies YAP signaling as a key regulator of intestinal epithelium maturation.

During intestinal organogenesis, equipotent epithelial progenitors mature into phenotypically distinct stem cells that are responsible for lifelong maintenance of the tissue. While the morphological changes associated with the transition are well characterized, the molecular mechanisms underpinning the maturation process are not fully understood. Here, we leverage intestinal organoid cultures to profile transcriptional, chromatin accessibility, DNA methylation, and three-dimensional (3D) chromatin conformation landscapes in fetal and adult epithelial cells. We observed prominent differences in gene expression and enhancer activity, which are accompanied by local changes in 3D organization, DNA accessibility, and methylation between the two cellular states. Using integrative analyses, we identified sustained Yes-Associated Protein (YAP) transcriptional activity as a major gatekeeper of the immature fetal state. We found the YAP-associated transcriptional network to be regulated at various levels of chromatin organization and likely to be coordinated by changes in extracellular matrix composition. Together, our work highlights the value of unbiased profiling of regulatory landscapes for the identification of key mechanisms underlying tissue maturation.

Positive Effect of Parathyroidectomy Compared to Observation on BMD in a Randomized Controlled Trial of Mild Primary Hyperparathyroidism.

Mild or asymptomatic disease is now the dominating presentation of primary hyperparathyroidism (PHPT). However, bone involvement with decreased bone mineral density (BMD) and an increased risk of fractures has been demonstrated. Indications for parathyroidectomy (PTX) in mild PHPT have been debated for years. There is a need of long-term randomized studies comparing PTX with observation without intervention (OBS). Here, we present bone health data from the Scandinavian Investigation of Primary Hyperparathyroidism (SIPH), a randomized controlled trial, comparing PTX to OBS. This study included 191 patients (96 OBS/95 PTX), and 129 patients (64 OBS/65 PTX) were followed for 10 years to the end of study (EOS). BMD was measured with dual-energy X-ray absorptiometry (DXA), peripheral fractures were noted, and spine radiographs were obtained for vertebral fracture assessment. There was a significant treatment effect of PTX on BMD compared with OBS for all analyzed compartments, most explicit for the lumbar spine (LS) and femoral neck (FN) (p < 0.001). The mean changes in T-score from baseline to 10 years were from 0.41 for radius 33% (Rad33) to 0.58 for LS greater in the PTX group than in the OBS group. There was a significant decrease in BMD for all compartments in the OBS group, most pronounced for FN, Rad33, and ultradistal radius (UDR) (p < 0.001). Even though there was a significant treatment effect of PTX compared with OBS, there was only a significant increase in BMD over time for LS (p < 0.001). We found no difference between groups in fracture frequency in the 10-year cohort, neither with modified intention-to-treat (mITT) analysis nor per protocol analysis. Because BMD is only a surrogate endpoint of bone health and PTX did not reduce fracture risk, observation could be considered a safe option for many patients with mild PHPT regarding bone health in a 10-year perspective. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Psychometric properties of the Marschak Interaction Method of Psychometrics and the Assessment of Parent-Child Interaction within residential care and non-referred settings.

Introduction: Assessment and identification of children with developmental needs and their interaction with primary caregivers are critical for emotional and social development. However, to the best of our knowledge, there is a scarcity of valid observation-based tools that guide the work with family communication, which is essential for the child's healthy development. Method: The Marschak Interaction Method of Psychometrics (MIM-P) and Assessment of Parent-Child Interaction (APCI) are both interaction and observation-based assessment tool, and they were explored for their validity and reliability in assessing caregiver-child interaction. The study included 30 trained and certified professionals who recruited referred and non-referred caregiver-child dyads over 11 months. Assessment data was collected from 139 caregiver-child dyads for the MIM-P with 278 individuals (100 referred and 178 non-referred) and 129 caregiver-child dyads for the APCI with 257 individuals (95 referred and 162 non-referred). Results: The psychometric analyses show that both the MIM-P and APCI presents relevant sources of reliability and validity for assessing caregiver-child interaction including interrater reliability, internal consistency, test re-test reliability as well as concurrent and construct validity. Discussion and conclusion: The study highlights the need for observation-based assessment tools within social work and contributes to the understanding of the importance of relationships and interaction in children's emotional and social development. However, further research is needed to explore norms and further strengthen implementation and quality of the tools.

Music, families and interaction (MUFASA): a protocol article for an RCT study.

BACKGROUND: General mental health and interpersonal skills of families are crucial to children's development and future. Research suggests a link between participation in music activities and individuals' own sense of mental health, as well as an effect in objective measures of health such as quality of life, social skills, and rehabilitation of various kinds. However, in Denmark there are not many services for families with school-aged children (7-12 years) that focus on prevention and strengthening family interaction hereby minimising the risk of children not thriving and developing optimally in terms of social and emotional skills and competences. The purpose of this study is to investigate the effect of shared music activities on family interaction, parental stress, and child well-being. METHODS: The study is a controlled effect study where 40 family dyads from Aalborg and Aarhus are randomized into 4 different groups including music therapy activities, community music, family concerts and a control group. Measurements of family interaction (Assessment of Parent Child Interaction, APCI) and mental health (Parental Stress Scale, PSS, and the Strengths and Difficulties Questionnaire, SDQ) will be made at baseline/pre intervention, at post intervention and a follow-up measurement 12 month after baseline (month 1, 3 and 12) 3 times in total. Furthermore, data about the family dyad's regular participation in music events as part of every-day life at the same measure points (month 1, 3 and 12). Since this is a planned prospective study, results are not yet available, but clinical experience from a feasibility study in 2021 were promising and expected challenges and changes are discussed in the article. DISCUSSION: Findings of this study will be relevant for all fields where music is applied for families, such as education, mental health, social work and for basic research on the study of music and interaction. Music used as a resource for families is crucial in understanding how different modalities can influence the family interaction including emotional communication and attachment. TRIAL REGISTRATION: ISRCTN, ISRCTN17290015, Registered 03 March 2022, https://www.isrctn.com/ISRCTN17290015 DATE AND PROTOCOL VERSION: July 2022, version 1. Protocol is planned to be updated after finalized recruitment during second data collection point and again after the third and last data collection point (see Additional file 1: SPIRIT Checklist).

Maternal thyroid disease in pregnancy and timing of pubertal development in sons and daughters.

OBJECTIVE: To study whether maternal thyroid disease in pregnancy is associated with pubertal timing in sons and daughters. DESIGN: Cohort study. SETTING: National birth cohort and health registers. PATIENT(S): A total of 15,763 mothers and children from the Danish National Birth Cohort and its Puberty Cohort. INTERVENTION(S): Register-based and self-reported information on maternal thyroid diseases during pregnancy (hyperthyroidism, hypothyroidism, benign goiter, or no thyroid disease [reference group]). MAIN OUTCOME MEASURE(S): The adjusted mean age difference (months) at attaining several self-reported pubertal milestones collected every 6 months using an interval-censored regression and the average difference in age at attaining all pubertal milestones using the Huber-White robust variance estimation (primary outcome). RESULT(S): Sons of mothers with hyperthyroidism had earlier pubertal development (average difference, -2.9 [95% confidence interval (CI), -5.0 to -0.7] months) than unexposed sons. Maternal hypothyroidism was not associated with pubertal development in sons (average difference, -1.2 [95% CI, -5.1 to 2.7] months). We observed nonstatistically significant indications of earlier pubertal development in sons of mothers with benign goiter (average difference, -1.9 [95% CI, -4.6 to 0.9] months). Maternal thyroid disease was not associated with pubertal development in daughters (average difference (months), hyperthyroidism, -0.8 [95% CI, -2.8 to 1.2]; hypothyroidism, 0.3 [95% CI, -3.1 to 3.8]; and benign goiter, 0.7 [95% CI, -2.0 to 3.4]). CONCLUSION(S): We found indications of earlier pubertal development in sons of mothers with hyperthyroidism. More research is needed to further investigate the observed sex-specific association.

Mortality and Morbidity in Mild Primary Hyperparathyroidism: Results From a 10-Year Prospective Randomized Controlled Trial of Parathyroidectomy Versus Observation.

BACKGROUND: Primary hyperparathyroidism (PHPT) is a common endocrine disorder associated with increased risk for fractures, cardiovascular disease, kidney disease, and cancer and increased mortality. In mild PHPT with modest hypercalcemia and without known morbidities, parathyroidectomy (PTX) is debated because no long-term randomized trials have been performed. OBJECTIVE: To examine the effect of PTX on mild PHPT with regard to mortality (primary end point) and key morbidities (secondary end point). DESIGN: Prospective randomized controlled trial. (ClinicalTrials.gov: NCT00522028). SETTING: Eight Scandinavian referral centers. PATIENTS: From 1998 to 2005, 191 patients with mild PHPT were included. INTERVENTION: Ninety-five patients were randomly assigned to PTX, and 96 were assigned to observation without intervention (OBS). MEASUREMENTS: Date and causes of death were obtained from the Swedish and Norwegian Cause of Death Registries 10 years after randomization and after an extended observation period lasting until 2018. Morbidity events were prospectively registered annually. RESULTS: After 10 years, 15 patients had died (8 in the PTX group and 7 in the OBS group). Within the extended observation period, 44 deaths occurred, which were evenly distributed between groups (24 in the PTX group and 20 in the OBS group). A total of 101 morbidity events (cardiovascular events, cerebrovascular events, cancer, peripheral fractures, and renal stones) were also similarly distributed between groups (52 in the PTX group and 49 in the OBS group). During the study, a total of 16 vertebral fractures occurred in 14 patients (7 in each group). LIMITATION: During the study period, 23 patients in the PTX group and 27 in the OBS group withdrew. CONCLUSION: Parathyroidectomy does not appear to reduce morbidity or mortality in mild PHPT. Thus, no evidence of adverse effects of observation was seen for at least a decade with respect to mortality, fractures, cancer, cardiovascular and cerebrovascular events, or renal morbidities. PRIMARY FUNDING SOURCE: Swedish government, Norwegian Research Council, and South-Eastern Norway Regional Health Authority.

Raised mortality in old adults with a history of hyperthyroidism following iodine fortification.

OBJECTIVE: A transient rise in the occurrence of hyperthyroidism ensued the introduction of iodine fortification (IF) of salt in Denmark. Older adults are at risk of complications to hyperthyroidism that could prove fatal to vulnerable individuals. We evaluated the association between thyroid function and mortality in older adults before and after nationwide implementation of IF. DESIGN: Retrospective cohort study. PATIENTS: All 68-year-olds from the general population in the city of Randers were invited to participate in a clinical study in 1988 and followed until death, emigration or end of study (31 December 2017) using Danish registries. MEASUREMENTS: Baseline measures comprised of a questionnaire, physical examination and blood and urine samples. Kaplan-Meier survival curves and Cox regression were used to determine the association between thyroid function and death before and after IF. Time-stratification of results before and after IF was employed due to violation of proportional hazards assumptions in Cox regression. RESULTS: Median urinary iodine concentration was 42 µg/L at baseline consistent with moderate iodine deficiency. Hyperthyroidism (thyrotropin < 0.4 mIU/L) occurred in 37 (9.1%) participants. Kaplan-Meier survival curves showed an increase in mortality among participants with hyperthyroidism after IF. There was no significant association between hyperthyroidism and mortality before IF compared to euthyroid participants, but after IF hyperthyroid subjects had an increased mortality (adjusted hazard ratio: 2.22, 95% confidence interval: 1.44-3.44). CONCLUSIONS: IF was associated with raised mortality among older adults with a history of hyperthyroidism and moderate iodine deficiency. Our results highlight the need for cautious iodine supplementation and for monitoring of IF.

Long-term iodine nutrition is associated with longevity in older adults: a 20 years' follow-up of the Randers-Skagen study.

Iodine intake affects the occurrence of thyroid disorders. However, the association of iodine intake with longevity remains to be described. This led us to perform a 20 years' follow-up on participants from the Randers-Skagen (RaSk) study. Residents in Randers born in 1920 (n 210) and Skagen born in 1918-1923 (n 218) were included in a clinical study in 1997-1998. Mean iodine content in drinking water was 2 µg/l in Randers and 139 µg/l in Skagen. We collected baseline data through questionnaires, performed physical examinations and measured iodine concentrations in spot urine samples. Income data were retrieved from Danish registries. We performed follow-up on mortality until 31 December 2017 using Danish registries. Complete follow-up data were available on 428 out of 430 of participants (99·5 %). At baseline, the median urinary iodine concentration was 55 µg/l in Randers and 160 µg/l in Skagen residents. Participants were long-term residents with 72·8 and 92·7 % residing for more than 25 years in Randers and Skagen, respectively. Cox regression showed that living in Skagen compared with Randers was associated with a lower hazard ratio (HR) of death in both age- and sex-adjusted analyses (HR 0·60, 95 % CI 0·41, 0·87, P = 0·006), but also after adjustment for age, sex, number of drugs, Charlson co-morbidity index, smoking, alcohol and income (HR 0·60, 95 % CI 0·41, 0·87, P = 0·008). Residing in iodine-replete Skagen was associated with increased longevity. This indicates that long-term residency in an iodine-replete environment may be associated with increased longevity compared with residency in an iodine-deficient environment.

Effects of Parathyroidectomy on Quality of Life: 10 Years of Data From a Prospective Randomized Controlled Trial on Primary Hyperparathyroidism (the SIPH-Study).

Primary hyperparathyroidism (PHPT) was previously considered a disease presenting with multiorgan involvement and a wide range of symptoms. Today, the disease presents with no symptoms or mild symptomatology in most patients. Data regarding nonspecific symptoms such as pain, fatigue, memory loss, depression, and other neuropsychiatric signs have been ambiguous, and results from prospective long-term randomized control trials are lacking. The Scandinavian Investigation on Primary Hyperparathyroidism (SIPH) is a prospective randomized controlled trial (RCT) with 10-year follow up, comparing parathyroidectomy (PTX) to observation without any treatment (OBS). From 1998 to 2005, 191 patients with mild PHPT were included from Sweden, Norway, and Denmark. A total of 95 patients were randomized to PTX and 96 to OBS. The generic Short Form-36 survey (SF-36) and the Comprehensive Psychopathological Rating Scale (CPRS) were studied at baseline, 2, 5, and 10 years after randomization. After 10 years, the PTX group scored significantly better on vitality (PTX 65.1 ± 20.2 versus OBS 57.4 ± 22.7; p = .017) compared to the OBS group in SF-36. We found no differences between the groups in the physical subscales. The OBS group had no significant change in any of the SF-36 scores throughout the study. The CPRS showed an improvement of symptoms in both groups for single items and sum scores after 10 years compared to baseline. There were, however, no significant differences between the two groups in the CPRS data. The results of this large and long-term RCT indicate improvement in some of the mental domains of SF-36 following PTX. However, the treatment effects between the groups were subtle with uncertain clinical significance. The observation group had stable SF-36 values and improvement in CPRS symptom-scores. Thus, in considering only quality of life (QoL) and in the absence of declines in renal and skeletal parameters, it may be safe to observe patients with mild PHPT for a decade. © 2020 American Society for Bone and Mineral Research (ASBMR).

DNA-dependent protein kinase regulates lysosomal AMP-dependent protein kinase activation and autophagy.

Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNA-dependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMP-dependent protein kinase (AMPK) complex and ULK1 kinase. In normal culture conditions, PRKDC interacted with the AMPK complex and phosphorylated its nucleotide-sensing γ1 subunit PRKAG1/AMPKγ1 at Ser192 and Thr284, both events being significantly reduced upon the activation of the AMPK complex. Alanine substitutions of PRKDC phosphorylation sites in PRKAG1 reduced AMPK complex activation without affecting its nucleotide sensing capacity. Instead, the disturbance of PRKDC-mediated phosphorylation of PRKAG1 inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with STK11 (serine/threonine kinase 11). Taken together, our data suggest that PRKDC-mediated phosphorylation of PRKAG1 primes AMPK complex to the lysosomal activation by STK11 in cancer cells thereby linking DNA damage response to autophagy and cellular metabolism. Abbreviations: AXIN1: axin 1; 3-MA: 3-methyladenine; 5-FU: 5-fluorouracil; AA mutant: double alanine mutant (S192A, T284A) of PRKAG1; ACACA: acetyl-CoA carboxylase alpha; AICAR: 5-Aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ATM: ataxia telangiectasia mutated; ATR: ATM serine/threonine kinase; AV: autophagic vacuole; AVd: degradative autophagic vacuole; AVi: initial autophagic vacuole; BECN1: beclin 1; BSA: bovine serum albumin; CBS: cystathionine beta-synthase; CDK7: cyclin dependent kinase 7; CDKN1A: cyclin dependent kinase inhibitor 1A; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S transferase; H2AX/H2AFX: H2A.X variant histone; HBSS: Hanks balanced salt solution; IP: immunopurification; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K9: mitogen-activated protein kinase kinase kinase 9; mRFP: monomeric red fluorescent protein; mCh: mCherry; MCM7: minichromosome maintenance complex component 7; MTORC1: mechanistic target of rapamycin kinase complex 1; NHEJ: non-homologous end joining; NRBP2: nuclear receptor binding protein 2; NTC: non-targeting control; NUAK1: NUAK family kinase 1; PBS: phosphate-buffered saline; PIK3AP1: phosphoinositide-3-kinase adaptor protein 1; PIK3CA: phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha; PIKK: phosphatidylinositol 3-kinase-related kinase; PRKAA: protein kinase AMP-activated catalytic subunit alpha; PRKAB: protein kinase AMP-activated non-catalytic subunit beta; PRKAG: protein kinase AMP-activated non-catalytic subunit gamma; PRKDC: protein kinase, DNA-activated, catalytic subunit; RLuc: Renilla luciferase; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TP53: tumor protein p53; TSKS: testis specific serine kinase substrate; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.

Long-term treatment with human immunoglobulin for antisynthetase syndrome-associated interstitial lung disease.

BACKGROUND: Interstitial lung disease-associated antisynthetase syndrome (AS-ILD) carries significant morbidity and mortality. Corticosteroids and immunosuppressive drugs are the mainstay of treatment. Human immunoglobulin (IVIg), an immunomodulator without immunosuppressive properties, is effective in myositis but the evidence supporting its use in ILD is scarce. OBJECTIVE: To describe clinical outcomes of AS-ILD patients receiving IVIg. METHODS: Retrospective analysis of AS-ILD patients. Linear mixed models using restricted maximum likelihood estimation was used to estimate the change in lung function and corticosteroid dose over time. RESULTS: Data from 17 patients was analyzed. Median follow-up was 24.6 months. Fourteen patients had refractory disease. The mean percent-predicted forced vital capacity (FVC%) (p = 0.048) and percent-predicted diffusing capacity of the lung for carbon monoxide (DLCO%) (p = 0.0223) increased over time, while the mean prednisone dose (p < 0.001) decreased over time. Seven patients achieved a >10% increase in FVC%, including two who used IVIg as initial treatment. Five patients showed a >10% increase in DLCO% and TLC%. Nine (53%) patients experienced side effects. CONCLUSIONS: IVIg may be a useful complementary therapy in active progressive AS-ILD but is associated with potential side effects. Fssssurther investigation is required to determine the value of IVIg as an initial treatment in AS-ILD.

Curvature- and Phase-Induced Protein Sorting Quantified in Transfected Cell-Derived Giant Vesicles.

Eukaryotic cells possess a dynamic network of membranes that vary in lipid composition. To perform numerous biological functions, cells modulate their shape and the lateral organization of proteins associated with membranes. The modulation is generally facilitated by physical cues that recruit proteins to specific regions of the membrane. Analyzing these cues is difficult due to the complexity of the membrane conformations that exist in cells. Here, we examine how different types of membrane proteins respond to changes in curvature and to lipid phases found in the plasma membrane. By using giant plasma membrane vesicles derived from transfected cells, the proteins were positioned in the correct orientation and the analysis was performed in plasma membranes with a biological composition. Nanoscale membrane curvatures were generated by extracting nanotubes from these vesicles with an optical trap. The viral membrane protein neuraminidase was not sensitive to curvature, but it did exhibit strong partitioning (coefficient of K = 0.16) disordered membrane regions. In contrast, the membrane repair protein annexin 5 showed a preference for nanotubes with a density up to 10-15 times higher than that on the more flat vesicle membrane. The investigation of nanoscale effects in isolated plasma membranes provides a quantitative platform for studying peripheral and integral membrane proteins in their natural environment.

ICAM1 expression is induced by proinflammatory cytokines and associated with TLS formation in aggressive breast cancer subtypes.

Intratumoral formation of tertiary lymphoid structures (TLS) within the tumor microenvironment is considered to be a consequence of antigen challenge during anti-tumor responses. Intracellular adhesion molecule 1 (ICAM1) has been implicated in a variety of immune and inflammatory responses, in addition to associate with triple negative breast cancer (TNBC). In this study, we detected TLS in the aggressive tumor phenotypes TNBC, HER2+ and luminal B, whereas the TLS negative group contained solely tumors of the luminal A subtype. We show that ICAM1 is exclusively expressed in TNBC and HER2 enriched subtypes known to be associated with inflammation and the formation of TLS. Furthermore, cell from normal mammary epithelium and breast cancer cell lines expressed ICAM1 upon stimulation with the proinflammatory cytokines TNFα, IL1ß and IFNγ. ICAM1 overexpression was induced in MCF7, MDA-MB-468 and SK-BR-3 cells regardless of hormone receptor status. Taken together, our findings show that ICAM1 is expressed in aggressive subtypes of breast cancer and its expression is inducible by well-known proinflammatory cytokines. ICAM1 may be an attractive molecular target for TNBC, but further investigations elucidating the role of ICAM1 in targeted therapies have to take into consideration selective subtypes of breast cancer.

Mesenchymal stem cells and T cells in the formation of Tertiary Lymphoid Structures in Lupus Nephritis.

Tertiary lymphoid structures (TLS) develop in the kidneys of lupus-prone mice and systemic lupus erythematosus (SLE) patients with lupus nephritis (LN). Here we investigated the presence of mesenchymal stem cells (MSCs) in the development of TLS in murine LN, as well as the role of human MSCs as lymphoid tissue organizer (LTo) cells on the activation of CD4+ T cells from three groups of donors including Healthy, SLE and LN patients. Mesenchymal stem like cells were detected within the pelvic wall and TLS in kidneys of lupus-prone mice. An increase in LTß, CXCL13, CCL19, VCAM1 and ICAM1 gene expressions were detected during the development of murine LN. Human MSCs stimulated with the pro-inflammatory cytokines TNF-α and IL-1ß significantly increased the expression of CCL19, VCAM1, ICAM1, TNF-α, and IL-1ß. Stimulated MSCs induced proliferation of CD4+ T cells, but an inhibitory effect was observed when in co-culture with non-stimulated MSCs. A contact dependent increase in Th2 and Th17 subsets were observed for T cells from the Healthy group after co-culture with stimulated MSCs. Our data suggest that tissue-specific or/and migratory MSCs could have pivotal roles as LTo cells in accelerating early inflammatory processes and initiating the formation of kidney specific TLS in chronic inflammatory conditions.

Selected reaction monitoring mass spectrometry of mastitis milk reveals pathogen-specific regulation of bovine host response proteins.

Mastitis is a major challenge to bovine health. The detection of sensitive markers for mastitis in dairy herds is of great demand. Suitable biomarkers should be measurable in milk and should report pathogen-specific changes at an early stage to support earlier diagnosis and more efficient treatment. However, the identification of sensitive biomarkers in milk has remained a challenge, in part due to their relatively low concentration in milk. In the present study, we used a selected reaction monitoring (SRM) mass spectrometry approach, which allowed the absolute quantitation of 13 host response proteins in milk for the first time. These proteins were measured over a 54-h period upon an in vivo challenge with cell wall components from either gram-negative (lipopolysaccharide from Escherichia coli; LPS) or gram-positive bacteria (peptidoglycan from Staphylococcus aureus; PGN). Whereas our data clearly demonstrate that all challenged animals have consistent upregulation of innate immune response proteins after both LPS and PGN challenge, the data also reveal clearly that LPS challenge unleashes faster and shows a more intense host response compared with PGN challenge. Biomarker candidates that may distinguish between gram-negative and gram-positive bacteria include α-2 macroglobulin, α-1 antitrypsin, haptoglobin, serum amyloid A3, cluster of differentiation 14, calgranulin B, cathepsin C, vanin-1, galectin 1, galectin 3, and IL-8. Our approach can support further studies of large cohorts of animals with natural occurring mastitis, to validate the relevance of these suggested biomarkers in dairy production.

Pilot randomized controlled trial of Tuning Relationships with Music: Intervention for parents with a trauma history and their adolescent.

For parents who have experienced childhood interpersonal trauma, the challenges of parenting an adolescent may trigger memories of abuse, intensifying conflict, resulting in negative cycles of relating and poorer responsiveness to emotions when parenting. This study examined whether Tuning Relationships with Music, a dyadic therapy for parents and adolescents, increased responsive parent-adolescent interactions and parent emotion coaching whilst reducing conflict and adolescent mental health difficulties. Twenty-six parent-adolescent dyads were recruited if parents had a trauma history and the dyad were currently having high levels of conflict. Dyads were randomly allocated into intervention or wait-list control and completed questionnaires and observation assessments at baseline and 4-month post-baseline follow-up. Those allocated to the intervention condition participated in 8 sessions of Tuning Relationships with Music. TRIAL REGISTRATION: ANZCTR: 12615000814572. Parents and adolescents reported significant reductions in conflict. Parents in the intervention condition were observed to significantly improve their nonverbal communication, emotional responsiveness and non-reactivity toward their adolescent. Although parents reported they were less dismissive and punitive, and more encouraging of their adolescent's emotions, and both parents and adolescents reported improvements in the adolescent's mental health, these were not statistically significant. Findings suggest Tuning Relationships with Music may assist parents with a history of childhood interpersonal trauma and their adolescent to reduce conflict and increase responsive ways of relating that may positively impact the young person's mental health. Future trials with a larger sample are warranted.